Science Of Oncology Research Articles

Abstract B11: Label-free hyperspectral microscopy detects alterations in nanoscale cellular structure with high temporal resolution

Abstract We have previously shown that analysis of the spectral variance of a reflected light microscope image allows direct study of cellular nanoscale organization through a technique called Partial Wave Spectroscopic (PWS) microscopy. This information is produced by measuring the spectral variations in light scattering to quantify the refractive index fluctuations, which reflect the distribution of macromolecular structures, within a biological sample with length scale sensitivity on the order of 20-200nm. Previously, this approach has been used for the early detection of tumors and the structure-function study of chromatin changes in cells. Here we present a state-of-the-art system utilizing this spectroscopic principle to develop a label-free, live-cell light microscopy imaging method capable of sensing temporal changes in the nanoscale structural organization of live cells called Live-cell PWS microscopy. This system is built into a commercial epi-fluorescence live-cell microscope and data is acquired using a hyperspectral camera (IMEC), which simultaneously collects 16 spectral bands in the visible spectrum with each exposure at rates up to ~100 frames per second. In addition, the design of this instrument allows simultaneous phase contrast and hyperspectral nanoscopic measurements. Phase contrast data can be acquired simultaneously with the hyperspectral data by providing phase contrast transmission illumination within one of the cameras 16 collection bands and using a corresponding notch filter for that band on the epi-illumination source. Calculating the spectral variance of each hyperspectral data cube yields images with label-free contrast of the cells where the intensity corresponds to the heterogeneity of nanoscale macromolecular organization within each diffraction limited voxel. Using this configuration, we can simultaneously acquire both phase contrast and the nanoscale sensitive hyperspectral videos of rapidly evolving structural changes in live cells without the need of any exogenous labels. To demonstrate the unique capabilities of this system, we perform a range of experiments to induce changes in cellular ultrastructure at rapid time-scales. In this work, we will show the changes in the cellular nanoarchitecture upon (1) addition of a glucose to live HeLa cells, (2) during chemical fixation by paraformaldehyde, and (3) upon cell exposure to UV-radiation. In these experiments, we observed immediate (<100ms) changes in nanoscale structural organization throughout the cell, with distinct structural changes differing between the cytoplasm and nucleus. For instance, incubation of glucose results in immediate transformation of the nuclear organization, causing a rapid decrease in nanoscale heterogeneity, which then quickly returned to pretreatment levels. Comparatively in the cytoplasm, addition of glucose results in significantly increased macromolecular mass-density heterogeneity upon treatment, which remains elevated even after the nucleus had recovered to pre-treatment levels. Likewise, we also recorded the rapid changes induced by chemical fixation with 4% paraformaldehyde solution in HeLa cells. In this case, we observe a gradual homogenization of the nanoscale structure as we lose clear delineation of the nucleus from the cytoplasm after fixation. This observation can be attributed to the randomized cross-linking process of the fixative yielding a more homogenous nanoscale structural organization than the non-random structures present prior to fixation in the cells. Finally, we explore the real-time effects of UV-light exposure on cellular behavior and the chromatin nanoarchitecture. These experiments demonstrate that rapidly evolving changes in the native unlabeled nanostructure of cells can be studied with high temporal resolution making this a unique tool for studying the nanoscale cellular structure-function relationship in real-time. Citation Format: John E. Chandler, Luay M. Almassalha, Vadim Backman. Label-free hyperspectral microscopy detects alterations in nanoscale cellular structure with high temporal resolution. [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 B11.

Abstract A53: Probing forces and modulation of cancer cell mechanical properties during transendothelial migration

Abstract For all types of cancer, the survival rate while the tumor is still localized is significantly higher than when cancer has metastasized. Though different therapeutic methods can be applied to successfully treat primary tumors, treatment of metastasised cancer is a great challenge due to its complex and systemic nature. A deeper understanding of how cancer initiates, grows and migrates is essential for creating successful therapies. Particularly, extravasation — a process during which streaming tumor cells (TCs) adhere to the blood vessels and traverse through the vascular endothelium into the surrounding tissue — is one of the crucial steps of cancer metastasis. While cancer research has mostly focused on the initial processes of metastasis, little is known about mechanisms of transmigration of tumor cells through the vascular wall. We found that during transendothelial migration (TEM), cancers cells undergo substantial shape changes transforming from semi-spherical (with a 2D contact with the endothelium) to a fully spread 3D morphology inside the ECM. This significant morphological change occurs due to forces generated intracellularly and distributed externally. During these morphological changes in transmigration, the cytoskeleton provides the force for shape change but the rate at which shape change occurs is dictated by the degree of deformability of the tumor cell, endothelial cells (ECs) and subendothelial tissue. Hence, the strength and distribution of forces, as well as mechanical properties of the cells and ECM, play a central role in the process of TEM. In this project, we investigate modulation of TCs and ECs rheological properties and the role of forces generated by the cytoskeleton and their transmission to adjacent cells and the ECM during extravasation. To address the role of mechanical modulation during TEM, we have developed in vitro assays and combed them with optical/confocal microscopy, Traction Force Microscopy (TFM) and Brillouin Confocal Microscopy (BCM). In particular, we recently designed a simple system in which ECs are cultured on a thin layer of collagen. Using a combination of different coating treatments on glass to produce surface hydrophobicity and maintain strong adhesion, we created a thin (~80 μm) layer of gel. Following seeding by ECs (HUVECs) to produce a tight and uniform EC monolayer we introduced TCs on top of the EC monolayer and monitored extravasation in real-time with high spatiotemporal resolution. Measurements of HUVEC monolayer permeability in this system show physiologically relevant values (8±3 10-8 cm2/s). We further tested our system by using different cancer cells with different invasiveness potentials and investigated their extravasation efficiencies. A major advantage of this platform is that it allows integration of TFM and BCM so it enabled us to characterize forces and modulation of cell mechanical properties during TEM. In particular, we demonstrated the feasibility of our system for application of BCM on cancer cells extravasating through a monolayer. BCM is an optical technique in which the mechanical information can be read out optically via spectral analysis of the scattered light; thanks to the interaction of light with intrinsic mechanical vibrations (phonons) of the material. Our BCM measurements indicate a significant decrease in elasticity of TCs at different stages; from 260±20 Pa for TCs that were in the process of transmigration to 195±17 Pa for those that were fully transmigrated. Our results shed a new light on the fundamental understanding of the extravasation mechanics. Furthermore, we anticipate that these studies will potentially enhance our ability to identify and screen for new therapies to inhibit the tendency for metastatic spread of disease. Citation Format: Emad Moeendarbary, Roger Kamm, Giuliano Scarcelli. Probing forces and modulation of cancer cell mechanical properties during transendothelial migration. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A53.

Abstract A11: Calibration, selection and validation of tumor growth models

Abstract As a result of the highly heterogeneous tumor microenvironment and a series of complex phenomena that occur at different scales, several modeling approaches have emerged. The tumor growth models can be categorized by the behaviors they aim to capture, the hallmarks of cancer that are being considered, and the temporal and biological scales represented. Moreover, these models can also be differentiated according to the mathematical framework that is being employed, being either discrete models, continuum models, or hybrid models. Therefore, with this large set of possible models, and with each model choice having many unknown parameters, it is important to develop an adaptive model selection and validation framework in order to choose the best model for the desired application. In this work, we employ the Occam Plausibility Algorithm (OPAL) framework [1] for model selection and validation of a large body of tumor growth models. We select several continuum tumor growth models, half of them using the phase-field modeling approach, derived from the models developed in [2], and the other half being reaction-diffusion type models derived from [3]. The OPAL framework brings together model calibration, determination of sensitivities of outputs to parameter variances, and calculation of model plausibilities for model selection. Firstly, we identify the model classes and compute the sensitivity of the quantity of interest (QoI), here the tumor volume as a function of time. The models with parameters that do not influence the QoI are eliminated. The next step consists in dividing the models into Occam categories according to their number of parameters, where the models with fewer parameters are assembled to category 1. Following the principle of Occam's Razor, one of main goals of OPAL is to select the simplest model among the several models that leads to the same prediction. The models from the first category are calibrated using Bayes' rule for a specific calibration scenario. The next step is to compute the plausibilities for the models in this category and choose the most plausible model. Finally, with the most plausible model known, we solve the problem in the validation scenario. The model is valid depending on the accuracy with which the model predictions of the QoI agree with experimental data. If the model is not valid, we move to the next category; otherwise, we select this model as being the simplest and the most plausible valid model. We demonstrate that the OPAL is able to assist in the selection of the best tumor growth model from a defined set of models. The model selected is valid for particular scenarios. In this work, we consider avascular tumor growth models to reproduce the data obtained in a murine model of glioma growth. The brain data is generated with diffusion weighted magnetic resonance imaging over a period of ten days. The methodologies and results confirm that the OPAL strategy provides a powerful framework for model selection, parameters estimation, and model validation in the presence of uncertainties.

Abstract A51: The role of extracellular stiffness in metastatic cell invasion

Abstract Invasion is a critical step in the metastatic process during which cells dissociate from the primary tumor to breach the basement membrane and migrate through the stroma. Successful invasion can then lead to other steps in the metastatic cascade such as intravasation through the vascular system to secondary sites, ultimately resulting in the spread of tumors throughout the body as well as a dramatic decrease in patient survival. Efficient migration through the extracellular matrix is a key determinant of a tumor cell's ability to invade local tissue and eventually metastasize to secondary sites. Previous work has indicated that the ability of cancer cells to migrate and therefore invade efficiently through the stroma is dependent on the microenvironment, which differs from its normal state in the presence of non-cancerous cells. Alterations to matrix properties such as fiber alignment, stiffness, and density may facilitate this migration. We have focused on how these properties impact the migratory abilities of cancer cells in in vitro models of the tumor microenvironment. There is significant evidence to suggest that the molecular mechanisms governing 2D migration are significantly different than those dictating migration in 3D matrices. In recent work, we have shown that in vivo, cells also migrate in pseudo-3D environments where they utilize preformed tunnels and interstitial spaces in the tissue to navigate through the stroma. To recreate these tunnels in vitro, we created and utilized a micromolded 3D system of patterned collagen microtracks to gain a more complete understanding of invasive migration. The consequences of altering matrix properties on cell behavior were then examined using this system and compared to the more commonly utilized 3D collagen matrix model. Results indicate that increasing collagen density affected single cell migration in 3D matrices but did not have a significant impact on average cell velocity in the microtracks, suggesting these cells migrate independently of density. These findings then raise the question of how important extracellular cues are in determining the behavior of cancer cells during track migration and how this influence changes depending on the state of the microenvironment. Since changes in collagen density affected migration in 3D matrices but not microtracks, it is worth determining if a similar trend is seen for stiffness. To this end, we utilized the in vitro microtrack system described above to investigate how single cell migration through tracks is affected by changes in extracellular stiffness. Collagen gels of varying stiffness were made through non-enzymatic glycation by allowing the gels to incubate in ribose solutions of different concentrations. The migration of cells through the tracks was quantified based on average cell velocities across the various conditions. Interestingly, the data indicate there is no significant difference in velocity with increasing stiffness. This is in contrast to 2D migration, where a similar change in stiffness causes an increase in cell velocity. These results suggest that either the change in stiffness is too subtle for the cells to detect within the tracks or they behave as they did in response to density, insensitive to alterations to either of these properties. When taken together with our previous published findings, the data suggest that while the tumor microenvironment is important in mediating malignancy and metastasis, it varies in its influence. Cells moving in microtracks, which offer the least resistance as they navigate through the extracellular matrix, may migrate independently of a variety of mechanical cues from the microenvironment that more closely regulate their migration in other contexts. Citation Format: Marianne Lintz. The role of extracellular stiffness in metastatic cell invasion. [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 A51.

Abstract PR09: Polymeric mechanical amplifiers of tumor apoptosis

Abstract Regulation of receptor-mediated phenomenon at the nanoscale in a controlled manner remains a challenge for a broad range of fields, including cell biology, medicine, and bioengineering. Fluid forces play a crucial role in receptor-mediated signaling necessary for physiological function, while materials such as nanoparticles have demonstrated potential for spatiotemporal control of cellular signaling. Here, we have developed polymeric mechanical amplifiers that tether to the cell surface and increase receptor-mediated apoptosis in the presence of physiological fluid flow. The mechanical amplifiers consist of both non-degradable and biodegradable polymer nanoparticles that are tethered to the surface of tumor cells via polyethylene glycol (PEG) linkers. Tethered nanoparticles, in the presence of fluid shear forces observed in vivo, increased the therapeutic effect of a TNF superfamily death ligand (TRAIL) on colon and prostate tumor cells by as much as 50% compared to soluble TRAIL treatment alone, while exerting negligible toxic effects on normal cells. Increasing the number of nanoparticles conjugated to the tumor cell surface enhanced TRAIL-mediated apoptosis under fluid shear exposure, while the therapeutic effect under static conditions was not altered. Our results suggest that the enhanced therapeutic response using polymeric nanoparticles is both receptor-mediated apoptosis-specific and does not affect doxorubicin efficacy. Inhibition studies indicate the response is caspase signaling-dependent, and increased tumor cell death receptor expression in the presence of shear forces is also observed. We show that targeted polymeric nanoparticles delivered to tumor cells in vivo mechanically amplified a subsequent treatment of soluble TRAIL, and reduced both circulating tumor cells in blood and overall tumor cell burden in vivo by over 90%. We conclude that surface-bound polymeric nanoparticles enhance receptor-mediated apoptosis in the presence of physiological shear forces, and represents a potentially new application for a broad range of nanotechnologies to maximize the capacity of receptor-mediated signaling and function in the presence of a ligand. This abstract is also being presented as Poster B35. Citation Format: Michael John Mitchell, Robert Langer. Polymeric mechanical amplifiers of tumor apoptosis. [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 PR09.

Abstract A22: A window into 3D culture: A multi-modal imaging compatible bioreactor for developing tumor growth models

Abstract Introduction: We aim to use clinical imaging data to initialize and constrain tumor-specific biophysical models of in situ cell proliferation, motility, and therapy response. Towards this goal, we have developed a 3D in vitro cell culture system that can be non-invasively interrogated by both microscopy and magnetic resonance imaging (MRI). The system supports long-term cell growth and allows for control over microenvironmental factors such as drug delivery and extracellular matrix. Cellular and subcellular level data obtained from confocal microscopy define the cell state and local environment and improve interpretation of the lower resolution, clinically relevant information provided by MRI. Here, we report system design and initial data collection. Apparatus Design: The system consists of the bioreactor body, a reservoir with a filter enabling CO2 equilibration in the incubator, and a peristaltic pump to drive media flow throughout the tubing and bioreactor. The bioreactor body has a cavity for cells embedded in extracellular matrix, a hollow-fiber for delivery of nutrients (FiberCell Systems, Frederick, MD), and is sealed using a gasket and lid with a confocal microscopy window. The removable lid makes the bioreactor reusable after sterilization. Data Collection: As a simplified model of 3D tumor growth, we selected the triple negative breast cancer cell line MDA-MB-231 embedded in 0.4% agarose. MDA-MB-231 cells were labeled with H2B-RFP and FUCCI-GFP to image cell nuclei and entry into the cell cycle, respectively, enabling measurements of cell number and cell division. Fluorescence microscopy images were collected on a Zeiss spinning disk confocal microscope using a 20x objective. Sixty 200 μm z -stacks using the green and red channels can be collected in approximately 90 minutes. The bioreactor is secured into a custom microscope mount using screws, ensuring it is placed in the same location every time. A reference point is marked on the first imaging day, to enable longitudinal alignment at subsequent imaging sessions. The bioreactor was also designed for use on a 9.4 T Agilent Technologies (Palo Alto, CA) MRI scanner equipped with a 38-mm quadrature RF coil, which is conventionally used for small animal studies. Diffusion-weighted (DW) images have been acquired using a standard pulsed gradient spin echo sequence with four b values (67, 200, 400, and 800 s/mm2) and gradients applied simultaneously along three orthogonal directions (x , y , and z ). The apparent diffusion coefficient obtained from the DW-MRI data provides an indirect measure of cell number. Images were acquired at a resolution of 100x100x1000 μm3. Conclusion: Overall, we have designed and fabricated a multi-modal imaging compatible bioreactor that supports long-term 3D cell growth under controlled conditions. We have successfully collected both MRI and confocal microscopy data on the bioreactor system at multiple time points. Currently, we are working towards co-registering these two imaging data types and extending the life of the bioreactor. Our novel multi-modal imaging compatible bioreactor is ideal for iterative biophysical tumor model refinement, providing a bridge to predictive models exclusively populated with non-invasive clinical imaging data. Citation Format: Abigail M. Searfoss, Matthew T. McKenna, Vito Quaranta, Thomas E. Yankeelov, Erin C. Rericha. A window into 3D culture: A multi-modal imaging compatible bioreactor for developing tumor growth models. [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 A22.

Abstract A49: Tumor-associated interstitial flow promotes macrophage migration and pro-metastatic M2 phenotype in 3D ECM

Abstract The growth of solid tumor is often accompanied by an increase in the flow of interstitial fluid (interstitial flow) from the center of the tumor to the surrounding stroma. Recent studies have shown that elevated interstitial flow can influence the migration of cancer cells and fibroblasts. Despite these findings, the effects of interstitial flow on macrophages, one of the key metastasis-promoting tumor-associated stromal cell types, have not been explored. We used microfluidic and transwell flow assays to investigate how high level of interstitial flow (~3 μm/s) often observed in tumor affect macrophage migration and protein expression. We found that interstitial flow not only enhanced macrophage migration speed, but also directed macrophages to migrate against the flow in 3D extracellular matrix. Moreover, we discovered that interstitial flow activated Akt and FAK, two kinases that are crucial for cell migration. Interestingly, interstitial flow also up-regulated macrophage expression of M2 markers Arg I and TGFβ through the activation of JAK1-STAT3/6 pathway. Finally, macrophages treated with interstitial flow were shown to have enhanced abilities to promote cancer cell migration and protrusion formation. Taken together, these results suggest that interstitial flow can promote metastasis through its effects on macrophages. Specifically, since macrophages migrate against the flow, interstitial flow, which emanates from the tumor, can act as a mechanical stimulus to recruit macrophages into tumor tissues. Moreover, interstitial flow can activate macrophages and polarize them toward pro-metastatic M2 phenotype to further promote tumor metastasis and immunosuppression. Citation Format: Ran Li, Hao Xing, Tara A. Lee, Hesham Azizgolshani, Roger D. Kamm. Tumor-associated interstitial flow promotes macrophage migration and pro-metastatic M2 phenotype in 3D ECM. [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 A49.

Abstract A42: Systematic modulation of the physical microenvironment for characterization of cancer cell lines and primary tissue

Abstract Introduction: The tumor microenvironment includes a range of physical, chemical, and cellular components such as pressure, hypoxia, acidosis, the extracellular matrix (ECM), stromal, and immune cells. These factors can influence the gene and protein expression of the various types of cells that make up the tumor as well as influence the selection of cells that can thrive in that environment; however, typical cancer cell culture rarely uses hypoxia and pressure nor utilizes substrates similar to the native ECM. We designed a system study the influence of hypoxia, pressure, and native ECM conditions on cancer cell lines and primary cells, with the goal of creating a more relevant culture environment for translational studies. Methods: Here, we comprehensively studied how hypoxia, pressure, and ECM composition and organization influences cell biology and gene expression, using transcriptome profiling across a range of physiologically-relevant culturing conditions to mimic various tumor microenvironments found within the body. To do this, we utilized Xcell's primary cell culture platform which includes a custom bioreactor that allows us to control both oxygen concentration (0.1%-20% O2) and hydrostatic pressure levels (26 to 260 mmHg / 0.5 psig to 5 psig). Additionally, we studied the influence of biomimetic substrates by controlling ECM composition and organization (aligned or unaligned collagen at concentrations from 1-2.5 mg/ml +/- fibronectin 0.1-10 microgram/ml) as both 2D ECM coatings and 3D hydrogels. Cell lines studied included models for brain cancer (U-87, A172), pancreatic cancer (PANC10.05), and prostate cancer (DU-145, PC-3, 22Rv1, LNCaP). We performed high-resolution immunofluorescence imaging and western blot protein expression analysis of key targets, including immunotherapy targets CTLA-4, PD-1, and PD-L1. Additionally, we applied our ability to tune culturing conditions to represent physiological conditions in the body for the successful culture of fresh primary tissues from normal volunteers as well as cancer patients for downstream analysis studies focused on biomarker discovery and as a clinical tool for disease monitoring and treatment decision-making. Conclusions: We identified both common and unique gene signatures across these different cells lines, with the hypoxic conditions activating HIF1 pathway activation having common across the cells studied whereas pressure results in more restricted signatures. Some cancer cell lines and PBMCs differentially express immunotherapeutic targets, at low oxygen and high pressure culturing conditions, resulting in reduced expression of key targets. Moreover, biomimetic ECM studies revealed modulation of ECM to represent a “tumor”-like ECM dramatically altered cell growth, morphology, and focal adhesion organization. In a 3D hydrogel context, a “tumor”-like ECM and a hypoxic environment dramatically enhanced tumor cell invasion. Consistently, analysis of mRNA-seq data revealed alterations in gene expression profiles of immunotherapeutic and drug-target pathways involving CTLA-4 and AR signaling as well as EMT targets. In contrast, we observed increased CD47 and CD44 expression at low oxygen and high pressure culturing conditions in cancer cell lines and PBMCs. Thus, supporting the notion that these are possible drug targets for tumors that are characterized by low oxygen levels and high interstitial fluid pressure. Citation Format: Luke Cassereau, Bruce Adams, Tianna Chow, Ajuni Sohota, Charles Ryan, James Lim. Systematic modulation of the physical microenvironment for characterization of cancer cell lines and primary tissue. [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 A42.

Abstract A25: Systems biology approach to delineating DNA damage response and R-loop dependencies in Ewing sarcoma

Abstract Ewing sarcoma is a rare, yet highly aggressive family of bone and soft tissue tumors that afflicts children and young adults. It is the prototypical example of mesenchymal tumors driven by a fusion oncogene involving the EWSR1 gene, most frequently EWS-FLI1. Ewing sarcoma is exquisitely sensitive to genotoxic agents, and more recently PARP1 inhibitors, predominantly when detected at an early stage. However, the molecular basis for this sensitivity is relatively under-explored. Further, the long term side effects of these drugs pose a critical barrier to effective treatment. In this study, we proposed to delineate the pathways dictating Ewing sarcoma chemosensitivity and identify alternative avenues for targeted therapy. R-loops are three nucleic acid DNA structures consisting of an RNA:DNA hybrid and a displaced complementary DNA loop. The importance of R-loops in both being causative as well as disruptive in DNA damage as well as DNA damage response has emerged through recent studies. Our previous work has demonstrated that Ewing sarcoma cell lines have high levels of endogenous damage as well as replication stress. We observe that Ewing cell lines have elevated levels of persistent R-loops and impaired homologous recombination repair of double strand breaks. In the present study, we use a multimodal, systems biology approach involving high-throughput RNAi based survival data, time-course RNA-seq in response to two chemotherapeutics, Chromatin immunoprecipitation-seq and DNA:RNA hybrid immunoprecipitation-seq datasets across multiple Ewing sarcoma and control cell lines. Our data led to the identification of a subset of genes whose expression patterns in response to DNA damage is dysregulated in Ewing sarcoma. Predominantly, among these genes is a network of RNA binding proteins that highlights the importance of RNA metabolism in DNA damage response. We also observe a concomitant alteration in damage-dependent transcription. Finally, we identified several compensatory mechanisms that are upregulated in Ewing sarcoma (RNASEH2, FEN1, Fanconi Anemia, etc.) in order to manage the exacerbated genomic stress conditions and targeting any of these pathways leads to severe tumor cell cytotoxicity. We therefore propose the use of these pathways as potential candidates for targeted therapy. Citation Format: Aparna Gorthi, Eva Loranc, Cao Lin, Alexander JR Bishop. Systems biology approach to delineating DNA damage response and R-loop dependencies in Ewing sarcoma. [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 A25.

Abstract PR06: VEGF-targeted therapy induces extracellular matrix remodeling and increases mechanical barriers to therapy in colorectal cancer liver metastases

Abstract Introduction: The anti-VEGF antibody bevacizumab in combination with chemotherapy is a standard treatment for metastatic colorectal cancer (mCRC) today, based on an overall survival improvement. However, this survival benefit is modest and mCRC ultimately progresses. The underlying mechanism of resistance to anti-angiogenic therapy remains unclear. Recent preclinical studies have shown that anti-angiogenic therapy increases collagen expression in tumors as a consequence of hypoxia. The extracellular matrix (ECM) play an important role in solid stress-induced blood vessel collapse as they transmit the mechanical stress created by proliferating cells within the confined space of a tumor. In this study, we investigated the effects of anti-angiogenic therapy on extracellular matrix expression, both collagenous and non-collagenous and perfusion as a novel mechanism of acquired resistance to anti-angiogenic therapy in liver metastases from CRC. Experimental Design: We used C57BL/6 and BALB/c, as well as CCR2-/- and ATR1-/- mice for this study. To generate liver metastasis, we exteriorized and transected spleen, inject syngenic CRC SL4 or CT26 cells (0.1 million cells) in one hemispleen and then, removed it. The other hemispleen remained intact. We monitored tumor burden by measuring blood Gaussia luciferase (Gluc) activity from Gluc transduced tumors and/or by high-frequency ultrasound imaging. Treatments include anti-VEGF monoclonal antibody B20.4-1.1 (5 mg/kg or 1 mg/kg i.p. 2x/week, Genentech), an anti-Ly6G antibody (5 mg/kg i.p. every 2days), pegylated hyaluronidase (PEGHAse, 4.5 mg/kg i.v. 2x/week 24 hour prior to administration of chemotherapy), 5-Fluorouracil (50 mg/kg i.v. 2x/week). We determined ECM–hyaluronic acid (HA), sulfated glycosaminoglycan (sGAG), and collagen contents—by ELISA, the Blyscan Proteoglycan and Glycosaminoglycan assay, and hydroxyproline assay, respectively. We further determined ECM, activated fibroblasts, vasculature, perfusion (Hechst33342), and hypoxia (pimonidazole) by immunohistochemistry, and immune cell profile using flow cytometry. We then, determined stiffness (Young's modulus) of tumors by unconfined compression tests (Cancer Res 60: 2497-503, 2000) and solid stress using a recently established method (PNAS 109: 15101-8, 2012). We obtained patient samples from the Heidelberg University Hospital and analyzed surgical specimens of patients who underwent liver resection for colorectal liver metastases without preoperative chemotherapy, after preoperative chemotherapy without bevacizumab or after preoperative chemotherapy with bevacizumab. Results: Here, we show that anti-VEGF therapy induces remodeling of the extracellular matrix with subsequent alteration of physical properties of liver metastases. We find that preoperative treatment with bevacizumab in patients with colorectal liver metastases resulted in a significant increase in HA deposition within the tumor. Moreover, in two syngeneic mouse models of mCRC, we show that anti-VEGF therapy markedly increased expression of HA and sGAG, without significant changes in collagen deposition. The density of these matrix components correlates with tumor stiffness; the latter was also significantly increased after anti-VEGF therapy. In time-course and immunofluorescence studies, treatment-induced hypoxia appeared as the driving force for enhanced extracellular matrix expression. Enzymatic depletion of HA partially reversed compromised perfusion in liver metastases after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and systemic chemotherapy. Conclusion: These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy. This abstract is also being presented as Poster B27. Citation Format: Nuh N. Rahbari, Dmitriy Kedrin, Joao Incio, Hao Liu, William T. Ho, Hadi T. Nia, Christina M. Edrich, Keehoon Jung, Julien Daubriac, Ivy Chen, Takahiro Heishi, John Martin, Yuhui Huang, Nir Maimon, Christoph Reissfelder, Juergen Weitz, Yves Boucher, Jeffrey W. Clark, Alan J. Grodzinsky, Dan G. Duda, Rakesh K. Jain, Dai Fukumura. VEGF-targeted therapy induces extracellular matrix remodeling and increases mechanical barriers to therapy in colorectal cancer liver metastases. [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 PR06.

Abstract A02: Multiple spatio-temporal scale modeling with application to brain cancer

Abstract Introduction: We aim to develop a multiple spatio-temporal scale (MSTS) framework capable of bridging tissue, cellular, and subcellular scales. Our overarching hypothesis is that an accurate and precise MSTS model, initialized with patient-specific information, can dramatically outperform current standard-of-care for diagnosis and selecting therapy for the individual cancer patient. Methodology: We developed a MSTS framework for tumor growth and treatment outcome prediction. Our model incorporated chemotherapy so that we can predict changes in tumor volume due to various therapeutic regimens. The MSTS framework consists of a tissue scale model, a cellular activity and growth model, and a subcellular signaling pathway model. To predict tissue growth at the tissue scale, a continuum model is constructed where the biological tissue is represented as a mixture of multiple constituents with each of which represents either a volume fraction or concentration. The constituents interact with each other through mass and momentum exchange so that the governing equations are based on both mass and momentum conservation laws. The constitutive equations account for tissue heterogeneity, nonlinear behavior, and thermodynamic consistency. The system of partial differential equations is solved using finite element techniques. To bridge the tissue and cell scales, each finite element is discretized into virtual cell clusters to model cellular growth and proliferation by using an agent-based model to determine various cellular scale activities including cell division, cell death, and phenotypical alteration. The cellular scale is further discretized temporally to model the effects of selected signaling pathways (e.g., PI3K/AKT/mTOR pathway). In many cancers, mTOR pathway becomes hyperactive and promotes abnormal cell proliferation. The mechanism and effects of a mTOR inhibiting drug known as rapamycin are tested in silico. The subcellular scale is modeled using a set of ordinary differential equations. A statistical inverse algorithm is used to calibrate and validate the results using magnetic resonance imaging (MRI) data, and a Bayesian inference method is employed to account for the uncertainties in model parameters. Results: The 3D simulation results recapitulate, at various physical and biological conditions, different tumor characteristics including morphological instability, changes in the spatio-temporal distribution of different cell phenotypes and density, and the reduction of growth rate due to drug delivery. Thus, this model has the potential to be used for patient-specific prediction of tumor growth as well as personalized treatment planning. The novelties of this modeling framework are that (1) the events at each scale are modeled using different numerical techniques suitable for that specific scale in terms of both space and time, (2) a novel numerical technique is developed to bridge the scales and solved simultaneously, (3) the tissue scale model is thermodynamically consistent with consideration of tissue heterogeneity and nonlinear behaviors, (4) various cellular events such as the phenotypical alteration, apoptosis, necrosis, mitosis, etc. are modeled at the cellular scale, and (5) the mTOR signaling pathway, an important pathway for many different cancers, is modeled at the subcellular scale. Conclusions: Our MSTS model is capable of predicting tumor growth with considerations of various factors at different spatio-temporal scales. It can be potentially used as a patient-specific tumor growth model for in silico drug testing, treatment planning, and prognosis. A future version of this model can include cancer stem cells, angiogenesis, and metastasis. Citation Format: Mohammad Rahman, Yusheng Feng, Thomas E. Yankeelov, J. Tinsley Oden. Multiple spatio-temporal scale modeling with application to brain cancer. [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 A02.

Abstract A65: Topographical guidance of angiogenesis at an interface of collagen densities

Abstract Tumor vasculature is characterized by leaky, tortuous, and dilated blood vessels that create heterogeneous blood flow and diminish the transport of oxygen. Pioneered by Jain, an emerging cancer treatment strategy seeks to normalize tumor vasculature in order to increase the delivery and efficacy of therapeutics. Overexpression of vascular endothelial growth factor (VEGF) and other pro-angiogenic factors within the tumor microenvironment have been considerably investigated as the major contributing factor to abnormal tumor vasculature, but less is understood about the influence of the physical environment and the underlying molecular mechanisms. During tumor progression, increased extracellular matrix (ECM) deposition occurs resulting in mechanically heterogeneous tissue defined by local alterations in density. Such changes to ECM density and composition can significantly disrupt cell behaviors. Our previous work has demonstrated that the presence of an interface produces directional migration and net cell migration is a function of collagen matrix density. Here, we utilize a similar sequential polymerization technique to create a distinct low-to-high collagen density interface, in order to replicate the heterogeneity found in tumor tissue and investigate the role of tumor matrix mechanics in promoting abnormal tumor vasculature. Specifically, Human Umbilical Vein Endothelial Cells (HUVECs) were encapsulated in a low-density collagen matrix, cultured for three days until capillary-like networks were formed, and a second, high-density collagen matrix supplemented with VEGF and basic fibroblast growth factor (bFGF) was added to create a 3D interface. Angiogenesis across and capillary-like network organization at the collagen density interface was then evaluated by time-lapse microscopy and immunofluorescence. Preliminary data indicates that the presence of a low-to-high density interface increased HUVEC capillary-like network branching and length. We demonstrate that the number of branches per network, total number of branches, and total network length all increased as a function of the density interface, where the larger difference in density between the two regions produced more branching points and increased network length. Together, our data demonstrate how ECM heterogeneities, in the form of interfacial boundaries, affect capillary-like network architecture and promote abnormal branching. This work provides further insight into cellular sensing during tumor angiogenesis, as well as helps to identify novel methods for tumor vasculature normalization, as a means to improve the efficacy of many chemotherapeutic and radiation treatments. Citation Format: Matthew R. Zanotelli, Francois Bordeleau, Cynthia A. Reinhart-King. Topographical guidance of angiogenesis at an interface of collagen densities. [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 A65.

Abstract B16: The detection of ductal carcinoma using noninvasive hyperspectral imaging

Abstract Introduction: Hyperspectral imaging (HSI) technology is an optical medical imaging modality that has great potential for noninvasive tissue analysis in breast cancer. This imaging method may be able to enhance the detection of cancer tissue and lead to advances in the optical diagnosis of patients. Objective: The goal of this study was to use high resolution hyperspectral imaging microscopy to differentiate marked biopsies of normal and breast cancer tissues. Methods: For our hyperspectral imaging platform, we obtained stained (n=10) and unstained (n=10) pathology slides from ductal carcinomas of the breast and compared them. To capture the HSI, we used a hyperspectral camera that generates a spectral cube containing the spectra of each pixel between 460 to 640 nm and normalized the data to a gray scale to maximize data stability. The spectral signatures were graphed using MATLAB software. Results: The system was able to detect a clear spectral difference between cancer and normal tissues in hematoxylin and eosin stained slides. The spectrum of cancer tissue had a relatively lower intensity than that of normal tissue in the wavelength range between 480 and 610 nm. Conclusions: Our preliminary study showed a clear hyperspectral snapshot differentiating normal breast tissue from ductal carcinoma. Further HSI and quantitative analysis will demonstrate the feasibility of using this new imaging technique in breast cancer. Citation Format: Yasser Khouj, Jeremy Dawson, James Coad, Linda Vona-Davis. The detection of ductal carcinoma using noninvasive hyperspectral imaging. [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 B16.

Abstract B59: Mathematical Model of Oxygen Transport in Tuberculosis Granulomas

Abstract Pulmonary granulomas—the hallmark of Mycobacterium tuberculosis (MTB) infection—are dense cellular lesions that, like solid tumors, often feature regions of hypoxia and necrosis partially due to limited transport of oxygen. Low oxygen in granulomas can impair the host immune response, while MTB are able to adapt and persist in hypoxic environments. Here, we used a physiologically based mathematical model of oxygen diffusion and consumption, based on models originally derived for avascular tumors, to calculate oxygen profiles within the granuloma, assuming Michaelis-Menten kinetics. An approximate analytical solution—using a priori and newly estimated parameters from experimental data in a rabbit model of tuberculosis—was able to predict the size of hypoxic and necrotic regions in agreement with experimental results from the animal model. Such quantitative understanding of transport limitations can inform future tuberculosis therapeutic strategies that may include adjunct host-directed therapies that facilitate oxygen and drug delivery for more effective treatment. Citation Format: Meenal Datta, Laura Via, Wei Chen, James Baish, Lei Xu, Clfton Barry, Rakesh Jain. Mathematical Model of Oxygen Transport in Tuberculosis Granulomas. [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 B59.

Abstract B62: Role of the blood microenvironment in the upregulation of oncoproteins in circulating pancreatic cancer cells

Abstract Background: Platelets are promoters of cancer cell proliferation at the metastatic niche, however the exact mechanism(s) by which platelets drive cancer cell proliferation remain ill-defined. The oncoprotein c-MYC, which governs neoplastic proliferation, is overexpressed in pancreatic tumors at the metastatic site as compared to primary tumors, with the overexpression of c-MYC indicative of a poor clinical outcome for patients with cancer. At present, the oncogenic pathways mediating c-MYC upregulation in metastatic cancer cells are largely unknown. Our exciting preliminary data demonstrate for the first time that platelets upregulate c-MYC protein levels in PANC-1 pancreatic cancer cells, suggesting a novel prometastatic role for platelets in pancreatic cancer. Aims: To define the mechanisms by which platelets upregulate c-MYC protein levels in PANC-1 pancreatic cancer cells. Methods: We utilized a co-culture platform to examine the effect of resting and thrombin-stimulated human platelets as well as platelet fractions (platelet-derived membrane bound molecules and soluble molecules) on c-MYC expression in PANC-1 cancer cells. Platelet inhibitors, including the GPIIbIIIa blocker Integrilin, were used to study the molecular mechanisms by which platelets upregulate c-MYC expression in cancer cells. Changes in c-MYC oncoprotein were detected by Western Blotting. The physical association between platelets and PANC-1 cancer cells was quantified by immunoblotting with an anti-GPIIb antibody against the platelet-specific integrin GPIIbIIIa. Results: Resting and activated platelets as well as platelet fractions induced an upregulation of c-MYC expression in PANC-1 cancer cells. The effect of platelets on c-MYC expression was largely dependent on direct contact with the cancer cells, as the preincubation of platelets with Integrilin reduced platelet-induced c-MYC expression. Conclusion: We show that the expression of c-MYC in PANC-1 cancer cells can be upregulated by platelets, revealing a novel mechanism of platelet action in the progression of cancer and suggesting that the overexpression of c-MYC at the metastatic niche may be inhibited through the use of platelet inhibitors. We are currently characterizing the molecular underpinnings of platelet-mediated c-MYC regulation with respect to the contribution of platelets to cancer cell proliferation and growth. Citation Format: Annachiara Mitrugno, Katherine Pelz, Rosalie Sears, Owen McCarty. Role of the blood microenvironment in the upregulation of oncoproteins in circulating pancreatic cancer cells. [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 B62.

Abstract A37: ‘Touch-and-go' behavior of cancer cells in spatially-confined, fiber-like microenvironments

Abstract In a crowded, fibrillar tumor microenvironment (TMEN), invading cancer cells encounter and interact with many other cells and cell types. Whether cancer cells stop migrating or reverse direction at every encounter with another cell, or circumnavigate and slide around the encounter, will affect the efficiency of the invasion process. We find that breast cancer cells migrating along fiber-like micropatterns (FLMs) respond differently to cell-cell encounters than what is expected based on the classically-established phenotype of contact-inhibition of locomotion (CIL). Cancer cells are remarkable efficient at sliding around other cancer cells, even along FLMs much narrower than their own cell body. In contrast, non-transformed breast epithelial cells reverse direction upon encountering other normal cells. Differences in cell size fail to predict these outcomes. Interestingly, the participation of normal cells in invasive sliding behavior increases when interacting with a cancer cell rather than another a normal cell. This induction of cancer-like behavior in normal cells suggests that heterotypic interactions between disparate cell types in the TMEN may produce synergistic, disease-promoting collective behaviors. Furthermore, using a series of individual and combinatorial genetic perturbations, we show that genetic factors (ErbB2, E-cadherin, PARD3, TGFbeta) quantitatively shift the ability of normal cells to slide. Specifically, successive genetic perturbations enable normal cells to slide on progressively narrower FLMs. These results suggest that as fibers align and mature during cancer progression, coincident genetic perturbations enable cells to take advantage of the widening fibrillar tracks to execute slides and achieve more efficient dispersion in a crowded microenvironment. Citation Format: Daniel Milano, Senthil Muthuswamy, Anand Asthagiri. ‘Touch-and-go' behavior of cancer cells in spatially-confined, fiber-like microenvironments. [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 A37.

Abstract A57: Novel 3D tumor-stromal model highlights the importance of ECM composition and biophysical properties in pancreatic cancer EMT and drug resistance

Abstract Introduction: Epithelial to mesenchymal transition (EMT), where cells detach from basement membrane (BM) and invade interstitial matrix (IM), plays a significant role in tumor metastasis and drug resistance. Mechanobiological mechanisms of such tumor-stroma interactions are poorly understood largely due to inherent limitations of current preclinical tumor models (e.g. 2D cell culture and spheroids). The objective of this work was to apply a novel in-vitro 3D tumor-stromal model to define how extracellular matrix (ECM) composition and biophysical properties modulate EMT. Methods: This work involved pancreatic cancer cells representing different EMT phenotypes (BxPc-3: epithelial; Panc-1: intermediate; MiaPaCa-2: mesenchymal). Matrigel was used to represent BM, while tunable type I collagen Oligomers represented IM. Cells were encapsulated within 3D matrices composed of Matrigel, Oligomer of varied fibril density (matrix stiffness), or Oligomer and Matrigel combined at varied ratios. Cell phenotype was characterized based on morphology and EMT protein immunostaining (i.e., E-cadherin and vimentin). Dose response curves to gemcitabine (standard of care) were used to define relative IC50. ECM microstructure was visualized using confocal reflection microscopy, and matrix stiffness (G') was determined using rheometric oscillatory shear testing. Results: Oligomer enhanced the mesenchymal phenotype with decreasing stiffness (vimentin expression; spindle-shaped cells), while Matrigel downregulated mesenchymal properties and encouraged an epithelial phenotype (E-cadherin expression; rounded, grouped cells). While maintaining vimentin expression in all matrices, Panc-1 and MiaPaCa-2 grew as tight clusters within Matrigel and the highest stiffness Oligomer, where they showed the greatest gemcitabine resistance. BxPc-3 were most sensitive in intermediate stiffness Oligomer where they appeared most proliferative. They also showed relatively high drug resistance in Matrigel and matched-stiffness Oligomer despite each matrix supporting opposite phenotypes. Cells were also cultured within matrices of varied Oligomer:Matrigel ratio to define how BM and IM engagement guides EMT. In matrices with high Oligomer content (>50%), MiaPaCa-2 generated a notably more invasive, mesenchymal phenotype, while BxPc-3 only transitioned to a mesenchymal phenotype in pure Oligomer. Since stiffness and fibril density were similar in the 75:25 and 100:0 ratios, the presence of BM components appears to mediate the phenotypic transition between these matrices. Collectively, these results emphasize the importance of ECM composition and biophysical properties in guiding EMT. Findings suggest that epithelial tumor cells (BxPc-3) may require engagement with a low stiffness IM to undergo EMT and reduce drug sensitivity. For already transitioned cells (Panc-1 and MiaPaCa-2), decreasing stiffness enhances their mesenchymal nature but increases drug sensitivity. Together these observations align with the idea that EMT increases drug resistance, yet challenge the view that increasing matrix stiffness mediates this transition. Also, engagement with both matrix types promotes invasiveness of mesenchymal cells while exposure to BM suppresses EMT of epithelial cells. Finally, Matrigel promotes a drug resistant, epithelial phenotype which highlights its limitations for studying EMT mechanisms. Conclusion: This work represents the first use of type I collagen oligomers for mechanistic study of EMT mechanobiology and provides new insight into how IM fibril microstructure and mechanical properties guide EMT. These results challenge correlations between stromal properties and EMT established using conventional preclinical models and help identify critical parameters for engineering pathophysiologically relevant tumor-stromal models. Citation Format: TJ Puls, Xiaohong Tan, Catherine Whittington, Sherry Voytik-Harbin. Novel 3D tumor-stromal model highlights the importance of ECM composition and biophysical properties in pancreatic cancer EMT and drug resistance. [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 A57.

Abstract B57: RNAi Nanotechnology for Cancer Target Validation and Therapy

Abstract RNA interference (RNAi) represents a promising strategy for identification and validation of putative therapeutic targets, and for treatment of a myriad of important human diseases including cancer. The ubiquitous application of RNAi in cancer research and therapy is nevertheless hindered by the challenge of effective systemic in vivo delivery of RNAi agents (e.g., siRNA) to solid tumors, which requires overcoming of multiple physiological barriers, such as enzymatic degradation, rapid elimination by renal excretion or by the mononuclear phagocyte system (MPS), poor tumor penetration, and insufficient cellular uptake and endosomal escape. Here we will describe the rational design and development of unique nanoparticle platforms to address these obstacles. These siRNA nanoparticles have been successfully applied to evaluate the therapeutic role of different targets (e.g., prohibitin1) in treating solid tumors, and have demonstrated significant potential for the clinical development of novel cancer therapies. Note: This abstract was not presented at the conference. Citation Format: Jinjun Shi. RNAi Nanotechnology for Cancer Target Validation and Therapy. [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 B57.

Abstract A21: 3D tumor models in bioreactors recapitulate microenvironment and disease progression

Abstract The high attrition rates observed in cancer drug discovery (up to 95% of failure of drugs tested in phase I trials) have raised the awareness of the scientific and industrial communities towards the need for more predictive pre-clinical models. These models should be more representative of the disease and consequently help to eliminate at pre-clinical stages drug candidates that lack efficacy or safety. Tumor microenvironment is composed by a network of fibroblasts, endothelial cells, immune-competent cells within the extracellular matrix (ECM). Interactions between these components are critical for tumor initiation, proliferation, migration and metastasis. The design of in vitro cancer cell models that recapitulate the tumor microenvironment and 3D architecture provides higher physiological relevance as they more closely resemble the in vivo cellular context. We have established methodologies for scalable generation of 3D cancer cell models in stirred-tank culture systems, and applied these to a large panel of tumor cell lines from different pathologies, including breast, colon, hepatic and lung tumor cell lines. Large numbers of spheroids were obtained per culture (typically 1000-1500 spheroids/mL) with representative characteristics of native tumors, such as morphology, proliferation and hypoxia gradients, in a cell-line dependent mode. With the aim of increasing the relevance of spheroids as tumor cell models, several aspects of tumor microenvironment were incorporated, such as the presence of stromal cells (fibroblasts and monocytes) and specific physical parameters, namely by embedding cells in a polymeric matrix. Heterotypic 3D breast and Non-Small Cell Lung Carcinoma (NSCLC) cancer models, based on co-cultures of tumor cells with stromal cells were established by using an alginate matrix to provide physical support to cells. Tumor spheroids were microencapsulated alone or with fibroblasts and monocytes, thus allowing the establishment of an epithelial tumor compartment and a stromal compartment of increasing complexity. Cultures were performed in stirred-tank vessels for 15 days with continuous monitoring. In both breast and lung tumor models, the presence of fibroblasts was associated with secretion of pro-inflammatory cytokines and accumulation of collagen in the microcapsules. Long-term culture (up to 15 days) resulted in phenotypic alterations in co-cultured breast tumor spheroids, such as loss of cell polarity, reduced cell-cell adhesions, collective cell migration and increased angiogenic potential. In contrast, the effects of fibroblasts were not as significant in NSCLC co-cultures using H1650, H1437 and H157 cell lines suggesting that the effect of tumor-stroma cross-talk is cell line dependent. Moreover, these models have also been shown as feasible tools for drug screening by assessing the effect of chemotherapeutic and specific inhibitors compounds on mono- and co-cultures. In conclusion, we have developed scalable, robust and versatile methodologies for the generation and culture of 3D cancer models, enabling long-term in vitro recapitulation of tumor-stroma crosstalk, via reconstruction of key aspects of the tumor microenvironment, allowing continuous monitoring of disease progression events in vitro. In addition, it is easily transferable to industry for feeding high-throughput systems or miniaturized bioreactors used in drug development, target validation and target identification. Acknowledgments: We acknowledge support from the Innovative Medicines Initiative Joint Undertaking (IMI grant agreement n° 115188), resources composed of financial contribution from EU - FP7 and EFPIA companies in kind contribution. iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofunded by Fundação para a Ciência e Tecnologia / Ministério da Ciência e do Ensino Superior, through national funds, and by FEDER under the PT2020 Partnership Agreement, is acknowledged. This research has also received support from Fundação para a Ciência e Tecnologia, Portugal—PTDC/BBB-BIO/1240/2012. MFE, SA and CP are the recipients of PhD fellowships from FCT (SFRH/BD/52208/2013, PD/BD/105768/2014 and SFRH/BD/52202/2013, respectively). Citation Format: Vitor E Santo, Marta F Estrada, Sofia P Rebelo, Sofia Abreu, Catarina Pinto, Elizabeth Anderson, Wolfgang Sommergruber, Paula Alves, Erwin Boghaert, Catarina Brito. 3D tumor models in bioreactors recapitulate microenvironment and disease progression. [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 A21.

Abstract A40: A novel, biosynthetic 3D hydrogel system for breast cancer mechanobiology studies

Abstract Breast tumor cell-extracellular matrix (ECM) interactions drive malignant transformation, invasion, and metastasis of breast cancer cells and contribute to treatment resistance. The tumor microenvironment, composed of multiple ECM components, activate mechanochemical pathways associated with breast cancer progression and signaling of runt-related transcription factor 2 (Runx2). There is an urgent need to model these interactions in physiologically relevant three-dimensional (3D) models in vitro. We recently demonstrated that we can create 3D PEG hydrogel systems, containing type I collagen, Arg-Gly-Asp (RGD), and hyaluronic acid, whose stiffness can be tuned independent of the number of ECM components incorporated. In this study, we demonstrate that these hydrogels can be utilized to mimic the in vivo tumor microenvironment. We examined the behavior of breast cancer cell lines MDA-MB-231 (invasive) and MCF7 (non-invasive) as well as MCF10A (non-tumorigenic mammary epithelial cells) in our novel, biosynthetic hydrogel systems. We showed that these hydrogels supported growth for each cell line and formation of acinar structures over a 23-day period in vitro. In future studies, we will assess the migratory behaviors of these cell lines in vitro using microfluidic devices as well as Runx2 expression and activity to determine the influence of matrix physiochemical properties on the invasive potential of breast tumor cells. This novel, biosynthetic system represents a reproducible framework where tumor cell behavior may be quantified as a function of a complex system in which combined microenvironmental parameters, including matrix stiffness, ECM composition, and their interactions, are systematically varied. Citation Format: Amy Elissa Blatt, Bita Carrion, Rhima M. Coleman. A novel, biosynthetic 3D hydrogel system for breast cancer mechanobiology studies. [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 A40.