Innate immunity in the edible ascidian Halocynthia roretzi developing soft tunic syndrome: Hemolymph can eliminate the causative flagellates and discriminate allogeneic hemocytes.

The infection of the kinetoplastid flagellate Azumiobodo hoyamushi causes soft tunic syndrome that often results in mass mortality in the aquaculture of the edible ascidian Halocynthia roretzi. In the diseased ascidian individuals, the flagellates are exclusively found in the tunic matrix that entirely cover the epidermis, and never invade into internal tissues, such as a mantle. The present study for the first time demonstrated that the ascidian blood plasma and hemolymph have an activity to agglutinate and disintegrate the flagellates, suggesting the innate immunity protects the internal tissue from the invasion of A. hoyamushi. This activity is indifferent between the healthy and the diseased individuals. Allo-specific recognition and cytotoxic reaction among ascidian hemocytes, so-called contact reaction, occur among the individuals of healthy-healthy, healthy-diseased, and diseased-diseased combination, and therefore, the hemocytes from diseased individuals still retain the allo-reactivity. Moreover, the allo-reactive combinations are not changed under the presence of the flagellates, indicating the flagellates neither suppress nor induce the effector system of the contact reaction. These results suggest that the infection of A. hoyamushi does not impair the innate immunity in the ascidian hemolymph.

Similarities and differences in metabolites of tongue cancer cells among two- and three-dimensional cultures and xenografts.

Metabolic programming of cancer cells is an essential step in transformation and tumor growth. We established two-dimensional (2D) monolayer and three-dimensional (3D) cultures, the latter called a "tissueoid cell culture system", using four types of tongue cancer cell lines. We also undertook a comprehensive metabolome analysis of three groups that included xenografts created by transplanting the cell lines into nude mice. In addition, we undertook a functional analysis of the mitochondria, which plays a key role in cancer metabolism. Principal component analysis revealed the plots of the four cell lines to be much narrower in 2D culture than in 3D culture and xenograft groups. Moreover, compared to xenografts, the 2D culture had significantly lower levels of most metabolites. These results suggest that the unique characteristics of each cell disappeared in 2D culture, and a type of metabolism unique to monolayer culture took over. Conversely, ATP production, biomass synthesis, and maintenance of redox balance were shown in 3D culture using sufficient nutrients, which closely resembled the metabolic activity in the xenografts. However, there were several differences between the metabolic activity in the 3D culture and xenografts. In vivo, the cancer tissue had blood flow with stromal cells present around the cancer cells. In the xenografts, we detected metabolized and degraded products in the liver and other organs of the host mice. Furthermore, the 3D system did not show impairment of mitochondrial function in the cancer cells, suggesting that cancer cells produce energy simultaneously through mitochondria, as well as aerobic glycolysis.

The circadian clock gene Per1 modulates context fear memory formation within the retrosplenial cortex in a sex-specific manner.

Context memory formation is a complex process that requires transcription in many subregions of the brain including the dorsal hippocampus and retrosplenial cortex. One critical gene necessary for memory formation is the circadian gene Period1 (Per1), which has been shown to function in the dorsal hippocampus to modulate spatial memory in addition to its well-documented role in regulating the diurnal clock within the suprachiasmatic nucleus (SCN). We recently found that alterations in Per1 expression in the dorsal hippocampus can modulate spatial memory formation, with reduced hippocampal Per1 impairing memory and overexpression of Per1 ameliorating age-related impairments in spatial memory. Whether Per1 similarly functions within other memory-relevant brain regions is currently unknown. Here, to test whether Per1 is a general mechanism that modulates memory across the brain, we tested the role of Per1 in the retrosplenial cortex (RSC), a brain region necessary for context memory formation. First, we demonstrate that context fear conditioning drives a transient increase in Per1 mRNA expression within the anterior RSC that peaks 60 m after training. Next, using HSV-CRISPRi-mediated knockdown of Per1, we show that reducing Per1 within the anterior RSC before context fear acquisition impairs memory in both male and female mice. In contrast, overexpressing Per1 with either HSV-CRISPRa or HSV-Per1 before context fear acquisition drives a sex-specific memory impairment; males show impaired context fear memory whereas females are not affected by Per1 overexpression. Finally, as Per1 levels are known to rhythmically oscillate across the day/night cycle, we tested the possibility that Per1 overexpression might have different effects on memory depending on the time of day. In contrast to the impairment in memory we observed during the daytime, Per1 overexpression has no effect on context fear memory during the night in either male or female mice. Together, our results indicate that Per1 modulates memory in the anterior retrosplenial cortex in addition to its documented role in regulating memory within the dorsal hippocampus, although this role may differ between males and females.

Differences in the Central Energy Metabolism of Cancer Cells between Conventional 2D and Novel 3D Culture Systems.

The conventional two-dimensional (2D) culture is available as an in vitro experimental model. However, the culture system reportedly does not recapitulate the in vivo cancer microenvironment. We recently developed a tissueoid cell culture system using Cellbed, which resembles the loose connective tissue in living organisms. The present study performed 2D and three-dimensional (3D) culture using prostate and bladder cancer cell lines and a comprehensive metabolome analysis. Compared to 3D, the 2D culture had significantly lower levels of most metabolites. The 3D culture system did not impair mitochondrial function in the cancer cells and produce energy through the mitochondria simultaneously with aerobic glycolysis. Conversely, ATP production, biomass (nucleotides, amino acids, lipids and NADPH) synthesis and redox balance maintenance were conducted in 3D culture. In contrast, in 2D culture, biomass production was delayed due to the suppression of metabolic activity. The 3D metabolome analysis using the tissueoid cell culture system capable of in vivo cancer cell culture yielded results consistent with previously reported cancer metabolism theories. This system is expected to be an essential experimental tool in a wide range of cancer research fields, especially in preclinical stages while transitioning from in vitro to in vivo.

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.

The Absence of DHHC3 Affects Primary and Latent Herpes Simplex Virus 1 Infection.

UL20, an essential herpes simplex virus 1 (HSV-1) protein, is involved in cytoplasmic envelopment of virions and virus egress. We reported recently that UL20 can bind to a host protein encoded by the zinc finger DHHC-type containing 3 (ZDHHC3) gene (also known as Golgi-specific DHHC zinc finger protein [GODZ]). Here, we show for the first time that HSV-1 replication is compromised in murine embryonic fibroblasts (MEFs) isolated from GODZ-/- mice. The absence of GODZ resulted in blocking palmitoylation of UL20 and altered localization and expression of UL20 and glycoprotein K (gK); the expression of gB and gC; and the localization and expression of tegument and capsid proteins within HSV-1-infected MEFs. Electron microscopy revealed that the absence of GODZ limited the maturation of virions at multiple steps and affected the localization of virus and endoplasmic reticulum morphology. Virus replication in the eyes of ocularly HSV-1-infected GODZ-/- mice was significantly lower than in HSV-1-infected wild-type (WT) mice. The levels of UL20, gK, and gB transcripts in the corneas of HSV-1-infected GODZ-/- mice on day 5 postinfection were markedly lower than in WT mice, whereas only UL20 transcripts were reduced in trigeminal ganglia (TG). In addition, HSV-1-infected GODZ-/- mice showed notably lower levels of corneal scarring, and HSV-1 latency reactivation was also reduced. Thus, normal HSV-1 infectivity and viral pathogenesis are critically dependent on GODZ-mediated palmitoylation of viral UL20.IMPORTANCE HSV-1 infection is widespread. Ocular infection can cause corneal blindness; however, approximately 70 to 90% of American adults exposed to the virus show no clinical symptoms. In this study, we show for the first time that the absence of a zinc finger protein called GODZ affects primary and latent infection, as well as reactivation, in ocularly infected mice. The reduced virus infectivity is due to the absence of the GODZ interaction with HSV-1 UL20. These results strongly suggest that binding of UL20 to GODZ promotes virus infectivity in vitro and viral pathogenesis in vivo.

DHHC7 Palmitoylates Glucose Transporter 4 (Glut4) and Regulates Glut4 Membrane Translocation.

Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane plays a key role in the dynamic regulation of glucose homeostasis. We recently showed that this process is critically dependent on palmitoylation of Glut4 at Cys-223. To gain further insights into the regulation of Glut4 palmitoylation, we set out to identify the palmitoyl acyltransferase (PAT) involved. Here we report that among 23 mammalian DHHC proteins, DHHC7 is the major Glut4 PAT, based on evidence that ectopic expression of DHHC7 increased Glut4 palmitoylation, whereas DHHC7 knockdown in 3T3-L1 adipocytes and DHHC7 KO in adipose tissue and muscle decreased Glut4 palmitoylation. Moreover, inactivation of DHHC7 suppressed insulin-dependent Glut4 membrane translocation in both 3T3-L1 adipocytes and primary adipocytes. Finally, DHHC7 KO mice developed hyperglycemia and glucose intolerance, thereby confirming that DHHC7 represents the principal PAT for Glut4 and that this mechanism is essential for insulin-regulated glucose homeostasis.

Dissociation of Golgi-associated DHHC-type Zinc Finger Protein (GODZ)- and Sertoli Cell Gene with a Zinc Finger Domain-ß (SERZ-ß)-mediated Palmitoylation by Loss of Function Analyses in Knock-out Mice.

The γ2 subunit of GABA type A receptors (GABAARs) is thought to be subject to palmitoylation by both Golgi-associated DHHC-type zinc finger protein (GODZ; also known as DHHC3) and its paralog Sertoli cell gene with a zinc finger domain-ß (SERZ-ß; DHHC7) based on overexpression of enzymes and substrates in heterologous cells. Here we have further investigated the substrate specificity of these enzymes by characterization of GODZ and SERZ-ß knock-out (KO) mice as well as double KO (DKO) neurons. Palmitoylation of γ2 and a second substrate, growth-associated protein of 43 kDa, that is independently implicated in trafficking of GABAARs was significantly reduced in brain of GODZ KO versus wild-type (WT) mice but unaltered in SERZ-ß KO mice. Accumulation of GABAARs at synapses, GABAergic innervation, and synaptic function were reduced in GODZ KO and DKO neurons to a similar extent, indicating that SERZ-ß does not contribute to palmitoylation or trafficking of GABAARs even in the absence of GODZ. Notably, these effects were seen only when mutant neurons were grown in competition with WT neurons, thereby mimicking conditions of shRNA-transfected neurons previously used to characterize GODZ. However, GABA-evoked whole-cell currents of DKO neurons and the GABAAR cell surface expression in DKO neurons and GODZ or SERZ-ß KO brain slices were unaltered, indicating that GODZ-mediated palmitoylation selectively controls the pool of receptors at synapses. The different substrate specificities of GODZ and SERZ-ß in vivo were correlated with their differential localization to cis- versus trans-Golgi compartment, a mechanism that was compromised by overexpression of GODZ.

Roles of hypertension in the rupture of intracranial aneurysms.

BACKGROUND AND PURPOSE: Systemic hypertension has long been considered a risk factor of aneurysmal rupture. However, a causal link between systemic hypertension and the development of aneurysmal rupture has not been established. In this study, using a mouse model of intracranial aneurysm rupture, we examined the roles of systemic hypertension in the development of aneurysmal rupture. METHODS: Aneurysms were induced by a combination of deoxycorticosterone acetate (DOCA)-salt and a single injection of elastase into the cerebrospinal fluid in mice. Antihypertensive treatment was started 6 days after aneurysm induction. Aneurysmal rupture was detected by neurological symptoms and confirmed by the presence of intracranial aneurysm with subarachnoid hemorrhage. Hydralazine (direct vasodilator) or discontinuation of DOCA-salt treatment was used to assess the roles of systemic hypertension. Captopril (angiotensin-converting enzyme inhibitor) or losartan (angiotensin II type 1 receptor antagonist) was used to assess the roles of the local renin-angiotensin system in the vascular wall. RESULTS: Normalization of blood pressure by hydralazine significantly reduced the incidence of ruptured aneurysms and the rupture rate. There was a dose-dependent relationship between reduction of blood pressure and prevention of aneurysmal rupture. Captopril and losartan were able to reduce rupture rate without affecting systemic hypertension induced by DOCA-salt treatment. CONCLUSIONS: Normalization of blood pressure after aneurysm formation prevented aneurysmal rupture in mice. In addition, we found that the inhibition of the local renin-angiotensin system independent from the reduction of blood pressure can prevent aneurysmal rupture.

The clock gene Per1 may exert diurnal control over hippocampal memory consolidation.

The circadian system influences many different biological processes, including memory performance. While the suprachiasmatic nucleus (SCN) functions as the brain's central pacemaker, downstream "satellite clocks" may also regulate local functions based on the time of day. Within the dorsal hippocampus (DH), for example, local molecular oscillations may contribute to time-of-day effects on memory. Here, we used the hippocampus-dependent Object Location Memory task to determine how memory is regulated across the day/night cycle in mice. First, we systematically determined which phase of memory (acquisition, consolidation, or retrieval) is modulated across the 24 h day. We found that mice show better long-term memory performance during the day than at night, an effect that was specifically attributed to diurnal changes in memory consolidation, as neither memory acquisition nor memory retrieval fluctuated across the day/night cycle. Using RNA-sequencing we identified the circadian clock gene Period1 (Per1) as a key mechanism capable of supporting this diurnal fluctuation in memory consolidation, as learning-induced Per1 oscillates in tandem with memory performance in the hippocampus. We then show that local knockdown of Per1 within the DH impairs spatial memory without affecting either the circadian rhythm or sleep behavior. Thus, Per1 may independently function within the DH to regulate memory in addition to its known role in regulating the circadian system within the SCN. Per1 may therefore exert local diurnal control over memory consolidation within the DH.

The clock gene Per1 is necessary in the retrosplenial cortex-but not in the suprachiasmatic nucleus-for incidental learning in young and aging male mice.

Aging impairs both circadian rhythms and memory, though the relationship between these impairments is not fully understood. Circadian rhythms are largely dictated by clock genes within the body's central pacemaker, the suprachiasmatic nucleus (SCN), though these genes are also expressed in local clocks throughout the body. As circadian rhythms can directly affect memory performance, one possibility is that memory deficits observed with age are downstream of global circadian rhythm disruptions stemming from the SCN. Here, we demonstrate that expression of clock gene Period1 within a memory-relevant cortical structure, the retrosplenial cortex (RSC), is necessary for incidental learning, and that age-related disruption of Period1 within the RSC-but not necessarily the SCN-contributes to cognitive decline. These data expand the known functions of clock genes beyond maintaining circadian rhythms and suggests that age-associated changes in clock gene expression modulates circadian rhythms and memory performance in a brain region-dependent manner.

The relation between ambulance transports stratified by heat stroke and air temperature in all 47 prefectures of Japan in August, 2009: ecological study.

OBJECTIVE: Our aim was to investigate the link between ambulance transports stratified by heat stroke and air temperature in August 2009 in Japan. METHODS: Monthly observations for ambulance transports stratified by heat stroke in August 2009, in all 47 prefectures of Japan were obtained from the Ministry of Internal Affairs and Communications. Daily air temperature data from the Japan Meteorological Agency in August 2009 for all 47 prefectures in Japan were also used. The effect of high air temperatures on ambulance transports stratified by heat stroke was analyzed in an ecological study. RESULTS: Various air temperature parameters , i.e., the mean of the mean air temperature, mean of the highest air temperature, mean of the lowest air temperature, the highest air temperature, and the lowest air temperature in August 2009 were significantly and positively correlated with ambulance transports stratified by heat stroke (per total number of ambulance transports and per 10,000 persons) in the 47 prefectures of Japan. The correlation coefficient between the mean of the highest air temperature in August 2009 and ambulance transports stratified by heat stroke (per 10 000 persons) was the highest among the examined parameters (r = 0.799, p < 0.0001). CONCLUSION: Higher air temperatures were closely associated with higher numbers of ambulance transports stratified by heat stroke in August 2009 in Japan.

Inhaled corticosteroids do not affect the antibody titer against the SARS-CoV-2 spike protein in BNT162b2 mRNA vaccinated patients.

OBJECTIVES: Oral corticosteroids reduce the antibody titer of the BNT162b2 mRNA vaccine against SARS-CoV-2. To date, the effect of inhaled corticosteroids on antibody titers is unknown. STUDY DESIGN: The design of this study is retrospective study. METHODS: We analyzed the relationship between the clinical features and total antibody titers against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein in 320 subjects who had never been infected with Coronavirus disease 2019 (COVID-19) and were vaccinated the second time with the BNT162b2 mRNA vaccine between October 1 to December 28, 2021. RESULTS: Of the 320 subjects, 205 were treated with inhaled corticosteroids. The median antibody titer of patients treated with inhaled corticosteroids was 572 U/mL, which was significantly higher than that of patients treated without inhaled corticosteroids (454U/mL, P = 0.00258). The median antibody titers of smokers, men, and patients aged 65 years and over, were 315.5 U/mL, 385 U/mL, and 425.5 U/mL, respectively. These results are significantly lower than those of patients who never smoked, women, and patients aged less than 64 years (582 U/mL [P < 0.0001], 682.5 U/mL [P < 0.0001], and 717 U/mL [P < 0.0001], respectively). The multivariate analysis revealed that females and age were independent antibody titer-reducing factors (P = 0.0001 and P < 0.0001, respectively). CONCLUSIONS: The use of inhaled corticosteroids did not reduce the antibody titer against SARS-CoV-2 spike protein. Clinicians should continue treatment with inhaled corticosteroids if indicated.

Comparison of the changes in temperatures among rural, urban and metropolitan areas around the Inland Sea in Japan.

We investigated the changes in temperatures in Takamatsu, Tadotsu and Osaka areas in Japan. Data of temperatures in Takamatsu (urban area: 1942-2009), Tadotsu (rural area: 1893-2009) and Osaka (metropolitan area: 1883-2009) areas around the Inland Sea in Japan was obtained from Japan Meteorological Agency. The changes in temperatures in three areas in Japan were analyzed and compared. By using data from 1942 to 2009, various parameters of temperatures were significantly correlated with years. The changes in mean temperature in August and in a year in Takamatsu area for 50 years were highest (August: 1.6°C, Year: 1.8°C) among three areas. In addition, correlation coefficient rate between years and the number of days over the level of 34°C in August in Takamatsu area was highest (r = 0.567, p < 0.0001). Parameters of temperatures were gradually increased with years in all three areas. The heat island effect in Takamatsu area was similar to Osaka area in Japan.

Surgical repair of hepatic hydrothorax caused by diaphragmatic fistula.

A 65-year-old woman visited our hospital complaining of dyspnea several days before admission. A chest X-ray showed massive right-sided pleural effusion, which was not observed 1 month previously. Although the patient had never been diagnosed with cirrhosis at regular visits, the patient was diagnosed with primary biliary cholangitis at admission. Hepatic hydrothorax was suspected because pleural effusion was transudative. A diaphragmatic fistula was confirmed and closed by thoracoscopy. Pleural effusion did not reappear after this procedure. Existence of a diaphragmatic defect should be confirmed under direct vision if pleural effusion accumulates acutely or becomes beyond control.

Wobbly hedgehog syndrome with disseminated histiocytic sarcoma and lateral ventricular meningioma in an African pygmy hedgehog.

An 8-mo-old male African pygmy hedgehog was anorectic and ataxic; physical examination revealed tetraparesis and a gangrenous left hindlimb. Analgesic and supportive care were administered, but the animal died 3 d after presentation. Postmortem examination revealed a histiocytic sarcoma in a mesenteric lymph node with metastasis to several organs, multifocal vacuolation in the cerebral and cerebellar white matter, and a meningioma in the left lateral ventricle. We diagnosed wobbly hedgehog syndrome (WHS) with disseminated histiocytic sarcoma and lateral ventricular meningioma. Ventricular meningioma, a rare neoplasm in veterinary and human patients, has not been reported previously in hedgehogs, to our knowledge. The neurologic signs in our case were probably caused by the WHS-related vacuolar lesions and are consistent with those of reported WHS cases. Duration of illness was shorter than is typical of WHS cases, which might be related to the disseminated histiocytic sarcoma. Clinical relevance of the lateral ventricular meningioma was not evident because the ventricular mass was localized and not invasive.

Expressions of ezrin, ERK, STAT3, and AKT in tongue cancer and association with tumor characteristics and patient survival.

BACKGROUND: Ezrin, ERK, STAT3, and AKT are proteins that are overexpressed in various types of cancer, although their expressions in tongue cancer has received less focus. This study aimed to address associations between the expression levels of these proteins and with characteristics of the tumor and patient survival. METHODS: We performed immunohistochemical staining of ezrin, ERK, STAT3, and AKT in tumors from patients with tongue carcinoma in situ (CIS, n = 17) and tongue squamous cell carcinoma (SCC, n = 46). Statistical differences between the SCC versus the CIS cohorts were estimated by calculations of bivariate odds ratios of low versus high expression of the proteins. Fisher's exact tests were used to appraise interassociations between the proteins, as well as expression levels versus patient and tumor characteristics. Survival based on Kaplan-Meier statistics in combination log-rank tests were used to address potential effects of the patient and tumor characteristics versus 5-year survival rate. RESULTS: The relative high: low expression of all four proteins in the two cohorts differed, and particularly ERK was markedly overexpressed in the SCC versus the CIS cohort (odds ratio = 45.3, p < .01). The relative high: low expression each protein versus patient and tumor characteristics; showed associations between AKT expression and T stage (p = .002) plus node metastases (p = .12), and between ERK expression and drinking (p = .01) and smoking history (p = .01). There was no significant difference observed between ERK and the three other molecules, nor any significant difference between the degree of expression of each protein and the 5-year disease-specific survival rate. CONCLUSION: Ezrin, ERK, STAT3, and AKT appear to be involved in the progress from carcinoma in situ in the tongue into squamous cell carcinoma. ERK in particular is overexpressed, suggesting that ERK may be a novel therapeutic target for preventing tongue cancer.

Tenosynovitis Induced by an Immune Checkpoint Inhibitor: A Case Report and Literature Review.

A 51-year-old man underwent second-line treatment for non-small-cell lung cancer (NSCLC) with the immune checkpoint inhibitor (ICI) pembrolizumab. On day 2 after two cycles of pembrolizumab, he presented with edema limited to the left third, fourth, and fifth fingers. Based on symptoms, laboratory results, and contrast-enhanced magnetic resonance imaging (MRI) findings, we diagnosed him with tenosynovitis. We prescribed oral prednisolone (0.5 mg/kg/day), and pembrolizumab was continued. Prednisolone immediately relieved the symptoms, and the tumor was still shrinking on day 21 after eight cycles of pembrolizumab. ICI-induced tenosynovitis was managed while continuing ICI usage, suggesting that 0.5 mg/kg/day prednisone might be effective for tenosynovitis without ICI cessation.

Mandibular vascular hamartoma in a cat.

A 10-year-old cat presented for evaluation with a 1-month history of salivation and oral bleeding. A right mandibular mass was palpated and computed tomography examination revealed entire bone proliferation. Mandibular bone biopsy was performed, and histopathological diagnosis was vascular hamartoma. The cat suddenly died on day 140.

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.