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The fibril orientation of type I collagen has been shown to contribute to tumor invasion and metabolic changes. Yet, there is limited information about its impact on tumor cells' behavior in a restrictive growth environment. Restrictive growth environments are generated by the inhibition of a proliferation stimulus during therapy or as an inflammatory response to suppress tumor expansion. In this study, the impact of a type I collagen matrix orientation and fibrous architecture on cell proliferation and response to estrogen receptor (ER) therapy were examined using estrogen-dependent breast tumor cells (MCF-7 and T-47D) cultured in a hormone-restricted environment. The use of hormone-free culture media, as well as pharmacological inhibitors of ER, Tamoxifen, and Fulvestrant, were investigated as hormone restrictive conditions. Examination of cultures at 72 h showed that tumor cell proliferation was significantly stimulated (1.8-fold) in the absence of hormones on collagen fibrous substrates, but not on polycaprolactone fibrous substrates of equivalent orientation. ER inhibitors did not suppress cell proliferation on collagen fibrous substrates. The examination of reporter cells for ER signaling showed a lack of activity, thus confirming a shift toward an ER-independent proliferation mechanism. Examination of two selective inhibitors of α2β1 and α1β1 integrins showed that cell proliferation is suppressed in the presence of the α2β1 integrin inhibitor only, thereby indicating that the observed changes in tumor cell behavior are caused by a combination of integrin signaling and/or an intrinsic structural motif that is uniquely present in the collagen fibrils. Adjacent coculture studies on collagen substrates showed that tumor cells on collagen can stimulate the proliferation of cells on tissue culture plastic through soluble factors. The magnitude of this effect correlated with the increased surface anisotropy of the substrate. This sensing in fibril orientation was further supported by a differential expression pattern of secreted proteins that were identified on random and aligned orientation substrates. Overall, this study shows a new role for electrospun collagen I fibrous substrates by supporting a shift toward an ER-independent tumor cell proliferation mechanism in ER+ breast tumor cells.

Cells can respond to different topographical cues in their natural microenvironment. Hence, scientists have employed microfabrication techniques and materials to generate culture substrates containing topographies for cell-based assays. However, one of the limitations of custom topographical platforms is the lack of adoption by the broad research community. These techniques and materials have high costs, require high technical expertise, and can leach components that may introduce artifacts. In this study, we developed an array of culture surfaces on polystyrene using razor printing and sanding methods to examine the impact of microscale topographies on cell behavior. The proposed technology consists of culture substrates of defined roughness, depth, and curvature on polystyrene films bound to the bottom of a culture well using double-sided medical-grade tape. Human monocytes and adult mesenchymal stem cells (hMSCs) were used as test beds to demonstrate the applicability of the array for cell-based assays. An increase in cell elongation and Arg-1 expression was detected in macrophages cultured in grooves and on rough substrates as compared to flat surfaces. Also, substrates with enhanced roughness stimulated the proliferation of hMSCs. This effect correlated with the secretion of proteins involved in cell proliferation and the downregulation of those associated with cell differentiation. Our results showed that the polystyrene topography sticker array supports cellular changes guided by microscale surface roughness and geometries. Consequently, microscale surface topographies on polished and razor-printed polystyrene films could leverage the endogenous mechanisms of cells to stimulate cellular changes at the functional level for cell-based assays.

Human adult mesenchymal stem or stromal cells (h-MSC) therapy has gained considerable attention due to the potential to treat or cure diseases given their immunosuppressive properties and tissue regeneration capabilities. Researchers have explored diverse strategies to promote high h-MSC production without losing functional characteristics or properties. Physical stimulus including stiffness, geometry, and topography, chemical stimulus, like varying the surface chemistry, and biochemical stimuli such as cytokines, hormones, small molecules, and herbal extracts have been studied but have yet to be translated to industrial manufacturing practice. In this review, we describe the role of those stimuli on h-MSC manufacturing, and how these stimuli positively promote h-MSC properties, impacting the cell manufacturing field for cell-based therapies. In addition, we discuss other process considerations such as bioreactor design, good manufacturing practice, and the importance of the cell donor and ethics factors for manufacturing potent h-MSC.

Cell culture technologies have provided biomedical researchers with fast and accessible tools to probe the breast tumor microenvironment. Exponential progress in fabrication methods combined with multiparametric approaches have enabled the development of cell culture model systems with enhanced biological complexity to identify key aspects that regulate breast cancer (BC) progression and therapeutic response. Yet, the culture parameters and conditions employed influence the behavior of tumor cells, thereby affecting its tissue biomimetic capabilities. In this chapter we review the wide range of culture platforms employed for the generation of breast tumor models and summarize their biomimetic capabilities, advantages, disadvantages and specific applications.

Pancreatic cancer is an aggressive cancer that is diagnosed early in only 10% of cases and has a 5-year survival rate of less than 9%. The process in determining the optimal treatment for each patient is both long and tedious - and current strategies do not enable therapy optimization at the individual level. The incidence rate for pancreatic cancer in Puerto Rico between 2013 and 2017 was 8.2%, which is lower than the rate reported for non-Hispanic whites and blacks (16% and 12.9%, respectively). The causes for such health disparity are unknown but it is very likely associated with changes at the genomic and tissue microenvironment level at the sub-population level. To identify pathological markers in a more patient-specific manner, patient-derived models need to be integrated into studies of drug screening and efficacy. Patient-derived organoids (PDO) are becoming a potential model for diagnosis and the study of disease progression due to their ability to recreate the pathology of the tumor in the patients. Although several PDO studies have highlighted their potential for therapeutic applications, the ability to successfully culture and propagate these using standard methods has not been shown in samples collected from the Hispanic population. We assessed the feasibility of establishing PDOs from tissue specimens of a pancreatic mass lesion from Puerto Rican patients using standard methods for pancreatic organoids. A total of 9 samples were collected from endoscopic ultrasound fine-needle biopsies to examine the pathological features of the sample and the retention of the parental tumor characteristics in culture. All tissue specimens were cultured in Matrigel domes within 24 hours of extraction and organoid establishment was monitored for 7-8 days. All samples were positive for pancreatic adenocarcinoma but only around 50% of them were successfully cultured in Matrigel. High ATP levels were observed in tumor organoids after one week in culture which is indicative of a high cell viability. Examination of pathological markers indicated that all samples were negative for epidermal growth factor (EGFR), 5/6 were positive for smooth muscle actin (SMA), 4/6 were positive for Vimentin, and 1/6 were positive for CA 19.9. Absence of EGFR was unexpected as this marker is highly expressed and abundant in 40-70% of pancreatic cancers. Samples with high levels of SMA, a fibroblast marker, resulted in a higher organoid yield than samples with no SMA present. Overall, the data suggests that the presence of fibroblasts is supportive of organoid establishment in culture and that standard culture methods need to be optimized to increase organoid yields. Future work will seek to increase the sample size to test alternative propagation methods and examine drug sensitivity in patient-derived pancreas organoids of the Puerto Rican population. Citation Format: Andrea S. Flores Perez, Janet Mendez Vega, Ana M. Reyes Ramos, Carlos Micames, Madeline Torres Lugo, Maribella Domenech. EUS-guided biopsy of pancreatic mass lesions for the development of patient-derived organoids in Puerto Rico [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3075.

Silicones (i.e. crosslinked poly(dimethylsiloxane), PDMS) are commonly used material for microfluidic device fabrication. Nonetheless, due to the uncontrollable absorption of small hydrophobic molecules (1 kDa) into the bulk, its applicability to cell-based drug assays and sensing applications has been limited. Here, we demonstrate the use of substrates made of silicones bulk modified with a poly(ethylene oxide) silane amphiphile (PEO-SA) to reduce hydrophobic small molecule sequestration for cell-based assays. Modified silicone substrates were generated with concentrations of 2 wt.%, 9 wt.% and, 14 wt.% PEO-SA. Incorporation of PEO-SA into the silicone bulk was assessed by FTIR analysis in addition to water contact angle analysis to evaluate surface hydrophobicity. Cell toxicity, absorption of small hydrophobic drugs, and cell response to hydrophobic molecules were also evaluated. Results showed that the incorporation of the PEO-SA into the silicone led to a reduction in water contact angle from 114° to as low as 16° that was stable for at least three months. The modified silicones showed viability values above 85% for NIH-3T3, MCF7, MDA-MB-468, and MDA-MB-231 cell lines. A drug response assay using tamoxifen and the MCF7 cell line showed full recovery of cell toxicity response when exposed to PDMS modified with 9 wt.% or 14 wt.% PEO-SA compared to tissue culture plastic. Therefore, our study supports the use of PEO-SA at concentrations of 9 wt.% or higher for enhanced surface wettability and reduced absorption of small hydrophobic molecules in PDMS-based platforms.

The therapeutic potential of human mesenchymal stromal cells (h-MSC) is dependent on the viability and secretory capacity of cells both modulated by the culture environment. Our previous studies introduced heparin and collagen I (HEP/COL) alternating stacked layers as a potential substrate to enhance the secretion of immunosuppressive factors of h-MSCs. Herein, we examined the impact of HEP/COL multilayers on the growth, morphology, and secretome of bone marrow and adipose-derived h-MSCs. The physicochemical properties and stability of the HEP/COL coatings were confirmed at 0 and 30 days. Cell growth was examined using cell culture media supplemented with 2 and 10% serum for 5 days. Results showed that HEP/COL multilayers supported h-MSC growth in 2% serum at levels equivalent to 10% serum. COL and HEP as single component coatings had limited impact on cell growth. Senescent studies performed over three sequential passages showed that HEP/COL multilayers did not impair the replicative capacity of h-MSCs. Examination of 27 cytokines showed significant enhancements in eight factors, including intracellular indoleamine 2, 3-dioxygenase, on HEP/COL multilayers when stimulated with interferon-gamma (IFN-γ). Image-based analysis of cell micrographs showed that serum influences h-MSC morphology; however, HEP-ended multilayers generated distinct morphological changes in response to IFN-γ, suggesting an optical detectable assessment of h-MSCs immunosuppressive potency. This study supports HEP/COL multilayers as a culture substrate for undifferentiated h-MSCs cultured in reduced serum conditions.

Tape-based razor-printing is a flexible and affordable ultra-rapid prototyping approach for microscale device fabrication. However, integration of this prototyping approach into cell-based assay development has been limited to proof of principle demonstrations. This is in large part due to lack of an established or well-characterized option for biocompatible adhesive tape. Without such an option, integration of these areas will remain unexplored. Therefore, to address this critical hurdle, we characterized microscale devices made using a potentially biocompatible double-sided adhesive, ARCare 90106. We validated tape-based device performance against 96-well plates and PDMS microdevices with respect to cell viability, hydrophobic small molecule sequestration, the potential for leaching compounds, use in fluorescence microscopy, and outgassing (bubble formation). Results supported the tape as a promising tool for future cell-based assay development. Therefore, we subsequently demonstrated specific strengths enabled by the ultra-rapid (< 1hr per prototype) and affordable (~$1,200 cutting plotter, < $0.05 per prototype) approach. Specifically, data demonstrate the ability to integrate disparate materials for advanced sticker-device functionality such as bonding of polystyrene devices to glass substrates for microscopy applications, inclusion of membranes, and incorporation of different electrospun biomaterials into a single device. Likewise, the approach allowed rapid adoption by uninitiated users. Overall, this study provides a necessary and unique contribution to the largely separate fields of tape-based razor-printing and cell-based microscale assay development by addressing a critical barrier to widespread integration and adoption while also demonstrating the potential for new and future applications.

The study of multi-cell-type (MCT) interactions has the potential to significantly impact our understanding of tissue and disease biology. Such studies require innovative culture tools for unraveling the contributions of each cell type. Micro- and macro-scale platforms for MCT culture each have different advantages and disadvantages owing to their widely different capabilities, availability, and ease-of-use. However, as evidenced in the literature, there are very few examples of MCT studies and culture platforms, suggesting both biological and technical barriers. We have developed an open multi-culture platform to promote more rapid progress by integrating advantages of both micro- and macro-scale culture devices. The proposed open multi-culture platform addresses technical barriers by allowing easy customization, independent control of basic physical culture parameters, and incorporation of multiple culture modalities (e.g., 2D, 3D, transwell, and spheroid). The design also permits the user to obtain independent endpoints for each culture region. We demonstrate use of the platform in two example studies where we evaluated how cell ratio and cell types influence the response of triple negative breast cancer cells to heat damage and Hedgehog signaling. We also show that the platform can improve soluble factor transport between cell types compared to compartmentalized macro- and micro-scale alternatives. Last, we examine current and future challenges of the platform. We envision simple, yet flexible and customizable, platforms such as this will be important for advancing in vitro study of tissue and tumor biology.

Pancreatic cancer is an aggressive cancer that is diagnosed early in only 10% of cases and has a 5-year survival rate of less than 9%. The process in determining the optimal treatment for each patient is both long and tedious – and current strategies do not enable therapy optimization at the individual level. The incidence rate for pancreatic cancer in Puerto Rico between 2013 and 2017 was 8.2%, which is lower than the rate reported for non-Hispanic whites and blacks (16% and 12.9%, respectively). The causes for such health disparity are unknown but it is very likely associated with changes at the genomic and tissue microenvironment level. To identify patient and sub-population specific markers, patient-derived models need to be integrated into studies of drug screening and efficacy. Patient-derived organoids (PDO) are becoming a potential model for diagnosis and the study of disease progression due to their ability to recreate the pathology of the tumor in patients. Although several PDO studies have highlighted their potential for therapeutic applications, the ability to successfully culture and propagate these using standard methods has not been established in samples collected from the Hispanic population. We assessed the feasibility of establishing PDOs from tissue specimens of a pancreatic mass lesion in Puerto Rican patients using standard methods for pancreatic organoids. A total of 10 samples were collected from endoscopic ultrasound (EUS) fine-needle biopsies to examine the pathological features of the tissue and the retention of the parental tumor characteristics in culture. All tissue specimens were cultured in Matrigel domes within 24 hours of extraction and organoid establishment was monitored for 7-8 days. All samples were positive for pancreatic adenocarcinoma but only 50% of them were successfully cultured in Matrigel. High ATP levels were observed in tumor organoids after one week in culture which is indicative of a high cell viability. Examination of pathological markers indicate that all samples were negative for epidermal growth factor (EGFR), 5/6 were positive for smooth muscle actin (SMA), 4/6 were positive for Vimentin, and 1/6 were positive for CA 19.9. Absence of EGFR was unexpected as this marker is highly expressed and abundant in 40-70% of pancreatic cancers. Samples with high levels of SMA, a fibroblast marker, resulted in a higher organoid yield than samples with no SMA present. Overall, the data suggests that the presence of fibroblasts is supportive of organoid establishment in culture and that standard culture methods need to be optimized to increase organoid yields. Future work will seek to increase the sample size to test alternative propagation methods and examine drug sensitivity in patient-derived pancreas organoids of the Puerto Rican population. Citation Format: Andrea S. Flores Pérez, Janet Mendez Vega, Ana M. Reyes Ramos, Carlos Micames, Madeline Torres-Lugo, Maribella Domenech. EUS-guided biopsy of pancreatic mass lesions for the development of patient-derived organoids in Puerto Rico [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-062.

Heart disease remains one of the leading causes of death in industrialized nations with myocardial infarction (MI) contributing to at least one fifth of the reported deaths. The hypoxic environment eventually leads to cellular death and scar tissue formation. The scar tissue that forms is not mechanically functional and often leads to myocardial remodeling and eventual heart failure. Tissue engineering and regenerative medicine principles provide an alternative approach to restoring myocardial function by designing constructs that will restore the mechanical function of the heart. In this review, we will describe the cellular events that take place after an MI and describe current treatments. We will also describe how biomaterials, alone or in combination with a cellular component, have been used to engineer suitable myocardium replacement constructs and how new advanced culture systems will be required to achieve clinical success.

Phosphodiesterase 4D (PDE4D) has recently been implicated as a proliferation-promoting factor in prostate cancer and is overexpressed in human prostate carcinoma. However, the effects of PDE4D inhibition using pharmacologic inhibitors have not been examined in prostate cancer. These studies examined the effects of selective PDE4D inhibitors, NVP-ABE171 and cilomilast, as anti–prostate cancer therapies in both in vitro and in vivo models. The effects of PDE4D inhibitors on pathways that are critical in prostate cancer and/or downstream of cyclic AMP (cAMP) were examined. Both NVP-ABE171 and cilomilast decreased cell growth. In vitro, PDE4D inhibitors lead to decreased signaling of the sonic hedgehog (SHH), androgen receptor (AR), and MAPK pathways, but growth inhibition was best correlated to the SHH pathway. PDE4D inhibition also reduced proliferation of epithelial cells induced by paracrine signaling from cocultured stromal cells that had activated hedgehog signaling. In addition, PDE4D inhibitors decreased the weight of the prostate in wild-type mice. Prostate cancer xenografts grown in nude mice that were treated with cilomilast or NVP-ABE171 had decreased wet weight and increased apoptosis compared with vehicle-treated controls. These studies suggest the pharmacologic inhibition of PDE4D using small-molecule inhibitors is an effective option for prostate cancer therapy. Implications: PDE4D inhibitors decrease the growth of prostate cancer cells in vivo and in vitro, and PDE4D inhibition has therapeutic potential in prostate cancer. Mol Cancer Res; 13(1); 149–60. ©2014 AACR.

Hedgehog signaling (Hh) has been shown to be hyper-activated in several cancers. However, active Hh signaling can promote or inhibit tumor growth, thus identification of markers beyond main canonical Hh target genes are needed to improve patient selection and clinical outcome in response to Hh inhibitors. Cancer-associated fibroblast (CAFs) have been linked with tumor progression and beneficial response to Hh inhibitors. Thus, we hypothesized that genes associated with Hh-activated CAFs can be used for stratification of tumors that will benefit from Hh inhibitors. In this work, we evaluated a 15-gene fingerprint that combines Hh and mesenchymal genes associated to CAFs phenotype to profile breast cancer sub-types based on gene expression patterns among clustered groups. About 3,800 cancer samples were evaluated using random forest models and linear discriminant analysis to sort breast cancer by subtypes and therapeutic approach. Results showed that the Hh-mesenchyme gene fingerprint has a highly sensitive and differential expression pattern among basal and luminal A sub-groups. Basal samples with high levels of Hh target genes had better prognosis than Luminal A samples. Luminal A samples with tendency towards Hh signaling suppression had higher overall and disease-free survival rates particularly if deprived of hormone therapy. Hh transcriptional repressor GLI3 and signaling activator SMO were the top 2 genes to discriminate among samples with active Hh signaling in human breast cancer subtypes and Hh-inhibitor resistant tumors. Caveolin-1 (CAV1), a gene associated to low expression in CAFs, shows strong correlation with active Hh signaling and discrimination among survival curves in Luminal A patients with active or inactive Hh signaling. Our data suggests that CAV1 is an important gene for monitoring Hh inhibition in tumors and supports further stratification by hormone therapy status prior to use of Hh inhibitors.

The paracrine interaction between tumor cells and adjacent stroma has been associated with the oncogenic activity of the Hedgehog (Hh) pathway in triple-negative breast tumors. The present study developed a model of paracrine Hh signaling and examined the impact of mesenchymal cell sources and culture modalities in the oncogenicity of the Hh pathway in breast tumor cells. Studies consisted of tumor cell monocultures and co-cultures with cancer-associated and normal fibroblasts, tumor cells that undergo epithelial&ndash, mesenchymal transition (EMT), or adipose-derived mesenchymal stem cells (ADMSCs). Hh ligand and pathway inhibitors, GANT61 and NVP-LDE225 (NVP), were evaluated in both cell cultures and a mouse xenograft model. Results in monocultures show that tumor cell viability and Hh transcriptional activity were not affected by Hh inhibitors. In co-cultures, down-regulation of GLI1, SMO, and PTCH1 in the stroma correlated with reduced tumor growth rates in xenografted tumors and cell cultures, confirming a paracrine interaction. Fibroblasts and EMT cells supported Hh transcriptional activity and enhanced tumor cell growth. Mixed and adjacent culture modalities indicate that tumor growth is supported via fibroblast-secreted soluble factors, whereas enriched tumor stemness requires close proximity between tumor and fibroblasts. Overall this study provides a tumor&ndash, mesenchymal model of Hh signaling and highlights the therapeutic value of mesenchymal cells in the oncogenic activity of the Hh pathway.

High degree of collagen I fiber orientation has been associated with guided tumor cell invasion, but little is known about its impact in therapeutic response. Anisotropic substrates have been shown to influence the phenotype of stem cells which suggests that it can influence tumor cell behavior beyond cell migration. We hypothesized that the anisotropy of collagen I matrix impacts therapeutic response by modulating the secretome of breast cancer cells and this effect is mediated by integrin signaling. To test our hypothesis, the cell secretome, response to hormone ablation therapy and integrin inhibitors were evaluated in breast tumor cells cultured in collagen type 1 substrates of defined orientation. The orientation of electrospun collagen I fibrils was regulated by adjusting the syringe flow rate and collector velocity to obtain random (80%) oriented fibrils. Average porosity (45± 3.4 %), matrix stiffness (0.8 ±0.24 MPa) and fiber diameter (1.6 ± 0.86μm) were kept constant across collagen substrates. Estrogen-dependent Luminal A cells (MCF-7 and T-47D) cells were cultured on collagen substrates in a hormone-deprived environment for 3-6 days. Polycaprolactone (PCL) fibrous substrates of aligned and random orientation were used as negative fibrous controls for integrin signaling. Evaluation of cell growth showed that a fibrillar structure in combination with a high degree of fibril orientation significantly increased cell proliferation (>75%) as compared to gelatin-coated tissue culture plastic (TCP). Collagen fibers significantly supported hormone-independent tumor growth at levels equivalent to or above those observed in the estrogen-treated control. Increased proliferation levels were supported on collagen fibrous substrates treated with Tamoxifen and Fulvestrant, and this effect was enhanced in aligned collagen I. Inhibition of α2β1 integrin resulted in enhanced proliferation rates in random fibers and suppression of cell growth in aligned fibers. PCL aligned substrates where not sufficient to support hormone-independent cell growth. MCF-7 cells on collagen substrates significantly stimulated the growth (>7 fold) of adjacent cells on TCP in a dose-response manner as follows: aligned > random > gelatin. Expression levels of 1000 factors were quantified in conditioned medium to identify differences in the secretome signatures associated with cell-matrix signaling. MCF-7 cells cultured on collagen fibrous substrates displayed secretome signatures that were different across all substrates. Fourteen factors were identified to be upregulated in fibrous substrates by more than ten folds including apoptotic regulators RalA and BCL-2. Our results show for the first time the influence of collagen I fibril orientation in supporting hormone-independent growth and as a novel regulator therapeutic response in luminal A cells. Citation Format: Ana M. Reyes, Jorge Almodovar, Elaine Alarid, Maribella Domenech. Anisotropy of collagen I matrix influences therapeutic response and secretome of Luminal A 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 1984.

Collagen matrix architecture is an important player in tumor transition to therapeutic resistance. Several studies have shown that high level of fiber orientation and density of collagen type I matrix enhances tumor cell invasion, decrease drug response and influence tumor metabolism, but the mechanisms of action that support transition to therapeutic resistance are not fully understood. We developed a 3D culture platform to evaluate the effects of collagen matrix architecture in the sensitivity of tumor and stromal cells to tumor promoting ligands and pharmacological inhibitors. Estrogen (E2) and Hedgehog (Hh) signaling pathways were selected for evaluation due to their relevance in breast cancer prognosis and tumor progression. In this 3D culture platform, electrospun collagen fiber sheets of random or aligned orientation are bound to double-sided medical grade tape to generate collagen fiber stickers. Automated cutting plotter allows razor-printing for precisely cutting shapes from collagen fiber sticker sheets. Round shapes were applied like a sticker to the bottom of culture well plates prior to cell seeding. The percentage of matrix porosity fiber orientation and diameter was quantified by Scanning Electron Microscopy (SEM) and Image J analysis. For estrogen signaling studies, MCF-7 cells were seeded in collagen fibers of random or aligned orientation using hormone free-culture conditions. Cells were treated +/- E2 (10nM) ligand and +/- estrogen receptor signaling inhibitors (Fulvestrant and Tamoxifen). Tumor cell proliferation was quantified at 48hrs via Image Cytometry. For Hh signaling studies, expression levels of GLI1, PTCH1 and SMO Hh target genes were monitored in triple negative breast cancer (MDA-MB-231 and MDA-MB-468) and mesenchymal cells. Cells were seeded on collagen fibers and treated with sonic hedgehog ligand for 24hrs. Tumor and mesenchymal cells were harvest for viability and qRT-PCR analysis respectively. Evaluation of cell growth in MCF-7 cultures shows that a fibrillar structure of collagen matrix in combination with high degree of fiber orientation can significantly increase cell proliferation as compared to gelatin or tissue culture plastic substrates. Collagen fibers significantly supported hormone-independent tumor growth at similar levels to those observed in E2 treatment. Increased proliferation levels were maintained in ER inhibition groups particularly when cultured in aligned collagen fibers. In Hh signaling studies, overexpression of GLI1 and SMO genes were observed for collagen fibers matrixes as compared to gelatin and tissue culture plastic. Overall our results show for the first time that collagen fiber architecture can promote hormone-independent growth dependence in breast cancer cells and activation of hedgehog signaling in tumor and mesenchymal cells. Citation Format: Ana M. Reyes, Jorge Almodovar, Maribella Domenech. Collagen fiber orientation influences tumor cell growth and Hedgehog signaling in the breast tumor microenvironment [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 174.

Lysosomal death pathways are being explored as alternatives of overcoming cancer tumor resistance to traditional forms of treatment. Nanotechnologies that can selectively target and induce permeabilization of lysosomal compartments in cells could become powerful medical tools. Here we demonstrate that iron oxide magnetic nanoparticles (MNPs) targeted to the epidermal growth factor receptor (EGFR) can selectively induce lysosomal membrane permeabilization (LMP) in cancer cells overexpressing the EGFR under the action of an alternating magnetic field (AMF). LMP was observed to correlate with the production of reactive oxygen species (ROS) and a decrease in tumor cell viability. Confocal microscopy images showed an increase in the cytosolic activity of the lysosomal protease cathepsin B. These observations suggest the possibility of remotely triggering lysosomal death pathways in cancer cells through the administration of MNPs which target lysosomal internalization pathways and the application of AMFs.

The development and progression of tumors are modulated by the interaction among multiple cell types present at the tumor adjacent tissue. Few in vitro cell culture platforms allow one to test multi-cellular interactions and those suitable require high numbers of cells limiting studies to cell lines and excluding heterogeneous patient samples, and fail to provide tunable discrete adjacent compartments for the retrieval of independent cell population readouts. Mixing of cells in adjacent compartments and cell sorting after mixed culture represent a costly and time consuming procedure that often results is lost, reduction and/or damage of the recovered sample. To overcome some of these limitations we have developed an open multi-microwell array that enables co-culture of up to 4 cell populations in discrete adjacent wells using either 2- or 3-dimension culture conditions. The circular microwells are made of poly-dimethyl siloxane (PDMS) and placed on top of glass or tissue culture plastic. The open-well format allows cells and culture media to be manually loaded and replaced using a pipette independently of passive pumping method, and without the need of vacuum-assisted filling or syringe pumps. The microwell array is aligned to a 96well plate format for automated fluorescent readouts. The cell culture surface area of each microwell is 5.7mm2 (5ul) and can accommodate 5,000-10,000 cells but the design of each microwell can be scale-down to accommodate smaller cell samples. The adjacent microwells are contained within an outer-ring of 8mm diameter and 750um height. After cell seeding, adjacent compartments can be connected by dispensing a volume of 30-40ul inside the outer ring. A model of tumor-mesenchyme paracrine Hedgehog signaling was developed and characterized to show the feasibility of this array for multi-cell soluble factor signaling studies. Activation of Hedgehog (Hh) signaling in the mesenchyme promotes proliferation of tumor cells via secreted factors. As a source of mesenchymal cell sub-types we evaluated TGF-b treated fibroblasts and tumor epithelial cells that undergo epithelial-mesenchymal transition. Myofibroblast phenotype was confirmed based on overexpression of smooth muscle actin, vimentin and fibroblast activating protein, and reduce levels of caveolin-1. Active Hh signaling and pharmacological inhibition with cyclopamine were confirmed by up- and down- regulation of canonical Hh target genes Gli1 and Patch1. Active hedgehog signaling and cell viability was maintained over 5-7days 2-dimensional and collagen type I embedded mesenchymal cells. Breast-derived human epithelial and mesenchymal cells co-cultured in microwells and transwells showed similar increase in tumor cell proliferation rates but with a reduction of 80% or more in the total cells needed compared to transwells. Flow cytometer read-outs of proliferation and tumor cell distribution of CD44/CD24 cell surface receptors were similar among transwells and microwells. Adjacent culture of mouse derived fibroblasts (NIH-3T3) and human tumor cells showed recovery of genomic sample without cell-population mixing using primers for specific for human and mouse GAPDH. Doubling of the number of microwells with mesenchymal cells modulated the observed proliferative effect in tumor cells suggesting that mesenchymal cell ratios modulate tumor response to paracrine Hh signaling. This observation could not be replicated in macro-scale platforms as active Hh signaling in the mesenchyme is achieved via cell growth arrest using a combination of low serum and cell confluence. The open multi-microwell array represents a new user-friendly multi-culture microscale array with optimized capabilities for the study of paracrine signaling among discrete cell populations compared to macroscale culture technology. Citation Format: Karla P. Ramos, Fernando Boria, Maribella Domenech. Open multi-microwell array for the study of paracrine signaling in tumors. [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 B02.

Triple negative breast cancer (TNBC) is a clinical therapeutic challenge due to the lack of receptors for estrogen, progesterone, and human epidermal growth factor receptor 2 which limits treatment options to chemotherapy and radiation. With current standard therapy less than 30% will survive the 5-year remission rate, especially Hispanic and African-American women where TNBC is more frequent and has the lowest (500 samples) show a strong correlation among TNBC patients and mesenchymal-driven Hh signaling in >80% of samples. We evaluated the distribution of mesenchymal cell-sub-types and stem cells among the sub-set of basal TNBC samples (82 samples). Gene markers associated to myofibroblasts, EMT, mesenchymal stem cell and cancer stem cell were found in samples with active Hh signaling and co-expressed in groups of two or more which represented the 45.6% of the total samples. The most abundant cell sub-type were cancer stem cells with 43% of the total samples and alone by itself in 13.9% of samples. Together, these results indicate that the Hh-pathway and adjacent tumor microenvironment are key players in the progression of TNBC. Mesenchymal cell component can modulate tumor growth behavior induced by Hh signaling and is a potential new clinical target for TNBC. Citation Format: Maribella Domenech, Karla P. Ramos, Wandaliz Torres, Fernando Boria. Mesenchymal-driven hedgehog signaling drives tumor cell growth and is a potential new target for triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B05.

Despite strong evidence for the involvement of the stroma in Hedgehog signaling, little is known about the identity of the stromal cells and the signaling mechanisms that mediate the growth promoting effect of Hh signaling. We developed an in vitro co-culture model using microchannel technology to examine the effect of paracrine Hh signaling on proliferation of prostate cancer cells. We show here that activation of Hh signaling in myofibroblasts is sufficient to accelerate tumor cell growth. This effect was independent of any direct effect of Hh ligand on tumor cells or other cellular components of the tumor stroma. Further, the trophic effect of Hh pathway activation in myofibroblasts does not require collaboration of other elements of the stroma or direct physical interaction with the cancer cells. By isolating the tropic effect of Hh pathway activation in prostate stroma, we have taken the first step toward identifying cell-specific mechanisms that mediate the effect of paracrine Hh signaling on tumor growth.

Polydimethylsiloxane (PDMS) has become a staple of the microfluidics community by virtue of its simple fabrication process and material attributes, such as gas permeability, optical transparency, and flexibility. As microfluidic systems are put toward biological problems and increasingly utilized as cell culture platforms, the material properties of PDMS must be considered in a biological context. Two properties of PDMS were addressed in this study: the leaching of uncured oligomers from the polymer network into microchannel media, and the absorption of small, hydrophobic molecules (i.e. estrogen) from serum-containing media into the polymer bulk. Uncured PDMS oligomers were detectable via MALDI-MS in microchannel media both before and after Soxhlet extraction of PDMS devices in ethanol. Additionally, PDMS oligomers were identified in the plasma membranes of NMuMG cells cultured in PDMS microchannels for 24 hours. Cells cultured in extracted microchannels also contained a detectable amount of uncured PDMS. It was shown that MCF-7 cells seeded directly on PDMS inserts were responsive to hydrophilic prolactin but not hydrophobic estrogen, reflecting its specificity for absorbing small, hydrophobic molecules; and the presence of PDMS floating in wells significantly reduced cellular response to estrogen in a serum-dependent manner. Quantification of estrogen via ELISA revealed that microchannel estrogen partitioned rapidly into the surrounding PDMS to a ratio of approximately 9:1. Pretreatments such as blocking with serum or pre-absorbing estrogen for 24 hours did not affect estrogen loss from PDMS-based microchannels. These findings highlight the importance of careful consideration of culture system properties when determining an appropriate environment for biological experiments.

Phosphodiesterase 4D (PDE4D) was implicated as a candidate gene in prostate cancer through a transposon-mutagenesis screen in mice. Functionally, PDE4D regulates cellular levels of cAMP, which is critical for multiple signaling pathways including protein kinase A and mTor. PDE4D expression was localized to the prostate epithelial cells and increased in the earliest stages of human prostate cancer (prostate intraepithelial neoplasia) through aggressive metastatic prostate cancer. Based on its function, PDE4D may also function to regulate signaling between epithelial and stromal cells. The purpose of this study was to examine the role of PDE4D signaling in prostate cancer and how it is influenced by the microenvironment. The role of PDE4D in prostate cancer was assessed both in vivo, using genetically modified mouse and xenograft models, and in vitro, using prostate cancer cell lines and novel microchannel cultures. Castration resistant C4 prostate cancer cells grown as xenografts under the kidney capsules of nude mice and were treated with control or PDE4D inhibitors cilomilast and NVP-ABE171. Treatment with cilomilast or NVP-ABE171 decreased the overall size of the C4 xenografts and increased apoptotic cells. For in vitro studies a novel microchannel culture system was used to examine to examine the effect of the microenvironment on PDE4D signaling. In microchannels distinct cell types can be cultured in small volumes (2-4 microliters), which concentrates secreted factors and allows for either culture with direct contact between the cells or separation between multiple cell types but permits the diffusion of soluble signaling factors. The effect of co-culturing prostate stromal and epithelial cells on growth, PDE4D signaling, growth factor secretion, and gene expression was examined. In this model prostate epithelial cells were co-cultured with prostate stromal cells. Proliferation of the epithelial cells increased when they were co-cultured with stromal cells. This proliferation occurred when the cells are cultured in chambers separated by diffusion ports that allow exchange of soluble factors. Preliminary studies showed differential secretion of growth factors and cytokines, including transforming growth factor beta (TGFβ) and vascular endothelial growth factor (VEGF), between cells in monoculture and co-culture. Initial studies also suggested that PDE4D inhibition might have differential effects on the prostate epithelial and stromal cells in monoculture and co-culture. The in vitro data demonstrated that co-culture of prostate epithelial and stromal cells can lead to increased proliferation, differential expression of secreted factors and genes, as well as response to PDE4D inhibitor treatment. Results from the in vivo studies suggested that PDE4D inhibitors may be a useful strategy for prostate cancer treatment as they decreases the growth and increased apoptosis of prostate cancer xenografts in vivo. Overall the results of these studies indicated that PDE4D might have a role in signaling between prostate epithelial and stromal cells and that some of the anti-tumor effects of PDE4D inhibitors are due to their impact on paracrine signaling. Citation Format: Ginny L. Powers, Kimberly DP Hammer, Xiaojing Su, Maribella Domenech, David J. Beebe, Paul C. Marker. The role of phosphodiesterase 4D and the microenvironment in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B22.

Our laboratory used an insertional somatic mutagenesis screen in mice to identify novel genes that may drive prostate cancer initiation and progression. Using this screen, we have identified candidate prostate cancer genes including the candidate oncogene phosphodiesterase 4D (PDE4D). Investigation of PDE4D showed that it was over-expressed in human prostate cancer cell lines and in human prostate cancers. Stable knockdown of PDE4D by shRNA reduced the growth and migration of prostate cancer cells in vitro and in vivo. PDE4D has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Several small molecule PDE4D selective inhibitors have been developed and tested in clinical trials for COPD. Although these studies showed that the inhibitors had modest efficacy as a therapy for COPD, they confirmed that the inhibitors were safe for use in humans at doses that inhibited PDE4D in vivo. Based on our previous studies with PDE4D, we postulated that PDE4D small molecule inhibitors would be candidate anti-prostate cancer drugs. Daily oral administration of PDE4D inhibitors, NVP-ABE171 or cilomilast, for 6 weeks significantly reduced the growth of LNCaP-C4 prostate cancer xenografts in immune-compromised mice. Under in vitro conditions in which prostate cancer cell growth was inhibited by either NVP-ABE171 or cilomilast, we also observed changes in the activation of potential downstream molecular targets of PDE4D inhibition. Affected pathways included the sonic hedgehog and mitogen-activated protein kinase pathways. We also tested the potential role of the PDE4D pathway in prostate cancer stromal-epithelial interactions by modeling these interactions using novel microchannel co-culture devices. In this context, PDE4D inhibitors reduced the activation of Shh pathway target genes in mesenchymal cells and reduced the proliferation of epithelial cancer cell lines, supporting the potential importance of the PDE4D pathway for prostate cancer stromal-epithelial interactions. We are continuing in vitro and in vivo studies with these candidate drugs to further support the use of NVP-ABE171 and cilomilast as anti-prostate cancer drugs and to understand their mechanism of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3046. doi:10.1158/1538-7445.AM2011-3046

The cellular microenvironment plays a critical role in shaping and directing the process of communication between the cells. Soluble signals are responsible for many cellular behaviors such as cell survival, proliferation and differentiation. Despite the importance of soluble signals, canonical methods are not well suited to the study of soluble factor interactions between multiple cell types. Macro-scale technology often puts cells into a convective environment that can wash away and dilute soluble signals from their targets, minimizing local concentrations of important factors. In addition, current methods such as transwells, require large numbers of cells and are limited to studying just two cell types. Here, we present data supporting the use of microchannels to study soluble factor signaling providing improved sensitivity as well as the ability to move beyond existing co-culture and conditioned medium paradigms. In addition, we present data suggesting that microculture can be used to unmask effects of population demographics. In this example the data support the hypothesis that a growth promoting subpopulation of cells exists in the mouse mammary gland.