Arginine deprivation reshapes tumor and immune transcriptional programs and potentiates IFN?–JAK–STAT signaling in 4T1 murine breast tumors

Arginine availability is a key metabolic regulator of tumor and immune cell function. We performed single-cell RNA sequencing of orthotopic 4T1 murine triple-negative breast tumors to define cell type–specific transcriptional adaptations to arginine deprivation. We demonstrate that arginine depletion induces widespread transcriptional reprogramming across both stromal and immune compartments, with particularly pronounced effects in cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). In tumor cells, cancer cell–intrinsic (via Ass1 knockdown), systemic (via an arginine-free diet), or combined restriction of arginine availability resulted in elevated inflammatory responses, including activation of interferon-alpha (IFNa) and interferon-gamma (IFN?) signaling, and, to a lesser extent, IL6–JAK–STAT3 signaling. These changes, mediated by tumor microenvironment (TME)–supportive interactions, sustain tumor cell survival under arginine-deprived conditions. Overall design: Orthotopic 4T1 mammary tumors were established in female BALB/c mice to assess the effects of tumor-intrinsic and systemic arginine restriction. Mice were divided into four groups: control (Ctrl; shGfp + regular diet), tumor-intrinsic restriction (KD; shAss1 + regular diet), systemic restriction (AFD; shGfp + arginine-free diet), and combined restriction (KD + AFD; shAss1 + arginine-free diet). Tumors were collected on day 16 post-implantation, dissociated into single-cell suspensions, and pooled by treatment group by combining equal numbers of cells from 4–5 tumors per condition.