Introduction

Esophageal cancer (EC) is the ninth common cancer and the sixth leading cause of cancer-related mortality.1 2 In 2020, EC was responsible for the deaths of more than 500 000 individuals worldwide.1 3 Traditionally, EC has been classified into esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) based on pathologic diagnosis. ESCC represents the predominant subtype of EC, comprising 80% of all EC cases, and can arise anywhere along the esophagus.4 The progression of ESCC is associated with various risk factors, including tobacco, alcohol, heat injuries, and micronutrient deficiencies.2 Nowadays, the therapeutic approaches for ESCC predominantly encompass surgical excision, supplemented with radiotherapy and chemotherapy. However, the prognosis for patients remains dismal, with low 5-year survival rate, frequent local recurrence, and rapid lymph node metastasis.5 6 Therefore, it is an urgent need to find a new therapeutic target and enhance the treatment efficacy for ESCC.

In recent years, immunotherapy has been employed in the treatment of various tumors, including melanoma, lung cancer, head and neck squamous cell carcinoma, gastric cancer, ovarian cancer, and ESCC.7–9 Immune checkpoint inhibitors (ICIs), such as anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) and anti-programmed cell death protein 1 (anti-PD-1), have been considered as promising therapeutic targets in cancer treatment.10 In several clinical studies focused on immunotherapy, ICIs have been recognized for enhancing the survival rates of patients afflicted with ESCC.11 12 However, multiple clinical trials demonstrated that many patients with ESCC experienced no therapeutic advantages from immunotherapy,13–15 and only 10%–20% patients with ESCC exhibited a response to ICI monotherapy.16 Therefore, a profound exploration into the mechanisms of immunotherapy resistance and the identification of potential biomarkers for those patients with ESCC are imperative. B7-H3 (also known as CD276) is an immune checkpoint molecule prominently expressed in various tumor tissues, including triple-negative breast cancer, non-small cell lung cancer, and head and neck squamous cell cancers but rarely expressed in normal tissues.17–19 The mechanisms through which CD276 mediates tumor immune evasion are multifaceted, including the reduction of CD8+ T cells and natural killer (NK) cells and suppression of T helper type 1-mediated immune responses.20 21 However, the mechanism by which CD276 modulates immune escape in ESCC remains elusive, lacking in-depth mechanistic study.

In this study, we identified that CD276 was highly expressed in ESCC and promotes tumorigenesis. Mechanistically, overexpression of CD276 promoted tumorigenesis through inducing the formation of neutrophil extracellular trap networks (NETs) and reducing the number of NK cells. Importantly, inhibition of CD276 markedly reduced the expression of CXCL1, diminished the formation of NETs through the CXCL1–CXCR2 axis, and increased NK cells.

ResultsOverexpression of CD276 is associated with worse survival of patients with ESCC

We performed immunohistochemical (IHC) staining to detect the CD276 expression in ESCC via two independent cohorts. The Sun Yat-sen University Cancer Center (SYSUCC) cohort, including 297 patients, was evaluated alongside The First Affiliated Hospital Sun Yat-sen University (FAH-SYSU) cohort that comprised 143 patients. Clinical characteristics of the patients from both cohorts are shown in online supplemental tables S1 and S2. IHC staining results showed that CD276 was predominantly expressed on the membrane of tumor cells (figure 1A,G). In the SYSUCC cohort, CD276 expression in para-tumor tissues was significantly lower than that in ESCC tissues (figure 1B). In both cohorts, the positive ratio of CD276 expression gradually increased from grade I and II to grade III ESCC (figure 1C,H), and the expression of CD276 was significantly elevated in patients with ESCC with late stages (stage3+4) compared with whom with early stages (stage1+2) (figure 1D,I). These findings indicated that CD276 protein was stepwisely upregulated during the malignant transformation of ESCC. Of note, CD276 expression in the recurrent group was significantly higher than the primary group in the SYSUCC cohort (figure 1E). At the same time, we found that patients with better first-line therapy outcomes displayed lower CD276 expression in FAH-SYSU cohort (figure 1J). In addition, the impact of CD276 on the survival of patients with ESCC was also analyzed. Patients with ESCC with high CD276 expression have worse overall survival (OS) than those with low CD276 expression (figure 1F,K). It is noteworthy that among patients with grade II and III ESCC, those with high CD276 expression exhibit worse OS, whereas there is a comparable OS among patients with grade I ESCC (online supplemental figure S1A–F). The median progression-free survival at first-line therapy was also shorter in patients with high versus low CD276 expression in FAH-SYSU cohort (figure 1L). Taken together, CD276 was highly expressed in ESCC and associated with worse survival.

Figure 1

CD276 upregulated expression is associated with clinical parameters and overall survival in patients with ESCC. (A) Representative IHC staining images for CD276 expression in para-tumor and ESCC chips in the SYSUCC patients’ cohort. Scale bar, 100 µm. (B) Statistical analysis of CD276 IHC scores from para-tumor (n=53) and ESCC tissues (n=297) in the SYSUCC patients’ cohort. (C) CD276 IHC score representation in the different grades of ESCC in the SYSUCC patients’ cohort. (D) CD276 IHC score representation in the different stages of ESCC in the SYSUCC patients’ cohort. (E) CD276 IHC score representation in the recurrence and non-recurrence tumors of ESCC in the SYSUCC patients’ cohort. (F) Kaplan-Meier survival curves stratified by CD276 IHC score based on the median in ESCC tissues from the SYSUCC patients’ cohort. (G) Representative IHC staining images for high (upper) and low (lower) CD276 expression of ESCC tissues in the FAH-SYSU patients’ cohort. Scale bar, 100 µm. (H, I) CD276 IHC score representation in the different grades (H) and stages (I) of ESCC in the FAH-SYSU patients’ cohort. (J) CD276 IHC score representation in the complete remission/partial remission (PR/CR) and progressive disease/stable disease (PD/SD) group of ESCC in the FAH-SYSU patients’ cohort. (K, L) Overall survival curve (K) and Kaplan-Meier progression-free survival (L) stratified by CD276 IHC score based on the median in ESCC tissues from the FAH-SYSU patients’ cohort. ESCC, esophageal squamous cell carcinoma; FAH-SYSU, The First Affiliated Hospital Sun Yat-sen University; IHC, immunohistochemistry; SYSUCC, Sun Yat-sen University Cancer Center.

CD276 whole body knockout inhibits ESCC tumorigenesis in vivo

To explore the role of CD276 in ESCC, we generated CD276 whole body knockout (CD276-wKO) mice (figure 2A). Consistent with the previous study,21 CD276-wKO mice were born at the expected Mendelian ratios and developed normally. We then used 4-nitroquinoline 1-oxide (4NQO) to induce ESCC in CD276-wKO mice and their control littermates (CD276-CTL). Knockout of CD276 was validated by immunofluorescence staining, which showed that CD276 protein expression was detected in wild type (WT) mice but absent in CD276-wKO mice (figure 2B). In comparison to the control group, which exhibited thick and palpable protuberances in most of the cases, the esophageal tissues in CD276-wKO mice appeared less severe on gross examination (figure 2C). In addition, CD276-wKO mice showed smaller size and lower number of esophageal lesions compared with control mice (figure 2D,E). Global depletion of CD276 greatly reduced the formation of high-grade ESCC (figure 2F,G). Moreover, we found decrease of cell proliferation (Ki67) and increase of cell apoptosis (active-Caspase3) in tumor area of CD276-wKO mice compared with that of the WT mice (figure 2H–K). Murine CD276 is known as a negative regulator of T cells and NK cells.20 22 Hence, we characterized the infiltration of CD4+, CD8+ T cells, and NK cells in ESCC after CD276 depletion. WT and CD276-wKO mice have comparable numbers of intratumoral CD4+ T cells (figure 2L,M). However, the intratumoral infiltration of CD8+ T cells increased significantly in CD276-wKO compared with WT mice (figure 2N,O). As shown in figure 2P,Q, the tumor-infiltrated NK cells greatly expanded in CD276-wKO compared with WT mice (5.5%±1.4 in CD276-wKO mice vs 1.9%±0.5 in WT mice). Taken together, these findings suggest that whole body knockout CD276 inhibits the ESCC development and progression in vivo.

Figure 2

CD276 whole body knockout and blockade inhibits tumorigenesis. (A) Construction of CD276-whole body knockout (wKO) mice. (B) Experimental strategy for the 4NQO-induced ESCC mice model and immunofluorescence images of CD276 in wild-type (WT) and wKO group. Scale bar, 50 µm. (C) Representative esophagi image from 4NQO-induced WT mice and mice with systemic knockdown of CD276 (wKO). The control (CTL) represents the WT group. (D, E) Quantification of lesion area (D) and lesion number (E) of the WT and wKO group. Data are presented as mean±SD (n=8). (F) Quantification of ESCC tumor grade in the WT and wKO group. (G) Representative images of H&E staining in the WT and wKO group. Scale bar, 100 µm. (H–K) Representative images of Ki67 (H) and Caspase3 (J) IHC staining and quantification of percentage of Ki67+ (I) and Caspase3+ (K) cells in WT and wKO group. Scale bar, 50 µm. (L–Q) Representative images of CD4 (L), CD8 (N), and NCR1 (P) IHC staining and quantification of percentage of CD4+ (M), CD8+ (O), and NCR1+ (Q) cells in WT and wKO group. Scale bar, 50 µm. ESCC, esophageal squamous cell carcinoma; IHC, immunohistochemistry; 4NQO, 4-nitroquinoline 1-oxide.

Epithelial conditional knockout of CD276 suppresses ESCC progression in vivo

CD276 is expressed in both tumor and immune cells.22 We wondered whether the tumorous CD276 is the major driver for ESCC tumorigenesis. To test that, we generated CD276flox mice and crossed them with K14CreER mice to obtain K14CreER; CD276flox/flox mice (CD276-cKO). We then used 4NQO to induce ESCC in cKO and their control littermates (CD276-CTL). We found epithelial depletion of CD276 led to a significant decrease in lesion size and lesion number (figure 3A–C). In addition, no CD276 expression was detected in the epithelial tissue of CD276-cKO mice (figure 3D). Similarly, the invasiveness and malignancy of the tumor tissues in the cKO mice were significantly mitigated compared with that in the CD276-CTL mice (figure 3E,F). Furthermore, Ki67 expression was decreased and active-Caspase3 expression was increased in cKO mice compared with the CD276-CTL mice (figure 3G–J). To investigate the alterations in the immune microenvironment following the depletion of CD276 in ESCC, we conducted flow cytometry (FCM) to examine changes in immune cell populations. Following the epithelial conditional knockout of CD276 in ESCC, the cKO mice exhibited an increase in intratumoral activated CD8+ T cells, whereas neutrophils were reduced. Moreover, the number of CD4+ regulatory T cells and macrophages in WT mice was comparable to those in cKO mice (online supplemental figure S2B–M).

Figure 3

Epithelial conditional knockdown of CD276 inhibits tumorigenesis. (A) Experimental strategy for the 4NQO-induced ESCC mice model and representative esophagi image from wild-type mice (WT) and mice with conditional knockdown of CD276 (cKO). (B, C) Quantification of lesion area (B) and lesion number (C) of the WT and cKO group. Data are presented as mean±SD (n=8). (D) Immunofluorescence images of CD276 in WT and cKO groups. Scale bar, 50 µm. (E) Representative images of H&E staining in the WT and cKO groups. Scale bar, 100 µm. (F) Quantification of ESCC tumor grade in the WT and cKO groups. (G–J) Representative images of Ki67 (G) and Caspase3 (I) IHC staining and quantification of percentage of Ki67+ (H) and Caspase3+ (J) cells in WT and cKO group. Scale bar, 100 µm. (K) Bubble diagram showing the top KEGG pathways enriched in WT group. (L–O) Representative images of PI3K (L) and AKT (N) IHC staining and quantification of percentage of PI3K+ (M) and AKT+ (O) cells in WT and cKO groups. Scale bar, 100 µm. AGE, advanced glycation end products; AKT, protein kinase B; ECM, extracellular matrix; ESCC, esophageal squamous cell carcinoma; IHC, immunohistochemistry; IL, interleukin; PI3K, phosphatidylinositol 3-kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; RAGE, receptor for advanced glycation end products; TGF, transforming growth factor; TNF, tumor necrosis factor; 4NQO, 4-nitroquinoline 1-oxide.

Next, to investigate the mechanism of CD276 on the development of ESCC, we resected both control and tumor tissues CD276-cKO for RNA sequencing (RNA-seq). The Kyoto Encyclopedia of Genes and Genomes analysis showed that the PI3K–Akt signaling pathway was significantly downregulated in cKO group compared with WT group (figure 3K). As shown in figure 3L–O, PI3K and AKT expression were reduced in cKO mice compared with the CD276-CTL mice. These results indicate that CD276 depletion suppresses the progression of murine ESCC and inhibits the effects of the PI3K–Akt signaling pathway in vivo.

CD276 deficiency inhibits tumorigenesis through sharply downregulating CXCL1 expression in vivo

To address the role of tumor cell CD276 on the immune microenvironment of ESCC, we dissected the cancer tissues from one control and two CD276-cKO samples for single-cell RNA sequencing (scRNA-seq). We were able to extract a total of 21 588 high-quality cells (6937 cells in CTL, 7971 cells in cKO-1, and 6680 cells in cKO-2) (figure 4A and online supplemental figure S3A,B). Based on canonical marker expression, all single cells were divided into seven major categories, including tumor epithelial cells (Krt5), T cells (Cd3e), MOMΦDC cells (Cd74), fibroblasts (Col1a1), neutrophils (Csf3r), NK cells (Klrd1), and endothelial cells (Vwf) (figure 4A,B). It was worth noting that the proportion of neutrophils was lower while the proportion of T cells and NK cells was higher in the CD276-cKO compared with CD276-CTL sample (figure 4C). We also compared our mouse scRNA-seq dataset with online human ESCC scRNA-seq data comprising 64 ESCC samples.23 We identified seven major cell populations based on marker expression (online supplemental figure S3C,D). Markedly, we were able to detect B cell population in the human dataset but not in the mouse dataset (figure 4A and online supplemental figure S3C). More importantly, the cell types detected both in mouse ESCC scRNA-seq and human ESCC scRNA-seq data displayed high similarity in transcriptome (online supplemental figure S3E,F).

Figure 4

Epithelial-specific CD276 knockdown yielded a significant reduction in CXCL1 expression. (A) UMAP plot displaying identified cell clusters including tumor epithelial cells, fibroblasts, endothelial cells, neutrophils, T cells, NK cells, and MOMΦDC cells. (B) UMAP plot showing the expression of cell-type marker genes. (C) Distribution of the seven major cell clusters in the WT and cKO groups. (D) Volcano plot showing the differential gene in the epithelial cells between WT and cKO groups. (E) Violin plot showing the expression of Cxcl1 between WT and cKO groups. (F, G) Representative images of CXCL1 (F) IHC staining and quantification of percentage of CXCL1+ (G) cells in WT and cKO groups. Scale bar, 100 µm. (H) Volcano plot showing the differential expressed gene from RNA-seq data between WT and cKO groups. (I) Experimental strategy for the treatment by anti-CXCL1 antibodies and representative esophagi image in mice treated with IgG control (IgG) or anti-CXCL1 antibody group. (J, K) Quantification of lesion area (J) and lesion number (K) of the IgG and anti-CXCL1 group. Data are presented as mean±SD (n=8). (L) Quantification of ESCC tumor grade in the IgG and anti-CXCL1 group. ESCC, esophageal squamous cell carcinoma; FDR, false discovery rate; MOMΦDC, monocyte-macrophage-dendritic cells; IHC, immunohistochemistry; NK, natural killer; UMAP, uniform manifold approximation and projection; WT, wild type.

To specifically study the effects of CD276 deficiency on epithelial cells, all the epithelial cells were analyzed separately (online supplemental figure S4A). As shown by the volcano plot of the differential expression genes (DEGs) in the scRNA-seq data, Cxcl1 chemokines were most significantly downregulated by CD276 knockdown (figure 4D). In addition, violin plots also showed that, compared with WT mice, the expression of Cxcl1 chemokine was significantly lower in the cKO mice (figure 4E). The IHC results further verified the differential expression of CXCL1 between cKO mice and WT mice (figure 4F,G). Interestingly, the DEGs in the RNA-seq data also demonstrated that Cxcl1 chemokines were markedly downregulated in CD276-cKO group (figure 4H).

To further analyze the effect of CXCL1 on ESCC, we first used 4NQO to induce ESCC in WT mice, and then half of the ESCC mice were treated with CXCL1 blocking antibody for 1 month, while the other half were treated with anti-IgG antibody. The tumor numbers and sizes were significantly decreased after treatment with CXCL1 blocking antibody (figure 4I–K). In addition, the proportion of high-grade ESCC was also reduced (figure 4L and online supplemental figure S4B). Noticeably, Ki67 expression was decreased and active-Caspase3 expression was increased in the mice treated with CXCL1 blocking antibody (online supplemental figure S4C–F). These results indicated that CXCL1 blocking antibody effectively inhibited tumorigenesis of ESCC in mice.

CD276 mediates tumor–neutrophil interactions and induces the formation of NETs via CXCL1/CXCR2 axis

To study whether epithelial cell-specific deficiency of CD276 affects cellular interactions in the tumor microenvironment. Cellchat R package24 was performed to analyze the interactions between tumor epithelial cells and immune cells. The numbers and the intensity of cell interactions were downregulated between tumor cells and T cells/neutrophils in the cKO mice, while compared with the WT group (figure 5A,B). In addition, among the interactions between tumor epithelial cells and various types of immune cells, the Cxcl1–Cxcr2 interactions between tumor cells and neutrophils were the highest intensity (figure 5C). It is noteworthy that the Cxcr2 was mainly expressed in neutrophils, while almost no expression observed in other types of immune cells (figure 5D). Consequently, neutrophil activity was inhibited by employing an anti-Ly6G antibody (online supplemental figure S5A). As shown in figure 5E–G, single depletion of neutrophil activity also affected the numbers and sizes of ESCC and concurrently potentiated the efficacy of the CD276 deficiency.

Figure 5

CD276 mediates tumor–neutrophil interactions via CXCL1/CXCR2. (A, B) Circle plots showing the differential interaction number (A) and strength (B) between the tumor cell and immune cell clusters based on CellChat analysis. (C) Dot plot showing the expression of receptor–ligand pairs between tumor cell and immune cell clusters in WT and cKO groups. (D) Violin plot showing the expression of Cxcr2 in different clusters. (E) Experimental strategy for the treatment by anti-Ly6G antibodies and representative esophagi image in mice treated with IgG control (IgG), cKO, anti-Ly6G, or cKO+anti-Ly6G group. (F, G) Quantification of lesion area (F) and lesion number (G) of IgG control (IgG), cKO, anti-Ly6G, or cKO+anti-Ly6G group. Data are presented as mean±SD (n=8). (H) Quantification of ESCC tumor grade in IgG control (IgG), cKO, anti-Ly6G, or cKO+anti-Ly6G group. (I, J) Quantification of percentage of Ki67+ (I) and Caspase3+ (J) cells in IgG control (IgG), cKO, anti-Ly6G, or cKO+anti-Ly6G group. (K, L) Quantification of percentage of CD8+ (K) and NCR1+ (L) cells in IgG control (IgG), cKO, anti-Ly6G, or cKO+anti-Ly6G group. (M) Quantification of percentage H3cit+ cells of ESCC in control (CTL) and cKO group. (N) Experimental strategy for the treatment by GSK484 inhibitors and representative esophagi image in mice treated with control or GSK484 group. (O, P) Quantification of lesion area (O) and lesion number (P) of the control and GSK484 group. Data are presented as mean±SD (n=8). (Q) Quantification of ESCC tumor grade in the control and GSK484 group. Scale bar, 100 µm. (R) Quantification of percentage H3cit+ cells of ESCC in the IgG and anti-CXCL1 group. ESCC, esophageal squamous cell carcinoma; NK, natural killer; 4NQO, 4-nitroquinoline 1-oxide; WT, wild type.

Subsequently, the pathologic grade of ESCC was also diminished following treatment of the anti-Ly6G antibody (figure 5H and online supplemental figure S5B). Following treatment with the anti-Ly6G antibody, the expression of Ki67 and active-Caspase3 paralleled that observed in the CD276 deficiency mice, and additionally, it augmented the efficacy of the knockout of CD276 (figure 5I,J and online supplemental figure S5C). Interestingly, the numbers of CD8+ T cells remained unaltered following treatment with the anti-Ly6G antibody; however, there was a significant augmentation in the number of NK cells after the anti-Ly6G therapy (figure 5K,L, and online supplemental figure S5D). Moreover, FCM analysis was performed to further verify the effect of the anti-Ly6G antibody on T cells and NK cells. In contrast with WT mice, the numbers of CD8+ T cells and CD4+ T cells remained unaltered in the anti-Ly6G group, while NK cells were elevated, and neutrophils were diminished (online supplemental figure S5E–L). These results indicated that the suppression of neutrophil activity mitigates the progression of ESCC by enhancing NK cell function.

Nonetheless, the mechanism by which neutrophils regulate NK cells remains elusive. Recently, CXCR2 was identified to induce the formation of NETs,25–28 which was significantly increased in the cancer progression. By means of fluorescence colocalization, the generation of NETs and neutrophils in cKO mice was significantly lower than that in WT mice (figure 5M and online supplemental figure S6A). To further elucidate the impact of NETs on ESCC, we administered a 1-month treatment with GSK484, a PAD4 inhibitor, to ESCC mice. The results showed that the tumor numbers and sizes were significantly decreased on GSK484 treatment group (figure 5N–P). Particularly, the high grade ESCC was also reduced after treatment with GSK484 inhibitor (figure 5Q and online supplemental figure S6B). As expected, Ki67 expression diminished, whereas Caspase3 expression escalated in tumor cells following treatment with GSK484 inhibitor (online supplemental figure S6C–F). Notably, the NCR1 expression in the cKO mice was significantly higher than in WT mice, and the NETs were generated around NK cells (online supplemental figure S6G–I). Furthermore, the quantity of NETs exhibited a significant reduction following treatment with anti-CXCL1 antibodies (figure 5R and online supplemental figure S6J). To sum up, these results indicate that CD276 orchestrates interactions between tumors and neutrophils, prompting the formation of NETs via CXCL1/CXCR2 axis. Furthermore, NETs could exert pro-tumor effects by suppressing the activity of NK cells.

Depleting NK cells impedes the efficacy of CD276 deficiency in suppressing the progression of ESCC

To ascertain whether the attenuation of ESCC progression by CD276 depletion is contingent on NK cell activity, NK cells were neutralized with the administration of an anti-NK1.1 antibody. The ablation of NK cells notably reduced the suppressive effect on tumor proliferation observed in CD276-deficient mice, as determined by analyses of tumor count and size (figure 6A–C). Histopathologic examination disclosed that the depletion of NK cells markedly reverted the pathologic grading of ESCC in mice lacking CD276 expression (figure 6D,E). Furthermore, both Ki67 and active-caspase3 expression were also restored to the levels in WT mice in CD276-deficient mice following the administration of the anti-NK1.1 antibody (figure 6F–H). As shown in figure 6I,J, the population of CD8+ T cells remained consistent following the administration of the anti-NK1.1 antibody in CD276-deficient mice. As expected, NK cell infiltration was elevated in CD276-deficient mice yet was eliminated with the administration of the anti-NK1.1 treatment (figure 6I–K). Importantly, we explored the association between CD276 expression and NK cells in ESCC samples. IHC assays confirmed that CD276 expression exhibited an inverse correlation with infiltration of NK cells in human ESCC samples (figure 6L). To explore the influence of CD8+ T cells in ESCC, the anti-CD8 antibodies were used to explore the functional role of CD8+ T cells in ESCC progression following CD276 depletion. After the ablation of CD8+ T cells, there was a slight augmentation in tumor numbers and size, resulting in a reduction of the suppressive effect on tumor progression in CD276-deficient mice (online supplemental figure S7A–C). These results revealed that the suppression of ESCC progression by CD276 depletion is primarily mediated through the inhibition of NK cell activity.

Figure 6

Depleting NK cells impedes the efficacy of CD276 deficiency in suppressing the progression of ESCC. (A) Experimental strategy for the treatment by anti-NK1.1 antibodies and representative esophagi image in mice treated with IgG control (IgG), cKO, anti-NK1.1, or cKO+anti-NK1.1 group. (B, C) Quantification of lesion area (B) and lesion number (C) of IgG control (IgG), cKO, anti-NK1.1, or cKO+anti-NK1.1 group. Data are presented as mean±SD (n=8). (D) Representative images of H&E staining in IgG control (IgG), cKO, anti-NK1.1, or cKO+anti-NK1.1 group. Scale bar, 100 µm. (E) Quantification of ESCC tumor grade in IgG control (IgG), cKO, anti-NK1.1, or cKO+anti-NK1.1 group. (F–H) Representative images of Ki67 (F) and Caspase3 (F) IHC staining and quantification of percentage of Ki67+ (G) and Caspase3+ (H) cells in IgG control (IgG), cKO, anti-NK1.1, or cKO+anti-NK1.1 group. Scale bar, 100 µm. (I–K) Representative images of CD8 and NCR1 (I) IHC staining and quantification of percentage of CD8+ (J) and NCR1+ (K) cells in IgG control (IgG), cKO, anti-NK1.1, or cKO+anti-NK1.1 group. Scale bar, 100 µm. (L) Representative images of CD276 and NCR1 IHC staining of ESCC in FAH-SYSU patients’ cohort; Scale bar, 100 µm (left panel). The correlation between the expression of CD276 and NCR1 was assessed, ***p<0.001 by Pearson (right panel). ESCC, esophageal squamous cell carcinoma; FAH-SYSU, The First Affiliated Hospital Sun Yat-sen University; IHC, immunohistochemistry; NK, natural killer.

CD276 overexpression promotes tumorigenesis in vivo

To further clarify the function of CD276 in ESCC, CD276 overexpression mice (cKI) was constructed. After 4NQO treatment as described above, the esophageal tissues appeared more severe on gross examination in cKI mice compared with the WT mice (figure 7A). In addition, cKI mice developed higher number and larger size of esophageal lesions, respectively, compared with WT mice (figure 7B,C). CD276 overexpression greatly increased the proportion of high-grade ESCC (figure 7D,E). Moreover, we found increased expression of Ki67 and decreased expression of active-Caspase3 in cKI mice compared with WT mice (figure 7F–I). To further verify the effect of CD276 overexpression on neutrophil and NK cells infiltration, we performed FCM and immunofluorescence staining analysis as previously mentioned. Compared with WT mice, the numbers of neutrophils and NETs in the cKI mice were significantly higher (figure 7J, K, N, and O), while NK cells were lower (figure 7L,M).

Figure 7

Overexpression of CD276 facilitates tumor progression. (A) Experimental strategy for the 4NQO-induced ESCC mice model and representative esophagi image from 4NQO-induced wild-type mice (WT) and mice with conditional knock-in of CD276 (cKI). (B, C) Quantification of lesion number (B) and lesion area (C) of the WT and cKI group. Data are presented as mean±SD (n=8). (D) Representative images of H&E staining in the WT and cKI group. Scale bar, 100 µm. (E) Quantification of ESCC tumor grade in the WT and cKI group. (F–I) Representative images of Ki67 (F) and Caspase3 (H) IHC staining and quantification of percentage of Ki67+ (G) and Caspase3+ (I) cells in WT and cKI group. Scale bar, 100 µm. (J–M) Repre