Gastric Cancer-associated Fibroblasts Research Articles

Helicobacter pylori-induced fibroblast-derived Serpin E1 promotes gastric cancer growth and peritoneal dissemination through p38 MAPK/VEGFA-mediated angiogenesis

BackgroundFibroblasts, especially cancer-associated fibroblasts (CAFs), represent the predominant stromal cell population in the tumor microenvironment and have an important function in tumorigenesis by interacting with tumor cells. However, their interaction remains elusive in an inflammatory tumor microenvironment induced by Helicobacter pylori (H. pylori).MethodsThe expression of Serpin family E member 1 (Serpin E1) was measured in fibroblasts with or without H. pylori infection, and primary gastric cancer (GC) cells. Serpin E1 knockdown and overexpression fibroblasts were generated using Serpin E1 siRNA or lentivirus carrying Serpin E1. Co-culture models of fibroblasts and GC cells or human umbilical vein endothelial cells (HUVECs) were established with direct contact or the Transwell system. In vitro functional experiments and in vivo tumorigenesis assay were employed to study the malignant behaviors of GC cells interacting with fibroblasts. ELISA was used for quantifying the levels of Serpin E1 and VEGFA in the culture supernatant. The tube formation capacity of HUVECs was assessed using a tube formation assay. Recombinant human Serpin E1 (recSerpin E1), anti-Serpin E1 antibody, and a MAPK pathway inhibitor were utilized to treat HUVECs for elucidating the underlying molecular mechanisms.ResultsSerpin E1 was predominantly expressed in gastric CAFs. H. pylori infection significantly enhanced the expression and secretion of Serpin E1 by CAFs. Both fibroblast-derived Serpin E1 and recSerpin E1 enhanced the growth, invasion, and migration of GC cells, along with increased VEGFA expression and tube formation in HUVECs. Furthermore, the co-inoculation of GC cells and fibroblasts overexpressing Serpin E1 triggered the expression of Serpin E1 in cancer cells, which facilitated together xenograft tumor growth and peritoneal dissemination of GC cells in nude mice, with an increased expression of Ki67, Serpin E1, CD31 and/or VEGFA. These processes may be mediated by Serpin E1-induced migration and p38 MAPK/VEGFA-mediated angiogenesis of HUVECs.ConclusionH. pylori infection induces Serpin E1 expression in fibroblasts, subsequently triggering its expression in GC cells through their interaction. Serpin E1 derived from these cells promotes the migration and p38 MAPK/VEGFA-mediated angiogenesis of HUVECs, thereby facilitating GC growth and peritoneal metastasis. Targeting Serpin E1 signaling is a potential therapy strategy for H. pylori-induced GC.

Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts through regulation of the HOXA6/ZBTB12 axis.

Cancer-associated fibroblasts (CAFs) play critical roles in the tumor microenvironment and exert tumor-promoting or tumor-retarding effects on cancer development. Astragaloside IV has been suggested to rescue the pathological impact of CAFs in gastric cancer. This study aimed to investigate the potential mechanism of astragaloside IV in the regulation of CAF pathological functions in gastric cancer development. Homeobox A6 (HOXA6), and Zinc Finger and BTB Domain Containing 12 (ZBTB12) are highly expressed in gastric CAFs compared with normal fibroblasts (NFs) based on the GSE62740 dataset. We found that astragaloside IV-stimulated CAFs suppressed cell growth, migration, and invasiveness of gastric cancer cells. HOXA6 and ZBTB12 were downregulated after astragaloside IV treatment in CAFs. Further analysis revealed that HOXA6 or ZBTB12 knockdown in CAFs also exerted inhibitory effects on the malignant phenotypes of gastric cells. Additionally, HOXA6 or ZBTB12 overexpression in CAFs enhanced gastric cancer cell malignancy, which was reversed after astragaloside IV treatment. Moreover, based on the hTFtarget database, ZBTB12 is a target gene that may be transcriptionally regulated by HOXA6. The binding between HOXA6 and ZBTB12 promoter in 293T cells and CAFs was further confirmed. HOXA6 silencing also induced the downregulation of ZBTB12 mRNA and protein in CAFs. Astragaloside IV was demonstrated to regulate the expression of ZBTB12 by mediating the transcriptional activity of HOXA6. Our findings shed light on the therapeutic value of astragaloside IV for gastric cancer.

Tumor-derived IL-8 facilitates lymph node metastasis of gastric cancer via PD-1 up-regulation in CD8+ T cells

BackgroundThe pretherapeutic serum interleukin-8 (sIL-8) levels have been revealed to be increased in about half of patients with locally advanced gastric cancer. However, the roles of IL-8 in lymph node metastasis (LNM) and the underlying mechanisms remain unclear.Methods146 patients with primary gastric carcinoma were enrolled in this study. ELISA was used to measure IL-8 levels. The CD4/CD8 ratio and programmed cell death-1 (PD-1) expression of T cells in primary tumor tissues, tumor-draining lymph nodes (TDLNs) and non-draining lymph nodes (NDLNs) were assayed with flow cytometry. Protein expression of the molecules was determined with immunohistochemistry, western blotting or immunoprecipitation. The gastric cancer mouse tumor model with LNM was utilized to determine the role of IL-8 in regulation of tumor metastasis and progression.ResultsThe elevated sIL-8 levels were associated with LNM and poor prognosis in gastric cancer. Furthermore, sIL-8 was identified to be prominently produced by gastric cancer-associated fibroblasts (CAFs). Elevated IL-8 can up-regulate PD-1 expression in CD8+ T cells, resulting in immunosuppression in primary tumors and TDLNs, which enhances LNM of gastric cancer. Molecularly, IL-8 increases PD-1 expression through JAK2/STAT3 signaling activation, and inhibits its ubiquitination via Fbxo38 down-regulation. In addition, the in vivo studies in mouse gastric cancer model demonstrated that IL-8 promotes LNM via PD-1 up-regulation in CD8+ T cells.ConclusionThe present study elucidates the pro-metastatic role of elevated IL-8 in gastric cancer, and provides novel insights to enhance immune checkpoint blockade therapy for anti-PD-1 in gastric cancer.

SAA1 is upregulated in gastric cancer-associated fibroblasts possibly by its enhancer activation.

Cancer-associated fibroblasts (CAFs) tend to have tumor-promoting capacity, and can provide therapeutic targets. Even without cancer cells, CAF phenotypes are stably maintained, and DNA methylation and H3K27me3 changes have been shown to be involved. Here, we searched for a potential therapeutic target in primary CAFs from gastric cancer and a mechanism for its dysregulation. Expression microarray using eight CAFs and seven non-CAFs (NCAFs) revealed that serum amyloid A1 (SAA1), which encodes an acute phase secreted protein, was second most upregulated in CAFs, following IGF2. Conditioned medium (CM) derived from SAA1-overexpressing NCAFs was shown to increase migration of gastric cancer cells compared with that from control NCAFs, and its tumor-promoting effect was comparable to that of CM from CAFs. In addition, increased migration of cancer cells by CM from CAFs was mostly canceled with CM from CAFs with SAA1 knockdown. Chromatin immunoprecipitation (ChIP)-quantitative PCR showed that CAFs had higher levels of H3K27ac, an active enhancer mark, in the promoter and the two far upstream regions of SAA1 than NCAFs. Also, BET bromodomain inhibitors, JQ1 and mivebresib, decreased SAA1 expression and tumor-promoting effects in CAFs, suggesting SAA1 upregulation by enhancer activation in CAFs. Our present data showed that SAA1 is a candidate therapeutic target from gastric CAFs and indicated that increased enhancer acetylation is important for its overexpression.

Abstract 5112: Interaction between RHOA mutated gastric cancer cells and cancer associated fibroblasts promotes tumorigenesis

Abstract Background: We previously observed that RHOA-mutated gastric cancer has desmoplastic morphological feature. Although desmoplastic reaction is known to consist mainly of cancer associated fibroblasts (CAF), the relationship cancer cells with RHOA mutation and CAF have not been reported. Thus, in this study, we investigated the interaction between RHOA-mutated gastric cancer cells and CAF and its underlying mechanism. Methods: We established stable gastric cell lines (AGS, MKN-45) expressing RHOA wild type (WT) of mutation (G17E, T37I, E40K, Y42C) using lentiviral vector and cocultured these cells with CAF using transwell. And proliferation and migration of RHOA-mutated gastric cancer cells were examined. To understand molecular mechanism of RHOA-mutated gastric cancer cells proliferation and migration, RNA-sequencing was conducted and the results were analyzed and validated. Results: Cocultivation of gastric cancer cells expressing RHOA WT or mutation (G17E, T37I, E40K, Y42C) with CAF increased proliferation relative to cells expressing an empty vector (EV). In addition, migration of RHOA mutant cells, except for cells with RHOA WT and Y42C, was increased by CAF relative to EV cells. Genes related to IL-17, IL-15, and IGF-1 signaling pathways were differentially expressed between RHOA WT or mutant cells and EV cells, suggesting that proliferation and migration of RHOA mutant cells promoted by CAF through these pathways. Furthermore, we identified CXCL1 and IGFBP2 as mediator of interaction between RHOA mutated gastric cancer cells and CAF. Conclusion: In summary, we found that gastric cancer cells with RHOA mutation have tumor-promoting activity by interaction with CAF. Thus, molecules for impairing their interactions could be potential therapeutic targets in gastric cancer patients with RHOA mutation. Citation Format: Daye Jeon, Yuri Choi, Seon Jin Lee, Kyung Un Choi, Do Youn Park, Chae Hwa Kwon. Interaction between RHOA mutated gastric cancer cells and cancer associated fibroblasts promotes tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5112.

Estrogen facilitates gastric cancer cell proliferation and invasion through promoting the secretion of interleukin-6 by cancer-associated fibroblasts

As a common disease, gastric cancer (GC) has influenced over 1 million people worldwide. Despite of prevention and optimal treatments, GC still has a high mortality. The role of cancer-associated fibroblasts (CAFs) in tumor progression has recently attracted attention, yet few studies have focused on GC. Estrogen has been reported to relate to the poor prognosis of GC. Therefore, we investigated whether estrogen can stimulate CAFs to produce tumor promoting factors in this study. Gastric CAFs were isolated from GC tissues and treated with estrogen. ELISA results suggested that CAFs produced interleukin-6 (IL-6) after estrogen treatment in a dose-dependent manner. The cell culture supernatant for estrogen-treated CAFs was collected and used as conditioned medium (CM) for GC cells. After cultured in CM, increased cell proliferation and alteration of cell cycle were detected by CCK-8 assay, BrdU assay, and flow cytometry. Western blot and gelatin zymography were used to determine cancer invasion-associated proteins. Results indicated that the expression of matrix metalloproteinase 2 (MMP2) and MMP9 were enhanced by Estrogen-CAFs-CM. Additional transwell assay showed that cell invasion and migration were promoted after cultured in CM. Lastly, western blot and immunofluorescence results demonstrated that the level of phosphorylated signal transducer and activator of transcription 3 (STAT3) in GC cells increased after cultured in CM. The effect was neutralized by IL-6 neutralizing antibody and STAT3 siRNA. Conclusively speaking, estrogen can activate CAFs to produce IL-6, ending up with promotion of GC cell proliferation and invasion. This result may suggest a new therapeutic target for GC.

Low expression of SPARC in gastric cancer-associated fibroblasts leads to stemness transformation and 5-fluorouracil resistance in gastric cancer

BackgroundThe aim of the present study was to clarify the correlations between SPARC expression in gastric cancer-associated fibroblasts (GCAFs) and the prognosis of patients with gastric cancer and to elucidate the role of GCAF-derived SPARC in stemness transformation and 5-fluorouracil resistance in gastric cancer.MethodsOne hundred ninety-two patients were enrolled in the present study. SPARC expression levels were evaluated by immunohistochemical staining. Primary GCAFs were obtained and cultured from cancer patients for in vitro study, and a lentivirus infection method was employed to knock down SPARC expression in GCAFs. The stemness phenotype and 5-fluorouracil (5-FU) response of gastric cancer cells were assessed via a 3D co-culture model. The apoptotic status and stemness alterations were monitored by flow cytometry and western blotting. Additionally, label-free quantification proteomics was used to identify the differentially expressed proteins and potential pathways in gastric cancer cells treated with GCAF-derived SPARC.ResultsLow expression of GCAF-derived SPARC was associated with decreased differentiation and reduced 5-year overall survival and was an independent predictive factor for prognosis in gastric cancer. The 3D tumour growth and 5-FU resistance abilities of gastric cancer cells were elevated after treatment with GCAFs with SPARC knockdown relative to these abilities in negative control cells. Additionally, suppressing SPARC expression in GCAFs facilitated the phenotypic alteration of gastric cancer cells towards CD44+/CD24− cancer stem cell (CSC)-like cells. Quantification proteomics analysis revealed that the differentially expressed proteins in gastric cancer cells were mainly involved in the AKT/mTOR and MEK/ERK signalling pathways.ConclusionsSPARC expression in GCAFs is a useful prognostic factor in patients with gastric cancer. Low expression of GCAF-derived SPARC can lead to CSC transformation and 5-FU resistance. Additionally, the AKT/mTOR and MEK/ERK signalling pathways may participate in the malignant process.

Paeoniflorin Inhibits Migration- and Invasion-Promoting Capacities of Gastric Cancer Associated Fibroblasts.

To investigate the inhibitory effects of paeoniflorin on migration- and invasion-promoting capacities of gastric cancer associated fibroblasts (GCAFs) and to explore the molecular mechanism underlying the effects. Paired gastric normal fifbroblast (GNF) and GCAF cultures were established from resected tissues. GCAFs were treated with control medium, or 2.5, 5 or 10 μg/mL paeoniflorin. Conditioned media were prepared from GNFs, GCAFs, control-treated GCAFs and paeoniflorin-treated GCAFs, and used to culture AGS human gastric cancer cells. The migration and invasion capacities of AGS cells were determined with wound healing test and transwell invasion assay, respectively. The interleukin 6 (IL-6) mRNA and microRNA-149 expression in GCAFs were detected by reverse transcription-quantitative polymerase chain reaction. The IL-6 protein expression and secretion by GCAFs were measured with Western blot and enzyme-linked immunosorbent assay analysis, respectively. The protein levels of phosphorylated signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase (MMP) and MMP9 in AGS cells were examined by Western blot. GCAFs displayed enhanced capacities to induce AGS cell migration and invasion as compared with GNFs. Paeoniflorin treatment significantly inhibited the migration- and invasion-promoting capacities of GCAFs (P<0.05). GCAFs produced and secreted more IL-6 into the conditioned medium than GNFs, leading to over-activation of STAT3-MMP signaling in AGS cells. Paeoniflorin suppressed IL-6 production and secretion by up-regulating microRNA149 expression in GCAFs, and subsequently prevented GCAFs from activating IL-6-STAT3-MMP signaling of AGS cells. Paeoniflorin inhibits the migration- and invasion-promoting capacities of GCAFs by targeting microRNA-149 and IL-6. Paeoniflorin is potentially a novel therapeutic agent against cancer microenvironment.

Activated gastric cancer-associated fibroblasts contribute to the malignant phenotype and 5-FU resistance via paracrine action in gastric cancer

BackgroundCancer-associated fibroblasts (CAFs) play important roles in tumor progression. However, the behaviors of activated CAFs in gastric cancer remain to be determined. The aim of the present study was to investigate the correlations between activated gastric CAFs and the prognosis of patients with gastric cancer, and to determine the effects of activated CAFs on the malignant phenotype and 5-fluorouracil resistance in this cancer.MethodsNinety-five patients with primary gastric cancer were enrolled in this study. Activation states of gastric CAFs were evaluated by immunohistochemistry. A modified method for the primary culture of gastric CAFs was employed. Types of CAFs and activation states were identified by immunocytochemical and immunofluorescent staining. Cell co-culture and gastric CAF conditioned medium transfer models were established to investigate the paracrine effects of activated CAFs on the migration and invasion of gastric cell lines. The half maximal inhibitory concentration of 5-fluorouracil and levels of cell apoptosis were examined using cell viability assay and flow cytometry, respectively. Protein expression levels of associated molecules were measured by Western blotting.ResultsKaplan–Meier survival curves showed that activated gastric CAFs identified via fibroblast activation protein were significantly related to poorer cumulative survival in gastric cancer patients. Five strains of CAFs were successfully cultured via the modified culture method, and three gastric CAFs strains were identified as activated gastric CAFs. The migration and invasion abilities of gastric cells were significantly enhanced in both the co-culture group and the conditioned medium group. The half maximal inhibitory concentration for 5-fluorouracil in BGC-823 cells was elevated after treatment with conditioned medium, and early apoptosis was inhibited. Additionally, an obvious elevation of epithelial–mesenchymal transition level was observed in the conditioned medium group.ConclusionsActivated gastric CAFs correlate with a poor prognosis of cancer patients and may contribute to the malignant phenotype and the development of resistance to 5-fluorouracil via paracrine action in gastric cancer. Gastric CAFs with a specific activation state might be used as a tumor biomarker within the microenvironment for prognosis and as a new therapeutic target for chemoresistant gastric cancer.

Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts.

AIMTo investigate the inhibitory effect of astragaloside IV on the pathological functions of cancer-associated fibroblasts, and to explore the underlying mechanism.METHODSPaired gastric normal fibroblast (GNF) and gastric cancer-associated fibroblast (GCAF) cultures were established from resected tissues. GCAFs were treated with vehicle control or different concentrations of astragaloside IV. Conditioned media were prepared from GNFs, GCAFs, control-treated GCAFs, and astragaloside IV-treated GCAFs, and used to culture BGC-823 human gastric cancer cells. Proliferation, migration and invasion capacities of BGC-823 cells were determined by MTT, wound healing, and Transwell invasion assays, respectively. The action mechanism of astragaloside IV was investigated by detecting the expression of microRNAs and the expression and secretion of the oncogenic factor, macrophage colony-stimulating factor (M-CSF), and the tumor suppressive factor, tissue inhibitor of metalloproteinase 2 (TIMP2), in different groups of GCAFs. The expression of the oncogenic pluripotency factors SOX2 and NANOG in BGC-823 cells cultured with different conditioned media was also examined.RESULTSGCAFs displayed higher capacities to induce BGC-823 cell proliferation, migration, and invasion than GNFs (P < 0.01). Astragaloside IV treatment strongly inhibited the proliferation-, migration- and invasion-promoting capacities of GCAFs (P < 0.05 for 10 μmol/L, P < 0.01 for 20 μmol/L and 40 μmol/L). Compared with GNFs, GCAFs expressed a lower level of microRNA-214 (P < 0.01) and a higher level of microRNA-301a (P < 0.01). Astragaloside IV treatment significantly up-regulated microRNA-214 expression (P < 0.01) and down-regulated microRNA-301a expression (P < 0.01) in GCAFs. Reestablishing the microRNA expression balance subsequently suppressed M-CSF production (P < 0.01) and secretion (P < 0.05), and elevated TIMP2 production (P < 0.01) and secretion (P < 0.05). Consequently, the ability of GCAFs to increase SOX2 and NANOG expression in BGC-823 cells was abolished by astragaloside IV.CONCLUSIONAstragaloside IV can inhibit the pathological functions of GCAFs by correcting their dysregulation of microRNA expression, and it is promisingly a potent therapeutic agent regulating tumor microenvironment.

Triptonide inhibits the pathological functions of gastric cancer-associated fibroblasts

Direct attacks on tumour cells with chemotherapeutic drugs have the drawbacks of accelerating tumour metastasis and inducing tumour stem cell phenotypes. Inhibition of tumour-associated fibroblasts, which provide nourishment and support to tumour cells, is a novel and promising anti-tumour strategy. However, effective drugs against tumour-associated fibroblasts are currently lacking. In the present study, we explored the possibility of inhibiting the pathological functions of tumour-associated fibroblasts with triptonide. Paired gastric normal fibroblasts (GNFs) and gastric cancer-associated fibroblasts (GCAFs) were obtained from resected tissues. GCAFs showed higher capacities to induce colony formation, migration, and invasion of gastric cancer cells than GNFs. Triptonide treatment strongly inhibited the colony formation-, migration-, and invasion-promoting capacities of GCAFs. The expression of microRNA-301a was higher and that of microRNA-149 was lower in GCAFs than in GNFs. Triptonide treatment significantly down-regulated microRNA-301a expression and up-regulated microRNA-149 expression in GCAFs. Re-establishment of microRNA expression balance increased the production and secretion of tissue inhibitor of metalloproteinase 2, a tumour suppressive factor, and suppressed the production and secretion of IL-6, an oncogenic factor, in GCAFs. Moreover, triptonide treatment abolished the ability of GCAFs to induce epithelial–mesenchymal transition in gastric cancer cells. These results indicate that triptonide inhibits the malignancy-promoting capacity of GCAFs by correcting abnormalities in microRNA expression. Thus, triptonide is a promisingly therapeutic agent for gastric cancer treatment, and traditional herbs may be a valuable source for developing new drugs that can regulate the tumour microenvironment.

Down-regulation of KLF5 in cancer-associated fibroblasts inhibit gastric cancer cells progression by CCL5/CCR5 axis

ABSTRACTIt was well known that cancer-associated fibroblasts (CAFs) were an essential factor in tumor progression. However, the actual mechanism of stromal fibroblasts activation and tumor promoting effects remain unclear. Here, we showed that KLF5 expression was more frequently observed in gastric cancer-associated fibroblasts compared with normal mucosal fibroblasts. Moreover, KLF5 expression in tumor stroma was closely associated with clinicopathological features such as tumor size, invasion depth, cell grade and lymph node metastasis, as well as poor prognosis in patients with gastric cancer. In addition, we further demonstrated that KLF5-regulating CAFs affect gastric cancer cells progression by CCL5 secretion and activation of CCR5. Taken together, we concluded that KLF5 expression in gastric cancer-associated fibroblasts contribute to poor survival and promote cancer cells progression by activation of CCL5/CCR5 axis, which suggesting that KLF5 in CAFs might be considered as a promising target for the treatment of gastric cancer.

Activation of Transforming Growth Factor Beta 1 Signaling in Gastric Cancer-associated Fibroblasts Increases Their Motility, via Expression of Rhomboid 5 Homolog 2, and Ability to Induce Invasiveness of Gastric Cancer Cells

Fibroblasts that interact with cancer cells are called cancer-associated fibroblasts (CAFs), which promote progression of different tumor types. We investigated the characteristics and functions of CAFs in diffuse-type gastric cancers (DGCs) by analyzing features of their genome and gene expression patterns. We isolated CAFs and adjacent non-cancer fibroblasts (NFs) from 110 gastric cancer (GC) tissues from patients who underwent gastrectomy in Japan from 2008 through 2016. Cells were identified using specific markers of various cell types by immunoblot and flow cytometry. We selected pairs of CAFs and NFs for whole-exome and RNA sequencing analyses, and compared expression of specific genes using quantitative reverse transcription PCR. Protein levels and phosphorylation were compared by immunoblot and immunofluorescence analyses. Rhomboid 5 homolog 2 (RHBDF2) was overexpressed from a transgene in fibroblasts or knocked down using small interfering RNAs. Motility and invasiveness of isolated fibroblasts and GC cell lines (AGS, KATOIII, MKN45, NUGC3, NUGC4, OCUM-2MD3 and OCUM-12 cell lines) were quantified by real-time imaging analyses. Weanalyzed 7 independent sets of DNA microarray data frompatients with GC and associated expression levels of specific genes with patient survival times. Nude mice were given injections of OCUM-2MD3 in the stomach wall; tumors and metastases were collected and analyzed by immunohistochemistry. Many of the genes with increased expression in CAFs compared with NFs were associated with transforming growth factor beta 1 (TGFB1) activity. When CAFs were cultured in extracellular matrix, they became more motile than NFs; DGC cells incubated with CAFs were also more motile and invasive invitro than DGC cells not incubated with CAFs. When injected into nude mice, CAF-incubated DGC cells invaded a greater number of lymphatic vessels than NF-incubated DGC cells. We identified RHBDF2 as a gene overexpressed in CAFs compared with NFs. Knockdown of RHBDF2 in CAFs reduced their elongation and motility in response to TGFB1, whereas overexpression of RHBDF2 in NFs increased their motility in extracellular matrix. RHBDF2 appeared to regulate oncogenic and non-canonical TGFB1 signaling. Knockdown of RHBDF2 in CAFs reduced cleavage of the TGFB receptor 1 (TGFBR1) by ADAM metallopeptidase domain 17 (ADAM17 or TACE) and reduced expression of genes that regulate motility. Incubation of NFs with in interleukin 1 alpha (IL1A), IL1B or tumor necrosis factor, secreted by DGCs, increased fibroblast expression of RHBDF2. Simultaneous high expression of these cytokines in GC samples was associated with shorter survival times of patients. In CAFs isolated from human DGCs, we observed increased expression of RHBDF2, which regulates TGFB1 signaling. Expression of RHBDF2 in fibroblasts is induced by inflammatory cytokines (such as IL1A, IL1B, and tumor necrosis factor) secreted by DGCs. RHBDF2 promotes cleavage of TGFBR1 by activating TACE and motility of CAFs in response to TGFB1. These highly motile CAFs induce DGCs to invade extracellular matrix and lymphatic vessels in nude mice.

Abstract 5069: IL-33 secreted by cancer-associated fibroblasts mediates epithelial-stromal interactions and promotes gastric cancer invasion via ERK signaling pathway

Abstract Background: Cancer-associated fibroblasts (CAFs) are important modifiers of tumor progression, which can actively communicate with cancer cells through soluble factors. Interleukin-33 (IL-33) is a regulator of immune and inflammatory responses and has also been associated with certain epithelial tumors. Although the importance of CAFs in gastric cancer has been documented in experimental studies, the mechanisms by which CAFs and IL-33 promote gastric cancer remain unclear. Objectives: The aim of this study was to determine the role of IL-33 in the epithelial-stromal interaction in gastric cancer and if CAFs can promote the invasion of gastric cancer cells via ERK pathway through IL-33. Methods: CAFs and normal fibroblasts (NFs) were isolated from gastric cancer tissues and adjacent non-cancerous tissues respectively. The identification of CAFs was detected by immunofluorescence (IF) and qRT-PCR. IL-33 expression in CAFs and gastric cancer cells was detected by qRT-PCR. CAFs was co-cultured with gastric cancer cells in the presence of exogenous TNF-α, IL-1β and/or their corresponding neutralizing antibodies, the expression of IL-33 in CAFs was detected using qRT-PCR. EMT changes and activation of ERK pathway in gastric cancer cells after co-cultured with CAF-conditioned media were detected by qRT-PCR and western blot, and cell invasion assays were performed by using 8 μm transwell chambers. Results: Gastric CAFs expressed more α-SMA, a marker of myofibroblasts, and more fibroblast activation protein (FAP) when compared with patient-matched NFs. In comparison to gastric cancer cells and NFs, IL-33 was highly expressed in CAFs. Upregulation of IL-33 in CAFs can be induced by co-culture with gastric cancer cells or in the presence of TNF-α and IL-1β, while deprivation of TNF-α and IL-1β using their neutralizing antibodies inhibited the upregulation of IL-33. CAFs induced EMT changes of gastric cancer cells, which was demonstrated by decreased expression of E-cadherin and increased expression of Twist1, MMP2, ZEB1 and ZEB2. However, deprivation of IL-33 using a neutralizing antibody impaired the EMT changes of gastric cancer cells. Gastric cancer cells co-cultured with CAFs or CAFs conditioned media showed enhanced ability of invasion than gastric cancer cells alone. However, adding neutralizing IL-33 antibody or IL-33-siRNA-CAFs conditioned media or U0126 into the culture system led to significantly decreased invasion (P &amp;lt; 0.05) of gastric cancer cells. Moreover, IL-33 secreted from CAFs activated ERK pathway in gastric cancer cells, however, the invasion of gastric cancer cells was attenuated when IL-33-ERK pathway was blocked. Conclusions: TNF-α and IL-1β derived from gastric cancer cells can significantly enhance the expression of IL-33 in CAFs, and IL-33 secreted by CAFs promotes gastric cancer invasion via the ERK signaling pathway. Note: This abstract was not presented at the meeting. Citation Format: Liping Su, Quan Zhou, Chenchen Wang, Xiongyan Wu, Xiaofeng Wang, Jianfang Li, Zhenggang Zhu, Bingya Liu. IL-33 secreted by cancer-associated fibroblasts mediates epithelial-stromal interactions and promotes gastric cancer invasion via ERK signaling pathway. [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 5069. doi:10.1158/1538-7445.AM2015-5069

Caveolin-1 is a modulator of fibroblast activation and a potential biomarker for gastric cancer.

Stromal fibroblasts play an important role in chronic cancer-related inflammation and the development as well as progression of malignant diseases. However, the difference and relationship between inflammation-associated fibroblasts (IAFs) and cancer-associated fibroblasts (CAFs) are poorly understood. In this study, gastric cancer-associated fibroblasts (GCAFs) and their corresponding inflammation-associated fibroblasts (GIAFs) were isolated from gastric cancer (GC) with chronic gastritis and cultured in vitro. These activated fibroblasts exhibited distinct secretion and tumor-promoting behaviors in vitro. Using proteomics and bioinformatics techniques, caveolin-1 (Cav-1) was identified as a major network-centric protein of a sub-network consisting of 121 differentially expressed proteins between GIAFs and GCAFs. Furthermore, immunohistochemistry in a GC cohort showed significant difference in Cav-1 expression score between GIAFs and GCAFs and among patients with different grades of chronic gastritis. Moreover, silencing of Cav-1 in GIAFs and GCAFs using small interfering RNA increased the production of pro-inflammatory and tumor-enhancing cytokines and chemokines in conditioned mediums that elevated cell proliferation and migration when added to GC cell lines AGS and MKN45 in vitro. In addition, Cav-1 status in GIAFs and GCAFs independently predicted the prognosis of GC. Our findings indicate that Cav-1 loss contributes to the distinct activation statuses of fibroblasts in GC microenvironment and gastritis mucosa, and Cav-1 expression in both GCAFs and GIAFs may serve as a potential biomarker for GC progression.

Effects of cancer-associated fibroblasts on the migration and invasion abilities of SGC-7901 gastric cancer cells

The aim of the current study was to investigate the correlation of cancer-associated fibroblasts (CAFs) with the migration and invasion abilities of gastric cancer cells. Gastric CAFs were grown in primary cultures. The in vitro model of interaction of SGC7901 gastric cancer cells with gastric CAFs was established by the use of Transwell co-culture cells to analyze the influence of CAFs on the migration and invasion abilities of SGC7901 cells. The results revealed that i) human gastric CAFs highly expressed vimentin, fibroblast-activated protein and smooth muscle actin in the in vitro passage culture process; ii) the migration and invasion ability of SGC-7901 cells in the CAF-conditioned medium group (98.67±13.49, 34.40±4.63) were significantly higher compared to those of the DMEM group without serum (78.47±10.59, 26.93±3.99; P<0.01). The interactions of CAFs and the extracellular matrix with SGC-7901 cells may significantly increase the migration and invasion abilities of SGC-7901 cells.

Abstract 40: Targeting MLCK expression in cancer-associated fibroblasts disrupts tumor-stromal interactions in gastric cancer

Abstract Aim: Accumulating evidence has indicated that microenvironment consisting of stromal affects cancer progression. However, the mechanism by which cancer cells and fibroblasts, the major cell type in stromal, interact with each other during tumor development remains to be elucidated. Our aim is to identify the molecular mechanism involved in tumor-stromal cell interactions in human gastric cancer. Methods: The expression of Myosin Light Chain Kinase (MLCK) in gastric cancer-associated fibroblasts (CAF) was detected by quantitative RT-PCR. The effects of CAF, which was interfered with MLCK, on cell proliferation, apoptosis, migration and invasion in vitro were measured. Results: Primary fibroblast cultures were established from human gastric cancer. MLCK was upregulated in CAF compared with NF (fibroblasts from patient matching non-cancerous tissues). Small interfering RNA knockdown of MLCK in CAF impaired the augmenting effect of CAF on gastric cancer cell proliferation, and induced the apoptosis of gastric cancer cells (9.1+0.8 vs 22.3+1.4, p&amp;lt;0.05). Moreover, the ability of stimulating gastric cancer cells migration (77.7+4.3 vs 61+4.2, p&amp;lt;0.05) and invasion (60.9+1.5 vs 48.3+1.4, p&amp;lt;0.05) in vitro by CAF was also inhibited when MLCK was knocked down. Furthermore, gastric cancer cells can also activate CAF in a MLCK-dependent manner. Conclusions: These findings indicate that MLCK mediates the cross-talk between tumor and stromal in gastric cancer, and suggest that targeting stromal cells for MLCK interference may be a therapeutic target in human gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 40. doi:1538-7445.AM2012-40

Twist1 Is Up-Regulated in Gastric Cancer-Associated Fibroblasts with Poor Clinical Outcomes

Stromal fibroblasts perform important roles in cancer development and progression. Overexpression of Twist1, a basic helix-loop-helix transcription factor, is often associated with aggressive behavior in many tumors. In this study, we investigated Twist1 expression patterns in gastric stromal fibroblasts and cancer cells using a monoclonal Twist1 antibody after validating the effectiveness of four commercial Twist1-specific antibodies. Twist1 expression was more frequently observed in gastric cancer-associated fibroblasts (CAFs) than in other cancer cells but was otherwise rarely expressed in noncancerous tissue. In laser capture microdissection of stromal fibroblasts, Twist1 immunopositive fibroblasts exhibited significantly increased Twist1, fibroblast-specific protein 1, and CXCL14 mRNA expression. Furthermore, Twist1 mRNA expression showed a significant linear correlation with that of platelet-derived growth factor receptors β and α. We found that conditioned media from Twist1-expressing skin and lung fibroblasts significantly promote invasion of gastric cancer cells in vitro. In 195 gastric cancer samples, CAF Twist1 expression was associated with tumor size, invasion depth, and lymph node metastasis. Twist1 was also associated with poor prognosis in patients with gastric cancer, particularly in those with the diffuse type. In conclusion, CAFs in gastric cancer frequently have altered Twist1 expression, and increased Twist1 expression in fibroblasts contributes to the progression of cancer cells and poor patient survival.

Abstract 5200: Effect of cancer-associated fibroblast on the progression of gastric cancer stem cells

Abstract Introduction: Cancer stem cells (CSCs) are thought to possess the ability to initiate tumor growth and sustain self-renewal, which explains the tumorigenicity and the maintenance of bulk. There is an increasing evidence of the importance of the host microenvironment in conditioning the CSCs status, such as the stemness and the tumor initiation. Recently, cancer-associated fibroblasts in the cancer microenvironment have been suggested to be associated with the tumor progression. Then, the aim of this study is to clarify the relation between CSCs and cancer-associated fibroblasts. Material and Methods: A gastric cancer cell line, OCUM-12, and cancer-associated gastric fibroblasts, CaF-37, were used. Side population (SP) cells, known as CSCs rich population, were isolated from OCUM-12 cells by flowcytometry using Hoechst 33342, and were named as OCUM-12/SP. OCUM-12/SP cells or OCUM-12 cells were cultured with or without CaF-37 fibroblasts. The percentage of SP cells was evaluated by flowcytometry, and the growth of cancer cells was examined by cell number count. In vivo tumorigenicity of OCUM-12/SP cells was evaluated by subcutaneous inoculation in the presence or absence of CaF-37 fibroblasts. Results: The percentage of SP fraction of OCUM-12/SP cells was increased in the presence of CaF-37 fibroblasts. In contrast, CaF-37 fibroblasts significantly decreased the proliferation of OCUM-12/SP cells, while that of OCUM-12 cells was significantly increased by CaF-37 cells. The inoculation of 3×105, 1×105, and 3×104 OCUM-12/SP cells with 1×106 CaF-37 fibroblasts resulted in tumor formation in 5 (100%) of 5 mice, 4 (80%) of mice, 5 (100%) of 5 mice, respectively. However, the inoculation of 3×105, 1×105, and 3×104 OCUM-12/SP cells alone results in poor tumor formation at 3 (60%) of 5 mice, 0 (0%) of 5 mice, and 0 (0%) of 5 mice, respectively.Conclusion: Cancer-associated fibroblast might sustain the CSCs phenotype of stemness, and might increase the tumor initiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5200. doi:10.1158/1538-7445.AM2011-5200

Comparison of biological features of gastric cancer-associated fibroblasts and human gastric mucosa fibroblasts

Objective To investigate the biologic characteristic differences between gastric cancerassociated fibroblasts (GCAF) and human gastric mucosa fibroblasts (HGMF). Methods The fibroblasts were cultured primarily by outgrowth method and identified by immunohistochemistry. Third-generation cells were chosen to be analyzed. Their differences in biological features were compared by light microscopy, electron microscopy, and CCK-8 assay. The ability to synthesize proteins and cell cycle were detected by BCA Protein Assay Kit and flow cytometry respectively. Each experiment was repeated three times and the average was calculated. Results Compared with HGMF, the both of GCAF was larger and irregular with conjugate nuclei or multinucleate cells under the light microscopy. Deformed nuclei and multiple nuclei could be found in GCAF under the electron microscopy with elevated karyoplasmic ratio and increased mitochondria smooth endoplasmic reticulum. The ability of proliferation and protein synthesis in GCAF was significantly enhanced as compared with HGMF (P ＜0. 01 ), and both proliferation index (P ＜0. 05 ) and percentage of S phase cells ( P ＜ 0. 01 ) in GCAF were significantly higher than HGMF. Conclusion There were not only obviously differences in morphology, but also ability of proliferation, protein synthesis and cell cycle activity in GCAF were significantly higher than HGMF. Key words: Gastric carcinoma; Cancer-associated fibroblast