Zenkoh, J. | Search bioRxiv

bioRxiv2026

Single-cell spatial multiomics identifies POSTN+ CAFs mediating chemoradiotherapy resistance in rectal cancer

Neoadjuvant chemoradiotherapy (CRT) is standard for locally advanced rectal cancer (LARC), yet many patients retain residual disease. To resolve CRT-associated remodeling of the tumor microenvironment, we generated a multimodal spatial atlas from serial sections of paired pretreatment and post-treatment specimens from 24 patients using Xenium single-cell spatial transcriptomics and PhenoCycler multiplex proteomics, profiling 2.8 million cells; matched Visium HD datasets were generated on adjacent serial sections. Resistance was most strongly associated with fibroblast and myeloid programs adjacent to residual tumor. We identify a periostin (POSTN)-expressing CAF subset selectively enriched around residual tumor cells in non-responders, displaying a myofibroblastic phenotype and activating extracellular matrix remodeling, noncanonical WNT signaling, and immunosuppressive pathways. Tumor cells neighboring POSTN+ CAFs show consistent epithelial-mesenchymal transition signatures. Together, this atlas enables interrogation of CRT-induced spatial remodeling and nominates POSTN+ CAFs as key mediators and targets of CRT resistance, with direct relevance to CRT-based combination strategies.

cancer biology↗

bioRxiv2026

Spatial profiling of CAR protein organization reveals in vivo remodeling during CAR-T therapy

Chimeric antigen receptor (CAR) T cell therapy utilizes genetically engineered patient-derived T cells to target cancer cells. Despite its clinical successes in multiple cancer types, the underlying molecular mechanisms by which molecules on CAR-T cells and surrounding cells interact with other proteins and collectively determine treatment efficacy remain elusive. Most previous studies have relied on transcriptome profiling, which does not fully reflect protein-level organization and interactions. In this study, we developed an antibody-oligonucleotide conjugate targeting the FMC63 region of CAR and integrated it into molecular pixelation (MPX). This approach enabled profiling of the dynamics of CAR molecules on cell surfaces as well as their colocalization with other proteins at the single-cell level. By applying MPX to longitudinal samples from three patients undergoing CAR-T cell therapy, we characterized the dynamic changes in CAR-associated protein organization in both pre-infusion CAR products and post-infusion peripheral blood. While CAR protein abundance and polarization showed limited variation across clinical courses, remodeling of a CAR-centered co-localization network was observed over time, including different retentions of specific molecular associations between patients with different clinical outcomes. Although derived from a limited cohort, our study identifies insights from this methodological framework beyond those gained by conventional omics analyses and offers results of a systematic investigation to predict and enhance CAR therapeutic outcomes. Key pointsO_LIMolecular pixelation was applied for chimeric antigen receptor (CAR) profiling at single-molecule and single-cell resolutions. C_LIO_LIProtein and transcriptome analyses of the CAR molecule showed dynamic remodeling during CAR-T therapy in patients with non-Hodgkin lymphoma. C_LI

genomics↗