Tumor-associated fibroblasts expression in esophageal squamous cell carcinoma and may descend from mesenchymal stem cells

Tumor-associated fibroblasts (TAFs) play a pivotal role in cancer proliferation, invasion and metastasis. The expression of TAFs in esophageal squamous cell carcinoma (ESCC) tissues remain unclear. We investigated the relevance of TAFs in ESCC, and then culture mesenchymal stem cells (MSC) by ESCC microenvironment induced their conversion into TAFs to assess whether MSC are precursors of TAFs. We detected the expression of TAFs surface markers, including α-SMA, and S100A4 in esophageal squamous cell cancer tissues by using immunohistochemistry, and evaluated associations with clinicopathological features. In order to verify whether MSC is one of the TAFs sources, we used culture supernatant from ESCC tumor cells to culture and induce MSC to transform into TAFs. TAFs surface markers, α-SMA and S100A4, were expressed in ESCC tissues, and their expressions correlated with tumor differentiation and clinical TNM stage. Real-time-PCR analysis and Western-Blot analysis confirmed MSC conversion into TAFs. Our study demonstrated that TAFs existence in esophageal carcinoma, and their expression was also closely related to metastasis and poor prognosis. Furthermore, we confirmed the conversion from MSC to TAFs and surmised MSC may be the precursors of TAFs. Total RNAs was isolated from supernatant of cancer cells using the tri-reagent TRIzol®, according to the manufacturer’s protocol, followed by the determination of its concentration and treatment with RNase-free DNase I for complete removal of genomic DNA. Reverse transcription of total RNAs was carried out using the M-MLV reverse transcriptase according to the manufacturer’s protocol. Later, the reaction mixture was incubated at 42°C for 60 min and 70°C for 10 min. After completion, cDNA was stored at – 20°C. The cDNA was then subjected to real-time quantitative PCR for the evaluation of the relative mRNA levels of β-actin, α-SMA and S100A4 with the corresponding primer pairs: β-actin The amplification conditions were 95°C (5 min) and 45 cycles of 95°C (30 sec), 60°C (30 sec) and 72°C


Introduction
Esophageal squamous cell carcinoma (ESCC) is an aggressive type of epithelial cancer characterized by scarce overall survival [1]. In Asian countries, ESCC accounts for over 90% of esophageal cancers [2] [3]. Apart from the nature inherent to the cancer cells, this poor outcome is also impacted by stromal cells, such as mesenchymal stem cells (MSC) and tumor-associated fibroblasts (TAFs), associated with cancer initiation and progression.
TAFs in cancer stroma, showing characteristics of myofibroblasts with a modified phenotype, play a "pivotal" role in cancer proliferation, invasion and metastasis [4] [5].
Spindle-shaped TAFs secrete growth factors which in turn alter the extracellular matrix (ECM) to create a tumor niche and enhance tumorigenesis, tumor cell proliferation, migration and thus cancer metastasis. Tumor progression is partly a result of evolving crosstalk between different cell types within the tumor and its surrounding supportive tissue or tumor stroma [6]. Several in vitro studies in ESCC cell lines have reported the dependency of cancer cell proliferation, angiogenesis, and mobility on the presence of activated fibroblasts [7].
Common cell surface markers of TAF include among others, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), Thy-1 and S100A4. The expression levels of these markers are different in various tumor tissues, and α-SMA is usually used to act as an important marker of TAF. [8] Our study assessed the expression of TAFs markers α-SMA and S100A in ESCC tissues using immunohistochemistry (IHC). They aim was to evaluate their relationship with patients clinicopathological features and look into the clinical relevance of TAFs in ESCC.
Furthermore, we simulated tumor microenvironment using ESCC cells culture supernatant (Eca-109 and TE-1 cells), to culture mesenchymal stem cells (MSC) and induce their conversion into TAFs, to observe the behaviour and expression of α-SMA and S100A during the conversion and assess whether MSC are precursors of TAFs. Immunohistochemical staining Esophageal cancer sections were heated at 65°C for 2-3 hours and deparaffinized with xylene. They were then hydrated using 100%, 95%, 85%, and 70% graded alcohol series (five minutes each) and immersed for five minutes in deionized water. After antigen retrieval followed by heat mediated antigen retrieval, the sections were cooled at room temperature and soaked into 0.01M PBS for 3×5 min. Drops of 0.3% hydrogen peroxide were added to cover the whole section, followed by incubation (room temperature for 20 min). The sections were rinsed 3×5 min in 0.01M PBS, blocked by normal goat serum for 10 min, dripped with corresponding primary antibodies (α-SMA at 1:800 and S100A4 at 1:800), and incubated overnight at 4 °C. After 30 min cooling at room temperature, the section were dipped in 0.01M PBS (3 changes of 5 min each), dripped and covered with secondary antibodies and incubated (room temperature for 30 min). Later, they were rinsed with 0.01M PBS (3 changes of 5 min each), dripped with tertiary antibodies, and washed again with 0.01M PBS 3×5 min after incubation (room temperature for 10min).
After soaking the sections in deionized water, the sections were treated with DAB, counterstained with hematoxylin before being dehydrated in graded ethanol (70%, 85%, 95%, 100%) and mounted on neutral resin for microscopic observation and photography.
Qualitative appreciation of the immunohistochemical staining for α-SMA and S100A4 was performed by assigning a score according to the staining intensity, where 3 is strong staining, 2 moderate staining, 1 weak staining and 0 is no staining. Quantitatively, after choosing 12 non-overlapping random visual fields on the stained slide at x400 magnification, the total number of stromal fibroblasts was counted as well as the total number of stromal fibroblasts for every staining intensity. The final immunohistochemistry score was calculated using the formula Western blotting for Protein samples from tissues and cell cultures For total protein extraction, the cells were floated in ice-cold lysing buffer, harvested by scraping and fragmented by ultrasonic waves three times in 4 seconds at 4℃. After a short centrifugation and boiling at 100℃ for 5 min, the extraction mixture was then centrifuged (12000rpm at 4℃ for 3 min), aliquoted and stored at -80°C. The samples were electrophoresed on a SDS-polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane. Membranes were then washed in TBST (25ml) for 10 min and blocked with 5% skimmed milk in 1×TBST. Next, the membranes were cut into strips and incubated in anti-GAPDH overnight at 4℃ on a vibrating platform. After rinsing 3 times in TBST for 5 min each, the strips were incubated at room temperature with goat anti-mouse or anti-rabbit secondary antibodies conjugated with Horseradish Peroxidase (HRP Labelled, 1:5000) for an hour on a vibrating platform, followed by three rinses in 1×TBST for 10 min each. The signal was visualized using an enhanced chemiluminescence solution followed by exposure to X-ray films.

Statistical analyses
All statistical analysis were carried out using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). They included the student's t-test to compare means, the logistic regression for multivariate analysis and the Chi-square test for univariate analysis. Data were expressed as mean±SD and the level of statistical significance was defined for P<0.05.

Expression of α-SMA
α-SMA expressed positively in 32 cases (47.0%), with a high expression recorded in tumor stroma, mainly within the cytoplasm of stromal fibroblasts (Figure 1 A, B). However, α-SMA was not expressed in cancer cells and normal esophageal stromal fibroblasts (Figure 1 C, D).
1.3. Expression of S100A4 S100A4 expressed positively in 29 cases (42.64), with a high expression recorded in tumor stroma, mainly within the cytoplasm of stromal fibroblasts (Figure 2 A, B). S100A4 was expressed scarcely in cancer cells, and was not expressed at all in normal esophageal stromal fibroblasts (Figure 2

Induction of MSC conversion into TAF in cancer cells culture supernatant
Variation of mRNA levels during MSC conversion into TAF Real-time-PCR analysis showed that mRNA expression of α-SMA and S100A4 were increased (figure 4) during MSC conversion into TAFs using supernatant medium of TE-1 and Eca-109 ESCC cell lines. The increase was more remarkable in TE-1 medium.

Variation of proteins levels during MSC conversion into TAF
Western-Blot analysis demonstrated that protein expressions of α-SMA and S100A4 were remarkably increased ( figure 5 A and figure 5 B), and therefore confirmed MSC conversion into TAFs. The increase was also more remarkable in TE-1 medium.

Discussion
Myofibroblasts can be found in the microenvironment of various tumors and extensive fibroblast-activation could be linked to rapid disease progression in malignant disease.
They have an established biological impact on tumorigenesis as matrix synthesizing or matrix degrading cells and play a critical role in cancer aggressiveness and metastasis [8] [9]. α-SMA and S100A4 expressions are recognized not only in the TAFs but also in smooth muscle cells, thus suggesting limitations for interpretation of their stromal positivity [10] [11].
In our study, we detected α-SMA and S100A4 expression in TAFs within the ESCC stroma of all 68 clinical specimens using immunohistochemistry staining, confirming the location of TAFs within the matrix of cancer cells as stated by other authors [12]. Two important findings are worth pointing out. First, α-SMA and S100A4 are mainly expressed within the cytoplasm of stromal fibroblasts, but not expressed in normal esophageal stromal fibroblasts. In addition, S100A4 was expressed scarcely in cancer cells, while α-SMA was not at all. Second, both TAFs markers were expressed in the tumor stroma in form of fibroblast-like cells. The present study therefore demonstrates that α-SMA and S100A4 expressions as surface markers of TAFs could be a useful marker for the biologic behavior of ESCC and prediction of the outcome. Their expression raised was detected by immunohistochemical analysis to various extents. However, the mechanism whereby α-SMA and S100A4 expressions in TAFs influence tumor aggressiveness is still not clear.
The existence of TAFs in esophageal squamous cell carcinoma was closely related to the differentiation level of tumor cells. According to the literature, the TAFs secrete an array of soluble factors including, among others, epidermal growth factor (EGF) and transforming growth factor (TGF) which promote paracrine cancer signaling pathways involved in proliferation, survival, angiogenesis, and metastasis of epithelial tumor [13] [ 14], and this phenomenon is correlated with tumor malignancy according to some published literatures [15] [16]. Similarly in our study, expression levels of α-SMA and S100A4 in ESCC cell matrixes from specimen of ESCC staged III or IV according to the TNM staging system correlated with tumor differentiation and TNM staging in univariate and multivariate statistical analysis, with higher expression linked to a poorly differentiation and poorer outcome, therefore making them important independent prognosticator for ESCC (Table 3-2, 3-3). These findings are in accordance with reports from other researchers. [15,16,17] Several studies show a preponderant role of TAFs during cancer metastasis [18] [19], by means of autocrine secretion of cytokines and enzymes. Lymph nodes metastasis is an important route of esophageal cancer metastasis [20,21]. Results from our experiment showed that expression levels of α-SMA and S100A4 and lymph nodes metastasis status were not significantly related (p>0.5), due probably to the small size of our sample.
Nevertheless, our results suggest that the degree and localization of α-SMA and S100A4 expressions in CAFs may be some of the key element of the cancer stromal microenvironment responsible for promoting cancer invasion and metastasis in ESCC.
More importantly, results from our study also demonstrated that MSCs can be converted  Tables   Tables   Table 1. Association of α-SMA and S100A4 with clinicopathologic features.    mRNA expression of α-SMA and S100A4. Figure 5 (A, B). Protein expression of α-SMA, S100A4 and β-actin.