Loss of MMP-27 Predicts Mandibular Bone Invasion in Oral Squamous Cell Carcinoma.

Invasion of the mandibular bone is frequent in oral squamous cell carcinoma (OSCC), which often results in extensive ablative and reconstructive procedures for the patient. The purpose of this single-center, retrospective study was to identify and evaluate potential biomarkers and risk factors for bone invasion in OSCC. Initially, in silico gene expression analysis was performed for different HNSCC tumor T-stages to find factors associated with invasive (T4a) tumor growth. Afterwards, the protein expression of bone-metabolizing MMP-27, TNFRSF11B (Osteoprotegerin, OPG), and TNFSF11 (RANKL) was investigated via Tissue Microarrays (TMAs) for their impact on mandibular bone invasion. TMAs were assembled from the bone-tumor interface of primary OSCCs of the floor of the mouth and gingiva from 119 patients. Sixty-four carcinomas with patho-histological jaw invasion (pT4a) were compared to 55 carcinomas growing along the mandible without invasion (pT2, pT3). Tissue samples were additionally evaluated for patterns of invasion using the WPOI grading system. Statistical analysis of in silico data revealed decreased MMP-27 mRNA expression to be strongly associated with the pT4a-stage in OSCC, indicating invasive tumor growth with infiltration of adjacent anatomical structures. Our own clinico-pathological data on OSCCs presented a significant decrease of MMP-27 in tumors invading the nearby mandible (pT4a), compared to pT2 and pT3 tumors without bone invasion. Loss of MMP27 evolved as the strongest predictor of mandibular bone invasion in binary logistic regression analysis. To our knowledge, this is the first study investigating the role of MMP-27 expression in OSCC and demonstrating the importance of the loss of MMP-27 in mandibular bone invasion.

PD-L1 Influences Cell Spreading, Migration and Invasion in Head and Neck Cancer Cells.

The programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis blockade has been implemented in advanced-stage tumor therapy for various entities, including head and neck squamous cell carcinoma (HNSCC). Despite a promising tumor response in a subgroup of HNSCC patients, the majority suffer from disease progression. PD-L1 is known to influence several intrinsic mechanisms in cancer cells, such as proliferation, apoptosis, migration and invasion. Here, we modulated PD-L1 expression in three HNSCC cell lines with differential intrinsic PD-L1 expression. In addition to an alteration in the epithelial-to-mesenchymal transition (EMT) marker expression, we observed PD-L1-dependent cell spreading, migration and invasion in a spheroid spreading assay on four different coatings (poly-L-lysine, collagen type I, fibronectin and Matrigel®) and a chemotactic transwell migration/invasion assay. Furthermore, the overexpression of PD-L1 led to increased gene expression and small interfering ribonucleic acid (siRNA) knockdown and decreased gene expression of Rho-GTPases and related proteins in a RT2 Profiler™ PCR Array. Rac1 and Rho-GTPase pulldown assays revealed a change in the activation state concordantly with PD-L1 expression. In summary, our results suggest a major role for PD-L1 in favoring cell motility, including cell spreading, migration and invasion. This is presumably caused by altered N-cadherin expression and changes in the activation states of small Rho-GTPases Rho and Rac1.

Evaluation of surgical outcome and influencing risk factors in patients with medication-related osteonecrosis of the jaws.

PURPOSE: Surgical treatment of the medication-related osteonecrosis of the jaw (MRONJ) is still challenging. We examined the outcome of the resection of osteonecrotic lesions and the influence of potential risk factors on the operative success. METHODS: Seventy six surgical interventions on 40 patients were evaluated in a prospective design with a mean follow-up of 55 weeks. Primary endpoints were: (i) maintenance of the mucosal closure and (ii) decrease of MRONJ stage. Influential variables included preoperative duration, location and diameter of MRONJ, duration and change of antiresorptive therapy, presence of actinomyces species. RESULTS: Only in 27.6% of cases long-term maintenance of the mucosal closure was achieved. However, stage II patients decreased to stage I in 81% after surgery (p < 0.01) and stage III patients improved in 83% of cases (OR = 8.08; p = 0.07). Stage I patients profited only in 38% by surgical intervention. MRONJ recurrence after surgery was associated with extended preoperative MRONJ duration (p = 0.015). There was no significance of further influential variables, but MRONJ of the upper jaw seems prognostically more favorable. CONCLUSION: Advanced stages of MRONJ benefit from surgical treatment, whereas stage I diseases may also be treated conservatively. An early intervention reduces the risk of recurrence.

Comparison of human dental follicle cells (DFCs) and stem cells from human exfoliated deciduous teeth (SHED) after neural differentiation in vitro.

Dental stem cells from human exfoliated deciduous teeth (SHED) and dental follicle cells (DFCs) are neural crest-derived stem cells from human dental tissues. Interestingly, SHED and DFCs can successfully differentiate into neuron-like cells. We hypothesized that SHED and DFCs have the same neural cell differentiation potentials. To evaluate neural cell differentiation, we cultivated SHED and DFCs in four different serum-replacement media (SRMs) and analyzed cell morphology, cell proliferation, and gene expression patterns before and after differentiation. In a standard cell culture medium, SHED and DFCs have not only similar cell morphologies, but they also have similar gene expression patterns for known stem cell markers. However, only SHED expressed the neural stem cell marker Pax6. After cultivation in SRMs, cell proliferations of DFCs and SHED were reduced and the cell morphology was spindle-like with long processes. However, differentiated DFCs and SHED had different neural cell marker expression patterns. For example, gene expression of the late neural cell marker microtubule-associated protein 2 was upregulated in DFCs and downregulated in SHED in SRM with the B27 supplement. In contrast, SHED formed neurosphere-like cell clusters in SRM with the B27 supplement, epidermal growth factor, and fibroblast growth factor-2. Moreover, SHED differentially expressed the glial cell marker glial fibrillary acidic protein, which in contrast was weakly or not expressed in DFCs. In conclusion, SHED and DFCs have different neural differentiation potentials under the same cell culture conditions.

Gene expression profiles of dental follicle cells before and after osteogenic differentiation in vitro.

Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast-like cells under in vitro conditions after the induction with dexamethasone or insulin. However, mechanisms for osteogenic differentiation are not understood in detail. In a previous study, real-time RT-PCR results demonstrated molecular mechanisms in dental follicle cells (DFCs) during osteogenic differentiation that are different from those in bone-marrow-derived mesenchymal stem cells. We analysed gene expression profiles in DFCs before and after osteogenic differentiation with the Affymetrix GeneChip(R) Human Gene 1.0 ST Array. Transcripts of 98 genes were up-regulated after differentiation. These genes could be clustered into subcategories such as cell differentiation, cell morphogenesis, and skeletal development. Osteoblast-specific transcription factors like osterix and runx2 were constitutively expressed in differentiated DFCs. In contrast, the transcription factor ZBTB16, which promotes the osteoblastic differentiation of mesenchymal stem cells as an up-stream regulator of runx2, was differentially expressed after differentiation. Transcription factors NR4A3, KLF9 and TSC22D3, involved in the regulation of cellular development, were up-regulated as well. In conclusion, we present the first transcriptome of human DFCs before and after osteogenic differentiation. This study sheds new light on the complex mechanism of osteogenic differentiation in DFCs.

Historical development of alloplastic temporomandibular joint replacement before 1945.

Resections of the temporomandibular joint (TMJ) have been carried out for about 150 years. This article reviews the beginning of TMJ surgery technique before 1945 by carrying out extensive inquiries in public and private libraries and collections. Before 1945 the technique of alloplastic reconstruction of the TMJ was mainly influenced by German and French surgeons. Reconstruction was limited to replacement of the condyle. The role of the TMJ within the orofacial system was not considered. Interposition of alloplastic implants, resection dressings and prostheses were the dominant technique. The main concerns were sterilisation, biocompatibility and implant fixation. No evidence-based data on outcomes are available from that time. By 1945 reconstruction of the TMJ involved the close cooperation of surgeons and dentists.

The buccal minor salivary glands as starting point for a metastasizing adenocarcinoma--report of a case.

BACKGROUND: With the 2005 WHO classification of salivary gland tumours and its increasingly recognized diagnostic entities, the frequency of adenocarcinoma (NOS) has decreased significantly. CASE PRESENTATION: This paper describes a fast growing adenocarcinoma (NOS), originating from the minor salivary glands of the left buccal mucosa with a rapid onset of multiple local and distant metastases, especially in the lung. A lung primary was unlikely as the tumour was characterized by positivity for cytokeratin 20 and negativity for the thyroid transcription factor-1 protein (TTF-1) in immunohistochemistry. CONCLUSION: A rare case of an adenocarcinoma (NOS) of the minor salivary glands with a rapid development and an unfavourable clinical course is reported. It shows that additional immunohistochemical analysis can decisively contribute to determine the site of the primary tumour in cases with unknown primary.

Intralesional corticosteroid therapy for mandibular Langerhans cell histiocytosis preserving the intralesional tooth germ.

BACKGROUND: Langerhans cell histiocytosis (LCH, histiocytosis X, ICD-O 9751/1) refers to a neoplastic proliferation of Langerhans cells. The course of the disease determines the treatment and prognosis. Solitary forms (eosinophilic granuloma) and limited multilocational lesions may be treated successfully with local surgical intervention and intralesional corticosteroid injection. PURPOSE: Presentation of our own case will review LCH, a very rare disease entity in oral-facial surgery and will document that intralesional corticosteroid injection is a less invasive alternative to the classical surgical curettage and local radiation therapy. CASE REPORT: In a 10-year-old boy, a progressive, pressure-sensitive swelling had developed within 1 week in the left paramandibular and submandibular area. Dental status was good. Diagnostic imaging demonstrated a diffusely contoured osteolysis caudal to tooth germ 37 with infiltration of the adjacent chewing muscles. Histological evaluation of the intraoral biopsy sample established an LCH. Having excluded a multifocal form of LCH, treatment with intralesional injection of methylprednisolone 200 mg was chosen. Symptoms of pain were quickly relieved and the swelling receded. Follow-up visits 6 weeks, 3 months, and 6 months after corticosteroid injection revealed continuous regression of mandibular osteolysis. OPG and MRI after 17 months demonstrated a good osseous consolidation in the left mandibular angle area, but a still discernible bone marrow edema. Development of the intralesional tooth germ 37 appeared normal. CONCLUSION: Local surgical interventions continue to be central to the range of accepted therapeutic measures. However, the increasing numbers of reports on the successful treatment of solitary LCH by intralesional corticosteroid injection suggest that this treatment option should be considered especially in children to preserve tooth germs.

Gene expression of nestin, collagen type I and type III in human dental follicle cells after cultivation in serum-free medium.

BACKGROUND: Undifferentiated human dental cells and especially human dental follicle cells are interesting for potential dental treatments. These somatic stem cells are cultured usually in cell culture medium containing bovine serum. In the age of bovine spongiform encephalopathy (BSE), a serum-free cell culture system for dental follicle cells are recommended, if these cells will be applied in dentistry. PURPOSE: However, less is known about the cultivation of dental follicle cells in serum-replacement medium. In this study, we cultivated dental follicle cells in serum-free cell culture medium, which is normally applied for neuronal stem/progenitor cells. MATERIALS AND METHODS: Dental follicle cells were cultivated in both serum-free and serum-containing cell culture media, and gene expression profiles were recorded for connective tissue markers collagen type I and type III and for the human dental follicle cell marker nestin. RESULTS: It is interesting to note that the gene expressions of collagens and nestin were similar after applying both cell culture conditions. CONCLUSION: Although the gene expression of dental follicle cell markers was unchanged, a more appropriate serum-free cell culture medium is recommended for cell proliferation of dental follicle cells.

Somatic stem cells for regenerative dentistry.

Complex human tissues harbour stem cells and/or precursor cells, which are responsible for tissue development or repair. Recently, dental tissues such as periodontal ligament (PDL), dental papilla or dental follicle have been identified as easily accessible sources of undifferentiated cells. The dental stem cell biology might provide meaningful insights into the development of dental tissues and cellular differentiation processes. Dental stem cells could also be feasible tools for dental tissue engineering. Constructing complex structures like a periodontium, which provides the functional connection between a tooth or an implant and the surrounding jaw, could effectively improve modern dentistry. Dental precursor cells are attractive for novel approaches to treat diseases like periodontitis, dental caries or to improve dental pulp healing and the regeneration of craniofacial bone and teeth. These cells are easily accessible and, in contrast to bone-marrow-derived mesenchymal stem cells, are more closely related to dental tissues. This review gives a short overview of stem cells of dental origin.

Loss of Maspin expression is a negative prognostic factor in common salivary gland tumors.

Maspin, a 42kDa protein, belongs to the serine protease inhibitor (serpin) family and is suggested to have inhibitory effects on tumor-induced angiogenesis, tumor cell motility, invasion and metastasis and influences prognosis of tumor patients. The aim of the study was to analyze Maspin expression in salivary gland cancer as well as its prognostic impact on survival in comparison to clinical parameters. Immunohistochemical staining was carried out in 73 cases of salivary gland malignancies. High proportions of Maspin expression were observed in adenoid cystic carcinomas, mucoepidermoid carcinomas and carcinomas ex pleomorphic adenoma, low proportions were seen in salivary duct carcinomas. Acinic cell carcinomas did not show any Maspin expression. Analysis of the prognostic impact of Maspin expression was restricted to salivary gland carcinoma types of intermediate malignancy grade (adenoid cystic carcinoma, mucoepidermoid carcinoma and carcinoma ex pleomorphic adenoma). For these tumors, univariate analyses revealed that T-stage (p=0.025), age70 (p=0.0065), loss of Maspin (p=0.0016) and presence of residual tumor (p<0.001) correlated with poor prognosis. In multivariate analysis age70 (p=0.005) and loss of Maspin (p=0.036) were significant prognostic factors. Moreover, negative Maspin staining was associated with lymph node metastasis and residual tumor. According to these findings, Maspin might be useful as a new prognostic marker in adenoid cystic carcinoma and in salivary gland carcinomas with intermediate grade of malignancy where grading systems are still under debate.

[Immunohistochemical study on collagen I content in the gingiva in cleidocranial dysplasia]. / Immunhistochemische Untersuchung des Kollagen-Typ-I-Gehalts der Gingiva bei Dysostosis cleidocranialis.

BACKGROUND: Patients with cleidocranial dysplasia (CCD) present a thickend and fibrotic gingiva. PURPOSE: To the best of our knowledge it was analysed for the first time, whether this is correlated with an increased rate of collagen I in oral mucosa. PATIENTS AND METHODS: 27 soft tissue biopsies of six CCD-patients and 17 tissue samples of 12 healthy persons were labled with a monoclonal antibody against collagen I and the bound antibodies were detected with alkaline phosphatase-anti-alkaline phophatase-kit. The histological slices were analysed by a digital image recognition software under a fully automated microscope and the rate of collagen I was converted into amounts of grey tones. RESULTS: The amount of grey tones reached from 11.909 to 15.319 in the CCD-group, and from 2752 to 12.556 in the control group. The U-Test of Mann, Whitney and Wilcoxon for two independent samples generated a rank sum of 91,50 for CCD-patients, and of 79,50 for the control group. The Z-value was 3,246, the p-value 0,005. "Fisher's exact test" identified a p-value of 0,0003. CONCLUSIONS: The rate of collagen I in the oral mucosa seems to be increased significantly in CCD. This could explain the typical thick and fibrotic consistency of the gingiva and could be one reason for the delayed or missing dentition.

[Identification of a recurrent oral squamous cell carcinoma by brush cytology]. / Identifikation eines rezidivierenden, oralen Plattenepithelkarzinoms mittels Bürstenzytologie.

BACKGROUND: Large oral lesions comprise the risk that an incisional biopsy does not reveal the most aggressive site in spite of carefully selecting the place of biopsy. CASE REPORT: The first incisional biopsy of a large, clinically suspect, oral lesion could not identify the subepidermal spread of a recurrent oral squamous cell carcinoma. Atypical cells obtained by a simultaneous brush biopsy prompted a renewed, incisional biopsy which finally established the diagnosis of a recurrent, oral squamous cell carcinoma. CONCLUSIONS: The presented case emphasizes the value of brush biopsy in the follow-up of oral squamous cell carcinoma, especially in examination of oral lesions covering a large area.

[The state of the art in human dental stem cell research]. / Stand der humanen dentalen Stammzellforschung.

This review article arranges the current results of stem cell biology for their use in dentistry. There are different types of stem cells, which are applicable for dental treatments. The use of embryonic stem cells, whose possibilities for breeding an artificial tooth were hardly evaluated, is however ethically precarious. On the other side the ethically harmless adult stem cells, which were isolated for example from bone marrow, were little examined for their capability of differentiation into dental tissues. Therefore their forthcoming use in dentistry is rather improbable. However, dental ectomesenchymal stem cells are more promising for dentistry in future. For example dental pulp stem cells (DPSCs) are capable to differentiate into dentin under in vitro conditions. Moreover it is possible to use periodontal ligament (PDL) stem cells and dental follicle precursors for periodontal tissue differentiations in vitro. Recently new populations of stem cells were isolated from the dental pulp and the PDL. These cells distinguish from the initially isolated DPSCs and PDL stem cells in growth and cell differentiation. Therefore stem cell markers are very important for the characterization of dental stem cells. A significant marker for dental stem cells is STRO-1, which is also a marker for bone marrow derived mesenchymal stem cells. Nonetheless dental stem cells are CD45 negative and they express rarely hematopoietic stem cell markers. These research results plead for the participation of dental stem cells in dental practice in future.

[Comparison of clinical immunohistochemical findings in keratocystic odontogenic tumours and ameloblastomas considering their risk of recurrence]. / Vergleichende klinische und immunhistochemische Charakterisierung keratozystischer odontogener Tumoren und Ameloblastome im Hinblick auf das Rezidivrisiko.

BACKGROUND: With the new term "keratocystic odontogenic tumour" (KCOT) keratocyts are even in the nomenclature a close differential diagnosis to ameloblastomas (A). PURPOSE: Recurrence of KCOT and A were retrospectively compared with regard to treatment and immunohistochemical markers of cell cycle and migration and cell architecture. PATIENTS AND METHODS: Biopsies harvested over a period of 22 years of 101 patients (86 KCOT, 15 A) were examined. The histopathological slides were stained with H&E and with the immunohistochemical markers: Cyclin D1, Collagen IV, p16, Cox-2-Laminin-5 and Tenascin-C. RESULTS: Mean age KCOT 47 years (range 14-80 years), A 41 years (range 16-79 years). Gender KCOT: m:f =2:1; A: m:f = 3:2. Region of origin mandible with predilection of the angle and the ramus: KCOT: 76; A: 12. Maxilla: KCOT: 18; A: 3. Multiple lesions were found in 5 KCOT patients. Treatment primary KCOT: cystectomy (46), cystostomy (6), cystectomy and curettage (17), cystectomy and marginal ostectomy (14), resection (11). A: resection (10), enucleation (5). Recurrence rate KCOT: 11,7% after 5,5 years. Recurrence after: cystostomy (4), cystectomy (6), cystectomy and curettage (3), cystectomy and marginal ostectomy (2). A: no recurrences. Immunohistochemistry Cell cycle associated and extracellular matrix proteins did not differ in quantity in KCOT and A, and did also not differ in recurrent and non-recurrent KCOT. CONCLUSIONS: 1. KCOT are in the own cohort more likely recurrent than A. 2. Recurrence rate of KCOT can not be predicted by the used (most common) markers of cell cycle, migration and modulation of architecture. 3. Higher recurrence rate of KCOT in the patients examined is proposed due to less extensive resection.

[Allergic fungal sinusitis, fungus ball and invasive sinonasal mycosis - three fungal-related diseases]. / Allergische Pilzsinusitis, Fungusball und invasive Mykose der Nasennebenhöhlen - drei distinkte, pilzinduzierte Krankheitsbilder.

BACKGROUND: Three different fungal-related clinical pictures have to be differentiated in the paranasal sinuses: allergic fungal sinusitis, fungus ball and invasive sinonasal mycosis. PURPOSE: A morphological reevaluation of fungal-related diseases of the paranasal sinuses as well as a retrospective analysis of their clinical parameters was performed. PATIENTS AND METHODS: 86 patients with patho-histological proven fungal-related disease of the nasal sinuses were enclosed in this study. Reevaluation and correlation of clinical and histological parameters were conducted on routine material (HE, PAS and Grocott) according to the modern morphological definitions. RESULTS: Invasive sinonasal mycosis was seen in 22 cases, eleven male and eleven female, mean age 57 years (22 to 84 years). It was significantly related (nine out of 22 patients, 41%) to immunocompromising conditions: three patients had diabetes mellitus type II, five had have a radiation therapy due to carcinoma and one patient suffered from bacterial endocarditis. A fungus ball was diagnosed in 60 patients, 26 male, 34 female, mean age 54 years (22-88 years). An immunocompromising condition was seen in nine out of 60 patients (15%). Causes for immune impairment were diabetes mellitus (two patients), radiation therapy due to carcinoma (four patients), myocarditis (one patient) and chronic hepatitis (two patients). Allergic fungal sinusitis was recorded in four patients, three male, one female, mean age 43 years (17-63 years). No immunosuppression was diagnosed. CONCLUSIONS: Despite the fact that allergic fungal sinusitis is the most common fungal disease of the paranasal sinuses, it is not well known among physicians and pathologists and therefore underrepresented within the diagnoses of paranasal infections. The term "aspergilloma" is imprecise and does not represent a clear diagnosis. A further differentiation in "fungus ball" (without invasion) and "invasive sinonasal mycosis" is required. The three groups of fungal-related sinusitis occur at different ages. Allergic fungal sinusitis is common among young adults. An immunocompromising condition is a prerequisite for an invasive sinonasal mycosis.

Protein profiling of oral brush biopsies: S100A8 and S100A9 can differentiate between normal, premalignant, and tumor cells.

In oral mucosa lesions it is frequently difficult to differentiate between precursor lesions and already manifest oral squamous cell carcinoma. Therefore, multiple scalpel biopsies are necessary to detect tumor cells already in early stages and to guarantee an accurate follow-up. We analyzed oral brush biopsies (n = 49) of normal mucosa, inflammatory and hyperproliferative lesions, and oral squamous cell carcinoma with ProteinChip Arrays (SELDI) as a non-invasive method to characterize putative tumor cells. Three proteins were found that differentiated between these three stages. These three proteins are able to distinguish between normal cells and tumor cells with a sensitivity of 100% and specificity of 91% and can distinguish inflammatory/hyperproliferative lesions from tumor cells with a sensitivity of up to 91% and specificity of up to 90%. Two of these proteins have been identified by immunodepletion as S100A8 and S100A9 and this identification was confirmed by immunocytochemistry. For the first time, brush biopsies have been successfully used for proteomic biomarker discovery. The identified protein markers are highly specific for the distinction of the three analyzed stages and therewith reflect the progression from normal to premalignant non-dysplastic and finally to tumor tissue. This knowledge could be used as a first diagnostic step in the monitoring of mucosal lesions.

Dual role of VEGF family members in the pathogenesis of head and neck cancer (HNSCC): possible link between angiogenesis and immune tolerance.

BACKGROUND: Antigen presenting cells, in particular dendritic cells (DCs), are critical elements in antitumor immunity induction. Some of the angiogenic factors released by tumor and stroma cells, including vascular endothelial growth factor (VEGF), are thought to affect DC function. MATERIAL/METHODS: The expression of Vascular Endothelial Growth Factor (VEGF) isoforms VEGF-A (121, 145,165, 189, 206), VEGF-C and VEGF-D were determined by immunohistochemistry, Western blotting and ELISA in 46 patients with Head and Neck Squamous Cell Carcinoma (HNSCC), 30 healthy donors, and two HNSCC tumor cell lines (PCI-1 and PCI-13). RESULTS: Increased expression of VEGF-A and VEGF-C was found in tumor tissues compared to normal epithelium (P=0.001). However, VEGF-D levels were decreased in patients with cervical nodal metastasis as compared to patients with negative lymph node status. VEGF-A plasma levels were increased in patients with lymph node metastasis (266 pg/ml) compared to patients with negative lymph node status (19.8 pg/ml). Multivariate analysis demonstrated that VEGF-A correlated with microvessel density (P=0.01), disease progression (P=0.038), a reduced number of local and peripheral mature dendritic cells (DC) (P=0.015) and an increased number of peripheral immature DCs (P=0.05). DCs incubated with tumor supernatant or VEGF-A differentiated into immature DCs and did not develop full allostimulatory activity. Allogenic T cells, when co-cultured with these immature DCs, expressed the T regulatory cell marker CD25, CTLA-4, and CD45Ro, and secreted TGF-beta, VEGF-A and IL-10. CONCLUSIONS: Taken together, our results identify VEGF-A as a multifunctional factor involved in angiogenesis, tumor progression, immunosuppression and immune tolerance.