Patient-derived tumor organoids (PDTOs) have become relevant pre-clinical models for therapeutic modeling since they highly recapitulate patients' responses to treatment. Nevertheless, their value for immunotherapy modeling has not been fully explored. We developed a tumor processing protocol that enables the establishment of PDTOs and tumor-infiltrating lymphocytes (TILs) isolation. By the optimization of functional assays, we compared the T-cells effector functions of matching PBMCs and TILs, demonstrating that PBMCs after co-culture and TILs after initial expansion display similar responses. In addition, the evaluation of cytokine production by fluorospot in combination with an image-based killing assay enables the screening of anti-PD-1 combinations with alternative immune checkpoint inhibitors as well as its combination with target inhibitors. Our proof-of-concept functional assays showed the potential and versatility of PDTOs and T-cell co-culture systems for immunotherapy screening. The optimization of scalable functional assays downstream co-culture represents a significant step forward to increase the value of PDTOs as pre-clinical models for immunotherapeutic screens.

No abstract

Focal radiation therapy (RT) can increase tumor immunogenicity and T cell-mediated tumor rejection when combined with immune checkpoint blockade (ICB). Our prior findings in patients and in preclinical studies suggest that one mechanism whereby RT increases tumor immunogenicity is by enhancing the expression of neoantigens.1,2 In response to RT, cells rapidly transcribe and translate hundreds of genes associated with the DNA damage response (DDR), protein turnover and cellular stress. Genes in these pathways are frequently mutated in cancer. Thus, we hypothesized that the presence of immunogenic mutations in genes upregulated by radiation could help predict the benefits of RT used in combination with ICB. In order to develop such a predictor, we built an in vitro system to study the RT-induced transcriptional response of primary human tumors cultured as patient-derived tumor organoids (PDO). PDOs were established from 2 breast cancer (BC), 4 non-small cell lung cancer (NSCLC) and 3 colorectal cancer (CRC) patients. PDOs were irradiated with doses of 5 to 8 Gy daily for 3 days (n=4) or left untreated (UT, n=4) and cultured for 24h prior to RNA extraction. RNA sequencing was carried out using a NovaSeq6000 sequencer (Illumina) to a depth of 30 million reads. Differential expression analysis between RT and UT samples was performed using DESeq2 and significantly perturbed genes were defined as genes with fold change greater than 1.5 and adjusted p-value cutoff of 0.05. In total we detected an average of 15,000+/-800 protein coding genes per sample and the number of genes modulated by RT was found to vary with cancer type. This ranged from an average of 3.9+/-1.6% genes upregulated by RT in NSCLC to 12.1+/-2.5% in CRC organoids. There were 104 and 490 genes commonly upregulated by RT in NSCLC and CRC, respectively. One of the BC PDOs is a triple-negative BC and the other HR+ and despite this difference, we identified 24 commonly upregulated genes. Gene Set Enrichment Analysis (GSEA), revealed enrichments in DDR and pro-inflammatory signaling pathways among the upregulated genes. Whole exome sequencing and paired normal tissue was used to determine the variants and predicted neoantigens for these PDOs. In one of the EGFR mutated NSCLC PDOs we found that among the genes transcriptionally upregulated by RT, 18 carried somatic mutations predicted to be antigenic. The presentation of these neoantigens is being investigated by mass spectrometry analysis of the immunopeptidome eluted from surface MHC-I molecules. Similar investigations are being carried out in the other models. Overall, results support the feasibility of identifying a common signature of RT upregulated genes in PDOs. This will provide a system to predict RT- exposed neoantigens. [1] Formenti et al. Nat. Med. 2018;24(12):1845-1851, [2] Lhuillier et al. J. Clin. Invest. 2021;131(5):e138740 Citation Format: Samantha J. Van Nest, Jared Capuano, Bhavneet Bhinder, Martin G. Klatt, Tuo Zhang, M. Laura Martin, Silvia C. Formenti, Olivier Elemento, Nils-Petter Rudqvist, Sandra Demaria. Patient-derived tumor organoids as a platform to study radiation-exposed neoantigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1309.