Abstract 6923: Deconvolution of breast cancer dissemination and therapy resistance via single-cell transcriptional lineage tracing

Abstract Chemotherapy remains the most used systemic treatment option for triple-negative breast cancer (TNBC). Lineage tracing studies demonstrated that treatment does not significantly alter clonal composition of primary tumors (PTs). The specific transcriptional phenotype (s) conferring chemoresistance in PTs and metastasis, however, are still largely undetermined, and have not been analyzed at single-clone resolution. We first modelled clinically relevant contexts of chemoresistance in our recently published single-cell lineage tracing approach (DOI: 10. 1158/0008-5472. CAN-22-2717), using the standard-of-care combination of adriamycin and cyclophosphamide (A+C) in TNBC xenografts. Treatment was administered in neoadjuvant or adjuvant settings, or both together. In all cases, surgical PT resection was followed by metastatic disease. The neoadjuvant treatment resulted into a stable disease, thus resembling a significant fraction of the treated patients. To investigate clonal and transcriptional dynamics we performed scRNAseq analysis of 12 replicates (3 matched PT-lung replicates for each condition, for a total of 24 samples). Clustering analyses revealed a separation of treatments and tissue of origin. We retrieved a large number of longitudinal clones, including rare clones with high metastatic potential, in all the experimental conditions. A+C did not significantly alter clonal abundances, neither in PTs nor in metastases. Pseudobulk transcriptional analyses revealed key transcriptional traits associated with chemoresistance. Differential expression on longitudinal clones gave insights about transcriptional features of chemoresistant and pro-metastatic clones under chemotherapy. Integration at single-clone level of cluster analysis, pseudobulk and differential expression revealed cystatin (CST) family genes as highly-enriched in the chemoresistant and pro-metastatic phenotype. We are currently validating the role of CST genes in tumor growth, dissemination and chemoresistance in vivo using a panel of specific shRNAs. In parallel, using the same experimental model, we successfully deconvoluted the metastatic cascade through a single-cell lineage tracing approach on matched PT-CTC (circulating tumor cells) -lung replicates. scRNAseq analyses of pro-metastatic, circulating and metastatic clones is ongoing. Our preliminary data showed that all the dominant clones in the metastatic lungs are also found as CTCs in the bloodstream, thus suggesting that all pro-metastatic cells are also capable of surviving in the bloodstream and reinforcing our concept of pro-metastatic clone. This approach led us to gain a complete overview around clonal and transcriptional evolution of breast cancer dissemination. Citation Format: Alberto Dalmasso, Andrea Cossa, Giulia Bertolini, Niccolò Roda, Ilaria Servidio, Francesco Antonio Tucci, Salvatore Pece, Gabriella Sozzi, Pier Giuseppe Pelicci. Deconvolution of breast cancer dissemination and therapy resistance via single-cell transcriptional lineage tracing abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 6923.