Spatiotemporal commonality of the TCR repertoire in a T-cell memory murine model and in metastatic human colorectal cancer.

Immune checkpoint inhibitors (ICIs) have shown superior clinical responses and significantly prolong overall survival (OS) for many types of cancer. However, some patients exhibit long-term OS, whereas others do not respond to ICI therapy at all. To develop more effective and long-lasting ICI therapy, understanding the host immune response to tumors and the development of biomarkers are imperative. In this study, we established an MC38 immunological memory mouse model by administering an anti-PD-L1 antibody and evaluating the detailed characteristics of the immune microenvironment including the T cell receptor (TCR) repertoire. In addition, we found that the memory mouse can be established by surgical resection of residual tumor following anti-PD-L1 antibody treatment with a success rate of > 40%. In this model, specific depletion of CD8 T cells revealed that they were responsible for the rejection of reinoculated MC38 cells. Analysis of the tumor microenvironment (TME) of memory mice using RNA-seq and flow cytometry revealed that memory mice had a quick and robust immune response to MC38 cells compared with naïve mice. A TCR repertoire analysis indicated that T cells with a specific TCR repertoire were expanded in the TME, systemically distributed, and preserved in the host for a long time period. We also identified shared TCR clonotypes between serially resected tumors in patients with colorectal cancer (CRC). Our results suggest that memory T cells are widely preserved in patients with CRC, and the MC38 memory model is potentially useful for the analysis of systemic memory T-cell behavior.

Fibroblast growth factor receptor 3 overexpression mediates ALK inhibitor resistance in ALK-rearranged non-small cell lung cancer.

The rearrangement of anaplastic lymphoma kinase (ALK) occurs in 3%-5% of patients with non-small cell lung cancer (NSCLC) and confers sensitivity to ALK-tyrosine kinase inhibitors (TKIs). For the treatment of patients with ALK-rearranged NSCLC, various additional ALK-TKIs have been developed. Ceritinib is a second-generation ALK-TKI and has shown great efficacy in the treatment of patients with both newly diagnosed and crizotinib (a first-generation ALK-TKI)-refractory ALK-rearranged NSCLC. However, tumors can also develop ceritinib resistance. This may result from secondary ALK mutations, but other mechanisms responsible for this have not been fully elucidated. In this study, we explored the mechanisms of ceritinib resistance by establishing ceritinib-resistant, echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive H3122 cells and ceritinib-resistant patient-derived cells. We identified a mechanism of ceritinib resistance induced by bypass signals that is mediated by the overexpression and activation of fibroblast growth factor receptor 3 (FGFR3). FGFR3 knockdown by small hairpin RNA or treatment with FGFR inhibitors was found to resensitize the resistant cells to ceritinib in vitro and in vivo. FGFR ligands from either human serum or fetal bovine serum were able to activate FGFR3 and induce ceritinib resistance. A detailed analysis of ceritinib-resistant patient-derived specimens confirmed that tyrosine-protein kinase Met (cMET) amplification induces ceritinib resistance. Amplified cMET counteractivated EGFR and/or Her3 and induced ceritinib resistance. These results reveal multiple ceritinib resistance mechanisms and suggest that ceritinib resistance might be overcome by identifying precise resistance mechanisms.

Claw Toe: Anatomic Guide for Injection of Botulinum Toxin into Foot Muscles.

BACKGROUND: Poor response to injection of botulinum toxin (BoNT) into the flexor digitorum longus (FDL) muscle has been reported especially in patients with claw foot deformity. We previously advocated BoNT injection into the flexor hallucis longus (FHL) muscle in such patients. Here, we determined the functional and anatomical relationships between FHL and FDL. METHODS: Toe flexion pattern was observed during electrical stimulation of FHL and FDL muscles in 31 post-stroke patients with claw-foot deformity treated with BoNT. The FHL and FDL tendon arrangement was also studied in five limbs of three cadavers. RESULTS: Electrical stimulation of the FHL muscle elicited big toe flexion in all 28 cases examined and second toe in 25, but the response was limited to the big toe in 3. FDL muscle stimulation in 29 patients elicited weak big toe flexion in 1 and flexion of four toes (2nd to 5th) in 16 patients. Cadaver studies showed division of the FHL tendon with branches fusing with the FDL tendon in all five limbs examined; none of the tendons was inserted only in the first toe. No branches of the FDL tendon merged with the FHL tendon. CONCLUSION: Our results showed coupling of FHL and FDL tendons in most subjects. Movements of the second and third toes are controlled by both the FDL and FHL muscles. The findings highlight the need for BoNT injection in both the FDL and FHL muscles for the treatment of claw-toe deformity.

Computed angiographic variations in hepatic venous vasculature in dogs.

OBJECTIVE: To identify the number of hepatic veins draining directly or indirectly into the caudal vena cava Thank you (CVC) using computed tomography angiography (CTA) in dogs. STUDY DESIGN: Retrospective clinical study. ANIMALS: Client-owned dogs (n = 77). METHODS: Abdominal CTA images were analyzed. Retrospective convenience sampling was performed using archived clinical cases to determine the number of hepatic veins in each liver lobe. RESULTS: A median of 2 vessels from the right lateral lobe (range: 1-4) and the caudate process of the caudate lobe (range: 1-5) drained directly into the CVC. In the quadrate lobe, most common patterns consisted of 1 vessel directly draining to the CVC or indirectly via the left hepatic vein (LHV), and a vessel from quadrate lobe and right medial lobe merging into 1 vessel draining into the CVC or the LHV. A median of 3 vessels in the left lateral lobe (range: 2-8) and a median of 1 vessel in the left medial lobe (range: 1-3) drained into the LHV. In the papillary process of the caudate lobe, a median of 1 (range: 1-2) vessel drained directly into the CVC or the LHV. CONCLUSION: The draining pattern of hepatic veins varied widely in all liver lobes, especially the left lateral liver lobe. CLINICAL SIGNIFICANCE: Veterinary surgeons should consider the potential presence of multiple hepatic veins and their draining pattern when performing hilar liver lobe resection. Attentive evaluation of a preoperative CTA is recommended for surgical planning.

Novel knock-in mouse model for the evaluation of the therapeutic efficacy and toxicity of human podoplanin-targeting agents.

Podoplanin is a key molecule for enhancing tumor-induced platelet aggregation. Podoplanin interacts with CLEC-2 on platelets via PLatelet Aggregation-inducing domains (PLAGs). Among our generated antibodies, those targeting the fourth PLAG domain (PLAG4) strongly suppress podoplanin-CLEC-2 binding and podoplanin-expressing tumor growth and metastasis. We previously performed a single-dose toxicity study of PLAG4-targeting anti-podoplanin-neutralizing antibodies and found no acute toxicity in cynomolgus monkeys. To confirm the therapeutic efficacy and toxicity of podoplanin-targeting antibodies, a syngeneic mouse model that enables repeated dose toxicity tests is needed. Replacement of mouse PLAG1-PLAG4 domains with human homologous domains drastically decreased the platelet-aggregating activity. Therefore, we searched the critical domain of the platelet-aggregating activity in mouse podoplanin and found that the mouse PLAG4 domain played a critical role in platelet aggregation, similar to the human PLAG4 domain. Human/mouse chimeric podoplanin, in which a limited region containing mouse PLAG4 was replaced with human homologous region, exhibited a similar platelet-aggregating activity to wild-type mouse podoplanin. Thus, we generated knock-in mice with human/mouse chimeric podoplanin expression (PdpnKI/KI mice). Our previously established PLAG4-targeting antibodies could suppress human/mouse chimeric podoplanin-mediated platelet aggregation and tumor growth in PdpnKI/KI mice. Repeated treatment of PdpnKI/KI mice with antibody-dependent cell-mediated cytotoxicity activity-possessing PG4D2 antibody did not result in toxicity or changes in hematological and biochemical parameters. Our results suggest that anti-podoplanin-neutralizing antibodies could be used safely as novel anti-tumor agents. Our generated PdpnKI/KI mice are useful for investigating the efficacy and toxicity of human podoplanin-targeting drugs.

[Paclitaxel plus Bevacizumab Therapy plus Surgical Resection Results in Local Control of Stage â ¢C Breast Cancer].

The case was a 40-year-old female who was aware of a right breast mass for 1 year before her first visit. She had visited her previous doctor because the mass was gradually increasing in size. After close examination, she was diagnosed with Stage ⅢC triple-negative breast cancer. She underwent 4 courses of EC therapy and 7 courses of paclitaxel(PTX)plus bevacizumab( Bev)therapy and was then, referred to our hospital for resection. We instituted a 2-month break from Bev to prevent postoperative complications. She underwent a right mastectomy, combined chest wall resection, lymph node dissection, and chest wall reconstruction. Because the postoperative course was good and quality of life improved, the multidisciplinary treatment with surgery and pharmacotherapy was considered effective in locally advanced breast cancer.

Dose reduction and image quality improvement of chest radiography by using bone-suppression technique and low tube voltage: a phantom study.

OBJECTIVE: To clarify the relationship between entrance surface dose (ESD) and physical image quality of original and bone-suppressed chest radiographs acquired using high and low tube voltages. METHODS: An anthropomorphic chest phantom and a 12-mm diameter spherical simulated nodule with a CT value of approximately + 100 HU were used. The lung field in the chest radiograph was divided into seven areas, and the nodule was set in a total of 66 positions. A total of 264 chest radiographs were acquired using four ESD conditions: approximately 0.3 mGy at 140 and 70 kVp and approximately 0.2 and 0.1 mGy at 70 kVp. The radiographs were processed to produce bone-suppressed images. Differences in contrast and contrast-to-noise ratio (CNR) values of the nodule between each condition and between the original and bone-suppressed images were analyzed by a two-sided Wilcoxon signed-rank test. RESULTS: In the areas not overlapping with the ribs, both contrast and CNR values were significantly increased with the bone-suppression technique (p < 0.01). In the bone-suppressed images, these values of the three conditions at 70 kVp were equal to or significantly higher than those of the condition at 140 kVp. There was no apparent decrease in these values between the ESD of approximately 0.3 and 0.1 mGy at 70 kVp. CONCLUSION: By using the shortest exposure time and the lowest tube voltage possible not to increase in blurring artifact and image noise, it is possible to improve the image quality of bone-suppressed images and reduce the patient dose. KEY POINTS: • The effectiveness of bone-suppression techniques differs in areas of lung field. • Image quality of bone-suppressed chest radiographs is improved by lower tube voltage. • Applying lower tube voltage to bone-suppressed chest radiographs leads to dose reduction.

Assessment of tumor enhancement by contrast-enhanced CT in solid tumor-bearing dogs treated with toceranib phosphate.

In humans, contrast-enhanced CT (CECT) has been used to indirectly assess the antiangiogenic effects demonstrated by a number of tyrosine kinase inhibitors. This retrospective, cross-sectional study aimed to quantitatively evaluate changes in tumor contrast-enhancement (CE) using CECT in solid tumor-bearing dogs treated with toceranib phosphate (TOC). The changes in tumor size and CE were measured using the Hounsfield unit (HU) scale in CECT images before TOC treatment and between 30 and 90 days after initiating the treatment. Among the 36 dogs treated with TOC, eight (22.2%) showed a partial response, 22 (61.1%) showed stable disease, and six (16.7%) showed progressive disease. Thirty (83.3%) of 36 dogs showed a decrease in tumor CE (median: -20%, range: -1% to -48%) after initiating the treatment. The results indicated that tumor CE and size changes were observed in tumor-bearing dogs that were treated with TOC; however, tumor CE was not significantly correlated with tumor regression. We suggest that these results could serve as pilot data to evaluate the antiangiogenic effects associated with TOC.

Acceleration of Skin Wound-Healing Reactions by Autologous Micrograft Tissue Suspension.

Background and objectives: Skin grafting is a method usually used in reconstructive surgery to accelerate skin regeneration. This method results frequently in unexpected scar formations. We previously showed that cutaneous wound-healing in normal mice is accelerated by a micrograft (MG) technique. Presently, clinical trials have been performed utilizing this technology; however, the driving mechanisms behind the beneficial effects of this approach remain unclear. In the present study, we focused on five major tissue reactions in wound-healing, namely, regeneration, migration, granulation, neovascularization and contraction. Methods: Morphometrical analysis was performed using tissue samples from the dorsal wounds of mice. Granulation tissue formation, neovascularization and epithelial healing were examined. Results: The wound area correlated well with granulation sizes and neovascularization densities in the granulation tissue. Vascular distribution analysis in the granulation tissue indicated that neovessels extended and reached the subepidermal area in the MG group but was only halfway developed in the control group. Moreover, epithelialization with regeneration and migration was augmented by MG. Myofibroblast is a known machinery for wound contraction that uses α-smooth muscle actin filaments. Their distribution in the granulation tissue was primarily found beneath the regenerated epithelium and was significantly progressed in the MG group. Conclusions: These findings indicated that MG accelerated a series of wound-healing reactions and could be useful for treating intractable wounds in clinical situations.

Rezoning Free Muscle-Sparing Transverse Rectus Abdominis Myocutaneous Flaps Based on Perforasome Groupings and a New Understanding of the Vascular Architecture of the Deep Inferior Epigastric Artery-Based Flaps.

BACKGROUND: We compare the vascular territory of free muscle-sparing transverse rectus abdominis myocutaneous (MS-TRAM) flaps, deep inferior epigastric perforator (DIEP) flaps, and crossover anastomosis (CA) flaps using intraoperative ex vivo angiography. We also use ex vivo angiography to analyze the vascular architecture of the MS-TRAM flap. METHODS: Our study includes 84 lower abdominal free flaps: MS-TRAM, DIEP-1 (1 perforator), DIEP-2 (2 perforators), and CA. We compare the arterial perfusion area and vascular territory pattern in each group. We also analyze the vascular architecture in MS-TRAM flaps and determine the number and location of their dominant perforators and the direction of the axial arteries connecting them. RESULTS: The CA's arterial perfusion area is the largest, and the DIEP-1's, the smallest of our groups; there is no statistically significant difference between MS-TRAM and DIEP-2. In all groups, average arterial perfusion area in the vascular pedicle's ipsilateral side is larger than in its contralateral side. The MS-TRAM and DIEP-2 flaps have homologous perfusion patterns and the same arterial perfusion areas. The DIEP-1 perfusion pattern varies with perforator location. Ex vivo angiograms show the MS-TRAM flap's axial arteries heading laterally to be larger and longer than those heading medially. CONCLUSIONS: Two dominant perforators are preferable in DIEP flap breast reconstruction. Lateral perforators play a more important role in flap perfusion than do medial ones. Crossover anastomosis is an effective technology for increasing arterial perfusion areas. Our rezoning shows which areas are better for surgery and which have a high risk of complications-valuable information for a surgeon designing a flap for breast reconstruction.

Characteristics of Bone Strength and Metabolism in Type 2 Diabetic Model Nagoya Shibata Yasuda Mice.

We evaluated the suitability of Nagoya Shibata Yasuda (NSY) mice as an animal model for examining the influence of a glucose metabolism disorder on bone integrity, using Institute of Cancer Research (ICR) mice as controls. We selected six NSY and ICR mice each that were matched for weight, and measured serum glucose levels, serum insulin levels, and conducted an oral glucose tolerance test. Histological sections of the femurs of both mouse lines were prepared, and the bone strength, mass, and microstructure of the femur were compared, along with bone metabolism. Serum glucose levels were significantly higher in the NSY mice than in the control mice, but body weight and serum insulin levels did not differ between the groups. Bone mass, microstructure, and strength of the femur, and bone metabolism were lower in the NSY mice than in the control mice. In the cortical bone of the femur in the NSY mice, several parts were not stained with eosin, demonstrating a strong negative correlation between serum glucose levels and bone mineral density; however, there was a negative correlation between serum glucose levels and bone metabolic markers. The bone turnover rate in the NSY mice was decreased by hyperglycemia, resulting in a thinner and shorter femur, reduced cortical and trabecular areas, and lower bone mass compared to those of the control mice. Collectively, these results suggest deteriorated bone strength of the femur in NSY mice, serving as a useful model for studying the link between glucose metabolism and bone integrity.

Germline factor DDX4 functions in blood-derived cancer cell phenotypes.

DDX4 (the human ortholog of Drosophila Vasa) is an RNA helicase and is present in the germ lines of all metazoans tested. It was historically thought to be expressed specifically in germline, but with additional organisms studied, it is now clear that in some animals DDX4/Vasa functions outside of the germline, in a variety of somatic cells in the embryo and in the adult. In this report, we document that DDX4 is widely expressed in soma-derived cancer cell lines, including myeloma (IM-9) and leukemia (THP-1) cells. In these cells, the DDX4 protein localized to the mitotic spindle, consistent with findings in other somatic cell functions, and its knockout in IM-9 cells compromised cell proliferation and migration activities, and downregulated several cell cycle/oncogene factors such as CyclinB and the transcription factor E2F1. These results suggest that DDX4 positively regulates cell cycle progression of diverse somatic-derived blood cancer cells, implying its broad contributions to the cancer cell phenotype and serves as a potential new target for chemotherapy.

Podoplanin promotes progression of malignant pleural mesothelioma by regulating motility and focus formation.

Malignant pleural mesothelioma (MPM) is characterized by dissemination and aggressive growth in the thoracic cavity. Podoplanin (PDPN) is an established diagnostic marker for MPM, but the function of PDPN in MPM is not fully understood. The purpose of this study was to determine the pathogenetic function of PDPN in MPM. Forty-seven of 52 tumors (90%) from Japanese patients with MPM and 3/6 (50%) MPM cell lines tested positive for PDPN. Knocking down PDPN in PDPN-high expressing MPM cells resulted in decreased cell motility. In contrast, overexpression of PDPN in PDPN-low expressing MPM cells enhanced cell motility. PDPN stimulated motility was mediated by activation of the RhoA/ROCK pathway. Moreover, knocking down PDPN with short hairpin (sh) RNA in PDPN-high expressing MPM cells resulted in decreased development of a thoracic tumor in mice with severe combined immune deficiency (SCID). In sharp contrast, transfection of PDPN in PDPN-low expressing MPM cells resulted in an increase in the number of Ki-67-positive proliferating tumor cells and it promoted progression of a thoracic tumor in SCID mice. Interestingly, PDPN promoted focus formation in vitro, and a low level of E-cadherin expression and YAP1 activation was observed in PDPN-high MPM tumors. These findings indicate that PDPN is a diagnostic marker as well as a pathogenetic regulator that promotes MPM progression by increasing cell motility and inducing focus formation. Therefore, PDPN might be a pathogenetic determinant of MPM dissemination and aggressive growth and may thus be an ideal therapeutic target.

Does a Treadmill Running Exercise Contribute to Preventing Deterioration of Bone Mineral Density and Bone Quality of the Femur in KK-Ay Mice, a Type 2 Diabetic Animal Model?

Although it has been recently shown that type 2 diabetics have an increased risk of hip fracture, the effects of exercise therapy to prevent this have not been clarified. We examined whether a treadmill running exercise contributes to the bone mineral density (BMD) and bone microarchitecture of the femur and what kind of exercise intensity and duration are optimum in type 2 diabetes mellitus using KK-Ay diabetic mice. The mice were divided into two running groups, one fast speed and short duration (FS), the other slow speed and long duration (SL), and a group of controls with no running (CO). The running exercise was started when the mice were 8 weeks of age, and continued once a day 5 days per week for 10 weeks. Ten weeks after the start of the running exercise, the BMD of the proximal region and mid-diaphysis in the SL were significantly higher in comparison with that in the CO, whereas there was no difference in bone microarchitecture among the three groups. Blood glucose, insulin levels, and visceral fat contents in the SL were significantly lower than those in the CO and FS. Bone resorption protein and C-reactive protein levels in the SL were significantly lower than those in the CO. These results suggest that slow, long duration loading is better for both bone and glycemic control than fast, short duration loading in type 2 diabetes.

A novel in vivo model for studying conditional dual loss of BLIMP-1 and p53 in B-cells, leading to tumor transformation.

The tumor suppressors B-lymphocyte-induced maturation protein-1 (BLIMP-1) and p53 play a crucial role in B-cell lymphomas, and their inactivation contributes to the pathogenesis of a wide spectrum of lymphoid malignancies, including diffuse large B-cell lymphomas (DLBCLs). Patients with activated B-cell-like (ABC) DLBCL may present with loss of BLIMP-1, c-Myc over-expression, decreased p53, and poor prognosis. Nevertheless, there is a lack of in vivo models recapitulating the biology of high-grade ABC DLBCL. We therefore aimed to develop an in vivo model aiming to recapitulate the phenotype observed in this cohort of patients. A Cre-Lox approach was used to achieve inactivation of both p53 and BLIMP-1 in murine B-cells. Contextual ablation of BLIMP-1 and p53 led to development of IgM-positive B-cell lymphoma with an aggressive phenotype, supported by c-Myc up-regulation, and accumulation of somatic mutations, as demonstrated by whole exome sequencing. Sensitivity of B-tumor cells to BTK inhibition was demonstrated. This model mirrors what reported in patients with ABC DLBLC, and therefore represents a novel model for studying the biology of ABC-DLBCL harboring the dual loss of BLIMP-1/p53 and c-Myc over-expression.

Computer-Based Radiographic Quantification of Joint Space Narrowing Progression Using Sequential Hand Radiographs: Validation Study in Rheumatoid Arthritis Patients from Multiple Institutions.

We have developed a refined computer-based method to detect joint space narrowing (JSN) progression with the joint space narrowing progression index (JSNPI) by superimposing sequential hand radiographs. The purpose of this study is to assess the validity of a computer-based method using images obtained from multiple institutions in rheumatoid arthritis (RA) patients. Sequential hand radiographs of 42 patients (37 females and 5 males) with RA from two institutions were analyzed by a computer-based method and visual scoring systems as a standard of reference. The JSNPI above the smallest detectable difference (SDD) defined JSN progression on the joint level. The sensitivity and specificity of the computer-based method for JSN progression was calculated using the SDD and a receiver operating characteristic (ROC) curve. Out of 314 metacarpophalangeal joints, 34 joints progressed based on the SDD, while 11 joints widened. Twenty-one joints progressed in the computer-based method, 11 joints in the scoring systems, and 13 joints in both methods. Based on the SDD, we found lower sensitivity and higher specificity with 54.2 and 92.8%, respectively. At the most discriminant cutoff point according to the ROC curve, the sensitivity and specificity was 70.8 and 81.7%, respectively. The proposed computer-based method provides quantitative measurement of JSN progression using sequential hand radiographs and may be a useful tool in follow-up assessment of joint damage in RA patients.

Relationship between the contrast effects of raw data projection images from three-dimensional digital subtraction angiography and the optimal volume rendering parameters.

Volume rendering (VR) is a technique commonly used for the reconstruction of three-dimensional (3D) digital subtraction angiography (DSA) images, and the rendering parameters greatly affect the characteristics of the 3D image. This study aimed to test whether the optimal VR parameters for 3D DSA could be estimated from the contrast effects in rotational two-dimensional (2D) DSA images acquired using 3D DSA. Simulated blood vessels filled with various concentrations of contrast medium were scanned, and the 3D DSA data sets were reconstructed. The syngo AX vessel analysis software that was able to analyze 3D DSA VR image was used for objective measures. Raw data projection images of the 3D DSA data sets in which the mean diameter was calculated as a true value by the software at nine different thresholds for vessel segmentation were selected. In each image set, five images of all 133 rotational 2D DSA images were selected, and the contrast-enhanced area was extracted using a region-growing algorithm. Mean values and standard deviations of each contrast-enhanced area were calculated, and as the thresholds for vessel segmentation of the software increased by 500 every time, significant differences were observed in the mean values (P < 0.01). This optimal threshold can be applied to the window settings of the VR technique. Therefore, the optimal VR parameters for 3D DSA may be determined by analyzing the contrast effects of the raw data projection images, and user-dependent over- and underestimations of 3D DSA VR images also may be prevented.

Contrast-enhanced ultrasonographic findings in three dogs with pancreatic insulinoma.

Abdominal ultrasonography is one of the most common diagnostic imaging modalities used for dogs with suspected insulinoma; however, pancreatic masses are clearly identified in fewer than half of affected dogs and benign pancreatic nodules can be difficult to differentiate from malignant ones. The purpose of this prospective study was to describe contrast-enhanced ultrasonography (CEUS) characteristics of confirmed pancreatic insulinoma in a group of dogs. Inclusion criteria were as follows: (1) repeated hypoglycemia (blood glucose levels <60 mg/dl, twice or more); (2) elevated blood insulin levels with hypoglycemia; (3) pancreatic nodules detected with conventional ultrasonography; and (4) histological confirmation of pancreatic islet cell carcinoma. Immediately following conventional ultrasonography of the entire abdomen, CEUS of the pancreatic nodule and adjacent parenchyma was performed using contrast-specific technology pulse inversion imaging and perflubutane microbubble contrast agent. Three dogs met inclusion criteria. Pancreatic nodules in all the three dogs became more clearly demarcated after injection of the contrast agent. Each nodule showed different enhancement patterns: markedly hyperechoic for 5 s, slightly hyperechoic for 1 s, and clearly hypoechoic for over 30 s. These results were not in complete agreement with previously reported CEUS findings in human patients with insulinoma. All nodules were surgically resected and histopathologically confirmed as malignant insulinomas. Findings from the current study indicated that contrast-enhanced ultrasound may help to increase conspicuity of pancreatic insulinomas in dogs and that enhancement characteristics may be more variable in dogs than in humans.

Expression of Aggrus/podoplanin in bladder cancer and its role in pulmonary metastasis.

Platelet aggregation-inducing factor Aggrus, also known as podoplanin, is associated with tumor malignancy by promoting hematogenous metastasis. Aggrus overexpression has been reported in some tumor tissues including lung, esophagus, head and neck and brain. We here found the frequent upregulation of aggrus mRNA in urinary bladder cancers using cancer tissue panels from various organs. Immunohistochemical analysis confirmed Aggrus protein expression in urinary bladder cancers and suggested a positive correlation between Aggrus expression and metastatic tendency in bladder cancers. Endogenous expression of Aggrus protein on the cell surface was found in the mouse bladder cancer MBT-2 cell line and human bladder cancer SCaBER cell lines. Knockdown of Aggrus expression in MBT-2 cells decreased their ability to induce platelet aggregation and form pulmonary metastasis in syngeneic mouse models. Knockdown of Aggrus expression in the human bladder cancer SCaBER cells also attenuated their ability to induce platelet aggregation and form pulmonary metastasis in mice. Moreover, pulmonary metastasis of SCaBER cells was prevented by prior administration of our generated anti-Aggrus neutralizing monoclonal antibodies by attenuating their retention in lung. These results indicate that Aggrus plays an important role in bladder cancer metastasis. Thus, anti-Aggrus neutralizing antibodies would be useful for the prevention of hematogenous metastasis of Aggrus-positive bladder cancer.