Abstract C044: Single cell RNA-sequencing coupled with lineage tracing identifies novel clonal populations associated with immunotherapy resistance
Authors: Saleh Reem , Patel Riyaben , Vassiliadis Dane , Hammal Fayrouz , Blyth Benjamin , Du Xin , Fennell Katie , Dawson Mark A. , McArthur Grant A. , Sheppard Karen 
Published: November 2024

Although immune checkpoint inhibitors (ICIs) have revolutionized melanoma treatment, many individuals either show no response or eventually develop acquired resistance. This variability could be linked to differences in the tumor immune microenvironment, pre-existing drug-resistant cells or treatment induced changes in melanoma cell states. Using single cell RNA sequencing (scRNA-seq) coupled with heritable barcodes, this study aims to lineage trace changes in melanoma transcriptional cell states and map changes in the tumor immune microenvironment to uncover if preexisting and/or therapy-induced melanoma transcriptional cell states lead to resistance to ICIs. In this study, we used YUMMER1.7 mouse melanoma cells which were transduced with barcodes under conditions that allow delivery of a single unique barcode to each cell, this served as a cell lineage tag. Each barcode is heritable and stably transcribed into RNA molecules so that individual barcoded cells can be matched with a gene expression profile from scRNA-seq outputs. The barcoded cells were subcutaneously injected into C57BL/6 mice, and once tumors were established, mice were treated with 3 cycles of anti-PD-1 and anti-CTLA-4 therapy. Lineage tracing and scRNA-seq analyses were performed on tumors harvested prior to treatment, early on treatment (Day 6), during minimal residual disease (Day 13) and upon relapse. Early on treatment (Day 6)- two tumour groups were harvested, those that responded well and others that only partially responded. Our results showed that during the early on treatment phase (Day 6) tumors that responded well and those that only partially responded displayed distinct barcodes. Additionally, prevalent barcoded cells identified at the minimal residual disease phase were also dominant upon relapse. Analysis of the transcriptional states is underway to determine if ICI therapy induces transcriptional heterogeneity in sensitive and tolerant cells and if a particular or several transcriptional states lead to relapse. This study will potentially identify predictive response biomarkers and vulnerabilities of ICI naïve and resistant cells that could be targeted to improve outcomes for melanoma patients. We will also identify neoadjuvant approaches, to remove the inherent heterogeneity within the tumor cell population, facilitating a more uniform sensitivity to ICIs.

Citation Format: Reem Saleh, Riyaben Patel, Dane Vassiliadis, Fayrouz Hammal, Benjamin Blyth, Xin Du, Katie Fennell, Mark A. Dawson, Grant A. McArthur, Karen Sheppard. Single cell RNA-sequencing coupled with lineage tracing identifies novel clonal populations associated with immunotherapy resistance [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C044.