Miki Yamaguchi

Fibroblastic foci, covered with alveolar epithelia exhibiting epithelial–mesenchymal transition, destroy alveolar septa by disrupting blood flow in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown cause. IPF has a distinct histopathological pattern of usual interstitial pneumonia in which fibroblastic foci (FF) represent the leading edge of fibrotic destruction of the lung. Currently there are three major hypotheses for how FF are generated: (1) from resident fibroblasts, (2) from bone marrow-derived progenitors of fibroblasts, and (3) from alveolar epithelial cells that have undergone epithelial–mesenchymal transition (EMT). We found that FF dissociated capillary vessels from the alveolar epithelia, the basement membranes of which are fused in normal physiological conditions, and pushed the capillaries and elastic fibers down ~100 μm below the alveolar epithelia. Furthermore, the alveolar epithelial cells covering the FF exhibited a partial EMT phenotype. In addition, normal human alveolar epithelial cells in vitro underwent dynamic EMT in response to transforming growth factor-β signaling within 72 h. Because it seems that resident fibroblasts or bone marrow-derived cells cannot easily infiltrate and form FF between the alveolar epithelia and capillaries in tight contact with each other, FF are more likely to be derived from the epithelial-to-mesenchymal transitioned alveolar epithelia located over them. Moreover, histology and immunohistochemistry suggested that the FF formed in the lung parenchyma disrupt blood flow to the alveolar septa, thus destroying them. Consequently, collapse of the alveolar septa is likely to be the first step toward honeycombing in the lung during late stage IPF. On the basis of these findings, inhibition of transforming growth factor-β signaling, which can suppress EMT of the alveolar epithelial cells in vitro, is a potential strategy for treating IPF.

Generation of a Considerable Number of Functional Mast Cells with a High Basal Level of FcɛRI Expression from Cord Blood CD34+ Cells by Co-culturing Them with Bone Marrow Stromal Cell Line Under Serum-free Conditions

The number of mast cells (MC) that can be obtained from tissue is limited, making it difficult to study the role of MC. Cultured MC derived from cord blood (CB)‐CD34+ cells proliferate well compared with those derived from adult CD34+ cells; however, they have been reported to be phenotypically or functionally immature regardless of culture system. For example, very few cells express FcɛRI. To resolve this problem, we addressed the effect of human bone marrow stromal cell line on the development of cultured MC. CB‐CD34+ (1 × 104) cells were cultured for 8 weeks in a serum‐free medium containing rhIL‐6 and rhSCF with or without a human bone marrow stromal cell line, namely, co‐culture and liquid culture, and were compared in various regards. MC were basically determined by metachromatic staining of granules. The number of MC obtained (60.3 ± 15.8 × 105 versus 2.0 ± 1.0 × 105), percentage of FcɛRI+ cells (29.3 ± 9.4% versus 1.9 ± 0.8%), histamine content (9.7 ± 2.8 pg/cell versus 5.8 ± 2.3 pg/cell), and IgE‐mediated histamine release (46 ± 10% versus 17 ± 7%) were higher (P < 0.01 and P < 0.05) in the co‐culture than in the liquid culture. When CB‐CD34+ cells were developed in liquid culture with the co‐culture supernatant (CM), a significant increase (P < 0.01) in the percentage of FcɛRI+ cells and in cell number was observed but these values were lower than those of co‐cultured MC. We concluded that this co‐culture system was useful for obtaining a considerable number of mature MC with a high basal level of functional FcɛRI expression from CB‐CD34+ cells. Yet unknown humoral factors in CM may partly mediate this effect.

A newly developed anti-Mucin 13 monoclonal antibody targets pancreatic ductal adenocarcinoma cells

Pancreatic cancer is one of the most severe forms of malignancy. Patients with unresectable or metastatic pancreatic cancer usually receive chemotherapy that causes various adverse effects. Antibody-drug conjugates (ADCs), drugs developed by conjugating an anticancer agent to a monoclonal antibody (mAb), can alleviate the side effects of chemotherapy because ADCs selectively bind to cancer cells expressing a particular antigen. We recently developed the recombinant protein DT3C comprising diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C). The mAb-DT3C conjugates can be used to select mAbs that are internalized by cells, because the conjugates decrease cell viability only when they are internalized by cells through Ab-antigen reactions. We developed a new mAb to be internalized by TCC-PAN2 cells, a pancreatic carcinoma cell line. The mAb, designated TCC56, recognized Mucin 13 (MUC13), while TCC56‑DT3C conjugates induced cell death in TCC-PAN2 cells expressing MUC13. We found that MUC13 was expressed, at least partially, in all 40 pancreatic ductal carcinoma tissues and adjacent non-cancerous tissues analyzed. The expression levels of MUC13 in pancreatic cancer tissues were greater than those in normal tissues. Our findings suggest that MUC13 can be a target molecule for pancreatic cancer treatment. ADCs, including mAb TCC56, could be promising anticancer agents to alleviate the adverse effects of chemotherapy.

Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells.

Antibody-drug conjugates (ADCs), drugs developed by conjugation of an anticancer agent to a monoclonal antibody (mAb), have lately attracted attention in cancer therapy because ADCs can directly bind cancer cells and kill them. Although mAbs for ADCs must be internalized by the target cells, few methods are available for screening mAbs for their ability to be internalized by cells. We have developed a recombinant protein, termed DT3C, which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C). When a mAb-DT3C conjugate, which functions in vitro like an ADC, reduces the viability of cancer cells, the mAb being tested must have been internalized by the target cells. DT3C can thus be a tool to identify efficiently and easily mAbs that can be internalized by cells, thereby enhancing the development of promising ADCs.

Development of an oncolytic HSV vector fully retargeted specifically to cellular EpCAM for virus entry and cell-to-cell spread

Oncolytic herpes simplex virus (HSV) vectors have attracted increasing attention as novel anti-cancer agents. HSV entry is triggered by the binding of glycoprotein D (gD) to its receptors, such as herpesvirus entry mediator or nectin-1. We have recently reported the construction of a fully retargeted HSV platform that incorporates single-chain antibodies (scFv) into gD to mediate entry exclusively via tumor-associated antigens. In this study, we created an scFv directed against epithelial cell adhesion molecule (EpCAM), a recognized carcinoma-associated antigen, and inserted it into the retargeted HSV platform that is ablated for gD recognition of its canonical receptors and contains the entry-enhancing mutations in gB we previously identified. We observed that both initial entry and subsequent cell-to-cell spread of the retargeted virus were stringently dependent on cellular EpCAM expression. Interestingly, the retargeted virus developed larger plaques on some of the human tumor lines tested than the control virus bearing wild-type gD. Intratumoral injection of the retargeted virus revealed antitumor activity in a mouse xenograft model. These observations illustrate the versatility of our retargeted HSV platform as it allows expansion of the oncolytic virus toolbox for the treatment of diverse cancers.

Prolyl isomerase Pin1 promotes survival in EGFR -mutant lung adenocarcinoma cells with an epithelial–mesenchymal transition phenotype

The secondary epidermal growth factor receptor (EGFR) T790M mutation is the most prominent mechanism that confers resistance to first- or second-generation EGFR tyrosine kinase inhibitors (TKIs) in lung cancer treatment. Although third-generation EGFR TKIs can suppress the kinase activity of T790M-positive EGFR, they still cannot eradicate EGFR-mutated cancer cells. We previously reported that a subpopulation of EGFR-mutant lung adenocarcinomas depends on enhanced autophagy, instead of EGFR, for survival, and in this study we explore another mechanism that contributes to TKI resistance. We demonstrate here that an EGFR-mutant lung adenocarcinoma cell line, H1975 (L858R+T790M), has a subset of cells that exhibits an epithelial–mesenchymal transition (EMT) phenotype and can thrive in the presence of third-generation EGFR TKIs. These cells depend on not only autophagy but also on the isomerase Pin1 for survival in vitro, unlike their parental cells. The Pin1 protein was expressed in an EGFR-mutant lung cancer tissue that has undergone partial EMT and acquired resistance to EGFR TKIs, but not its primary tumor. These findings suggest that inhibition of Pin1 activity can be a novel strategy in lung cancer treatment.

Bone Marrow Stromal Cell Line Promotes the Proliferation of Mast Cell Progenitors Derived from Cord Blood CD34+ Cells under Serum-free Conditions with a Combination of Both Cell-cell Interaction and Soluble Factors

Abstract A higher production of functional mast cells (MCs) can be generated by co-culturing cord blood-derived CD34+ cells with a human bone marrow stromal cell line under serum-free conditions supplemented with stem cell factor and IL-6. We addressed the question of whether the higher proliferation of MCs in this co-culture system might be due to the higher proliferation of MC progenitors. The stromal cell line increased the cell numbers of MC progenitors derived from cord blood-derived CD34+ cells, in a combination of cell-cell interactions between stromal cells and CD34+ cells, and as yet unidentified soluble factors derived from stromal cells.

Survivin knockdown induces senescence in TTF‑1-expressing, KRAS-mutant lung adenocarcinomas

Survivin plays a key role in regulating the cell cycle and apoptosis, and is highly expressed in the majority of malignant tumors. However, little is known about the roles of survivin in KRAS-mutant lung adenocarcinomas. In the present study, we examined 28 KRAS-mutant lung adenocarcinoma tissues and two KRAS-mutant lung adenocarcinoma cell lines, H358 and H441, in order to elucidate the potential of survivin as a therapeutic target. We found that 19 (68%) of the 28 KRAS-mutant lung adenocarcinomas were differentiated tumors expressing thyroid transcription factor-1 (TTF-1) and E-cadherin. Patients with tumors immunohistochemically positive for survivin (n=18) had poorer outcomes than those with survivin-negative tumors (n=10). In the H358 and H441 cells, which expressed TTF-1 and E-cadherin, survivin knockdown alone induced senescence, not apoptosis. However, in monolayer culture, the H358 cells and H441 cells in which survivin was silenced, underwent significant apoptosis following combined treatment with ABT-263, a Bcl-2 inhibitor, and trametinib, a MEK inhibitor. Importantly, the triple combination of survivin knockdown with ABT-263 and trametinib treatment, clearly induced cell death in a three-dimensional cell culture model and in an in vivo tumor xenograft model. We also observed that the growth of the H358 and H441 cells was slightly, yet significantly suppressed in vitro when TTF-1 was silenced. These findings collectively suggest that the triple combination of survivin knockdown with ABT-263 and trametinib treatment, may be a potential strategy for the treatment of KRAS-mutant lung adenocarcinoma. Furthermore, our findings indicate that the well-differentiated type of KRAS-mutant lung tumors depends, at least in part, on TTF-1 for growth.

Syncytial Mutations Do not Impair the Specificity of Entry and Spread of a Glycoprotein D Receptor-Retargeted Herpes Simplex Virus

ABSTRACT Membrane fusion, which is the key process for both initial cell entry and subsequent lateral spread of herpes simplex virus (HSV), requires the four envelope glycoproteins gB, gD, gH, and gL. Syncytial mutations, predominantly mapped to the gB and gK genes, confer hyperfusogenicity on HSV and cause multinucleated giant cells, termed syncytia. Here we asked whether interaction of gD with a cognate entry receptor remains indispensable for initiating membrane fusion of syncytial strains. To address this question, we took advantage of mutant viruses whose viral entry into cells relies on the uniquely specific interaction of an engineered gD with epidermal growth factor receptor (EGFR). We introduced selected syncytial mutations into gB and/or gK of the EGFR-retargeted HSV and found that these mutations, especially when combined, enabled formation of extensive syncytia by human cancer cell lines that express the target receptor; these syncytia were substantially larger than the plaques formed by the parental retargeted HSV strain. We assessed the EGFR dependence of entry and spread separately by using direct entry and infectious center assays, respectively, and we found that the syncytial mutations did not override the receptor specificity of the retargeted viruses at either stage. We discuss the implications of these results for the development of more effective targeted oncolytic HSV vectors. IMPORTANCE Herpes simplex virus (HSV) is investigated not only as a human pathogen but also as a promising agent for oncolytic virotherapy. We previously showed that both the initial entry and subsequent lateral spread of HSV can be retargeted to cells expressing tumor-associated antigens by single-chain antibodies fused to a receptor-binding-deficient envelope glycoprotein D (gD). Here we introduced syncytial mutations into the gB and/or gK gene of gD-retargeted HSVs to determine whether viral tropism remained dependent on the interaction of gD with the target receptor. Entry and spread profiles of the recombinant viruses indicated that gD retargeting does not abolish the hyperfusogenic activity of syncytial mutations and that these mutations do not eliminate the dependence of HSV entry and spread on a specific gD-receptor interaction. These observations suggest that syncytial mutations may be valuable for increasing the tumor-specific spreading of retargeted oncolytic HSV vectors.

Characterization of distal airway stem-like cells expressing N-terminally truncated p63 and thyroid transcription factor-1 in the human lung

Abstract Distal airway stem cells (DASCs) in the mouse lung can differentiate into bronchioles and alveoli. However, it remains unclear whether the same stem cells exist in the human lung. Here, we found that human lung epithelial (HuL) cells, derived from normal, peripheral lung tissue, in monolayer, mostly express both the N‐terminally truncated isoform of p63 (&Dgr;Np63), a marker for airway basal cells, and thyroid transcription factor‐1 (TTF‐1), a marker for alveolar epithelial cells, even though these two molecules are usually expressed in a mutually exclusive way. Three‐dimensionally cultured HuL cells differentiated to form bronchiole‐like and alveolus‐like organoids. We also uncovered a few bronchiolar epithelial cells expressing both &Dgr;Np63 and TTF‐1 in the human lung, suggesting that these cells are the cells of origin for HuL cells. Taken together, &Dgr;Np63+ TTF‐1+ peripheral airway epithelial cells are possibly the human counterpart of mouse DASCs and may offer potential for future regenerative medicine. Graphical abstract Figure. No Caption available. HighlightsSome bronchiolar epithelial basal cells express both &Dgr;Np63 and TTF‐1 in the lung.These specific cells can form bronchiole‐like or alveolus‐like organoids.The dual‐positive cells can survive and differentiate in the mouse lung.They are a distinct type of peripheral lung epithelial stem cell in the human lung.