Abstract B030: Identification of tumor microenvironment informed exosome biomarkers for predicting response to anti-PD1 therapy

Abstract On-treatment biomarkers for assessing the response to immune-oncology (I/O) therapy have a better predictive value than baseline markers, prior to treatment1. The Farcast TruTumor histoculture platform that preserves the near native tumor microenvironment has the potential to identify such biomarkers post treatment with I/O therapy2. Accessing fresh tumor sample is often challenging, limiting the clinical utility of such ex-vivo platforms. To address this limitation, we assessed exosomes from explant culture supernatants post-treatment, for identifying response biomarkers in the culture supernatant that mimic the TME response and will seek to validate these in exosomes from matched patient plasma. Head and Neck Squamous Cell Carcinoma (HNSCC) surgical excess samples along with matched blood were used for this study. Explants generated from these samples treated with anti-PD1, Nivolumab (132µg/ml) for 72 hours, and response evaluated using flow cytometry and cytokine release analysis. Exosomes were isolated from culture supernatant and plasma using a combination of precipitation and CD63/81/9 coated magnetic bead-based purification. Exosome size was verified using nano particle tracking and further characterized using multicolor flowcytometry analysis. We observed similar distribution patterns of exosomes from the culture supernatant and matched plasma, with highest proportion of CD63+ exosomes. We evaluated exosomal composition of two samples (S1 and S2) that responded differentially to anti-PD1 treatment. S1 displayed strong anti-PD1 treatment driven T cell reinvigoration phenotype along with tumor cytolytic activity, while S2 did not. There was a treatment induced increase in T cell derived exosome (CD3+, TCex) and Interferon gamma (IFNg) positive TCex in S1 but a decrease of both in S2, mirroring the strong response phenotype in S1. We observed a decrease in PD1/PDL1 ratio in exosomes from the culture supernatant of S1 (∼1.5 fold) indicative of decreased exhaustion. This correlated with increase in IFNg secretion on treatment. The PD1/PDL1 ratio remained unchanged in S2. Additionally, TCex/ M2 like macrophage derived exosome (CD206+, MPex) ratio was higher in S1 (1.8) compared to S2 (0.7) upon treatment, indicative of an M2 like macrophage driven immunosuppressive TME that explained the non-responsive phenotype in S2. A similar picture emerged on immune cell profiling, wherein, on treatment S1 had a higher T cell/ M2 macrophage ratio (18.4) compared to S2 (1.9). The data presented from the TruTumor ex vivo platform illustrates good concordance between treatment induced changes in immune cell derived exosome composition and intra-tumoral immune cell profile. This platform presents a unique opportunity for identification of exosomal biomarkers that could be potentially tracked in periphery. 1. Chen, P.L et al. Cancer Discov. 6, 827–837 (2016) 2. Basak, NP et al. Nat Commun 15, 1585 (2024). Citation Format: Kowshik Jaganathan, Moumita Nath, Biswajit Das, Méhul Kapur, Christopher Davitt, Ritu Malhotra, Mohit Malhotra, Nandini Pal Basak, Satish Sankaran. Identification of tumor microenvironment informed exosome biomarkers for predicting response to anti-PD1 therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B030.