Identification and characterization of a proliferative cell population in estrogen receptor-positive metastatic breast cancer through spatial and single-cell transcriptomics.

Background: Intratumor heterogeneity is a hallmark of most solid tumors, including breast cancers. We applied spatial transcriptomics and single-cell RNA-sequencing technologies to profile spatially resolved cell populations within estrogen receptor-positive (ER ) metastatic breast cancers and elucidate their importance in estrogen-dependent tumor growth.

Methods: Spatial transcriptomics and single-cell RNA-sequencing were performed on two patient-derived xenografts (PDXs) of "ER-high" metastatic breast cancers with opposite estrogen-mediated growth responses: estrogen-suppressed GS3 (80-100% ER) and estrogen-stimulated SC31 (30-75% ER) models. The analyses included samples treated with and without 17β-estradiol. The findings were validated via scRNA-seq analyses on "ER-low" estrogen-accelerating PDX, GS1 (5% ER). The results from our spatial and single-cell analyses were further supported by the analysis of a publicly available single cell dataset and a protein-based dual immunohistochemical (IHC) evaluation using three important clinical markers [i.e., ER, progesterone receptor (PR), and Ki67]. The translational implication of these results was assessed by clinical outcome analyses on public breast cancer cohorts.

Results: Our novel space-gene-function study revealed a "proliferative" cell population in addition to three major spatially distinct compartments within ER metastatic breast cancers. These compartments showed functional diversity (i.e., estrogen-responsive, proliferative, hypoxia-induced, and inflammation-related). The "proliferative ( )" population, not "estrogen-responsive" compartment, was crucial for estrogen-dependent tumor growth, leading to the acquisition of luminal B features. The cells with induction of typical estrogen-responsive genes such as were not directly linked to estrogen-dependent proliferation. Additionally, the dual IHC analyses demonstrated the distinct contribution of the Ki67 proliferative cells toward estrogen-mediated growth and their response to palbociclib, a CDK4/6 inhibitor. The gene signatures developed from the proliferative, hypoxia-induced, and inflammation-related compartments were significantly correlated with worse clinical outcomes, while patients with the high estrogen-responsive scores showed better prognosis, confirming that the estrogen-responsive compartment would not be directly associated with estrogen-dependent tumor progression.

Conclusions: For the first time, our study elucidated a "proliferative" cell population distinctly distributed in ER metastatic breast cancers. They contribute differently toward progression of these cancers, and the gene signature in the "proliferative" compartment is an important determinant of luminal cancer subtypes.