Soichiro Funaki

IL-6 Secreted from Cancer-Associated Fibroblasts Mediates Chemoresistance in NSCLC by Increasing Epithelial-Mesenchymal Transition Signaling

Introduction: The tumor microenvironment is composed of different types of stromal cells that represent a key component of tumor progression. Cancer‐associated fibroblasts (CAFs) secrete several factors that promote tumorigenesis. The purpose of this study was to clarify the role of the interleukin‐6 (IL‐6) secreted from CAFs in the communication between CAFs and NSCLC cells that modulates chemoresistance. Methods: We used standard NSCLC cell lines as well as NSCLC cells, lung normal fibroblasts, and CAFs obtained from specimens from patients with NSCLC to evaluate phenotypic changes. Immunohistochemical analysis was also utilized to examine the stromal changes in tumor specimens obtained from patients with NSCLC who had undergone chemotherapy. Results: IL‐6 significantly increased transforming growth factor‐&bgr;1–induced epithelial‐to‐mesenchymal transition (EMT) changes in cancer cells. Cisplatin treatment increased expression of transforming growth factor‐&bgr; in cancer cells, and the conditioned media from cancer cells activated fibroblasts and increased their IL‐6 production. Expression of IL‐6 was increased in CAFs compared with in lung normal fibroblasts. The conditioned media from CAFs induced EMT and resistance to cisplatin in NSCLC cells through IL‐6 signaling. Immunohistochemical analysis showed that stromal IL‐6 expression was correlated with EMT changes in cancer cells as well as with a diffuse distribution of smooth muscle actin–stained fibroblasts. Univariate and multivariate analyses indicated that stromal IL‐6 expression was an independent prognostic factor in patients with NSCLC. Conclusions: IL‐6 from CAFs enhanced EMT in NSCLC cells. IL‐6 may contribute to maintenance of a paracrine loop that functions as part of the communication between CAFs and NSCLC cells, resulting in chemoresistance.

Atrial natriuretic peptide prevents cancer metastasis through vascular endothelial cells

Significance Postoperative cancer recurrence is a major problem following curative cancer surgery. Perioperative systemic inflammation induces the adhesion of circulating tumor cells released from the primary tumor to the vascular endothelium of distant organs, which is the first step in hematogenous metastasis. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here, we demonstrate that cancer recurrence after lung cancer surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). We show that ANP prevents cancer metastasis by suppressing the inflammatory reaction of endothelial cells, thereby inhibiting cancer cell adhesion to vascular endothelial cells. Most patients suffering from cancer die of metastatic disease. Surgical removal of solid tumors is performed as an initial attempt to cure patients; however, surgery is often accompanied with trauma, which can promote early recurrence by provoking detachment of tumor cells into the blood stream or inducing systemic inflammation or both. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). ANP is known to bind specifically to NPR1 [also called guanylyl cyclase-A (GC-A) receptor]. In mouse models, we found that metastasis of GC-A–nonexpressing tumor cells (i.e., B16 mouse melanoma cells) to the lung was increased in vascular endothelium-specific GC-A knockout mice and decreased in vascular endothelium-specific GC-A transgenic mice compared with control mice. We examined the effect of ANP on tumor metastasis in mice treated with lipopolysaccharide, which mimics systemic inflammation induced by surgical stress. ANP inhibited the adhesion of cancer cells to pulmonary arterial and micro-vascular endothelial cells by suppressing the E-selectin expression that is promoted by inflammation. These results suggest that ANP prevents cancer metastasis by inhibiting the adhesion of tumor cells to inflamed endothelial cells.

Interaction between non-small-cell lung cancer cells and fibroblasts via enhancement of TGF-β signaling by IL-6

INTRODUCTION Fibroblasts are key components of the tumor microenvironment. We clarified the role of transforming growth factor (TGF)-β and interleukin (IL)-6 in the interaction between fibroblasts and non-small-cell lung cancer (NSCLC) cells. METHODS We used NSCLC cells (A549, NCI-H358) and normal human lung fibroblast (NHLF) cells to evaluate phenotypic changes in the presence of human IL-6, TGF-β1, and conditioned media (CM) from these cells. Possible pathways were evaluated with SB431542, a TGF-β receptor inhibitor, or an anti-human IL-6 receptor neutralizing antibody (IL-6R-Ab). RESULTS A549 and NCI-H358 cells incubated with IL-6 (50 ng/mL) and TGF-β1 (2 ng/mL) showed significantly increased epithelial-mesenchymal transition (EMT) signaling compared to those treated with TGF-β1 alone. Furthermore, NHLF cells were synergistically activated by IL-6 and TGF-β1. IL-6 increased the expression of TGF-β type I receptors on the surface of A549, NCI-H358 and NHLF cells and enhanced TGF-β signaling. TGF-β1 induced phenotypic changes were attenuated by IL-6R-Ab. NHLF cells were activated and A549 cells showed induction of EMT in response to CM from the other cell type. These activities were attenuated by SB431542 or IL-6R-Ab, suggesting that interplay between NSCLC cells and NHLF may lead to increased EMT signaling in NSCLC cells and activation of NHLF cells through TGF-β and IL-6 signaling. Subcutaneous co-injection of A549 and NHLF cells into mice resulted in a high rate of tumor formation compared with injection of A549 cells without NHLF cells. SB431542 or IL-6R-Ab also attenuated the tumor formation enhanced by co-injection of the two cell types. CONCLUSION IL-6 enhanced epithelial cell EMT and stimulated tumor progression by enhancing TGF-β signaling. IL-6 and TGF-β may play a contributing role in maintenance of the paracrine loop between these two cytokines in the communication between fibroblasts and NSCLC cells for tumor progression.

The oncological feasibility and limitations of video-assisted thoracoscopic thymectomy for early-stage thymomas

OBJECTIVES Although video-assisted thoracoscopic thymectomy (VATS-Tx) for thymoma has been introduced, its oncological outcome remains unclear. Our institutional experience with early-stage thymoma was retrospectively reviewed to evaluate the oncological feasibility of thoracoscopic thymectomy. METHODS A retrospective review consisting of 74 patients with Masaoka Stage I and II thymomas who had undergone thymectomy was performed. Forty-five patients underwent thoracoscopic thymectomy, while 29 underwent thymectomy through the open sternotomy approach. The clinical factors associated with the surgical outcome, including tumour recurrence, were investigated. RESULTS Neither operative death nor major postoperative complications were observed. The median intraoperative blood loss and operative time of thoracoscopic thymectomy were 50 ml and 180 min, respectively. Among the patients with thymomas >5 cm, the number of patients with operative time >4 h was 9 of 26 (34.6%) in the thoracoscopic thymectomy and 1 of 21 (4.8%) in the open sternotomy groups. Pleural recurrence was observed in 3 (6.7%) patients with thymoma >5 cm only in the thoracoscopic thymectomy group. Tumour capsule injury by manipulation during the operation was recorded in 2 of these 3 patients. In 2 of the 3 cases who had tumours with cystic portions on computed tomography, a tumour capsule injury occurred due to manipulation during thoracoscopic thymectomy. CONCLUSIONS VATS-Tx for early-stage thymomas is feasible, while the indications should be carefully considered in patients with large or cystic tumours. The conventional open sternotomy approach could be recommended in patients with thymomas >5 cm to avoid capsule injury.

Pulmonary Fibroblasts Induce Epithelial Mesenchymal Transition and Some Characteristics of Stem Cells in Non-Small Cell Lung Cancer

BACKGROUND Fibroblasts are key components of the tumor microenvironment. The purpose of this study was to clarify the role of fibroblasts in tumor progression in non-small cell lung cancer (NSCLC). METHODS Fibroblasts isolated from surgical exploration were co-cultured with human lung adenocarcinoma cell lines. We defined fibroblasts obtained from tumors as cancer associated fibroblasts (CAFs) and those from normal lung tissue as lung normal fibroblasts (LNFs). RESULTS Expression levels of myofibroblast markers were higher in CAFs than LNFs within 5 passages in the absence of continuing interaction with carcinoma cells. Thus, we used at least 2 pairs of these CAFs and LNFs in the following experiments; conditioned medium (CM) from fibroblast-induced epithelial mesenchymal transition and acquisition of cancer stem cell-like qualities in lung cancer cells (A549 and NCI-H358), indicating that CM from fibroblasts was biologically active. Furthermore, the concentration of the transforming growth factor (TGF)-β1 was higher in CM from CAFs as compared with that from LNFs, and phenotypic changes of cancer cells by CM from CAFs were greater than those induced by CM from LNFs. These CAF-induced changes were inhibited by addition of the TGF-β inhibitor SB431542. Subcutaneous co-injection of lung cancer cells and CAFs in mice enhanced tumor growth when compared with cancer cells alone, which was attenuated by administration of SB431542. CONCLUSIONS Fibroblasts were associated with increased malignant potential and the acquisition of stem cell-like properties in NSCLC tumors. Targeting CAFs as a therapeutic strategy against cancer is an intriguing concept that would benefit from further study.

Novel approach for detection of isolated tumor cells in pulmonary vein using negative selection method: morphological classification and clinical implications

OBJECTIVE The presence of isolated tumor cells (ITCs) in the pulmonary vein (PV) of a lung resected for lung cancer has been reported to be a prognostic factor. Previous investigations noted correlations between prognosis and the presence or amount of ITCs, although few studies have investigated the clinical implications of the morphological characteristics of those cells. We assessed the clinical implications of ITCs in the PV using a novel enrichment approach that maintained their morphological characteristics. METHODS Ninety-four consecutive patients with primary non-small-cell lung cancer (NSCLC) without preoperative chemo- and/or radiation therapy (p-stage I in 75, II in 13, III or IV in six) were studied. Blood samples were drawn from the PV draining the lung just after pulmonary resection, and ITCs were enriched using a CD45-negative selection method and density-gradient centrifugation, followed by Papanicolaou staining using 1ml of PV blood and immunohistochemical staining for cytokeratin in cases with an additional available blood sample. The ITCs were classified into four types based on patterns of cluster formation: no tumor cells (N), singular tumor cells (S), clustered cells (≤ 0.2mm) (CSs), and bulky clustered cells (> 0.2mm) (BCSs). We evaluated the correlations between ITC morphology and clinical results. RESULTS ITCs were detected in 68 of 94 patients (72%), of which the BCS type was observed in two, CS in 33, S in 33, and N in 26. Over a median follow-up period of 13 months (range 6-22 months), cancer recurrence occurred in 16 cases (17%): 14 in the combined CS/BCS group, one in S, and one in N. Log-rank analysis revealed that the disease-free survival rate was exclusively worse in patients with clustered ITCs as compared with the other two groups (p < 0.01). CONCLUSIONS The present method was useful to detect and enrich ITCs from the PV, and showed the clinical relevance of their morphology in lung cancer cases. The presence of ITC clusters may be a prognostic biomarker for patients with resected NSCLC.

Pluripotent Stem Cells Derived From Mouse Primordial Germ Cells by Small Molecule Compounds

Primordial germ cells (PGCs) can give rise to pluripotent stem cells known as embryonic germ cells (EGCs) when cultured with basic fibroblast growth factor (bFGF), stem cell factor (SCF), and leukemia inhibitory factor. Somatic cells can give rise to induced pluripotent stem cells (iPSCs) by introduction of the reprogramming transcription factors Oct4, Sox2, and Klf4. The effects of Sox2 and Klf4 on somatic cell reprogramming can be reproduced using the small molecule compounds, transforming growth factor‐β receptor (TGFβR) inhibitor and Kempaullone, respectively. Here we examined the effects of TGFβR inhibitor and Kempaullone on EGC derivation from PGCs. Treatment of PGCs with TGFβR inhibitor and/or Kempaullone generated pluripotent stem cells under standard embryonic stem cell (ESC) culture conditions without bFGF and SCF, which we termed induced EGCs (iEGCs). The derivation efficiency of iEGCs was dependent on the differentiation stage and sex. DNA methylation levels of imprinted genes in iEGCs were reduced, with the exception of the H19 gene. The promoters of genes involved in germline development were generally hypomethylated in PGCs, but three germline genes showed comparable DNA methylation levels among iEGs, ESCs, and iPSCs. These results show that PGCs can be reprogrammed into pluripotent state using small molecule compounds, and that DNA methylation of these germline genes is not maintained in iEGCs. Stem Cells 2015;33:45–55

Inhibition of maintenance DNA methylation by Stella.

DNA methylation is a key epigenetic regulator in mammals, and the dynamic balance between methylation and demethylation impacts various processes, from development to disease. DNA methylation is erased during replication when DNA methyltransferase 1 (DNMT1) fails to methylate the daughter strand, in a process known as passive DNA demethylation. We found that the enforced expression of Stella (also known as PGC7, Dppa3), a maternal factor required for the maintenance of DNA methylation in early embryos, induced global DNA demethylation in NIH3T3 cells. This demethylation was caused by the binding of Stella to Np95 (also known as Uhrf1, ICBP90) and the subsequent inhibition of DNMT1 recruitment. Considering that impaired DNA methylation profiles are associated with various developmental or disease phenomena, Stella may be a powerful tool with which to study the biological effects of global DNA hypomethylation.

Experience with thoracoscopic resection for mediastinal mature teratoma: a retrospective analysis of 15 patients

OBJECTIVES Although video-assisted thoracoscopic surgery (VATS) is widely used for the resection of a mediastinal mass, it is converted to an open resection in some patients with a mature teratoma because of dense adhesions. We reviewed cases with a mature teratoma removed by VATS and investigated the indications for that procedure for this tumour. METHODS We retrospectively investigated 15 patients with a benign mediastinal mature teratoma who underwent a thoracoscopic procedure. RESULTS The mean tumour diameter was 5.3 cm (range 3.2-8.5). The mean operative time was 188 min (78-430), and intraoperative blood loss was 138 ml (10-450). Thoracoscopic resection was completed in all except 3 patients with larger tumours, which presented the most difficult problems with dissection. Each of those 3 had severe preoperative chest pain and a tumour larger than 5.5 cm. No mortality or postoperative complications were recorded, except for postoperative chylothorax. Tumour recurrence did not develop in any patient during the mean follow-up period of 4.6 years. CONCLUSIONS For selected patients with a mediastinal teratoma, VATS may be considered standard care, as most are benign. In contrast, an open approach may be more appropriate for patients with a large tumour or preoperative symptoms.

Significance of tumour vessel invasion in determining the morphology of isolated tumour cells in the pulmonary vein in non-small-cell lung cancer

OBJECTIVES The existence of clustered isolated tumour cells (ITCs) in the pulmonary vein (PV) of the lungs of patients with lung cancer has been reported to be a prognostic factor. However, the clinical-pathological characteristics related to their presence in the PV remain unclear. METHODS We analysed the surgical results and clinical-pathological findings of 130 patients who underwent surgery for non-small-cell lung cancer in regard to blood vessel invasion (BVI), serum carcinoembryonic antigen (CEA) level, maximum standardized uptake value (SUV-max), size of the solid region in computed tomography findings and pathological stage according to an ITC type, i.e. no tumour (N), singular tumour cells (S) and clustered tumour cells (C). RESULTS ITCs were detected in 96 (74%) of the patients, with C observed in 43, S in 53 and N in 34. Recurrence was seen in 33 (26%) cases, 21 of which were classified as C, 9 as S and 3 as N. The disease-free survival rate was significantly worse in C cases when compared with the others (P < 0.01). The rate of C was high in cases with high serum CEA, advanced p-staging and positive BVI ratio. Furthermore, BVI positive and ITC morphology were strongly related (BVI positive; 79 in C, 40 in S, 9% in N; P < 0.01). CONCLUSIONS Clustered ITCs were shown to be a prognostic indicator and strongly related to BVI. Our results suggest that determination of BVI has prognostic value, as clustered ITCs with metastatic potential are disseminated from the invaded vein.