Spatial transcriptomic analysis of immune checkpoint blockade response in triple negative breast cancers with tertiary lymphoid structures

Tertiary lymphoid structures (TLSs) are associated with improved cancer immunotherapy responses. However, TLSs vary in their ability to elicit anticancer immune activity, so it is important to develop databases that allow study of variables that regulate their function. We applied single RNA molecule resolution imaging to longitudinal biopsies taken from women with TLS-enriched triple negative breast cancers prior to therapy, after pembrolizumab and after pembrolizumab plus radiation therapy. We developed a computational framework to align and analyze spatial trajectories between TLSs and tumor beds. Tumors with higher T cell infiltration rates were eradicated after pembrolizumab. In contrast, those with lower malignant cell and T cell interaction rates at baseline showed CXCL9⁺ macrophage infiltration after pembrolizumab, and infiltration of T cells expressing CXCL9-associated programs prior to cancer cell removal after radiation therapy. This manuscript describes single RNA molecule resolution profiling of breast tumors bearing tertiary lymphoid structures throughout an immunotherapy response.