Double-positive T cells form heterotypic clusters with circulating tumor cells to foster cancer metastasis
David Scholten, Lamiaa El-Shennawy, Yuzhi Jia, Youbin Zhang, Elizabeth Hyun, Carolina Reduzzi, Andrew D. Hoffmann, Hannah F. Almubarak, Fangjia Tong, Nurmaa K. Dashzeveg, Yuanfei Sun, Joshua R. Squires, Janice Lu, Leonidas C. Platanias, Clive H. Wasserfall, William J. Gradishar, Massimo Cristofanilli, Deyu Fang, Huiping Liu
David Scholten, Lamiaa El-Shennawy, Yuzhi Jia, Youbin Zhang, Elizabeth Hyun, Carolina Reduzzi, Andrew D. Hoffmann, Hannah F. Almubarak, Fangjia Tong, Nurmaa K. Dashzeveg, Yuanfei Sun, Joshua R. Squires, Janice Lu, Leonidas C. Platanias, Clive H. Wasserfall, William J. Gradishar, Massimo Cristofanilli, Deyu Fang, Huiping Liu
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Research Article Clinical Research Immunology Oncology

Double-positive T cells form heterotypic clusters with circulating tumor cells to foster cancer metastasis

Abstract

The immune ecosystem is central to maintaining effective defensive responses. However, it remains largely understudied how immune cells in the peripheral blood interact with circulating tumor cells (CTCs) in metastasis. Here, blood analysis of patients with advanced breast cancer revealed that over 75% of CTC-positive blood specimens contained heterotypic CTC clusters with CD45+ white blood cells (WBCs), which correlates with breast cancer subtypes, racial groups, and decreased survival. CTC-WBC clusters included overrepresented T cells and underrepresented neutrophils. Specifically, a rare subset of CD4 and CD8 double-positive T (DPT) cells was 140-fold enriched in CTC clusters versus their frequency in WBCs. DPT cells shared properties with CD4+ and CD8+ T cells but exhibited unique features of T cell exhaustion and immune suppression. Mechanistically, the integrin heterodimer α4β1, also named very late antigen 4 (VLA-4), in DPT cells and its ligand, VCAM1, in tumor cells are essential mediators of DPT-CTC clusters. Neoadjuvant administration of anti-VLA-4 neutralizing antibodies markedly blocked CTC–DPT clusters, inhibited metastasis, and extended mouse survival. These findings highlight a pivotal role of rare DPT cells in fostering cancer dissemination through CTC clustering. It lays a foundation for developing innovative biomarker-guided therapeutic strategies to prevent and target cancer metastasis.

Authors

David Scholten, Lamiaa El-Shennawy, Yuzhi Jia, Youbin Zhang, Elizabeth Hyun, Carolina Reduzzi, Andrew D. Hoffmann, Hannah F. Almubarak, Fangjia Tong, Nurmaa K. Dashzeveg, Yuanfei Sun, Joshua R. Squires, Janice Lu, Leonidas C. Platanias, Clive H. Wasserfall, William J. Gradishar, Massimo Cristofanilli, Deyu Fang, Huiping Liu

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Figure 4

scRNA-Seq reveals enrichment of rare T cell subsets (DPTs) in the blood of breast cancer patients, dependent on CTC status.

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scRNA-Seq reveals enrichment of rare T cell subsets (DPTs) in the blood ...
(A) Schematic depicting the isolation of human WBCs and subsequent scRNA-Seq. (B) UMAP plots of 47,234 single WBCs from 19 breast cancer patients (n = 35,401 cells) and 12 healthy control liquid biopsies (n = 11,833 cells), with broad immune cell subsets annotated. (C) Correlation plots depicting χ2 test residuals to determine over- or underenrichment of each immune cell subset from the WBCs of breast cancer patients versus healthy controls (top) and by CTC status (bottom). Dot size corresponds to the absolute value of correlation coefficients, and color corresponds to χ2 residuals. (D) UMAP plots of T cell subsets (n = 20,930 cells). (E) Correlation plots of over- or underenrichment of each T cell subset from the WBCs of CTC-positive, -low, and -negative cancer patients and healthy controls. (F) DPT cells highlighted on T cell subset UMAP plots, split by CTC status. (G) UMAP plot of mouse T cells, including single-positive and DPT cells collected from BALB/c mouse splenocytes. (H) Volcano plot depicting most differentially expressed genes in DPT cells versus all other T cells in mouse splenic T cells. (I) Venn diagram showing the overlap between adhesion molecule genes expressed in total human T, mouse T, human DPT, and mouse DPT cells and a list of 32 genes shared among 4 groups. (J) Violin plots of mouse Itgb1 mRNA expression in mouse T cells isolated from splenocytes, as measured by scRNA-Seq. Kruskal-Wallis test P values are provided. (K) Bar graphs of Cd29 (encoded by Itgb1) and Cd49d (encoded by Itga4) expression (MFI) in mouse DPT, CD4+, and CD8+ cells isolated from BALB/c mouse splenocytes. One-way ANOVA with Tukey’s multiple-comparison test P values provided. N = 6 mice.