BACKGROUND

Pulmonary squamous cell carcinoma has a poor prognosis, and new therapeutic targets are needed. The aberrant expression of the immunomodulatory proteins B7-H1 and B7-H3 by malignant cells may contribute to tumoral immune evasion. Data about the expression of these proteins by squamous cell carcinoma of the lung are limited.

METHODS

Immunohistochemistry for B7-H1 and B7-H3 was performed on 214 resected pulmonary squamous cell carcinoma specimens.

RESULTS

At the last follow-up, 171 of 214 (80%) of patients were deceased (median survival time, 3.76 years). Forty-two (19.6%) of 214 cases showed positivity with B7-H1, with a range of 5% to 60% of cells that stained positively. A total of 189 (88.3%) of 214 cases showed positivity with B7-H3, with a range of 5% to 80% of cells staining positively. By using multivariate analysis, no degree of B7-H1 or B7-H3 positivity was significantly associated with patient outcome.

CONCLUSIONS

Although B7-H1 and B7-H3 are not of independent prognostic value, they are commonly expressed on a subset of tumor cells in pulmonary squamous cell carcinomas. Known interaction of the B7-H proteins with cytotoxic T-lymphocyte antigen-4 may make them attractive candidate biomarkers for response to immunomodulatory therapeutics, eg, ipilimumab, and warrants further study.

B7-H3 (CD276) is an important immune checkpoint member of the B7 and CD28 families. Induced on antigen-presenting cells, B7-H3 plays an important role in the inhibition of T-cell function. Importantly, B7-H3 is highly overexpressed on a wide range of human solid cancers and often correlates with both negative prognosis and poor clinical outcome in patients. Challenges remain to identify the receptor(s) of B7-H3 and thus better elucidate the role of the B7-H3 pathway in immune responses and tumor evasion. With a preferential expression on tumor cells, B7-H3 is an attractive target for cancer immunotherapy. Based on the clinical success of inhibitory immune checkpoint blockade (CTLA-4, PD-1, and PD-L1), mAbs against B7-H3 appear to be a promising therapeutic strategy worthy of development. An unconventional mAb against B7-H3 with antibody-dependent cell-mediated cytotoxicity is currently being evaluated in a phase I clinical trial and has shown encouraging preliminary results. Additional therapeutic approaches in targeting B7-H3, such as blocking mAbs, bispecific mAbs, chimeric antigen receptor T cells, small-molecule inhibitors, and combination therapies, should be evaluated, as these technologies have already shown positive results in various cancer settings. A better understanding of the B7-H3 pathway in humans will surely help to further optimize associated cancer immunotherapies. Clin Cancer Res; 22(14); 3425-31. ©2016 AACR.

CONCLUSIONS

B7-H3 and B7-H4 belong to a new class of immune regulatory molecules, which primarily execute their functions in peripheral tissues to fine tune immune responses in target organs. In normal circumstances, while the mRNA for both molecules is broadly distributed, tight control at the post-transcriptional level is imposed. Under a pathogenic environment, such as inflammation and cancer, the control is often aberrant. Upon engaging their receptors, these molecules regulate the immune response in positive or negative ways depending on the expression and type of cells bearing the receptors. Thus, manipulation of the expression of these molecules and/or their receptors may represent a realistic opportunity to fine tune immune responses and to design new immunotherapeutic approaches.

B7-H3 is a B7 family molecule with T cell costimulatory function in vitro. The in vivo role of B7-H3 in the stimulation of tumor immunity is unclear. We report here that expression of B7-H3 by transfection of the mouse P815 tumor line enhances its immunogenicity, leading to the regression of tumors and amplification of a tumor-specific CD8+ CTL response in syngeneic mice. Tumor cells engineered to express B7-H3 elicit a rapid clonal expansion of P1A tumor Ag-specific CD8+ CTL in lymphoid organs in vivo and acquire the ability to directly stimulate T cell growth, division, and development of cytolytic activity in vitro. Our results thus establish a role for B7-H3 in the costimulation of T cell immune responses in vivo.

OBJECTIVE

The goal of this research was to harness a monoclonal antibody (mAb) discovery platform to identify cell-surface antigens highly expressed on cancer and develop, through Fc optimization, potent mAb therapies toward these tumor-specific antigens.

METHODS

Fifty independent mAbs targeting the cell-surface immunoregulatory B7-H3 protein were obtained through independent intact cell-based immunizations using human tissue progenitor cells, cancer cell lines, or cell lines displaying cancer stem cell properties. Binding studies revealed this natively reactive B7-H3 mAb panel to bind a range of independent B7-H3 epitopes. Immunohistochemical analyses showed that a subset displayed strong reactivity to a broad range of human cancers while exhibiting limited binding to normal human tissues. A B7-H3 mAb displaying exquisite tumor/normal differential binding was selected for humanization and incorporation of an Fc domain modified to enhance effector-mediated antitumor function via increased affinity for the activating receptor CD16A and decreased binding to the inhibitory receptor CD32B.

RESULTS

MGA271, the resulting engineered anti-B7-H3 mAb, mediates potent antibody-dependent cellular cytotoxicity against a broad range of tumor cell types. Furthermore, in human CD16A-bearing transgenic mice, MGA271 exhibited potent antitumor activity in B7-H3-expressing xenograft models of renal cell and bladder carcinoma. Toxicology studies carried out in cynomolgus monkeys revealed no significant test article-related safety findings.

CONCLUSIONS

This data supports evaluation of MGA271 clinical utility in B7-H3-expressing cancer, while validating a combination of a nontarget biased approach of intact cell immunizations and immunohistochemistry to identify novel cancer antigens with Fc-based mAb engineering to enable potent antitumor activity.

B7-H3, a member of the B7-family molecules, plays an important role in adaptive immune responses, and was shown to either promote or inhibit T-cell responses in various experimental systems. B7-H3 was expressed in some human cancers and correlated with poor outcome of cancer patients. However, its exact role in cancer is not known. In the present study, we studied the expression of B7-H3 in the pathologic specimens of 102 patients treated for colorectal carcinoma (CRC) by immunohistochemistry. Strong B7-H3 expression was found in cancer tissues from 54.3% CRC patients, while minimal expression was found in adjacent normal colorectal tissues. Higher B7-H3 expression in tumor positively correlated with a more advanced tumor grade. In addition, consistent with a role of B7-H3 in suppressing tumor immune surveillance, the expression of B7-H3 in cancer cells negatively correlated with the intensity of tumor infiltrating T lymphocytes in both tumor nest and tumor stroma. Furthermore, we found that the level of soluble B7-H3 in sera from CRC patients was higher than healthy donors. TNF-alpha, an important cancer-promoting inflammatory molecule, was subsequently found to significantly increase the release of soluble B7-H3 in colon cancer cell lines. Therefore, our data suggest that both soluble and membranous B7-H3 proteins are involved in colon cancer progression and evasion of cancer immune surveillance.

Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies.

The B7 family consists of structurally related, cell-surface proteins that regulate immune responses by delivering costimulatory or coinhibitory signals through their ligands. Eight family members have been identified to date including CD80 (B7-1), CD86 (B7-2), CD274 (programmed cell death-1 ligand [PD-L1]), CD273 (programmed cell death-2 ligand [PD-L2]), CD275 (inducible costimulator ligand [ICOS-L]), CD276 (B7-H3), B7-H4, and B7-H6. B7 ligands are expressed on both lymphoid and nonlymphoid tissues. The importance of the B7 family in regulating immune responses is clear from their demonstrated role in the development of immunodeficiency and autoimmune diseases. Manipulation of the signals delivered by B7 ligands shows great potential in the treatment of cancers including leukemias and lymphomas and in regulating allogeneic T-cell responses after stem cell transplantation.

B7-H3 is a novel protein structurally related to the B7 family of ligands by the presence of a single set of immunoglobulin-V-like and immunoglobulin-C-like (VC) domains. By multiplex PCR, the dominantly expressed form of human B7-H3 was found to be a splice variant containing tandemly duplicated VC domains (VCVC). In contrast, mouse B7-H3 cDNA contained only one single VC form due to an exon structure corresponding to V-(pseudoexon C)-(pseudoexon V)-C. Comparisons of human, monkey, mouse, and hamster genomic B7-H3 reveal that primates, but not rodents, exhibited a higher degree of intramolecular sequence similarity between VC duplications than between molecules. Both VC and VCVC forms of human B7-H3 inhibited CD4(+) T cell proliferation and downregulated cytokine production upon TCR activation. These results suggest independent, but convergent, paths of B7-H3 active domain duplication followed by divergent histories of exon degeneration in rodents and exon maintenance by humans.

OBJECTIVE

To investigate the expression of co-stimulatory molecule B7-H3 in gastric carcinoma and adenoma tissue as well as normal gastric tissue and to explore the relationship between B7-H3 expression and pathological features and prognosis of gastric carcinoma.

METHODS

B7-H3 expression was detected in 102 samples of human gastric carcinoma and 10 samples of gastric adenoma and 10 samples of normal gastric tissue by immunohistochemical assay. Correlation between the expression of B7-H3 and the patients' age, sex, gastric carcinoma locus, tumor size, tissue type, tumor infiltration depth, differentiation degree, lymph node metastasis, and survival time was analyzed.

RESULTS

B7-H3 was expressed in all gastric adenoma samples and in 58.8% samples of gastric carcinoma. B7-H3 expression in gastric carcinoma samples was not related with the patients' age, sex, lymph node metastasis, and tumor size (P>0.05), but with the survival time, infiltration depth of tumor and tissue type.

CONCLUSIONS

Detection of B7-H3 expression in gastric carcinoma tissue is beneficial to the judgment of the prognosis of gastric carcinoma patients and the choice of treatment.

BACKGROUND

B7-H3 is a new member of the B7 ligand family and regulates T-cell responses in various conditions. However, the role of B7-H3 in tumour immunity is largely unknown. The purpose of this study was to evaluate the clinical significance of B7-H3 expression in human pancreatic cancer and the therapeutic potential for cancer immunotherapy.

METHODS

We investigated B7-H3 expression in 59 patients with pancreatic cancer by immunohistochemistry and real-time PCR. Furthermore, we examined the anti-tumour effect of B7-H3-blocking monoclonal antibody in vivo in a murine pancreatic cancer model.

RESULTS

Tumour-related B7-H3 expression was abundant in most human pancreatic cancer tissues and was significantly higher compared with that in non-cancer tissue or normal pancreas. Moreover, its expression was significantly more intense in cases with lymph node metastasis and advanced pathological stage. B7-H3 blockade promoted CD8(+) T-cell infiltration into the tumour and induced a substantial anti-tumour effect on murine pancreatic cancer. In addition, the combination of gemcitabine with B7-H3 blockade showed a synergistic anti-tumour effect without overt toxicity.

CONCLUSIONS

Our data show for the first time that B7-H3 may have a critical role in pancreatic cancer and provide the rationale for developing a novel cancer immunotherapy against this fatal disease.

B7-H3 is a member of the B7-family of co-stimulatory molecules, which has been shown to be broadly expressed in various tumor tissues, and which plays an important role in adaptive immune responses. The role of B7-H3 in osteosarcoma, however, remains unknown. In this study we used immunohistochemistry to analyze B7-H3 expression in 61 primary osteosarcoma tissues with case-matched adjacent normal tissues, and 37 osteochondroma and 20 bone fibrous dysplasia tissues. B7-H3 expression was expressed in 91.8% (56/61) of the osteosarcoma lesions, and the intensity of B7-H3 expression in osteosarcoma was significantly increased compared with adjacent normal tissues, osteochondroma and bone fibrous dysplasia tissues (p<0.001). Patients with high tumor B7-H3 levels had a significantly shorter survival time and recurrence time than patients with low tumor B7-H3 levels (p<0.001). Moreover, tumor B7-H3 expression inversely correlated with the number of tumor-infiltrating CD8(+) T cells (p<0.05). In vitro, increasing expression of B7-H3 promotes osteosarcoma cell invasion, at least in part by upregulating matrix metalloproteinase-2 (MMP-2). In conclusion, our study provides the first evidence of B7-H3 expression in osteosarcoma cells as a potential mechanism controlling tumor immunity and invasive malignancy, and which is correlated with patients' survival and metastasis.

Although immunotherapeutics targeting the inhibitory receptors (IRs) CTLA-4, PD-1 or PD-L1 have made substantial clinical progress in cancer, a considerable proportion of patients remain unresponsive to treatment. Targeting novel IR-ligand pathways in combination with current immunotherapies may improve clinical outcomes. New clinical immunotherapeutics target T cell-expressed IRs (LAG-3, TIM-3 and TIGIT) as well as inhibitory ligands in the B7 family (B7-H3, B7-H4 and B7-H5), although many of these targets have complex biologies and unclear mechanisms of action. With only modest clinical success in targeting these IRs, current immunotherapeutic design may not be optimal. This Review covers the biology of targeting novel IR-ligand pathways and the current clinical status of their immunotherapeutics, either as monotherapy or in combination with antibody to PD-1 or to its ligand PD-L1. Further understanding of the basic biology of these targets is imperative to the development of effective cancer immunotherapies.

BACKGROUND

Despite high bacterial colonization, acute infections are rare in the oral mucosa, implicating tolerogenic predominance. Bacterial antigens like LPSs are recognized by innate immunity receptors such as Toll-like receptor 4 (TLR4), associated with LPS receptor (CD14).

OBJECTIVE

Toll-like receptor 4 agonist monosphoryl lipid A has been successfully used as adjuvant in subcutaneous immunotherapy, suggesting reinforcement of allergen-specific tolerance. Recently sublingual immunotherapy (SLIT) has been shown to be an effective alternative to subcutaneous immunotherapy. We observed CD14 expression on human oral Langerhans cells (oLCs), representing a major target of SLIT. However, not much is known about TLR4 expression and its effect on oLCs.

METHODS

Cell suspensions were obtained by trypsinization of human oral mucosa and analyzed by flow cytometry, RT-PCR, cytometric bead arrays, ELISA, and mixed lymphocyte reactions.

RESULTS

We could show that oLCs express TLR4, and its ligation by monosphoryl lipid A upregulated expression of coinhibitory molecules B7-H1 and B7-H3 while surface expression of costimulatory molecule CD86 was concomitantly decreased. Furthermore, TLR4 ligation on oLCs increased their release of the anti-inflammatory cytokine IL-10 and decreased their stimulatory capacity toward T cells. Moreover, TLR4-ligation on oLCs induced IL-10, TGF-beta1, Forkhead box protein 3, IFN-gamma, and IL-2 production in T cells.

CONCLUSIONS

In view of these data, TLR4-ligation on oLCs might not only play a role in pathogen recognition for efficient immunity but also contribute to the tolerogenic state predominating in the oral cavity.

Expression of membrane CD276 (mB7-H3) has been reported on dendritic cells (DCs), monocytes, activated T cells, and various carcinoma cells. However, reports concerning its in vivo function have been inconsistent. Moreover, whether there is a soluble form of this protein is not known. In this study, using a sensitive dual monoclonal antibody sandwich enzyme-linked immunosorbent assay (ELISA) to detect the soluble form of B7-H3 (sB7-H3), we demonstrated the release of sB7-H3 by monocytes, DCs, activated T cells, and various mB7-H3+ but not mB7-H3- carcinoma cells. Release from cells was blocked by addition of a matrix metalloproteinase inhibitor (MMPI), which concomitantly caused the accumulation of B7-H3 on the cell surface. To determine the level of circulating sB7-H3, more than 200 serum samples were included in the study. The results indicated that sB7-H3 was present at high levels in all serum samples. Western blotting of sB7-H3 from cell culture supernatants or sera of healthy donors indicated that the molecular size was approximately 16 kDa. Soluble B7-H3 was able to bind to the B7-H3 receptor (B7-H3R) on activated T cells, which showed that sB7-H3 is a functionally active form. These results indicate that release of sB7-H3 from the cell surface is mediated by a matrix metalloproteinase and probably regulates B7-H3R/B7-H3 interactions in vivo. Cleavage of sB7-H3 to an active soluble form would alter both proximal and distal cellular responses.

The first generation of immune checkpoint inhibitors (anti-CTLA-4 and anti-PD-1/PD-L1) targeted natural immune homeostasis pathways, co-opted by cancers, to drive anti-tumor immune responses. These agents led to unprecedented results in patients with previously incurable metastatic disease and may become first-line therapies for some advanced cancers. However, these agents are efficacious in only a minority of patients. Newer strategies are becoming available that target additional immunomodulatory mechanisms to activate patients' own anti-tumor immune responses. Herein, we present a succinct summary of emerging immune targets with reported pre-clinical efficacy that have progressed to active investigation in clinical trials. These emerging targets include co-inhibitory and co-stimulatory markers of the innate and adaptive immune system. In this review, we discuss: 1) T lymphocyte markers: Lymphocyte Activation Gene 3 [LAG-3], T-cell Immunoglobulin- and Mucin-domain-containing molecule 3 [TIM-3], V-domain containing Ig Suppressor of T cell Activation [VISTA], T cell ImmunoGlobulin and ITIM domain [TIGIT], B7-H3, Inducible T-cell Co-stimulator [ICOS/ICOS-L], CD27/CD70, and Glucocorticoid-Induced TNF Receptor [GITR]; 2) macrophage markers: CD47/Signal-Regulatory Protein alpha [SIRPα] and Indoleamine-2,3-Dioxygenase [IDO]; and 3) natural killer cell markers: CD94/NKG2A and the Killer Immunoglobulin-like receptor [KIR] family. Finally, we briefly highlight combination strategies and potential biomarkers of response and resistance to these cancer immunotherapies.

B7-H3 (CD276), a newly identified member of the B7 family of molecules, is often induced in human tumors and its overexpression is closely correlated with survival, prognosis or tumor grade. Although cancer immunotherapy has not been completely translated into clinical successes, interest has been further enhanced by the realization of these costimulatory molecules' potential as targets to modulate clinical immune responses. Despite ample evidence implicating B7-H3 in tumor immune escape, a steady flow of reports have suggested that it may also have antitumor effects under certain circumstances. The safety and efficacy of targeting B7-H3 with a monoclonal antibody for the treatment of advanced-stage central nervous system cancer in children has been proven, making B7-H3 an attractive therapeutic target for this kind of tumor. In addition, B7-H3 was shown to promote invasion and accelerate carcinogenesis in tumor progression according to its nonimmunological regulatory roles. In this review, we discuss current understanding of the diverse functions of B7-H3 in carcinogenesis and cancer progression, and consider future directions for designing cancer immunotherapeutic agents targeting B7-H3.

The interaction between tumor and the immune system is still poorly understood. Significant clinical responses have been achieved in cancer patients treated with antibodies against the CTLA4 and PD-1/PD-L1 checkpoints; however, only a small portion of patients responded to the therapies, indicating a need to explore additional co-inhibitory molecules for cancer treatment. B7-H3, a member of the B7 superfamily, was previously shown by us to inhibit T-cell activation and autoimmunity. In this study, we have analyzed the function of B7-H3 in tumor immunity. Expression of B7-H3 was found in multiple tumor lines, tumor-infiltrating dendritic cells, and macrophages. B7-H3-deficient mice or mice treated with an antagonistic antibody to B7-H3 showed reduced growth of multiple tumors, which depended on NK and CD8+ T cells. With a putative receptor expressed by cytotoxic lymphocytes, B7-H3 inhibited their activation, and its deficiency resulted in increased cytotoxic lymphocyte function in tumor-bearing mice. Combining blockades of B7-H3 and PD-1 resulted in further enhanced therapeutic control of late-stage tumors. Taken together, our results indicate that the B7-H3 checkpoint may serve as a novel target for immunotherapy against cancer.

The semiallogenic fetus is tolerated by the maternal immune system through control of innate and adaptive immune responses. Trophoblast cells secrete nanometer scale membranous particles called exosomes, which have been implicated in modulation of the local and systemic maternal immune system. Here we investigate the possibility that exosomes secreted from the first trimester and term placenta carry HLA-G and B7 family immunomodulators. Confocal microscopy of placental sections revealed intracellular co-localization of B7-H1 with CD63, suggesting that B7-H1 associates with subcellular vesicles that give rise to exosomes. First trimester and term placental explants were then cultured for 24 h. B7H-1 (CD274), B7-H3 (CD276) and HLA-G5 were abundant in pelleted supernatants of these cultures that contained microparticles and exosomes; the latter, however, was observed only in first trimester pellets and was nearly undetectable in term explant-derived pellets. Further purification of exosomes by sucrose density fractionation confirmed the association of these proteins specifically with exosomes. Finally, culture of purified trophoblast cells in the presence or absence of EGF suggested that despite the absence of HLA-G5 association with term explant-derived exosomes, it is present in exosomes secreted from mononuclear cytotrophoblast cells. Further, differentiation of cytotrophoblast cells reduced the presence of HLA-G5 in secreted exosomes. Together, the results suggest that the immunomodulatory proteins HLA-G5, B7-H1 and B7-H3, are secreted from early and term placenta, and have important implications in the mechanisms by which trophoblast immunomodulators modify the maternal immunological environment.

T-cell costimulation and coinhibition generated by engagement of the B7 family and their receptor CD28 family are of central importance in regulating the T-cell response, making these pathways very attractive therapeutic targets. Here we describe HERV-H LTR-associating protein 2 (HHLA2) as a member of the B7 family that shares 10-18% amino acid identity and 23-33% similarity to other human B7 proteins and phylogenetically forms a subfamily with B7x and B7-H3 within the family. HHLA2 is expressed in humans but not in mice, which is unique within the B7 and CD28 families. HHLA2 protein is constitutively expressed on the surface of human monocytes and is induced on B cells after stimulation with LPS and IFN-γ. HHLA2 does not interact with other known members of the CD28 family or the B7 family, but does bind a putative receptor that is constitutively expressed not only on resting and activated CD4 and CD8 T cells but also on antigen-presenting cells. HHLA2 inhibits proliferation of both CD4 and CD8 T cells in the presence of T-cell receptor signaling. In addition, HHLA2 significantly reduces cytokine production by T cells including IFN-γ, TNF-α, IL-5, IL-10, IL-13, IL-17A, and IL-22. Thus, we have identified a unique B7 pathway that is able to inhibit human CD4 and CD8 T-cell proliferation and cytokine production. This unique human T-cell coinhibitory pathway may afford unique strategies for the treatment of human cancers, autoimmune disorders, infection, and transplant rejection and may help to design better vaccines.

OBJECTIVE

: B7 ligand family members have been shown to be important immunoregulatory factors in host tumor immune responses. We hypothesized that B7-H3, a coinhibitory factor, is expressed by primary breast cancer cells and associated with metastasis to regional tumor-draining lymph nodes.

METHODS

: American Joint Committee on Cancer stage I to III primary breast cancers (n = 82) and normal breast specimens (n = 17) were assessed for B7-H3 expression using paraffin-embedded archival tissues. B7-H3 expression by breast cancer cells was assessed by a quantitative real-time reverse transcription-polymerase chain reaction, and B7-H3 protein expression was evaluated using immunohistochemistry.

RESULTS

: B7-H3 mRNA expression was detected in 32 of 82 (39%) primary breast tumors but not in normal breast tissues (P = 0.0029). B7-H3 expression in primary tumors significantly correlated with increasing tumor size, American Joint Committee on Cancer stage, and lymphovascular invasion (P < 0.0001, P < 0.0001, P = 0.0071). B7-H3 expression was highly correlated to sentinel lymph node and overall number of lymph nodes with metastasis P = 0.003, and P = 0.004, respectively). In a multivariate analysis, B7-H3 mRNA expression of the primary tumor significantly predicted metastasis to regional lymph nodes (P = 0.021, and P = 0.023, respectively). Antibody staining analysis of paraffin-embedded archival tissue breast tumors and flow cytometry of breast cancer cell lines demonstrated B7-H3 protein expression.

CONCLUSIONS

: B7-H3 protein expressed by primary breast cancer cells is a tumor progression factor and is associated with extent of regional nodal metastasis.

B7-H3, an immunoregulatory protein, is known to play a role in tumor progression. In many cancer types, observed correlations between high B7-H3 expression and poor prognosis have been attributed to involvement in antitumor immunity. However, here we demonstrate a nonimmunological alternative function of B7-H3 in cancer metastasis. Since advanced malignant melanoma is a disease with a poor survival rate and a broad pattern of metastasis, we used this disease as a model in our studies. We found that shRNA silencing of B7-H3 reduced the in vitro migratory potential and matrigel invasiveness of MDA-MB-435 and FEMX-I melanoma cells. In an experimental metastasis model in vivo, B7-H3 silencing of MDA-MB-435 cells resulted in reduced metastatic capacity and significantly increased the median symptom-free survival of nude mice (147 vs. 65 days, p < 0.001) and rats (53 vs. 42 days, p = 0.025) injected with MDA-MB-435 cells. Furthermore, a smaller fraction of mice had microscopically detectable metastases compared to control animals, and the pattern of metastases was slightly different between the two groups but with the brain as the predominant organ. Immunohistochemistry on samples from two melanoma patients showed strong B7-H3 staining in both a primary tumor and metastases. Notably, the metastasis-associated proteins, matrix metalloproteinase (MMP)-2, signal transducer and activator of transcription 3 (Stat3), and the level of secreted interleukin-8 (IL-8) were reduced in the B7-H3 knock-down cell variants, whereas tissue inhibitor of metalloproteinase (TIMP)-1 and-2 levels were increased. Taken together, our findings indicate a novel role for B7-H3 in the regulation of the metastatic capacity of melanoma cells and it might be a potential therapeutic target for anti-metastasis therapy.