The bioinformatics and experimental analysis of CD276 for prognosis and immune infiltrates in colon adenocarcinoma

doi:10.21203/rs.3.rs-1987050/v1

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The bioinformatics and experimental analysis of CD276 for prognosis and immune infiltrates in colon adenocarcinoma

https://doi.org/10.21203/rs.3.rs-1987050/v1

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Background

Colon adenocarcinoma (COAD), although the third-most common type of gastrointestinal tumors, still lacks specific biomarkers for early diagnosis, treatment, and prognosis. CD276, an immune checkpoint, is upregulated in various cancers.This study aimed to evaluate the role of CD276 in tumorigenesis, prognosis and immunity for COAD.

Methods

The expression profile of CD276 gene in COAD was established by using RNA-sequencing transcriptomic data of The Cancer Genome Atlas (TCGA) databases. The biological functions of CD276 were evaluated using the Metascape database and Gene Set Enrichment Analysis (GSEA). The association between CD276 and immune cell infiltration was investigated by TIMER website. Correlation analysis was performed between CD276 expression and clinicopathological characteristics. Cox proportional hazard regression and Kaplan-Meier final analysis were applied for identifying the prognostic role of CD276.

Results

CD276 expression was significantly elevated in COAD tumor (P < 0.0001). Functional annotation revealed many enriched GO-terms among which the activity of the growth factor, cell adhesion and corresponding cancer-related pathways were highly represented in high CD276 expression phenotype. High CD276 was associated with microsatellite instability (MSI) status, patients’ survival, and disease progression. Cox regression analysis revealed that CD276 was a risk factor for overall survival [hazard ratio (HR): 1.848, P = 2.64E−03], disease-specific survival (HR: 2.406, P = 5.35E−04), and progression-free interval (HR: 1.772, P = 2.04E−03). Moreover, CD276 level was significantly associated with tumor immune cell infiltration, biomarkers of immune cells, and immune checkpoint expression.

Conclusions

Our analyses indicated that increased CD276 may contribute to COAD development by activing tumor‐promoting signal pathways and altering the immune microenvironment. It is believed that abnormal expression of CD276 has clinical prognostic value in COAD patients.

CD276

colon adenocarcinoma

clinical outcome

prognosis

immune infiltration

Recent advances in cancer immunotherapy have revolutionized the treatment of advanced cancers, in particular, antibodies targeting programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4(CTLA-4)have produced durable remissions in various human solid tumors.[1, 2]. Unfortunately, the remarkable therapeutic benefit of anti-PD-1/PD-L1 treatment are only achieved in a minor of patients including high PD-L1 expression in Non-Small Cell Lung Cancer (NSCLC) and a moderate-high tumor mutational burden (TMB). A substantial number of patients do not response to immune checkpoint inhibitor (ICI) [3], which underscore the critical need for biomarkers to predict response to ICI and identify new ICI agents. These findings have garnered our strong interest in immune checkpoints as potential novel therapeutic targets in colon carcinoma (COAD). Herein, we performed an explorative analysis of public databases containing data regarding B7-CD28 family members in COAD.

Immune checkpoint molecule B7 homolog 3 (B7-H3,CD276 or B7RP-2 ), a type I transmembrane protein beloning to the Ig superfamily and a member of B7/CD28 family, has immune modulatory functions [4]. The molecular characteristics of its receptor remain still unclear, but overexpression of CD276 is widely observed in numerous human malignancies and seems to be associated with a poorer prognosis [5–7]. CD276 was initially considered as a co-stimulatory molecule for T cell activation, nevertheless, but subsequent research revealed that CD276 likely has a predominantly inhibitory role in adaptive immunity, suppressing T-cell activation and proliferation. Some relevant studies present important evidence for the CD276 are involved in tumor initiation and progression via immunological or non-immunological mechanisms [8, 9].Of note, CD276 highly expression might promoted tumor cell growth, invasion and eventually metastasis, which is proved to be a new independent prognostic indicator for many solid tumors [10]. However, on more in-depth analyses regarding CD276 expression and its function in COAD are still lacking; therefore, in the present study, we conducted a comprehensive analysis of CD276 in COAD.

Data Processing and Ethics Statement

We downloaded high-throughput sequencing RNA data [fragments per kilobase per million (FPKM) format] and corresponding clinicopathological information from the colon adenocarcinomas project (COAD) on the TCGA database. Excluding these patients with incomplete clinicopathological information and intact survival data, a total of 453 COAD patients (COAD=453) were enrolled. RNA sequencing data were transformed from. FPKM format to transcripts per million reads for this study. As the TCGA database is open to the public under specific guidelines, it confirms that all written informed consents were obtained before data collection.

Differentially Expressed Genes in Colon adenocarcinoma

In total, 453 COAD patients were separated into high- and low-CD276 expression groups according to CD276 median value. The “DESeq2”[11] was used to screen for differentially expressed genes (DEGs) by two-tailed hypothesis test based on negative binomial generalized linear models, where an adjusted P-value <0.05 and |log2 fold change (FC)| ≥1 were used as a threshold for significance. The results were illustrated in the form of heatmaps and volcano plots by use of the R packages “pheatmap”[12] and “EnhancedVolcano” [13].

Functional Annotation of CD276-Associated Differentially Expressed Genes in Colon adenocarcinoma

Gene set enrichment analysis (GSEA) was applied to analyze cancer-related signaling pathways regulated by CD276 in the TCGA-COAD dataset. Besides, the reference gene sets in this study was the c2.cp.kegg.v7.5.1.symbols.gmt, and the NES (normalized enrichment score) was calculated. If the normal p value and FDR (false discovery rate) q value were both less than 0.05, the gene set was significantly enriched.

Correlation Analyses for CD276 Expression and Clinicopathological Characteristics of Colon adenocarcinoma

Differences in the clinicopathological characteristics between high- and low-CD276 expression groups were undertaken using Wilcoxon rank sum test, Pearson’s Chi-square test and Cox regression.

Clinical Significance of CD276 Expression in Colon adenocarcinoma

CD276 expression was compared between COAD tumors and pericarcinous tissues by receiver operating characteristic (ROC) analysis to test the predictive value of CD276 for COAD diagnosis.Information on COAD patients’ clinical outcome was obtained from TCGA dataset, including overall survival, disease-specific survival, disease-free interval and progression-free interval. Kaplan–Meier (K-M) analysis, univariate, and multivariate Cox regression analysis were employed for prognosis analysis. The R package “rms” was used to construct nomograms and calibration plots. The above statistical analyses were all carried out by R (v4.0.5), with P-values less than 0.05 considered significant.

Investigation of CD276 Biological Function in Vitro

Human normal colonic mucosal epithelial cell line NCM-460 and cancer cell lines (including SW480, SW620, HCT116 and HT-29) were obtained from the American Type Culture Collection (ATCC). Cell lines were cultured according to the instructions. The shRNA of CD276 was synthesized by Shanghai GeneChem Co., Ltd. Colon cell lines (including SW480 and HCT116) were transduced with sh-CD276 lentiviral vectors and selected with puromycin for 10 days. The detailed procedures were referred to operation manual provided by Shanghai GeneChem Co., Ltd. QT-PCR and Western blotting were applied to verify the knockout efficiency of the lentivirus. The cell viability was detected by using CCK-8 (Cell Counting Kit-8). The cell migration and invasion ability were evaluated by Scratch assay experiment and transwell migration assay. All experiments above were carried out in three replications and repeated three times.

GEPIA database analysis

GEPIA (http://gepia.cancer-pku.cn/) is a web-based tool that provides gene expression profiling and interactive analyses based on TCGA data [14]. GEPIA was employed to conduct expression analysis for CD276 in 11 various cancer types. P value <0.05 was considered as statistically significant. In addition, expression correlation of CD276 with immune checkpoints in COAD was also evaluated using GEPIA database. |R| >0.1 and p value <0.05 were set as selection criteria for identifying as statistically significant.

TIMER database analysis

Estimates of immune cell infiltration was performed using the TIMER (https://cistrome.shinyapps.io/timer/) web server [15]. We estimated the correlations between CD276 expression and tumor immune-infiltrating cells in COAD from TIMER. Statistic p value <0.05 was considered as statistically significant.

Statistical Analysis

Differences in the expression of the CD276 in the public data sets were compared by one-way ANOVA, and differences in clinical information and immune checkpoint inhibitor response between the two different subgroups were compared by the chisquared test. Differences in overall survival (OS) and progression free interval (PFI) between the two subgroups were compared by the Kaplan–Meier method and log-rank rest. The hazard ratios (HRs) were calculated by univariate Cox regression and multiple Cox regression analysis. GraphPad Prism 8 (GraphPad Software) was used for all the image analysis of this study. All p-values were two-sided, with p-value of less than 0.05 was considered to be statistically significant. R 4.0.4 software was used for some data processing, statistical analysis, and mapping.

CD276 was Upregulated in COAD Tissues Compared with Normal Colon Tissues

Based on TCGA database, we determined the expression of CD276 mRNA in different cancers. As shown in Figure 1A, among 33 cancer types, the CD276 was significantly highly expressed in 15 cancers, especially in tumors located in gastrointestinal, urogenital tracts and system of department of gynaecology. More specifically, CD276 expression was much higher in COAD tumors than in pericarcinous tissues (P < 0.001, Figure 1B). Interestingly, in none of the investigated cancer profiles was CD276 expression significantly decreased.

According to the median expression of CD276 in COAD, 494 patients were stratified into the high and low-CD276 expression groups. We next compared mRNA expression between the two groups. Finally, 3833 mRNAs (2093 upregulated and 1740 downregulated, Figure 1C) was recognized as DEGs (absolute value of fold change >1.5, P < 0.05) in the high-CD276 group. Representative DEGs were also illustrated by heatmaps. (Figure 1D).

Functional Annotation of CD276-Associated Differentially Expressed Genes in Colon adenocarcinoma

In order to evaluate the function of CD276-associated DEGs in COAD patients, the software “Metascape” was applied. As presented in Figures 2A-C, we found that several COAD-related pathways were enriched, including extracellular matrix disassembly (GO: 0022617, P < 0.001, enrichment factor = 0.243), epithelial to mesenchymal transition in colorectal cancer (WP4239, P < 0.001, enrichment factor = 0.873), and Wnt signaling pathway (KEGG hsa04310, P = 0.004, enrichment factor = 0.884). Moreover, the GSEA showed CD276-associated DEGs significantly enriched in cancer pathways (Figure 2D), especially the cell cycle-related Hedgehog signaling pathway (NES = 2.104, adjusted P = 0.000, FDR = 0.0001), MAPK signaling pathway(NES = 1.860, adjusted P = 0.000, FDR = 0.0027), Notch signaling pathway(NES = 1.843, adjusted P = 0.000, FDR = 0.0034), VEGF signaling pathway (NES = 1.744, adjusted P = 0.002, FDR = 0.0080), JAK_STAT signaling pathway(NES = 1.739, adjusted P = 0.000, FDR = 0.0081) and WNT signaling pathway (NES = 1.526, adjusted P = 0.003, FDR = 0.0441) (Figure 2E-J). More interestingly, CD276-associated DEGs were associated with the activity of the growth factor and cell adhesion (Figures 2K, L), which are usually involved in oncogenesis.

Association of CD276 Expression and Clinicopathological Characteristics in Colon adenocarcinoma Patients

We investigated the clinicopathological characteristics of COAD patients with differential CD276 expression, as shown in Table 1. Compared with the low-CD276 group, patients in the high-CD276 group manifested a higher proportion of microsatellite instability high (MSI-H), death, and progression. However, there was no remarkable distinction with regards to age, gender, TNM stages, cea level, residual_tumor, vasucular invasion, lymph_node invasion, perineural invasion, radiation therapy, mismatch repair protein (MMR) status, MSI status, Kras, neoadjuvant_treatment, or recurrence between high-CD276 and low-CD276 groups.

Further, we analyzed CD276 expression in patients with different clinicopathological characteristics. CD276 expression was significantly reduced in patients of non MSI-H (Figure 3A) and disease specific alive (Figure 3B). As shown in Table 1, patients with progression status (Figure 3C) and alive (Figure 3D) both shared similar CD276 expression levels.

Predictive Value of CD276 for Colon adenocarcinoma Patients Diagnosis and Prognosis

To investigate the influence of CD276 on clinical outcomes, a ROC curve was constructed to assess its value on discriminating COAD diagnosis. As the area under the curve (AUC) was 0.912, CD276 showed significant high sensitivity and specificity for COAD diagnosis (Figure 4A). Next, K-M analyses were applied to verify the prediction of CD276 on clinical outcomes. As illustrated in Figures 4B–D, patients in high CD276 expression group had worse overall survival [hazard ratio (HR): 1.848, P = 2.64E−03)], disease-specific survival [hazard ratio (HR): 2.406, P = 5.35E−04)], shorter time to progression [hazard ratio (HR): 1.772, P = 2.04E−03)] than in low-CD276 group.

Moreover, we performed the Cox regression analysis to further evaluate the predictive value of CD276 on clinical outcomes. As shown in Table 2, in the univariate model, Tstage, N stage, M stage, TNM stage, CEA level, Residual_tumor, Vasucular invasion, Lymph_node invasion, MMR status and CD276 expression were significantly related to overall survival (OS), disease-specific survival (DSS) and progression-free interval (PFS) in COAD (all P < 0.05). The following multivariate analyses did not provide any significant predictive ability for OS, DSS and PFS (all P < 0.05) after adjusting other prognostic (Tables 2).Conversely, residual_tumor also showed predictive advantages for OS and DSS in multivariate Cox regression analyses. Susequently, additional K-M analyses for overall survival and disease-specific survival in the subgroups based on residual_tumor were also conducted given the heterogeneity between the 2 groups (Figure 5A-F). For R2 group, all the results demonstrated significantly better clinical outcomes in the low-CD276 expression groups.

Validation of the Prognostic Value of CD276 in COAD Based on Nomograms

To further validate the prognostic value of CD276, nomograms were constructed based on CD276 mRNA expression and residual_tumor, which were identified as independent indicators in terms of OS and DSS via multivariate analyses. A calibration curve was drawn to test the efficiency of the nomogram. residual_tumor, as well as CD276, were included in the nomogram to predict overall survival, which had a C-index of 0.543 (Figure 6A). Residual_tumor and CD276 were also evaluated in a nomogram as predictors of disease specific survival, with a C-index of 0.556 (Figure 6C). A predictive nomogram for progression-free interval was constructed using only CD276 with a C-index of 0.570 (Figure 6E). The calibration curves all presented desirable prediction of the three nomograms for the 1-, 3-, and 5-year clinical outcomes, with the exception of the 1-year prediction for clinical outcomes, which was accurately estimated (Figures 6B, D, F).

CD276 positively correlates with immune cell infiltration in COAD

CD276 plays a critical role in the regulation of immune responses from CD28:B7 immunoglobulin family. As shown in Figure 7A, significant change of B cells, CD8+ T cells, Neutrophil, and Dendritic cells were detected under various copy numbers of CD276 in COAD. Correlation analysis could provide key clues for the studies of CD276 function and mechanism. Whereas, our analysis of human COAD has suggested a correlation between CD276 and immune cell infiltrates. As presented in Figures 7B–7G, TIMER revealed a significant correlation between CD276 and immune cells infiltrations, including B cell, CD8+ T cell, CD4+ T cell, macrophage, neutrophil, and dendritic cell in COAD.

Expression correlation of CD276 and biomarkers of immune cells in COAD

To determine if CD276 in plays an important role in tumor immune, we detected the correlation between CD276 and immune cell biomarkers in COAD by GEPIA database. As listed in Table 3, CD276 correlated significantly with the measured biomarkers of immune cells , including B cell’s biomarkers (CD79A), CD8+ T cell’s biomarkers (CD8A and CD8B), CD4+ T cell’s biomarker (CD4), M1 macrophage’s biomarkers ( IRF5 and PTGS2), M2 macrophage’s biomarkers (CD163, VSIG4, and MS4A4A), neutrophil’s biomarkers (ITGAM and CCR7), and dendritic cell’s biomarkers (HLA-DPB1, HLA-DQB1, HLA-DRA, HLA-DPA1, CD1C, NRP1, and ITGAX) in COAD. These findings partially support that CD276 is positively linked to immune cell infiltration.

Relationship between CD276 and immune checkpoints in COAD

PD1/PD-L1 and CTLA-4 are important immune checkpoints that are responsible for tumor immune escape. Considering CD276 has the potential oncogenic role in COAD, we further explore the relationship of CD276 with PD1, PD-L1, or CTLA-4. As suggested in Figures 8A–8C, CD276 expression was significantly positively correlated with PD1, PD-L1, and CTLA-4 in COAD, which was adjusted by purity. Similar to TIMER data analysis, we also found that there was significant positive correlation of CD276 with PD1, PD-L1, or CTLA-4 in COAD (Figures 8D–8F). These results demonstrate that tumor immune escape might be involved in CD276-mediated carcinogenesis of COAD.

Knockdown of CD276 Expression Reduced Cell Proliferation, Migration, and Invasion of COAD Cells

Firstly, we knocked down the CD276 protein level by shCD276 in both SW480 and HCT116 cell lines, which obtained the higher level of CD276 as compared to control NCM-460 and confirmed the knockdown by western blot (WB). Compared with SW480/ sh-NC cells, the cell viability of SW480/sh-CD276 cells at 48 and 72 h was significantly decreased. The same trend was observed in HCT116 cell (Figure 9A). Cell migration and cell invasion assay were also performed to compare the cell adhesion and migration; Wound healing assay showed that knockdown of CD276 suppressed significantly the migration of SW480 and HCT116 cells. (Figure 9B). Furthermore, we also evaluated the impaction of cell invasion in sh-NC and sh-CD276 subgroups.The results showed that knockdown of CD276 can decrease about 10%–20% cell invasion (Figure 9C); therefore, the target therapy to CD276 might be a potential new strategy for COAD patients.

CD276 Expression Elevated in Tumor Tissues as Compared With Normal Tissues

In order to further explore our findings from TCGA, we examined the expression of CD276 by immunohistochemistry (IHC) in 26 paired COAD tumors and para-tumor tissue samples. The results showed that CD276 staining in COAD tumor tissues (TTs) was mainly located in the cell membrane, and the corresponding para-tumor tissues showed extremely weak staining. In contrast, CD276 expression was relatively high in COAD TTs; among the COAD TT samples, 3 had negative staining (15.1%, Figure 10A), 6 had weak positive staining (23.9%, Figure 10B), 10 had moderate positive staining(42.8%, Figure 10C), and 7 had strong positive staining (18.2%, Figure 10D).Overall, the B7-H3 expression levels of nontumor tissue samples were significantly lower than those of their paired colon tumor tissue samples (p < 0.01, Figure 10E-F )

In the present study, we investigated expression profiles, the association of clinicopathological parameters, and the clinical significance of an important immune checkpoint factor, CD276, by analyzing the COAD dataset from TCGA. In colon cancer, we first observed prominent increased CD276 expression compared with compared with that in normal tissue. In our study, we further found that CD276 expression was upregulated in the COAD tissues at the protein levels from patients’ samples, and IHC staining revealed CD276 protein expression in most of the COAD samples. Differentially expressed genes (DEGs) regulated by higher CD276 levels were specifically enriched in cell adhesion- and growth factor-associated pathways, as well as signal pathways in cancer. We also revealed a novel association of CD276 expression with MSI status. Our reports showed the prognostic value of CD276 for overall survival, disease specific survival, and progression-free interval in COAD patients. Finally, we also conducted the knockdown studies of CD276 in two COAD cell lines, SW480 and HCT116. Results showed in colon tumor, CD276 knockdown not only inhibit tumor cell migration/invasion, but also cell proliferation. Steven S et al [16] found that tumor cells and tumor vessels frenquently expressed high levels of CD276. Nevertheless, our results are in line with the findings of Steven S et al. who suggested that high CD276 levels correlate with poor prognosis, increased pathological grade and metastasis, drug resistence, and short survival. Zhan et al [17]indicated that CD276 was aberrantly expressed not only on tumor cells but also on adjacent stromal cells of gastric adenocarcinomas (GACs); inhibition of CD276 via small interfering RNA leads to reducing the potency of invasion and migration in cancer-associated fibroblasts (CAFs) and suppression of cytokine secretion.

Further, we attempted to delineate the biological significance and mechanism through which CD276 is involved in colon cancer. Studies have found that the downregulation of CD276 lead to a reduced cell adhesion to fibronectin and the capacity of cells to migrate and invade the matrigel in vitro. However, since the matrigel invasion capacity of these cells was significantly decreased by CD276 silencing, the symptom-free survival time of mice and rats was prolonged after injection with CD276-knockout cells [18]. Study reported that CD276 stimulated CAFs secrete hepatocyte growth factor (HGF), which mediate tumor cell proliferation, invasion, and migration[19].In our study, based on functional annotation of CD276-associated DEGs, the activity of the growth factor and cell adhesion were closely associated with CD276 expression. Moreover, CD276 was associated with epithelial-mesenchymal transition (EMT) in COAD. Based on additional GSEA, several cancer-related pathways were enriched in the high-CD276 group. The above data all provided evidence that CD276 functions as a critical cancerogenic factor in COAD. CD276 activates ROS/Src/c-Cbl signal pathway in multiple myeloma by integrating redox regulation and persistently activates JAK2/STAT3 at the level of STAT3 suppressor degradation[20]. CD276 promotes metastasis of breast cancer to lung via activation of Raf/MEK/ERK/MAPK cascade[21]. CD276 induces VEGFA expression and promotes angiogenesis in colorectal cancer through activation of the NF-kappaB pathway [22].Therefore, CD276 expression might be of great importance in COAD tumorigenesis by affecting function of cancer-associated signal pathways.

However, the clinical significance of CD276 in COAD is another issue of great interest. The high diagnostic value of CD276 was demonstrated by a ROC curve with an AUC of 0.912 for COAD. Because the existing serum biomarkers lacked high sensitivity and specificity for the surveillance of metastatic or recurrent disease in COAD, it may be significant to determine the cut-off value of CD276 and use as a biomarker for early alarming tumor recurrence after surgery [23–25]. In addition, the clinical implication of CD276 upregulation in COAD was evaluated. Our study demonstrated that high CD276 expression was strongly associated with MSI status, death, disease specific survival, and progression free interval, highlighting that CD276 might lead to the progression and poor prognosis of COAD. Meanwhile, KM analyses showed that COAD patients with high CD276 expression had relatively poor OS, DSS and PFI (all P < 0.05). So far, significant heterogeneity in prognostic factors has been considered among individuals with COAD and the existing clinical prognostic predictors are not precise enough [26, 27], it is extremely necessary to establish a genomic–clinicopathologic nomogram to predict the long-term outcomes of patients with advanced colorectal cancer. We created nomograms models with the independent prognostic parameters (including CD276 expression and residual_tumor) for OS and DSS of patients.The results showed that the genomic–clinicopathologic nomograms represented more precise prognostic models compared with the other clinical predictors alone. Our analysis have important implications that CD276 will be clinically useful for monitoring postoperative recurrence and guiding treatment decision making.

A multitude of studies have confirmed that the combination of immunotherapy with chemotherapy and radiotherapy could recruti tumor infiltrating immune cell into the TME, protect the body from tumor reattack and ultimately prolong the survival of patients [28–30]. Our work confirmed that CD276 was positively correlated with various immune cells, including B cell, CD8 + T cell, CD4 + T cell, macrophage, neutrophil, and dendritic cell in COAD. Moreover, CD276 was also markedly positively associated with biomarkers of these infiltrated immune cells. These findings indicated that tumor immune infiltration might partially account for CD276-mediated oncogenic roles in COAD. Also, the immune cells infiltration and the expression of immune checkpoints in tumor microenvironment correlate with response to immunotherapy [31, 32]. Thus, we also assessed the relationship between CD276 and immune checkpoints. The results demonstrated that high expression of CD276 was strongly linked to PD1, PD-L1, or CTLA-4 in COAD.

It is well-known that the molecular expression of immune chenkpoint such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, IDO1, and IL-10 could impact the infiltration of immune cells in the tumor microenvironment, thereby regulating the immune state of the tumor microenvironment [33–35].several promising results have been achieved with checkpoint inhibitors such as nivolumab, pembrolizumab and ipilimumab, resulting in U.S. FDA’s approval for the treatment of patients with MSI-H advanced COAD [36–38]. Our results revealed that abnormal CD276 expression in COADs was closely correlated with distinct patterns of MSI status. These findings provide evidence of a potential mechanistic link between MSI, the immunocyte infiltration, immunotherapy, and increased CD276 expression, and the combination therapy of anti–CD276 monoclonal antibodies with other immune checkpoint inhibitors may yield superior curative effects for COAD patients in future.

Although we uncovered a potential mechanism for CD276 activity in COAD tumorigenesis and its predictive value in COAD clinical outcomes, our study still presented several limitations.First, because of the incomplete information about treatments and corresponding responses, we could not evaluate a specific role for CD276 in COAD treatment. Second, we mainly focused on the RNA sequencing data from the TCGA database and information on the relative protein levels or downstream pathways of CD276 was lacking. Thus, further studies will focus on the direct mechanism of CD276 as an oncogenic factor leading to the development and progression of COAD.

In this study, we found a certain association between CD276 and colon adenocarcinoma, suggesting that CD276 may play an important role in colon cancer pathogenesis. Morover, the overexpression of CD276 is also highly correlated with some biomarker genes in immune infiltrating cells (such as CD79A, CD8, IRF5, CD163, and HLA), which may also become a new research target in the future. However, our results were obtained through various bioinformatics analyses and simple tests in vitro, which can only provide some preliminary theoretical basis. Future researches based on large samples need to be carried out to clarify the biological functions of CD276 and its impact on immune response.

B7-H3: B7 homolog 3

CAFs: cancer-associated fibroblasts

COAD: colon adenocarcinoma

CTLA-4: cytotoxic T-lymphocyte-associated protein 4

DEGs: differentially expressed genes

DSS: disease-specific survival

EMT: epithelial-mesenchymal transition

FDR: false discovery rate

FPKM: fragments per kilobase per million

GACs: gastric adenocarcinomas

GSEA: Gene Set Enrichment Analysis

HGF: hepatocyte growth factor

HRs: hazard ratios

ICI: immune checkpoint inhibitors

IHC: immunohistochemistry

K-M: Kaplan–Meier

MSI: microsatellite instability

NES: normalized enrichment score

OS: overall survival

PD‐1: programmed cell death protein 1

PD‐L1: programmed death ligand 1

PFI: progress-free interval

ROC: receiver operating characteristic

TCGA: The Cancer Genome Atlas

TTs: tumor tissues

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The data used to support the findings of this study are available from the corresponding author upon request.

Competing interests

The authors declare that they have no competing interests

Funding

This study was supported by the Science Innovation Fund of People's Hospital of Suzhou New District (SGY2020C03), and the Higher Education Innovation Fund of Gansu Province (2021B-001).

Authors' contributions

R C and C C were responsible for the conception and design.QL G and DJ W were responsible for the Administrative support.QL G and DJ W were responsible for the provision of study materials or patients. SC M and DJ W were responsible for the collection and assembly of data. R C and C C were responsible for the data analysis and interpretation.

All authors were responsible for manuscript writing. All authors were responsible for the final approval of manuscript.All authors have read and agreed to the published version of the manuscript. Rui Chen and Chen Chai contributed equally to this work.

Acknowledgments

We are very thankful to Dr. Fei Su for design in whole paper and Dr Tao Zhang advice in statistical analysis.

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Table 1

Clinicopathological characteristics of COAD patients with differential CD276 expression.

		CD276 Expression
Characteristic	Total (N=433)	High (N=130)	Low (N=303)	P-value*
Age (year)
< 65	166 (38.3%)	48 (36.9%)	118 (38.9%)	0.924
≧ 65	267 (61.7%)	82 (63.1%)	185 (61.1%)
Gender
Male	232 (53.6%)	66 (50.8%)	166 (54.8%)	0.745
Female	201 (46.4%)	64 (49.2%)	137 (45.2%)
T stage
T3-T4	319 (73.7%)	102 (78.5%)	217 (71.6%)	0.622
Tis-T2	87 (20.1%)	20 (15.4%)	67 (22.1%)
Unknown	27 (6.2%)	8 (6.2%)	19 (6.3%)
N stage
N0	254 (58.7%)	73 (56.2%)	181 (59.7%)	0.82
N1-N2	126 (29.1%)	43 (33.1%)	83 (27.4%)
Unknown	53 (12.2%)	14 (10.8%)	39 (12.9%)
M stage
M0	319 (73.7%)	100 (76.9%)	219 (72.3%)	0.565
M1	48 (11.1%)	16 (12.3%)	32 (10.6%)
Unknown	66 (15.2%)	14 (10.8%)	52 (17.2%)
TNM stage
I	73 (16.9%)	18 (13.8%)	55 (18.2%)	0.983
II	167 (38.6%)	54 (41.5%)	113 (37.3%)
III	123 (28.4%)	38 (29.2%)	85 (28.1%)
IV	60 (13.9%)	18 (13.8%)	42 (13.9%)
Unknown	10 (2.3%)	2 (1.5%)	8 (2.6%)
CEA level
abnormal	98 (22.6%)	27 (20.8%)	71 (23.4%)	0.985
normal	178 (41.1%)	55 (42.3%)	123 (40.6%)
Unknown	157 (36.3%)	48 (36.9%)	109 (36.0%)
Residual_tumor
R0	313 (72.3%)	100 (76.9%)	213 (70.3%)	0.668
R1-R2	25 (5.8%)	5 (3.8%)	20 (6.6%)
Unknown	95 (21.9%)	25 (19.2%)	70 (23.1%)
Vasucular invasion
YES	90 (20.8%)	31 (23.8%)	59 (19.5%)	0.901
NO	287 (66.3%)	83 (63.8%)	204 (67.3%)
Unknown	56 (12.9%)	16 (12.3%)	40 (13.2%)
Lymph_node invasion
YES	151 (34.9%)	53 (40.8%)	98 (32.3%)	0.552
NO	241 (55.7%)	67 (51.5%)	174 (57.4%)
Unknown	41 (9.5%)	10 (7.7%)	31 (10.2%)
Perineural invasion
YES	45 (10.4%)	17 (13.1%)	28 (9.2%)	0.831
NO	131 (30.3%)	39 (30.0%)	92 (30.4%)
Unknown	257 (59.4%)	74 (56.9%)	183 (60.4%)
Radiation therapy
YES	9 (2.1%)	1 (0.8%)	8 (2.6%)	0.612
NO	354 (81.8%)	104 (80.0%)	250 (82.5%)
Unknown	70 (16.2%)	25 (19.2%)	45 (14.9%)
MMR status
dMMR	55 (12.7%)	20 (15.4%)	35 (11.6%)	0.822
pMMR	267 (61.7%)	80 (61.5%)	187 (61.7%)
Unknown	111 (25.6%)	30 (23.1%)	81 (26.7%)
MSI status
non MSI-H	345 (79.7%)	95 (73.1%)	250 (82.5%)	0.0142*
MSI-H	75 (17.3%)	34 (26.2%)	41 (13.5%)
Unknown	13 (3.0%)	1 (0.8%)	12 (4.0%)
Kras
Mut	22 (5.1%)	7 (5.4%)	15 (5.0%)	0.904
Wild	24 (5.5%)	5 (3.8%)	19 (6.3%)
Unknown	387 (89.4%)	118 (90.8%)	269 (88.8%)
Neoadjuvant_treatment
Yes	3 (0.7%)	2 (1.5%)	1 (0.3%)	0.381
No	430 (99.3%)	128 (98.5%)	302 (99.7%)
Living status
Alive	337 (77.8%)	90 (69.2%)	247 (81.5%)	0.0187*
Dead	96 (22.2%)	40 (30.8%)	56 (18.5%)
Disease specific living status
Alive	358 (82.7%)	97 (74.6%)	261 (86.1%)	0.0403*
Dead	59 (13.6%)	28 (21.5%)	31 (10.2%)
Unknown	16 (3.7%)	5 (3.8%)	11 (3.6%)
Recurrence status
NO	163 (37.6%)	40 (30.8%)	123 (40.6%)	0.387
YES	23 (5.3%)	9 (6.9%)	14 (4.6%)
Unknown	247 (57.0%)	81 (62.3%)	166 (54.8%)
Progression status
NO	317 (73.2%)	84 (64.6%)	233 (76.9%)	0.0302*
YES	116 (26.8%)	46 (35.4%)	70 (23.1%)

*Statistically significant p values are given in bold, P < 0.05

COAD, Colon adenocarcinoma; MMR, mismatch repair; dMMR, MMR absent; Pmmr, MMR proficient; MSI, microsatellite instability; MSI-H, MSI-high.

Table 2 is available in the Supplementary Files section

Table 3

Correlation analysis between CD276 and biomarkers of immune cells in COAD determined by GEPIA database.

Immune cell	Biomarker	R value	p value
B cell	CD19	0.079	0.11
B cell	CD79A	0.13a	0.032∗ a
CD8+ T cell	CD8A	0.13a	0.027∗ a
CD8+ T cell	CD8B	0.13a	0.038∗ a
CD4+ T cell	CD4	0.51a	0∗∗∗ a
M1 macrophage	NOS2	-0.015	0.8
	IRF5	0.18a	0.0035∗∗ a
	PTGS2	0.29a	6.8e−07∗∗∗ a
M2 macrophage	CD163	0.56a	0∗∗∗ a
	VSIG4	0.53a	0∗∗∗ a
	MS4A4A	0.55a	0∗∗∗ a
Neutrophil	CEACAM8	0.029	0.63
	ITGAM	0.61a	0∗∗∗ a
	CCR7	0.2a	0.00063∗∗∗ a
Dendritic cell	HLA-DPB1	0.33a	1.4e−08∗∗∗ a
	HLA-DQB1	0.22a	0.00025∗∗∗ a
	HLA-DRA	0.34a	1e−08∗∗∗ a
	HLA-DPA1	0.27a	6.6e−06∗∗∗ a
	CD1C	0.25a	2.2e−05∗∗∗ a
	NRP1	0.7a	0∗∗∗ a
	ITGAX	0.56a	0∗∗∗ a

a These results are statistically significant.

∗p value < 0.05; ∗∗p value < 0.01; ∗∗∗p value < 0.001.

No competing interests reported.

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The bioinformatics and experimental analysis of CD276 for prognosis and immune infiltrates in colon adenocarcinoma

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Abstract

Figures

Introduction

Materials And Methods

Data Processing and Ethics Statement

Differentially Expressed Genes in Colon adenocarcinoma

Functional Annotation of CD276-Associated Differentially Expressed Genes in Colon adenocarcinoma

Correlation Analyses for CD276 Expression and Clinicopathological Characteristics of Colon adenocarcinoma

Clinical Significance of CD276 Expression in Colon adenocarcinoma

Investigation of CD276 Biological Function in Vitro

GEPIA database analysis

TIMER database analysis

Statistical Analysis

Results

CD276 was Upregulated in COAD Tissues Compared with Normal Colon Tissues

Functional Annotation of CD276-Associated Differentially Expressed Genes in Colon adenocarcinoma

Association of CD276 Expression and Clinicopathological Characteristics in Colon adenocarcinoma Patients

Predictive Value of CD276 for Colon adenocarcinoma Patients Diagnosis and Prognosis

Validation of the Prognostic Value of CD276 in COAD Based on Nomograms

CD276 positively correlates with immune cell infiltration in COAD

Expression correlation of CD276 and biomarkers of immune cells in COAD

Relationship between CD276 and immune checkpoints in COAD

Knockdown of CD276 Expression Reduced Cell Proliferation, Migration, and Invasion of COAD Cells

CD276 Expression Elevated in Tumor Tissues as Compared With Normal Tissues

Discussion

Conclusion

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors' contributions

Acknowledgments

References

Tables

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Supplementary Files

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