bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.08.18.670793

Spatial and Single-Cell Dissection of Fibroblast Subpopulation Reprogramming Driving Stromal Collapse in Breast Cancer Lymph Node Metastasis

Rastad, H.·Seifi Alan, M.·Pandey, S.
View original source ↗Export metadata ↓

Abstract

ObjectiveThis study aimed to define conserved molecular drivers of breast cancer lymph node metastasis (LNM) through an unbiased, multi-omics approach, resolving spatial and cellular mechanisms of stromal reprogramming. MethodsWe integrated three bulk RNA-seq datasets (GSE110590, GSE193103, GSE209998) to identify differentially expressed genes (DEGs) in LNMs versus primary tumors. Tissue-specific genes were excluded where possible. Single-cell (scRNA-seq; GSE225600) and spatial transcriptomics (Visium) from primary/metastatic tissues were analyzed using Seurat and spacexr. Fibroblast subclusters were isolated for functional enrichment. Clinical validation included genetic alteration profiling (cBioPortal), survival analysis (KM Plotter), and stage-specific expression dynamics (TNMplot). Therapeutic candidates were screened via CTD. FindingsBulk RNA-seq identified three genes consistently downregulated in lymph node metastases compared to primary tumors: MAB21L1, F2RL2, and COL6A6 (log2FC [&le;] -2.62; adj. p < 0.05). Single-cell RNA-seq analysis revealed that all three DEGs converged exclusively within specific fibroblast subpopulations in primary tumors; these DEGs were not enriched in other cell types. DEG-expressing fibroblasts orchestrated complementary protective barriers: F2RL2+ fibroblasts mediated immune crosstalk (via CXCL14, C1S) and TGF-{beta} balance; COL6A6+ fibroblasts reinforced ECM structure (COL6A6/COL6A3) and inhibited Wnt signaling (SFRP2); MAB21L1+ fibroblasts suppressed SMAD signaling (through NR2F1 upregulation) and blocked cell migration. Metastatic niches exhibited significant depletion of protective fibroblast subpopulations (F2RL2+: reduced from 1.9% to 0.1%; COL6A6+: reduced from 3.1% to 1.3%); this depletion coincided with ECM dissolution and disrupted spatial coordination of immune markers (proximity-dependent HLA-DRA/CXCL9 expression). High expression of these DEGs correlated with improved recurrence-free survival (e.g., COL6A6: HR = 0.19, p = 0.002); genomic deletions of the DEGs were significantly more frequent in metastases versus primary tumors (e.g., MAB21L1 deep deletion: 9.13% vs. 0.57%). ConclusionAn unbiased transcriptomic approach revealed specialized fibroblast subpopulations as central gatekeepers against LNM via ECM, immune, and signaling barriers. Their erosion drives stromal collapse in metastasis. Epigenetic modulators (Valproic Acid, Vorinostat) were prioritized for therapeutic restoration of suppressive stroma.

Explore related subjects

geneticsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Rastad, H., Seifi Alan, M., Pandey, S.. 2025-08-22. Spatial and Single-Cell Dissection of Fibroblast Subpopulation Reprogramming Driving Stromal Collapse in Breast Cancer Lymph Node Metastasis. https://doi.org/10.1101/2025.08.18.670793

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Synergistic Variants in C-terminal Binding Protein 1 and Alkaline Phosphatase Lead to Mandibular Hypoplasia Through Impaired Wnt Signaling: An Oligogenic Model

Craniofacial malformations account for one third of all congenital anomalies. Genetic factors play a vital role, yet the list of causal genes and their mechanisms are far from complete. As part of a larger effort to sequence patients with micrognathia and Pierre-Robin sequence, we identified two candidate pathogenic missense variants in C-terminal binding protein 1 (CTBP1) along with a heterozygous early stop missense variant in alkaline phosphatase (ALPL) in a proband with mandibular hypoplasia. Ctbp1 has been shown to regulate Wnt/{beta}-Catenin signaling but it has not yet been implicated in craniofacial development. Here we generated two orthologous variants of Ctbp1 mimicking the patient variants using genome editing in mice and explored the micrognathia phenotype in combination with a previously reported Alpl null allele. Ctbp1Q148H/G238S; Alplnull/Wt complex heterozygous mutants have smaller mandibles recapitulating the human mandibular hypoplasia. We identified that a reduction in cell proliferation and active {beta}-Catenin levels could possibly account for the micrognathia phenotype in the Ctbp1; Alpl complex heterozygous. These data uncover a novel role for Ctbp1 in craniofacial development and highlight the complex genetic and molecular signaling in the pathogenesis of craniofacial malformations.

genetics↗
bioRxiv2026

Intergenerational instability of the C9orf72 hexanucleotide repeat

The C9orf72 hexanucleotide repeat expansion (HRE) is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It follows autosomal dominant inheritance in families, however, a high proportion of cases are sporadic, raising the possibility of parental premutation. We have demonstrated that intermediate-length alleles (IAs) with >18 repeats (allele frequency ~1%) belong to the same pool of haplotypes as the HRE, suggesting shared ancestry. Here, we tested whether alleles with >18 repeats expand in parental transmission. We used two repeat-primed PCR methods to analyze allele lengths in 539 genetically unselected parent-offspring pairs and in 152 pairs known to carry the SNP (rs139185008*C) that tags >18 repeat IAs and the HRE in Finland. We discovered intergenerational repeat length changes only in >20 repeat alleles. A significant (P = 0.0059) sex bias in 6-40 repeat alleles was noted using a logistic regression model. In this allele range, 12 out of 16 expansions were paternally inherited and 6 out of 7 contractions were maternally inherited. The expansion rate of 20-40 repeat alleles was 34 % in paternal and 11 % in maternal transmissions. In the 20-40 repeat range, most intergenerational expansions were 1-4 repeats in size (15/16), but one larger jump, a paternal expansion from 27 to 73 repeats, was observed. These results demonstrate that alleles with >20 repeats have an increased likelihood of instability, that a paternal expansion bias is observed in alleles with 20-40 repeats, and that expansion events are predominantly 1-4 repeats in size.

genetics↗
bioRxiv2026

Unravelling the role of IRX4 variants in non-syndromic and Down syndrome associated congenital heart disease

IRX4 is a TALE- homeodomain transcription factor which is essential for cardiac development. In murine models, Irx4 deficiency leads to impaired ventricular function and results in cardiomyopathy. To elucidate the role of IRX4 in human congenital heart disease (CHD), Sanger sequencing of the IRX4 gene was performed in 205 individuals with non-syndromic CHD, 24 Down syndrome (DS) cases with CHD, 27 DS cases without CHD, and 150 healthy control individuals. Two novel (p.Ser24Asn and p.Thr217Iso) and one reported variant (rs2232376) were identified in non-syndromic CHD. Concurrently, rs2232376 was also detected in DS with CHD. The first novel (p.Ser24Asn) and reported (rs2232376) variants lie in the N-terminal region while the second novel (Thr217Iso) variant lies within the TALE homeodomain. In silico structural modelling suggested that both the novel variants (p.Ser24Asn and Thr217Iso) induce conformational changes in the IRX4 protein, potentially altering its DNA-binding affinity. A significant reduced expression of IRX4 muteins was noted in Western blotting by both variants (p.Ser24Asn and Thr217Iso). Furthermore, luciferase reporter assays demonstrated decline in the activity of Nanog promoter and HEY2 enhancer in response to both the variants which was further corroborated by decrease mRNA expression in qRT-PCR. Additional downstream targets, including Nfyc, Nppa, and Bmp10, also exhibited anomalous expression due to both the variants (p.Ser24Asn and Thr217Iso). Altogether, the aberrant expression of muteins as well as downstream target genes along with compromised activities of promoters substantiate the pathogenic potential of the identified IRX4 variants and underscore the critical role of IRX4 in regulating multiple stages of cardiogenesis.

genetics↗