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Objective: The current study aimed to investigate the characteristics of tumor-associated macrophages (TAMs) and their association with microvascular density (MVD) in tumor progression in different grades of orofacial squamous cell carcinoma (OSCC) in the Pakistani population., Study Design: This prospective study included 234 patients with oral cancer reported at different hospitals in Pakistan diagnosed with OSCC. Tumors were graded on the Anneroth grading system and the association between the frequency of TAMs and MVD was examined in vivo. The macrophages visible through immunohistochemistry for CD68 and the microvessels observed through immunohistochemistry for CD34 were manually counted in 3 high-power fields., Results: The CD68 and CD34 counts were significantly lower in well-differentiated squamous cell carcinoma compared to poorly differentiated squamous cell carcinoma. Linear regression analysis revealed a positive correlation between the area percentage of CD68 immunoreactivity and the grade of the tumor (r = 0.776). Vice versa, a positive correlation also existed between the area percentage of CD34 immunoreactivity and the grade of the tumor (r = 0.690). Pearson correlation revealed a positive association between the TAMs and MVD (r = 0.680; P < .001)., Conclusions: There was an increased population of tumor-associated macrophages and tumor angiogenesis with the increasing grade of orofacial squamous cell carcinoma. (Oral Surg Oral Med Oral Pathol Oral Radiol YEAR;VOL:page range)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

At present, the molecular mechanisms driving the progression and metastasis of oral squamous cell carcinoma (OSCC) remain largely uncharacterized. The activation of transforming growth factor-β (TGF-β) signaling in the tumor microenvironment has been observed in various types of cancer and has been implicated their progression by enhancing the migration and invasion of epithelial cancer cells. However, its specific roles in the oral cancer progression remain unexplored. In this study, we examined the effects of TGF-β signaling on the murine squamous cell carcinoma, SCCVII cells in vitro and in vivo. The incubation of SCCVII cells with TGF-β induced the activation of TGF-β signals and epithelial-mesenchymal transition (EMT). Notably, the motility of SCCVII cells was increased upon the activation of the TGF-β signaling. RNA sequencing revealed upregulation of genes related to EMT and angiogenesis. Consistent with these in vitro results, the inhibition of TGF-β signals in SCCVII cell-derived primary tumors resulted in suppressed angiogenesis. Furthermore, we identified six candidate factors (ANKRD1, CCBE1, FSTL3, uPA, TSP-1 and integrin β3), whose expression was induced by TGF-β in SCCVII cells, and associated with poor prognosis for patients with head and neck squamous cell carcinoma. These results highlight the role of TGF-β signals in the progression of OSCC via multiple mechanisms, including EMT and angiogenesis, and suggest novel therapeutic targets for the treatment of OSCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background/aim: Oral squamous cell carcinoma (OSCC) is characterized by early metastasis, clinical resistance and poor prognosis. Recently, we showed that aggressive OSCC cells co-express endothelial cell markers and can form tube-like structures, known as vasculogenic mimicry (VM), a process associated with poor prognosis in head and neck cancers. Given the limited success of current antiangiogenic therapy in treating OSCC, this study sought to explore the efficiency of these drugs in targeting an ex vivo model of VM., Materials and Methods: OSCC cell lines from the tongue and floor of the mouth in addition to human endothelial cells were used. The treatments comprised a set of clinically relevant antiangiogenic drugs: sorafenib, sunitinib, and axitinib, which were administered in different doses. Multiple ex vivo approaches including cell tubulogenesis, proliferation, apoptosis, and migration assays were used., Results: Although these drugs inhibited the formation of endothelial cell capillaries, they showed clear differential effects on OSCC cell-derived VM and cell morphology. Sorafenib inhibited the tubulogenesis of aggressive OSCC cells compared with the limited effect of sunitinib and axitinib. Furthermore, our data consistently demonstrated a preferential efficacy of certain drugs over others. Sorafenib and sunitinib exhibited anti-cancer effects on tumor cell proliferation, apoptosis, and cell migration, compared with the limited effect of axitinib., Conclusion: The antiangiogenic drugs, except sorafenib, had limited effect on VM formation in vitro and exhibited varying anti-cancer effects on OSCC cells. These data support the notion that VM formation may in part explain the development of drug resistance in OSCC cells., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objectives: This study aimed to investigate the role of blood and lymphatic microvascular density in the progression of oral squamous cell carcinoma (OSCC)., Materials and Methods: The sample was composed of 54 cases of OSCC. The immunoexpression to anti-alpha-smooth muscle actin (α-SMA) and to anti-endoglin (CD105) was used to determine the microvessel density (MVD); anti-podoplanin (D2-40) was used to assess the lymphatic vessel density (LVD); vascular endothelial growth factor (VEGF) was evaluated in malignant cells. The histological differentiation, the worst pattern of invasion (WPOI), tumour thickness and tumour budding (TB) intensity were assessed using haematoxylin-eosin and anti-pan-cytokeratin (AE1/AE3). Patients' age and sex, TNM classification and follow-up time were collected from the medical records., Results: MVD markers presented a similar pattern of expression in blood vessels. However, only α-SMA + MVD was significantly higher among women and in tumours ≤4 cm. LVD was lower in tumours with lymph node metastasis. Regarding the histological parameters, high TB intensity was associated with histological differentiation, advanced clinical stage, greater tumour thickness and reduced disease-free survival. No difference was found in VEGF., Conclusions: The decrease in OSCC LVD could be related to pathological node involvement, whereas high TB intensity could indicate OSCC progression and worse patient outcomes., (© 2023 Wiley Periodicals LLC.)

Targeting tumor angiogenesis is an important approach in advanced tumor therapy. Here we investigated the effect of the suppressor of variegation 3-9 homolog 1 (SUV39H1) on tumor angiogenesis in oral squamous cell carcinoma (OSCC). The GEPIA database was used to analyze the expression of SUV39H1 in various cancer tissues. The expression of SUV39H1 in OSCC was detected by immunohistochemistry, and the correlation between SUV39H1 and Notch1 and microvascular density (MVD) was analyzed. The effect of SUV39H1 inhibition on OSCC was investigated in vivo by chaetocin treatment. The migration and tube formation of vascular endothelial cells by conditioned culture-medium of different treatments of oral squamous cell cells were measured. The transcriptional level of SUV39H1 is elevated in various cancer tissues. The transcription level of SUV39H1 in head and neck squamous cell carcinoma was significantly higher than that in control. Immunohistochemistry result showed increased SUV39H1 expression in OSCC, which was significantly correlated with T staging. The expression of SUV39H1 was significantly correlated with Notch1 and CD31. In vivo experiment chaetocin treatment significantly inhibit the growth of tumor, and reduce SUV39H1, Notch1, CD31 expression. The decreased expression of SUV39H1 in OSCC cells lead to the decreased expression of Notch1 and VEGF proteins, as well as the decreased migration and tube formation ability of vascular endothelial cells. Inhibition of Notch1 further enhance this effect. Our results suggest inhibition of SUV39H1 may affect angiogenesis by regulating Notch1 expression. This study provides a foundation for SUV39H1 as a potential therapeutic target for OSCC., Competing Interests: The authors declare that they have no competing interests., (© 2024 Chen et al.)

Objective: Treatment of orofacial tumors in dogs is associated with high morbidity and reliable prognostic factors are lacking. Dynamic contrast-enhanced computed tomography (DCECT) can be used to assess tumor perfusion. The objectives of this study were to describe the perfusion parameters of different types of orofacial tumors and to describe the changes in perfusion parameters during radiotherapy (RT) in a subset of them., Animals: 11 dogs with orofacial tumors prospectively recruited., Clinical Presentation and Procedures: All dogs had baseline DCECT to assess blood volume (BV), blood flow (BF), and transit time (TT). Five dogs had repeat DCECT during megavoltage RT., Results: 5 squamous cell carcinomas, 3 sarcomas, 1 melanoma, 1 histiocytic sarcoma, and 1 acanthomatous ameloblastoma were included. Blood volume and BF were higher in squamous cell carcinomas than in sarcomas, although no statistical analysis was performed. At repeat DCECT, 4 dogs showed a reduction in the size of their tumor during RT. Among these dogs, 3 showed an increase in BV and BF and 1 a decrease in these parameters between the baseline and the follow-up DCECT. The only dog whose tumor increased in size between the first and the second DCECT showed a decrease in BV and BF., Clinical Relevance: Perfusion parameters derived from DCECT were described in a series of dogs with various types of orofacial tumors. The results suggest that epithelial tumors could have higher BV and BF than mesenchymal tumors, although larger sample sizes are needed to support these preliminary findings.

Background: The incidence of oral squamous cell carcinoma (OSCC) is very high in South Asia and Vascular endothelial growth factor (VEGF) is one of the key factors essential for cancer growth. The importance of VEGF-A and VEGF Receptor 2(VEGFR-2) in oral cancer pathophysiology is yet to be decided. Vascular Endothelial Growth Factor A (VEGF-A) is the main factor concerned in angiogenesis in tumors, but its role in Oral Squamous Cell Carcinoma (OSCC) is still debatable. Our study aimed to determine the role of VEGF-A and VEGFR-2 in OSCC., Methods: Blood from 30 patients with primary OSCC and 1:1 age-sex-matched controls was subjected to qPCR and ELISA to detect VEGF-A gene expression and serum level. Tumors of the 30 patients were investigated for VEGF Receptor-2 (VEGFR-2) expression and were analyzed using Image J software version 1.52 for DAB percentage (DAB-P) area and optical density (OD)., Results: VEGF-A relative gene expression among patients was 2.43-fold higher compared to the healthy control group. Well-differentiated had a 1.98-fold increment, while poorly differentiated had a 3.58-fold increment. Serum VEGF-A was significantly elevated among the patients compared to controls (458.7 vs 253.2, p=0.0225). Poorly differentiated had a higher serum VEGF concentration (1262.0±354.7pg/ml) compared with other two. Mean VEGFR-2 DAB-P level in OSCC was 42.41±5.61(p=0.15). Well-differentiated had a DAB-P of 41.20±5.32 while poorly differentiated had DAB-P 46.21±3.78. The mean OD in OSCC was 0.54±0.16. VEGFR-2 OD in well and poorly differentiated OSCC were 0.48±0.12 and 0.68±0.17, respectively., Conclusions: VEGF-A gene expression, serum levels, and tissue VEGFR-2 levels correlated linearly with the stage and grade of the tumor. This study justifies the value of VEGF-A as a potential biomarker in OSCC in early detection of OSCC. More studies are needed to accept the use of VEGF-A.

Aim: To investigate the computed tomography (CT) and integrated positron-emission tomography (PET)/CT findings of primary pulmonary lymphoepithelioma-like carcinoma (PLELC)., Materials and Methods: The imaging and histopathological data of 215 patients with PLELC confirmed at histopathology were analysed retrospectively. All patients underwent CT, and 70 underwent PET/CT. None of the cohort had nasopharyngeal lymphoepithelioma-like carcinoma., Results: The PLELC was demonstrated as a solitary nodule/mass in 188 cases (188/215, 87%), multiple nodules/masses in 12 cases (12/215, 6%), lobar or segmental consolidation in 15 cases (15/215, 7%). The tumour showed a well-defined margin in 171 cases (171/215, 80%), lobular sign in 177 cases (177/215, 82%), and spicule sign in 91 cases (91/215, 42%). Most of the cases showed homogeneous density in unenhanced CT (128/215, 60%), and vascular shadows inside the tumour in the arterial stage were found in 105 cases (105/158, 66%). Involvement of the bronchus was found in 154 cases (154/215, 72%). Hilar or mediastinal lymph nodes were enlarged in 160 patients (160/215, 74%). Seventy cases demonstrated avid 2-[ 18 F]-fluoro-2-deoxy-d-glucose (FDG) uptake on PET/CT. The range of maximum standardised uptake values (SUVmax) was 2.1-28.5 (14 ± 5.93). Microscopic pathological classification of 124 resected specimens included 87 cases of the Regaud type and 37 cases of the Schmincke type. Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) was positive in all 215 cases., Conclusion: PLELC should be suspected when a large, lobulate, well-defined lung tumour with homogeneous density, vascular encasement, and high 18 F-FDG uptake is found. Moreover, EBERs are helpful in patients with suspected PLELC., (Copyright © 2021 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.)

We evaluated cervical volume and spectral Doppler parameters: peak systolic velocity (PSV), resistance index (RI) and pulsatility index (PI) in the tumour dominant vessel of 50 patients with cervical squamous cell carcinoma (SCC) staged IIB and IIIB and their changes during treatment. The patients underwent transvaginal Doppler ultrasonography prior to treatment, after external beam radiation therapy (EBRT) and 6 weeks after brachytherapy. Radiotherapy decreased cervical volume and PSV values of the tumour dominant vessel. The PSV values before EBRT in G1 + G2 tumours were higher than in G3 tumours. No correlations between cervical volume, PSV, RI and PI values with disease-free survival (DFS) and overall survival (OS) were found. We concluded, that sonographic assessment of changes in cervical volume of patients with locally advanced cervical SCC during treatment did not allow to predict treatment results. Spectral Doppler parameters PSV, RI and PI of tumour dominant vessel did not predict prognosis for these patients.Impact Statement What is already known on this subject? Transvaginal Doppler sonography is considered as a useful diagnostic method in patients with cervical carcinoma. However, despite numerous studies, the value of spectral Doppler parameters in tumour dominant vessel and cervical volume of patients with locally advanced cervical SCC is still not well defined. What the results of this study add? In our prospective study, we found that sonographic assessment of changes in cervical volume of patients with locally advanced cervical SCC during treatment did not allow to predict treatment results and spectral Doppler parameters of tumour dominant vessel did not predict prognosis for these patients. What the implications are of these findings for clinical practice and/or further research? Our study underlines the limited value of spectral Doppler technique in patients with cervical carcinoma. Further research should be focussed on identifying and validating novel prognostic and predictive factors.

Cylindromatosis (CYLD) is a deubiquitinase (DUB) enzyme that was initially characterized as a tumor suppressor of adnexal skin tumors in patients with CYLD syndrome. Later, it was also shown that the expression of functionally inactive mutated forms of CYLD promoted tumor development and progression of non-melanoma skin cancer (NMSC). However, the ability of wild-type CYLD to inhibit skin tumorigenesis in vivo in immunocompetent mice has not been proved. Herein, we generated transgenic mice that express the wild type form of CYLD under the control of the keratin 5 (K5) promoter (K5-CYLDwt mice) and analyzed the skin properties of these transgenic mice by WB and immunohistochemistry, studied the survival and proliferating characteristics of primary keratinocytes, and performed chemical skin carcinogenesis experiments. As a result, we found a reduced activation of the nuclear factor kappa B (NF-κB) pathway in the skin of K5-CYLDwt mice in response to tumor necrosis factor-α (TNF-α); accordingly, when subjected to insults, K5-CYLDwt keratinocytes are prone to apoptosis and are protected from excessive hyperproliferation. Skin carcinogenesis assays showed inhibition of tumor development in K5-CYLDwt mice. As a mechanism of this tumor suppressor activity, we found that a moderate increase in CYLD expression levels reduced NF-κB activation, which favored the differentiation of tumor epidermal cells and inhibited its proliferation; moreover, it decreased tumor angiogenesis and inflammation. Altogether, our results suggest that increased levels of CYLD may be useful for anti-skin cancer therapy.

Cancer-associated fibroblast (CAF) secretes extracellular vesicle (EV)-encapsulated microRNAs (miRNAs) which have been underlined great promise for therapeutic target for diseases and cancers. Our study aimed to explore the role of EV-encapsulated miR-10a-5p from CAFs in angiogenesis in cervical cancer. Expression of miR-10a-5p in clinical samples of cervical cancer and cancer cells was detected by in situ hybridization and RT-qPCR. Results demonstrated that miR-10a-5p expression was upregulated in both cancer tissues and cells. CAFs and normal fibroblasts (NFs) from cervical cancer patient tissues were characterized under transmission electron microscopy, followed by EV isolation from CAFs. The EVs labeled with PKH67 were cultured with cervical squamous cell carcinoma (CSCC) cell line (SiHa) and HUVECs. Data indicated that CAF-EVs were internalized by cancer cells and promoted cell proliferation and tube formation. CAF-EVs then were transfected with miR-10a-5p inhibitor and then injected into nude mice. While injection of CAF-EVs promoted tumor growth and increased VEGFR and CD31 expression level, miR-10a-5p inhibitor-treated CAF-EVs resulted in decreased tumor volume and amount of vessel around tumor. Of note, dual-luciferase reporter gene assay and bioinformatic analysis indicated TBX5 as a target gene of miR-10a-5p. Moreover, EV-derived miR-10a-5p promoted angiogenesis in vivo and in vitro through activation of Hedgehog signaling via downregulation of TBX5. Taken altogether, miR-10a-5p in CAF-EVs promoted CSCC cell angiogenesis and tumorigenicity via activation of Hh signaling by inhibition of TBX5, providing insight into novel treatment based on miR-10a-5p against CSCC.

Objective: To investigate the effects of Semaphorin 4A (Sema4A) on the angiogenesis, migration and invasion of oral squamous cell carcinoma (OSCC) cells., Methods: Sema4A expression in OSCC patients was detected by Immunohistochemistry, and its relationship with clinicopathological features and prognosis of patients was analyzed. The mRNA and protein expression of Sema4A in primary human oral keratinocytes (HOKs) and OSCC cells (SCC-25, HSC-3, CAL-27) were determined by Western blotting and qRT-PCR. After HOKs, HSC-3 cells and SCC-25 cells transfected with Control/Sema4A CRISPR activation plasmid, the migration and invasion abilities were detected by Wound healing and Transwell invasion. Tube formation assay was also performed on endothelial cells and the contents of VEGF and bFGF were quantified using qRT-PCR and ELISA., Results: Cytoplasmic Sema4A expression was related to T classification, clinical stage and nodal metastasis of OSCC patients. Patients with low cytoplasmic Sema4A expression showed the higher microvessel density (MVD) and the poorer prognosis in OSCC. Compared with HOK, OSCC cells (SCC-25, HSC-3, CAL-27) declined apparently in Sema4A expression, which was much more significant in metastatic HSC-3 and SCC-25 cells. After HOKs, HSC-3 cells and SCC-25 cells transfected with Sema4A over-expression plasmid, the invasion and migration abilities were decreased. Besides, overexpression of Sema4A could significantly inhibit the tube formation of HUVEC induced by OSCC cells with reductions of angiogenic factors (VEGF and bFGF)., Conclusion: Over-expression of Sema4A could restrict tumor progression through inhibiting the angiogenesis, invasion and migration of OSCC cells., (Copyright © 2021. Published by Elsevier Ltd.)

Background: The E6 oncoproteins of human papillomavirus (HPV) 16/18 are the critical drivers of cervical cancer (CC) progression. Extracellular vesicles (EVs) are emerging as critical mediators of cancer-tumor microenvironment (TME) communication. However, whether EVs contribute to HPV 16/18 E6-mediated impacts on CC progression remains unclear., Methods: A series of in vitro and in vivo assays were performed to elucidate the roles and mechanism of EV-Wnt7b in HPV E6-induced CC angiogenesis. The prognostic value of serum EV-Wnt7b was determined and a predictive nomogram model was established., Results: HPV 16/18 E6 upregulated Wnt7b mRNA expression in four HPV 16/18-positive CC cell lines and their EVs. In vitro and in vivo experiments demonstrated that EV-Wnt7b mRNA was transferred to and modulated human umbilical vein endothelial cells (HUVECs) toward more proliferative and proangiogenic behaviors by impacting β-catenin signaling. Clinically, serum EV-Wnt7b levels were elevated in CC patients and significantly correlated with an aggressive phenotype. Serum EV-Wnt7b was determined to be an independent prognostic factor for CC overall survival (OS) and recurrence-free survival (RFS). Notably, we successfully established a novel predictive nomogram model using serum EV-Wnt7b, which showed good prediction of 1- and 3-year OS and RFS., Conclusions: Our results illustrate a potential crosstalk between HPV 16/18-positive CC cells and HUVECs via EVs in the TME and highlight the potential of circulating EV-Wnt7b as a novel predictive biomarker for CC prognosis.

Objective: The aim of this study was to predict response to neoadjuvant chemotherapy (NAC) in patients with locally advanced hypopharyngeal cancer by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Methods: A retrospective study enrolled 46 diagnosed locally advanced hypopharyngeal cancer. DCE-MRI were performed prior to and after two cycles of NAC. The volume transfer constant (K trans ), extracellular extravascular volume fraction (V e ), and plasma volume fraction (K ep ) were computed from primary tumors. DCE-MRI parameters were used to measure tumor response according to the Response Evaluation Criteria in Solid Tumors criteria (RECIST). Results： After 2 NAC cycles, 30 out of 46 patients were categorized into the responder group, whereas the other 16 were categorized into non-responder group. Compared with the pretreatment value, the post-treatment K trans and K ep was significantly lower ( P < 0.05), but no significant change in V e ( P > 0.05). Compared with non-responders, a notably higher pretreatment K trans , K ep , lower post-treatment K trans , higher ΔK trans and ΔK ep were observed in responders (all P < 0.05). While the pretreatment V e , post-treatment V e , and ΔV e did not differ significantly ( P> 0.05) between the two groups. The receiver operating characteristic curve analysis revealed that pretreatment K trans of 0.202/min is the most optimal cut-off in predicting response to chemotherapy, resulting in an AUC of 0.837 and corresponding sensitivity and specificity of 76.7%, and 81.1%, respectively. Conclusion: DCE-MRI especially pretreatment K trans can potentially predict the treatment response to neoadjuvant chemotherapy for hypopharyngeal cancer. Advances in knowledge: Few studies of DCE-MRI on hypopharyngeal cancer treated with chemoradiation reported. The results demonstrate that DCE-MRI especially pretreatment K trans may be more potential value in predicting the treatment response to neoadjuvant chemotherapy for hypopharyngeal cancer.

Squamous cell carcinoma of the uterine cervix is the second most common malignancy in women worldwide. We describe an unusual telangiectatic variant of squamous cell carcinoma in a 53 yr old woman. The tumor showed the usual morphologic features of a poorly differentiated keratinizing squamous cell carcinoma with >75% tumor area showing cavernous hemangioma like ectatic spaces filled with blood. The blood-filled spaces lacked an endothelial lining as evidenced by negativity for CD31 and CD34. This unusual variant has not been reported previously. Awareness of this entity is necessary for avoiding confusion with vascular tumors such as hemangiomas and angiosarcoma.

The establishment of a proangiogenic phenotype and epithelial-to-mesenchymal transition (EMT) are considered as critical events that promote the induction of invasive growth in epithelial tumors, and stimulation of lymphangiogenesis is believed to confer the capacity for early dissemination to cancer cells. Recent research has revealed substantial interdependence between these processes at the molecular level as they rely on common signaling networks. Of great interest are the molecular mechanisms of (lymph-)angiogenesis and EMT associated with the earliest stages of transition from intraepithelial development to invasive growth, as they could provide the source of potentially valuable tools for targeting tumor metastasis. However, in the case of early-stage cervical cancer, the players of (lymph-)angiogenesis and EMT processes still remain substantially uncharacterized. In this study, we used RNA sequencing to compare transcriptomes of HPV(+) preinvasive neoplastic lesions and early-stage invasive carcinoma of the cervix and to identify (lymph-)angiogenesis- and EMT-related genes and pathways that may underlie early acquisition of invasive phenotype and metastatic properties by cervical cancer cells. Second, we applied flow cytometric analysis to evaluate the expression of three key lymphangiogenesis/EMT markers (VEGFR3, MET, and SLUG) in epithelial cells derived from enzymatically treated tissue specimens. Overall, among 201 differentially expressed genes, a considerable number of (lymph-)angiogenesis and EMT regulatory factors were identified, including genes encoding cytokines, growth factor receptors, transcription factors, and adhesion molecules. Pathway analysis confirmed enrichment for angiogenesis, epithelial differentiation, and cell guidance pathways at transition from intraepithelial neoplasia to invasive carcinoma and suggested immune-regulatory/inflammatory pathways to be implicated in initiation of invasive growth of cervical cancer. Flow cytometry showed cell phenotype-specific expression pattern for VEGFR3, MET, and SLUG and revealed correlation with the amount of tumor-infiltrating lymphocytes at the early stages of cervical cancer progression. Taken together, these results extend our understanding of driving forces of angiogenesis and metastasis in HPV-associated cervical cancer and may be useful for developing new treatments.

Background: The clinicopathological distinctness of oral squamous cell carcinoma arising in the background of oral submucous fibrosis (OSCC-OSF) is well known; however, the molecular distinctness of this unique OSCC-OSF has not been investigated to date. With this in mind, we compared the expression of Ki67, CD105, and α-SMA between OSCC-OSF and oral squamous cell carcinoma (OSCC)., Methods: Formalin-fixed paraffin-embedded tissues of 105 OSCC-OSF and 112 OSCC cases were subjected to immunohistochemistry for evaluation of Ki67, CD105, and α-SMA expression., Results: Ki67 (labeling index) LI, MVD and α-SMA expression were significantly higher in OSCC compared to OSCC-OSF. Ki67 LI and MVD was significantly higher in OSCC compared to OSCC-OSF in parameters such as well-differentiated, early TNM stage, non-metastatic, and more than 3-year survival. α-SMA expression was significantly higher in OSCC compared to OSCC-OSF in parameters such as moderate differentiation, metastatic lesions, and survival less than 3 years. Ki67 LI, MVD and α-SMA showed significant positive correlation with each other in OSCC and OSCC-OSF., Conclusion: Proliferation, neoangiogenesis and myofibroblast differentiation were significantly higher in the OSCC group compared to the OSCC-OSF group. This suggests the biological distinctness of OSCC-OSF, which could help the future development of targeted therapies.

Background: Anti-angiogenic therapy represents a promising strategy for non-small-cell lung cancer (NSCLC) but its application in lung squamous cell carcinoma (SQC) is limited due to the high-risk adverse effects. Accumulating evidence indicates that long noncoding RNAs (lncRNAs) mediate in tumor progression by participating in the regulation of VEGF in NSCLC, which might guide the development of new antiangiogenic strategies., Methods: Differential lncRNA expression in SQC was analyzed in AE-meta and TCGA datasets, and further confirmed in lung cancer tissues and adjacent normal tissues with RT-qPCR and in-situ hybridization. Statistical analysis was performed to evaluate the clinical correlation between LINC00173.v1 expression and survival characteristics. A tube formation assay, chick embryo chorioallantoic membrane assay and animal experiments were conducted to detect the effect of LINC00173.v1 on the proliferation and migration of vascular endothelial cells and tumorigenesis of SQC in vivo. Bioinformatics analysis, RNA immunoprecipitation and luciferase reporter assays were performed to elucidate the downstream target of LINC00173.v1. The therapeutic efficacy of antisense oligonucleotide (ASO) against LINC00173.v1 was further investigated in vivo. Chromatin immunoprecipitation and high throughput data processing and visualization were performed to identify the cause of LINC00173.v1 overexpression in SQC., Results: LINC00173.v1 was specifically upregulated in SQC tissues, which predicted poorer overall and progression-free survival in SQC patients. Overexpression of LINC00173.v1 promoted, while silencing LINC00173.v1 inhibited the proliferation and migration of vascular endothelial cells and the tumorigenesis of SQC cells in vitro and in vivo. Our results further revealed that LINC00173.v1 promoted the proliferation and migration of vascular endothelial cells and the tumorigenesis of SQC cells by upregulating VEGFA expression by sponging miR-511-5p. Importantly, inhibition of LINC00173.v1 via the ASO strategy reduced the tumor growth of SQC cells, and enhanced the therapeutic sensitivity of SQC cells to cisplatin in vivo. Moreover, our results showed that squamous cell carcinoma-specific factor ΔNp63α contributed to LINC00173.v1 overexpression in SQC., Conclusion: Our findings clarify the underlying mechanism by which LINC00173.v1 promotes the proliferation and migration of vascular endothelial cells and the tumorigenesis of SQC, demonstrating that LINC00173.v1-targeted drug in combination with cisplatin may serve as a rational regimen against SQC.

Background: Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have longterm effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model., Objective: In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors., Materials and Methods: Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis., Results: All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins., Conclusion: These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis.

Background: Quantification of pharmacokinetic parameters in dynamic contrast enhanced (DCE) MRI is heavily dependent on the arterial input function (AIF). In the present patient study on advanced stage head and neck squamous cell carcinoma (HNSCC) we have acquired DCE-MR images before and during chemo radiotherapy. We determined the repeatability of image-derived AIFs and of the obtained kinetic parameters in muscle and compared the repeatability of muscle kinetic parameters obtained with image-derived AIF's versus a population-based AIF., Materials and Methods: We compared image-derived AIFs obtained from the internal carotid, external carotid and vertebral arteries. Pharmacokinetic parameters (v e , K trans , k ep ) in muscle-located outside the radiation area-were obtained using the Tofts model with the image-derived AIFs and a population averaged AIF. Parameter values and repeatability were compared. Repeatability was calculated with the pre- and post-treatment data with the assumption of no DCE-MRI measurable biological changes between the scans., Results: Several parameters describing magnitude and shape of the image-derived AIFs from the different arteries in the head and neck were significantly different. Use of image-derived AIFs led to higher pharmacokinetic parameters compared to use of a population averaged AIF. Median muscle pharmacokinetic parameters values obtained with AIFs in external carotids, internal carotids, vertebral arteries and with a population averaged AIF were respectively: v e (0.65, 0.74, 0.58, 0.32), K trans (0.30, 0.21, 0.13, 0.06), k ep (0.41, 0.32, 0.24, 0.18). Repeatability of pharmacokinetic parameters was highest when a population averaged AIF was used; however, this repeatability was not significantly different from image-derived AIFs., Conclusion: Image-derived AIFs in the neck region showed significant variations in the AIFs obtained from different arteries, and did not improve repeatability of the resulting pharmacokinetic parameters compared with the use of a population averaged AIF. Therefore, use of a population averaged AIF seems to be preferable for pharmacokinetic analysis using DCE-MRI in the head and neck area., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Amsterdam University Medical Centres. Published by Elsevier Inc. All rights reserved.)

Background: The objectives of the present study were to evaluate angiogenesis and mast cell density in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC)., Materials and Methods: This was an observational, retrospective and quantitative study. The samples consisted of 60 tissue specimens from patients with squamous cell carcinoma, epithelial dysplasia and controls (n=20/group). Immunohistochemistry was performed using an anti-tryptase antibody to mast cells and anti-CD31 and anti-CD34 for blood vessels and we count the number of mast cells and determine the percentage of CD31 and CD34 antibody staining (vascular density)., Results: The mast cells had lower density in OSCC compared to control and dysplasia (p = 0.009). In angiogenesis, the expression of CD31 showed a higher percentage of blood vessels in OSCC (p < 0.001), however, CD34 showed no difference between groups (p=0.092). The CD31 antibody presented as a high immunostaining in oral mucosa than CD34., Conclusions: The increased vascularity in squamous cell carcinoma suggests that angiogenesis begins when malignant transformation starts that seems to be inversely associated with the number of mast cells.

Objectives: The goals of the present study were to prospectively analyze salvage surgery with microvascular reconstruction in recurrent squamous cell carcinoma of the oral cavity (OSCC) in terms of oncological outcome and quality of life., Patients and Methods: From 2012 to 2015, 28 patients underwent salvage surgery due to recurrent OSCC or second primary OSCC without the option of curative re-irradiation. Endpoints were disease-specific survival and progression-free survival after 12 months. The survival was estimated by using the Kaplan-Meier blotting. Quality of life data (European Organization for Research and Treatment of Cancer - EORTC: QLQ-C30 and QLQ-H&N35) was assessed at baseline and subsequently every 3 months up to one year., Results: Estimated 1-year-survival was 68.4% and progression-free survival was 38.5%. Overall quality of life was significantly reduced three months after salvage surgery [baseline (mean 64.15) versus time 1 (mean 53.04); p = 0.002]. However, the patients experienced a recovery within the first year [baseline (mean 64.15) versus time 4 (mean 70.33); p = 0.176]. Furthermore, the sensation of pain is significantly reduced after salvage surgery [baseline (mean 47.53) versus time 2 (mean 31.25); p = 0.036]. Microvascular reconstruction success rate was 93.1%., Conclusion: Salvage surgery is a curative treatment option in recurrent and intensively pretreated OSCC. Microvascular reconstruction is feasible with acceptable morbidity and high success rates. Quality of life can be preserved. Further studies combining checkpoint inhibition with salvage surgery are justified., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The natriuretic peptide (NP) system comprises of three ligands, the Atrial Natriuretic Peptide (ANP), Brain Natriuretic peptide (BNP) and C-type Natriuretic peptide (CNP), and three natriuretic peptide receptors, NPRA, NPRB and NPRC. Here we present a comprehensive study of the natriuretic peptide system in healthy murine and human submandibular salivary glands (SMGs). We show CNP is the dominant NP in mouse and human SMG and is expressed together with NP receptors in ducts, autonomic nerves and the microvasculature of the gland, suggesting CNP autocrine signalling may take place in some of these glandular structures. These data suggest the NP system may control salivary gland function during homeostasis through the regulation of electrolyte re-absorption, neural stimulation and/or blood vessel wall contraction/relaxation. We also show abnormal expression of NPRA in the stroma of a subset of human SMGs resected from patients diagnosed with oral squamous cell carcinoma (OSCC) of non-salivary gland origin. This finding warrants further research to investigate a possible correlation between early OSCC invasion and NPRA overexpression.

It is well known that Prospero homeobox 1 (PROX1) is a crucial regulator of lymphangiogenesis, that reprograms blood endothelial cells to lymphatic phenotype. However, the role of PROX1 in tumor progression, especially in angiogenesis remains controversial. Herein, we studied the role of PROX1 in angiogenesis in cell lines derived from follicular thyroid cancer (FTC: FTC-133) and squamous cell carcinoma of the thyroid gland (SCT: CGTH-W-1) upon PROX1 knockdown. The genes involved in angiogenesis were selected by RNA-seq, and the impact of PROX1 on vascularization potential was investigated using human umbilical vein endothelial cells (HUVECs) cultured in conditioned medium collected from FTC- or SCT-derived cancer cell lines after PROX1 silencing. The angiogenic phenotype was examined in connection with the analysis of focal adhesion and correlated with fibroblast growth factor 2 (FGF2) levels. Additionally, the expression of selected genes involved in angiogenesis was detected in human FTC tissues. As a result, we demonstrated that PROX1 knockdown resulted in upregulation of factors associated with vascularization, such as metalloproteinases (MMP1 and 3), FGF2, vascular endothelial growth factors C (VEGFC), BAI1 associated protein 2 (BAIAP2), nudix hydrolase 6 (NUDT6), angiopoietin 1 (ANGPT1), and vascular endothelial growth factor receptor 2 (KDR). The observed molecular changes resulted in the enhanced formation of capillary-like structures by HUVECs and upregulated focal adhesion in FTC-133 and CGTH-W-1 cells. The signature of selected angiogenic genes' expression in a series of FTC specimens varied depending on the case. Interestingly, PROX1 and FGF2 showed opposing expression levels in FTC tissues and seven thyroid tumor-derived cell lines. In summary, our data revealed that PROX1 is involved in the spreading of thyroid cancer cells by regulation of angiogenesis.

Various local flaps have been defined for small skin defects of the nose. However, the repair of large nasal defects is only possible with flaps allowing a large tissue transfer, such as a free flap, forehead flap, and nasolabial flap. In this study, large nasal defects were reconstructed with extended central artery perforator propeller (CAPP) flaps in an attempt to describe a single-stage procedure as an alternative technique to the median forehead flap. Thirteen large nasal skin defects, including dorsum and nasal sidewall and/or dome, were repaired with a CAPP flap between January 2015 and March 2018. A total of 13 patients aged 19 to 92 years were included. The mean follow-up period was 14.9 months. Pathological diagnoses were basal cell carcinoma in 5 patients, squamous cell carcinoma in 6 patients, and trauma in 2 patients. Defect size ranged between 3 × 3 and 4 × 5 cm. Flap size ranged between 3 × 7 and 5 × 10 cm. No major complications including total flap failure, hematoma, or infection were observed. However, a partial flap necrosis occurred in 1 patient. In 3 patients, scar revision surgery was performed at the postoperative period. In conclusion, CAPP flap use is a safe and reliable option to repair large nasal defects. This flap is able to cover large nasal defects including dorsal, dome, and nasal sidewall defects in a single-stage procedure. Requiring no pedicle separation, this flap is an alternative option to the conventional median forehead flap.

Aims: Recently, cancer-derived exosomes were shown to have pro-metastasis function in cancer, but the mechanism remains unclear. Angiogenesis is essential for tumor progression and is a great promising therapeutic target for advanced cervical cancer. Here, we investigated the role of cervical cancer cell-secreted exosomal miR-221-3p in tumor angiogenesis., Methods and Results: miR-221-3p was found to be closely correlated with microvascular density in cervical squamous cell carcinoma (CSCC) by evaluating the microvascular density with immunohistochemistry and miR-221-3p expression with in situ hybridization in clinical specimens. Using the groups of CSCC cell lines (SiHa and C33A) with miR-221-3p overexpression and silencing, the CSCC exosomes were characterized by electron microscopy, western blotting, and fluorescence microscopy. The enrichment of miR-221-3p in CSCC exosomes and its transfer into human umbilical vein endothelial cells (HUVECs) were confirmed by qRT-PCR. CSCC exosomal miR-221-3p promoted angiogenesis in vitro in Matrigel tube formation assay, spheroid sprouting assay, migration assay, and wound healing assay. Then, exosome intratumoral injection indicated that CSCC exosomal miR-221-3p promoted tumor growth in vivo. Thrombospondin-2 (THBS2) was bioinformatically predicted to be a direct target of miR-221-3p, and this was verified by using the in vitro and in vivo experiments described above. Additionally, overexpression of THBS2 in HUVECs rescued the angiogenic function of miR-221-3p., Conclusions: Our results suggest that CSCC exosomes transport miR-221-3p from cancer cells to vessel endothelial cells and promote angiogenesis by downregulating THBS2. Therefore, CSCC-derived exosomal miR-221-3p could be a possible novel diagnostic biomarker and therapeutic target for CSCC progression.

Rationale and Objectives: To explore the imaging features of whole uterus volume CT perfusion (vCTP) and the influence factors of blood supply in cervical squamous carcinoma (CSC)., Materials and Methods: vCTP was performed on a 640-slice computed tomography system in 43 patients with CSC diagnosed by biopsy, and 24 cases of them underwent magnetic resonance imaging. The size of the tumor was measured on vCTP and magnetic resonance (MR) images. Perfusion parameters, including arterial blood flow (AF), blood volume, and permeability surface (PS), were measured by two radiologists, using interclass correlation coefficient to evaluate the interobserver reliability. The difference of tumor size and perfusion data was analyzed by paired t test and rank sum test. The correlation of perfusion parameters with some factors was analyzed by Pearson or Spearman correlation analysis., Results: Tumor sizes were not significantly different between vCTP and MR images. The interclass correlation coefficient of each parameter was 0.818-0.945. The AF value of CSC was significantly higher than normal uterine body, and the blood volume and PS values of CSC were not statistically different compared with those of normal uterine body. There was no significant difference in AF value of CSC among different FIGO stages and pathological grades. The AF and PS values of CSC were negatively correlated with the age of the patients., Conclusion: The vCTP could accurately shows the size of the CSC with use of MR as the reference standard, and its perfusion parameters have good measurement stability; the CSC was hypervascular, but this trend was less pronounced in older women., (Copyright © 2018. Published by Elsevier Inc.)

Objective: To evaluate the ability of pulsatility index (PI), resistance index (RI), and hypoxia inducible factor-1α (HIF-1α) expression in predicting the clinical response after radiation in patients with cervical cancer. Methods: A prospective cohort was carried on in Department of Obstetric and Gynecology Dr. Hasan Sadikin Hospital/ Faculty of Medicine, Padjadjaran University, during the period of July 2017 to March 2018 which include 51 samples with stage IIB to IVA cervical cancer. Tumor perfusion and oxygenation were evaluated using color Doppler ultrasound indices (pulsatility index and resistance index) and the expression of hypoxia inducible factor-1α (HIF-1α). The clinical response was assessed 2 months after external radiation. Result: Among 51 patients, 31 patients demonstrated good response and 20 patients demonstrated poor response to radiation. The mean value of PI was significantly lower in patients who demonstrated good response as compared to patients with poor response (0.84±0.916 vs. 1.70±1.260, p = 0.004). The mean value of RI did not differ significantly (0.29±0.112 vs. to 0.36±0.189 p =0.173). HIF-1α expression was significantly lower in patients who demonstrated good response as compared to patients with poor response (1.83±1.529 vs. 6.55±2.625, p = 0.0001). In multivariate model, PI and HIF-1α expression both predicted the clinical response after radiation. Conclusion: PI and HIF-1α expression predict the clinical response after radiation in patients with cervical cancer.

The highly malignant phenotype of oral squamous cell carcinoma (OSCC), including the presence of nodal and distant metastasis, reduces patient survival. High-mobility group A protein 2 (HMGA2) is a non-histone chromatin factor that is involved in advanced malignant phenotypes and poor prognosis in several human cancers. However, its biological role in OSCC remains to be elucidated. The purpose of this study was to determine the clinical significance and role of HMGA2 in the malignant potential of OSCC. We first investigated the expression pattern of HMGA2 and its clinical relevance in 110 OSCC specimens using immunohistochemical staining. In addition, we examined the effects HMGA2 on the regulation of vascular endothelial growth factor (VEGF)-A, VEGF-C, and fibroblast growth factor (FGF)-2, which are related to angiogenesis, in vitro. High expression of HMGA2 was significantly correlated with distant metastasis and poor prognosis. Further, HMGA2 depletion in OSCC cells reduced the expression of angiogenesis genes. In OSCC tissues with high HMGA2 expression, angiogenesis genes were increased and a high proportion of blood vessels was observed. These findings suggest that HMGA2 plays a significant role in the regulation of angiogenesis and might be a potential biomarker to predict distant metastasis and prognosis in OSCC.

Hematopoietic stem cell transplantation (HSCT) recipients have been reported to have an increased risk of chronic graft versus host disease (cGVHD) and hematological and solid cancers. Oral manifestations are the first signs of cGVHD observed in the majority of patients, and oropharyngeal cancer is the most frequent secondary malignancy occurred after HSCT. In this study, we have evaluated the inflammatory infiltrate cell content and correlated with the vascular density in patients affected by primary oral squamous cell carcinoma (OSCC) from previous healthy controls and OSCC after cGVHD. Results have demonstrated that patients with OSCC after GVHD show a more consistent inflammatory infiltrate as compared with the OSCC ones. In detail, the inflammatory background composed of CD3-positive T cells, tryptase-positive mast cells, CD31-positive endothelial cells, and CD68-positive macrophages may be more pronounced in the setting of GVHD + OSCC than in the control group. By contrast, CD20-positive B cells and CD1a-positive dendritic cells were more abundant in the latter population. Finally, a positive correlation was found as between vascular density and inflammatory cell infiltration in both GVHD + OSCC and OSCC groups. Overall, these results confirm the role played by immune cells in enhancing tumor progression and angiogenesis and suggest a potential therapeutic strategy involving inhibition of recruitment of immune cells to the tumor microenvironment and blockade of pro-tumoral effects and pro-angiogenic functions.

Objective: Despite the existence of detailed consensus guidelines, challenges remain regarding the role angiogenetic factors on non-small cell lung cancer (NSCLC). This study was conducted to determine the role of the vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) and angiopoietin2 (Ang2) in patients with NSCLC. Methods: This study included 64 consecutive patients with non-small cell lung cancer, who admitted to clinic. Pre-treatment serum VEGF, IL-8 and Ang2 levels were evaluated. Patients were treated according to internationally accepted guidelines. Results: VEGF and IL-8 serum levels of patients with both squamous cell carcinoma and adenocarcinoma were significantly higher than controls (p<0.05). In addition, IL-8 levels were lower among treatment-responders than non-responders (p:0.031). Impact of elevated or decreased levels of VEGF, Ang2 and IL-8 on survival was evaluated, accepting median level as reference. There was no correlation between the serum levels of VEGF, Ang2, IL-8 and survival. Conclusion: We found that the levels of angiogenic markers were significantly different between non-small cell lung cancer patients and controls. These markers could elicit more information related to stage and prognosis., (Creative Commons Attribution License)

Objective: The study was to explore the roles of Nck1 in the angiogenesis of cervical squamous cell carcinoma (CSCC)., Methods: mRNA and protein levels were evaluated with real-time quantitative PCR and immunohistochemisty/western blotting respectively. The cancer microvessel density (MVD) was assayed with CD34 endothelial labeling. Nck1 gene knock-in (SiHa-Nck1+) and knock-down (SiHa-Nck1-) were achieved by gene transfection and siRNA respectively. Protein level from cellular supernatant was measured with ELISA. Proliferation, migration and tube formation of the Human Umbilical Vein Endothelial cells (HUVECs) were evaluated by CCK-8 cell viability assay, transwell chamber assay and in vitro Matrigel tubulation assay respectively., Results: Nck1 level gradually increased from normal cervical epithelia to high-grade CIN, overexpressed in CSCC and was associated with cancer MVD. The ability of proliferation, migration and tube formation of HUVECs was enhanced in SiHa-Nck1+-treated while decreased in SiHa-NcK1--treated cells compared to SiHa-control-treated cells. Mechanistically, RAC1-GTP, p-PAK1 and MMP2 were increased in SiHa-NCK1+ cells and pretreatment with the Rac1 inhibitor (NSC23766) significantly decreased their levels. Furthermore, inhibition of PAK1 reduced MMP2 level in SiHa-Nck1+ cells whereas the level of Rac1-GTP was unaltered. Also, inhibition of Rac1 or PAK1 impaired angiogenesis-inducing capacity of cancer cells., Conclusions: Nck1 promotes the angiogenesis-inducing capacity of CSCC via the Rac1/PAK1/MMP2 signal pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recently, exosomes secreted by menstrual mesenchymal stem cells have been identified as inhibitory agents of tumor angiogenesis and modulators of the tumor cell secretome in prostate and breast cancer. However, their direct effect on endothelial cells and paracrine mediators have not yet been investigated. Using a carrier-based cell culture system to test the scalability for exosome production, we showed that different types of endothelial cells present specific kinetics for exosomes internalization. Exosome-treatment of endothelial cells increased cytotoxicity and reduced VEGF secretion and angiogenesis in a dose-dependent manner. Using the hamster buccal pouch carcinoma as a preclinical model for human oral squamous cell carcinoma, we demonstrated a significant antitumor effect of intra-tumoral injection of exosomes associated with a loss of tumor vasculature. These results address up-scaling of exosome production, a relevant issue for their clinical application, and also assess menstrual stem cell exosomes as potential anti-angiogenic agents for the treatment of neoplastic conditions.

Objectives: Evaluate spectral Doppler parameters peak systolic velocity (PSV), end diastolic velocity (EDV), resistance index(RI) and pulsatility index (PI) in infiltrated and non-infiltrated uterine arteries of patients with locally advanced (stages II B,III B) squamous cell cervical cancer and their changes during treatment., Material and Methods: the study group included 36 patients aged 35-78 years old. At diagnosis, PSV, EDV, RI and PI inuterine arteries were examined and compared with MRI findings. All patients underwent transvaginal doppler ultrasonographyprior to the treatment, after external beam radiation therapy and six weeks after the last application of brachytherapy., Results: The median PSV value in the first examination was higher in infiltrated uterine arteries than compared to non-infiltratedones (p = 0.001). The PSV values for all vessels decreased between the first and the third observation (p < 0.001).There was a significant difference in PI values between infiltrated and non-infiltrated uterine arteries between the first andthe third examination (p = 0.027)., Conclusions: In patients with locally advanced cervical cancer of uterine arteries, assessment of PSV but not EDV, RI or PIcan be helpful in differentiating infiltrated from non-infiltrated vessels. In this group of patients, radiotherapy decreasesPSV, but not EDV, RI or PI values in uterine arteries. An observation conducted from the onset of radiotherapy to end ofthe follow-up in uterine arteries reveals that PI, but not RI, PSV or EDV, is different in infiltrated and non-infiltrated vessels.

Cyclin E and hepatocyte growth factor (HGF) have been observed as a multifaceted factor in many cancers, and the assessment of microvascular density (MVD) and micro-lymphatic vessel density (MLVD) has been used to quantify tumor angiogenesis and lymphangiogenesis. The aim of this study was to explore the association between expression of cyclin E, HGF, MVD, and MLVD, and clinicopathologic parameters in esophageal squamous cell carcinoma (ESCC). The expression of cyclin E, HGF, MVD, and MLVD were detected using immunohistochemically anticyclin E, HGF, CD34, and lymphatic vessel endothelial hyaluronan receptor 1 in 168 surgically resected ESCC cases and 30 normal esophageal mucosal samples. The expression levels of cyclin E, HGF, MVD, and MLVD were higher compared to controls. High cyclin E and HGF expression was found more frequently in the tumors larger than 5 cm ( P < .001), with poorer differentiation ( P = .034) and higher tumor node metastasis (TNM) staging ( P = .009) compared to their counterparts. Both MVD and MLVD values were found to be higher in the tumors larger than 5 cm ( P < .001), with poorer differentiation ( P < .001) and higher TNM staging ( P < .001) compared to their counterparts. Furthermore, the expression of MVD and MLVD in both the high cyclin E and high HGF expression groups was significantly higher compared to the low cyclin E and HGF expression groups ( P < .001). This study demonstrated that high cyclin E and HGF expression is closely correlated with tumor size, tumor differentiation degree, and TNM stage in patients with ESCC. These findings proposed that cyclin E and HGF could serve as novel molecular markers for preoperational evaluation of ESCC.

The nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC) is a heterodimeric enzyme with an α and β subunit. NO binds to heme of the β 1 -subunit of sGC, activates the enzyme in the reduced heme iron state in vascular smooth muscle cells (VSMCs), and generates cGMP-inducing vasodilatation and suppression of VSMC proliferation. In the complex tumor milieu with higher levels of reactive oxygen species (ROS), sGC heme iron may become oxidized and insensitive to NO. To change sGC from an NO-insensitive to NO-sensitive state or NO-independent manner, protein expression of sGC in VSMC is required. Whether sGCα 1 β 1 exists at the protein level in arterial VSMCs of oropharyngeal squamous cell carcinoma (OPSCC) is unknown. In addition, whether differences in the genetic profile between human papillomavirus (HPV)-positive and HPV-negative OPSCC contributes to the regulation of sGCα 1 β 1 is unclear. Therefore, we compared the effects of HPV-positive and HPV-negative OPSCC on the expression of sGCα 1 β 1 in arterial VSMCs from tumor-free and tumor-containing regions of human tissue sections using quantitative immunohistochemistry. In comparison to the tumor-free region, we found a decrease in expression of both α 1 - and β 1 -subunits in the arterial VSMC layer of the tumor-containing areas. The OPSCC-induced significant downregulation of the α 1 - and β 1 -subunits of sGC in arterial VSMC was HPV-independent. We conclude that the response of sGC to NO in tumor arterial VSMCs may be impaired by oxidation of the heme of the β 1 -subunit, and thus, α 1 - and β 1 -subunits of sGC could be targeted to degradation under oxidative stress in OPSCC in an HPV-independent manner. The degradation of sGCα 1 β 1 in VSMCs may result in increased proliferation of VSMCs, promoting tumor arteriogenesis in OPSCC. This can be interrupted by preserving the active heterodimer sGCα 1 β 1 in arterial VSMCs.

Background: Tumor-associated neutrophils (TAN), matrix metalloproteinase-9 (MMP-9), interleukin-17 (IL-17), and angiogenesis have been proposed as prognostic biomarkers of malignant tumors. The purpose of this study was to investigate these inflammatory markers as prognostic factors for oral squamous cell carcinoma (OSCC)., Methods: Specimens of OSCC (n = 30), healthy oral mucosa (negative control, n = 10), oral leukoplakia (n = 10), and apical granuloma with abscess (positive inflammatory controls, n = 10) were immunostained for CD66b (neutrophils), MMP-9, IL-17, and CD105 (neoformed microvessels). Semiquantitative (IL-17) and quantitative (CD66b, IL-17, MMP-9, and CD105) analyses were performed. Clinical information (TNM stage, metastasis, recurrence, and survival) and tumor histological grade were also obtained., Results: Positivity for TAN, MMP-9, IL-17, and CD105 was higher in OSCC than in the negative control (P < 0.05) and oral leukoplakia, but similar to the positive inflammatory control. Coincident high counts of inflammatory markers (CD66b, MMP-9, IL-17, and CD105) were associated with lymph node metastasis of OSCC. Associations between high numbers of neoformed microvessels and advanced clinical stage and a higher degree of malignancy were also demonstrated., Conclusions: Combined positivity for TAN, MMP-9, IL-17, and CD105 appears to be associated with the metastasis-prone phenotype of OSCC., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Angiogenesis is crucial for the progression of esophageal squamous cell carcinoma (ESCC). Anti‑angiogenesis by targeting important molecules has been considered as one of the most promising and efficient strategy for cancer therapy. Recent studies have demonstrated that the IQ‑domain GTPase activating protein 1 (IQGAP1) plays critical roles in tumorigenesis and cancer progression. We previously reported that IQGAP1 is overexpressed in ESCC, and IQGAP1 knockdown can decrease cell proliferation and metastasis ability in vitro and in vivo. However, the effects of IQGAP1 on the angiogenesis of ESCC and its underlying mechanisms remain unknown. In the present study, we found that IQGAP1 overexpression promoted tumor angiogenesis confirmed by human umbilical vascular endothelial cell (HUVEC) tube formation in vitro and chicken embryo chorioallantoic membrane (CAM) assay in vivo. Moreover, IQGAP1 overexpression in ESCC cells increased expression of vascular endothelial growth factor (VEGF) and phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). Meanwhile, we found that levels of AKT and ERK phosphorylation were upregulated in IQGAP1‑overexpressing cells. Importantly, IQGAP1‑knockdown cells showed the opposing results. Furthermore, AKT and ERK inhibitors not only significantly decreased VEGF expression and VEGFR2 phosphorylation in IQGAP1‑overexpressing cells, but also abolished the pro‑angiogenic effect of IQGAP1 overexpression on angiogenesis in the HUVEC tube formation and chicken embryo CAM assay. Taken together, this evidence confirms that IQGAP1 overexpression promotes tumor angiogenesis via the AKT and ERK‑mediated VEGF‑VEGFR2 signaling pathway in ESCC, and IQGAP1 may be an attractive therapeutic target for cancer anti‑angiogenesis treatment.

Objective: Squamous cell carcinoma is often associated with the deletion or mutation of zinc finger protein 750 (ZNF750), its deletion or mutation is associated with squamous epithelial malignant biological characteristics. The present study is to explore the mechanism of ZNF750 to suppress the tumor malignant process by regulation tumor microenvironment., Methods: To evaluate the changes of tumor microenvironment in oral squamous cells carcinoma cell line CAL-27 cell, the expression of angiogenin, vascular endothelial growth factor (VEGF), prolyl hydroxylase 2 (PHD2), G protein signal regulated protein 5 (RGS5), integrin A5 (ITGA5), integrin B1 (ITGB1) and CD44 were detected by Western-blot. The changes of platelet derived growth factor (PDGFB) and tumor vascular marker CD105 (Endoglin) mRNA were estimated by qPCR. The effect of over-expressed ZNF750 on cell viability and lateral migration capacity was investigated by CCK-8 and cell scratch assay in three oral squamous cells carcinoma., Results: ZNF750 could effectively inhibit the protein or mRNA expression of angiogenin, VEGF, RGS5 and CD105, repressed the cell adhesion molecules ITGA5, ITGB1 and CD44, but up-regulate the protein or mRNA expression of PHD2 and PDGFB. The cell viability and lateral migration ability of three oral squamous cells carcinoma were reduced by over-expression of ZNF750., Conclusion: ZNF750 could modulate the tumor vascular microenvironment to inhibit the oral squamous cells carcinoma malignant progression., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

PA28γ promotes tumor development and progression and is suggested to play a role in tumor angiogenesis, but the molecular mechanisms have not been investigated. Here, we found that PA28γ enhanced the ability of OSCC cells to promote the migration, invasion, and tube formation of HUVECs and promoted tumor-induced angiogenesis in xenograft mice models in vivo. Then, a mechanism study revealed that the expression and secretion of IL-6 and CCL2 were dependent on PA28γ expression. Furthermore, blocking IL-6 or CCL2 or the transcription factor NF-κB induced the inhibition of tube formation in HUVECs co-cultured with PA28γ-overexpression OSCC cell supernatants. Moreover, we revealed that p-STAT3 and p-AKT, which are downstream of the IL-6 and CCL2 signaling axis, were downregulated in HUVECs co-cultured with the PA28γ-silenced supernatant and were upregulated with the PA28γ-overexpressing supernatant. In addition, IL-6, CCL2 and PA28γ expressions were correlated in a clinical OSCC cohort. Collectively, our study indicates that PA28γ contributes to tumor angiogenesis by regulating IL-6 and CCL2. PA28γ may be a novel therapeutic target as a dual regulator of IL-6 and CCL2 for treating PA28γ-positive OSCC., (Copyright © 2018. Published by Elsevier B.V.)

Background: Angiogenic activity is an important event in oral carcinogenesis. During transition of normal oral mucosa to different grades of dysplasia and to invasive carcinoma, significant increase of vascularity occurs. Angiogenesis can be determined by immunohistochemical assessment of several endothelial cell markers like Endogelin (CD 105), expressed in activated endothelial cells and associated with neovasculature, and the vascular endothelial growth factor (VEGF). This study was conducted to evaluate angiogenic activity in oral precancerous lesions compared with oral invasive carcinomas by immunohistochemical staining of VEGF and CD 105 proteins. Methods: In the present cross-sectional study, 20 normal, 20 pre-cancerous mucosa and 20 oral invasive carcinoma samples were immunohistochemically stained. Positive cells were counted in each section and micro vessel density (MVD) was determined. The data were statistically analyzed by Mann-Whitney and Kruskal-Wallis tests, with a P-value ≤0.05 considered significant. Results: The mean expression value for VEGF was 24.6 in oral invasive carcinoma, 16.4 in precancerous mucosa and 15.5 in normal mucosa, with no significant differences between the latter two. Endoglin was negative in all normal mucosa samples, but had scores of 7.58 for precancerous mucosa and 19.4 in oral invasive carcinoma specimens. MVD was significantly higher in SCC than in dysplastic mucosa. Conclusion: Oral invasive carcinoma has more angiogenic activity in comparison with pre-cancerous lesions and normal mucosa. Given the high expression of CD105 positive vessels in malignant lesions, we can argue that determination of mean vessel density (MVD) by application of the CD105 marker could be a useful parameter to differentiate cancerous from pre-cancerous lesions., (Creative Commons Attribution License)

Purpose: Microvessel density (MVD) corresponds to the intensity of neo-angiogenesis. MVD assessments are based on the expression levels of the vascular endothelium markers such as, e.g., CD34 or CD105. The goal of this study was to assess MVD among patients with head and neck squamous cell carcinoma (HNSCC), and to evaluate the predictive value of MVD in head and neck cancers., Methods: The study included 49 patients treated for HNSCC and 11 patients with dysplasia of the upper respiratory tract epithelium. Control tissues consisted of 12 normal mucous membranes of the throat. Expression levels of MVD markers were assessed by immunohistochemistry (IHC) using tissue microarrays (TMA). Clinicopathological factors and patients' survival over the 5-year follow-up period were analyzed., Results: The MVD/CD34 values were found to be significantly elevated in the HNSCCs compared to the non-malignant control tissues (p = 0.001) and to dysplastic tissues. (p = 0.02). Significantly higher MVD/CD105 values were also seen in the tumor compared to the control tissues (p = 0.001) or the dysplastic tissues (p = 0.001). Unexpectedly, significantly lower MVD/CD34 values were seen in the tumor tissues of patients with the T3-T4 tumors compared to those with T1-T2 tumors (p = 0.01)., Conclusions: HNSCCs have statistically higher MVD values compared to dysplasia of the upper respiratory tract epithelium. However, the MVD/CD34 values did not correlate with local invasiveness (the T feature) of HNSCCs. This counterintuitive observation suggests that assessments of MVD as performed on TMA by IHC using anti-CD34 or anti-CD105 antibodies considered to be specific for endothelial cell markers might underestimate the extent of the tumor vascularity in HNSCC.

Background/aim: One of the most important factors concerning cancer growth is angiogenesis. The purpose of this study was to clarify the relationship of maturation of tumor vessels and prognosis of lung cancer., Materials and Methods: Immunohistochemical stainings of 125 lung cancers for VEGF, CD31 and α-smooth muscle actin (αSMA) were scored by multiplying the intensity and the frequency from 0 to 12., Results: Adenocarcinomas showed significantly higher staining scores of both VEGF and αSMA than squamous cell carcinomas did. In 42 cases of high CD31 score, five-year survival rate (87%) of patients with lung cancer showing mature tumor vessels was significantly better than that (69%) of patients with immature tumor vessels., Conclusion: Not the number of tumor vessels but their maturation may be a prognostic factor of patients with lung cancer. VEGF may not only stimulate proliferation of endothelial cells but also their maturation in differentiated lung cancers., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Aidi injection, a proprietary Chinese medicine, has been widely used for the treatment of cancer. However, its effects and potential mechanism in esophageal squamous cell carcinoma (ESCC) have not yet been characterized. The aim of the present study was to identify the mechanism underlying the anti‑metastatic effects of treatment with Aidi. To test the effects and mechanism, EC9706 and KYSE70 cells were selected for in vitro experiments. In vivo, the anti‑metastatic effects of Aidi injection on a nude mouse peritoneal metastasis model were examined, and the mechanisms were assessed with immunohistochemical staining. A cell proliferation assay demonstrated that treatment with more than 3 mg/ml Aidi for 24 or 48 h significantly inhibited the proliferation of EC9706 (P<0.01) and KYSE70 cells (P<0.05, P<0.01). Subsequent experiments assessed cell migration, invasion and vasculogenic mimicry (VM) formation, with 5‑fluorouracil serving as a positive control. It was observed that treatment with Aidi inhibited cell migration, invasion and VM formation. Furthermore, it was identified that treatment with Aidi inhibited epithelial‑mesenchymal transition (EMT) signaling and the expression of vascular endothelial growth factor A (VEGF‑A) in EC9706 and KYSE70 cells, using western blotting. In the in vivo experiments, Aidi injection effectively suppressed tumor metastasis in the mouse tumor model. Additionally, the expression of vimentin and vascular endothelial growth factor was decreased, and the expression of cadherin‑1 was increased in the tumor tissue. The present results suggested that treatment with Aidi may inhibit tumor metastasis in ESCC through the inhibition of EMT signaling and angiogenesis.

To explore the clinical value of superselective lingual artery embolization in treating the severe hemorrhage in patients with advanced carcinoma of tongue. Four patients with advanced tongue cancer hemorrhage from March 2014 to February 2016 were enrolled in this study. T3N2M0 (2 cases) and T4N1M0 (2 cases) were diagnosed preoperatively. Two cases of advanced tongue carcinoma tumors had severe bleeding and the other 2 cases of hemorrhage were after radiotherapy. All cases including 3 squamous cell carcinoma and 1 adenocarcinoma were firstly demonstrated by arterigraphy under seldinger technique with digtial subtraction angiogarphy to ensure the rupture site and then all cases were followed by superselective artery embolization. The efficacy and complications of interventional embolizationg were observed. There was no serious complication of central nervous system injury such as hemorrhage and hemiplegia during follow-up. Superselective lingual artery embolization can accurately locate the responsibility of blood vessels, and the injury is small, significant effect, fewer complications.

Background: While lung adenocarcinoma patients can somewhat benefit from anti-angiogenic therapies, patients with squamous cell lung carcinoma (SQLC) cannot. The reasons for this discrepancy remain largely unknown. Soluble VEGF receptor-1, namely sVEGFR1-i13, is a truncated splice variant of the cell membrane-spanning VEGFR1 that has no transmembrane or tyrosine kinase domain. sVEGFR1-i13 is mainly viewed as an anti-angiogenic factor which counteracts VEGF-A/VEGFR signalling in endothelial cells. However, its role in tumour cells is poorly known., Methods: mRNA and protein status were analysed by Real-Time qPCR, western blotting, ELISA assay, proximity ligation assay or immunohistochemistry in human tumour cell lines, murine tumourgrafts and non small cell lung carcinoma patients samples., Results: We show that anti-angiogenic therapies specifically increase the levels of sVEGFR1-i13 in SQLC cell lines and chemically induced SQLC murine tumourgrafts. At the molecular level, we characterise a sVEGFR1-i13/β1 integrin/VEGFR autocrine loop which determines whether SQLC cells proliferate or go into apoptosis, in response to anti-angiogenic therapies. Furthermore, we show that high levels of both sVEGFR1-i13 and β1 integrin mRNAs and proteins are associated with advanced stages in SQLC patients and with a poor clinical outcome in patients with early stage SQLC., Conclusions: Overall, these results reveal an unexpected pro-tumoural function of sVEGFR1-i13 in SQLC tumour cells, which contributes to their progression and escape from anti-angiogenic therapies. These data might help to understand why some SQLC patients do not respond to anti-angiogenic therapies.

Background: The antidiabetic drug metformin (Met) is believed to inhibit tumor proliferation by altering the metabolism of cancer cells. In this study, we examined the effects of Met on tumor oxygenation, metabolism, and growth in head and neck squamous cell carcinoma (HNSCC) using non-invasive multimodal imaging., Materials and Methods: Severe combined immunodeficient (SCID) mice bearing orthotopic FaDu HNSCC xenografts were treated with Met (200 mg/kg, ip) once daily for 5 days. Tumor oxygen saturation (%sO 2 ) and hemoglobin concentration (HbT) were measured using photoacoustic imaging (PAI). Fluorescence imaging was employed to measure intratumoral uptake of 2-deoxyglucosone (2-DG) following Met treatment while magnetic resonance imaging (MRI) was utilized to measure tumor volume. Correlative immunostaining of tumor sections for markers of proliferation (Ki67) and vascularity (CD31) was also performed., Results: At 5 days post-Met treatment, PAI revealed a significant increase (P < .05) in %sO 2 and HbT levels in treated tumors compared to untreated controls. Fluorescence imaging at this time point revealed a 46% decrease in mean 2-DG uptake compared to controls. No changes in hemodynamic parameters were observed in mouse salivary gland tissue. A significant decrease in Ki-67 staining (P < .001) and MR-based tumor volume was also observed in Met-treated tumors compared to controls with no change in CD31 + vessel count following Met therapy., Conclusion: Our results provide, for the first time, direct in vivo evidence of Met-induced changes in tumor microenvironmental parameters in HNSCC xenografts. Our findings highlight the utility of multimodal functional imaging for non-invasive mapping of the effects of Met in HNSCC., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)