Aging-associated differences in mammary tumor-initiating populations and immune evasion pathways in breast cancer [bulk_RNA_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 collected and snap frozen in liquid nitrogen. RNA was isolated using RNeasy Mini Kit (Qiagen) with on-column DNA digestion. Two tumors (1m-4 and 6m-1) were excluded from library preparation due to extended postmortem intervals, as the animals were discovered several hours after death. Libraries preparation with poly(A) selection and 150-bp paired-end sequencing on an Illumina HiSeq platform were performed by GENEWIZ (USA; www.GENEWIZ.com).