NicheSphere reveals SPP1⁺ myeloid cells as central hubs of fibrotic remodeling in myeloproliferative neoplasms

Zenodo (CERN European Organization for Nuclear Research)

Abstract

Bone marrow fibrosis in myeloproliferative neoplasms arises through reciprocal interactions between mutant hematopoietic cells and fibrosis-associated stromal populations. Using dual lineage tracing, single-cell and multiplet RNA sequencing, spatial proteomics, and NicheSphere, a framework integrating condition-specific co-localization with ligand–receptor inference, we identified Spp1⁺ macrophages as central cellular communication hubs within a “Fibrosis Interacting core” comprising osteoCAR cells, fibroblasts, megakaryocytes, and additional hematopoietic populations. NicheSphere distinguished macrophage–vascular compartments enriched in inflammatory signaling from this Fibrosis Interacting core characterized by TGF-β, NF-κB, and extracellular-matrix programs. CODEX imaging confirmed increased proximity of SPP1⁺ immune cells, activated stromal cells, and megakaryocytes. Genetic deletion of SPP1 demonstrated complementary contributions of stromal- and hematopoietic-derived Spp1 to fibrosis, inflammation, and myeloproliferative features. Human myelofibrosis single-cell analysis identified SPP1⁺ cells as a prominent predicted communication hub, while elevated circulating SPP1 was associated with adverse clinical outcome. Together, these findings establish SPP1-centered multicellular communication as a conserved driver of myelofibrosis and a therapeutic vulnerability of the fibrotic niche. This repository contains the following datasets: Physically Interacting Cells sequencing (PIC-seq) - /data/PICseq/ CODEX - /data/CODEX/ Human myelofibrosis (MF) scRNA-seq JAK2 subset from [https://doi.org/10.5281/zenodo.17210242] - /data/MF/ and the code used to produce the presented results (also available in GitHub: https://github.com/CostaLab/NicheSphere_paper_analysis ). Final data files are in the "final_data" folders: /data/PICseq/final_data/ adata_singlets_final.h5ad Singlets data with final cell type names in the metadata "annotation" column (processing done in Python in PIC_data.ipynb) adata_singlets_final_imp.h5ad (MAGIC) Imputated singlets data with final cell type names in the metadata "annotation" column (processing done in Python in PIC_data.ipynb) adata_doublets_final.h5ad Doublets data with final doublet cluster annotation in in the metadata "max_pair" column (processing done in Python in PIC_data.ipynb) adata_doublets_final.rds Sceasy converted adata_doublets_final.h5ad (employed for ridge plots with doublet cluster names) adata_doublets_final_imp.h5ad (MAGIC) Imputated doublets data with final doublet cluster annotation in the metadata "max_pair" column (processing done in Python in PIC_data.ipynb) Phenotype (TPO or EV) is indicated in the metadata "stage" columns for these datasets. /data/CODEX/final_data/ CODEX_run45_anndata_final.h5ad Final CODEX dataset with final cell type names in the metadata "clusters_named" column, ohenotype (Thpo or EV) is in the "condition" column and sample and run are indicated in the columns with those names. zarr/ Folder containing zarr files with cell shapes for each sample /data/MF/ MF_pre_treatment_Amplicon_object.h5ad JAK2 subset of human MF scRNA-seq dataset from [https://doi.org/10.5281/zenodo.17210242] MF_subcluster_subset_metadata.csv Metadata related to JAK2 scRNA-seq human data MF_JAK2_LR_data_final.Rds Resulting data from CrossTalkeR cell - cell communication analysis on JAK2 scRNA-seq human data Intermediate data files which were used in the scripts can be found in the "intermediate_steps" folders ( eg: /data/PICseq/intermediate_steps/ ). Analysis code is in the "code" folder and resulting data files and images are located in folders "res_data" and "figures" respectively. Database related files are in the "csv" folder.

Authors 2

1. Mayra L. Ruiz Tejada Segura Aachen

   RWTH Aachen University

   Affiliation as printed

   RWTH Aachen University

2. James Shiniti Nagai Aachen

   RWTH Aachen University

   Affiliation as printed

   Rheinisch-Westfälische Technische Hochschule Aachen Medizinische Fakultät