Efficacy of Cisplatin-CXCR4 Antagonist Combination Therapy in Oral Cancer.

Cisplatin is a platinum-based compound that is widely used for treating inoperable oral squamous cell carcinoma (OSCC) in Japan; however, resistance to cisplatin presents a challenge and innovative approaches are required. We aimed to investigate the therapeutic potential of targeting the chemokine receptor CXCR4, which is involved in angiogenesis and tumor progression, using the CXCR4 inhibitor AMD3100, in combination with cisplatin. AMD3100 induced necrosis and bleeding in OSCC xenografts by inhibiting angiogenesis. We investigated the combined ability of AMD3100 plus cisplatin to enhance the antitumor effect in cisplatin-resistant OSCC. An MTS assay identified HSC-2 cells as cisplatin-resistant cells in vitro. Mice treated with the cisplatin-AMD combination exhibited the most significant reduction in tumor volume, accompanied by extensive hemorrhage and necrosis. Histological examination indicated thin and short tumor vessels in the AMD and cisplatin-AMD groups. These results indicated that cisplatin and AMD3100 had synergistic antitumor effects, highlighting their potential for vascular therapy of refractory OSCC. Antitumor vascular therapy using cisplatin combined with a CXCR4 inhibitor provides a novel strategy for addressing cisplatin-resistant OSCC.

Central dentinogenic ghost cell tumor of the maxilla: a case report with new imaging findings and review of the literature.

A dentinogenic ghost cell tumor (DGCT) is a rare benign odontogenic tumor that commonly shows characteristics of solid proliferation and has a relatively high risk of recurrence after surgical treatment. We herein report a case of a central DGCT that occurred in the maxilla and resulted in bone expansion. This study highlights new imaging findings (particularly magnetic resonance imaging) along with histopathological observations. In addition, we conducted a review of the existing literature on this rare tumor. A 37-year-old man developed swelling around the right cheek. A benign odontogenic tumor such as ameloblastoma was suspected based on the imaging examination findings (including bone expansion and the internal characteristics of the tumor) on panoramic imaging, computed tomography, and magnetic resonance imaging. The lesion was surgically excised from the right maxilla. Postoperative histopathological examination led to a definitive diagnosis of central DGCT. The tumor comprised epithelial neoplastic islands, resembling ameloblastoma, inside tight fibroconnective tissue; masses of ghost cells and formation of dentin were also observed. We had suspected that the minute high-density region around the molars on the imaging examinations represented alveolar bone change; however, it represented dentin formation. This led to difficulty diagnosing the lesion. Although DGCT may present characteristic findings on imaging examinations, its occurrence is infrequent, and in some cases, the findings may include the presence or absence of an impacted tooth without obvious calcification. The present case suggests that we should consider the possibility of an odontogenic tumor with calcification when high-density structures are observed inside the lesion.

Prediction Formula for Pathological Depth of Invasion From Clinical Depth of Invasion in Tongue Squamous Cell Carcinoma (SCC) Stage I/II Cases.

BACKGROUND:  The depth of invasion (DOI) of tongue squamous cell carcinoma (SCC) is an important prognostic factor. The definition is clear for pathological DOI (pDOI), but the treatment strategy is determined by the preoperative clinical DOI (cDOI). Few studies have investigated the difference between these DOIs. The purpose of this study was to obtain the correlation equation between cDOI and pDOI for Stage I/II tongue SCC and to consider the points to be noted in actual clinical practice. METHODS:  In this retrospective study, 58 patients with clinical stage I/II tongue SCC were included. Correlations between cDOI and pDOI were obtained for all 58 cases, as well as for 39 cases which excluded superficial and exophytic lesions. RESULTS:  The overall cDOI and pDOI median values were 8.0 and 5.5 mm, respectively; the 2.5 mm reduction was significant (p < 0.01). The correlation equation was pDOI = 0.81 × cDOI-0.23 (r = 0.73). Furthermore, re-analysis of the 39 cases revealed that pDOI = 0.84 × cDOI-0.37 (r = 0.62). Hence, a derived equation pDOI = 0.84 × (cDOI-0.44) was obtained to predict pDOI from cDOI. CONCLUSIONS:  This study indicated that it is necessary to consider contraction due to specimen fixation by subtracting the thickness of the mucosal epithelium. Clinical T1 cases with a cDOI of 5 mm or less had a pDOI of 4 mm or less, and it would be expected to have low positive rate of neck lymph node metastasis.

Ultrasound procedure for the diagnosis of mass lesions in the oral region.

OBJECTIVES: To examine the diagnostic usefulness and procedures of ultrasonography (US) for mass lesions in the soft tissue of the oral region. METHODS: This study involved patients with mass lesions (tumorous lesions and cysts) who had undergone US and histopathological examinations from January 2017 to December 2019. The following points were evaluated by two observers using an evaluation scale: vascularity, echo intensity level, boundary, margin shape, distribution of internal echoes, and capsule. The usefulness of each point for differential diagnosis of tumorous lesions and cysts was statistically analyzed. RESULTS: Forty-five mass lesions in the soft tissue of the oral region (33 tumorous lesions and 12 cysts) were analyzed. There were significant differences in four evaluation points between the tumorous lesions and cysts: vascularity, echo intensity level, boundary, and margin shape. Cysts were almost completely excluded diagnostically, especially when vascularity was observed. There were also significant differences in two evaluation points between nonvascular tumorous lesions and cysts: echo intensity level and boundary. CONCLUSIONS: In US examination for mass lesions in the oral region, it was possible to diagnose tumorous lesions and exclude cysts when vascularity was observed. When vascularity was not observed, however, tumorous lesions and cysts could be identified using two evaluation points: echo intensity level and boundary.

Guidance on the need for contraception related to use of pharmaceuticals: the Japan Agency for Medical Research and Development Study Group for providing information on the proper use of pharmaceuticals in patients with reproductive potential.

BACKGROUND: The U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) have published guidelines on the use of cancer treatments in young people of reproductive potential. However, no such guideline is available in Japan. Therefore, this project aimed to gather relevant data and draft a respective guidance paper. METHODS: From April 2019 to March 2021, the Study Group for Providing Information on the Proper Use of Pharmaceuticals in Patients with Reproductive Potential at the Japan Agency for Medical Research and Development gathered opinions from experts in reproductive medicine, toxicology, and drug safety measures. The group considered these opinions, the FDA and EMA guidelines, and relevant Japanese guidelines and prepared a guidance paper, which they sent to 19 related organizations for comment. RESULTS: By November 2020, the draft guidance paper was completed and sent to the related organizations, 17 of which provided a total of 156 comments. The study group finalized the guidance paper in March 2021. CONCLUSIONS: The "Guidance on the Need for Contraception Related to Use of Pharmaceuticals" (The report of the Study Group for Providing Information on the Proper Use of Pharmaceuticals in Patients with Reproductive Potential, Research on Regulatory Science of Pharmaceuticals and Medical Devices, Japan Agency for Medical Research and Development: JP20mk0101139) is expected to help Japanese healthcare professionals provide fertility-related care and advice to adolescents, and young adults with cancer and their families.

Resident stroma-secreted chemokine CCL2 governs myeloid-derived suppressor cells in the tumor microenvironment.

Accumulating evidence has shown that cancer stroma and BM-derived cells (BMDCs) in the tumor microenvironment (TME) play vital roles in tumor progression. However, the mechanism by which oral cancer stroma recruits any particular subset of BMDCs remains largely unknown. Here, we sought to identify the subset of BMDCs that is recruited by cancer stroma. We established a sequential transplantation model in BALB/c nude mice, including (a) BM transplantation of GFP-expressing cells and (b) coxenografting of patient-derived stroma (PDS; 2 cases, designated PDS1 and PDS2) with oral cancer cells (HSC-2). As controls, xenografting was performed with HSC-2 alone or in combination with normal human dermal fibroblasts (HDF). PDS1, PDS2, and HDF all promoted BMDC migration in vitro and recruitment in vivo. Multicolor immunofluorescence revealed that the PDS coxenografts recruited Arginase-1+CD11b+GR1+GFP+ cells, which are myeloid-derived suppressor cells (MDSCs), to the TME, whereas the HDF coxenograft did not. Screening using microarrays revealed that PDS1 and PDS2 expressed CCL2 mRNA (encoding C-C motif chemokine ligand 2) at higher levels than did HDF. Indeed, PDS xenografts contained significantly higher proportions of CCL2+ stromal cells and CCR2+Arginase-1+CD11b+GR1+ MDSCs (as receiver cells) than the HDF coxenograft. Consistently, a CCL2 synthesis inhibitor and a CCR2 antagonist significantly inhibited the PDS-driven migration of BM cells in vitro. Furthermore, i.p. injection of the CCR2 antagonist to the PDS xenograft models significantly reduced the CCR2+Arginase-1+CD11b+GR1+ MDSC infiltration to the TME. In conclusion, oral cancer stroma-secreted CCL2 is a key signal for recruiting CCR2+ MDSCs from BM to the TME.

Application of "Tissueoid Cell Culture System" Using a Silicate Fiber Scaffold for Cancer Research.

BACKGROUND: We developed a 3-dimensional (3D) culture system using a high-purity silica fiber scaffold of unwoven sheets called CellbedTM. METHODS: We used adherent colon and esophagogastric junction adenocarcinoma cells, tongue squamous cell carcinoma (SqCC) cells, and nonadherent gastric cancer cells. These cells were subjected to staining with various substances and observed by electron microscopy. To evaluate the effects of extracellular matrix in carcinoma tissues, SqCC cells were cultured in Cellbed coated with collagens I, III, and IV. RESULTS: Especially well-differentiated carcinoma cells cultured in this 3D system showed their own unique characteristics: luminal formation in adenocarcinoma cells and cell stratification and keratinization in SqCC cells. Scanning electron microscopy revealed the proliferation of cancer cells with cytoplasm entwined in Cellbed. Intercellular desmosomes in squamous epithelia were detected by transmission electron microscopy of vertical cross sections. SqCC cells cultured in Cellbed coated with collagen IV showed enhanced invasive and proliferative abilities. CONCLUSION: Because the morphology of cancer cells cultured in this 3D culture system is similar to that in living organisms, we called the system a "tissueoid cell culture system." Coating with collagen IV enables the modification of cell-matrix interactions as well as recapitulation of the in vivo microenvironment.

Differentiation and roles of bone marrow-derived cells on the tumor microenvironment of oral squamous cell carcinoma.

The stroma affects the properties and dynamics of the tumor. Previous studies have demonstrated that bone marrow-derived cells (BMDCs) possess the capability of differentiating into stromal cells. However, the characteristics and roles of BMDCs in oral squamous cell carcinoma remain unclear. The current study therefore investigated their locations and features by tracing green fluorescent protein (GFP)-labeled BMDCs in a transplantation mouse model. After irradiation, BALB-c nu-nu mice were injected with bone marrow cells from C57BL/6-BALB-C-nu/nu-GFP transgenic mice. These recipient mice were then injected subcutaneously in the head with human squamous cell carcinoma-2 cells. Immunohistochemistry for GFP, Vimentin, CD11b, CD31 and α-smooth muscle actin (SMA), and double-fluorescent immunohistochemistry for GFP-Vimentin, GFP-CD11b, GFP-CD31 and GFP-α-SMA was subsequently performed. Many round-shaped GFP-positive cells were observed in the cancer stroma, which indicated that BMDCs served a predominant role in tumorigenesis. Vimentin(+) GFP(+) cells may also be a member of the cancer-associated stroma, originating from bone marrow. Round or spindle-shaped CD11b(+) GFP(+) cells identified in the present study may be macrophages derived from bone marrow. CD31(+)GFP(+) cells exhibited a high tendency towards bone marrow-derived angioblasts. The results also indicated that spindle-shaped α-SMA(+) GFP(+) cells were not likely to represent bone marrow-derived cancer-associated fibroblasts. BMDCs gathering within the tumor microenvironment exhibited multilineage potency and participated in several important processes, such as tumorigenesis, tumor invasion and angiogenesis.

The Role of Sonic Hedgehog Signaling in the Tumor Microenvironment of Oral Squamous Cell Carcinoma.

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of SHH expression appear to correlate with cancer progression. However, the role of SHH in the tumor microenvironment (TME) of oral squamous cell carcinoma (OSCC) is still unclear. No studies have compared the expression of SHH in different subtypes of OSCC and focused on the relationship between the tumor parenchyma and stroma. In this study, we analyzed SHH and expression of its receptor, Patched-1 (PTCH), in the TME of different subtypes of OSCC. Fifteen endophytic-type cases (ED type) and 15 exophytic-type cases (EX type) of OSCC were used. H&E staining, immunohistochemistry (IHC), double IHC, and double-fluorescent IHC were performed on these samples. ED-type parenchyma more strongly expressed both SHH and PTCH than EX-type parenchyma. In OSCC stroma, CD31-positive cancer blood vessels, CD68- and CD11b-positive macrophages, and α-smooth muscle actin-positive cancer-associated fibroblasts partially expressed PTCH. On the other hand, in EX-type stroma, almost no double-positive cells were observed. These results suggest that autocrine effects of SHH induce cancer invasion, and paracrine effects of SHH govern parenchyma-stromal interactions of OSCC. The role of the SHH pathway is to promote growth and invasion.

Tumor Angiogenic Inhibition Triggered Necrosis (TAITN) in Oral Cancer.

CXCR4 is a chemokine receptor crucial in tumor progression, although the angiogenic role of CXCR4 in oral squamous cell carcinoma (OSCC) has not been investigated. Here we show that CXCR4 is crucial for tumor angiogenesis, thereby supporting tumor survival in OSCC. Immunohistochemistry on human clinical specimens revealed that CXCR4 and a tumor vasculature marker CD34 were co-distributed in tumor vessels in human OSCC specimens. To uncover the effects of CXCR4 inhibition, we treated the OSCC-xenografted mice with AMD3100, so-called plerixafor, an antagonist of CXCR4. Notably, we found a unique pathophysiological structure defined as tumor angiogenic inhibition triggered necrosis (TAITN), which was induced by the CXCR4 antagonism. Treatment with AMD3100 increased necrotic areas with the induction of hypoxia-inducible factor-1α in the xenografted tumors, suggesting that AMD3100-induced TAITN was involved in hypoxia and ischemia. Taken together, we demonstrated that CXCR4 plays a crucial role in tumor angiogenesis required for OSCC progression, whereas TAITN induced by CXCR4 antagonism could be an effective anti-angiogenic therapeutic strategy in OSCC treatment.

Antitumor activity of potent pyruvate dehydrogenase kinase 4 inhibitors from plants in pancreatic cancer.

Phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase 4 (PDK4) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is frequently upregulated in various cancer tissues, with its elevation being critical for the induction of the Warburg effect. PDK4 is an attractive target for cancer therapy given its effect on shifting glucose metabolism. Previous research has highlighted the necessity of identifying a potent compound to suppress PDK4 activity at the submicromolar concentrations. Here we identified natural diterpene quinones (KIS compounds) that inhibit PDK4 at low micromolar concentrations. KIS37 (cryptotanshinone) inhibited anchorage-independent growth in three-dimensional spheroid and soft agar colony formation assays of KRAS-activated human pancreatic (MIAPaCa-2 and Panc-1) and colorectal (DLD-1 and HCT116) cancer cell lines. KIS37 also suppressed KRAS protein expression in such cell lines. Furthermore, KIS37 suppressed phosphorylation of Rb protein and cyclin D1 protein expression via the PI3K-Akt-mTOR signaling pathway under nonadherent culture conditions and suppressed the expression of cancer stem cell markers CD44, EpCAM, and ALDH1A1 in MIAPaCa-2 cells. KIS37 also suppressed pancreatic cancer cell growth in both subcutaneous xenograft and orthotopic pancreatic tumor models in nude mice at 40 mg/kg (intraperitoneal dose) without any evident toxicity. Reduced ALDH1A1 expression was observed in KIS37-treated pancreatic tumors, suggesting that cancer cell stemness was also suppressed in the orthotopic tumor model. The aforementioned results indicate that KIS37 administration is a novel therapeutic strategy for targeting PDK4 in KRAS-activated intractable human pancreatic cancer.

Notch Signaling Affects Oral Neoplasm Cell Differentiation and Acquisition of Tumor-Specific Characteristics.

Histopathological findings of oral neoplasm cell differentiation and metaplasia suggest that tumor cells induce their own dedifferentiation and re-differentiation and may lead to the formation of tumor-specific histological features. Notch signaling is involved in the maintenance of tissue stem cell nature and regulation of differentiation and is responsible for the cytological regulation of cell fate, morphogenesis, and/or development. In our previous study, immunohistochemistry was used to examine Notch expression using cases of odontogenic tumors and pleomorphic adenoma as oral neoplasms. According to our results, Notch signaling was specifically associated with tumor cell differentiation and metaplastic cells of developmental tissues. Notch signaling was involved in the differentiation of the ductal epithelial cells of salivary gland tumors and ameloblast-like cells of odontogenic tumors. However, Notch signaling was also involved in squamous metaplasia, irrespective of the type of developmental tissue. In odontogenic tumors, Notch signaling was involved in epithelial-mesenchymal interactions and may be related to tumor development and tumorigenesis. This signaling may also be associated with the malignant transformation of ameloblastomas. Overall, Notch signaling appears to play a major role in the formation of the characteristic cellular composition and histological features of oral neoplasms, and this involvement has been reviewed here.

Host factors influence Barrett's carcinogenesis: findings from a mouse gastroduodenal reflux model.

BACKGROUND: Rat gastroduodenal reflux models have been used for analyzing Barrett's carcinogenesis. Mice seem to be more useful than rats for studies targeting genes. METHODS: We induced gastroduodenal contents reflux by esophagojejunostomy using C57BL/6J mice. Mice were divided into a standard diet and high-fat diet groups and kept for 60 weeks. Bile was sampled from the gallbladder to analyze bile acid fractions, and the esophagus was removed for a histological investigation. Human esophagogastric junction adenocarcinoma cells (OE19) were exposed to taurocholic acid (TCA), after which cell proliferative activity was measured. Rat esophageal cancer cell lines, ESCC-DR and ESCC-DRtca with higher malignant potential induced by continuous TCA exposure, were used to perform comprehensive genetic analysis (CGH). RESULTS: Barrett's epithelium onset occurred in all mice, and no differences in histological changes were noted between the standard diet and high-fat diet groups. However, no development of adenocarcinoma was noted. Most of the mouse bile acid was taurine conjugates. In the experiment using OE-19 cells, TCA promotes cell proliferation in a dose-dependent manner. Array CGH analysis revealed a large number of chromosomal abnormalities in the ESCC-DR, in addition to genetic abnormalities such as in the UGT2B gene, the substrate of which is bile acid. TCA administration resulted in more chromosomal abnormalities being detected. CONCLUSIONS: We showed the effects of TCA in cancer progression in vitro. However, Barrett's adenocarcinoma onset rates differ between mice and rats despite undergoing similar reflux stimulation including taurine-conjugated bile acids being detected in mouse bile juice. These results suggest that host factors seem to influence Barrett's carcinogenesis.

Importance of tumor budding grade as independent prognostic factor for early tongue squamous cell carcinoma.

BACKGROUND: Factors involved in neck lymph node metastasis (NLM) and prognosis of early tongue squamous cell carcinoma (SCC) remain unknown. METHODS: We analyzed disease-specific survival (DSS) and NLM including tumor budding grade (TBG) among 64 patients with cT1/2N0 tongue SCC. RESULTS: Univariate analysis of DSS of primary lesions uncovered significant differences in new cT, pT, new pT, pDiameter, venous infiltration, and TBG. Multivariate analysis selected only TBG3 as a predictor of NLM (odds ratio, 9.55; 95% confidence interval [CI], 1.80-50.8; P = .008), and a prognostic factor for DSS (hazard ratio, 4.41; 95% CI, 1.34-14.5; P = .02). CONCLUSION: The sole predictor of NLM and the prognosis of early tongue SCC was TBG, indicating that it might help to select overwhelming risk patients.

Expression and localization of aromatase in human gastric mucosa : Immunohistochemical study using biopsy materials.

Parietal cells in the gastric mucosa are known not only as cells playing major roles in food digestion but also as cells bearing endocrine function. In addition to their production of gastrin and ghrelin, it has been recently revealed that these cells are also involved in the synthesis and secretion of estrogens with their expression of aromatase in experimental animals. Although aromatase activity has been detected in human gastric cancer cells and related cell lines, much less study has been done to ascertain the expression of the enzymatic activity in normal gastric mucosa. It has not been established which cell type is responsible for estrogen production in human gastric glands consisting of epithelial cells of several types. The aim of this study is to define the expression of aromatase by parietal cells in human gastric glands using immunohistochemical techniques. We retrieved formalin-fixed paraffin embedded materials of gastric biopsies from 16 patients (nine men, seven women). Colocalization of aromatase and H+/K+-ATPase ß-subunit indicated that positive cells are parietal cells, but not chief cells and mucous cells. Furthermore, immunoreactivity of aromatase was detected within gastric glands irrespective of age or sex. These results suggest that human parietal cells synthesize estrogens within gastric mucosa and subsequently secrete them to the portal vein via gastric vein, as they do in rats. These estrogens might influence liver functions in humans. The estrogenic effects related to liver dysfunction might also be attributed to them.

Significance of PD-L1 Expression in Tongue Cancer Development.

Aims: Immunohistochemistry of PD-L1 has been recently established as a surrogate method to predict if immunotherapy targeting PD-L1/PD-1 has a significant effect on suppression of cancers such as lung non-small cell carcinoma, melanoma, and renal cell carcinoma. Here we performed immunohistochemistry for PD-L1 expression in squamous cell carcinoma (SCC) of the tongue to investigate the potential correlation between PD-L1 expression and clinicopathological factors and whether PD-L1 expression would be associated with prognosis. Methods: Tissue microarray cores of paraffin-embedded blocks from 135 cases with surgically resected tongue SCC were immunohistochemically analysed for PD-L1 expression. Results: We observed a positive correlation between PD-L1 expression and tongue SCC pT1 and pT2 tumours, but a negative correlation with pT2, pT3 and pT4 tumours. We also observed a positive correlation with lymph node metastasis. However, no positive correlation was demonstrated between PD-L1 expression and overall survival. Conclusions: PD-L1 tends to be overexpressed at the early stage of tongue SCC, showing a close correlation with initial development of tongue. However, PD-L1 expression may not affect prognosis.

Immune checkpoint inhibitors of CTLA4 and PD-1 for malignant melanoma arising in ovarian cystic teratoma: A case report.

RATIONALE: Malignant melanoma (MM) arising in ovarian cystic teratoma (OCT) is a rare disease with poor prognosis. Recently, immune checkpoint inhibitors of cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and programmed death 1 (PD-1) have shown promising results in MM. Herein we report a case of MM arising in OCT. PATIENT CONCERNS: A 63-year-old Japanese primigravida had lower abdominal pain. Magnetic resonance imaging revealed the presence of an 85-mm mass at the right ovary. DIAGNOSES: The patient underwent right salpingo-oophorectomy for right ovarian tumor, and histopathological examinations revealed MM arising in OCT. On immunohistochemical analysis, the tumor cells were positive for HMB-45, Melan A, and S-100 protein, and negative for programmed death-ligand 1 (PD-L1). BRAF gene mutations were not detected by the Real-Time PCR. Two months after surgery, liver metastasis was detected. INTERVENTIONS: The patient underwent immune checkpoint inhibitors of CTLA4 (ipilimumab) and PD-1 (pembrolizumab and nivolumab). She had interstitial pneumonia associated with ipilimumab, but she safely underwent the immune checkpoint inhibitors therapy along with oral prednisolone. Pembrolizumab, ipilimumab, and nivolumab therapies had poor effect on the tumor. OUTCOMES: Now, the present case has had tumor-bearing survival for 14 months since the initial diagnosis and 12 months since the detection of liver metastasis. LESSONS: This is the first case of MM arising in OCT treated by immune checkpoint inhibitors, with information of PD-L1 immunohistochemical expression and adverse events. The present case is the longest survivor following the detection of recurrence among all the previous reports. The long survival and slow-growing tumor in the present case may be associated with no PD-L1 expressions.

ERK phosphorylation functions in invadopodia formation in tongue cancer cells in a novel silicate fibre-based 3D cell culture system.

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase (ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet CellbedTM with a structure resembling the loose connective tissue morphology in a novel 3D culture system. We confirmed that the 3D system using CellbedTM accurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3D-cultured tongue cancer cell lines than in 2D cultures. Typically, under conventional 2D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells. However, in the 3D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3D culture systems using Cellbed™ are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

Morphological analysis of articularis genus and involvement in muscle synchronicity with vastus intermedius / Análisis morfológico del músculo articular de la rodilla y su participación en la sincronicidad muscular con el músculo vasto intermedio

The articularis genus, which lies under the vastus intermedius, has been regarded as part of the quadriceps femoris. However, they are well known to have different function because their respective origins and insertions are mutually distinct. These muscles are considered to work almost simultaneously when the knee is extended. The electromyogram has been used to demonstrate muscle co-contraction. However, the articularis genus is deeper than other muscles. Moreover, it is difficult to analyze it by surface electromyogram. The relative proportions of muscle fiber types and the characteristics of these fiber types are important determinants of the surface electromyogram. Furthermore, biomechanical analysis of AG has remained unclear. This study investigated the ratio of muscle fiber types in these muscles. Muscle samples from seven human cadaveric specimens were used with application of immunofluorescence double staining. Results show that in the vastus intermedius and articularis genus, the percentage of Type I fibers was significantly higher than that of Type II fibers. No significant difference was found in the mean percentages of Type I and Type II fiber types. The percentages of Type I and Type II fibers in articularis genus muscle were correlated positively to the percentage in the vastus intermedius. These results suggest that similar muscle fiber compositions of these muscles might reflect their contraction during the same active phase of knee extension, despite their different functions.

El músculo articular de la rodilla, que se encuentra cubierto por el músculo vasto intermedio, se ha considerado como parte del músculo cuádriceps femoral. Sin embargo, es sabido que tienen diferentes funciones debido a que sus respectivos orígenes e inserciones son mutuamente distintas. Se considera que estos músculos trabajan de forma casi simultánea cuando la rodilla está extendida. El electromiograma se ha usado para demostrar la contracción muscular. Sin embargo, el músculo articular de la rodillas es más profundo que otros músculos. Además, es difícil analizarlo por electromiograma de superficie. Las proporciones relativas de los tipos de fibras musculares y las características de estos tipos de fibras son importantes determinantes del electromiograma de superficie. Además, el análisis biomecánico de músculo articular de la rodilla no ha sido claro. Este estudio investigó la proporción de tipos de fibras musculares en estos músculos. Se usaron muestras musculares de siete cadáveres humanos con la aplicación de doble tinción de inmunofluorescencia. Los resultados muestran que en los músculos articular de la rodilla y vasto intermedio, el porcentaje de fibras de Tipo I fue significativamente mayor que el de las fibras de Tipo II. No se encontraron diferencias significativas en los porcentajes medios de los Tipo I y Tipo II. Los porcentajes de fibras Tipo I y Tipo II en el músculo articular de la rodilla se correlacionaron positivamente con el porcentaje en el músculo vasto intermediario. Estos resultados sugieren que las composiciones de las fibras musculares similares de estos músculos podrían reflejar su contracción durante la misma fase activa de la extensión de la rodilla, a pesar de sus diferentes funciones.

Effects of ß-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats.

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is associated with sensitivity to cold sensation in mammals. A previous study demonstrated that TRPM8 was overexpressed in the skin of ovariectomized (OVX) rats due to the loss of estrogen. In the present study, we investigated whether estrogen replacement restricts overexpression of the TRPM8 channel in the skin of OVX rats. We divided 15 Sprague Dawley rats into three groups: a non-operated group (NON-OPE), an ovariectomy group (OVX), and a group subjected to estrogen replacement during 4 weeks beginning 7 days after ovariectomy (OVX + E2). Five weeks later, TRPM8 channel mRNA and protein in lumbar skin were quantified by real-time RT-PCR, protein ELISA, and immunohistochemistry. The OVX + E2 group exhibited a trend for decreased expression of the TRPM8 channel in the lumbar skin in comparison with the OVX group, whereas ELISA data and immunohistochemistry data and immunohistochemistry graphs relating to TRPM8 protein did not show any obvious differences between the OVX group and the OVX + E2 group. Estrogen replacement may restrict the overexpression of TRPM8 in the dermis of OVX rats.