JAK/STAT3 represents a therapeutic target for colorectal cancer patients with stromal-rich tumors.

Colorectal cancer (CRC) is a heterogenous malignancy underpinned by dysregulation of cellular signaling pathways. Previous literature has implicated aberrant JAK/STAT3 signal transduction in the development and progression of solid tumors. In this study we investigate the effectiveness of inhibiting JAK/STAT3 in diverse CRC models, establish in which contexts high pathway expression is prognostic and perform in depth analysis underlying phenotypes. In this study we investigated the use of JAK inhibitors for anti-cancer activity in CRC cell lines, mouse model organoids and patient-derived organoids. Immunohistochemical staining of the TransSCOT clinical trial cohort, and 2 independent large retrospective CRC patient cohorts was performed to assess the prognostic value of JAK/STAT3 expression. We performed mutational profiling, bulk RNASeq and NanoString GeoMx® spatial transcriptomics to unravel the underlying biology of aberrant signaling. Inhibition of signal transduction with JAK1/2 but not JAK2/3 inhibitors reduced cell viability in CRC cell lines, mouse, and patient derived organoids (PDOs). In PDOs, reduced Ki67 expression was observed post-treatment. A highly significant association between high JAK/STAT3 expression within tumor cells and reduced cancer-specific survival in patients with high stromal invasion (TSPhigh) was identified across 3 independent CRC patient cohorts, including the TrasnSCOT clinical trial cohort. Patients with high phosphorylated STAT3 (pSTAT3) within the TSPhigh group had higher influx of CD66b + cells and higher tumoral expression of PDL1. Bulk RNAseq of full section tumors showed enrichment of NFκB signaling and hypoxia in these cases. Spatial deconvolution through GeoMx® demonstrated higher expression of checkpoint and hypoxia-associated genes in the tumor (pan-cytokeratin positive) regions, and reduced lymphocyte receptor signaling in the TME (pan-cytokeratin- and αSMA-) and αSMA (pan-cytokeratin- and αSMA +) areas. Non-classical fibroblast signatures were detected across αSMA + regions in cases with high pSTAT3. Therefore, in this study we have shown that inhibition of JAK/STAT3 represents a promising therapeutic strategy for patients with stromal-rich CRC tumors. High expression of JAK/STAT3 proteins within both tumor and stromal cells predicts poor outcomes in CRC, and aberrant signaling is associated with distinct spatially-dependant differential gene expression.

Vascular mechanisms of post-COVID-19 conditions: Rho-kinase is a novel target for therapy.

BACKGROUND: In post-coronavirus disease-19 (post-COVID-19) conditions (long COVID), systemic vascular dysfunction is implicated, but the mechanisms are uncertain, and the treatment is imprecise. METHODS AND RESULTS: Patients convalescing after hospitalization for COVID-19 and risk factor matched controls underwent multisystem phenotyping using blood biomarkers, cardiorenal and pulmonary imaging, and gluteal subcutaneous biopsy (NCT04403607). Small resistance arteries were isolated and examined using wire myography, histopathology, immunohistochemistry, and spatial transcriptomics. Endothelium-independent (sodium nitroprusside) and -dependent (acetylcholine) vasorelaxation and vasoconstriction to the thromboxane A2 receptor agonist, U46619, and endothelin-1 (ET-1) in the presence or absence of a RhoA/Rho-kinase inhibitor (fasudil), were investigated. Thirty-seven patients, including 27 (mean age 57 years, 48% women, 41% cardiovascular disease) 3 months post-COVID-19 and 10 controls (mean age 57 years, 20% women, 30% cardiovascular disease), were included. Compared with control responses, U46619-induced constriction was increased (P = 0.002) and endothelium-independent vasorelaxation was reduced in arteries from COVID-19 patients (P < 0.001). This difference was abolished by fasudil. Histopathology revealed greater collagen abundance in COVID-19 arteries {Masson's trichrome (MT) 69.7% [95% confidence interval (CI): 67.8-71.7]; picrosirius red 68.6% [95% CI: 64.4-72.8]} vs. controls [MT 64.9% (95% CI: 59.4-70.3) (P = 0.028); picrosirius red 60.1% (95% CI: 55.4-64.8), (P = 0.029)]. Greater phosphorylated myosin light chain antibody-positive staining in vascular smooth muscle cells was observed in COVID-19 arteries (40.1%; 95% CI: 30.9-49.3) vs. controls (10.0%; 95% CI: 4.4-15.6) (P < 0.001). In proof-of-concept studies, gene pathways associated with extracellular matrix alteration, proteoglycan synthesis, and viral mRNA replication appeared to be upregulated. CONCLUSION: Patients with post-COVID-19 conditions have enhanced vascular fibrosis and myosin light change phosphorylation. Rho-kinase activation represents a novel therapeutic target for clinical trials.

Spatially Resolved Transcriptomics Deconvolutes Prognostic Histological Subgroups in Patients with Colorectal Cancer and Synchronous Liver Metastases.

Strong immune responses in primary colorectal cancer correspond with better patient survival following surgery compared with tumors with predominantly stromal microenvironments. However, biomarkers to identify patients with colorectal cancer liver metastases (CRLM) with good prognosis following surgery for oligometastatic disease remain elusive. The aim of this study was to determine the practical application of a simple histological assessment of immune cell infiltration and stromal content in predicting outcome following synchronous resection of primary colorectal cancer and CRLM and to interrogate the underlying functional biology that drives disease progression. Samples from patients undergoing synchronous resection of primary colorectal cancer and CRLM were evaluated in detail through histological assessment, panel genomic and bulk transcriptomic assessment, IHC, and GeoMx spatial transcriptomics (ST) analysis. High immune infiltration of metastases was associated with improved cancer-specific survival. Bulk transcriptomic analysis was confounded by stromal content, but ST demonstrated that the invasive edge of the metastases of long-term survivors was characterized by adaptive immune cell populations enriched for type II IFN signaling and MHC-class II antigen presentation. In contrast, patients with poor prognosis demonstrated increased abundance of regulatory T cells and neutrophils with enrichment of Notch and TGFß signaling pathways at the metastatic tumor center. In summary, histological assessment can stratify outcomes in patients undergoing synchronous resection of CRLM, suggesting that it has potential as a prognostic biomarker. Furthermore, ST analysis has revealed significant intratumoral and interlesional heterogeneity and identified the underlying transcriptomic programs driving each phenotype. SIGNIFICANCE: Spatial transcriptomics uncovers heterogeneity between patients, between matched lesions in the same patient, and within individual lesions and identifies drivers of metastatic progression in colorectal cancer with reactive and suppressed immune microenvironments.

Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data.

PURPOSE: Precise mechanism-based gene expression signatures (GES) have been developed in appropriate in vitro and in vivo model systems, to identify important cancer-related signaling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumor samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumor stroma percentage can directly influence GESs, undermining the intended mechanistic signaling. EXPERIMENTAL DESIGN: Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single-cell RNA sequencing and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely used GESs to be influenced, or confounded, by stromal content in tumor tissue. To complement this work, we generated a freely-available resource, ConfoundR; https://confoundr.qub.ac.uk/, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets. RESULTS: Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumor models not fully recapitulating the stromal and immune microenvironment. CONCLUSIONS: Efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples.