Quiescent cancer cells resist T cell attack by forming an immunosuppressive niche (PADMEseq)

Immunotherapy is a promising treatment for Triple-Negative Breast Cancer (TNBC), but patients recur, arising the need to understand mechanisms of resistance. We discovered that in primary breast cancer, tumor cells that survive T-cell attack are in a quiescent state. Quiescent Cancer Cells (QCCs) are found clustering together forming regions with reduced immune infiltration. QCCs display superior tumorigenic capacity and higher expression of stemness genes than their proliferative counterparts. We adapted single-cell-RNA-sequencing with precise spatial resolution to profile infiltrating cells inside and outside QCC niches. This transcriptomic analysis revealed hypoxia-induced programs and identified more exhausted T-cells, tumor-protective fibroblasts, and dysfunctional dendritic cells inside clusters of QCCs. This uncovered differential phenotypes in infiltrating cells based on their specific intra-tumor location. Thus, QCCs constitute immunotherapy-resistant reservoirs by orchestrating a local hypoxic immune-suppressive milieu that blocks T-cell function. Eliminating QCCs holds the promise to counteract resistance to immunotherapy and prevent disease recurrence in TNBC. tdTomato-p27K-expressing 4T07 cells were injected into the mammary fat pad of Kaede x Balb/cJ F1 females. Tumors were embedded in 2% low melting agarose and cut into 300μm slices by the Compresstome VF 310-0Z vibrating microtome (Precisionary Instruments). . Consecutive slices were used to photoconvert cells infiltrating tdTomato-negative areas and then cells in regions with tdTomato-p27KHigh cells.