Knockdown of CSF1R molecules enhances the anti-tumor effects of CD8 + T lymphocytes in bladder cancer

Knockdown of CSF1R molecules enhances the anti-tumor effects of CD8 + T lymphocytes in bladder cancer | 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 Research Article Knockdown of CSF1R molecules enhances the anti-tumor effects of CD8 + T lymphocytes in bladder cancer Jingxuan peng, Zhongyi Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4562641/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 Objective: In the study, we preliminary investigated the role of CSF1R molecules on CD8 + T cells in bladder cancer (BLCA) and its molecular mechanism. Methods: The effect of CSF1R on BLCA cell proliferation，migration and invasion was determined by cell-killing assay and transwell. In vivo experiments, tumor tissues were divided into four portions, one for histological staining, one for flow cytometry detection, one for mRNA gene sequencing analysis, and one for qPCR validation. Information related to 161 bladder cancer patients in Xiangya Hospital of Central South University in the past five years was collected and stained with relevant pathology. By calculating the Jordon index, CSF1R was included in the high expression group by expressing 0.39-fold or more in CD8 + T cells. Results: Knockdown of CSF1R molecule inhibited bladder cancer proliferation, migration, and invasion, and flow cytometry revealed that it could lead to increased infiltration of immune cells. mRNA sequencing and qPCR results suggested that Ckm was significantly decreased after CSF1R knockdown. Conclusion:Our study provides novel insights into the roles of CSF1R in BLCA progression and the underlying crosstalk of tumor metabolism and immune microenvironment. CSF1R CD8+ T cells bladder cancer immune Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Bladder cancer is a common urologic tumor with high incidence and recurrence rates. [1] According to China's tumor patient statistics in 2020, there were 66,242 men with new bladder cancer, accounting for 15.03% of new cases globally. [2] According to the degree of tumor cell infiltration, bladder cancer is classified into muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC). [3,4] Currently, the main treatment modalities for bladder cancer include surgery, chemotherapy, radiotherapy, etc. Despite the various treatment modalities, the 5-year survival rate of bladder cancer patients has not improved significantly over the past 10 years, and recurrence and metastasis of bladder cancer are still the main reasons for treatment failure. [5, 6] Immune imbalance in the tumor microenvironment is one of the important features of tumors. Adaptive immune responses mediated by immune cells play an extremely important role in tumor development [7, 8] .The bladder tumor microenvironment consists of various components such as immune cells, mesenchymal stromal cells, endothelial cells, ECM molecules and inflammatory mediators [9] . T-lymphocytes are important immune cells in the human body, CD8 + T-cells recognize endogenous antigenic peptides presented by MHC class I molecules, and the activated and differentiated effector cells can specifically kill the target cells, which are the main effector cells of cellular immunity. CD8 + T-cells play a key role in anti-tumor activity, and they can directly kill the tumor cells that are sensitized to antigenic peptide-MHC, which is proved in the clinical observation that the prognosis of many kinds of tumors is correlated with the infiltration of intra-tumor CD8 + T-cells. Clinical observations have shown that the prognosis of many tumors is positively correlated with the infiltration of intratumoral CD8 + T cells. [10-12] The human Colony stimulating factor 1 receptor (CSF1R) gene, located on chromosome 5q33-q35, is a single-channel type III transmembrane receptor tyrosine kinase (RTK) encoded by the c-fms proto-oncogene.CSF1R has been identified as a proto-oncogene in a variety of cancers, such as CRC, peripheral T-cell lymphoma and gliomas [13,14] . However, CSF1R expression in BLCA and its prognostic value are not fully understood.The CSF1/CSF1R signaling pathway is critical in the differentiation of monocytes, especially macrophages, and maintains the immunosuppressive and pro-tumorigenic functions of tumor-associated macrophages (TAMs). [15] TAMs are abundant in the tumor microenvironment to suppress cytotoxic T lymphocytes [16, 17] . In addition, CSF1R is also characterized by inhibition of CSF1R overexpression in dendritic cells, neutrophils, and myeloid lineage-derived inhibitory CSF1R overexpression promotes tumor growth and metastasis, cells (MDSCs), and its expression tends to correlate with reduced survival in cancer patients [18, 19] . Therefore, blocking CSF1/CSF1R with CSF1R inhibitors is a promising anticancer immunotherapy strategy [20] . The combination of Magnetic hyperthermia and CSF1R inhibitor (BLZ945) repolarizes M2 macrophages in the tumor microenvironment, relieves immunosuppression, normalizes tumor vasculature, and increases the number of anti-tumor effector CD8+ T cells [21] . CSF1R is closely related to the tumor microenvironment and its expression can affect CD8 + T cell infiltration. However, whether the expression of CSF1R on CD8 + T cells can affect the function of CD8 + T cells and thus the progression of bladder cancer is unknown. We hypothesized that the expression of CSF1R on CD8 + T lymphocytes could influence the function of CD8 + T cells and thus regulate the changes in the immune microenvironment, thus exerting its role in promoting bladder cancer progression. In this study, we collected data from bladder cancer patients at Xiangya Hospital and analyzed them together with pathological information of bladder cancer patients at Xiangya Hospital of Central South University, and found that patients with high expression of CSF1R on CD8 + T cells had a worse prognosis. We further performed subcutaneous tumor formation with MBT2 cells and injected CD8 + T cells knocking down CSF1R through tail vein to observe the changes of tumor size and immune-related indexes. And further analyzed by mRNA sequencing, we found that Ckm may be the key molecule that plays a role. Materials And Methods 2.1 Cell culture and transfection MB49 cells were purchased from Starfish Biologicals (TCM-C794), and MBT2 cells were purchased from Starfish Biologicals (TCM-C802), and cultured in DMEM medium (Shanghai Yuanpei Bio-technology Co., Ltd.) containing 10% fetal bovine serum (FBS, Gibco, NY, United States) at a constant temperature of 37℃. The plasmids of sh-NC and three different sh-CSF1R were purchased from Genechem, Shanghai, China. Cell transfection was performed using Lipofectamine 2000 according to the standard protocol. 2.2 Study population A statement to confirm that all experimental protocols were approved by a named institutional and/or licensing committee and the study approval by the Medical Ethics Sub-committee for Clinical Research of Central South University (NO.202112653-2). All experiments were performed in accordance with ARRIVE guidelines and other relevant guidelines. We checked the electronic medical record system of Xiangya Hospital and collected a total of 161 patients in the last five years with relevant information such as gender, age, history of the disease, grading and staging of the tumor, and contact information. After discharge from the hospital, we tracked the outpatient and re-hospitalization information and followed up the patients by telephone. If we were unable to contact the patients for three consecutive times or the patients and their families refused to provide the relevant information, they were defined as lost patients. We borrowed sections from the Department of Pathology of Xiangya Hospital of Central South University for staining of relevant pathology, and obtained informed consent from the patients for all the above operations. The datasets generated and/or analysed during the current study are not publicly available due patient privacy but are available from the corresponding author on reasonable request 2.3 Transwell assay 24 wells transwell chambers (costar, Corning, US) coated with or without Matrigel (Corning, USA) were used to assess cell migration and invasion. Then, 5 x 10 4 cells were seeded in the upper chamber in medium without serum, while the lower chamber contained medium enriched with 5% serum. After 24 h, the cells were fixed with 4% formaldehyde and stained with crystal violet. 2.4 Cell-killing assay 5 x 10 5 cells were seeded in the six-well plates after 6 hours the cells were divided into 3 groups for intervention. After 24 h, the cells were fixed with 4% formaldehyde and stained with crystal violet. 2.5 CD8 + Tcells isolation CD8 + T cells isolated from mouse spleen using Dynabeads® FlowComp™ Mouse CD8 (11462D thermofisher). 2.6 Quantitative polymerase chain reaction (qPCR) Total RNA was extracted using the TransZol Up Plus RNA Kit (ER501) and then reversed into cDNA. Briefly, the quantitative real-time (qRT)-PCR was performed under a 20 μL system. SyBrGreen PCR system was utilized for qRT-PCR detection. The following primers (synthesized by Beijing Tsingke Biotech) were used: Acta1-F: 5’ -CCCAAAGCTAACCGGGAGAAG-3’; Acta1-R: 5’ -GACAGCACCGCCTGGATAG-3’; Ckm-F: 5’ -GGCAACACCCACAACAAGTTC-3’; Ckm-R: 5’ -CCTTGAAGACCGTGTAGGACT-3’; Gzmf-F: 5’ -TTCCTGCCTACGAGAGGCTC-3’; Gzmf-R: 5’ -CCTGGACCGATTGTCCTGTTTA-3’; Gzmg-F: 5’ -AAGGCCAAGAGAACTAAAGCTG-3’; Gzmg-R: 5’ -CACACTGCACACATCCCCT-3’; Gzmd-F: 5’ -AAGGGGAACAGGATATACTGTGG-3’; Gzmd-R: 5’ -TTGCATAGGCGAAAAGTCCAT-3’; Plet1-F: 5’ -ATCTACACCTCCGACATCTTGG-3’; Plet1-R: 5’ -GGGACCGTCACTGTATAGGTTA-3’; GAPDH-F: 5’ -AGGTCGGTGTGAACGGATTTG-3’; GAPDH-R: 5’ -GGGGTCGTTGATGGCAACA-3’; 2.7 Tumor xenograft experiments All experimental animals were purchased from Changsha Tianqin Biotechnology Co. 10 6-8 weeks BALB/c male mice were kept in SPF grade environment and divided into two groups, experimental group mice were injected with 100ul of PBS containing 5 X 106 knockdown of CSF1R in MB49 cells, and the control group was injected with 100ul of PBS containing 5 X 106 in MB49 cells. daily. The size of subcutaneous tumors in mice was observed and measured. Animals were kept in the Experimental Animal Center of Central South University, and this animal experimental study was approved by the Ethical Review of Experimental Animal Welfare of Xiangya Hospital of Central South University (Ethics No.: NO: 202112653-2) and all methods were performed in accordance with the relevant guidelines and regulations. After 2 weeks of subcutaneous tumor formation, the mice were anesthetized with sodium pentobarbital 1%, the subcutaneous tumor tissues were peeled off, and the tumor tissues were weighed separately and the tumor sizes were compared. 2.8 Immunohistochemistry Immunohistochemistry (IHC) was performed using an immunohistochemical kit (Solarbio, Beijing, China). Paraffin sections were dehydrated and antigen retrieval was performed using EDTA thermal repair. Tissue samples were then incubated with primary antibodies overnight, followed by incubation with a secondary antibody at room temperature for 1 h. The sample was stained with DAB after washing with PBS three times. 2.9 Statistical analysis The results are expressed as the means ± SD (all data were obtained from at least three separate experiments) and statistical analysis was performed using Prism 9.0 (GraphPad Software, La Jolla CA). Data analysis in Table 1 was carried out by Pearson's χ 2 test. Kaplan–Meier curve was draw to explore the survival of OC patients. By calculating the Jordon index, CSF1R was included in the high expression group by expressing 0.39-fold or more in CD8 + T cells. The intergroup differences were evaluated using Student's t-test or one-way ANOVA. p < .05 was considered significant difference. Results 3.1 BLCA patients with high CSF1R expression in CD8+ T cells have a poorer prognosis We performed immunohistofluorescence staining of pathology sections from 161 bladder cancer patients (Fig1 A), and according to the results of staining, they were divided into CD8 + T-cell high-expression and low-expression CSF1R groups By calculating the Jordon's index, CSF1Rs with 0.39-fold or more expression of CD8 + T were included in the high-expression group (Fig1 B). The clinical data of 161 Bca patients included the patients’ T stage, N stage, M stage, pathologic stage, gender, age, histologic grade, subtype and OS event. A total of 110 males and 51 females were analyzed in the present study. The Fisher’s exact test result showed that the CSF1R expression in CD8 + T cells was signifificantly correlated with subtype (p < 0.001) and OS event ( p = 0.030); the chi-square test result revealed that CSF1R expression in CD8 + T cells had a trend of correlation with T stage ( p =0.007), pathological stage ( p = 0.031), and histological stage ( p = 0.047). CSF1R expression in CD8 + T cells was not signifificantly correlated with other clinicopathologic features. Table 1. Demographic and clinicopathologic data of bladder cancer patients with high and low CSF1R expression in CD8 + T cells at Xiangya Hospital. Characteristics Low expression of CSF1R in CD8 + T cells High expression of CSF1R in CD8 + T cells P value n 79 82 Pathologic T stage, n (%) 0.007 T1 42 (26.1%) 23 (14.3%) T2 19 (11.8%) 26 (16.1%) T3 9 (5.6%) 22 (13.7%) T4 9 (5.6%) 11 (6.8%) Pathologic N stage, n (%) 0.175 N0 66 (41.0%) 64 (39.8%) N1 9 (5.6%) 11 (6.8%) N2 2 (1.2%) 7 (4.3%) N3 2 (1.2%) 0 (0%) Pathologic M stage, n (%) 0.582 M0 78 (48.4%) 80 (49.7%) M1 1 (0.6%) 2 (1.2%) Pathologic stage, n (%) 0.031 Stage I 31 (19.3%) 17 (10.6%) Stage II 23 (14.3%) 24 (14.9%) Stage III 16 (9.9%) 21 (13.0%) Stage IV 9 (5.6%) 20 (12.4%) Gender, n (%) 0.173 Female 21 (13.0%) 30 (18.6%) Male 58 (36.0%) 52 (32.3%) Age, n (%) 0.580 > 70 12 (7.5%) 10 (6.2%) <= 70 67 (41.6%) 72 (44.7%) Histologic grade, n (%) 0.047 High grade 55 (34.2%) 68 (42.2%) Low grade 24 (14.9%) 14 (8.7%) Subtype, n (%) < 0.001 Non-Papillary 41 (25.5%) 65 (40.4%) Papillary 38 (23.6%) 17 (10.6%) OS event, n (%) 0.030 Alive 76 (47.2%) 71 (44.1%) Dead 3 (1.9%) 11 (6.8%) 3.2 Knockdown of CSF1R in CD8+ T cells significantly inhibits proliferation, migration and invasion of MB49/MBT2 cells In in vitro experiments, the number of tumor cells was significantly decreased after co-culture of tumor cells and CD8 + T cells and knockdown of CSF1R (Fig2 A, B). Similarly, tumor cell migration (Fig2 C, D)and invasion (Fig2 E, F) abilities were significantly decreased after co-culture of tumor cells and CD8 + T cells and knockdown of CSF1R. 3.3 In vivo experiments, knockdown of CSF1R significantly inhibited tumor growth after tumor cells in nude mice To determine whether knocking down CSF1R could inhibit tumor growth in animals, we established a subcutaneous tumor xenograft model based on MBT2 cell nude mice using thymus-free nude mice, and performed in vivo experiments when the tumor volume was about 100 mm 3 (Fig3 A), which showed that knocking down CSF1R did not cause any significant alteration of body weight of nude mice (Fig3 B). As expected, tumor proliferation was significantly inhibited after knockdown of CSF1R compared to the con-treated group (Fig3 C, D). 3.4 Detection of tumor tissue and spleen by flow cytometry After stripping the tumor tissues and spleens, flow cytometry assay (Fig4 A, B)revealed that CD4 and PD1 were significantly elevated on CD8 + T cells in the tumors after CSF1R knockdown; GZMB and PERFORIN were significantly elevated on CD8 + T cells in the spleens and tumors of mice. 3.5 Tumor tissue mRNA sequencing and initial validation Tumor tissues were subjected to mRNA sequencing (Figure 5A), and qPCR (Figure 5B) and immunofluorescence assays (Figure 5C) based on the results revealed a significant decrease in Ckm after CSF1R knockdown. Discussion In recent years, although much progress has been made in the clinical treatment of BLCA as well as in the study of tumor characterization, its high incidence and high recurrence rate have imposed a heavy economic burden on patients and society. Therefore, the identification of more accurate biomarkers for improved disease surveillance and prediction is considered to be of great clinical and social significance. [22] With the wide application of immunotherapy, ICIs have been increasingly studied in cancer treatment. However, only 20-30% of BLCA patients can benefit from immunotherapy due to the complex regulatory mechanisms between various immune cells in TME. Current studies based on CD8 + T cell-associated immune checkpoints are still far from achieving the expected therapeutic efficacy. [23] Complex interactions of immunomodulatory synergistic inhibition and synergistic stimulation exist in the tumor microenvironment, regulating T cell activity through a complex regulatory network. However, with prolonged duration of antigenic stimulation, these effector functions of T gradually diminish to the point of loss and inability to convert into non-antigen-dependent memory T cells, a dysfunctional state that represents a unique state of T-cell differentiation known as T-cell depletion. [24] Defective T cell function and depletion of their numbers as well as co-expression of immunosuppressive molecules such as CTLA-4, PD-1, IDO-1, Tim3 and other immunosuppressive molecules in the tumor microenvironment may be responsible for the poor anti-tumor immune response of patients. [25] CD8 + T cells are key players in the fight against cancer, and there is growing evidence that the total number of T cells found within a tumor, as well as their ability to distribute and localize within the tumor, influences the outcome of tumor development and responsiveness to cancer immunotherapy. [26] CSF1R is expressed by myeloid cells, so it is expected that inhibition of CSF1R would not only affect microglia, but also have potential targeting effects in circulating and tissue-resident myeloid populations, with possible downstream immunosuppressive effects.CSF1R signaling is required for the development, survival, recruitment, and proliferation of mononuclear phagocytes, including microglia and macrophages. Tumor-associated macrophage (TAMs) recruitment and survival are regulated by colony-stimulating factor 1 (CSF1) binding to the CSF1 receptor ; thus, blockade of TAM suppression and depletion by CSF1R may enhance T-cell responses against tumors when coupled with PD-1 inhibition. CSF1R is a member of class III RTKs, which also include PDGFRα, PDGFRβ, c- Kit, and FLT3. These receptors have been shown to be implicated in the development of a variety of cancers [13] . The data presented in this study validate the growing evidence that CSF1R is a new potential immunotherapeutic target for BLCA. In the study,we firstly analyzed the expression level of CSF1R in CD8 + T cells in different pathological types of BLCA by based on the clinical data of 161 BLCA patients in the database of Xiangya Hospital of Central South University. We performed immunohistofluorescence staining on the pathology sections of 161 bladder cancer patients, and according to the results of staining, they were divided into CD8 + T-cell high-expression and low-expression CSF1R groups By calculating the Jordon's index, we included CSF1R in the high-expression group at a level of 0.39-fold or more of CD8 + T expression. Patients with high CSF1R expression in CD8 + T cells were found to have worse pathologic stage and shorter overall survival time. In in vitro experiments, CD8 + T cells and knockdown of CSF1R expression were added to tumor cells for co-culture, respectively, and it was found that knockdown of CSF1R expression significantly inhibited the proliferation, migration and metastasis of tumor cells. In in vivo experiments, we found that the input of tumor cells with knocked-down CSF1R into mice enhanced the anti-tumor immunity of mice, increased the expression level of CD8 + T cells and their anti-tumor ability in spleen and tumor tissues, and significantly inhibited the development of tumors. Inhibition of CSF1R expression on tumor cells was found to promote the immune microenvironment by flow cytometry on tumor tissues and spleen. In conclusion, this article initially identified the function of CSF1R in the progression of BLCA (Fig6). CSF1R expression was over-expressed in CD8 + T cells, relating to poor clinical outcome. CSF1R knockdown suppressed the proliferation, invasion and metastasis. Moreover, CSF1R silencing could repress MBT2 cells growth in vivo. Thus, CSF1R might be used as a novel target for the target treatment of BLCA in the clinical practice. Of course, more relevant studies are yet to be implemented in the future in order to provide sufficient molecular basis for the clinical use of CSF1R in BLCA-targeted therapy. Declarations FUNDING INFORMATION This work was supported by the Hunan Provincial Natural Science Foundation (2024JJ7554) References WOŁĄCEWICZ M, HRYNKIEWICZ R, GRYWALSKA E, et al. Immunotherapy in Bladder Cancer: Current Methods and Future Perspectives [J]. Cancers, 2020, 12(5). CAO W, CHEN H, YU Y, et al. Changing profiles of cancer burden worldwide and in China: a secondary analysis of the global cancer statistics 2020 [J]. Chinese medical journal, 2021, 134(7): 783-91. WOLDU S, BAGRODIA A, LOTAN Y. Guideline of guidelines: non-muscle-invasive bladder cancer [J]. BJU international, 2017, 119(3): 371-80. 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Derivation and external validation of dendritic cell-related gene signatures for predicting prognosis and immunotherapy efficacy in bladder urothelial carcinoma [J]. Front Immunol, 2022, 13: 1080947. BLACKBURN S D, SHIN H, HAINING W N, et al. Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection [J]. Nat Immunol, 2009, 10(1): 29-37. JIANG X, WANG J, DENG X, et al. Role of the tumor microenvironment in PD-L1/PD-1-mediated tumor immune escape [J]. Mol Cancer, 2019, 18(1): 10. PERANZONI E, LEMOINE J, VIMEUX L, et al. Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti-PD-1 treatment [J]. Proc Natl Acad Sci U S A, 2018, 115(17): E4041-e50. Additional Declarations No competing interests reported. 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Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACPmYgkcBjI8fP3tj48AMxWtjAWmTSjCV7DjcbSxClBUzaHE7ccCO9TYCHKC3s7M8kHuQwJzbcfNjGIMFgJ6fbQNhhaRIJZ9iMG2cntj0oYEg2NjtAWMsxicQeHtlm6cR2AwmGA4nbCGthbJNI/CfB2CZ5sE2ChzgtzGwSCTwGij1AXcRqYWO2SOBJMJbgSQQGsgERfuHnP/7w5g+e/3L2x48/fPihwk6OoBYgYEGKQAPCykGAmahkMgpGwSgYBSMYAAA5SjsKrpY1RQAAAABJRU5ErkJggg==","orcid":"","institution":"Central South University","correspondingAuthor":true,"prefix":"","firstName":"Zhongyi","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-06-11 08:51:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4562641/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4562641/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59870888,"identity":"c47b1ffe-fb73-49e5-81b5-902aac5fc2a1","added_by":"auto","created_at":"2024-07-08 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3","display":"","copyAsset":false,"role":"figure","size":546694,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4562641/v1/d243ce3baf2db0adc7ce7c54.png"},{"id":59870887,"identity":"a269b62e-137d-4560-aafb-c684451a8ae9","added_by":"auto","created_at":"2024-07-08 17:01:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1487795,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4562641/v1/4e4d4eb4914a5efec44254ab.png"},{"id":59870885,"identity":"0538f68a-7274-4c1e-bf0f-f851905d358d","added_by":"auto","created_at":"2024-07-08 17:01:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1712795,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4562641/v1/3b8d9ae5e05262b1a59b7c6a.png"},{"id":59870886,"identity":"1e51ce0a-9de5-4bfc-8ccb-e0e8510f6edf","added_by":"auto","created_at":"2024-07-08 17:01:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":309654,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4562641/v1/fa22d9abd495c678ab12734a.png"},{"id":60451392,"identity":"100b04fe-1004-4cda-927a-ff847a217c1a","added_by":"auto","created_at":"2024-07-17 00:16:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10380237,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4562641/v1/f13b2b8e-7926-4b30-bbf8-8f0f6a3acbc1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Knockdown of CSF1R molecules enhances the anti-tumor effects of CD8 + T lymphocytes in bladder cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBladder cancer is a common urologic tumor with high incidence and recurrence rates.\u0026nbsp;\u003csup\u003e[1]\u003c/sup\u003e According to China\u0026apos;s tumor patient statistics in 2020, there were 66,242 men with new bladder cancer, accounting for 15.03% of new cases globally.\u003csup\u003e[2]\u003c/sup\u003e According to the degree of tumor cell infiltration, bladder cancer is classified into muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC).\u003csup\u003e[3,4]\u003c/sup\u003e Currently, the main treatment modalities for bladder cancer include surgery, chemotherapy, radiotherapy, etc. Despite the various treatment modalities, the 5-year survival rate of bladder cancer patients has not improved significantly over the past 10 years, and recurrence and metastasis of bladder cancer are still the main reasons for treatment failure.\u003csup\u003e[5, 6]\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eImmune imbalance in the tumor microenvironment is one of the important features of tumors. Adaptive immune responses mediated by immune cells play an extremely important role in tumor development\u003csup\u003e[7, 8]\u003c/sup\u003e.The bladder tumor microenvironment consists of various components such as immune cells, mesenchymal stromal cells, endothelial cells, ECM molecules and inflammatory mediators\u003csup\u003e[9]\u003c/sup\u003e. T-lymphocytes are important immune cells in the human body, CD8\u003csup\u003e+\u003c/sup\u003e T-cells recognize endogenous antigenic peptides presented by MHC class I molecules, and the activated and differentiated effector cells can specifically kill the target cells, which are the main effector cells of cellular immunity. CD8\u003csup\u003e+\u003c/sup\u003e T-cells play a key role in anti-tumor activity, and they can directly kill the tumor cells that are sensitized to antigenic peptide-MHC, which is proved in the clinical observation that the prognosis of many kinds of tumors is correlated with the infiltration of intra-tumor CD8\u003csup\u003e+\u003c/sup\u003e T-cells. Clinical observations have shown that the prognosis of many tumors is positively correlated with the infiltration of intratumoral CD8\u003csup\u003e+\u003c/sup\u003e T cells.\u003csup\u003e[10-12]\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe human Colony stimulating factor 1 receptor (CSF1R) gene, located on chromosome 5q33-q35, is a single-channel type III transmembrane receptor tyrosine kinase (RTK) encoded by the c-fms proto-oncogene.CSF1R has been identified as a proto-oncogene in a variety of cancers, such as CRC, peripheral T-cell lymphoma and gliomas\u003csup\u003e[13,14]\u003c/sup\u003e. However, CSF1R expression in BLCA and its prognostic value are not fully understood.The CSF1/CSF1R signaling pathway is critical in the differentiation of monocytes, especially macrophages, and maintains the immunosuppressive and pro-tumorigenic functions of tumor-associated macrophages (TAMs).\u0026nbsp;\u003csup\u003e[15]\u003c/sup\u003e TAMs are abundant in the tumor microenvironment to suppress cytotoxic T lymphocytes\u003csup\u003e[16, 17]\u003c/sup\u003e. In addition, CSF1R is also characterized by inhibition of CSF1R overexpression in dendritic cells, neutrophils, and myeloid lineage-derived inhibitory CSF1R overexpression promotes tumor growth and metastasis, cells (MDSCs), and its expression tends to correlate with reduced survival in cancer patients\u003csup\u003e[18, 19]\u003c/sup\u003e. Therefore, blocking CSF1/CSF1R with CSF1R inhibitors is a promising anticancer immunotherapy strategy\u003csup\u003e[20]\u003c/sup\u003e. The combination of Magnetic hyperthermia and CSF1R inhibitor (BLZ945) repolarizes M2 macrophages in the tumor microenvironment, relieves immunosuppression, normalizes tumor vasculature, and increases the number of anti-tumor effector CD8+ T cells\u003csup\u003e[21]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCSF1R is closely related to the tumor microenvironment and its expression can affect CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration. However, whether the expression of CSF1R on CD8\u003csup\u003e+\u003c/sup\u003e T cells can affect the function of CD8\u003csup\u003e+\u003c/sup\u003e T cells and thus the progression of bladder cancer is unknown. We hypothesized that the expression of CSF1R on CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes could influence the function of CD8\u003csup\u003e+\u003c/sup\u003e T cells and thus regulate the changes in the immune microenvironment, thus exerting its role in promoting bladder cancer progression. In this study, we collected data from bladder cancer patients at Xiangya Hospital and analyzed them together with pathological information of bladder cancer patients at Xiangya Hospital of Central South University, and found that patients with high expression of CSF1R on CD8\u003csup\u003e+\u003c/sup\u003e T cells had a worse prognosis. We further performed subcutaneous tumor formation with MBT2 cells and injected CD8\u003csup\u003e+\u003c/sup\u003e T cells knocking down CSF1R through tail vein to observe the changes of tumor size and immune-related indexes. And further analyzed by mRNA sequencing, we found that Ckm may be the key molecule that plays a role.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Cell culture and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMB49 cells were purchased from Starfish Biologicals (TCM-C794), and MBT2 cells were purchased from Starfish Biologicals (TCM-C802), and cultured in DMEM medium (Shanghai Yuanpei Bio-technology Co., Ltd.) containing 10% fetal bovine serum (FBS, Gibco, NY, United States) at a constant temperature of 37℃.\u0026nbsp;The plasmids of sh-NC and three different sh-CSF1R were purchased from Genechem, Shanghai, China. Cell transfection was performed using Lipofectamine 2000 according to the standard protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Study population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA statement to confirm that all experimental protocols were approved by a named institutional and/or licensing committee and the study approval by the Medical Ethics Sub-committee for Clinical Research of Central South University (NO.202112653-2). All experiments were performed in accordance with ARRIVE guidelines and other relevant guidelines. We checked the electronic medical record system of Xiangya Hospital and collected a total of 161 patients in the last five years with relevant information such as gender, age, history of the disease, grading and staging of the tumor, and contact information. After discharge from the hospital, we tracked the outpatient and re-hospitalization information and followed up the patients by telephone. If we were unable to contact the patients for three consecutive times or the patients and their families refused to provide the relevant information, they were defined as lost patients. We borrowed sections from the Department of Pathology of Xiangya Hospital of Central South University for staining of relevant pathology, and obtained informed consent from the patients for all the above operations. The datasets generated and/or analysed during the current study are not publicly available due patient privacy but are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Transwell assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e24 wells transwell chambers (costar, Corning, US) coated with or without Matrigel (Corning, USA) were used to assess cell migration and invasion. Then, 5 x 10\u003csup\u003e4\u003c/sup\u003e cells were seeded in the upper chamber in medium without serum, while the lower chamber contained medium enriched with 5% serum. After 24 h, the cells were fixed with 4% formaldehyde and stained with crystal violet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Cell-killing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e5 x 10\u003csup\u003e5\u003c/sup\u003e cells were seeded in the six-well plates after 6 hours the cells were divided into 3 groups for intervention. After 24 h, the cells were fixed with 4% formaldehyde and stained with crystal violet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 CD8\u003csup\u003e+\u003c/sup\u003e Tcells isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells isolated from mouse spleen using Dynabeads\u0026reg; FlowComp\u0026trade; Mouse CD8 (11462D thermofisher).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Quantitative polymerase chain reaction (qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using the TransZol Up Plus RNA Kit (ER501) and then reversed into cDNA. Briefly, the quantitative real-time (qRT)-PCR was performed under a 20 \u0026mu;L system. SyBrGreen PCR system was utilized for qRT-PCR detection. The following primers (synthesized by Beijing Tsingke Biotech) were used:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eActa1-F: 5\u0026rsquo; -CCCAAAGCTAACCGGGAGAAG-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003eActa1-R: 5\u0026rsquo; -GACAGCACCGCCTGGATAG-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003eCkm-F: 5\u0026rsquo; -GGCAACACCCACAACAAGTTC-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003eCkm-R: 5\u0026rsquo; -CCTTGAAGACCGTGTAGGACT-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003eGzmf-F: 5\u0026rsquo; -TTCCTGCCTACGAGAGGCTC-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003eGzmf-R: 5\u0026rsquo; -CCTGGACCGATTGTCCTGTTTA-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003eGzmg-F: 5\u0026rsquo; -AAGGCCAAGAGAACTAAAGCTG-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003eGzmg-R: 5\u0026rsquo; -CACACTGCACACATCCCCT-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003eGzmd-F: 5\u0026rsquo; -AAGGGGAACAGGATATACTGTGG-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003eGzmd-R: 5\u0026rsquo; -TTGCATAGGCGAAAAGTCCAT-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003ePlet1-F: 5\u0026rsquo; -ATCTACACCTCCGACATCTTGG-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003ePlet1-R: 5\u0026rsquo; -GGGACCGTCACTGTATAGGTTA-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003eGAPDH-F: 5\u0026rsquo; -AGGTCGGTGTGAACGGATTTG-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003eGAPDH-R: 5\u0026rsquo; -GGGGTCGTTGATGGCAACA-3\u0026rsquo;;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Tumor xenograft experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental animals were purchased from Changsha Tianqin Biotechnology Co. 10 6-8 weeks BALB/c male mice were kept in SPF grade environment and divided into two groups, experimental group mice were injected with 100ul of PBS containing 5 X 106 knockdown of CSF1R in MB49 cells, and the control group was injected with 100ul of PBS containing 5 X 106 in MB49 cells. daily. The size of subcutaneous tumors in mice was observed and measured. Animals were kept in the Experimental Animal Center of Central South University, and this animal experimental study was approved by the Ethical Review of Experimental Animal Welfare of Xiangya Hospital of Central South University (Ethics No.: NO: 202112653-2) and all methods were performed in accordance with the relevant guidelines and regulations. After 2 weeks of subcutaneous tumor formation, the mice were anesthetized with sodium pentobarbital 1%, the subcutaneous tumor tissues were peeled off, and the tumor tissues were weighed separately and the tumor sizes were compared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry (IHC) was performed using an immunohistochemical kit (Solarbio, Beijing, China). Paraffin sections were dehydrated and antigen retrieval was performed using EDTA thermal repair. Tissue samples were then incubated with primary antibodies overnight, followed by incubation with a secondary antibody at room temperature for 1 h. The sample was stained with DAB after washing with PBS three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results are expressed as the means \u0026plusmn; SD (all data were obtained from at least three separate experiments) and statistical analysis was performed using Prism 9.0 (GraphPad Software, La Jolla CA). Data analysis in Table 1 was carried out by Pearson\u0026apos;s \u0026chi; \u003csup\u003e2\u003c/sup\u003e test. Kaplan\u0026ndash;Meier curve was draw to explore the survival of OC patients.\u0026nbsp;By calculating the Jordon index, CSF1R was included in the high expression group by expressing 0.39-fold or more in CD8\u003csup\u003e+\u003c/sup\u003e T cells.\u0026nbsp;The intergroup differences were evaluated using Student\u0026apos;s t-test or one-way ANOVA. p \u0026lt; .05 was considered significant difference.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 BLCA patients with high CSF1R expression in CD8+ T cells have a poorer prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed immunohistofluorescence staining of pathology sections from 161 bladder cancer patients (Fig1 A), and according to the results of staining, they were divided into CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT-cell high-expression and low-expression CSF1R groups By calculating the Jordon\u0026apos;s index, CSF1Rs with 0.39-fold or more expression of CD8\u003csup\u003e+\u003c/sup\u003e T were included in the high-expression group (Fig1 B).\u003c/p\u003e\n\u003cp\u003eThe clinical data of 161 Bca patients included the patients\u0026rsquo; T stage, N stage, M stage, pathologic stage, gender, age, histologic grade, subtype and OS event. A total of 110 males and 51 females were analyzed in the present study. The Fisher\u0026rsquo;s exact test result showed that the CSF1R expression in CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells was signifificantly correlated with subtype (p\u0026nbsp;\u0026lt; 0.001) and OS event (\u003cem\u003ep\u003c/em\u003e = 0.030); the chi-square test result revealed that CSF1R expression in CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells had a trend of correlation with T stage (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e=0.007), pathological stage (\u003cem\u003ep\u003c/em\u003e = 0.031), and histological stage (\u003cem\u003ep\u003c/em\u003e = 0.047). CSF1R expression in CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells was not signifificantly correlated with other clinicopathologic features.\u003c/p\u003e\n\u003cp\u003eTable 1. Demographic and clinicopathologic data of bladder cancer patients with high and low CSF1R expression in CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells at Xiangya Hospital.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"601\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003eLow expression of CSF1R in CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003eHigh expression of CSF1R in CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003ePathologic T stage, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003cp\u003e\u0026nbsp;0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e42\u0026nbsp;(26.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e23\u0026nbsp;(14.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e19\u0026nbsp;(11.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e26\u0026nbsp;(16.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e9\u0026nbsp;(5.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e22\u0026nbsp;(13.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e9\u0026nbsp;(5.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e11\u0026nbsp;(6.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003ePathologic N stage, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003cp\u003e0.175\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e66\u0026nbsp;(41.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e64\u0026nbsp;(39.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eN1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e9\u0026nbsp;(5.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e11\u0026nbsp;(6.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eN2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e2\u0026nbsp;(1.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e7\u0026nbsp;(4.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eN3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e2\u0026nbsp;(1.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e0\u0026nbsp;(0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003ePathologic M stage, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003cp\u003e0.582\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eM0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e78\u0026nbsp;(48.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e80 (49.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e1\u0026nbsp;(0.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e2\u0026nbsp;(1.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003ePathologic stage, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003cp\u003e0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eStage I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e31 (19.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e17\u0026nbsp;(10.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eStage II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e23\u0026nbsp;(14.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e24\u0026nbsp;(14.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eStage III\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e16\u0026nbsp;(9.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e21\u0026nbsp;(13.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eStage IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e9\u0026nbsp;(5.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e20\u0026nbsp;(12.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eGender, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003cp\u003e0.173\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e21\u0026nbsp;(13.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e30\u0026nbsp;(18.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e58\u0026nbsp;(36.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e52\u0026nbsp;(32.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eAge, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003cp\u003e0.580\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003e\u0026gt; 70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e12\u0026nbsp;(7.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e10\u0026nbsp;(6.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003e\u0026lt;= 70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e67\u0026nbsp;(41.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e72\u0026nbsp;(44.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eHistologic grade, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003cp\u003e0.047\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eHigh grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e55\u0026nbsp;(34.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e68\u0026nbsp;(42.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eLow grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e24\u0026nbsp;(14.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e14\u0026nbsp;(8.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eSubtype, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eNon-Papillary\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e41\u0026nbsp;(25.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e65\u0026nbsp;(40.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003ePapillary\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e38\u0026nbsp;(23.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e17\u0026nbsp;(10.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eOS event, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003cp\u003e0.030\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eAlive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e76\u0026nbsp;(47.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e71\u0026nbsp;(44.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.77870216306157%\"\u003e\n \u003cp\u003eDead\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.78702163061564%\"\u003e\n \u003cp\u003e3\u0026nbsp;(1.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.119800332778702%\"\u003e\n \u003cp\u003e11\u0026nbsp;(6.8%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.314475873544094%\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Knockdown of CSF1R in CD8+ T cells significantly inhibits proliferation, migration and invasion of MB49/MBT2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn in vitro experiments, the number of tumor cells was significantly decreased after co-culture of tumor cells and CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells and knockdown of CSF1R (Fig2 A, B). Similarly, tumor cell migration (Fig2 C, D)and invasion (Fig2 E, F) abilities were significantly decreased after co-culture of tumor cells and CD8\u003csup\u003e+\u003c/sup\u003e T cells and knockdown of CSF1R.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 In vivo experiments, knockdown of CSF1R significantly inhibited tumor growth after tumor cells in nude mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether knocking down CSF1R could inhibit tumor growth in animals, we established a subcutaneous tumor xenograft model based on MBT2 cell nude mice using thymus-free nude mice, and performed in vivo experiments when the tumor volume was about 100 mm\u003csup\u003e3\u0026nbsp;\u003c/sup\u003e(Fig3 A), which showed that knocking down CSF1R did not cause any significant alteration of body weight of nude mice (Fig3 B). As expected, tumor proliferation was significantly inhibited after knockdown of CSF1R compared to the con-treated group (Fig3 C, D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Detection of tumor tissue and spleen by flow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter stripping the tumor tissues and spleens, flow cytometry assay (Fig4 A, B)revealed that CD4 and PD1 were significantly elevated on CD8\u003csup\u003e+\u003c/sup\u003e T cells in the tumors after CSF1R knockdown; GZMB and PERFORIN were significantly elevated on CD8\u003csup\u003e+\u003c/sup\u003e T cells in the spleens and tumors of mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Tumor tissue mRNA sequencing and initial validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor tissues were subjected to mRNA sequencing (Figure 5A), and qPCR (Figure 5B) and immunofluorescence assays (Figure 5C) based on the results revealed a significant decrease in Ckm after CSF1R knockdown.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, although much progress has been made in the clinical treatment of BLCA as well as in the study of tumor characterization, its high incidence and high recurrence rate have imposed a heavy economic burden on patients and society. Therefore, the identification of more accurate biomarkers for improved disease surveillance and prediction is considered to be of great clinical and social significance.\u003csup\u003e[22]\u003c/sup\u003e With the wide application of immunotherapy, ICIs have been increasingly studied in cancer treatment. However, only 20-30% of BLCA patients can benefit from immunotherapy due to the complex regulatory mechanisms between various immune cells in TME. Current studies based on CD8\u003csup\u003e+\u003c/sup\u003e T cell-associated immune checkpoints are still far from achieving the expected therapeutic efficacy.\u003csup\u003e[23]\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eComplex interactions of immunomodulatory synergistic inhibition and synergistic stimulation exist in the tumor microenvironment, regulating T cell activity through a complex regulatory network. However, with prolonged duration of antigenic stimulation, these effector functions of T gradually diminish to the point of loss and inability to convert into non-antigen-dependent memory T cells, a dysfunctional state that represents a unique state of T-cell differentiation known as T-cell depletion.\u003csup\u003e[24]\u003c/sup\u003e Defective T cell function and depletion of their numbers as well as co-expression of immunosuppressive molecules such as CTLA-4, PD-1, IDO-1, Tim3 and other immunosuppressive molecules in the tumor microenvironment may be responsible for the poor anti-tumor immune response of patients.\u003csup\u003e[25]\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells are key players in the fight against cancer, and there is growing evidence that the total number of T cells found within a tumor, as well as their ability to distribute and localize within the tumor, influences the outcome of tumor development and responsiveness to cancer immunotherapy.\u003csup\u003e[26]\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eCSF1R is expressed by myeloid cells, so it is expected that inhibition of CSF1R would not only affect microglia, but also have potential targeting effects in circulating and tissue-resident myeloid populations, with possible downstream immunosuppressive effects.CSF1R signaling is required for the development, survival, recruitment, and proliferation of mononuclear phagocytes, including microglia and macrophages. Tumor-associated macrophage (TAMs) recruitment and survival are regulated by colony-stimulating factor 1 (CSF1) binding to the CSF1 receptor ; thus, blockade of TAM suppression and depletion by CSF1R may enhance T-cell responses against tumors when coupled with PD-1 inhibition. CSF1R is a member of class III RTKs, which also include PDGFR\u0026alpha;, PDGFR\u0026beta;, c- Kit, and FLT3. These receptors have been shown to be implicated in the development of a variety of cancers\u003csup\u003e[13]\u003c/sup\u003e. The data presented in this study validate the growing evidence that CSF1R is a new potential immunotherapeutic target for BLCA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the study,we firstly analyzed the expression level of CSF1R in CD8\u003csup\u003e+\u003c/sup\u003e T cells in different pathological types of BLCA by based on the clinical data of 161 BLCA patients in the database of Xiangya Hospital of Central South University. We performed immunohistofluorescence staining on the pathology sections of 161 bladder cancer patients, and according to the results of staining, they were divided into CD8\u003csup\u003e+\u003c/sup\u003e T-cell high-expression and low-expression CSF1R groups By calculating the Jordon\u0026apos;s index, we included CSF1R in the high-expression group at a level of 0.39-fold or more of CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT expression. Patients with high CSF1R expression in CD8\u003csup\u003e+\u003c/sup\u003e T cells were found to have worse pathologic stage and shorter overall survival time. In in vitro experiments, CD8\u003csup\u003e+\u003c/sup\u003e T cells and knockdown of CSF1R expression were added to tumor cells for co-culture, respectively, and it was found that knockdown of CSF1R expression significantly inhibited the proliferation, migration and metastasis of tumor cells. In in vivo experiments, we found that the input of tumor cells with knocked-down CSF1R into mice enhanced the anti-tumor immunity of mice, increased the expression level of CD8\u003csup\u003e+\u003c/sup\u003e T cells and their anti-tumor ability in spleen and tumor tissues, and significantly inhibited the development of tumors. Inhibition of CSF1R expression on tumor cells was found to promote the immune microenvironment by flow cytometry on tumor tissues and spleen.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this article initially identified the function of CSF1R in the progression of BLCA (Fig6). CSF1R expression was over-expressed in CD8\u003csup\u003e+\u003c/sup\u003e T cells, relating to poor clinical outcome. CSF1R knockdown suppressed the proliferation, invasion and metastasis. Moreover, CSF1R silencing could repress MBT2 cells growth in vivo. Thus, CSF1R might be used as a novel target for the target treatment of BLCA in the clinical practice. Of course, more relevant studies are yet to be implemented in the future in order to provide sufficient molecular basis for the clinical use of CSF1R in BLCA-targeted therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFUNDING INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Hunan Provincial Natural Science Foundation (2024JJ7554)\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWOŁĄCEWICZ M, HRYNKIEWICZ R, GRYWALSKA E, et al. Immunotherapy in Bladder Cancer: Current Methods and Future Perspectives [J]. Cancers, 2020, 12(5).\u003c/li\u003e\n\u003cli\u003eCAO W, CHEN H, YU Y, et al. Changing profiles of cancer burden worldwide and in China: a secondary analysis of the global cancer statistics 2020 [J]. Chinese medical journal, 2021, 134(7): 783-91.\u003c/li\u003e\n\u003cli\u003eWOLDU S, BAGRODIA A, LOTAN Y. Guideline of guidelines: non-muscle-invasive bladder cancer [J]. BJU international, 2017, 119(3): 371-80.\u003c/li\u003e\n\u003cli\u003eMONTIRONI R, CHENG L, SCARPELLI M, et al. Pathology and Genetics: Tumours of the Urinary System and Male Genital System: Clinical Implications of the 4th Edition of the WHO Classification and Beyond [J]. European urology, 2016, 70(1): 120-3.\u003c/li\u003e\n\u003cli\u003eCAO R, YUAN L, MA B, et al. An EMT-related gene signature for the prognosis of human bladder cancer [J]. Journal of cellular and molecular medicine, 2020, 24(1): 605-17.\u003c/li\u003e\n\u003cli\u003eZHANG P, LIU Z, WANG D, et al. Identification of Survival and Therapeutic Response-Related Ferroptosis Regulators in Bladder Cancer through Data Mining and Experimental Validation [J]. Cancers, 2021, 13(23).\u003c/li\u003e\n\u003cli\u003eSPEISER D E, HO P-C, VERDEIL G. Regulatory circuits of T cell function in cancer [J]. Nat Rev Immunol, 2016, 16(10): 599-611.\u003c/li\u003e\n\u003cli\u003eTHOMMEN D S, SCHUMACHER T N. T Cell Dysfunction in Cancer [J]. Cancer Cell, 2018, 33(4): 547-62.\u003c/li\u003e\n\u003cli\u003ePICHLER R, FRITZ J, ZAVADIL C, et al. Tumor-infiltrating immune cell subpopulations influence the oncologic outcome after intravesical Bacillus Calmette-Gu\u0026eacute;rin therapy in bladder cancer [J]. Oncotarget, 2016, 7(26): 39916-30.\u003c/li\u003e\n\u003cli\u003eOSHIKIRI T, MIYAMOTO M, SHICHINOHE T, et al. Prognostic value of intratumoral CD8+ T lymphocyte in extrahepatic bile duct carcinoma as essential immune response [J]. J Surg Oncol, 2003, 84(4): 224-8.\u003c/li\u003e\n\u003cli\u003eKONDRATIEV S, SABO E, YAKIREVICH E, et al. Intratumoral CD8+ T lymphocytes as a prognostic factor of survival in endometrial carcinoma [J]. Clin Cancer Res, 2004, 10(13): 4450-6.\u003c/li\u003e\n\u003cli\u003eANRAKU M, CUNNINGHAM K S, YUN Z, et al. Impact of tumor-infiltrating T cells on survival in patients with malignant pleural mesothelioma [J]. J Thorac Cardiovasc Surg, 2008, 135(4): 823-9.\u003c/li\u003e\n\u003cli\u003eZHU M, BAI L, LIU X, et al. Silence of a dependence receptor CSF1R in colorectal cancer cells activates tumor-associated macrophages [J]. J Immunother Cancer, 2022, 10(12).\u003c/li\u003e\n\u003cli\u003eWANG X, ZHANG J, HU B, et al. High Expression of CSF-1R Predicts Poor Prognosis and CSF-1R(high) Tumor-Associated Macrophages Inhibit Anti-Tumor Immunity in Colon Adenocarcinoma [J]. Front Oncol, 2022, 12: 850767.\u003c/li\u003e\n\u003cli\u003eJEANNIN P, PAOLINI L, ADAM C, et al. The roles of CSFs on the functional polarization of tumor-associated macrophages [J]. Febs j, 2018, 285(4): 680-99.\u003c/li\u003e\n\u003cli\u003eDENG Y, HU J C, HE S H, et al. Sphingomyelin synthase 2 facilitates M2-like macrophage polarization and tumor progression in a mouse model of triple-negative breast cancer [J]. Acta Pharmacol Sin, 2021, 42(1): 149-59.\u003c/li\u003e\n\u003cli\u003eCHEN Y, SONG Y, DU W, et al. Tumor-associated macrophages: an accomplice in solid tumor progression [J]. J Biomed Sci, 2019, 26(1): 78.\u003c/li\u003e\n\u003cli\u003eBAGHDADI M, ENDO H, TAKANO A, et al. High co-expression of IL-34 and M-CSF correlates with tumor progression and poor survival in lung cancers [J]. Sci Rep, 2018, 8(1): 418.\u003c/li\u003e\n\u003cli\u003eSTRACHAN D C, RUFFELL B, OEI Y, et al. CSF1R inhibition delays cervical and mammary tumor growth in murine models by attenuating the turnover of tumor-associated macrophages and enhancing infiltration by CD8(+) T cells [J]. Oncoimmunology, 2013, 2(12): e26968.\u003c/li\u003e\n\u003cli\u003eCANNARILE M A, WEISSER M, JACOB W, et al. Colony-stimulating factor 1 receptor (CSF1R) inhibitors in cancer therapy [J]. J Immunother Cancer, 2017, 5(1): 53.\u003c/li\u003e\n\u003cli\u003eFANG Y, HE Y, WU C, et al. Magnetism-mediated targeting hyperthermia-immunotherapy in \u0026quot;cold\u0026quot; tumor with CSF1R inhibitor [J]. Theranostics, 2021, 11(14): 6860-72.\u003c/li\u003e\n\u003cli\u003eMONTEIRO-REIS S, MIRANDA-GON\u0026ccedil;ALVES V, GUIMAR\u0026atilde;ES-TEIXEIRA C, et al. Vimentin epigenetic deregulation in Bladder Cancer associates with acquisition of invasive and metastatic phenotype through epithelial-to-mesenchymal transition [J]. Int J Biol Sci, 2023, 19(1): 1-12.\u003c/li\u003e\n\u003cli\u003eAN B, GUO Z, WANG J, et al. Derivation and external validation of dendritic cell-related gene signatures for predicting prognosis and immunotherapy efficacy in bladder urothelial carcinoma [J]. Front Immunol, 2022, 13: 1080947.\u003c/li\u003e\n\u003cli\u003eBLACKBURN S D, SHIN H, HAINING W N, et al. Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection [J]. Nat Immunol, 2009, 10(1): 29-37.\u003c/li\u003e\n\u003cli\u003eJIANG X, WANG J, DENG X, et al. Role of the tumor microenvironment in PD-L1/PD-1-mediated tumor immune escape [J]. Mol Cancer, 2019, 18(1): 10.\u003c/li\u003e\n\u003cli\u003ePERANZONI E, LEMOINE J, VIMEUX L, et al. Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti-PD-1 treatment [J]. Proc Natl Acad Sci U S A, 2018, 115(17): E4041-e50.\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":"CSF1R, CD8+ T cells, bladder cancer, immune","lastPublishedDoi":"10.21203/rs.3.rs-4562641/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4562641/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eObjective: In the study, we preliminary investigated the role of CSF1R molecules on CD8\u003csup\u003e+\u003c/sup\u003e T cells in bladder cancer (BLCA) and its molecular mechanism.\u003c/p\u003e\n\u003cp\u003eMethods: The effect of CSF1R on BLCA cell proliferation，migration and invasion was determined by cell-killing assay and transwell. In vivo experiments, tumor tissues were divided into four portions, one for histological staining, one for flow cytometry detection, one for mRNA gene sequencing analysis, and one for qPCR validation. Information related to 161 bladder cancer patients in Xiangya Hospital of Central South University in the past five years was collected and stained with relevant pathology. By calculating the Jordon index, CSF1R was included in the high expression group by expressing 0.39-fold or more in CD8\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e\n\u003cp\u003eResults: Knockdown of CSF1R molecule inhibited bladder cancer proliferation, migration, and invasion, and flow cytometry revealed that it could lead to increased infiltration of immune cells. mRNA sequencing and qPCR results suggested that Ckm was significantly decreased after CSF1R knockdown.\u003c/p\u003e\n\u003cp\u003eConclusion:Our study provides novel insights into the roles of CSF1R in BLCA progression and the underlying crosstalk of tumor metabolism and immune microenvironment.\u003c/p\u003e","manuscriptTitle":"Knockdown of CSF1R molecules enhances the anti-tumor effects of CD8 + T lymphocytes in bladder cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-08 17:01:40","doi":"10.21203/rs.3.rs-4562641/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":"ff054a3e-0199-4716-aa2b-17df848ad1e4","owner":[],"postedDate":"July 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-12T05:38:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-08 17:01:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4562641","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4562641","identity":"rs-4562641","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}