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The interaction between various components in the tumor environment plays a pivotal role in tumor development, migration and metastasis. While the 2-dimensional (2D) in vitro culture methods fail to assimilate the complexity of tumor microenvironment, the 3D multicellular tumor spheroids support co-culture conditions and allow a more physiologically relevant environment. We are working to establish such 3D model to investigate the molecular mechanisms regulating the interactions between human cancer cells such as melanoma or breast cancer, myeloid cells and T cells. Spheroids were developed using hanging drop technique with multiple cancer cell lines, dermal fibroblasts and immune cells. Preliminary experiments were carried out to determine the optimum conditions for spheroid formation. Spheroid formation occurred within 72 hours and their integrity was maintained throughout the experiments. Immunofluorescence analysis of spheroid cryosections showed a homogeneous distribution of fibroblasts in spheroids. Addition of purified blood CD14+ blood monocytes to the mixture of cancer cells and dermal fibroblasts enabled monocyte integration into spheroids. Adding melanoma specific CD8+ T cells to tumor spheroids formed by melanoma cell line (Me275), dermal fibroblasts and monocytes resulted in homogeneous infiltration and increased number of cell death. Addition of PBMCs to the mix of breast cancer cells and fibroblasts enabled the discrimination of spheroids into two groups, tumor spheroids with immune cells at the margin preferentially located at tumor margin and tumor spheroids with infiltrating immune cells. These preliminary observations resemble those from our in vivo models of humanized mice, where different tumor xenografts show different level of immune cell infiltration. Thus, our 3D model might allow the classification of tumor cells based on immune cell infiltration and could potentially be used to assess the distribution of immune infiltrates and the interaction of cancer cells with immune compartments. Citation Format: Elaheh Ahmadzadeh, Jan Martinek, Florentina Marches, Gabby Aucello, Chun I Yu, Karolina Palucka. Multicellular spheroids to dissect the interplay between human immune system and cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-04-02.

Introduction: Conventional 2D monolayer cell culture methods have distinct weaknesses in mimicking in vivo tumors as they ignore major phenotypic physical tumor microenvironment (TME) factors. 3D multicellular tumor spheroid (MTS) systems have been developed to study cancer cell growth in the presence of such TME factors, including cell-cell and cell-extracellular matrix (ECM) mechanical interactions. However, the development of analysis methodologies for these complex culture systems has considerably lagged. We hypothesize that a coupled experimental-computational framework using a microscopy image-driven biophysical mathematical model of MTS systems can accurately estimate phenotypic growth and biophysical TME properties, revealing significantly more mechanistic information than traditional morphometric analysis methods. Materials and Methods: MDA-MB-231 triple-negative breast cancer cells were used to generate spheroids. A single 3D MTS embedded in Collagen I ECM was used as a 3D culture tumor model system as they exhibit cell-cell and cell-ECM interactions that mimic in vivo tumors. Fluorescent fiducial microspheres were placed in the ECM to track displacement induced by the MTS. Time-lapse fluorescence microscopy images were taken of the MTS and microspheres for 72 hours on an automated fluorescent microscope every 12-hours. Images were compiled using a customized fully-automated tiling/stitching/image processing software in MATLAB. Time-lapse images of the MTS and microspheres are used in conjunction with subsequent mathematical modeling analysis for biophysical parameter estimation. We assess MTS using a mathematical model based on two coupled partial differential equations describing growth/motility and mechanical equilibrium in response to cellular-induced forces. Parameters describing cell proliferation rate (k), cell diffusion (D), and cellular mechanical forces (λ) are estimated based on fitting acquired microscopy images to the model via an iterative least-squares parameter estimation. Experiments and model-based analyses were performed in triplicate to define the reproducibility of biophysical parameter estimation. Results and Discussion: In this preliminary study, our modeling framework is able to estimate biophysical parameters of cell diffusion, proliferation rate, and cellular mechanical force in a 3D breast cancer cell culture system of invading MTS embedded in ECM with coefficients of variation across all parameters less than 15% on average. The model predictions accurately represent changes in cellular density and ECM deformation induced by cellular traction forces throughout the observed time series, demonstrating that this model is capable of representing MTS/ECM growth behavior and parameterizing the driving biophysical properties. Conclusions: These results indicate that our imaging-driven mathematical modeling framework for estimating biophysical model parameters has potential to reliably characterize 3D MTS growth and invasion. With reproducibility established, we plan to extend the method to investigate these phenotypic biophysical TME properties in a range of culture and treatment condition interventions. These biophysical properties could be utilized in developing anti-neoplastic drug sensitivity assays to improve cancer therapy by enabling patient-specific estimation/prediction of response to agents and dosing conditions. Acknowledgements: NIH-NCI K25CA204599 and P30CA012197 Citation Format: Haley Brooke Johnson, Emily E. Fannin, Alexandra Thomas, Jared A Weis. Image driven biophysical mathematical modeling of multicellular breast tumor spheroids [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-06-05.

Background: Breast cancer (BC) is a heterogeneous disease comprising different clinical, histopathological, and molecular subtypes. Hormone receptor-positive (HR+) advanced BC cancer, in particular the inflammatory BC (IBC) represents a significant clinical challenge because of the development of endocrine resistance during either adjuvant or metastatic treatment translating to poor outcome. We aim to develop unique PDX tumor models and 3D spheroid/organoid cultures that recapture advanced IBC using pleural effusions or/and circulating tumor cells (CTCs) as tumor resource and that can help understand the mechanism of IBC metastasis for new drug development. Methods: Five samples of pleural effusion-derived tumor cells or circulating tumor cells (CTCs) from three advanced IBC patients were obtained from Northwestern Memorial Hospital to establish IBC PDX tumor model in immunodeficient NSG female mice via subcutaneous or breast fat pad xenografting and develop the derived 3D organoid/spheroid cultures for anti-cancer drug evaluation. STR profiling against with original patient tumor DNA was conducted to confirm the tumor authentication. Results: Three developed IBC PDX tumor models (one HR+-IBC and two triple negative (TN)-IBC) were demonstrated by pathology to have highly heterogeneous characteristics and metastatic features similar to the original patient tumor. NSG mice xenografted with original TN-IBC pleural effusion, or derived spheroid cultures with varying passage numbers (6 passages) manifested different inflammatory clinical symptoms. Mice xenografted with one case of original TN-IBC pleural effusion cells (~40% of tumor-associated macrophages and ~60% of tumor cells) developed palpable tumors and was shown to have the most IBC-like characteristics including reddish skin and ulcerative lesions. They were sacrificed 2 months after xenograft due to declining health. Mice xenografted with >6 passages or Citation Format: Wenan Qiang, Zhangfeng Zhong, Pamela Monahan, Kristy Tse, Youbin Zhang, Qiang Zhang, Lorenzo Gerratana, Andrew Davis, Demirkan Gursel, Reiner Bleher, Jian-Jun Wei, Charles D. James, Thomas V. O’Halloran, Massimo Cristofanilli. Development of patient-derived xenograft tumor models and 3D spheroid culture from advanced hormone receptor-positive inflammatory breast cancer patients for evaluation of new therapeutics [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-06.

Ovarian cancer is the deadliest gynecological cancer in women, with a survival rate of less than 30% when the cancer has spread throughout the peritoneal cavity. Aggregation of cancer cells increases their viability and metastatic potential; however, there are limited studies that correlate these functional changes to specific phenotypic alterations. In this study, we investigated how changes in media composition affects the metastatic potential of ovarian cancer cells. This was conducted by using aggregated ovarian cancer cells incubated in different media conditions supplemented with different levels of glucose, glutamine and serum. Additionally, we identified the adherence capacity through examining secretions and expression of extracellular matrix proteins as well as mitochondrial localization and quality control protein content that could support the viability of these aggregates. We determined that spheroid formation requires more glutamine and limited serum and glucose to allow for successful aggregation. Additionally, this media composition supports a more proliferative phenotype that closely resembles the high glucose controls. Low glucose and hypoxia also limit the ability of spheroids to adhere due to limited expression and secretion of collagen type I and fibronectin proteins. Further, we find that mitochondrial localization is promptly more affiliated with the adherence site of spheroids incubated in high glucose, high oxygen media in comparison to low glucose, hypoxic spheroids where mitochondrial localization is heavily dispersed. Finally, we find that mitophagy and mitobiogenesis are codependent in regulating mitochondrial content to support viability in cancer spheroids in peritoneal media conditions. Altogether, these data indicate that ovarian cancer spheroids adapt to non-permissive conditions through limiting their adherence capacity and upregulating mitochondrial quality control functions to support viability during dissemination. Citation Format: Joseph Grieco, Stephanie Edwards Compton, James Smyth, Eva Schmelz. Dysregulated metastatic capacity of ovarian cancer spheroids in response to physiologically relevant media conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2932.

DNA-damaging agents are important anticancer therapeutics but are rarely curative. The current study combines the DNA-damaging agents trabectedin and temozolomide with varied DNA repair inhibitors to identify combinations that increase malignant cell kill and therefore might improve therapeutic benefit. Twenty-seven tumor cell lines focused on sarcoma and small cell lung cancer (https://pdmr.cancer.gov/models/database.htm) were grown as complex spheroids containing 60% tumor cells, 25% endothelial cells and 15% mesenchymal stem cells as an in vitro model for human solid tumors. After 3 days of growth, the complex spheroids were screened using a 3-log concentration range with the highest concentration at the clinical Cmax for each agent. The spheroids were treated with temozolomide or trabectedin as single agents or in combination with a PARP inhibitor (olaparib or talazoparib), an ATM inhibitor (AZD-1390), an ATR inhibitor (berzosertib (M6620) or BAY-1895344), a DNA-PK inhibitor (nedisertib (M3814) or VX984) or a BET bromodomain inhibitor (birabresib). Seven days after drug exposure, ATP was measured with CellTiter-Glo 3D. Olaparib or talazoparib produced additive to greater than additive cell killing in combination with either trabectedin or temozolomide. Whereas cell killing with temozolomide alone was modest, greater than 1 log of cell killing was achieved in combination with either olaparib or talazoparib. The ATM inhibitor AZD-1390 in combination with trabectedin showed greater than additive responses in most models and approximately 3 logs of cell killing in sarcoma and small cell lung cancer models at higher trabectedin concentrations. The ATR inhibitors BAY-1895344 and berzosertib had additive and greater than additive responses in combination with temozolomide or trabectedin among the small cell lung cancer models tested and produced greater than 2 logs of cell kill in many models when combined with temozolomide. When combined with trabectedin, the DNA-PK inhibitor VX-984 had additive responses, whereas nedisertib had greater than additive responses in most models. The BET inhibitor birabresib had greater than additive responses in many models when combined with trabectedin and additive responses with temozolomide. Overall, greater than additive responses were observed more frequently with trabectedin combinations containing the ATM inhibitor AZD-1390 or the DNA-PK inhibitor nedisertib, as well as temozolomide combinations with the PARP inhibitors olaparib or talazoparib or the ATR inhibitor BAY-1895344, particularly among sarcoma and small cell lung cancer models. These results might inform the prioritization of drug combinations for further investigation. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E. Citation Format: Nathan P. Coussens, Ralph E. Parchment, Jeffrey A. Moscow, L. Austin Doyle, René Delosh, Julie Laudeman, Russell Reinhart, Chad Ogle, Thomas Silvers, Thomas S. Dexheimer, Joel Morris, Beverly A. Teicher, James H. Doroshow. Combination screening of DNA damaging agents temozolomide and trabectedin with inhibitors of DNA repair using a complex spheroid model with a panel of patient-derived and established tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1072.

Colorectal cancer (CRC) is one of the most frequent cancers accounting for 12% of all deaths from cancer in Europe (2016). Furthermore, CRC patients are prone to develop metastases and have a poor prognosis underpinning the need for new targeted and broadly applicable therapeutic strategies. Tumor cell secretion contributes to hallmarks of cancer e.g. hyperproliferation, evasion of growth suppression and resistance to cell death. Dysregulated secretion is thus a driver of cancer progression and therefore holds promise as a general therapeutic target for the treatment of cancers. The overall aim of this study is to gain a quantitative understanding of the cancer secretome on a systems level to elucidate the influence of the microenvironment on selected players and vice versa the influence of the secretome in vitro and later on in vivo. In a first attempt to elucidate the role of secreted factors in colon cancer progression, we cultured 20 CRC cell lines (five PDX derived and 15 commercial lines) in 3D spheroid cultures and compared the secreted cytokine profile with the 2D culture conditions of the same CRC cell line panel. We analyzed 45 cytokines in duplicates by using a human specific magnetic bead-based assay. Of the 45 cytokines, 41 were detected at relevant levels in at least one cell line in either 2D or 3D. Four cytokines were exclusively secreted in a 3D setting: IFN alpha (tumorigenic), IL-17A (pro-inflammatory), TNF alpha (pro-inflammatory and tumorigenic) and VEGF-D (pro-metastatic). Every cell line displayed a distinct cytokine profile depending on the culture conditions. 13 lines showed more cytokines upregulated in 3D, four lines displayed more cytokines upregulated in 2D and 3 lines exhibited an equal number of cytokines up-regulated in the two culture conditions. Eleven cytokines were specifically higher secreted when the cells were cultured in 3D across the majority of the 20 cell lines. Most of them with pro-inflammatory, pro-angiogenic and tumorigenic capabilities. A cluster analysis helped to identify four clusters, two comprising mainly 3D cultured cell line and two primarily consisting of 2D datasets. The main discriminators for these four clusters were the secretion levels of VEGF-A, IL-1Ralpha and HGF. The thorough characterization of the secretome in different in vitro conditions revealed the influence of the tumor microenvironment on basic tumor characteristics like proliferation and invasiveness. Based on this data set we gained a quantitative understanding of the cancer secretome on a systems level. From here on we will further elucidate the influence of the microenvironment on selected players of the secretome and vice versa the influence of the secretome on tumor biology in vitro and later on in vivo. Citation Format: Jacqueline Bersano, Katharina Schaich, Kanstantsin Lashuk, Julia Schüler. Culturing colorectal cancer cell lines in 3D spheroid cultures drives their cytokine profile towards a pro-inflammatory, tumorigenic and highly metastatic phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2009.

Tumor permeability is a critical determinant of drug delivery and sensitivity, but systematic methods to identify factors that perform permeability barrier functions in the tumor microenvironment are not yet available. Multicellular tumor spheroids have become tractable in vitro models to study the impact of a three-dimensional (3D) environment on cellular behavior. In this study, we characterized the spheroid-forming potential of cancer cells and correlated the resulting spheroid morphologies with genetic information to identify conserved cellular processes associated with spheroid structure. Spheroids generated from 100 different cancer cell lines were classified into four distinct groups based on morphology. In particular, round and compact spheroids exhibited highly hypoxic inner cores and permeability barriers against anticancer drugs. Through systematic and correlative analysis, we reveal JAK–STAT signaling as one of the signature pathways activated in round spheroids. Accordingly, STAT3 inhibition in spheroids generated from the established cancer cells and primary glioblastoma patient–derived cells altered the rounded morphology and increased drug sensitivity. Furthermore, combined administration of the STAT3 inhibitor and 5-fluorouracil to a mouse xenograft model markedly reduced tumor growth compared with monotherapy. Collectively, our findings demonstrate the ability to integrate 3D culture and genetic profiling to determine the factors underlying the integrity of the permeability barrier in the tumor microenvironment, and may help to identify and exploit novel mechanisms of drug resistance. Cancer Res; 76(5); 1044–54. ©2015 AACR.

Background: Leucine-rich repeat containing 15 (LRRC15), a transmembrane protein, has been found to be highly expressed in stromal cells in multiple solid tumors especially tumors of mesenchymal origin (e.g. sarcoma, glioma, melanoma) [1]. LRRC15 has been nominated as a candidate target for immunotherapy with antibody drug conjugates in cancers, notably osteosarcomas that strongly express this surface marker. In addition, LRRC15 has been found to be significantly upregulated during osteogenic induction [2] and TGFβ induction in mesenchymal stem cells [3]. The present study explores the functional role of LRRC15 in osteosarcoma (OS) cells. Methods: To delineate the role of LRRC15 in OS cells, we generated inducible shRNA knockdown derivatives of high LRRC15 expressing OS cells (SAOS2/HuO9) and induced LRRC15 in low expressing OS cells (HOS & U2OS). We explored various cell-cell and cell-ECM interactions using invasion, migration, adhesion, 3D colony formation (Matrigel), spheroid formation and spheroid migration assays. Results: LRRC15 expression and knockdown in different OS cells were confirmed by transcriptional, flow cytometric and western blot analysis We found that expression of LRRC15 varies significantly among osteosarcoma (OS) cell-lines. Here we report that inducible knockdown of LRRC15 in high expressing OS cells significantly reduces collagen type 1 based adhesion (using extracellular matrix screening array), migration, invasion and 3D colony formation using Matrigel. Enlarged colonies were observed in TGFβ treated HOS cells in Matrigel. Spheroids were formed using ultra low attachment plates with SaOS2/HuO9/HOS & U2OS cells. Knockdown of LRRC15 significantly affects the spheroid compaction in high LRRC15 expressing cells (SaOS2 & HuO9). A spheroid-based migration assay on collagen type 1 coated plates revealed significantly reduced migratory phenotype in LRRC15-knockdown SAOS2 cells. Conclusion: Taken together, these results suggest that LRRC15 plays a key role in cell-cell and cell-ECM interactions of OS cells providing insights relevant to biological function of LRRC15 in osteosarcoma cells. 1. Satoh, K., M. Hata, and H. Yokota, A novel member of the leucine-rich repeat superfamily induced in rat astrocytes by beta-amyloid. Biochem Biophys Res Commun, 2002. 290(2): p. 756-62. 2. Wang, Y., et al., LRRC15 promotes osteogenic differentiation of mesenchymal stem cells by modulating p65 cytoplasmic/nuclear translocation. Stem Cell Res Ther, 2018. 9(1): p. 65. 3. Purcell, J.W., et al., LRRC15 Is a Novel Mesenchymal Protein and Stromal Target for Antibody-Drug Conjugates. Cancer Res, 2018. 78(14): p. 4059-4072. Citation Format: Sanjit Mukherjee, Konrad Huppi, Robert L. Walker, Jack Zhu, Marbin Pineda, James W. Purcell, Lee J. Helman, Paul S. Meltzer. Role of LRRC15, a candidate immunotherapy target, in cell adhesion, migration and spheroid formation in osteosarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2635.

Introduction: Progression into metastatic disease is a critical step in most malignancies. Cancer cells may enter circulation very early (circulating tumor cells or CTCs), but distant metastases are disseminated only by cancer stem cells (CSCs) that survive circulation. CSCs are known to form cell clusters in vitro (spheroids/tumorspheres). We reported that tumorspheres also circulate in blood in vivo. We found that they carry cancer stem cells and strongly correlate with metastatic disease. We collected tumorspheres from blood by filtration. This project aims to test the metastatic potential of tumorspheres by developing an assay using in vivo circulation models. The goal is to establish a novel prognostic test for metastatic predisposition in cancer patients. Methods: To develop an in vivo assay for metastasis we use the chorioallantoic circulation in the chicken embryo, with several advantages: easy access to circulation, lack of immune response, rapid turnaround time (5 to 10 days) and inexpensive and simple technique. For clinical applications we introduced three technical innovations: (i) to make fertilized chicken embryos easily available we worked out a method to store viable embryos beyond 30 days; (ii) we developed a new coverglass chamber technique for micromanipulations; and (iii) for microscopic analysis new over-drained chorioallantois preparations can accommodate high resolution objectives. To test tumorspheres in embryonic circulation we used both highly metastatic (e.g. MDA-MB-436) and non-metastatic (e.g. MCF7) cell lines with GFP label, DiI membrane dye and CFDA-SA dye. For molecular studies DNA was extracted 5 days after cancer spheroid implantation and used qPCR to detect human sequences. Results: In traditional storage few embryos survive beyond 6 days. As fertilized eggs carry pluripotent stem cells, we optimized temperature, gas and humidity combinations to match known stem cell optimums. By suppressing metabolism, blocking embryo adhesion and controlling gas and humidity exchange we extended viable embryo storage beyond 30 days. We modified the traditional window method by forming visually clear microscopic coverglass chambers, and the new over-draining approach lowered the chorioallantois membranes and allowed high resolution imaging. The results showed invasive and hematogenic dissemination of the cancer spheroids. Molecular studies of human markers detected metastatic progression and generated quantitative data. Conclusions: We optimized the in vivo embryonic metastasis assay. We show that it has the potential for a sensitive and quantitative method to diagnose metastatic predisposition in cancer patients. The molecular approach (DNA extraction, PCR markers) is easily up-scalable, the instrumentation is available in most hospitals and offers cost effective and critical early diagnosis of cancer progression. Citation Format: Peter Geck, James Kubilus, Andrew Makarovskiy, Kathleen Marinelli, Christine Kyritsis, Viktoria Denes. The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2714.

Gastroenteropancreatic neuroendocrine tumors (GEP-NET) are the second most common form of gastrointestinal cancer, and account for roughly 60% of all neuroendocrine tumors. Unfortunately, no animal models or cell lines exist that reflect the in vivo characteristics of human GEP-NETs. Specifically, human GEP-NETs have low proliferation rates that animal models and cell lines fail to capture. Therefore, drug testing for GEP-NETs often begins in patients without rigorous pre-clinical validation. There is a need to predict the most effective treatments prior to clinical trials for GEP-NET patients, to increase treatment efficacy and reduce toxicities. Here we show that a three-dimensional spheroid model of primary GEP-NET patient tumors is representative of the original patient tumors including histological features and markers of proliferation (Ki67). Furthermore, this study uses the spheroid model to test a novel drug combination on GEP-NET spheroids, specifically ABT263, a Bcl-2 family inhibitor, and everolimus, a standard GEP-NET treatment that inhibits mTOR. Previous studies have shown that ABT263 induces apoptosis in cancer cells when used in combination with everolimus, making this a promising drug combination for GEP-NET patients. Treatment response to ABT263, everolimus, and the combination, was tested in three-dimensional spheroids derived from seven primary GEP-NET patients, using two-photon optical metabolic imaging (OMI). OMI captures the fluorescence lifetime (τm) and intensity of metabolic cofactors NAD(P)H and FAD. These metrics can be combined into a single number, the OMI index, which was developed to separate responsive and non-responsive cells. The OMI index is the linear combination of the norm-centered redox ratio (NAD(P)H intensity / FAD intensity) + NAD(P)H τm - FAD τm. The OMI index also provides single cell response information from intact spheroids, so that heterogeneity in response can be quantified. Altogether, two-photon OMI in primary patient GEP-NET spheroids provides a novel tool to measure metabolic response to drug treatment. At 72 hours post-treatment, all patients except two showed a decrease in OMI index, corresponding with a response, to the combination treatment. Heterogeneous response was resolved with two distinct subpopulations of cell metabolism in one of the nonresponsive patients. No changes in spheroid diameter were observed with any treatment, due to the slow growth rate of the GEP-NET tumors and spheroids, further highlighting a need for molecular measures of drug response in these samples. We have shown that representative GEP-NET models can be generated in patient-derived tumor spheroids. Additionally, ABT263 plus everolimus is a promising combination treatment for GEP-NETs. Overall, this work shows that patient-derived tumor spheroids can be established from GEP-NETs, and that OMI provides single-cell metabolic measurements of drug response to guide drug development for patients. Citation Format: Amani Gillette, Christopher Babiarz, Cheri Pasch, Dustin Deming, Melissa Skala. Neuroendocrine tumor spheroid growth and drug response assessment using optical metabolic imaging [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2782.

Engineering tumor-homing bacteria to locally deliver therapeutics is an emerging approach for next-generation living therapy for cancer. However, the lack of physiologically-relevant in vitro testing environments to rapidly and quantitatively characterize complex multicellular interactions of bacteria and cancer cells is a major bottleneck for clinical translation. We recently developed a 3D multicellular coculture platform that grow various bacteria within the necrotic core of multicellular spheroids, recapitulating selective bacterial growth in solid tumor in vivo. Tumor spheroids were generated from CT26 colorectal cell lines in a round-bottom low-adhesion 96 well plate. Bacteria were inoculated into the media containing tumor spheroids and gentamicin, a poorly diffusible antimicrobial agent, were added to confine bacterial growth to spheroid cores. Time-lapse microscopy was utilized to track growth dynamics and spatial profiles. To validate efficacy, mice with hind flank tumors were treated with engineered bacteria and measured for the therapeutic effect on tumor size. The multicellular coculture platform allowed for parallel and continuous culture of bacteria in tumor spheroids. Notably, bacteria stably grew within the spheroid necrotic cores, representing tumor colonization in animals and patients. Leveraging the high throughput nature of the platform, we screened a library of ten cytotoxic molecules delivered by clinically relevant tumor-homing S. typhimurium. We validated similarities in efficacies using a syngeneic mouse model and identified novel therapeutic candidates that reduced tumor growth significantly. Additionally, we utilized the tumor spheroid to construct bacterial biosensors. As tumor microenvironment such as oxygen, pH, and metabolite gradients are recapitulated in the 3D spheroid setting, we tested hypoxia, acid and lactate sensors and confirmed their activity in tumor environment. Constructing an AND-gate logic circuit for bacterial growth using synthetic biology tools, we rapidly tested the bacterial containment capability to the spheroid cores. This strain demonstrated a 100-fold reduction in off-target colonization compared to the wildtype strain in an animal model. Rapid and quantitative characterization of dynamical bacteria-cancer cell interactions can predict therapeutic performance more accurately. We hope this scalable approach serves to accelerate future clinical development of bacterial applications for cancer therapy. Citation Format: Tetsuhiro Harimoto, Zakary Singer, Oscar Velazquez, Joanna Zhang, Samuel Castro, Taylor Hinchliffe, William Mather, Tal Danino. 3D multicellular coculture platform enables rapid engineering of tumor-homing bacteria [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6317.

Epithelial to mesenchymal transition (EMT) of cancer cells during tumor progression has been implicated in tumor initiation, growth, invasion, metastasis, colonization and resistance to therapy. In building a successful in vitro tumor model, it is critical to recapitulate in vivo cancer cell heterogeneity inclusive of both cell types and transition state present. This study investigates the EMT hybrid states of mouse triple negative breast cancer cells during ex vivo culturing across multiple methodologies. Mouse mammary carcinoma (4T1) tumors were harvested from syngeneic, orthotopic mice and subsequently cultured using various methods over the course of 12 days. Ex vivo cultures were first assessed for retention of tumor cellular heterogeneity using endogenous Thy1.1 (CD90.1) expression via flow cytometry to distinguish 4T1 cancer cells from stromal derived cells. Cancer cell populations rapidly became the majority of cells in culture. After 3 days of ex vivo culture, we found the cancer cell population to have significantly expanded ~2-3 fold compared to the original tumor population, while other stromal cellular subtypes decreased. Additionally, monolayer culture of ex vivo cells contained significantly more cancer cells relative to the spheroid or tumoroid (tumor fragment) culturing techniques. Cancer cells from each culturing technique were also evaluated for loss of Epcam expression and mesenchymal cell fate in reference to the initial EMT distribution at time of isolation. 4T1 cells cast into hydrogel retained high proportions of cells undergoing EMT, evidenced by fewer cells expressing Epcam. However, all other in vitro conditions favored the expansion of cancer cells in an epithelial state compared to in vivo tumors. Subsequent analysis of EMT populations for transitional hybrid states based on CD51, CD61, and CD106 expression was conducted using flow cytometric analysis. We found that in vitro culturing promoted mesenchymal character and this selection was time dependent. Additionally, we found that spheroids cultured in hydrogel for 7 days most closely resembled early hybrid EMT cell states ratios found in vivo Future research aims to optimize ex vivo culturing methodologies to best retain cancer cell characteristics and behavior of tumors obtained from human biopsies. Alterations in growth conditions and identifying critical stromal populations of interest will be critical in development of optimized preclinical ex vivo tumor culture models for drug discovery or personalized treatment. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM: LLNL-ABS-798441 Citation Format: Nicholas R. Hum, Kelly A. Martin, Elizabeth Wheeler, Matthew A. Coleman, Gabriela G. Loots. Epithelial-mesenchymal hybrid population changes from monolayer, spheroid, and tumoroid ex vivo culture of syngeneic murine mammary tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2810.

Reproducible, scalable, and patient-specific 3D models of the tumor microenvironment are required to study tumor growth, metastasis and dormancy. In this study, patient-derived cancer cells, mesenchymal stem cells (MSCs), and optimization of the oxygen level and extracellular matrix (ECM) were incorporated in the development of a spheroid system using 5 cancer cell models (pancreatic [Panc], lung adenocarcinoma [LA], colon adenocarcinoma [CA], endometrioid ovarian [EO] and high-grade serous carcinoma [HGSC]). The cell models were isolated and scaled up using a feeder-free culture medium in either ambient oxygen (approximately 21% O2) or hypoxic (5% O2) conditions, depending on the individual model requirements. Cell migration was assessed via monolayer scratch assays, and the relative rates were compared to the T stage of the tumor from which the models were derived. To assess spheroid formation, the 5 models were cultured in ECM-containing medium. The Panc and HGSC spheroids were also generated with the addition of MSCs at ratios of 1-100 MSCs per 100 cancer cells. The relative rates of migration of the cell models in ambient oxygen (CA > EO ≈ Panc > HGSC ≈ LA) differed significantly from that in 5% O2 (CA = Panc = HGSC > EO > LA). Interestingly, the T stage of the original tumors (CA = Panc = HGSC > LA > EO) correlated more closely to the relative rate of cell migration when the cell models were cultured in 5% compared to 21% O2. When cultured in a 3D (spheroid) format containing ECM, the Panc and EO models demonstrated increased invasion into the surrounding ECM compared to the LA, HGSC, and CA models. The addition of MSCs into the LA model dramatically increased the invasive phenotype of the LA model. A seeding ratio of 1:100 MSC:LA cells enabled spheroid formation and invasion into the ECM, previously unobserved for the LA model in this format. The Panc model became extremely aggressive with respect to invasion into the ECM, at a ratio as low as 1:100 MSCs to cancer cells. In pilot studies with the Panc and HGSC models, spheroid size and invasion into the ECM increased upon adding the MSCs. Spheroid diameter increased by approximately 30%, and there was a notable increase in invasiveness of the already mobile and invasive Panc cancer cells into the surrounding ECM. We present here an efficient, patient-specific, spheroid model system representing dormant and metastatic tumor states that is suitable for studying antitumor drug response, personalized therapy, and disease mechanisms. The relative rates of cell migration positively correlated with the T stage of the original tumors in the more physiologically-relevant oxygen condition (5%), underscoring the need to optimize this parameter during model development. Continued optimization will include incorporating tissue-specific ECMs from lung, liver and pancreas, and assessing the robustness and scalability of these models for high-throughput drug screens. Citation Format: Dmitry Shvartsman, Amy Pepicelli, Liam Deems, David Deems, Elin S. Agoston, Amit Shahar. A metastatic phenotype is reproduced in spheroids containing patient-specific cancer cells and mesenchymal stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 337.

While 3D cell cultures have been used as suitable representatives of in vivo conditions compared to 2D systems, scalability and flexibility in designing such platforms has been a major challenge1. Micron-sized 3D culture platforms offer the possibility of high throughput analysis, however major challenges exist with respect to their overall design. Technical obstacles include difficulties in supporting multiple cell types (e.g. co-cultures of cancer associated fibroblasts (CAFs) and tumor cells) as well as enabling monitoring of important parameters, such as proliferation and metabolism, in real time. Our 3D co-culture systems are based on pancreatic ductal adenocarcinoma (PDAC) spheroid models in microgels, generated by coaxial droplet microfluidics. This allows for co-culturing of cancer cells with the supporting cell types that constitute the major portion of the actual tumor. Initial optimization experiments utilized co-culture of commercial pancreatic cancer cell lines such, as L3.6pl cells, and stromal CAFs. In addition to optimizing the 3D microcultures using commercial PDAC cell lines, we are also modifying the system to take advantage of patient-matched CAFs and conditionally reprogrammed (CR) primary PDAC cells. Both drug sensitive and isogenic nab-paclitaxel resistant PDAC CR cells will be used, as recently published2. Our preliminary data showed that our platform supports rapid generation of numerous identical spheroids inside the microgel. Using microscopy under different treatment conditions (e.g the presence/absence of drugs) we can quantify spheroid growth. In addition, spatio-temporal mapping of the microenvironmental parameters such as dissolved O2, pH and K+ during growth is performed by time lapse fluorescence microscopy using fluorescent silica microparticles embedded in the matrix. This process will yield an in-depth understanding of the kinetics of growth and associated biochemical and metabolic activity for each cell type (e.g. CAF and PDAC) under different treatment conditions. Our T3D platform offers a rapid, quantifiable assessment of the behavior of patient-derived cells in providing additional details related to the changing microenvironment and may advance our understanding of treatment response and failures. The research leading to these results received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 759959, ERC-StG “INTERCELLMED”). Reference (1) Nunes et al. Biotechnol. Bioeng. 2019, 116 (1), 206-226. (2) Parasido et al. Mol. Cancer Res. 2019, 17 (9), 1815-1827. Citation Format: Anil Chandra, Saumya Prasad, Francesco Alemanno, Adriano Barra, Enza Lonardo, Erika Parasido, Christopher Albanese, Loretta L. del Mercato. Microgel-based in vitro tumoroid platform for real time assessment of drug sensitivity and resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2967.

Breast cancer (BC) tumors originate in epithelial cells and rank second as a cause of cancer-related death in women (in 2020, ~270,000 women will be diagnosed, and 42,000 women will die from this disease in the USA). While important progress has been made on new treatments there is an urgent need to discover new therapeutics. We aim to discover therapeutics that elicits differentiation of BC tumor cells towards a phenotype that ceases proliferation and/or becomes more sensitive to therapeutic drugs. Also, since current high-throughput breast cancer drug discovery screens use conventional 2D cell cultures that do not fully represent the complex microenvironments of tumors in patients and often fail to predict clinical efficacy we are using 3D spheroids to better represent patient tumors. To identify potential therapeutic agents, we will quantify effects of test compounds on epigenetic markers (histone methylation/acetylation) within the nuclei of the tumor cells utilizing the Microscopic Imaging of Epigenetic Landscape (MIEL) assay developed by Alexey Terskikh's laboratory at SBP. The changes in epigenetics occur more rapidly than phenotypic changes resulting from differentiation which enables more rapid screening to identify potential therapeutic agents. Here we show that treating 2D cell cultures of the breast cancer cell line MCF7 with Tamoxifen, DPPE or E2 elicit changes in the epigenetic landscape. These epigenetic changes concur with increase in lipid droplet formation (a feature that is linked to milk-producing breast epithelium), and demonstrates that MIEL can be used as a readout of differentiation status. We are developing protocols to image breast cancer 3D spheroids on Vala Sciences' IC200 Structured Illumination Microscopy (IC200-SIM™ workstation). The SIM™ uses a multi-million array of micro-mirrors to achieve confocal resolution with rapid image acquisition, suitable for high throughput screening of spheroids cultured in the 96- or 384-well format. We envision that when developed, the MIEL-Breast Cancer Differentiation (MIEL-BCD) assay will be used to screen large chemical libraries (e.g., libraries with 100,000 different chemical entities) to identify compounds that promote differentiation of breast cancer cells to non-tumorigenic cells. Citation Format: Christine Gjerdrum Rines, Kara Gordon, Ranor Basa, Alyson Smith, Robert Oshima, Chen Farhy, Alexey Terskikh, Patrick McDonough, Jeffrey Price. A human breast cancer 3D spheroid platform for microscopy based high throughput screening [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-180.

Tumor spheroids, organoids, and cell culture models demand defined cell-cell and cell-matrix interactions, ideally with specific cell attachment on adhesion sites for multicellular spheroids or single cells. Our bioinert based micropatterning technology allows for the creation of adhesion spots while the surrounding passivated area is biologically inert, not interfering with the assay.We use this micro-patterning of adhesion ligands on highly passivated surfaces to generate homogenously distributed spheroids within a microfluidic channel system which can be perfused. Spheroids can either be directly generated by a simple injection of a large number of cells and a self-sorting process or by the addition of pre-formed spheroids, which will bind to the patterned adhesion sites. Perfusion of the spheroid chip enables the homogenous supply of all spheroids with nutrients and oxygen, and also the application of a defined shear stress, metabolite analysis together with toxicological screenings or even co-culture of multiple spheroid types. Applying a flow increases the overall spheroid growth, but condenses the cell aggregate, as well as influences the drug triggered protein expression response towards a more in vivo-like reaction.We also use this technique to generate arrays of homogenously distributed single cancer cells to evaluate cytotoxic T cell activity on a single cell level. By optical analysis combined with machine-learning based image processing of cell / spheroid arrays we obtained efficient label-free analysis of each cancer cell - T cell interaction over time. Citation Format: Elias Horn, Miriam Balles, Angelika J. Fischbeck, Jan Schwarz, Dane A. Thyssen, David W. Drell, Elfriede Noessner, Roman Zantl. Micropatterning supported method for cancer spheroid assays under flow and high throughput CAR-T cell potency assay with single cell resolution [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 314.

High-throughput single-cell RNA-seq (scRNA-seq) is used to describe complex tissues by characterizing transcriptional states of individual cells. Defining a cell's position, both in regard to tissue margins and its social context, is essential for understanding the intrinsic and extrinsic variables that effect the transcriptional identity of individual cells. Conventional high-throughput scRNA-seq assays, however, decouple cells from their original locations within tissues. In situ hybridization readouts of gene expression and in situ sequencing preserve spatial information in tissues, but currently have a lower total read threshold than NextGen sequencing – imposing a restriction on either cell throughput or transcriptional breadth. Combining the above methods or using regional barcodes to define 2D positions have spatially-reconstructed tissue regions but are seldom employed in unspecialized laboratories. We describe SEgmentation by Exogenous Perfusion (SEEP), a rapid, integrated, method for providing 3D spatial-segmentation to scRNA-seq data. Tissues are divided into layers based on accessibility of a fluorescent dye allowing sorted cells to be characterized by transcriptomic and regional identity. We use SEEP to explore how the transcriptional states of cells in high-grade serous ovarian cancer vary with respect to intratumoral position. We describe an integrated method for correlating 3D radial-spatial cell positions with scRNA-seq data. By employing a basic stain-and-sort method using an off-the-shelf live-dead stain, we are able to correlate transcriptional profiles with radial-spatial positions in both radially symmetric tissues (e.g., spheroids, organoids, spherical tumor masses) and linear tissue samples (e.g., punch biopsies). SEEP uses an imaging-based calibration step to inform the parameters of a FACS and sequencing based measurement step. Across spheroid, organoid, and PDX models of high-grade serous ovarian cancer, we find common positional transcriptomic profiles, ranging from single-gene proclivities to multi-gene-set enrichments across tissue layers. Citation Format: David B. Morse, Michele Ceribelli, Joachim De Jonghe, Aleksandra Michalowski, Christoph Muus, Maria Vias, Samantha Boyle, David A. Weitz, James Brenton, Jason D. Buenrostro, Craig Thomas, Tuomas P. Knowles. Spatially-segmented single-cell transcriptomics by diffusional accessibility to a small-molecule dye [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5879.

Purpose of this study is to explore, in vivo and within tridimensional (3D) models, a novel CAR- based adoptive immunotherapy against incurable soft tissue sarcomas (STS). The strategy focuses on Cytokine-Induced Killer (CIK) lymphocytes redirected against the Chondroitin Sulfate Proteoglycan 4 (CSPG4) target, associated with tumorigenesis, tumor aggressiveness and negative clinical outcome. CIK are ex vivo expanded T-NK lymphocytes endowed with intrinsic HLA-independent antitumor activity. Experimental procedures. The experimental platform is based on patient-derived CAR.CIK and multiple histotypes of STS. CAR.CIK were generated by transduction of PBMC with a retroviral vector encoding for a 2nd generation anti-CSPG4 CAR with 4-1BB costimulation. 3D in vitro essays were based on STS spheroids generated in ultralow attachment conditions. In vivo experiments included 3 different STS xenograft models (fibrosarcoma, leiomyosarcoma, UPS). Results. CAR.CIK were efficiently generated by STS patients (n=5). Mean expression of anti-CSPG4 was 44%±6.2, rates of ex vivo expansion (29 fold, range 27-348) and phenotype (CD8: 65%±4; CD56: 38%±6, NKG2D: 66%±7) were comparable with unmodified controls (NTD.CIK.). The CSPG4 target resulted expressed in 16/17 STS (leiomyosarcoma n=2, fibrosarcoma n=1, UPS n=6, GIST n=5, liposarcoma n=3), with variable membrane density per cell (300±47). CAR.CIK efficiently killed all STS in vitro regardless of their histotype. Mean STS-specific killing by CAR.CIK was significantly higher compared with NTD.CIK (E:T 1:1: 71% vs 34%, p Conclusions. We report the intense activity of anti-CSPG4 CAR.CIK against multiple histotypes of currently incurable STS. CIK lymphocytes, considering their intrinsic antitumor activity, may be a favorable platform for the translation of CAR-based strategies against solid tumors. Our findings support anti-CSPG4 CAR.CIK as a promising therapeutic strategy warranting clinical exploration in the challenging field of advanced STS. Citation Format: Chiara Donini, Valeria Leuci, Ramona Rotolo, Giovanni Grignani, Giulia Mesiano, Erika Fiorino, Loretta Gammaitoni, Lorenzo D'Ambrosio, Soldano Ferrone, Massimo Aglietta, Gianpietro Dotti, Dario Sangiolo. Anti-CSPG4 CAR.CIK lymphocytes are effective against advanced sarcomas in 3D spheroid and xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 885.

Although cancer stem cells (CSC) are thought to be responsible for tumor recurrence and resistance to chemotherapy, CSC-related research and drug development have been hampered by the limited supply of diverse, patient-derived CSC. Here, we present a functional polymer thin film (PTF) platform that promotes conversion of cancer cells to highly tumorigenic three-dimensional (3D) spheroids without the use of biochemical or genetic manipulations. Culturing various human cancer cells on the specific PTF, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4), gave rise to numerous multicellular tumor spheroids within 24 hours with high efficiency and reproducibility. Cancer cells in the resulting spheroids showed a significant increase in the expression of CSC-associated genes and acquired increased drug resistance compared with two-dimensional monolayer-cultured controls. These spheroids also exhibited enhanced xenograft tumor-forming ability and metastatic capacity in nude mice. By enabling the generation of tumorigenic spheroids from diverse cancer cells, the surface platform described here harbors the potential to contribute to CSC-related basic research and drug development. Significance: A new cell culture technology enables highly tumorigenic 3D spheroids to be easily generated from various cancer cell sources in the common laboratory.

Alternative treatment schedules, such as low-dose, high-frequency (metronomic; METRO) of some chemotherapeutics enhance efficacy and reduce toxicity in some cancers when compared to chemotherapy given conventionally (CONV). Previously, we demonstrated that low-dose, prolonged exposure to topotecan (30-day infusion 0.1 mg/kg/day via Alzet pump) improved survival and reduced tumor volume ~120%, compared to topotecan given CONV (4 mg/kg, every 4 days) in xenograft model of aggressive prostate cancer in athymic mice. Yet, detailed examination of the mechanisms underlying this enhanced activity and optimization of treatment schedules is limited with existing 2-dimension (2D) cell culture models that do not adequately reflect in vivo reality. Therefore, we developed and applied a 3D spheroid model that allows for the evaluation of longer drug exposures. We used PC-3-Luc-GFP, an aggressive, metastatic castration-resistant prostate cancer that expresses luciferase (Luc) and green fluorescent (GFP) that support bioluminescent and fluorescence imaging. Cell growth and cytotoxicity studies were performed using 2D or 3D models. Briefly, 3D spheroids were formed by seeding cells in a poly-HEMA coated 96-well plate with 2.5% recombinant basement membrane followed by centrifugation (5 min at 1000 g) to encourage spheroid generation. The 2D cell cultures reached confluency in 72-96 hr. While we previously demonstrated enhanced activity in vivo, the 2D model showed no benefit (improved potency) over this time frame, ie, METRO vs CONV dosing (logIC50 2.278 (177nM), 95% CI [1.781-2.776] vs logIC50 2.248 (189 nM) 95% CI [1.723-2.773]. Attempts to reduce seeding density to increase study duration resulted in poor and inconsistent cell growth and greater variability. 3D spheroids yielded reproducible round spheroids of similar size (~800 μm) that grew for up to 28 days, thus permitting evaluation of long treatment exposures. Tumor stromal cells are also known to alter tumor pathology, drug delivery and responsiveness to therapy. Therefore, 3D spheroids with PC-3-Luc-GFP and macrophages and endothelial cells in different cell proportions (1:1:1, 1:2:1, etc.,) were examined. Growth of 3D spheroids was altered based on cell type and cell proportion. Further, differences in rates of growth and cellular morphology were observed for tumor activated vs wild macrophages. Overall, we developed a high-throughput, liquid overlay, scaffold-based 3D co-culture spheroid that can be kept for 28 days. This model will permit evaluation of single agent and concomitant therapy over prolonged periods better mimicking clinical practice. Further, this model will allow for more detailed molecular analysis and ability to discern the effect of tumor microenvironment on alternate treatment schedules. Citation Format: Joshua Davis, Taraswi Mitra Ghosh, Lani Jasper, Matthew Eggert, Jeff Warner, Brian Cummings, Robert Arnold. Development of a three-dimensional spheroid model system to more accurately evaluate schedule dependent mechanistic differences between metronomic and conventional dosing of topotecan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 32.

Modeling tissues in vitro has long consisted of two-dimensional (2D) monolayer cultures grown in a dish, flask, or multi-well plate. While these methods remain adequate for some applications, the advent of personalized cancer therapies has necessitated the development of more complex models that more accurately represent the in vivo environment. Attempting to characterize tumor cell biology and response to candidate pharmaceuticals in 2D cultures has led to results that fail to translate to clinical therapies. A key source of this failure is the loss of cell diversity due to selective pressure resulting in cell populations that favor adhering to a rigid substrate. Three-dimensional (3D) cell culture methods have produced models that capture many of the dynamics of the in vivo tissue environment. Patient-derived organoids (PDOs) are quickly becoming the most attractive solution for creating predictive, patient-specific tumor models in vitro. Typically grown as spheroids, these models still present limitations in terms of cell survival and recapitulation of tumor development. Lena Biosciences has previously developed complex, vascularized tissue models using patented 3D scaffolds and a perfusion system. Hepatocytes grown in this system demonstrated increased cell viability and recovery of cytochrome P450 enzyme activity compared to 2D controls. Integral to the perfusion system is a pure, synthetic liquid that is twice as dense as and immiscible with cell culture medium and reagents. It is permissive to atmospheric gases, providing a gas-rich, liquid-liquid interface below 3D cultures maintained in the system. We hypothesized that PDOs could be cultured without a 3D scaffold, directly at the interface between the cell culture medium and this synthetic liquid. A specific preparation of this interface resulted in lung-derived PDOs that spontaneously formed dense, 3D, tissue-like structures that showed increased viability (as measured by a 50% reduction in LDH release) and cellular respiration (three-fold increase as measured by an alamarBlue assay). Ongoing studies are focused on characterizing PDOs from other patients and other tissues as they develop in long-term in vitro studies. Should this PDO model prove to be a faithful representation of in vivo tumor development, this system would be ideal for applications such as testing drug efficacy and studying mechanisms of tumor metastasis. Citation Format: James T. Shoemaker, Katherine R. Richardson, Jamie Arnst, Adam Marcus, Jelena Vukasinovic. Characterization of patient-derived organoids cultured on a gas-rich, liquid-liquid interface [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 54.

Ovarian cancer is typically diagnosed at late stages with a poor prognosis due to high recurrence. Exfoliation from the primary tumor results in aggregation of ovarian cancer cells into spheroids in the peritoneal cavity, which may increase survival of cancer and metastasis to other tissues. We have previously reported metabolic changes in adherent ovarian cancer cells to rely more heavily on glycolysis. However, metabolic changes in cancer cell aggregates are poorly understood. In this study, we investigated differences in spheroid metabolism in normoxic and hypoxic conditions as compared to adherent cells. We examined late-stage and tumor-initiating murine ovarian cancer monolayers and spheroids. Using a Seahorse XF extracellular flux analyzer, we investigated changes in metabolism over time in the presence of mitochondrial inhibitors (oligomycin and rotenone) or uncouplers (FCCP). Data show a reduction in basal respiration, maximal oxygen consumption rate, and spare respiratory capacity of spheroids compared to adherent monolayers. This suggests a reduction in energetic demand and metabolic flexibility in spheroids, which may confer increased survival capabilities. The identification of these metabolic changes that may increase survival and metastasis of spheroids provide targets for treatment to control the ability of spheroids to survive in the peritoneal cavity. Citation Format: Stephanie Edwards, Emily Pyne, Lu Lui, Jack Guinan, Madlyn Frisard, Eva Schmelz. Aggregation alters the metabolism of serous ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 808.

The accumulation of hyaluronan (HA) in the solid tumor microenvironment (TME) can be associated with poor prognosis in several human cancers. Accumulation of HA in the TME can increase interstitial pressure that compresses local blood vessels and impairs the access of anti-cancer therapy. In preclinical models, enzymatic degradation of HA by pegvorhyaluronidase alfa (PEGPH20; PVHA), a PEGylated form of recombinant human hyaluronidase PH20 (rHuPH20), is associated with decreased tumor interstitial pressure, improved tumor vascular perfusion, and increased access and anti-tumor efficacy of cytotoxic and immunotherapies. The functional effect of HA degradation on the TME has been mostly characterized in animal tumor models. Here, we describe the utilization of 3D tumor spheroid models to evaluate the effects of HA degradation on the cytotoxic effect of anti-cancer therapies. A number of commonly used human colorectal cancer (CRC) cell lines were assessed for HA accumulation and tumor spheroid morphology: HCT 116, HT-29 parental, and HT-29/HAS3 (HT-29 cells engineered to over-express hyaluronan synthase 3). CRC 3D tumor spheroid models were developed to evaluate the uptake and cytotoxic activity of liposome-encapsulated doxorubicin (DOX) using the IncuCyte® S3 Live-Cell Analysis System. The effect of HA degradation on DOX uptake and cytotoxic activity was initially tested with rHuPH20 treatment in all three models, and further evaluated with PVHA treatment in HT-29 parental and HT-29/HAS3 models. A dose-dependent uptake and cytotoxic activity of DOX was observed in HCT 116 tumor spheroids. HA degradation after rHuPH20 treatment enhanced DOX uptake by an average of 6.1% (up to 7.7%) and cytotoxicity by an average of 37.9% (up to 58.2%) over the 5-day duration of the experiment compared with DOX alone. Dose-dependent uptake and cytotoxic activity of DOX in HT-29/HAS3 tumor spheroids were enhanced with rHuPH20 treatment to a greater extent than in HT-29 parental tumor spheroids. Specifically, rHuPH20 treatment led to an average increase in DOX uptake of 9.2% (up to 14.6%) with HT-29/HAS3 spheroids and 2.6% (up to 5.1%) with HT-29 parental spheroids. The addition of rHuPH20 resulted in an average increase in DOX cytotoxicity of 101% (up to 227.6%) with HT-29/HAS3 spheroids and 24.9% (up to 63.9%) with HT-29 parental spheroids. Enhanced uptake and cytotoxic activity of DOX was also observed with PVHA treatment in HT-29 and HT-29/HAS3 models. These data corroborate our previous findings with in vivo tumor models, in which the combination of PVHA-mediated HA degradation and other anti-cancer therapies display enhanced anti-tumor activity compared with either agent alone. Further, 3D tumor spheroid models may be used to evaluate the effect of extracellular matrix remodeling such as HA degradation and serve as a bridge between 2D cell-based assays and in vivo tumor models. Citation Format: Feng Gao, Susan Zimmerman, Kelly Chen, Daniel C. Maneval, Chunmei Zhao. The effect of hyaluronan degradation on the uptake and cytotoxic activity of liposome-encapsulated doxorubicin in 3D tumor spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 97.

Clinical studies using TCR-transgenic T cells for adoptive T cell therapy revealed the efficacy of this therapeutic approach but also uncovered possible toxic effects against healthy tissues, such as neuronal or cardiac cells, caused by on-target/off-tumor or off-target toxicity (Morgan et al., J Immunother. 2013; Linette et al., Blood 2013). Due to the lack of adequate in vivo models for prediction of TCR efficacy or potential TCR-mediated toxicity against healthy tissues, physiologically relevant in vitro models need to be developed. The usage of 3D spheroids as targets for TCR-transduced T cells represents a powerful strategy to test killing capacity or possible safety issues of TCRs, which could fill the gap between 2D co-cultures and animal models. Compared to 2D cell layers, cells within a 3D spheroid structure more closely resemble the physiological in vivo situation regarding cell-cell-interactions, proliferation rates, hypoxia, and gene expression. Here we describe the use of 3D spheroids generated from tumor cell lines for assessment of TCR efficacy or healthy primary cells/iPSC-derived cells for evaluation of potential TCR-derived toxicity. HLA-A2-positive cells served as target cells in functional co-culture assays with CD8+ T cells transduced with an HLA-A2-restricted TCR. 3D spheroids have been generated using ULA (ultra-low attachment) plates or via the hanging-drop culture method. Spheroids were co-cultured with TCR-transduced T cells and killing of spheroids was analyzed in real-time using the IncuCyte live cell imaging system. For better visualization of spheroid killing, tumor cell lines were transduced using a lentivirus encoding a nuclear-restricted red fluorescent protein or by adding Annexin V red fluorescent reagent for detection of apoptotic cells. Thereby, spheroid killing could be quantified via decrease or increase in red fluorescence over time. Killing of 3D spheroids derived from healthy cells was analyzed via phase contrast and evaluation of spheroid morphology. TCR-transduced T cells showed efficient killing of tumor cell spheroids, confirming the results generated in a 2D system. Furthermore, 3D spheroids generated from healthy cells (e.g. normal human lung fibroblasts) or iPSC-derived cells (e.g. cardiomyocytes or neurons) were not killed via TCR-transduced T cells, whereas exogenous loading of the spheroids with the specific peptide led to efficient killing, showing the general susceptibility to TCR-mediated killing of healthy cell 3D spheroids.In summary, we showed that functional in vitro assays using 3D spheroid structures using tumor cell lines or healthy cells represent an elegant approach to assess both efficacy and potential toxicity of a given TCR in vitro. The analyzed TCR showed potent efficacy as well as a favorable safety pattern in 3D in vitro cultures. Citation Format: Maja Buerdek, Kathrin Mutze, Kai Pinkernell, Dolores J. Schendel. In vitro evaluation of TCR efficacy and toxicity using 3D spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2303.

The Golgi apparatus plays an essential role in the transport, processing, and sorting of cell surface proteins. We previously demonstrated that a Golgi disrupting agent, 2-methylcoprophilinamide (M-COPA), exhibited antitumor effect in human cancer cells in vitro and in vivo; however, in vivo antitumor efficacy of M-COPA did not simply reflect its in vitro efficacy, probably due to the lack of microenvironmental features of tumor tissues in the monolayer culture conditions. To bridge the gap between in vitro and in vivo efficacies, we exploited 3-dimensional (3D) spheroid culture models, which can recapitulate physiological characteristics of tumor tissues; i.e., nutrient/oxygen gradient, cell-cell interaction and cell-extracellular matrix (ECM) interaction, if cancer cells synthesize their own ECM molecules. Interestingly, human gastric cancer MKN1 cells displayed hypersensitivity to M-COPA under the 3D culture conditions compared to those in the monolayer culture conditions. Especially, we found that M-COPA caused loss of spheroid integrity. We previously demonstrated that M-COPA exerted antitumor effects via inhibiting intracellular transport of receptor tyrosine kinases onto the cell surface. Therefore, we examined the involvement of cell adhesion molecules in the antitumor effect of M-COPA. As a result, we found that specific integrins, which serve as cell surface receptors responsible for cell-ECM adhesion, were overexpressed and their downstream signaling molecules such as Src, Akt and MEK/ERK were highly phosphorylated in the cells under the 3D culture conditions compared to those under the monolayer culture conditions. Upon exposure to M-COPA, cell surface expression of these integrins was efficiently decreased and the downstream signaling molecules were dephosphorylated along with loss of spheroid integrity. Moreover, expression knockdown or neutralizing antibody of the integrins clearly suppressed sphere formation of MKN1 cells. These results suggest that M-COPA exerted in vivo antitumor effects via targeting specific integrins that mediate cell-matrix adhesion and its downstream intracellular signaling pathway observed in 3D spheroid culture models. Citation Format: Yoshimi Ohashi, Kazuma Takeuchi, Mutsumi Okamura, Akinobu Akatsuka, Isamu Shiina, Kentaro Yoshimatsu, Shingo Dan. Potential antitumor effects of a Golgi disrupting agent, M-COPA, via targeting cell-extracellular matrix interaction under the spheroid culture conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1299.

Nanoparticles offer potential as drug delivery systems for chemotherapeutics based on certain advantages of molecular drugs. In this study, we report that particle size exerts great influence on the penetration and retention behavior of nanoparticles entering tumors. On comparing gold-coated Au@tiopronin nanoparticles that were prepared with identical coating and surface properties, we found that 50 nanoparticles were more effective in all in vitro, ex vivo, and in vivo assays conducted using MCF-7 breast cells as a model system. Beyond superior penetration in cultured cell monolayers, 50 nm Au@tiopronin nanoparticles also penetrated more deeply into tumor spheroids ex vivo and accumulated more effectively in tumor xenografts in vivo after a single intravenous dose. In contrast, larger gold-coated nanoparticles were primarily localized in the periphery of the tumor spheroid and around blood vessels, hindering deep penetration into tumors. We found multicellular spheroids to offer a simple ex vivo tumor model to simulate tumor tissue for screening the nanoparticle penetration behavior. Taken together, our findings define an optimal smaller size for nanoparticles that maximizes their effective accumulation in tumor tissue. Cancer Res; 73(1); 319–30. ©2012 AACR.

The tumor protein p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 (p21) are two proteins with an intriguing and a paradoxical role in cancer. Through p53-dependent signals, p21 negatively controls cell cycle progression to protect cells against deleterious effects of DNA damage. Although p53 is a tumor suppressor protein, recent evidence shows that some patients with wild-type (wt) p53 develop aggressive and drug-resistant cancers. Similarly, recent studies also suggest the oncogenic role of p21. In a p53 deficient background, p21 results in the development of a subset of highly proliferative and resistant population of cancer cells. In this study, we examined the role of p53 and p21 in inducing cellular alterations using a panel of colorectal carcinoma cell lines with different p53 and p21 background and spheroid and spheroid-derived cell cultures. First, our data show that despite the presence of proliferating cells, spheroid-derived cultures of HCT116/p53wt cells continue to display an elevated p21 level even after 6 days of culture as a monolayer. These p21-overexpressing proliferating cells show increased resistance to doxorubicin and paclitaxel in comparison to their parental monolayer counterpart. In contrast to HCT116/p53wt cells, DLD-1/p53mut cells did not show elevated p21 levels following reversal from spheroid to monolayer. The increased p21 level in spheroids and spheroid-derived cultures of HCT116/p53wt cells led us to examine the role of p53 and p21 in drug penetration. Spheroids of HCT116/p53wt cells were less permeable to doxorubicin and showed increased expression of intracellular junctional (IJ) proteins (adherens and tight junctions). Knocking-out p21 in HCT116/p53wt cells decreased the level of IJ proteins, resulting in an increased doxorubicin penetration. Re-expression of p21 in p21-knocked-out HCT116/p53wt cells restored IJ protein levels, reducing doxorubicin penetration and the subsequent effect of doxorubicin on spheroids. Interestingly, however, knocking-out p21 in DLD-1/p53mut cells neither altered intracellular junctions nor affected doxorubicin penetration. These data indicate that the effect of p21 on IJ proteins and drug penetration is pertinent only in cells with wt p53. Overall, the data from our study suggest an intricate interplay between wt p53 and p21 in inducing cellular alterations that affect the sensitivity of cells to anti-cancer drugs in spheroids. Citation Format: Viswanath Das, Narendran Annadurai, Marián Hajdúch. An intricate role of p53 and p21 in cellular alterations and drug penetration in spheroids of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4474.

Three-dimensional (3D) cell culture models provide more physiologically relevant drug and toxicity screening platforms than traditional 2D platforms (1). Phenotypic analysis is an informative endpoint for high-throughput screens; however, variability in 3D culture systems can make statistically sound identification of good candidate compounds from a small number of replicates difficult. To minimize this variability we tested AggreWell™, a microwell-based 3D cell culture device that is a prototype system for 3D culture including drug and toxicity screening. The geometry of this system provides precise localization, segmentation and uniformity of spheroids derived from various cell types, and is predicted to minimize experimental variability. To test this directly, estrogen receptor-expressing MCF-7 breast cancer cells were seeded into AggreWell™400 plates in MammoCult™ medium in triplicate, and 24 hours after seeding, the resulting spheroids were treated with a single drug or combination of tamoxifen (TMX), the HER2 and EGFR inhibitor lapatinib (LTB), and the γ-secretase and Notch inhibitor DAPT. Three days later, the spheroids were imaged for the following parameters: size, brightness, shape, and specific morphologic features. Spheroids were also dissociated for viable cell counts. The IC50 values for drug effects for TMX and LTB are 21.6 and 6.3 uM, respectively, whereas DAPT had no effect on the spheroids at doses up to 100 uM. These results are consistent with previous published results and allow for correlation of observed changes in morphologic and cell viability post-treatment. For combinatorial drug treatments, concentrations of 20 uM TMX, 5 uM LTB, and 10 uM DAPT were used. TMX treatment exhibited the greatest reduction of cell reduction (52%). Combination of TMX with DAPT, LTB, or both, resulted in synergistic reductions of viability of 64%, 74% and 83%, respectively. Morphologic features of these cultures analyzed by principal component analysis reveal that parameters including circularity and gray values are associated with untreated cultures, whereas spheroid perimeter and area are associated with treated cultures. Graphing combinations of these parameters, for example, area vs. integrated density, delineates a clear decision plane for classifying treated versus untreated conditions. Analysis of variability in aggregate morphology demonstrates that measurement of multiple spheroids per well in AggreWell™ increased experimental power (e.g., power of 0.8 for 16 spheroids versus 0.3 for 4 spheroids, respectively; α = 0.05) and reduced experimental variance. These results confirm that morphologic analysis of spheroids grown on AggreWell™ plates is highly suited for high-throughput 3D drug screening.Reference: 1. Lee GY et al. Nat. Methods 2007. Citation Format: Michael Hiatt, Marta Mroczek, Eric Jervis, Terry E. Thomas, Allen C. Eaves, Sharon Louis. Drug screening and phenotypic analysis in a microwell-based 3D cell culture system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5015.

Background: Standard two-dimensional cell cultures do not retain the key characteristics of the human cancers from which they are derived and treatment effects are not always able to be replicated in vivo, making the development of alternative culturing systems paramount. Specifically, commercially available cell lines do not fully represent the mutation profiles seen in human cancers. Here we investigate the feasibility of three-dimensional PDOCS to more accurately represent the cancers from which they are derived and to predict treatment sensitivity in a clinically meaningful time frame. Methods: Surgical resection, core needle biopsies, paracentesis or thoracentesis samples from patients with various types of cancer were obtained under an approved IRB protocol, digested and spheroid cultures grown suspended in Matrigel. PDOCS were grown for up to two weeks and passaged at least once prior to treatment. PDOCS were imaged using brightfield imaging (4X) prior to treatment with vehicle or 5-fluorouracil (5-FU; 1, 10, or 100 µM) and/or radiation (2 or 5 Gy). After 2 days of treatment, the 5-FU was removed and the cultures were allowed to grow for an additional 2 days. PDOCS were re-imaged and the relative change in diameter was calculated using ImageJ software and compared to untreated controls. Optical metabolic imaging (OMI) was performed with a multiphoton microscope to probe the fluorescence lifetime and optical redox ratio of metabolic co-enzymes NAD(P)H and FAD. Single-cell analysis of each image was completed using Cell Profiler software to parse resistant cell populations in each PDOCS sample tested. DNA for mutation profile analysis was isolated and sequenced using a QIAseq targeted panel. Results: PDOCS were successfully isolated from a variety of cancers including colorectal (CRC), pancreas, lung, neuroendocrine, liver, breast, and ovarian. Key phenotypic characteristics of the tumors were retained in PDOCS cultured including crypt-like structures, mucin production and Ki67 proliferation rates. Cancer hot spot sequencing was performed comparing PDOCS and the tumors from which they were derived. Over 90% of the nonsynonymous mutations were identical, except in the setting of microsatellite instability. All driver mutations were identical (i.e., APC, KRAS, PIK3CA, TP53). Differential sensitivity to chemoradiation was observed among 4 different colorectal PDOCS treated with 5-FU and radiation (Median % PDOCS diameter change vs control: Patient A 17.1, p=0.64; Patient B -3.05, p=0.02; Patient C -19.4, p=2x10-5; Patient D -31.3, p=0.002). Similar response data were determined using OMI; however, single-cell analyses identified potentially resistant cell populations. Conclusions: PDOCS retain key characteristics of the cancers from which they are derived and can be utilized for treatment sensitivity testing in a clinically meaningful time frame. Citation Format: Cheri A. Pasch, Peter F. Favreau, Alex E. Yueh, Kwang P. Nickel, Christopher P. Babiarz, Philip B. Emmerich, Rosabella T. Pitera, Susan N. Payne, Demetra P. Korkos, Joseph T. Sharick, Carley M. Sprackling, Linda Clipson, Kristina A. Matkowskyj, Michael A. Newton, Melissa C. Skala, Michael F. Bassetti, Randall J. Kimple, Dustin A. Deming. Patient-derived organotypic cancer spheroids (PDOCS) as predictive models for the treatment of cancer in a clinically meaningful time frame [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5011.

3D bioprinting which is a significant improvement over 2D models offers layer by layer positioning of various cells which can provide superior cell-matrix interactions in the tumor microenvironment in vitro. The objective of the present study was to develop a co-culture model using Non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) cells and lung cancer associated fibroblasts (CAFs) in sodium alginate/ gelatin hydrogel using a 3D bioprinter. Various concentrations of sodium alginate/gelatin hydrogels (w/w) were prepared and rheological properties were evaluated. PDX cells (EGFR T790M, obtained from Dr. Rishi's Laboratory, Wayne State University) and CAFs (AA0022, obtained from Dr. Noyes Laboratory, Moffit Cancer Center) were mixed with the hydrogel and printed usingINKREDIBLE bioprinter (Cellink, Sweden). A two layered cube was designed in Slic3r software, converted to g-code file and printed through a 22G needle. Printed scaffolds were cross linked by Crosslinking Agent (Cellink, Sweden) for 5 minutes, washed with HBSS and maintained with media at 37°C and 5% CO2. Spheroid formation was observed using an inverted microscope (Olympus IX70). Live/Dead and NucBlue/ActinGreen staining was performed to determine cell viability and spheroid formation. Immuno-fluorescence staining was performed for E-cadherin, α-SMA and vimentin to study cellular crosstalk within the spheroid. Printable hydrogels were prepared by adding 3.25% sodium alginate to 4% gelatin which was found to be the most optimal mixture. Decrease in storage modulus (G') from 995.29± 30.69 Pa to 12.16± 8.83 Pa and loss modulus (G”) from 314.10± 17.54 Pa to 22.22± 12.69 Pa was observed from 18°C to 37°C when using a temperature sweep test. At lowest shear rate (1s-1), the viscosity of the hydrogel was 2.75± 0.37 Pa.s and at 100s-1 the viscosity was 0.50± 0.12 Pa.s. Decrease in hydrogel viscosity with increasing shear rate confirmed shear thinning. The height, width and length of the printed scaffolds were found as 1.45± 0.06 mm, 9.56± 0.15 mm and10.67± 0.13 mm respectively. Formation of spheroids was observed after 4 days and after 15 days, 59.88± 14.81% small (0-400µm2), 15.43± 4.86% medium (401-1000 µm2) and 24.67± 10.14% large size spheroids (1001 µm2-above) were observed which indicated proliferation of individual cells within the scaffolds. LIVE/ DEAD assay showed approximately 82% cells were viable in the printed scaffold. Up-regulation of vimentin and α-SMA and concomitant loss of E-cadherin was observed in the printed co-cultures indicating cross talk between the two cells. This study demonstrates the preparation and characterization of NSCLC lung PDX and CAF co-culture spheroids in 3D bio-printed sodium alginate/ gelatin scaffold. This model can be further used for high throughput drug screening platform. Citation Format: Arindam Mondal, Aragaw Gebeyehu, Ramakrishnan Subramanian, Arun Rishi, Mandip Singh. Bioprinted (3D) co-cultured spheroids with NSCLC PDX cells and cancer associated fibroblasts (CAFs) using alginate/gelatin hydrogel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5018.

The tumor-associated extracellular matrix (ECM) provides critical biochemical micro-environment cues, as well as an essential structural scaffold, for solid tumors to survive and grow (see Pickup et al. 2014 for review). With a view to enabling more translational and turnkey 3D in vitro assays for cancer biology, we have developed and optimized techniques for seeding, growing and automatically quantifying the properties of multiple tumor spheroids in ECMs in 96-well format using real-time live-cell analysis. Matrigel (Corning) was dispensed across a range of volumes (20 - 50 μL) and concentrations (1 - 5 mg mL-1) into flat-bottomed 96-well TC micro-plates to form a solidified base layer. Tumor cells (A549, MCF-7, SKOV-3, MDA-MB-231) were seeded on top (1 - 2K cells per well), and in some experiments a full ECM sandwich was created by addition of a further volume of Matrigel (2 - 25%, 0.2 - 5 mg mL-1). Using a custom autofocusing method, phase contrast, bright-field and fluorescence images (10x) were captured every 6h for 7 days from within the cell incubator (IncuCyte S3 live-cell analysis system). Typically, 20 - 80 spheroids were analyzed in each well. All four cell types formed multiple cell aggregates within the first 3 days, ranging in diameter from 30 - 80 μM. A549, SKOV-3 and MCF-7 multi-spheroids grew as round aggregates while MDA-MB-231 spheroids displayed stellate branching characteristic of an invasive morphology. At Matrigel volumes less than 40 μL or concentrations less than 3 mg mL-1, cells penetrated to the base of the plate and grew as ‘flat 2D' cultures. Using a novel bright-field image analysis algorithm, the number, area and average size of the spheroids could be computed over time non-invasively and without the use of fluorescent labels. Once formed, A549, SKOV-3, MCF-7 and MDA-MB-231 multi-spheroids increased 3.0-, 1.6-, 3.8- and 3.3-fold in size over 4 days, respectively. Treatment of A549 multi-spheroids with the DNA enzyme topo-isomerase inhibitor camptothecin (1μM) inhibited growth with comparable spheroid size at day 0 and day 7 post treatment (average brightfield area 1.4 x104 μM2). Using fluorescent protein reporters for apoptosis (Annexin V) and cell viability (IncuCyte CytoTox Green) we could verify camptothecin-induced cell death (fluorescence values 149±16% of control (Annexin V) and 243±51% of control (CytoTox). A concomitant decrease of stably expressed RFP (to 3±1% of control) was observed. The combination of protocol developments, novel image acquisition/analysis algorithms and cell health reporters creates an integrated solution for measuring growth and vitality of multiple small spheroids in a relevant and 3D bio-matrix over time. This approach should be applicable to primary- and patient-derived organoid tumor samples as well as cancer cell lines. Pickup, MW, Muow, JK, Weaver, MW (2014), EMBO Rep. 15(12): 1243-1253 Citation Format: Kalpana Patel, Miniver Oliver, Nevine Holtz, Tim Jackson, Nicholas Dana, Gillian Lovell, Nicola J. Bevan, Tim J. Dale, Derek J. Trezise. Development and optimization of Matrigel-based multi-spheroid 3D tumor assays using real-time live-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5030.

American Cancer Society predicts over 160,000 new prostate cancer cases in 2017, which amounts to about 20 percent of all cancer diagnoses in men. Despite several treatment options being available, poor prognosis of high-risk and metastasis remains a major concern for prostate cancer (PCa) patients. Therefore, it is important to understand molecular mechanisms of PCa progression to aggressive disease. Our previous research identified Abelson interactor 1 (Abi1), a member of WAVE complex, as a tumor suppressor in human prostate. Not only did we find mutations and deletions in the gene in prostate tumor patient samples, but WAVE complex levels were also found to be inversely correlated to disease recurrence and castration resistance. Moreover, Abi1 is downregulated in majority of prostate cancer cell lines, and patient organoid cultures derived from metastatic bone and lymph node biopsies. These data point to the clinical significance of defining Abi1-dependent tumor suppression mechanisms. To understand these mechanisms we generated CRISPR-mediated Abi1 KO in the RWPE-1 non-tumor human prostate cell line. When plated in 3D cultures in matrigel, we observed striking phenotypic differences in the appearance and behavior of the Abi1 KO spheroids compared to the parental cells. While the parental RWPE-1 grew into spherical organoids with tight cell-cell boundaries, the Abi1 KO cells grew as loose irregular-shaped spheres, with many cells migrating out of the organoids. Hence, the loss of Abi1 in these non-tumor cells lead to gain of an invasive phenotype. Western Blots of Abi1-KO cells demonstrated concurrent disruption of WAVE complex and deregulation of WAVE complex protein levels. Moreover, cell-cell adhesion proteins such as E-cadherin and β-catenin showed modest decrease and/or a substantial loss of membrane localization. Rescue experiments of Abi1 re-expression in the KO cells reversed the phenotype indicating that the observed phenotype is Abi1-dependent. Analyses of RNA sequencing of cells obtained from 3D cultures demonstrated differences in expression signatures characteristic of altered cell-cell and cell-matrix adhesion pathways and indicated upregulation of pathways associated with the invasive phenotype of cancer cells. We propose that Abi1 loss and WAVE complex deregulation represents key mediators of invasive phenotype in PCa. [Supported by NCI R01 CA161018 and NYS Department of Health Prostate Cancer Hypothesis Development RFA #1410200115] Citation Format: Disharee Das, Ming Chen, Claudia Mondragon, Dawn Post, Alexander Nappi, Heidi Hehnly, Anita Hryniewicz-Jankowska, Pier Paolo Pandolfi, Gennady Bratslavsky, Leszek Kotula. Modeling of Abi1 loss using spheroid cultures to investigate mechanisms of prostate tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1403.

Introduction: 2D cells lack cell-cell and cell-extracellular matrix interactions, critical for regulating cell functions. 3D cells in matrigel, hydrogels, hanging drops, and micropatterned plates can overcome these drawbacks but suffer from long spheroid formation times with variable efficiency. Magnetic 3D bioprinting (M3DB) can circumvent these issues by utilizing nanoparticles-magnetized cells for spheroid formation. M3DB spheroids can emulate tissue microenvironments and exhibit higher resistance to toxic agents than their 2D counterparts. It is, however, unclear how M3DB spheroids influence toxic responses in cells via metabolic perturbations. We employed Stable Isotope-Resolved Metabolomics (SIRM) with 13C6-glucose to map metabolic networks in 2D cells and M3DB spheroids formed from lung (A549) and pancreatic (PANC1) adenocarcinoma cells ± anti-cancer Na2SeO3. Experimental: A549 and PANC1 cells were grown in 2D culture in DMEM to ca. 70% confluence for loading with magnetic nanoparticles (Nanoshuttle, N3D Biosciences), detached, and placed in cell repellent plates for spheroid formation. Spheroids were then treated in [U-13C]-glucose ± Na2SeO3 for 24 h. Metabolites were extracted in cold 70% methanol and analyzed by IC-FTMS and NMR 1,2 to determine the 13C isotopologue and isotopomer distributions for reconstructing metabolic networks. Findings: We found that spheroids remained metabolically active in 10-day cultures. 13C label incorporation into glycolytic, Krebs cycle, and nucleotide metabolites was similar between spheroids and their 2D counterparts. The exceptions were higher 13C-ribose incorporation into uracil nucleotides in 2D than M3DB cultures of A549 cells and activation of gluconeogenesis in PANC1 M3DB spheroids. Selenite inhibited these pathways less in spheroids than 2D counterparts for both cell lines, consistent with the growth changes. Thus, the increased resistance of cancer spheroids to selenite may be mediated by these metabolic pathways central to growth and survival. Conclusions: 3D spheroids offer convenient models with more relevant 3-D architectures for preclinical studies on metabolic reprogramming and drug response of human cancer cells and cancer-stromal cell co-cultures. Spheroid formation in the M3DB system is fast, high throughput without matrix interferences for metabolic analyses. Supported by NCI P01CA163223-01A1, P30 CA177558 and NIDDK 1U24DK097215-01A1 1. Sun RC, Fan TW-M, Deng P, et al. Liquid diet delivery of stable isotopes into PDX mice for deep metabolic pathway tracing. Nat. Commun. 2017;9:1646. 2. Fan TW-M, Warmoes MO, Sun Q, et al. Distinctly perturbed metabolic networks underlie differential tumor tissue damages induced by immune modulator β-glucan in a two-case ex vivo non-small cell lung cancer study. CSH Molec Case Studies 2016;2:a000893. Citation Format: Teresa W. Fan, Salim S. El-Amouri, Jessica K. Macedo, Qiushi Sun, Andrew N. Lane. Mapping metabolic networks and drug resistance in 3D spheroids using stable isotope-resolved metabolomics (SIRM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5028.

Three-dimensional (3D) cancer cell models, such as 3D multicellular spheroids, are superior models of in vivo systems than the more popular two-dimensional (2D) cell culture. 3D cancer spheroids provide a closer reflection of tumor gene expression and biology than 2D cultures. This is particularly relevant for drug development and precision medicine programs were cell culture conditions that better reflect the 3D tumor environments, including matrix stiffness, would be highly advantageous. A key challenge for the routine application of embedded 3D cancer spheroids in precision and drug programs is the lack of high throughput and uniform production of spheroids in a biocompatible matrix. To address this, we have developed a high-throughput method of producing 3D multicellular cancer spheroids embedded inside a tissue-like matrix using a custom-built drop-on-demand 3D bioprinter. The 3D bioprinter is capable of printing a cell-suspension ink with up to 300 million cells/mL and a cell viability greater than 95%. We have validated the 3D bioprinting using glioblastoma, neuroblastoma and lung cancer cells. The 3D bioprinted spheroids were confirmed to maintain all the biological characteristics typically found in a cancer spheroid. In particular, the 3D bioprinted cancer spheroids were shown to be viable and proliferating, preserved the apoptotic and cancer-stemness characteristics and using super-resolution lattice-light sheet microscopy, we showed the spheroids maintained the same structural integrity as manually formed spheroids. The potential application of the 3D bioprinted spheroids for high-throughput drug screening in 3D environments was demonstrated using doxorubicin as the model drug. 3D-bioprinting of patient-derived cancer cells for precision medicine applications is now underway. High-throughput embedded 3D bioprinting has enormous potential to accelerate precision medicine, drug discovery and cancer biology. Citation Format: Lakmali Atapattu, Robert Utama, Aiden O'Mahony, Christopher Fife, Jongho Baek, Théophile Allard, Kieran O'Mahony, Julio Ribeiro, Katharina Gaus, Justin Gooding, Maria Kavallaris. Precision medicine: High-throughput 3D bioprinting of embedded multicellular cancer spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5022.

Metastasis is the main cause of death in cancer patients and one of the most complex biological processes in human diseases. The development of therapies designed to forestall the metastatic activity of tumors has been met with multiple challenges. The first is the initial focus on single target remedies. As many types of cancers develop multiple mutations during tumor progression, individual cancers are often little affected by this type of drug. The advancement of novel methods that allow for the discernment of the effect a potential therapy has on the invasive phenotype of a particular type of cancer has proven invaluable to circumventing these early failures. A second hurdle is the choice of an appropriate cell model. Tumors in vivo exist as a three-dimensional (3D) mass of multiple cell types, including cancer and stromal cells. Therefore, incorporating a 3D spheroid-type cellular structure that includes co-cultured cell types forming a tumoroid, provides a more predictive model than the use of individual cancer cells cultured on the bottom of a well in traditional two-dimensional (2D) format. A third impediment is the inclusion of proper incubation time. Traditionally in vitro assessments of anti-cancer drugs have included short-term incubations, sometimes limited to 48 hours. In vivo treatments, however, can consist of a multi-week regimen. Pre-clinical tests must also, then, extend a similar length of time. A final hindrance which is necessary to overcome is the proper capture and analysis of microscopic images. The ability to monitor the entire tumoroid with a degree of sensitivity across multiple z-planes as cells invade into the matrix, as well as quantify changes over time, is critical. The information presented here will demonstrate a combined procedure for the generation of 3D spheroidal tumoroid structures, creation of a suitable invasion matrix, automated kinetic image-based monitoring of tumor invasion, and cellular analysis of captured z-stacked images. Citation Format: Brad Larson. Automated monitoring and analysis of kinetic live cell 3D spheroid-based tumor invasion within a hydrogel matrix [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-318.

The establishment of cancer stem-like cell (CSC) culture systems may be instrumental in devising strategies to fight refractory cancers. Inhibition of the Rho kinase ROCK has been shown to favorably affect CSC spheroid cultures. In this study, we show how ROCK inhibition in human serous ovarian cancer (SOC) cells can help establish a CSC system, which illuminates cancer pathophysiology and its treatment in this setting. In the presence of a ROCK kinase inhibitor, spheroid cultures of SOC cells expressed characteristic CSC markers including ALDH1A1, CD133, and SOX2, along with differentiation and tumorigenic capabilities in mouse xenograft models of human SOC. High expression levels of ALDH, but not CD133, correlated with spheroid formation CSC marker expression and tumor forming capability. In clinical specimens of SOC, high levels of ALDH1A1 correlated with advanced stage and poor prognosis. Pharmacologic or genetic blockade of ALDH blocked cell proliferation and reduced expression of SOX2, the genetic ablation of which abolished spheroid formation, whereas SOX2 overexpression inhibited ALDH1A1 expression and blocked spheroid proliferation. Taken together, our findings illustrated a new method to culture human ovarian CSC, and they defined a reciprocal regulatory relationship between ALDH1A1 and SOX2, which impacts ovarian CSC proliferation and malignant progression. Cancer Res; 76(1); 150–60. ©2015 AACR.

The intracellular and extracellular dynamics that govern tumor growth and invasiveness in vivo remain poorly understood. Cell genotype and phenotype, and nutrient, oxygen, and growth factor concentrations are key variables. In previous work, using a reaction-diffusion mathematical model based on variables that directly describe tumor cell cycle and biology, we formulated the hypothesis that tumor morphology is determined by the competition between heterogeneous cell proliferation caused by spatial diffusion gradients, e.g., of cell nutrients, driving shape instability and invasive tumor morphologies, and stabilizing mechanical forces, e.g., cell-to-cell and cell-to-matrix adhesion. To test this hypothesis, we here obtain variable-based statistics for input to the mathematical model from in vitro human and rat glioblastoma cultures. A linear stability analysis of the model predicts that glioma spheroid morphology is marginally stable. In agreement with this prediction, for a range of variable values, unbounded growth of the tumor mass and invasion of the environment are observed in vitro. The mechanism of invasion is recursive subspheroid component development at the tumor viable rim and separation from the parent spheroid. Results of computer simulations of the mathematical model closely resemble the morphologies and spatial arrangement of tumor cells from the in vitro model. We propose that tumor morphogenesis in vivo may be a function of marginally stable environmental conditions caused by spatial variations in cell nutrients, oxygen, and growth factors, and that controlling these conditions by decreasing spatial gradients could benefit treatment outcomes, whereas current treatment, and especially antiangiogenic therapy, may trigger spatial heterogeneity (e.g., local hypoxia), thus causing invasive instability. (Cancer Res 2006; 66(3): 1597-604)

To reach cancer cells in optimal quantities, therapeutic agents must be delivered to tumors through their imperfect blood vascular system, cross vessel walls into the interstitium, and penetrate multiple layers of tissue. Strategies to enhance drug penetration have potential to improve therapeutic outcome. The development of multicellular layers (MCLs), in which tumor cells are grown on a semipermeable Teflon support membrane, has facilitated quantification of drug penetration through solid tissue. The goals of the present study were to quantify the penetration of anticancer drugs as a function of cellular adhesion and packing density and to determine the effects of variable penetration on therapeutic efficacy in this model system. We compared the properties of MCLs grown from two epithelioid and round subclones of a colon carcinoma cell line. One pair of epithelioid and round sublines differed in expression of α-E-catenin, and both pairs generated MCLs with different packing density. The penetration of commonly used anticancer agents (paclitaxel, doxorubicin, methotrexate, and 5-fluorouracil) through MCLs derived from these cell lines was significantly greater through the round (loosely packed) than through the epithelioid (tightly packed) sublines. In MCLs treated with doxorubicin, we observed greater survival in the tightly packed cell lines than in the loosely packed cell lines. Impaired penetration of anticancer agents through MCLs derived from the tightly packed cell lines and relative resistance to killing of cells within them by doxorubicin treatment strengthen the role of tumor cell adhesion and packing density as contributing to drug resistance. (Cancer Res 2006; 66(2): 1033-9)

There have been 1600 FDA approved drugs since their inception in 1938. Currently it costs approximately 883 million dollars to bring a drug candidate to the market, and often times the potential candidate fails at the beginning of the clinical phase. Therefore, it is important to develop drug screening methods that are more clinically relevant or predictable. The current 2D methods for cancer drug discovery have had some difficulty in identifying potential drug candidates that can be used for clinical testing. To overcome this challenge, there has been an increase in research of 3D tissue culture that facilitated the development of new in-vitro tumor model assays. Traditional 3D spheroid analysis method relied heavily on visual observation using standard microscopy, which is time-consuming and has high operator variations. In the recent years, high-throughput image-based cytometers, such as Celigo, have demonstrated the ability to perform bright-field and fluorescence cell-based assays. Celigo imaging cytometer can be employed to rapidly analyze and characterize 3D tumor spheroids, which can be used to generate both quantitative and qualitative results. In this work, we demonstrate a high-throughput 3D tumor spheroid screening method using the Celigo imaging cytometer to screen the effects of 14 drug compounds (NIH/NCAT) on U87MG spheroid size, matrigel invasion, and tumor spheroid viability. First, a dose response experiment is performed to screen the growth inhibitory effects of the drug compounds. In addition to direct spheroid size analysis, dose inhibitory responses of tumor invasion into the matrigel are also examined. Finally, the use of specific fluorescent dyes such as Calcein AM, PI, and Caspase 3/7 were used to screen drug induced cytotoxicity on the tumor spheroids. The results showed that Celigo imaging cytometer can quickly generate accurate growth inhibitory data to identify potential drug candidates. Furthermore, tumor invasion were clearly observed and quantified in the captured images, as well as fluorescent analysis of tumor spheroid viability. By utilizing the 3D spheroid screening method, researchers can rapidly characterize and quantify drug effects on tumor spheroids in a high-throughput format, which can improve the efficiency of identifying more qualified cancer drug candidates. Citation Format: Leo Li-Ying Chan, Scott Cribbes, Sarah Kessel, Scott McMenemy, Jean Qiu. A high-throughput 3D tumor spheroid screening method for drug discovery using imaging cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4206. doi:10.1158/1538-7445.AM2017-4206

Introductory Sentence Real-time metabolic flux can be measured non-invasively using hyperpolarized magnetic resonance spectroscopy in cancer spheroids as well as tumor biopsies. Experimental Procedures LnCAP cells were resuspended in a 1:1 mixture of Matrigel:alginate (1x105 cells/ml) and grown in a porous hollow fiber (A/G Technology Corporation, NY). [1-13C] pyruvate was polarized in a SpinLab (General Electric, NY) before dissolving with appropriate buffers. NMR studies were performed on a 1 Tesla Magritek Spectrometer (Magritek, CA). HP spectra were acquired (10o degree flip angle with repetition time of 4s for 25 scans in total). Cell tracking used the manual tracking function in ImageJ. Xenograft biopsies were obtained from LnCAP tumors using a 2mm punch. Data Summary LnCAP cells seeded sparsely enable cancer spheroid formation remain viable as assessed by Live/DEAD fluorescent staining. Histological sectioning revealed spheroids of approximately 200 µm in diameter. Direct deposition of these threads into a 5mm NMR tube allows measurement of metabolic flux using hyperpolarized [1-13C] pyruvate resulting in the formation of [1-13C] lactate. To modulate metabolism, we used an allosteric AKT inhibitor, MK2206. This drug resulted in relocalization of AKT from the plasma membrane as well as a reduction in cell motility from 0.72 ± 0.04 to 0.46 ± 0.03 μm/s (n=5, p0.05) 24 hr after treatment. To ensure that these methods are applicable clinical settings, LnCAP xenograft biopsies (~60mg) were deposited directly into an NMR tube to measure hyperpolarized [1-13C] pyruvate metabolism. Line-width of the pyruvate peak was measured to be 1.72 ± 0.19 Hz (n=3) in the first spectra, demonstrating good homogeneity of the magnetic field and conversion to lactate was observed. Future experiments will focus on measuring the metabolism of patient-derived xenografts and effects of treatment response. Conclusions Metabolic measurements using hyperpolarized pyruvate can be performed on cancer spheroids as well as biopsies, allowing quantification of metabolism to complement on-going clinical trials utilizing hyperpolarized MR imaging. Citation Format: Sui Seng Tee. Measuring tumor metabolism in cancer spheroids and biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2856. doi:10.1158/1538-7445.AM2017-2856

The purpose of this study was to examine the induction of epithelial to mesenchymal transition (EMT) in a prostate cancer cell line by measuring classical biomarker expression in three-dimensional (3D) spheroid cultures compared to traditional 2D monolayers in an effort to develop a more biologically relevant assay. Using Ultra-Low Attachment (ULA) microplates, we grew spheroids from a human prostate cancer cell line (DU 145). Numerous studies have implicated a role for EMT in carcinoma invasion and metastasis. EMT is characterized by rearrangement of the extracellular matrix (ECM) and differential regulation of ECM proteins. We induced EMT using TGF-beta and phorbol-12-myristate-13-acetate (PMA) and compared expression levels of specific biomarkers, such as E-cadherin, fibronectin, and IL-6, using AlphaLISA and LANCE (TR-FRET) assay technologies. We confirmed that treatment of DU 145 cells with TGF-beta is sufficient for inducing changes in both EMT biomarker expression and characteristic cellular morphology in monolayer cultures. However, in 3D spheroid cultures, we observed only a partial EMT response to the same TGF-beta treatment as demonstrated by changes in the expected biomarker expression pattern. Using the small molecule, PMA, we see significant differences in the levels of IL-6 secretion after EMT induction between cells grown in monolayer and those grown in spheroids. Cellular proliferation, growth and vitality were assessed using ATPlite luminescence assays and confocal microscopy of live-stained cells with a high content imaging system. Though we observe increased proliferation in monolayer cultures compared to 3D spheroids, the changes observed in protein expression patterns cannot be sufficiently explained by differences in cell number or viability. These data illustrate the differences in protein expression levels and in cellular tolerance for compound treatment between a human prostate cancer cell line grown in monolayers and those same cells grown in 3D spheroids. Citation Format: Jen Carlstrom, Jeanine M. Hinterneder, Lindsay Nelson, Stephen Hurt. Comparison of EMT biomarker expression in 2D monolayer and 3D spheroid cultures in a prostate cancer cell model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5793. doi:10.1158/1538-7445.AM2017-5793

Background: Hypoxia in solid tumors acts as the major barrier to anti-cancer therapies and promotes tumor progression. Hypoxia-activated prodrugs (HAPs) are designed to not only eliminate these resistant cells, but also to exploit hypoxia as a basis of tumor selectivity. A key concept in exploiting hypoxia with HAPs is that active drug metabolite should be able to diffuse from hypoxic zones to maximize effect in surrounding aerobic tumor tissue. There is some evidence that these ‘bystander effects’ contribute to the antitumor activity of HAPs, but currently there is a lack of robust methods for their detection. Objectives: To investigate bystander effects from HAPs in a novel spheroid co-culture system using PR104A which releases a diffusible DNA crosslinking mustard and the tirapazamine analogue SN30000 which is converted to a short lived radical that is not expected to diffuse out of cells. Methods: A new method for measuring the bystander effect with multicellular spheroids under hypoxic conditions was developed using co-cultures of HCT116 colon cancer cells with high metabolic capacity for HAP metabolism (activators, transfected to overexpress cytochrome P450 reductase [POR]) and cells that cannot activate HAPs efficiently (targets, with POR knocked out by zinc finger nuclease gene targeting) along with differential antibiotic resistance and fluorescent protein markers to enable their quantitation in mixed cultures. Sensitivity of activators and targets to PR104A and SN30000 was determined in spheroid co-cultures by dissociation and clonogenic cell survival assay. Results were compared to output of a novel agent-based mathematical model (ABM) of spheroid co-cultures. Results: In monolayer cultures, activator cells showed markedly higher sensitivity to the HAPs than did targets. Tagging of activators and targets with fluorescent proteins allowed visualization of their spatial distribution in spheroid co-cultures. When spheroids were treated with PR104A, killing of targets markedly increased with increasing proportion of activators, confirming a bystander effect. However, increasing proportions of activators reduced cell killing of targets after SN30000 treatment, suggesting that rapid metabolism by activators inhibits its penetration, which was confirmed by flux experiments in multicellular layer co-cultures. ABM simulations of cell killing by PR104A and SN30000 in spheroids using our published transport parameters agreed with experimental results indicating diffusion of active metabolites of PR104A but not SN30000. Discussion: Using this novel spheroid co-culture model, a bystander effect of PR104A was demonstrated while SN30000 showed no bystander effect. This is the most direct demonstration of a hypoxic bystander effect for PR104A to date, and demonstrates the utility of the novel spheroid ABM in simulating active metabolite diffusion in 3D co-cultures. Citation Format: Cho Rong Hong, Gib Bogle, William R. Wilson, Kevin O. Hicks. Investigation of bystander effects of hypoxia activated prodrugs using three dimensional cell cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4513. doi:10.1158/1538-7445.AM2017-4513

Patient-derived Xenografts (PDXs) are considered as relevant preclinical model for anticancer drug development due to original recapitulation of patient genetic profile, gene expression patterns and tissue histology. In this study, we investigated combination efficacy of CDODA-Me (Methyl 2-cyano-3,11-dioxo-18-olean-1,12-dien-30-oate) and TKI inhibitor Erlotinib (ERL) against Lung NSCLC PDX spheroids and 3D bio-printed PDX cells. NSCLC PDX cells (EGFR T790M mutants) were obtained from Dr. Rishi’s Laboratory. PDX spheroids were grown in DMEM/ F12 media supplemented with L-glutamine, B27 supplement, recombinant human epidermal growth factor (EGF) and recombinant human basic fibroblast growth factor (bFGF). Spheroids were treated with CDODA-Me, ERL alone and in combination. Cell viability was measured by MTT assay. Western blot analysis was used to study the modulation of Bcl-xL, MDR1 and ABCG2 in treated PDX spheroids. For 3D bio-printing of PDX cells, hydrogels were prepared by partial cross-linking of sodium alginate (4.5% w/v) and gelatin (1% w/v) mixture with 40mM CaCl2 solution. PDX Cells were mixed with partially cross-linked hydrogel and printed with Inkredible 3D bio-printer (CELLINK, Sweden). Bio-printed scaffolds were fully cross-linked by 160 mM CaCl2 solution and then incubated overnight with cell culture media. The scaffolds were treated with CDODA-Me and ERL alone and in combination. After 48 h cell viabilities were determined by live/dead assay using fluorescence microscopy. MTT assay showed that approximately 65% and 74% viability was observed at 10 µM ERL and 2.5 µM CDODA-Me respectively. Decreased spheroid cell viability was observed in ERL and CDODA-Me combination treatment. Our western blot studies showed down-regulation of Bcl-xL, MDR1 and ABCG2 in combination group. Further, 81.04 ± 5.65, 78.65 ± 3.98 and 74.35 ± 4.24 percent viable PDX cells were observed in the bio-printed scaffolds after 48, 72 and 96 h respectively. Higher percentage of dead cells (52.62 ± 1.66) were found in the combination group than CDODA-Me (28.39 ± 1.60) and ERL (29.62 ± 4.91) alone. In conclusion, CDODA-Me in combination with ERL was found to be effective against human lung PDX spheroids and bio-printed PDX cells by decreasing the cell viability and overcoming drug resistance. Partially cross-linked sodium alginate-gelatin hydrogel enhances the possibility of PDX cell bio-printing with high cell survival rate. CDODA-Me can be considered as an effective neo-adjuvant to improve ERL efficacy in human NSCLC. Citation Format: Arindam Mondal, Aragaw Gebeyehu, Ebony Nottingham, Arvind Bagde, Subramanian Ramakrishnan, Arun K. Rishi, Mandip Singh. Combination of CDODA-Me, a glycyrrhetinic acid derivative, and Erlotinib overcomes chemo-resistance in NSCLC PDX spheroids and 3D bio-printed cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2072. doi:10.1158/1538-7445.AM2017-2072

Interaction between cancer cells and stromal components in the tumor microenvironment is well known for their significant roles in tumor progression and subsequent treatment failure. Hepatic stellate cells (HSCs), as a predominant cell type in the microenvironment of hepatocellular carcinoma (HCC), are involved in creating desmoplastic and chemoresistance-inducing microenvironment. HSCs secrete various paracrine factors that modify the HCC tumor microenvironment leading to tumor growth, drug resistance and metastasis via promoting epithelial-to-mesenchymal transition (EMT). Although several studies using 2D co-culture system have shown bidirectional cross-talk between cancer cells and HSCs, data obtained remain limited in their clinical relevance due to lack of in vivo tumor-like characteristics. We developed a mixed-cell spheroid model which recapitulates direct 3D tumor-HSCs interactions in paracrine and contact-mediated manners. Huh-7, human HCC cells, were mixed co-cultured with LX-2 cells, immortalized human HSC at 1:3 ratio using liquid overlay technique in 96-well plates. Electron microscopy was used to examine subcellular structural changes. Cellular distribution within spheroids was observed by labeling LX-2 cells with fluorescent tracer. The expression of pro-fibrotic and EMT markers was detected by immunohistochemistry or immunofluorescence staining on paraffin embedded sections. Invasion ability was determined using migration assay into 3D matrigel matrix. Dose-response curve was obtained by using APH assay and Ki-67 detection. Mixed co-culture of Huh-7 and LX-2 cells showed a spontaneous self-organization forming highly compact mixed-cell spheroids with well-defined contour. Heterotypic cell-to-cell contact increased as shown by gap junction and desmosomes in the mixed-cell spheroids. LX-2 cells were uniformly distributed within the spheroids. Expression of fibroblast-associated factors such as α-SMA, collagen I, TGF-β and CTGF showed similar patterns of distribution to that of LX-2 cells, but at an elevated level in the mixed-cell spheroids compared to that of cancer cell-alone spheroids. An increased expression of EMT-related factors was observed along with enhanced invasion into 3D matrix. Differential drug sensitivity was shown; mixed-cell spheroids were sensitive to sorafenib but not to other agents including oxaliplatin, gemcitabine, and 5-FU. Based on these results, the mixed cell-spheroids of HCC cells-HSCs may be proposed as a useful 3D model for in vitro therapeutic screening of targets and agents as well as for the study of malignant progression in HCC. Citation Format: Hyo-Jeong Kuh, Iftikhar Ali Khawar. Characterization of mixed-cell spheroid as an in vitro model of hepatocellular carcinoma for epithelial-to-mesenchymal transition and chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5789. doi:10.1158/1538-7445.AM2017-5789

Solid tumors grow in a three-dimensional (3D) spatial conformation, resulting in a heterogeneous exposure to oxygen and nutrients as well as to other physical and chemical stresses. To mimic the 3D spatial conformation, 3D in vitro culture models have been used in cancer research since the diffusion-limited distribution of oxygen (hypoxia), nutrients, metabolites, and signaling molecules is not mimicked in conventional two-dimensional (2D) monolayer cultures. One of the 3D in vitro culture models, the tumorsphere culture is an emerging model for studying and expanding the cancer stem cell (CSC) population. E006AA cell line is a spontaneously immortalized cell line derived from a Gleason 6 localized prostate cancer in a hormone-naive prostate cancer patient of African American descent. This cell line expresses androgen receptor and was repeatedly reported to be non-tumorigenic in nude mice. However this cell line forms continuously growing tumor in NOG-SCID triple-deficient mice (i.e., NOG-SCID mice not having NK, B, and T-cells) and an establishment of highly tumorgenic subline E006AA-hT was reported suggesting this cell line has a potential for CSC proliferation by 3D sphere cultures. Here we report tumorsphere cultures of E006AA cells by a new 3D spheroid culture media system. The cells showed continuous proliferation supported during serial passage of 3D tumorsphere cultures. In addition to the stable proliferation, increases of Aldehyde Dehydrogenase (ALDH) expressing CSC population were observed with the increase of passages of 3D tumorsphere cultures. Citation Format: Nick Asbrock, Vi Chu, Kan Saito. Cancer stem cell proliferation in human prostate cancer cells utilizing a new defined 3D spheroid culture system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 927. doi:10.1158/1538-7445.AM2017-927

The transcription factor MYC is associated with the regulation of over 15% of human gene expression. Deregulation of MYC is found in up to 70% of human cancers. As MYC is responsible for the reprogramming of cell metabolism, growth and survival, it is an attractive therapeutic target, yet effective drug development has been slow. As the Bromodomain/BET proteins epigenetically regulate MYC, BET inhibitors like JQ1 have been developed as MYC therapeutics. We used the Ex Vivo Analysis of Programmed Cell Death (EVA/PCD) previously shown to correlate with response, time to progression and survival, to examine JQ1 activity in 46 human tumor specimens isolated from surgical biopsies. Methods: Surgical biopsies were mechanically and enzymatically disaggregated, with micro spheroids of desired size isolated by density centrifugation. Five-Point dose response curves were interpolated to provide LC50 values for comparison by Z-score. Synergy was conducted by median-effect. Results: JQ1 revealed activity in hematologic, breast, glial & gastrointestinal (GI) tumors. By rank order, JQ1 activity favored hematologic (AML & NHL) & breast tumors over Lung, Ovary and GI with inter-patient differences observed within tumor types. MYC association with histone acetylation and aurora kinase (AURK) activity, led to an exploration of JQ1 synergy with SAHA (HDACi), MLN 8054 & Tozasertib (both AURKi). JQ1 synergy analysis revealed SAHA > Tozasertib > MLN 8054. Conclusions: Based upon JQ1 activity, several tumor types appear to be candidates for agents that target MYC-mediated cell survival, offering potential treatment options for patients with advanced malignancies. JQ1 synergy with HDACi & AURKi suggests novel drug combinations for future development. As MYC signaling influences thousands of gene targets, phenotypic analyses like the EVA/PCD platform can offer insights into cellular cell death events that serve as surrogate markers for clinical response to MYC inhibition. Supported in part by the Vanguard Cancer Foundation, the Nagourney Institute and the Malcolm C. Todd Cancer Institute. Citation Format: Robert Alan Nagourney, Steven S. Evans, Alexander J. Nagourney, Paula J. Bernard, Federico R. Francisco, Milan Sheth, Nilesh Vora, Eknath Deo. MYC inhibition in human malignancy by BET-Bromodomain inhibitor JQ1, using a 3-D human tumor primary culture micro-spheroid platform to explore clinically relevant observations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4684. doi:10.1158/1538-7445.AM2017-4684

The OncoPanel™ service has established 100 genomically-characterized human tumor cell lines as targets for the screening of compounds in 3D spheroid models. We sought to (1) expand the cell line capability of the service and (2) validate the cell lines in a long-term (10-day) assay. For the cell line expansion, cells were plated at two densities in 384-well, spheroid-promoting plates, and were incubated for 3 days to allow formation of spheroids. The growth media were then removed and replaced with media containing the hypoxia probe, Lox-1. Twenty-four hours later, the ability to form spheroids was determined by bright-field, high-content imaging, and fluorescent detection of the Lox-1 probe in the hypoxic core of the spheroids. Cells were then seeded in assay plates at pre-determined densities, along with a time zero (T0) plate, and were incubated for 4 days to allow spheroid formation. Spheroid formation was verified by bright-field, high-content imaging, as previously mentioned. The growth media were removed and replaced with fresh media, followed by the addition of staurosporine over a range of 10 concentrations, using acoustic liquid transfer. At this time, the T0 plate was processed for subsequent doubling calculations. The assay plates were incubated for 10 days, re-imaged, and the spheroids were lysed using CellTiter-Glo® 3D viability reagent. Data were analyzed as the relative cell count, where the measured bioluminescence intensity was transformed to percent of control, as compared with a vehicle-treated control. Cellular response parameters were calculated using nonlinear regression to a sigmoidal single-site dose response model. A vehicle CV of Citation Format: Jesse J. Parry, Steven M. Garner, Lee R. Cavedine, Alyssa M. Cracchiolo, Brogan A. Epkins, Samantha C. Burkart, Timothy J. Sindelar, Charles R. Wageman, Usha Warrior, Alastair J. King. Validation of spheroids with long-term incubation for high-throughput screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5787. doi:10.1158/1538-7445.AM2017-5787

The inability of most systemically delivered therapeutics to cross the blood-brain-barrier (BBB) is considered a major barrier to effective brain cancer treatment. In malignant glioma the tumor neovasculature is known to be leaky, however invasive cells remain protected behind an intact BBB, and continue to thrive within the brain, ultimately leading to tumor regrowth and patient death. This leaves us with an urgent unmet need for the development of next-generation therapeutics with improved brain delivery. For the first time, we describe here the utility of 3D multicellular BBB spheroids made of human brain endothelial cells (ECs), pericytes and astrocytes as a screening tool for brain-penetrating agents. We show that the outer surface of the spheroids, composed primarily of ECs and pericytes, form a tight barrier which is permeabilized in the presence of VEGF. The barrier is characterized by the presence of intact tight junctions and efflux-pump activity (i.e., P-glycoprotein). Furthermore, we have used this model to successfully demonstrate the transport of angiopep-2 (a well-known brain delivery vector) and its conjugates (containing cargoes of various sizes such as peptide, protein and affibody), thereby displaying the versatility of this model to screen and study a wide range of therapeutic agents. We demonstrate that this model is superior to the conventional transwell model in maintaining essential BBB characteristics (i.e., tight/adherens junctions and P-glycoprotein expression) and as a drug-screening tool. We have utilized the spheroid model to screen a panel of cell-penetrating peptides (CPPs) to identify several candidates with high brain-penetration potential. We then verified the ability of the top 4 candidate CPPs to cross the BBB in mice. This high-throughput model can lead to better design and analysis of first-in-class glioma therapeutics, and improve prediction of drug penetration in a living model, paving the way for breakthrough discoveries in brain cancer. Citation Format: Choi-Fong Cho. Blood-brain-barrier spheroids: Next-generation screening platform for brain-penetrating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4014. doi:10.1158/1538-7445.AM2017-4014

Robust, scalable, and quantitative in vitro tumor models that recapitulate the complex multicellular in vivo tumor microenvironment are lacking. Most in vitro tumor models fail to recapitulate the abnormal tissue architecture characteristic of solid tumors, which has been shown to promote tumor progression through forced-depolarization, enhanced cell-cell contacts, and loss of tensional homeostasis. Here, we used an in vitro tumor model that effectively mimics the in vivo multicellular architecture, which we refer to as the 3D collagen embedded spheroid model, to quantitatively investigate the response of breast cancer cells to chemotherapeutic intervention and assess the presence of the highly tumorigenic subpopulation of cancer cells known as cancer stem cells (CSCs). We compared our embedded spheroid model to two other in vitro models—a 2D monolayer culture and a 3D diffusely embedded single-cell model—and show that our 3D embedded spheroid model exhibits a more robust drug response as well as an enriched CSC subpopulation. In this study, we investigated the efficacy of two front-line chemotherapeutics (paclitaxel and cisplatin) against cells from the post-metastatic and triple-negative breast cancer cell line MDA.MB.231 cultured within the aforementioned models. In the 3D diffuse model, cells were embedded within 4 mg/mL collagen gels at a seeding density of 105 cells/mL. For the embedded spheroid model, spheroids composed of 104 cells were embedded within a 4 mg/mL collagen gel. The treatment regimen for all models consisted of 72 hours of drug exposure—10 ng/mL for paclitaxel and 1.5 μg/mL for cisplatin—followed by removal of drug and an additional 72 hours of culture. Upon completion of the experiment, cells were harvested and drug efficacy was quantified via cell viability measurements using an MTS assay. Moreover, for the embedded spheroid condition, collagenase treatment was used to isolate two distinct populations: 1) cells that remained within the spheroid structure, termed the “core population”; and 2) cells that had invaded into the surrounding collagen, termed the “periphery population”. Our results showed that both drugs effectively reduced viability to about 15% in the 2D monolayer, and 30% in the 3D diffusely embedded and 3D spheroid periphery conditions. However, cells in the core population exhibited a highly robust response against the agents with significantly higher viabilities of 91 and 97% for paclitaxel and cisplatin, respectively. Additionally, we sought to quantify the CSC content across the three models to determine whether the aforementioned differences in drug efficacy correlated with an enriched CSC content. Here, we used three independent techniques to quantify CSC content within our untreated conditions: 1) Aldefluor assay, 2) Mammosphere assay, and 3) TaqMan RT-qPCR assay of two known CSC markers (ALDH1A3 and SOX2). The Aldefluor assay revealed CSCs to be preferentially located in the spheroid core compared to the spheroid periphery, which was independently corroborated by both the mammosphere and RT-qPCR assays. In addition, the high sensitivity of the TaqMan assay provided us with the means to assess ALDH1A3 and SOX2 expression across our in vitro models following treatment with either paclitaxel or cisplatin. Specifically, treatment with either paclitaxel or cisplatin led to a statistically significant increase in ALDH1A3 and SOX2 expression across all models, compared to the untreated 2D monolayer. These findings indicate that paclitaxel and cisplatin efficacy is inversely related to CSC content and that these therapies can even enrich for the highly malignant CSC subpopulation. In conclusion, the 3D embedded spheroid model described herein recreates a phenotypic landscape possessing an enriched and spatially-dependent CSC population that reflects the response of CSCs to clinical treatment. Citation Format: Daniel S. Reynolds, Kristie M. Tevis, Mark W. Grinstaff, Muhammad H. Zaman. Chemotherapeutic treatment enriches for cancer stem cell content in breast cancer spheroids. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A19.

Traditional understanding holds that metastasis begins as tumor cells undergo epithelial-to-mesenchymal transition (EMT) to migrate and escape. Recent studies suggest that EMT is not necessary for metastasis, however. Tumor cells may retain their epithelial phenotype to migrate collectively during metastasis. But if EMT is not the crucial permissive step, what is? Our group recently discovered cellular jamming; a process by which an epithelial layer is dormant, non-migratory, and solid-like, corresponding to the jammed state, but in other circumstances becomes active, migratory, and fluid-like, corresponding to the unjammed state. Therefore, based on this phenomenon, do breast cancer cells unjam before they can collectively migrate and metastasize? Spheroidal aggregates of MCF10A and MDA-MB-231 cells spreading on collagen-coated polyacrylamide gels, modeling normal and malignant breast epithelium, respectively, were used to investigate cell jamming, cell-matrix adhesion, and associated traction forces. Compared with MCF10A spheroids, MDA-MB-231 spheroids permeated more rapidly onto gels, generated largely localized traction forces, and dissociated to spread more rapidly outwards. These results suggest MDA-MB-231 cells are more susceptible to unjamming and collective migration. This work will provide a new framework by which to explain collective cellular behavior of normal and malignant breast epithelium. Citation Format: Karin Wang, Jeffrey J. Fredberg. Unjamming and spreading of a cellular aggregate as a model of breast cancer migration. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A62.