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Breast cancer (BCa) proliferates within a complex, three-dimensional microenvironment amid heterogeneous biochemical and biophysical cues. Understanding how mechanical forces within the tumor microenvironment (TME) regulate BCa phenotype is of great interest. We demonstrate that mechanical strain enhanced the proliferation and migration of both estrogen receptor (ER)+ and triple-negative (TNBC) human and mouse BCa cells. Furthermore, a critical role for exosomes derived from cells subjected to mechanical strain in these pro-tumorigenic effects was identified. Exosome production by TNBC cells increased upon exposure to oscillatory strain (OS), which correlated with elevated cell proliferation. Using a syngeneic, orthotopic mouse model of TNBC, we identified that preconditioning BCa cells with OS significantly increased tumor growth and myeloid-derived suppressor cells (MDSCs) and M2 macrophages at the TME. This pro-tumorigenic myeloid cell enrichment also correlated with a decrease in CD8+ T cells. An increase in PD-L1+ exosome release from BCa cells following OS supported additive T cell inhibitory functions in the TME. The role of exosomes in MDSC and M2 macrophage was confirmed in vivo by cytotracking fluorescent exosomes, derived from labeled 4T1.2 cells, preconditioned with oscillatory strain. Additionally, in vivo internalization and intratumoral localization of tumor-cell derived exosomes was observed within MDSCs, M2 macrophages, and CD45-negative cell populations following direct injection of fluorescently-labeled exosomes. Our data demonstrate that exposure to mechanical strain promotes invasive and pro-tumorigenic phenotypes in BCa cells, indicating that mechanical strains can impact the growth and proliferation of cancer cell, alters exosome production by BCa, and induces immunosuppression in the TME by dampening anti-tumor immunity.

Among gynecologic malignancies, ovarian cancer (OC) has the poorest survival rate, and its clinical management remains challenging due to the high rate of recurrence and chemoresistance. Improving survival for these patients is critical, although this requires the ability to translate preclinical studies to actual patient care: bench to bedside and back. Our objective was to develop a preclinical model that accurately represents tumor biology and its microenvironment. We utilized SKOV-3, OVCAR-8, and CS-99 cell lines to show that this model was suitable for in vitro assessment of cell proliferation. We tested OC cells independently and in co-culture with cancer associated fibroblasts (CAFs) or immune cells. Additionally, we used patient-derived ovarian carcinoma and carcinosarcoma samples to show that the system maintains the histologic morphology of the primary tissue after 7 days. Moreover, we tested the response to chemotherapy using both cell lines and patient-derived tumor specimens and confirmed that cell death was significantly higher in the treated group compared to the vehicle group. Finally, we immune profiled the 3-D model containing patient tissue after several days in the bioreactor system and revealed that the immune populations are still present. Our data suggest that this model is a suitable preclinical model to aid in research that will ultimately impact the treatment of patients with gynecologic cancer.

The tumor microenvironment is a key regulator of tumor biology and response to therapeutic intervention, with intercellular communication between tumor and stromal cells regulating growth and progression. While two dimensional cultures are commonly utilized for in vitro preclinical studies, they do not recapitulate the tissue microenvironment or three dimensional tissue architecture. Herein, we utilize tissue engineering strategies and patient-derived specimen to develop ex vivo non-small cell lung tumor models. These models allow for evaluation of tumor-stromal interactions and response to immune directed therapies while keeping the native tissue microenvironment intact. For this study, tumor models were generated utilizing remnant lung tumor specimen from consented patients undergoing surgical tumor resection. 5 mm diameter tissue cores were placed in a volume of extracellular matrix within a perfusion bioreactor platform, and through-channels were generated to provide nutrient circulation during ex vivo culture. Spatial profiling using the Nanostring GeoMx platform, multiplex cytokine analysis, histologic and flow cytometric analyses were performed following culture. Primary human tumor specimens cultured ex vivo maintain histologic architecture and representative cell populations following 14 days culture. Tissues treated with an anti-programmed cell death protein 1 (PD-1) blocking antibody showed reduced IL-6 (380.8 ±133.9 pg/mL) levels within the circulating media when compared to IgG control treated tissues (1119 ±382.9 pg/mL, p=0.08). Spatial profiling showed increased proportions of dividing T cells (2.27 ± 1.02 IgG vs. 8.28 ± 1.171 anti-PD-1; p=0.02) and CD8+ memory T cells (0.09 ± 0.09 IgG vs. 1.74 ± 0.64 anti-PD-1; p=0.04) and a trend towards more natural killer cells (3.88 ± 0.73 IgG vs. 6.56 ± 3.14 anti-PD-1; p=0.067), along with decreased proportions of macrophages (14.33 ± 2.16 IgG vs. 6.44 ± 0.59 anti-PD-1; p=0.004) near the tumor in tissues treated with anti-PD-1 when compared to control. Additionally, genes within the tumor immune signature associated with programmed cell death ligand 1 suppression, anti-tumor cytotoxicity and T cell response, including CXCR6, CCL3, NKG7, CMKLR1, CD27 & PSMB10, were upregulated and genes associated with immune suppression, including IDO and HLA-E, were downregulated with anti-PD-1 treatment when compared to IgG control. Moving forward, this platform will allow for extensive characterization of tumor-stromal interactions, response to therapeutic intervention, and therapeutic resistance in a patient specific manner. Funding: Nanostring DSP Cancer Transcriptome Atlas Award; 1R21 CA263365-01A1; Respiratory Health Association Lung Cancer Award (RHA2022-01-LC). Citation Format: Kayla F. Goliwas, Anthony M. Wood, Young-il Kim, Joel L. Berry, James M. Donahue, Jessy S. Deshane. Tumor-stromal response to immune checkpoint blockade within patient tissue derived three-dimensional lung tumor models [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B33.

On March 1 and 2, 2018, the National Institutes of Health 2018 Progenitor Cell Translational Consortium (PCTC) and Cardiovascular Bioengineering Symposium (CVBE) was held at the University of Alabama at Birmingham. Convergence of life sciences and engineering to advance the understanding and treatment of heart failure was the theme of the meeting. Over 150 attendees were present for more than 40 scientists presenting their latest works on engineering human functional myocardium for disease modeling, drug development, and heart failure research. The scientists, engineers and physicians in the field of cardiovascular sciences, met and discussed on the most recent advances in their works and propose future strategies in overcoming the major roadblocks of cardiovascular bioengineering and therapy. Particular emphasis was given for manipulation and using of stem/progenitor cells, biomaterials, and methods to provide molecular, chemical and mechanical cues to cells in order to influence their identity and fate in vitro and in vivo. Collectively, these works are profoundly impacting and progressing toward deciphering the mechanisms and developing novel treatments for left ventricular dysfunction of failing hearts. Here we present some important perspectives that emerged from this meeting.

Tumor-stromal interactions within the tumor microenvironment (TME) influence lung cancer progression and response to therapeutic interventions, yet traditional in vitro studies fail to replicate the complexity of these interactions. Herein, we developed three-dimensional (3D) lung tumor models that mimic the human TME and demonstrate tumor-stromal crosstalk mediated by extracellular vesicles (EVs). EVs released by tumor cells, independent of p53 status, and fibroblasts within the TME mediate immunomodulatory effects; specifically, monocyte/macrophage polarization to a tumor-promoting M2 phenotype within this 3D-TME. Additionally, immune checkpoint inhibition in a 3D model that included T cells showed an inhibition of tumor growth and reduced hypoxia within the TME. Thus, perfused 3D tumor models incorporating diverse cell types provide novel insights into EV-mediated tumor-immune interactions and immune-modulation for existing and emerging cancer therapies.

Cellular exosome-mediated crosstalk in tumor microenvironment (TME) is a critical component of anti-tumor immune responses. In addition to particle size, exosome transport and uptake by target cells is influenced by physical and physiological factors, including interstitial fluid pressure, and exosome concentration. These variables differ under both normal and pathological conditions, including cancer. The transport of exosomes in TME is governed by interstitial flow and diffusion. Based on these determinants, mathematical models were adapted to simulate the transport of exosomes in the TME with specified exosome release rates from the tumor cells. In this study, the significance of spatial relationship in exosome-mediated intercellular communication was established by treating their movement in the TME as a continuum using a transport equation, with advection due to interstitial flow and diffusion due to concentration gradients. To quantify the rate of release of exosomes by biomechanical forces acting on the tumor cells, we used a transwell platform with confluent triple negative breast cancer cells 4T1.2 seeded in BioFlex plates exposed to an oscillatory force. Exosome release rates were quantified from 4T1.2 cells seeded at the bottom of the well following the application of either no force or an oscillatory force, and these rates were used to model exosome transport in the transwell. The simulations predicted that a larger number of exosomes reached the membrane of the transwell for 4T1.2 cells exposed to the oscillatory force when compared to controls. Additionally, we simulated the interstitial fluid flow and exosome transport in a 2-dimensional TME with macrophages, T cells, and mixtures of these two populations at two different stages of a tumor growth. Computational simulations were carried out using the commercial computational simulation package, ANSYS/Fluent. The results of this study indicated higher exosome concentrations and larger interstitial fluid pressure at the later stages of the tumor growth. Quantifying the release of exosomes by cancer cells, their transport through the TME, and their concentration in TME will afford a deeper understanding of the mechanisms of these interactions and aid in deriving predictive models for therapeutic intervention.

Engineered cardiac tissues fabricated from human induced pluripotent stem cells (hiPSCs) show promise for ameliorating damage from myocardial infarction, while also restoring function to the damaged left ventricular (LV) myocardium. For these constructs to reach their clinical potential, they need to be of a clinically relevant volume and thickness, and capable of generating synchronous and forceful contraction to assist the pumping action of the recipient heart. Design prerequisites include a structure thickness sufficient to produce a beneficial contractile force, prevascularization to overcome diffusion limitations and sufficient structural development to allow for maximal cell communication. Previous attempts to meet these prerequisites have been hindered by lack of oxygen and nutrient transport due to diffusion limits (100–200 μm) resulting in necrosis. This study employs a layer-by-layer (LbL) fabrication method to produce cardiac tissue constructs that meet these design prerequisites and mimic normal myocardium in form and function. Thick (>2 mm) cardiac tissues created from hiPSC-derived cardiomyocytes, -endothelial cells (ECs) and -fibroblasts (FBs) were assessed,in vitro, over a 4-week period for viability (t-tubule network development, gap junction communication as well as previously unseen, physiologically relevant conduction velocities (CVs) (>30 cm/s). These results demonstrate that LbL fabrication can be utilized successfully to create prevascularized, functional cardiac tissue constructs from hiPSCs for potential therapeutic applications.

Objective Mechanical forces including tension, compression, and shear stress are increasingly implicated in tumor progression and metastasis. Understanding the mechanisms behind epithelial ovarian cancer (EOC) progression and metastasis is critical, and this study aimed to elucidate the effect of oscillatory and constant tension on EOC. Methods SKOV-3 and OVCAR-8 EOC cell lines were placed under oscillatory tension for 3 days and compared to cells placed under no tension. Cell proliferation, migration, and invasion were analyzed while RNAseq and Western Blots helped investigate the biological mechanisms underlying the increasingly aggressive state of the experimental cells. Finally, in vivo experiments using SCID mice assisted in confirming the in vitro results. Results Oscillatory tension (OT) and constant tension (CT) significantly increased SKOV-3 proliferation, while OT caused a significant increase in proliferative genes, migration, and invasion in this cell line. CT did not cause significant increases in these areas. Neither OT nor CT increased proliferation or invasion in OVCAR-8 cells, while both tension types significantly increased cellular migration. Two proteins involved in metastasis, E-cadherin and Snail, were both significantly affected by OT in both cell lines, with E-cadherin levels decreasing and Snail levels increasing. In vivo, tumor growth and weight for both cell types were significantly increased, and ascites development was significantly higher in the experimental OVCAR-8 group than in the control group. Conclusions This study found that mechanical forces are influential in EOC progression and metastasis. Further analysis of downstream mechanisms involved in EOC metastasis will be critical for improvements in EOC treatment.

Cardiac tissue surrogates show promise for restoring mechanical and electrical function in infarcted left ventricular (LV) myocardium. For these cardiac surrogates to be useful in vivo, they are required to support synchronous and forceful contraction over the infarcted region. These design requirements necessitate a thickness sufficient to produce a useful contractile force, an area large enough to cover an infarcted region, and prevascularization to overcome diffusion limitations. Attempts to meet these requirements have been hampered by diffusion limits of oxygen and nutrients (100–200 µm) leading to necrotic regions. This study demonstrates a novel layer-by-layer (LbL) fabrication method used to produce tissue surrogates that meet these requirements and mimic normal myocardium in form and function. Thick (1.5–2 mm) LbL cardiac tissues created from human induced pluripotent stem cell-derived cardiomyocytes and endothelial cells were assessed, in vitro, over a 4-week period for viability (R 2 < 0.98). Functional performance assessment showed enhanced t-tubule network development, gap junction communication as well as conduction velocity (16.9 ± 2.3 cm s−1). These results demonstrate that LbL fabrication can be utilized successfully in creating complex, functional cardiac surrogates for potential therapeutic applications.

Current strategies for engineering cardiovascular cells and tissues have yielded a variety of sophisticated tools for studying disease mechanisms, for development of drug therapies, and for fabrication of tissue equivalents that may have application in future clinical use. These efforts are motivated by the need to extend traditional 2-dimensional (2D) cell culture systems into 3D to more accurately replicate in vivo cell and tissue function of cardiovascular structures. Developments in microscale devices and bioprinted 3D tissues are beginning to supplant traditional 2D cell cultures and preclinical animal studies that have historically been the standard for drug and tissue development. These new approaches lend themselves to patient-specific diagnostics, therapeutics, and tissue regeneration. The emergence of these technologies also carries technical challenges to be met before traditional cell culture and animal testing become obsolete. Successful development and validation of 3D human tissue constructs will provide powerful new paradigms for more cost effective and timely translation of cardiovascular tissue equivalents.

Functional myocardium derived from human induced pluripotent stem cells (hiPSCs) can be impactful for cardiac disease modeling, drug testing, and the repair of injured myocardium. However, when hiPSCs are differentiated into cardiomyocytes, they do not possess characteristics of mature myocytes which limits their application in these endeavors. We hypothesized that mechanical and electrical stimuli would enhance the maturation of hiPSC-derived cardiomyocyte (hiPSC-CM) spheroids on both a structural and functional level, potentially leading to a better model for drug testing as well as cell therapy. Spheroids were generated with hiPSC-CM. For inducing mechanical stimulation, they were placed in a custom-made device with PDMS channels and exposed to cyclic, uniaxial stretch. Spheroids were electrically stimulated in the C-Pace EP from IONOptix for 7 days. Following the stimulations, the spheroids were then analyzed for cardiomyocyte maturation. Both stimulated groups of spheroids possessed enhanced transcript and protein expressions for key maturation markers, such as cTnI, MLC2v, and MLC2a, along with improved ultrastructure of the hiPSC-CMs in both groups with enhanced Z-band/Z-body formation, fibril alignment, and fiber number. Optical mapping showed that spheroids exposed to electrical stimulation were able to capture signals at increasing rates of pacing up to 4 Hz, which failed in unstimulated spheroids. Our results clearly indicate that a significantly improved myocyte maturation can be achieved by culturing iPSC-CMs as spheroids and exposing them to cyclic, uniaxial stretch and electrical stimulation.

At the University of Alabama at Birmingham, a partnership formed between faculty in the School of Nursing, the School of Engineering, the Honors College, and UAB Hospital to facilitate interaction between students of biomedical engineering and clinicians. Clinicians needed a way to address their unsolved problems in delivery of health care and students needed a way to engage in meaningful real-world clinical experiences. The authors identified barriers to clinical innovation, created formal freshman and sophomore level courses to lower those barriers, and created a web-based tool to facilitate bringing together domains of expertise. The courses provide a formal mechanism for students to engage in clinical innovation. The web tool creates a collaborative innovation hub that provides a pathway for clinicians at UAB Hospital to enter their problems into a viewable database. The web tool also allows users to form project teams and use built-in functionality to enhance the possibility that they will successfully solve significant problems. Results to date have demonstrated that clinicians are invested in students' efforts to improve approaches to patient care, students are empowered to seek challenging clinical innovation opportunities, and interacting in a digital space represents a viable way to improve collaboration.

Background As patient-derived xenografts and other preclinical models of neuroendocrine tumors for testing personalized therapeutics are lacking, we have developed a perfused, 3D bioreactor model to culture tumor surrogates from patient-derived neuroendocrine tumors. This work evaluates the duration of surrogate culture and surrogate response to a novel antibody-drug conjugate. Methods Twenty-seven patient-derived neuroendocrine tumors were cultured. Histologic sections of a pancreatic neuroendocrine tumor xenograft (BON-1) tumor were assessed for SSTR2 expression before tumor implantation into 2 bioreactors. One surrogate was treated with an antibody-drug conjugate composed of an anti-mitotic Monomethyl auristatin-E linked to a somatostatin receptor 2 antibody. Viability and therapeutic response were assessed by pre-imaging incubation with IR-783 and the RealTime-Glo AnnexinV Apoptosis and Necrosis Assay (Promega Corporation, Madison, WI) over 6 days. A primary human pancreatic neuroendocrine tumor was evaluated similarly. Results Mean surrogate growth duration was 34.8 days. Treated BON-1 surrogates exhibited less proliferation (1.2 vs 1.9-fold) and greater apoptosis (1.5 vs 1.1-fold) than controls, whereas treated patient-derived neuroendocrine tumor bioreactors exhibited greater degrees of apoptosis (13- vs 9-fold) and necrosis (2.5- vs 1.6-fold). Conclusion Patient-derived neuroendocrine tumor surrogates can be cultured reliably within the bioreactor. This model can be used to evaluate the efficacy of antibody-guided chemotherapy ex vivo and may be useful for predicting clinical responses.

It is well documented that electrospun tissue engineering scaffolds can be fabricated with variable degrees of fiber alignment to produce scaffolds with anisotropic mechanical properties. Several attempts have been made to quantify the degree of fiber alignment within an electrospun scaffold using image-based methods. However, these methods are limited by the inability to produce a quantitative measure of alignment that can be used to make comparisons across publications. Therefore, we have developed a new approach to quantifying the alignment present within a scaffold from scanning electron microscopic (SEM) images. The alignment is determined by using the Sobel approximation of the image gradient to determine the distribution of gradient angles with an image. This data was fit to a Von Mises distribution to find the dispersion parameter κ, which was used as a quantitative measure of fiber alignment. We fabricated four groups of electrospun polycaprolactone (PCL) + Gelatin scaffolds with alignments ranging from κ = 1.9 (aligned) to κ = 0.25 (random) and tested our alignment quantification method on these scaffolds. It was found that our alignment quantification method could distinguish between scaffolds of different alignments more accurately than two other published methods. Additionally, the alignment parameter κ was found to be a good predictor the mechanical anisotropy of our electrospun scaffolds. The ability to quantify fiber alignment within and make direct comparisons of scaffold fiber alignment across publications can reduce ambiguity between published results where cells are cultured on "highly aligned" fibrous scaffolds. This could have important implications for characterizing mechanics and cellular behavior on aligned tissue engineering scaffolds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1680-1686, 2016.

Introduction: Breast cancer (BCa) occurs with a complex, three-dimensional microenvironment that involves heterogeneous biochemical and biophysical cues. Understanding how mechanical properties within the tumor microenvironment (TME) regulate breast cancer phenotype and immunosuppression is of great interest. Materials and Methods: BCa cells (MCF-7, MDA-MB-231 or 4T1.2) cultured to confluence on collagen coated FlexCell culture plates were subjected to 10% uniaxial cyclic/oscillatory strain at 0.3 Hz, or 10% constant strain, or no strain for 48 hours. They were isolated for analysis of proliferation (MTT assay and cell count by trypan blue), and migration (transwell and wound healing assay). Exosomes from conditioned media were isolated by differential centrifugation or using the Total Exosome Isolation kit. The purified exosomes were quantified by NanoSight and characterized by ImageStream. 5 × 105 4T1.2 cells or PKH67-labeled strained or control cells were injected into the mammary fat pad of BALB/c mice. Tumor volume was measured at the indicated time points after injection. Tumor-infiltrating immune cells and the internalization of exosomes were analyzed by flow cytometry on day 14 post implantation. In some experiments, on day 6 after tumor injection, 7.5 × 108 PHK67-labeled tumor cell-derived exosomes or PBS were injected into the tumor nodule. Tumor tissues were harvested for analysis of the internalization of exosomes by immune cells and tumor cells on days 2 and 8 after exosome injection. Results: We show that mechanical strain enhanced the proliferation and migration of BCa cells in vitro. Exosome concentrations produced by triple negative breast cancer (TNBC) cells were increased following exposure to oscillatory strain. Phenotyping exosomes by ImageStream showed that the percentages of CD81+PD-L1+ and CD63+PD-L1+ exosomes were increased after exposure to oscillatory strain. Using a syngeneic orthotopic mouse model of TNBC, we showed that preconditioning with mechanical strain increased tumor growth. The percentages of tumor-infiltrating monocytic myeloid-derived suppressor cells (M-MDSC) and recruited macrophages were increased while CD8+ T cells decreased in the TME of mice implanted with 4T1.2 cells preconditioned with oscillatory strain. Further, exosome internalizations by M-MDSC and recruited macrophages were elevated when tumor cells were preconditioned with oscillatory strain. Moreover, exosomes internalization by immune cells and tumor cells in TME were identified by PKH67 positive signals on days 2 and 8 after injection of PKH67-labeled exosomes into tumor nodules by flow cytometry analyses and confocal microscope imaging. Conclusions: Our data indicate that exposure to mechanical strain promotes invasive and pro-tumorigenic phenotypes in BCa, alters exosome production by BCa and induces immunosuppression in the TME. Citation Format: Yong Wang, Kayla F. Goliwas, Paige E. Severino, Kenneth Hough, Derek Van Vessem, Hong Wang, Sultan Tousif, Roy P. Koomullil, Andra R. Frost, Selvarangan Ponnazhagen, Joel L. Berry, Jessy S. Deshane. Mechanical strain induces phenotypic changes in breast cancer cells and promotes immunosuppression in the tumor microenvironment [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 1711.

Introduction: Epithelial ovarian cancer (EOC) is the most common cause of death among gynecologic malignancies. This is a result of challenging clinical management due to the high rate of recurrence and the development of chemoresistance in EOC patients. Therefore, the development of new therapeutics is crucial. Unfortunately, the success rate of establishing new drugs is very low. A major factor contributing to this is the lack of preclinical models that accurately predict in vivo efficacy of candidate therapeutics. Preclinical models do not accurately mimic the biomechanical regulation of the tumor microenvironment (TME) and cannot predict in vivo efficacy of candidate therapeutics. We have developed an EOC 3D perfused bioreactor system that recapitulates EOC tumor biology; better predicts the clinical response of new drug candidates, thereby eliminating ineffective candidates prior to clinical trials; and incorporates tumor biomechanical regulation. Materials and Methods: EOC cell lines (luciferase-tagged SKOV-3 and OVCAR-8) were embedded in a relevant ECM (5.25 × 105 total cells/100 uL ECM) containing 90% bovine collagen I + 10% basement membrane (Matrigel) and injected into a polydimethylsiloxane bioreactor (PDMS). The volume was then perforated by five wires and the bioreactor was placed in the incubator (37C, 5%CO2) for polymerization. After that, the wires were removed to create microchannels so that the cell culture media (RPMI 10% FBS, 1% penicillin-streptomycin-gentamicin) can flow through the perfusion bioreactor system and provide nutrient delivery and gas exchange, which helps maintain viability and function of surrounding cells. Finally, the bioreactors were connected to a peristaltic pump that allows the cell culture media to run over 14 days, along the channels and through the matrix. We monitored cell growth over 14 days using bioluminescence (BLI) and these results were validated with histologic analysis of the cell density. In order to prove that the bioreactor keeps EOC biology, by immunohistochemical (IHC) analysis with p53 and PAX8, clinically used ovarian cancer markers. Results and Discussion: For our preliminary data utilizing the 3D perfusion bioreactor system in ovarian cancer, we first used the luciferase-tagged OVCAR-8 cell line to evaluate cell growth. The bioluminescence signal showed a linear increase over 14 days. These results were validated histologically with cell density by measuring the number of nucleated cells per micron2. Graphical representation of the region of interest (ROI) showed a high correlation between manual determination of cell number and bioluminescence score. In our second experiment, we used the luciferase-tagged SKOV-3 cell line (high-grade serous origin) and we also monitored cell growth over 14 days using BLI and histologic analysis of the cell density as described for OVCAR-8 using the same conditions in the bioreactor. IHC analysis with p53 and PAX8 was positive and proved that the perfusion bioreactor system keeps EOC biology. Citation Format: Alba Martinez, Molly Buckley, Joel Berry, Rebecca Arend, Michael Birrer. 3D perfusion bioreactor system as a model for studying cell biology of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B12.

The tumor microenvironment is a key regulator of tumor biology and response to therapeutic intervention, with intercellular communication between tumor and stromal cells known to regulate growth and progression. While two-dimensional cell culture is the most common method utilized for in vitro preclinical studies, it creates an artificial environment that does not take into account critical tissue components, including the tissue microenvironment (i.e., matrix proteins and stromal cell populations) and the three-dimensional tissue architecture/dimensionality. Recently, in vitro engineered tissues have been implemented as surrogates of human pathophysiology in biomedical and pharmaceutical research. Herein we utilize tissue engineering strategies to develop in vitro non-small cell lung tumor mimics that include key tissue components, including matrix proteins and stromal cell populations, utilizing patient-derived specimen. With the inclusion of stromal cell populations, these tumor mimics allow for evaluation of tumor-stromal interactions and could be utilized to determine the impact of these interactions on therapeutic response. Engineered tumor mimics were generated utilizing freshly isolated lung tumor specimen from consented patients undergoing surgical tumor resection. 5-mm-diameter tissue cores were produced and placed in a volume of extracellular matrix (ECM) within the bioreactor platform and maintained via a closed perfusion platform to provide nutrient circulation. Histologic and flow cytometric analyses were performed to evaluate cellular heterogeneity within the tumor mimics following culture. Primary human lung tumor specimens cultured ex vivo in the engineered tumor mimic platform maintain histologic architecture and representative cell populations, including endothelial cells (CD31+,10.46%), epithelial cells (EpCAM+,25.15%), fibroblasts (CD90+, 7.23%), CD8+ T cells (CD3+CD8+, 2.95%), and macrophages (CD14+CD64+CD11b+, 9.25%) following 14 days’ culture. Additionally, a tumor-promotive microenvironment is supported by this platform with maintenance of monocytic and granulocytic myeloid-derived suppressor cells (8.59% and 0.83 %, respectively) and tumor-promotive macrophages (M2 like, 81.3% of macrophages). Furthermore, the hypoxic biochemical microenvironment is recapitulated with carbonic anhydrase 9-positive cell populations maintained during culture. Moving forward, this platform will allow for extensive characterization of tumor-stromal interactions, response to therapeutic intervention, and therapeutic resistance in a patient-specific manner. Citation Format: Kayla F. Goliwas, Yong Wang, Joel L. Berry, Victor J. Thannickal, James M. Donahue, Jessy S. Deshane. Engineered three-dimensional lung tumor mimics maintain tissue heterogeneity allowing for investigation of tumor-stromal interactions [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B23.

There is a need for preclinical testing systems that predict the efficacy, safety and pharmacokinetics of cancer therapies better than existing in vitro and in vivo animal models. An approach to the development of predictive in vitro systems is to more closely recapitulate the cellular and spatial complexity of human cancers. One limitation of using current in vitro systems to model cancers is the lack of an appropriately large volume to accommodate the development of this complexity over time. To address this limitation, we have designed and constructed a novel flow-perfusion bioreactor system that can support large-volume, engineered tissue comprised of multicellular cancer surrogates by modifying current microfluidic devices. Key features of this technology are a three-dimensional (3D) volume (1.2 cm3 ) that has greater tissue thickness than is utilized in existing microfluidic systems and the ability to perfuse the volume, enabling the development of realistic tumour geometry. The constructs were fabricated by infiltrating porous carbon foams with an extracellular matrix (ECM) hydrogel and engineering through-microchannels. The carbon foam structurally supported the hydrogel and microchannel patency for up to 161 h. The ECM hydrogel was shown to adhere to the carbon foam and polydimethylsiloxane flow chamber, which housed the hydrogel-foam construct, when surfaces were coated with glutaraldehyde (carbon foam) and nitric acid (polydimethylsiloxane). Additionally, the viability of breast cancer cells and fibroblasts was higher in the presence of perfused microchannels in comparison to similar preparations without microchannels or perfusion. Therefore, the flow-perfusion bioreactor system supports cell viability in volume and stromal contexts that are physiologically-relevant. Copyright © 2015 John Wiley & Sons, Ltd.

The use of in vitro, engineered surrogates in the field of cancer research is of interest for studies involving mechanisms of growth and metastasis, and response to therapeutic intervention. While biomimetic surrogates better model human disease, their complex composition and dimensionality make them challenging to evaluate in a real-time manner. This feature has hindered the broad implementation of these models, particularly in drug discovery. Herein, several methods and approaches for the real-time, non-invasive analysis of cell growth and response to treatment in tissue-engineered, three-dimensional models of breast cancer are presented. The tissue-engineered surrogates used to demonstrate these methods consist of breast cancer epithelial cells and fibroblasts within a three dimensional volume of extracellular matrix and are continuously perfused with nutrients via a bioreactor system. Growth of the surrogates over time was measured using optical in vivo (IVIS) imaging. Morphologic changes in specific cell populations were evaluated by multi-photon confocal microscopy. Response of the surrogates to treatment with paclitaxel was measured by optical imaging and by analysis of lactate dehydrogenase and caspase-cleaved cytokeratin 18 in the perfused medium. Each method described can be repeatedly performed during culture, allowing for real-time, longitudinal analysis of cell populations within engineered tumor models.

The purpose of the work is to investigate whether the electromagnetic properties of multi-walled carbon nanotubes (MWCNT) in the presence of radiofrequency (RF) energy is (1) safe, and (2) improves the precision of the therapeutic efficiency of the RF-ablation (RFA) procedure. An in vitro phantom was created for evaluating temperature near RF treated nanotubes. For the in vivo study, three baboons and six pigs were submitted for RFA procedure in superior/inferior kidney poles embolized with a non-adherent, lipophilic embolic agent (marsembol) with or without MWCNT. Tissue damage in the surrounding kill zone was assayed through caspase-3 activation. The in vitro results showed marked heat increase only in the region of the nanotubes. In vivo, necrosis/ischemic damage resulted from RFA therapy alone, RFA plus marsembol only. In marsembol + MWCNT condition, dramatic disruption of cell membranes and sub-cellular organelles was found whereas the nuclear membranes and basal cell membranes remained largely intact. The marsembol vaporized under RFA and tissue fluid filled the space. This caused the MWCNT to cluster within the new aqueous environment. RFA plus marsembol + MWCNT created a well-defined demarcation between healthy and apoptotic cells as evidenced by a marked reduction of caspase-3 expression. By contrast, there was a much less defined ablation zone in the absence of MWCNT. In conclusion, the combination of RFA plus marsembol + MWCNT embolization delineated the kill zone in vitro and in vivo. We demonstrate that MWCNTs remain in the ablation region thus minimizing their migration to the systemic circulation.

Functional myocardium derived from human induced pluripotent stem cells (hiPSCs) can be impactful for cardiac disease modeling, drug testing, and the repair of injured myocardium. However, when hiPSCs are differentiated into cardiomyocytes, they do not possess characteristics of mature myocytes which limits their application in these endeavors. We hypothesized that mechanical and electrical stimuli would enhance the maturation of hiPSC-derived cardiomyocyte (hiPSC-CM) spheroids on both a structural and functional level, potentially leading to a better model for drug testing as well as cell therapy. Spheroids were generated with hiPSC-CM. For inducing mechanical stimulation, they were placed in a custom-made device with PDMS channels and exposed to cyclic, uniaxial stretch. Spheroids were electrically stimulated in the C-Pace EP from IONOptix for 7 days. Following the stimulations, the spheroids were then analyzed for cardiomyocyte maturation. Both stimulated groups of spheroids possessed enhanced transcript and protein expressions for key maturation markers, such as cTnI, MLC2v, and MLC2a, along with improved ultrastructure of the hiPSC-CMs in both groups with enhanced Z-band/Z-body formation, fibril alignment, and fiber number. Optical mapping showed that spheroids exposed to electrical stimulation were able to capture signals at increasing rates of pacing up to 4 Hz, which failed in unstimulated spheroids. Our results clearly indicate that a significantly improved myocyte maturation can be achieved by culturing iPSC-CMs as spheroids and exposing them to cyclic, uniaxial stretch and electrical stimulation.

Introduction: Cells within the breast tumor mass are met with a variety of biophysical or mechanical signals that is associated with elevated compression or solid stress within the tumor interior, tension at the tumor periphery, and altered interstitial fluid pressure. Understanding how the mechanical stress within the tumor microenvironment (TME) regulates cancer cell phenotype is of interest, yet the response of breast cancer (BCa) cells to these forces is largely unknown. Our study aims to identify the impact of mechanical stress on BCa cell phenotype by mimicking the tension at the tumor periphery. Materials and Methods: BCa cells (MCF-7 or 4T1.2) were cultured to confluence on collagen coated FlexCell culture plates. These plates were then subjected to 10% uniaxial cyclic/oscillatory strain at 0.3 Hz, or 10% constant strain, or no strain for 48 hours. Strained or control cells were isolated for analysis of proliferation (MTT assay), and migration (8.0 μm pore transwell). Exosomes from conditioned media were isolated via differential centrifugation and purified exosomes were characterized by ImageStream. 5x105 4T1.2 cells or PKH-labeled strained or control cells were injected into the mammary fat pad of BALB/c mice. Tumor volume was measured over 14 days. Tumor-infiltrating immune cells and the uptake of exosomes were analyzed by flow cytometry on day-14 post implantation. Results: We determined significant increases in proliferation and migration of 4T1.2 and MCF-7 cells in vitro following exposure to oscillatory forces. The populations of CD63+, CD63+CD24+ and CD63+PD-L1+ exosomes were increased when 4T1.2 cells were exposed to oscillatory strain compared to unstrained control cells. Further, we investigated how oscillatory forces affect tumor growth and tumor-immune cell interactions in vivo by using a syngeneic, orthotopic mouse model of BCa. Mice implanted with 4T1.2 cells that were pre-exposed to oscillatory forces showed a significant increase in primary tumor growth at 8 and 11 days post tumor challenge. The percentages of tumor-infiltrating monocytic myeloid-derived suppressor cells (M-MDSC) and recruited macrophages were increased in the TME of mice implanted with 4T1.2 cells that were pre-exposed to oscillatory forces, while the granulocytic MDSC subset was not significantly different between the two groups. A marginal decrease in the percentage of CD8+ T cells was noted in the TME of mice implanted with strained 4T1.2 when compared to controls, suggesting immune suppression in the TME. Furthermore, exosome uptakes by M-MDSC and recruited macrophages were increased in the TME of mice implanted with PKH-labeled 4T1.2 cells, exposed to oscillatory strain. Conclusion: Together, these data indicate that exposure to mechanical stress changes BCa cell phenotype to an invasive and protumorigenic phenotype that promotes immunosuppressive effects in the TME. Citation Format: Yong Wang, Paige E. Severino, Kayla Goliwas, Kenneth Hough, Derek Van Vessem, Hong Wang, Andra R. Frost, Selvarangan Ponnazhagen, Joel L. Berry, Jessy S. Deshane. Mechanical strain induces phenotypic changes in breast cancer cells and promotes immunosuppression in the tumor microenvironment [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 1177.

Breast carcinomas are complex, three-dimensional tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix. In vitro models that more faithfully recapitulate this dimensionality and stromal microenvironment should more accurately elucidate the processes driving carcinogenesis, tumor progression, and therapeutic response. Herein, novel in vitro breast carcinoma surrogates, distinguished by a relevant dimensionality and stromal microenvironment, are described and characterized. A perfusion bioreactor system was used to deliver medium to surrogates containing engineered microchannels and the effects of perfusion, medium composition, and the method of cell incorporation and density of initial cell seeding on the growth and morphology of surrogates were assessed. Perfused surrogates demonstrated significantly greater cell density and proliferation and were more histologically recapitulative of human breast carcinoma than surrogates maintained without perfusion. Although other parameters of the surrogate system, such as medium composition and cell seeding density, affected cell growth, perfusion was the most influential parameter.

A preclinical testing model for cancer therapeutics that replicates in vivo physiology is needed to accurately describe drug delivery and efficacy prior to clinical trials. To develop an in vitro model of breast cancer that mimics in vivo drug/nutrient delivery as well as physiological size and bio-composition, it is essential to describe the mass transport quantitatively. The objective of the present study was to develop in vitro and computational models to measure mass transport from a perfusion system into a 3D extracellular matrix (ECM). A perfusion-flow bioreactor system was used to control and quantify the mass transport of a macromolecule within an ECM hydrogel with embedded through-channels. The material properties, fluid mechanics, and structure of the construct quantified in the in vitro model were input into, and served as validation of, the computational fluid dynamics (CFD) simulation. Results showed that advection and diffusion played a complementary role in mass transport. As the CFD simulation becomes more complex with embedded blood vessels and cancer cells, it will become more recapitulative of in vivo breast cancers. This study is a step toward development of a preclinical testing platform that will be more predictive of patient response to therapeutics than two-dimensional cell culture.

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

The bacterium Gluconacetobacter xylinus (previously known as Acetobacter xylinum) has been shown to produce cellulose fibrils of 10–30 nm in diameter. Through careful control of their motion, it is possible to direct them to produce well-defined three-dimensional (3D) scaffolds for tissue reconstruction. This chapter reviews the state of the art in fabrication of tissue engineered structures and biomedical implants using bacterial nanocellulose (BNC). Of particular focus is the use of electric fields to produce highly oriented cellulose networks and also the development 198of a microvascular network within BNC structures. The movement of G. xylinus at the nanoscale can be controlled by electric fields to create custom cellulose networks for engineered tissues and biomedical implants. This is the first attempt to control a bottom-up biofabrication process in three dimensions. The manipulation of electrokinetic forces acting upon a bacterial cell can produce complex cellulose patterns on the nanoscale not achievable in static culture. The ability to control the direction of fiber orientation could be readily expanded to weave structures of multiple fiber layers by changing the orientation of the applied electric field for each layer. Using this method, these structures could be tailored to have desired mechanical properties for a variety of applications, including tissue engineering, microelectromechanical systems (MEMS), textiles, and electronics.

To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes.

Due to their low immunogenicity, biodegradability and native cell-binding domains, fibrinogen fibers may be good candidates for tissue engineering scaffolds, drug delivery vehicles and other medical devices. We used a combined atomic force microscope (AFM)/optical microscope technique to study the mechanical properties of individual, electrospun fibrinogen fibers in dry, ambient conditions. The AFM was used to stretch individual fibers suspended over 13.5 μm wide grooves in a transparent substrate. The optical microscope, located below the sample, was used to monitor the stretching process. Electrospun fibrinogen fibers (diameter, 30–200 nm) can stretch to 74% beyond their original length before rupturing at a stress of 2.1 GPa. They can stretch elastically up to 15% beyond their original length. Using incremental stress–strain curves the viscoelastic behavior of these fibers was determined. The total stretch modulus was 4.2 GPa while the relaxed elastic modulus was 3.7 GPa. When held at constant strain, fibrinogen fibers display stress relaxation with a fast and slow relaxation time of 1.2 s and 11 s. In comparison to native and electrospun collagen fibers, dry electrospun fibrinogen fibers are significantly more extensible and elastic. In comparison to wet electrospun fibrinogen fibers, dry fibers are about 1000 times stiffer.

Current cardiovascular therapies are limited by the loss of endothelium, restenosis and thrombosis. The goal of this study was to develop a biomimetic hybrid nanomatrix that combined the unique properties of electrospun polycaprolactone (ePCL) nanofibers with self-assembled peptide amphiphiles (PAs). ePCL nanofibers have interconnected nanoporous structures, but are hampered by a lack of surface bioactivity to control cellular behavior. It has been hypothesized that PAs could self-assemble onto the surface of ePCL nanofibers and endow them with the characteristic properties of native endothelium. The PAs, which comprised hydrophobic alkyl tails attached to functional hydrophilic peptide sequences, contained enzyme-mediated degradable sites coupled to either endothelial cell-adhesive ligands (YIGSR) or polylysine (KKKKK) nitric oxide (NO) donors. Two different PAs (PA–YIGSR and PA–KKKKK) were successfully synthesized and mixed in a 90:10 (YK) ratio to obtain PA–YK. PA–YK was reacted with pure NO to develop PA–YK–NO, which was then self-assembled onto ePCL nanofibers to generate a hybrid nanomatrix, ePCL–PA–YK–NO. Uniform coating of self-assembled PA nanofibers on ePCL was confirmed by transmission electron microscopy. Successful NO release from ePCL–PA–YK–NO was observed. ePCL–YK and ePCL–PA–YK–NO showed significantly increased adhesion of human umbilical vein endothelial cells (HUVECs). ePCL–PA–YK–NO also showed significantly increased proliferation of HUVECs and reduced smooth muscle cell proliferation. ePCL–PA–YK–NO also displayed significantly reduced platelet adhesion compared with ePCL, ePCL–PA–YK and a collagen control. These results indicate that this hybrid nanomatrix has great potential application in cardiovascular implants.

Millions of people worldwide are diagnosed each year with valvular heart disease, resulting in hundreds of thousands of valve replacement operations. Prosthetic valve replacements are designed to correct narrowing or backflow through the valvular orifice. Although commonly used, these therapies have serious disadvantages including morbidity associated with long-term anticoagulation and limited durability necessitating repeat operations. The ideal substitute would be widely available and technically implantable for most cardiac surgeons, have normal hemodynamic performance, low risk for structural degeneration, thrombo-embolism and endocarditis, and growth potential for pediatric patients. Tissue engineered heart valves hold promise as a viable substitute to outperform existing valve replacements. An essential component to the development of tissue engineered heart valves is a bioreactor. It is inside the bioreactor that the scaffold and cells are gradually conditioned to the biochemical and mechanical environment of the valve to be replaced.

BACKGROUND AND PURPOSE: Novel stratagems to improve the efficacy of platinum coils in occluding cerebral aneurysms have primarily involved coating coils with materials thought likely to provoke more desirable histologic reactions. No investigations to date, however, have evaluated the utility of gold or vitronectin coatings, despite known endovascular histologic effects of these agents, which may be favorable for treating cerebral aneurysms. This study was conducted to evaluate the degree of endovascular histologic change associated with ultrathin gold- or vitronectin-coated platinum coils. It was hypothesized that such coatings would increase intra-aneurysmal intimal hyperplasia and the degree of luminal occlusion compared with standard platinum coils. MATERIALS AND METHODS: The ligated carotid artery rat model was used to study 4 different aneurysm coil conditions: no coil (sham-surgery controls), uncoated platinum coil, and gold- or vitronectin-coated platinum coil. Two weeks postimplantation, the aneurysms were harvested and stained with hematoxylin-eosin. Slides were evaluated for the degree of neointimal response by a pathologist blinded to treatment. Additional quantitative evaluation was performed blindly by using the ratio of intimal-to-luminal cross-sectional area. RESULTS: A gold- or vitronectin-coated platinum aneurysm coil produced a statistically significant increase in neointimal response compared with a sham (no coil). Arterial segments treated with gold-coated platinum coils also demonstrated a statistically significant 100% increase in neointimal response compared with those treated with bare platinum coils. CONCLUSIONS: In concordance with our hypothesis, ultrathin coatings of gold provoked a neointimal response and degree of luminal occlusion greater than that of plain platinum aneurysm coils in a rat arterial occlusion model.

Small-caliber synthetic grafts used for coronary bypass artery grafting are compromised by thrombogenicity and accelerated intimal thickening, resulting in early graft occlusion. Herein we describe the fabrication and physical properties of small-caliber blood vessels using decellularized porcine aortic segments. These vessels were further coated with human saphenous vein endothelial cells (HSVECs) for future clinical applications. Chemical staining of decellularized vessels showed that they preserved their native matrix architecture, including several collagen layers in between internal and external elastin layers. The burst pressure for the decellularized vessels was higher than 1,000 mmHg. HSVECs, seeded on the luminal side, adhered to the matrix and formed a uniform monolayer. HSVEC-seeded vessels produced prostaglandin I2 and released vasoactive agents in response to the calcium ionophore A23187. These results show that engineered blood vessels coated with the host endothelial cells possess morphologic and functional characteristics of human small-caliber vessels. Tissue-engineered vessels may potentially be useful clinically as vascular grafts.

Autologous and synthetic vessel grafts have been used as a vascular substitute for cardiovascular bypass procedures. However, these materials are limited by the availability of appropriate caliber autologous vessels, increased susceptibility to thrombosis and intimal hyperplasia following surgery. Electrospinning technology offers the potential for controlling composition, structure and mechanical properties of biomaterials. Vascular graft scaffolds have been fabricated using electrospun polymer blends of Type I collagen, elastin from ligamentum nuchae, and poly (d,l-lactide-co-glycolide). This study demonstrates improved electrospinning characteristics versus previous studies by increasing polymer concentration and adding PLGA to the polymer blend. Additionally, new in vitro biocompatibility and mechanical testing data is presented. The scaffolds possess tissue composition and mechanical properties similar to native vessels. The electrospun vessel matrix is biocompatible and does not elicit local or systemic toxic effects when implanted in vivo. This study demonstrates the promise of electrospinning as a fabrication process for a functional vascular graft for clinical use.

Vascular stents influence the post-procedural hemodynamic environment in ways that may encourage restenosis. Understanding how stents influence flow patterns may lead to more hemodynamically compatible stent designs that alleviate thrombus formation and promote endothelialization. This study employed time-resolved Digital Particle Image Velocimetry (DPIV) to compare the hemodynamic performance of two stents in a compliant vessel. The first stent was a rigid insert, representing an extreme compliance mismatch. The second stent was a commercially available nitinol stent with some flexural characteristics. DPIV showed that compliance mismatch promotes the formation of a ring vortex in the vicinity of the stent. Larger compliance mismatch increased both the size and residence time of the ring vortex, and introduced in-flow stagnation points. These results provide detailed quantitative evidence of the hemodynamic effect of stent mechanical properties. Better understanding of these characteristics will provide valuable information for modifying stent design in order to promote long-term patency.

Introduction: Tumor dimensionality creates a dynamic three dimensional (3D) architecture that is influenced by the associated microenvironment, including stromal cells and the extracellular matrix. These paracrine interactions impact therapeutic efficacy and can alter drug response in vivo , yet most current in vitro models do not accurately recapitulate the dimensionality or the stromal microenvironment of human tumors. In vitro models that are more recapitulative of the human tumor microenvironment have broad applicability in evaluation of signaling pathways driving cancer progression, therapeutic efficacy, and mechanisms involved in therapeutic resistance and tumor recurrence. There is a great need to adapt traditional analytical methods, developed for two dimensional cell culture, for use in 3D tissue models. Herein, a novel perfusion bioreactor system is used to support the multi-week growth and development of 3D breast carcinoma tissue surrogates (measuring 1.0 cm in maximum dimension) consisting of breast carcinoma epithelial cell lines and cancer associated fibroblasts (CAF) in a supportive extracellular matrix. Further, non-invasive imaging techniques, commonly employed to evaluate in vivo animal model systems, were used to measure growth of the surrogates overtime. Methods: 3D breast carcinoma surrogates were generated by incorporating MDA-MB-231 cells (tagged with GFP and luciferase) or MCF-7 cells (tagged with GFP and luciferase), with or without CAF, into an extracellular matrix. Surrogates were cultured in a perfusion bioreactor system for up to 3 weeks. Cell growth was measured on histologic sections of surrogates by counting the number of nucleated cells per surrogate cross-sectional area (cell density). Growth and viability were also determined in the same surrogates over time by using non-invasive fluorescence and luminescence imaging (IVIS 100 system). Results: The use of a flow perfusion bioreactor system resulted in a marked increase in the cell density of surrogates compared to non-perfused surrogates (perfused: 93.3 nucleated cells/area vs. non-perfused: 32.1 nucleated cells/area) at 21 days culture. Fluorescence and luminescence imaging of surrogates, containing increasing concentrations of breast cancer epithelial cells, were imaged at day 0 to confirm a correlation between signal intensity and cell number using each imaging modality (GFP: R2=0.97, p Conclusions: The presence of perfusion allows the growth and development of a recapitulative breast carcinoma surrogate with a size similar to human breast carcinomas at the time of detection and with an appropriate tumor microenvironment. Non-invasive imaging methods have successfully been adapted to evaluate growth of the breast carcinoma surrogates throughout multi-week culture. Future Directions: The use of these perfused breast carcinoma surrogates and imaging modalities in the evaluation of established and candidate cancer therapeutics will be assessed. Citation Format: Kayla F. Goliwas, Jillian R. Richter, Lauren E. Marshall, Joel L. Berry, Andra R. Frost. Evaluation of in vitro three dimensional breast cancer surrogates using histologic morphology and non-invasive imaging to monitor growth and viability throughout culture. [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 A05.

The project plan is to develop a three-dimensional (3D) microvascularized in vitro model system for breast cancer that could be used to culture individual cancer cells from patients and permit testing of a panel of chemotherapeutics for drug development. The research project will develop a new approach for drug efficacy testing that can better predict in vivo responses to therapeutics than current in vitro preclinical models. There are two aims. In the first, a microphysiologic, microvascularized 3D breast cancer model system that incorporates breast cancer associated fibroblasts (CAFs), microvascular endothelial cells (ECs), and cancer epithelial cells, embedded in extracellular matrix (ECM) supported by a novel carbon foam (reticulate vitreous carbon [RVC]) scaffold that contains EC-lined microchannels, will be assembled using breast cancer cell lines and characterized. In Aim 2, epithelial and stromal cells from cancer tissues from patients will be incorporated into the model system and parameters adjusted to achieve a microscopic architecture and representative molecular subtype of the originating cancer.

There is a need for preclinical testing systems that predict the efficacy, safety and pharmacokinetics of cancer therapies better than existing in vitro and in vivo animal models. An approach to the development of predictive in vitro systems is to more closely recapitulate the cellular and spatial complexity of human cancers. One limitation of using current in vitro systems to model cancers is the lack of an appropriately large volume to accommodate the development of this complexity over time. To address this limitation, we have designed and constructed a novel flow–perfusion bioreactor system that can support large-volume, engineered tissue comprised of multicellular cancer surrogates by modifying current microfluidic devices. Key features of this technology are a three-dimensional (3D) volume (1.2 cm3) that has greater tissue thickness than is utilized in existing microfluidic systems and the ability to perfuse the volume, enabling the development of realistic tumour geometry. The constructs were fabricated by infiltrating porous carbon foams with an extracellular matrix (ECM) hydrogel and engineering through-microchannels. The carbon foam structurally supported the hydrogel and microchannel patency for up to 161 h. The ECM hydrogel was shown to adhere to the carbon foam and polydimethylsiloxane flow chamber, which housed the hydrogel–foam construct, when surfaces were coated with glutaraldehyde (carbon foam) and nitric acid (polydimethyl-siloxane). Additionally, the viability of breast cancer cells and fibroblasts was higher in the presence of perfused microchannels in comparison to similar preparations without microchannels or perfusion. Therefore, the flow–perfusion bioreactor system supports cell viability in volume and stromal contexts that are physiologically-relevant.

Idiopathic Pulmonary Fibrosis is a devastating condition characterized by excessive localized production of collagen in the lungs. Over 131,000 people are living with IPF in America (1, 2). There is currently no known treatment or cure for the disease. It has recently been shown that IPF myofibroblasts are sensitive to the stiffness of their substrate. Specifically, alpha Smooth Muscle Actin (alpha-SMA), a known indicator of IPF activity, was differentially produced on soft vs. stiff substrates (3). This suggests a mechanotransduction pathway within the IPF myofibroblasts.Copyright © 2013 by ASME

Drug development platforms such as two-dimensional (2D) in vitro cell culture systems and in vivo animal studies do not accurately predict human in vivo effectiveness of candidate therapeutics [1]. Cell culture systems have limited similarities to primary human cells and tissues as only one cell type is employed and animal studies have a generally limited ability to recapitulate human drug response as different species have differences in metabolism, physiology, and behavior. Mike Leavitt, a former U.S. Secretary of Health and Human Services, has stated that “currently, nine out of ten experimental drugs fail in clinical studies because we cannot accurately predict how they will behave in people based on laboratory and animal studies” [2]. Therefore, this research project is focused on developing an in vitro platform to test candidate therapeutics for more efficacious predictions of human response. We have fabricated a three-dimensional (3D) breast cancer tissue volume containing a vascular network. This vascular network is necessary because current in vitro systems (e.g., rotating bioreactors, suspension of spheroids, and growth on a porous scaffold) are limited in size (1–2 mm) by their absence of micrometer-scale blood flow micro-channels that allow for oxygen and nutrient diffusion into the tissue [4]. The extracellular matrix scaffold has been developed to mimic the native extracellular matrix and includes relevant cell types (e.g., human breast cancer epithelial cells and human breast fibroblasts) along with the prefabricated vascular network (prevascularization). These systems are intended to support long-term growth, recapitulate physiological tissue function, and accurately model response to treatment. It is hypothesized that the development of reproducible tissue volumes will transform breast cancer drug development by providing reliable, cost-effective models that can more accurately predict therapeutic efficacy than current preclinical in vivo and in vitro models.Copyright © 2013 by ASME

The objective of this work was to expose dried trabecular bone material to a decalcifying environment and to quantify the change in the spatial distribution of the bone with a fractal measure. Digitized radiographic images were produced from four separate slices of human vertebral bone as they dissolved within a solution of nitric acid. Pixel data from a region of interest (ROI) within the trabecular bone were used to estimate the time-dependent change in fractal dimension of the ROI as the bone dissolved. Results demonstrated that a change in the spatial distribution of trabecular material may be expressed in terms of a concurrently changing estimate of the fractal dimension.

Purpose To develop a simple method for determining the elastic behavior of vascular stents. Materials and Methods An experimental apparatus was constructed to determine the elastic behavior of four different vascular stents. Each stent was expanded within an artificial compliant vessel and was subjected to an increasing external pressure. The cross-sectional area of each stent was recorded at incremental changes in pressure. Compliance was estimated from the slope of a linear regression analysis fit to the pressure-area data in the elastic range of deformation. Results The self-expandable stents were the most compliant, and balloon-expandable stents exhibited the least compliance. The balloon-expandable stents initially deformed in an elastic manner and then yielded irrecoverably at higher pressures. Conclusion A simple method has been devised that allows the elastic behavior of stents to be assessed. Quantification of stent compliance with this method is important as a predictor of stent resistance to compression in vivo.

Mechanical deformation of cell-seeded electrospun matrices plays an important role in cell signaling. However, electrospun biomaterials have inherently complex geometries due to the random deposition of fibers during the electrospinning process. This confounds attempts at quantifying strains exerted on adherent cells during electrospun matrix deformation. We have developed a novel mechanical test platform that allows deposition and tensile testing of electrospun fibers in a highly parallel arrangement to simplify mechanical analysis of the fibers alone and with adherent cells. The device is capable of optically recording fiber strain in a cell culture environment. Here we report on the mechanical and viscoelastic properties of highly parallel electrospun poly(ε-caprolactone) fibers. Force-strain data derived from this device will drive the development of cellular mechanotransduction studies as well as the customization of electrospun matrices for specific engineered tissue applications.

In the United States, chronic kidney disease affects 26 million adults.1 Chronic kidney disease is a progressive disease that takes months or years to develop, with diabetes and high blood pressure the most common causes. Patients with end-stage renal disease need life-sustaining dialysis or renal transplantation. They are frequently treated with regular hemodialysis to remove toxins and fluid. Reliable vascular access is critical for adequate hemodialysis and must provide high blood flow (≈400–500 mL/min) to the dialysis circuit.2 Among the 3 types of vascular access (arteriovenous fistula, synthetic graft, and catheter), the arteriovenous fistula is preferred because of its longevity and low frequency of complications. An arteriovenous fistula is placed surgically by creating a direct anastomosis between a native artery and vein and is the preferred method for reliable vascular access.3 However, the feeding artery of a successful arteriovenous fistula first needs to dilate to supply the estimated 500 mL/min necessary for a mature arteriovenous fistula.4,5 For the arteriovenous fistula draining vein to withstand arterial pressure and to accommodate greater blood flow, it also needs to enlarge and mature before use.6 When these necessities are met to yield a mature arteriovenous fistula, advantages of a fistula compared to other vascular access options include lower infection rates and a lower incidence of thrombosis.4 Preoperative vascular ultrasound (US) mapping is routinely used to assess the upper extremity arteries and veins, identifying suitable conduits for arteriovenous fistula creation.7,8 To optimize fistula maturation, minimum vascular diameter criteria aid in selection of vessels that are likely to support successful fistula maturation, and veins with stenosis or thrombosis are excluded. Although preoperative vascular mapping has had positive impact on increasing fistula placement, it has not decreased the fistula nonmaturation rate.4,7,9 With institutions recording 20% to 60% of fistulas failing to mature,9–13 other potential etiologies for arteriovenous fistula nonmaturation need to be investigated. There are currently some known technical causes of fistula nonmaturation that contribute to this failure rate, such as thrombosis, anatomic lesions, accessory veins, early vascular stenosis, and poor venous outflow.13 Although these circumstances can occur, they do not explain all of the failures, and the remaining reasons for arteriovenous fistula failure remain unknown. This disappointing observation suggests the existence of functional vascular parameters that may impair fistula maturation despite vessel selection by adequate structural measurement. Evaluation of the vessel diameter alone provided by preoperative vascular mapping is not sufficient to determine vascular properties such as distensibility and capacity. Thus, for example, a heavily calcified inflow artery may not be able to dilate sufficiently to supply a mature arteriovenous fistula.4,5,14 Elasticity has been shown to identify abnormalities within biological tissues, and imaging these elastic properties has become a pivotal point of many research efforts.15,16 Tissue characterization can estimate elastic properties of tissue, which is known to change with disease. The high temporal resolution of US and its ability to image small changes in vessel motion have proven useful for visualizing abnormalities in the vascular system.17–21 Stress is naturally applied to the arterial vessels by the cardiovascular pulsations from each individual.16,22–24 For a given stress field, one can attain high strain for lower moduli due to collagen fibers engaging to maintain the wall shape.25,26 Basic theory from engineering mechanics points us to the conclusion that the stiffer the material, the less deformation will occur under an applied stress. Ultrasound can be used to track vessel wall motion and overall vessel deformation.27 Thus, US-based noninvasive elasticity measurements may be a useful addition to current predictors, yielding supplemental information on the biomechanical properties of arteries. The ability to adapt algorithms to image data currently being collected clinically helps minimize cost and learning curves associated with adopting new imaging technology. The long-term goal of this research is to investigate the use of US and image processing to noninvasively measure the elasticity of human brachial arteries in patients with chronic kidney disease. This investigation presents the results of a pilot study to assess the feasibility of this approach.

Electrospun biomaterials are gaining popularity as scaffolding for engineered tissues. This fibrous scaffolding of natural or synthetic polymers can mimic properties of the natural extra-cellular matrix. Moreover, undifferentiated cells seeded onto and within an electrospun matrix may be directed to differentiate into a desired tissue type through the application of the appropriate biochemical and mechanical conditions. It is becoming clear that the mechanical deformation of any electrospun matrix plays an important role in cell signaling. However, electrospun biomaterials have inherently complex geometries due to the random deposition of fibers during the electrospinning process. Even “aligned” electrospun matrices generate off-axis forces under load. This complex fiber geometry complicates any attempt at quantifying forces exerted on adherent cells during electrospun matrix deformation.Copyright © 2012 by ASME

The objective of this study was to create 3D engineered tissue models to accelerate identification of safe and efficacious breast cancer drug therapies. It is expected that this platform will dramatically reduce the time and costs associated with development and regulatory approval of anti-cancer therapies, currently a multi-billion dollar endeavor [1]. Existing two-dimensional (2D) in vitro and in vivo animal studies required for identification of effective cancer therapies account for much of the high costs of anti-cancer medications and health insurance premiums borne by patients, many of whom cannot afford it. An emerging paradigm in pharmaceutical drug development is the use of three-dimensional (3D) cell/biomaterial models that will accurately screen novel therapeutic compounds, repurpose existing compounds and terminate ineffective ones. In particular, identification of effective chemotherapies for breast cancer are anticipated to occur more quickly in 3D in vitro models than 2D in vitro environments and in vivo animal models, neither of which accurately mimic natural human tumor environments [2]. Moreover, these 3D models can be multi-cellular and designed with extracellular matrix (ECM) function and mechanical properties similar to that of natural in vivo cancer environments [3].Copyright © 2012 by ASME