Involvement of matricellular proteins and immune receptors in the dynamic interaction between tumour and immune cells

Abstract

Background: Osteosarcoma (OS) is the most common primary malignant bone tumour in children and young adults, characterised by high metastatic potential and poor prognosis in advanced stages. Despite recent therapeutic advances, metastatic OS remains challenging to treat. Increasing evidence highlights the tumour microenvironment (TME), particularly immune cell components such as macrophages, as key modulators of tumour progression. Ovarian cancer (OC) is a significant cause of cancer-related deaths among women worldwide. It is a highly metastatic malignancy that shares similar molecular and immunological features with OS, including late-stage diagnosis, immune evasion, and extracellular matrix (ECM) remodelling. Matricellular proteins (MCPs) are non-structural extracellular matrix proteins that regulate cell– matrix interactions and signalling rather than providing structural support. They influence cell adhesion, migration, proliferation, and differentiation, and are often upregulated in cancer, promoting tumour growth, angiogenesis, and metastasis. Comparative analysis of OS and OC may reveal shared immune–metabolic pathways that contribute to tumour aggressiveness. This project investigates how MCPs and the interactions with immune cells influence tumour behaviour, survival, and metastatic progression within the TME. Using OC and OS as model systems, it integrates bioinformatic analyses, experimental validation, and functional assays to elucidate the molecular and immune mechanisms underpinning cancer progression and metastasis. Methods: Publicly available transcriptomic datasets for OS (GSE42352, GSE12865) and ovarian cancer (GSE18520, GSE54388, GSE14407) were analysed to identify differentially expressed genes (DEGs). The UniProt tool was used for functional annotation, with an emphasis on immune receptor (IR), cell adhesion (CAD), and ECM pathways. Survival analyses were performed using the KM Plotter. Candidate genes were validated in vitro using RT-qPCR and Western blotting in OS cell lines (MG63, SaOS-2, MNNG, 143B) and THP-1 monocytes differentiated into M0, M1, and M2 macrophages via PMA and cytokine stimulation. In addition, MNNG and 143B cells were treated with the FPR3 agonist WKYMVm and antagonist WRW4 (10 μM and 50 μM) for 24 h to directly assess the functional impact of FPR3 signalling on OS cell motility. Proliferation and scratch migration assays were performed using tumour- and macrophage-conditioned media. Secretomes were subjected to ultracentrifugation to assess the roles of soluble and vesicle-bound factors. Co-culture experiments were conducted to examine the bidirectional effects of macrophage-tumour signalling.

Results: Initial bioinformatics analyses revealed a significant enrichment of DEGs associated with ECM remodelling, immune signalling, and poor survival in both OS and OC datasets. In OC, D24 (Cluster of Differentiation 24), DCN (Decorin), and OGN (Osteoglycin/Mimecan) were associated with adverse prognosis. In OS, SPP1 (osteopontin) and FPR3 (formyl peptide receptor 3) were upregulated, whereas LEPR (leptin receptor) was consistently downregulated across patient tissues, and it was a common gene among OC and OS. Functional assays demonstrated that M2- conditioned media enhanced the migration and proliferation of OS cells. Ultracentrifugation experiments suggested that vesicle-bound factors mediate these effects on the target cells. The reciprocal communication between osteosarcoma cells and macrophages establishes a pro-metastatic feedback loop. In vitro experiments using RT-qPCR and Western blotting revealed the differential expression of key genes involved in tumour progression and immune regulation across various osteosarcoma cell lines. Genes such as FPR3, LEPR, and SPP1 displayed variable mRNA and protein levels, indicating distinct molecular profiles between the cell lines. These differences highlight the heterogeneity of osteosarcoma and suggest that specific gene expression patterns may be linked to variations in tumour behaviour, immune interactions, and metastatic potential. Treatment of MNNG and 143B cells with the FPR3 agonist WKYMVm significantly increased migration velocity at both 10 μM and 50 μM, while the antagonist WRW4 effectively reduced migration, particularly at 50 μM. M2 macrophages showed high expression of CD163, FPR3, LEPR, and SPP1, reflecting their tumour-promoting, anti-inflammatory phenotype.THP-1 cells treated with OS-conditioned media upregulated CD163, consistent with M2 polarisation, whereas OS cell lines treated with macrophage supernatants displayed increased SPP1 and LEPR, particularly under M2 stimulation.

Conclusion: This study identified SPP1, FPR3, and LEPR as key immune-metabolic mediators in OS, highlighting their roles in tumour–macrophage crosstalk and metastasis. The combination of in silico and in vitro findings supports a model in which the TME, particularly M2 macrophages, enhances OS progression via dynamic signalling pathways. These findings provide a foundation for targeting tumour–immune interactions in future OS therapies and suggest new directions for precision medicine strategies in metastatic cancers. Notably, LEPR was also consistently downregulated in OC, indicating a shared immunometabolism mechanism that may contribute to metastatic progression across tumour types.