The anti-apoptotic protein lifeguard is expressed in osteosarcoma, chondrosarcoma and soft tissue sarcoma

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Vogt, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4301676/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The success of modern chemotherapy in overall survival of patients with advanced stages of osteosarcoma and soft tissue sarcoma has reached a plateau. Therefore a deeper understanding of molecular mechanisms behind deregulated apoptosis in sarcoma is essential for the cure of patients with advanced stages of osteosarcoma and soft tissue sarcoma. Lifeguard (LFG) is a member of the Bax Inhibitor-1 (BI-1) protein family and has anti-apoptotic effects by inhibiting Fas-mediated cell death signaling. Although LFG has been proven to be expressed in several breast cancer tissues, the expression and function of LFG regarding apoptosis in different subtypes of sarcoma remains unclear. In the present study, the expression of LFG in osteosarcoma (50 samples), chondrosarcoma (28 samples) and soft tissue sarcoma (total 55 samples) with different tumor stages for each sarcoma subtype were analyzed. For each subtype, clinical TNM-classification and pathological grading were determined and compared to healthy corresponding tissues. Soft tissue sarcoma subtypes included liposarcoma, dermatofibrosarcoma, angiosarcoma, leiomyosarcoma, malignant schwannoma and synovial cell sarcoma. In this study, significantly higher expressions of anti-apoptotic LFG protein in osteosarcoma, chondrosarcoma and many different subtypes of soft tissue sarcoma were found, compared to corresponding healthy tissues. More importantly a positive correlation between LFG expression and tumor stage for osteosarcoma and chondrosarcoma was found. In conclusion, LFG protein might play an important role in inhibition of Fas-mediated apoptosis in osteosarcoma cells, with possible potential for targeted tumor therapy in osteosarcoma. Lifeguard apoptosis osteosarcoma chondrosarcoma soft tissue sarcoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Sarcoma are a heterogeneous group of tumors derived from mesenchymal origin with a distinct tendency of hematogenous metastasis [1]. They can be roughly divided into osteosarcoma, chondrosarcoma and the heterogenous group of soft tissue sarcoma. Although sarcoma only contribute to 1% of human malignancies, they have a high burden in childhood cancer with approximately 14% of all malignancies among children [2]. The prevalence of osteosarcoma, the most common type of malignant bone tumor, depends on age, race, sex, and a number of other factors, and has a peak during the adolescent years [3]. In addition, the prognosis of both, osteosarcoma and soft tissue sarcoma, measured by the 10-year survival, improved over the last decades, but is still not satisfactory [4]. One possible explanation could be, that the enormous pathological diversity of more than 100 different sarcoma subtypes is not jet adequately addressed in standardized sarcoma chemotherapy [4]. Furthermore, secondary cardiac, pulmonary and neurocognitive disorders as well as secondary malignancies have been described in sarcoma long-time survivors after successful chemotherapeutic treatment [5]. Current research on osteosarcoma identified more possible therapeutic targets with a high potential for future targeted tumor therapy and immunotherapy [6], based on a more precise understanding of the molecular mechanisms behind cell-growth signaling, sarcoma microenvironment, and the apoptosis evading strategies of different sarcoma subtypes. One possibility of apoptosis activation is receptor-mediated cell death, such as the Fas/Fas ligand pathway [7]. The human osteosarcoma cell line LM7 showed a high metastatic potential in an experimental nude mouse model based on low Fas expression [8]. In a rodent model, transfection of LM7 cells with Fas led to a significantly reduced incidence, size and weight of tumor nodules compared to the controls [8]. Accordingly, Fas expression was lower in human osteosarcoma lung metastases compared to controls [8]. These findings suggest that the reduction of Fas expression, leading to Fas-mediated cell death, may be a mechanism of increased osteosarcoma metastasis. It remains still unclear how Fas-mediated cell death inactivation contributes to the growth and metastasis of different sarcoma subtypes and its regulation through tumorigenesis. The protein lifeguard (LFG) is a member of the Bax-Inhibitor (BI-1) family that is highly conserved and inhibits programmed cell death via different anti-apoptotic mechanisms that are not completely understood yet [9]. LFG is a hydrophobic, multi-pass transmembrane protein that is located in the plasma membrane and intracellular membranes [10]. The suspected protein structure of lifeguard consists of seven transmembrane domains, a three residues long extracellular C-terminus and a small cytoplasmic domain at the N-terminus [11]. Fernandez et al. indicated that LFG is also present on the lipid rafts of SH-SY5Y human neuroblastoma cells, assuming a mechanistic interaction between LFG and the major side of death inducing signaling complex and caspase-8 [12]. Somia et al. isolated LFG in 1999 for the first time and reported that it uniquely provided protection from Fas-mediated cell death, but not from the tumor necrosis factor α signal pathway [10]. LFG interacts with Fas, without reducing Fas expression or Fas-associated protein with death domain (FADD) binding [12]. The distribution of LFG in healthy human tissues is diverse, ranging from ubiquitous occurrence in almost all tissues, except spleen and placenta [11,12], to a high expression in cerebral and cerebellar tissue [13,14]. Furthermore, LFG is highly expressed in breast cancer cell lines MCF-7, MDA-MB-231 and T47D and in native breast cancer tissue [15]. The level of LFG expression correlated with high tumor grades in primary breast tumors [15]. A high LFG expression could be related to decreased Fas sensitivity in breast cancer cell lines [15]. LFG might even be a novel therapeutic target in breast cancer and sarcoma, as down-regulation of LFG sensitized solid breast cancer cell line MCF-7 and sarcoma cell line SW872 to perifosine-induced cell death activation caused by an agonistic Fas antibody [16]. However, the exact molecular regulation of LFG in human malignancies remains unclear. In breast cancer cell lines the expression of LFG correlated with glycogen synthase kinase-3 (GSK3β) and LEF-1 activation [16]. Further evidence for the role of the Akt-lymphoid enhancer-binding factor-1 (LEF-1) pathway for the regulation of LFG in human breast cancer cells could be seen in small interfering RNA (siRNA) based on transfection experiments [16]. LFG messenger RNA (mRNA) was down regulated after transfection with siRNA against LEF1 in MDA-MB-231 cells [16]. The opportunity of osteosarcoma to gain resistance to adjuvant chemotherapy suggests that a reduced sensitivity regarding Fas-mediated apoptosis could play an important role in sarcoma cells as well [6]. Moreover current research showed that osteosarcoma cells could increase their viability during intravasation and dedifferentiation by inducing their expression of LFG/FAIM2 triggered by higher serum albumin concentration in the angiorrhea region [17]. Based on these findings, a higher expression of LFG in osteosarcoma is postulated to promote early pulmonary metastasis, leading to a prevalence of 10 to 20% when being diagnosed [4]. But the exact anti-apoptotic function of LFG in osteosarcoma, chondrosarcoma and soft tissue sarcoma remains unclear. The aim of the present study was the analysis of LFG expression in human osteosarcoma, chondrosarcoma and soft tissue sarcoma samples using immunohistochemistry. Samples were compared to healthy human tissue as controls. Materials and Methods LFG protein structure The three-dimensional (3D) model of the protein structure of LFG was created with the help of SWISS-MODEL® software (page accessed on March 10, .2023). SWISS-MODEL® is an automated protein homology server. The LFG protein structure was based on comparative modeling and needed a given amino acid sequence, deposited in the protein data bank “uniprot” (uniprot PDB) for the modeling process. The structural template was identified at uniprot and added to SWISS-MODEL® server. The alignment of target sequence and template structures, model building and model quality evaluation were done automatically by the server. The modeling results of the predicted LFG protein structure were compared to other modeling processes and evaluated using global model quality estimate (GMQE) values. Osteosarcoma and chondrosarcoma samples For LFG expression analysis in osteosarcoma and chondrosarcoma the commercially available tissue array (OS802c, hematoxylin-eosin stain, Biomax US, Derwood, MD, USA), containing 80 different patient samples (1 core/case), was used. According to the manufactures’ specifications 50 samples of osteosarcoma, 28 samples of chondrosarcoma and 2 samples of healthy human bone tissue were included in the analysis and were prepared for immunohistochemistry. For each tumor sample, pathological grading and clinical findings, such as TNM-classification, were included in the analysis. The healthy human bone samples served as controls. Soft tissue sarcoma samples For LFG expression analysis in soft tissue sarcoma the commercially available soft tissue sarcoma array (SO208a, hematoxylin-eosin stain, Biomax US, Derwood, MD, USA) was used. According to the manufacturers’ specifications, 55 samples of different soft tissue sarcoma subtypes, 4 samples of healthy human smooth muscle tissue and 5 samples of healthy human skeletal muscle tissue, were analyzed and prepared for immunohistochemistry. For each tumor sample, pathological grading and clinical findings such as TNM-classification were included in the analysis. The healthy human skeletal and smooth muscle samples served as controls. Sample preparation The osteosarcoma and the soft tissue sarcoma tissue arrays were deparaffinized in xylen (Lab Alley, Spicewood,TX, USA) and rinsed with phosphate buffered saline (PBS) (Thermo Scientific, Schwerte, Germany) for 5 minutes (min) each. The slides were immersed in a descending alcohol concentration row (100% ethanol, 80% ethanol, 70% ethanol) for 2 min each. Immunohistochemistry For antigen retrieval the slides were pressure cooked in 6.5 mM sodium citrate (pH 6.0) (Thermo Scientific, Schwerte, Germany) and washed thrice with PBS (Thermo Scientific, Schwerte, Germany). Specific background staining was reduced by incubating the slides in 2.5% bovine serum albumin/1X Tris-buffered saline (Lab Alley, Spicewood, TX, USA) for 60 min. To optimize primary antibody binding, the slides were incubated at 4ºC overnight with rabbit anti-hLFG primary antibody (Santa Cruz Biotechnology, Dallas, TX, USA; final dilution 1:100) dissolved in 1% bovine serum albumin/PBS solution (Thermo Scientific, Schwerte, Germany). After incubation, the slides were washed with PBS thrice for 5 min. For secondary antibody binding the slides were incubated with goat anti-rabbit 800CW conjugated secondary antibody (IRDye® Li-Cor, Lincoln, USA; final dilution, 1:100) at room temperature for 30 min. The Li-Cor infrared imaging System Odyssey® (Li-Cor Biosiences, Lincoln, NE, USA) was used to detect the signals. For further analysis the Image Studio Software Odyssey® (Li-Cor Biosiences, Lincoln, NE, USA) was used to visualize LFG protein expression. The relative fluorescent units, measured by Li-Cor Infra-Red Imaging System, were used to quantify LFG protein expression. Statistical analysis The LFG protein expression was observed for each subtype of sarcoma in different tumor stages, matched to healthy tissue controls. The tumor stages were based on clinical findings, specified according TNM-classification (World Health Organization 2017) and pathological grading. The means of LFG expression and the confidence intervals were determined on basis of measured LFG fluorescent units for each sarcoma subtype and stage. p<0,05 was determined significant. The results were calculated using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and visualized with Datatab software (Datatab, Seiersberg, Austria). Results LFG protein simulation To visualize the 3D structure of the LFG protein, the sequence based SWISS-MODEL® software was used (Figure 1). Figure 1A shows the expected LFG monomer protein structure that consists of seven transmembrane domains (blue), that pass multiple times through the plasma membrane and are linked to a seperate protein tail (orange). Figure 1B shows the template alignment of LFG, as given by the uniprot protein data bank. The high expression of LFG protein in osteosarcoma tissues As above described, osteosarcoma samples were analyzed using commercially available immunofluorescence arrays. Table 1 gives an overview of the different anatomical localizations of the samples, the corresponding TNM classification, tumor grading, tumor stage, number of samples and mean LFG intensity. Osteosarcoma tissues from various anatomical regions were included, predominantly coming from femur and tibia (Table 1). The mean LFG expression in osteosarcoma stage IIA (54,7; CI=47,1; 62,4) (p<0,05) was significantly higher than in normal bone marrow tissue (20,7; CI= 18,8; 22,8) (p<0,05). In stage IIB osteosarcoma, the mean LFG expression was calculated to 61,1 (CI=56,9; 65,3), being significantly higher compared to controls (p<0,05). Interestingly, the LFG expression seemed to correlate with the tumor stage, being higher in osteosarcoma stage IIB than in stage IIA as indicated by the relative fluorescent units (stage IIA 54,7 ± 7,7; stage IIB 61,1 ±4,2) (p<0,05) (Fig. 2, I). Samples with advanced osteosarcoma stage IIB had a larger tumor size, compared to IIA, and had the highest mean LFG expression of all osteosarcoma samples in the analysis. Both samples with osteosarcoma stage IIA and IIB had grade 3 tumors in which the sarcoma cells grew more aggressively and showed abnormal cell morphology. Figure 2, II shows a representative selection of LFG antibody stained fluorescent images of osteosarcomas. The increase in the LFG intensity in osteosarcoma stage IIB (Figure 2, IIC) , compared to stage IIA (Figure 2, IIB) and the controls (Figure 2, IIA) can be seen in the constant increase of green coloring from Figure 2, IIA to Figure 2, IIC. The high expression of LFG protein in chondrosarcoma Table 2 gives an overview of the different tumor localizations, tumor grading, tumor stage and mean LFG intensity of chondrosarcoma samples. The mean LFG expression in chondrosarcoma stage IA was significantly higher compared to normal bone marrow tissue (stage IA=44,3; controls=20,7) with a p-value <0,05 (Fig. 3,I). A significant higher LFG expression in chondrosarcoma stage IIA was oberserved (53,4; CI=42,4; 64,5) (p<0,05) compared to chondrosarcoma stage IA (36,1; CI=32,4; 39,8) (p<0,05) (Fig. 3,I). Although a significant increase in LFG expression could not be seen in all stages, the data assume a constant positive correlation between LFG expression and chondrosarcoma stages. The LFG expression level detected in stage IIB chondrosarcoma (49,3; CI=39,5; 59,1) was lower than in stage IIA (53,4; CI=42,4; 64,5) (p>0,05). Figure 3,II,A-E shows fluorescent images of chondrosarcoma samples with different tumor stages. The LFG intensity is expressed by green immunofluorescence. Healthy bone marrow tissue showed the lowest LFG expression compared to all subtypes of chondrosarcoma (Figure 3, IIA). The LFG expression increases from chondrosarcoma stage IA (Figure 3, IIB) to stage IB (Figure 3, IIC), as shown by an increase in intensitiy of the green immunoflourescence. Figure 2, IID shows the high LFG expression in chondrosarcoma stage IIA, significantly higher compared to controls (Figure 3, IIA) and stage IA chondrosarcoma (Figure 3, IIB). Figure 3, IIE shows the LFG expression of chondrosarcoma stage IIB being significantly higher compared to controls (Figure 3, IIA), but lower compared to stage IIA (Figure 3, IID). The high expression of LFG protein in soft tissue sarcoma Taking into consideration that the anti-apoptotic protein LFG showed high expression levels in osteosarcoma and chondrosarcoma, the expression of LFG in different subtypes of soft tissue sarcoma was analyzed. Table 3 shows the different soft tissue sarcoma subtypes that were investigated regarding LFG expression. A total number of 55 soft tissue sarcoma tissue samples were analyzed, using 5 skeletal muscle samples and 4 endometrium smooth muscle samples as unmatched controls. Table 3 gives an overview of the different soft tissue subtypes, the corresponding TNM classification, tumor grading, tumor stage and mean LFG intensity of the samples. The means of LFG expression were calculated for each soft tissue subtype as a whole by summarizing the data of all corresponding stages for each subtype (Table 3). Overall, the LFG expression variated strongly dependening on the soft tissue subtype (Figure 4A). Liposarcoma showed low LFG expression (47,4 ±4,2) (p<0,05), not noticeable compared to skeletal muscle control (45,8 ±3,9) (p<0,05), whereas malignant schwannoma (86,3 ±28,5) (p<0,05), dermatofibrosarcoma (65,9 ±9) (p<0,05) and leiomyosarcoma (90,6 ±25,4) (p<0,05) had a significantly higher LFG expression compared to skeletal muscle control (45,8 ±3,9) (p<0,05) and smooth muscle control (49,3 ±5,8) (p0,05) (Figure 4A). Figure 4B shows that LFG expression in dermatofibrosarcoma stage IA (63,5 ±8,8) (p<0,05) that was significantly higher compared to the controls (p<0,05), with an increase in LFG expression from stage IA (63,5 ±8,8) (p<0,05) to more advanced stage IB (82,3; no CI). The LFG expression correlated with malignant schwannoma tumor stage, increasing from stage IA (56,2; no CI) to stage IIA (74,7; no CI) and stage IIB (124,9; no CI) (Figure 4C). The LFG expression of malignant schwannoma stage III (89,6; no CI) was observed lower compared to stage IIB (124,9; no CI) (Figure 4C). The highest LFG expression of all specimen was detected in leiomyosarcoma stage IA (139,3; no CI) (Figure 4D). The LFG expression in leiomysarcoma stage IIB (77,0 ±15,4) (p<0,05) showed to be significantly higher compared to both controls (p<0,05) (Figure 4D).The LFG expression in leiomyosarcoma is exemplarily shown in Figure 5. Figure 5A shows an immunofluorescent image of endometrium smooth muscle as control with low LFG expression. The highest LFG expression of all specimen was detected in leiomyosarcoma stage IA presenting with multiple, widely distributed fluorescent signals (Figure 5B). Figure 5C shows the high LFG expression of leiomyosarcoma stage IIB, being significantly higher (p<0,05) compared to endometrium smooth muscle control (Figure 5A). Figure 5D shows an image of the high LFG expression that was observed in leiomyosarcoma stage III. Discussion The present study is among the first to describe LFG expression in different types of sarcoma. The data presented show convincing evidence that LFG expression is significantly higher in advanced stages of osteosarcoma and chondrosarcoma compared to localized tumor stages and their original tissue. More importantly LFG expression was found to increase with osteosarcoma and chondrosarcoma tumor stage. In addition a significantly higher expression of anti-apoptotic LFG was observed in soft tissue sarcoma subtypes, including dermatofibrosarcoma, malignant schwannoma and leiomyosarcoma. The correlation between LFG expression and tumor stage was reduced in subtypes of soft tissue sarcoma, compared to osteosarcoma and chondrosarcoma. Although sarcoma are a rare tumor entitiy, osteosarcoma are the most common malignant bone tumors in pediatrics, with approximately 4-5 cases per 1 million children in developed countries [3]. Additionally to the rareness of sarcoma, their pathologic heterogeneity with more than 100 different subtypes, makes modern multimodal treatment in specialized sarcoma centers very challenging [18]. For optimal treatment, an early image guided biopsy in specialized centers for diagnosis followed by a multi-disciplinary therapy approach is mandatory [19]. The multimodal and patient specific treatment, includes an individualized concept with surgery, standardized chemotherapy, targeted tumor therapy, immunotherapy and/or radiation [6]. Early hematogenous metastasis of even localized osteosarcoma often require a patient specific combination chemotherapy that improved the 5-year survival rates from 20% in the 1970s to approximately 60-80% in the last decades [20]. Overall, 10% of all sarcoma patients show metastatic lesions at the time of diagnosis [20]. When patients are diagnosed with metastatic disease, the 5-year survival rate drops from 75% to 20% [2]. Therefore a deeper understanding of the molecular mechanisms behind tumor growth and metastasis of osteosarcoma and soft tissue sarcoma as well as their ability to reduce sensitivity to standardized chemotherapy is crucial for future targeted tumor therapy approaches [6]. One key point of tumor progression and resistance to chemotherapy in osteosarcoma is the inhibition of apoptosis by multiple different mechanisms, including mutations in extrinsic and intrinsic apoptotic pathways [21]. Various apoptotic agents are reported to contribute to programmed cell death in osteosarcoma cell lines in vitro such as micro RNA (miRNA) [22], natural compounds like chimaphilin [23] and icaritin [24] and numerous apoptotic proteins [21]. Pro-apoptotic proteins, for example inhibitor of growth protein 4 (ING4), induced the apoptosis of osteosarcoma cells by the blockage of the NF-kB signaling pathway and activation of the intrinsic pathway trough decreasing the ratio of Bcl-2/Bax [25]. The Runx2 gene, an important transcription factor that is involved in osteoblast maturation and bone development, turned out to be overexpressed in many osteosarcoma cells [21]. Furthermore the loss of Runx2 in osteosarcoma sensitized the cells to doxo-induced apoptosis in vitro and in vivo [26]. Another regulator of apoptosis in osteosarcoma can be found in the enhancer of zeste homolog 2 (EZH2) protein, which corresponds to the catalytic subunit of polycomb repressive complex 2 and showed higher expression in patients with advanced tumor stages [27]. Moreover silencing of EZH2 in osteosarcoma by siRNA reduced osteosarcoma cell growth, invasion and lung metastasis [28]. Another pathway resulting in extrinsic programmed cell death is the Fas/Fas ligand system, which results in activation of caspase-8 and subsequent caspase cascades, initialized by ligation of Fas to its agonistic antibody and formation of death-inducing signaling complex [29,30]. In addition to the intrinsic mitochondria pathway, Fas-mediated programmed cell death inhibition seems to be not only highly relevant for apoptosis, but also for metastasis in human osteosarcoma, shown by reduced Fas expression levels in LM7 cell lines with high metastatic potential [8]. The studies of Koshkina et. al give further evidence to the idea, that osteosarcoma cells that reduce their Fas expression, get promoted and positively selected during the process of metastasis in the Fas-Ligand (FasL)-positive microenvironment of the lung [31,32]. Osteosarcoma cells with higher Fas expression, might be eliminated by FasL induced apoptosis in the lung, resulting in the selection of Fas-negative cell populations [31]. It was shown that Fas/FasL-mediated apoptosis signaling in osteosarcoma during lung metastasis did not only depended on the amount of Fas expression [32]. In addition, down regulation of Fas-signaling might serve as a gatekeeper for metastasis [32]. Supporting this hypothesis, osteosarcoma K7 cells were transfected with FADD-dominant negative (FDN), resulting in K7/FDN cells, that showed the ability to form Fas-positive metastases in a mice model with higher metastatic potential than untransfected K7 cells [32]. The expression of FasL, although being lower than in carcinoma, was detected in many subtypes of sarcoma, being predominantly high in rhabdomyosarcoma, malignant schwannoma and Ewing sarcoma [33]. Taking these findings together, Fas-mediated cell death seems to be essential for both evading apoptosis and enabling metastasis of osteosarcoma by altering Fas expression and signaling [31-33]. Therefore it is necessary to examine the expression of more anti-apoptotic proteins in different sarcoma tissues that interfere with Fas-mediated apoptosis, such as LFG also known as FAS apoptosis inhibitory molecule 2 (FAIM2) [10]. The endogenous expression of LFG in human cancer tissue was already shown for many breast cancer tissues and cell lines, being positively correlated with tumor stage and causing a reduced sensitivity to Fas-signaling without changing the levels of Fas expression [15]. In contrast to past research, that indicated a physical association between LFG and Fas in healthy human tissue [10,13], results in Fas-antibody immunofluorescence showed no co-localization between LFG and Fas in MCF-7 breast cancer cell lines, supposing there could be an altered function of LFG in human cancer tissues [15,16]. Currently, the data regarding LFG in sarcoma tissue is scarce. Therefore the expression and exact function of LFG in clinical related phenotypes of sarcoma needs to be investigated. The results of the present study show that LFG is significantly higher expressed in tissues of osteosarcoma and chondrosarcoma than in healthy bone tissue (p<0,05). Additionally the level of LFG expression was different in several subtypes of soft tissue sarcoma, with the highest expression in dermatofibrosarcoma, malignant schwannoma and leiomyosarcoma. Furthermore, LFG expression positively correlated with tumor stages of osteosarcoma (IIB>IIA, p>0,05) and was significantly higher in advanced stages of chondrosarcoma (IIA>IA, p<0,05). The high LFG expression in osteosarcoma can be seen as another possible explanation for the decrease of Fas-mediated apoptosis in advanced tumor diseases [33]. The correlation of LFG expression in osteosarcoma to the clinical stages underlines the importance of LFG for osteosarcoma cell growth and supports the concept of LFG guided cell dedifferentiation within metastatic cell populations [31-33]. In addition, Pan et. al. stated that LFG overexpression enabled lower malignant osteosarcoma to survive in lower serum albumin environments, that might act as a defense system against growing tumor lesions during hematogenous metastasis [17]. However, only higher levels of serum albumin, occurring in nearby neovascularization, empower osteosarcoma cells with high expression of LFG via calcium mediated interactions to enter the circulatory system and form metastasis in distant organs [17]. Unlike with Bax/Bcl-xL, for which high expression rates have been demonstrated in chondrosarcoma [34-36] this is the first time that high LFG expression could be correlated to chondrosarcoma and special subtypes of soft tissue sarcoma. The anti-apoptotic properties of LFG in advanced stages of chondrosarcoma might represent another explanation that chemotherapy resistance added to a higher expression of anti-apoptotic Bcl-2 proteins and the presence of multidrug resistance (MDR) pumps [36]. Nevertheless a more detailed research on LFG expression and function, compared to related BI-1 expression in osteosarcoma and chondrosarcoma is needed. Concluding the LFG expression analysis of the present study, significantly higher levels of LFG expression in leiomyosarcoma might be linked to the aggressive tumor growth and high resistance rates to chemotherapy of this sarcoma subtype [37]. Although no continuous correlation could be found between tumor stage and LFG expression in soft tissue sarcoma, significant higher LFG expression rates, compared to adjacent healthy tissue, were observed in all soft tissue sarcoma subtypes except liposarcoma and synovial cell sarcoma. More research, including higher numbers of soft tissue sarcoma specimen and cell lines, is needed to clarify the expression of LFG and its role regarding tumorigenesis in soft tissue sarcoma. In summary, high LFG expression could be found in osteosarcoma, chondrosarcoma and several soft tissue sarcoma subtypes. LFG expression even correlated with tumor stage in osteosarcoma and chondrosarcoma and might function as a predictive marker for tumor progression. Further research LFG expression and function, including apoptosis related changes in tumor gene expression and LFG knockout experiments in vitro and in vivo will help to evaluate the potential of LFG as a novel target for sarcoma therapy. Declarations Acknowledgement : The authors are grateful to Andrea Lazaridis for her excellent technical assistance. Author Contributions: Conceptualization: Sebastian Senger, Sarah Strauß, Peter M. Vogt, Frederik Schlottmann, Vesna Bucan. Data curation: Sebastian Senger, Vesna Bucan. Formal analysis: Sebastian Senger, Vesna Bucan. Funding acquisition: Peter M. Vogt, Vesna Bucan. Investigation: Sebastian Senger, Andrea Lazaridis, Vesna Bucan. Methodology: Sebastian Senger, Sarah Strauß, Vesna Bucan. Project administration: Sarah Strauß, Peter M. Vogt, Vesna Bucan. Resources: Sarah Strauß, Vesna Bucan. Supervision: Sarah Strauß, Peter M. Vogt, Vesna Bucan. Validation: Sarah Strauß, Vesna Bucan. Visualization: Sebastian Senger, Vesna Bucan. Writing – original draft: Sebastian Senger. Writing – review & editing: Sebastian Senger, Sarah Strauß, Frederik Schlottmann, Peter M. Vogt, Vesna Bucan. Competing Interests: We have no conflicts of interest to disclose. Ethical statement: This research received no external funding. This publication is funded by the Deutsche Forschungsgemeinschaft (DFG) as part of the “Open Access Publikationskosten” program. References Miwa S, Yamamoto N, Tsuchiya H. Sarcoma: Molecular Pathology, Diagnostics, and Therapeutics. Int J Mol Sci 2023 Mar 19;24(6):5833. doi: 10.3390/ijms24065833. HaDuong JH, Martin AA, Skapek SX, Mascarenhas L. Sarcomas. Pediatr Clin North Am 2015 Feb;62(1):179-200. Sadykova LR, Ntekim AI, Muyangwa-Semenova M, Rutland CS, Jeyapalan JN, Blatt N, et al. Epidemiology and Risk Factors of Osteosarcoma. Cancer Invest 2020 May;38(5):259-269. 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Chimaphilin inhibits human osteosarcoma cell invasion and metastasis through suppressing the TGF-β1-induced epithelial-to-mesenchymal transition markers via PI-3K/Akt, ERK1/2, and Smad signaling pathways. Can J Physiol Pharmacol 2018 Jan;96(1):1-7. Wang X, Wang J. Icaritin suppresses the proliferation of human osteosarcoma cells in vitro by increasing apoptosis and decreasing MMP expression. Acta Pharmacol Sin 2014 Apr;35(4):531-539. Li M, Zhu Y, Zhang H, Li L, He P, Xia H, et al. Delivery of inhibitor of growth 4 (ING4) gene significantly inhibits proliferation and invasion and promotes apoptosis of human osteosarcoma cells. Sci Rep 2014 Dec 9;4:7380. Roos A, Satterfield L, Zhao S, Fuja D, Shuck R, Hicks MJ, et al. Loss of Runx2 sensitises osteosarcoma to chemotherapy-induced apoptosis. Br J Cancer 2015 Nov 3;113(9):1289-1297. Sun R, Shen J, Gao Y, Zhou Y, Yu Z, Hornicek F, et al. Overexpression of EZH2 is associated with the poor prognosis in osteosarcoma and function analysis indicates a therapeutic potential. Oncotarget 2016 Jun 21;7(25):38333-38346. Lv Y, Yan G, Meng G, Zhang X, Guo Q. Enhancer of zeste homolog 2 silencing inhibits tumor growth and lung metastasis in osteosarcoma. Sci Rep 2015 Aug 12;5:12999. Hengartner MO. The biochemistry of apoptosis. Nature 2000 Oct 12;407(6805):770-776. Nagata S, Golstein P. The Fas death factor. Science 1995 Mar 10;267(5203):1449-1456. Huang G, Nishimoto K, Yang Y, Kleinerman ES. Participation of the Fas/FasL signaling pathway and the lung microenvironment in the development of osteosarcoma lung metastases. Adv Exp Med Biol 2014;804:203-217. Koshkina N, Yang Y, Kleinerman ES. The Fas/FasL Signaling Pathway: Its Role in the Metastatic Process and as a Target for Treating Osteosarcoma Lung Metastases. Adv Exp Med Biol 2020;1258:177-187. Lee SH, Jang JJ, Lee JY, Kim SY, Park WS, Kim CS, et al. Immunohistochemical analysis of Fas ligand expression in sarcomas. Sarcomas express high level of FasL in vivo. APMIS 1998 Nov;106(11):1035-1040. Chen C, Zhou H, Wei F, Jiang L, Liu X, Liu Z, et al. Increased levels of hypoxia-inducible factor-1α are associated with Bcl-xL expression, tumor apoptosis, and clinical outcome in chondrosarcoma. J Orthop Res 2011 Jan;29(1):143-151. Shen Z, Nishida K, Doi H, Oohashi T, Hirohata S, Ozaki T, et al. Suppression of chondrosarcoma cells by 15-deoxy-Delta 12,14-prostaglandin J2 is associated with altered expression of Bax/Bcl-xL and p21. Biochem Biophys Res Commun 2005 Mar 11;328(2):375-382. van Oosterwijk JG, Herpers B, Meijer D, Briaire-de Bruijn IH, Cleton-Jansen AM, Gelderblom H, et al. Restoration of chemosensitivity for doxorubicin and cisplatin in chondrosarcoma in vitro: BCL-2 family members cause chemoresistance. Ann Oncol 2012 Jun;23(6):1617-1626. Bourcier K, Le Cesne A, Tselikas L, Adam J, Mir O, Honore C, et al. Basic Knowledge in Soft Tissue Sarcoma. Cardiovasc Intervent Radiol 2019 Sep;42(9):1255-1261. Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.tiff Table 1: LFG expression in human osteosarcoma Table2.tiff Table 2: LFG expression in human chondrosarcoma subtypes Table3.tiff Table 3: LFG expression in human soft tissue sarcoma subtypes Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-4301676","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":295033790,"identity":"b0c3f4a0-1bd2-48a8-b107-d0d047ae3f19","order_by":0,"name":"Sebastian Senger","email":"data:image/png;base64,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","orcid":"","institution":"Hannover Medical School","correspondingAuthor":true,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Senger","suffix":""},{"id":295033791,"identity":"4b02cce4-19fd-4e00-bb3b-0c314b2e29ed","order_by":1,"name":"Frederik Schlottmann","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Frederik","middleName":"","lastName":"Schlottmann","suffix":""},{"id":295033792,"identity":"e007f6c8-de75-43d8-acfc-e745cd979399","order_by":2,"name":"Sarah Strauß","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Strauß","suffix":""},{"id":295033793,"identity":"22eff29c-1403-4b9e-9537-3c016157dbe7","order_by":3,"name":"Peter M. Vogt","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"M.","lastName":"Vogt","suffix":""},{"id":295033794,"identity":"e43e49fe-aed2-48a2-a9ee-5651f77fa047","order_by":4,"name":"Vesna Bucan","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Vesna","middleName":"","lastName":"Bucan","suffix":""}],"badges":[],"createdAt":"2024-04-21 17:25:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4301676/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4301676/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55553190,"identity":"82906480-4325-4a5b-b34e-853f2aacd698","added_by":"auto","created_at":"2024-04-29 22:21:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":243538,"visible":true,"origin":"","legend":"\u003cp\u003eHuman lifeguard/FAIM2 protein model created with Swiss Model software. \u003cstrong\u003eA\u003c/strong\u003e – Resulting model of LFG monomer protein structure based on amino acid template. Seven transmembrane domains (blue) pass through plasma membrane (grey) (created: 03-25-2023) \u003cstrong\u003eB – \u003c/strong\u003eTemplate alignment Q1LZ71.1.A. for LFG, added from uniprot protein data bank (created: 03-25-2023). Colors represent the confidence gradient of the model (blue=high confidence) and (orange=low confidence).\u003c/p\u003e","description":"","filename":"EntwurfFigure114.11.23.png","url":"https://assets-eu.researchsquare.com/files/rs-4301676/v1/ef423381163563d9de996955.png"},{"id":55553191,"identity":"f6b7010d-328e-4ead-90b5-44bcd7dd5cf5","added_by":"auto","created_at":"2024-04-29 22:21:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":384375,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of LFG in human osteosarcoma \u003cstrong\u003eI\u003c/strong\u003e – The fluorescence was quantified and normalized to cdc6 expression. Graphical visualization of LFG expression, measured by relative fluorescent units, in different osteosarcoma tumor stages (IIA, IIB in green) compared to normal bone marrow as control in orange. The data is presented as means ± 95%-confidence interval, * represents p\u0026lt;0,05; ** represents p\u0026lt;0,01. \u003cstrong\u003eII \u003c/strong\u003e– Immunofluorescent images of LFG expression in different stages of osteosarcoma IIA and IIB compared to normal bone marrow as control. \u003cstrong\u003eIIA\u003c/strong\u003e – LFG expression in healthy bone marrow tissue. \u003cstrong\u003eIIB\u003c/strong\u003e – LFG expression in stage IIA osteosarcoma tissue. \u003cstrong\u003eIIC\u003c/strong\u003e– LFG expression in stage IIB osteosarcoma tissue.\u003c/p\u003e","description":"","filename":"EntwurfFigure214.11.23.png","url":"https://assets-eu.researchsquare.com/files/rs-4301676/v1/0cf170fa65894a33e303a8d8.png"},{"id":55552179,"identity":"698781c1-54e5-4551-b4a2-d304ebb2834f","added_by":"auto","created_at":"2024-04-29 22:13:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":358985,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of LFG in human chondrosarcoma. \u003cstrong\u003eI\u003c/strong\u003e– The fluorescence was quantified and normalized to cdc6 expression. Graphical visualization of LFG expression, measured by relative fluorescent units, in different chondrosarcoma tumor stages IA, IB (green); IIA, IIB (blue) compared normal bone marrow as control (orange). The data are presented as means ± 95%-confidence intervals, * represents p\u0026lt;0,05; ** represents p\u0026lt;0,01. \u003cstrong\u003eII \u003c/strong\u003e– Immunofluorescent images of LFG expression in different stages of chondrosarcoma IA, IB, IIA and IIB compared to normal bone marrow as control. \u003cstrong\u003eIIA\u003c/strong\u003e – LFG expression in healthy bone marrow. \u003cstrong\u003e\u0026nbsp;IIB\u003c/strong\u003e – LFG expression in stage IA chondrosarcoma. \u003cstrong\u003eIIC\u003c/strong\u003e – LFG expression in stage IB chondrosarcoma. \u003cstrong\u003eIID\u003c/strong\u003e– LFG expression in stage IIA chondrosarcoma. \u003cstrong\u003eIIE\u003c/strong\u003e – LFG expression in stage IIB chondrosarcoma.\u003c/p\u003e","description":"","filename":"EntwurfFigure314.11.23.png","url":"https://assets-eu.researchsquare.com/files/rs-4301676/v1/37a4beb167ead96ff25472bb.png"},{"id":55552183,"identity":"9f944d15-0b31-4b38-be24-9fb809471beb","added_by":"auto","created_at":"2024-04-29 22:13:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":183485,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of LFG in human soft tissue sarcoma. \u003cstrong\u003eA – \u003c/strong\u003eGraphical visualization of LFG expression in different subtypes of soft tissue sarcoma, including liposarcoma, dermatofibrosarcoma, angiosarcoma, leiomyosarcoma, malignant schwannoma and synovial cell sarcoma, compared with skeletal muscle and smooth muscle as control. The data are presented as means ± 95%-confidence intervals, * represents p\u0026lt;0,05; ** represents p\u0026lt;0,01. \u003cstrong\u003eB – \u003c/strong\u003eGraphical visualization of LFG expression in different stages of dermatofibrosarcoma stage IA and stage IB (green), compared to controls (orange); \u003cstrong\u003eC\u003c/strong\u003e – LFG expression in different stages of malignant schwannoma stage IA (green), stage IIA, stage IIB (blue) and stage III (red), compared to controls (orange) \u003cstrong\u003eD\u003c/strong\u003e– LFG expression in different stages of leiomyosarcoma stage IA (green), stage IIB (blue) and stage III (red), compared to controls.\u003c/p\u003e","description":"","filename":"EntwurfFigure414.11.23.png","url":"https://assets-eu.researchsquare.com/files/rs-4301676/v1/25e2172e1292ef99d9880881.png"},{"id":55552187,"identity":"27128a88-63e7-4a2c-a122-6c9828576500","added_by":"auto","created_at":"2024-04-29 22:13:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":475402,"visible":true,"origin":"","legend":"\u003cp\u003eLFG expression in different stages of leiomyosarcoma (IA, IIB, III) compared to endometrium smooth muscle as control. \u003cstrong\u003eA – \u003c/strong\u003eLFG expression in healthy endometrium smooth muscle control\u003cstrong\u003e. B – \u003c/strong\u003eLFG expression in leiomyosarcoma stage IA\u003cstrong\u003e. C – \u003c/strong\u003eLFG expression in leiomyosarcoma stage IIB\u003cstrong\u003e. D – \u003c/strong\u003eLFG expression in leiomyosarcoma stage III.\u003cstrong\u003e \u003c/strong\u003eThe slides were stained with anti-hLFG rabbit primary antibodies followed by secondary antibody 800CW (pseudocoloured green). The images were detected by Li-Cor infra-red imaging system. Relative fluorescent units were used to quantify LFG protein expression.\u003c/p\u003e","description":"","filename":"EntwurfFigure514.11.23.png","url":"https://assets-eu.researchsquare.com/files/rs-4301676/v1/1b4773302e0bff29bfbd8f58.png"},{"id":59477165,"identity":"8b0fd21c-ca67-436b-adf2-6dc18df8d8ce","added_by":"auto","created_at":"2024-07-02 09:12:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2591554,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4301676/v1/ebed2590-72e4-44a1-90c1-73f03c00d13e.pdf"},{"id":55553192,"identity":"375c6de1-052c-4bcc-8647-b925b76e5ce7","added_by":"auto","created_at":"2024-04-29 22:21:42","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":95242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e LFG expression in human osteosarcoma\u003c/p\u003e","description":"","filename":"Table1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4301676/v1/212e6e8e25f1074fd63c6684.tiff"},{"id":55552181,"identity":"4a0a6b64-000b-4292-b98c-84a3fd2f3bfe","added_by":"auto","created_at":"2024-04-29 22:13:42","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":147044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e LFG expression in human chondrosarcoma subtypes\u003c/p\u003e","description":"","filename":"Table2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4301676/v1/578ba49a83886fd069ef8eb6.tiff"},{"id":55552184,"identity":"d5f00f18-8986-449f-9f77-b407db14a08b","added_by":"auto","created_at":"2024-04-29 22:13:42","extension":"tiff","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":89790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 3:\u003c/strong\u003e LFG expression in human soft tissue sarcoma subtypes\u003c/p\u003e","description":"","filename":"Table3.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4301676/v1/99c8b7e25697620c0aba5459.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"The anti-apoptotic protein lifeguard is expressed in osteosarcoma, chondrosarcoma and soft tissue sarcoma","fulltext":[{"header":"Background","content":"\u003cp\u003eSarcoma are a heterogeneous group of tumors derived from mesenchymal origin with a distinct tendency of hematogenous metastasis [1]. They can be roughly divided into osteosarcoma, chondrosarcoma and the heterogenous group of soft tissue sarcoma. Although sarcoma only contribute to 1% of human malignancies, they have a high burden in childhood cancer with approximately 14% of all malignancies among children [2]. The prevalence of osteosarcoma, the most common type of malignant bone tumor, depends on age, race, sex, and a number of other factors, and has a peak during the adolescent years [3]. In addition, the prognosis of both, osteosarcoma and soft tissue sarcoma, measured by the 10-year survival, improved over the last decades, but is still not satisfactory [4]. One possible explanation could be, that the enormous pathological diversity of more than 100 different sarcoma subtypes is not jet adequately addressed in standardized sarcoma chemotherapy [4]. Furthermore, secondary cardiac, pulmonary and neurocognitive disorders as well as secondary malignancies have been described in sarcoma long-time survivors after successful chemotherapeutic treatment [5]. Current research on osteosarcoma identified more possible therapeutic targets with a high potential for future targeted tumor therapy and immunotherapy [6], based on a more precise understanding of the molecular mechanisms behind cell-growth signaling, sarcoma microenvironment, and the apoptosis evading strategies of different sarcoma subtypes. One possibility of apoptosis activation is receptor-mediated cell death, such as the Fas/Fas ligand pathway [7]. The human osteosarcoma cell line LM7 showed a high metastatic potential in an experimental nude mouse model based on low Fas expression [8]. In a rodent model, transfection of LM7 cells with Fas led to a significantly reduced incidence, size and weight of tumor nodules compared to the controls [8]. Accordingly, Fas expression was lower in human osteosarcoma lung metastases compared to controls [8]. These findings suggest that the reduction of Fas expression, leading to Fas-mediated cell death, may be a mechanism of increased osteosarcoma metastasis. It remains still unclear how Fas-mediated cell death inactivation contributes to the growth and metastasis of different sarcoma subtypes and its regulation through tumorigenesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe protein lifeguard (LFG) is a member of the Bax-Inhibitor (BI-1) family that is highly conserved and inhibits programmed cell death via different anti-apoptotic mechanisms that are not completely understood yet [9]. LFG is a hydrophobic, multi-pass transmembrane protein that is located in the plasma membrane and intracellular membranes [10]. The suspected protein structure of lifeguard consists of seven transmembrane domains, a three residues long extracellular C-terminus and a small cytoplasmic domain at the N-terminus [11]. Fernandez et al. indicated that LFG is also present on the lipid rafts of SH-SY5Y human neuroblastoma cells, assuming a mechanistic interaction between LFG and the major side of death inducing signaling complex and caspase-8 [12]. Somia et al. isolated LFG in 1999 for the first time and reported that it uniquely provided protection from Fas-mediated cell death, but not from the tumor necrosis factor \u0026alpha; signal pathway [10]. LFG interacts with Fas, without reducing Fas expression or Fas-associated protein with death domain (FADD) binding [12]. The distribution of LFG in healthy human tissues is diverse, ranging from ubiquitous occurrence in almost all tissues, except spleen and placenta [11,12], to a high expression in cerebral and cerebellar tissue [13,14]. Furthermore, LFG is highly expressed in breast cancer cell lines MCF-7, MDA-MB-231 and T47D and in native breast cancer tissue [15]. The level of LFG expression correlated with high tumor grades in primary breast tumors [15]. A high LFG expression could be related to decreased Fas sensitivity in breast cancer cell lines [15]. LFG might even be a novel therapeutic target in breast cancer and sarcoma, as down-regulation of LFG sensitized solid breast cancer cell line MCF-7 and sarcoma cell line SW872 to perifosine-induced cell death activation caused by an agonistic Fas antibody [16]. However, the exact molecular regulation of LFG in human malignancies remains unclear. In breast cancer cell lines the expression of LFG correlated with glycogen synthase kinase-3 (GSK3\u0026beta;) and LEF-1 activation [16]. Further evidence for the role of the Akt-lymphoid enhancer-binding factor-1 (LEF-1) pathway for the regulation of LFG in human breast cancer cells could be seen in small interfering RNA (siRNA) based on transfection experiments [16]. LFG messenger RNA (mRNA) was down regulated after transfection with siRNA against LEF1 in MDA-MB-231 cells [16]. The opportunity of osteosarcoma to gain resistance to adjuvant chemotherapy suggests that a reduced sensitivity regarding Fas-mediated apoptosis could play an important role in sarcoma cells as well [6]. Moreover current research showed that osteosarcoma cells could increase their viability during intravasation and dedifferentiation by inducing their expression of LFG/FAIM2 triggered by higher serum albumin concentration in the angiorrhea region [17]. Based on these findings, a higher expression of LFG in osteosarcoma is postulated to promote early pulmonary metastasis, leading to a prevalence of 10 to 20% when being diagnosed [4]. But the exact anti-apoptotic function of LFG in osteosarcoma, chondrosarcoma and soft tissue sarcoma remains unclear. The aim of the present study was the analysis of LFG expression in human osteosarcoma, chondrosarcoma and soft tissue sarcoma samples using immunohistochemistry. Samples were compared to healthy human tissue as controls.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eLFG protein structure\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe three-dimensional (3D) model of the protein structure of LFG was created with the help of SWISS-MODEL\u0026reg; software (page accessed on March 10, .2023). SWISS-MODEL\u0026reg; is an automated protein homology server. The LFG protein structure was based on comparative modeling and needed a given amino acid sequence, deposited in the protein data bank \u0026ldquo;uniprot\u0026rdquo; (uniprot PDB) for the modeling process. The structural template was identified at uniprot and added to SWISS-MODEL\u0026reg; server. The alignment of target sequence and template structures, model building and model quality evaluation were done automatically by the server. The modeling results of the predicted LFG protein structure were compared to other modeling processes and evaluated using global model quality estimate (GMQE) values.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsteosarcoma and chondrosarcoma samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor LFG expression analysis in osteosarcoma and chondrosarcoma the commercially available tissue array (OS802c, hematoxylin-eosin stain, Biomax US, Derwood, MD, USA), containing 80 different patient samples (1 core/case), was used. According to the manufactures\u0026rsquo; specifications 50 samples of osteosarcoma, 28 samples of chondrosarcoma and 2 samples of healthy human bone tissue were included in the analysis and were prepared for immunohistochemistry. For each tumor sample, pathological grading and clinical findings, such as TNM-classification, were included in the analysis. The healthy human bone samples served as controls.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoft tissue sarcoma samples\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor LFG expression analysis in soft tissue sarcoma the commercially available soft tissue sarcoma array (SO208a, hematoxylin-eosin stain, Biomax US, Derwood, MD, USA) was used. According to the manufacturers\u0026rsquo; specifications, 55 samples of different soft tissue sarcoma subtypes, 4 samples of healthy human smooth muscle tissue and 5 samples of healthy human skeletal muscle tissue, were analyzed and prepared for immunohistochemistry. For each tumor sample, pathological grading and clinical findings such as TNM-classification were included in the analysis. The healthy human skeletal and smooth muscle samples served as controls. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe osteosarcoma and the soft tissue sarcoma tissue arrays were deparaffinized in xylen (Lab Alley, Spicewood,TX, USA) and rinsed with phosphate buffered saline (PBS) (Thermo Scientific, Schwerte, Germany) for 5 minutes (min) each. The slides were immersed in a descending alcohol concentration row (100% ethanol, 80% ethanol, 70% ethanol) for 2 min each.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor antigen retrieval the slides were pressure cooked in 6.5 mM sodium citrate (pH 6.0) (Thermo Scientific, Schwerte, Germany) and washed thrice with PBS (Thermo Scientific, Schwerte, Germany).\u0026nbsp;Specific background staining was reduced by incubating the slides in 2.5% bovine serum albumin/1X Tris-buffered saline (Lab Alley, Spicewood, TX, USA) for 60 min. To optimize primary antibody binding, the slides were incubated at 4\u0026ordm;C overnight with rabbit anti-hLFG primary antibody (Santa Cruz Biotechnology, Dallas, TX, USA; final dilution 1:100) dissolved in 1% bovine serum albumin/PBS solution (Thermo Scientific, Schwerte, Germany). After incubation, the slides were washed with PBS thrice for 5 min. For secondary antibody binding the slides were incubated with goat anti-rabbit 800CW conjugated secondary antibody (IRDye\u0026reg; Li-Cor, Lincoln, USA; final dilution, 1:100) at room temperature for 30 min. The Li-Cor infrared imaging System Odyssey\u0026reg; (Li-Cor Biosiences, Lincoln, NE, USA) was used to detect the signals. For further analysis the Image Studio Software Odyssey\u0026reg; (Li-Cor Biosiences, Lincoln, NE, USA) was used to visualize LFG protein expression. The relative fluorescent units, measured by Li-Cor Infra-Red Imaging System, were used to quantify LFG protein expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LFG protein expression was observed for each subtype of sarcoma in different tumor stages, matched to healthy tissue controls. The tumor stages were based on clinical findings, specified according TNM-classification (World Health Organization 2017) and pathological grading. The means of LFG expression and the confidence intervals were determined on basis of measured LFG fluorescent units for each sarcoma subtype and stage. p\u0026lt;0,05 was determined significant. The results were calculated using Microsoft Excel \u0026nbsp;(Microsoft Corporation, Redmond, WA, USA) and visualized with Datatab software (Datatab, Seiersberg, Austria).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLFG protein simulation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo visualize the 3D structure of the LFG protein, the sequence based SWISS-MODEL\u0026reg; software was used (Figure 1). Figure 1A shows the expected LFG monomer protein structure that consists of seven transmembrane domains (blue), that pass multiple times through the plasma membrane and are linked to a seperate protein tail (orange). Figure 1B shows the template alignment of LFG, as given by the uniprot protein data bank.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe high expression of LFG protein in osteosarcoma tissues\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs above described, osteosarcoma samples were analyzed using commercially available immunofluorescence arrays. Table 1 gives an overview of the different anatomical localizations of the samples, the corresponding TNM classification, \u0026nbsp;tumor grading, tumor stage, number of samples and mean LFG intensity. Osteosarcoma tissues from various anatomical regions were included, predominantly coming from femur and tibia (Table 1).\u003c/p\u003e\n\u003cp\u003eThe mean LFG expression in osteosarcoma stage IIA (54,7; CI=47,1; 62,4) \u0026nbsp;(p\u0026lt;0,05) was significantly higher than in normal bone marrow tissue (20,7; CI= 18,8; 22,8) (p\u0026lt;0,05). In stage IIB osteosarcoma, the mean LFG expression was calculated to 61,1 (CI=56,9; 65,3), being significantly higher compared to controls (p\u0026lt;0,05). Interestingly, the LFG expression seemed to correlate with the tumor stage, being higher in osteosarcoma stage IIB than in stage IIA as indicated by the relative fluorescent units (stage IIA 54,7 \u0026plusmn; 7,7; stage IIB 61,1 \u0026plusmn;4,2) (p\u0026lt;0,05) (Fig. 2, I). Samples with advanced osteosarcoma stage IIB had a larger tumor size, compared to IIA, and had the highest mean LFG expression of all osteosarcoma samples in the analysis. Both samples with osteosarcoma stage IIA and IIB had grade 3 tumors in which the sarcoma cells grew more aggressively and showed abnormal cell morphology. Figure 2, II shows a representative selection of LFG antibody stained fluorescent images of osteosarcomas. The increase in the LFG intensity in osteosarcoma stage IIB (Figure 2, IIC) , compared to stage IIA (Figure 2, IIB) and the controls (Figure 2, IIA) can be seen in the constant increase of green coloring from Figure 2, IIA to Figure 2, IIC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe high expression of LFG protein in chondrosarcoma\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 2 gives an overview of the different tumor localizations, tumor grading, tumor stage and mean LFG intensity of chondrosarcoma samples. The mean LFG expression in chondrosarcoma stage IA was significantly higher compared to normal bone marrow tissue (stage IA=44,3; controls=20,7) with a p-value \u0026lt;0,05 (Fig. 3,I). A significant higher LFG expression in chondrosarcoma stage IIA was oberserved (53,4; CI=42,4; 64,5) (p\u0026lt;0,05) compared to chondrosarcoma stage IA (36,1; CI=32,4; 39,8) (p\u0026lt;0,05) (Fig. 3,I). Although a significant increase in LFG expression could not be seen in all stages, the data assume a constant positive correlation between LFG expression and chondrosarcoma stages. The LFG expression level detected in stage IIB chondrosarcoma (49,3; CI=39,5; 59,1) was lower than in stage IIA (53,4; CI=42,4; 64,5) (p\u0026gt;0,05). Figure 3,II,A-E shows fluorescent images of chondrosarcoma samples with different tumor stages. The LFG intensity is expressed by green immunofluorescence. Healthy bone marrow tissue showed the lowest LFG expression compared to all subtypes of chondrosarcoma (Figure 3, IIA). The LFG expression increases from chondrosarcoma stage IA (Figure 3, IIB) to stage IB (Figure 3, IIC), as shown by an increase in intensitiy of the green immunoflourescence. Figure 2, IID shows the high LFG expression in chondrosarcoma stage IIA, significantly higher compared to controls (Figure 3, IIA) and stage IA chondrosarcoma (Figure 3, IIB). Figure 3, IIE shows the LFG expression of chondrosarcoma stage IIB being significantly higher compared to controls (Figure 3, IIA), but lower compared to stage IIA (Figure 3, IID).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe high expression of LFG protein in soft tissue sarcoma\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTaking into consideration that the anti-apoptotic protein LFG showed high expression levels in osteosarcoma and chondrosarcoma, the expression of LFG in different subtypes of soft tissue sarcoma was analyzed. Table 3 shows the different soft tissue sarcoma subtypes that were investigated regarding LFG expression. A total number of 55 soft tissue sarcoma tissue samples were analyzed, using 5 skeletal muscle samples and 4 endometrium smooth muscle samples as unmatched controls. Table 3 gives an overview of the different soft tissue subtypes, the corresponding TNM classification, tumor grading, tumor stage and mean LFG intensity of the samples. The means of LFG expression were calculated for each soft tissue subtype as a whole by summarizing the data of all corresponding stages for each subtype (Table 3). Overall, the LFG expression variated strongly dependening on the soft tissue subtype (Figure 4A). Liposarcoma showed low LFG expression (47,4 \u0026plusmn;4,2) (p\u0026lt;0,05), not noticeable compared to skeletal muscle control (45,8 \u0026plusmn;3,9) (p\u0026lt;0,05), whereas malignant schwannoma (86,3 \u0026plusmn;28,5) (p\u0026lt;0,05), dermatofibrosarcoma (65,9 \u0026plusmn;9) (p\u0026lt;0,05) and leiomyosarcoma (90,6 \u0026plusmn;25,4) (p\u0026lt;0,05) had a significantly higher LFG expression compared to skeletal muscle control (45,8 \u0026plusmn;3,9) (p\u0026lt;0,05) and \u0026nbsp;smooth muscle control (49,3 \u0026plusmn;5,8) (p\u0026lt;0,05) (Figure 4A). The LFG expression in angiosarcoma (60,6 \u0026plusmn;5,9) and synovial cell sarcoma (59,9) was higher compared to controls with the results not being significant (p\u0026gt;0,05) (Figure 4A). Figure 4B shows that LFG expression in dermatofibrosarcoma stage IA (63,5 \u0026plusmn;8,8) (p\u0026lt;0,05) that was significantly higher compared to the controls (p\u0026lt;0,05), with an increase in LFG expression from stage IA (63,5 \u0026plusmn;8,8) (p\u0026lt;0,05) to more advanced stage IB (82,3; no CI). The LFG expression correlated with malignant schwannoma tumor stage, increasing from stage IA (56,2; no CI) to stage IIA (74,7; no CI) and stage IIB (124,9; no CI) (Figure 4C). The LFG expression of malignant schwannoma stage III (89,6; no CI) was observed lower compared to stage IIB (124,9; no CI) (Figure 4C). The highest LFG expression of all specimen was detected in leiomyosarcoma stage IA (139,3; no CI) (Figure 4D). The LFG expression in leiomysarcoma stage IIB (77,0 \u0026plusmn;15,4) (p\u0026lt;0,05) showed to be significantly higher compared to both controls (p\u0026lt;0,05) (Figure 4D).The LFG expression in leiomyosarcoma is exemplarily shown in Figure 5. Figure 5A shows an immunofluorescent image of endometrium smooth muscle as control with low LFG expression. The highest LFG expression of all specimen was detected in leiomyosarcoma stage IA presenting with multiple, widely distributed fluorescent signals (Figure 5B). Figure 5C shows the high LFG expression of leiomyosarcoma stage IIB, being significantly higher (p\u0026lt;0,05) compared to endometrium smooth muscle control (Figure 5A). Figure 5D shows an image of the high LFG expression that was observed in leiomyosarcoma stage III.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study is among the first to describe LFG expression in different types of sarcoma. The data presented show convincing evidence that LFG expression is significantly higher in advanced stages of osteosarcoma and chondrosarcoma compared to localized tumor stages and their original tissue. More importantly LFG expression was found to increase with osteosarcoma and chondrosarcoma tumor stage. In addition a significantly higher expression of anti-apoptotic LFG was observed in soft tissue sarcoma subtypes, including dermatofibrosarcoma, malignant schwannoma and leiomyosarcoma. The correlation between LFG expression and tumor stage was reduced in subtypes of soft tissue sarcoma, compared to osteosarcoma and chondrosarcoma.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough sarcoma are a rare tumor entitiy, osteosarcoma are the most common malignant bone tumors in pediatrics, with approximately 4-5 cases per 1 million children in developed countries [3]. Additionally to the rareness of sarcoma, their pathologic heterogeneity with more than 100 different subtypes, makes modern multimodal treatment in specialized sarcoma centers very challenging [18]. For optimal treatment, an early image guided biopsy in specialized centers for diagnosis followed by a multi-disciplinary therapy approach is mandatory [19]. The multimodal and patient specific treatment, includes an individualized concept with surgery, standardized chemotherapy, targeted tumor therapy, immunotherapy and/or radiation [6]. Early hematogenous metastasis of even localized osteosarcoma often require a patient specific combination chemotherapy that improved the 5-year survival rates from 20% in the 1970s to approximately 60-80% in the last decades [20]. Overall, 10% of all sarcoma patients show metastatic lesions at the time of diagnosis [20]. When patients are diagnosed with metastatic disease, the 5-year survival rate drops from 75% to 20% [2]. Therefore a deeper understanding of the molecular mechanisms behind tumor growth and metastasis of osteosarcoma and soft tissue sarcoma as well as their ability to reduce sensitivity to standardized chemotherapy is crucial for future targeted tumor therapy approaches [6]. One key point of tumor progression and resistance to chemotherapy in osteosarcoma is the inhibition of apoptosis by multiple different mechanisms, including mutations in extrinsic and intrinsic apoptotic pathways [21]. Various apoptotic agents are reported to contribute to programmed cell death in osteosarcoma cell lines \u003cem\u003ein vitro\u003c/em\u003e such as micro RNA (miRNA) [22], natural compounds like chimaphilin [23] and icaritin [24] and numerous apoptotic proteins [21]. Pro-apoptotic proteins, for example inhibitor of growth protein 4 (ING4), induced the apoptosis of osteosarcoma cells by the blockage of the NF-kB signaling pathway and activation of the intrinsic pathway trough decreasing the ratio of Bcl-2/Bax [25]. The Runx2 gene, an important transcription factor that is involved in osteoblast maturation and bone development, turned out to be overexpressed in many osteosarcoma cells [21]. Furthermore the loss of Runx2 in osteosarcoma sensitized the cells to doxo-induced apoptosis\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e and \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003e[26]. Another regulator of apoptosis in osteosarcoma can be found in the enhancer of zeste homolog 2 (EZH2) protein, which corresponds to the catalytic subunit of polycomb repressive complex 2 and showed higher expression in patients with advanced tumor stages\u0026nbsp;[27]. Moreover silencing of EZH2 in osteosarcoma by siRNA reduced osteosarcoma cell growth, invasion and lung metastasis [28]. Another pathway resulting in extrinsic programmed cell death is the Fas/Fas ligand system, which results in activation of caspase-8 and subsequent caspase cascades, initialized by ligation of Fas to its agonistic antibody and formation of death-inducing signaling complex [29,30]. In addition to the intrinsic mitochondria pathway, Fas-mediated programmed cell death inhibition seems to be not only highly relevant for apoptosis, but also for metastasis in human osteosarcoma, shown by reduced Fas expression levels in LM7 cell lines with high metastatic potential [8]. The studies of Koshkina et. al give further evidence to the idea, that osteosarcoma cells that reduce their Fas expression, get promoted and positively selected during the process of metastasis in the Fas-Ligand (FasL)-positive microenvironment of the lung [31,32]. Osteosarcoma cells with higher Fas expression, might be eliminated by FasL induced apoptosis in the lung, resulting in the selection of Fas-negative cell populations [31]. It was shown that Fas/FasL-mediated apoptosis signaling in osteosarcoma during lung metastasis did not only depended on the amount of Fas expression [32]. In addition, down regulation of Fas-signaling might serve as a gatekeeper for metastasis [32]. Supporting this hypothesis, osteosarcoma K7 cells were transfected with FADD-dominant negative (FDN), resulting in K7/FDN cells, that showed the ability to form Fas-positive metastases in a mice model with higher metastatic potential than untransfected K7 cells [32]. The expression of FasL, although being lower than in carcinoma, was detected in many subtypes of sarcoma, being predominantly high in rhabdomyosarcoma, malignant schwannoma and Ewing sarcoma [33]. Taking these findings together, Fas-mediated cell death seems to be essential for both evading apoptosis and enabling metastasis of osteosarcoma by altering Fas expression and signaling [31-33]. Therefore it is necessary to examine the expression of more anti-apoptotic proteins in different sarcoma tissues that interfere with Fas-mediated apoptosis, such as LFG also known as FAS apoptosis inhibitory molecule 2 (FAIM2) [10]. The endogenous expression of LFG in human cancer tissue was already shown for many breast cancer tissues and cell lines, being positively correlated with tumor stage and causing a reduced sensitivity to Fas-signaling without changing the levels of Fas expression [15]. In contrast to past research, that indicated a physical association between LFG and Fas in healthy human tissue [10,13], results in Fas-antibody immunofluorescence showed no co-localization between LFG and Fas in MCF-7 breast cancer cell lines, supposing there could be an altered function of LFG in human cancer tissues [15,16]. Currently, the data regarding LFG in sarcoma tissue is scarce. Therefore the expression and exact function of LFG in clinical related phenotypes of sarcoma needs to be investigated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results of the present study show that LFG is significantly higher expressed in tissues of osteosarcoma and chondrosarcoma than in healthy bone tissue (p\u0026lt;0,05). Additionally the level of LFG expression was different in several subtypes of soft tissue sarcoma, with the highest expression in dermatofibrosarcoma, malignant schwannoma and leiomyosarcoma. Furthermore, LFG expression positively correlated with tumor stages of osteosarcoma (IIB\u0026gt;IIA, p\u0026gt;0,05) and was significantly higher in advanced stages of chondrosarcoma (IIA\u0026gt;IA, p\u0026lt;0,05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe high LFG expression in osteosarcoma can be seen as another possible explanation for the decrease of Fas-mediated apoptosis in advanced tumor diseases [33]. The correlation of LFG expression in osteosarcoma to the clinical stages underlines the importance of LFG for osteosarcoma cell growth and supports the concept of LFG guided cell dedifferentiation within metastatic cell populations [31-33]. In addition, Pan et. al. stated that LFG overexpression enabled lower malignant osteosarcoma to survive in lower serum albumin environments, that might act as a defense system against growing tumor lesions during hematogenous metastasis [17]. However, only higher levels of serum albumin, occurring in nearby neovascularization, empower osteosarcoma cells with high expression of LFG via calcium mediated interactions to enter the circulatory system and form metastasis in distant organs [17].\u003c/p\u003e\n\u003cp\u003eUnlike with Bax/Bcl-xL, for which high expression rates have been demonstrated in chondrosarcoma [34-36] this is the first time that high LFG expression could be correlated to chondrosarcoma and special subtypes of soft tissue sarcoma. The anti-apoptotic properties of LFG in advanced stages of chondrosarcoma might represent another explanation that chemotherapy resistance added to a higher expression of anti-apoptotic Bcl-2 proteins and the presence of multidrug resistance (MDR) pumps [36]. Nevertheless a more detailed research on LFG expression and function, compared to related BI-1 expression in osteosarcoma and chondrosarcoma is needed.\u003c/p\u003e\n\u003cp\u003eConcluding the LFG expression analysis of the present study, significantly higher levels of LFG expression in leiomyosarcoma might be linked to the aggressive tumor growth and high resistance rates to chemotherapy of this sarcoma subtype [37]. Although no continuous correlation could be found between tumor stage and LFG expression in soft tissue sarcoma, significant higher LFG expression rates, compared to adjacent healthy tissue, were observed in all soft tissue sarcoma subtypes except liposarcoma and synovial cell sarcoma. More research, including higher numbers of soft tissue sarcoma specimen and cell lines, is needed to clarify the expression of LFG and its role regarding tumorigenesis in soft tissue sarcoma.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, high LFG expression could be found in osteosarcoma, chondrosarcoma and several soft tissue sarcoma subtypes. LFG expression even correlated with tumor stage in osteosarcoma and chondrosarcoma and might function as a predictive marker for tumor progression. Further research LFG expression and function, including apoptosis related changes in tumor gene expression and LFG knockout experiments \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e will help to evaluate the potential of LFG as a novel target for sarcoma therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to Andrea Lazaridis for her excellent technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u003c/strong\u003e Sebastian Senger, Sarah Strau\u0026szlig;, Peter M. Vogt, Frederik Schlottmann, Vesna Bucan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData curation:\u003c/strong\u003e Sebastian Senger, Vesna Bucan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFormal analysis:\u003c/strong\u003e Sebastian Senger, Vesna Bucan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding acquisition:\u003c/strong\u003e Peter M. Vogt, Vesna Bucan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation:\u003c/strong\u003e Sebastian Senger, Andrea Lazaridis, Vesna Bucan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology:\u003c/strong\u003e Sebastian Senger, Sarah Strau\u0026szlig;, Vesna Bucan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProject administration:\u003c/strong\u003e Sarah Strau\u0026szlig;, Peter M. Vogt, Vesna Bucan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResources:\u003c/strong\u003e Sarah Strau\u0026szlig;, Vesna Bucan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupervision:\u003c/strong\u003e Sarah Strau\u0026szlig;, Peter M. Vogt, Vesna Bucan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation:\u003c/strong\u003e Sarah Strau\u0026szlig;, Vesna Bucan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisualization:\u003c/strong\u003e Sebastian Senger, Vesna Bucan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting\u003c/strong\u003e \u0026ndash; \u003cstrong\u003eoriginal draft:\u003c/strong\u003e Sebastian Senger.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting \u0026ndash; review \u0026amp; editing:\u003c/strong\u003e Sebastian Senger, Sarah Strau\u0026szlig;, Frederik Schlottmann, Peter M. Vogt, Vesna Bucan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have no conflicts of interest to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding. This publication is funded by the Deutsche Forschungsgemeinschaft (DFG) as part of the \u0026ldquo;Open Access Publikationskosten\u0026rdquo; program.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMiwa S, Yamamoto N, Tsuchiya H. Sarcoma: Molecular Pathology, Diagnostics, and Therapeutics. Int J Mol Sci 2023 Mar 19;24(6):5833. doi: 10.3390/ijms24065833.\u003c/li\u003e\n\u003cli\u003eHaDuong JH, Martin AA, Skapek SX, Mascarenhas L. Sarcomas. Pediatr Clin North Am 2015 Feb;62(1):179-200.\u003c/li\u003e\n\u003cli\u003eSadykova LR, Ntekim AI, Muyangwa-Semenova M, Rutland CS, Jeyapalan JN, Blatt N, et al. 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Cardiovasc Intervent Radiol 2019 Sep;42(9):1255-1261.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lifeguard, apoptosis, osteosarcoma, chondrosarcoma, soft tissue sarcoma","lastPublishedDoi":"10.21203/rs.3.rs-4301676/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4301676/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The success of modern chemotherapy in overall survival of patients with advanced stages of osteosarcoma and soft tissue sarcoma has reached a plateau. Therefore a deeper understanding of molecular mechanisms behind deregulated apoptosis in sarcoma is essential for the cure of patients with advanced stages of osteosarcoma and soft tissue sarcoma. Lifeguard (LFG) is a member of the Bax Inhibitor-1 (BI-1) protein family and has anti-apoptotic effects by inhibiting Fas-mediated cell death signaling. Although LFG has been proven to be expressed in several breast cancer tissues, the expression and function of LFG regarding apoptosis in different subtypes of sarcoma remains unclear. In the present study, the expression of LFG in osteosarcoma (50 samples), chondrosarcoma (28 samples) and soft tissue sarcoma (total 55 samples) with different tumor stages for each sarcoma subtype were analyzed. For each subtype, clinical TNM-classification and pathological grading were determined and compared to healthy corresponding tissues. Soft tissue sarcoma subtypes included liposarcoma, dermatofibrosarcoma, angiosarcoma, leiomyosarcoma, malignant schwannoma and synovial cell sarcoma. In this study, significantly higher expressions of anti-apoptotic LFG protein in osteosarcoma, chondrosarcoma and many different subtypes of soft tissue sarcoma were found, compared to corresponding healthy tissues. More importantly a positive correlation between LFG expression and tumor stage for osteosarcoma and chondrosarcoma was found. In conclusion, LFG protein might play an important role in inhibition of Fas-mediated apoptosis in osteosarcoma cells, with possible potential for targeted tumor therapy in osteosarcoma.","manuscriptTitle":"The anti-apoptotic protein lifeguard is expressed in osteosarcoma, chondrosarcoma and soft tissue sarcoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-29 22:13:37","doi":"10.21203/rs.3.rs-4301676/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"298189ef-2a0c-40cd-8510-ed6b53a6857d","owner":[],"postedDate":"April 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-02T09:04:02+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-29 22:13:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4301676","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4301676","identity":"rs-4301676","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}