Tumor Microtissues Research Articles

Abstract 52: PDX-derived 3D InSightTM tumor microtissues as ex-vivo human experimental models for evaluating therapeutic responses

Abstract Background The selection of appropriate preclinical models comes always with the major question on how accurately and robustly they can represent the complexity of human disease. Patient-derived xenograft (PDX) models faithfully preserve the biological features and the genetic expression profile of human tumor specimens. However, one limiting aspect of patient-derived models is the replacement of the human host microenvironment by murine stroma within the tumor. Lack of cross-species compatibility compromises the induction of a broad range of signaling pathways that cannot be entirely recapitulated. With our in vitro 3D InSightTMTumor Microtissues derived from PDX lines, we provide a relevant physiological environment and a strategy to assess candidate drugs for novel therapeutic approaches. Aim Development of in vitro 3D InSightTM Tumor Microtissues from PDX lines aimed to retain the cellular heterogeneity found in the original human tumor tissue. Material & Methods and Results PDX cell suspensions of lung, breast and melanoma origin were successfully used to assess 3D aggregation in 96-well format and characterized over 10 days in culture. After careful removal of mouse cell contaminants in each PDX sample, 3D PDX cell cultures were supplied with exogenous normal human dermal fibroblasts (nHDF). Furthermore, to provide a more physiological cancer niche, PDX cells were also co-cultured with tumor-matched cancer-associated fibroblasts (CAFs). 3D in vitro tumors were analyzed histologically and cancer phenotypic alterations were evaluated through the analysis of epithelial-to-mesenchymal transition (EMT) markers. The morphology, viability and growth rate of PDX-derived microtumors were assessed by size analysis (cell scanner) and ATP assay. To assess the distribution of various cell populations within the tumor, 3D PDX samples were screened for standard stromal vs. epithelial-tumor cells markers (e.g. FAP, pan-CK, E-Cadherin), and diagnostic cancer type-specific biomarkers. 3D PDX samples were also employed to investigate the efficacy of specific targeted therapies based on distinct molecular signatures of PDX tumor models. Immunohistochemistry assessment of 3D microtumors validated the resemblance with their respective PDX tumor models. 3D tumor growth rate and cell behavior observations reflected the diversity of disease progression in vivo. Conclusion Further efforts will focus on employing this platform to establish more complex co-cultures with integration of additional relevant stromal and immune cells, to enable a reliable preclinical translational research of tumor/immune cell interactions. We suggest that in vitro 3D PDX models offer a more suitable and robust approach to expedite faithful efficacy assessment of immunomodulators and approval of optimal drug candidates. Citation Format: Francesca Chiovaro, Irina Agarkova, Nicole Buschmann, Chloe' Pichon, Teresa Langova, Armin Maier, Julia Schueler, Patrick Guye. PDX-derived 3D InSightTM tumor microtissues as ex-vivo human experimental models for evaluating therapeutic responses [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 52.

Abstract 2887: Patient-derived 3-dimensional models of pediatric tumors for in situ study of cancer biology, immunotherapy, and drug treatment

Abstract The 2D culture systems are currently the mainstay of pre-clinical testing of potential therapeutics, however, these models fail to recapitulate the dynamic and heterogenous conditions found within the in vivo tumor microenvironment and are poor predictors of clinical activity. Patient derived xenograft models of cancer excel recapitulating these complexities but are not the most tractable approaches for high throughput efficacy testing, particularly of immuno-oncologic therapies. In addition, small animal imaging techniques to noninvasively and longitudinally monitor PDX tumor growth require costly equipment and a high level of technical expertise. Thus, there is a tremendous need for in vitro model systems with improved diagnostic accuracy to better predict therapeutic responses earlier in drug development. This need is even more acute for pediatric cancers such as osteosarcoma (OS) and glioblastoma (GB), for which there are few or no viable non-clinical models to investigate basic science or for pre-clinical drug efficacy studies.We have developed a 3D culture system integrating 3D printing in Liquid Like Solids (LLS) materials to enable the precise arrangement of delicate and highly detailed assemblies of cells, that include, fibroblasts, vascular endothelial cells, and/or activated T-cells and extra-cellular matrix components-with a modular perfusion system that uses a negative pressure chamber to draw fluid through the culture chamber and assay workspace without disturbing the positioning of the experiments. Real-time imaging in these 3D assays is performed in situ using confocal microscopy under controlled perfusion. In this study we present preliminary results from in situ studies of drug cytotoxicity, angiogenesis, and fibroblast activation, as well as, T cell migration and infiltration in bio-fabricated patient derived 3D tumor microtissues of OS and GB. We assessed the impact of oxygen tension, DNA damage, ER stress, and stromal composition (matrix and cell types) on response to frontline therapies, tumor angiogenesis, and T cell activation. Secretion of cytokines, pro-inflammatory factors and the release of damage-associated molecular patterns by microtumor constructs cultured under each condition was measured by ELISA. These data highlight the unique capabilities of our integrated confocal microscope and 3D passive perfusion culture system for studies of cancer biology, immunotherapy, and drug treatment regimens to provide in situ measurements of tumor-stroma interactions and immune cell invasion dynamics in precision fabricated OS and GB patient derived 3D microtissues. Citation Format: Padraic P. Levings, Juan M. Urueña, Eric O. McGhee, Alex J. McGhee, Derek L. Hood, Kylie E. Van Meter, Steve H. Ghivizzani, Colin J. Anderson, Wallace G. Sawyer. Patient-derived 3-dimensional models of pediatric tumors for in situ study of cancer biology, immunotherapy, and drug treatment [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 2887.

High-Frequency Mechanical Properties of Tumors Measured by Brillouin Light Scattering.

The structure of tumors can be recapitulated as an elastic frame formed by the connected cytoskeletons of the cells invaded by interstitial and intracellular fluids. The low-frequency mechanics of this poroelastic system, dictated by the elastic skeleton only, control tumor growth, penetration of therapeutic agents, and invasiveness. The high-frequency mechanical properties containing the additional contribution of the internal fluids have also been posited to participate in tumor progression and drug resistance, but they remain largely unexplored. Here we use Brillouin light scattering to produce label-free images of tumor microtissues based on the high-frequency viscoelastic modulus as a contrast mechanism. In this regime, we demonstrate that the modulus discriminates between tissues with altered tumorigenic properties. Our micrometric maps also reveal that the modulus is heterogeneously altered across the tissue by drug therapy, revealing a lag of efficacy in the core of the tumor. Exploiting high-frequency poromechanics should advance present theories based on viscoelasticity and lead to integrated descriptions of tumor response to drugs.

Probing tumor microtissue formation and epithelial-mesenchymal transition on a well-mesh microchip.

Three-dimensional cultures of tumor microtissues and biomimetic simulation of tumor microenvironments are of great significance in the study of tumorigenesis and development processes. In this study, a well-mesh microchip was developed to realize the formation and culture of tumor microtissues in vitro. Human lung adenocarcinoma HCC827 cells and large-cell lung cancer NCI-H460 cells were used. The size and morphology of the microtissues have been observed. In addition, we constructed an in situ three-dimensional co-culture model with tumor cell microtissues (HCC827 or NCI-H460 cells), extracellular matrix (Matrigel), and human umbilical vein endothelial cells. HCC827 microtissue epithelial-mesenchymal transition (EMT) in the established well-mesh microchip also was investigated, and the results showed that recombinant transforming growth factor could activate the Snail and Akt gene and promote migration and EMT with the decrease of E-cadherin expression for HCC827. This well-mesh microchip features simple operation and easy observation, and could provide a new method for the study of tumor cells and tumor microenvironments in vitro. Therefore, this model has potential application value in organ-on-chip technology, tissue engineering, and drug evaluation.

Beyond the on/off chip trade-off: A reversibly sealed microfluidic platform for 3D tumor microtissue analysis

Nowadays, microfluidic 3D cell culture is widely used to mimic complex microtissue and dynamic environment, performing more realistic in vitro assays for drug testing. Herein, we developed a novel microfluidic platform for tumor microtissue culture, drug response analysis and versatile microscopic characterization. By reversibly bonding the chip, we go beyond the on/off chip tradeoff, which allows us to perform both fluorescence and SEM characterization of tumor microtissues on a simple platform. The microfluidic chip consists of spherical microwells connected via microchannels, bonded through a magnetic system. Colorectal cancer HT-29 cells were cultured as spherical microtissues on chip and their growth kinetics monitored. The cytotoxic activity of Camptothecin was evaluated by in situ live/dead fluorescence staining and quantification of morphology parameters. Finally, we demonstrated the possibility to collect the 3D tumor microtissues and characterize their surface damaged by the drug using scanning electron microscopy. This reversibly sealed microfluidic platform thus enables to grow sets of 3D tumor microtissues in a controlled dynamic microenvinroment, and subsequently to retrieve the 3D tumor microtissues after chemotherapeutic treatment for in-depth analysis.

Abstract LB-034: Development and characterization of PDX-derived 3D tumor microtissues as platform for screening targeted molecular therapeutics

Abstract Background A major issue for improving the overall success rate in drug development is the lack of accurate experimental human in-vitro models. While there is a significant and increasing need for such better ex-vivo cell-based models, the question on how closely these systems resemble and recapitulate the original tumors is of great importance. Patient-derived xenograft (PDX) models act as vehicle systems to propagate human tumor specimens, faithfully preserving the biological features and the genetic expression profile. The retainment of such criteria in in vitro 3D InSightTM Tumor Microtissues derived from PDX lines is crucial to provide a relevant physiological environment and strategy to assess candidate drugs for novel therapeutic approaches. Aim Development and characterization of in vitro 3D InSightTM Tumor Microtissues from patient-derived xenograft models. Material & Methods and Results PDX cell suspensions of Lung, Breast and Melanoma origin were successfully used to assess 3D aggregation in 96 well format and characterized over 10 days in culture. After careful removal of mouse cell contaminants in each in vitro 3D PDX sample, PDX cell cultures were supplied with exogenous normal human dermal fibroblasts (nHDF). The morphology, biomarker phenotype (IHC) as well as cell proliferation were assessed by histological analysis. In addition to screening for standard diagnostic marker such as proliferating vs. dead cells (e.g. Ki67, ClCasp3) and stromal vs. epithelial-tumor cells (e.g. FAP, pan-CK, E-Cadherin), we also assessed the expression of cancer type-specific biomarkers. Moreover, to monitor the dynamics of cancer phenotypic alterations, epithelial-to-mesenchymal transition (EMT) marker were evaluated and scored. Immunohistochemistry assessment of 3D microtumors validated the resemblance with their respective PDX tumor models. The viability and growth rate of PDX-derived microtumors were assessed by size analysis (cell scanner) and ATP assay. 3D tumor growth rate and cell behavior observations reflected the diversity of disease progression in vivo. Further efforts will focus on employing this platform to investigate the efficacy of specific targeted therapies based on the distinct molecular signatures of PDX tumor models. Conclusion Development and characterization of In vitro 3D InSightTM Tumor Microtissues from patient-derived xenograft (PDX) lines demonstrated that the morphological and molecular features of the parental tumors are well retained. We suggest that in vitro 3D PDX models offer a more suitable and robust approach to expedite faithful efficacy assessment and approval of optimal drug candidates. Citation Format: Francesca Chiovaro, Irina Agarkova, Armin Maier, Simon Messner, Julia Schueler, Patrick Guye. Development and characterization of PDX-derived 3D tumor microtissues as platform for screening targeted molecular therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-034.

Recapitulating spatiotemporal tumor heterogeneity in vitro through engineered breast cancer microtissues

Tumor and microenvironmental heterogeneity hinders the study of breast cancer biology and the assessment of therapeutic strategies, being associated with high variability and drug resistance. In this context, it is mandatory to develop three-dimensional breast tumor models able to reproduce this heterogeneity and the dynamic interaction occurring between tumor cells and microenvironment. Here we show a new breast cancer microtissue model (T-µTP) uniquely able to present intra-tumor morphological heterogeneity in a dynamic and responsive endogenous matrix. T-µTP consists of adenocarcinoma cells, endothelial cells and stromal fibroblasts. These three kinds of cells are totally embedded into an endogenous matrix which is rich in collagen and hyaluronic acid and it is directly produced by human fibroblasts. In this highly physiologically relevant environment, tumor cells evolve in different cluster morphologies recapitulating tumor spatiotemporal heterogeneity. Moreover they activate the desmoplastic and vascular reaction with affected collagen content, assembly and organization and the presence of aberrant capillary-like structures (CLS). Thus, T-µTP allows to outline main crucial events involved in breast cancer progression into a single model overcoming the limit of artificial extra cellular matrix surrogates. We strongly believe that T-µTP is a suitable model for the study of breast cancer and for drug screening assays following key parameters of clinical interest. Statement of SignificanceTumor and microenvironmental heterogeneity makes very hurdle to find a way to study and treat breast cancer. Here we develop an innovative 3D tumor microtissue model recapitulating in vitro tumor heterogeneity. Tumor microtissues are characterized by the activation of the stromal and vascular reaction too. We underline the importance to mimic different microenvironmental tumor features in the same time and in a single tissue in order to obtain a model of spatiotemporal tumor genesis and progression, suitable for the study of tumor treatment and resistance.

A high-content image analysis approach for quantitative measurements of chemosensitivity in patient-derived tumor microtissues

Organotypic, three-dimensional (3D) cancer models have enabled investigations of complex microtissues in increasingly realistic conditions. However, a drawback of these advanced models remains the poor biological relevance of cancer cell lines, while higher clinical significance would be obtainable with patient-derived cell cultures. Here, we describe the generation and data analysis of 3D microtissue models from patient-derived xenografts (PDX) of non-small cell lung carcinoma (NSCLC). Standard of care anti-cancer drugs were applied and the altered multicellular morphologies were captured by confocal microscopy, followed by automated image analyses to quantitatively measure phenotypic features for high-content chemosensitivity tests. The obtained image data were thresholded using a local entropy filter after which the image foreground was split into local regions, for a supervised classification into tumor or fibroblast cell types. Robust statistical methods were applied to evaluate treatment effects on growth and morphology. Both novel and existing computational approaches were compared at each step, while prioritizing high experimental throughput. Docetaxel was found to be the most effective drug that blocked both tumor growth and invasion. These effects were also validated in PDX tumors in vivo. Our research opens new avenues for high-content drug screening based on patient-derived cell cultures, and for personalized chemosensitivity testing.

Abstract 5775: A unique 3D tissue-engineered human melanoma model combining lymphatic and blood microvasculature to study cancer cell dissemination

Abstract INTRODUCTION: Melanoma is amongst malignancies with constantly increasing incidence in developed countries. One of the underlying causes of death for patients diagnosed with melanoma is metastasis, which can spread through lymphatic or blood vessels. Mechanisms controlling the dissemination paths are poorly understood and relevant models for studying metastasis physiopathology are often inadequate. To address this, we hypothesized that the human tumor microenvironment can be mimicked in vitro by combining tissue-engineered microvascularized skin and melanoma microtissues. METHODS: Tissue-engineered skin was produced using primary dermal, epidermal and microvascular endothelial cells by the self-assembly technique without any exogenous biomaterial. Tumor microtissues were produced using the hanging drop method. Six melanoma cell lines were used originating from primary tumor sites (A375, SK-MEL 28 and WM983a) and from metastatic sites (RPMI 7951, Malme 3M and WM983b). Tumor development and growth were assessed by histology, immunofluorescence and confocal microscopy, while cytokine secretion profiles were determined by ELISA. WM983a and WM983b models were treated for 11 days with vemurafenib. Response to treatment was assessed by counting the ratio of tumor cells positive for Ki67, representative of the tumor proliferation. RESULTS: We obtained a tissue-engineered skin displaying two distinct microvascular networks: a VE-cadherin+ CD31+ blood network, and a PDPN+ LYVE-1+ CD31+ lymphatic network. Blood capillaries were thin and highly connected whereas lymphatic capillaries were larger and presented a distinct morphology. Histological analyses revealed tumor microtissue integration at the dermoepidermal junction within the reconstructed skin. The pro-lymphangiogenic factor and tumor-secreted VEGF-C was detected in conditioned media from the melanoma microtissues (662 pg/ml). Furthermore, CCL21, a chemoattractant known to be secreted by lymphatic endothelial cells, displayed secretion levels that were 10-fold higher in microvascularized tissues compared to the non-microvascularized skin (P ≤ 0.001). Both of these cytokines are involved in the cross-talk between tumor cells and capillaries, and thus in tumor dissemination. The 3D melanoma model responded to vemurafenib with up to a 5-fold decrease of tumor cell proliferation and a partial pigmentation of the tumor. CONCLUSION: This unique 3D in vitro melanoma model mimics tumor microenvironment by combining blood and lymphatic capillaries with melanoma microtissues in a reconstructed skin. Being responsive to treatment such as vemurafenib, it represents a valuable tool for studying mechanisms of metastasis and drug response in a fully human cell and matrix microenvironment, and thus testing anti-metastatic compounds could better predict their safety and efficacy. Citation Format: Jennifer Bourland, Julie Fradette, François A. Auger. A unique 3D tissue-engineered human melanoma model combining lymphatic and blood microvasculature to study cancer cell dissemination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5775. doi:10.1158/1538-7445.AM2017-5775

The influence of stromal cells and tumor-microenvironment-derived cytokines and chemokines on CD3+CD8+ tumor infiltrating lymphocyte subpopulations

ABSTRACT The tumor microenvironment has been identified as a major mediator of immunological processes in solid tumors. In particular, tumor-associated fibroblasts are known to interact with tumor infiltrating immune cells. We describe the influence of fibroblasts and tumor-microenvironment-derived cytokines on the infiltration capacity of CD3+CD8+ cytotoxic T lymphocyte subpopulations using a multicellular 3D co-culture system. 3D tumor microtissues were cultivated using a hanging drop system. Human A549 and Calu-6 cancer cell lines were incubated alone or together with the human fibroblast cell line SV80 for 10 d to form microtissues. On day 10, peripheral blood mononuclear cells (PBMC) were added with or without cytokine stimulation for 24 h. Infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of the microtissues and the infiltration of the PBMCs were analyzed by immunohistochemistry, and endogenous cytokine and chemokine expression was analyzed with a multi-cytokine immunoassay. Secretion of chemokines is increased in microtissues consisting of cancer cells and fibroblasts. PBMC infiltrate the whole spheroid in cancer cell monocultures, whereas in co-cultures of cancer cells and fibroblasts, PBMCs are rather localized at the margin. Activated CD69+ and CD49d+ T lymphocytes show an increased microtissue infiltration in the presence of fibroblasts. We demonstrate that the stromal component of cancer microtissues significantly influences immune cell infiltration. The presence of fibroblasts in cancer microtissues induces a shift of T lymphocyte infiltration toward activated T lymphocytes.

3D tumor microtissues as an in vitro testing platform for microenvironmentally-triggered drug delivery systems.

A major issue of modern cancer research is the development of accurate and predictive experimental models of human tumors consistent with tumor microenvironment and applicable as screening platforms for novel therapeutic strategies. In this work, we developed and validated a new 3D microtissue model of human breast tumor as a testing platform of anti-cancer drug delivery systems. To this aim, biodegradable nanoparticles responsive to physiological changes specifically occurring in tumor microenvironment were used. Our findings clearly demonstrate that the breast tumor microtissue well recapitulates in vivo physiological features of tumor tissue and elicits a specific response to microenvironmentally-responsive nanoparticles compared to healthy tissue. We believe this study is of particular interest for cancer research and paves the way to exploit tumor microtissues for several testing purposes.

Bioengineered tumoral microtissues recapitulate desmoplastic reaction of pancreatic cancer.

Tumor microenvironment is extremely complex and its organization is due to the interaction between different kind of cells and the extracellular matrix. Tissue engineering could give the answer to the increasing need of 3D culture model that better recapitulate the tumor features at cellular and extracellular level. We aimed in this work at developing a microtissue tumor model by mean of seeding together cancer cells and fibroblasts on gelatin microsphere in order to monitor the crosstalk between the two cell populations and the endogenous extracellular matrix deposition. Results are of particular interest because of the need of heterotypic cancer model that can replicate the complexity of the tumor microenvironment and could be used as drug screening platform.

Abstract 358: In vitro assessment of tumor relapse in a 3D tumor microtissue model

Abstract One of the most pressing problems in the treatment of cancer is tumor relapse. Although many tumors regress in response to neoadjuvant chemotherapy, residual tumor cells are detected in most cancer patients post-treatment. These residual tumor cells are thought to remain dormant for years before resuming growth, resulting in tumor recurrence. Considering that recurrent tumors are most often responsible for patient mortality, there exists an urgent need to study signaling pathways that drive tumor dormancy/recurrence. Current in vitro assays based on monolayer cultures do not provide the required time frame to study tumor recurrence. We developed a new method to study tumor relapse in a 3D in vitro tumor microtissue model. HCT116 colorectal tumor microtissues were produced and dosed with 3 reference compounds, Taxol (cell division inhibitor), Staurosporine (protein kinase inhibitor) and Doxorubicin (DNA synthesis inhibitor). Based on an initial range finding to determine IC20 and IC50 values, tumor growth was monitored via size profiling and viability (intra-tissue ATP content) over 10 days. Three different conditions were tested: (i) continuous treated tumor microtissues, (ii) microtissues which were supplemented with the drug for 5 days and (iii) non-treated tumor microtissues. Growth differences couldn't be observed comparing IC20 concentrations, however growth profiles significantly varied comparing IC50 concentrations. Whereas a 5-day treatment of Taxol was leading to a complete growth inhibition after removing the drug, Staurosporine treated tumor microtissues relapsed even with a similar growth kinetic as the untreated control group. Pulsed treatment of Doxorubicin was leading to a slower growth kinetic as compared to the control but not to a complete reminiscence. Based on the individual growth profiles relative to the continuous treated and non-treated groups we calculated a relapse index (ReI, patent pending) to enable classification of compounds and compound combinations according to their risk for tumor relapse. Here we present a novel in vitro tumor relapse assay method based on a 3-dimensional tumor microtissue model, to enable compound classification based on their efficiency to prevent tumor relapse. Citation Format: Sumeer Dhar. In vitro assessment of tumor relapse in a 3D tumor microtissue model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 358.

Abstract 771: Multi-parametric 3D tumor microtissue-based phenotypic compound classification

Abstract Here, we report on the development of homo- and heterotypic ovarian and pancreatic microtissue tumor models for a screening of up to 40 compounds selected from the NCATS Oncology focused library of small molecules. The goal of this study was to develop a high throughput compatible drug screening platform that could leverage the best of high throughput and high content screening assays to generate a pharmacological profile of activities that we hope will help better predict activity in vivo .This platform utilizes 3D multicellular spheroids from ovarian (HEY and SKOV) and pancreatic (Panc-1) cancer and enables the discrimination of tumor-specific efficacy and nonspecific cytotoxicity of a drug candidate with subsequent identification and validation of the molecular mechanism of action (MMOA). The biological responses measured over a 10-day drug exposure period included (i) growth kinetic (microtissue size), (ii) potency (IC50ATP_10_days) and efficacy (max. response ATP and size). The biological response of the compounds was compared between the different cell culture formats tested, 2D, 3D homotypic and 3D heterotypic. The comparison of IC50 values among the different ovarian cancer cell cultures showed that 21 out of the 38 compounds tested were more potent in 3D than in 2D. Within the pancreatic models, 13 out of 20 compounds tested were more potent in 3D than in 2D. Interestingly, most of the compounds which showed stronger potency in 3D were targeted small molecule agents. Comparing drug responses of homo vs heterotypic ovarian tumor model systems, 3 compounds were effective only in the heterotypic model including the WNT inhibitor PNU-74654 and GABA uptake inhibitor (Gat-1) SK&F-89976A. We also evaluated nonspecific cytotoxic effects on the incorporated NIH3T3 fibroblasts by quantifying a secreted reporter over time. The assay system allowed us to discriminate between acute cytotoxic effects (3-day exposure) and sub-chronic toxicity (7-day exposure). Whereas only a very limited number of compounds had acute cytotoxic effects, there were considerable more compounds with sub-chronic cytotoxicity. Based on the single endpoint classifications, we chose an mTOR inhibitor, Torin-2, to further assess molecular mechanism of drug action for all three tumor microtissue models used in this study, applying reverse phase protein array (RPPA) and transcriptome analysis for signaling pathway profiling. Preliminary results of RPPA analysis revealed a clear down-regulation in the cell cycle and PI3K/Akt/mTOR signaling pathway, especially for S6 ribosomal protein phosphorylated at Ser 235, 236, 240 and 244, indicating a strong inhibitory effect on translation and cell growth control. In summary, we present a 3D tumor microtissue-based phenotypic drug discovery testing scheme which allows a multi-parametric endpoint analysis paired with subsequent molecular pathway identification. Citation Format: Madhu A. Lal, Zoe Weydert, Jens Kelm, Marc Ferrer. Multi-parametric 3D tumor microtissue-based phenotypic compound classification. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 771.

Infiltration of lymphocyte subpopulations into cancer microtissues as a tool for the exploration of immunomodulatory agents and biomarkers

IntroductionThe interaction between the immune system and malignant diseases is a proven key target for cancer therapy. We describe an innovative 3D cell culture system comprising both immune and cancer cells to evaluate their interaction and immune cell infiltration to provide an innovative in vitro screening of immunomodulatory agents and biomarkers. Methods3D tumor microtissues were cultivated using a hanging drops system. Human non-small-cell lung cancer cell lines were incubated for 7 days to form microtissues. On day 5, peripheral blood mononuclear cells (PBMC) were added with or without interleukin-2 (IL-2) for 24 or 48h. Viability of cancer cells and the infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of tumor cells and PBMC and the infiltration of the PBMC into the tumor microtissues were analyzed by immunohistochemistry. Quantification of infiltration was measured by applying the TissueFAXS system. ResultsImmunohistochemistry revealed PBMC infiltration in all cell lines which increased under IL-2 stimulation. Analysis of infiltrating populations showed both lymphocyte subpopulations and monocytes within the tumor microtissues. In all three co-cultures, CD3+CD8+ and CD3+CD8+CD45R0+CD28+ lymphocytes were increased with IL-2, whereas CD3+CD8+CD45R0-CD28+ PBMCs were decreased with and without IL-2 stimulation. ConclusionIn summary, we present a novel cell culture system to study the interaction between cancer cells and immune cells in 3-dimensional microtissues. In addition, we report for the first time an in vitro infiltration assay based on 3D microtissues. This model has the potential to provide a tool for ex-vivo drug testing and biomarker screening of immunomodulatory agents.

Abstract B75: High-throughput, bright-field imaging of 3D tumor spheroid growth and morphology for therapeutic and efficacy screening: A study using chemotherapeutic compounds

Abstract Background 3D tumor microtissues or spheroids are widely accepted as a more representative biological model in phenotypic drug discovery. Size and morphology are important determinants to evaluate the biological behavior of 3D spheroids, particularly in development of anti-cancer drugs where monitoring cell growth is a particularly critical endpoint. The increasing number of applications of 3D tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including high-throughput image analysis. In this study, we assessed the growth and morphology changes of HCT116 colorectal cancer spheroids in response to two chemotherapy drugs-gemcitabine and docetaxel- using a high throughput, bright-field spheroid imager (Cell3iMager, SCREEN Holdings Ltd., Inc.). Methods Tumor spheroids consisting of the HCT116 colorectal cancer cell line were aggregated in InSphero's GravityPLUS™ plate (a scaffold-free hanging drop platform). The aggregated spheroids were transferred into a GravityTRAP™ plate prior to dosing. Spheroids were dosed with anti-cancer drugs-gemcitabine and docetaxel (0.8-20nM) at day 0 and re-dosed at day 3. The plates were scanned and spheroid size and morphology were analyzed daily for 7 days. Endpoint analysis of ATP content was carried out at day 7 utilizing a luminescence-based ATP assay (CellTiter-Glo® 2.0 Assay, Promega) Results HCT116 spheroids were measured daily for 7 days to determine dose-response effects of gemcitabine and docetaxel treatment (0.8-20nM) on spheroid area (um2). A size increase was observed for the control and the lowest compound concentration treated spheroids whereas the spheroid growth was inhibited at the higher concentrations tested (>4nM). Among the tested compounds, HCT116 spheroids showed less sensitivity to docetaxel compared to gemcitabine. At day 7 the treatment with 20nM gemcitabine resulted in more than 50% reduction in tumor area (um2) which was positively correlated with the decrease in cell viability, determined by ATP content. Using HCT116 tumor microtissues cultured in InSphero's GravityTRAP™ plates, we demonstrate that the measurement of tumor size with the Cell3iMager could be a suitable endpoint compared to that of ATP content for drug induced cytotoxicity in tumor spheroids, without lysing cells or otherwise interfering with long term culture of spheroids. Conclusions Label-free measurement of changes in 3D microtissue size and morphology using the Cell3iMager enables efficient assessment of phenotypic endpoints without disrupting tumor growth, and can be used to focus selection of therapeutic targets and treatment strategies before costly and tedious testing in animal models. Citation Format: Leena Mol Thuruthippallil, Jens M. Kelm, David Fluri. High-throughput, bright-field imaging of 3D tumor spheroid growth and morphology for therapeutic and efficacy screening: A study using chemotherapeutic compounds. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B75.

Pro-drug activation in dynamic microphysiological fluidic systems interconnecting liver and tumor microtissues

Pro-drug activation in dynamic microphysiological fluidic systems interconnecting liver and tumor microtissues

Abstract 324: High throughput triculture: A breast cancer spheroid model for drug screening

Abstract Many drugs that show promise in preclinical evaluation fail miserably during clinical trials, and the inability of current preclinical models to properly predict drug response may be attributed to oversimplified or non-existent tumor microenvironments. To provide a more physiologically predictive model for drug screening, we have developed an in vitro, 384 well, triculture system that presents key chemical (i.e. low pH, low glucose, low oxygen), extracellular matrix (ECM), and cellular (tumor cells, stromal cells and endothelial cells) attributes of tumor tissues. By using ECM proteins, we can promote the proper physiology for each of these cell types in triculture. To evaluate interactions between each cell type, they are fluorescently labeled with fluorophores with different excitation and emission spectra; MCF7 and MDA-MB-231 human breast cancer cell lines express a red fluorescence protein, while human umbilical vein endothelial cells (HUVECs) and human adipose-derived mesenchymal stem cells (hMSCs) are labeled with stable lipophilic membrane dyes. Breast tumor microtissues spontaneously assemble when breast cancer cells are co-cultured with hMSCs under low adhesion conditions. Once formed, these microtissues are deposited onto preformed tubule networks comprised of HUVECs and hMSCs and are embedded within a hydrogel composed of ECM proteins. Cellular interactions and dissemination are monitored via fluorescence microscopy, and cell proliferation of the breast cancer cells is quantified using a fluorescence plate reader. The breast cancer cells exhibit cell-cell interactions with endothelial tubules and stromal cells, forming microtissues. Furthermore, the response to anti-cancer drugs, paclitaxel and fluorouracil, in an in vitro triculture system is similar to the response for the same drugs in xenografts model, unlike 2D or 3D monoculture. This high throughput triculture cancer model will provide more insight into pathological mechanisms and can better predict response to anti-cancer drug treatment. Citation Format: Gabriel J. Benton, Gerald DeGray, Irina Arnaoutova, Hynda K. Kleinman, Jay George. High throughput triculture: A breast cancer spheroid model for drug screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 324. doi:10.1158/1538-7445.AM2015-324

Isothermal microcalorimetry accurately detects bacteria, tumorous microtissues, and parasitic worms in a label-free well-plate assay.

Isothermal microcalorimetry is a label-free assay that allows monitoring of enzymatic and metabolic activities. The technique has strengths, but most instruments have a low throughput, which has limited their use for bioassays. Here, an isothermal microcalorimeter, equipped with a vessel holder similar to a 48-well plate, was used. The increased throughput of this microcalorimeter makes it valuable for biomedical and pharmaceutical applications. Our results show that the sensitivity of the instrument allows the detection of 3 × 104 bacteria per vial. Growth of P. mirabilis in Luria Broth medium was detected between 2 and 9 h with decreasing inoculum. The culture released 2.1J with a maximum thermal power of 76 μW. The growth rate calculated using calorimetric and spectrophotometric data were 0.60 and 0.57 h–1, respectively. Additional insight on protease activities of P. mirabilis matching the last peak in heat production could be gathered as well. Growth of tumor microtissues releasing a maximum thermal power of 2.1 μW was also monitored and corresponds to a diameter increase of the microtissues from ca. 100 to 428 μm. This opens new research avenues in cancer research, diagnostics, and development of new antitumor drugs. For parasitic worms, the technique allows assessment of parasite survival using motor and metabolic activities even with a single worm.

3D spherical microtissues and microfluidic technology for multi-tissue experiments and analysis

Rational development of more physiologic in vitro models includes the design of robust and flexible 3D-microtissue-based multi-tissue devices, which allow for tissue–tissue interactions. The developed device consists of multiple microchambers interconnected by microchannels. Pre-formed spherical microtissues are loaded into the microchambers and cultured under continuous perfusion. Gravity-driven flow is generated from on-chip reservoirs through automated chip-tilting without any need for additional tubing and external pumps. This tilting concept allows for operating up to 48 devices in parallel in order to test various drug concentrations with a sufficient number of replicates. For a proof of concept, rat liver and colorectal tumor microtissues were interconnected on the chip and cultured during 8 days in the presence of the pro-drug cyclophosphamide. Cyclophosphamide has a significant impact on tumor growth but only after bio-activation by the liver. This effect was only observed in the perfused and interconnected co-cultures of different microtissue types on-chip, whereas the discontinuous transfer of supernatant via pipetting from static liver microtissues that have been treated with cyclophosphamide did not significantly affect tumor growth. The results indicate the utility and multi-tissue functionality of this platform. The importance of continuous medium circulation and tissue interaction is highlighted.