The importin beta superfamily member RanBP17 exhibits a role in cell proliferation and is associated with improved survival of patients with HPV+ HNSCC.

BACKGROUND: More than twenty years after its discovery, the role of the importin beta superfamily member Ran GTP-binding protein (RanBP) 17 is still ill defined. Previously, we observed notable RanBP17 RNA expression levels in head and neck squamous cell carcinoma (HNSCC) cell lines with disruptive TP53 mutations. METHODS: We deployed HNSCC cell lines as well as cell lines from other tumor entities such as HCT116, MDA-MB-231 and H460, which were derived from colon, breast and lung cancers respectively. RNAi was used to evaluate the effect of RanBP17 on cell proliferation. FACS analysis was used for cell sorting according to their respective cell cycle phase and for BrdU assays. Immunocytochemistry was deployed for colocalization studies of RanBP17 with Nucleolin and SC35 (nuclear speckles) domains. TCGA analysis was performed for prognostic assessment and correlation analysis of RanBP17 in HNSCC patients. RESULTS: RNAi knockdown of RanBP17, significantly reduced cell proliferation in HNSCC cell lines. This effect was also seen in the HNSCC unrelated cell lines HCT116 and MDA-MB-231. Similarly, inhibiting cell proliferation with cisplatin reduced RanBP17 in keratinocytes but lead to induction in tumor cell lines. A similar observation was made in tumor cell lines after treatment with the EGFR kinase inhibitor AG1478. In addition to previous reports, showing colocalization of RanBP17 with SC35 domains, we observed colocalization of RanBP17 to nuclear bodies that are distinct from nucleoli and SC35 domains. Interestingly, for HPV positive but not HPV negative HNSCC, TCGA data base analysis revealed a strong positive correlation of RanBP17 RNA with patient survival and CDKN2A. CONCLUSIONS: Our data point to a role of RanBP17 in proliferation of HNSCC and other epithelial cells. Furthermore, RanBP17 could potentially serve as a novel prognostic marker for HNSCC patients. However, we noted a major discrepancy between RanBP17 RNA and protein expression levels with the used antibodies. These observations could be explained by the presence of additional RanBP17 splice isoforms and more so of non-coding circular RanBP17 RNA species. These aspects need to be addressed in more detail by future studies.

Improving cardiotoxicity prediction in cancer treatment: integration of conventional circulating biomarkers and novel exploratory tools.

Early detection strategies and improvements in cancer treatment have dramatically reduced the cancer mortality rate in the United States (US). However, cardiovascular (CV) side effects of cancer therapy are frequent among the 17 million cancer survivors in the US today, and cardiovascular disease (CVD) has become the second leading cause of morbidity and mortality among cancer survivors. Circulating biomarkers are ideal for detecting and monitoring CV side effects of cancer therapy. Here, we summarize the current state of clinical studies on conventional serum and plasma CVD biomarkers to detect and prevent cardiac injury during cancer treatment. We also review how novel exploratory tools such as genetic testing, human stem cell-derived cardiomyocytes, Omics technologies, and artificial intelligence can elucidate underlying molecular and genetic mechanisms of CV injury and to improve predicting cancer therapy-related cardiotoxicity (CTRC). Current regulatory requirements for biomarker qualifications are also addressed. We present generally applicable lessons learned from published studies, particularly on how to improve reproducibility. The combination of conventional circulating biomarkers and novel exploratory tools will pave the way for precision medicine and improve the clinical practice of prediction, detection, and management of CTRC.

Real-time monitoring of circulating tumor cell (CTC) release after nanodrug or tumor radiotherapy using in vivo flow cytometry.

Noninvasive biological readouts of tumor metastatic risk and therapeutic efficacy are needed as healthcare costs rise. CTCs are the source of metastasis in distant organs that are responsible for the majority of cancer-related deaths. Here we demonstrate the acute and long-term effect of vascular disrupting therapies (high-dose radiotherapy and tumor necrosis factor-alpha (TNF)) on CTCs released from the primary tumor with a non-invasive real-time in vivo flow cytometry system. Using our innovative flow cytometry platform, we show here that radiation and nanodrug treatment can lead to short term release of CTC from the primary tumor. There was no increase in metastasis frequency or extent between control and TNF-treated mice; however, a significant reduction in lung metastasis was noted in the radiotherapy alone group. Mice treated with both TNF and radiotherapy had a slightly elevated metastatic profile between that of radiation alone and control (untreated) tumors. Possible mechanisms based on therapy specific vessel disruption and cell death are discussed. Overall, CTCs correlated with tumor progression and suggest CTC enumeration described herein may be useful in clinical management of solid tumor malignancies.

In vivo noninvasive analysis of graphene nanomaterial pharmacokinetics using photoacoustic flow cytometry.

Graphene-based nanomaterials (GBNs) are quickly revolutionizing modern electronics, energy generation and storage, clothing and biomedical devices. Due to GBN's variety of physical and chemical parameters that define their toxicity and their aggregation in suspension, interpreting its toxicology without accurate information on graphene's distribution and behavior in live organisms is challenging. In this work, we present a laser-based optical detection methodology for noninvasive detection and pharmacokinetics analysis of GBNs directly in blood flow in mice using in vivo photoacoustic (PA) flow cytometry (PAFC). PAFC provides unique insight on how chemical modifications of GBNs affect their distribution in blood circulation and how quickly they are eliminated from the flow. Overall, PAFC provided unique data crucial for understanding GBN toxicity through real-time detection of GBNs using their intrinsic light absorption contrast. Copyright © 2017 John Wiley & Sons, Ltd.

MicroRNA-3666 Regulates Thyroid Carcinoma Cell Proliferation via MET.

BACKGROUND/AIMS: Thyroid carcinoma (TC) is a highly lethal malignant cancer and its carcinogenesis remains undetermined. Dysregulation of microRNAs (miRNAs) is well known to be involved in the development of various cancers, including TC, whereas a role of miR-3666 in the pathogenesis of TC has not been appreciated. METHODS: We analyzed the levels of MET and miR-3666 in TC tissue and the relationship of miR-3666 levels with patients' prognosis. We then overexpressed miR-3666 by miRNA mimics transfection and inhibited miR-3666 by miRNA antisense transfection in TC cells. Cell survival and growth were analyzed by CCK-8 assay and MTT assay, respectively. Cell apoptosis and proliferation were analyzed by flow cytometry. Bioinformatics analyses were applied to predict miR-3666 targets, which was then confirmed using luciferase reporter assay. RESULTS: We detected significantly higher levels of MET, and significantly lower levels of miR-3666 in TC tissue, compared to the adjacent non-tumor tissue. Moreover, the low miR-3666 levels were associated with poor survival of the patients. Overexpression of miR-3666 significantly inhibited cell growth, while depletion of miR-3666 increased cell growth in TC cells. Moreover, the effects of miR-3666 on cell growth appeared to result from alteration in cell proliferation, rather than changes in cell apoptosis. MiR-3666 was found to bind to the 3'-UTR of MET mRNA to inhibit its translation in TC cells. CONCLUSION: Reduced miR-3666 levels in TC tissue may promotes TC growth, possibly through MET-mediated cell proliferation.

In vivo photoacoustic flow cytometry for early malaria diagnosis.

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry.

Anatomical study of surgical approaches for minimally invasive transoral thyroidectomy: eMIT and TOPP.

OBJECTIVE: Anatomical study of surgical approaches of endoscopic minimally invasive thyroidectomy (eMIT) and transoral partial parathyroidectomy (TOPP) was conducted to evaluate their safety and feasibility. MATERIAL AND METHODS: After performing an eMIT- and TOPP-procedure on fresh frozen human cadavers, a layer-by-layer dissection of the floor of the mouth and the anterior cervical region was carried out in five specimens. The blood vessels, nerves and muscles related to the surgical approach were exposed. RESULTS: The anterior region of the neck can be reached through the midline of the mouth floor and the suprahyoid muscles. No important nerves and vessels were found in the approach of eMIT. TOPP set up the space at the dorsal side of the thyroid gland and adjacent to the trachea. The hypoglossal nerve and the lingual nerve as well as their accompanying blood vessels were anatomically related to the approach and could be injured during the procedure. The surgical space is much limited in TOPP (<20 mm in diameter) and current surgical instruments still did not match the requirement of this technique. CONCLUSIONS: This study demonstrated that the transoral approach of eMIT is anatomically safer and more feasible than that of TOPP.

Predicting oncology drug-induced cardiotoxicity with donor-specific iPSC-CMs-a proof-of-concept study with doxorubicin.

Many oncology drugs have been found to induce cardiotoxicity in a subset of patients, which significantly limits their clinical use and impedes the benefit of lifesaving anticancer treatments. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carry donor-specific genetic information and have been proposed for exploring the interindividual difference in oncology drug-induced cardiotoxicity. Herein, we evaluated the inter- and intraindividual variability of iPSC-CM-related assays and presented a proof of concept to prospectively predict doxorubicin (DOX)-induced cardiotoxicity (DIC) using donor-specific iPSC-CMs. Our findings demonstrated that donor-specific iPSC-CMs exhibited greater line-to-line variability than the intraindividual variability in impedance cytotoxicity and transcriptome assays. The variable and dose-dependent cytotoxic responses of iPSC-CMs resembled those observed in clinical practice and largely replicated the reported mechanisms. By categorizing iPSC-CMs into resistant and sensitive cell lines based on their time- and concentration-related phenotypic responses to DOX, we found that the sensitivity of donor-specific iPSC-CMs to DOX may predict in vivo DIC risk. Furthermore, we identified a differentially expressed gene, DND microRNA-mediated repression inhibitor 1 (DND1), between the DOX-resistant and DOX-sensitive iPSC-CMs. Our results support the utilization of donor-specific iPSC-CMs in assessing interindividual differences in DIC. Further studies will encompass a large panel of donor-specific iPSC-CMs to identify potential novel molecular and genetic biomarkers for predicting DOX and other oncology drug-induced cardiotoxicity.

Epidermal growth factor-induced modulation of cytokeratin expression levels influences the morphological phenotype of head and neck squamous cell carcinoma cells.

The migratory ability of tumor cells requires cytoskeletal rearrangement processes. Epidermal growth factor receptor (EGFR)-signaling tightly correlates with tumor progression in head and neck squamous cell carcinomas (HNSCCs), and has previously been implicated in the regulation of cytokeratin (CK) expression. In this study, HNSCC cell lines were treated with EGF, and CK expression levels were monitored by Western blot analysis. Changes in cellular morphology were documented by fluorescence- and atomic force microscopy. Some of the cell lines demonstrated an EGF-dependent modulation of CK expression levels. Interestingly, regression of some CK subtypes or initial up-regulation followed by downregulation at higher EGF-levels could also be observed in the tested cell lines. Overall, the influence of EGF on CK expression levels appeared variable and cell-type-dependent. Real-time cellular analysis of EGF-treated and -untreated HNSCC cell lines demonstrated a rise over time in cellular impedance. In three of the EGF-treated HNSCC cell lines, this rise was markedly higher than in untreated controls, whereas in one of the cell lines the gain of cellular impedance was paradoxically reduced after EGF treatment, which was found to correlate with changes in cellular morphology rather than with relevant changes in cellular viability or proliferation. After treating HNSCC cells with EGF, CK filaments frequently appeared diffusely distributed throughout the cytoplasm, and in some cases were found in a perinuclear localization, the latter being reminiscent to observations by other groups. In summary, the data points to a possible role of EGFR in modulating HNSCC cell morphology.

Sex-Related Differences in Drug-Induced QT Prolongation and Torsades de Pointes: A New Model System with Human iPSC-CMs.

Numerous drugs have the potential to prolong the QT interval and may cause accidental cardiac arrest (torsades de pointes [TdP]). Women are at a higher risk than men for experiencing drug-induced TdP. Due to the lack of appropriate tools, few studies have investigated whether genetic differences between men and women have any effects on drug-induced proarrhythmia. Sex hormones are believed to play a predominant role in the induction of TdP. Recently, progress in induced pluripotent stem cell (iPSC) technologies has made it possible to utilize human iPSC-derived cardiomyocytes (hiPSC-CMs) to investigate the influence of both genetics and sex hormones on cardiac ion channel gene expression and cardiomyocyte function. In this study, we investigated genetic and hormonal effects on sex differences of drug-induced QT prolongation and TdP with hiPSC-CMs from healthy male and female donors. We found that despite batch variations in beating rates and field potential durations (FPD), female-derived hiPSC-CMs showed steeper slopes of FPD to interspike interval ratios and were more sensitive to IKr blocker-induced FPD prolongation. 17ß-estradiol increased FPD and 5α-dihydrotestosterone shortened FPD, but the addition of sex hormones had limited effect on the responses of hiPSC-CMs to IKr blockades. The differential expression of KCNE1 gene and reduced repolarization reserve in female-derived hiPSC-CMs compared with male-derived hiPSC-CMs may partially explain why females are more susceptible to proarrhythmias. Human iPSC-CMs can be a useful new model to study mechanisms of sex differences in cardiomyocyte repolarization processes and aid in the prediction of drug-induced proarrhythmias in both men and women.

Photoacoustic in vitro flow cytometry for nanomaterial research.

Conventional flow cytometry is a versatile tool for drug research and cell characterization. However, it is poorly suited for quantification of non-fluorescent proteins and artificial nanomaterials without the use of additional labeling. The rapid growth of biomedical applications for small non-fluorescent nanoparticles (NPs) for drug delivery and contrast and therapy enhancement, as well as research focused on natural cell pigments and chromophores, demands high-throughput quantification methods for the non-fluorescent components. In this work, we present a novel photoacoustic (PA) fluorescence flow cytometry (PAFFC) platform that combines NP quantification though PA detection with conventional in vitro flow cytometry sample characterization using fluorescence labeling. PAFFC simplifies high-throughput analysis of cell-NP interactions, optimization of targeted nanodrugs, and NP toxicity assessment, providing a direct correlation between NP uptake and characterization of toxicity markers for every cell.

Photoacoustic Flow Cytometry for Single Sickle Cell Detection In Vitro and In Vivo.

Control of sickle cell disease (SCD) stage and treatment efficiency are still time-consuming which makes well-timed prevention of SCD crisis difficult. We show here that in vivo photoacoustic (PA) flow cytometry (PAFC) has a potential for real-time monitoring of circulating sickled cells in mouse model. In vivo data were verified by in vitro PAFC and photothermal (PT) and PA spectral imaging of sickle red blood cells (sRBCs) expressing SCD-associated hemoglobin (HbS) compared to normal red blood cells (nRBCs). We discovered that PT and PA signal amplitudes from sRBCs in linear mode were 2-4-fold lower than those from nRBCs. PT and PA imaging revealed more profound spatial hemoglobin heterogeneity in sRBCs than in nRBCs, which can be associated with the presence of HbS clusters with high local absorption. This hypothesis was confirmed in nonlinear mode through nanobubble formation around overheated HbS clusters accompanied by spatially selective signal amplification. More profound differences in absorption of sRBCs than in nRBCs led to notable increase in PA signal fluctuation (fluctuation PAFC mode) as an indicator of SCD. The obtained data suggest that noninvasive label-free fluctuation PAFC has a potential for real-time enumeration of sRBCs both in vitro and in vivo.

In Vivo Long-Term Monitoring of Circulating Tumor Cells Fluctuation during Medical Interventions.

The goal of this research was to study the long-term impact of medical interventions on circulating tumor cell (CTC) dynamics. We have explored whether tumor compression, punch biopsy or tumor resection cause dissemination of CTCs into peripheral blood circulation using in vivo fluorescent flow cytometry and breast cancer-bearing mouse model inoculated with MDA-MB-231-Luc2-GFP cells in the mammary gland. Two weeks after tumor inoculation, three groups of mice were the subject of the following interventions: (1) tumor compression for 15 minutes using 400 g weight to approximate the pressure during mammography; (2) punch biopsy; or (3) surgery. The CTC dynamics were determined before, during and six weeks after these interventions. An additional group of tumor-bearing mice was used as control and did not receive an intervention. The CTC dynamics in all mice were monitored weekly for eight weeks after tumor inoculation. We determined that tumor compression did not significantly affect CTC dynamics, either during the procedure itself (P = 0.28), or during the 6-week follow-up. In the punch biopsy group, we observed a significant increase in CTC immediately after the biopsy (P = 0.02), and the rate stayed elevated up to six weeks after the procedure in comparison to the tumor control group. The CTCs in the group of mice that received a tumor resection disappeared immediately after the surgery (P = 0.03). However, CTC recurrence in small numbers was detected during six weeks after the surgery. In the future, to prevent these side effects of medical interventions, the defined dynamics of intervention-induced CTCs may be used as a basis for initiation of aggressive anti-CTC therapy at time-points of increasing CTC number.

A cisplatin-resistant head and neck cancer cell line with cytoplasmic p53(mut) exhibits ATP-binding cassette transporter upregulation and high glutathione levels.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) cell lines with cytoplasmically sequestered mutant p53 (p53(mut_c)) are frequently more resistant to cisplatin (CDDP) than cells with mutant but nuclear p53 (p53(mut_n)). The aim of the study was to identify underlying mechanisms implicated in CDDP resistance of HNSCC cells carrying cytoplasmic p53(mut). METHODS: Microarray analysis, quantitative reverse transcription polymerase chain reaction, Western blot analysis and immunocytochemistry were used to identify and evaluate candidate genes involved in CDDP resistance of p53(mut_c) cells. RNAi knockdown or treatment with inhibitors together with flow cytometry-based methods was used for functional assessment of the identified candidate genes. Cellular metabolic activity was assessed with the XTT assay, and the redox capacity of cells was evaluated by measuring cellular glutathione (GSH) levels. RESULTS: Upregulation of ABCC2 and ABCG2 transporters was observed in CDDP-resistant p53(mut_c) HNSCC cells. Furthermore, p53(mut_c) cells exhibited a pronounced side population that could be suppressed by RNAi knockdown of ABCG2 as well as treatment with the ATP-binding-cassette transporter inhibitors imatinib, MK571 and tariquidar. Metabolic activity and cellular GSH levels were higher in CDDP-resistant p53(mut_c) cells, consistent with a higher capacity to fend off cytotoxic oxidative effects such as those caused by CDDP treatment. Finally, ABCC2/G2 inhibition of HNSCC cells with MK571 markedly enhanced CDDP sensitivity of HNSCC cells. CONCLUSIONS: The observations in this study point to a major role of p53(mut_c) in conferring a stem cell like phenotype to HNSCC cells that is associated with ABCC2/G2 overexpression, high GSH and metabolic activity levels as well as CDDP resistance.

SIVmac239-Nef down-regulates cell surface expression of CXCR4 in tumor cells and inhibits proliferation, migration and angiogenesis.

AIM: To evaluate if the lentiviral accessory protein Nef can down-regulate the C-X-C chemokine receptor type 4 (CXCR4) in tumor cells and affect tumor cell proliferation, migration and angiogenesis. MATERIALS AND METHODS: HeLa-(ACC) cells, which according to genotype analysis are virtually identical to the cervical cancer-derived HeLa cell line, were transfected with Nef from SIV(mac239) and expression levels of cell surface CXCR4 were monitored by flow cytometry. Real-time proliferation and migration of cells was measured with the xCELLigence system or with the in vitro scratch assay. In vitro tube formation was deployed to assess the effect of Nef on angiogenesis. RESULTS: Cell surface down-regulation of CXCR4 was observed in HeLa-(ACC) cells after Nef transfection, as well as in the monkey kidney-derived COS-7 cell line after co-transfection of CXCR4 and Nef. Proliferation, as well as migration, of Nef-transfected HeLa-(ACC) cells appeared to be significantly reduced. In vitro tube formation was markedly lowered after Nef transfection, and CXCR4 knockdown with siRNA. CONCLUSION: SIV-Nef could serve as an interesting tool to study the biological behavior of CXCR4-expressing tumor cells and could be helpful in the discovery of new therapeutic approaches for the treatment of CXCR4-positive tumors.

Runx3 expression in lymph nodes with metastasis is associated with the outcome of gastric cancer patients.

Accumulating evidence shows that runt-related transcription factor 3 (Runx3) is a putative tumor suppressor in various types of cancer, the lower levels of which are associated with a less favorable cancer outcome. However, these studies were restricted to primary cancer lesions. Lymph node metastasis (LNM) is a significant factor in determining the prognosis of patients with gastric cancer and is a frequent target of chemotherapy. In the present study, we investigated the expression of Runx3 in the lymph nodes (LNs) of stomach carcinoma and the association of Runx3 expression with the prognosis of patients. The expression of Runx3 in LNs with and without metastasis was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The positive rate of Runx3 mRNA in LNM specimens was significantly lower (28.4%, 21 out of 74) compared to that of the non-metastatic samples (33.3%, 9 out of 27, P<0.05). Similar findings were obtained by Western blotting. Univariate analysis revealed that the loss of Runx3 expression in LNs was not only associated with poor clinicopathological factors, such as LNM, distant organ metastasis, later clinicopathological stages and deep infiltration, but also with a lower 5-year survival rate and poorer prognosis. These results strongly suggest a potential diagnostic value of Runx3 expression in LNs and multiple pathways contributing to the outcome of patients with gastric cancer.

Nef from SIV(mac239) decreases proliferation and migration of adenoid-cystic carcinoma cells and inhibits angiogenesis.

The HIV/SIV accessory protein Nef is known to down-modulate cell surface receptors that are required for virus entry such as CD4, CCR5 and CXCR4 to block lethal viral superinfection of the infected cell. The chemokine receptor CXCR4 also plays an important role in promoting cell proliferation, metastasis and tumor angiogenesis. Therefore it was of interest to evaluate if Nef can down-regulate CXCR4 in tumor cells since this could affect these critical prognostic parameters. The CXCR4-expressing cell line ACC3 that was derived from a salivary gland adenoid cystic carcinoma (ACC) of the head and neck was transfected with Nef from SIV(mac239) and cell surface expression of the receptor was monitored by FACS analysis. Real time proliferation of cells was measured with the xCELLigence system (Roche, Mannheim, Germany). Cell migration was detected by an in vitro scratch assay. Similarly, COS-7 cells were co-transfected with CXCR4 and Nef and were treated as described for ACC3. In vitro tube formation was deployed to assess the effect of Nef on angiogenesis. siRNA was used for CXCR4 knockdown. Cell surface down-modulation of endogenous CXCR4 could be observed in ACC3 cells after Nef-transfection as well as in COS-7 cells after co-transfection of CXCR4 and Nef. Proliferation as well as migration of Nef-transfected ACC3 tumor cells appeared significantly reduced. In vitro tube formation was significantly lowered after Nef-transfection or CXCR4 knockdown with siRNA. SIV-Nef could serve as an interesting tool to study the biologic behavior of CXCR4-expressing tumors such as ACC. Deploying SIV-Nef thereby could help in the discovery of new therapeutic approaches for the treatment of ACC and other CXCR4-expressing tumors.

Differential expression of VEGF121, VEGF165 and VEGF189 in angiomas and squamous cell carcinoma cell lines of the head and neck.

Vascular endothelial growth factor A (VEGF) is one of the major regulators of angiogenesis. It plays an important role during the process of physiological and pathological neovascularization. Variant VEGF isoforms are generated as a result of alternative pre-mRNA splicing. To determine the expression of VEGF isoforms in angioma and head and neck squamous cell carcinoma (HNSCC), both being dependent on pathological neovascularization, we included 11 HNSCC cell lines, 4 hemangiomas and 5 vascular malformations (VMs) in the study. Tonsil mucosa served as normal control. Using reverse transcription polymerase chain reaction (RT-PCR) sequencing, the VEGF isoforms VEGF(189), VEGF(165) and VEGF(121) were regularly detected in all tested samples. VEGF(121) was the most abundant isoform in all tested tissues, whereas VEGF(165) exhibited lower levels, and VEGF(189) only very small amounts of transcript. Interestingly, VMs expressed significantly higher (p=0.0286) amounts of VEGF(121) compared with hemangiomas, which had levels similar to normal control mucosa. HNSCC cell lines demonstrated on-average higher levels of all three isoforms compared with the controls. Consistent with the clinical staging, a trend for VEGF overexpression was observed in tumor cells derived from N+ tumors compared to those derived from N0 tumors. One drawback of this study was the small number of specimens available, particularly since VMs and hemangiomas are relatively rare diseases. Future studies need to follow-up on these observations and further evaluate the potential role of specific VEGF isoforms in the pathogenesis of hemangioma and VM.