Spatial Characterization of Molecular Pathways Suggests the Dysregulation of LGR5 Expressing Fibroblasts in Patients with sclerodermatous Chronic Graft-Vs-Host Disease

Chronic graft-vs-host disease (cGVHD) is a common and often morbid immune related complication of allogeneic hematopoietic cell transplantation (HCT), and a major obstacle to the success of this cellular therapy. Its presentation is heterogeneous and the pathophysiology of cGVHD is incompletely understood. Sclerodermatous GVHD (Scl GVHD) is a severe form of cGVHD and is associated with poor prognosis and low sensitivity to immune suppression which is the current first line therapy. Here with the spatial transcriptomics analysis using the skin biopsy tissues demonstrated the dysregulation of LGR5+ fibroblast cells in cGVHD patient skins. LGR5 gene is known for functioning in excessive ECM degradation and hence reduced in cGVHD patients. We demonstrated the upregulation of ECM components deposition in the patient skins. In addition, we expanded our cohort and profiled 20 patient and 6 control skin tissue biopsies using next-generation spatial molecular profiling to gain deep insights into the immune landscape and pathogenesis of Scl cGVHD. In summary, we have demonstrated a distinct LGR5-expressing fibroblast subset as the central axis of perturbation in SSc patients. Targeted therapies directed against this pathway might begin a new era for SSc therapies.

Introduction: Chronic graft-vs-host disease (cGVHD) is a major obstacle to the success of allogeneic hematopoietic stem cell transplantation (HCT) in patients. This debilitating condition is characterized by chronic inflammation, cell-mediated and humoral immunity, and ultimately tissue fibrosis. There is currently little or no understanding of the molecular pathogenesis of chronic cGVHD resulting in poor effective treatment strategies. Sclerodermatous GVHD (sclGVHD) is one of the more severe forms of cGVHD associated with poor prognosis and low sensitivity to immune suppressive therapy.

Methods: We used the Nanostring CosMx platform to analyze biopsies of skin lesions from twenty patients and six controls to obtain in situ, single cell transcriptomic and protein expression profiles. Samples were prepared as follows: First, a slide (~5 µm thickness) of FFPE lesional tissue was permeabilized for target retrieval and hybridized with RNA-specific probes and antibodies to bind to the targets. The slide was then processed within the flow cell of the CoxMX SMI which images through cycles of in situ hybridization, fluorescence detection, UV photocleavage, and finally washes to remove probes. Immediately following transcriptomics, the protein panel will be run to detect fluorescence from protein targets using tagged antibodies.

The 1000-plex Human Universal Cell Typing and Cell State Panel was used, which contains 950 target probes, as well as negative controls and Segmentation Markers. This panel provides robust tissue mapping, cell typing, and analysis of cell states and a wide range of signaling interactions including our immunotherapy targets of interest, fibroblast markers, as well as other markers of pathogenesis pathways. Segmentation Markers were used for field of view (FOV) selection in instrument software and provide vital input for allocating transcripts to individual cells. For instance, DAPI and CD298/B2M are used to segmentalize the nuclear and cell membrane compartments, respectively.

Data Analysis: Data were analyzed using the R-programming and CosMx software analysis interface (AtoMx), which includes QC normalization, graphical visualization, cell typing (with marker identification) and spatial clustering. Data was compared to publicly available reference libraries of gene expression.

Results: To investigate cGVHD immunopathogenesis, in the context of tissue architecture we integrated spatial RNA sequencing on a tissue microarray assembled with total 20 skin biopsies of GVHD patients and 6 skin biopsies from normal controls. We demonstrated a tight correlation of dysregulation of LGR5+ fibroblast in Sclerodermatous cGVHD patient compared to control skin biopsies. We also demonstrated the ECM components (Col1A1, COL3A1, COL6A2) in sclerodermatous cGVHD skin biopsies. We also observed the macrophages switching from M1(Tissue resident macrophages, TRMs) to M2 (Tumor associated macrophages, TAMs) in diseased skin biopsies.

Conclusion: We spatially profiled 26 skin biopsies with 20 sclerodermatous cGVHD patients and identified a novel biomarker that can be targeted for the treatment of disease.