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

Cancer progression is an evolutionary process driven by the selection of cells adapted to gain growth advantage. We present the first formal study on the adaptation of gene expression in subclonal evolution. We model evolutionary changes in gene expression as stochastic Ornstein-Uhlenbeck processes, jointly leveraging the evolutionary history of subclones and single-cell expression data. Applying our model to sublines derived from single cells of a mouse melanoma revealed that sublines with distinct phenotypes are underlined by different patterns of gene expression adaptation, indicating non-genetic mechanisms of cancer evolution. Interestingly, sublines previously observed to be resistant to anti-CTLA-4 treatment showed adaptive expression of genes related to invasion and non-canonical Wnt signaling, whereas sublines that responded to treatment showed adaptive expression of genes related to proliferation and canonical Wnt signaling. Our results suggest that clonal phenotypes emerge as the result of specific adaptivity patterns of gene expression.

Melanoma clonal subline analysis uncovers heterogeneity-driven immunotherapy resistance mechanisms.

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.

Studying the History of Tumor Evolution from Single-Cell Sequencing Data by Exploring the Space of Binary Matrices.

Single-cell sequencing (SCS) data have great potential in reconstructing the evolutionary history of tumors. Rapid advances in SCS technology in the past decade were followed by the design of various computational methods for inferring trees of tumor evolution. Some of the earliest methods were based on the direct search in the space of trees with the goal of finding the maximum likelihood tree. However, it can be shown that instead of searching directly in the tree space, we can perform a search in the space of binary matrices and obtain maximum likelihood tree directly from the maximum likelihood matrix. The potential of the latter tree search strategy has recently been recognized by different research groups and several related methods were published in the past 2 years. Here we provide a review of the theoretical background of these methods and a detailed discussion, which are largely missing in the available publications, of the correlation between the two tree search strategies. We also discuss each of the existing methods based on the search in the space of binary matrices and summarize the best-known single-cell DNA sequencing data sets, which can be used in the future for assessing performance on real data of newly developed methods.

PhISCS: a combinatorial approach for subperfect tumor phylogeny reconstruction via integrative use of single-cell and bulk sequencing data.

Available computational methods for tumor phylogeny inference via single-cell sequencing (SCS) data typically aim to identify the most likely perfect phylogeny tree satisfying the infinite sites assumption (ISA). However, the limitations of SCS technologies including frequent allele dropout and variable sequence coverage may prohibit a perfect phylogeny. In addition, ISA violations are commonly observed in tumor phylogenies due to the loss of heterozygosity, deletions, and convergent evolution. In order to address such limitations, we introduce the optimal subperfect phylogeny problem which asks to integrate SCS data with matching bulk sequencing data by minimizing a linear combination of potential false negatives (due to allele dropout or variance in sequence coverage), false positives (due to read errors) among mutation calls, and the number of mutations that violate ISA (real or because of incorrect copy number estimation). We then describe a combinatorial formulation to solve this problem which ensures that several lineage constraints imposed by the use of variant allele frequencies (VAFs, derived from bulk sequence data) are satisfied. We express our formulation both in the form of an integer linear program (ILP) and-as a first in tumor phylogeny reconstruction-a Boolean constraint satisfaction problem (CSP) and solve them by leveraging state-of-the-art ILP/CSP solvers. The resulting method, which we name PhISCS, is the first to integrate SCS and bulk sequencing data while accounting for ISA violating mutations. In contrast to the alternative methods, typically based on probabilistic approaches, PhISCS provides a guarantee of optimality in reported solutions. Using simulated and real data sets, we demonstrate that PhISCS is more general and accurate than all available approaches.