Gradishar, W. J. | Search bioRxiv

bioRxiv2026

AI-enabled Spatial Profiling of Circulating Tumor-Immune Ecosystems Predicts Patient Outcomes Across Cancers

Circulating tumor cells (CTCs) and immune cells form dynamic multicellular ecosystems in blood, but their spatial organization and clinical relevance have not been systematically characterized. We developed the Cell and Cluster Identification Program (CCIP), an artificial intelligence-based framework that analyzes routine multiplex immunofluorescence blood scans to segment cells, identify CTCs and five immune lineages with high accuracy, and quantify multicellular clusters and tumor-immune interactions. Applying CCIP to 2,693 blood scans from 1,399 patients, we profiled over 60 million cells (>7 million multi-cell clusters) and linked imaging-derived features to patient outcomes. Correlated with circulating-tumor DNA mutation burdens, a 14-feature image model predicted overall survival in breast cancer, outperformed clinicopathologic variables and CTC enumeration, and generalized to prostate cancer. Prognostic imaging signatures were also associated with therapy response-related progression-free survival as well as with single-cell RNA sequencing-derived immune suppression states, connecting circulating tumor-immune architecture with systemic immune dysfunction.

cancer biology↗

bioRxiv2025

Rare Subset of T Cells Form Heterotypic Clusters with Circulating Tumor Cells to Foster Cancer Metastasis

The immune ecosystem is central to maintaining effective defensive responses. However, how immune cells in the periphery blood interact with circulating tumor cells (CTCs) - seeds of metastasis - remains largely understudied. Here, our analysis of the blood specimens (N=1,529) from patients with advanced breast cancer revealed that over 75% of the CTC-positive blood specimens contained heterotypic CTC clusters with CD45+ white blood cells (WBCs). Detection of CTC-WBC clusters correlates with breast cancer subtypes (triple negative and luminal B), racial groups (Black), and decreased survival rates. Flow cytometry and ImageStream analyses revealed diverse WBC composition of heterotypic CTC-WBC clusters, including 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 versus its frequency in WBCs. DPT cells shared part of the profiles with CD4+ T cells and others with CD8+ T cells but exhibited unique features of T cell exhaustion and immune suppression with higher expression of TIM-3 and PD-1. Single-cell RNA sequencing and genetic perturbation studies further pinpointed the integrin VLA4 (4{beta}1) in DPT cells and its ligand VCAM1 in tumor cells as essential mediators of heterotypic WBC-CTC clusters. Neoadjuvant administration of anti-4 (VLA4) neutralizing antibodies markedly blocked CTC-DPT cell clustering and inhibited metastasis for extended survival in preclinical mouse models in vivo. These findings uncover a pivotal role of rare DPT cells with immune suppressive features in fostering cancer dissemination through direct interactive clustering with CTCs. It lays a foundation for developing innovative biomarkers and therapeutic strategies to prevent and target cancer metastasis, ultimately benefiting cancer care. Brief summaryOur findings uncover a fostering role of immune-suppressive T cells in contact with circulating tumor cells and identify therapeutic approaches to eliminate devastating cancer metastasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/646421v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@13b7862org.highwire.dtl.DTLVardef@15c97b2org.highwire.dtl.DTLVardef@1eb4fd2org.highwire.dtl.DTLVardef@1c588e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗