Minoru Toyoda

Immunosuppressive activity of CD14+ HLA-DR- cells in squamous cell carcinoma of the head and neck.

Myeloid‐derived suppressor cells (MDSC) represent a heterogeneous population and have the potential to suppress immune responses via diverse mechanisms. In recent studies, a new subset of MDSC was identified by the markers CD14+ and HLA‐DR− in the peripheral blood from cancer patients. In this study, we investigated the proportions and characteristics of CD14+ HLA‐DR− cells in patients with squamous cell carcinoma of the head and neck (SCCHN). As expected, the percentage of CD14+ HLA‐DR− cells was significantly elevated in patients relative to healthy donors and the sorted CD14+ HLA‐DR− cells were able to suppress effectively both the proliferation and IFN‐γ production of anti‐CD3/anti‐CD28 stimulated T cells, suggesting that CD14+ HLA‐DR− cells in patients with SCCHN contribute to the immune suppressive status. Furthermore, CD14+ HLA‐DR− cells revealed a higher level of CD86 and PD‐L1 expression and transforming growth factor (TGF)‐β production than CD14+ HLA‐DR+ cells. Addition of anti‐CD86 mAb, anti‐PD‐L1 mAb and anti‐TGF‐β mAb partially restored T‐cell proliferation and IFN‐γ production, respectively, indicating that the suppressive effects of CD14+ HLA‐DR− cells appear to be mediated by various molecules, including coinhibitory molecules and cytokines. Our data suggest that CD14+ HLA‐DR− cells act as potent immunosuppressive cells and particularly contribute to tumor escape from the host immune system in patients with SCCHN. (Cancer Sci 2012; 103: 976–983)

Prognostic significance of amino-acid transporter expression (LAT1, ASCT2, and xCT) in surgically resected tongue cancer.

Background:Amino-acid transporters are necessary for the tumour cell growth and survival, and have a crucial role in the development and invasiveness of cancer cells. But, it remains unclear about the prognostic significance of L-type amino-acid transporter 1 (LAT1), system ASC amino-acid transporter-2 (ASCT2), and xCT expression in patients with tongue cancer. We conducted the clinicopathological study to investigate the protein expression of these amino-acid transporters in tongue cancer.Methods:Eighty-five patients with surgically resected tongue cancer were evaluated. Tumour sections were stained by immunohistochemistry for LAT1, ASCT2, xCT, 4F2hc/CD98hc (4F2hc), Ki-67, and microvessel density (MVD) determined by CD34, and p53.Results:L-type amino-acid transporter 1 and 4F2hc were highly expressed in 61% (52 out of 85) and 45% (38 out of 47), respectively. ASC amino-acid transporter-2 and xCT were positively expressed in 59% (50 out of 85) and 21% (18 out of 85), respectively. The expression of both LAT1 and ASCT2 was significantly associated with disease staging, lymph-node metastasis, lymphatic permeation, 4F2hc expression and cell proliferation (Ki-67). xCT expression indicated a significant association with advanced stage and tumour factor. By univariate analysis, disease staging, lymphatic permeation, vascular invasion, LAT1, ASCT2, 4F2hc, and Ki-67 had a significant relationship with overall survival. Multivariate analysis confirmed that LAT1 was an independent prognostic factor for predicting poor prognosis.Conclusions:L-type amino-acid transporter 1 and ASCT2 can serve as a significant prognostic factor for predicting worse outcome after surgical treatment and may have an important role in the development and aggressiveness of tongue cancer.

Alteration of cancer stem cell‐like phenotype by histone deacetylase inhibitors in squamous cell carcinoma of the head and neck

Recent progression in the understanding of stem cell biology has greatly facilitated the identification and characterization of cancer stem cells (CSCs). Moreover, evidence has accumulated indicating that conventional cancer treatments are potentially ineffective against CSCs. Histone deacetylase inhibitors (HDACi) have multiple biologic effects consequent to alterations in the patterns of acetylation of histones and are a promising new group of anticancer agents. In this study, we investigated the effects of two HDACi, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), on two CD44+ cancer stem‐like cell lines from squamous cell carcinoma of the head and neck (SCCHN) cultured in serum‐free medium containing epidermal growth factor and basic fibroblast growth factor. Histone deacetylase inhibitors inhibited the growth of SCCHN cell lines in a dose‐dependent manner as measured by MTS assays. Moreover, HDACi induced cell cycle arrest and apoptosis in these SCCHN cell lines. Interestingly, the expression of cancer stem cell markers, CD44 and ABCG2, on SCCHN cell lines was decreased by HDACi treatment. In addition, HDACi decreased mRNA expression levels of stemness‐related genes and suppressed the epithelial‐mesencymal transition phenotype of CSCs. As expected, the combination of HDACi and chemotherapeutic agents, including cisplatin and docetaxel, had a synergistic effect on SCCHN cell lines. Taken together, our data indicate that HDACi not only inhibit the growth of SCCHN cell lines by inducing apoptosis and cell cycle arrest, but also alter the cancer stem cell phenotype in SCCHN, raising the possibility that HDACi may have therapeutic potential for cancer stem cells of SCCHN.

Immunological significance of the accumulation of autophagy components in oral squamous cell carcinoma

The immunological significance of autophagy in the tumor microenvironment remains unclear. To explore the relationship between autophagy and anti‐tumor immune responses, we investigated the expression of autophagy‐related proteins and infiltration of immune cells using immunohistochemistry (IHC). The expression of three representative autophagy components, LC3, Beclin‐1 and p62/SQSTM1, as well as the number of dendritic cells (DC), T cells and NK cells were examined by IHC in 74 patients with oral squamous cell carcinoma (OSCC). The relationship between the expression of autophagy‐associated molecules and various clinicopathological parameters was also evaluated. The expression of both LC3 and p62/SQSTM1 in the peripheral site significantly correlated with an increase in the infiltration of T cells. Furthermore, the expression of p62/SQSTM1 and Beclin‐1 correlated with that of HLA class I and class II in tumor cells, respectively. In addition, several unfavorable clinicopathological parameters correlated with an increase in the expression of LC3 in the peripheral site. The correlation observed between LC3 or p62/SQSTM1 and the infiltration of T cells suggests that autophagy may actively mobilize immune cells toward the cancer bed. Meanwhile, the three autophagy‐associated proteins examined were linked to malignant potential and an unfavorable prognosis.

Cancer-associated fibroblasts promote an immunosuppressive microenvironment through the induction and accumulation of protumoral macrophages

Stromal cells in the tumor microenvironment (TME) closely interact with tumor cells and affect tumor cell behavior in diverse manners. We herein investigated the mechanisms by which cancer-associated fibroblasts (CAFs) affect the functional polarization of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC) in vitro and in human cancer samples. The expression of CD68, CD14, CD163, CD200R, CD206, HLA-G, CD80, and CD86 was higher in CD14-positive cells co-cultured with the culture supernatants of CAFs established from OSCC specimens (CAF-educated cells) than in control cells. The gene expression level of ARG1, IL10, and TGFB1 was increased in CAF-educated cells. CAF-educated cells suppressed T cell proliferation more strongly than control cells, and the neutralization of TGF-β IL-10, or arginase I significantly restored T cell proliferation. We then investigated the relationship between the infiltration of CAFs and TAMs using tissue samples obtained from patients with OSCC. The infiltration of CAFs was associated with the numbers of CD68-positive and CD163-positive macrophages. It also correlated with lymphatic invasion, vascular invasion, lymph node involvement, and the TNM stage. The infiltration of CAFs was identified as an independent prognostic factor in OSCC. Our results indicate that CAFs play important roles in shaping the tumor immunosuppressive microenvironment in OSCC by inducing the protumoral phenotype of TAMs. Therapeutic strategies to reverse CAF-mediated immunosuppression need to be considered.

Immunosuppressive activity of cancer-associated fibroblasts in head and neck squamous cell carcinoma

Abstract Cancer-associated fibroblasts (CAFs) have been shown to play an important role in angiogenesis, invasion, and metastasis. In the present study, we determined whether CAFs within the tumor microenvironment (TME) in head and neck squamous cell carcinoma (HNSCC) contributed to promoting immunosuppression and evasion from immune surveillance. Six pairs of CAFs and normal fibroblasts (NFs) were established from the resected tumor tissues of patients with HNSCC. The effects of CAFs and NFs on the functions of T cells were comparatively analyzed. CAFs expressed the co-regulatory molecules, B7H1 and B7DC, whereas NFs did not. The expression levels of cytokine genes, including those for IL6, CXCL8, TNF, TGFB1, and VEGFA, were higher in CAFs. T cell proliferation was suppressed more by CAFs or their supernatants than by NFs. Moreover, PBMCs co-cultured with the supernatants of CAFs preferentially induced T cell apoptosis and regulatory T cells over those co-cultured with the supernatants of NFs. A microarray analysis revealed that the level of genes related to the leukocyte extravasation and paxillin signaling pathways was higher in CAFs than in NFs. These results demonstrated that CAFs collaborated with tumor cells in the TME to establish an immunosuppressive network that facilitated tumor evasion from immunological destruction.

Simple and new surgical procedure for laryngotracheal separation in pediatrics.

Objectives: In children with severe physical and mental disabilities who repeatedly develop aspiration pneumonia due to intractable aspiration, laryngotracheal separation/tracheoesophageal anastomosis or laryngotracheal separation has been performed in many institutions for the prevention of aspiration, and good results have been reported. However, families sometimes show a marked reluctance to give consent to these surgical techniques because of tracheal transection. A purpose of this study is to evaluate a new surgical procedure for laryngotracheal separation without tracheal transection.

Congenital laryngeal anomalies presenting as chronic stridor: a retrospective study of 55 patients.

OBJECTIVES Congenital laryngeal anomalies are less frequent, but their causes are surprisingly variable. In addition, a variety of synchronous airway lesions as well as comorbidities are accompanied. The objective of this study was to review of patients with congenital laryngeal anomalies presenting as chronic stridor in our experiences. METHODS Fifty-five patients, 30 male (54.5%) and 25 female (45.4%), were enrolled in this study, and their hospital records were retrospectively reviewed. RESULTS The most frequent diagnosis was laryngomalacia (36.4%), followed by subglottic stenosis (30.9%) and vocal cord paralysis (29.1%). Twenty-six (47.3%) of the 55 patients had synchronous airway lesions, whereas thirty-one (56.4%) had various comorbidities. Further analysis was performed in patients diagnosed with laryngomalacia, subglottic stenosis, or vocal cord paralysis, which are major causes of congenital laryngeal stridor. The frequency of synchronous airway lesions was not different significantly in these three groups. On the other hand, the frequency of establishment of airway in patients with laryngomalacia was significantly lower compared to those with subglottic stenosis or vocal cord paralysis. Moreover, median duration of the symptoms and the proportion of patients with poor outcome and decease in laryngomalacia were shorter and lower than that in subglottic stenosis or vocal cord paralysis. CONCLUSIONS While a variety of congenital airway anomalies were causes of chronic stridor, laryngomalacia was the most frequent diagnosis. Severe condition and progression of symptoms should increase suspicion of the synchronous airway lesions and/or comorbidities, which may be important factors for outcome as well as indication of surgical intervention.

Expression of ER stress markers (GRP78/BiP and PERK) in adenoid cystic carcinoma

Abstract Conclusion: A high GRP78/BiP expression was proved to be a significant marker for predicting poor outcome after surgery. GRP78/BiP may be a promising molecular target for treatment of ACC. Background: The glucose-regulated protein GRP78/BiP plays a crucial role in the endoplasmic reticulum (ER) stress. The level of GRP78 is highly elevated in various human cancers, but the clinicopathological significance of GRP78/BiP remains controversial in patients with adenoid cystic carcinoma (ACC). Methods: A total of 26 ACC patients were analyzed, and tumor specimens were stained by immunohistochemistry for GRP78/BiP, PERK, Ki-67, and microvessel density (MVD) determined by CD34. Results: GRP78/BiP and PERK were highly expressed in 58% (15/26) and 35% (9/26), respectively. The high expression of GRP78/BiP was significantly associated with PERK, cell proliferation and angiogenesis.

Decreasing expression of glucose-regulated protein GRP78/BiP as a significant prognostic predictor in patients with advanced laryngeal squamous cell carcinoma.

The immunoglobulin heavy chain binding protein (BiP)/glucose‐regulated protein 78 (GRP78) is important in the endoplasmic reticulum stress, and is highly expressed in various human cancers. The clinical and pathological features of GRP78/BiP are unclear in patients with advanced laryngeal squamous cell carcinoma (SCC). The purpose of this study was to investigate the clinicopathological significance of GRP78/BiP as a prognostic marker for laryngeal SCC.