Schwann cell derived pleiotrophin stimulates fibroblast proliferation and excessive collagen deposition in plexiform neurofibroma

Neurology Medical Laboratory Science Oral Sciences Medicine Cell Biology Biochemistry Oncology Life Sciences Immunology
Schwann cells pleiotrophin fibroblasts plexiform neurofibroma collagen deposition signaling pathway

This study explores the interactions between Schwann cells and fibroblasts in neurofibromatosis type 1 (NF1) related plexiform neurofibroma (PNF). The background of the research is based on the high incidence of NF1, which affects about 1 in 3000 newborns worldwide and is associated with a series of unique clinical manifestations. PNF is a common peripheral nerve sheath tumor in NF1 patients, affecting approximately 50% of patients and severely impacting their quality of life. Despite decades of research, PNF remains incurable. Current treatments primarily target Schwann cells associated with NF1 and have achieved some clinical efficacy. However, many treatments, such as the selective MEK inhibitor Selumetinib, are only effective in some patients, and long-term use can lead to drug resistance. This has prompted researchers to focus on the role of fibroblasts in PNF and their abnormal proliferation and collagen deposition in tumors.

Research Origins

This study was conducted by the following authors and institutions: Zhu Weitian, Zhong Du, Guo Bai, Gong Qiyu, You Yuanhe, Xu Guisong, Liu Jialiang, Xiao Meng, Wang Yanan, and He Yue. The research units include the Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People’s Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai Stomatology Hospital, and other institutions. The study was published in Volume 31 of Cancer Gene Therapy in 2024.

Research Process

Research Methods

  1. Collection and Processing of Tumor Specimens: Fresh specimens were obtained from three NF1-related PNF patients, and cell dispersion and single-cell suspension were performed.

  2. Single-cell RNA Sequencing: Single-cell RNA sequencing was conducted using the 10x Genomics platform and data alignment and processing were performed using Cell Ranger software. Quality control steps included detecting the number of features per cell and mitochondrial gene proportion.

  3. Clustering and Annotation: Principal component analysis and unsupervised clustering algorithms were used with the Seurat package to divide the sample cells into approximately 10 primary clusters, annotating cell types. Clustering results were validated using known cell type-specific marker genes.

  4. Functional Comparison and Trajectory Analysis: Unsupervised clustering of Schwann cells and fibroblasts was conducted, and differential expression genes were obtained using the MAST package for GO enrichment analysis. The Monocle package was used for pseudo-time trajectory analysis, and data from the Seurat package were employed for typing and prediction.

  5. Annotation of Cell Interactions: Nichenet and CellChat tools were used to analyze ligand-receptor interactions between Schwann cells and fibroblasts.

Experimental Methods

  1. Immunohistochemical Staining: Antibodies targeting human collagen type I and VI, among others, were used for staining, employing biotinylated secondary antibodies and a DAB kit for detection.

  2. Cell Culture and Isolation: Schwann cell lines were cultured, and NF-related fibroblasts (NFAFs) were isolated and cultured using the explant growth method.

  3. Real-time Quantitative PCR (qRT-PCR) and Western Blot Analysis: Total RNA was extracted, and protein expression levels were detected using Western blotting.

  4. Enzyme-linked Immunosorbent Assay (ELISA) and Cell Proliferation Assay: Supernatants from culture media were collected for ELISA, and CCK-8 and EdU kits were used to detect cell proliferation.

  5. Cell Cycle Detection and Collagen Synthesis Detection: Flow cytometry was used to detect the cell cycle, and a hydroxyproline detection kit was used to assess collagen synthesis.

Data Processing and Statistical Analysis

All statistical analyses were performed using SPSS and GraphPad Prism software. Student’s t-test was used to determine significant differences between groups, and data were presented as mean ± standard deviation.

Research Results

Single-cell RNA sequencing of specimens from three NF1 patients revealed various cell types in PNF, with fibroblasts being the major component and a smaller proportion of Schwann cells. Further analysis showed that Schwann cells could be divided into five subgroups with significant differences in gene expression and function. Among these, one subgroup of Schwann cells was associated with the extracellular matrix or collagen deposition.

Ptn Promotes Fibroblast Proliferation and Collagen Synthesis

The study demonstrated that Ptn secreted by Schwann cells promotes fibroblast proliferation and collagen synthesis in NFAFs through a specific PtN/Ncl ligand-receptor pair, including collagen types I, III, and VI. By disrupting Ptn or Ncl, fibroblast collagen synthesis could be significantly reversed to normal levels, indicating the critical role of this pathway in PNF and its potential as a future therapeutic target.

Conclusion

This study reveals the molecular mechanism by which multifunctional protein secreted by Schwann cells promotes fibroblast proliferation and collagen deposition in PNF through the Ptn/Ncl/Pras40 axis. This pathway may serve as a novel therapeutic target, offering effective treatment options for PNF patients in the future.

Research Highlights

  1. New Findings in Ligand-Receptor Interactions: The core role of the Ptn/NCL axis in the interaction between Schwann cells and NFAFs was discovered and verified.
  2. Fibroblast Subgroup Typing and Functional Status: Gene expression and functional differences between fibroblast and Schwann cell subgroups in PNF were revealed.
  3. Key Role of Multifunctional Protein PTN: PTN can independently promote fibroblast proliferation and collagen synthesis, as well as synergize with TGF-β1.

Clinical Significance and Research Value

The findings of this study provide new insights into the pathological mechanisms of PNF, revealing the interactions between Schwann cells and NFAFs and their regulatory effects on collagen deposition. Especially, the Ptn/NCL/Pras40 pathway as a key regulatory mechanism in PNF occurrence and development offers an important theoretical basis for developing therapeutic strategies targeting this pathway in the future.

This research not only enriches the molecular mechanism knowledge of PNF but also provides new potential targets for treating this disease, with significant clinical application value.