In vivo selective pressure drives phenotypic convergence and elicits CD95L-mediated immune adaptation in breast cancer subpopulations

BACKGROUND: Breast cancer stem cells (CSCs) are fundamentally implicated in tumor initiation, metastasis, and therapeutic recalcitrance. While their intrinsic traits are well-documented in vitro, the spatial-temporal evolution of their immune-escape mechanisms under the selective pressure of an intact in vivo tumor microenvironment (TME) remains poorly elucidated. This study aimed to dissect the transcriptomic adaptation of immune-escape genes in CSC and non-CSC subpopulations when transitioning from static cultures to a dynamic in vivo syngeneic model.METHODS: Utilizing the highly aggressive murine 4T1 breast cancer model, cells were strictly sorted via flow cytometry into putative CSC (CD44+CD24-/low) and bulk non-CSC subpopulations. Both fractions were evaluated in vitro and subsequently subcutaneously transplanted into the mammary fat pads of immunocompetent, syngeneic female BALB/c mice. Tumor progression was longitudinally monitored over 28 days. The transcriptional architectures of a targeted panel of immune-modulatory genes (including CD95L, ADAM-17, CCL22, and CXCL12) were quantified via qRT-PCR to map environmentally driven phenotypic shifts. RESULTS: In vitro baseline profiling revealed minimal intrinsic transcriptomic divergence between the subpopulations, with only CCL22 exhibiting statistical significance (P