Expression and prognostic significance of CD93 in blood vessels in colorectal cancer: an immunohistochemical analysis of 134 cases
Colorectal cancer (CRC) is the third most common malignancy and second leading cause of cancer-related deaths . GLOBOCAN estimated that there were more than 1.9 million new CRC (including anus) cases and 935,000 deaths in 2020, representing about one in 10 cancer cases and deaths . There are various treatment options for CRC, including different surgical approaches, radiotherapy, adjuvant chemotherapy, targeted therapy, and immunotherapy; however, the overall survival (OS) of patients with advanced disease is just 3 years . Given that the disease only becomes symptomatic at an advanced stage, the prognosis is thus likely to be poor. It is therefore critical to understand the molecular mechanisms and to identify molecularly based prognostic markers underlying CRC progression to allow the development of effective therapeutic strategies. Emerging evidence suggests that tumor blood vessels are tortuous, dilated, and leaky, and contribute to the aberrant tumor microenvironment (TME) by impairing blood flow and limiting the delivery of oxygen and nutrients, thereby reducing the efficacy of chemotherapy, radiotherapy, and immunotherapy . Since the discovery that vascular endothelial growth factor (VEGF) is a critical driver of tumor angiogenesis, a primary focus of cancer therapy has been the development of drugs that inhibit VEGF, intending to shrink tumors by depriving them of essential oxygen and nutrients. However, recent findings challenge the effectiveness of this approach when used in isolation. Anti-VEGF monotherapy, surprisingly, has shown limited success in patients with solid tumors. On the other hand, combining anti-VEGF treatments with standard chemotherapy has yielded a notable improvement in survival rates compared to chemotherapy alone. This apparent contradiction—the increased efficacy of combination therapy versus monotherapy—may be clarified by the concept of “tumor vasculature normalization” induced by anti-VEGF therapy. Preclinical studies indicate that anti-VEGF treatment fosters a shift in tumor blood vessels toward a “mature” or “normalized” state. This change involves a reduction in hyperpermeability, a strengthened basement membrane, and, consequently, a decrease in both tumor hypoxia and interstitial fluid pressure. In essence, rather than simply starving tumors, anti-VEGF therapy may be transforming the tumor microenvironment itself, creating vascular conditions that enhance the effectiveness of chemotherapy. This dual approach, by altering the pathological characteristics of the tumor’s blood vessels, may offer a more robust and sustainable treatment pathway . The detection of vascular normalization in tumor tissues is thus important in terms of treatment selection and the prediction of prognosis. Tumor angiogenesis is inextricably linked to the tumor microenvironment(TME). Recent studies have found that a variety of immune cells in the tumor mesenchyme can secrete VEGF to promote the formation of tumor blood vessels, leading to an abnormal TME. This tumor microenvironment induces T-cell dysfunction and depletion. In addition, abnormal tumor vascular endothelial cells may be associated with cytotoxic T-cell suppression and ultimately lead to immunosuppression in patients. Therefore, the search for effective tumor vascular markers may reduce the negative effects of VEGF and restore the patient’s immune function by altering TME to provide a good prognosis for the patient . CD93 is an epidermal growth factor-like domain-containing transmembrane protein that is predominantly expressed in the vascular endothelium . CD93 was recently identified as one of the most important genes for angiogenesis in human primary tumors . The immature and dysfunctional vascular network within solid tumors poses a substantial obstacle to immunotherapy by creating a hypoxic tumor microenvironment that actively limits immune cell infiltration . Bao et al. showed that high vascular expression of CD93 was indicative of poor survival in patients with nasopharyngeal carcinoma . Interestingly however, high CD93 expression does not always suggest a poor prognosis, and Tong et al. suggested that elevated CD93 expression was associated with better OS in patients with kidney clear cell carcinoma . We speculated that this apparently contradictory result may be related to the expression of CD93. Available studies demonstrate that downregulation of CD 93 will lead to disruption of Vascular endothelial calreticulin (VE-cadherin) in endothelial junctions, which then increases vascular permeability . VE-cadherin is one of the major signaling and structural proteins associated with adhesion junctions. When vascular permeability is increased, solid tumors release factors that promote the growth of disorganized and leaky vascular networks. These vascular networks show disturbed blood flow, inflammatory cell infiltration and tumor cell extravasation leading to poor prognosis . Existing studies have found that this abnormal hyperpermeability of the tumor vasculature is ameliorated when normalization of the tumor vasculature occurs. So we hypothesized that its low expression indicates the formation of abnormal tumor blood vessels, while its high expression indicates normalization of these tumor blood vessels. Different expression levels of CD93 in different tumor types may thus lead to different prognoses. However, the prognostic significance of CD93 expression levels in CRC remains unclear. Existing studies on CD93 have focused more on other cancers such as nasopharyngeal carcinoma (NPC) and bladder carcinoma (BC), and little research has been done in GI tumors.Renate S. et al. found that CD93 expression was significantly higher in CRC tumor tissues compared to normal colorectal tissues.Wu et al. found that CD93 was highly expressed in stomach adenocarcinoma (STAD), as compared to adjacent normal tissues . Wu et al. found that CD93 was more highly expressed in CRC tumor tissues compared to normal tissues. They found that CD93 was highly expressed in STAD compared to neighboring normal tissues. Furthermore, CD93 was significantly correlated with the abundance of multiple immune cell infiltration levels in STAD . In contrast, there has been no study on the correlation between CD93 and immune cell infiltration levels in CRC. Since both STAD and CRC are GI tumors, we conjecture that CD93 has a similar biological role in these two diseases. In the present study, we investigated the clinical significance of CD93 in CRC. We also analyzed the correlations between CD93 and infiltrating immune cells, including CD4 + T cells, CD8 + T cells, and macrophages. The results of this study suggest that CD93 might be a prognostic factor and might predict immune infiltration in CRC.   Patients and samples Fresh surgically resected tissue samples, including CRC tissues and matched adjacent normal tissues, were obtained from 134 patients who underwent complete surgical excision at Shanxi Province Cancer Hospital, Taiyuan, China, between June 2016 and June 2018. All the CRC tissues were primary neoplastic lesions. None of the patients had received any preoperative therapies, including radiotherapy, chemotherapy, or immunotherapy. Patients with familial adenomatous polyposis or any other relevant genetic history were excluded. The diagnosis of CRC was confirmed independently by two pathologists. Clinicopathological data for all patients were collected retrospectively. TNM stages were evaluated according to AJCC 8th. Tissue microarray (TMA) analysis TMAs can provide an unprecedented degree of standardization, speed, and cost efficiency. We obtained all the slides from these tumors from the archive. For each case of tumor tissue we selected the center of the tumor, the deepest part of the tumor infiltration and the colorectal mesenchyme next to the deepest part of the tumor infiltration as tissue cores. All the sections were reviewed by one pathologist who selected the optimal slide for each specimen, and marked the tissue areas for subsequent punching. We then prepared recipient blocks (diameter of the premade hole, 2 mm), defined the structure (outline) of the TMA, and created a file containing the identification numbers of the tissues, together with their locations and real coordinates. Finally, a cylindrical tissue sample was removed from each donor paraffin block and placed in the premade hole in the recipient block, and the identification numbers of the tissues were recorded in the file. After the paraffin embedded tissue was placed into the recipient block, the block was heated to 65 °C and then cooled down at room temperature for 7 min, until the tissues were fully fused with the paraffin block . Histology and immunohistochemistry (IHC) staining TMA blocks were cut into 3 μm sections and used for hematoxylin and eosin staining and IHC. CD93 protein expression in 134 CRC samples was evaluated by IHC. The slides were blocked and incubated with primary antibodies against CD93 (Invitrogen, USA; polyclonal antibody, diluted 1:400), CD4 (Genetech, China; machine working fluid), CD8 (Genetech; machine working fluid), CD68 (Maxim, China; machine working fluid), and CD80 (Abcam, United Kingdom; diluted 1:1000). Among them, CD4 and CD8 were used to count T cells. CD68 and CD80 were used to count macrophages and dendritic cells respectively. IHC score CD93 expression, determined by the staining intensity and relative percentage of vascular endothelial cells, was analyzed by two pathologists blinded to the data. Positive staining was scored according to the color intensity and whether the vascular endothelium cells were continuously positive as follows: 0 = no staining; 1 = light yellow staining and discontinuously positive vascular endothelial cells; 2 = yellow or orange staining and vascular endothelial cells continuously positive; and 3 = brown staining and vascular endothelial cells continuously positive. The percentage of positive vessels was based on the following criteria: 0–25% = 0.5 points; 26–50% = 1 point; 51–75% = 1.5 points; and > 75% = 2 points. The IHC score was calculated by multiplying the staining intensity by the percentage of positive vessels. Scores ≥ 3 were classified as high and scores < 3 were classified as low. For assessment of the immune cells, cells were counted in three selected areas in each tissue cores. These three areas include the tumor mesenchyme, the mesenchyme at the front of tumor cell infiltration, and the mesenchymal region where no tumor cell infiltration is seen, respectively. All of these regions are hotspots for immune cell infiltration, and they are not continuous with each other. This counting method allows the data obtained to be as close as possible to the amount of immune cell infiltration in the patient’s tumor tissue. The cut-off value is obtained by plotting the ROC curve. We calculate the sensitivity and specificity of the data. The two are added and subtracted by 1 and the value corresponding to the maximum value of the result is chosen as the cut-off value. Samples with more cells than the cut-off value were classified as high expression, and vice versa . Statistical analysis Statistical analysis was performed using SPSS software (ver. 21, SPSS Inc., Chicago, IL, USA). The relationships between vascular CD93 expression and clinicopathological features of CRC and the infiltration of immune cells were analyzed by χ 2 tests. Survival curves were estimated by Kaplan–Meier analysis and compared by log-rank test. Values of p < 0.05 were considered statistically significant with 95% confidence.   Fresh surgically resected tissue samples, including CRC tissues and matched adjacent normal tissues, were obtained from 134 patients who underwent complete surgical excision at Shanxi Province Cancer Hospital, Taiyuan, China, between June 2016 and June 2018. All the CRC tissues were primary neoplastic lesions. None of the patients had received any preoperative therapies, including radiotherapy, chemotherapy, or immunotherapy. Patients with familial adenomatous polyposis or any other relevant genetic history were excluded. The diagnosis of CRC was confirmed independently by two pathologists. Clinicopathological data for all patients were collected retrospectively. TNM stages were evaluated according to AJCC 8th.   TMAs can provide an unprecedented degree of standardization, speed, and cost efficiency. We obtained all the slides from these tumors from the archive. For each case of tumor tissue we selected the center of the tumor, the deepest part of the tumor infiltration and the colorectal mesenchyme next to the deepest part of the tumor infiltration as tissue cores. All the sections were reviewed by one pathologist who selected the optimal slide for each specimen, and marked the tissue areas for subsequent punching. We then prepared recipient blocks (diameter of the premade hole, 2 mm), defined the structure (outline) of the TMA, and created a file containing the identification numbers of the tissues, together with their locations and real coordinates. Finally, a cylindrical tissue sample was removed from each donor paraffin block and placed in the premade hole in the recipient block, and the identification numbers of the tissues were recorded in the file. After the paraffin embedded tissue was placed into the recipient block, the block was heated to 65 °C and then cooled down at room temperature for 7 min, until the tissues were fully fused with the paraffin block .   TMA blocks were cut into 3 μm sections and used for hematoxylin and eosin staining and IHC. CD93 protein expression in 134 CRC samples was evaluated by IHC. The slides were blocked and incubated with primary antibodies against CD93 (Invitrogen, USA; polyclonal antibody, diluted 1:400), CD4 (Genetech, China; machine working fluid), CD8 (Genetech; machine working fluid), CD68 (Maxim, China; machine working fluid), and CD80 (Abcam, United Kingdom; diluted 1:1000). Among them, CD4 and CD8 were used to count T cells. CD68 and CD80 were used to count macrophages and dendritic cells respectively.   CD93 expression, determined by the staining intensity and relative percentage of vascular endothelial cells, was analyzed by two pathologists blinded to the data. Positive staining was scored according to the color intensity and whether the vascular endothelium cells were continuously positive as follows: 0 = no staining; 1 = light yellow staining and discontinuously positive vascular endothelial cells; 2 = yellow or orange staining and vascular endothelial cells continuously positive; and 3 = brown staining and vascular endothelial cells continuously positive. The percentage of positive vessels was based on the following criteria: 0–25% = 0.5 points; 26–50% = 1 point; 51–75% = 1.5 points; and > 75% = 2 points. The IHC score was calculated by multiplying the staining intensity by the percentage of positive vessels. Scores ≥ 3 were classified as high and scores < 3 were classified as low. For assessment of the immune cells, cells were counted in three selected areas in each tissue cores. These three areas include the tumor mesenchyme, the mesenchyme at the front of tumor cell infiltration, and the mesenchymal region where no tumor cell infiltration is seen, respectively. All of these regions are hotspots for immune cell infiltration, and they are not continuous with each other. This counting method allows the data obtained to be as close as possible to the amount of immune cell infiltration in the patient’s tumor tissue. The cut-off value is obtained by plotting the ROC curve. We calculate the sensitivity and specificity of the data. The two are added and subtracted by 1 and the value corresponding to the maximum value of the result is chosen as the cut-off value. Samples with more cells than the cut-off value were classified as high expression, and vice versa .   Statistical analysis was performed using SPSS software (ver. 21, SPSS Inc., Chicago, IL, USA). The relationships between vascular CD93 expression and clinicopathological features of CRC and the infiltration of immune cells were analyzed by χ 2 tests. Survival curves were estimated by Kaplan–Meier analysis and compared by log-rank test. Values of p < 0.05 were considered statistically significant with 95% confidence.   CD93 expression was upregulated in clinical CRC tissue samples After immunohistochemical staining of colorectal cancer tissues, we found that CD93 was expressed in vascular endothelial cells in tumor tissues, and CD93 was also detected in some immune cells. We believe that the immunohistochemical method can be more intuitive to observe the differential expression of CD93 in vascular endothelial cells in colorectal cancer tumour tissues and normal tissues. We compared the expression levels of CD93 in 134 clinical CRC tissues and paired normal tissues by IHC. CD93 expression levels were significantly higher in CRC tissues and were almost negative in the corresponding normal tissues (Fig. ). Correlation between expression of CD93 and clinicopathological factors of CRC We analyzed the expression of CD93 in 134 CRC patients by IHC. The expression characteristics and clinicopathological data of the included patients are presented in Table . CD93 expression levels differed significantly in relation to tumor site and microsatellite instability (MSI). CD93 expression was positively related to macrophages in CRC We determined if CD93 expression correlated with the infiltration of immune cells, including lymphocytes and macrophages. Lymphocytes were labelled with CD4 and CD8, whereas the macrophages labelled with CD68 and CD80, which identified macrophages and dendritic cells respectively. The results are summarized in Table ; Fig. . Notably, patients with high CD93 expression showed greater infiltration of macrophages and dendritic cells. Also we obtained consistent results when we used TIMER ( http://timer.cistrome.org/ ) to analyse the relationship between CD93 expression and immune cell infiltration (Fig. ). Overexpression of CD93 promoted survival Differentially expressed genes are often associated with patient survival. We analyzed the association between CD93 gene expression and OS using GEPIA ( http://gepia.cancer-pku.cn/ ), which showed no significant association between CD93 expression and OS ( p > 0.05). We also obtained the clinical data and analyzed the prognostic value of CD93 in CRC using Kaplan–Meier survival analysis (Fig. ). The results confirmed that CRC patients with high CD93 protein expression in tumor vessels had better outcomes in terms of OS ( p < 0.05). Moreover, the results of univariate analysis and multivariate analysis about prognostic factors were shown in Table . And they suggested that CD93 expression was an independent prognostic factor in CRC patients.   After immunohistochemical staining of colorectal cancer tissues, we found that CD93 was expressed in vascular endothelial cells in tumor tissues, and CD93 was also detected in some immune cells. We believe that the immunohistochemical method can be more intuitive to observe the differential expression of CD93 in vascular endothelial cells in colorectal cancer tumour tissues and normal tissues. We compared the expression levels of CD93 in 134 clinical CRC tissues and paired normal tissues by IHC. CD93 expression levels were significantly higher in CRC tissues and were almost negative in the corresponding normal tissues (Fig. ).   We analyzed the expression of CD93 in 134 CRC patients by IHC. The expression characteristics and clinicopathological data of the included patients are presented in Table . CD93 expression levels differed significantly in relation to tumor site and microsatellite instability (MSI).   We determined if CD93 expression correlated with the infiltration of immune cells, including lymphocytes and macrophages. Lymphocytes were labelled with CD4 and CD8, whereas the macrophages labelled with CD68 and CD80, which identified macrophages and dendritic cells respectively. The results are summarized in Table ; Fig. . Notably, patients with high CD93 expression showed greater infiltration of macrophages and dendritic cells. Also we obtained consistent results when we used TIMER ( http://timer.cistrome.org/ ) to analyse the relationship between CD93 expression and immune cell infiltration (Fig. ).   Differentially expressed genes are often associated with patient survival. We analyzed the association between CD93 gene expression and OS using GEPIA ( http://gepia.cancer-pku.cn/ ), which showed no significant association between CD93 expression and OS ( p > 0.05). We also obtained the clinical data and analyzed the prognostic value of CD93 in CRC using Kaplan–Meier survival analysis (Fig. ). The results confirmed that CRC patients with high CD93 protein expression in tumor vessels had better outcomes in terms of OS ( p < 0.05). Moreover, the results of univariate analysis and multivariate analysis about prognostic factors were shown in Table . And they suggested that CD93 expression was an independent prognostic factor in CRC patients.   CRC ranks third in terms of cancer incidence but second in terms of cancer-related mortality. Cancer biomarkers are substances or molecules that are objectively detectable in cells, body fluids, or tissues, and which manifest the existence of cancer or predict survival . It is therefore important to identify biomarkers that can indicate patient prognosis. In the present study, we demonstrated that CD93 expression was correlated with clinicopathological features and immunological regulation in patients with CRC, and that CD93 overexpression was a protective factor in CRC. In solid tumors, endothelial cells form the inner lining of blood vessels, contributing to the tumor’s expansion. Tumor-derived endothelial cells (TECs) display distinct phenotypes and morphologies, diverging from normal endothelial cells on both cellular and molecular fronts. Unlike their normal counterparts, TECs are characterized by heightened proliferative capacity and stem cell-like traits. This transformation is accompanied by altered gene expression profiles, resulting in increased secretion of immunomodulatory cytokines and modifications in cell-surface receptor expression . Expression of CD93 in tumor vessels has previously been shown to be a potential malignant biomarker in several cancers and to play a role in tumor angiogenesis, resulting in poorly perfused tumors . However, Tong et al. found that CD93 mRNA expression levels were significantly downregulated in CRC, whereas Zhang et al. showed that mRNA expression levels were increased in CRC . Considering the complicated molecular mechanism of tumors and the controversial results for CD93 mRNA, we chose to detect CD93 protein levels in CRC by IHC. We found that expression levels of CD93 were higher in CRC vessels than in vessels in paired adjacent tissues, suggesting that CD93 was present in the TME. CD93 has been reported as a high-risk factor that regulates tumor growth in several tumors. Bao et al. revealed that CD93 was highly expressed in NPC and that vascular CD93 expression was associated with clinical progression and poor prognosis . Sun et al. indicated that CD93 protein was up-regulated in pancreatic adenocarcinoma and primitive neuroectodermal tumor. Insulin-like growth factor binding protein 7 (IGFBP7) acted as a ligand for CD93, and overexpression of the IGFBP7/CD93 pathway was associated with a poor response to therapy . Conversely, Guo et al. reported that low CD93 expression was associated with shorter OS in patients with KIRC and other tumors, suggesting that CD93 may act as a protective factor . The current survival analysis confirmed our findings that vessels with high CD93 protein levels were associated with better OS. Studies of CD93 in different cancers have been more limited to cell line and mouse experiments as well as methods such as raw letter analysis, but few studies have been done on the relevance of CD93 in the tissues of tumor patients. Therefore, the reason for the different prognostic significance of CD93 in various cancer types remains to be further investigated. This may be related to the complex TME in different cancer types.There are a variety of stromal cells in the TME and these stromal cells play a key role in tumor progression, metastasis, immune infiltration, and chemoresistance through the production of growth factors, cytokines, chemokines, pro- and anti-tumorigenic factors. Through complex biological effects, this ultimately results in CD93 having different roles for patients with different tumors. In addition to this, this study found a correlation between CD93 and MSI in CRC. This may be related to the fact that MSI tumors have a better prognosis and are less likely to have lymph node spread and metastasis than MSS tumors . However, MSI is rarely detected in other tumors, which may partly explain the different role of CD93 in colorectal cancer from its role in other cancers. In this study, we determined high CD93 expression by continuous expression of CD93 in endothelial cells combined with staining intensity, which differed from the methods used to measure CD93 expression in previous studies. The abnormal architecture of tumor blood vessels engenders a microenvironment rife with heightened interstitial hydrostatic pressure, hypoxia, and acidosis. This increased pressure hampers the delivery of therapeutic agents to tumor tissues, while hypoxia fosters resistance to chemoradiotherapy and induces genetic instability, allowing malignant cells with heightened metastatic potential to thrive. Together, these vascular irregularities and their resulting microenvironment propel tumor growth and metastasis, posing formidable challenges to the effectiveness of cancer therapies. In response, angiogenesis inhibitors aim to repair these aberrant vessels, creating “normalized” blood vessels akin to those in healthy colorectal tissue. These vessels are marked by uniform diameters, fewer collateral branches, and reduced permeability, effectively normalizing the tumor microenvironment and enhancing the delivery of drugs and oxygen to tumor cells. Consequently, vascular normalization is pivotal for improving patient prognosis; however, existing studies lack a comprehensive mechanism and evaluation framework. Current evaluation metrics, such as peripheral cell coverage, endothelial cell permeability, and basement membrane integrity, fall short of fully capturing this complexity. Previous studies showed that CD93 protein was highly expressed in tumor tissue and tumor-related vascular endothelial cells but not in non-proliferating endothelial cells . Notably, our experiments indicate that high CD93 expression correlates with enhanced tumor blood vessel proliferation and lumen integrity, positioning CD93 as a promising new marker for assessing vascular normalization in future research. Molecular alterations within tumor cells intricately reshape the tumor microenvironment, catalyzing the formation of blood vessels that nourish tumors. Yet, these newly formed vessels are not mere conduits; they morph and release cytokines that significantly impact tumor initiation and progression. Thus, probing the relationship between endothelial cell phenotypes and the molecular mutations in tumor cells becomes paramount. This exploration unveils the dynamic interplay between tumor vasculature and immune microenvironments, ultimately offering critical insights into how these vessels influence tumorigenesis and its advancement. Loss of p53 leads to the downregulation of miR-107 and enhances hypoxia-inducible factor-1β(HIF-1β) expression and hypoxic signaling in human colon cancer cells, as well as induces tumor angiogenesis . In our preliminary investigation, we scrutinized mutations in BRAF, KRAS, MSI, and P53 among tumor patients, aiming to identify mutation sites linked to the varied expression of CD93. Unfortunately, we found a scarcity of KRAS and BRAF mutations in colorectal cancer patients. Consequently, we pivoted our focus to MSI and P53 for our formal experiments. MSI has also been identified as an independent prognostic factor in several tumor types . Our findings revealed a correlation between MSI status and CD93 expression, leading us to hypothesize that elevated CD93 levels could potentially enhance the prognosis for patients exhibiting MSI status. We therefore speculate that the combination of MSI with CD93 expression may affect the prognosis in CRC. CD93, also known as C1q receptor, is a type-1 transmembrane glycoprotein . A previous study reported on the immune characteristics of CD93 in the tumor microenvironment. Gasqu et al. detected CD93 on the vascular endothelium in human tissue sections using a recombinant human CD93-Fc fusion protein, raising the possibility that CD93 might mediate leukocyte–endothelial interactions . Greenlee et al. suggested that CD93 functions in the modulation of phagocytosis and adhesion, and revealed that endothelial cell CD93 modulated gene transcription to regulate inflammatory processes . Guo et al. revealed that elevated CD93 levels strongly correlate with the infiltration of a diverse array of cells within the tumor microenvironment (TME). This includes endothelial cells and immune cells of various types, such as CD8 + and CD4 + T cells, neutrophils, myeloid dendritic cells, macrophages (both M1 and M2 phenotypes), regulatory T cells, monocytes, B cells, and even hematopoietic stem cells across multiple cancer types. The TME itself is a complex landscape, composed of both cellular and non-cellular elements. Among the cellular residents are not only fibroblasts, endothelial cells, neurons, and adipocytes, but also a range of adaptive and innate immune cells. Meanwhile, the non-cellular components add layers of biochemical complexity, including the extracellular matrix and a myriad of soluble factors—chemokines, cytokines, growth factors, and extracellular vesicles—that continuously shape and influence tumor dynamics . The interaction of various components of the immune microenvironment can influence tumour development. Existing studies suggest new ideas for enhancing cancer immunotherapy using anti-angiogenic drugs . Research has shown that targeting only tumor cells or isolated elements within the TME rarely yields sufficient therapeutic outcomes. This limitation underscores the potential of investigating the intricate interactions among TME components to unlock new avenues in cancer therapy. One prominent feature of many solid tumors is vascular abnormality—a condition that facilitates immune evasion and resistance. These vascular anomalies arise from elevated levels of proangiogenic factors, notably VEGF and angiopoietin-2. Strategic targeting of these molecules can enhance treatment efficacy by partially normalizing tumor vasculature, which allows for greater infiltration of immune effector cells. This shift not only breaks down the immune-resistant barriers of the TME but also fosters an immunosupportive environment. Cancer immunotherapy, meanwhile, aims to activate cytotoxic T lymphocytes (CTLs) directly within the tumor, while also promoting the effective priming of tumor-specific CTLs in lymphoid tissues to establish robust, long-lasting antitumor immunity. During this critical priming phase, CD4 + T cells communicate activation signals to CD8 + T cells via specialized dendritic cells, refining both the strength and quality of the CTL response for maximum therapeutic impact . Immune cell infiltration is the basis of immunotherapy. Therefore, it is crucial to explore the specific effects of tumour vascular normalisation in the tumour microenvironment on the production of immune cell infiltration. In the current study, we evaluated the association between CD93 expression and the numbers of T cells (CD8 + and CD4+), macrophages and dendritic cells. The results suggested that high CD93 protein levels promoted infiltration of macrophages and dendritic cells, which could explain why CD93 improved patient prognosis, and illustrated the pivotal role of CD93 in tumor immunity. Forssell et al. stained 446 colorectal cancer specimens with CD 68 and found that survival increased incrementally with increasing mean infiltration grade of CD68, regardless of surgical outcome and site. This may be due to the fact that the degree of antitumorigenic capacity depends in part on the ability of macrophages to directly contact tumor cells. In vitro experiments have been shown to reach a similar conclusion, with colon cancer cell survival decreasing as the macrophage-to-cancer cell ratio increases . Relevant studies have shown that increased infiltration of activated and mature DCs in CRC is associated with prolonged OS and DFS. DC recruitment may be positively associating with cytotoxic T-lymphocyte infiltration, which may promote the host immune response to the tumor . Unfortunately, our experiment did not find a direct relationship between CD93 and CD4 + and CD8 + T cell infiltration, which may be related to the small sample size. It was found that high infiltration of CD8 + T cells tended to be accompanied by high infiltration of CD68, a phenomenon that suggests that lymphocytes are involved in anti-tumor responses along with other immune cells despite the fact that the correlation between CD93 expression and lymphocyte infiltration is not statistically willing . The correlation between CD93 and T cell infiltration and whether CD93 can participate in the evaluation of the effectiveness of immunotherapy can be explored through further experiments in the future. The normalisation of blood vessels is an important basis for detecting the effectiveness of anti-angiogenic drugs. Since most of the existing studies on effective indicators of vascular normalisation focus on the structural integrity of blood vessels, but cannot demonstrate the correlation between such blood vessels and immune cell infiltration to provide a basis for immunotherapy, CD93 can better compensate for the limitations of the existing indicators. This suggests that we can use CD93 as a more complete indicator of vascular normalisation in future studies. Unfortunately, however, very few of the patients had undergone immunotherapy, so this paper is limited to detecting the relationship between CD93 expression and immune cell infiltration. Future studies could collect more patients who have received anti-VEGF therapy and immunotherapy for CD93 testing to draw more convincing conclusions. This study had some potential limitations. First, all patients with CRC were enrolled from Shanxi Province, and further multicenter studies are therefore needed. Second, we only detected CD93 protein expression levels using IHC, and the results should be confirmed by other methods, such as immunofluorescence staining. Third, the molecular mechanism of CD93 in colorectal cancer remains unclear and needs to be explored further in cell experiments. In conclusion, we analyzed the prognostic value and immune characteristics of CD93 in CRC. CD93 was present in blood vessels in the TME in CRC tumors and was correlated with the infiltration of immune cells. CD93 was also associated with clinicopathological factors and OS. These results indicate that CD93 is involved in tumor immunity and may act as a novel vascular marker to detect vascular normalization in CRC.   Below is the link to the electronic supplementary material. Supplementary Material 1   