This study utilized a two-sample MR analysis to explore the potential causal relationship between 91 circulating inflammatory proteins and the risk of OS. According to literature review, this study is the first comprehensive and in-depth investigation into the causal relationship between circulating inflammatory proteins and OS based on publicly available GWAS data. The results indicated potential associations between increased risk of OS and GCST90274776 (Macrophage colony-stimulating factor 1 levels, CSF-1), CST90274822 (Monocyte chemoattractant protein 2 levels, MCP-1/CCL2), and GCST90274847 (Urokinase-type plasminogen activator levels, uPA). Conversely, GCST90274844 (TNF-related activation-induced cytokine levels, TRANCE) was found to be potentially associated with a decreased risk of OS. Additionally, in the sensitivity analyses, neither MR-Egger intercept nor MRPRESSO tests revealed evidence of horizontal pleiotropy. No significant heterogeneity was observed in the Cochran’s Q test results (P > 0.05). Furthermore, the leave-one-out test did not identify any SNP that significantly influenced the results, thereby enhancing the reliability of the study data. These findings suggest that circulating inflammatory proteins may be important factors influencing OS and play a crucial role in its pathogenesis.

CSF-1, a highly pleiotropic cytokine, exhibits remarkably elevated expression levels in OS compared to normal osteoblasts, strongly hinting at its crucial role in initiating and driving the progression of this aggressive malignancy [12]. Further investigation has unveiled a profound connection between CSF-1 and tumor angiogenesis in OS [13], suggesting that CSF-1 facilitates the development of new blood vessels within tumors via specific molecular mechanisms, thereby providing vital oxygen and nutrients to OS cells and accelerating their growth and dissemination. Notably, studies have confirmed that CSF-1 promotes angiogenesis by activating the AIF-1/Rac-1 signaling pathway [14, 15]. AIF-1, a protein closely linked to cytoskeletal rearrangements and inflammation, works in tandem with Rac-1, a small GTPase regulating cytoskeletal dynamics and cell migration [16]. This synergistic action promotes endothelial cell proliferation, migration, and lumen formation—all essential stages in the angiogenesis process. Experimental evidence underscores the significance of CSF-1 in OS by demonstrating that inhibiting its expression or activity through genetic knockout, antibody neutralization, or small molecule inhibitors drastically reduces OS cell proliferation, migration, and angiogenesis capabilities [17]. These findings not only highlight CSF-1 as a promising biomarker for early diagnosis and prognosis assessment of OS but also position it as a potential therapeutic target. By targeting CSF-1 and its downstream signaling pathways, researchers have been able to uncover a novel perspective on disrupting the intricate processes that sustain osteosarcoma growth and spread. This offers crucial insights and potential avenues for developing more effective treatment strategies for this aggressive cancer, ultimately paving the way for better patient outcomes.

MCP-1/CCL2, a highly pleiotropic immunoregulatory factor, plays a crucial role in the progression of OS [18]. Its unique biological functions not only directly involve in the proliferation, invasion, and metastasis of OS cells but also profoundly influence the construction and remodeling of the tumor microenvironment, thereby indirectly accelerating cancer deterioration [19]. Recent research has delved into the intricate interplay between OS cells and indispensable cellular components within the tumor microenvironment, such as fibroblasts and endothelial cells, forming a vital secretory network for CCL2. This discovery underscores the regulatory role of complex intercellular interactions within the tumor microenvironment on CCL2 production and release, further elucidating the multidimensional mechanisms underlying OS tumorigenesis and development [20]. As a core chemokine, CCL2 demonstrates potent cell recruitment capabilities, guiding macrophages precisely into the tumor microenvironment, initiating a series of intricate cascade reactions that profoundly impact OS progression. Macrophage enrichment within the tumor microenvironment augments immune infiltration and provides essential support for rapid OS cell proliferation and survival through the release of growth factors, inflammatory mediators, and angiogenic factors.

uPA plays a pivotal role in OS biology by activating plasminogen into plasmin, leading to the degradation of the extracellular matrix (ECM), which serves as a natural barrier during OS cell invasion and metastasis [23, 24]. uPA activation further promotes OS cell infiltration and proliferation, enhancing their malignant behavior and making tumors more aggressive [25, 26]. In the prognostic assessment of OS, the detection of soluble uPA levels in plasma has demonstrated its significance [27]. Given uPA’s central role in OS invasion and metastasis mechanisms, it can be reasonably speculated that uPA levels may emerge as an important indicator for predicting OS patient prognosis. Furthermore, several uPA inhibitors have shown significant inhibitory effects on OS growth in both in vitro and in vivo experiments by blocking uPA activity, reducing ECM degradation and angiogenesis, and effectively suppressing OS cell invasion and metastasis [28, 29].

TRANCE, also known as Receptor Activator of NF-κB Ligand (RANKL) or TNF superfamily member 11 (TNFSF11), is a central member of the tumor necrosis factor family, exerting profound effects on bone metabolism and the immune system [30]. RANKL regulates the differentiation of monocytes/macrophages into osteoclasts and accelerates osteoclast precursor maturation, crucial for bone renewal and remodeling [31]. It also extensively regulates osteoblast activity, influencing protease expression and humoral immunity balance, playing an indispensable role in bone modeling, maintenance, and remodeling [32]. The dynamic balance between RANKL and Osteoprotegerin (OPG) maintains bone metabolic homeostasis [33]. This discovery not only reveals a novel perspective on bone metabolic regulation but also provides potential drug targets for treating OS. A significant negative correlation between TNFSF11 (RANKL) and OS risk uncovered in this study offers new insights into OS pathogenesis and emphasizes the critical role of the RANKL/RANK/OPG system in maintaining bone health and preventing malignant bone tumors.

In the TARGET osteosarcoma cohort, our analysis revealed no significant association between the expression levels of inflammatory factors CSF-1, CCL2, uPA, and TRANCE and patient survival outcomes, consistent across both survival analysis and ROC evaluation. However, our MR study provided robust evidence for a significant causal relationship between these genes and osteosarcoma susceptibility. This apparent discrepancy between the observational prognostic findings and causal MR results is primarily due to inherent limitations of observational studies, where unmeasured confounding factors—such as treatment heterogeneity, comorbidities, and tumor microenvironment complexity—likely obscured true associations in the Target cohort. In contrast, MR’s use of genetic instrumental variables effectively mitigates such confounding to reveal genuine causal effects. Furthermore, the fundamental distinction between the research questions addressed contributes to the divergence: MR assesses lifetime genetic risk (etiology), while survival analysis examines the prognostic impact of baseline expression in already-diagnosed patients. Thus, while MR solidly implicates these factors as key causal drivers in osteosarcoma pathogenesis and highlights their therapeutic potential, their utility as standalone prognostic biomarkers within the Target cohort appears limited. Future investigations should explore dynamic expression profiling and integrated modeling in larger cohorts to better evaluate their prognostic and predictive roles.

Collectively, CSF-1, CCL2, uPA, and TRANCE (RANKL) provide important clues for developing novel therapeutic strategies for OS. These proteins serve not only as biomarkers for early diagnosis and prognosis assessment but also as potential therapeutic targets. The preservation of their reference numbers ensures the accuracy and traceability of the scientific findings presented.

The study is the first to assess the causal relationship between 91 circulating inflammatory proteins and OS using MR techniques, offering several notable advantages. Firstly, it employs two-sample MR techniques to evaluate the relationship between these 91 circulating inflammatory proteins and OS from a genetic perspective. These techniques help eliminate the influence of confounding factors and enhance the robustness of the research findings through sensitive analysis. Secondly, the exposure and outcome data are sourced from distinct datasets, minimizing potential biases arising from sample overlap. Lastly, the comprehensive examination of 91 diverse circulating inflammatory proteins enables a thorough detection of the broader inflammatory network in OS. However, this study acknowledges certain limitations in accordance with the reviewer’s comments: Firstly, the GWAS data utilized are primarily from European populations, which may lack standardization across cohorts. Thus, the consistency of the final research findings across other populations remains to be investigated. Secondly, the study relies on proxy SNPs for certain proteins, which may reduce the accuracy of causal estimates. Consequently, further experimental validation of the identified proteins’ roles in OS pathogenesis is necessary.To address these limitations, follow-up studies are essential. These studies should involve diverse populations or experimental approaches to validate the findings and delve deeper into the impact of specific circulating inflammatory proteins, such as CSF-1, CCL2, uPA, and TRANCE, on OS. Additionally, conducting further investigations to evaluate the potential of these factors as therapeutic targets for OS is also crucial.