Yoshinori Doki

Podoplanin Expression in Cancerous Stroma Induces Lymphangiogenesis and Predicts Lymphatic Spread and Patient Survival

CONTEXT Podoplanin is a mucin-type glycoprotein and a lymphatic endothelial marker. Immunohistochemical staining for podoplanin is currently used as a routine pathologic diagnosis tool in Japan to identify lymphatic invasion of cancer cells. Recent reports suggest that podoplanin and other proangiogenic molecules are expressed in stromal fibroblasts and myofibroblasts. OBJECTIVE To analyze the distribution of podoplanin expression in tumor stroma and its clinical and biologic significance. DESIGN We performed immunohistochemistry for podoplanin on tissue microarrays from 1350 cases of 14 common cancer types. RESULTS Two hundred eighty-seven of 662 cases (43%) showed podoplanin expression in the stromal cells within cancer nests. Stromal podoplanin expression in 14 common cancer types was significantly associated with tumor stage (P < .001), lymph node metastases (P < .001), lymphatic invasion (P  =  .02), and venous invasion (P < .001). The stromal cells positive for podoplanin were also positive for α-smooth muscle actin but negative for desmin, confirming a myofibroblasts phenotype. In contrast, myofibroblasts in inflammatory fibrotic lung diseases were podoplanin negative. Lymphatic vessel density was greater in the stromas with podoplanin expression than in the stroma lacking podoplanin-expressing stromal cells (P  =  .01). Survival data were available for non-small cell lung cancer. Stromal podoplanin expression was associated with poorer prognosis in adenocarcinoma (P < .001) and remains statistically significant after adjustment for sex, age, and stage (P  =  .01). CONCLUSION Our data indicate that podoplanin expression in stromal myofibroblasts may function as a proangiogenic biomarker and may serve as a predictive marker of lymphatic/vascular spread of cancer cells and a prognostic marker of patient survival.

Regulation of activator protein-1 activity in the mediastinal lymph node metastasis of lung cancer.

Orthotopic implantation of a metastatic cell line of Lewis lung carcinoma (LLC-MLN), which was isolated by an in vivo selection method, resulted in greater metastatic growth in mediastinal lymph nodes as compared with that of the original LLC cells. LLC-MLN cells also had increased invasive ability and activator protein-1 (AP-1) transcriptional activity as compared with the original LLC cells. This is well consistent with the previously reported finding that overexpression of AP-1 is associated with lymphatic metastasis in lung cancer patients. Oral administration of curcumin, which downregulates AP-1 transcription, significantly inhibited the mediastinal lymph node metastasis of orthotopically implanted LLC cells in a dose-dependent manner, but did not affect the tumor growth at the implantation site. Combined treatment with curcumin and an anti-cancer drug, cis-diamine-dichloroplatinum (CDDP), resulted in a marked inhibition of tumor growth at the implanted site and of lymphatic metastasis, and a significant prolongation of the survival time. The downregulation of transcriptional AP-1 activity by curcumin as seen in the dual luciferase assay caused inhibition of LLC cell invasion through the repression of expression of the mRNAs for urokinase-type plasminogen activator (u-PA) and its receptor (u-PAR). Inhibition of AP-1 transcriptional activity may offer improved therapeutic efficacy for lung cancer patients with lymphatic metastasis.

Scheduled autologous blood donation at the time of cardiac catheterization in infants and children

Preoperative autologous blood donation is commonly performed to avoid homologous blood transfusion during cardiac operations in adult patients. 1 However, autologous blood donation for children is hampered by technical problems including lack of an adequate blood collection system and acute anemia after blood collection. Given the life span of children, it is most important to avoid the complications of homologous transfusion. 2 We describe a technique of scheduled autologous blood donation during preoperative cardiac catheterization and examine the efficacy and safety of this method for use in infants and children. Technique. Cardiac catheterization was performed about 2 weeks before elective operations in infants and children weighing at least 5 kg. Autologous blood donation was performed in patients with a hematocrit value of 33% or more and a hemoglobin value of 11 gm/dl or more. Sedation was achieved with thiopental sodium and local anesthesia was achieved with lidocaine hydrochloride. Sheaths were inserted into the femoral artery and vein. After hemodynamic measurements, but before contrast angiography, 10 ml/kg of blood was collected via the arterial sheath. The same volume of lactated Ringers solution was infused at the same rate through the venous sheath. Collected blood was stored as packed red cells and plasma or as whole blood. Blood erythropoietin concentrations were measured before and after blood collection, and recombinant human erythropoietin (100 U/kg) was administered intravenously to acyanotic patients on the first and seventh days after blood collection. Cyanotic patients were not treated with recombinant human erythropoietin. Each patient was given ferrous sulfate (2 mg/ kg) orally every day. Results. From October 1995 through September 1997, preoperative autologous blood donation was performed in 27 children, including 13 infants (16 boys and 11 girls). Their ages ranged from 6 months to 6 years, 8 months (average 1.9 -+ 2.1 years). Their body weights ranged from 5.8 kg to 20.2 kg (average 9.7 -+ 5.5 kg). The patients

Critical contribution of MCL-1 in EMT-associated chemo-resistance in A549 non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of death in all lung cancer patients due to its metastatic spread. Even though cisplatin treatment after surgical resection of the primary tumor has been established as a standard chemotherapy for residual disease including metastatic spread, NSCLC often acquires a resistance against chemotherapy, and metastatic disease is often observed. Amongst many potential mechanisms, epithelial-to-mesenchymal transition (EMT) has been considered as an important process in acquiring both metastatic spread and chemo-resistance of NSCLC. In this study, we identified MCL-1 as a critical molecule for chemo-resistance in A549 cells associated with TGF-β-induced EMT. Importantly, downregulation of MCL-1 by siRNA or inhibition of MCL-1 with pan-BCL2 inhibitor to inhibit MCL-1 was able to overcome the EMT-associated chemo-resistance in A549 cells. Collectively, MCL-1 can be a new therapeutic target for overcoming EMT-associated chemo-resistance in NSCLC patients in the context of post-operative chemotherapies.

Profiling of phospho-AKT, phospho-mTOR, phospho-MAPK and EGFR in non-small cell lung cancer.

Activation of numerous pathways has been documented in non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) has emerged as a common therapeutic target. The mitogen-activated protein kinase (MAPK) and AKT signaling pathways are downstream of EGFR and deregulated via genetic and epigenetic mechanisms in many human cancers. We evaluated selected markers in the EGFR pathway with reference to outcome. Tissues from 220 cases of NSCLC patients presented in a tissue microarray were assayed with immunohistochemistry for phosphorylated AKT, phosphorylated MAPK, phosphorylated mTOR, and EGFR and then quantified by automated image analysis. Individually, the biomarkers did not predict. Combined as ratios, p-mTOR/p-AKT, and p-MAPK/EGFR function as prognostic markers of survival (p=0.008 and p=0.029, respectively), however, no significance was found after adjustment (p=0.221, p=0.103). The sum of these ratios demonstrates a stronger correlation with survival (p<0.001) and remained statistically significant after adjustment (p=0.026). The algebraic combination of biomarkers offer the capacity to understand factors that predict outcome better than current approaches of evaluating biomarkers individually or in pairs. Our results show the sum of p-mTOR/p-AKT and p-MAPK/EGFR is a potential predictive marker of survival in NSCLC patients.

Inducible Capillary Formation in Lymphatic Endothelial Cells by Blocking Lipid Phosphate Phosphatase-3 Activity

Lymphangiogenesis plays critical roles under normal and/or pathological conditions; however, the molecular contributors to this event were unknown until recently. In the present study, we first employed gene chip analysis and confirmed that lipid phosphate phosphatase-3 (LPP3) expression was increased until capillary formation in the conditionally immortalized rat lymphatic endothelial cell line. Signaling responses occur when several lipids induce acute biological functions; further, lipid phosphate phosphatases (LPPs) control their functions via dephosphorylation; however, there is no report on the association between LPP3 and lymphangiogenesis. siRNA-targeted LPP3 significantly increased capillary formation of human lymphatic endothelial cells; in contrast, it decreased cell adhesion to the basement membrane matrix. Furthermore, the inducible effect of the LPP inhibitor on capillary formation was observed. For the first time, we report that LPP3 abolishes accelerated abnormal lymphangiogenesis. Blocking LPP3 activities may aid in the development of novel therapy for lymph vessel defects.

Survival of Lung Adenocarcinoma Patients Predicted from Expression of PD-L1, Galectin-9, and XAGE1 (GAGED2a) on Tumor Cells and Tumor-Infiltrating T Cells.

The survival of lung adenocarcinoma patients could be predicted with the use of a discriminant function using as parameters tumor cell expression of PD-L1, Galectin-9, and XAGE1 (GAGED2a), and CD4 and CD8 T-cell infiltration. The immune status of tumors varies, and this may affect the overall survival (OS) of patients. We examined tumors from 120 patients with lung adenocarcinomas with a tissue microarray for T-cell infiltration and the expression of PD-L1 and Galectin-9 (both ligands for inhibitory receptors on T cells), and cancer/testis (CT) antigen XAGE1 (GAGED2a; a tumor antigen often found on lung tumors) expression, to determine their relevance to OS. Patients defined as pStage I–IIIA could be grouped, based on the expression profiles of PD-L1, Galectin-9, and XAGE1, into cluster A, who had prolonged survival, and cluster B, who had shorter survival. The difference in survival of the clusters was confirmed separately for pStage I and pStage II–IIIA patients. Cluster A patients who also had CD4 and CD8 T-cell infiltration showed even better survival, as expected. The findings were confirmed by examining an independent validation cohort of 68 pStage I lung adenocarcinoma patients. Our data showed that PD-L1 expression was a positive indicator, whereas Galectin-9 and XAGE1 expression was negative. In vitro analyses suggested that PD-L1 expression was upregulated by IFNγ secreted from activated T cells in the tumor and Galectin-9 expression was counteracting those T cells. Thus, use of these immune markers enables the creation of a discriminant function with which to classify tumors and predict survival. Cancer Immunol Res; 4(12); 1049–60. ©2016 AACR.

Calpain 1 and -2 play opposite roles in cord formation of lymphatic endothelial cells via eNOS regulation

Calpains are a family of calcium-dependent proteases. Two isoforms, calpain 1 and 2, have been implicated in angiogenesis and endothelial cell adhesion and migration. Calpains regulate the function of eNOS; however, the relation of calpains and eNOS to lymphangiogenesis is still unclear. In the present study, we evaluated the role of calpain and eNOS in the formation of cords by lymphatic endothelial cells on Matrigel. Human lymphatic microvascular dermal-derived endothelial cells were transfected with siRNA against calpain 1 or 2. Calpain 2 knockdown, but not calpain 1 knockdown, significantly reduced cord formation, adhesion, and migration on Matrigel. These decreases correlated with a reduction in eNOS, and phosphorylated eNOS and Hsp90 levels, as assayed by immunoprecipitation and western blotting. In contrast, the knockdown of calpain 1, but not calpain 2, increased cell adhesion, enhanced migration, and stabilized late-stage cord formation by increasing cord length compared to the control. These differences correlated with an increase in the level of phosphorylated eNOS. The results indicated that the functions of calpains and eNOS are important for cord formation by lymphatic endothelial cells. For the first time, we have found different functions of calpain 1 and 2. Calpain 1 is involved in the degradation of eNOS and Hsp90 and the phosphorylation of eNOS, while calpain 2 regulates eNOS phosphorylation during cord formation by lymphatic endothelial cells on Matrigel.

Differential distribution of lymphatic clearance between upper and lower regions of the lung.

Many lung diseases arise as the consequence of inhalational injury. When pathogenic materials are inhaled, it is possible that their clearance routes become the main focus of injury in the lung. Lymphatic clearance is important in the removal from the lung of small inhaled particles. The leak of toxic agents from the lymphatic flow potentially explains the topographic distribution of diffuse lung diseases triggered by inhaled materials, for example asbestosis. The aim of this study was to evaluate the differences in lymphatic distribution across various craniocaudal levels of the lung by using carbon dust deposition (CDD) as a tracing marker.

Early Surgeryfor Treatment of Spontaneous Hemopneumothorax

Purpose: Spontaneous hemopneumothorax (SHP) is a rare life threatening disorder. We retrospectively investigated patients with SHP who were treated with video- assisted thoracic surgery (VATS), and report our results. Methods: From January 1993 to July 2006, 239 patients with spontaneous pneumothorax were treated, among whom 11 (4.6%) were diagnosed with SHP. Results: All 11 patients had a collapsed lung condition worse than moderate and a chest tube inserted, of whom 10 underwent an emergency operation. The points of hemorrhaging, each of which were in the apical portion of the lung, were easily revealed during VATS, and we were able to distinguish between brisk flow and seepage. Hemostasis was acquired using VATS in all surgery cases, while the other was treated with tube drainage. The single patient who did not undergo surgical treatment had recurrent spontaneous pneumothorax 3 months later. Conclusion: It is important to perform surgery for SHP at the appropriate time. VATS was found to be an easily performed and safe procedure for initial treatment in patients with active hemorrhaging and massive blood clotting in the thorax. The long-term outcome of our patients with early surgical indication was excellent and we recommend early surgical treatment for SHP.