Abstract 136: Single cell lineage analysis reveals clonal evolution and dynamic intra-clonal gene expression states during colorectal cancer progression
Authors: Kasem M Haidar , Jády Attila , Mallm Jan-Philipp , Glimm Hanno , Dieter Sebastian M. , Herbst Friederike , Ball Claudia R. 
Published: April 2023

Abstract
Colorectal cancer harbors heterogeneous cell populations that contribute to tumor progression. Clones with long term tumor-initiating cell activity are thought to fuel colorectal cancer (CRC) metastasis formation and recent reports suggest that drug tolerant persister cells are endowed with the ability to replenish the tumor during relapse upon therapy. To decipher gene expression heterogeneity of sub-populations within individual clones, we link gene expression states to clonal dynamics in metastatic and therapy resistant sub-populations. We transduced CRC patient-derived organoids with CloneTracker XP™ library, which bears stable genetic barcodes expressed on the RNA level. 300,000 uniquely barcoded cells were either passaged with and without drugs treatment (5-fluorouracil, oxaliplatin and irinotecan) in vitro or serially transplanted orthotopically into immune deficient NSG mice. At determined endpoints, cells or xenograft tumors were harvested and analyzed for clonal dynamics and sub-clonal gene expression programs associated to tumor progression and therapy resistance, respectively using single cell RNA and barcode DNA sequencing.Integrated bioinfomatic analyses of scRNA data from more than 35,000 cells before, during and after recovery from in vitro drug treatment linked clonal barcodes to gene expression of individual cells. The clonal complexity score, i.e number of distinct clones/number of cells, remained stable before and after treatment, suggesting that the clonal composition of the cell population was marginally affected by chemotherapy. After recovery, single cell gene expression data reflected that individual clones contain cells in distinct transcriptional states, with an elevated proportion expressing the intestinal stem-like program. In addition, single cell and bulk barcode analysis of transplanted xenografts revealed that the bulk of tumor and metastasis in liver and lung is formed by a subfraction of 0.006% of all transplanted clones, indicating a strong clonal selection. In serial transplantations of metastasis, only 4 to 5 clones constituted more than 90% of cancer cell bulk. Regarding gene expression in tumor, individual cell subpopulations within the same clone were distributed within 12 different gene expression clusters, which were maintained in metastasis. Overall, our results indicate that multiple clones contribute to both metastasis and therapy resistance and suggest that intra-tumoral heterogeneity is driven by modulation of subclonal gene expression programs. Here, we provide insights into subclonal gene expression heterogeneity and identify transcriptional states which may be potentially relevant for tumor progression and therapy resistance. This may provide the basis for the future development and testing of innovative novel treatment strategies directed against these cellular states.
Citation Format: M Haidar Kasem, Attila Jády, Jan-Philipp Mallm, Hanno Glimm, Sebastian M. Dieter, Friederike Herbst, Claudia R. Ball. Single cell lineage analysis reveals clonal evolution and dynamic intra-clonal gene expression states during colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 136.