Non-immune functions of B7-H3: bridging tumor cells and the tumor vasculature

B7-H3 (CD276), an immune checkpoint molecule, is overexpressed in various types of cancer and their tumor vasculature, demonstrating significant associations with adverse clinical outcomes. In addition to its well-known immune functions, B7-H3 exhibits dual co-stimulatory/co-inhibitory roles in normal physiology and the tumor microenvironment. The non-immune functions of B7-H3 in tumor cells and the tumor vasculature, including promoting tumor cell anti-apoptosis, proliferation, invasion, migration, drug resistance, radioresistance, as well as affecting cellular metabolism and angiogenesis, have increasingly gained attention from researchers. Particularly, the co-expression of B7-H3 in both tumor cells and tumor endothelial cells highlights the higher potential and clinical utility of therapeutic strategies targeting B7-H3. This review aims to summarize the recent advances in understanding the non-immune functions of B7-H3 in tumors and provide insights into therapeutic approaches targeting B7-H3, focusing on its co-expression in tumor cells and endothelial cells. The aim is to establish a theoretical foundation and practical reference for the development and optimization of B7-H3-targeted therapies.


Introduction
CD276, also known as B7 homolog 3 (B7-H3), is a type I transmembrane protein consisting of 316 amino acids.It is encoded by the gene located on chromosome 9 in mice and the human 15q24 region.The primary structure of B7-H3 includes an extracellular Iglike domain, a transmembrane region, and a short cytoplasmic tail (1).Based on the number of extracellular Ig-like domains, membrane-bound B7-H3 can be classified into two isoforms: 2IgB7-H3 and 4IgB7-H3.The former contains one IgV (variable) and one IgC (constant) domain, while the latter possesses tandem IgV and IgC domains due to exon duplication (2).
malignancies, and gynecologic tumors, B7-H3 has been found to promote cancer cell proliferation by regulating different signaling pathways (46).B7-H3 can also enhance cancer cell migration by modulating the expression of E-cadherin, increasing their migratory capacity (20).Additionally, B7-H3 can augment cancer cell invasion into surrounding tissues by regulating the activity of matrix metalloproteinases (MMPs) (22).These findings provide us with new insights and suggest that targeting B7-H3 and its associated signaling pathways could be an effective strategy for the treatment of these cancers.The summary of signaling pathways mediating the proliferation, invasion, and migration effects of B7-H3 is presented in Table 1.

Drug resistance
Drug resistance is a reflection of tumor evolution and is a major cause of cancer recurrence and patient mortality (47).Studies have shown that overexpression of B7-H3 often leads to drug resistance in cancer cells.Different mechanisms of resistance exist in different tumor cells.For example, the high expression of the B7-H3 immune checkpoint protein in neuroblastoma is involved in carcinogenic signaling, tumor cell plasticity, and the development of drug resistance (48).However, inhibiting the expression of B7-H3 or blocking its associated signaling pathways can reverse this resistance.One approach is to use a combination of paclitaxel and MEK inhibitors to block the B7-H3 signal, which has been demonstrated to effectively reverse chemotherapy resistance (16).Another approach is to use the specific inhibitor LY294002 to block the PI3K signaling pathway, which can also effectively inhibit this resistance (49).Another study found that bromodomain and extra-terminal domain inhibitors could be used to reduce the levels of B7-H3 protein and mRNA in pancreatic cancer cells, providing a new therapeutic strategy to overcome immune and chemotherapy resistance in pancreatic cancer (21).In related studies on traditional Chinese medicine, researchers found that artemisinin-mediated inhibition of B7-H3 may help enhance neuroblastoma cell sensitivity to doxorubicin (50).Additionally, Astragaloside IV enhanced the chemosensitivity to cisplatin by inhibiting B7-H3, suggesting that a combination therapy using Astragaloside IV and a B7-H3 inhibitor may be a potential treatment method for lung cancer patients (51).The summary of signaling pathways mediating the drug resistance effects of B7-H3 is presented in Table 1.

Radiation resistance
Radiation therapy is a common treatment modality for solid tumors; however, radiation resistance is a major factor contributing to treatment failure in cancer (52).A study by Ma et al. reported an interesting phenomenon: after X-ray irradiation, the expression of B7-H3 was upregulated in colorectal cancer cells, and this upregulation depended on the upregulation of KIF15 via the NF-kB signaling pathway.Further research revealed that B7-H3/KIF15, through activation of the ERK1/2 signaling pathway, promoted radiation resistance in colorectal cancer (31).Additionally, Zhou et al. found that B7-H3 increased the radiation resistance of gastric cancer cells by regulating baseline levels of cellular autophagy.In cells with high B7-H3 expression, increasing the baseline level of cellular autophagy using rapamycin enhanced their sensitivity to radiation (32).These studies all point to the important role of B7-H3 in regulating tumor cell resistance to radiation therapy.Therefore, finding effective ways to inhibit or modulate the expression and function of B7-H3 may provide new strategies to improve the effectiveness of radiation therapy.

Metabolism
The Warburg effect is one of the characteristic features of tumor cell metabolism, where cancer cells tend to produce energy through glycolysis even in the presence of abundant oxygen, which is significantly different from the metabolic mechanism of normal cells (53).Research by Lim et al. found that B7-H3 plays an important role in the metabolic reprogramming of cancer cells.In cells expressing B7-H3, glucose uptake and lactate production are increased (34).On the other hand, there is an interaction between the glycolytic enzyme ENO1 and B7-H3.Downregulation of B7-H3 in cervical cancer HeLa cells leads to decreased levels of ATP, lactate, c-Myc, and lactate dehydrogenase A (54).Furthermore, overexpression of B7-H3 effectively increases glucose consumption and lactate production rate, while knockout of B7-H3 has the opposite effect.Liu et al. demonstrated that using the anti-B7-H3 antibody 8H9 can shift cellular metabolism from glycolysis to oxidative phosphorylation, and they suggested that anti-B7-H3 blockade may alter tumor glucose metabolism through reactive oxygen species-mediated pathways by measuring cellular ROS levels in A549 cells treated with the anti-B7-H3 antibody using fluorescence probes (55).In addition to changes in glucose metabolism, cancer cells often possess altered fatty acid metabolism properties.In glioma cells, genetic risk features associated with fatty acid catabolism are significantly correlated with the immune checkpoint molecule B7-H3 (56).These research findings further emphasize the important role of B7-H3 in the metabolic regulation of cancer cells and may provide new therapeutic strategies for targeting this process.The summary of signaling pathways mediating the metabolism effects of B7-H3 is presented in Table 1.
3 The role of B7-H3 in tumor vasculature and clinical treatment

The role of B7-H3 in tumor vasculature
In cancer development, angiogenesis plays a crucial role by providing oxygen and nutrients to tumors while allowing their spread to other parts of the body.Thus, inhibiting or reducing the blood supply in the tumor microenvironment to restrict tumor angiogenesis has become a key strategy against various solid tumors (57).B7-H3 is a highly expressed protein in many types of cancer, including lung, breast, colorectal, endometrial, renal, ovarian, hepatocellular carcinoma, and melanoma.However, this protein is not expressed in normal vascular tissues (4,5).Furthermore, in hepatocellular carcinoma, colorectal cancer, renal cell carcinoma, and melanoma, the expression rate of B7-H3 in the tumor vasculature reaches 86% to 98% (58).In ovarian cancer, although the expression rate of B7-H3 is only 44%, it is undetectable in normal ovarian vascular tissues (5).
Under normal physiological conditions, the process of angiogenesis is strictly regulated by factors that promote and inhibit angiogenesis to maintain a stable internal environment and prevent uncontrolled blood vessel growth.However, B7-H3 protein can disrupt this balance by enhancing the activity of several pro-angiogenic factors, favoring tumor development (9).It mainly activates multiple signaling pathways, such as MMP-2, MMP-9, NF-kB, AKT1/mTOR/VEGFA, and Tie-2, thus promoting tumor angiogenesis and further facilitating tumor growth and spread (38)(39)(40)(41).By inhibiting key molecules in these signaling pathways, we can effectively inhibit angiogenesis.For example, in neuroblastoma, overexpression of miR-29 can activate the JAK/STAT1 signaling pathway, weakening the regulatory effect of MYC-B7-H3 and inhibiting tumor angiogenesis (38).Additionally, Wang et al. found that recombinant VEGFA can eliminate the inhibitory effect of shB7-H3 on human umbilical vein endothelial cell angiogenesis in conditioned medium from colorectal cancer cells.Furthermore, siRNA against VEGFA or neutralizing antibodies against VEGFA can reverse the effects of B7-H3 in overexpressed colorectal cancer cell-conditioned medium on human umbilical vein endothelial cell angiogenesis (39).These studies further elucidate the potential role of B7-H3 in tumor angiogenesis and may serve as a crucial target for future anti-angiogenic therapies.
Moreover, B7-H3 also plays an important role in vasculogenic mimicry (VM).VM is an alternative microcirculation pattern independent of angiogenesis, where cancer cells form microvascular-like channels to provide nutrients and oxygen to tumors (59).Recent studies have found that B7-H3 expressed in tumors promotes VM formation in non-small cell lung cancer cells through the PI3K/AKT signaling pathway without affecting normal angiogenesis, providing a new strategy for future anticancer therapies (42).The summary of signaling pathways mediating B7-H3-induced tumor angiogenesis is presented in Table 1.

Clinical treatment
In numerous types of tumors, the widespread overexpression of B7-H3 on cancer cells and tumor-associated blood vessels makes it a potential ideal dual-target for therapy.CD276-ADC, utilizing pyrrolobenzodiazepine as a linker, demonstrates significant cytotoxicity against cancer cells while also exhibiting remarkable destructive effects on the tumor vascular system.This therapy shows great potential in eradicating large existing tumors and metastatic lesions, with the possibility of significantly improving long-term overall survival rates for patients (5).To target the overexpression of B7-H3 in tumor cells and tumor vasculature, along with its limited expression in normal tissue vasculature, researchers have employed an innovative strategy by conjugating the Fab fragment of anti-CD276 antibody with the photosensitizer IRDye700 to form a novel CD276-targeted agent.The findings show that combining photodynamic therapy using this agent with inhibition strategies targeting the PD-L1/PD-1 axis can induce strong local and systemic anti-tumor responses, effectively eliminating primary and metastatic tumors (60).Chen et al. reported the development of pH-responsive drug release using B7H3-targeted doxorubicin-conjugated gold nano-cages (B7H3/ Dox@GNCs), which represents a selective, precise, and synergistic chemotherapy-photothermal therapy for NSCLC, capable of simultaneously destroying B7H3-positive tumor cells, tumorassociated vasculature, and cancer-associated fibroblasts (61).The expression of B7-H3 in the tumor vasculature can also be utilized to significantly improve the diagnostic accuracy of breast cancer, which is undoubtedly an important discovery (62).
Furthermore, as a promising immunotherapy target, B7-H3 has been applied in combination treatment strategies.When used as a key element in T cell co-stimulation and anti-angiogenesis therapy for hepatocellular carcinoma, the effect of B7-H3 is significantly superior to using these treatments alone.Injecting plasmids expressing B7-H3 into subcutaneous tumors in mice resulted in complete eradication of these tumors within 24 hours, which was not achieved when using angiostatin or B7-H3 treatment alone (63).This process revealed the ability of angiostatin gene transfer to inhibit tumor angiogenesis and enhance NK cell infiltration, as well as the critical role of B7-H3 in activating CD8 + tumor infiltration, increasing circulating IFN-c levels, and even completely regressing distant tumor nodules (63).However, although B7-H3 plays an important role in the aforementioned combination treatment strategies, its primary role in the broader field of immunotherapy is still as an immune checkpoint inhibitor.For example, in the treatment of triplenegative breast cancer, Cheng et al. found that blocking B7-H3 with anti-B7-H3 antibodies improved the abnormal state of the tumor vascular system, thereby enhancing the effects of chemotherapy and PD-1 treatment (64).These research findings further emphasize that B7-H3 can serve as an important therapeutic target in anticancer treatment strategies, whether used alone or in combination with other treatment modalities.More clinical trials targeting B7-H3 for cancer treatment are listed in Table 2.

Conclusion
Compared to other immune checkpoints, the uniqueness of B7-H3 lies in its ability to regulate both innate and adaptive immune responses.Furthermore, it promotes tumor development by stimulating various non-immune-related functions, making it an important prognostic marker for adverse outcomes.B7-H3 influences the life processes of tumor cells, including apoptosis, proliferation, invasion, migration, drug resistance, resistance to radiotherapy, metabolic processes, and abnormal angiogenesis, through the regulation of various signaling pathways.Therefore, inhibiting B7-H3 expression or blocking related signaling pathways may be an effective approach to prevent tumor progression.
Considering the high expression of B7-H3 in tumor cells and tumor vasculature, along with its low expression in normal vasculature, it becomes an ideal dual-target in cancer therapy.By considering B7-H3 as a bridge between tumor cells and the tumor vascular system, we can achieve dual therapeutic effects by targeting B7-H3.However, since the role and signaling pathways of B7-H3 may vary among different tumors and even within the vascular systems of different tumors, gaining a deeper understanding of the specific functions of B7-H3 in particular tumors will help us design more targeted treatment plans for patients, thus effectively controlling tumor development.This could be an important direction for future research in cancer therapy.

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TABLE 1 Signaling
Pathways Mediating the Diverse Functions of B7-H3.