CXCL7 plays a crucial role in facilitating extramedullary invasion and osteolytic damage in multiple myeloma

CXCL7 plays a crucial role in facilitating extramedullary invasion and osteolytic damage in multiple myeloma | 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 CXCL7 plays a crucial role in facilitating extramedullary invasion and osteolytic damage in multiple myeloma Peng Liu, Yue Wang, Tianwei Lan, Chi Zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4489552/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 primary distinction between multiple myeloma (MM) and its precursor conditions lies in the deterioration of the biological behavior of tumor cells. In MM, a type of mature B-cell tumor, chemokines may serve as pivotal regulatory genes. Through exploration of GEO database and single-cell RNA-seq data from our laboratory, we have identified chemokines CXCL7 as a potential key regulator of the cellular biological behavior in MM. Subsets of MM cells with high CXCL7 exhibit heightened malignant potential. Elevated CXCL7 is associated with extramedullary invasion and pathological fractures in patients. In vitro, CXCL7 promoted the proliferation, invasion and migration of MM cells. Leveraging the homing ability of plasma cell, we established a mouse xenograft tumor model through vein injection of a CXCL7-overexpressing cell line. We found that MM cells with elevated CXCL7 exhibited enhanced engraftment in bone marrow, induced extramedullary lesions and increased susceptibility to leg fractures. Through exploration of intracellular signaling pathways and subsequent experiments, we observed that CXCL7 can modulate the biological behavior of MM cells by activating the IL-2-STAT5 pathway in the absence of exogenous IL-2. Our findings provide new insights into understanding the pathogenesis mechanisms underlying MM, suggesting that targeting CXCL7 may offer promising therapeutic opportunities. Biological sciences/Cancer/Haematological cancer/Myeloma Biological sciences/Cancer/Oncogenes multiple myeloma CXCL7 extramedullary invasion osteolytic damage IL2-STAT5 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Multiple myeloma (MM) is a difficult-to-cure malignant tumor with a relatively slow progression and high intra-tumor heterogeneity[ 1 ]. Multiple myeloma presents with diverse clinical manifestations, target organ damage, and prognosis among different patients [ 2 ]. Furthermore, despite the minimal variance in the morphology of monoclonal plasma cells between myeloma and its precursor disease (including monoclonal gammopathy of unknown significance (MGUS) and smoldering myeloma (SMM))[ 3 ], their biological behavior differs significantly[ 4 ]. The deterioration of biological behavior in multiple myeloma may be the fundamental reason why it differs from its precursor diseases. Being a mature B-cell tumor, its genetic regulation of biological behavior may fundamentally differ from other types of tumors. The CRAB symptoms (hypercalcemia, renal insufficiency, anemia and osteolytic lesions) of myeloma are primarily a result of the high proliferation rate of tumor cells. The extramedullary infiltration and activation of osteoclast signaling are attributed to the malignant biological potential of tumor cells[ 3 , 5 ]. Indeed, modulating the biological behavior of myeloma cells to achieve prolonged tumor-bearing survival while managing the extent of target organ damage may represent a more efficacious therapeutic approach than complete depletion of monoclonal plasma cells. Therefore, it is imperative to identify the key genes that impact the biological behavior of myeloma cells. By analyzing the RNA-seq data from the myeloma database and single-cell sequencing data from our center, we found that CXCL7 may be an important factor in the deterioration of myeloma cell biological behavior, which can promote myeloma proliferation, osteolytic bone destruction and extramedullary (EMD) invasion in myeloma. The chemokine CXCL7 belongs to a class of chemotactic cytokines known as chemokines, which play a crucial role in regulating the migration of immune cells. Chemokines serve as signaling molecules that coordinate, recruit, and facilitate the entry and exit of immune cells within tissues, while also guiding their spatial distribution and facilitating cell-to-cell interactions[ 6 ]. In the context of tumors, chemokines are essential for directing the migration of immune cells to elicit effective anti-tumor immune responses. Chemokines in solid tumors have been studied mainly for their effects on immune cell infiltration and stromal cell function[ 7 ], and are unlikely to be the main carcinogenic factors of tumor cells. However, plasma cells are mature B lymphocytes whose recruitment, infiltration, migration, and proliferation in tissues may all be influenced by chemokines. In other words, chemokines may play a decisive role in immune cell tumors. Previous studies have shown that CXCL7 promotes proliferation, facilitates invasion, and is associated with poor prognosis in malignancies such as breast cancer, lung cancer, and renal cancer[ 8 – 10 ]. However, the role of CXCL7 in myeloma remains unclear. The aim of this study is to explore the role and associated mechanisms of CXCL7 in myeloma and provide a theoretical basis for new therapeutic targets. Results CXCL7 plays a pivotal role in governing the biological activities of myeloma cells The objective of our study was to identify the crucial genes influencing the biological behavior of MM cells. Proliferation, osteoclast activity, invasion, and metastasis are the most significant indicators of MM cell deterioration. However, there is no established signaling pathway for extramedullary invasion and migration of plasma cell tumors. Therefore, we utilized proliferation and osteoclast signaling activity as features to identify the key genes. We analyzed RNA-seq cohort data from MM patients in the GSE24080 dataset, using the TT2 cohort as the training set and the TT3 cohort as the validation set. The WGCNA analysis divided 23517 genes into 33 modules in TT2 set (Fig. 1 a). Subsequently, we used the ssGSEA method to quantify the activation of "B cell proliferation" and "Osteoclast signaling pathway" in all patients of the TT2 cohort. We then analyzed the correlation between coexpression gene modules calculated by WGCNA and the activation of these pathways. The results showed that the darkmagenta module significantly promoted activation in both "B cell proliferation" (P = 5.6e-11) and "Osteoclast signaling" (P = 9.0e-17) pathways. The relationships between different modules and traits are shown in Fig. 1 b. And then, all genes within the darkmagenta module were calculated for their correlation with the two traits (Fig. 1 c and 1 d). Based the cut-off criteria (|MM| > 0.78), two genes with high connectivity in the clinically significant module were identified as hub genes. The two hub genes are CXCL7 (MM = 0.787, P = 6.6e-74) and S100A8 (MM = 0.811, P = 5.6e-82). Considering the potential involvement of chemokines in MM as mentioned above, CXCL7 was chosen for further investigation. We confirmed that the expression level of CXCL7 was positively correlated with "B cell proliferation" (r = 0.32, P < 0.0001) (Fig. 1 e) and "Osteoclast signaling" (r = 0.36, P < 0.0001) (Fig. 1 f) in the TT2 cohort. In addition, we also confirmed that CXCL7 was closely related to the two traits in the TT3 validation cohort (Fig. 1 g and 1 h). We also examined the expression of CXCL7 and assessed drug sensitivity using the CellMiner database[ 11 ]. Our findings revealed a significant correlation between elevated levels of CXCL7 expression and resistance to multiple anti-tumor agents, including ixazomib, a proteasome inhibitor commonly employed in the treatment of myeloma (Supplementary Fig. 1). Single-cell RNA-seq analysis revealed that MM cell subsets with elevated expression level of CXCL7 exhibited aggressive biological features Owing to the high intratumor heterogeneity of myeloma, the biological behavior of distinct subsets varies significantly. We aim to further investigate the characteristics of MM cell subsets exhibiting elevated expression level of CXCL7. Therefore, bone marrow specimens were obtained from three newly diagnosed MM patients with International Staging System (ISS) III for single-cell RNA-seq analysis. All three patients presented with pathological fractures at the time of diagnosis. The resulting data from individual cells across multiple patients were integrated for UMAP downscaling analysis which revealed distinct subgroups consisting of MM cells along with monocytes, erythrocytes, and T-cell subsets (Fig. 2 a). In addition to the high expression level of CXCL7 in monocytes, we observed a significant upregulation of CXCL7 in some monoclonal plasma cells as well (Fig. 2 b-c) By utilizing CD38 and CD138 (SDC1) as markers for the isolation of plasma cells in subsequent downscaling analysis, we successfully classified MM cells into 11 clonal subpopulations (Fig. 2 d). The expression of CXCL7 was predominantly observed in the MM03 subgroup (Fig. 2 e). We selected "B cell proliferation" and "osteoclast signaling" as target pathways and conducted signaling pathway enrichment analysis for all MM cell subsets (Fig. 2 f-g). The results revealed that the MM03 subset exhibited the strongest correlation (lowest p-value) with the "B cell proliferation" and "osteoclast signaling" pathways (Fig. 2 h). Moreover, MM03 was found to be the most activated subgroup in both pathways (Fig. 2 f-g). CXCL7 is closely related to OS and PFS of patients, and is also related to pathological fracture and extramedullary invasion We collected bone marrow samples from 108 newly diagnosed MM patients, assessed the expression levels of CXCL7 through qPCR. By employing the "cut-off" function in the "Survival" package, we identified the optimal cut-off value for CXCL7 and compared prognosis differences among patients with different expression levels of CXCL7 (Fig. 3 a-b). The findings indicated that elevated levels of CXCL7 in myeloma cells were correlated with reduced overall survival (OS) (P = 0.016) and progression-free survival (PFS) (P = 0.008) (Fig. 3 c-d). Additionally, we compared clinicopathological variances between the high CXCL7 expression group and low CXCL7 expression group. The outcomes demonstrated that patients in the high CXCL7 expression group exhibited a higher likelihood of extramedullary invasion (P = 0.010) and pathological fracture (P = 0.048) (Fig. 3 e-f). However, no substantial correlation was observed between CXCL7 and ISS stage or high-risk cytogenetics. The clinical baseline characteristics of the patients are shown in Table 1 . These findings indicate that CXCL7 may facilitate the proliferation of myeloma cells, the activation of the osteoclast signaling pathway and the invasion of MM cells. Table 1 Baseline characteristics of 108 patients with multiple myeloma. Variable Total CXCL7_High CXCL7_Low Patinets(No) 108 69 39 Gender(No,%) Male 74(68.5) 46(66.7) 28(71.8) Female 34(31.5) 23(33.3) 11(28.2) Age(year, (No,%)) <60 30(27.8) 15(21.7) 15(38.5) ≥60 78(72.2) 54(78.3) 24(61.5) ISS stage(No,%) I 43(39.8) 29(42.0) 14(35.9) II 28(25.9) 16(23.2) 12(30.8) III 37(34.3) 24(34.8) 13(33.3) FISH high-risk(No,%) Detected 34(31.5) 23(33.3) 11(28.2) Not detected 74(68.5) 46(66.6) 28(71.8) Follow-up time (month, (median,range)) 48.7(2.1–103.0) 46.3(2.1–77.6) 52.2(5.4–103.0) Progress(No,%) 53(49.1) 41(68.1) 12(30,8) Dead(No,%) 34(31.5) 27(39.1) 7(17.9) Construction of MM cells with CXCL7 overexpression and knockdown We utilized the CCLE database to assess the expression of CXCL7 in MM cell lines. Our findings revealed that CXCL7 is expressed at low levels in the NCI-H929 cell line, moderate levels in the RPMI 8226 cell line, and high levels in the U266 cell line (Fig. 4 a). Lentiviruses carrying CXCL7-overexpression (CXCL7-OE) and Normal-Control (NC) were used to infect NCI-H929 and RPMI 8226 cell lines. Meanwhile, siRNA was employed for CXCL7 knockdown (CXCL7-KD) and NC transfection in U266 and RPMI 8226 cell lines. The RNA levels of CXCL7 in these four cells were assessed by qPCR (Fig. 4 b-e). Additionally, WB (Fig. 4 f-i) and ELISA (Fig. 4 j-m) techniques were utilized to detect intracellular protein levels as well as secreted protein levels of CXCL7. CXCL7 promotes the proliferation of MM cells The proliferative viability of the cells was assessed using a CCK-8 assay. The results demonstrated that the proliferation rate of NCI-H929 and RPMI 8226 cell lines in the CXCL7-OE group was significantly higher (Fig. 5 a-b). Conversely, the proliferation rate of U266 and RPMI 8226 cell lines in the CXCL7-KD group was slower (Fig. 5 c-d). Although we failed to find that CXCL7 was able to impression the cell cycle of MM cells (Supplementary Fig. 2). To further investigate the impact of CXCL7 on the proliferation of MM cells in vivo, subcutaneous xenograft tumor models were established in BNGD mice using the RPMI 8226 cell line with overexpression of CXCL7 and a normal control group. Subsequent analysis revealed faster tumor growth in the CXCL7-OE group (Fig. 5 e-g). Immunohistochemistry performed on excised tumors showed elevated levels of Ki-67 proliferation index in CXCL7-OE group (Fig. 5 h). CXCL7 significantly enhances the invasion and migration of MM cells The migratory and invasive capacities of MM cell lines were then evaluated using transwell assays. In the experiments assessing migratory capabilities, a significantly higher number of cells were found to penetrate the lower chamber surface in the NCI-H929 CXCL7-OE group (P = 0.0024) and in the RPMI 8226 CXCL7-OE group (P = 0.0066) (Fig. 5 i), indicating that up-regulation of CXCL7 enhances the migratory ability of MM cells. Furthermore, assessment of invasive ability demonstrated a significantly higher number of protruding cells in both NCI-H929 and RPMI 8226 CXCL7-OE groups (P = 0.0026 and P = 0.0006, respectively), suggesting that up-regulation of CXCL7 enhances the invasive ability of MM cells (Fig. 5 j). Then, transwell assays were also performed in CXCL7-KD cell lines, revealing a significant reduction in the migration (Fig. 5 k) and invasion (Fig. 5 l) capabilities of MM cells (P < 0.05). CXCL7 can significantly promote the extramedullary invasion of myeloma cells and enhance their colonization ability in bone marrow Subsequently, we established a mouse xenograft model by intravenously injecting MM cells to investigate the ability of CXCL7-OE MM cells to colonize the bone marrow through homing effects and to generate extramedullary lesions through migration and infiltration (Fig. 6 a). This approach effectively mimics the activity of plasma cells in vivo, allowing us to explore the impact of CXCL7 on the biological behavior of myeloma cells in a more physiological context. At 7 weeks, it was observed that some mice in the CXCL7-OE group exhibited symptoms indicative of long bone fractures (three out of five). No such phenomenon was observed in the control group. Concurrently, we conducted regular venous blood collection on mice to quantify serum free light chain levels and assess tumor burden. The results revealed a significantly higher tumor burden in the CXCL7-OE group (Fig. 6 b). After the mice were euthanized and dissected, more than two extramedullary plasmacytomas were identified in the subcutaneous soft tissues of all the mice in the CXCL7-OE group. Additionally, an increased number of intraperitoneal tumors were observed. A small number of peritoneal colonization lesions were also noted in the NC group, but no colonization of the subcutaneous soft tissues was found in any of the mice in the NC group. Previous studies have indicated that subcutaneous soft tissue is the most common site for extramedullary invasion (Bone independent EMD) in MM[ 12 ]. Our findings suggest that overexpression of CXCL7 enhances MM cells' ability to infiltrate and colonize subcutaneous soft tissue in a mouse xenograft model, which is consistent with clinical observations. For further analysis, tissue samples from the femur, subcutaneous metastases, liver, lungs, and kidneys were collected for histopathological examination using hematoxylin and eosin (HE) staining as well as immunohistochemical staining (Fig. 6 c-d). The sections of the mouse femur revealed that myeloma cells in the group with high expression level of CXCL7 exhibited enhanced colonization and homing in the bone marrow. The sections of mice visceral and subcutaneous soft tissue tumors revealed that the CXCL7-OE MM cell line did not exhibit significant metastasis to the lung, liver, and kidney. However, it did result in increased infiltration and generation of subcutaneous soft tissue tumors. Following the identification of leg bone fracture in the CXCL7-OE group, we proceeded to conduct micro-CT bone scan imaging on the femur of mice (Fig. 6 e). It was evident that the CXCL7-OE group exhibited more significant deterioration in femur bone compared to the NC group, as indicated by a reduction in bone mineral density (BMD) (P = 0.0068) and a decrease in average bone trabeculae thickness (Tb.Th) (P = 0.0371) (Fig. 6 f-g). Osteoclast markers tartrate resistant acid phosphatase (TRAP) and matrix metalloproteinases-9 (MMP9), along with bone marrow stromal cell markers matrix metalloproteinases-2 (MMP2) and matrix metalloproteinases-13 (MMP13) were used for staining of the bone marrow (Fig. 6 h). The findings indicated a significant increase in osteoclast markers within the CXCL7-OE group, with a predominant concentration observed near the interface of myeloma lesions and bone marrow. Amongst the osteoclast signaling activators investigated, MMP13 exhibited the highest expression levels. CXCL7 is capable of modulating the biological behavior of MM cells via the IL2-STAT5 pathway We conducted transcriptome sequencing (RNA-seq) on CXCL7-OE and NC RPMI 8226 cell lines. GSEA methods were employed to identify significant activation and enrichment pathways. The results indicated a substantial up-regulation of four hallmark gene sets in the CXCL7-OE group (Fig. 7 a-b). Among these pathways, it was observed that the IL-2-STAT5 pathway plays a critical role in immune cell regulation. Studies have demonstrated that activation of the IL-2-STAT5 pathway promotes B lymphocyte proliferation, regulates their differentiation, and enhances their capacity for antibody and light chain secretion[ 13 ]. Given that MM is characterized by the secretion of light chains and monoclonal immunoglobulins, we have chosen to further investigate the IL2-STAT5 signaling pathway. Intracellular transduction of the IL2-STAT5 signaling pathway is facilitated by the heterodimerization of the IL-2R cytoplasmic domain, leading to activation of the JAK1-STAT5 pathway. To investigate the IL-2R/JAK1/STAT5 signaling pathway, we conducted western blot analysis to assess the expression of IL-2R, phosphorylated JAK1 protein (p-JAK1), and phosphorylated STAT5 (p-STAT5) as well as their total protein. The results revealed that in NCI-H929 and RPMI 8226 cell lines, the expression level of JAK1 and STAT5 were similar between the CXCL7-OE group and NC group; however, the expression level of IL-2R, p-JAK1, and p-STAT5 were elevated in CXCL7-OE group (Fig. 7 c-d), indicating that upregulation of CXCL7 activates the IL-2R/JAK1/STAT5 signaling pathway. In U266 and RPMI 8226 cell lines, there was no significant difference in the expression level of JAK1 and STAT5 between CXCL7-KD group and NC group; nevertheless, the expression level of IL-2R, p-JAK1, and p-STAT5 were reduced in the CXCL7-KD group (Fig. 7 e-f), suggesting that inhibition of CXCL7 leads to suppression of the IL-2R/JAK1/STAT5 signaling pathway. Immunohistochemical staining of tumor sections from the aforementioned mouse xenograft model also revealed a significant increase in IL-2R/JAK1/STAT5 levels in the CXCL7-OE group (Fig. 7 g). Additionally, analysis of single cell-based pathways revealed a significant augmentation in the IL-2-STAT5 pathway in MM03, providing further evidence for activation of this pathway in a subset of highly CXCL7-expressing MM cells (Fig. 2 i). To further investigate the role of CXCL7, RPMI 8226 CXCL7-OE cells were treated with Ruxolitinib[ 14 ], a JAK1/JAK2 inhibitor (Supplementary Fig. 3). Cell proliferation was assessed using CCK-8 assay. The results demonstrated that cells in the CXCL7-OE group exhibited accelerated proliferation compared to the NC group, whereas cells in the CXCL7-OE + Ruxolitinib group showed attenuated proliferation. Notably, there was no significant difference between the CXCL7-OE + Ruxolitinib group and the NC group, suggesting that Ruxolitinib effectively inhibited MM cell proliferation induced by CXCL7 (Fig. 7 h). Additionally, a transwell assay was performed to evaluate the migratory and invasive capabilities of MM cells. The results indicated that the migratory and invasive capability of cells in the CXCL7-OE group exceeded that of NC group, whereas it was diminished in the CXCL7-OE + Ruxolitinib group compared to both CXCL7-OE and NC groups (Fig. 7 i-j). Our study has demonstrated that a subset of MM cells exhibiting high expression level of CXCL7 is capable activating IL2-STAT5 pathway in absence exogenous IL-2. Discussion Multiple myeloma is a distinct neoplasm characterized by the presence of monoclonal plasma cells in precursor states such as MGUS and SMM[ 3 ]. In most instances, MGUS and SMM are typically managed with observation as they generally do not exert a substantial impact on the patient's quality of life[ 15 , 16 ]. However, myeloma presents a distinct scenario in which the altered biological behavior of neoplastic cells results in multi-organ dysfunction. Genomic variations contribute to the unique biological characteristics exhibited by these plasma cells[ 17 ]. The high degree of intratumor heterogeneity in multiple myeloma results in distinct biological characteristics among different tumor cell subsets, posing challenges for effective treatment and leading to diverse target organ damage and clinical manifestations in patients[ 18 , 19 ]. Our study has identified CXCL7 as a potential key gene influencing the biological behavior of tumors in MM. We observed that a subset of myeloma cells exhibiting high expression level of CXCL7 displayed highly malignant biological behavior. Through genomic exploration in a large sample cohort, we have confirmed the significant impact of CXCL7 on lytic bone lesions and extramedullary invasion in MM. Furthermore, we have conducted cytological experiments and utilized a mouse xenograft model to investigate the function and potential regulatory mechanisms of CXCL7. It is noteworthy that CXCL7 belongs to the chemokine family, which plays a role in directing chemotaxis and mediating immune cell function in humans[ 20 , 21 ]. While numerous chemokines are involved in promoting tumorigenesis and progression in solid tumors, their primary role is to modulate tumor-associated immune responses, rather than serving as key oncogenes. In the context of multiple myeloma, a malignant plasma cell tumor, chemokines may indeed act as central oncogenes. CXCL7 has been identified as the primary activator of MMP13 in MM, a crucial pathway for osteolytic damage in the bone marrow that may be secreted by MSCs[ 22 ]. The differentiation and maturation of immune cells are intricately linked to chemokine activity, as they can govern the activation, recruitment, phenotype, and function of immune cells by reprogramming chemokine and cytokine receptors and modulating signaling pathway activation[ 7 ]. As mature B lymphocytes with secretory function, plasma cells not only produce immunoglobulins but also secrete a variety of cytokines and chemokines. Our study revealed that a subset of monoclonal plasma cells is capable of secreting CXCL7. Plasma cells with high expression levels of CXCL7 can stimulate their own proliferation, enhance the invasion and infiltration capabilities of plasma cells, and significantly increase the likelihood of extramedullary invasion. Furthermore, CXCL7 can induce the activation of MMP13 in stromal cells through paracrine effects to promote osteoclast signaling pathway activation. Moreover, our study successfully established a mouse model of myeloma transplantation through tail vein injection. By harnessing the inherent homing ability of plasma cells, we have demonstrated that monoclonal plasma cells display enhanced colonization in the bone marrow and rapid proliferation upon overexpression of CXCL7. Upon high expression levels of CXCL7, plasma cells also exhibited improved colonization in subcutaneous tissues, leading to extramedullary invasion lesions. Consistent with previous findings, diffuse MM lesions were observed in the NC RPMI8226 cell line injected via tail vein in mice, while subcutaneous soft tissue nodules were not detected[ 23 ]. Subcutaneous soft tissue is the most common site of extramedullary invasion in MM patients[ 12 , 24 ], and the high expression level of CXCL7 makes this phenomenon replicated in the mouse model. The IL2-STAT5 pathway plays a critical role in regulating immune cell function[ 25 ], including stimulating T cell proliferation, modulating T cell apoptosis, and enhancing NK cell cytolytic activity[ 26 ]. In B cells, the IL2-STAT5 pathway promotes B cell proliferation, enhances light chain and immunoglobulin secretion, and regulates B cell function[ 13 ]. The activation of the IL2-STAT5 pathway in lymphocytes is not solely dependent on high doses of IL-2. For instance, some regulatory T cells are capable of robustly activating downstream STAT5 even stimulated with low doses or even in the absence of exogenous IL-2 due to the signal amplification effect mediated by Hippo kinases Mst1 and Mst2[ 27 , 28 ]. Our study has also demonstrated that CXCL7 can activate intracellular IL2R-STAT5 signaling independently of exogenous IL-2; however, it is noteworthy that CXCL7 does not directly bind to IL-2R. We hypothesize that this phenomenon may be attributed to two potential reasons: firstly, CXCL7 may trigger signal amplifiers similar to Mst1 and Mst2 within MM cells; secondly, it is possible that CXCL7 binds to surface receptors on MM cells forming a complex multi-binding complex leading to activation of intracellular IL2R-STAT5 signaling pathways. Further investigations are warranted in the future. Methods and Materials Weighted correlation network analysis (WGCNA) First, we utilized the gene expression profiles of patients in the GEO database. We chose the GSE24080 cohort and designated the TT2 patient group as the training set[ 29 ], while assigning the TT3 patient group as the validation set[ 30 ]. The R package “WGCNA” was used to construct a scale-free co-expression network [ 31 ]. To classify genes with similar expression profiles into gene modules, we conducted average linkage hierarchical clustering according to the TOM-based dissimilarity measure with a minimum gene group size of 30 for the genes dendrogram. Additionally, we merged modules with a distance less than 0.25. Single sample Gene Set Enrichment Analysis (GSEA) Single sample GSEA (ssGSEA) analysis was performed to determine the degree of activation for each pathway, which was recorded as the Enrichment Score (ES)[ 32 ]. The score ranged from 0 to 1, indicating minimal to maximal activation. The gene sets for these pathways were obtained from the MsiGDB database[ 33 ]. The expression level of CXCL7 and clinicopathological features of samples A total of 108 clinical samples of newly diagnosed MM patients were included in this study, which were collected from patients who visited Zhongshan Hospital of Fudan University from 2019 to 2020. In order to further observe the relationship between CXCL7 and the prognosis and drug treatment response of patients, only patients who had not undergone autologous hematopoietic stem cell transplantation were included. All patients received an initial treatment of a three-drug combination of protease inhibitor, lenalidomide, and dexamethasone. The bone marrow samples of patients were collected at the time of diagnosis. All bone marrow fluid samples were enriched with nucleated cells and sorted with CD138 magnetic beads. The expression level of CXCL7 is detected by the qPCR. Single cell mRNA sequencing A diverse array of bone marrow samples was collected from Zhongshan Hospital affiliated with Fudan University, following approval from the Institutional Review Board of Zhongshan Hospital. Bone marrow mononuclear cells (BMMCs) were isolated and employed for single-cell RNA sequencing (scRNAseq). Single-cell libraries were generated from unsorted BMMCs using a 10× Genomics Chromium Single Cell 5′ Library Kit and Chromium instrument, followed by sequencing on an Illumina sequencer. The obtained FASTQ data was aligned to the reference genome (GRCh38) using Cell Ranger (v5.0.1)[ 34 ]. Seurat v4.0.1 software was utilized for cell clustering and dimension reduction. For pathway activation analysis in different cell subsets, we employed the irGSEA package along with AUCell and UCell methods to score pathway activation levels based on gene sets obtained from the MSigDB database[ 33 ]. Establishment of lentiviral stabilized cell lines U266 and NCI-H929 cell lines are purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and RPMI-8226 cell line is obtained from the cell bank of the Chinese Academy of Sciences for use in this investigation. The vector components used in the construction of the CXCL7 overexpression lentivirus are Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin. Small interfering RNA transfection Small interfering RNA (siRNA) targeting CXCL7, as shown in Supplementary Table 1, is transfected into MM cells in logarithmic growth phase using a transfection complex consisting of RPMI 1640 medium, Lipo3000, and siRNA. Enzyme-linked immunosorbent assay (ELISA assay) The concentration of human CXCL7 cytokine in the culture medium was measured using an ELISA kit (Abcam, England). All procedures were performed according to the manufacturer's instructions. Transwell migration and invasion assays A transwell chamber with 8.0-µm pore size (Corning, USA) was used to measure cell migration, and a Matrigel transwell chamber (Corning, USA) was used for cell invasion tests. Cells were initially inoculated onto top-perforated membranes and incubated for 24 hours (migration experiment) or 48 hours (invasion experiment). After incubation, cells in the upper part of the insert were removed and cells adhering to the lower part were fixed, stained with crystal violet, photographed at three locations, and counted using Image J (NIH, US). Quantitative RT-PCR analysis Quantitative RT-PCR analyses were performed using the EZ Bioscience RNA Extraction Kit to extract RNA from control or treated cells, followed by cDNA synthesis with the Takara cDNA Synthesis Kit and real-time PCR using the Takara TB Green Premix Ex Taq (Tli RNaseH Plus) quantitative PCR kit. All primer sequences used in this study are shown in Supplementary Table 2. Western blot analysis To lyse the cells, use 1X RIPA buffer containing Tris buffer (50mM, pH 7.4), NaCl (150mM), Triton X-100 (1%), PMSF (1mM), SDS (0.1%), and protease inhibitor mixture from Beyotime Biotechnology. Cell lysates were mixed with Loading Buffer, separated by electrophoresis in a 10–15% SDS-PAGE gel, and transferred to a PVDF membrane from Millipore. Membranes were blocked with QuickBlock Blocking Buffer for Western Blot from Biotechnology. Membranes were washed three times with PBST and incubated with enzyme-linked secondary antibodies for 1 h at room temperature. Following PBST cleaning, luminescence was recorded with Lumin4000 from GE, USA and ECL reagent from BioSharp. All antibodies used are listed in Supplementary Table 3. Subcutaneous Transplantation Tumor Cells were harvested during logarithmic growth, washed with PBS, and resuspended in the appropriate volume of PBS to achieve a density of f 5×10 6 /100 µL. The cells were then subcutaneously injected into B-NDG mice. Five randomly assigned 5-week-old female mice were divided into NC group and CXCL7-OE group. Xenograft tumor model in mice through intravenous injection Cells were collected during logarithmic growth, washed with PBS, and resuspended in the appropriate amount of PBS at a density of 10 7 /100µL before being injected into the tail vein of B-NDG mice within 2.5 hours. Each group of five 5-week-old female mice was randomly assigned to either the NC group or the CXCL7-OE group. Tumor development in the mice was regularly monitored. Immunohistochemistry (IHC) analysis Mouse tumors were sampled and then embedded in paraffin and sectioned by a microtome. Immunohistochemistry was performed according to the steps provided by the manufacturer of the immunohistochemistry kit (Sevier, China), in which the primary antibodies used are also listed in Supplementary Table 3. Statistical analysis. Statistical methods were all analyzed by R version 4.0.3 ( http://cran.r-project.org ). P values were all two-sided and statistical significance was set at P < 0.05. Declarations Acknowledgments None applicable. Ethics approval and consent to participate The study was approved by the Ethics Committee of Fudan University Zhong Hospital and complied with the principles of the Helsinki Accord. The real-world patient data included in this study was a retrospective study and patient consent was therefore deemed unnecessary. However, bone marrow samples were collected from all subjects, with patient consent, and stored in the tissue bank at Fudan University Zhongshan Hospital. Data and materials availability The single-cell RNA-seq processed gene expression data reported in this paper have been deposited into the CNGB Sequence Archive (CNSA)[35] of the China National GeneBank DataBase (CNGBdb)[36] with accession number CNP0005613, and raw sequencing data have been deposited in the OMIX, China National Center for Bioinformation, Chinese Academy of Sciences (accession no. OMIX006258)[37]. To request access to raw sequencing data, please apply at the Human Genetic Resources Service System of the Ministry of Science and Technology (https://apply.hgrg.net/) in accordance with the Regulations on the Management of Human Genetic Resources of China. The multiple myeloma RNA-seq data GSE24080 were downloaded from the GEO database. Declaration of interests None declared. Author contributions Conceptualization: YW, TL, PL Methodology: YW, TL Assistant: CZ Data collection: YW, TL, CZ Writing—original draft: YW, TL Writing—review & editing: PL Funding Science and Technology Innovation Action Plan of Shanghai (21YF1406300) Youth Foundation of Zhongshan hospital Fudan University (2021ZSQNN61) National Natural Science Foundation of China (81570123) Natural Science Foundation of Shanghai (22ZR1411400). References Rajkumar SV. Treatment of multiple myeloma. Nat Rev Clin Oncol 2011; 8: 479-491. Chng WJ, Dispenzieri A, Chim CS, Fonseca R, Goldschmidt H, Lentzsch S et al . IMWG consensus on risk stratification in multiple myeloma. Leukemia 2014; 28: 269-277. van de Donk N, Pawlyn C, Yong KL. Multiple myeloma. Lancet 2021; 397: 410-427. Mann H, Katiyar V, Varga C, Comenzo RL. Smoldering multiple myeloma - Past, present, and future. Blood Rev 2022; 52: 100869. Bansal R, Rakshit S, Kumar S. Extramedullary disease in multiple myeloma. Blood Cancer J 2021; 11: 161. Mempel TR, Lill JK, Altenburger LM. How chemokines organize the tumour microenvironment. Nat Rev Cancer 2024; 24: 28-50. Ozga AJ, Chow MT, Luster AD. Chemokines and the immune response to cancer. Immunity 2021; 54: 859-874. Wang YH, Shen CY, Lin SC, Kuo WH, Kuo YT, Hsu YL et al . Monocytes secrete CXCL7 to promote breast cancer progression. Cell Death Dis 2021; 12: 1090. Fang W, You J, Xu Q, Jiang Y, Hu H, Chen F et al . Plasma Exosomal CXCL7 is a Potential Biomarker for Lung Adenocarcinoma. Clin Lab 2022; 68. Kinouchi T, Uemura M, Wang C, Ishizuya Y, Yamamoto Y, Hayashi T et al . Expression level of CXCL7 in peripheral blood cells is a potential biomarker for the diagnosis of renal cell carcinoma. Cancer Sci 2017; 108: 2495-2502. Reinhold WC, Varma S, Sunshine M, Elloumi F, Ofori-Atta K, Lee S et al . RNA Sequencing of the NCI-60: Integration into CellMiner and CellMiner CDB. Cancer Res 2019; 79: 3514-3524. Beksac M, Seval GC, Kanellias N, Coriu D, Rosinol L, Ozet G et al . A real world multicenter retrospective study on extramedullary disease from Balkan Myeloma Study Group and Barcelona University: analysis of parameters that improve outcome. Haematologica 2020; 105: 201-208. Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol 2012; 12: 180-190. Cervantes F, Vannucchi AM, Kiladjian J-J, et al. Three-year efficacy, safety, and survival findings from COMFORT-II, a phase 3 study comparing ruxolitinib with best available therapy for myelofibrosis. Blood. 2013;122(25):4047-4053. Blood 2016; 128: 3013. Thomsen H, Chattopadhyay S, Weinhold N, Vodicka P, Vodickova L, Hoffmann P et al . Genome-wide association study of monoclonal gammopathy of unknown significance (MGUS): comparison with multiple myeloma. Leukemia 2019; 33: 1817-1821. Sun F, Cheng Y, Ying J, Mery D, Al Hadidi S, Wanchai V et al . A gene signature can predict risk of MGUS progressing to multiple myeloma. J Hematol Oncol 2023; 16: 70. Chen M, Wan Y, Li X, Xiang J, Chen X, Jiang J et al . Dynamic single-cell RNA-seq analysis reveals distinct tumor program associated with microenvironmental remodeling and drug sensitivity in multiple myeloma. Cell Biosci 2023; 13: 19. Terpos E, Christoulas D, Gavriatopoulou M, Dimopoulos MA. Mechanisms of bone destruction in multiple myeloma. Eur J Cancer Care (Engl) 2017; 26. Matsumoto T, Abe M. Bone destruction in multiple myeloma. Ann N Y Acad Sci 2006; 1068: 319-326. Bikfalvi A, Billottet C. The CC and CXC chemokines: major regulators of tumor progression and the tumor microenvironment. Am J Physiol Cell Physiol 2020; 318: C542-C554. Marcuzzi E, Angioni R, Molon B, Cali B. Chemokines and Chemokine Receptors: Orchestrating Tumor Metastasization. Int J Mol Sci 2018; 20. Lo CH, Shay G, McGuire JJ, Li T, Shain KH, Choi JY et al . Host-Derived Matrix Metalloproteinase-13 Activity Promotes Multiple Myeloma-Induced Osteolysis and Reduces Overall Survival. Cancer Res 2021; 81: 2415-2428. Mitsiades CS, Mitsiades NS, Bronson RT, Chauhan D, Munshi N, Treon SP et al . Fluorescence imaging of multiple myeloma cells in a clinically relevant SCID/NOD in vivo model: biologic and clinical implications. Cancer Res 2003; 63: 6689-6696. Blade J, Beksac M, Caers J, Jurczyszyn A, von Lilienfeld-Toal M, Moreau P et al . Extramedullary disease in multiple myeloma: a systematic literature review. Blood Cancer J 2022; 12: 45. Chen E, Staudt LM, Green AR. Janus kinase deregulation in leukemia and lymphoma. Immunity 2012; 36: 529-541. Waldmann TA. Cytokines in Cancer Immunotherapy. Cold Spring Harb Perspect Biol 2018; 10. Shi H, Liu C, Tan H, Li Y, Nguyen TM, Dhungana Y et al . Hippo Kinases Mst1 and Mst2 Sense and Amplify IL-2R-STAT5 Signaling in Regulatory T Cells to Establish Stable Regulatory Activity. Immunity 2018; 49: 899-914 e896. Abbas AK, Trotta E, D RS, Marson A, Bluestone JA. Revisiting IL-2: Biology and therapeutic prospects. Sci Immunol 2018; 3. Mehta J. Total therapy 2 in treatment of multiple myeloma: questions about gene expression profiling and treatment-related mortality. J Clin Oncol 2011; 29: e124; author reply e125-126. Pineda-Roman M, Zangari M, Haessler J, Anaissie E, Tricot G, van Rhee F et al . Sustained complete remissions in multiple myeloma linked to bortezomib in total therapy 3: comparison with total therapy 2. Br J Haematol 2008; 140: 625-634. Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 2008; 9: 559. Hanzelmann S, Castelo R, Guinney J. GSVA: gene set variation analysis for microarray and RNA-seq data. BMC Bioinformatics 2013; 14: 7. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdottir H, Tamayo P, Mesirov JP. Molecular signatures database (MSigDB) 3.0. Bioinformatics 2011; 27: 1739-1740. Zheng GX, Terry JM, Belgrader P, Ryvkin P, Bent ZW, Wilson R et al . Massively parallel digital transcriptional profiling of single cells. Nat Commun 2017; 8: 14049. Guo X, Chen F, Gao F, Li L, Liu K, You L et al . CNSA: a data repository for archiving omics data. Database (Oxford) 2020; 2020. Chen FZ, You LJ, Yang F, Wang LN, Guo XQ, Gao F et al . CNGBdb: China National GeneBank DataBase. Yi Chuan 2020; 42: 799-809. Members C-N, Partners. Database Resources of the National Genomics Data Center, China National Center for Bioinformation in 2024. Nucleic Acids Res 2024; 52: D18-D32. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupplementaryFigure1.tif Supplementary Figure 1. The relationship between CXCL7 and drug sensitivity in the CellMiner database. SupplementaryFigure2.tif Supplementary Figure 2. Effect of CXCL7 on MM cell apoptosis. (The apoptotic ability of MM cells was detected by flow cytometry. a-b. The apoptotic ratio of NCI-H929 and RPMI 8226 CXCL7-OE group and control group. c-d. Apoptosis ratio in U266 and RPMI 8226 cells in CXCL7 -siRNA knockdown group and control group). SupplementaryFigure3.tif Supplementary Figure 3. Inhibitory effect of Ruxolitinib on JAK1 protein in MM cells. (The half maximal inhibitory concentration (IC50) of Ruxolitinib to inhibit JAK1 was 3.3 nM, and the concentration gradient of 0, 3.3 nM, 6.6 nM, 13.2 nM, 26.4 nM, 53.8 nM was set based on this concentration, which was added into RPMI 8226 cells, and then cultured for 24 h. The level of JAK1 protein was measured by WB. After 24 h, the level of JAK1 protein was detected by WB, and it can be seen that the amount of JAK1 protein was significantly reduced at a concentration of 26.4 nM). SupplementaryTable1.docx Supplementary Table 1. Sense primer sequences of CXCL7-siRNA. SupplementaryTable2.docx Supplementary Table 2. Primer sequences used in qPCR. SupplementaryTable3.docx Supplementary Table 3. Antibodies used in Western blot and IHC. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4489552","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":309003087,"identity":"858b4706-02ef-4ad1-9f1b-1de15d34e060","order_by":0,"name":"Peng Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIie2RsWrDMBCGzxjOixKtztK8giDQMXkVB68lGLJkKEFg8BToauhThEJmhYNmMXTt4MEQyJy9IfTiWJvtrIXqA4lfoE86nQAcjj/I0G+C5FFnBPAMgOlU0CojbRWs9/coNijTKPBQCYTyk1U5/fg6VBzWi+E4rQxcSpDvuqMwVvLiFO9McQu0RERlvOwEYdl+U60MMoqf95vgZ5CZeYZcpKcJVBj1KFeKJ2lQsbtmJThzYY8UTVPFh7PisyK4FdinYEL5J0Vhwa6o3/KScHkkwu92RUraHpNXmsk37pjgjo3Tw7Y6X+hJ5u3KDeIx180isrPo3A/3H5zZRffJDofD8W/5BR0vVsUaZtDLAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9639-6606","institution":"Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Peng","middleName":"","lastName":"Liu","suffix":""},{"id":309003088,"identity":"bb16dc90-e78f-4305-8405-c15715ebc4b8","order_by":1,"name":"Yue Wang","email":"","orcid":"","institution":"[email protected]","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Wang","suffix":""},{"id":309003089,"identity":"52898fba-f57a-44c4-905a-04e8ef31b4d4","order_by":2,"name":"Tianwei Lan","email":"","orcid":"","institution":"Zhongshan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Tianwei","middleName":"","lastName":"Lan","suffix":""},{"id":309003090,"identity":"0602e1cf-1143-4b0e-a210-90f0c631ddc0","order_by":3,"name":"Chi Zhou","email":"","orcid":"","institution":"Zhongshan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Chi","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2024-05-28 09:01:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4489552/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4489552/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58317168,"identity":"0aa61f01-d339-4e24-9007-ce9856fa86c8","added_by":"auto","created_at":"2024-06-13 21:16:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3572642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCXCL7 plays a crucial role in the proliferation of multiple myeloma and activation of the osteoclast signaling pathway in TT2 and TT3 patients. \u003c/strong\u003e(a. Results of weighted gene co-expression network analysis of RNA-seq data in the TT2 validation set. b. Correlation between different co-expression modules and \"Osteolysis signaling \" and \"B cell proliferation\". c. The relationship between genes in the darkmagenta co-expression module and signaling \"Osteolysis signaling\" pathway. d. The relationship between genes in the darkmagenta co-expression module and signaling \"B cell proliferation\" pathway. e. The expression level of CXCL7 and the activation of \" Osteolysis signaling\" pathway in TT2 training set. f. The expression level of CXCL7 and the activation of \"B cell proliferation\" pathway in TT2 training set. g. The expression level of CXCL7 and the activation of \"Osteolysis signaling\" pathway in TT3 validation set. h. The expression level of CXCL7 and the activation of \"B cell proliferation\" pathway in TT3 validation set.)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/8763bb253ecbd2639715dc3c.png"},{"id":58316609,"identity":"6f25e9b3-2ae0-43fa-b08d-e9943ea47afa","added_by":"auto","created_at":"2024-06-13 21:08:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1578529,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe results of single-cell RNA-seq confirmed that the subpopulation of MM cells exhibiting high CXCL7 expression displayed a more aggressive biological phenotype. \u003c/strong\u003e(a. Cell clustering results from single-cell RNA-seq in three newly diagnosed MM patients. b. Expression of CXCL7 in CD38+ cells. c. Expression of CXCL7 in all cells. d. Cell clustering results from single-cell RNA-seq of MM cells. e. CXCL7 was mainly expressed in MM03. f. Activation level of the \"B-cell proliferation\" pathway in various MM subsets. g. Activation level of the \"osteoclast signaling\" pathway in various MM subsets. h. MM03 subset exhibited the strongest correlation with the \"B cell proliferation\" and \"osteoclast signaling\" pathways. i. Activation status of pathways in distinct subsets of multiple myeloma cells.)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/f0504fcb6b2372450ded0ccc.png"},{"id":58316639,"identity":"6929ba91-46d4-4fe3-af8d-a496accbff3a","added_by":"auto","created_at":"2024-06-13 21:08:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4315685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrognostic implications and clinicopathological characteristics of patients stratified based on varying levels of CXCL7 expression. \u003c/strong\u003e(a. The optimal cut-off value of the relative expression level of CXCL7. b. The scatter plot illustrates the distribution of patient groups in relation to the gene expression levels of CXCL7. c. Survival curves for overall survival time of patients in different CXCL7 groups. d. Survival curves for progression-free-survival time of patients in different CXCL7 groups. e.Probability of extramedullary invasion in patients with different CXCL7 expression levels. f. Probability of pathological fracture in patients with different CXCL7 expression levels.)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/f93b2661dc8f61c42f9d4144.png"},{"id":58316645,"identity":"f3318391-05a7-48c6-9132-8fdca0cbc4e5","added_by":"auto","created_at":"2024-06-13 21:08:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1017502,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelection of cell lines used to establish CXCL7 overexpression and knockdown and validation of the efficiency of the operation.\u003c/strong\u003e (a. The expression level of CXCL7 in MM cell lines. b-c. qPCR was employed to assess the differential expression of CXCL7 between the overexpression and control groups in NCI-H929 and RPMI 8226 cells. d-e. In U266 and RPMI 8226 cell lines, the knockdown of SiRNA1 and SiRNA2 resulted in a significant difference in CXCL7 expression, which was assessed using qRCR to compare with the control group. f-g. Western blot analysis was performed to compare the levels of CXCL7 protein in NCI-H929 and RPMI 8226 cell lines between the overexpression group and the control group. h-i. Western blot analysis was performed to compare the levels of CXCL7 protein in NCI- U266 and RPMI 8226 cell lines between the knockdown group and the control group. j-k. ELISA was used to detect the secretion of CXCL7 protein between the overexpression group and the control group in NCI-H929 and RPMI 8226 cells. l-m. ELISA was used to detect the secretion of CXCL7 protein between the knockdown group and the control group in NCI-H929 and RPMI 8226 cells.)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/a7cd50324398d64bc3e01eaf.png"},{"id":58316642,"identity":"9f5b3130-044c-4082-a5c0-d6cd5485edf1","added_by":"auto","created_at":"2024-06-13 21:08:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3271702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe impact of CXCL7 on the proliferation, migratory and invasive of MM cell lines. \u003c/strong\u003e(a-d. The CCK8 assay was employed to assess the differences in cell proliferation rates among the CXCL7 overexpression group, knockdown group, and control group. e-f. Subcutaneous xenograft tumors of mice in CXCL7-OE group and NC group. g. The mean growth rate of subcutaneous xenograft tumors in CXCL7-OE and NC mice. h. The expression levels of CXCL7 and Ki-67 in the subcutaneous xenograft tumors of the CXCL7-OE group and NC group were detected using immunohistochemical staining. i-j. Transwell assay was conducted to evaluate the invasion and migration abilities of NCI-H929 and RPMI 8226 cell lines in the CXCL7-OE and NC groups. k-l. Transwell assay was conducted to evaluate the invasion and migration abilities of U266 and RPMI 8226 cell lines in the CXCL7- knockdown and NC groups.)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/046ee194b261ad9c52e0a059.png"},{"id":58316629,"identity":"c9da92c3-8c5e-418a-a0bf-1883d6193b59","added_by":"auto","created_at":"2024-06-13 21:08:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1932586,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of CXCL7 on the metastatic potential of MM cells in vivo. \u003c/strong\u003e(a. Schematic diagram of subcutaneous xenograft model and tail vein injection xenograft model in mice. b. Quantitative levels of free λ light chain in the veins of CXCL7-OE and NC group. c. The presence of tumor colonization in the femur was confirmed through HE staining in both the CXCL7-OE group and the NC group. d. HE staining was employed to identify the presence of lung, liver, kidney, and subcutaneous tumor colonization in mice from the CXCL7-OE group and NC group. e-g. MicroCT imaging was utilized to assess bone destruction in the femur of mice in the CXCL7-OE group and NC group, with a focus on differences in bone mineral density (BMD) and trabecular thickness (Tb). h. Immunohistochemistry was employed to detect TRAP, MMP2, MMP9, and MMP13 expression levels in CXCL7-OE and NC groups.)\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/195fdb0aa2989a2aa6ff7ce6.png"},{"id":58316606,"identity":"0a7dde02-69ae-4ed9-8bce-c9126b8d5544","added_by":"auto","created_at":"2024-06-13 21:08:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3017963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe impact of the CXCL7 gene on the IL2-STAT5 signaling pathway.\u003c/strong\u003e (a. Gene sets exhibiting statistically significant differences between CXCL7-OE cell lines and control cell lines were subjected to GSEA enrichment analysis. b. Gene sets enrichment analysis of the IL2-STAT5 signaling pathway. c-f. Western blot analysis was performed to assess the protein expression levels of JAK1, p-JAK1, STAT5, p-STAT5 and IL-2R in H929, U266 and RPMI 8226 cells within the CXCL7-OE group, CXCL7 knockdown group and NC group. g. Immunohistochemistry was used to detect the expression of IL-2R, p-JAK1 and pSTAT5 in the transplanted tumor of mice. h. CCK8 assay was employed to assess the influence of CXCL7 on the proliferation of MM cells following the administration of ruxolitinib. i-j. Transwell assay was employed to assess the impact of CXCL7 on the migratory and invasive capabilities of MM cells following treatment with ruxolitinib.)\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/da5098ccea00e27216ffa28d.png"},{"id":58810127,"identity":"8b4d6713-c6dd-42d1-9f79-001bf0b500d0","added_by":"auto","created_at":"2024-06-21 11:44:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17710949,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/4dd74e6a-142f-4886-9ffc-559b8589d974.pdf"},{"id":58316640,"identity":"5a9d8c1c-5140-4355-b5b9-0dc6fb3ddc5f","added_by":"auto","created_at":"2024-06-13 21:08:36","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3114912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. The relationship between CXCL7 and drug sensitivity in the CellMiner database.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/83be57b4372d4045a332f18d.tif"},{"id":58316604,"identity":"b6e7db43-8fdb-4d0e-91ef-d89ed5bb3307","added_by":"auto","created_at":"2024-06-13 21:08:34","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12928632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Effect of CXCL7 on MM cell apoptosis.\u003c/strong\u003e (The apoptotic ability of MM cells was detected by flow cytometry. a-b. The apoptotic ratio of NCI-H929 and RPMI 8226 CXCL7-OE group and control group. c-d. Apoptosis ratio in U266 and RPMI 8226 cells in CXCL7 -siRNA knockdown group and control group).\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/534f42103c8af56fe07a7080.tif"},{"id":58316638,"identity":"c0fb1b67-4a15-4d6a-82bc-cc259ce152af","added_by":"auto","created_at":"2024-06-13 21:08:36","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3772256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. Inhibitory effect of Ruxolitinib on JAK1 protein in MM cells. \u003c/strong\u003e(The half maximal inhibitory concentration (IC50) of Ruxolitinib to inhibit JAK1 was 3.3 nM, and the concentration gradient of 0, 3.3 nM, 6.6 nM, 13.2 nM, 26.4 nM, 53.8 nM was set based on this concentration, which was added into RPMI 8226 cells, and then cultured for 24 h. The level of JAK1 protein was measured by WB. After 24 h, the level of JAK1 protein was detected by WB, and it can be seen that the amount of JAK1 protein was significantly reduced at a concentration of 26.4 nM).\u003c/p\u003e","description":"","filename":"SupplementaryFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/a95be78702f38872d0462608.tif"},{"id":58317169,"identity":"03128740-ccd6-4468-81f6-4011c8403c04","added_by":"auto","created_at":"2024-06-13 21:16:36","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1. Sense primer sequences of CXCL7-siRNA.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/e514fceeb7526156565d7c06.docx"},{"id":58316641,"identity":"925f82da-c446-4dab-8c02-f98eaf9a1f65","added_by":"auto","created_at":"2024-06-13 21:08:36","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 2. Primer sequences used in qPCR.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/cdd0cd2ec308c504f3c422eb.docx"},{"id":58317341,"identity":"5e35381b-3c07-47a7-96da-a037bbd464cc","added_by":"auto","created_at":"2024-06-13 21:24:36","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":16590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 3. Antibodies used in Western blot and IHC.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryTable3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4489552/v1/cea5393d455d06b17d1f98bb.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"CXCL7 plays a crucial role in facilitating extramedullary invasion and osteolytic damage in multiple myeloma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMultiple myeloma (MM) is a difficult-to-cure malignant tumor with a relatively slow progression and high intra-tumor heterogeneity[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Multiple myeloma presents with diverse clinical manifestations, target organ damage, and prognosis among different patients [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Furthermore, despite the minimal variance in the morphology of monoclonal plasma cells between myeloma and its precursor disease (including monoclonal gammopathy of unknown significance (MGUS) and smoldering myeloma (SMM))[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], their biological behavior differs significantly[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The deterioration of biological behavior in multiple myeloma may be the fundamental reason why it differs from its precursor diseases. Being a mature B-cell tumor, its genetic regulation of biological behavior may fundamentally differ from other types of tumors. The CRAB symptoms (hypercalcemia, renal insufficiency, anemia and osteolytic lesions) of myeloma are primarily a result of the high proliferation rate of tumor cells. The extramedullary infiltration and activation of osteoclast signaling are attributed to the malignant biological potential of tumor cells[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Indeed, modulating the biological behavior of myeloma cells to achieve prolonged tumor-bearing survival while managing the extent of target organ damage may represent a more efficacious therapeutic approach than complete depletion of monoclonal plasma cells. Therefore, it is imperative to identify the key genes that impact the biological behavior of myeloma cells.\u003c/p\u003e \u003cp\u003eBy analyzing the RNA-seq data from the myeloma database and single-cell sequencing data from our center, we found that CXCL7 may be an important factor in the deterioration of myeloma cell biological behavior, which can promote myeloma proliferation, osteolytic bone destruction and extramedullary (EMD) invasion in myeloma. The chemokine CXCL7 belongs to a class of chemotactic cytokines known as chemokines, which play a crucial role in regulating the migration of immune cells. Chemokines serve as signaling molecules that coordinate, recruit, and facilitate the entry and exit of immune cells within tissues, while also guiding their spatial distribution and facilitating cell-to-cell interactions[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the context of tumors, chemokines are essential for directing the migration of immune cells to elicit effective anti-tumor immune responses. Chemokines in solid tumors have been studied mainly for their effects on immune cell infiltration and stromal cell function[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and are unlikely to be the main carcinogenic factors of tumor cells. However, plasma cells are mature B lymphocytes whose recruitment, infiltration, migration, and proliferation in tissues may all be influenced by chemokines. In other words, chemokines may play a decisive role in immune cell tumors. Previous studies have shown that CXCL7 promotes proliferation, facilitates invasion, and is associated with poor prognosis in malignancies such as breast cancer, lung cancer, and renal cancer[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the role of CXCL7 in myeloma remains unclear. The aim of this study is to explore the role and associated mechanisms of CXCL7 in myeloma and provide a theoretical basis for new therapeutic targets.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCXCL7 plays a pivotal role in governing the biological activities of myeloma cells\u003c/h2\u003e \u003cp\u003eThe objective of our study was to identify the crucial genes influencing the biological behavior of MM cells. Proliferation, osteoclast activity, invasion, and metastasis are the most significant indicators of MM cell deterioration. However, there is no established signaling pathway for extramedullary invasion and migration of plasma cell tumors. Therefore, we utilized proliferation and osteoclast signaling activity as features to identify the key genes. We analyzed RNA-seq cohort data from MM patients in the GSE24080 dataset, using the TT2 cohort as the training set and the TT3 cohort as the validation set. The WGCNA analysis divided 23517 genes into 33 modules in TT2 set (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Subsequently, we used the ssGSEA method to quantify the activation of \"B cell proliferation\" and \"Osteoclast signaling pathway\" in all patients of the TT2 cohort. We then analyzed the correlation between coexpression gene modules calculated by WGCNA and the activation of these pathways. The results showed that the darkmagenta module significantly promoted activation in both \"B cell proliferation\" (P\u0026thinsp;=\u0026thinsp;5.6e-11) and \"Osteoclast signaling\" (P\u0026thinsp;=\u0026thinsp;9.0e-17) pathways. The relationships between different modules and traits are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb. And then, all genes within the darkmagenta module were calculated for their correlation with the two traits (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Based the cut-off criteria (|MM| \u0026gt; 0.78), two genes with high connectivity in the clinically significant module were identified as hub genes. The two hub genes are CXCL7 (MM\u0026thinsp;=\u0026thinsp;0.787, P\u0026thinsp;=\u0026thinsp;6.6e-74) and S100A8 (MM\u0026thinsp;=\u0026thinsp;0.811, P\u0026thinsp;=\u0026thinsp;5.6e-82). Considering the potential involvement of chemokines in MM as mentioned above, CXCL7 was chosen for further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe confirmed that the expression level of CXCL7 was positively correlated with \"B cell proliferation\" (r\u0026thinsp;=\u0026thinsp;0.32, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) and \"Osteoclast signaling\" (r\u0026thinsp;=\u0026thinsp;0.36, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef) in the TT2 cohort. In addition, we also confirmed that CXCL7 was closely related to the two traits in the TT3 validation cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eWe also examined the expression of CXCL7 and assessed drug sensitivity using the CellMiner database[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Our findings revealed a significant correlation between elevated levels of CXCL7 expression and resistance to multiple anti-tumor agents, including ixazomib, a proteasome inhibitor commonly employed in the treatment of myeloma (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSingle-cell RNA-seq analysis revealed that MM cell subsets with elevated expression level of CXCL7 exhibited aggressive biological features\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOwing to the high intratumor heterogeneity of myeloma, the biological behavior of distinct subsets varies significantly. We aim to further investigate the characteristics of MM cell subsets exhibiting elevated expression level of CXCL7. Therefore, bone marrow specimens were obtained from three newly diagnosed MM patients with International Staging System (ISS) III for single-cell RNA-seq analysis. All three patients presented with pathological fractures at the time of diagnosis. The resulting data from individual cells across multiple patients were integrated for UMAP downscaling analysis which revealed distinct subgroups consisting of MM cells along with monocytes, erythrocytes, and T-cell subsets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In addition to the high expression level of CXCL7 in monocytes, we observed a significant upregulation of CXCL7 in some monoclonal plasma cells as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c) By utilizing CD38 and CD138 (SDC1) as markers for the isolation of plasma cells in subsequent downscaling analysis, we successfully classified MM cells into 11 clonal subpopulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The expression of CXCL7 was predominantly observed in the MM03 subgroup (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe selected \"B cell proliferation\" and \"osteoclast signaling\" as target pathways and conducted signaling pathway enrichment analysis for all MM cell subsets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-g). The results revealed that the MM03 subset exhibited the strongest correlation (lowest p-value) with the \"B cell proliferation\" and \"osteoclast signaling\" pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Moreover, MM03 was found to be the most activated subgroup in both pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-g).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCXCL7 is closely related to OS and PFS of patients, and is also related to pathological fracture and extramedullary invasion\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe collected bone marrow samples from 108 newly diagnosed MM patients, assessed the expression levels of CXCL7 through qPCR. By employing the \"cut-off\" function in the \"Survival\" package, we identified the optimal cut-off value for CXCL7 and compared prognosis differences among patients with different expression levels of CXCL7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b). The findings indicated that elevated levels of CXCL7 in myeloma cells were correlated with reduced overall survival (OS) (P\u0026thinsp;=\u0026thinsp;0.016) and progression-free survival (PFS) (P\u0026thinsp;=\u0026thinsp;0.008) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d). Additionally, we compared clinicopathological variances between the high CXCL7 expression group and low CXCL7 expression group. The outcomes demonstrated that patients in the high CXCL7 expression group exhibited a higher likelihood of extramedullary invasion (P\u0026thinsp;=\u0026thinsp;0.010) and pathological fracture (P\u0026thinsp;=\u0026thinsp;0.048) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-f). However, no substantial correlation was observed between CXCL7 and ISS stage or high-risk cytogenetics. The clinical baseline characteristics of the patients are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These findings indicate that CXCL7 may facilitate the proliferation of myeloma cells, the activation of the osteoclast signaling pathway and the invasion of MM cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline characteristics of 108 patients with multiple myeloma.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCXCL7_High\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCXCL7_Low\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatinets(No)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender(No,%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74(68.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46(66.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28(71.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34(31.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23(33.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11(28.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge(year, (No,%))\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30(27.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15(21.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15(38.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78(72.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54(78.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24(61.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eISS stage(No,%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43(39.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29(42.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14(35.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28(25.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16(23.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12(30.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37(34.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24(34.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13(33.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFISH high-risk(No,%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetected\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34(31.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23(33.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11(28.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNot detected\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74(68.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46(66.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28(71.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFollow-up time (month, (median,range))\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48.7(2.1\u0026ndash;103.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.3(2.1\u0026ndash;77.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.2(5.4\u0026ndash;103.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProgress(No,%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53(49.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41(68.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12(30,8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDead(No,%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34(31.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27(39.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7(17.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of MM cells with CXCL7 overexpression and knockdown\u003c/h2\u003e \u003cp\u003eWe utilized the CCLE database to assess the expression of CXCL7 in MM cell lines. Our findings revealed that CXCL7 is expressed at low levels in the NCI-H929 cell line, moderate levels in the RPMI 8226 cell line, and high levels in the U266 cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Lentiviruses carrying CXCL7-overexpression (CXCL7-OE) and Normal-Control (NC) were used to infect NCI-H929 and RPMI 8226 cell lines. Meanwhile, siRNA was employed for CXCL7 knockdown (CXCL7-KD) and NC transfection in U266 and RPMI 8226 cell lines. The RNA levels of CXCL7 in these four cells were assessed by qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-e). Additionally, WB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-i) and ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej-m) techniques were utilized to detect intracellular protein levels as well as secreted protein levels of CXCL7.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCXCL7 promotes the proliferation of MM cells\u003c/h2\u003e \u003cp\u003eThe proliferative viability of the cells was assessed using a CCK-8 assay. The results demonstrated that the proliferation rate of NCI-H929 and RPMI 8226 cell lines in the CXCL7-OE group was significantly higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). Conversely, the proliferation rate of U266 and RPMI 8226 cell lines in the CXCL7-KD group was slower (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-d). Although we failed to find that CXCL7 was able to impression the cell cycle of MM cells (Supplementary Fig.\u0026nbsp;2). To further investigate the impact of CXCL7 on the proliferation of MM cells in vivo, subcutaneous xenograft tumor models were established in BNGD mice using the RPMI 8226 cell line with overexpression of CXCL7 and a normal control group. Subsequent analysis revealed faster tumor growth in the CXCL7-OE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-g). Immunohistochemistry performed on excised tumors showed elevated levels of Ki-67 proliferation index in CXCL7-OE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCXCL7 significantly enhances the invasion and migration of MM cells\u003c/h2\u003e \u003cp\u003eThe migratory and invasive capacities of MM cell lines were then evaluated using transwell assays. In the experiments assessing migratory capabilities, a significantly higher number of cells were found to penetrate the lower chamber surface in the NCI-H929 CXCL7-OE group (P\u0026thinsp;=\u0026thinsp;0.0024) and in the RPMI 8226 CXCL7-OE group (P\u0026thinsp;=\u0026thinsp;0.0066) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei), indicating that up-regulation of CXCL7 enhances the migratory ability of MM cells. Furthermore, assessment of invasive ability demonstrated a significantly higher number of protruding cells in both NCI-H929 and RPMI 8226 CXCL7-OE groups (P\u0026thinsp;=\u0026thinsp;0.0026 and P\u0026thinsp;=\u0026thinsp;0.0006, respectively), suggesting that up-regulation of CXCL7 enhances the invasive ability of MM cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej). Then, transwell assays were also performed in CXCL7-KD cell lines, revealing a significant reduction in the migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek) and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003el) capabilities of MM cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCXCL7 can significantly promote the extramedullary invasion of myeloma cells and enhance their colonization ability in bone marrow\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we established a mouse xenograft model by intravenously injecting MM cells to investigate the ability of CXCL7-OE MM cells to colonize the bone marrow through homing effects and to generate extramedullary lesions through migration and infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). This approach effectively mimics the activity of plasma cells in vivo, allowing us to explore the impact of CXCL7 on the biological behavior of myeloma cells in a more physiological context. At 7 weeks, it was observed that some mice in the CXCL7-OE group exhibited symptoms indicative of long bone fractures (three out of five). No such phenomenon was observed in the control group. Concurrently, we conducted regular venous blood collection on mice to quantify serum free light chain levels and assess tumor burden. The results revealed a significantly higher tumor burden in the CXCL7-OE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter the mice were euthanized and dissected, more than two extramedullary plasmacytomas were identified in the subcutaneous soft tissues of all the mice in the CXCL7-OE group. Additionally, an increased number of intraperitoneal tumors were observed. A small number of peritoneal colonization lesions were also noted in the NC group, but no colonization of the subcutaneous soft tissues was found in any of the mice in the NC group. Previous studies have indicated that subcutaneous soft tissue is the most common site for extramedullary invasion (Bone independent EMD) in MM[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Our findings suggest that overexpression of CXCL7 enhances MM cells' ability to infiltrate and colonize subcutaneous soft tissue in a mouse xenograft model, which is consistent with clinical observations.\u003c/p\u003e \u003cp\u003eFor further analysis, tissue samples from the femur, subcutaneous metastases, liver, lungs, and kidneys were collected for histopathological examination using hematoxylin and eosin (HE) staining as well as immunohistochemical staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-d). The sections of the mouse femur revealed that myeloma cells in the group with high expression level of CXCL7 exhibited enhanced colonization and homing in the bone marrow. The sections of mice visceral and subcutaneous soft tissue tumors revealed that the CXCL7-OE MM cell line did not exhibit significant metastasis to the lung, liver, and kidney. However, it did result in increased infiltration and generation of subcutaneous soft tissue tumors.\u003c/p\u003e \u003cp\u003eFollowing the identification of leg bone fracture in the CXCL7-OE group, we proceeded to conduct micro-CT bone scan imaging on the femur of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). It was evident that the CXCL7-OE group exhibited more significant deterioration in femur bone compared to the NC group, as indicated by a reduction in bone mineral density (BMD) (P\u0026thinsp;=\u0026thinsp;0.0068) and a decrease in average bone trabeculae thickness (Tb.Th) (P\u0026thinsp;=\u0026thinsp;0.0371) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef-g). Osteoclast markers tartrate resistant acid phosphatase (TRAP) and matrix metalloproteinases-9 (MMP9), along with bone marrow stromal cell markers matrix metalloproteinases-2 (MMP2) and matrix metalloproteinases-13 (MMP13) were used for staining of the bone marrow (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). The findings indicated a significant increase in osteoclast markers within the CXCL7-OE group, with a predominant concentration observed near the interface of myeloma lesions and bone marrow. Amongst the osteoclast signaling activators investigated, MMP13 exhibited the highest expression levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCXCL7 is capable of modulating the biological behavior of MM cells via the IL2-STAT5 pathway\u003c/h2\u003e \u003cp\u003eWe conducted transcriptome sequencing (RNA-seq) on CXCL7-OE and NC RPMI 8226 cell lines. GSEA methods were employed to identify significant activation and enrichment pathways. The results indicated a substantial up-regulation of four hallmark gene sets in the CXCL7-OE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-b). Among these pathways, it was observed that the IL-2-STAT5 pathway plays a critical role in immune cell regulation. Studies have demonstrated that activation of the IL-2-STAT5 pathway promotes B lymphocyte proliferation, regulates their differentiation, and enhances their capacity for antibody and light chain secretion[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Given that MM is characterized by the secretion of light chains and monoclonal immunoglobulins, we have chosen to further investigate the IL2-STAT5 signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIntracellular transduction of the IL2-STAT5 signaling pathway is facilitated by the heterodimerization of the IL-2R cytoplasmic domain, leading to activation of the JAK1-STAT5 pathway. To investigate the IL-2R/JAK1/STAT5 signaling pathway, we conducted western blot analysis to assess the expression of IL-2R, phosphorylated JAK1 protein (p-JAK1), and phosphorylated STAT5 (p-STAT5) as well as their total protein. The results revealed that in NCI-H929 and RPMI 8226 cell lines, the expression level of JAK1 and STAT5 were similar between the CXCL7-OE group and NC group; however, the expression level of IL-2R, p-JAK1, and p-STAT5 were elevated in CXCL7-OE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec-d), indicating that upregulation of CXCL7 activates the IL-2R/JAK1/STAT5 signaling pathway. In U266 and RPMI 8226 cell lines, there was no significant difference in the expression level of JAK1 and STAT5 between CXCL7-KD group and NC group; nevertheless, the expression level of IL-2R, p-JAK1, and p-STAT5 were reduced in the CXCL7-KD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee-f), suggesting that inhibition of CXCL7 leads to suppression of the IL-2R/JAK1/STAT5 signaling pathway. Immunohistochemical staining of tumor sections from the aforementioned mouse xenograft model also revealed a significant increase in IL-2R/JAK1/STAT5 levels in the CXCL7-OE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). Additionally, analysis of single cell-based pathways revealed a significant augmentation in the IL-2-STAT5 pathway in MM03, providing further evidence for activation of this pathway in a subset of highly CXCL7-expressing MM cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003eTo further investigate the role of CXCL7, RPMI 8226 CXCL7-OE cells were treated with Ruxolitinib[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], a JAK1/JAK2 inhibitor (Supplementary Fig.\u0026nbsp;3). Cell proliferation was assessed using CCK-8 assay. The results demonstrated that cells in the CXCL7-OE group exhibited accelerated proliferation compared to the NC group, whereas cells in the CXCL7-OE\u0026thinsp;+\u0026thinsp;Ruxolitinib group showed attenuated proliferation. Notably, there was no significant difference between the CXCL7-OE\u0026thinsp;+\u0026thinsp;Ruxolitinib group and the NC group, suggesting that Ruxolitinib effectively inhibited MM cell proliferation induced by CXCL7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh). Additionally, a transwell assay was performed to evaluate the migratory and invasive capabilities of MM cells. The results indicated that the migratory and invasive capability of cells in the CXCL7-OE group exceeded that of NC group, whereas it was diminished in the CXCL7-OE\u0026thinsp;+\u0026thinsp;Ruxolitinib group compared to both CXCL7-OE and NC groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei-j). Our study has demonstrated that a subset of MM cells exhibiting high expression level of CXCL7 is capable activating IL2-STAT5 pathway in absence exogenous IL-2.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMultiple myeloma is a distinct neoplasm characterized by the presence of monoclonal plasma cells in precursor states such as MGUS and SMM[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In most instances, MGUS and SMM are typically managed with observation as they generally do not exert a substantial impact on the patient's quality of life[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, myeloma presents a distinct scenario in which the altered biological behavior of neoplastic cells results in multi-organ dysfunction. Genomic variations contribute to the unique biological characteristics exhibited by these plasma cells[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The high degree of intratumor heterogeneity in multiple myeloma results in distinct biological characteristics among different tumor cell subsets, posing challenges for effective treatment and leading to diverse target organ damage and clinical manifestations in patients[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our study has identified CXCL7 as a potential key gene influencing the biological behavior of tumors in MM. We observed that a subset of myeloma cells exhibiting high expression level of CXCL7 displayed highly malignant biological behavior. Through genomic exploration in a large sample cohort, we have confirmed the significant impact of CXCL7 on lytic bone lesions and extramedullary invasion in MM. Furthermore, we have conducted cytological experiments and utilized a mouse xenograft model to investigate the function and potential regulatory mechanisms of CXCL7. It is noteworthy that CXCL7 belongs to the chemokine family, which plays a role in directing chemotaxis and mediating immune cell function in humans[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. While numerous chemokines are involved in promoting tumorigenesis and progression in solid tumors, their primary role is to modulate tumor-associated immune responses, rather than serving as key oncogenes. In the context of multiple myeloma, a malignant plasma cell tumor, chemokines may indeed act as central oncogenes.\u003c/p\u003e \u003cp\u003eCXCL7 has been identified as the primary activator of MMP13 in MM, a crucial pathway for osteolytic damage in the bone marrow that may be secreted by MSCs[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The differentiation and maturation of immune cells are intricately linked to chemokine activity, as they can govern the activation, recruitment, phenotype, and function of immune cells by reprogramming chemokine and cytokine receptors and modulating signaling pathway activation[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As mature B lymphocytes with secretory function, plasma cells not only produce immunoglobulins but also secrete a variety of cytokines and chemokines. Our study revealed that a subset of monoclonal plasma cells is capable of secreting CXCL7. Plasma cells with high expression levels of CXCL7 can stimulate their own proliferation, enhance the invasion and infiltration capabilities of plasma cells, and significantly increase the likelihood of extramedullary invasion. Furthermore, CXCL7 can induce the activation of MMP13 in stromal cells through paracrine effects to promote osteoclast signaling pathway activation.\u003c/p\u003e \u003cp\u003eMoreover, our study successfully established a mouse model of myeloma transplantation through tail vein injection. By harnessing the inherent homing ability of plasma cells, we have demonstrated that monoclonal plasma cells display enhanced colonization in the bone marrow and rapid proliferation upon overexpression of CXCL7. Upon high expression levels of CXCL7, plasma cells also exhibited improved colonization in subcutaneous tissues, leading to extramedullary invasion lesions. Consistent with previous findings, diffuse MM lesions were observed in the NC RPMI8226 cell line injected via tail vein in mice, while subcutaneous soft tissue nodules were not detected[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Subcutaneous soft tissue is the most common site of extramedullary invasion in MM patients[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and the high expression level of CXCL7 makes this phenomenon replicated in the mouse model.\u003c/p\u003e \u003cp\u003eThe IL2-STAT5 pathway plays a critical role in regulating immune cell function[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], including stimulating T cell proliferation, modulating T cell apoptosis, and enhancing NK cell cytolytic activity[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In B cells, the IL2-STAT5 pathway promotes B cell proliferation, enhances light chain and immunoglobulin secretion, and regulates B cell function[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The activation of the IL2-STAT5 pathway in lymphocytes is not solely dependent on high doses of IL-2. For instance, some regulatory T cells are capable of robustly activating downstream STAT5 even stimulated with low doses or even in the absence of exogenous IL-2 due to the signal amplification effect mediated by Hippo kinases Mst1 and Mst2[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our study has also demonstrated that CXCL7 can activate intracellular IL2R-STAT5 signaling independently of exogenous IL-2; however, it is noteworthy that CXCL7 does not directly bind to IL-2R. We hypothesize that this phenomenon may be attributed to two potential reasons: firstly, CXCL7 may trigger signal amplifiers similar to Mst1 and Mst2 within MM cells; secondly, it is possible that CXCL7 binds to surface receptors on MM cells forming a complex multi-binding complex leading to activation of intracellular IL2R-STAT5 signaling pathways. Further investigations are warranted in the future.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWeighted correlation network analysis (WGCNA)\u003c/h2\u003e \u003cp\u003eFirst, we utilized the gene expression profiles of patients in the GEO database. We chose the GSE24080 cohort and designated the TT2 patient group as the training set[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], while assigning the TT3 patient group as the validation set[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The R package \u0026ldquo;WGCNA\u0026rdquo; was used to construct a scale-free co-expression network [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. To classify genes with similar expression profiles into gene modules, we conducted average linkage hierarchical clustering according to the TOM-based dissimilarity measure with a minimum gene group size of 30 for the genes dendrogram. Additionally, we merged modules with a distance less than 0.25.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSingle sample Gene Set Enrichment Analysis (GSEA)\u003c/h2\u003e \u003cp\u003eSingle sample GSEA (ssGSEA) analysis was performed to determine the degree of activation for each pathway, which was recorded as the Enrichment Score (ES)[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The score ranged from 0 to 1, indicating minimal to maximal activation. The gene sets for these pathways were obtained from the MsiGDB database[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe expression level of CXCL7 and clinicopathological features of samples\u003c/h2\u003e \u003cp\u003eA total of 108 clinical samples of newly diagnosed MM patients were included in this study, which were collected from patients who visited Zhongshan Hospital of Fudan University from 2019 to 2020. In order to further observe the relationship between CXCL7 and the prognosis and drug treatment response of patients, only patients who had not undergone autologous hematopoietic stem cell transplantation were included. All patients received an initial treatment of a three-drug combination of protease inhibitor, lenalidomide, and dexamethasone. The bone marrow samples of patients were collected at the time of diagnosis. All bone marrow fluid samples were enriched with nucleated cells and sorted with CD138 magnetic beads. The expression level of CXCL7 is detected by the qPCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSingle cell mRNA sequencing\u003c/h2\u003e \u003cp\u003e A diverse array of bone marrow samples was collected from Zhongshan Hospital affiliated with Fudan University, following approval from the Institutional Review Board of Zhongshan Hospital. Bone marrow mononuclear cells (BMMCs) were isolated and employed for single-cell RNA sequencing (scRNAseq). Single-cell libraries were generated from unsorted BMMCs using a 10\u0026times; Genomics Chromium Single Cell 5\u0026prime; Library Kit and Chromium instrument, followed by sequencing on an Illumina sequencer. The obtained FASTQ data was aligned to the reference genome (GRCh38) using Cell Ranger (v5.0.1)[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Seurat v4.0.1 software was utilized for cell clustering and dimension reduction. For pathway activation analysis in different cell subsets, we employed the irGSEA package along with AUCell and UCell methods to score pathway activation levels based on gene sets obtained from the MSigDB database[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of lentiviral stabilized cell lines\u003c/h2\u003e \u003cp\u003eU266 and NCI-H929 cell lines are purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and RPMI-8226 cell line is obtained from the cell bank of the Chinese Academy of Sciences for use in this investigation. The vector components used in the construction of the CXCL7 overexpression lentivirus are Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSmall interfering RNA transfection\u003c/h2\u003e \u003cp\u003eSmall interfering RNA (siRNA) targeting CXCL7, as shown in Supplementary Table\u0026nbsp;1, is transfected into MM cells in logarithmic growth phase using a transfection complex consisting of RPMI 1640 medium, Lipo3000, and siRNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA assay)\u003c/h2\u003e \u003cp\u003eThe concentration of human CXCL7 cytokine in the culture medium was measured using an ELISA kit (Abcam, England). All procedures were performed according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTranswell migration and invasion assays\u003c/h2\u003e \u003cp\u003eA transwell chamber with 8.0-\u0026micro;m pore size (Corning, USA) was used to measure cell migration, and a Matrigel transwell chamber (Corning, USA) was used for cell invasion tests. Cells were initially inoculated onto top-perforated membranes and incubated for 24 hours (migration experiment) or 48 hours (invasion experiment). After incubation, cells in the upper part of the insert were removed and cells adhering to the lower part were fixed, stained with crystal violet, photographed at three locations, and counted using Image J (NIH, US).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT-PCR analysis\u003c/h2\u003e \u003cp\u003e Quantitative RT-PCR analyses were performed using the EZ Bioscience RNA Extraction Kit to extract RNA from control or treated cells, followed by cDNA synthesis with the Takara cDNA Synthesis Kit and real-time PCR using the Takara TB Green Premix Ex Taq (Tli RNaseH Plus) quantitative PCR kit. All primer sequences used in this study are shown in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eTo lyse the cells, use 1X RIPA buffer containing Tris buffer (50mM, pH 7.4), NaCl (150mM), Triton X-100 (1%), PMSF (1mM), SDS (0.1%), and protease inhibitor mixture from Beyotime Biotechnology. Cell lysates were mixed with Loading Buffer, separated by electrophoresis in a 10\u0026ndash;15% SDS-PAGE gel, and transferred to a PVDF membrane from Millipore. Membranes were blocked with QuickBlock Blocking Buffer for Western Blot from Biotechnology. Membranes were washed three times with PBST and incubated with enzyme-linked secondary antibodies for 1 h at room temperature. Following PBST cleaning, luminescence was recorded with Lumin4000 from GE, USA and ECL reagent from BioSharp. All antibodies used are listed in Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSubcutaneous Transplantation Tumor\u003c/h2\u003e \u003cp\u003eCells were harvested during logarithmic growth, washed with PBS, and resuspended in the appropriate volume of PBS to achieve a density of f 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e/100 \u0026micro;L. The cells were then subcutaneously injected into B-NDG mice. Five randomly assigned 5-week-old female mice were divided into NC group and CXCL7-OE group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eXenograft tumor model in mice through intravenous injection\u003c/h2\u003e \u003cp\u003eCells were collected during logarithmic growth, washed with PBS, and resuspended in the appropriate amount of PBS at a density of 10\u003csup\u003e7\u003c/sup\u003e/100\u0026micro;L before being injected into the tail vein of B-NDG mice within 2.5 hours. Each group of five 5-week-old female mice was randomly assigned to either the NC group or the CXCL7-OE group. Tumor development in the mice was regularly monitored.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC) analysis\u003c/h2\u003e \u003cp\u003eMouse tumors were sampled and then embedded in paraffin and sectioned by a microtome. Immunohistochemistry was performed according to the steps provided by the manufacturer of the immunohistochemistry kit (Sevier, China), in which the primary antibodies used are also listed in Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis.\u003c/h2\u003e \u003cp\u003eStatistical methods were all analyzed by R version 4.0.3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cran.r-project.org\u003c/span\u003e\u003cspan address=\"http://cran.r-project.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). P values were all two-sided and statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of Fudan University Zhong Hospital and complied with the principles of the Helsinki Accord. The real-world patient data included in this study was a retrospective study and patient consent was therefore deemed unnecessary. However, bone marrow samples were collected from all subjects, with patient consent, and stored in the tissue bank at Fudan University Zhongshan Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe single-cell RNA-seq processed gene expression data reported in this paper have been deposited into the CNGB Sequence Archive (CNSA)[35]\u0026nbsp;of the China National GeneBank DataBase (CNGBdb)[36]\u0026nbsp;with accession number CNP0005613, and raw sequencing data have been deposited in the OMIX, China National Center for Bioinformation, Chinese Academy of Sciences (accession no. OMIX006258)[37]. To request access to raw sequencing data, please apply at the Human Genetic Resources Service System of the Ministry of Science and Technology (https://apply.hgrg.net/) in accordance with the Regulations on the Management of Human Genetic Resources of China. The multiple myeloma RNA-seq data GSE24080 were downloaded from the GEO database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone declared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: YW, TL, PL\u003c/p\u003e\n\u003cp\u003eMethodology: YW, TL\u003c/p\u003e\n\u003cp\u003eAssistant: CZ\u003c/p\u003e\n\u003cp\u003eData collection: YW, TL, CZ\u003c/p\u003e\n\u003cp\u003eWriting—original draft: YW, TL\u003c/p\u003e\n\u003cp\u003eWriting—review \u0026amp; editing: PL\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScience and Technology Innovation Action Plan of Shanghai (21YF1406300)\u003c/p\u003e\n\u003cp\u003eYouth Foundation of Zhongshan hospital Fudan University (2021ZSQNN61)\u003c/p\u003e\n\u003cp\u003eNational Natural Science Foundation of China (81570123)\u003c/p\u003e\n\u003cp\u003eNatural Science Foundation of Shanghai (22ZR1411400).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRajkumar SV. 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Database Resources of the National Genomics Data Center, China National Center for Bioinformation in 2024. \u003cem\u003eNucleic Acids Res\u003c/em\u003e 2024; 52: D18-D32.\u003c/li\u003e\n\u003c/ol\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":"multiple myeloma, CXCL7, extramedullary invasion, osteolytic damage, IL2-STAT5","lastPublishedDoi":"10.21203/rs.3.rs-4489552/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4489552/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe primary distinction between multiple myeloma (MM) and its precursor conditions lies in the deterioration of the biological behavior of tumor cells. In MM, a type of mature B-cell tumor, chemokines may serve as pivotal regulatory genes. Through exploration of GEO database and single-cell RNA-seq data from our laboratory, we have identified chemokines CXCL7 as a potential key regulator of the cellular biological behavior in MM. Subsets of MM cells with high CXCL7 exhibit heightened malignant potential. Elevated CXCL7 is associated with extramedullary invasion and pathological fractures in patients. In vitro, CXCL7 promoted the proliferation, invasion and migration of MM cells. Leveraging the homing ability of plasma cell, we established a mouse xenograft tumor model through vein injection of a CXCL7-overexpressing cell line. We found that MM cells with elevated CXCL7 exhibited enhanced engraftment in bone marrow, induced extramedullary lesions and increased susceptibility to leg fractures. Through exploration of intracellular signaling pathways and subsequent experiments, we observed that CXCL7 can modulate the biological behavior of MM cells by activating the IL-2-STAT5 pathway in the absence of exogenous IL-2. Our findings provide new insights into understanding the pathogenesis mechanisms underlying MM, suggesting that targeting CXCL7 may offer promising therapeutic opportunities.\u003c/p\u003e","manuscriptTitle":"CXCL7 plays a crucial role in facilitating extramedullary invasion and osteolytic damage in multiple myeloma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-13 21:08:21","doi":"10.21203/rs.3.rs-4489552/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":"96796b94-3690-48a4-8ea1-8fd8cabf0253","owner":[],"postedDate":"June 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32648462,"name":"Biological sciences/Cancer/Haematological cancer/Myeloma"},{"id":32648463,"name":"Biological sciences/Cancer/Oncogenes"}],"tags":[],"updatedAt":"2024-06-21T11:35:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-13 21:08:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4489552","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4489552","identity":"rs-4489552","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}