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Quantitative microscopy is a powerful method for performing phenotypic screens, from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments. Here, we examine the performance of the Cell Painting assay across multiple high-throughput microscope systems and find that all are compatible with this assay. Furthermore, we determine independently for each microscope system the best performing settings, providing those who wish to adopt this assay an ideal starting point for their own assays. We also explore the impact of microscopy setting changes in the Cell Painting assay and find that few dramatically reduce the quality of a Cell Painting profile, regardless of the microscope used.

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Immunotherapy is now recognized as a powerful therapeutic approach to treat and cure cancer. However, the objective response rates for most solid tumors are low (below 30%). Accumulating evidence has shown that chemotherapy can increase the efficacy of immune checkpoint blockade and improve cancer outcomes. We have investigated if chemotherapy can modulate anti-PDL1 enhanced T cell cytotoxicity in breast cancer using novel in vitro 3D co-culture assay system. 3D co-culture of cancer and immune cells is a powerful platform for disease modeling and therapies testing because it can mimic the tumor micro-environment and complex cellular interactions. In this study we evaluated the immunomodulatory effect of chemotherapy on spheroid-T cells interactions in response to PD-L1 inhibition in triple-negative breast cancer. MCF7 and MDA231 cell lines with differential PD-L1 status were formed into spheroids and used as a tumor model. T cells were activated from PBMCs using ImmunoCult CD3/CD28 T cell Activator. Co-culture assays were performed over 72 hr in a Pu·MA System. The Pu·MA System is an automated microfluidic platform that enables phenotypic and functional assays using physiologically relevant 3D cell models. Cancer spheroids and T cells were cultured, manipulated, and measured in a single well of a microfluidic flowchip. The platform integrates 1) 3D cell model with delivery of immune cells, 2) drug delivery, 3) imaging of phenotypic features and 4) functional profiling for cytokine secretion and viability. T cells-spheroid interaction, invasion and cell viability was assessed using high-content fluorescence imaging CellVoyager CQ1 system. IL2, IFN-γ and TNF-α secretion was measured in collected supernatants using Lumit immunoassays. After viability determination, co-cultures were fixed and stained for cancer and T cell markers (E-Cadherin, F-Actin, CD3, CD8) using automated IF staining protocol and then imaged. Image analysis revealed colocalization and infiltration of the T cells into the spheroids. Disintegration of spheroid, loss of circular shape, and increased number of dead cancer cells indicated T cell mediated cancer cell death. To evaluate the immunomodulatory effect of chemotherapy on spheroid-T cells interactions in response to PD-L1 inhibition, co-cultures were exposed to PD-L1 inhibitor Atezolizumab in the presence or absence of Cisplatin/Pemetrexed. We have analyzed a shift in T cell-mediated killing activity and function in the presence or absence of chemotherapeutics. The proposed co-culture platform can be further extended to a more complex patient-derived 3D models using different cell types. Our functional immune-oncology 3D platform allows to study the crosstalk between immune, cancer and other cell interactions, evaluate new drug candidates and assess individual therapeutic approaches to advance precision medicine. Citation Format: Ekaterina Moroz Nikolov, Anthony Thai, Lila Cooper, Mahomi Suzuki, Arvonn Tully, Rashmi Rajendra, Evan F. Cromwell. Evaluation of the chemotherapy potential to improve the anti-cancer efficacy of PD-L1 inhibition using novel 3D microfluidic co-culture platform. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4559.

Background: Immunotherapy has revolutionized cancer research, however limited clinical success to date underscores the need for novel therapeutic targets. Assessment of new targets has been challenging due to limitations of ex vivo animal platforms that only partially recapitulate tumor-intrinsic features and cell interactions. Here, we present a live-cell imaging approach for assessing efficacy of immune-targeting therapies using a 3D in vitro oncology platform in combination with high-content imaging and analysis (HCA) techniques. This study shows how effects of immune-targeting agents on tumor infiltration and killing by peripheral blood mononuclear cells (PBMCs) can be visualized and quantified with high-content endpoints. Aim: To develop and test a robust live-cell-imaging methodology for assessing efficacy of immuno-modulatory cancer therapies. Material & Methods: Lung carcinoma cell-line models were generated using GFP-A549 cells in coculture with dermal fibroblasts and PBMCs. PDX cell suspensions of lung, breast and melanoma origin were self-aggregated with cancer-associated fibroblasts (CAFs) and labeled with live-cell dye for specific monitoring of tumor cell viability. Tumor killing activity and T-cell effector function were evaluated by measuring tumor model size using automatic stage fluorescence microscopy or confocal HCA instrument. Release of inflammatory cytokines IL-6, TNF, IFNγ and GM-CSF was measured with a multiplex cytokine panel. T cell infiltration into tumor models was quantified using fluorescent staining of CD3+ cells. To generate a pro-inflammatory tumor microenvironment, PBMCs were stimulated prior to coculture either with cytokines or anti-CD3/CD28. We monitored immune cell attack on tumors in realtime (56 timepoints) and in 3 dimensions without any discernable phototoxicity. Using cell centroid tools, we mapped physical locations of individual cells belonging to each population. Finally, we calculated tumor and immune cell volumes from individual image stacks to evaluate tumor killing and proliferation/infiltration of immune cells. Results: Our realtime confocal analysis of tumor-immune cell cocultures showed activation of PBMCs with either cytokines or CD3/CD28 increased proliferation/infiltration of immune cells and enhanced tumor killing. Conclusion: Proinflammatory tumor models combined with our novel, high-content imaging analysis endpoints offer a versatile solution for preclinical translational research and a promising approach for high-content screening of immune-targeting therapeutic agents and I-O studies involving engineered T cells (e.g., CAR-T) as well as for testing immunomodulatory agents, such as bispecific antibodies and immune checkpoint inhibitors. Citation Format: Irina Agarkova, Mahomi Suzuki, Silvan Strebel, Armin Wolf, Judith Wardwell-Swanson, Francesca Chiovaro. A live-cell imaging approach for assessing efficacy of immune-targeting therapies using high content imaging and analysis of 3D in vitro tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1449.