Protocol to isolate live single cells while retaining spatial information by combining cell photolabeling and FACS.

Single-cell techniques have revolutionized biology; however, the required sample processing inherently implies the loss of spatial localization. Here, using an approach called photoconversion of areas to dissect micro-environments (PADME), we detail steps to isolate live single cells from a primary breast tumor while retaining spatial information by combining cell photolabeling and FACS (fluorescence-activated cell sorting). These live cells can be subsequently used for myriad techniques, from flow cytometry to single-cell RNA sequencing or other single cell "omics" approach. For complete details on the use and execution of this protocol, please refer to Baldominos et al. (2022).

Quiescent cancer cells resist T cell attack by forming an immunosuppressive niche.

Immunotherapy is a promising treatment for triple-negative breast cancer (TNBC), but patients relapse, highlighting the need to understand the mechanisms of resistance. We discovered that in primary breast cancer, tumor cells that resist T cell attack are quiescent. Quiescent cancer cells (QCCs) form clusters with reduced immune infiltration. They also display superior tumorigenic capacity and higher expression of chemotherapy resistance and stemness genes. We adapted single-cell RNA-sequencing with precise spatial resolution to profile infiltrating cells inside and outside the QCC niche. 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 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 immunotherapy resistance and prevent disease recurrence in TNBC.