Aging-associated differences in mammary tumor-initiating populations and immune evasion pathways in breast cancer [scRNA-Seq]

Aging is a major risk factor for breast cancer, yet how it shapes tumor development, molecular phenotype, and immune evasion remains incompletely understood. Deciphering how aging influences cancer development is critical for improving risk assessment, prevention, and treatment. Here, using a N-nitroso-N-methylurea (NMU)-induced rat mammary tumor model that recapitulates key features of human breast cancer, we integrated bulk and single-cell transcriptomics, whole-exome sequencing, and histopathological analysis to dissect age-associated differences in tumorigenesis. We found that age at NMU exposure critically influences tumor incidence, mutational burden, subtype, and immune microenvironment. Tumors arising in aged rats originate from luminal progenitor-like cells with increased genomic instability, suppressed immune infiltration, and impaired antigen presentation linked to loss of heterozygosity at Chr 20p. These age-associated epithelial and immune changes were conserved in human breast cancers, where loss of the homologous Chr 6p region correlated with reduced lymphocyte infiltration and shorter relapse-free survival. These findings reveal that aging alters the tumor-initiating cell population and promotes immune-evasive tumor states through chromosomal instability-driven antigen presentation defects. Our work provides mechanistic insight into the reduced efficacy of immunotherapy in older patients. Overall design: To induce mammary tumors, rats of different ages (1-, 3-, 6-, 12-, 20-month-old) received two intraperitoneal injections of 50 mg/kg N-Nitroso-N-methylurea (NMU) at a one-week interval. Tumor growth was monitored regularly using caliper measurements. The maximum allowable tumor burden of 4 cm was not exceeded in any experiment. Rats were euthanized by CO2 inhalation, and tumors were harvested as previously described. Briefly, tumors were enzymatically digested in DMEM/F12 medium containing 2 mg/mL collagenase IV, 2 mg/mL hyaluronidase, and 2 mg/mL bovine serum albumin (BSA) at 37ºC for 1–2 hours, followed by freezing in cryopreservation medium (90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide) in liquid nitrogen. For mammary tumor scRNA-seq, viable cells were resuspended in PBS with 0.04% BSA at a cell concentration of 1000 cells/µL. 10,000 cells were loaded onto a 10x Genomics Chromium™ instrument (10x Genomics) according to the manufacturer's instructions. The scRNA-seq libraries were prepared according to the Chromium Next GEM Single Cell 5' HT v2 protocol (10x Genomics). Quality control for amplified cDNA libraries and final sequencing libraries were performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). The pooled libraries were sequenced on Illumina NovaSeq X plus platform.