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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE294399 | GEO | 2025/04/18

REPOSITORIES: GEO

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Project description:About 230 clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but no assays enable comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for CTC enrichment and isolation with 100% purity with a non-random whole genome amplifiation method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 breast cancer patients. We defined a genome integrity index (GII) to identify cells suited for molecular chracterization by different molecular assay in more than 90% of single cells, such as diagnostic profiling for point mutations, gene amplifications and whole genomes of single cells. The high reliability on clinical samples enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified therapeutically relevant genomic disparity between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered preexisting cells reistsant to ERBB2 targeted therapies suggesting ongoing microevolution at late stage disease whose exploration may provide essential information for personalized treatment decisions. The analysis aimed to indentify profiles of copy number changes in genomic DNA of single circulating tumor cells (CTCs). For this, CTCs were enrched using the FDA approved CellSearch System and single-cell were isolated using the DEPArray System. Subsequently, single-cell DNA was amplified using the Ampli1 WGA Kits and subjected to single-cell aCGH analysis according to previously published protocol (Czyz ZT et al., PLoS One. 2014 Jan 21;9(1):e85907). The analysis included 38 single CTCs and 10 white blood cells (WBCs) obtained from 16 breast cancer patient. WBCs were used as controls for the analysis. In addition, four CTC cell pools were included in the analysis. This was done to show the discrepancies between the profiles of individual cells and corresponing average genomic profile of CTCs in a patient material (cell pools), thereby demonstrating the importance of the analysis on the cell-by-cell basis. The reference sample used for all aCGH experiments consisted of a pool of four single-cell WGA products generated from WBCs of a healthy female donor.

Project description:Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Highly parallel architecture of the chip allows deterministic screening of clinically relevant volumes of blood samples at slow, and hence, non-damaging flow rates. Using the Cluster-Chip, we identify CTC-clusters in 30-40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as being tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis. We used the Cluster-Chip to capture CTC-clusters from the blood of a breast cancer patient with high CTC counts, released CTC-clusters in solution, stained them with TexasRed-conjugated antibodies against the leukocyte cell surface markers CD45, CD14 and CD16, and then isolated intact CTC-clusters individually using a micromanipulator. From a single time point, we retrieved 15 CTC-clusters, and each of those clusters was individually subjected to RNA-sequencing analysis using a next generation platform (SOLiD 5500). In addition, two leukocytes were isolated from the blood of a healthy donor were individually subjected to RNA-sequencing analysis using the same platform.

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

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