Genome profiles of lymphovascular breast cancer cells reveal multiple clonally differentiated outcomes with multi-regional LCM and G&T-seq

Abstract

The authors declare that there is no conflict of interest regarding the publication of this article.Character count: 52,408 (60,000 characters MAX including spaces and main figure legends but excluding STAR Methods text, supplemental item legends, and References section): Summary (Currently 120 words): 120 words MAXWe comprehensively analyzed the genomic and transcriptional profiles of 97 laser capture microdissection (LCM) isolated cell clusters from a single patient with triple negative breast cancer (TNBC). By performing simultaneous genome and transcriptome amplification and sequencing (G&T-seq) on these clusters, we identified four RNA cancer cell clones that were directly linked to DNA clone counterparts. We also detected the presence of a lymphovascular invasive (LVI) clone characterized by chromosome instability (CIN), and a specific genomic and transcriptional pattern shared with several primary cancer cell clusters. Furthermore, combination phylogeny analysis pointed to three evolutionarily distinct metastatic pathways in this patient, and hub gene evaluation found the down regulation of ribosomal protein mRNA to be a potential prognostic marker for breast cancer.Keywords: Triple negative breast cancer (TNBC), lymphovascular invasion (LVI), metastasis, lymphatics, laser capture microdissection (LCM), G&T-seq (genome and transcriptome sequencing) Significance (Currently 120 words): 120 words MAXThe lymphatic system is a unidirectional vascular network responsible for trafficking immune cells and lymphatic fluid. However, many cancer cell types use this system as a primary means of metastatic spread to regional lymph nodes. We describe the use of laser capture microdissection in the isolation of lymphovascular invasive (LVI) cancer cell clusters from within lymphatic vessels of a single breast cancer patient. Genomic and transcriptional profiling of LVI associated cancer cells, and cancer cell clusters derived from the primary tumor and lymph nodes of this patient, was performed using G&T-seq. Our findings improve our understanding of LVI metastatic mechanisms, and reveal that poor breast cancer survival outcomes may be related to the extensive down-regulation of transcripts encoding ribosomal proteins. Highlights:1. LVI clone shares a specific genome and transcriptome pattern with a small proportion of primary cancer cell clusters.2. G&T sequencing revealed RNA cancer cell clones to be highly correlative to DNA clones.3. Three evolutionarily and transcriptionally distinct pathways of tumor clone development and metastasis were found in one TNBC patient using combination phylogeny analysis. 4.Extensive ribosomal protein down-regulation may be a potential marker of poor prognosis in breast cancer patients.

Into the multiverse: advances in single-cell multiomic profiling

“…Recently G&T-seq [13] was coupled with laser capture microdissection (LCM) to generate spatially resolved genomic and transcriptional profiles of cancer cells with the potential for lymphovascular invasion in a patient with triple-negative breast cancer [76].…”

Exploiting Single-Cell Tools in Gene and Cell Therapy

“…Whole genome sequencing (WGS) approaches provide a crucial tool for characterizing genomic abnormalities in primary tumors (270). Zhu et al recently applied G&T-seq to a subset of lymphovascular invasive cells, isolated from a breast cancer patient (271), describing the relationship between RNA and CNV clones and outlining multiple functionally distinct clones and their role in metastatic dynamics. This illustrates the power of G&T-seq to uniquely integrate genomic abnormalities with transcriptional consequences, potentially of substantial utility in deciphering tumor heterogeneity and intra-tumoral clonal dynamics post CAR T therapy.…”