Takayuki Ninomiya

Visualized Evaluation of Blood Flow to the Gastric Conduit and Complications in Esophageal Reconstruction

BACKGROUND Evaluation of the blood supply to gastric conduits is critically important to avoid complications after esophagectomy. We began visual evaluation of blood flow using indocyanine green (ICG) fluorescent imaging in July 2015, to reduce reconstructive complications. In this study, we aimed to statistically verify the efficacy of blood flow evaluation using our simplified ICG method. STUDY DESIGN A total of 285 consecutive patients who underwent esophagectomy and gastric conduit reconstruction were reviewed and divided into 2 groups: before and after introduction of ICG evaluation. The entire cohort and 68 patient pairs after propensity score matching (PS-M) were evaluated for clinical outcomes and the effect of visualized evaluation on reducing the risk of complication. RESULTS The leakage rate in the ICG group was significantly lower than in the non-ICG group for each severity grade, both in the entire cohort (285 subjects) and after PS-M; the rates of other major complications, including recurrent laryngeal nerve palsy and pneumonia, were not different. The duration of postoperative ICU stay was approximately 1 day shorter in the ICG group than in the non-ICG group in the entire cohort, and approximately 2 days shorter after PS-M. Visualized evaluation of blood flow with ICG methods significantly reduced the rate of anastomotic complications of all Clavien-Dindo (CD) grades. Odds ratios for ICG evaluation decreased with CD grade (0.3419 for CD ≥ 1; 0.241 for CD ≥ 2; and 0.2153 for CD ≥ 3). CONCLUSIONS Objective evaluation of blood supply to the reconstructed conduit using ICG fluorescent imaging reduces the risk and degree of anastomotic complication.

Iron depletion enhances the effect of sorafenib in hepatocarcinoma

ABSTACT Human hepatocellular carcinoma (HCC) is known to have a poor prognosis. Sorafenib, a molecular targeted drug, is most commonly used for HCC treatment. However, its effect on HCC is limited in clinical use and therefore new strategies regarding sorafenib treatment are required. Iron overload is known to be associated with progression of chronic hepatitis and increased risk of HCC. We previously reported that iron depletion inhibited cancer cell proliferation and conversely induced angiogenesis. Indeed iron depletion therapy including iron chelator needs to be combined with anti-angiogenic drug for its anti-cancer effect. Since sorafenib has an anti-angiogenic effect by its inhibitory targeting VEGFR, we hypothesized that sorafenib could complement the anti-cancer effect of iron depletion. We retrospectively analyzed the relationship between the efficacy of sorafenib and serum iron-related markers in clinical HCC patients. In clinical cases, overall survival was prolonged in total iron binding capacity (TIBC) high- and ferritin low-patients. This result suggested that the low iron-pooled patients, who could have a potential of more angiogenic properties in/around HCC tumors, could be adequate for sorafenib treatment. We determined the effect of sorafenib (Nexavar®) and/or deferasirox (EXJADE®) on cancer cell viability, and on cell signaling of human hepatocarcinoma HepG2 and HLE cells. Both iron depletion by deferasirox and sorafenib revealed insufficient cytotoxic effect by each monotherapy, however, on the basis of increased angiogenesis by iron depletion, the addition of deferasirox enhanced anti-proliferative effect of sorafenib. Deferasirox was confirmed to increase vascular endothelial growth factor (VEGF) secretion into cellular supernatants by ELISA analysis. In in vivo study sorafenib combined with deferasirox also enhanced sorafenib-induced apoptosis. These results suggested that sorafenib combined with deferasirox could be a novel combination chemotherapy for HCC.

Cancer-Associated Fibroblasts Affect Intratumoral CD8+ and FoxP3+ T Cells Via IL6 in the Tumor Microenvironment

Purpose: Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) play a central role in tumor progression. We investigated whether CAFs can regulate tumor-infiltrating lymphocytes (TILs) and their role in tumor immunosuppression. Experimental Design: A total of 140 cases of esophageal cancer were analyzed for CAFs and CD8+ or forkhead box protein 3 (FoxP3+) TILs by IHC. We analyzed cytokines using murine or human fibroblasts and cancer cells. Murine-derived fibroblasts and cancer cells were also inoculated into BALB/c or BALB/c-nu/nu mice and the tumors treated with recombinant IL6 or anti-IL6 antibody. Results: CD8+ TILs and CAFs were negatively correlated in intratumoral tissues (P < 0.001), whereas FoxP3+ TILs were positively correlated (P < 0.001) in esophageal cancers. Cocultured Colon26 cancer cells and fibroblasts resulted in accelerated tumor growth in BALB/c mice, along with decreased CD8+ and increased FoxP3+ TILs, compared with cancer cells alone. In vitro, IL6 was highly secreted in both murine and human cancer cell/fibroblast cocultures. IL6 significantly increased Colon26 tumor growth in immune-competent BALB/c (P < 0.001) with fewer CD8+ TILs than untreated tumors (P < 0.001), whereas no difference in BALB/c-nu/nu mice. In contrast, FoxP3+ TILs increased in IL6-treated tumors (P < 0.001). IL6 antibody blockade of tumors cocultured with fibroblasts resulted not only in regression of tumor growth but also in the accumulation of CD8+ TILs in intratumoral tissues. Conclusions: CAFs regulate immunosuppressive TIL populations in the TME via IL6. IL6 blockade, or targeting CAFs, may improve preexisting tumor immunity and enhance the efficacy of conventional immunotherapies. Clin Cancer Res; 24(19); 4820–33. ©2018 AACR.

Iron depletion is a novel therapeutic strategy to target cancer stem cells

Adequate iron levels are essential for human health. However, iron overload can act as catalyst for the formation of free radicals, which may cause cancer. Cancer stem cells (CSCs), which maintain the hallmark stem cell characteristics of self-renewal and differentiation capacity, have been proposed as a driving force of tumorigenesis and metastases. In the present study, we investigated the role of iron in the proliferation and stemness of CSCs, using the miPS-LLCcm cell model. Although the anti-cancer agents fluorouracil and cisplatin suppressed the proliferation of miPS-LLCcm cells, these drugs did not alter the expression of stemness markers, including Nanog, SOX2, c-Myc, Oct3/4 and Klf4. In contrast, iron depletion by the iron chelators deferasirox and deferoxamine suppressed the proliferation of miPS-LLCcm cells and the expression of stemness markers. In an allograft model, deferasirox inhibited the growth of miPS-LLCcm implants, which was associated with decreased expression of Nanog and Sox2. Altogether, iron appears to be crucial for the proliferation and maintenance of stemness of CSCs, and iron depletion may be a novel therapeutic strategy to target CSCs.

Abstract 4160: Prognostic correlation of tumor-infiltrating lymphocytes (TILs) and cancer associated fibroblasts (CAFs) in patients with human esophageal carcinoma

Backgrounds: Cancer associated fibroblasts (CAFs) are thought to play an essential role in cancer invasion, migration, metastasis, and tumor immunosuppression. However, there has been still a little evidence of the correlation of tumor immunosuppression and CAFs in human esophageal carcinoma. The other hand, as like rising of PD-1 antibody targeting therapy to tumor immunosuppression have been shown dramatic cure responses in melanoma and now ongoing to other malignancies. Tumor-infiltrating lymphocytes (TILs) are considered typically to represent a host immune response against carcinoma. In many types of tumors TILs have been shown their strong correlation to patient9s clinicopathological features. In this study, we evaluated the prognostic correlation of CAFs and TILs, which are classified respectively in tumor-associated CD8+ cytotoxic T lymphocytes (CTL) and FoxP3+ regulatory T cells (Treg) in surgically resected esophageal carcinoma. Materials and methods: Total 58 cases with esophageal carcinoma in our institution were evaluated for the presence of CAFs and TILs by immunohistochemistry. TILs of CTL and Treg were calculated each in the intratumoral and the peripheral tissues, and the cutoff for subgroups was defined at the median value. CAFs were defined as fibroblasts expressing alpha smooth muscle actin (α-SMA), and evaluated with the α-SMA scoring by using “Area Index”, which is calculated by imageJ. TILs and CAFs were assessed for the associations with pathological invasion depth (pT), lymph node metastases (pN), histological types, and disease-free survival (DFS) or overall survival (OS). Result: In intratumoral tissues, Treg was significantly associated with advanced T stages, and Treg and a CTL/Treg ratio were associated with lymph node metastasis. Higher CTL, higher CTL/Treg ratio and lower Treg were significantly associated with improved DFS and OS in univariate analysis. Furthermore, multivariate analysis demonstrated selected higher CTL as an independent prognostic factor (P = 0.010). On the other hand, in peripheral tissues, CTL, Treg, and CTL/Treg ratio were not correlated with clinicopathological factors or the any prognosis. Additionally the overexpression of CAFs was strongly associated with poor prognosis (P = 0.002) and the “Area Index” of α-SMA was inversely correlated with the CTL and CTL/Treg ratio in intratumoral tissues (P = 0.029, 0.017). It suggests that CAFs accumulation in tumor tissue is correlated to status of intratumoral immunesuppression. Conclusion: The CTL in intratumoral tissues are independent prognostic factors, and are considered to play an essential role in tumor immunity. Our results demonstrate that CAFs are significant correlation of immunosuppression in esophageal carcinoma. In the future, targeting CAFs therapy itself might improve tumor immunosuppression, and there is possibility to prolong a prognosis. Citation Format: Takuya Kato, Kazuhiro Noma, Yuki Katsura, Hajime Kashima, Takayuki Ninomiya, Toshiaki Ohara, Hiroshi Tazawa, Shunsuke Kagawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Prognostic correlation of tumor-infiltrating lymphocytes (TILs) and cancer associated fibroblasts (CAFs) in patients with human esophageal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4160.

Cancer-associated fibroblasts (CAFs) promote the lymph node metastasis of esophageal squamous cell carcinoma: Cancer-associated fibroblasts (CAFs) promote the lymph node metastasis of esophageal squamous cell carcinoma

Lymph node metastasis is a pathognomonic feature of spreading tumors, and overcoming metastasis is a challenge in attaining more favorable clinical outcomes. Esophageal cancer is an aggressive tumor for which lymph node metastasis is a strong poor prognostic factor, and the tumor microenvironment (TME), and cancer‐associated fibroblasts (CAFs) in particular, has been implicated in esophageal cancer progression. CAFs play a central role in the TME and have been reported to provide suitable conditions for the progression of esophageal cancer, similar to their role in other malignancies. However, little is known concerning the relevance of CAFs to the lymph node metastasis of esophageal cancer. Here, we used clinical samples of esophageal cancer to reveal that CAFs promote lymph node metastasis and subsequently verified the intercellular relationships in vitro and in vivo using an orthotopic metastatic mouse model. In the analysis of clinical samples, FAP+ CAFs were strongly associated with lymph node metastasis rather than with other prognostic factors. Furthermore, CAFs affected the ability of esophageal cancer cells to acquire metastatic phenotypes in vitro; this finding was confirmed by data from an in vivo orthotopic metastatic mouse model showing that the number of lymph node metastases increased upon injection of cocultured cancer cells and CAFs. In summary, we verified in vitro and in vivo that the accumulation of CAFs enhances the lymph node metastasis of ESCC. Our data suggest that CAF targeted therapy can reduce lymph node metastasis and improve the prognosis of patients with esophageal cancer in the future.

Clinical characteristics and management of gastric tube cancer after esophagectomy

BackgroundGastric cancer is the second most common malignancy, overlapping with thoracic esophageal cancer (TEC). Among them, metachronous gastric tube cancers after TEC surgery have been increasing. The aims of this study were to examine the clinicopathological factors and treatment outcomes of gastric tube cancer (GTC) after TEC surgery.MethodsThirty-three GTCs in 30 cases after TEC treated between 1997 and 2016 were investigated retrospectively.ResultsMost cases were males. The median interval from TEC surgery to GTC occurrence was 57 (6.5–107.5) months. Almost 2/3 lesions occurred in the lower third of the gastric tube (21/33); 29 lesions (in 26 cases) were superficial cancers, and 4 lesions were advanced cancers. Twenty-two lesions of superficial cancer were differentiated type, and the remaining seven lesions were undifferentiated type. Treatment for superficial cancer had previously been performed with partial gastric tube resection (10 lesions), and the number of cases undergoing endoscopic submucosal dissection (ESD) had increased recently (19 lesions). Most cases with superficial cancer survived without relapse. Four lesions of advanced cancer were found after a relatively long interval following TEC surgery. Most lesions of advanced cancer were scirrhous, undifferentiated type, and they died due to GTC.ConclusionGTCs may occur late in the postoperative course following TEC surgery. If they are discovered at an early stage, these lesions can be cured with ESD. Long-term periodic endoscopic examinations after TEC surgery are important.

Abstract 1560: Correlation of FAP(fibroblast activation protein)-expressing cancer associated fibroblasts (CAFs) and tumor metastasis in esophageal carcinoma

Background: The tumor and its microenvironment have dynamic interactions and signaling that strongly affect tumor progression. Cancer associated fibroblasts (CAFs) are activated fibroblasts and thought to be an important player in the tumor microenvironment. Although there are several indicators of CAF, fibroblast activation protein (FAP) is unique in its selectivity for CAFs in comparison with other markers. The aim of this study is to investigate CAFs expressing FAP and its relationship to cancer metastasis in esophageal cancer. Methods: Sections of paraffin-embedded resected primary human esophageal cancer specimens from 2008 through 2010 in Okayama University hospital were stained with antibody directed against FAP. Overall percentage of stromal FAP staining of the primary tumor was assessed semi-quantitatively (0, 1, 2, 3, 4, 5) and staining intensity was also graded (none, weak, intermediate, strong). Survival time and time to recurrence data were analyzed using Kaplan-Meier plots, log-rank tests, and Cox proportional hazards models. Results: 49 patients with resected specimens were available for study with 26 (53.1%) stage I, 8 (16.3%) stage II, 14 (28.6%) stage III, and 1 (2.0%) stage IV patients. All patients were divided to 2 groups, FAP high group and FAP low group, which are 25 patients (51%) and 24 (49%) respectively. The cutoff for subgroups was defined at the median value. FAP (immune) expression at tumoral stroma was a significant predictive factor for lymph node metastasis (p Conclusions: Our clinicopathological analysis indicates that patients whose esophageal carcinoma have high levels of stromal FAP expression are more likely to have an aggressive disease progression and a potential development of metastases and recurrence. Although therapeutic strategies targeting the tumor cells have been generally inadequate in esophageal carcinomas yet, here we announce that a stroma-targeted therapy should be considered. Now we are ongoing to evaluate metastatic potential of CAFs in vitro and vivo, in order to elucidate the function of FAP expressing CAFs and, in future to establish a novel therapeutic strategy. Citation Format: Hajime Kashima, Kazuhiro Noma, Yuki Katsura, Takuya Kato, Ryoichi Katsube, Takayuki Ninomiya, Toshiaki Ohara, Hiroshi Tazawa, Shunsuke Kagawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Correlation of FAP(fibroblast activation protein)-expressing cancer associated fibroblasts (CAFs) and tumor metastasis in esophageal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1560.

Abstract 4243: Iron control is a novel therapeutic target of cancer stem cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Iron overload is known to cause cancer in animal models. Several studies have shown that iron deprivation treatment has a strong anti-cancer effect. However, it is unclear whether iron deprivation treatment suppresses cancer stem cells or not. A cancer stem cell model, miPS-LLCcm, was recently epigenetically established from murine induced pluripotent stem cells (miPS cells) in Okayama University. In this model, the green fluorescent protein (GFP) and the puromycin resistant gene were inserted into the 59-bp untranslated region of the Nanog gene of miPS cells. By this procedure, undifferentiated cancer progenitor cells are recognized as GFP positive cells. We then examined the iron dependency of these cancer stem cells both in in vitro and in vivo studies and we used this model to examine the possibility of cancer stem cells by iron deprivation. Materials and Methods: In in vitro studies, the miPS-LLCcm cells were used as cancer stem cells and colon26 and 4T1 cells were used as differentiated murine cancer cells. Puromycin was used to purify the cancer stem cells before seeding of the miPS-LLCcm cells. The dependency on iron for cell proliferation was examined following transferrin addition. Transferrin (Holo) was used to simulate an iron rich condition. Iron free medium and the iron chelators, Deferasirox and Deferoxamine, plus 1% fetal bovine serum (FBS) were used to simulate iron depletion conditions. Cell proliferation assays and flow cytometric analyses were performed 48 hours after adjustment of the iron concentration level. In in vivo studies, an iron depleted diet was used to simulate iron depleted conditions. Nude mice were divided into normal diet and iron depleted diet groups. The mice were fed with these diets for three weeks and then a suspension of miPS-LLCcm cells was injected into the backs of the nude mice. Tumor size was measured and the tumors were immunohistologically examined. Results: In the in vitro studies, transferrin strongly promoted the proliferation of cancer stem cells under iron depletion conditions compared to no transferrin(p<0.001). However, transferrin did not promote the proliferation of the differentiated 4T1 and colon26 cancer cells. The number of GFP-expressing cancer stem cells decreased as the iron concentration was decreased. In the vivo studies, iron depletion significantly suppressed the tumor growth of the cancer stem cells (p<0.05). Immunohistological analysis indicated that Nanog expression was also decreased in the tumors of the iron depletion diet group. Conclusions: Iron is a key element for the proliferation and differentiation of cancer stem cells. Iron controlling therapy including iron chelators is a novel therapeutic target of cancer stem cells. Citation Format: Takayuki Ninomiya, Toshiaki Ohara, Hajime Kashima, Ryoichi Katsube, Kazuhiro Noma, Yasuko Tomono, Akifumi Mizutani, Tomonari Kasai, Masaharu Seno, Shinji Kuroda, Hiroyuki Kishimoto, Hiroshi Tazawa, Yasuhiro Shirakawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. Iron control is a novel therapeutic target of cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4243. doi:10.1158/1538-7445.AM2015-4243

Abstract 401: A novel photoimmunotherapy targeting cancer-associated fibroblasts (CAFs) overcomes therapeutic resistance in human esophageal cancer

Cancer associated fibroblasts (CAFs) are thought to play an essential role in cancer invasion, migration, metastasis and resistance to anticancer drugs. CAFs were originally defined as fibroblasts expressing α-SMA in cancer tissue; however, fibroblast activation protein (FAP) has been recently identified as a more functional and specific surface protein of CAFs. The photoimmunotherapy (PIT) is a new molecular targeted therapy, which is based on a near-infrared (NIR) photosensitizer IR700, conjugated to monoclonal antibody (mAb) targeting particular molecules. PIT induces the selective destruction of targeted cells. The aim of this study is to analyze the influence of CAFs on human esophageal cancer and to develop a novel therapeutic strategy to eliminate CAFs by using FAP-targeting PIT. We produced the IR700-FAP antibody for this study. Human esophageal cancer cell lines (TE-4 and OE-19) and FEF3 human fibroblasts were used in this study. TE-4 and OE-19 cells stimulated with conditioned medium (CM) of CAFs exhibited malignant phenotypes confirmed by invasion assay, migration assay, and colony formation assay. Tumor cells were significantly more malignant when directly co-cultured with CAFs. We further examined whether tumor cells stimulated by CM (CAFs) could acquire the resistance to chemotherapy or radiotherapy. Cell viability assay showed that tumor cells stimulated with CAFs was more resistant to conventional chemotherapy or radiation than unstimulated tumor cells. Western blot analysis demonstrated that E-cadherin expression was decreased and that of vimentin was increased in CAF-stimulated tumor cells, indicating the epithelial mesenchymal transition (EMT) induction. Moreover, OE-19 cells stimulated with CAFs contained more CD133-positive cancer stem-like cells. In vivo experiments demonstrated that subcutaneous TE4 tumors were more refractory to chemotherapy in the presence of co-inoculated CAFs as observed in vitro. Finally, we investigated whether elimination of CAFs by FAP-targeting PIT could affect the resistance to chemotherapy in vitro and in vivo. In vivo PIT following intratumoral anti-FAP mAb-conjugated IR700 suppressed the growth of subcutaneous tumors co-inoculated with CAFs. These results demonstrated that human tumor cells became more malignant and resistant in the presence of CAFs, which could be reversed by using CAF-specific PIT. Targeting fibroblast itself is a unique strategy and can be clinically promising as combination targeting cancer cells and their fundamental microenvironment. Citation Format: Ryoichi Katsube, Kazuhiro Noma, Shinichiro Watanabe, Shinichi Urano, Takayuki Ninomiya, Toshiaki Ohara, Hiroshi Tazawa, Shunsuke Kagawa, Hisataka Kobayashi, Toshiyoshi Fujiwara. A novel photoimmunotherapy targeting cancer-associated fibroblasts (CAFs) overcomes therapeutic resistance in human esophageal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 401. doi:10.1158/1538-7445.AM2015-401