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Hepatocyte growth factor activates tumor stromal fibroblasts to promote tumorigenesis in gastric cancer
Cancer Letters 335 (2013) 128–135
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Hepatocyte growth factor activates tumor stromal fibroblasts to promote tumorigenesis in gastric cancer Xiongyan Wu a,1, Xuehua Chen a,1, Quan Zhou a, Pu Li a, Beiqin Yu a, Jianfang Li a, Ying Qu a, Jun Yan b, Yingyan Yu a, Min Yan a, Zhenggang Zhu a, Bingya Liu a,⇑, Liping Su a,⇑ a
Shanghai Key Laboratory of Gastric Neoplasms, Department of Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, PR China James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA
b
a r t i c l e
i n f o
Article history: Received 29 November 2012 Received in revised form 1 February 2013 Accepted 2 February 2013
Keywords: HGF Cancer-associated fibroblasts Gastric cancer
a b s t r a c t Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in tumor stroma, are important modifiers of tumor progression. However, the mechanisms underlying stromal fibroblast activation and their promotion of tumor growth remain largely unknown in gastric cancer. Here, we show that normal fibroblasts (NFs) from non-cancerous regions of gastric cancer exhibit the traits of CAFs when grown together with gastric cancer cells in vivo. Activation of NFs can be induced by co-culture with gastric cancer cells, while deprivation of hepatocyte growth factor (HGF) using a neutralizing antibody inhibits the activation of NFs. Moreover, we identify HGF as an important factor from CAFs that acts in a paracrine manner to promote tumorigenesis in vitro and in vivo. Taken together, these results suggest that HGF may play a pivotal role in the regulatory circuit between gastric cancer cells and stromal fibroblasts, and neutralization of HGF inhibits both activation and tumor-promoting properties of CAFs. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Gastric cancer is one of the most common malignancies and is a leading cause of cancer death worldwide, especially in Eastern Asia, with an estimated 988,000 new cases and 736,000 deaths occurring in 2008 [1,2]. Surgical resection, the only curative therapy for gastric cancer, is possible in less than 25% of the patients at time of diagnosis, and the overall prognosis of non-resected gastric cancer is dismal [3]. Therefore, achieving a better understanding of molecular aberrations of gastric cancer carcinogenesis may lead to identification of novel diagnostic and therapeutic targets for this disease. Accumulating evidence has indicated that interactions between tumor and stromal cells create a unique microenvironment that is essential for tumor growth, invasion, and metastasis [4,5]. Cancerassociated fibroblasts (CAFs) are the major constituent of the tumor stroma and are distinguishable from their normal counterparts in their increased rate of proliferation and in the activated myofibroblastic phenotype, which includes a prominent contractile ability and expression of a-smooth muscle actin (a-SMA) [4,6]. Other markers found to be expressed by CAFs are platelet derived growth factor receptor-b (PDGFRb), fibroblast specific ⇑ Corresponding authors. Tel.: +86 21 64674654. 1
E-mail addresses: [email protected] (B. Liu), [email protected] (L. Su). These authors contributed equally.
0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.02.002
protein-1 (FSP-1), vimentin, and fibroblast activation protein (FAP) [7,8]. The contribution of CAFs to tumor development and progression has been reported in multiple cancer models, and the prevalence of CAFs in tumor bearing desmoplastic stroma is recognized to have a prognostic value for many solid tumors [9– 11]. Recent studies have shown that CAFs also frequently accumulate in gastric cancer tissues, and that the prevalence of CAFs correlates with the size, depth and metastasis of the tumor [12]. CAFs actively communicate with cancer cells, and contribute to tumor progression, via a direct cell–cell interaction and by modification of extracellular matrix components, or through soluble factors such as growth factors and chemokines [5,13,14]. Among the many factors that can contribute to such a phenotype modulation of cancer cells, hepatocyte growth factor (HGF), a stromal-derived functional growth factor, has been associated with metastases in many types of human cancers [15–18]. HGF binds to the receptor tyrosine kinase Met that is expressed in the epithelium, which contributes to oncogenesis and tumor progression [19]. Although it has been shown that CAFs are mainly converted from the local-residing fibroblasts, the mechanisms underlying stromal fibroblast activation and their promotion of tumor growth remain largely unknown in gastric cancer. To address this question, we examined the epithelial-stromal interaction in gastric cancer xenograft model using CAFs and adjacent normal fibroblasts (NFs). Here, we find that NFs exhibit the traits of CAFs when they are grown together with gastric cancer cells in vivo at an advanced
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stage, and identify HGF as an important inducer of activated fibroblasts from adjacent NFs that are in a quiescent status in gastric cancer. Moreover, we show that the expression of HGF is elevated in all CAFs, acting in a paracrine manner to promote gastric cancer tumorigenesis in vitro and in vivo. These data collectively indicate that HGF may play a pivotal role in the regulatory circuit between gastric cancer cells and stromal fibroblasts, and blocking this crosstalk by interrupting HGF signaling may provide an alternative treatment strategy for gastric cancer.
2.7. Cell migration assay
2. Materials and methods
2.8. RNA interference
2.1. Cell lines
Human HGF siRNA and scrambled siRNA as the control (Santa Cruz, CA, USA) at the final concentration of 100 nmol/L were transfected into CAFs cells with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Cells were collected for further assay at 24 h, 48 h and 72 h after transfection.
Cell migration assays were performed by using 8 lm transwell chambers (Corning Life Science, Acton, MA, USA). Cells were incubated in serum-free medium for 24 h, and then 2 104 MKN-45 cells in 200 ll serum-free medium were added to the upper chamber, RPMI 1640 medium containing 10% FBS were added to the lower chamber. Non-migrating cells from the interior of the inserts were removed with cotton-tipped swabs 48 h later, and cells that migrated to the bottom of the membranes were stained with 0.1% crystal violet for 20 min. The stained cells were counted and photographed with 100 magnification. At least three randomly selected fields were counted and the average number was presented.
Gastric cancer cell line MKN45 was obtained from the Japanese Cancer Research Resources Bank and was cultured at 37 °C in a humidified atmosphere of 5% CO2 with RPMI-1640 medium containing 10% fetal calf serum with 100 U/ml penicillin and 100 lg/ml streptomycin. Fibroblasts were isolated from 4 independent gastric cancer patients during radical gastric resection at the Department of Surgery, Ruijin hospital, School of Medicine, Shanghai Jiaotong University. Tumor tissues and adjacent non-tumor tissues located at least over 6 cm apart from the margin of the resection were collected and minced into organoids of approximately 1 mm3 and seeded onto 15 cm petri dishes in above-described medium. These conditions produced a homogenous fibroblastic cell population after 7 days of culture. Each fibroblast culture was then expanded into two 15 cm petri dishes and stored as cells passaged for 2–3 population doublings. We used fibroblasts passaged for up to 10 population doublings for subsequent experiments, in order to minimize clonal selection and culture stress, which could occur during extended culture.
Male BALB/c nu/nu nude mice at the age of 4–5 weeks (Institute of Zoology Chinese Academy of Sciences), were housed at a specific pathogen-free environment in the Animal Laboratory Unit, Shanghai Jiao Tong University School of Medicine, China. Fibroblasts and MKN-45 cells were mixed at the ratio of 1:4 within 0.1 ml PBS and injected subcutaneously. Five mice per group were used in all experiments. Tumor growth was monitored weekly and tumor volume was assessed by measuring tumor size with digital calipers using the following formula: volume = ab2p/6. All animal studies were conducted with the approval of the Committee on Animal Care in Shanghai Jiao Tong University School of Medicine.
2.2. Immunofluorescence
2.10. Immunohistochemistry (IHC)
Cells were fixed for 30 min with 1% paraformaldehyde and rinsed with phosphate buffered saline (PBS). Cells were then blocked with normal nonimmune goat serum for 30 min prior to being incubated with anti-vimentin, anti-CD31, anti-asmooth-muscle actin, anti-pan-cytokeratin or anti-FAP (Abcam, Cambridge, USA) at 37 °C for 2 h. After 5 rinses with PBS, cells were stained with appropriate Alexa dye-conjugated secondary immune reagents, Alexa dye-conjugated phalloidin, and Hoechst 33342 (Invitrogen, Carlsbad, CA, USA). Negative control staining was performed by omission of the primary antibody.
Tissues were fixed in 10% neutralized formalin and embedded in paraffin blocks. Sections (4 lm) were dewaxed with xylene and rehydrated with gradient ethanol. Antigen retrieval was performed by boiling in 10 mmol/l of citrate buffer (pH 6.0) for 10 min. Endogenous peroxidase was blocked by incubation in 0.3% H2O2 in methanol for 30 min. Nonspecific binding was then blocked with 2% bovine serum albumin in PBS for 30 min. The sections were incubated with rabbit anti-a-SMA antibody (Abcam, Cambridge, MA, USA) overnight at 4 °C. The immune complex was visualized with the Dako REAL™EnVision™ Detection System, Perox-idase/ DAB, Rabbit/Mouse (Dako, Denmark), according to the manufacturers’ manual. The nuclei were counterstained with hematoxylin.
2.3. Quantitative real-time PCR (QRT-PCR) Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s manual. RNA (1 lg) was reverse transcribed into cDNA using Reverse Transcription system (Promega, Madison, WI, USA). QRT-PCR was performed to quantify the mRNA levels of HGF, a-SMA, FAP, and SDF-1 with the SYBR Green PCR core Reagent kit (Applied Biosystems, Foster City, CA, USA). HPRT1 was used as the endogenous reference. Data were analyzed by using the comparative Ct method. Specificity of resulting PCR products was confirmed by melting curves. The primers used in this assay were: HGF: 50 -TACAGGGGCACTGT CAATACC-30 (upper) and 50 -GGATACTGAGAATCCCAACGC-30 (lower); a-SMA: 50 -CAGGGCTGTTTTCCCATCCAT-30 (upper), 50 -GCCATGTTCTATCGGGTACTT C-30 (lower); FAP: 50 -AATGAGAGCACTCACACTGAAG -30 (upper), 50 -CCGATCA GGTGATAAGCCGTAAT-30 (lower); SDF-1: 50 -GTTTGTGCTGTGGTGTGTC C-30 (upper), 50 -ATACTAAGGTTGGGGGAGGTG-30 (lower); HPRT1: 50 -GACCAG TA ACAGGGGACAT-30 , 50 -GTCCTTTTCACCAGCAAGCT-30 (lower). 2.4. Enzyme-linked immunosorbent assay (ELISA) The protein levels of HGF and SDF-1 in supernatants were measured by an ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. 2.5. Cell proliferation assay Cell proliferation was assessed by water-soluble tetrazolium salt (WST) assay using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Briefly, 1 103 cells were seeded into 96-well plates in triplicate and incubated for 4 days. CCK-8 solution (20 ll) was added into plates, and absorbance at 450 nm was measured after 4 h incubation. 2.6. Soft agar colony formation assay Cells were resuspended with 0.3% soft agar in RPMI 1640 containing 10% FBS and layered onto 0.6% solidified agar in RPMI 1640 containing 10% FBS in 6-well plates (1 103 cells/well) at 24 h post-transfection. The plates were incubated for 2 weeks. Colonies containing at least 50 cells were counted.
2.9. Tumor xenograft model and tumorigenicity assay
2.11. Statistical analysis Results were summarized as means ± SEM. Student t test and one-way analysis of variance (ANOVA) was used to analyze the data and the significance level was set at P < 0.05.
3. Results 3.1. CAFs are more competent in enhancing tumor growth than NFs at the early stage of tumor growth To illustrate the role of CAFs in promoting tumor growth of gastric cancer, we isolated CAFs from human gastric cancer tissue and NFs from adjacent non-cancerous tissue of the same patients. We then verified these fibroblasts by immunostaining. As shown in Fig. 1A, both gastric CAFs and NFs strongly expressed the fibroblastic marker vimentin but negative for cytokeratin and CD31. Moreover, gastric CAFs expressed more a-smooth muscle actin (a-SMA), a marker of myofibroblasts, and more fibroblast activation protein (FAP) when compared with patient-matched NFs (Fig. 1A and B). In addition, we verified the purity of various fibroblast populations by flow cytometry, which indicated that no more than 0.1% of the cells in each fibroblast population were positive for CD45, CD11b, CD68 and Gr-1 (data not shown). These primary CAFs and NFs could be maintained in culture for up to 20 population doublings. Thus, we used these CAFs and NFs for subsequent experiments. To assess the contribution of both fibroblast populations to tumor growth in vivo, we mixed CAFs or NFs with human gastric cancer cells MKN45 in a 1:4 ratio and inoculated them subcutaneously
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into immunodeficient nude mice. Interestingly, we found that MKN45 cells mixed with CAFs generated tumors with a greater volume (Fig. 1C) at the initial stage than did MKN45 cells mixed with NFs, 6.5-fold larger after 7 days (0.07 ± 0.01 cm3 vs 0.01 ± 0.01 cm3), 2.0-fold larger after 14 days (0.19 ± 0.07 cm3 vs 0.15 ± 0.02 cm3) (Fig. 1C, Table 1). However, at an advanced stage MKN45 cells mixed with CAFs did not have such a large differential size compared with those mixed with NFs, only 1.7-fold larger after 21 days (0.75 ± 0.09 cm3 vs 0.44 ± 0.06 cm3) and 1.3-fold larger after 28 days (1.47 ± 0.21 cm3 vs 1.10 ± 0.15 cm3), respectively (Fig. 1C, Table 1). These observations indicated that CAFs have an increased ability to foster tumor formation when compared to NFs, especially at the initial stages of tumor growth. 3.2. Activation of NFs induced by gastric cancer cells is dependent on the presence of HGF Intriguingly, mixing NFs with gastric cancer cells resulted in tumor growth that, although slower than that of tumors mixed with CAFs, was significantly greater than that of tumor cells alone (Fig. 1C, Table 1). Meanwhile, tumor growth from MKN45 cells coinjected with NFs was almost identical to that from MKN45 cells coinjected with CAFs at 28 days (Fig. 2A and B). These results indicated that gastric cancer cells can educate their surrounding fibroblasts to acquire a stronger tumor-promoting ability to support their own progression. To verify that NFs, when grown together with gastric cancer cells in vivo, were educated into CAFs, we detected a-SMA expression, the characteristic signature of CAFs, by IHC in the tumors generated from MKN45 cells with NFs or CAFs, as shown in Fig. 2C. When NFs were coinjected with MKN45 cells,
Table 1 The tumor volume with each group at days 7, 14, 21 and 28.
MKN45 group MKN45 + NFs group MKN45 + CAFs group
7 days
14 days
21 days
28 days
0.00 ± 0.00 0.01 ± 0.01 0.07 ± 0.01
0.01 ± 0.01 0.15 ± 0.02 0.29 ± 0.07
0.05 ± 0.05 0.44 ± 0.06 0.75 ± 0.09
0.37 ± 0.10 1.10 ± 0.15 1.47 ± 0.21
Note: Unit of the tumor volume: cm3.
fibroblasts in the resultant tumor expressed similar a-SMA to that in the tumor generated from MKN45 cells with CAFs. To define the molecule responsible for the activation of CAFs from NFs induced by gastric cancer cells, we established an in vitro transwell co-culture system (Fig. 2D). Using a cytokine/ growth factor antibody array, we found that HGF was up-regulated in both NFs and CAFs after co-culture with gastric cancer cells (data not shown). To validate the up-expression trend of HGF obtained from the cytokine/growth factor antibody array analysis, qRT-PCR was performed to detect the HGF expression in both NFs and CAFs after co-culture with MKN45 cells. As shown in Fig. 2E, HGF was significantly up-regulated in NFs (P < 0.05), while there was a slight increase in CAFs after co-culture with MKN45 cells. To test whether activation of CAFs could be induced by HGF, we treated NFs with exogenous HGF, the results showed that HGF upregulated FAP, a-SMA, and SDF-1 mRNA expression, which collectively is a signature of CAFs (Fig. 2F). Furthermore, the CAFs’ signature was also largely induced in NFs co-cultured with MKN45 cells in a transwell system, as assessed by ELISA; expression of SDF-1 in the culture supernatant from NFs was significantly up-regulated (Fig. 2G). However, induction of SDF-1 was inhibited when a neu-
Fig. 1. Enhanced tumor growth kinetics of MKN45 gastric cancer cells commingled with CAFs. (A) Primary fibroblastic population was isolated from human gastric cancer tissue and adjacent non-cancerous tissue from the same patients, and immunostained by anti-vimentin, anti-cytokeratin, anti-CD31, anti-a-SMA, and anti-FAP antibodies. Scale bar, 20 lm. (B) a-SMA and FAP mRNA levels in both CAFs and NFs were quantified by qRT-PCR. (C) MKN45 cells were injected alone or coinjected with either CAFs or NFs subcutaneously into nude mice (N = 5). Tumor volume was monitored weekly. Data are representative of three independent experiments. P < 0.05.
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Fig. 2. Activation of NFs induced by gastric cancer cells is dependent on the presence of HGF. (A) Photographs of tumors derived from MKN45 cells alone or MKN45 cells coinjected with either CAFs or NFs subcutanously in nude mice. (B) Average weight of tumors of each group (N = 5). (C) Representative images of immunohistochemical staining for a-SMA expression in the tumors generated from MKN45 cells with NFs or CAFs. Magnification, 200. (D) In vitro transwell co-culture system. (E) qRT-PCR detection of HGF mRNA level in both NFs and CAFs after co-culture with MKN45 cells. (F) qRT-PCR detection of FAP, a-SMA, and SDF-1 mRNA levels in NFs treated with HGF for 48 h. (G) Secretion of SDF-1, measured by ELISA, in the culture supernatant from NFs after co-culture with MKN45 cells in the presence of HGF neutralizing antibody or IgG isotype control antibody. Data are representative of three independent experiments. P < 0.05.
tralizing antibody to HGF was added to the transwell system (Fig. 2G), suggesting that activation of CAFs from NFs by gastric cancer cells is mediated by HGF in an autocrine manner. 3.3. HGF was highly expressed in CAFs in the tumor microenvironment of gastric cancer To further identify factors contributing to the tumor-promoting effect of CAF relative to NFs, we also compared the profiles of se-
creted proteins from CAFs and NFs using the cytokine/growth factor antibody arrays. These analyses showed an elevation of HGF expression in CAFs (data not shown). We further quantified expression levels of HGF in four paired CAFs/NFs and in gastric cancer cells. Using qRT-PCR, we found HGF mRNA levels were significantly higher in all the CAFs compared to the levels in the NFs. However, gastric cancer cells MKN45 cells showed a far lower level of HGF mRNA when compared to CAFs (Fig. 3A). In addition, we performed an ELISA assay on the medium conditioned by CAFs, NFs and
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MKN45 cells respectively. As shown in Fig. 3B, there were also higher levels of HGF protein in the medium conditioned by CAFs when compared to NFs, and HGF was almost undetectable in conditioned medium from MKN45 cells. Together, these findings indicate that CAFs produce higher levels of HGF in vivo than do NFs present in non-cancerous stroma, and CAFs is one of the main cellular sources for producing HGF in the tumor microenvironment of gastric cancer. 3.4. HGF neutralizing antibody inhibits tumor promotion by CAFs in vitro To determine whether HGF directly contributes to the tumorpromoting effect of CAFs in gastric cancer, we detected the effect of CAFs on GC cell growth and migration after HGF neutralization. In a WST assay, MKN45 cells co-cultured with CAFs proliferated faster than MKN45 alone. However, after we added 100 ng/ml of HGF neutralizing antibody into the co-culture system, MKN45 cells grew more slowly than the isotype control group (Fig. 4A). To further validate the proliferative effect of HGF from CAFs on GC cells, a colony formation assay was performed. The colony number of the MKN45 cells in the CAF co-culture was significantly reduced by the addition of 100 ng/ml of the anti-HGF antibody (neutralizing HGF group, 65.5 ± 5.5 cells per well; isotype control group, 92.0 ± 6.0 cells per well; P < 0.05) (Fig. 4B). We further assessed the effects of HGF secreted by gastric CAFs on cell migration, a key determinant of malignant progression and metastasis. As shown in Fig. 4C and D, adding neutralizing HGF antibody into the co-culture system led to significantly decreased migration (neutralizing HGF group, 180 ± 15 cells per field; isotype control group, 308 ± 25 cells per field; P < 0.05) of MKN45 cells. Overall, although neutralizing HGF cannot completely inhibit the promoting effects of CAF on gastric cancer cell growth and migration, our results suggest that HGF is the major factor contributing to the tumor-promoting properties of CAFs. 3.5. Blocking HGF impairs tumor growth in nude mice We further tested whether down-regulation of HGF could repress tumor growth mediated by CAFs in vivo. To generate a HGF loss-of-function model, gastric CAFs expressing endogenous HGF were used for silencing experiments. CAFs were transiently transfected with human HGF siRNA (siHGF) or with control scrambled siRNA (siNC). HGF was knocked down to 16.5% of the basal level as confirmed by qRT-PCR (Fig. 5A). ELISA assay also showed a 2.6-fold decrease in HGF secretion by CAFs transfected with HGF siRNA (Fig. 5B). We next examined whether HGF released by CAFs did indeed play an essential role in enhancing tumor growth in vivo.
Coinjection of fibroblasts with MKN45 cells was performed. MKN45 cells mixed with CAFs-siHGF generated tumors of smaller volume (Fig. 5C) and weight (Fig. 5D and E) than those generated by MKN45 cells mixed with CAFs-siNC prior to injection into nude mice. These results indicate that HGF secreted by CAFs promote gastric cancer growth in vivo, consistent with the data obtained from proliferation and migration assays in vitro. 4. Discussion Accumulating evidence has indicated that CAFs, as the activated fibroblasts in cancer stroma, are important modifiers of tumor progression. A better understanding of the molecular mechanism for the activation of stromal fibroblasts and the tumor-promoting prosperities of CAFs in gastric cancer is of obvious importance for understanding tumor growth. In this study, we determined the differential contribution of CAFs and patient-matched NFs to gastric tumorigenesis. We find that CAFs can better promote tumor formation of MKN45 cells in vivo than NFs. Moreover, mixing NFs with gastric cancer cells also results in a greater tumor growth in animals, compared with cancer cells alone, but the growth ability is almost similar to that of the MKN45 cells mixed with CAFs at an advanced stage. This observation suggests that gastric cancer cells can educate their surrounding fibroblasts to acquire stronger tumor-promoting properties to support their own progression. Actually, we find activation of CAFs from NFs can be induced by co-culture with gastric cancer cells in a transwell system, while deprivation of HGF using a neutralizing antibody inhibits the activation of NFs. We further show that HGF secretion from CAFs promotes gastric cancer cell growth. Deprivation of HGF using a neutralizing antibody or knockdown of HGF with siRNA in CAFs significantly reduced CAF-mediated proliferation, colony formation, migration of gastric cancer cells in vitro as well as tumor growth in vivo, indicating that the enhancement of tumor growth and progression by CAFs are mainly exerted through HGF. CAFs are a cell population identifiable by their expression of aSMA and FAP. CAFs originate from at least three main sources, of which activation of resident NFs is the most dominant one [4]. In addition, epithelial and endothelial cells, especially those within the tumor, can also differentiate into CAFs through epithelial or endothelial to mesenchymal transition (EMT or EnMT) [20,21]. Moreover, bone-marrow derived progenitor cells and mesenchymal stem cells can be recruited from distant sites and have been shown to be a source of CAFs [22,23]. It has been previously proposed that NFs can be induced in a reversibly ‘‘primed state’’ by tumor cells, which in turn promote tumorigenesis even though they do not have all the characteristics of the irreversibly modified CAFs [24]. In this report, we have shown that NFs manifest the characteristics of CAFs and gain their tumor-promoting properties when
Fig. 3. High HGF expression in CAFs in the tumor microenvironment of gastric cancer. (A) qRT-PCR detection of HGF mRNA levels in four pairs of fibroblasts and in MKN45 gastric cancer cells. (B) ELISA detection of HGF protein concentration in the medium conditioned by these cells. Data are representative of four independent experiments. P < 0.05.
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Fig. 4. In vitro inhibition of tumor-promoting effect of CAFs by a HGF neutralizing antibody. (A) WST assay of the effect of CAFs on cell growth in the presence of HGF neutralizing antibody or IgG isotype control antibody. (B) Colony formation assay of the effect of CAFs on cell proliferation in the presence of HGF neutralizing antibody or IgG isotype control antibody. The number of colonies was counted on the 14th day after seeding. (C) Transwell migration assay of the effect of CAFs on cell migration in the presence of HGF neutralizing antibody or IgG isotype control antibody. Representative photographs of migratory cells on the membrane (magnification, 100) are shown. (D) Morphometric analysis of migratory cells. Values are represented as mean ± SEM of three independent experiments. P < 0.05.
they are grown together with gastric cancer cells in vivo at the advanced stage. In a transwell system, we demonstrate that co-culture with gastric cancer cells induces the activation signatures in NFs at least partly through HGF in an autocrine manner. The induction of activation signature CAFs is dependent on NF-kB pathway activation with secretion IL-6, IL-8, SDF-1 [25,26]. Among the paracrine factors that act on fibroblasts, members of the transforming growth factor-b (TGF-b) and platelet-derived growth factor (PDGF) families have also been recognized as major regulators of CAFs [27,28]. We here identify HGF as an important inducer of activation of CAFs from adjacent NFs, which has not been reported previously. Our data present functional evidence that gastric cancer cells have the ability to instruct their surrounding fibroblasts to reprogram their expression profiles and to take advantage of the plastic nature of fibroblasts to generate a tumor-enhancing microenvironment. It has been reported that several growth factors secreted by cancer cells themselves, in particular IL-1b, IL-6, prostaglandins, PDGF-b, or FGF, act on neighboring CAFs leading them to secretion of HGF [29–31]. We cannot exclude the possibility that gastric cancer cells secrete these growth factors and induce HGF. However, the molecular mechanisms involved in the induction of HGF secretion by NFs or CAFs after co-culture with gastric cancer cells need further exploration in the future. CAFs are a major component of the tumor environment in many cancers and have been shown to promote cancer cell proliferation, invasion, angiogenesis and tumor growth [4,5,13,32]. The tumor– stroma interactions during tumor progression are likely significant, and therefore paracrine mediators of this interaction in tumor
microenvironment are rational candidates. CAFs are known to secrete multiple growth factors and chemokines such as SDF-1, VEGF, FGF, and CXCL14 into the tumor microenvironment that can promote tumorigenesis [26,33,34]. In the present study, by using cytokine/growth factor antibody array to screen factors that promote gastric cancer growth, we focus on HGF that is significantly up-regulated in CAFs when compared with NFs. HGF has been associated with tumor progression in many types of human cancers [15–18]. Genetic modification of human mammary fibroblasts to express HGF before xenograft with clinically normal mammary epithelial cells results in outgrowth of malignant lesions [35]. It has been demonstrated that HGF specifically binds to its receptor c-Met, activating a complex set of intracellular pathways, and the HGF/ Met signaling pathway has been found to be activated in numerous human tumor types [36], including gastric cancer where elevated HGF serum levels correlate with disease severity [37]. Although we cannot preclude the likely involvement of other growth factors and/or cytokines, the studies of neutralizing HGF or knockdown of HGF expression by siRNA reveal that HGF is an important mediator in tumor-promoting effects of gastric CAFs. Therefore, the crosstalk between gastric cancer cells and their surrounding fibroblasts result in a high level of HGF in the microenvironment of gastric cancer, which partly induces many more CAFs to support the gastric cancer progression through HGF signaling in a paracrine manner. In summary, we found that the crosstalk between gastric cancer cells and their stromal cells-CAFs contributes to tumor growth through HGF signaling, and neutralization of HGF inhibits activation of CAFs from NFs and the tumor-promoting properties of
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Fig. 5. Blocking HGF inhibits the subcutaneous tumor growth in nude mice. (A) qRT-PCR detection of HGF mRNA levels in CAFs treated with siRNA. (B) ELISA detection of HGF protein concentration in the medium conditioned by CAFs. (C) Tumor in nude mice (N = 5) from MKN45 cells that were injected alone or coinjected with CAFs transfected with HGF siRNA (siHGF) or with scrambled control siRNA (siNC). Tumor volume was monitored weekly. (D) Photographs of tumors in nude mice (N = 5) from MKN45 cells or MKN45 cells coinjected with CAFs that were transfected with HGF siRNA (siHGF) or with control siRNA (siNC). (E) Average weight of tumors from nude mice (N = 5) from MKN45 cells or MKN45 cells coinjected with CAFs that were transfected with HGF siRNA (siHGF) or with control siRNA (siNC). Data are representative of three independent experiments. P < 0.05.
CAF. Thus, targeting this pathway to prevent the stromal support of tumor progression could be a complementary approach to conventional treatment strategies for gastric cancer.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.canlet.2013. 02.002.
Acknowledgements We would like to thank Fred Bogott, M.D., Ph.D., at Austin Medical Center, Austin of, Minnesota, USA, and Dezhong Liao, M.D., Ph.D., at University of Minnesota, for their excellent English editing of this manuscript. This study was supported by grants from National Natural Science Foundation of China (Nos. 30900670, 81272749, 81072012, 81172324 and 91229106), Science and Technology Commission of Shanghai Municipality (Nos. 11jc1407602, 10140904200, 10jc1411100, 09DZ1950100 and 09DZ2260200), Shanghai Key Discipline (No. S30204), Key Projects in the National Science & Technology Pillar Program of China (Nos. 2008BA152B03 and 2011BA203191), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Hepatocyte growth factor activates tumor stromal fibroblasts to promote tumorigenesis in gastric cancer Xiongyan Wu a,1, Xuehua Chen a,1, Quan Zhou a, Pu Li a, Beiqin Yu a, Jianfang Li a, Ying Qu a, Jun Yan b, Yingyan Yu a, Min Yan a, Zhenggang Zhu a, Bingya Liu a,⇑, Liping Su a,⇑ a
Shanghai Key Laboratory of Gastric Neoplasms, Department of Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, PR China James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA
b
a r t i c l e
i n f o
Article history: Received 29 November 2012 Received in revised form 1 February 2013 Accepted 2 February 2013
Keywords: HGF Cancer-associated fibroblasts Gastric cancer
a b s t r a c t Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in tumor stroma, are important modifiers of tumor progression. However, the mechanisms underlying stromal fibroblast activation and their promotion of tumor growth remain largely unknown in gastric cancer. Here, we show that normal fibroblasts (NFs) from non-cancerous regions of gastric cancer exhibit the traits of CAFs when grown together with gastric cancer cells in vivo. Activation of NFs can be induced by co-culture with gastric cancer cells, while deprivation of hepatocyte growth factor (HGF) using a neutralizing antibody inhibits the activation of NFs. Moreover, we identify HGF as an important factor from CAFs that acts in a paracrine manner to promote tumorigenesis in vitro and in vivo. Taken together, these results suggest that HGF may play a pivotal role in the regulatory circuit between gastric cancer cells and stromal fibroblasts, and neutralization of HGF inhibits both activation and tumor-promoting properties of CAFs. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Gastric cancer is one of the most common malignancies and is a leading cause of cancer death worldwide, especially in Eastern Asia, with an estimated 988,000 new cases and 736,000 deaths occurring in 2008 [1,2]. Surgical resection, the only curative therapy for gastric cancer, is possible in less than 25% of the patients at time of diagnosis, and the overall prognosis of non-resected gastric cancer is dismal [3]. Therefore, achieving a better understanding of molecular aberrations of gastric cancer carcinogenesis may lead to identification of novel diagnostic and therapeutic targets for this disease. Accumulating evidence has indicated that interactions between tumor and stromal cells create a unique microenvironment that is essential for tumor growth, invasion, and metastasis [4,5]. Cancerassociated fibroblasts (CAFs) are the major constituent of the tumor stroma and are distinguishable from their normal counterparts in their increased rate of proliferation and in the activated myofibroblastic phenotype, which includes a prominent contractile ability and expression of a-smooth muscle actin (a-SMA) [4,6]. Other markers found to be expressed by CAFs are platelet derived growth factor receptor-b (PDGFRb), fibroblast specific ⇑ Corresponding authors. Tel.: +86 21 64674654. 1
E-mail addresses: [email protected] (B. Liu), [email protected] (L. Su). These authors contributed equally.
0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.02.002
protein-1 (FSP-1), vimentin, and fibroblast activation protein (FAP) [7,8]. The contribution of CAFs to tumor development and progression has been reported in multiple cancer models, and the prevalence of CAFs in tumor bearing desmoplastic stroma is recognized to have a prognostic value for many solid tumors [9– 11]. Recent studies have shown that CAFs also frequently accumulate in gastric cancer tissues, and that the prevalence of CAFs correlates with the size, depth and metastasis of the tumor [12]. CAFs actively communicate with cancer cells, and contribute to tumor progression, via a direct cell–cell interaction and by modification of extracellular matrix components, or through soluble factors such as growth factors and chemokines [5,13,14]. Among the many factors that can contribute to such a phenotype modulation of cancer cells, hepatocyte growth factor (HGF), a stromal-derived functional growth factor, has been associated with metastases in many types of human cancers [15–18]. HGF binds to the receptor tyrosine kinase Met that is expressed in the epithelium, which contributes to oncogenesis and tumor progression [19]. Although it has been shown that CAFs are mainly converted from the local-residing fibroblasts, the mechanisms underlying stromal fibroblast activation and their promotion of tumor growth remain largely unknown in gastric cancer. To address this question, we examined the epithelial-stromal interaction in gastric cancer xenograft model using CAFs and adjacent normal fibroblasts (NFs). Here, we find that NFs exhibit the traits of CAFs when they are grown together with gastric cancer cells in vivo at an advanced
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stage, and identify HGF as an important inducer of activated fibroblasts from adjacent NFs that are in a quiescent status in gastric cancer. Moreover, we show that the expression of HGF is elevated in all CAFs, acting in a paracrine manner to promote gastric cancer tumorigenesis in vitro and in vivo. These data collectively indicate that HGF may play a pivotal role in the regulatory circuit between gastric cancer cells and stromal fibroblasts, and blocking this crosstalk by interrupting HGF signaling may provide an alternative treatment strategy for gastric cancer.
2.7. Cell migration assay
2. Materials and methods
2.8. RNA interference
2.1. Cell lines
Human HGF siRNA and scrambled siRNA as the control (Santa Cruz, CA, USA) at the final concentration of 100 nmol/L were transfected into CAFs cells with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Cells were collected for further assay at 24 h, 48 h and 72 h after transfection.
Cell migration assays were performed by using 8 lm transwell chambers (Corning Life Science, Acton, MA, USA). Cells were incubated in serum-free medium for 24 h, and then 2 104 MKN-45 cells in 200 ll serum-free medium were added to the upper chamber, RPMI 1640 medium containing 10% FBS were added to the lower chamber. Non-migrating cells from the interior of the inserts were removed with cotton-tipped swabs 48 h later, and cells that migrated to the bottom of the membranes were stained with 0.1% crystal violet for 20 min. The stained cells were counted and photographed with 100 magnification. At least three randomly selected fields were counted and the average number was presented.
Gastric cancer cell line MKN45 was obtained from the Japanese Cancer Research Resources Bank and was cultured at 37 °C in a humidified atmosphere of 5% CO2 with RPMI-1640 medium containing 10% fetal calf serum with 100 U/ml penicillin and 100 lg/ml streptomycin. Fibroblasts were isolated from 4 independent gastric cancer patients during radical gastric resection at the Department of Surgery, Ruijin hospital, School of Medicine, Shanghai Jiaotong University. Tumor tissues and adjacent non-tumor tissues located at least over 6 cm apart from the margin of the resection were collected and minced into organoids of approximately 1 mm3 and seeded onto 15 cm petri dishes in above-described medium. These conditions produced a homogenous fibroblastic cell population after 7 days of culture. Each fibroblast culture was then expanded into two 15 cm petri dishes and stored as cells passaged for 2–3 population doublings. We used fibroblasts passaged for up to 10 population doublings for subsequent experiments, in order to minimize clonal selection and culture stress, which could occur during extended culture.
Male BALB/c nu/nu nude mice at the age of 4–5 weeks (Institute of Zoology Chinese Academy of Sciences), were housed at a specific pathogen-free environment in the Animal Laboratory Unit, Shanghai Jiao Tong University School of Medicine, China. Fibroblasts and MKN-45 cells were mixed at the ratio of 1:4 within 0.1 ml PBS and injected subcutaneously. Five mice per group were used in all experiments. Tumor growth was monitored weekly and tumor volume was assessed by measuring tumor size with digital calipers using the following formula: volume = ab2p/6. All animal studies were conducted with the approval of the Committee on Animal Care in Shanghai Jiao Tong University School of Medicine.
2.2. Immunofluorescence
2.10. Immunohistochemistry (IHC)
Cells were fixed for 30 min with 1% paraformaldehyde and rinsed with phosphate buffered saline (PBS). Cells were then blocked with normal nonimmune goat serum for 30 min prior to being incubated with anti-vimentin, anti-CD31, anti-asmooth-muscle actin, anti-pan-cytokeratin or anti-FAP (Abcam, Cambridge, USA) at 37 °C for 2 h. After 5 rinses with PBS, cells were stained with appropriate Alexa dye-conjugated secondary immune reagents, Alexa dye-conjugated phalloidin, and Hoechst 33342 (Invitrogen, Carlsbad, CA, USA). Negative control staining was performed by omission of the primary antibody.
Tissues were fixed in 10% neutralized formalin and embedded in paraffin blocks. Sections (4 lm) were dewaxed with xylene and rehydrated with gradient ethanol. Antigen retrieval was performed by boiling in 10 mmol/l of citrate buffer (pH 6.0) for 10 min. Endogenous peroxidase was blocked by incubation in 0.3% H2O2 in methanol for 30 min. Nonspecific binding was then blocked with 2% bovine serum albumin in PBS for 30 min. The sections were incubated with rabbit anti-a-SMA antibody (Abcam, Cambridge, MA, USA) overnight at 4 °C. The immune complex was visualized with the Dako REAL™EnVision™ Detection System, Perox-idase/ DAB, Rabbit/Mouse (Dako, Denmark), according to the manufacturers’ manual. The nuclei were counterstained with hematoxylin.
2.3. Quantitative real-time PCR (QRT-PCR) Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s manual. RNA (1 lg) was reverse transcribed into cDNA using Reverse Transcription system (Promega, Madison, WI, USA). QRT-PCR was performed to quantify the mRNA levels of HGF, a-SMA, FAP, and SDF-1 with the SYBR Green PCR core Reagent kit (Applied Biosystems, Foster City, CA, USA). HPRT1 was used as the endogenous reference. Data were analyzed by using the comparative Ct method. Specificity of resulting PCR products was confirmed by melting curves. The primers used in this assay were: HGF: 50 -TACAGGGGCACTGT CAATACC-30 (upper) and 50 -GGATACTGAGAATCCCAACGC-30 (lower); a-SMA: 50 -CAGGGCTGTTTTCCCATCCAT-30 (upper), 50 -GCCATGTTCTATCGGGTACTT C-30 (lower); FAP: 50 -AATGAGAGCACTCACACTGAAG -30 (upper), 50 -CCGATCA GGTGATAAGCCGTAAT-30 (lower); SDF-1: 50 -GTTTGTGCTGTGGTGTGTC C-30 (upper), 50 -ATACTAAGGTTGGGGGAGGTG-30 (lower); HPRT1: 50 -GACCAG TA ACAGGGGACAT-30 , 50 -GTCCTTTTCACCAGCAAGCT-30 (lower). 2.4. Enzyme-linked immunosorbent assay (ELISA) The protein levels of HGF and SDF-1 in supernatants were measured by an ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. 2.5. Cell proliferation assay Cell proliferation was assessed by water-soluble tetrazolium salt (WST) assay using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Briefly, 1 103 cells were seeded into 96-well plates in triplicate and incubated for 4 days. CCK-8 solution (20 ll) was added into plates, and absorbance at 450 nm was measured after 4 h incubation. 2.6. Soft agar colony formation assay Cells were resuspended with 0.3% soft agar in RPMI 1640 containing 10% FBS and layered onto 0.6% solidified agar in RPMI 1640 containing 10% FBS in 6-well plates (1 103 cells/well) at 24 h post-transfection. The plates were incubated for 2 weeks. Colonies containing at least 50 cells were counted.
2.9. Tumor xenograft model and tumorigenicity assay
2.11. Statistical analysis Results were summarized as means ± SEM. Student t test and one-way analysis of variance (ANOVA) was used to analyze the data and the significance level was set at P < 0.05.
3. Results 3.1. CAFs are more competent in enhancing tumor growth than NFs at the early stage of tumor growth To illustrate the role of CAFs in promoting tumor growth of gastric cancer, we isolated CAFs from human gastric cancer tissue and NFs from adjacent non-cancerous tissue of the same patients. We then verified these fibroblasts by immunostaining. As shown in Fig. 1A, both gastric CAFs and NFs strongly expressed the fibroblastic marker vimentin but negative for cytokeratin and CD31. Moreover, gastric CAFs expressed more a-smooth muscle actin (a-SMA), a marker of myofibroblasts, and more fibroblast activation protein (FAP) when compared with patient-matched NFs (Fig. 1A and B). In addition, we verified the purity of various fibroblast populations by flow cytometry, which indicated that no more than 0.1% of the cells in each fibroblast population were positive for CD45, CD11b, CD68 and Gr-1 (data not shown). These primary CAFs and NFs could be maintained in culture for up to 20 population doublings. Thus, we used these CAFs and NFs for subsequent experiments. To assess the contribution of both fibroblast populations to tumor growth in vivo, we mixed CAFs or NFs with human gastric cancer cells MKN45 in a 1:4 ratio and inoculated them subcutaneously
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into immunodeficient nude mice. Interestingly, we found that MKN45 cells mixed with CAFs generated tumors with a greater volume (Fig. 1C) at the initial stage than did MKN45 cells mixed with NFs, 6.5-fold larger after 7 days (0.07 ± 0.01 cm3 vs 0.01 ± 0.01 cm3), 2.0-fold larger after 14 days (0.19 ± 0.07 cm3 vs 0.15 ± 0.02 cm3) (Fig. 1C, Table 1). However, at an advanced stage MKN45 cells mixed with CAFs did not have such a large differential size compared with those mixed with NFs, only 1.7-fold larger after 21 days (0.75 ± 0.09 cm3 vs 0.44 ± 0.06 cm3) and 1.3-fold larger after 28 days (1.47 ± 0.21 cm3 vs 1.10 ± 0.15 cm3), respectively (Fig. 1C, Table 1). These observations indicated that CAFs have an increased ability to foster tumor formation when compared to NFs, especially at the initial stages of tumor growth. 3.2. Activation of NFs induced by gastric cancer cells is dependent on the presence of HGF Intriguingly, mixing NFs with gastric cancer cells resulted in tumor growth that, although slower than that of tumors mixed with CAFs, was significantly greater than that of tumor cells alone (Fig. 1C, Table 1). Meanwhile, tumor growth from MKN45 cells coinjected with NFs was almost identical to that from MKN45 cells coinjected with CAFs at 28 days (Fig. 2A and B). These results indicated that gastric cancer cells can educate their surrounding fibroblasts to acquire a stronger tumor-promoting ability to support their own progression. To verify that NFs, when grown together with gastric cancer cells in vivo, were educated into CAFs, we detected a-SMA expression, the characteristic signature of CAFs, by IHC in the tumors generated from MKN45 cells with NFs or CAFs, as shown in Fig. 2C. When NFs were coinjected with MKN45 cells,
Table 1 The tumor volume with each group at days 7, 14, 21 and 28.
MKN45 group MKN45 + NFs group MKN45 + CAFs group
7 days
14 days
21 days
28 days
0.00 ± 0.00 0.01 ± 0.01 0.07 ± 0.01
0.01 ± 0.01 0.15 ± 0.02 0.29 ± 0.07
0.05 ± 0.05 0.44 ± 0.06 0.75 ± 0.09
0.37 ± 0.10 1.10 ± 0.15 1.47 ± 0.21
Note: Unit of the tumor volume: cm3.
fibroblasts in the resultant tumor expressed similar a-SMA to that in the tumor generated from MKN45 cells with CAFs. To define the molecule responsible for the activation of CAFs from NFs induced by gastric cancer cells, we established an in vitro transwell co-culture system (Fig. 2D). Using a cytokine/ growth factor antibody array, we found that HGF was up-regulated in both NFs and CAFs after co-culture with gastric cancer cells (data not shown). To validate the up-expression trend of HGF obtained from the cytokine/growth factor antibody array analysis, qRT-PCR was performed to detect the HGF expression in both NFs and CAFs after co-culture with MKN45 cells. As shown in Fig. 2E, HGF was significantly up-regulated in NFs (P < 0.05), while there was a slight increase in CAFs after co-culture with MKN45 cells. To test whether activation of CAFs could be induced by HGF, we treated NFs with exogenous HGF, the results showed that HGF upregulated FAP, a-SMA, and SDF-1 mRNA expression, which collectively is a signature of CAFs (Fig. 2F). Furthermore, the CAFs’ signature was also largely induced in NFs co-cultured with MKN45 cells in a transwell system, as assessed by ELISA; expression of SDF-1 in the culture supernatant from NFs was significantly up-regulated (Fig. 2G). However, induction of SDF-1 was inhibited when a neu-
Fig. 1. Enhanced tumor growth kinetics of MKN45 gastric cancer cells commingled with CAFs. (A) Primary fibroblastic population was isolated from human gastric cancer tissue and adjacent non-cancerous tissue from the same patients, and immunostained by anti-vimentin, anti-cytokeratin, anti-CD31, anti-a-SMA, and anti-FAP antibodies. Scale bar, 20 lm. (B) a-SMA and FAP mRNA levels in both CAFs and NFs were quantified by qRT-PCR. (C) MKN45 cells were injected alone or coinjected with either CAFs or NFs subcutaneously into nude mice (N = 5). Tumor volume was monitored weekly. Data are representative of three independent experiments. P < 0.05.
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Fig. 2. Activation of NFs induced by gastric cancer cells is dependent on the presence of HGF. (A) Photographs of tumors derived from MKN45 cells alone or MKN45 cells coinjected with either CAFs or NFs subcutanously in nude mice. (B) Average weight of tumors of each group (N = 5). (C) Representative images of immunohistochemical staining for a-SMA expression in the tumors generated from MKN45 cells with NFs or CAFs. Magnification, 200. (D) In vitro transwell co-culture system. (E) qRT-PCR detection of HGF mRNA level in both NFs and CAFs after co-culture with MKN45 cells. (F) qRT-PCR detection of FAP, a-SMA, and SDF-1 mRNA levels in NFs treated with HGF for 48 h. (G) Secretion of SDF-1, measured by ELISA, in the culture supernatant from NFs after co-culture with MKN45 cells in the presence of HGF neutralizing antibody or IgG isotype control antibody. Data are representative of three independent experiments. P < 0.05.
tralizing antibody to HGF was added to the transwell system (Fig. 2G), suggesting that activation of CAFs from NFs by gastric cancer cells is mediated by HGF in an autocrine manner. 3.3. HGF was highly expressed in CAFs in the tumor microenvironment of gastric cancer To further identify factors contributing to the tumor-promoting effect of CAF relative to NFs, we also compared the profiles of se-
creted proteins from CAFs and NFs using the cytokine/growth factor antibody arrays. These analyses showed an elevation of HGF expression in CAFs (data not shown). We further quantified expression levels of HGF in four paired CAFs/NFs and in gastric cancer cells. Using qRT-PCR, we found HGF mRNA levels were significantly higher in all the CAFs compared to the levels in the NFs. However, gastric cancer cells MKN45 cells showed a far lower level of HGF mRNA when compared to CAFs (Fig. 3A). In addition, we performed an ELISA assay on the medium conditioned by CAFs, NFs and
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MKN45 cells respectively. As shown in Fig. 3B, there were also higher levels of HGF protein in the medium conditioned by CAFs when compared to NFs, and HGF was almost undetectable in conditioned medium from MKN45 cells. Together, these findings indicate that CAFs produce higher levels of HGF in vivo than do NFs present in non-cancerous stroma, and CAFs is one of the main cellular sources for producing HGF in the tumor microenvironment of gastric cancer. 3.4. HGF neutralizing antibody inhibits tumor promotion by CAFs in vitro To determine whether HGF directly contributes to the tumorpromoting effect of CAFs in gastric cancer, we detected the effect of CAFs on GC cell growth and migration after HGF neutralization. In a WST assay, MKN45 cells co-cultured with CAFs proliferated faster than MKN45 alone. However, after we added 100 ng/ml of HGF neutralizing antibody into the co-culture system, MKN45 cells grew more slowly than the isotype control group (Fig. 4A). To further validate the proliferative effect of HGF from CAFs on GC cells, a colony formation assay was performed. The colony number of the MKN45 cells in the CAF co-culture was significantly reduced by the addition of 100 ng/ml of the anti-HGF antibody (neutralizing HGF group, 65.5 ± 5.5 cells per well; isotype control group, 92.0 ± 6.0 cells per well; P < 0.05) (Fig. 4B). We further assessed the effects of HGF secreted by gastric CAFs on cell migration, a key determinant of malignant progression and metastasis. As shown in Fig. 4C and D, adding neutralizing HGF antibody into the co-culture system led to significantly decreased migration (neutralizing HGF group, 180 ± 15 cells per field; isotype control group, 308 ± 25 cells per field; P < 0.05) of MKN45 cells. Overall, although neutralizing HGF cannot completely inhibit the promoting effects of CAF on gastric cancer cell growth and migration, our results suggest that HGF is the major factor contributing to the tumor-promoting properties of CAFs. 3.5. Blocking HGF impairs tumor growth in nude mice We further tested whether down-regulation of HGF could repress tumor growth mediated by CAFs in vivo. To generate a HGF loss-of-function model, gastric CAFs expressing endogenous HGF were used for silencing experiments. CAFs were transiently transfected with human HGF siRNA (siHGF) or with control scrambled siRNA (siNC). HGF was knocked down to 16.5% of the basal level as confirmed by qRT-PCR (Fig. 5A). ELISA assay also showed a 2.6-fold decrease in HGF secretion by CAFs transfected with HGF siRNA (Fig. 5B). We next examined whether HGF released by CAFs did indeed play an essential role in enhancing tumor growth in vivo.
Coinjection of fibroblasts with MKN45 cells was performed. MKN45 cells mixed with CAFs-siHGF generated tumors of smaller volume (Fig. 5C) and weight (Fig. 5D and E) than those generated by MKN45 cells mixed with CAFs-siNC prior to injection into nude mice. These results indicate that HGF secreted by CAFs promote gastric cancer growth in vivo, consistent with the data obtained from proliferation and migration assays in vitro. 4. Discussion Accumulating evidence has indicated that CAFs, as the activated fibroblasts in cancer stroma, are important modifiers of tumor progression. A better understanding of the molecular mechanism for the activation of stromal fibroblasts and the tumor-promoting prosperities of CAFs in gastric cancer is of obvious importance for understanding tumor growth. In this study, we determined the differential contribution of CAFs and patient-matched NFs to gastric tumorigenesis. We find that CAFs can better promote tumor formation of MKN45 cells in vivo than NFs. Moreover, mixing NFs with gastric cancer cells also results in a greater tumor growth in animals, compared with cancer cells alone, but the growth ability is almost similar to that of the MKN45 cells mixed with CAFs at an advanced stage. This observation suggests that gastric cancer cells can educate their surrounding fibroblasts to acquire stronger tumor-promoting properties to support their own progression. Actually, we find activation of CAFs from NFs can be induced by co-culture with gastric cancer cells in a transwell system, while deprivation of HGF using a neutralizing antibody inhibits the activation of NFs. We further show that HGF secretion from CAFs promotes gastric cancer cell growth. Deprivation of HGF using a neutralizing antibody or knockdown of HGF with siRNA in CAFs significantly reduced CAF-mediated proliferation, colony formation, migration of gastric cancer cells in vitro as well as tumor growth in vivo, indicating that the enhancement of tumor growth and progression by CAFs are mainly exerted through HGF. CAFs are a cell population identifiable by their expression of aSMA and FAP. CAFs originate from at least three main sources, of which activation of resident NFs is the most dominant one [4]. In addition, epithelial and endothelial cells, especially those within the tumor, can also differentiate into CAFs through epithelial or endothelial to mesenchymal transition (EMT or EnMT) [20,21]. Moreover, bone-marrow derived progenitor cells and mesenchymal stem cells can be recruited from distant sites and have been shown to be a source of CAFs [22,23]. It has been previously proposed that NFs can be induced in a reversibly ‘‘primed state’’ by tumor cells, which in turn promote tumorigenesis even though they do not have all the characteristics of the irreversibly modified CAFs [24]. In this report, we have shown that NFs manifest the characteristics of CAFs and gain their tumor-promoting properties when
Fig. 3. High HGF expression in CAFs in the tumor microenvironment of gastric cancer. (A) qRT-PCR detection of HGF mRNA levels in four pairs of fibroblasts and in MKN45 gastric cancer cells. (B) ELISA detection of HGF protein concentration in the medium conditioned by these cells. Data are representative of four independent experiments. P < 0.05.
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Fig. 4. In vitro inhibition of tumor-promoting effect of CAFs by a HGF neutralizing antibody. (A) WST assay of the effect of CAFs on cell growth in the presence of HGF neutralizing antibody or IgG isotype control antibody. (B) Colony formation assay of the effect of CAFs on cell proliferation in the presence of HGF neutralizing antibody or IgG isotype control antibody. The number of colonies was counted on the 14th day after seeding. (C) Transwell migration assay of the effect of CAFs on cell migration in the presence of HGF neutralizing antibody or IgG isotype control antibody. Representative photographs of migratory cells on the membrane (magnification, 100) are shown. (D) Morphometric analysis of migratory cells. Values are represented as mean ± SEM of three independent experiments. P < 0.05.
they are grown together with gastric cancer cells in vivo at the advanced stage. In a transwell system, we demonstrate that co-culture with gastric cancer cells induces the activation signatures in NFs at least partly through HGF in an autocrine manner. The induction of activation signature CAFs is dependent on NF-kB pathway activation with secretion IL-6, IL-8, SDF-1 [25,26]. Among the paracrine factors that act on fibroblasts, members of the transforming growth factor-b (TGF-b) and platelet-derived growth factor (PDGF) families have also been recognized as major regulators of CAFs [27,28]. We here identify HGF as an important inducer of activation of CAFs from adjacent NFs, which has not been reported previously. Our data present functional evidence that gastric cancer cells have the ability to instruct their surrounding fibroblasts to reprogram their expression profiles and to take advantage of the plastic nature of fibroblasts to generate a tumor-enhancing microenvironment. It has been reported that several growth factors secreted by cancer cells themselves, in particular IL-1b, IL-6, prostaglandins, PDGF-b, or FGF, act on neighboring CAFs leading them to secretion of HGF [29–31]. We cannot exclude the possibility that gastric cancer cells secrete these growth factors and induce HGF. However, the molecular mechanisms involved in the induction of HGF secretion by NFs or CAFs after co-culture with gastric cancer cells need further exploration in the future. CAFs are a major component of the tumor environment in many cancers and have been shown to promote cancer cell proliferation, invasion, angiogenesis and tumor growth [4,5,13,32]. The tumor– stroma interactions during tumor progression are likely significant, and therefore paracrine mediators of this interaction in tumor
microenvironment are rational candidates. CAFs are known to secrete multiple growth factors and chemokines such as SDF-1, VEGF, FGF, and CXCL14 into the tumor microenvironment that can promote tumorigenesis [26,33,34]. In the present study, by using cytokine/growth factor antibody array to screen factors that promote gastric cancer growth, we focus on HGF that is significantly up-regulated in CAFs when compared with NFs. HGF has been associated with tumor progression in many types of human cancers [15–18]. Genetic modification of human mammary fibroblasts to express HGF before xenograft with clinically normal mammary epithelial cells results in outgrowth of malignant lesions [35]. It has been demonstrated that HGF specifically binds to its receptor c-Met, activating a complex set of intracellular pathways, and the HGF/ Met signaling pathway has been found to be activated in numerous human tumor types [36], including gastric cancer where elevated HGF serum levels correlate with disease severity [37]. Although we cannot preclude the likely involvement of other growth factors and/or cytokines, the studies of neutralizing HGF or knockdown of HGF expression by siRNA reveal that HGF is an important mediator in tumor-promoting effects of gastric CAFs. Therefore, the crosstalk between gastric cancer cells and their surrounding fibroblasts result in a high level of HGF in the microenvironment of gastric cancer, which partly induces many more CAFs to support the gastric cancer progression through HGF signaling in a paracrine manner. In summary, we found that the crosstalk between gastric cancer cells and their stromal cells-CAFs contributes to tumor growth through HGF signaling, and neutralization of HGF inhibits activation of CAFs from NFs and the tumor-promoting properties of
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Fig. 5. Blocking HGF inhibits the subcutaneous tumor growth in nude mice. (A) qRT-PCR detection of HGF mRNA levels in CAFs treated with siRNA. (B) ELISA detection of HGF protein concentration in the medium conditioned by CAFs. (C) Tumor in nude mice (N = 5) from MKN45 cells that were injected alone or coinjected with CAFs transfected with HGF siRNA (siHGF) or with scrambled control siRNA (siNC). Tumor volume was monitored weekly. (D) Photographs of tumors in nude mice (N = 5) from MKN45 cells or MKN45 cells coinjected with CAFs that were transfected with HGF siRNA (siHGF) or with control siRNA (siNC). (E) Average weight of tumors from nude mice (N = 5) from MKN45 cells or MKN45 cells coinjected with CAFs that were transfected with HGF siRNA (siHGF) or with control siRNA (siNC). Data are representative of three independent experiments. P < 0.05.
CAF. Thus, targeting this pathway to prevent the stromal support of tumor progression could be a complementary approach to conventional treatment strategies for gastric cancer.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.canlet.2013. 02.002.
Acknowledgements We would like to thank Fred Bogott, M.D., Ph.D., at Austin Medical Center, Austin of, Minnesota, USA, and Dezhong Liao, M.D., Ph.D., at University of Minnesota, for their excellent English editing of this manuscript. This study was supported by grants from National Natural Science Foundation of China (Nos. 30900670, 81272749, 81072012, 81172324 and 91229106), Science and Technology Commission of Shanghai Municipality (Nos. 11jc1407602, 10140904200, 10jc1411100, 09DZ1950100 and 09DZ2260200), Shanghai Key Discipline (No. S30204), Key Projects in the National Science & Technology Pillar Program of China (Nos. 2008BA152B03 and 2011BA203191), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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