Explore tumor intrinsic factors regulating immunotherapy response in ovarian cancer

Despite approval of immunotherapy for the treatment of ovarian cancer patients, the majority of patients fail to respond to immunotherapy or relapse following initial response. These failures may be attributed to immunosuppressive mechanisms co-opted by tumor cells. However, it is challenging to use conventional methods to systematically evaluate the potential of tumor intrinsic factors to act as immune regulators in patients with cancer. To identify immunosuppressive mechanisms in non-responders to cancer immunotherapy in ovarian cancer , we firstly performed an unbiased genome-wide CRISPR immune screens to explore tumor intrinsic factors which regulates ovarian cancer cell lines response to T cell mediated killing. Then we integrated the in vitro CRISPR screen results with multi-omics clinical data to generate a sub-library including top candidates for in vivo CRISPR screen to further evaluate the role of potential tumor intrinsic factors in regulating ovarian cancer cell response to immunotherapy with using the immune checkpoint blocker. Collectively, the data provide a rich resource of novel targets for rational immuno-oncology combinations. Overall design: To explore the potential tumor intrinsic factors regulating T cell mediated killing, we performed both in vitro and in vivo immune screens. For in vitro screens: gRNA-expressing ID8/GC cells were cultured with or without gp100-specific T cells from pmel mice. Experimental groups are listed below. Equal amount of barcoded genomic DNA samples from all groups were pooled and sequenced into 4 individual sequencing lanes. 1.ID8/GC cells transduced with genome-wide gRNA library without T cell treatment: Ctrl 1_1,Ctrl1_2, Ctrl2_1 and Ctrl2_2; 2. ID8/GC cells transduced with genome-wide gRNA library with T cell treatment at ET ratio=0.2: ET02-2,ET02-3 and ET02-4; 3. ID8/GC cells transduced with genome-wide gRNA library with T cell treatment at ET ratio=1: ET1-2, ET1-3 and ET1-4; For in vitro screens: sub-library expressing ID8/GC cells(ID8/GCSL) were inoculated in NSG or C57BL/6 mice. Tumor bearing mice were treated with or without anti-PD-1. Experimental groups are listed below. Equal amount of barcoded genomic DNA samples from all groups were pooled and sequenced into 4 individual sequencing lanes. 1. ID8/GCSL tumor tissue which were from tumor-bearing NSG mice: NSG-1~5; 2. ID8/GCSL tumor tissue which were from tumor-bearing C57/BL6 mice receiving mock treatment: Mock-1~5; 3. ID8/GCSL tumor tissue which were from tumor-bearing C57/BL6 mice receiving anti-PD-1 treatment: PD1-1~10 4. Plasmid of for in vivo gRNA sub-library: plasmid_ICB sub-library; 5. ID8/GCSL cells after 2-day puromycin selection: Ref-1; 6. ID8/GCSL cells after 7-day puromycin selection: Ref-2;