Multi-omics analysis reveals inhibition of osteosarcoma progression by sporoderm-removed Ganoderma lucidum spores via targeting glycerophospholipid and fatty acid Metabolism

Multi-omics analysis reveals inhibition of osteosarcoma progression by sporoderm-removed Ganoderma lucidum spores via targeting glycerophospholipid and fatty acid Metabolism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Multi-omics analysis reveals inhibition of osteosarcoma progression by sporoderm-removed Ganoderma lucidum spores via targeting glycerophospholipid and fatty acid Metabolism Haitao Pan, Congshu Li, Mengyao Wang, Kang Ye, Xiaohui Fan, Yongping Fu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6046286/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 6 You are reading this latest preprint version Abstract Osteosarcoma (OS) is a severe malignancy affecting children and adolescents, with limited effective treatments leading to poor outcomes. This study explored the potential of sporoderm-removed Ganoderma lucidum spores (RGLS) as a novel therapeutic agent against OS and elucidated its mechanism of action. Our in vivo and in vitro experiments showed that RGLS significantly inhibited S-180 OS cell proliferation, colony formation, and migration, while simultaneously inducing apoptosis. Multi-omics analysis revealed that RGLS disrupted glycerophospholipid metabolism by upregulating lysophosphatidylcholine (LysoPC) levels through phospholipase A2 (PLA2)-mediated pathways. Co-culture assays further demonstrated that RGLS promoted the endocytosis of macrophage-derived Pla2g7 protein into S-180 cells, enhancing its anti-cancer effects. Additionally, RGLS modulated the cellular fatty acid profile and suppressed the β-oxidation of long-chain fatty acids, leading to energy depletion in OS cells. These findings provide a novel view of the multi-targeted mechanisms of RGLS, positioning it as a promising candidate for OS therapy. Biological sciences/Cancer Biological sciences/Computational biology and bioinformatics Biological sciences/Drug discovery Health sciences/Medical research Sporoderm-removed Ganoderma lucidum spores Osteosarcoma Glycerophospholipid metabolism fatty acid metabolism multi-omics analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Osteosarcoma (OS) is a prevalent primary malignant bone tumor of mesenchymal origin, predominantly affecting children and adolescents. Epidemiological studies have revealed that OS accounts for 15% of all solid extracranial cancers in adolescents aged 15–19 years 1 . Current therapeutic approaches for OS primarily revolve around surgical intervention followed by chemotherapy. However, the aggressive nature of OS, coupled with chemotherapy resistance, recurrence, and adverse side effects leading to reduced patient compliance, present significant challenges in achieving satisfactory treatment outcomes. Consequently, there is an urgent need to explore effective and safe agents for the prevention and treatment of OS. Metabolic reprogramming is a defining hallmark of cancer cells 2 , and in osteosarcoma (OS), fatty acids and glycerophospholipids emerge as critical metabolic drivers of progression. Fatty acids, central energy sources for tumor cells, fuel OS growth, invasion, and immunosuppressive microenvironment formation 3 , 4 . Recent studies show that inhibiting fatty acid β-oxidation—via pharmacological or genetic approaches—disrupts mitochondrial energy production, thereby reducing OS cell viability, migration, and invasion 5 , 6 . Beyond de novo fatty acid synthesis mediated by FASN, glycerophospholipids, the most abundant membrane phospholipids, are hydrolyzed by phospholipases (PLs) into bioactive metabolites such as lysophospholipids and fatty acids 7 , 8 . These metabolites regulate membrane dynamics, signaling, and proliferation, with aberrant glycerophospholipid metabolism linked to aggressive OS phenotypes. For instance, elevated phospholipase A2 group 16 (PLA2G16) correlates with lung metastasis and poor prognosis 9 , while phospholipase D (PLD) antagonizes tumor-suppressive miR-638 to drive OS proliferation 10 . Notably, disrupting phosphatidylcholine (PC) biosynthesis—a major glycerophospholipid subclass—suppresses OS cell proliferation and mineralization capacity 11 . Clinically, first-line OS chemotherapeutics like cisplatin and doxorubicin act partly by inducing membrane degradation or blocking lipid synthesis 12 . Collectively, targeting these interlinked metabolic pathways—notably glycerophospholipid metabolism and fatty acid β-oxidation—offers a promising therapeutic strategy to disrupt energy production and membrane synthesis critical for OS progression. Ganoderma lucidum , a medicinal mushroom with a historical usage spanning over 6800 years 13 . has been found to influence lipid metabolism. For instance, Ganoderma lucidum aqueous extract regulates the biological metabolic process of membrane components in tetrahymena thermophila 14 and Ganoderma lucidum polysaccharide-cadmium chelate regulates glycerophospholipid metabolism in liver 15 . Additionally, sporoderm-broken GLS has demonstrated anti-OS effects by inducing apoptosis and inhibiting the STAT3/PD-L1 signaling pathway 16 , 17 . However, there have been no reports on the specific role of Ganoderma lucidum or its extracts in regulating the metabolic processes of membrane components and phosphoglyceride metabolism to exert its anti-OS effects. The present study aimed to analyze the effects of sporoderm-removed GLS (RGLS), a highly purified GLS extract 18 , 19 on OS development both in vitro and in vivo , and to elucidate the potential mechanism of RGLS against OS based on the integrative analysis of transcriptomics, proteomics and metabolomics 20 , which has not been previously reported. Our data indicate that RGLS effectively suppresses OS development by inhibiting proliferation, and inducing cell apoptosis. Additionally, we observed that RGLS promotes PCs metabolism by enhancing endocytosis of macrophage-secreted Pla2g7 by S-180 cells. Furthermore, we found that RGLS regulates fatty acid pool composition and inhibits the conversion of long-chain acylcarnitine to long-chain acyl-CoA in mitochondria, and ultimately leading to fatty acid β-oxidation disorders and intracellular energy insufficiency. Results RGLS inhibits S-180 cells growth in vivo. In the present study, we administered RGLS prophylactically to BALB/c nude mice and then injected them subcutaneously with S-180 cells to evaluate RGLS's tumor-inhibiting effects (Fig. 1 A). The results showed that RGLS significantly reduced the weight of S-180 xenograft tumors compared with the model group (Fig. 1 B), and the tumor inhibition rate was high at 35.8% (Fig. 1 C). In addition, we analyzed the effect of therapeutic administration of RGLS on the growth of S-180 xenograft tumors. As shown in Fig. S1 A, in the absence of prophylactic administration, only 7 days of therapeutic administration also reduced the weight of S-180 xenograft tumors (Fig. S1 B), but the tumor inhibition rate was lower at 25.8% (Fig. S1 C). Moreover, the body weight of mice was not affected by both administration methods of RGLS (Fig. 1 D and S1D). These results indicated that RGLS effectively delays the growth of S-180 xenograft tumors. Apoptosis is the most classical mode of cell death in tumors. Next, we observed the effects of RGLS on the pathological changes in S-180 xenograft tumors using H&E staining. As shown in Fig. 1 E, RGLS greatly induced apoptosis in S-180 cells within xenograft tumors, which was mainly manifested as the loss of intercellular connections, increased cytoplasmic density, reduced cell volume, and nuclear contraction. In addition to apoptosis, inhibiting cell proliferation is also an effective strategy for tumor treatment. Furthermore, we examined the expression of Ki-67, a nuclear cell proliferation marker, in xenograft tumors using IHC staining. The results are shown in Fig. 1 F, the expression of Ki-67 was significantly decreased in the RGLS treatment groups compared with the model group. Taken together, RGLS delays S-180 xenograft tumor growth by inhibiting cell proliferation and inducing cell apoptosis. RGLS alleviates tumor-induced immune deficiency in mice. Immune deficiency is an important factor in tumorigenesis, and in turn, the development of tumors can further exacerbate the immune deficiency. Next, we analyzed the effects of RGLS on internal morphological structure of the thymus using H&E staining. As shown in Fig. S2 A, the boundary between cortex and medulla of thymus in control group was distinct and orderly arranged. However, compared to the control group, the cortex and medulla of the thymus in the model group underwent spatial rearrangement, and RGLS exhibited a mitigating effect on this phenomenon. The thymus is the site of T lymphocyte development, and damage to the thymus inevitably leads to abnormal development of T cells. Further, we analyzed the effects of RGLS on proportions of specific T cell subtypes in peripheral blood, including CD3 + , CD3 + CD4 + , and CD3 + CD8 + T cells, relative to total CD3 + T cells. Compared with the control group, the proportion of CD3 + T cells in the model group was significantly reduced, while RGLS increased the proportion of CD3 + T cells compared with the model group. However, the proportions of CD3 + CD4 + and CD3 + CD8 + T cells did not change significantly in both the model group and the RGLS group (Fig. S2 B and C). These results suggest that RGLS ameliorates tumor-induced immune decline, which may be related to the regulation of non-traditional T cell development, such as double negative (DN, CD4 − CD8 − ) iNKT cells 21 . RGLS inhibits S-180 cells growth in vitro . To further analyze the anti-OS effects of RGLS, we examined the impact of RGLS on S-180 cells growth in vitro . The CCK-8 assay showed that RGLS decreased the viability of S-180 cells in a dose-dependent manner (Fig. 2 A), and the half-maximal inhibitory concentration ( IC50 ) was 2.51 mg/ml. We then examined whether RGLS induced apoptosis in S-180 cells. As shown in Fig. 2 B and C, RGLS treatment significantly increased the ratio of apoptotic cells, and the late apoptotic (Annexin V − /PI + ) cells were predominant. Specifically, our results show that RGLS treatment significantly reduces the expression levels of pro-Casp3 and Bcl-xL in a concentration-dependent manner, but does not significantly affect the Bax/Bcl-xL ratio (Fig. 2 D). Collectively, these findings suggest that RGLS effectively inhibits S-180 cell proliferation by inducing apoptosis, potentially through a mechanism independent of the mitochondrial pathway. RGLS regulates glycerophospholipid metabolism in S-180 cells . Reprogramming of lipid metabolism is an emerging hallmark that distinguishes tumor cells from normal cells 2 . To further elucidate the molecular mechanism of RGLS in inhibiting S-180 cell growth, we performed a metabonomics analysis to assess the effects of RGLS on S-180 cell metabolism. Principal component analysis (PCA) revealed that RGLS significantly altered the metabolite composition in S-180 cells (Fig. 3 A). Compared with the control group, RGLS upregulated 49 metabolites and downregulated 47 metabolites (Fig. 3 B). Following KEGG metabolic pathway enrichment analysis of the differential metabolites (Fig. 3 C), nine metabolic pathways, namely Choline metabolism in cancer (ko05231), Efferocytosis (ko040148), Glycerophospholipid metabolism (ko00564), Thermogenesis (ko04714), Sulfur relay system (ko04122), Nucleotide metabolism (ko01232), Zeatin biosynthesis (ko00908), Cysteine and methionine metabolism (ko00270), and ABC transporters (ko02010) were enriched ( P ˂0.01). Among them, the number of targeted differential metabolites related to glycerophospholipid metabolic pathway is the largest. Focusing on glycerophospholipid metabolic, a heatmap analysis of the total 42 differential metabolites (Fig. 3 D) showed the degree of variation with various colors. Specifically, RGLS treatment significantly increased the levels of lysophosphatidylcholine (LysoPC, including LysoPC 16:1, LysoPC 18:2, LysoPC 18:3, and LysoPC 20:2), a metabolite of phosphatidylcholine (PC), which has been shown with anti-tumor activity 22 , 23 . In contrast, the RGLS treatment reduced the levels of phosphatidylinositol (PI, including PI 18:0/14:1, PI 16:1/18:2, PI 16:1/18:1, PI 18:2/20:4, PI 18:2/20:3, and PI 18:0/20:4), an important component for cell and organelle membrane biosynthesis and function 24 , and its metabolite LysoPI (including LysoPI 16:0, LysoPI 17:0, LysoPI 18:3, and LysoPI 20:1), which has been shown with pro-tumor activity 25 . Taken together, these results indicate that RGLS exerts bidirectional regulation on glycerolphospholipid metabolism to promote anti-tumor activity. RGLS regulates glycerophospholipid metabolism by enhancing the interaction between S-180 cells and macrophages . Phospholipases (PLs) are the rate-limiting enzymes in glycerolphospholipid metabolism, including PLA1, PLA2, PLC, and PLD. Among them, glycerolphospholipids are hydrolyzed by PLA1 or PLA2 and then lose a molecule of fatty acid to produce lyso-phospholipids (LP) (Fig. 4 A). To further elucidate the regulatory effect of RGLS on PC metabolism, transcriptomic and proteomic analyses were conducted to assess gene and protein expression changes in xenograft tumors. As shown in Fig. 4 B, PCA revealed that RGLS significantly altered the gene and protein expression in xenograft tumors. Compared with the control group, RGLS upregulated 1494 genes and 431 proteins, and downregulated 414 genes and 43 proteins. Initially, we verified the role of RGLS in inducing S-180 cell apoptosis. As shown in Fig. S3 A, RGLS treatment significantly upregulated the expression levels of apoptosis-related proteins, including the Casp family (Casp1, Casp3, Casp7, Casp8), Bcl-2 family (Bax, Bid), and Aif family (Aif1, Aifm1, Aifm2, Aifm3), suggesting that RGLS induced S-180 cell apoptosis in vivo . Next, gene ontology (GO) enrichment analysis showed that the upregulated genes and proteins are mainly enriched in cellular compartment terms related to membrane, such as lysosomal membrane (GO:0098574), microvillus membrane (GO:0031528), endosome membrane (GO:0010008), actin cytoskeleton (GO:0015629), mitochondrial inner membrane (GO0005743), and mitochondrial membrane (GO:0031966) (Fig. 4 C and D). This suggests that lysosome may be involved in the process of RGLS regulating PC metabolism 26 . Indeed, we found that RGLS significantly increased autophagy levels in S-180 cells in vitro , including increasing the number of autophagosomes (Fig. S3 B), upregulating LC3B protein levels and decreasing p62 protein levels (Fig. S3 C), and enhancing lysosomal acidity (Fig. S3 D). In addition, RGLS upregulated the expression levels of lysosomal functional proteins (Lamp1 and Lamp2) (Fig. S3 E) and lysosomal cathepsin (Fig. S3 F). Furthermore, we analyzed the effect of RGLS on PLA expression. As shown in Fig. 4 E, we found that RGLS upregulated the expression levels of multiple LPA members in S-180 xenograft tumors, especially Plaa (a PLA2 activating protein) and Pla2g7 (the seventh member of the PLA2 family), a secreted PL that metabolizes PC to LysoPC under the action of lysophosphatidylcholine acyltransferase (Lpcat) in the lysosome 27 . However, Pla2g7 is usually derived from macrophages 28 . Indeed, transcriptome data showed that S-180 xenograft tumor did not express Pla2g7 and RGLS did not affect Pla2g7 mRNA expression levels (Fig. S3 G). These results suggest that RGLS may promote PC metabolism by enhancing the interaction between S-180 cells and macrophages. Endocytosis is an important pathway for cellular uptake of biological macromolecules. Combined transcriptome and proteome analyses revealed that 46 regulatory factors, such as Smap1, Unc119 and Ubaln2, were upregulated simultaneously at both gene and protein levels (Fig. 4 F). Further GO enrichment analysis revealed a strong association between co-upregulated genes/proteins and regulation of clathrin-dependent endocytosis (GO:2000369) (Fig. 4 G). We also identified a series of ABC superfamily members whose expression levels were upregulated by RGLS (Fig. 4 H). Thus, we hypothesized that RGLS might enhance the uptake of Pla2g7 secreted by macrophages in S-180 cells. To test this hypothesis, we performed co-culture of S-180 cells with Raw264.7 cells (Fig. 4 I). Compared to S-180 cells cultured alone, the Pla2g7 protein levels (Fig. 4 J), but not mRNA levels (Fig. S3 G), were significantly higher in S-180 cells co-cultured with Raw264.7 cells. However, it was disappointing that RGLS (2 mg/ml) treatment did not further increase significantly the levels of Pla2g7 protein and mRNA (Fig. 4 J and Fig. S3 G), in co-cultured S-180 cells. This phenomenon may be explained by the Pla2g7 protein in S-180 cells in the form of enzyme-substrate binding or the Pla2g7 protein released by Raw264.7 cells reaching its limit. In addition, at non-toxicity concentrations (2 mg/ml) (Fig. S3 H), RGLS decreased significantly Pla2g7 protein levels in Raw264.7 cells (Fig. 4 K). Interestingly, RGLS did not reduce pla2g7 mRNA expression levels in Raw264.7 cells (Fig. S3 I). These results showed that RGLS promoted the release of Pla2g7 protein in Raw264.7 cells and the phagocytosis of Pla2g7 protein in S-180 cells, thus enhancing PC metabolism. RGLS regulates acylcarnitine and fatty acid metabolism . Fatty acids are intermediate metabolites of glycerolphospholipid, releasing ATP through β-oxidation. Surprisingly, treatment with RGLS significantly reduced intracellular ATP content in S-180 cells (Fig. 5 A). To investigate whether β-oxidation is compromised by RGLS, we analyzed its role in regulating fatty acid metabolism. It is well established that short- and medium-chain fatty acids can enter the mitochondria directly through diffusion, whereas long-chain fatty acids are converted to fatty acyl-CoA by long-chain acyl-CoA synthetase (Acsl) before entering the mitochondria. As shown in Fig. 5 B, RGLS treatment increased the expression levels of Acsl family members, particularly Acsl3. Subsequently, we analyzed the effect of RGLS on carnitine-acyl transferase 1 (Cat1), which facilitates the binding of acyl-CoA to carnitine to form acylcarnitine, thereby enabling entry into the mitochondria. As shown in Fig. 5 C, RGLS treatment significantly increased the protein expression level of carnitine-palmitoyl transferase 1a (Cpt1a), a member of the Cat1, suggesting that RGLS may promote the entry of long-chain fatty acids into the mitochondria. In the mitochondria, long-chain acylcarnitine reconverted to long-chain acyl-CoA under the action of Cat2 and L-carnitine is released. As shown in Fig. 5 D, RGLS treatment significantly decreased the protein expression level of Cat2, and reduced the L-carnitine content in S-180 cells (Fig. 5 E), suggesting that RGLS may inhibit long-chain acylcarnitine metabolism. Next, we analyzed the changes of acylcarnitine levels in S-180 cells. As illustrated in Fig. 5 F and 5 G, RGLS significantly increased the levels of long-chain acylcarnitine (C 16:1 and C16:0) and slightly decreased the levels of short- and medium-chain acylcarnitine (C 4:0, C 5:0, and C 9:1). Further, we analyzed the effect of RGLS on fatty acid composition in S-180 cells. As shown in 5H, RGLS treatment significantly reduced the levels of short- and medium-chain fatty acids while increasing the levels of various long-chain fatty acids. These results suggest that RGLS compromises energy supply by increasing the proportion of long-chain fatty acids and inhibiting long-chain acylcarnitine metabolism, thereby inhibiting fatty acid β-oxidation and promoting S-180 cell death. Discussion Among the myriad of TCM remedies, Ganoderma lucidum renowned for its health-promoting and life-extending properties, has shown considerable promise in managing cardiovascular diseases and brain injury 29 , 30 . While Ganoderma lucidum has been recognized for its significant therapeutic benefits, its potential in osteosarcoma prevention and treatment has remained relatively underexplored. Limited studies have been conducted to investigate the anti-OS effects of Ganoderma lucidum or its active component 16 , 17 . In our research, we evaluated the anti-OS effects of RGLS and its mechanisms (as summarized in Fig. 6 ). Encouragingly, our results showed that RGLS has a good inhibitory effect on tumor growth. This observation supports the notion that RGLS possesses a strong potential for tumor prevention and treatment. The efficacy of RGLS might be attributed to its ability to target specific molecular pathways or cellular processes involved in tumor initiation and progression. However, to fully comprehend the clinical applicability of RGLS in tumor prevention, further research is essential. Robust pre-clinical and clinical studies are necessary to validate the efficacy, safety, and dosage regimen of RGLS in preventing various types of tumors. Additionally, exploring the mechanistic basis of RGLS's tumor-preventive effects would provide critical insights into its mode of action and potential targets. Phospholipids are the material basis of cellular and organismal life activities, and they are not only the main components of cell and organelle membranes, but also involved in the regulation of countless signal pathways 31 . In tumor cells, the content of phospholipids, especially PC, PI and sphingomyelin, is sharply increased 32 , which suggests that phospholipid metabolism as a target may be an effective strategy for tumor treatment 33 . In our study, we found that RGLS significantly increased the content of LysoPCs. As an intermediate product of PC metabolism, LysoPCs are not only closely involved in atherosclerosis and diabetes 31 , 32 but also inhibits the growth of a variety of tumor cells, including bladder cancer, lung cancer, and melanoma 22 , 23 , 34 and are used to predict the efficacy and favorable prognosis of lenvatinib combined with programmed death-1 (PD-1) inhibitor in the clinical treatment of unresectable hepatocellular carcinoma (uHCC) 35 . In addition, LysoPC has been shown to continuously increase intracellular calcium ion concentration by activating calcium-permeable channels and contribute to of cell cytoskeletal 36 . Correspondingly, our study also found that RGLS regulates the biological processes of the cytoskeleton (GO: 0005856) (Fig. 4 C), which may be a potential mechanism by which RGLS regulates PC metabolism to exert anti-tumor effects. Lysosomes are essential for maintaining cell homeostasis and are involved in a variety of biological processes, such as immunity and intracellular pH regulation 37 . Recent studies have shown that lysosomes also participate in glycerophospholopid metabolism 27 . PLA2 is a ubiquitous enzyme uniquely characterized by a sub-cellular localization to the lysosome and late endosome 38 , which hydrolyzes PC to produce LysoPC and causes lysosome destabilization 39 . In our research, RGLS significantly increased Pla2g7 protein levels but not mRNA levels in S-180 cells, suggested that Pla2g7 protein in S-180 cells might be exogenous. Indeed, Pla2g7 is highly expressed in immune cells, such as B cells and macrophages 28 . Our study also found that RGLS increases the expression of CD47 (Fig. S3 J), an immune checkpoint that interacts with SIRPα expressed by macrophages 40 . These phenomena indicate that RGLS enhances the interaction between S-180 cells and macrophages. Our previous studies have shown that polysaccharide derived from RGLS inhibits the growth of hepatocellular carcinoma cells by promoting macrophages to M1 type polarization and inflammatory cytokines secretion such as TNF-α, IL-1β, IL-6 41 . However, studies have shown that in the course of COVID-19 infection, Pla2g7 is primarily produced by pro-inflammatory (M1 type) macrophages 42 , and macrophages with high expression of PLA2 showed a profound immunosuppressive phenotype 43 . The above background suggests that RGLS regulation of Pla2g7 release may be a novel mechanism to promote macrophage-mediated immunity, and the regulation of endocytosis of tumor cells on Pla2g7 may be related to lysosomal-dependent cell death induced by RGLS 44 , these speculations are worthy of further study. Fatty acids are important energy sources for tumor cells, and targeting fatty acid metabolism is considered an effective strategy for tumor (including cancer) management 2 . Fatty acid utilization is a delicately regulated process. Short- and medium-chain fatty acids can enter the mitochondria directly, but long-chain fatty acids require conversion to long-chain acyl-CoA and transport by L-carnitine 45 . However, this is not the end of the process; once inside the mitochondria, long-chain acyl-CoA must be re-converted to long-chain acylcarnitine to generate energy through β-oxidation 46 . If any of these stages are obstructed, the metabolism of fatty acids is compromised. In our study, RGLS promotes the entry of long-chain fatty acids into mitochondria by upregulating expression levels of Acsl family members and ABC transport families. However, RGLS treatment did not increase intracellular ATP levels. In contrast, RGLS treatment significantly increased the content of long-chain acylcarnitine and decreased the L-carnitine content. These results suggest that RGLS inhibits the re-conversion of acylcarnitine to acyl-CoA and the renewal of L-carnitine, which prevents new acylcarnitine from entering mitochondria and exacerbates mitochondrial metabolic disorders. Interestingly, in the fatty acid pool, RGLS significantly increased the content of long-chain fatty acids, which means that RGLS interferes with fatty acid metabolism by increasing the source of long-chain fatty acids and inhibiting the utilization of long-chain fatty acids. In addition, the depletion of short- and medium-chain acylcarnitine and accumulation of long-chain acylcarnitine are considered markers of mitochondrial stress or damaging to fatty acid metabolism 47 . In summary, our study has provided compelling evidence that RGLS effectively inhibits OS cell growth and induces apoptosis by targeting glycerophospholipid metabolism and fatty acid β-oxidation. These findings shed light on the intricate molecular mechanisms through which RGLS exerts its anti-cancer effects against OS. Importantly, our study offers a strong rationale for the potential clinical application of RGLS as a therapeutic agent for OS treatment. Materials and Methods Materials. Shouxiangu® sporoderm-removed Ganoderma lucidum spores were obtained from Jinhua Shouxiangu Pharmaceutical Co., Ltd (Wuyi, Zhejiang, China). Cell counting Kit-8 (CCK-8) was purchased from MedChemExpress Technology (Princeton, New Jersey, USA). Polyclonal Bax, Bcl-xL, LC3B, p62, β-Actin antibodies and HRP-conjugated goat anti-rabbit IgG (H + L) were purchased from Diagbio Biotechnology Co., Ltd (Hangzhou, Zhejiang, China). Polyclonal Casp3, Ki-67 antibodies were purchased from CST (Boston, USA). The Pla2g7 antibody was purchased from Proteintech (Wuhan, Hubei, China). The mouse osteosarcoma S-180 cells was purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and the mouse macrophage Raw264.7 were purchased from American Type Culture Collection (ATCC, VA, USA). Xenograft mouse model. The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the NIH guidelines. All animal experiments were approved by the Animal Ethics Committee of Zhejiang Research Institute of Traditional Chinese Medicine (Approved Number: 20190003). The specific pathogen-free (SPF) male BALB/c nude mice (18 ~ 20 g), provided by the Hangzhou Medical College, were raised in the Zhejiang Research Institute of Traditional Chinese Medicine. The mice were randomly divided by weight into three groups: con group (distilled water, n = 6), model group (distilled water, n = 12), RGLS group (0.7 g/kg, n = 12, the dosage, based on clinical use, was weekly adjusted to match the mice's weight changes). Continuous administration for 45 days and S-180 cells (4×10 6 cells in 200 µL PBS) were subcutaneously transplanted into mice on day 38 of administration, and all mice were sacrificed seven days later. We first excised tumors from six mice for each group, and these tumors were stored immediately in liquid nitrogen for omics study (three for proteomics and three for transcriptomics study). For another six mice in each group, we weighed body and tumor weight. Then, three tumors per group were placed in formalin solution for pathological detection, and another three per group were placed in 4% formaldehyde for hematoxylin and eosin (H&E) staining. Tumor tissue histological examination. The harvested tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-µm sections. Then, the sections were stained with H&E according to the previous description 48 . The morphological changes in tumor tissues were observed, and images were randomly captured by a microscope (DM4000, Leica Biosystems). Immunohistochemical analysis of tumor tissues. The expression level of Ki-67 in tumor tissues was detected by immunohistochemistry (IHC) referred to previous reports 49 . The images were randomly captured by a microscope (DM4000, Leica Biosystems). Preparation of RGLS solution for in vitro experiments. RGLS was dissolved in DMEM as stock solution of 10 mg/mL, and then passed through 0.22-µm filter and diluted to required concentrations. Cell culture. Mycoplasma-free S-180 cells and Raw264.7 were cultured with Dulbecco's Modified Eagle's Medium (DMEM, Bio-channel, Nanjing, China) supplemented with 10% fetal bovine serum (FBS, Genom, Hangzhou, China) and 1% penicillin-streptomycin (Bio-channel, Nanjing, China), and maintained in a humidified atmosphere of 5% CO 2 at 37°C. Cell viability assay. S-180 and Raw264.7 cells were seeded into 96-well plates at a density of 1×10 4 cells per well and incubated in DMEM medium containing 10% FBS until cells reach 50% confluence. Cells well then treated with different concentration of RGLS for 24 h. Then 10% CCK-8 solution was added and incubated for an additional 2 h. After incubation, the absorbance was measured at 450 nm using a multi-well plate reader (Molecular Devices Instrument Inc., California, USA). The relative cell viability was calculated according to the formula: relative cell viability (%) = ( A RGLS - A blank )/( A Con - A blank ) × 100%, where A is the absorbance. Western blotting. Total protein of S-180 and Raw264.7 cells were extracted using ice-cold RIPA buffer (Beyotime, Beijing, China), and total protein concentrations were measured by BCA protein assay kit (Thermo Scientific, Waltham, Massachusetts, USA), and the expression levels of Casp3, Bax, Bcl-xL, LC3B, p62, Pla2g7, and β-Actin were detected according to previous reports 49 . The antibodies concentrations as listed in table S1 , and the semi-quantitative analysis of optical density was done using ImageJ 1.41 software (Bethesda, MD, USA). RNA isolation and quantitative real-time PCR. Total RNA isolation was performed using EASYspin reagent Kit (Aidlab Biotech, Beijing, China) according to the manufacturer’s protocol. Both the quality and quantity of total RNA were analyzed by the NanoDrop™ one spectrophotometer (Thermo Scientific, Grand Island, NY, USA). First-strand cDNA was synthesized from 1 µg of total RNA using iScript Reverse transcription supermix (Vazyme Biotech, Nanjing, China). qRT-PCR analysis was performed using Faststart Essential DNA Green Master (Roche, Basel, Swit) on LightCycler96 Real-Time PCR system (Roche). Primers were designed with open-sourced software Primer3Plus (Cambridge, MA, USA). GAPDH was used as the reference gene. The relative expression of mRNA was calculated by following the previous literature 49 . qRT-PCR primer sequences are shown in table S2 . Flow cytometric analysis of apoptosis. S-180 cells were seeded into 6-well plates at a density of 2×10 5 cells per well and treated with different concentrations of RGLS for 24 h. Cells were then washed with PBS, harvested by trypsinization and washed twice with PBS, and cells were stained with propidium iodide (PI) and annexin V-FITC using Annexin V-FITC apoptosis detection kit (Beyotime, Beijing, China) according to the manufacturer's instructions. The stained cells were analyzed by C6 plus flow cytometer (BD, New Jersey, USA), and the percentage of early apoptosis (annexin V + /PI − ), late apoptosis (annexin V + /PI + ), and necrosis (annexin V − /PI + ) cells were analyzed. Data was represented as rate of total apoptosis cells with both early and late apoptotic rate indicated. ATP content detection. Intracellular ATP content detection was using ATP content assay kit (Solarbio life sciences, Beijing, China) according to the manufacturer's instructions. Label-free proteomic analysis. Total protein of tumor tissue was extracted, enzymatically digested, and desalted before separation using nanoliter reversed-phase liquid chromatography and detection by mass spectrometry. Protein abundance was quantified using a Proteome Discoverer library search. Before performing differential protein expression analysis, we used the mice (v3.16) package to perform multiple imputations and fill in missing values. Next, we took the logarithm of the protein expression matrix. We then used the edgeR (v3.42) package to identify differential proteins with adjusted P -values less than 0.05. Finally, we conducted functional enrichment using the clusterProfiler (v4.8) package. Transcriptomics analysis. Total RNA of tumor tissue was extracted, and libraries were constructed and sequenced to obtain mRNA sequence information. The sequences were aligned to the mouse mm10 genome using Bowtie2 and GENCODE database, and gene expression was quantified using HTSeq. Gene expression values were then normalized using the TMM method. Differential gene expression analysis was performed using edgeR, with genes having a P -value less than 0.05 and an absolute fold change greater than two considered as differentially expressed. Metabonomics analysis. Total metabolite of S-180 cells (about 1×10 7 cells) was extracted by 1000 µL methanol: acetonitrile: H 2 O (2:2:1), sonicated at 4℃ for 10 min, incubated at -40℃ for 60 min and centrifuged at 4000×g for 15 min at 4℃. The supernatant was collected and filtered through 0.22 µm PVDF membrane. Next, the detection, identification, and data analysis of metabolites in S-180 cells were referred to previous reports 50 . Statistical analysis. All data are presented as the mean ± standard error (SE) of at least three independent experiments. For comparisons between the two conditions, a student t -test was used. And for multiple comparisons, one-way analysis of variance (ANOVA) with Tukey's test was used. All analyzed were performed using Graphpad Prism 8.0 software (Graphpad Software, Inc., USA), and a value of P 0.05 was a considered to be statistically significant difference. Declarations Ethics Statement The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the NIH guidelines. All animal experiments were approved by the Animal Ethics Committee of Zhejiang Research Institute of Traditional Chinese Medicine (Approved Number: 20190003). Author Contribution Statement J.H.Y. and Z.H.L. designed the experiments. H.T.P., C.S.L., M.Y.W. and K.Y. performed the experiments. H.T.P. wrote the first draft of the manuscript. X.H.F., J.H.Y., Z.H.L. and Y.P.F. supervised the experiments and edited the manuscript. Competing interests: The authors declare no competing interests. Author Contribution J.H.Y. and Z.H.L. designed the experiments. H.T.P., C.S.L., M.Y.W. and K.Y. performed the experiments. H.T.P. wrote the first draft of the manuscript. X.H.F., J.H.Y., Z.H.L. and Y.P.F. supervised the experiments and edited the manuscript. Acknowledgments This study was supported by Zhejiang Science and Technology Major Program (Grant No. 2021C020732), National Natural Science Foundation of China (Grant No.32270028), Scientific and Technological Planning Project of Jilin Province (Grant No. YDZJ202302CXJD002 and No. 20220202109NC) and Central Guiding Local Science and Technology Development Fund Project (Grant No. 2024ZY01009). Zhejiang Research Institute of Traditional Chinese Medicine is acknowledged for its laboratory equipment. Data Availability The metabolomics data presented in the study are deposited in the open data repository for metabolomics (metaboLights, https://www.ebi.ac.uk/metabolights/), accession number MTBLS11629. The proteomics data presented in the study are deposited in the iProX repository, accession number PXD056264. The transcriptomics data in the study are deposited in the GEO repository, accession number GSE277633. References Basit, Q., Qazi, H. S. & Tanveer, S. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6046286","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":446689846,"identity":"caec3b5e-985e-42bf-8e9d-fa7d3c48068b","order_by":0,"name":"Haitao Pan","email":"","orcid":"","institution":"BoYu Intelligent Health Innovation Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Haitao","middleName":"","lastName":"Pan","suffix":""},{"id":446689847,"identity":"5f3daa3b-fa33-41d6-a867-7b09b83034ca","order_by":1,"name":"Congshu Li","email":"","orcid":"","institution":"BoYu Intelligent Health Innovation Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Congshu","middleName":"","lastName":"Li","suffix":""},{"id":446689848,"identity":"36647449-4e36-40b2-b5e9-609db27f133b","order_by":2,"name":"Mengyao Wang","email":"","orcid":"","institution":"BoYu Intelligent Health Innovation Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Mengyao","middleName":"","lastName":"Wang","suffix":""},{"id":446689849,"identity":"c20f927d-7b3a-4248-adda-27a7c7f51cf2","order_by":3,"name":"Kang Ye","email":"","orcid":"","institution":"Zhejiang Research Institute of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kang","middleName":"","lastName":"Ye","suffix":""},{"id":446689850,"identity":"1a0313fe-3daa-4126-85f8-1f09264b9b48","order_by":4,"name":"Xiaohui Fan","email":"","orcid":"","institution":"National Key Laboratory of Chinese Medicine Modernization, Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Xiaohui","middleName":"","lastName":"Fan","suffix":""},{"id":446689851,"identity":"b985f692-14be-419b-8437-bebcddcc8397","order_by":5,"name":"Yongping Fu","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yongping","middleName":"","lastName":"Fu","suffix":""},{"id":446689852,"identity":"ff1bef3c-4c53-40f9-ac43-6747f5cf1cd6","order_by":6,"name":"Jihong Yang","email":"","orcid":"","institution":"BoYu Intelligent Health Innovation Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Jihong","middleName":"","lastName":"Yang","suffix":""},{"id":446689853,"identity":"eda12841-118f-42b3-951e-d92d4c455d9f","order_by":7,"name":"Zhenhao Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYDACCQYGZiAlx8BwAEixkaDFmHQtiQ1gHjFa5Gc3H3tcUGOTPr/xjAHDh7LDDPyzG/BrYZxzLN14xrG03MaGMwaMM84dZpC4cwC/FmaJHDNpHrbDuc0MZwyYedsOMxhIJODXwiaR/02a59/hdDaQlr/EaOGRyGGTBhqewAPSwkiMFgmJNDNp3r40wxkMxwoO9pxL55G4QUCL/IzkZ9I832zk5Wcc3vjgR5m1HP8MAlqQ7DsAjkweYtUDAX8DCYpHwSgYBaNgRAEAu5A9D/SWpkUAAAAASUVORK5CYII=","orcid":"","institution":"BoYu Intelligent Health Innovation Laboratory","correspondingAuthor":true,"prefix":"","firstName":"Zhenhao","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-02-17 09:09:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6046286/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6046286/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-05890-5","type":"published","date":"2025-07-01T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81690047,"identity":"4dfd4e2c-8693-449e-b189-ddd3e9fc7d85","added_by":"auto","created_at":"2025-04-30 11:26:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":542435,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of RGLS on growth of S-180 xenograft tumor \u003cem\u003ein vivo\u003c/em\u003e. (A) Experimental protocol. (B-C) Effect of RGLS on tumor size (B) and weight (C). (D) Effect of RGLS on body weight of mice. (E) Effect of RGLS on pathological changes of tumor tissue. (F) Effect of RGLS on Ki-67 expression in tumor tissue. \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e˂0.001 compared with Model group, \u003cem\u003en\u003c/em\u003e = 6.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6046286/v1/0d6215b883a3113ca2c6f5dc.png"},{"id":81690048,"identity":"ade0b74b-87b7-4849-ae3e-05417254b28b","added_by":"auto","created_at":"2025-04-30 11:26:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":240628,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of RGLS on growth of S-180 cells \u003cem\u003ein vivo\u003c/em\u003e. (A) Effect of RGLS on S-180 cells proliferation. (B-C) Effect of RGLS on apoptosis rate of S-180 cells. (D) Effect of RGLS on apoptosis-related protein expression in S-180 cells. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e˂0.05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e˂0.001 compared with Con group, \u003cem\u003en\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6046286/v1/f620c5ba2a6ea089581be093.png"},{"id":81692516,"identity":"51e738bd-a58c-4677-adf4-ab35d7655206","added_by":"auto","created_at":"2025-04-30 11:42:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":462270,"visible":true,"origin":"","legend":"\u003cp\u003eMetabonomics analysis of S-180 cells. (A) Principal component analysis (PCA) of metabonomics data. (B) Differential expression analysis of metabonomicsdata. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) terms enriched by differentially metabolites. (D) Effect of RGLS on phospholipid and lysophospholipid content.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6046286/v1/97464c5c708be7bc6a1a70e6.png"},{"id":81690055,"identity":"260156f6-90a8-4bfb-b192-052418c46763","added_by":"auto","created_at":"2025-04-30 11:26:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":601119,"visible":true,"origin":"","legend":"\u003cp\u003eRGLS enhances PC metabolism. (A) Schematic diagram of phospholipid metabolic sites. (B) PCA analysis of transcriptomics and proteomics data. (C-D) Gene Ontology Biological Process (GOBP) terms enriched by upregulated genes (C) and proteins (D). (E) Effect of RGLS on the expression of phospholipase in tumor tissue. (F) Combined proteomic and transcriptomic analysis. (G) GOBP terms enrichment of genes and proteins upregulated by RGLS simultaneously. (H) Effect of RGLS on the expression of ABC families in tumor tissue. (I) Model diagram of co-culture of S-180 cells with Raw264.7 cells. (J) Effect of RGLS on the expression of Pla2g7 in S-180 cells co-cultured with Raw264.7 cells. (K) Effect of RGLS on the expression of Pla2g7 in Raw264.7 cells. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e˂0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e˂0.001 compared with Con group, \u003cem\u003en\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6046286/v1/948a49eddf9be90064a9fb92.png"},{"id":81691365,"identity":"103857fb-18f5-45f5-8341-08518a0eb10a","added_by":"auto","created_at":"2025-04-30 11:34:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":394468,"visible":true,"origin":"","legend":"\u003cp\u003eRGLS inhibits fatty acid metabolism. (A) Effect of RGLS on ATP content in S-180 cells. (B-D) Effect of RGLS on the expression of Acsl families (B), Cpt1a (C), and Cat2 (D) in tumor tissue. (E) Effect of RGLS on L-carnitine content in S-180 cells. (F-G) Effect of RGLS on long-chain Acylcarnitine (F) and short-, and medium-chain Acylcarnitine (G) content in S-180 cells. (H) Effect of RGLS on the composition of fatty acid pool in S-180 cells. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e˂0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e˂0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e˂0.001 compared with Con or Model group, \u003cem\u003en\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6046286/v1/d5b66559a131a8c219bf195f.png"},{"id":81690058,"identity":"01e3520e-e31c-46f1-8964-b6d0d89d8bed","added_by":"auto","created_at":"2025-04-30 11:26:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":516342,"visible":true,"origin":"","legend":"\u003cp\u003eWorking model of molecular mechanisms by which RGLS exerts its anticancer activity in osteosarcoma S-180 cells and xenograft tumors. On the one hand, RGLS promotes S-180 uptake of pla2g7 protein released by macrophages, which promotes the metabolism of PCs to produce LysoPCs with cytotoxicity. On the other hand, RGLS restructures fatty acid composition and compromises energy supply by inhibiting β-oxidation of long-chain fatty acid in S180 cells.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6046286/v1/af954afedd0a9b723fb770be.png"},{"id":86179724,"identity":"10cf7a79-3de4-4ff5-bc3b-d7af83f1c943","added_by":"auto","created_at":"2025-07-07 16:19:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3257188,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6046286/v1/7d46e8b0-1498-4065-a91c-2b0c77d02fc6.pdf"},{"id":81690054,"identity":"1e779d2a-1f8e-4906-98fa-0c7c3d6d8fb9","added_by":"auto","created_at":"2025-04-30 11:26:43","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":26651,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6046286/v1/a0d659151c69cb20ce12f613.docx"},{"id":81690080,"identity":"e6fa4c5c-2f45-4bbc-a4b4-a513fc87238c","added_by":"auto","created_at":"2025-04-30 11:26:45","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":34887878,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6046286/v1/2dbb323d509fcce48c856ed7.tif"},{"id":81690078,"identity":"06fe4f8a-039b-4269-aee7-71bc590eaeae","added_by":"auto","created_at":"2025-04-30 11:26:44","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":40444560,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6046286/v1/84a6f9831cbdf2b0d90a9df8.tif"},{"id":81690083,"identity":"493f366e-a958-4b7a-a4ff-22495fe3b992","added_by":"auto","created_at":"2025-04-30 11:26:46","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":58769394,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-6046286/v1/f883d50e7316085465c70ca9.tif"},{"id":81690085,"identity":"903b79ff-1dfd-46f6-9543-995ece36d6ef","added_by":"auto","created_at":"2025-04-30 11:26:47","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":81030048,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-6046286/v1/3f7a0776717e5a9bbd669fac.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Multi-omics analysis reveals inhibition of osteosarcoma progression by sporoderm-removed Ganoderma lucidum spores via targeting glycerophospholipid and fatty acid Metabolism","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteosarcoma (OS) is a prevalent primary malignant bone tumor of mesenchymal origin, predominantly affecting children and adolescents. Epidemiological studies have revealed that OS accounts for 15% of all solid extracranial cancers in adolescents aged 15\u0026ndash;19 years \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Current therapeutic approaches for OS primarily revolve around surgical intervention followed by chemotherapy. However, the aggressive nature of OS, coupled with chemotherapy resistance, recurrence, and adverse side effects leading to reduced patient compliance, present significant challenges in achieving satisfactory treatment outcomes. Consequently, there is an urgent need to explore effective and safe agents for the prevention and treatment of OS.\u003c/p\u003e \u003cp\u003eMetabolic reprogramming is a defining hallmark of cancer cells \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, and in osteosarcoma (OS), fatty acids and glycerophospholipids emerge as critical metabolic drivers of progression. Fatty acids, central energy sources for tumor cells, fuel OS growth, invasion, and immunosuppressive microenvironment formation \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Recent studies show that inhibiting fatty acid β-oxidation\u0026mdash;via pharmacological or genetic approaches\u0026mdash;disrupts mitochondrial energy production, thereby reducing OS cell viability, migration, and invasion \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Beyond \u003cem\u003ede novo\u003c/em\u003e fatty acid synthesis mediated by FASN, glycerophospholipids, the most abundant membrane phospholipids, are hydrolyzed by phospholipases (PLs) into bioactive metabolites such as lysophospholipids and fatty acids \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These metabolites regulate membrane dynamics, signaling, and proliferation, with aberrant glycerophospholipid metabolism linked to aggressive OS phenotypes. For instance, elevated phospholipase A2 group 16 (PLA2G16) correlates with lung metastasis and poor prognosis \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, while phospholipase D (PLD) antagonizes tumor-suppressive miR-638 to drive OS proliferation \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Notably, disrupting phosphatidylcholine (PC) biosynthesis\u0026mdash;a major glycerophospholipid subclass\u0026mdash;suppresses OS cell proliferation and mineralization capacity \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Clinically, first-line OS chemotherapeutics like cisplatin and doxorubicin act partly by inducing membrane degradation or blocking lipid synthesis \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Collectively, targeting these interlinked metabolic pathways\u0026mdash;notably glycerophospholipid metabolism and fatty acid β-oxidation\u0026mdash;offers a promising therapeutic strategy to disrupt energy production and membrane synthesis critical for OS progression.\u003c/p\u003e \u003cp\u003e \u003cem\u003eGanoderma lucidum\u003c/em\u003e, a medicinal mushroom with a historical usage spanning over 6800 years \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. has been found to influence lipid metabolism. For instance, \u003cem\u003eGanoderma lucidum\u003c/em\u003e aqueous extract regulates the biological metabolic process of membrane components in \u003cem\u003etetrahymena thermophila\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eGanoderma lucidum\u003c/em\u003e polysaccharide-cadmium chelate regulates glycerophospholipid metabolism in liver \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Additionally, sporoderm-broken GLS has demonstrated anti-OS effects by inducing apoptosis and inhibiting the STAT3/PD-L1 signaling pathway \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, there have been no reports on the specific role of \u003cem\u003eGanoderma lucidum\u003c/em\u003e or its extracts in regulating the metabolic processes of membrane components and phosphoglyceride metabolism to exert its anti-OS effects.\u003c/p\u003e \u003cp\u003eThe present study aimed to analyze the effects of sporoderm-removed GLS (RGLS), a highly purified GLS extract \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e on OS development both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, and to elucidate the potential mechanism of RGLS against OS based on the integrative analysis of transcriptomics, proteomics and metabolomics \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, which has not been previously reported. Our data indicate that RGLS effectively suppresses OS development by inhibiting proliferation, and inducing cell apoptosis. Additionally, we observed that RGLS promotes PCs metabolism by enhancing endocytosis of macrophage-secreted Pla2g7 by S-180 cells. Furthermore, we found that RGLS regulates fatty acid pool composition and inhibits the conversion of long-chain acylcarnitine to long-chain acyl-CoA in mitochondria, and ultimately leading to fatty acid β-oxidation disorders and intracellular energy insufficiency.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eRGLS inhibits S-180 cells growth in vivo.\u003c/b\u003e In the present study, we administered RGLS prophylactically to BALB/c nude mice and then injected them subcutaneously with S-180 cells to evaluate RGLS's tumor-inhibiting effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The results showed that RGLS significantly reduced the weight of S-180 xenograft tumors compared with the model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and the tumor inhibition rate was high at 35.8% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In addition, we analyzed the effect of therapeutic administration of RGLS on the growth of S-180 xenograft tumors. As shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, in the absence of prophylactic administration, only 7 days of therapeutic administration also reduced the weight of S-180 xenograft tumors (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB), but the tumor inhibition rate was lower at 25.8% (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). Moreover, the body weight of mice was not affected by both administration methods of RGLS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and S1D). These results indicated that RGLS effectively delays the growth of S-180 xenograft tumors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eApoptosis is the most classical mode of cell death in tumors. Next, we observed the effects of RGLS on the pathological changes in S-180 xenograft tumors using H\u0026amp;E staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, RGLS greatly induced apoptosis in S-180 cells within xenograft tumors, which was mainly manifested as the loss of intercellular connections, increased cytoplasmic density, reduced cell volume, and nuclear contraction. In addition to apoptosis, inhibiting cell proliferation is also an effective strategy for tumor treatment. Furthermore, we examined the expression of Ki-67, a nuclear cell proliferation marker, in xenograft tumors using IHC staining. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, the expression of Ki-67 was significantly decreased in the RGLS treatment groups compared with the model group. Taken together, RGLS delays S-180 xenograft tumor growth by inhibiting cell proliferation and inducing cell apoptosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRGLS alleviates tumor-induced immune deficiency in mice.\u003c/b\u003e Immune deficiency is an important factor in tumorigenesis, and in turn, the development of tumors can further exacerbate the immune deficiency. Next, we analyzed the effects of RGLS on internal morphological structure of the thymus using H\u0026amp;E staining. As shown in Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, the boundary between cortex and medulla of thymus in control group was distinct and orderly arranged. However, compared to the control group, the cortex and medulla of the thymus in the model group underwent spatial rearrangement, and RGLS exhibited a mitigating effect on this phenomenon.\u003c/p\u003e \u003cp\u003eThe thymus is the site of T lymphocyte development, and damage to the thymus inevitably leads to abnormal development of T cells. Further, we analyzed the effects of RGLS on proportions of specific T cell subtypes in peripheral blood, including CD3\u003csup\u003e+\u003c/sup\u003e, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e, and CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells, relative to total CD3\u003csup\u003e+\u003c/sup\u003e T cells. Compared with the control group, the proportion of CD3\u003csup\u003e+\u003c/sup\u003e T cells in the model group was significantly reduced, while RGLS increased the proportion of CD3\u003csup\u003e+\u003c/sup\u003e T cells compared with the model group. However, the proportions of CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells did not change significantly in both the model group and the RGLS group (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB and C). These results suggest that RGLS ameliorates tumor-induced immune decline, which may be related to the regulation of non-traditional T cell development, such as double negative (DN, CD4\u003csup\u003e\u0026minus;\u003c/sup\u003eCD8\u003csup\u003e\u0026minus;\u003c/sup\u003e ) iNKT cells \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRGLS inhibits S-180 cells growth in vitro\u003c/b\u003e. To further analyze the anti-OS effects of RGLS, we examined the impact of RGLS on S-180 cells growth \u003cem\u003ein vitro\u003c/em\u003e. The CCK-8 assay showed that RGLS decreased the viability of S-180 cells in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and the half-maximal inhibitory concentration (\u003cem\u003eIC50\u003c/em\u003e) was 2.51 mg/ml. We then examined whether RGLS induced apoptosis in S-180 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and C, RGLS treatment significantly increased the ratio of apoptotic cells, and the late apoptotic (Annexin V\u003csup\u003e\u0026minus;\u003c/sup\u003e/PI\u003csup\u003e+\u003c/sup\u003e) cells were predominant. Specifically, our results show that RGLS treatment significantly reduces the expression levels of pro-Casp3 and Bcl-xL in a concentration-dependent manner, but does not significantly affect the Bax/Bcl-xL ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Collectively, these findings suggest that RGLS effectively inhibits S-180 cell proliferation by inducing apoptosis, potentially through a mechanism independent of the mitochondrial pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRGLS regulates glycerophospholipid metabolism in S-180 cells\u003c/b\u003e. Reprogramming of lipid metabolism is an emerging hallmark that distinguishes tumor cells from normal cells \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. To further elucidate the molecular mechanism of RGLS in inhibiting S-180 cell growth, we performed a metabonomics analysis to assess the effects of RGLS on S-180 cell metabolism. Principal component analysis (PCA) revealed that RGLS significantly altered the metabolite composition in S-180 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Compared with the control group, RGLS upregulated 49 metabolites and downregulated 47 metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Following KEGG metabolic pathway enrichment analysis of the differential metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), nine metabolic pathways, namely Choline metabolism in cancer (ko05231), Efferocytosis (ko040148), Glycerophospholipid metabolism (ko00564), Thermogenesis (ko04714), Sulfur relay system (ko04122), Nucleotide metabolism (ko01232), Zeatin biosynthesis (ko00908), Cysteine and methionine metabolism (ko00270), and ABC transporters (ko02010) were enriched (\u003cem\u003eP\u003c/em\u003e˂0.01). Among them, the number of targeted differential metabolites related to glycerophospholipid metabolic pathway is the largest. Focusing on glycerophospholipid metabolic, a heatmap analysis of the total 42 differential metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) showed the degree of variation with various colors. Specifically, RGLS treatment significantly increased the levels of lysophosphatidylcholine (LysoPC, including LysoPC 16:1, LysoPC 18:2, LysoPC 18:3, and LysoPC 20:2), a metabolite of phosphatidylcholine (PC), which has been shown with anti-tumor activity \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In contrast, the RGLS treatment reduced the levels of phosphatidylinositol (PI, including PI 18:0/14:1, PI 16:1/18:2, PI 16:1/18:1, PI 18:2/20:4, PI 18:2/20:3, and PI 18:0/20:4), an important component for cell and organelle membrane biosynthesis and function \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and its metabolite LysoPI (including LysoPI 16:0, LysoPI 17:0, LysoPI 18:3, and LysoPI 20:1), which has been shown with pro-tumor activity \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Taken together, these results indicate that RGLS exerts bidirectional regulation on glycerolphospholipid metabolism to promote anti-tumor activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRGLS regulates glycerophospholipid metabolism by enhancing the interaction between S-180 cells and macrophages\u003c/b\u003e. Phospholipases (PLs) are the rate-limiting enzymes in glycerolphospholipid metabolism, including PLA1, PLA2, PLC, and PLD. Among them, glycerolphospholipids are hydrolyzed by PLA1 or PLA2 and then lose a molecule of fatty acid to produce lyso-phospholipids (LP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To further elucidate the regulatory effect of RGLS on PC metabolism, transcriptomic and proteomic analyses were conducted to assess gene and protein expression changes in xenograft tumors. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, PCA revealed that RGLS significantly altered the gene and protein expression in xenograft tumors. Compared with the control group, RGLS upregulated 1494 genes and 431 proteins, and downregulated 414 genes and 43 proteins. Initially, we verified the role of RGLS in inducing S-180 cell apoptosis. As shown in Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA, RGLS treatment significantly upregulated the expression levels of apoptosis-related proteins, including the Casp family (Casp1, Casp3, Casp7, Casp8), Bcl-2 family (Bax, Bid), and Aif family (Aif1, Aifm1, Aifm2, Aifm3), suggesting that RGLS induced S-180 cell apoptosis \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, gene ontology (GO) enrichment analysis showed that the upregulated genes and proteins are mainly enriched in cellular compartment terms related to membrane, such as lysosomal membrane (GO:0098574), microvillus membrane (GO:0031528), endosome membrane (GO:0010008), actin cytoskeleton (GO:0015629), mitochondrial inner membrane (GO0005743), and mitochondrial membrane (GO:0031966) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). This suggests that lysosome may be involved in the process of RGLS regulating PC metabolism \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Indeed, we found that RGLS significantly increased autophagy levels in S-180 cells \u003cem\u003ein vitro\u003c/em\u003e, including increasing the number of autophagosomes (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB), upregulating LC3B protein levels and decreasing p62 protein levels (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC), and enhancing lysosomal acidity (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD). In addition, RGLS upregulated the expression levels of lysosomal functional proteins (Lamp1 and Lamp2) (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eE) and lysosomal cathepsin (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eFurthermore, we analyzed the effect of RGLS on PLA expression. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, we found that RGLS upregulated the expression levels of multiple LPA members in S-180 xenograft tumors, especially Plaa (a PLA2 activating protein) and Pla2g7 (the seventh member of the PLA2 family), a secreted PL that metabolizes PC to LysoPC under the action of lysophosphatidylcholine acyltransferase (Lpcat) in the lysosome \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. However, Pla2g7 is usually derived from macrophages \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Indeed, transcriptome data showed that S-180 xenograft tumor did not express \u003cem\u003ePla2g7\u003c/em\u003e and RGLS did not affect \u003cem\u003ePla2g7\u003c/em\u003e mRNA expression levels (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eG). These results suggest that RGLS may promote PC metabolism by enhancing the interaction between S-180 cells and macrophages.\u003c/p\u003e \u003cp\u003eEndocytosis is an important pathway for cellular uptake of biological macromolecules. Combined transcriptome and proteome analyses revealed that 46 regulatory factors, such as Smap1, Unc119 and Ubaln2, were upregulated simultaneously at both gene and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Further GO enrichment analysis revealed a strong association between co-upregulated genes/proteins and regulation of clathrin-dependent endocytosis (GO:2000369) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). We also identified a series of ABC superfamily members whose expression levels were upregulated by RGLS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Thus, we hypothesized that RGLS might enhance the uptake of Pla2g7 secreted by macrophages in S-180 cells. To test this hypothesis, we performed co-culture of S-180 cells with Raw264.7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Compared to S-180 cells cultured alone, the Pla2g7 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ), but not mRNA levels (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eG), were significantly higher in S-180 cells co-cultured with Raw264.7 cells. However, it was disappointing that RGLS (2 mg/ml) treatment did not further increase significantly the levels of Pla2g7 protein and mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ and Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eG), in co-cultured S-180 cells. This phenomenon may be explained by the Pla2g7 protein in S-180 cells in the form of enzyme-substrate binding or the Pla2g7 protein released by Raw264.7 cells reaching its limit. In addition, at non-toxicity concentrations (2 mg/ml) (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eH), RGLS decreased significantly Pla2g7 protein levels in Raw264.7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). Interestingly, RGLS did not reduce \u003cem\u003epla2g7\u003c/em\u003e mRNA expression levels in Raw264.7 cells (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eI). These results showed that RGLS promoted the release of Pla2g7 protein in Raw264.7 cells and the phagocytosis of Pla2g7 protein in S-180 cells, thus enhancing PC metabolism.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRGLS regulates acylcarnitine and fatty acid metabolism\u003c/b\u003e. Fatty acids are intermediate metabolites of glycerolphospholipid, releasing ATP through β-oxidation. Surprisingly, treatment with RGLS significantly reduced intracellular ATP content in S-180 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To investigate whether β-oxidation is compromised by RGLS, we analyzed its role in regulating fatty acid metabolism. It is well established that short- and medium-chain fatty acids can enter the mitochondria directly through diffusion, whereas long-chain fatty acids are converted to fatty acyl-CoA by long-chain acyl-CoA synthetase (Acsl) before entering the mitochondria. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, RGLS treatment increased the expression levels of Acsl family members, particularly Acsl3. Subsequently, we analyzed the effect of RGLS on carnitine-acyl transferase 1 (Cat1), which facilitates the binding of acyl-CoA to carnitine to form acylcarnitine, thereby enabling entry into the mitochondria. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, RGLS treatment significantly increased the protein expression level of carnitine-palmitoyl transferase 1a (Cpt1a), a member of the Cat1, suggesting that RGLS may promote the entry of long-chain fatty acids into the mitochondria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the mitochondria, long-chain acylcarnitine reconverted to long-chain acyl-CoA under the action of Cat2 and L-carnitine is released. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, RGLS treatment significantly decreased the protein expression level of Cat2, and reduced the L-carnitine content in S-180 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), suggesting that RGLS may inhibit long-chain acylcarnitine metabolism. Next, we analyzed the changes of acylcarnitine levels in S-180 cells. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, RGLS significantly increased the levels of long-chain acylcarnitine (C 16:1 and C16:0) and slightly decreased the levels of short- and medium-chain acylcarnitine (C 4:0, C 5:0, and C 9:1). Further, we analyzed the effect of RGLS on fatty acid composition in S-180 cells. As shown in 5H, RGLS treatment significantly reduced the levels of short- and medium-chain fatty acids while increasing the levels of various long-chain fatty acids. These results suggest that RGLS compromises energy supply by increasing the proportion of long-chain fatty acids and inhibiting long-chain acylcarnitine metabolism, thereby inhibiting fatty acid β-oxidation and promoting S-180 cell death.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAmong the myriad of TCM remedies, \u003cem\u003eGanoderma lucidum\u003c/em\u003e renowned for its health-promoting and life-extending properties, has shown considerable promise in managing cardiovascular diseases and brain injury \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. While \u003cem\u003eGanoderma lucidum\u003c/em\u003e has been recognized for its significant therapeutic benefits, its potential in osteosarcoma prevention and treatment has remained relatively underexplored. Limited studies have been conducted to investigate the anti-OS effects of \u003cem\u003eGanoderma lucidum\u003c/em\u003e or its active component \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our research, we evaluated the anti-OS effects of RGLS and its mechanisms (as summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Encouragingly, our results showed that RGLS has a good inhibitory effect on tumor growth. This observation supports the notion that RGLS possesses a strong potential for tumor prevention and treatment. The efficacy of RGLS might be attributed to its ability to target specific molecular pathways or cellular processes involved in tumor initiation and progression. However, to fully comprehend the clinical applicability of RGLS in tumor prevention, further research is essential. Robust pre-clinical and clinical studies are necessary to validate the efficacy, safety, and dosage regimen of RGLS in preventing various types of tumors. Additionally, exploring the mechanistic basis of RGLS's tumor-preventive effects would provide critical insights into its mode of action and potential targets.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhospholipids are the material basis of cellular and organismal life activities, and they are not only the main components of cell and organelle membranes, but also involved in the regulation of countless signal pathways \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In tumor cells, the content of phospholipids, especially PC, PI and sphingomyelin, is sharply increased \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, which suggests that phospholipid metabolism as a target may be an effective strategy for tumor treatment \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In our study, we found that RGLS significantly increased the content of LysoPCs. As an intermediate product of PC metabolism, LysoPCs are not only closely involved in atherosclerosis and diabetes \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e but also inhibits the growth of a variety of tumor cells, including bladder cancer, lung cancer, and melanoma \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and are used to predict the efficacy and favorable prognosis of lenvatinib combined with programmed death-1 (PD-1) inhibitor in the clinical treatment of unresectable hepatocellular carcinoma (uHCC) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In addition, LysoPC has been shown to continuously increase intracellular calcium ion concentration by activating calcium-permeable channels and contribute to of cell cytoskeletal \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Correspondingly, our study also found that RGLS regulates the biological processes of the cytoskeleton (GO: 0005856) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), which may be a potential mechanism by which RGLS regulates PC metabolism to exert anti-tumor effects.\u003c/p\u003e \u003cp\u003eLysosomes are essential for maintaining cell homeostasis and are involved in a variety of biological processes, such as immunity and intracellular pH regulation \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Recent studies have shown that lysosomes also participate in glycerophospholopid metabolism \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. PLA2 is a ubiquitous enzyme uniquely characterized by a sub-cellular localization to the lysosome and late endosome \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, which hydrolyzes PC to produce LysoPC and causes lysosome destabilization \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In our research, RGLS significantly increased Pla2g7 protein levels but not mRNA levels in S-180 cells, suggested that Pla2g7 protein in S-180 cells might be exogenous. Indeed, Pla2g7 is highly expressed in immune cells, such as B cells and macrophages \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Our study also found that RGLS increases the expression of CD47 (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eJ), an immune checkpoint that interacts with SIRPα expressed by macrophages \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. These phenomena indicate that RGLS enhances the interaction between S-180 cells and macrophages. Our previous studies have shown that polysaccharide derived from RGLS inhibits the growth of hepatocellular carcinoma cells by promoting macrophages to M1 type polarization and inflammatory cytokines secretion such as TNF-α, IL-1β, IL-6 \u003csup\u003e41\u003c/sup\u003e. However, studies have shown that in the course of COVID-19 infection, Pla2g7 is primarily produced by pro-inflammatory (M1 type) macrophages \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, and macrophages with high expression of PLA2 showed a profound immunosuppressive phenotype \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The above background suggests that RGLS regulation of Pla2g7 release may be a novel mechanism to promote macrophage-mediated immunity, and the regulation of endocytosis of tumor cells on Pla2g7 may be related to lysosomal-dependent cell death induced by RGLS \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, these speculations are worthy of further study.\u003c/p\u003e \u003cp\u003eFatty acids are important energy sources for tumor cells, and targeting fatty acid metabolism is considered an effective strategy for tumor (including cancer) management \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Fatty acid utilization is a delicately regulated process. Short- and medium-chain fatty acids can enter the mitochondria directly, but long-chain fatty acids require conversion to long-chain acyl-CoA and transport by L-carnitine \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. However, this is not the end of the process; once inside the mitochondria, long-chain acyl-CoA must be re-converted to long-chain acylcarnitine to generate energy through β-oxidation \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. If any of these stages are obstructed, the metabolism of fatty acids is compromised. In our study, RGLS promotes the entry of long-chain fatty acids into mitochondria by upregulating expression levels of Acsl family members and ABC transport families. However, RGLS treatment did not increase intracellular ATP levels. In contrast, RGLS treatment significantly increased the content of long-chain acylcarnitine and decreased the L-carnitine content. These results suggest that RGLS inhibits the re-conversion of acylcarnitine to acyl-CoA and the renewal of L-carnitine, which prevents new acylcarnitine from entering mitochondria and exacerbates mitochondrial metabolic disorders. Interestingly, in the fatty acid pool, RGLS significantly increased the content of long-chain fatty acids, which means that RGLS interferes with fatty acid metabolism by increasing the source of long-chain fatty acids and inhibiting the utilization of long-chain fatty acids. In addition, the depletion of short- and medium-chain acylcarnitine and accumulation of long-chain acylcarnitine are considered markers of mitochondrial stress or damaging to fatty acid metabolism \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, our study has provided compelling evidence that RGLS effectively inhibits OS cell growth and induces apoptosis by targeting glycerophospholipid metabolism and fatty acid β-oxidation. These findings shed light on the intricate molecular mechanisms through which RGLS exerts its anti-cancer effects against OS. Importantly, our study offers a strong rationale for the potential clinical application of RGLS as a therapeutic agent for OS treatment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cem\u003eMaterials.\u003c/em\u003e Shouxiangu\u0026reg; sporoderm-removed \u003cem\u003eGanoderma lucidum\u003c/em\u003e spores were obtained from Jinhua Shouxiangu Pharmaceutical Co., Ltd (Wuyi, Zhejiang, China). Cell counting Kit-8 (CCK-8) was purchased from MedChemExpress Technology (Princeton, New Jersey, USA). Polyclonal Bax, Bcl-xL, LC3B, p62, β-Actin antibodies and HRP-conjugated goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) were purchased from Diagbio Biotechnology Co., Ltd (Hangzhou, Zhejiang, China). Polyclonal Casp3, Ki-67 antibodies were purchased from CST (Boston, USA). The Pla2g7 antibody was purchased from Proteintech (Wuhan, Hubei, China). The mouse osteosarcoma S-180 cells was purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and the mouse macrophage Raw264.7 were purchased from American Type Culture Collection (ATCC, VA, USA).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e\u003cem\u003eXenograft mouse model.\u003c/em\u003e The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the NIH guidelines. All animal experiments were approved by the Animal Ethics Committee of Zhejiang Research Institute of Traditional Chinese Medicine (Approved Number: 20190003). The specific pathogen-free (SPF) male BALB/c nude mice (18\u0026thinsp;~\u0026thinsp;20 g), provided by the Hangzhou Medical College, were raised in the Zhejiang Research Institute of Traditional Chinese Medicine.\u003c/p\u003e \u003cp\u003eThe mice were randomly divided by weight into three groups: con group (distilled water, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6), model group (distilled water, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12), RGLS group (0.7 g/kg, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12, the dosage, based on clinical use, was weekly adjusted to match the mice's weight changes). Continuous administration for 45 days and S-180 cells (4\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells in 200 \u0026micro;L PBS) were subcutaneously transplanted into mice on day 38 of administration, and all mice were sacrificed seven days later. We first excised tumors from six mice for each group, and these tumors were stored immediately in liquid nitrogen for omics study (three for proteomics and three for transcriptomics study). For another six mice in each group, we weighed body and tumor weight. Then, three tumors per group were placed in formalin solution for pathological detection, and another three per group were placed in 4% formaldehyde for hematoxylin and eosin (H\u0026amp;E) staining.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTumor tissue histological examination.\u003c/em\u003e The harvested tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-\u0026micro;m sections. Then, the sections were stained with H\u0026amp;E according to the previous description \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The morphological changes in tumor tissues were observed, and images were randomly captured by a microscope (DM4000, Leica Biosystems).\u003c/p\u003e \u003cp\u003e \u003cem\u003eImmunohistochemical analysis of tumor tissues.\u003c/em\u003e The expression level of Ki-67 in tumor tissues was detected by immunohistochemistry (IHC) referred to previous reports \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The images were randomly captured by a microscope (DM4000, Leica Biosystems).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePreparation of RGLS solution for in vitro experiments.\u003c/em\u003e RGLS was dissolved in DMEM as stock solution of 10 mg/mL, and then passed through 0.22-\u0026micro;m filter and diluted to required concentrations.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell culture.\u003c/em\u003e Mycoplasma-free S-180 cells and Raw264.7 were cultured with Dulbecco's Modified Eagle's Medium (DMEM, Bio-channel, Nanjing, China) supplemented with 10% fetal bovine serum (FBS, Genom, Hangzhou, China) and 1% penicillin-streptomycin (Bio-channel, Nanjing, China), and maintained in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell viability assay.\u003c/em\u003e S-180 and Raw264.7 cells were seeded into 96-well plates at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well and incubated in DMEM medium containing 10% FBS until cells reach 50% confluence. Cells well then treated with different concentration of RGLS for 24 h. Then 10% CCK-8 solution was added and incubated for an additional 2 h. After incubation, the absorbance was measured at 450 nm using a multi-well plate reader (Molecular Devices Instrument Inc., California, USA). The relative cell viability was calculated according to the formula: relative cell viability (%) = (\u003cem\u003eA\u003c/em\u003e\u003csub\u003eRGLS\u003c/sub\u003e-\u003cem\u003eA\u003c/em\u003e\u003csub\u003eblank\u003c/sub\u003e)/(\u003cem\u003eA\u003c/em\u003e\u003csub\u003eCon\u003c/sub\u003e-\u003cem\u003eA\u003c/em\u003e\u003csub\u003eblank\u003c/sub\u003e) \u0026times; 100%, where \u003cem\u003eA\u003c/em\u003e is the absorbance.\u003c/p\u003e \u003cp\u003e\u003cem\u003eWestern blotting.\u003c/em\u003e Total protein of S-180 and Raw264.7 cells were extracted using ice-cold RIPA buffer (Beyotime, Beijing, China), and total protein concentrations were measured by BCA protein assay kit (Thermo Scientific, Waltham, Massachusetts, USA), and the expression levels of Casp3, Bax, Bcl-xL, LC3B, p62, Pla2g7, and β-Actin were detected according to previous reports \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The antibodies concentrations as listed in table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, and the semi-quantitative analysis of optical density was done using ImageJ 1.41 software (Bethesda, MD, USA).\u003c/p\u003e \u003cp\u003e \u003cem\u003eRNA isolation and quantitative real-time PCR.\u003c/em\u003e Total RNA isolation was performed using EASYspin reagent Kit (Aidlab Biotech, Beijing, China) according to the manufacturer\u0026rsquo;s protocol. Both the quality and quantity of total RNA were analyzed by the NanoDrop\u0026trade; one spectrophotometer (Thermo Scientific, Grand Island, NY, USA). First-strand cDNA was synthesized from 1 \u0026micro;g of total RNA using iScript Reverse transcription supermix (Vazyme Biotech, Nanjing, China). qRT-PCR analysis was performed using Faststart Essential DNA Green Master (Roche, Basel, Swit) on LightCycler96 Real-Time PCR system (Roche). Primers were designed with open-sourced software Primer3Plus (Cambridge, MA, USA). GAPDH was used as the reference gene. The relative expression of mRNA was calculated by following the previous literature \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. qRT-PCR primer sequences are shown in table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eFlow cytometric analysis of apoptosis.\u003c/em\u003e S-180 cells were seeded into 6-well plates at a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and treated with different concentrations of RGLS for 24 h. Cells were then washed with PBS, harvested by trypsinization and washed twice with PBS, and cells were stained with propidium iodide (PI) and annexin V-FITC using Annexin V-FITC apoptosis detection kit (Beyotime, Beijing, China) according to the manufacturer's instructions. The stained cells were analyzed by C6 plus flow cytometer (BD, New Jersey, USA), and the percentage of early apoptosis (annexin V\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e\u0026minus;\u003c/sup\u003e), late apoptosis (annexin V\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e+\u003c/sup\u003e), and necrosis (annexin V\u003csup\u003e\u0026minus;\u003c/sup\u003e/PI\u003csup\u003e+\u003c/sup\u003e) cells were analyzed. Data was represented as rate of total apoptosis cells with both early and late apoptotic rate indicated.\u003c/p\u003e \u003cp\u003e \u003cem\u003eATP content detection.\u003c/em\u003e Intracellular ATP content detection was using ATP content assay kit (Solarbio life sciences, Beijing, China) according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLabel-free proteomic analysis.\u003c/em\u003e Total protein of tumor tissue was extracted, enzymatically digested, and desalted before separation using nanoliter reversed-phase liquid chromatography and detection by mass spectrometry. Protein abundance was quantified using a Proteome Discoverer library search. Before performing differential protein expression analysis, we used the mice (v3.16) package to perform multiple imputations and fill in missing values. Next, we took the logarithm of the protein expression matrix. We then used the edgeR (v3.42) package to identify differential proteins with adjusted \u003cem\u003eP\u003c/em\u003e-values less than 0.05. Finally, we conducted functional enrichment using the clusterProfiler (v4.8) package.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTranscriptomics analysis.\u003c/em\u003e Total RNA of tumor tissue was extracted, and libraries were constructed and sequenced to obtain mRNA sequence information. The sequences were aligned to the mouse mm10 genome using Bowtie2 and GENCODE database, and gene expression was quantified using HTSeq.\u0026nbsp;Gene expression values were then normalized using the TMM method. Differential gene expression analysis was performed using edgeR, with genes having a \u003cem\u003eP\u003c/em\u003e-value less than 0.05 and an absolute fold change greater than two considered as differentially expressed.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMetabonomics analysis.\u003c/em\u003e Total metabolite of S-180 cells (about 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells) was extracted by 1000 \u0026micro;L methanol: acetonitrile: H\u003csub\u003e2\u003c/sub\u003eO (2:2:1), sonicated at 4℃ for 10 min, incubated at -40℃ for 60 min and centrifuged at 4000\u0026times;g for 15 min at 4℃. The supernatant was collected and filtered through 0.22 \u0026micro;m PVDF membrane. Next, the detection, identification, and data analysis of metabolites in S-180 cells were referred to previous reports \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e All data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE) of at least three independent experiments. For comparisons between the two conditions, a student \u003cem\u003et\u003c/em\u003e-test was used. And for multiple comparisons, one-way analysis of variance (ANOVA) with Tukey's test was used. All analyzed were performed using Graphpad Prism 8.0 software (Graphpad Software, Inc., USA), and a value of \u003cem\u003eP\u003c/em\u003e0.05 was a considered to be statistically significant difference.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003eThe research was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the NIH guidelines. All animal experiments were approved by the Animal Ethics Committee of Zhejiang Research Institute of Traditional Chinese Medicine (Approved Number: 20190003).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e \u003cp\u003eJ.H.Y. and Z.H.L. designed the experiments. H.T.P., C.S.L., M.Y.W. and K.Y. performed the experiments. H.T.P. wrote the first draft of the manuscript. X.H.F., J.H.Y., Z.H.L. and Y.P.F. supervised the experiments and edited the manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.H.Y. and Z.H.L. designed the experiments. H.T.P., C.S.L., M.Y.W. and K.Y. performed the experiments. H.T.P. wrote the first draft of the manuscript. X.H.F., J.H.Y., Z.H.L. and Y.P.F. supervised the experiments and edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis study was supported by Zhejiang Science and Technology Major Program (Grant No. 2021C020732), National Natural Science Foundation of China (Grant No.32270028), Scientific and Technological Planning Project of Jilin Province (Grant No. YDZJ202302CXJD002 and No. 20220202109NC) and Central Guiding Local Science and Technology Development Fund Project (Grant No. 2024ZY01009). Zhejiang Research Institute of Traditional Chinese Medicine is acknowledged for its laboratory equipment.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe metabolomics data presented in the study are deposited in the open data repository for metabolomics (metaboLights, https://www.ebi.ac.uk/metabolights/), accession number MTBLS11629. The proteomics data presented in the study are deposited in the iProX repository, accession number PXD056264. The transcriptomics data in the study are deposited in the GEO repository, accession number GSE277633.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBasit, Q., Qazi, H. S. \u0026amp; Tanveer, S. Osteosarcoma and Its Advancement. \u003cem\u003eCancer Treat. Res.\u003c/em\u003e \u003cb\u003e185\u003c/b\u003e, 127\u0026ndash;139. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-031-27156-4_8\u003c/span\u003e\u003cspan address=\"10.1007/978-3-031-27156-4_8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaubert, B., Solmonson, A. \u0026amp; DeBerardinis, R. J. Metabolic reprogramming and cancer progression. \u003cem\u003eSci. 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Proteome Res.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 3158\u0026ndash;3166. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acs.jproteome.6b00356\u003c/span\u003e\u003cspan address=\"10.1021/acs.jproteome.6b00356\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sporoderm-removed Ganoderma lucidum spores, Osteosarcoma, Glycerophospholipid metabolism, fatty acid metabolism, multi-omics analysis","lastPublishedDoi":"10.21203/rs.3.rs-6046286/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6046286/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteosarcoma (OS) is a severe malignancy affecting children and adolescents, with limited effective treatments leading to poor outcomes. This study explored the potential of sporoderm-removed \u003cem\u003eGanoderma lucidum\u003c/em\u003e spores (RGLS) as a novel therapeutic agent against OS and elucidated its mechanism of action. Our \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments showed that RGLS significantly inhibited S-180 OS cell proliferation, colony formation, and migration, while simultaneously inducing apoptosis. Multi-omics analysis revealed that RGLS disrupted glycerophospholipid metabolism by upregulating lysophosphatidylcholine (LysoPC) levels through phospholipase A2 (PLA2)-mediated pathways. Co-culture assays further demonstrated that RGLS promoted the endocytosis of macrophage-derived Pla2g7 protein into S-180 cells, enhancing its anti-cancer effects. Additionally, RGLS modulated the cellular fatty acid profile and suppressed the β-oxidation of long-chain fatty acids, leading to energy depletion in OS cells. These findings provide a novel view of the multi-targeted mechanisms of RGLS, positioning it as a promising candidate for OS therapy.\u003c/p\u003e","manuscriptTitle":"Multi-omics analysis reveals inhibition of osteosarcoma progression by sporoderm-removed Ganoderma lucidum spores via targeting glycerophospholipid and fatty acid Metabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-30 11:26:37","doi":"10.21203/rs.3.rs-6046286/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-15T12:02:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-15T11:58:57+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"35729509359213627565923836367714630567","date":"2025-04-26T05:01:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-23T05:23:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-22T14:31:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-22T01:10:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3296ca19-1127-4573-94ce-93ea0790ad32","owner":[],"postedDate":"April 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47544402,"name":"Biological sciences/Cancer"},{"id":47544403,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":47544404,"name":"Biological sciences/Drug discovery"},{"id":47544405,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-07-07T16:10:24+00:00","versionOfRecord":{"articleIdentity":"rs-6046286","link":"https://doi.org/10.1038/s41598-025-05890-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-01 15:57:35","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-04-30 11:26:37","video":"","vorDoi":"10.1038/s41598-025-05890-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-05890-5","workflowStages":[]},"version":"v1","identity":"rs-6046286","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6046286","identity":"rs-6046286","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}