Abstract Background: Breast cancer incidence among young women has risen markedly over the past decade, particularly hormone receptor-positive (HR+) breast cancer (BC), yet the tumor microenvironment (TME) in this young population remains poorly characterized. Cancer-associated fibroblasts (CAFs) are abundant stromal cells that contribute to extracellular matrix remodeling, cytokine signaling, and immune regulation; their persistent activation and phenotype shift support tumor growth and treatment resistance. To delineate fibroblast heterogeneity and functional alterations in young HR+BC, we conducted spatially resolved single-cell analyses on matched tumor and adjacent normal tissue using image mass cytometry (IMC). Methods: We collected formalin-fixed, paraffin-embedded (FFPE) specimens from ten treatment-naïve, stage I-II HR+ BC patients diagnosed at age ≤40. Patients with germline pathogenic mutations such as BRCA1/2 and PALB2 were excluded. From each case, two regions of interest (ROIs)—one within the invasive tumor and one from adjacent normal tissue—were selected and imaged at 1 μm resolution using a 40-marker IMC panel targeting hormone receptors, stromal components, adhesion/migration, and immune populations. Key markers were used to identify different phenotypes of fibroblasts. Image preprocessing and cell segmentation were performed with imctools and DeepCell; single-cell feature extraction, clustering, and differential expression analyses employed Scanpy, while spatial neighborhood and interaction analyses utilized Squidpy. Results: Across 20 ROIs, we identified 109,097 individual cells, including 71,406 from tumor ROIs and 37,691 from normal ROIs. Cluster annotation was based on differential marker expression to identify cancer, stromal, and immune populations, and 5 distinct fibroblast subsets were identified. When comparing fibroblast phenotypes between tumor and normal ROIs, Ki67+PD-L1+ and MHCII+ fibroblasts in tumor tissue exhibited more active functional states, as indicated by higher expression of FAP, αSMA, and increased collagen secretion. Additionally, hormone receptors such as ERα and ERβ, were significantly upregulated in tumor-associated fibroblasts compared to those in normal tissue. MHCII+ fibroblasts in tumors also displayed a more pro-tumorigenic phenotype, with elevated expression of MMP9 and CXCL12. Spatial analysis revealed that both Ki67+PD-L1+ and MHCII+ fibroblasts were actively engaged in cell-cell interactions with T cells and epithelial/cancer cells. Specifically, Ki67+PD-L1+ fibroblasts showed increased interactions with CD8+ T cells in tumor ROIs, while MHCII+ fibroblasts were more involved with epithelial cells in normal tissue. Conclusions: Our study uncovers functionally activated fibroblast subsets in young HR+ BC compared to adjacent normal tissues. The distinct fibroblast phenotypes and their spatial interactions with immune and epithelial cells highlight stromal PD-L1 and MHCII pathways as potential therapeutic targets. Future studies with larger sample size, including older HR+ cases, are required to establish the age-associated specificity of these stromal alterations. Citation Format: J. Tan, H. Hackbart, Y. Li, N. Bercovici, X. Cui, Y. Yuan, J. Bitar. Comparing Functional Fibroblasts in Tumor vs. Adjacent Normal Tissue in Young Hormone Receptor-Positive Breast Cancer abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-09-29.
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