Biology subjects

Scholten, D.

Publications and source records attributed to Scholten, D..

6 recordsLinked to original sources

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↗

bioRxiv2024

Timing anti-PD-L1 checkpoint blockade immunotherapy to enhance tumor irradiation

BackgroundThe ability of radiotherapy (RT) to drive anti-tumor immunity is limited by adaptive resistance. While RT induces inflammation and recruits activated tumor-infiltrating lymphocytes (TILs) including cytotoxic T lymphocytes (CTLs), the resulting radiation- and IFN{gamma}-dependent PD-L1 expression restores an immunosuppressed tumor microenvironment. Unleashing an effective anti-tumor response may require precise sequencing of RT and checkpoint blockade immunotherapy (CBI) to block PD-L1 signaling before it can mediate its suppressive effects. MethodsFlank tumors formed in BALB/c mice with syngeneic CT26 colon or 4T1 mammary carcinoma cells were treated with otherwise ineffective doses of ionizing radiation (10 Gy) followed by CBI (0.2 mg anti-PD-L1, i.v.) after 0, 1, 3, 5 or 7 days, comparing tumor response. Anti-PD-L1 delivery was measured by fluorescence, TILs by flow cytometry and immunofluorescence, PD-L1 expression by immunohistochemistry, and tumor size by calipers. Results In both CT26 and 4T1 tumors, 10 Gy alone resulted in a transient growth delay associated with infiltrating CTLs peaking at 3 days and PD-L1 at 5 days. CTLs returned toward baseline by 7 days, consistent with adaptive resistance. Anti-PD-L1 failed to potentiate radiation except when injected 5 days after 10 Gy, which prevented CTL depletion and led to tumor elimination. Potentially contributing to compound effects, anti-PD-L1 penetrated tumors and bound PD-L1 more efficiently after irradiation. ConclusionsOptimal timing to exploit radiation-induced permeability to enhance CBI delivery and interrupt adaptive resistance by blocking PD-L1 as it peaks may offer a general strategy to enhance external beam radiotherapy by protecting activated TILs and potentiating anti-tumor immune response.

cancer biology↗

bioRxiv2023

Robust collection and processing for label-free single voxel proteomics

With advanced mass spectrometry (MS)-based proteomics, genome-scale proteome coverage can be achieved from bulk tissues. However, such bulk measurement lacks spatial resolution and obscures important tissue heterogeneity, which make it impossible for proteome mapping of tissue microenvironment. Here we report an integrated wet collection of single tissue voxel and Surfactant-assisted One-Pot voxel processing method termed wcSOP for robust label-free single voxel proteomics. wcSOP capitalizes on buffer droplet-assisted wet collection of single tissue voxel dissected by LCM into the PCR tube cap and MS-compatible surfactant-assisted one-pot voxel processing in the collection cap. This convenient method allows reproducible label-free quantification of [~]900 and [~]4,600 proteins for single voxel from fresh frozen human spleen tissue at 20 m x 20 m x 10 m (close to single cells) and 200 m x 200 m x 10 m ([~]100 cells), respectively. 100s-1000s of protein signatures with differential expression levels were identified to be spatially resolved between spleen red and white pulp regions depending on the voxel size. Region-specific signaling pathways were enriched from single voxel proteomics data. Antibody-based CODEX imaging was used to validate label-free MS quantitation for single voxel analysis. The wcSOP-MS method paves the way for routine robust single voxel proteomics and spatial proteomics.

molecular biology↗

bioRxiv2023

Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

Metastasis is the cause of over 90% of all deaths associated with breast cancer, yet the strategies to predict cancer spreading based on primary tumor profiles and therefore prevent metastasis are egregiously limited. As rare precursor cells to metastasis, circulating tumor cells (CTCs) in multicellular clusters in the blood are 20-50 times more likely to produce viable metastasis than single CTCs. However, the molecular mechanisms underlying various CTC clusters, such as homotypic tumor cell clusters and heterotypic tumor-immune cell clusters, are yet to be fully elucidated. Combining machine learning-assisted computational ranking with experimental demonstration to assess cell adhesion candidates, we identified a transmembrane protein Plexin- B2 (PB2) as a new therapeutic target that drives the formation of both homotypic and heterotypic CTC clusters. High PB2 expression in human primary tumors predicts an unfavorable distant metastasis-free survival and is enriched in CTC clusters compared to single CTCs in advanced breast cancers. Loss of PB2 reduces formation of homotypic tumor cell clusters as well as heterotypic tumor-myeloid cell clusters in triple-negative breast cancer. Interactions between PB2 and its ligand Sema4C on tumor cells promote homotypic cluster formation, and PB2 binding with Sema4A on myeloid cells (monocytes) drives heterotypic CTC cluster formation, suggesting that metastasizing tumor cells hijack the PB2/Sema family axis to promote lung metastasis in breast cancer. Additionally, using a global proteomic analysis, we identified novel downstream effectors of the PB2 pathway associated with cancer stemness, cell cycling, and tumor cell clustering in breast cancer. Thus, PB2 is a novel therapeutic target for preventing new metastasis.

cancer biology↗

bioRxiv2022

CD81 partners with CD44 in promoting exosome biogenesis, tumor cluster formation, and lung metastasis in triple negative breast cancer

Tumor-initiating cells with reprogramming plasticity are thought to be essential for cancer development and metastatic regeneration in many cancers; however, the molecular mechanisms are not fully understood. This study reports that CD81, a tetraspanin protein marker of small extracellular vesicles (exosomes), functions as a binding partner of CD44 and facilitates self-renewal of tumor initiating cells. Using machine learning-assisted protein structure modeling, co-immunoprecipitation, and mutagenesis approaches, we further demonstrate that CD81 interacts with CD44 on the cellular membrane through their extracellular regions. In-depth global and phosphoproteomic analyses of clustering tumor cells unveils endocytosis-related signature pathways of proteins and phosphorylation patterns regulated by CD81 and CD44 specifically or shared between two. Notably, CRISPR Cas9-mediated depletion of either CD44 or CD81 results in loss of both proteins in cancer cell-secreted exosomes, a state which abolishes exosome-induced self-renewal of recipient cells for mammosphere formation. CD81 is expressed in >80% of human circulating tumor cells (CTCs) and specifically enriched in clustered CTCs along with CD44 isolated from breast cancer patients. Mimicking the phenotypes of CD44 deficiency, loss of CD81 also inhibits tumor cluster aggregation, tumorigenesis, and lung metastasis of triple negative breast cancer (TNBC), supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights the novel role of CD81 and its partnership with CD44 in cancer exosomes, self-renewal, CTC clustering, and metastasis initiation of TNBC.

cancer biology↗