Influence of Estrogen Treatment On ESR1+ and ESR1- Cells In ER+ Breast Cancer: Insights From Single-Cell Analysis of Patient-Derived Xenograft Models

Abstract

Background: Estrogen typically promotes the progression of hormone-dependent breast cancer through activation of estrogen receptor (ER)-α encoded by ESR1. While estrogen-induced tumor suppression in ER+ breast cancer has been clinically observed as an unexpected outcome of aromatase inhibitor (AI)-resistance, the molecular mechanisms have not yet been fully defined. Characterization of estrogen regulation in two ER+ breast cancer patient-derived xenograft (PDX) models with opposite responses to estrogen offered us an unprecedented opportunity to assess how 17β-estradiol (E2) modulates ER+ cancer.Methods: We established two PDX breast cancer models in mice using ER+ tumors from patients that responded (SC31) or were suppressed (GS3) by exogenous estrogen. In vivo tumor promotion or suppression by estrogen were confirmed through experiments by implanting E2 pellets in mice carrying SC31 or GS3, and then single-cell analysis was performed.Results: E2 promoted SC31 tumor growth but suppressed growth of GS3 in vivo. The E2-mediated suppression of GS3 involves ERα, which was wild-type and not amplified. Single-cell RNA sequencing analysis showed that E2 treatment induced cell cycle promotion in SC31, while E2 induced cell cycle arrest in GS3. However, E2 treatment upregulated the expression of estrogen-regulated genes in both tumors. These gene-expression changes by E2 occurred in both ESR1+ cells and ESR1– cells within the same tumor, demonstrating for the first time the influence of estrogen on ESR1– cells in ER+ breast tumors. E2 also upregulated a tumor suppressor gene, IL24, only in GS3, and lower levels of IL24 were linked to estrogen independence, after three rounds of intermittent E2 treatment. More IL24+ cells were ESR1+ and in G1 phase than IL24– cells. Hallmark apoptosis gene sets were upregulated and the hallmark G2M checkpoint gene set was downregulated in IL24+ cells after E2 treatment.Conclusions: Our study has revealed the effects of estrogen treatment on both ESR1+ and ESR1– cells in ER+ tumors, but not all ER+ cancers respond the same manner to estrogen. SC31 is a tumor that is stimulated by E2, while GS3 is suppressed by E2 via cell cycle arrest. Our results indicate a potential role of IL24 in estrogen-suppressive tumors.

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

“…Two "ER-high" PDXs, which were previously established from metastatic lesions and possessed definitive and contrasting growth responses to estrogen, were used (Supplementary Table S1). GS3 (80-100% ER) was E2-suppressive and luminal B-like, while SC31 (30-75% ER) was E2-dependent and luminal A-like [5,6,8]. Tumor pieces of GS3 and SC31 were implanted into mammary fat pads of 8-10week-old NOD-SCID/IL2Rγ −/− (NSG) mice, respectively [5,6,8].…”

“…GS3 (80-100% ER) was E2-suppressive and luminal B-like, while SC31 (30-75% ER) was E2-dependent and luminal A-like [5,6,8]. Tumor pieces of GS3 and SC31 were implanted into mammary fat pads of 8-10week-old NOD-SCID/IL2Rγ −/− (NSG) mice, respectively [5,6,8]. Once the implanted tumors were established, placebo or 17β-estradiol (E2; 1 mg) pellet were implanted subcutaneously.…”

“…The copyright holder for this preprint this version posted February 3, 2023. ; https://doi.org/10.1101/2023.01.31.526403 doi: bioRxiv preprint weeks, as described in our previous scRNA-seq study [8]. After the treatment periods, the mice were euthanized, and tumor samples were collected.…”

“…The datasets prepared from GS3-Placebo/E2 and SC31-Placebo/E2 in our previous study were analyzed [8]. The "ER-low" GS1 dataset was prepared as described in Supplementary Methods.…”

“…Since current treatment approaches mostly target cancer as a homogeneous disease (e.g., endocrine therapy for ER + breast cancers), intratumor heterogeneity often contributes to the acquisition of therapy resistance through the expansion of pre-existing resistant cells [7]. We reported that ER + breast cancer PDXs contained both ESR1 + (encoding ER) and ESR1cells with molecular and functional differences using single-cell RNA sequencing (scRNA-seq) [8]. Nonetheless, the influence of estrogen on individual cell populations, as well as the importance of their responses toward the estrogen-dependent growth of ER + breast cancers, has yet to be better defined.…”