Michael G. Kattah

Imaging-based single-cell spatial transcriptomics (iSCST) on formalin-fixed, paraffin-embedded (FFPE) tissue enables comprehensive analysis of archived specimens while preserving spatial context, critical to an understanding of ulcerative colitis (UC) pathology. Here, we deployed a robust framework for applying iSCST to clinical FFPE mucosal biopsies from patients with UC, immune checkpoint inhibitor-induced (ICI) colitis and healthy controls. iSCST using custom Xenium gene panels enabled precise detection of diverse cell subsets and disease-specific genes. We mapped transcriptionally distinct fibroblast subsets within mucosal niches, including inflammation-associated fibroblasts (IAFs), and identified colitis-specific neighborhoods formed by IAFs, monocytes, and neutrophils. Transcriptional signatures and spatial neighborhoods uncovered through iSCST were associated with vedolizumab (VDZ) response, with non-responders exhibiting either an innate IAF-monocyte-neutrophil signature or adaptive gut-associated lymphoid tissue (GALT) signature, while responders showed enrichment of an epithelial cellular neighborhood. These signatures were validated in an internal and an external dataset, supporting the existence of two distinct archetypes of treatment resistance to VDZ in UC. This iSCST framework provides a powerful approach for analyzing FFPE tissues, offering insights into colitis-associated cellular networks and identifying biomarkers to enhance patient risk stratification in routine clinical workflows.

Group 2 innate lymphocytes (ILC2s) are prevalent in small intestine but their role during homeostasis is unclear. Here we show that thymic stromal lymphopoietin (TSLP)—a cytokine implicated in ILC2 activation—is expressed constitutively in subepithelial fibroblasts, including telocytes and crypt-associated trophocytes, which are specialized fibroblasts necessary to sustain epithelial identity. Feeding increases TSLP and induces ILC2 type 2 cytokines that are attenuated by deletion of TSLP in fibroblasts or TSLP receptor on ILC2s. Both mouse and human intestinal fibroblasts express receptors for glucagon-like peptide-2 (GLP-2)—an intestinotrophic growth factor released by enteroendocrine cells following food intake. GLP-2 promotes intestinal TSLP in mouse and human intestinal fibroblasts, and TSLP-dependent ILC2 activation and tuft cell hyperplasia in mice, thus linking nutrient detection with ILC2-mediated amplification of the tuft cell chemosensory circuit that promotes epithelial surveillance of ingested cargo.

High-throughput pooled screening has advanced functional genomics, but most existing methods rely on endpoint sequencing and are blind to dynamic, time-resolved phenotypes. We developed RainBar (Rainbow Barcodes), an optical barcoding system that supports pooled live-cell imaging with single-cell resolution. RainBar uses lentiviral co-delivery of spectrally distinct nuclear and cytoplasmic fluorescent proteins to encode up to 64 unique perturbations per well. To mitigate barcode recombination and improve decoding accuracy, we employed single-template viral production, codon diversification, and a ratio-based spectral unmixing pipeline tailored to overlapping fluorophores. An inverted cytoplasmic segmentation approach and multilayer perceptron classifier enabled accurate barcode identification in both arrayed and pooled formats. As a proof of concept, we applied RainBar to dissect NF-κB signaling dynamics in epithelial cells. Live imaging of RelA translocation uncovered stimulus-specific kinetics: IL-1β triggered rapid recovery, while TNF induced prolonged nuclear localization. In pooled CRISPRi screens, RainBar recovered known NF-κB regulators (e.g., IL1R1, MYD88, TNFRSF1A) and highlighted additional modulators, including the Ino80 chromatin remodeling complex subunits and KAT2A acetyltransferase. Together, these results position RainBar as a flexible platform for multiplexed, image-based functional genomics, with potential to reveal dynamic signaling architectures across diverse cellular contexts in live cells.

Background & Aims: Imaging-based, single-cell spatial transcriptomics (iSCST) using formalin-fixed, paraffin-embedded (FFPE) tissue could transform translational research by retaining all tissue cell subsets and spatial locations while enabling the analysis of archived specimens. We aimed to develop a robust framework for applying iSCST to archived clinical FFPE mucosal biopsies from patients with inflammatory bowel disease (IBD). Methods: We performed a comprehensive benchmarking comparison of three iSCST platforms capable of analyzing FFPE specimens. We analyzed FFPE mucosal biopsies (n=57) up to 5 years old from non-IBD controls (HC; n=9) and patients with ulcerative colitis (UC;n=11). After platform-specific cell segmentation, we applied a uniform data processing pipeline to all datasets, including transcript detection, cell annotation, differential gene expression, and neighborhood enrichment. Transcriptomic signatures identified with iSCST were validated using external, publicly available bulk transcriptomic datasets. Results: A custom 290-plex Xenium gene panel exhibited the highest sensitivity and specificity for transcript detection, enabling precise identification and quantification of diverse cell subsets and differentially expressed genes across cell types and disease states. We mapped transcriptionally distinct fibroblast subsets to discrete spatial locations and identified inflammation-associated fibroblasts (IAFs) and monocytes as a colitis-associated cellular neighborhood. We also identified signatures associated with Vedolizumab (VDZ) responsiveness. VDZ non-responders were characterized by an IAF-monocyte transcriptional signature, while responders exhibited enrichment of epithelial gene sets. Conclusions: Our optimized iSCST framework for archived FFPE biopsies provides unique advantages for assessing the role of colitis-associated cellular networks in routinely collected clinical samples. FFPE-based biomarkers could integrate with existing clinical workflows and potentially aid in risk-stratifying patients.

Lay Summary: Immune checkpoint inhibitor-induced (CPI) enterocolitis is a frequent complication of cancer immunotherapy. This case details the treatment of CPI enterocolitis with risankizumab (anti-IL23) and the use of intestinal ultrasound (IUS) to noninvasively monitor treatment response. This report is clinically relevant given the frequency of CPI enterocolitis and the expanding applications of IUS.

Background: Colitis caused by checkpoint inhibitors (CPI) is frequent and is treated with empiric steroids, but CPI colitis mechanisms in steroid-experienced or refractory disease are unclear.Methods: Using colon biopsies and blood from predominantly steroid-experienced CPI colitis patients, we performed multiplexed single-cell transcriptomics and proteomics to nominate contributing populations.Results: CPI colitis biopsies showed enrichment of CD4⁺resident memory (RM) T cells in addition to CD8⁺RM and cytotoxic CD8⁺T cells. Matching T cell receptor (TCR) clonotypes suggested that both RMs are progenitors that yield cytotoxic effectors. Activated, CD38⁺HLA-DR⁺CD4⁺RM and cytotoxic CD8⁺T cells were enriched in steroid-experienced and a validation data set of steroid-naïve CPI colitis, underscoring their pathogenic potential across steroid exposure. Distinct from ulcerative colitis, CPI colitis exhibited perturbed stromal metabolism (NAD⁺, tryptophan) impacting epithelial survival and inflammation. Endothelial cells in CPI colitis after anti-TNF and anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) upregulated the integrin α4β7 ligand molecular vascular addressin cell adhesion molecule 1 (MAdCAM-1), which may preferentially respond to vedolizumab (anti-α4β7).Conclusions: These findings nominate CD4⁺RM and MAdCAM-1⁺endothelial cells for targeting in specific subsets of CPI colitis patients.

Ulcerative colitis (UC) is driven by immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin antibody that is effective for treating UC. VDZ is known to inhibit lymphocyte trafficking to the intestine, but its broader effects on other cell subsets are less defined. To identify the inflammatory cells that contribute to colitis and are affected by VDZ, we perform single-cell transcriptomic and proteomic analyses of peripheral blood and colonic biopsies in healthy controls and patients with UC on VDZ or other therapies. Here we show that VDZ treatment is associated with alterations in circulating and tissue mononuclear phagocyte (MNP) subsets, along with modest shifts in lymphocytes. Spatial multi-omics of formalin-fixed biopsies demonstrates trends towards increased abundance and proximity of MNP and fibroblast subsets in active colitis. Spatial transcriptomics of archived specimens pre-treatment identifies epithelial-, MNP-, and fibroblast-enriched genes related to VDZ responsiveness, highlighting important roles for these subsets in UC.

Ulcerative colitis (UC) is driven by immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin antibody that is effective for treating UC. VDZ is known to inhibit lymphocyte trafficking to the intestine, but its broader effects on other cell subsets are less defined. To identify the inflammatory cells that contribute to colitis and are affected by VDZ, we performed single-cell transcriptomic and proteomic analyses of peripheral blood and colonic biopsies in healthy controls and patients with UC on VDZ or other therapies. Here we show that VDZ treatment is associated with alterations in circulating and tissue mononuclear phagocyte (MNP) subsets, along with modest shifts in lymphocytes. Spatial multi-omics of formalin-fixed biopsies demonstrates trends towards increased abundance and proximity of MNP and fibroblast subsets in active colitis. Spatial transcriptomics of archived specimens pre-treatment identifies epithelial-, MNP-, and fibroblast-enriched genes related to VDZ responsiveness, highlighting important roles for these subsets in UC.

Anti-α₄β₇ (Vedolizumab) treats inflammatory bowel disease (IBD) by blocking the interaction between integrin α₄β₇ on leukocytes and mucosal addressin cell-adhesion molecule-1 (MAdCAM-1) on the gut endothelium. Women with IBD often require continuing biologic therapy during pregnancy to avoid disease flare. To date, there have been no reports of an increase in adverse events with Vedolizumab use during pregnancy. Notably, integrins play a major role in human placental development during pregnancy. It is unknown whether Vedolizumab disrupts placental cell (cytotrophoblast) invasion and/or adhesion by blocking interactions with MAdCAM-1. We therefore investigated human placental expression of MAdCAM-1, the role of MAdCAM-1/α₄β₇ interactions in cytotrophoblast invasion/adhesion in vitro, and whether Vedolizumab administration in vivo alters the placental structure. Histological sections of placentas from normal pregnancies were evaluated for MAdCAM-1 expression by immunofluorescence. The impacts of Vedolizumab or anti-integrin β₇ on human cytotrophoblast invasion and adhesion were assessed. Histology results from term placentas of 2 patients with IBD receiving Vedolizumab were compared to those of untreated healthy controls. Placental MAdCAM-1 expression was predominantly associated with invading extravillous cytotrophoblasts at the maternal-fetal interface. Treatment of isolated primary cytotrophoblasts with Vedolizumab or anti-integrin β₇ significantly reduced Matrigel invasion, adherence to a MAdCAM-1-coated substrate, and interactions with HuT-78 cells. Placentas from 2 Vedolizumab-treated patients with IBD exhibited pronounced pathologic features as compared to healthy control specimens. This study revealed a previously unrecognized role for α₄β₇ and MAdCAM-1 in human placentation. More clinical and histological data from Vedolizumab-treated pregnant patients will be necessary to determine whether this medication poses any risk to the mother and fetus.