Characterizing the gene expression profiles of 4T1 and 4T1.2 cells at different stages of bone metastsis using SMART-Seq2 technology
Bone metastasis, a frequent and devastating occurrence in breast cancer, affects over 70% of patients over time. Intriguingly, cancer cells residing within the bone microenvironment can serve as "cancer seeds," disseminating to distant organs and driving extensive metastasis. Even post-successful primary tumor removal, quiescent cells can persist, contributing to bone recurrence. This phenomenon underscores the remarkably low efficiency of bone metastasis establishment and underscores the significance of comprehending the molecular underpinnings of this early bone-seeding phase. Deciphering the mechanisms orchestrating tumor cell dormancy and reawakening holds pivotal implications for designing effective anti-cancer therapies. Dormant tumor cells within the bone might be likened to being ensnared in an "extended seeding phase" prior to eventual expansion. Conceivably, shared signaling molecules could govern both proficient bone metastatic colonization and the re-emergence of indolent metastatic cells. Consequently, the molecular facilitators enabling bone colonization could also be integral to the reactivation of dormant tumor cells within the bone milieu. In this study, we conducted single-cell RNA sequencing (scRNA-seq) analysis on tumor cells procured from the initial stages of bone colonization within experimental bone metastasis models. Our investigation unearthed potential factors that foster tumor cell survival during this critical seeding phase. Overall design: Female BALB/c mice were subjected to intracardiac (IC) injections of 4T1 and 4T1.2 cells labeled with Firefly-luciferase and mCherry. At various time points, including Day 4 (early seeding phase, D4), Day 10 (progression phase, D10), and Day 16 (late-stage bone metastasis phase, D16), a subset of mice were sacrificed to recover metastatic tumor cells from the hindlimbs. The mCherry+ tumor cells were subsequently isolated using fluorescence-activated cell sorting (FACS). Utilizing single-cell cDNA amplification and next-generation sequencing (NGS) library construction, we conducted comprehensive analysis. Furthermore, we isolated in vitro cultured cells and primary tumor cells injected into the mammary fat pad to generate supplementary scRNA-seq data, serving as additional controls for our study.