Tamoxifen Response at Single-Cell Resolution in Estrogen Receptor-Positive Primary Human Breast Tumors

Abstract

Purpose: In estrogen receptor-positive (ER+)/HER2- breast cancer, multiple measures of intratumor heterogeneity are associated with a worse response to endocrine therapy. We sought to develop a novel experimental model to measure heterogeneity in response to tamoxifen treatment in primary breast tumors.

Experimental design: To investigate heterogeneity in response to treatment, we developed an operating room-to-laboratory pipeline for the collection of live normal breast specimens and human tumors immediately after surgical resection for processing into single-cell workflows for experimentation and genomic analyses. Live primary cell suspensions were treated ex vivo with tamoxifen (10 μmol/L) or control media for 12 hours, and single-cell RNA libraries were generated using the 10X Genomics droplet-based kit.

Results: In total, we obtained and processed normal breast tissue from two women undergoing reduction mammoplasty and tumor tissue from 10 women with ER+/HER2- invasive breast carcinoma. We demonstrate differences in tamoxifen response by cell type and identify distinctly responsive and resistant subpopulations within the malignant cell compartment of human tumors. Tamoxifen resistance signatures from resistant subpopulations predict poor outcomes in two large cohorts of ER+ breast cancer patients and are enriched in endocrine therapy-resistant tumors.

Conclusions: This novel ex vivo model system now provides the foundation to define responsive and resistant subpopulations within heterogeneous human tumors, which can be used to develop precise single cell-based predictors of response to therapy and to identify genes and pathways driving therapeutic resistance.

Figure 1.

Characterization of cell type specific response to tamoxifen in normal human breast tissue. A, Representation of the operating room to single-cell sequencing workflow. The breast schematic was created using BioRender.com. B, UMAP plot of all scRNA-seq cells from two normal breast tissue samples. Cells were color-coded by cell type. C, UMAP plot of scRNA-seq cells color coded by sample and treatment condition. D, Feature plots showing the expression of select epithelial markers in normal breast cells. E, Bar chart comparing the total number of normal breast cells sequenced per treatment condition, color coded by cell type. F, Heat map of gene sets enriched and depleted in differentially regulated genes in tamoxifen-treated cells relative to control cells by cell type, demonstrating distinct biologic activity of tamoxifen in different cell compartments. Gene set enrichment was determined using enricher, and P values were reported after correction for false discovery.

Figure 2.

T47D response to tamoxifen at a single-cell level. A, Illustration showing T47D scRNA-seq workflow. B, UMAP plot of T47D scRNA-seq cells color coded by groupA (luminal A–like subpopulation) or groupB (luminal B–like subpopulation). C, UMAP plot of T47D scRNA-seq cells color coded by treatment condition. D, Volcano plot showing enriched and depleted gene sets after tamoxifen treatment in T47D cells. Significant gene sets were defined as a gene ratio of >0.125 and a log₁₀q value of <‒4.5 or >4.5, and a thresholding limit of 10 was applied when log₁₀ q value >10 for visualization. E, Feature plots showing the expression of luminal epithelial markers and proliferation score in T47D cells.

Figure 3.

Tamoxifen response in 10 primary ER⁺/HER2⁻ breast tumors. A, UMAP plot of all scRNA-seq cells from 10 breast tumor samples. Cells were color coded by cell type. Malignant epithelial cells (Epi. Tumor) were distinguished from normal epithelial cells (Epi. Nontumor) by inferred copy-number changes using InferCNV. B, UMAP plot of all cells color coded by tumor and treatment condition. C, Feature plots showing the expression of luminal epithelial markers and proliferation score. D, Box-plots of early estrogen-response genes (CCND1, PGR, and GREB1) in the InferCNV+ malignant cells, comparing expression in control and tamoxifen-treated cells. Significance was determined using the Wilcoxon rank-sum test *, P < 0.05; **, P < 0.01. Sample PAM50 subtypes determined on bulk mRNA-seq are denoted by the color code. E, Gene set enrichment heat map showing distinct tamoxifen response within InferCNV+ malignant cells across tumors.

Figure 4.

Targeted analysis of four tamoxifen-responsive tumor pairs. A, UMAP plot of scRNA-seq cells from four tumors that demonstrated depletion of scTAM-response-T47D signature. Cells are color coded by cell type. B, UMAP plot of scRNA-seq cells from four tumor pairs, color coded by tumor and treatment condition. C, Heat map of upregulated and downregulated gene sets in tamoxifen-treated tumor cells relative to control cells. D, Volcano plot showing enriched and depleted gene sets after tamoxifen treatment in malignant cells from 4 ER⁺/HER2⁻ tumor pairs. Significant gene sets were defined as a gene ratio of > 0.085 and a log₁₀ q value of < ‒10 or > 10 for visualization. E, Kaplan–Meier (KM) curve for overall survival using two independent clinically annotated datasets with transcriptional data. ER⁺/HER2⁻ patients treated with endocrine therapy were assigned a centroid score of our malignant cell-specific tamoxifen resistance signature (scTAM-resistance-M) and stratified by high and low score. High signature score is associated with significantly worse overall survival in patients in METABRIC (HR, 1.63; P = 0.023) and SCAN-B (HR, 2.94; P = 0.002). Statistical significance was assessed by the log-rank test and the estimates of survival probabilities and cumulative hazard with a univariate Cox proportional hazards model.

Figure 5.

Identification and characterization of resistant tumor cell subpopulations in four tamoxifen-responsive tumor pairs. A, Individual cells were assigned a score from the T47D tamoxifen response signature compared with cluster matched untreated score. Application of response score to UMAP plot demonstrated three distinct clusters with enriched signature score on treatment (cluster 3, 12, 19) and one tamoxifen-sensitive cluster with depletion of response score (cluster 2). B, Abundance of cells from resistant clusters in each of the four tumors. C, Stacked bar chart showing distribution of cells within 22 distinct clusters, color coded by tumor and treatment condition. D, Kaplan–Meier (KM) curve of the cluster 19 signature (scTAM-resistance-C19), using ER⁺/HER2⁻ patients that received endocrine therapy from METABRIC. Higher signature score predicted worse overall survival (HR, 2.17; P < 0.001). Survival curve differences were calculated by the log-rank test and the estimates of survival probabilities and cumulative hazard with a univariate Cox proportional hazards model. E, We obtained data of clinically annotated endocrine therapy sensitive and resistant tumors from Xia and colleagues (47). All three tamoxifen resistance signatures were enriched in resistant tumors compared with sensitive. Significance was determined using the Wilcoxon rank-sum test, ***, P < 0.001.