Co-targeting CD47 and VEGF Elicited Potent Antitumor Effects in Gastric Cancer

Co-targeting CD47 and VEGF Elicited Potent Antitumor Effects in Gastric 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 Co-targeting CD47 and VEGF Elicited Potent Antitumor Effects in Gastric Cancer Kaiqi Zhang, Yuan Xu, Xusheng Chang, Caili Xu, Wenjing Xue, Dan Ding, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3698194/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background CD47, serving as an intrinsic immune checkpoint, has demonstrated efficacy as an antitumor target in hematologic malignancies. Nevertheless, the clinical relevance of CD47 in gastric cancer and its potential as a therapeutic target remains unclear. Methods The expression of CD47 in clinical gastric cancer tissues was assessed using immunohistochemistry and western blot. Patient-derived cells were obtained from gastric cancer tissues and co-cultured with macrophages derived from human peripheral blood mononuclear cells. Flow cytometry analyses were employed to evaluate the rate of phagocytosis. Humanized patient-derived xenografts (Hu-PDXs) models were established to assess the efficacy of anti-CD47 immunotherapy or the combination of anti-CD47 and anti-VEGF therapy in treating gastric cancer. The infiltrated immune cells in the xenograft were analyzed by immunohistochemistry. Results In this study, we have substantiated the high expression of CD47 in gastric cancer tissues, establishing a strong association with unfavorable prognosis. Through the utilization of SIRPα-Fc to target CD47, we have effectively enhanced macrophage phagocytosis of PDCs in vitro and impeded the growth of Hu-PDXs. It is noteworthy that anti-CD47 immunotherapy has been observed to sustain tumor angiogenic vasculature, with a positive correlation between the expression of VEGF and CD47 in gastric cancer. Furthermore, the successful implementation of anti-angiogenic treatment has further augmented the antitumor efficacy of anti-CD47 therapy. In addition, the potent suppression of tumor growth, prevention of cancer recurrence after surgery, and significant prolongation of overall survival in Hu-PDX models can be achieved through the simultaneous targeting of CD47 and VEGF using the bispecific fusion protein SIRPα-VEGFR1 or by combining the two single-targeted agents. Conclusions Our preclinical studies collectively offer substantiation that CD47 holds promise as a prospective target for gastric cancer, while also highlighting the potential of anti-angiogenic therapy to enhance tumor responsiveness to anti-CD47 immunotherapy. Anti-CD47 therapy Innate immunity Angiogenic vasculature VEGF Combinational therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Gastric cancer, being one of the most widespread and lethal forms of cancer, constitutes 5.6% of newly diagnosed cancer cases and 7.7% of cancer-related fatalities on a global scale ( 1 ). As a consequence of being frequently diagnosed at an advanced stage, patients often fail to seize the optimal surgical intervention window, resulting in a significant mortality rate ( 2 ). In recent times, there has been a growing trend wherein a greater proportion of patients diagnosed with gastric cancer have been deriving advantages from immunotherapies. Nevertheless, the majority of immunotherapeutic interventions that have obtained approval for clinical utilization primarily concentrate on activating the adaptive immune system, thereby overlooking the crucial contribution of the innate immune system in the context of cancer therapy ( 3 ). CD47, which has been recognized as an intrinsic immune checkpoint, exhibits extensive overexpression in diverse types of tumor cells ( 4 – 6 ). The interaction between CD47 and signal regulatory protein alpha (SIRPα) directly hinders the phagocytosis of tumor cells by macrophages, leading to a subsequent impairment in the presentation of tumor antigens to T lymphocytes( 7 – 9 ). CD47 exhibits a strong association with unfavorable prognosis in patients diagnosed with non-small cell lung cancer, melanoma, and acute myeloid leukemia ( 10 – 12 ). The augmentation of macrophage phagocytosis of tumor cells has been observed in various preclinical tumor models, such as small-cell lung cancer, anaplastic thyroid carcinoma, and glioblastoma, through the implementation of monoclonal antibodies or fusion proteins to obstruct the CD47/SIRPα axis ( 13 – 15 ). However, the clinical significance of CD47 expression in gastric cancer and the efficacy of targeting CD47 as a strategy for eradicating tumor cells have yet to be fully understood Although immune checkpoint inhibitors (ICIs) have demonstrated significant efficacy in various cancers, their benefits are limited to a small subset of patients, and their effectiveness is constrained in clinical settings. Tumors can evade immune system responses by establishing an immunosuppressive tumor microenvironment (TME) via local angiogenesis ( 16 – 18 ). Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) exhibit excessive activity in various tumor types, thereby stimulating endothelial cell proliferation and facilitating microangiogenesis ( 19 – 21 ). The aberrant formation of new blood vessels exacerbates the oxygen deficiency and acidic conditions within the tumor microenvironment (TME), thereby facilitating additional blood vessel growth and immune system suppression ( 22 – 24 ). An elevated concentration of vascular endothelial growth factor (VEGF) hampers the maturation process of dendritic cells, and impedes T-cell infiltration and cytotoxic activity, while concurrently promoting the recruitment and proliferation of pro-tumor M2-like macrophages ( 25 – 28 ). The administration of appropriate doses of Vascular Endothelial Growth Factor (VEGF) facilitates vascular normalization and transforms the immunosuppressive Tumor Microenvironment (TME) into an immunosupportive milieu ( 23 ). The evaluation of combining antiangiogenic agents and anti-PD-L1 ICIs has been conducted as a result of the influence of anti-angiogenesis therapy on the tumor immune microenvironment. It has been reported that antiangiogenic therapy enhances the efficacy of anti-PD-L1 therapy in pancreatic neuroendocrine tumors by promoting the development of intratumoral high endothelial venules, which in turn facilitates increased infiltration and activity of cytotoxic T cells ( 29 ). In the context of gastric cancer, antiangiogenic therapies have demonstrated efficacy, leading to the approval of ramucirumab for the treatment of advanced gastric cancer ( 30 ). Ongoing clinical trials are currently investigating the efficacy of combining VEGF/VEGFR blockade with PD-1/PD-L1 inhibitors, such as mepolizumab, pembrolizumab, and durvalumab, for the treatment of solid tumors, including gastric cancer ( 31 ). Nevertheless, there remains a lingering uncertainty regarding the role of angiogenesis in the application of innate immune checkpoint blockade therapy. In this context, a comprehensive analysis was conducted on a cohort of 89 patients diagnosed with gastric cancer, to elucidate the clinical relevance of CD47 in the context of gastric cancer. Subsequently, patient-derived cell (PDC) models and humanized patient-derived xenograft (Hu-PDX) models were utilized to evaluate the therapeutic efficacy of anti-CD47 immunotherapy in suppressing tumor growth. Additionally, the impact of anti-CD47 immunotherapy on the microvessel network within the tumor microenvironment (TME) was investigated, along with the potential correlation between anti-CD47 immunotherapy and anti-angiogenic therapy. Our study has shown that CD47 serves as a viable therapeutic target for gastric cancer, and it has also presented a potentially effective combination approach involving the inhibition of both the CD47/SIRPα axis and angiogenesis for the treatment of this disease. Materials and methods Reagents and antibodies Anti-CD80 monoclonal antibody (66406-1-Ig, Proteintech), anti-CD163 monoclonal antibody (GB13340, Servicebio), anti-CD8 monoclonal antibody (GB12068, Servicebio), anti-CD31 monoclonal antibody (GB113151, Servicebio), VEGFA Monoclonal antibody (19003-1-AP, Proteintech), anti-CD47 monoclonal antibody (ab218810, Abcam), carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (C0051, Beyotime), PerCP anti-CD45 (304027, BioLegend), PE anti-CD11b (101208, BioLegend), granulocyte-macrophage colony-stimulating factor (GM-CSF) (C003, novoprotein), FITC-labeled anti-CD47 (CC2C6, BioLegend), Human Lymphocyte separation medium (7111011, DAKEWE). The fusion protein SIRPα-Fc has been engineered based on the initial extracellular domain of SIRPα and is currently undergoing Phase I/II clinical trial (NCT05140811). SIRPα-VEGFR1 is constructed by combining the extracellular domain of SIRPα with that of VEGFR1 (GenBank accession number: MG920788). Patients Written informed consent was obtained from all patients according to the guidelines established by the Clinical Research Ethics Committee of Changhai Hospital, which approved the current study (CREC: CHEC-2021-119). The Department of Gastrointestinal Surgery at Changhai Hospital collected gastric cancer tissues and their corresponding adjacent tissues from a total of 89 patients between the years 2021 and 2022. None of the patients underwent pre-surgical radiotherapy. Clinicopathological characteristics were gathered, and the T classification, N classification, and TNM stage were determined following the American Joint Committee on Cancer (AJCC) TNM staging system. Immunohistochemical analysis The specimens were prepared and processed per the provided instructions. Subsequently, the slides underwent staining using primary antibody, secondary antibody, DAB, and hematoxylin, respectively. Pathologists assessed the expression abundance of CD47 and VEGF by evaluating the Immunohistochemical (IHC) score for each field. This score was determined based on the intensity of cytoplasm and membrane staining (strong = 3, moderate = 2, weak = 1, no staining = 0) as well as the proportion of stained cells (76–100% = 4, 51–75% = 3, 26–50% = 2, 5–25% = 1, 0–5% = 0). The composite score, which ranged from 0 to 12, was calculated by multiplying the stained cell range score and the intensity score. The final score was determined by taking the average of the composite scores obtained from 5 representative fields. Bioinformatics analysis The Kaplan-Meier plotter (KM-plotter, http//kmplot.com) was utilized to investigate the association between CD47 expression and the survival outcomes of patients with gastric cancer. Additionally, the Gene-Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn ) database was employed to examine the potential correlation between the expression of CD47 and VEGF in gastric cancer. Immunoblot The gastric cancer tissues were homogenized and combined with lysis buffer. Subsequently, the protein was separated using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane via electrical means. The membranes were then obstructed using 5% bovine serum albumin (BSA) and subjected to incubation with a specific primary antibody. On the subsequent day, the membrane was immersed in a solution containing a secondary antibody. Ultimately, the immunoreactive bands were observed to measure the protein content. Cell and Flow cytometry The gastric cancer cell lines PDC1 and PDC2 were isolated from freshly obtained gastric cancer tissues and subsequently cultured in RPMI1640 medium supplemented with 10% FBS (Gibco). To assess the expression of CD47, the cells were subjected to staining with FITC anti-CD47 antibody and subsequently analyzed using flow cytometry. Additionally, human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and subjected to incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) at a concentration of 50 ng/mL for a duration of 7 days, resulting in their differentiation into macrophages. Using flow cytometry, macrophages were identified as CD45 + CD11b + cells. Gastric cancer cells PDC1 and PDC2 were then labeled with Carboxyfluorescein diacetate succinimidyl ester (CFDA SE) and co-cultured with macrophages in serum-free medium with or without SIRPα-Fc at 37°C for 2 hours. The ratio of tumor cells to macrophages was 2:1. The cells were subsequently incubated with PE anti-human CD11b antibody and analyzed using flow cytometry. The phagocytosis index represents the proportion of CD11b + CFDA SE + macrophages among the total CD11b + macrophages. Hu-PDX models The mouse experiments were conducted in adherence to the protocols approved by the Animal Ethical Committee of the School of Pharmacy at Fudan University (AEC: 2020-12-SY-ZXY-01). A strain of NOD/ShiltJGpt mice (NCG mice), which had been genetically modified to lack the Prkdc and Il2rg genes and were severely immunodeficient, were procured from Gempharmatech Co., Ltd. These mice (6–8 weeks old) were housed in a controlled environment free from pathogens. To establish a Hu-PDX model, the NCG mice were subjected to intraperitoneal injection of Busulfan (25mg/kg) to suppress the bone marrow. After 24 hours, human CD34 + hematopoietic stem cells, obtained from neonate cord blood using the CD34 Positive Selection Kit (17897, STEMCELL Technologies), were administered to the NCG mice via the caudal vein. The reconstitution of the human immune system in the NCG mice was observed two weeks later. Subsequently, the gastric cancer patient-derived xenograft was implanted into the humanized NCG mice. Fresh gastric cancer tissues obtained from surgical procedures were aseptically divided into smaller fragments. NCG mice were anesthetized using 1-1.5% isoflurane. Subsequently, a 1 cm incision was made on the dorsal flank of the mice, and the tumor fragment was implanted. The implantation site was closed using subcutaneous sutures, and the wound was sterilized with iodine. Intraperitoneal injections of Signal regulatory protein α (SIRPα) -Fc (4.5 mg/kg), VEGFR1-Fc (3.5 mg/kg), SIRPα-Fc (4.5 mg/kg) + VEGFR1-Fc (3.5 mg/kg), and SIRPα-VEGFR1 (5mg/kg) were administered twice a week. In the gastric carcinoma recurrence model, fragments of fresh gastric cancer tissue were transplanted into NCG mice that had been humanized. Once the tumor volume reached 1000 mm3, the tumors were partially removed, with 1% of the original tumor mass remaining. Intraperitoneal injections of SIRPα-Fc (4.5 mg/kg) + VEGFR1-Fc (3.5 mg/kg) and VEGFR1-SIRPα (5mg/kg) were administered twice a week for a duration of 2 weeks. Statistical analysis The data analysis was performed using GraphPad Prism (version 6.01). The association between survival and the variables of interest was assessed using Kaplan-Meier curves. Statistical comparisons were conducted using a Student's t-test or One-Way Analysis of Variance (ANOVA). The correlation between the two variables was evaluated using Pearson's correlation test. A P value less than 0.05 was considered statistically significant. Results CD47 is overexpressed in gastric cancer and correlated with poor prognosis In light of the ongoing debate surrounding CD47 expression in gastric cancer, our study sought to assess the expression of CD47 in tumor tissues and their corresponding adjacent mucosae from a cohort of 89 gastric cancer patients. Immunohistochemical staining of both normal mucosae and tumor tissues revealed a significant upregulation of CD47 in gastric cancer tissues, irrespective of their differentiation stage (Fig. 1 A, C). The verification of the finding was additionally supported by the immunoblot results (Fig. 1 B). To investigate the association between CD47 expression and gastric cancer pathology (Supplemental Table S1 ), a further examination of the clinicopathologic variables was conducted, revealing elevated levels of CD47 expression in poorly differentiated and non-intestinal type gastric cancers, suggesting an unfavorable prognosis (Fig. 1 D, E). Furthermore, the analysis demonstrated a significant association between CD47 expression and both lymph node metastasis and advanced tumor stage (Fig. 1 F, G). Moreover, survival analysis conducted using the Kaplan-Meier plotter, which relied on RNA-seq data, further substantiated that elevated CD47 expression in individuals diagnosed with stomach adenocarcinoma generally indicated a diminished likelihood of relapse-free survival (Fig. 1 H). These findings suggest that CD47 is extensively upregulated in gastric cancer tissues and exhibits a significant correlation with unfavorable prognosis. Targeting CD47 promoted macrophage phagocytosis of PDCs Before examining the impact of targeting the CD47-SIRPα axis, we utilized CD47 + gastric cancer patient-derived cells (PDC1 and PDC2) and verified their identity through flow cytometry analysis (Fig. 2 A). Subsequently, CFDA SE labeling was applied to PDC1 and PDC2, which were then co-cultured with macrophages derived from human peripheral blood mononuclear cells (Fig. 2 B). The administration of SIRPα-Fc to co-cultured cells resulted in a notable augmentation in the phagocytic activity of macrophages towards PDC1 (from 5.84–25.10%) and PDC2 (from 7.91–25.37%) (Fig. 2 C, D). These findings provide compelling evidence that the inhibition of CD47 can effectively enhance the phagocytic capability of macrophages toward gastric cancer cells. Targeting CD47 significantly inhibited the growth of gastric cancer To accurately and authentically forecast the efficacy of anti-CD47 immunotherapy on human gastric cancer, we developed Hu-PDX models that maintained the attributes of the original tumors and provided a more accurate representation of the pertinent constituents and interactions within the tumor microenvironment (TME). Mice-bearing tumors were randomly assigned to specified groups and treated with either SIRPα-Fc fusion protein or isotype control IgG1-Fc twice weekly for a duration of 4 weeks. The findings indicated that the group treated with SIRPα-Fc exhibited reduced tumor volume and weight compared to the control group, as depicted in Fig. 3 A-D. Immunohistochemical analysis revealed an increase in both CD80 + M1 macrophages and CD8 + T cells infiltrating the tumor microenvironment (TME) following anti-CD47 therapy. However, it is important to note that there was also an increase in pro-tumor CD163 + M2 macrophages in the anti-CD47 treatment group (Fig. 3 E). The findings suggest that the inhibition of CD47 resulted in an antitumor response by promoting the polarization of M1 macrophages and enhancing macrophage phagocytosis. However, it was also observed that CD47 blockade facilitated the infiltration of M2 macrophages. CD47 is positively correlated with VEGF in gastric cancer Given the presence of an immunosuppressive tumor microenvironment in the context of CD47 blockade, it is imperative to elucidate the underlying mechanism and devise a strategy to enhance the efficacy of anti-CD47 therapy. The development of sustained tumor angiogenic vasculature frequently leads to the establishment of an immunosuppressive microenvironment. In this study, we conducted an analysis of intratumoral microvessel density within the tumor microenvironment (TME) and observed a significant increase in microvessel density following treatment with SIRPα-Fc (Fig. 4 A). In addition, an examination was conducted on the relationship between VEGF and CD47 in gastric cancer. Notably, an analysis of samples obtained from patients with gastric cancer using immunohistochemistry revealed a novel finding: VEGF levels were significantly elevated in samples exhibiting high CD47 expression compared to those with low CD47 levels (Fig. 4 B). This observation was further supported by the immunohistochemistry results obtained from our comprehensive collection of 89 gastric cancer specimens (Fig. 4 C). The results of this study indicate that the combination of CD47 and VEGF blockade may be a viable approach for treating gastric cancer. Furthermore, analysis of gene expression data from the GEPIA database supports the positive association between VEGF and CD47 in gastric cancer tissues (Fig. 4 D). These findings provide evidence that CD47 expression is positively linked to VEGF expression in gastric cancer samples. Consequently, we propose that targeting tumor neovascularization could potentially impact the efficacy of anti-CD47 therapy. CD47 blockade combined with antiangiogenetic therapy elicited enhanced anti-tumor effect Subsequently, an investigation was conducted to determine whether the inhibition of VEGF could augment the effectiveness of CD47 blockade in gastric cancer. The results depicted demonstrate that the combination of SIRPα-Fc and VEGFR1-Fc resulted in the complete suppression of tumor growth in the Hu-PDX1 model (Fig. 5 A, B). The tumor weights in the control group, SIRPα-Fc group, VEGFR1-Fc group, and SIRPα-Fc + VEGFR1-Fc group were recorded as 962.36 ± 171.10 mg, 514.50 ± 83.86 mg, 381.40 ± 95.65 mg, and 68.47 ± 7.53 mg, respectively. Similar findings were observed in the Hu-PDX2 model (Fig. 5 C, D). Moreover, an examination was conducted on the presence of macrophages, CD8 + T cells, and microvessel density within the tumor microenvironment (TME). Immunohistochemical (IHC) analysis revealed that the combination of antiangiogenic therapy and CD47 blockade resulted in a reduction of tumor angiogenic vasculature and infiltrated M2-like macrophages, in comparison to the use of anti-CD47 therapy alone. Furthermore, the combined treatment exhibited the greatest ability to induce infiltration of CD8 + T cells when compared to all other groups (Fig. 5 E). The data collectively indicate that the concurrent utilization of angiogenetic axis blockade and CD47/SIRPα axis inhibition can effectively stimulate both the innate and adaptive immune systems, resulting in a synergistic anti-tumor response. Dual blocking CD47 and VEGF elicited synergetic anti-tumor effect and prevented gastric cancer recurrence The evaluation of the anti-tumor effects of SIRPα-VEGFR1, a bispecific fusion protein that concurrently inhibits CD47 and VEGF, demonstrated significant tumor regression in the Hu-PDX1 model, comparable to the observed effects in the SIRPα-Fc + VEGFR1-Fc combination group (Fig. 6 A, B). This underscores the significance of employing bispecific fusion proteins as a method to enhance the targeting efficacy in vivo and improve patient adherence. Following a treatment duration of 27 days, the tumor weights in the control group, SIRPα-Fc + VEGFR1-Fc group, and SIRPα-VEGFR1 group were measured at 905.30 ± 167.60 mg, 72.82 ± 8.65 mg, and 83.30 ± 11.71 mg, respectively. In the Hu-PDX2 model, comparable outcomes were observed in terms of tumor weights among the control group (826.30 ± 71.52 mg), the combinational administration group (98.33 ± 4.379 mg), and the bispecific fusion protein group (80.78 ± 9.63 mg) (Fig. 6 C, D). Analysis of the tumor microenvironment (TME) revealed that the impact of SIRPα-VEGFR1 on macrophages, CD8 + T cells, and microvessel density is akin to that of the combination therapy (Fig. 6 E). In the clinical setting, the occurrence of recurrence plays a significant role in the high mortality rates observed in patients with gastric cancer. Consequently, we established a post-surgical gastric cancer model to assess the efficacy of SIRPα-VEGFR1 in preventing recurrence. The surgical procedure resulted in incomplete removal of the tumor. Subsequently, the mice were administered injections of IgG1-Fc, SIRPα-Fc + VEGFR1-Fc, or SIRPα-VEGFR1. In comparison to the control group, the co-targeting of CD47 and VEGF demonstrated effective inhibition of tumor recurrence (Fig. 6 F). Furthermore, all mice receiving SIRPα-VEGFR1 treatment remained alive for a period of 60 days following the surgery (Fig. 6 G). In summary, the simultaneous inhibition of CD47 and VEGF using SIRPα-VEGFR1 successfully stimulated the immune response of the host against cancer recurrence, leading to a significant extension in overall survival. Discussion There is mounting evidence that tumor cells increase the expression of CD47 to evade detection by the innate immune system( 4 ). Blocking CD47 not only directly enhances the ability of macrophages to engulf tumor cells, but also activates effector T cells and strengthens the adaptive immune response by facilitating the presentation of tumor antigens on macrophages ( 32 , 33 ). CD47 blocking agents, including anti-CD47 antibody, anti-SIRPα antibody, and SIRPα-Fc fusion protein, have demonstrated significant efficacy in suppressing the proliferation of diverse tumor types, encompassing colon cancer, breast cancer, pancreatic neuroendocrine tumors, ovarian cancer, leiomyosarcoma, glioblastoma, and small cell lung cancer ( 6 , 13 , 34 , 35 ). Nevertheless, the efficacy of CD47 as a viable therapeutic target for gastric cancer remains uncertain owing to contradictory outcomes reported in prior research. For instance, Yoshida et al. ( 36 ) have demonstrated that CD47 serves as an autonomous negative prognostic indicator in gastric cancer. In contrast, an alternative investigation has revealed no statistically substantial variation in CD47 mRNA levels between primary gastric cancer and healthy tissues, thereby concluding that CD47 in primary gastric tumors does not exhibit any correlation with clinicopathological factors or prognosis ( 37 ). In this study, clinical specimens were obtained from a cohort of 89 patients diagnosed with gastric cancer, and subsequent analysis was conducted to assess the expression of CD47 in both tumor tissues and corresponding normal tissues. The findings of our investigation substantiated the overexpression of CD47 in gastric cancer, establishing a significant association with unfavorable prognosis. Furthermore, we proceeded to investigate the therapeutic potential of anti-CD47 treatment in gastric cancer, revealing that the blockade of CD47 effectively impeded tumor growth by activating macrophage-mediated innate and adaptive immune responses. The application of immunotherapy in cancer treatment has revealed multiple instances of both intrinsic and acquired resistance ( 38 ). Despite the demonstrated antitumor effects of immune checkpoint inhibitors (ICIs) in various cancer types, eradicating tumor cells remains challenging, including in the case of CD47 blockade therapy ( 39 – 41 ). Consequently, several studies have been conducted to explore novel strategies aimed at achieving a more potent anti-tumor effect. For instance, the combination of anti-GD2 and anti-CD47 has been found to synergistically facilitate the eradication of neuroblastoma by altering both pro- and anti-phagocytic signals within the tumor microenvironment ( 42 ). The efficacy of immunotherapy against melanoma and colorectal cancer was enhanced by the simultaneous blocking of CD47 and PD-L1 using bispecific antibodies, which activated both innate and adaptive immune responses ( 43 – 45 ). The combination of autophagy inhibitors with anti-CD47 therapy further improved tumoricidal activity by regulating catabolic pathways and cellular homeostasis ( 15 , 46 ). In our study, we observed a significant presence of microvessels in gastric cancer following anti-CD47 treatment, leading us to discover that tumor angiogenesis plays a crucial role in limiting the effectiveness of anti-CD47 therapy for the first time. Tumor neovascularization hinders the infiltration of immune cells that react to tumors and fosters an immunosuppressive tumor microenvironment (TME) that enables tumors to resist immunotherapy. In the majority of cancer cases, vascular endothelial growth factor (VEGF) promotes neovascularization, impairs the interaction between endothelial cells and leukocytes, and limits the infiltration of immune cells into the TME by reducing adhesion molecules. Simultaneously, elevated levels of VEGF and angiogenesis in tumors impede the cytotoxic activity of CD8 + T cells and enhance the presence of immunosuppressive cells, such as regulatory T cells (Treg cells) and M2-like macrophages( 47 ). In the current investigation, it was observed that administration of anti-CD47 treatment resulted in an elevation in microvessel density within the tumor ( 48 ). This finding is significant as it aligns with previous research indicating that anti-VEGF agents can normalize tumor vasculature and transform the immunosuppressive tumor microenvironment (TME) into an immunosupportive one. Consequently, this alteration in the TME has been shown to enhance the efficacy of PD-1/PD-L1 inhibitors in various cancers, including gastric cancer. Furthermore, the combination of anti-VEGF/VEGFR and PD-1/PD-L1 inhibitors has been granted FDA approval for the treatment of hepatocellular carcinoma and renal cell carcinoma ( 49 – 51 ). It is noteworthy that the blockade of CD47 has been shown to alter the phenotype of macrophages towards the M1 subtype, which has antitumorigenic properties, as demonstrated in previous studies ( 52 ), However, our study also observed an increase in the presence of pro-tumorigenic M2 macrophages in the tumor microenvironment, potentially attributed to the influence of tumor angiogenesis on the recruitment and proliferation of M2 macrophages. Notably, our study also revealed a positive correlation between VEGF expression and CD47 in gastric cancer tissues, suggesting the potential efficacy of combining anti-CD47 and anti-angiogenesis therapies for the treatment of gastric cancer in clinical settings. To investigate the impact of tumor neovascularization on tumor growth and the potential interference with the anti-tumor effect of CD47 blockade within the tumor immune microenvironment, Hu-PDX models were subjected to the administration of SIRPα-Fc and VEGFR1-Fc, aiming to achieve a simultaneous blockade of CD47 and tumor angiogenesis. The findings revealed that this combined therapeutic approach resulted in a synergistic anti-tumor effect against gastric cancer, characterized by a reduction in microvessel density and M2 macrophages. The bispecific fusion protein SIRPα-VEGFR1 was employed to concurrently target CD47 and VEGF, resulting in significant suppression of tumor growth in the Hu-PDX model. Furthermore, SIRPα-VEGFR1 demonstrated a sustained immune response, effectively preventing the recurrence of gastric cancer and extending overall survival in a recurrence model. In summary, our research has validated the elevated expression of CD47 in human gastric cancer and its significant association with unfavorable prognosis. The utilization of SIRPα-Fc in anti-CD47 therapy has demonstrated remarkable efficacy in Hu-PDX models of gastric cancer. However, it is worth noting that the administration of anti-CD47 treatment led to an escalation in microvessel density within the tumor, potentially fostering an immunosuppressive microenvironment and constraining the effectiveness of CD47 blockade. Additionally, there exists a positive correlation between VEGF expression and CD47 expression in gastric cancer. The concurrent administration of anti-CD47 and anti-VEGF therapies, along with the utilization of a bispecific fusion protein, yielded notable enhancements in the tumor immune microenvironment and a substantial augmentation of the antitumor response. These findings strongly indicate that CD47 holds promise as a viable therapeutic target for gastric cancer, and underscore the considerable advantages of combining anti-CD47 treatment with anti-angiogenic therapy in the context of gastric cancer treatment. Abbreviations Hu-PDXs: humanized patient-derived xenografts SIRPα: signal regulatory protein alpha ICIs: immune checkpoint inhibitors TME: tumor microenvironment VEGF: vascular endothelial growth factor PDC: patient-derived cell Hu-PDX: patient-derived xenograft AJCC: American Joint Committee on Cancer KM-plotter: Kaplan-Meier plotter GEPIA: gene-expression profiling interactive analysis IHC: immunohistochemical ANOVA： analysis of variance SIRPα: signal regulatory protein α NCG mice: a severely immunodeficient strain obtained by knocking out the Prkdc and Il2rg genes in NOD/ShiltJGpt mice GM-CSF: granulocyte-macrophage colony-stimulating factor CFDA SE: carboxyfluorescein diacetate succinimidyl ester PBMCs: peripheral blood mononuclear cells BSA: bovine serum albumin Declarations Acknowledgments Author contributions LF, XZ, and KY designed the project and proofread the manuscript. KZ, YX, XC, CX, WX, YN, DD, MN, HC, JX, and LZ performed the experiments and prepared the manuscript. XZ, JF, JH, TC, and DJ provided material and analyzed the data. All authors approved the final manuscript. Funding This work was supported by grants from the National Key Research and Development Program of China (2023YFC3606600, 2023YFC3503403 and 2023YFC3404000), the National Natural Science Foundation of China (32200745, 82371781 and 82073752), Shanghai Sailing Program (21YF1401900), and Medical Innovation Research Program of Science and Technology Commission of Shanghai Municipality (21Y11913100). Song Li and Wenzhi Tian provided technical support for the experiments. Data Availability Statement The survival analysis data between relapse-free survival and stomach adenocarcinoma are available online through the Kaplan-Meier plotter based on RNA-seq. The correlation between CD47 and VEGF data is available online through Correlation Analysis in GEPIA based on the TCGA STAD tumor database. All other data that support the findings of this study are available from the corresponding author upon reasonable request. Conflict of Interest The other authors declare no conflicts of interest. Ethics declarations The studies involving human participants were reviewed and approved by the Clinical Research Ethics Committee of Changhai Hospital (CREC: CHEC-2021-119). All procedures followed were per the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions. All patients provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Animal Ethical Committee of the School of Pharmacy at Fudan University (AEC: 2020-12-SY-ZXY-01). All institutional and national guidelines for the care and use of laboratory animals were followed. 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Supplementary Files TableS1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 29 Jan, 2024 Reviews received at journal 02 Jan, 2024 Reviewers agreed at journal 17 Dec, 2023 Reviewers invited by journal 16 Dec, 2023 Editor assigned by journal 05 Dec, 2023 Submission checks completed at journal 03 Dec, 2023 First submitted to journal 02 Dec, 2023 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-3698194","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":255696746,"identity":"9aca9e37-ef0b-43c1-9276-25651b33addb","order_by":0,"name":"Kaiqi Zhang","email":"","orcid":"","institution":"Changhai Hospital, Naval Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kaiqi","middleName":"","lastName":"Zhang","suffix":""},{"id":255696748,"identity":"0e4c3b1d-d444-4b88-a675-0026bd800f5c","order_by":1,"name":"Yuan Xu","email":"","orcid":"","institution":"Changhai Hospital, Naval Medical 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Hospital, Naval Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Yin","suffix":""}],"badges":[],"createdAt":"2023-12-02 17:14:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3698194/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3698194/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":47718770,"identity":"ac950cc5-f3cc-4d7b-be37-74094136379a","added_by":"auto","created_at":"2023-12-06 14:05:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2568978,"visible":true,"origin":"","legend":"\u003cp\u003eCD47 is overexpressed in gastric cancer tissues and is closely correlated with poor prognosis. (\u003cstrong\u003eA\u003c/strong\u003e) Representative images of immunohistochemical CD47 staining of normal mucosa and the tumor tissue of the stomach. (\u003cstrong\u003eB\u003c/strong\u003e) CD47 expression in normal mucosa and the tumor tissue of the stomach by immunoblot (N, normal; T, tumor). (\u003cstrong\u003eC\u003c/strong\u003e) The expression of CD47 in tumor and adjacent normal tissues of gastric cancer patients without classification. (\u003cstrong\u003eD\u003c/strong\u003e) The expression of CD47 in tumors of gastric cancer patients under the classification of intestinal and non-intestinal. (\u003cstrong\u003eE\u003c/strong\u003e) non-poor differentiation and poor differentiation, (\u003cstrong\u003eF\u003c/strong\u003e) N0-1 and N2-3, and (\u003cstrong\u003eG\u003c/strong\u003e)TNM stage I, II, and III. (\u003cstrong\u003eH\u003c/strong\u003e) Relapse-free survival analysis conducted by Kaplan-Meier plotter according to CD47 expression of all patients. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3698194/v1/e2be073ac5b0442a79af0008.png"},{"id":47718768,"identity":"232fe102-c79d-475d-bb3c-7dd054ca3c26","added_by":"auto","created_at":"2023-12-06 14:05:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1073178,"visible":true,"origin":"","legend":"\u003cp\u003eTargeting CD47 promotes macrophage phagocytosis of gastric cancer patient-derived cells. (\u003cstrong\u003eA\u003c/strong\u003e) FACS analysis for cell surface CD47 expression in gastric cancer patients-derived cells PDC1 and PDC2. \u003cstrong\u003e(B) \u003c/strong\u003eFACS analysis for CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+ \u003c/sup\u003emacrophage derived from PBMC. (\u003cstrong\u003eC\u003c/strong\u003e-\u003cstrong\u003eD\u003c/strong\u003e) Representative flow cytometry plots depicting macrophage phagocytosis of PDC1 and PDC2 treated with SIRPα-Fc vs. control.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3698194/v1/44883e581addac94a5aa353d.png"},{"id":47719095,"identity":"1b583aff-2213-43b9-ab42-7488595078ef","added_by":"auto","created_at":"2023-12-06 14:13:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4421821,"visible":true,"origin":"","legend":"\u003cp\u003eTargeting CD47 significantly inhibited the growth of gastric cancer in Hu-PDX models. (\u003cstrong\u003eA\u003c/strong\u003e) In the Hu-PDX1 model, tumor volume was measured twice a week and presented as mean ± SD. After treatment with SIRPα-Fc for 4 weeks, tumor weight was presented. (n=6 for control group and n=8 for SIRPα-Fc group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (\u003cstrong\u003eB\u003c/strong\u003e) Each line represented the tumor volume from an independent mouse in the Hu-PDX1 model. (\u003cstrong\u003eC\u003c/strong\u003e) In the Hu-PDX2 model, tumor volume was measured twice a week and presented as mean ± SD. After treatment with SIRPα-Fc for 4 weeks, tumor weight was presented. (n=6 per group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). (\u003cstrong\u003eD\u003c/strong\u003e) Each line represented the tumor volume from an independent mouse in the Hu-PDX2 model. (\u003cstrong\u003eE\u003c/strong\u003e) Representative photographs of Immunohistochemical staining for CD80, CD163, and CD8 of tumor tissue sections and the number of CD80\u003csup\u003e+\u003c/sup\u003e, CD163\u003csup\u003e+,\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e cells in each group were normalized to the control group. The value of control was set to 1.0. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3698194/v1/7f0df9a2a190547cdd025eeb.png"},{"id":47716375,"identity":"8492566b-700a-43e5-80a0-05d62d55ac35","added_by":"auto","created_at":"2023-12-06 13:49:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7497287,"visible":true,"origin":"","legend":"\u003cp\u003eCD47 expression is positively correlated with VEGF expression in gastric cancer specimens. (\u003cstrong\u003eA\u003c/strong\u003e) Representative photographs of immunohistochemical staining for CD31 of tumor tissue sections and the relative vessel density in each group were normalized to the control group. The value of control was set to 1.0. (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). \u003cstrong\u003e(B)\u003c/strong\u003e Representative photograph of immunohistochemical staining for CD47 and VEGF of human gastric cancer tissue sections. \u003cstrong\u003e(C)\u003c/strong\u003e The VEGF expression in tumors of 89 gastric cancer patients under the classification of CD47 high and CD47 low and analysis of the correlation between CD47 and VEGF in the gastric cancer tissues.\u003cstrong\u003e (D\u003c/strong\u003e) Analysis of correlation between CD47 and VEGF in gastric cancer using the GEPIA database. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3698194/v1/e850498dc5d2b8058de9acf7.png"},{"id":47717904,"identity":"e8dd27a5-ac47-43cf-8e39-507de7690435","added_by":"auto","created_at":"2023-12-06 13:57:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4540060,"visible":true,"origin":"","legend":"\u003cp\u003eCD47 blockade combined with antiangiogenetic therapy elicited enhanced anti-tumor effect in Hu-PDX models of gastric cancer. (\u003cstrong\u003eA \u003c/strong\u003eand\u003cstrong\u003e B\u003c/strong\u003e) In the Hu-PDX1 model, tumor-bearing mice were treated with SIRPα-Fc and/or VEGFR1-Fc for 4 weeks, tumor volume and tumor weight were presented as mean ± SD. Each line represented the value of the tumor volume of a single mouse. (\u003cstrong\u003eC \u003c/strong\u003eand\u003cstrong\u003e D\u003c/strong\u003e) In the Hu-PDX2 model, tumor volume and tumor weight were presented after the same treatment in the Hu-PDX1 model. Each line represented the value of the tumor volume of a single mouse. (n=6 per group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). (\u003cstrong\u003eE\u003c/strong\u003e) Representative photographs of Immunohistochemical staining for CD80, CD163, CD8, and CD31 of tumor tissue sections and the number of CD80\u003csup\u003e+\u003c/sup\u003e, CD163\u003csup\u003e+\u003c/sup\u003e, and CD8\u003csup\u003e+\u003c/sup\u003e cells and the relative vessel density in each group were normalized to the control group. The value of control was set to 1.0. (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3698194/v1/58596cb0bfb1b0781958c5ff.png"},{"id":47716370,"identity":"4f774d48-52c5-4972-a180-bd45efe7f73a","added_by":"auto","created_at":"2023-12-06 13:49:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":803808,"visible":true,"origin":"","legend":"\u003cp\u003eBispecific fusion protein SIRPα-VEGFR1 elicited synergetic antitumor effect and prevented gastric cancer recurrence. (\u003cstrong\u003eA \u003c/strong\u003eand\u003cstrong\u003e B\u003c/strong\u003e) In the Hu-PDX1 model, tumor volume and tumor weight were measured and the data was presented as mean ± SD after treatment with SIRPα-Fc plus VEGFR1-Fc, and SIRPα-VEGFR1 for 4 weeks. Each line represented the value of the tumor volume of a single mouse. (\u003cstrong\u003eC \u003c/strong\u003eand\u003cstrong\u003e D\u003c/strong\u003e) In the Hu-PDX2 model, tumor volume and tumor weight were measured after the same treatment in the Hu-PDX1 model. (\u003cstrong\u003eE\u003c/strong\u003e) The number of CD80\u003csup\u003e+\u003c/sup\u003e, CD163\u003csup\u003e+\u003c/sup\u003e, and CD8\u003csup\u003e+\u003c/sup\u003e cells and the relative vessel density in each group were normalized to the control group. The value of control was set to 1.0. (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). (\u003cstrong\u003eF\u003c/strong\u003e) In the humanized tumor recurrence model, mice were treated with control, SIRPα-Fc + VEGFR1-Fc, SIRPα-VEGFR1 for 2 weeks, and tumor volume was measured. Each line represented the value of the tumor volume of a single mouse. (\u003cstrong\u003eG\u003c/strong\u003e) Survival curves for different treatment groups. (n=6 per group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3698194/v1/2e4ca0c48fee4af17cdc4a4a.png"},{"id":47720508,"identity":"aed3cdcc-6ac8-4365-9de2-1141fdceb424","added_by":"auto","created_at":"2023-12-06 14:21:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6315581,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3698194/v1/05a24707-0962-43d4-ba40-5fd9392e9c23.pdf"},{"id":47717901,"identity":"96d23c82-6b0e-4a79-b8b9-4c43587058e6","added_by":"auto","created_at":"2023-12-06 13:57:24","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11642,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3698194/v1/ef17ae3d985df9b7796a16e0.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Co-targeting CD47 and VEGF Elicited Potent Antitumor Effects in Gastric Cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastric cancer, being one of the most widespread and lethal forms of cancer, constitutes 5.6% of newly diagnosed cancer cases and 7.7% of cancer-related fatalities on a global scale (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). As a consequence of being frequently diagnosed at an advanced stage, patients often fail to seize the optimal surgical intervention window, resulting in a significant mortality rate (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In recent times, there has been a growing trend wherein a greater proportion of patients diagnosed with gastric cancer have been deriving advantages from immunotherapies. Nevertheless, the majority of immunotherapeutic interventions that have obtained approval for clinical utilization primarily concentrate on activating the adaptive immune system, thereby overlooking the crucial contribution of the innate immune system in the context of cancer therapy (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCD47, which has been recognized as an intrinsic immune checkpoint, exhibits extensive overexpression in diverse types of tumor cells (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The interaction between CD47 and signal regulatory protein alpha (SIRPα) directly hinders the phagocytosis of tumor cells by macrophages, leading to a subsequent impairment in the presentation of tumor antigens to T lymphocytes(\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). CD47 exhibits a strong association with unfavorable prognosis in patients diagnosed with non-small cell lung cancer, melanoma, and acute myeloid leukemia (\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The augmentation of macrophage phagocytosis of tumor cells has been observed in various preclinical tumor models, such as small-cell lung cancer, anaplastic thyroid carcinoma, and glioblastoma, through the implementation of monoclonal antibodies or fusion proteins to obstruct the CD47/SIRPα axis (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). However, the clinical significance of CD47 expression in gastric cancer and the efficacy of targeting CD47 as a strategy for eradicating tumor cells have yet to be fully understood\u003c/p\u003e \u003cp\u003eAlthough immune checkpoint inhibitors (ICIs) have demonstrated significant efficacy in various cancers, their benefits are limited to a small subset of patients, and their effectiveness is constrained in clinical settings. Tumors can evade immune system responses by establishing an immunosuppressive tumor microenvironment (TME) via local angiogenesis (\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) exhibit excessive activity in various tumor types, thereby stimulating endothelial cell proliferation and facilitating microangiogenesis (\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The aberrant formation of new blood vessels exacerbates the oxygen deficiency and acidic conditions within the tumor microenvironment (TME), thereby facilitating additional blood vessel growth and immune system suppression (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). An elevated concentration of vascular endothelial growth factor (VEGF) hampers the maturation process of dendritic cells, and impedes T-cell infiltration and cytotoxic activity, while concurrently promoting the recruitment and proliferation of pro-tumor M2-like macrophages (\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). The administration of appropriate doses of Vascular Endothelial Growth Factor (VEGF) facilitates vascular normalization and transforms the immunosuppressive Tumor Microenvironment (TME) into an immunosupportive milieu (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The evaluation of combining antiangiogenic agents and anti-PD-L1 ICIs has been conducted as a result of the influence of anti-angiogenesis therapy on the tumor immune microenvironment. It has been reported that antiangiogenic therapy enhances the efficacy of anti-PD-L1 therapy in pancreatic neuroendocrine tumors by promoting the development of intratumoral high endothelial venules, which in turn facilitates increased infiltration and activity of cytotoxic T cells (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In the context of gastric cancer, antiangiogenic therapies have demonstrated efficacy, leading to the approval of ramucirumab for the treatment of advanced gastric cancer (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Ongoing clinical trials are currently investigating the efficacy of combining VEGF/VEGFR blockade with PD-1/PD-L1 inhibitors, such as mepolizumab, pembrolizumab, and durvalumab, for the treatment of solid tumors, including gastric cancer (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Nevertheless, there remains a lingering uncertainty regarding the role of angiogenesis in the application of innate immune checkpoint blockade therapy.\u003c/p\u003e \u003cp\u003eIn this context, a comprehensive analysis was conducted on a cohort of 89 patients diagnosed with gastric cancer, to elucidate the clinical relevance of CD47 in the context of gastric cancer. Subsequently, patient-derived cell (PDC) models and humanized patient-derived xenograft (Hu-PDX) models were utilized to evaluate the therapeutic efficacy of anti-CD47 immunotherapy in suppressing tumor growth. Additionally, the impact of anti-CD47 immunotherapy on the microvessel network within the tumor microenvironment (TME) was investigated, along with the potential correlation between anti-CD47 immunotherapy and anti-angiogenic therapy. Our study has shown that CD47 serves as a viable therapeutic target for gastric cancer, and it has also presented a potentially effective combination approach involving the inhibition of both the CD47/SIRPα axis and angiogenesis for the treatment of this disease.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eReagents and antibodies\u003c/p\u003e\n\u003cp\u003eAnti-CD80 monoclonal antibody (66406-1-Ig, Proteintech), anti-CD163 monoclonal antibody (GB13340, Servicebio), anti-CD8 monoclonal antibody (GB12068, Servicebio), anti-CD31 monoclonal antibody (GB113151, Servicebio), VEGFA Monoclonal antibody (19003-1-AP, Proteintech), anti-CD47 monoclonal antibody (ab218810, Abcam), carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (C0051, Beyotime), PerCP anti-CD45 (304027, BioLegend), PE anti-CD11b (101208, BioLegend), granulocyte-macrophage colony-stimulating factor (GM-CSF) (C003, novoprotein), FITC-labeled anti-CD47 (CC2C6, BioLegend), Human Lymphocyte separation medium (7111011, DAKEWE). The fusion protein SIRP\u0026alpha;-Fc has been engineered based on the initial extracellular domain of SIRP\u0026alpha; and is currently undergoing Phase I/II clinical trial (NCT05140811). SIRP\u0026alpha;-VEGFR1 is constructed by combining the extracellular domain of SIRP\u0026alpha; with that of VEGFR1 (GenBank accession number: MG920788).\u003c/p\u003e\n\u003cp\u003ePatients\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all patients according to the guidelines established by the Clinical Research Ethics Committee of Changhai Hospital, which approved the current study (CREC: CHEC-2021-119). The Department of Gastrointestinal Surgery at Changhai Hospital collected gastric cancer tissues and their corresponding adjacent tissues from a total of 89 patients between the years 2021 and 2022. None of the patients underwent pre-surgical radiotherapy. Clinicopathological characteristics were gathered, and the T classification, N classification, and TNM stage were determined following the American Joint Committee on Cancer (AJCC) TNM staging system.\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analysis\u003c/p\u003e\n\u003cp\u003eThe specimens were prepared and processed per the provided instructions. Subsequently, the slides underwent staining using primary antibody, secondary antibody, DAB, and hematoxylin, respectively. Pathologists assessed the expression abundance of CD47 and VEGF by evaluating the Immunohistochemical (IHC) score for each field. This score was determined based on the intensity of cytoplasm and membrane staining (strong\u0026thinsp;=\u0026thinsp;3, moderate\u0026thinsp;=\u0026thinsp;2, weak\u0026thinsp;=\u0026thinsp;1, no staining\u0026thinsp;=\u0026thinsp;0) as well as the proportion of stained cells (76\u0026ndash;100% = 4, 51\u0026ndash;75% = 3, 26\u0026ndash;50% = 2, 5\u0026ndash;25% = 1, 0\u0026ndash;5% = 0). The composite score, which ranged from 0 to 12, was calculated by multiplying the stained cell range score and the intensity score. The final score was determined by taking the average of the composite scores obtained from 5 representative fields.\u003c/p\u003e\n\u003cp\u003eBioinformatics analysis\u003c/p\u003e\n\u003cp\u003eThe Kaplan-Meier plotter (KM-plotter, http//kmplot.com) was utilized to investigate the association between CD47 expression and the survival outcomes of patients with gastric cancer. Additionally, the Gene-Expression Profiling Interactive Analysis (GEPIA, \u003cspan\u003e\u003cspan\u003ehttp://gepia.cancer-pku.cn\u003c/span\u003e\u003c/span\u003e) database was employed to examine the potential correlation between the expression of CD47 and VEGF in gastric cancer.\u003c/p\u003e\n\u003cp\u003eImmunoblot\u003c/p\u003e\n\u003cp\u003eThe gastric cancer tissues were homogenized and combined with lysis buffer. Subsequently, the protein was separated using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane via electrical means. The membranes were then obstructed using 5% bovine serum albumin (BSA) and subjected to incubation with a specific primary antibody. On the subsequent day, the membrane was immersed in a solution containing a secondary antibody. Ultimately, the immunoreactive bands were observed to measure the protein content.\u003c/p\u003e\n\u003cp\u003eCell and Flow cytometry\u003c/p\u003e\n\u003cp\u003eThe gastric cancer cell lines PDC1 and PDC2 were isolated from freshly obtained gastric cancer tissues and subsequently cultured in RPMI1640 medium supplemented with 10% FBS (Gibco). To assess the expression of CD47, the cells were subjected to staining with FITC anti-CD47 antibody and subsequently analyzed using flow cytometry. Additionally, human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and subjected to incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) at a concentration of 50 ng/mL for a duration of 7 days, resulting in their differentiation into macrophages. Using flow cytometry, macrophages were identified as CD45\u0026thinsp;+\u0026thinsp;CD11b\u0026thinsp;+\u0026thinsp;cells. Gastric cancer cells PDC1 and PDC2 were then labeled with Carboxyfluorescein diacetate succinimidyl ester (CFDA SE) and co-cultured with macrophages in serum-free medium with or without SIRP\u0026alpha;-Fc at 37\u0026deg;C for 2 hours. The ratio of tumor cells to macrophages was 2:1. The cells were subsequently incubated with PE anti-human CD11b antibody and analyzed using flow cytometry. The phagocytosis index represents the proportion of CD11b\u0026thinsp;+\u0026thinsp;CFDA SE\u0026thinsp;+\u0026thinsp;macrophages among the total CD11b\u0026thinsp;+\u0026thinsp;macrophages.\u003c/p\u003e\n\u003cp\u003eHu-PDX models\u003c/p\u003e\n\u003cp\u003eThe mouse experiments were conducted in adherence to the protocols approved by the Animal Ethical Committee of the School of Pharmacy at Fudan University (AEC: 2020-12-SY-ZXY-01).\u003c/p\u003e\n\u003cp\u003eA strain of NOD/ShiltJGpt mice (NCG mice), which had been genetically modified to lack the Prkdc and Il2rg genes and were severely immunodeficient, were procured from Gempharmatech Co., Ltd. These mice (6\u0026ndash;8 weeks old) were housed in a controlled environment free from pathogens.\u003c/p\u003e\n\u003cp\u003eTo establish a Hu-PDX model, the NCG mice were subjected to intraperitoneal injection of Busulfan (25mg/kg) to suppress the bone marrow. After 24 hours, human CD34\u0026thinsp;+\u0026thinsp;hematopoietic stem cells, obtained from neonate cord blood using the CD34 Positive Selection Kit (17897, STEMCELL Technologies), were administered to the NCG mice via the caudal vein. The reconstitution of the human immune system in the NCG mice was observed two weeks later. Subsequently, the gastric cancer patient-derived xenograft was implanted into the humanized NCG mice. Fresh gastric cancer tissues obtained from surgical procedures were aseptically divided into smaller fragments. NCG mice were anesthetized using 1-1.5% isoflurane. Subsequently, a 1 cm incision was made on the dorsal flank of the mice, and the tumor fragment was implanted. The implantation site was closed using subcutaneous sutures, and the wound was sterilized with iodine. Intraperitoneal injections of Signal regulatory protein \u0026alpha; (SIRP\u0026alpha;) -Fc (4.5 mg/kg), VEGFR1-Fc (3.5 mg/kg), SIRP\u0026alpha;-Fc (4.5 mg/kg)\u0026thinsp;+\u0026thinsp;VEGFR1-Fc (3.5 mg/kg), and SIRP\u0026alpha;-VEGFR1 (5mg/kg) were administered twice a week. In the gastric carcinoma recurrence model, fragments of fresh gastric cancer tissue were transplanted into NCG mice that had been humanized. Once the tumor volume reached 1000 mm3, the tumors were partially removed, with 1% of the original tumor mass remaining. Intraperitoneal injections of SIRP\u0026alpha;-Fc (4.5 mg/kg)\u0026thinsp;+\u0026thinsp;VEGFR1-Fc (3.5 mg/kg) and VEGFR1-SIRP\u0026alpha; (5mg/kg) were administered twice a week for a duration of 2 weeks.\u003c/p\u003e\n\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eThe data analysis was performed using GraphPad Prism (version 6.01). The association between survival and the variables of interest was assessed using Kaplan-Meier curves. Statistical comparisons were conducted using a Student\u0026apos;s t-test or One-Way Analysis of Variance (ANOVA). The correlation between the two variables was evaluated using Pearson\u0026apos;s correlation test. A P value less than 0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eCD47 is overexpressed in gastric cancer and correlated with poor prognosis\u003c/p\u003e\n\u003cp\u003eIn light of the ongoing debate surrounding CD47 expression in gastric cancer, our study sought to assess the expression of CD47 in tumor tissues and their corresponding adjacent mucosae from a cohort of 89 gastric cancer patients. Immunohistochemical staining of both normal mucosae and tumor tissues revealed a significant upregulation of CD47 in gastric cancer tissues, irrespective of their differentiation stage (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, C). The verification of the finding was additionally supported by the immunoblot results (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). To investigate the association between CD47 expression and gastric cancer pathology (Supplemental Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e), a further examination of the clinicopathologic variables was conducted, revealing elevated levels of CD47 expression in poorly differentiated and non-intestinal type gastric cancers, suggesting an unfavorable prognosis (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Furthermore, the analysis demonstrated a significant association between CD47 expression and both lymph node metastasis and advanced tumor stage (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). Moreover, survival analysis conducted using the Kaplan-Meier plotter, which relied on RNA-seq data, further substantiated that elevated CD47 expression in individuals diagnosed with stomach adenocarcinoma generally indicated a diminished likelihood of relapse-free survival (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH). These findings suggest that CD47 is extensively upregulated in gastric cancer tissues and exhibits a significant correlation with unfavorable prognosis.\u003c/p\u003e\n\u003cp\u003eTargeting CD47 promoted macrophage phagocytosis of PDCs\u003c/p\u003e\n\u003cp\u003eBefore examining the impact of targeting the CD47-SIRP\u0026alpha; axis, we utilized CD47\u0026thinsp;+\u0026thinsp;gastric cancer patient-derived cells (PDC1 and PDC2) and verified their identity through flow cytometry analysis (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Subsequently, CFDA SE labeling was applied to PDC1 and PDC2, which were then co-cultured with macrophages derived from human peripheral blood mononuclear cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). The administration of SIRP\u0026alpha;-Fc to co-cultured cells resulted in a notable augmentation in the phagocytic activity of macrophages towards PDC1 (from 5.84\u0026ndash;25.10%) and PDC2 (from 7.91\u0026ndash;25.37%) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). These findings provide compelling evidence that the inhibition of CD47 can effectively enhance the phagocytic capability of macrophages toward gastric cancer cells.\u003c/p\u003e\n\u003cp\u003eTargeting CD47 significantly inhibited the growth of gastric cancer\u003c/p\u003e\n\u003cp\u003eTo accurately and authentically forecast the efficacy of anti-CD47 immunotherapy on human gastric cancer, we developed Hu-PDX models that maintained the attributes of the original tumors and provided a more accurate representation of the pertinent constituents and interactions within the tumor microenvironment (TME). Mice-bearing tumors were randomly assigned to specified groups and treated with either SIRP\u0026alpha;-Fc fusion protein or isotype control IgG1-Fc twice weekly for a duration of 4 weeks. The findings indicated that the group treated with SIRP\u0026alpha;-Fc exhibited reduced tumor volume and weight compared to the control group, as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-D. Immunohistochemical analysis revealed an increase in both CD80\u0026thinsp;+\u0026thinsp;M1 macrophages and CD8\u0026thinsp;+\u0026thinsp;T cells infiltrating the tumor microenvironment (TME) following anti-CD47 therapy. However, it is important to note that there was also an increase in pro-tumor CD163\u0026thinsp;+\u0026thinsp;M2 macrophages in the anti-CD47 treatment group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). The findings suggest that the inhibition of CD47 resulted in an antitumor response by promoting the polarization of M1 macrophages and enhancing macrophage phagocytosis. However, it was also observed that CD47 blockade facilitated the infiltration of M2 macrophages.\u003c/p\u003e\n\u003cp\u003eCD47 is positively correlated with VEGF in gastric cancer\u003c/p\u003e\n\u003cp\u003eGiven the presence of an immunosuppressive tumor microenvironment in the context of CD47 blockade, it is imperative to elucidate the underlying mechanism and devise a strategy to enhance the efficacy of anti-CD47 therapy. The development of sustained tumor angiogenic vasculature frequently leads to the establishment of an immunosuppressive microenvironment. In this study, we conducted an analysis of intratumoral microvessel density within the tumor microenvironment (TME) and observed a significant increase in microvessel density following treatment with SIRP\u0026alpha;-Fc (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, an examination was conducted on the relationship between VEGF and CD47 in gastric cancer. Notably, an analysis of samples obtained from patients with gastric cancer using immunohistochemistry revealed a novel finding: VEGF levels were significantly elevated in samples exhibiting high CD47 expression compared to those with low CD47 levels (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). This observation was further supported by the immunohistochemistry results obtained from our comprehensive collection of 89 gastric cancer specimens (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). The results of this study indicate that the combination of CD47 and VEGF blockade may be a viable approach for treating gastric cancer. Furthermore, analysis of gene expression data from the GEPIA database supports the positive association between VEGF and CD47 in gastric cancer tissues (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). These findings provide evidence that CD47 expression is positively linked to VEGF expression in gastric cancer samples. Consequently, we propose that targeting tumor neovascularization could potentially impact the efficacy of anti-CD47 therapy.\u003c/p\u003e\n\u003cp\u003eCD47 blockade combined with antiangiogenetic therapy elicited enhanced anti-tumor effect\u003c/p\u003e\n\u003cp\u003eSubsequently, an investigation was conducted to determine whether the inhibition of VEGF could augment the effectiveness of CD47 blockade in gastric cancer. The results depicted demonstrate that the combination of SIRP\u0026alpha;-Fc and VEGFR1-Fc resulted in the complete suppression of tumor growth in the Hu-PDX1 model (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). The tumor weights in the control group, SIRP\u0026alpha;-Fc group, VEGFR1-Fc group, and SIRP\u0026alpha;-Fc\u0026thinsp;+\u0026thinsp;VEGFR1-Fc group were recorded as 962.36\u0026thinsp;\u0026plusmn;\u0026thinsp;171.10 mg, 514.50\u0026thinsp;\u0026plusmn;\u0026thinsp;83.86 mg, 381.40\u0026thinsp;\u0026plusmn;\u0026thinsp;95.65 mg, and 68.47\u0026thinsp;\u0026plusmn;\u0026thinsp;7.53 mg, respectively. Similar findings were observed in the Hu-PDX2 model (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Moreover, an examination was conducted on the presence of macrophages, CD8\u0026thinsp;+\u0026thinsp;T cells, and microvessel density within the tumor microenvironment (TME). Immunohistochemical (IHC) analysis revealed that the combination of antiangiogenic therapy and CD47 blockade resulted in a reduction of tumor angiogenic vasculature and infiltrated M2-like macrophages, in comparison to the use of anti-CD47 therapy alone. Furthermore, the combined treatment exhibited the greatest ability to induce infiltration of CD8\u0026thinsp;+\u0026thinsp;T cells when compared to all other groups (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). The data collectively indicate that the concurrent utilization of angiogenetic axis blockade and CD47/SIRP\u0026alpha; axis inhibition can effectively stimulate both the innate and adaptive immune systems, resulting in a synergistic anti-tumor response.\u003c/p\u003e\n\u003cp\u003eDual blocking CD47 and VEGF elicited synergetic anti-tumor effect and prevented gastric cancer recurrence\u003c/p\u003e\n\u003cp\u003eThe evaluation of the anti-tumor effects of SIRP\u0026alpha;-VEGFR1, a bispecific fusion protein that concurrently inhibits CD47 and VEGF, demonstrated significant tumor regression in the Hu-PDX1 model, comparable to the observed effects in the SIRP\u0026alpha;-Fc\u0026thinsp;+\u0026thinsp;VEGFR1-Fc combination group (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). This underscores the significance of employing bispecific fusion proteins as a method to enhance the targeting efficacy in vivo and improve patient adherence. Following a treatment duration of 27 days, the tumor weights in the control group, SIRP\u0026alpha;-Fc\u0026thinsp;+\u0026thinsp;VEGFR1-Fc group, and SIRP\u0026alpha;-VEGFR1 group were measured at 905.30\u0026thinsp;\u0026plusmn;\u0026thinsp;167.60 mg, 72.82\u0026thinsp;\u0026plusmn;\u0026thinsp;8.65 mg, and 83.30\u0026thinsp;\u0026plusmn;\u0026thinsp;11.71 mg, respectively. In the Hu-PDX2 model, comparable outcomes were observed in terms of tumor weights among the control group (826.30\u0026thinsp;\u0026plusmn;\u0026thinsp;71.52 mg), the combinational administration group (98.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.379 mg), and the bispecific fusion protein group (80.78\u0026thinsp;\u0026plusmn;\u0026thinsp;9.63 mg) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). Analysis of the tumor microenvironment (TME) revealed that the impact of SIRP\u0026alpha;-VEGFR1 on macrophages, CD8\u0026thinsp;+\u0026thinsp;T cells, and microvessel density is akin to that of the combination therapy (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). In the clinical setting, the occurrence of recurrence plays a significant role in the high mortality rates observed in patients with gastric cancer. Consequently, we established a post-surgical gastric cancer model to assess the efficacy of SIRP\u0026alpha;-VEGFR1 in preventing recurrence. The surgical procedure resulted in incomplete removal of the tumor. Subsequently, the mice were administered injections of IgG1-Fc, SIRP\u0026alpha;-Fc\u0026thinsp;+\u0026thinsp;VEGFR1-Fc, or SIRP\u0026alpha;-VEGFR1. In comparison to the control group, the co-targeting of CD47 and VEGF demonstrated effective inhibition of tumor recurrence (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF). Furthermore, all mice receiving SIRP\u0026alpha;-VEGFR1 treatment remained alive for a period of 60 days following the surgery (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG). In summary, the simultaneous inhibition of CD47 and VEGF using SIRP\u0026alpha;-VEGFR1 successfully stimulated the immune response of the host against cancer recurrence, leading to a significant extension in overall survival.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere is mounting evidence that tumor cells increase the expression of CD47 to evade detection by the innate immune system(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Blocking CD47 not only directly enhances the ability of macrophages to engulf tumor cells, but also activates effector T cells and strengthens the adaptive immune response by facilitating the presentation of tumor antigens on macrophages (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). CD47 blocking agents, including anti-CD47 antibody, anti-SIRPα antibody, and SIRPα-Fc fusion protein, have demonstrated significant efficacy in suppressing the proliferation of diverse tumor types, encompassing colon cancer, breast cancer, pancreatic neuroendocrine tumors, ovarian cancer, leiomyosarcoma, glioblastoma, and small cell lung cancer (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Nevertheless, the efficacy of CD47 as a viable therapeutic target for gastric cancer remains uncertain owing to contradictory outcomes reported in prior research. For instance, Yoshida et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) have demonstrated that CD47 serves as an autonomous negative prognostic indicator in gastric cancer. In contrast, an alternative investigation has revealed no statistically substantial variation in CD47 mRNA levels between primary gastric cancer and healthy tissues, thereby concluding that CD47 in primary gastric tumors does not exhibit any correlation with clinicopathological factors or prognosis (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In this study, clinical specimens were obtained from a cohort of 89 patients diagnosed with gastric cancer, and subsequent analysis was conducted to assess the expression of CD47 in both tumor tissues and corresponding normal tissues. The findings of our investigation substantiated the overexpression of CD47 in gastric cancer, establishing a significant association with unfavorable prognosis. Furthermore, we proceeded to investigate the therapeutic potential of anti-CD47 treatment in gastric cancer, revealing that the blockade of CD47 effectively impeded tumor growth by activating macrophage-mediated innate and adaptive immune responses.\u003c/p\u003e \u003cp\u003eThe application of immunotherapy in cancer treatment has revealed multiple instances of both intrinsic and acquired resistance (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Despite the demonstrated antitumor effects of immune checkpoint inhibitors (ICIs) in various cancer types, eradicating tumor cells remains challenging, including in the case of CD47 blockade therapy (\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Consequently, several studies have been conducted to explore novel strategies aimed at achieving a more potent anti-tumor effect. For instance, the combination of anti-GD2 and anti-CD47 has been found to synergistically facilitate the eradication of neuroblastoma by altering both pro- and anti-phagocytic signals within the tumor microenvironment (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). The efficacy of immunotherapy against melanoma and colorectal cancer was enhanced by the simultaneous blocking of CD47 and PD-L1 using bispecific antibodies, which activated both innate and adaptive immune responses (\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). The combination of autophagy inhibitors with anti-CD47 therapy further improved tumoricidal activity by regulating catabolic pathways and cellular homeostasis (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). In our study, we observed a significant presence of microvessels in gastric cancer following anti-CD47 treatment, leading us to discover that tumor angiogenesis plays a crucial role in limiting the effectiveness of anti-CD47 therapy for the first time.\u003c/p\u003e \u003cp\u003eTumor neovascularization hinders the infiltration of immune cells that react to tumors and fosters an immunosuppressive tumor microenvironment (TME) that enables tumors to resist immunotherapy. In the majority of cancer cases, vascular endothelial growth factor (VEGF) promotes neovascularization, impairs the interaction between endothelial cells and leukocytes, and limits the infiltration of immune cells into the TME by reducing adhesion molecules. Simultaneously, elevated levels of VEGF and angiogenesis in tumors impede the cytotoxic activity of CD8\u0026thinsp;+\u0026thinsp;T cells and enhance the presence of immunosuppressive cells, such as regulatory T cells (Treg cells) and M2-like macrophages(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). In the current investigation, it was observed that administration of anti-CD47 treatment resulted in an elevation in microvessel density within the tumor (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). This finding is significant as it aligns with previous research indicating that anti-VEGF agents can normalize tumor vasculature and transform the immunosuppressive tumor microenvironment (TME) into an immunosupportive one. Consequently, this alteration in the TME has been shown to enhance the efficacy of PD-1/PD-L1 inhibitors in various cancers, including gastric cancer. Furthermore, the combination of anti-VEGF/VEGFR and PD-1/PD-L1 inhibitors has been granted FDA approval for the treatment of hepatocellular carcinoma and renal cell carcinoma (\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). It is noteworthy that the blockade of CD47 has been shown to alter the phenotype of macrophages towards the M1 subtype, which has antitumorigenic properties, as demonstrated in previous studies (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), However, our study also observed an increase in the presence of pro-tumorigenic M2 macrophages in the tumor microenvironment, potentially attributed to the influence of tumor angiogenesis on the recruitment and proliferation of M2 macrophages. Notably, our study also revealed a positive correlation between VEGF expression and CD47 in gastric cancer tissues, suggesting the potential efficacy of combining anti-CD47 and anti-angiogenesis therapies for the treatment of gastric cancer in clinical settings. To investigate the impact of tumor neovascularization on tumor growth and the potential interference with the anti-tumor effect of CD47 blockade within the tumor immune microenvironment, Hu-PDX models were subjected to the administration of SIRPα-Fc and VEGFR1-Fc, aiming to achieve a simultaneous blockade of CD47 and tumor angiogenesis. The findings revealed that this combined therapeutic approach resulted in a synergistic anti-tumor effect against gastric cancer, characterized by a reduction in microvessel density and M2 macrophages. The bispecific fusion protein SIRPα-VEGFR1 was employed to concurrently target CD47 and VEGF, resulting in significant suppression of tumor growth in the Hu-PDX model. Furthermore, SIRPα-VEGFR1 demonstrated a sustained immune response, effectively preventing the recurrence of gastric cancer and extending overall survival in a recurrence model.\u003c/p\u003e \u003cp\u003eIn summary, our research has validated the elevated expression of CD47 in human gastric cancer and its significant association with unfavorable prognosis. The utilization of SIRPα-Fc in anti-CD47 therapy has demonstrated remarkable efficacy in Hu-PDX models of gastric cancer. However, it is worth noting that the administration of anti-CD47 treatment led to an escalation in microvessel density within the tumor, potentially fostering an immunosuppressive microenvironment and constraining the effectiveness of CD47 blockade. Additionally, there exists a positive correlation between VEGF expression and CD47 expression in gastric cancer. The concurrent administration of anti-CD47 and anti-VEGF therapies, along with the utilization of a bispecific fusion protein, yielded notable enhancements in the tumor immune microenvironment and a substantial augmentation of the antitumor response. These findings strongly indicate that CD47 holds promise as a viable therapeutic target for gastric cancer, and underscore the considerable advantages of combining anti-CD47 treatment with anti-angiogenic therapy in the context of gastric cancer treatment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHu-PDXs: humanized patient-derived xenografts\u003c/p\u003e\n\u003cp\u003eSIRP\u0026alpha;: signal regulatory protein alpha\u003c/p\u003e\n\u003cp\u003eICIs: immune checkpoint inhibitors\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTME: tumor microenvironment\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVEGF: vascular endothelial growth factor\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePDC: patient-derived cell\u003c/p\u003e\n\u003cp\u003eHu-PDX: patient-derived xenograft\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAJCC: American Joint Committee on Cancer\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKM-plotter: Kaplan-Meier plotter\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGEPIA: gene-expression profiling interactive analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIHC: immunohistochemical\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eANOVA：\u0026nbsp;analysis of variance\u003c/p\u003e\n\u003cp\u003eSIRP\u0026alpha;: signal regulatory protein \u0026alpha;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNCG mice: a severely immunodeficient strain obtained by knocking out the Prkdc and Il2rg genes in NOD/ShiltJGpt mice\u003c/p\u003e\n\u003cp\u003eGM-CSF: granulocyte-macrophage colony-stimulating factor\u003c/p\u003e\n\u003cp\u003eCFDA SE: carboxyfluorescein diacetate succinimidyl ester\u003c/p\u003e\n\u003cp\u003ePBMCs: peripheral blood mononuclear cells\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBSA: bovine serum albumin\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e LF, XZ, and KY designed the project and proofread the manuscript. KZ, YX, XC, CX, WX, YN, DD, MN, HC, JX, and LZ performed the experiments and prepared the manuscript. XZ, JF, JH, TC, and DJ provided material and analyzed the data. All authors approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by grants from the National Key Research and Development Program of China (2023YFC3606600, 2023YFC3503403 and 2023YFC3404000), the National Natural Science Foundation of China (32200745, 82371781 and 82073752), Shanghai Sailing Program (21YF1401900), and Medical Innovation Research Program of Science and Technology Commission of Shanghai Municipality (21Y11913100). Song Li and Wenzhi Tian provided technical support for the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u0026nbsp;\u003c/strong\u003eThe survival analysis data between relapse-free survival and stomach adenocarcinoma are available online through the Kaplan-Meier plotter based on RNA-seq. The correlation between CD47 and VEGF data is available online through Correlation Analysis in GEPIA based on the TCGA STAD tumor database. All other data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe other authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u0026nbsp;\u003c/strong\u003eThe studies involving human participants were reviewed and approved by the Clinical Research Ethics Committee of Changhai Hospital (CREC: CHEC-2021-119). All procedures followed were per the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions. \u0026nbsp;All patients provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Animal Ethical Committee of the School of Pharmacy at Fudan University (AEC: 2020-12-SY-ZXY-01). All institutional and national guidelines for the care and use of laboratory animals were followed.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. Cancer J Clin 71:209\u0026ndash;249. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3322/caac.21660\u003c/span\u003e\u003cspan address=\"10.3322/caac.21660\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmyth EC, Nilsson M, Grabsch HI, van Grieken NC, Lordick F (2020) Gastric cancer. 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PLoS ONE 11:e0153550. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0153550\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0153550\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Anti-CD47 therapy, Innate immunity, Angiogenic vasculature, VEGF, Combinational therapy","lastPublishedDoi":"10.21203/rs.3.rs-3698194/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3698194/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCD47, serving as an intrinsic immune checkpoint, has demonstrated efficacy as an antitumor target in hematologic malignancies. Nevertheless, the clinical relevance of CD47 in gastric cancer and its potential as a therapeutic target remains unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe expression of CD47 in clinical gastric cancer tissues was assessed using immunohistochemistry and western blot. Patient-derived cells were obtained from gastric cancer tissues and co-cultured with macrophages derived from human peripheral blood mononuclear cells. Flow cytometry analyses were employed to evaluate the rate of phagocytosis. Humanized patient-derived xenografts (Hu-PDXs) models were established to assess the efficacy of anti-CD47 immunotherapy or the combination of anti-CD47 and anti-VEGF therapy in treating gastric cancer. The infiltrated immune cells in the xenograft were analyzed by immunohistochemistry.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we have substantiated the high expression of CD47 in gastric cancer tissues, establishing a strong association with unfavorable prognosis. Through the utilization of SIRPα-Fc to target CD47, we have effectively enhanced macrophage phagocytosis of PDCs in vitro and impeded the growth of Hu-PDXs. It is noteworthy that anti-CD47 immunotherapy has been observed to sustain tumor angiogenic vasculature, with a positive correlation between the expression of VEGF and CD47 in gastric cancer. Furthermore, the successful implementation of anti-angiogenic treatment has further augmented the antitumor efficacy of anti-CD47 therapy. In addition, the potent suppression of tumor growth, prevention of cancer recurrence after surgery, and significant prolongation of overall survival in Hu-PDX models can be achieved through the simultaneous targeting of CD47 and VEGF using the bispecific fusion protein SIRPα-VEGFR1 or by combining the two single-targeted agents.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur preclinical studies collectively offer substantiation that CD47 holds promise as a prospective target for gastric cancer, while also highlighting the potential of anti-angiogenic therapy to enhance tumor responsiveness to anti-CD47 immunotherapy.\u003c/p\u003e","manuscriptTitle":"Co-targeting CD47 and VEGF Elicited Potent Antitumor Effects in Gastric Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-06 13:49:19","doi":"10.21203/rs.3.rs-3698194/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-01-29T05:26:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-01-02T21:13:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0bdeded2-b592-47a4-9e52-dc19c2f9929c","date":"2023-12-17T12:32:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-12-17T02:31:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-05T19:24:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-12-03T14:29:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2023-12-02T17:08:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c9fde57c-6301-4205-a951-0eb2f00f7cb8","owner":[],"postedDate":"December 6th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-03-01T07:02:00+00:00","versionOfRecord":[],"versionCreatedAt":"2023-12-06 13:49:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3698194","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3698194","identity":"rs-3698194","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}