Background Immune checkpoint inhibitors (ICIs) have shifted the cancer treatment paradigm. Cancers such as melanoma and non-small cell lung cancer (NSCLC) demonstrate high tumor mutational burden and tumor neoantigen expression which renders them more responsive to checkpoint inhibitor blockade compared to other malignancies. Yet, 40–65% of metastatic melanoma patients do not have an initial response to ICI therapy1 and in NSCLC, PD-L1 expression, often a prerequisite for ICI treatment, does not definitively associate with ICI clinical response2. Mechanisms of resistance often result from aberrant interactions between tumor and immune cells. Development of pre-clinical models that mimic the complex interplay between cells within the tumor microenvironment in a patient-specific manner are critical for accurate ex vivo profiling of ICIs. To improve immunotherapy predictive testing, we present a 3D spheroid culture system for testing personalized ICI efficacy.
Methods Cell lines co-cultured with T-cells from healthy donor peripheral blood mononuclear cells were used to optimize assay conditions and confirm ICI binding to target proteins. For ex vivo testing, primary melanoma or NSCLC tumor tissue from treatment naïve patients was dissociated and cultured as 3D spheroids using autologous immune cells to profile ICI target expression and sensitivity to treatment. ICI enhanced T-cell killing of tumor cells was quantified via lactate dehydrogenase release. Changes in IFNγ were used as a metric for ICI induced immune cell activation. Ratios and activation status of T-cell subsets was determined using flow cytometry. Fluorescent imaging was used to confirm the mechanism of tumor cell killing.
Results ICI binding to target proteins was measured across six ICIs, and no significant differences in concentration-dependent site occupancy within drug target classes was observed. However, differences in drug induced cytotoxicity across different tumor samples was detected even within the same drug target class. The immune composition of tumor samples that responded to ICIs displayed increased T-cell activation and increased IFNγ production. Furthermore, changes in PD-L1 and MHC-class I expression were detected which reflected ICI response. Finally, T-cell-dependent induction of tumor cell apoptosis was confirmed to be the observed mechanism of cytotoxicity within the 3D spheroid system.
Conclusions This work demonstrates that differences in ICI induced cytotoxicity can accurately be detected using our ex vivo 3D spheroid platform. The differences in therapy sensitivity can be related back to cell composition and function to potentially predict patient-specific drug response. Future correlation to patient clinical outcomes will be necessary for true clinical applications.
Trial Registration N/A
Ethics Approval Tissue for this study was procured from commercial vendors who maintain strict ethical compliance, including fully de-identified materials and stringent IRB and Ethics Committee compliance.
Fenton SE, Sosman JA, Chandra S. Resistance mechanisms in melanoma to immuneoncologic therapy with checkpoint inhibitors. Cancer Drug Resistance. 2019;2(3):744–61.
Chiang AC, Herbst RS. Frontline immunotherapy for NSCLC - the tale of the tail. Nat Rev Clin Oncol 2020;17(2):73–4.
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