Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product.

Circulating Tumor Cells (CTCs) in blood encompass DNA, RNA, and protein biomarkers, but clinical utility is limited by their rarity. To enable tumor epitope-agnostic interrogation of large blood volumes, we developed a high-throughput microfluidic device, depleting hematopoietic cells through high-flow channels and force-amplifying magnetic lenses. Here, we apply this technology to analyze patient-derived leukapheresis products, interrogating a mean blood volume of 5.

A TROP2/Claudin Program Mediates Immune Exclusion to Impede Checkpoint Blockade in Breast Cancer.

Immune exclusion inhibits anti-tumor immunity and response to immunotherapy, but its mechanisms remain poorly defined. Here, we demonstrate that Trophoblast Cell-Surface Antigen 2 (TROP2), a key target of emerging anti-cancer Antibody Drug Conjugates (ADCs), controls barrier-mediated immune exclusion in Triple-Negative Breast Cancer (TNBC) through Claudin 7 association and tight junction regulation. TROP2 expression is inversely correlated with T cell infiltration and strongly associated with outcomes in TNBC.

Cancer cells restrict immunogenicity of retrotransposon expression via distinct mechanisms.

To thrive, cancer cells must navigate acute inflammatory signaling accompanying oncogenic transformation, such as via overexpression of repeat elements. We examined the relationship between immunostimulatory repeat expression, tumor evolution, and the tumor-immune microenvironment. Integration of multimodal data from a cohort of pancreatic ductal adenocarcinoma (PDAC) patients revealed expression of specific Alu repeats predicted to form double-stranded RNAs (dsRNAs) and trigger retinoic-acid-inducible gene I (RIG-I)-like-receptor (RLR)-associated type-I interferon (IFN) signaling.

Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product.

Circulating Tumor Cells (CTCs), interrogated by sampling blood from patients with cancer, contain multiple analytes, including intact RNA, high molecular weight DNA, proteins, and metabolic markers. However, the clinical utility of tumor cell-based liquid biopsy has been limited since CTCs are very rare, and current technologies cannot process the blood volumes required to isolate a sufficient number of tumor cells for in-depth assays. We previously described a high-throughput microfluidic prototype utilizing high-flow channels and amplification of cell sorting forces through magnetic lenses.

Mesothelin CAR T Cells Secreting Anti-FAP/Anti-CD3 Molecules Efficiently Target Pancreatic Adenocarcinoma and its Stroma.

Purpose: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells).

Dysregulated Repeat Element Viral-like Immune Response in Hepatocellular Carcinoma.

Purpose: Dysregulation of viral-like repeat RNAs are a common feature across many malignancies that are linked with immunological response, but the characterization of these in hepatocellular carcinoma (HCC) is understudied. In this study, we performed RNA hybridization (RNA-ISH) of different repeat RNAs, immunohistochemistry (IHC) for immune cell subpopulations, and spatial transcriptomics to understand the relationship of HCC repeat expression, immune response, and clinical outcomes.

Experimental Design: RNA-ISH for LINE1, HERV-K, HERV-H, and HSATII repeats and IHC for T-cell, Treg, B-cell, macrophage, and immune checkpoint markers were performed on 43 resected HCC specimens.

Spatial transcriptomics reveals distinct tissue niches linked with steroid responsiveness in acute gastrointestinal GVHD.

Severe acute graft-versus-host disease (aGVHD) is associated with significant mortality and morbidity, especially in steroid-resistant (SR) cases. Spatial transcriptomic technology can elucidate tissue-based interactions in vivo and possibly identify predictors of treatment response. Tissue sections from 32 treatment-naïve patients with biopsy-confirmed lower gastrointestinal (GI) aGVHD were obtained.

Spatial analysis of human lung cancer reveals organized immune hubs enriched for stem-like CD8 T cells and associated with immunotherapy response.

The organization of immune cells in human tumors is not well understood. Immunogenic tumors harbor spatially-localized multicellular 'immunity hubs' defined by expression of the T cell-attracting chemokines and abundant T cells. Here, we examined immunity hubs in human pre-immunotherapy lung cancer specimens, and found that they were associated with beneficial responses to PD-1-blockade.

TGF-β in the microenvironment induces a physiologically occurring immune-suppressive senescent state.

TGF-β induces senescence in embryonic tissues. Whether TGF-β in the hypoxic tumor microenvironment (TME) induces senescence in cancer and how the ensuing senescence-associated secretory phenotype (SASP) remodels the cellular TME to influence immune checkpoint inhibitor (ICI) responses are unknown. We show that TGF-β induces a deeper senescent state under hypoxia than under normoxia; deep senescence correlates with the degree of E2F suppression and is marked by multinucleation, reduced reentry into proliferation, and a distinct 14-gene SASP.

Satellite repeat RNA expression in epithelial ovarian cancer associates with a tumor-immunosuppressive phenotype.

Aberrant expression of viral-like repeat elements is a common feature of epithelial cancers, and the substantial diversity of repeat species provides a distinct view of the cancer transcriptome. Repeatome profiling across ovarian, pancreatic, and colorectal cell lines identifies distinct clustering independent of tissue origin that is seen with coding gene analysis. Deeper analysis of ovarian cancer cell lines demonstrated that human satellite II (HSATII) satellite repeat expression was highly associated with epithelial-mesenchymal transition (EMT) and anticorrelated with IFN-response genes indicative of a more aggressive phenotype.

Differential Kinase Activity Across Prostate Tumor Compartments Defines Sensitivity to Target Inhibition.

Unlabelled: Cancer therapy often results in heterogeneous responses in different metastatic lesions in the same patient. Inter- and intratumor heterogeneity in signaling within various tumor compartments and its impact on therapy are not well characterized due to the limited sensitivity of single-cell proteomic approaches. To overcome this barrier, we applied single-cell mass cytometry with a customized 26-antibody panel to PTEN-deleted orthotopic prostate cancer xenograft models to measure the evolution of kinase activities in different tumor compartments during metastasis or drug treatment.

Active RB causes visible changes in nuclear organization.

RB restricts G1/S progression by inhibiting E2F. Here, we show that sustained expression of active RB, and prolonged G1 arrest, causes visible changes in chromosome architecture that are not directly associated with E2F inhibition. Using FISH probes against two euchromatin RB-associated regions, two heterochromatin domains that lack RB-bound loci, and two whole-chromosome probes, we found that constitutively active RB (ΔCDK-RB) promoted a more diffuse, dispersed, and scattered chromatin organization.

Selective targeting of MYC mRNA by stabilized antisense oligonucleotides.

MYC is a prolific proto-oncogene driving the malignant behaviors of numerous common cancers, yet potent and selective cell-permeable inhibitors of MYC remain elusive. In order to ultimately realize the goal of therapeutic MYC inhibition in cancer, we have initiated discovery chemistry efforts aimed at inhibiting MYC translation. Here we describe a series of conformationally stabilized synthetic antisense oligonucleotides designed to target MYC mRNA (MYCASOs).

Spatially organized multicellular immune hubs in human colorectal cancer.

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors.

LGR5 in Barrett's Esophagus and its Utility in Predicting Patients at Increased Risk of Advanced Neoplasia.

Introduction: The expression of LGR5, a known stem cell marker, is poorly understood in Barrett's esophagus (BE) and related neoplasia. The aim of this study was to evaluate LGR5 in BE and related neoplasia and to evaluate its utility as a potential biomarker of progression to advanced neoplasia.

Methods: We evaluated total 137 patients, including 119 with BE and 18 with normal gastroesophageal mucosa for expression of LGR5 using RNA in situ hybridization; this also included 28 progressors and 30 nonprogressors.