Educational Session 5: Best of AACR

Y. Yan; Systems Biology, MD Anderson Cancer Center, Houston, TX.

Triple-negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer. The pillar of treatment is chemotherapy, but only half of the patients have a complete response and good survival. To resolve inter- and intra-tumoral heterogeneity and determine their clinical associations, we performed single-cell RNA-sequencing and spatial transcriptomics methods including Visium and Xenium on treatment-naïve samples of TNBC patients in the ARTEMIS clinical trial. We find that TNBC is classified into 4 major archetypes at patient level: luminal secretory-associated, basal-associated, immunoreactive, and luminal androgen receptor. At cell level, cancer cells exhibited intratumoral heterogeneity in 13 gene expression metaprograms. The TNBC tumor microenvironment (TME) consisted of 49 distinct immune and stromal cell states, many of which were reprogrammed relative to normal breast tissues from disease-free women. We further identified 8 ecotypes of cancer cells and TME cell states that co-occurred among patients and were associated with specific archetypes and chemotherapy response groups. In contrast to previous work on T-cells, our data showed the importance of macrophage cell states and cancer cell metaprograms for interferon signaling, HLA expression and cell cycle activity that were associated with chemotherapy response. To facilitate a clinical application, we developed a 13-gene-based model to predict response. Collectively, this study provides new insights into the natural biology of untreated TNBC tumors and their association with chemotherapy response.

K. Shimada¹, D. Michaud², Y. X. Cui³, K. Zheng², J. Goldberg³, Z. Ju⁴, S. J. Schnitt², R. Pastorello⁵, L. D. Kania², J. Hoffer¹, J. L. Muhlich¹, N. Hyun¹, R. Krueger⁶, A. Gottlieb², A. Nelson², C. W. Wanderley², G. Antonellis², S. S. McAllister², S. M. Tolaney³, A. G. Waks³, R. Jeselsohn³, P. K. Sorger¹, J. Agudo³, E. A. Mittendorf⁷, J. L. Guerriero²; ¹Harvard Medical School, Boston, MA, ²Brigham and Women’s Hospital, Boston, MA, ³Dana-Farber Cancer Institute, Boston, MA, ⁴MD Anderson Medical Center, Houston, TX, ⁵Hospital Sírio Libanês, São Paulo, BRAZIL, ⁶New York University, Brooklyn, NY, ⁷Beth Israel Deaconess Medical Center, Boston, MA.

T cells are sparse in hormone receptor-positive (HR⁺) breast cancer, potentially due to limited antigen presentation, but the mechanisms remain unclear. This study aimed to elucidate how estrogen receptor (ER) signaling (ERS) modulates immune pathways and the tumor microenvironment using bulk and single-cell RNA sequencing (scRNA-seq) data and spatial analysis via multiplexed cyclic immunofluorescence (CyCIF). Analysis of HR⁺/HER2^– patients from TCGA and METABRIC datasets identified co-expressed gene modules linked to ERS and three key immune pathways—TNFα-induced NF-κB (TNFα/NF-κB), type I interferon response (IFN-I), and antigen presentation machinery/T cell signaling (APM/TC)—all strongly inversely correlated with ERS. scRNA-seq of 34 breast cancer samples revealed ERS activity primarily in cancer cells, while immune pathways were enriched in non-cancer cells. A subset of immune-related modules expressed in CCLE cancer cell lines was inversely correlated with ERS, suggesting a cancer cell-intrinsic mechanism of immune suppression. Bulk and single-nucleus RNA sequencing and CyCIF in 29 HR+ BC samples showed that ERS-high tumors excluded all non-cancer cell types, including T cells, linked to reduced intrinsic IFN-I activity in cancer cells. In ERS-low tumors, TNFα/NF-κB activity was associated with angiogenesis, promoting sprouting microvessels in stroma distant from the tumor core, while IFN-I activity correlated with CD163⁺ macrophages. These macrophages were co-localized with CD4⁺/CD8⁺ T cells, predominantly in the peritumoral stroma, where elevated PD-1 expression indicated T cell exhaustion. T cell density positively correlated with MHC class I and II expression, while ERα expression in cancer cells inversely correlated with adjacent MHC expression. ERα-dependent regulation of MHC varied by cell type, with suppression in cancer cells and macrophages but not fibroblasts or T cells. Analysis of pre- and post-treatment samples from the ACOSOG Z1031B trial showed aromatase inhibitor (AI) therapy reduced ERS and increased TNFα/NF-κB and APM/TC activity in responsive tumors, though its effect on IFN-I was less pronounced. Altogether, our study suggests ERS suppresses immune activation and excludes T cells through cancer cell-intrinsic and microenvironmental mechanisms. Combining ER inhibition with therapies targeting TNFα/NF-κB and IFN-I pathways, along with immunotherapy to activate exhausted T cells, may offer a promising strategy to overcome immune suppression and improve outcomes in HR⁺ BC.