Rare Subset of T Cells Form Integrin-dependent Clustering with Circulating Tumor Cells to Foster Metastasis in Breast Cancer

The immune ecosystem, particularly T cells, is central to maintaining effective anti-tumor responses. However, how immune cells in the periphery blood interact with circulating tumor cells (CTCs) - critical mediators of metastatic spread 1-8 – remains largely unexplored. Here, our analysis of the blood specimens from patients with advanced breast cancer (N=1,529) revealed that over 75% of the CTC-positive blood specimens contained heterotypic CTC clusters with CD45+ white blood cells (WBCs). These CTC-WBC clusters correlate with breast cancer subtypes (triple negative and luminal B), racial groups (Black), and unfavorable clinical outcomes. Using flow cytometry and ImageStream, we characterized the diverse WBC composition in these heterotypic clusters, such as overrepresented T cells and underrepresented neutrophils. Most strikingly, a rare subset of CD4 and CD8 double positive T (DPT) cells showed an up to 140-fold enrichment in the CTC clusters (~14.2%) versus its frequency in WBCs (0.1%). DPT cells shared part of profiles with CD4+ T cells and others with CD8+ T cells but exhibited unique features of T cell exhaustion and immune suppression (TIM-3 and PD-1). Single-cell RNA sequencing further identified distinct cell adhesion profiles of DPT cells and CTCs. CRISPR/Cas9-mediated gene depletion pinpointed the integrin VLA4 (β1/α4) in DPT and its ligand VCAM1 in tumor cells as essential mediators of heterotypic CTC clusters. Anti-α4 (VLA4) neutralizing antibodies markedly reduced CTC–DPT cell clustering and blocked metastatic lesion formation in murine models in vivo. These findings uncover a pivotal role for a rare T cell subset in fostering cancer dissemination through heterotypic CTC cluster interactions. It lays a foundation for innovative therapeutic strategies aimed at preventing and targeting metastasis of breast cancer, thus benefiting Black and non-Black patients.