Garima Kaushik, PhD, Padma Kamineny, PhD, and Amy Wesa, PhD
Cancer progression and response to therapy are governed by complex cellular interactions in the tumor microenvironment (TME). Better understanding of TME and immune microenvironment requires robust experimental systems modeling patient specific immune interactions. Conventional 2D cultures fall short in recapitulating 3D morphological structures and resultant complex immune biology of native human tumors. A vastly heterogenous TME impacts patient treatment response, and there is a dearth of 2D or 3D in vitro models, that mimic in vivo diversity of TME, including infiltrating immune populations. 3D spheroid/organoid cultures typically contain neoplastic epithelium and fail to retain tumor- syngeneic tumor-infiltrating lymphocytes (TILs) interactions vital for exhibiting a clinically significant response to immune-therapeutics. Here we describe patient-derived xenograft (PDX) derived 3D organoid (PDXO) cultures with autologous TILs to avoid alloreactivity and simulate the tumor-specific immune response. The described model allows the evaluation of T cell infiltration and high throughput screening of immune-modulatory therapies.
Patient-derived xenografts from mice were enzymatically digested to establish PDXO. Established PDXO were characterized for expression of cancer markers. PBMCs from matching patients were isolated, and TILs were expanded in culture. TILs were characterized for expression of memory, inhibitory and proliferation markers. TILs and organoids from the same patient were fluorescent labeled and co-cultured for four days to evaluate tumor infiltration and drug cytotoxicity in 3D cultures. CellInsight CX7 high content imaging platform was used to track TILs and cancer cells. Immunofluorescence for T cell markers was used to confirm tumor infiltration. T cell infiltration and Tumor cell killing was evaluated in the presence and absence of immune-modulatory therapies. Luminex technology was used to quantify changes in the cytokine profile upon drug treatment.
PDXO were established to mimic in vivo tumor biology. Tumor-specific TILs were successfully expanded and characterized for expression of memory, inhibitory, proliferation and regulatory T cell markers. TIL infiltration in organoids was observed from day one in culture and maintained over four days. Immunofluorescence for T cell marker CD3 (cluster of differentiation 3) with in PDXO confirmed tumor infiltration. Infiltrating TILs with-in tumor organoids and tumor cell killing quantification using fluorescent dyes via high throughput imaging platform showed disease and patient specific response. PDXO treatment with immune-modulatory therapeutics led to changes in the secreted levels of cytokines IL2 (interleukin 2), IFNγ (Interferon gamma) and TNFα (Tumor Necrotic Factor alpha). Our patient autologous TILs – PDXO culture platform can be used to model IO therapeutic response with the tumor-specific immune microenvironment.