Melanoma clonal subline analysis uncovers heterogeneity-driven immunotherapy resistance mechanisms

Abstract

Intratumoral heterogeneity (ITH) can promote cancer progression and treatment failure, but the complexity of the regulatory programs and contextual factors involved complicates its study. To understand the specific contribution of ITH to immune checkpoint blockade (ICB) response, we generated single cell-derived clonal sublines from an ICB-sensitive and genetically and phenotypically heterogeneous mouse melanoma model, M4. Genomic and single cell transcriptomic analyses uncovered the diversity of the sublines and evidenced their plasticity. Moreover, a wide range of tumor growth kinetics were observed in vivo, in part associated with mutational profiles and dependent on T cell-response. Further inquiry into melanoma differentiation states and tumor microenvironment (TME) subtypes of untreated tumors from the clonal sublines demonstrated correlations between highly inflamed and differentiated phenotypes with the response to anti-CTLA-4 treatment. Our results demonstrate that M4 sublines generate intratumoral heterogeneity at both levels of intrinsic differentiation status and extrinsic TME profiles, thereby impacting tumor evolution during therapeutic treatment. These clonal sublines proved to be a valuable resource to study the complex determinants of response to ICB, and specifically the role of melanoma plasticity in immune evasion mechanisms.

Stochastic modelling of single-cell gene expression adaptation reveals non-genomic contribution to evolution of tumor subclones

“…The development of the 24 clonal sublines was described previously 24 . In brief, 24 single cells were randomly selected and isolated from the parental B2905 mouse melanoma line, which was generated 23 and cultured in our laboratory, by cell sorting without any labeling.…”

“…They were expanded in culture to become individual clonal sublines, C1 to C24. Their analyses of whole exome, whole transcriptomics, and single-cell full transcripts were also described by 24 .…”

“…Experimental Setting To study gene expression evolution in cancer, we analyze data that was previously generated in Gruen et al 24 ; here we briefly review the data generation protocol. When implanted in syngeneic C57BL/6 mice, B2905 melanoma cells form tumors that exhibit heterogeneous responses to immune checkpoint blockade (ICB) therapies among individual hosts 23 .…”

“…When implanted in syngeneic C57BL/6 mice, B2905 melanoma cells form tumors that exhibit heterogeneous responses to immune checkpoint blockade (ICB) therapies among individual hosts 23 . To reconstruct the subclonal evolutionary history of the melanoma, 24 single cells were first isolated from the parental B2905 cell line and expanded to become individual clonal sublines, C1 to C24 (Figure 2a, box 1), as described previously 24 . The sublines were then subjected to bulk whole-exome sequencing (Figure 2a, box 3).…”