Peritoneal Dissemination Of Gastric Cancer Research Articles

Anticancer effect of a Kampo preparation Daikenchuto.

No traditional Japanese and Chinese herbal preparations have been shown to be effective antitumor agents, and a Japanese herbal therapy (Kampo medicine) for cancer that causes fewer adverse drug reactions than orthodox pharmaceuticals is desired. Our present study demonstrated that a Kampo preparation Daikenchuto (DKT) exerts an antitumor effect against various cancer cells. We also discovered an antitumor factor in Japanese Zanthoxylum peel, which is an ingredient of DKT. Breast, esophageal, gastric, and colon cancer cell lines were individually incubated with DKT for 1-72h, followed by assessment of tumor growth inhibition by MTT assay. The cancer cells were also analyzed for apoptotic changes after DKT treatment. Nude mice were used to establish a model of gastric cancer tumor growth and peritoneal disseminated metastasis, in which the number of peritoneal disseminations was evaluated after oral administration of DKT for 4weeks. In addition, the antitumor effects of the individual DKT ingredients (viz., ginseng, Japanese Zanthoxylum peel, and processed ginger) and other Kampo preparations were also analyzed. The antitumor effect of DKT was demonstrated in gastric, breast, esophageal, and colon cancer cells. DKT treatment induced apoptosis in these cells. Oral administration of DKT had a tendency to reduce the growth and significantly reduced the peritoneal dissemination of gastric cancer in the nude mouse model compared with control. DKT exhibited a higher antitumor effect than other Kampo preparations. Furthermore, Japanese Zanthoxylum peel, an ingredient of DKT, showed a particularly potent antitumor effect. Our study indicated that DKT is useful as a Kampo preparation for cancer therapy. We also showed that Japanese Zanthoxylum peel, an ingredient of DKT, contains an antitumor factor.

Trastuzumab-Based Photoimmunotherapy Integrated with Viral HER2 Transduction Inhibits Peritoneally Disseminated HER2-Negative Cancer.

Peritoneal dissemination is the most frequent metastasis in gastric cancer and is associated with poor prognosis. The lack of particular target antigens in gastric cancer other than HER2 has hampered the development of treatments for peritoneal dissemination of gastric cancer. We hypothesized that HER2-extracellular domain (HER2-ECD) gene transduction combined with trastuzumab-based photoimmunotherapy (PIT) might provide excellent and selective antitumor effects for peritoneal dissemination of gastric cancer. In vitro, adenovirus/HER2-ECD (Ad/HER2-ECD) efficiently transduced HER2-ECD into HER2-negative gastric cancer cells. Trastuzumab-IR700 (Tra-IR700)-mediated PIT induced selective cell death of HER2-ECD-transduced tumor cells. Ad/HER2-ECD also induced homogenous expression of HER2 in heterogeneous gastric cancer cells, resulting in uniform sensitivity of the cells to Tra-IR700-mediated PIT. Anti-HER2 PIT integrated with adenoviral HER2-ECD gene transfer was applied in mice bearing peritoneal dissemination of HER2-negative gastric cancer. Intraperitoneal administration of Ad/HER2-ECD and Tra-IR700 with PIT inhibited peritoneal metastasis and prolonged the survival of mice bearing MKN45. Furthermore, minimal side effects allowed the integrated therapy to be used repeatedly, providing better control of peritoneal dissemination. In conclusion, the novel therapy of molecular-targeted PIT integrated with gene transfer technology is a promising approach for the treatment of peritoneal dissemination in gastric cancer.

Production of Cisplatin-Incorporating Hyaluronan Nanogels via Chelating Ligand-Metal Coordination.

Hyaluronan (HA) is a promising drug carrier for cancer therapy because of its CD44 targeting ability, good biocompatibility, and biodegradability. In this study, cisplatin (CDDP)-incorporating HA nanogels were fabricated through a chelating ligand-metal coordination cross-linking reaction. We conjugated chelating ligands, iminodiacetic acid or malonic acid, to HA and used them as a precursor polymer. By mixing the ligand-conjugated HA with CDDP, cross-linking occurred via coordination of the ligands with the platinum in CDDP, resulting in the spontaneous formation of CDDP-loaded HA nanogels. The nanogels showed pH-responsive release of CDDP, because the stability of the ligand-platinum complex decreases in an acidic environment. Cell viability assays for MKN45P human gastric cancer cells and Met-5A human mesothelial cells revealed that the HA nanogels selectively inhibited the growth of gastric cancer cells. In vivo experiments using a mouse model of peritoneal dissemination of gastric cancer demonstrated that HA nanogels specifically localized in peritoneal nodules after the intraperitoneal administration. Moreover, penetration assays using multicellular tumor spheroids indicated that HA nanogels had a significantly higher ability to penetrate tumors than conventional, linear HA. These results suggest that chelating-ligand conjugated HA nanogels will be useful for targeted cancer therapy.

Endoglin overexpression mediates gastric cancer peritoneal dissemination by inducing mesothelial cell senescence

Peritoneal dissemination (PD), which is highly frequent in gastric cancer (GC) patients, is the main cause of death in advanced GC. Senescence of human peritoneal mesothelial cells (HPMC) may contribute to GC peritoneal dissemination (GCPD). In this study of 126 patients, we investigated the association between Endoglin expression in GC peritoneum and the clinicopathological features. The prognosis of patients was evaluated according to Endoglin and ID1 expression. In vitro, GC cell (GCC)-HPMC coculture was established. Endoglin and ID1 expression was evaluated by Western blot. Cell cycle and HPMC senescence were analyzed after harvesting HPMC from the coculture. GCC adhesion and invasion to HPMC were also assayed. Our results showed that positive staining of Endoglin (38%) was associated with a higher TNM stage and higher incidence of GCPD (both P < .05). Kaplan-Meier analysis showed that the patients who were Endoglin positive had a shorter survival time compared with Endoglin-negative patients (P = .02). Using the HPMC and GCC adherence and invasion assay, we demonstrated that transforming growth factor beta 1 (TGF-β)1-induced HPMC senescence was attenuated by silencing the Endoglin expression, which also prevented GCC attachment and invasion. Our study indicated a positive correlation between Endoglin overexpression and GCPD. Up-regulated Endoglin expression induced HPMC senescence via TGF-β1 pathway. The findings suggest that Endoglin-induced HPMC senescence may contribute to peritoneal dissemination of GCCs.

Molecular mechanisms of peritoneal dissemination in gastric cancer.

Peritoneal dissemination represents a devastating form of gastric cancer (GC) progression with a dismal prognosis. There is no effective therapy for this condition. The 5-year survival rate of patients with peritoneal dissemination is 2%, even including patients with only microscopic free cancer cells without macroscopic peritoneal nodules. The mechanism of peritoneal dissemination of GC involves several steps: detachment of cancer cells from the primary tumor, survival in the free abdominal cavity, attachment to the distant peritoneum, invasion into the subperitoneal space and proliferation with angiogenesis. These steps are not mutually exclusive, and combinations of different molecular mechanisms can occur in each process of peritoneal dissemination. A comprehensive understanding of the molecular events involved in peritoneal dissemination is important and should be systematically pursued. It is crucial to identify novel strategies for the prevention of this condition and for identification of markers of prognosis and the development of molecular-targeted therapies. In this review, we provide an overview of recently published articles addressing the molecular mechanisms of peritoneal dissemination of GC to provide an update on what is currently known in this field and to propose novel promising candidates for use in diagnosis and as therapeutic targets.

腹膜播種に対し化学療法後根治手術をしえたが，術後8か月目に髄膜癌腫症を来したHER2陽性4型胃癌の1例

症例は71歳の男性で，HER2陽性4型胃癌に対し審査腹腔鏡検査を施行した．SE N1 M1 P1 CY1 Stage IVにてtrastuzumabを含む化学療法を行った．再度審査腹腔鏡検査で腹膜播種消失を確認後，胃全摘（D2），脾摘術を施行した．病理組織診はpor SM N0 M0 P0 CY0 Stage IAで，術後にS-1+trastuzumabを投与した．術後8か月より嘔気が出現し，CEA上昇を認めた．胸腹部造影CTで再発所見はなかった．意識レベルの低下で近医に緊急入院となった．頭部造影MRIで小脳，中脳，右側側頭葉内側の軟膜に異常濃染を認めた．感染所見はなく感染性髄膜炎は否定的で，経過より髄膜癌腫症と診断するも発症12病日に死亡した．胃癌の髄膜癌腫症はまれで極めて予後不良である．症状も多彩で診断に苦慮することが多い．脳症状・神経症状を認めた場合は髄膜癌腫症を念頭に精査する必要があると考えられた．

Tumor-associated macrophages of the M2 phenotype contribute to progression in gastric cancer with peritoneal dissemination.

BackgroundTumor-associated macrophages (TAMs) of the M2 phenotype are known to promote tumor proliferation and to be associated with a poor prognosis in numerous cancers. Here, we investigated whether M2 macrophages participate in the development of peritoneal dissemination in gastric cancer.MethodsThe characteristics of peritoneal macrophages in gastric cancer patients with or without peritoneal dissemination were examined by flow cytometry and the real-time quantitative polymerase chain reaction. The effects of M2 macrophages on phenotypic changes of the gastric cancer cell line MKN45 were assessed with a direct or indirect co-culture system in vitro and an in vivo mouse xenograft model.ResultsThe number of peritoneal macrophages with the M2 phenotype (CD68+CD163+ or CD68+CD204+) was significantly higher in gastric cancer patients with peritoneal dissemination than in those without peritoneal dissemination. Higher expression of the M2-related messenger RNAs (IL-10, vascular endothelial growth factor A, vascular endothelial growth factor C, matrix metalloproteinase 1, and amphiregulin) and lower expression of M1-related messenger RNAs (TNF-α, CD80, CD86, and IL-12p40) were also confirmed in the TAMs. Macrophage co-culture with gastric cancer cells converted M1 phenotype into M2 phenotype. Moreover, the coexistence of MKN45 cells with M2 macrophages resulted in cancer cell proliferation and an acceleration of tumor growth in the xenograft model.ConclusionsIntraperitoneal TAMs in gastric cancer patients with peritoneal dissemination were polarized to the M2 phenotype, and could contribute to tumor proliferation and progression. Therefore, intraperitoneal TAMs are expected to be a promising target in the treatment of peritoneal dissemination in gastric cancer.

Abstract 5169: Omics approach to identify driver genes for peritoneal dissemination of gastric cancer cells

Abstract Background Little is known about the molecular mechanisms of peritoneal dissemination in gastric cancer cells. In this study, to clear the molecular mechanisms of accelerating the peritoneal dissemination, we performed to identify significant genes which give rise to peritoneal dissemination and poor prognosis of gastric cancer. We conducted expression array analysis in mice model of peritoneal dissemination and in 200 cases of gastric cancer cases, and compared with each other to identify driver genes. Methods As described previously, three parental scirrhous gastric carcinoma-derived cell lines (HSC-39, HSC-44PE and HSC-58) were established from human scirrhous gastric carcinoma. Moreover, inoculating each parental cell lines into the gastric wall of nude mice, four matched highly peritoneal-metastatic cell lines (HSC-39As8, HSC-44As3, HSC-58As1 and HSC-58As9) were established after 12 cycles of stepwise selection. To identify gene expression signatures for peritoneal dissemination, we performed expression analysis for matched parental and each resultant cell line. Next, to examine the relationship between gene expression signatures and clinical behaviors, we performed synthetic analysis of gene expression signatures from in vivo peritoneal dissemination mouse model data and expression data set containing 200 gastric tumors from the Singapore cohort. Results (1) Our synthetic analysis, EEM analysis, which tested the functionality of the input genes sets on the basis of their coherence, showed that one gene set from the 58As9 cell line harbored a coherently expressed subset of significant size. (2) We found 57 genes in the coherent subset which had clinical correlation. (3) We found that peritoneal metastases patients tended to show higher expression of such genes, and patients prognosis with higher expression of such genes tended to be poor. (4) Among such genes, we focused on chemokine(C-X-C motif) receptor 7(CXCR7). CXCR7 expression was significantly higher in 58As9 than in HSC58(P&amp;lt;0.005). We also found that overall survival of patients with CXCR7 high expression gastric cancer tended to be poorer than that with CXCR7 low expression gastric cancer in both the Singapore and Japanese cohorts. Conclusions In conclusion, synthetic analysis from two data sets indicated several candidate driver genes for strongly accelerating gastric cancer peritoneal dissemination, the current approach to compare gene signatures in vivo and clinical samples with accurate clinical annotations enabled us to identify brand-new driver candidate genes. Among them, CXCR7 might play an important role in peritoneal dissemination and CXCR7 might be a therapeutic target for suppression of gastric cancer peritoneal dissemination in the near future. Note: This abstract was not presented at the meeting. Citation Format: Sho Nambara, Junji Kurashige, Tomoko Saito, Hisateru Komatsu, Masami Ueda, Shotaro Sakimura, Hidenari Hirata, Ryutaro Uchi, Yuki Takano, Yoshiaki Shinden, Tomohiro Iguchi, Hidetoshi Eguchi, Keishi Sugimachi, Yoshihiko Maehara, Koshi Mimori. Omics approach to identify driver genes for peritoneal dissemination of gastric cancer 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 5169. doi:10.1158/1538-7445.AM2015-5169

Exosomal miRNAs from Peritoneum Lavage Fluid as Potential Prognostic Biomarkers of Peritoneal Metastasis in Gastric Cancer

Peritoneal metastasis is the most frequent type of recurrence in patients with gastric cancer (GC) and is associated with poor prognosis. Peritoneal lavage cytology, used to evaluate the risk of peritoneal metastasis, has low sensitivity. Here, we assessed the diagnostic potential of exosomal miRNA profiles in peritoneal fluid for the prediction of peritoneal dissemination in GC. Total RNA was extracted from exosomes isolated from six gastric malignant ascites (MA) samples, 24 peritoneal lavage fluid (PLF) samples, and culture supernatants (CM) of two human gastric carcinoma cell lines that differ in their potential for peritoneal metastasis. Expression of exosomal miRNAs was evaluated with Agilent Human miRNA microarrays and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The microarray analysis indicated a low variability in the number and signal intensity of miRNAs detected among the samples. In the six MA fluids, miR-21 showed the highest signal intensity. We identified five miRNAs (miR-1225-5p, miR-320c, miR-1202, miR-1207-5p, and miR-4270) with high expression in MA samples, the PLF of serosa-invasive GC, and the CM of a highly metastatic GC cell line; these candidate miRNA species appear to be related to peritoneal dissemination. Differential expression of miR-21, miR-320c, and miR-1225-5p was validated in the PLF of serosa-invasive and non-invasive GC by qRT-PCR and miR-21 and miR-1225-5p were confirmed to be associated with serosal invasion in GC. PLF can be used to profile the expression of exosomal miRNAs. Our findings suggest that miR-21 and miR-1225-5p may serve as biomarkers of peritoneal recurrence after curative GC resection, thus providing a novel approach to early diagnosis of peritoneal dissemination of GC.

Photodynamic therapy using nanoparticle loaded with indocyanine green for experimental peritoneal dissemination of gastric cancer

Photodynamic therapy using nanoparticle loaded with indocyanine green for experimental peritoneal dissemination of gastric cancer

RNAi-mediated gene silencing of ST6GalNAc I suppresses the metastatic potential in gastric cancer cells.

ST6GalNAc I is a sialyltransferase controlling the expression of sialyl-Tn antigen (STn), which is overexpressed in several epithelial cancers, including gastric cancer, and is highly correlated with cancer metastasis. However, the functional contribution of ST6GalNAc I to development or progression of gastric cancer remains unclear. In this study, we investigated the effects of suppression of ST6GalNAc I on gastric cancer in vitro and in vivo. Gastric cancer cell lines were transfected with ST6GalNAc I siRNA and were examined by cell proliferation, migration, and invasion assays. We also evaluated the effect of ST6GalNAc I siRNA treatment in a peritoneal dissemination mouse model. The differences in mRNA levels of selected signaling molecules were analyzed by polymerase chain reaction (PCR) arrays associated with tumor metastasis in MKN45 cells. The signal transducer and activator of transcription 5b (STAT5b) signaling pathways that reportedly regulate the insulin-like growth factor-1 (IGF-1) were analyzed by Western blot. ST6GalNAc I siRNA inhibited gastric cancer cell growth, migration, and invasion in vitro. Furthermore, intraperitoneal administration of ST6GalNAc I siRNA- liposome significantly inhibited peritoneal dissemination and prolonged the survival of xenograft model mice with peritoneal dissemination of gastric cancer. PCR array confirmed that suppression of ST6GalNAc I caused a significant reduction in expression of IGF-1 mRNA. Decreased IGF-1 expression in MKN45 cells treated with ST6GalNAc I siRNA was accompanied by reduced phosphorylation of STAT5b. ST6GalNAc I may regulate the gene expression of IGF-1 through STAT5b activation in gastric cancer cells and may be a potential target for treatment of metastasizing gastric cancer.

Peritoneal milky spots serve as a hypoxic niche and favor gastric cancer stem/progenitor cell peritoneal dissemination through hypoxia-inducible factor 1α.

Peritoneal dissemination is the most common cause of death in gastric cancer patients. The hypoxic microenvironment plays a major role in controlling the tumor stem cell phenotype and is associated with patients' prognosis through hypoxia-inducible factor-1α (HIF-1α), a key transcriptional factor that responds to hypoxic stimuli. During the peritoneal dissemination process, gastric cancer stem/progenitor cells (GCSPCs) are thought to enter into and maintained in peritoneal milky spots (PMSs), which have hypoxic microenvironments. However, the mechanism through which the hypoxic environment of PMSs regulated GCSPC maintenance is still poorly understood. Here, we investigated whether hypoxic PMSs were an ideal cancer stem cell niche suitable for GCSPC engraftment. We also evaluated the mechanisms through which the HIF-1α-mediated hypoxic microenvironment regulated GCSPC fate. We observed a positive correlation between HIF-1α expression and gastric cancer peritoneal dissemination (GCPD) in gastric cancer patients. Furthermore, the GCSPC population expanded in primary gastric cancer cells under hypoxic condition in vitro, and hypoxic GCSPCs showed enhanced self-renewal ability, but reduced differentiation capacity, mediated by HIF-1α. In an animal model, GCSPCs preferentially resided in the hypoxic zone of PMSs; moreover, when the hypoxic microenvironment in PMSs was destroyed, GCPD was significantly alleviated. In conclusion, our results demonstrated that PMSs served as a hypoxic niche and favored GCSPCs peritoneal dissemination through HIF-1α both in vitro and in vivo. These results provided new insights into the GCPD process and may lead to advancements in the clinical treatment of gastric cancer. Stem Cells 2014;32:3062–3074

Epigenetic modulation and repression of miR-200b by cancer-associated fibroblasts contribute to cancer invasion and peritoneal dissemination in gastric cancer.

Cancer-associated fibroblasts (CAFs) have recently been linked to the invasion and metastasis of gastric cancer. In addition, the microRNA (miR)-200 family plays a central role in the regulation of the epithelial-mesenchymal transition process during cancer metastasis, and aberrant DNA methylation is one of the key mechanisms underlying regulation of the miR-200 family. In this study, we clarified whether epigenetic changes of miR-200b by CAFs stimulate cancer invasion and peritoneal dissemination in gastric cancer. We evaluated the relationship between miR-200b and CAFs using a coculture model. In addition, we established a peritoneal metastasis mouse model and investigated the expression and methylation status of miR-200b. We also investigated the expression and methylation status of miR-200b and CAFs expression in primary gastric cancer samples. CAFs (CAF-37 and CAF-50) contributed to epigenetic changes of miR-200b, reduced miR-200b expression and promoted tumor invasion and migration in NUGC3 and OCUM-2M cells in coculture. In the model mice, epigenetic changes of miR-200b were observed in the inoculated high-frequency peritoneal dissemination cells. In the 173 gastric cancer samples, the low miR-200b expression group demonstrated a significantly poorer prognosis compared with the high miR-200b expression group and was associated with peritoneal metastasis. In addition, downregulation of miR-200b in cancer cells was significantly correlated with alpha-smooth muscle actin expression. Our data provide evidence that CAFs reduce miR-200b expression and promote tumor invasion through epigenetic changes of miR-200b in gastric cancer. Thus, CAFs might be a therapeutic target for inhibition of gastric cancer.

Photodynamic therapy using nanoparticle loaded with indocyanine green for experimental peritoneal dissemination of gastric cancer.

Although there have been multiple advances in the development of novel anticancer agents and operative procedures, prognosis of patients with advanced gastric cancer remains poor, especially in patients with peritoneal metastasis. In this study, we established nanoparticles loaded with indocyanine green (ICG) derivatives: ICG loaded lactosomes (ICGm) and investigated the diagnostic and therapeutic value of photodynamic therapy (PDT) using ICGm for experimental peritoneal dissemination of gastric cancer. Experimental peritoneal disseminated xenografts of human gastric cancer were established in nude mice. Three weeks after intraperitoneal injection of the cancer cells, either ICGm (ICGm-treated mice) or ICG solution (ICG-treated mice) was injected through the tail vein. Forty-eight hours after injection of the photosensitizer, in vivo and ex vivo imaging was carried out. For PDT, 48 h after injection of the photosensitizer, other mice were irradiated through the abdominal wall, and the body weight and survival rate were monitored. In vivo imaging revealed that peritoneal tumors were visualized through the abdominal wall in ICGm-treated mice, whereas only non-specific fluorescence was observed in ICG-treated mice. The PDT reduced the total weight of the disseminated nodules and significantly improved weight loss and survival rate in ICGm-treated mice. In conclusion, ICGm can be used as a novel diagnostic and therapeutic nanodevice in peritoneal dissemination of gastric cancer.

Abstract 2096: HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer

Abstract Background The molecular mechanisms of peritoneal dissemination in gastric cancer still remain unclear. This study was designed to clarify the role of HIF-1α in the development of peritoneal dissemination of gastric cancer. Methods HIF-1α knockdown (KD) cells were established in the scirrhous gastric cancer cell line 58As9 by siRNA transfection. In western blot analysis, HIF-1α expression was assessed using KD and the control transfectant (SC) cells under normoxia (20%O2) as well as hypoxia (1%O2). A series of in vitro analysis were conducted and compared between the 2 transfectants. Using nude mice, peritoneal dissemination was evaluated by orthotopic implantation (o.i.) and intraperitoneal injection (i.p.) models. Finally, tumor angiogenesis of venous and lymph vessels was immunohistochemically analyzed. Results Deficient HIF-1α expression was observed in KD, but not SC cells under normoxia and hypoxia. In vitro proliferation and invasion was significantly reduced in KD compared with SC cells. On contrary, HIF-1α exerts unfavorable effects on anoikis resistance and adhesion ability. In o.i. model, stomach tumor was highly developed in both of KD (86.7%) and SC mice(100%). On the other hand, peritoneal dissemination was more frequently observed in the SC mice (93%) than in the KD mice (13%) (p &amp;lt; 0.001). In the i.p. model, the dissemination highly occurred in both mice (each 100%), while greater number of nodules was observed in the KD mice (p = 0.017). Angiogenesis with vascular invasion were more highly developed in the SC stomach tumors (venous vessels: p=0.001, lymph vessels: p=0.043). In the disseminated nodules, the vascular invasion was developed at intratumoral regions in the SC o.i. mice, however such appearance was not found in i.p. mice. Conclusions This study clearly showed that HIF-1α is crucial for the development of peritoneal dissemination in the spontaneous metastatic (o.i.) model. Moreover, in i.p. model, larger number of dissemination was found in KD than SC mice, indicating that the dissemination by o.i. would not be developed via seeding mechanism. Analysis of tumor angiogenesis in o.i. model demonstrated that HIF-1α activated venous as well as lymph angiogenesis in the stomach tumor. In peritoneal disseminated nodules, vascular invasion was observed at intratumoral area in o.i. SC mice, but not in the i.p. mice. This unique feature further suggests that the dissemination in o.i. mice might be developed by extravasation of cancer cells, which are micro-connected to the stomach tumor via trans-vessel route. Citation Format: Shuusuke Miyake, Yoshihiko Kitajima, Jun Nakamura, Keita Kai, Kazuyoshi Yanagihara, Tomokazu Tanaka, Masatsugu Hiraki, Kohji Miyazaki, Hirokazu Noshiro. HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2096. doi:10.1158/1538-7445.AM2014-2096

Tks5 activation in mesothelial cells creates invasion front of peritoneal carcinomatosis

Scirrhous gastric cancer is frequently associated with peritoneal dissemination, and the interaction of cancer cells with peritoneal mesothelial cells (PMCs) is crucial for the establishment of the metastasis in the peritoneum. Although cells derived from PMCs are detected within tumors of peritoneal carcinomatosis, how PMCs are incorporated into tumor architecture is not understood. The present study shows that PMCs create the invasion front of peritoneal carcinomatosis, which depends on activation of Tks5 in PMCs. In peritoneal tumor implants, PMCs represent majority of cells located at the invasive edge of the cancer tissue. Exogenously implanted PMCs and host PMCs aggressively invade into abdominal wall upon the peritoneal inoculation of cancer cells, and PMCs locate ahead of cancer cells in the direction of invasion. Tks5, a substrate of Src kinase, is predominantly expressed in the PMCs of cancer tissue, and promotes the invasion of PMCs and cancer cells. Expression and activation of Tks5 was induced in PMCs following their exposure to gastric cancer cells, and increased Tks5 expression was detected in PMCs located at the invasion front. Reduced Tks5 expression in PMCs blocked PMC invasion, which in turn prevents cancer cell invasion both in vitro and in vivo. The peritoneal dissemination of gastric cancer was significantly increased by mixing cancer cells and PMCs, and was suppressed by knockdown of Tks5 in PMCs. These results suggest that cancer-activated PMCs create invasion front by guiding cancer cells. Signaling leading to Tks5 activation in PMCs may be a suitable therapeutic target for prevention of peritoneal carcinomatosis.

Tumor-associated mesothelial cells are negative prognostic factors in gastric cancer and promote peritoneal dissemination of adherent gastric cancer cells by chemotaxis

Peritoneal dissemination is highly frequent in gastric cancer. Damage to human peritoneal mesothelial cell (HPMC) barriers provokes gastric cancer peritoneal dissemination (GCPD), the key events during GCPD, is characterized by fibroblastic development. In this study, we have studied the association between fibroblast activation protein (FAP) expression in peritoneum and the pathological features of the primary tumor. The clinical prognosis of gastric cancer patients was evaluated according to FAP expression. In a gastric cancer cell-HPMC co-culture system, expression of E-cadherin, α-smooth muscle actin, and FAP were evaluated by Western blotting. Gastric cancer cell migration and adhesion to HPMC were also assayed. Our results showed positive peritoneal staining of FAP in 36/86 cases (41.9 %), which was associated with a higher TNM stage in primary gastric cancer and higher incidence of GCPD (both p<0.05). Survival analysis showed FAP expression was an independent prognostic factor of poor survival (p=0.02). Peritoneum of FAP-positive expression exhibited a distinct fibrotic development and expressed higher level of the mesenchymal marker α-SMA, which was confirmed by the in vitro Western blot assay. In HPMC and gastric cancer cell adherence assay, SGC-7901 cells preferentially adhered to TA-HPMC at different cell densities (both p<0.05). Additionally, SGC-7901 cells were more prone to chemotaxis by FAP-expressed tumor-associated-human peritoneal mesothelial cells (TA-HPMC) compared with HPMC co-cultured with normal gastric glandular epithelial cells in a time-dependent manner (both p<0.05). Our study indicated a positive correlation between peritoneum FAP expression and GCPD. FAP-expressed TA-HPMC might be an important cellular component and instigator of GCPD.

Elevated Plasma D-Dimer Levels Correlate with Long Term Survival of Gastric Cancer Patients

BackgroundIncreasing evidence indicated plasma D-dimer could be regarded as a marker in cancers, however, its role in gastric cancer is still largely unknown.MethodsPlasma D-dimer levels were measured by enzyme linked fluorescent immunoassays and evaluated by receiver operating characteristic (ROC) curves for peritoneal dissemination in gastric cancer and healthy subjects. The overall survival (OS) characteristics were determined using Kaplan–Meier and Cox regression analyses.ResultsThe average of the plasma D-dimer levels for gastric cancer patients was significantly higher than the healthy subjects. A Spearman correlation analysis showed that plasma D-dimer levels correlated with the depth of invasion, lymph node metastasis, peritoneal dissemination, distant metastasis, tumor size and TNM stage. The mean plasma D-dimer level was 2.20±1.51 µg/mL in peritoneal dissemination patients and 1.01±0.79 µg/mL in non-peritoneal dissemination patients (P<0.001). Additionally, the mean plasma D-dimer concentration in patients alive at the final follow-up evaluation was 0.79±0.72 µg/mL,which was significantly lower than the amounts determined for the deceased patients (1.36±1.13 µg/mL) (P<0.001). The AUC of D-dimer was 0.833 (95%CI: 0.780–0.885). At a cut-off value of 1.465 µg/mL, the D-dimer measurement had a sensitivity of 78.00%, a specificity of 83.76% and an accuracy of 82.59%. The median OS was 48.10 months (95% CI: 43.88–52.31) in patients with plasma D-dimer levels less than 1.465 µg/mL and 22.39 months (95% CI: 16.95–27.82) in patients with plasma D-dimer levels exceeding 1.465 µg/mL (log-rank test, P<0.001). Importantly, plasma D-dimer levels exceeding 1.465 µg/mL were significantly associated with poor OS, as determined using a multivariate Cox regression analysis (hazard ratio [HR], 2.28; 95%CI: 1.36–3.81; P = 0.002).ConclusionsPlasma D-dimer levels are increased in gastric cancer patients and may be a valuable biomarker for peritoneal dissemination, with high D-dimer levels predicting poor outcomes for gastric cancer patients.

Intraperitoneal injection of in vitro expanded Vγ9Vδ2 T cells together with zoledronate for the treatment of malignant ascites due to gastric cancer

Malignant ascites caused by peritoneal dissemination of gastric cancer is chemotherapy-resistant and associated with poor prognosis. We conducted a pilot study to evaluate the safety of weekly intraperitoneal injections of in vitro expanded Vγ9Vδ2 T cells together with zoledronate for the treatment of such malignant ascites. Patient peripheral blood mononuclear cells were stimulated with zoledronate (5 μmol/L) and interleukin-2 (1000 IU/mL). After 14 days culture, Vγ9Vδ2 T-cells were harvested and administered intraperitoneally in four weekly infusions. The day before T-cell injection, patients received zoledronate (1 mg) to sensitize their tumor cells to Vγ9Vδ2 T-cell recognition. Seven patients were enrolled in this study. The number of Vγ9Vδ2 T-cells in each injection ranged from 0.6 to 69.8 × 108 (median 59.0 × 108). There were no severe adverse events related to the therapy. Intraperitoneal injection of Vγ9Vδ2 T cells allows them access to the tumor cells in the peritoneal cavity. The number of tumor cells in the ascites was significantly reduced even after the first round of therapy and remained substantially lower over the course of treatment. IFN-γ was detected in the ascites on treatment. Computed tomography revealed a significant reduction in volume of ascites in two of seven patients. Thus, injection of these antitumor Vγ9Vδ2 T-cells can result in local control of malignant ascites in patients for whom no standard therapy apart from paracentesis is available. Adoptively transferred Vγ9Vδ2 T-cells do indeed recognize tumor cells and exert antitumor effector activity in vivo, when they access to the tumor cells.

大腿ヘルニア嵌頓で発見された胃癌腹膜播種の1例

症例は69歳，女性．約1カ月前からの右鼠径部膨隆を主訴に当科外来を受診した．右鼠径靱帯の足側に用手的に還納できない母指頭大の硬い膨隆を認め大腿ヘルニア嵌頓と診断し，翌日，大腿ヘルニア根治術を施行した．手術所見ではヘルニア内容は大網であったが一部で硬結を認めたため，切除して病理検査に提出した．病理組織学的検査の結果，印環細胞癌の大網転移が疑われた．原発巣検索の目的で施行した上部消化管内視鏡検査で胃前庭部に3型病変を認め，同部位の生検から大網の結節と同様の病理所見が得られたため，胃癌の大網転移と診断した．CTでは胃周囲の多発リンパ節腫大・多発腹膜播種病変を認め，胃癌cT4aN2M1 Stage IVと診断した．その後，S-1+CDDPによる化学療法を施行したが，病状は徐々に悪化し，手術から約5カ月後に死亡した．大腿ヘルニア嵌頓を契機に発見された胃癌腹膜播種のまれな1例を経験したので報告する．