The clinicopathological significance of Thrombospondin-4 expression in the tumor microenvironment of gastric cancer.

INTRODUCTION: Thrombospondin-4 [1] is an extracellular glycoprotein involved in wound healing and tissue remodeling. Although THBS4 is reportedly frequently expressed in solid tumors, there are few reports of the clinicopathological features of carcinomas with THBS4 expression. We evaluated the clinicopathologic significance of THBS4 expression in gastric carcinoma (GC).
MATERIALS AND METHODS: We retrospectively analyzed the cases of 584 GC patients. The expression of THBS4 in each tumor was evaluated by immunohistochemistry. We then divided the patients into the THBS4-high (n = 223, 38.2%) group and THBS4-low (n = 361, 61.8%) group. THBS4 expression in cancer-associated fibroblasts (CAFs), normal-associated fibroblasts (NFs) and gastric cancer cell lines was examined by western blotting.
RESULTS: THBS4 is expressed on stromal cells with αSMA or Podoplanin expression in the GC microenvironment, but not expressed on cancer cells with cytokeratin expression. The western blot analysis results showed that CAFs (but not NFs and cancer cells) expressed THBS4. Compared to the THBS4-low expression status, the THBS4-high expression status was correlated with higher αSMA expression, higher invasion depth, lymph-node metastasis, lymphatic invasion, peritoneal cytology, peritoneal metastasis, larger tumor size, microscopic diffuse type, and the macroscopic diffuse infiltrating type. The THBS4-high group's 5-year overall survival rate was significantly poorer than that of the THBS4-low group. A multivariate analysis revealed that THBS4 expression was an independent prognostic factor.
CONCLUSION: THBS4 is expressed on CAFs in the gastric cancer microenvironment. THBS4 from CAFs is associated with the metastasis of cancer cells, and is a useful prognostic indicator for gastric cancer patients.

Introduction

Despite recent advances in diagnostic techniques and therapies for gastric cancer (GC), the prognosis of GC remains poor; GC is the fifth most common fatal carcinoma and the third leading morbidity in the world [2, 3]. To improve the prognosis of GC patients, new strategies based on its biological behavior are necessary. Tumor progression has been recognized as the product of evolving crosstalk between the cancer cells and the surrounding tissue or tumor stroma[4]. The elucidation of the characteristic features of tumor stroma could provide a new biological marker in the crosstalk.

Thrombospondin-4 (THBS4) is one of the extracellular secreted glycoproteins involved in wound healing and tissue remodeling by regulating the organization, repair, and remodeling of the extracellular matrix [5-7]. It was recently reported that THBS4 is frequently expressed in the tumor stroma of some types of solid cancers such as breast cancer and prostate cancer [8] [9]. By conducting a transcriptome-wide comparative analysis, Forester et al. [10] observed that THBS4 is a potent marker for diffuse-type gastric cancer, and they detected THBS4 expression in tumor stroma which showed positivity for α-Smooth muscle actin (αSMA) and vimentin.

Although it has been reported that THBS4 is highly expressed in cancer stroma, the significance of THBS4 in tumor stroma in the development of gastric cancer has been controversial. THBS4 was reported to stimulate the development of cancer [8, 10–13], whereas other studies found that THBS4 acts as a tumor suppressor [14, 15]. Few investigations have examined the clinicopathological significance of THBS4, and we conducted the present study to evaluate the clinicopathological significance of THBS4 expression in gastric cancer, especially in tumor stroma.

Materials & methods

Patients and clinical materials

This study was retrospective analysis of 585 gastric cancer patients who received gastrectomy at Osaka City University Hospital. Gastric cancer tissues were obtained from each patient. The pathological diagnoses and classifications were made according to the Japanese classification of gastric carcinoma (14th edition) [15]. This study was approved by the Osaka City University Ethics Committee (approval number 924). Written informed consent for research was obtained from patients.

Immunohistochemical determination of THBS4, Podoplanin, αSMA and Cytokeratin

The immunohistochemical determination of THBS4, Podoplanin, αSMA and cytokeratin were examined as the manufacturer’s instructions. Shortly, slides were deparaffinized and activated by heating. After blocking endogenous peroxidase activity, the samples were incubated with anti-human THBS4 (R&D Systems, Minneapolis, MN: MAB2390; 1:200), anti-Pan-Keratin (Proteintech, Rosemont, IL: 26411-1-AP; 1:3000), anti-Podoplanin (Santa Cruz Biotechnology, Dallas, TX: sc-376695; 1:300) or anti-human αSMA (Leica Biosystems Newcastle, Newcastle, UK: NCL-L-SMA; 1:200). The samples were incubated with biotinylated second antibody. The samples were treated with streptavidin-peroxidase reagent, and counterstaining with Mayer’s hematoxylin. THBS4 expression was evaluated by intensity of staining and percentage of stained stromal cells, respectively: intensity was given scores 0–3 (0 = no, 1 = weak, 2 = moderate, 3 = intense), and the percentage of immunopositive stromal cells in all stroma cells was given scores 0–3 (0 = 0%, 1 = 1%-10%, 2 = 11%-50%, 3 = 51%-100%). The two scores were added to obtain the final result of 0–6. Expressions were considered THBS4-high when scores were 3 or more and THBS4-low when scores were 2 or less. Expressions were considered αSMA-high when scores were 5 or more and αSMA-low when scores were 4 or less. Two double-blinded independent observers who were unaware of clinical data and outcome evaluated, and when a discrepant evaluation between the two independent observers was found, the result was rechecked and discussed.

Gastric fibroblast cell lines and cancer cell lines

Three pairs of cancer associated fibroblast (CAF82, CAF105) and normal tissue fibroblast (NF82, NF105) were established from tumoral gastric wall and non-tumoral gastric wall, respectively. CAF82 and NF82, CAF105 and NF105 were from same patients. These fibroblasts were used from third passage to fifth passage in culture. CAFs and NFs were examined by αSMA staining, as previously reported [16]. MKN45 and NUGC4, which are diffuse and intestinal type gastric cancer cell lines, respectively, were used.

Immunostaining of fibroblasts

Fibroblasts were incubated into Lab-Tek II Chamber Slide System (Nunc, Naperville, IL) for 3 days. After washing with PBS and fixing with methanol for 10 min, fibroblasts were incubated with anti-podoplanin antibody (Santa Cruz Biotechnology, Dallas, TX: sc-376695; 1:300). The samples were incubated with biotinylated second antibody.

Western blot analysis

Cell lysates were collected after different treatments. After the protein concentration of each sample was adjusted, electrophoresis was carried out using 10% Tris/Gly gels (Invitrogen, Inc., Gaithersburg, MD). The protein bands obtained were transferred to an Immobilon-P Transfer membrane (Amersham, Aylesbury, UK). The membrane was kept in PBS-T (10 mM PBS and 0.05% Tween 20) supplemented with 5% bovine albumin (Sigma, St. Louis, MO) at room temperature for 1 h. Then, the membrane was placed in PBS-T solution containing each primary antibody: THBS4 (R&D Systems), β-actin (1:1000; Cell Signaling), and allowed to react at room temperature for 2 h. The levels of specific proteins in each lysate were detected by enhanced chemiluminescence using ECL plus (Amersham) followed by autoradiography.

Statistical analysis

Statistical analysis was performed using R for Mac OS X (version 3. 5. 2). The chi-square test was used to determine the significance of the difference between the covariates. Survival was measured from the date of surgery. Overall survival was calculated using Kaplan-Meier method, and survival curves were compared by log-rank test. The Cox proportional hazards model was used for multivariate analysis. A p-value <0.05 was defined as being statistically significant.

Results

Immunostaining findings of THBS4

Fig 1 provides representative immunostaining patterns of THBS4. THBS4 was stained at the cytoplasm and the cell membrane of stroma cells, but cancer cells with cytokeratin expression did not express THBS4. Whereas, the THBS4 expression of the stromal cells was in agreement with the cells showing alpha-smooth muscle actin (αSMA) or Podoplanin expression. Of the total of 584 cases, 223 (38.2%) were THBS4-high.

Fig 1

Representative picture of THBS4 expression, H&E staining, αSMA expression, Podoplanin expression, and cytokeatin expression in diffuse and intestinal type of gastric cancer.

THBS4 was stained at the cytoplasm and cell membrane of stroma cells. The stromal cells with THBS4 expression were much to the cells showing αSMA or Podoplanin expression. cytokeratin was stained in cancer cells.

The relationship between clinicopathological features and the THBS4 expression of stromal cells

The clinicopathological characteristics of all cases according to their THBS4 expression in stroma are summarized in Table 1. Compared to the THBS4-low expression in stroma, THBS4-high expression in stroma was significantly associated with higher αSMA expression, macroscopic type 4 (p<0.001), greater tumor diameter (p<0.001), diffuse-type histology (p<0.001), microscopic undifferentiated-type, higher tumor invasion (p<0.001), lymph node metastasis (p<0.001), lymphatic invasion (p<0.001), peritoneal cytology (p<0.001), and peritoneal metastasis (p<0.001). Among Borrmann's type 4 cancer of 61 cases, 52 (85.2%) were THBS4-high. The results of subgroup analysis for type-4 or other type gastric cancer were shown in S1 and S2 Tables.

Table 1

Correlation between the expression of THBS4 in tumor stromal cells and clinicopathologic features in 584 patients with gastric carcinoma.

	THBS4
	High(n = 223, 38.2%)	Low(n = 361, 61.8%)	p value
Age (year-old)
< 65	105 (47.1%)	151 (41.8%)
≥ 65	118 (52.9%)	210 (58.2%)	0.247
Gender
Female	97 (43.5%)	162 (44.9%)
Male	126 (56.5%)	199 (55.1%)	0.810
αSMA expression
High	139 (62.3%)	136 (37.7%)
Low	84 (37.7%)	225 (62.3%)	<0.001
Macroscopic type
type 4	52 (23.3%)	9 (2.5%)
Other types	171 (76.7%)	352 (97.5%)	<0.001
Tumor diameter
< 50	103 (46.4%)	250 (69.3%)
≥ 50	120 (53.6%)	111 (30.7%)	<0.001
Microscopic type
Differentiated	81 (36.3%)	205 (56.8%)
Undifferentiated	142 (63.7%)	156 (43.2%)	<0.001
Depth of tumor invasion
T1-2	73 (32.7%)	264 (73.1%)
T3-4	150 (67.3%)	97 (26.9%)	<0.001
Lymph node metastasis
N0	83 (37.2%)	246 (68.1%)
N1-3	140 (62.8%)	115 (31.9%)	<0.001
Lymphatic invasion
Absent	56 (25.1%)	204 (56.5%)
Present	167 (74.9%)	157 (43.5%)	<0.001
Venous invasion
Absent	176 (84.2%)	305 (79.1%)
Present	47 (15.8%)	56 (20.9%)	0.109
Ascites cytology
Negative	174 (78.0%)	346 (95.8%)
Positive	49 (22.0%)	15 (4.2%)	<0.001
Peritoneal metastasis
Absent	200 (89.7%)	349 (96.7%)
Present	23 (10.3%)	12 (3.3%)	0.001
Hepatic metastasis
Negative	216 (96.9%)	354 (98.1%)
Positive	7 (3.1%)	7 (1.9%)	0.409
pStage
I, II	99 (44.4%)	285 (78.9%)
III, IV	124 (55.6%)	76 (21.1%)	<0.001

Survival outcomes

The 5-year overall survival rate of the patients in THBS4-high expression group (38.5%) was significantly poorer compared to that of the THBS4-low group (81.4%) (p<0.001; Fig 2). The results of subgroup analysis for type-4 or other type gastric cancer were shown in S1 Fig. Our analysis for each tumor stage revealed that the overall survival of the THBS4-high expression patients at Stage I and Stage III was significantly poorer than that of the patients with THBS4-low expression (p<0.001 and p = 0.001, respectively; Fig 2). The recurrence site according to pathological stages is shown in S2 Table. Hematogenous recurrence was frequent in patients at stage II patients, in compared with those at stage III.

Fig 2

The overall survival (OS) of the gastric cancer patients based on the THBS4 expression in their tumor stromal cells.

(A) The Kaplan-Meier survival curve indicates that the OS of all patients with high THBS4 expression in stromal cells was significantly worse than that of the patients with low THBS4 expression (p<0.001). (B–E) The Kaplan-Meier survival curve for each stage. The 5-year OS rates of the Stage I patients with high THBS4 expression were poorer than those of the Stage I patients with low THBS4 expression (p<0.001), and the same was true of the Stage III patients (p = 0.001).

Univariate and multivariate analyses

Table 2 provides the results of the univariate and multivariate analyses for overall survival. The univariate analysis revealed that poor survival was significantly correlated with high αSMA expression (p<0.001), high THBS4 expression (p<0.001), age ≥ 65 years old (p<0.001), macroscopic type 4 (p<0.001), microscopic undifferentiated type (p<0.001), high T stage (p<0.001), lymph node metastasis (p<0.001), lymphatic invasion (p<0.001), vascular invasion (p<0.001), ascites cytology positive (p<0.001), peritoneal metastasis (p<0.001), and hepatic metastasis (p<0.001).

Table 2

Univariate and multivariate Cox multiple regression analysis with respect to overall survival after surgery in 584 patients with gastric carcinoma.

	Univariate analysis		Multivariate analysis
	Hazard Ratio (95% CI)	p value	Hazard Ratio (95% CI)	p value
THBS4-high	3.43 (2.48–4.75)	<0.001	1.53 (1.05–2.25)	0.028
Age ≥65 year-old	1.61 (1.16–2.23)	0.004	1.74 (1.23–2.46)	0.002
Female (vs. Male)	1.29 (0.93–1.79)	0.123
αSMA-high	2.37 (1.70–3.31)	<0.001	1.27 (0.90–1.80)	0.180
Borrmann’s type 4	7.41 (5.22–10.5)	<0.001	1.62 (1.05–2.50)	0.042
Tumor diameter ≥50 mm	6.27 (4.37–9.00)	<0.001	1.49 (0.90–2.46)	0.088
Undifferentiated type	1.72 (1.24–2.37)	0.001	1.18 (0.81–1.70)	0.386
T3&4 (vs. T1&2)	6.72 (4.61–9.80)	<0.001	1.41 (0.82–2.40)	0.211
N1-3 (vs. N0)	8.29 (5.50–12.5)	<0.001	2.79 (1.62–4.80)	<0.001
Lymphatic invasion	5.22 (3.42–7.95)	<0.001	1.06 (0.61–1.83)	0.837
Vascular invasion	3.10 (2.22–4.32)	<0.001	1.06 (0.73–1.53)	0.772
Cytology positive	7.42 (5.23–10.5)	<0.001	1.91 (1.28–2.85)	0.001
Peritoneal metastasis	9.11 (5.97–13.9)	<0.001	2.60 (1.64–4.12)	<0.001
Hepatic metastasis	5.90 (3.18–11.0)	<0.001	3.64 (1.89–7.02)	<0.001

The multivariate analysis revealed that THBS4 expression, age ≥65 years, macroscopic type 4 (p<0.001), lymph node metastasis (p<0.001), cytology-positive status (p<0.001), peritoneal metastasis (p<0.001), and hepatic metastasis (p<0.001) were independent prognostic factors.

THBS4 expression on CAFs and NFs

Fig 3A shows established CAFs and NFs stained with Podoplanin. The results of the western blot analysis revealed that the CAF82 and CAF105 significantly expressed THBS4 compared with NF82 and NF105, respectively (t-test, p<0.001 and p<0.001, respectively). MKN45 and NUGC4 did not. (Fig 3B).

Fig 3

The picture of CAFs and NFs stained with Podoplanin and the western blot analysis of THBS4.

(A), Representative picture of Podoplanin staining. Podoplanin was mainly stained at the cytoplasm fibroblasts. The expression level of Podoplanin was higher in CAFs, in compared with that in NFs. (B), THBS4 expression. CAFs expressed THBS4, but NFs and cancer cells did not.

Discussion

THBS4 was reported to be expressed not only in normal tissues but also in some types of solid cancers [8, 10, 17]. In the present study, we observed that gastric tumors (especially the stromal cells in tumor microenvironment) also express THBS4. In addition, the stromal cells expressing THBS4 also expressed αSMA or podoplanin, which is a marker of CAFs. In fact, the western blotting indicated that THBS4 was expressed from CAFs, but not from NFs and cancer cell lines. In case analysis, THBS4-high expression status was correlated with high αSMA expression. These findings suggest that in gastric cancer, THBS4 may be expressed by the gastric cancer-associated fibroblasts.

We reported that CAFs play an important role in the progression, growth, and spread of gastric cancer [18]. In present study, we showed in the results that THBS4-high expression in stroma was significantly associated with higher αSMA expression, higher tumor invasion, lymph node metastasis, lymphatic invasion, peritoneal cytology, and peritoneal metastasis. We also found that high THBS4 expression in tumor stroma was significantly poorer than that of the patients with low THBS4 expression. Moreover, multivariate analysis for overall survival showed that not αSMA expression on CAFs but THBS4 expression on CAFs was an independent prognostic factor. These findings indicated that THBS4 from CAFs might be associated with the invasion of cancer cells, and THBS4 from CAFs is associated with the metastasis of cancer cells.

By targeting the protein coding gene Kruppel-like factor 9 (KLF9) using gastric cancer cell lines, Chen et al. observed that THBS4 had positive effects on gastric cancer cells' proliferation and metastasis [1]. However, little is known about the molecular mechanism of THBS4 remains unclear. Muppala S et al. reported that THBS4 is upregulated in response to TGF-β and promotes the effect of TGF-β on angiogenesis, mediating cell proliferation and survival [10]. Forester et al. showed THBS4 transcription on CAFs is stimulated by incubation with conditioned medium derived from human diffuse gastric cancer lines [4]. It is speculated that TGF-β derived from cancer cells stimulated THBS4 expression on CAFs resulting in angiogenesis and cancer progression.

In particular, the stage I and stage III patients with high THBS4 expression had significantly poorer prognosis than the stage I and III patients with low THBS4 expression, respectively. High THBS4 expression in stromal cells was revealed as an independent prognostic factor for gastric cancer patients. THBS4 from CAFs might be a useful prognostic indicator for gastric cancer patients, especially for those with stage I or III cancer.

In contrast, no significant difference in prognosis was shown between the THBS4-high group and THBS4-low group at stage II. Liver metastasis is a relatively frequent recurrence pattern among patients with stage II gastric cancer [19], and THBS4 expression is not correlated with vascular invasion or liver metastasis, which might be one of the reasons for the absence of a difference in the prognosis between our two THBS4 expression groups at stage II. Additionally, effects of adjuvant chemotherapy might be caused especially at stage II. It was difficult to compare adjuvant regimen according to the stage, because the standard regimen for adjuvant chemotherapy was not established for the enrolled patients at the time. However, the prognosis of patients with lymph node metastasis which was significantly correlated with THBS4-high might be improved.

To the best of our knowledge, the present study is the first to investigate the clinicopathological significance of THBS4 in the gastric cancer microenvironment. Macroscopic type 4 and the histologic diffuse type were significantly associated with a high expression of THBS4 in our patient series. Remarkably, THBS4 is expressed in most stroma of Borrmann's type 4 cancer. THBS4 is involved in tissue remodeling [6]. Macroscopic type 4, diffusely infiltrating carcinoma, and scirrhous gastric carcinoma (also known as linitis plastica-type carcinoma) are characterized by cancer cell infiltration and proliferation accompanied by extensive stromal fibrosis and abundant extracellular matrix [20]. This typical histological finding of macroscopic type 4 might be regulated in part by the tissue remodeling activity of THBS4.

FGFR2 is overexpressed on macroscopic type-4 gastric cancer [21]. The secretion of FGF7 (a ligand of fibroblast growth factor receptor 2 [FGFR]2) by gastric fibroblasts is likely to contribute in a paracrine manner to the remarkable cell proliferation seen in scirrhous gastric cancer with FGFR2 overexpression [22]. Whereas Huang et al. reported that the downregulation of THBS4 promotes the FGFR2 signal of gastric cancer progression via the PI3K-AKT-mTOR pathway using non-type-4 gastric cancer cell lines [23]. It is necessary in the future to examine the effects of THBS4 on the PI3K-AKT-mTOR pathway by using type-4 gastric cancer cell lines with FGFR2 overexpression.

In conclusion, THBS4 is expressed on CAFs in the microenvironment of gastric cancer, especially in macroscopic type-4 gastric cancer. THBS4 from CAFs might be associated with the invasion of cancer cells, and THBS4 is also a useful prognostic indicator for gastric cancer patients, especially for those with stage I or stage III cancer.

The Kaplan-Meier survival curve for Borrmann’s type 4 tumor and other type.

No significant difference in overall survival was shown between the THBS-high group and THBS-low group at Borrmann’s type 4. Whereas the prognosis of the patients with high THBS4 expression were poorer than that of the patients with low THBS4 expression at other type.

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Data of clinicopathologic factors in 584 patients.

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Original images for blots and gels.

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Correlation between the expression of THBS4 in tumor stromal cells and clinicopathologic features in Borrmann’s type 4.

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Correlation between the expression of THBS4 in tumor stromal cells and clinicopathologic features in other macroscopic type.

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Recurrence pattern according to the stage.

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6 Aug 2019

PONE-D-19-19270

The Clinicopathological Significance of Thrombospondin-4 Expression in the Tumor Microenvironment of Gastric Cancer

PLOS ONE

Dear Dr. Yashiro,

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(1) Immunostaining of Podoplanin (PDPN) as a widely accepted marker of cancer associated fibroblasts (CAFs)

(2) Detailed methodological description of CAFs and normal-associated fibroblasts (NAFs) and presentation of their images

(3) Explanation about THBS4 expression in stroll cells but not cancer cells in primary gastric cancer, as well as presentation of more clear images of THBS4 immunostaining, and results of THBS4 immunostaining

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(5) Refinement of Kaplan-Meier analysis

(6) Additional experiments or discussion on the functions of thrombospondin-4 (THBS4) in CAFs

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Reviewer #1: This manuscript is to evaluate that THBS4 expressed in CAFs is a prognostic marker to predict poor prognosis in gastric cancer. They proved that THBS4 was expressed in stromal cells expressing alpha-SMA in gastric cancer, and positive THBS4 expression is correlated with poor prognosis of gastric cancer patients. The results are clear, but for publication in Plos One, following points should be fixed.

1. Authors determined the expression of THBS4 in gastric cancer tissues according to the intensity and the percentage of stained cancer cells and stromal cells (Page 4, Line 71). However, they said that it was only expression in stromal cells. How did authors calculate the percentage of positive THBS cells? Was the percentage meaning the ratio of positive stromal cells in all cells including cancer cells and stromal cells?

2. In figure 1, the positive staining of THBS4 look like too diffuse in the entire tissues. Can authors show the clearer photo for THBS4 staining?

3. The gastric cancer generally showed histologic heterogeneity according to the classification. In figure 1, I recommend that authors show the representative photos for THBS4 expression in two different histological subtypes (diffuse and intestinal type).

4. In figure 1, the staining photo of cytokeratin, cancer cell specific maker, should be added to support that THBS4 was not stained in cancer cells.

5. In figure 3, authors just showed the expression of THBS4 in NAF and CAF. I recommend authors that the results should be added the expression of THBS of several gastric cancer cell lines.

6. Authors described in abstract and conclusion that “THBS4 from CAFs is associated with the metastasis of cancer cells” and “THBS4 from CAFs might be associated with the invasion of cancer cells”. However, they did not show the any data for proof of concept. Authors should add the experimental data to prove them, or the descriptions in abstract and conclusion should be toned down.

7. Authors emphasized that THBS4 is especially expression on CAFs of macroscopic type-4 gastric cancer. It would be better to add the results of subgroup analysis for type-4 or other type gastric cancer.

8. Authors insisted that non-significance of stage II would be explained as recurrent pattern and adjuvant chemotherapy. To supper this hypothesis, recurrent pattern or adjuvant regimen according to the stage should be added.

Reviewer #2: The authors investigated the clinicopathological significance of stromal thrombospondin-4 expression in the tumor microenvironment of gastric cancer, and found that THBS4 was expressed on CAFs. Stromal THBS4 was associated with the metastasis of cancer cells. The results are interesting and promising. However, this article suffers from several major flaws:

1. The authors used α-SMA to indicate the location of CAFs. However, α-SMA is a microfilament protein with contractile ability, mainly expressed at myoepithelial cells. Although α-SMA expression could also appear in the transformation of fibroblasts to myofibroblasts around cancer cells, the positive rate of α-SMA in CAFs is not high, and some of muscle tissues may be unexpectedly positive. I recommend the authors to add podoplanin (PDPN) in immunostaining assays, which is a widely accepted marker for CAFs with a relative high expression level.

2. Primary culture was performed to obtain CAFs and NAFs in this research. However, there was no demonstrations or photos to show the status of cells in order to verify the results. And the isolations and purifications of CAFs and NAFs were not mentioned either.

3. Inconsistent data were found between abstract and main text (e.g. positive rate of THBS4 expression).

4. For WB assay, the authors did not elucidate the sample size and necessary statistical analysis (t-test). A solitary result cannot clarify the real world.

5. According to the clinical data, THBS4 in stroma has obvious correlations with tumor invasion and lymph node metastasis. Since the primary CAFs have been cultured successfully, the authors should perform gene function experiments (e.g. migration & invasion) subsequently and explore the potential mechanisms to explain the phenomena they observed.

6. Are there any references to support the IHC evaluating method of THBS4 expression? If yes, please cite the articles, otherwise the authors should state the reason why score 3 was the watershed.

7. Terms were not used properly in this article. NAF was not common in the description of normal fibroblast. And definitions of positive and high were confused.

Reviewer #3: Dear editors,

In this study, the authors mainly investigated the clinicopathological value of THBS4

In gastric cancer based on the retrospective cohort from their own center. The research methods were statistical analyses of clinical data, including chi-square test, Cox proportional hazards regression models, as well as survival curves. The endpoint parameter was overall survival (OS). The article was well written, and focused on practical clinical aspects. While some problems still existed and should be improved.

1. The exact correlation of α-SMA or CAFs and THBS4 should be explained more apparently both in Introduction and Discussion.

2. The qualities of all K-M curves were poor. We believe this problem should be focused since key results of this study were reflected herein. Some normative forms could be referenced, for instance, ANNALS OF ONCOLOGY.

3. Information from Figure 3 was too simple to explain exact relation between the expression of THBS4 and α-SMA or CAFs.

4. References should be updated by using articles in recent 5 years.

Reviewer #4: The authors Kuroda et.al have identified THBS4 as a marker of CAFs in gastric cancer. IHC analysis on 584 GC patients specimen shows statistical significance when THBS4 expression is compared with the clinico-pathological characteristics of the patients. However, I have some comments with respect to the methodology of CAFs and NAFs isolation for western blot analysis. The authors have not elaborated on the specific markers that were used to identify these from the samples.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Kakoli Das

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26 Sep 2019

Reviewer #1

Thank you very much for the careful review of the Reviewer #1. We corrected several points according to the descriptions by the Reviewer #1, as described below. We indicated the changes point by point and highlighted them in the revised paper.

1. Authors determined the expression of THBS4 in gastric cancer tissues according to the intensity and the percentage of stained cancer cells and stromal cells (Page 4, Line 71). However, they said that it was only expression in stromal cells. How did authors calculate the percentage of positive THBS cells? Was the percentage meaning the ratio of positive stromal cells in all cells including cancer cells and stromal cells?

We calculated the percentage of positive THBS cells by the ratio of stained stromal cells in all stromal cells. We added the comments in the materials and methods. (on page4 line 22-26)

2. In figure 1, the positive staining of THBS4 look like too diffuse in the entire tissues. Can authors show the clearer photo for THBS4 staining?

We replaced the picture with the clearer photo for THBS4 staining in Figure 1.

3. The gastric cancer generally showed histologic heterogeneity according to the classification. In figure 1, I recommend that authors show the representative photos for THBS4 expression in two different histological subtypes (diffuse and intestinal type).

We added the picture of THBS4 expression in two different histological subtypes including diffuse type and intestinal type. We added some comments in the manuscript. (on page 7 line 12-13)

4. In figure 1, the staining photo of cytokeratin, cancer cell specific maker, should be added to support that THBS4 was not stained in cancer cells.

We evaluated the expression of cytokeratin as cancer cell specific maker in the gastric cancer tissues. Cancer cells were positive for cytokeratin staining, but negative for THBS4 staining. The pictures of cytokeratin were added in Figure 1. We added these comments in the manuscript. (on page 2 line 15) (on page 4 line 17) (on page 7 line 5-6) (on page 7 line 12) (on page 7 line 15)

5. In figure 3, authors just showed the expression of THBS4 in NAF and CAF. I recommend authors that the results should be added the expression of THBS4 of several gastric cancer cell lines.

In accordance with the Reviewer’s comment, we added western blot of THBS4 of two gastric cancer cell lines, MKN45 and NUGC4 in Figure 3B. We added some comments in the manuscript. (on page 2 line 13) (on page 2 line 17) (on page 5 line 9-10) (on page 11 line 12) (on page 11 line 18) (on page 12 line 3-7)

6. Authors described in abstract and conclusion that “THBS4 from CAFs is associated with the metastasis of cancer cells” and “THBS4 from CAFs might be associated with the invasion of cancer cells”. However, they did not show the any data for proof of concept. Authors should add the experimental data to prove them, or the descriptions in abstract and conclusion should be toned down.

We showed in the results that THBS4-high expression in stroma was significantly correlated with higher tumor invasion (p<0.001), lymph node metastasis (p<0.001), lymphatic invasion (p<0.001), peritoneal cytology (p<0.001), and peritoneal metastasis (p<0.001). Taken together, we found that the prognosis of patients with high THBS4 expression in tumor stroma was significantly poorer than that of patients with low THBS4 expression. These findings indicated that THBS4 from CAFs might be associated with the invasion and metastasis of cancer cells. We added these comments in the discussion. (on page 12 line 12-20)

7. Authors emphasized that THBS4 is especially expression on CAFs of macroscopic type-4 gastric cancer. It would be better to add the results of subgroup analysis for type-4 or other type gastric cancer.

We performed the subgroup analysis of type-4 or other types gastric cancer. No significant difference of clinic-pathological factors was found between THBS4-high group and THBS4-low group in Bormann’s type 4 cancer (S1 Fig and S1 Table). In contrast, the other types gastric cancer had significant differences between the two groups. (S1 Fig and S2 Table). THBS4 might be associated with Bormann’s type 4 cancer, but not associated with malignant progression of Bormann’s type 4 cancer. We added these comments in the manuscript. (on page 7 line 25-28) (on page 9 line 6-7)

8. Authors insisted that non-significance of stage II would be explained as recurrent pattern and adjuvant chemotherapy. To supper this hypothesis, recurrent pattern or adjuvant regimen according to the stage should be added.

As reviewer pointed out, we added S3 Table which shows recurrence site according to the stage. Hematogenous recurrence was frequent in patients at stage II patients, in compared with those at stage III. In contrast, it was difficult to compare adjuvant regimen according to the stage, because the standard regimen for adjuvant chemotherapy was not established for the enrolled patients at the time. We added these comments in the text.

(on page 9 line 10; on page 9 line 13)

Reviewer #2

Thank you very much for the careful review of the Reviewer #2. We correct several points according to the descriptions by the Reviewer #2, as follows.

1. The authors used α-SMA to indicate the location of CAFs. However, α-SMA is a microfilament protein with contractile ability, mainly expressed at myoepithelial cells. Although α-SMA expression could also appear in the transformation of fibroblasts to myofibroblasts around cancer cells, the positive rate of α-SMA in CAFs is not high, and some of muscle tissues may be unexpectedly positive. I recommend the authors to add podoplanin (PDPN) in immunostaining assays, which is a widely accepted marker for CAFs with a relative high expression level.

I agree with the Reviewer’s comment, we added the immunostaining experiment of podoplanin which evaluated the for CAF in tumor. The picture of podoplanin staining was added in Fig 1. And we commented the Podoplanin immunostaining in the text. (on page 2 line 15) (on page 4 line 11-20) (on page 7 line 8) (on page 7 line 15) (on page 12 line 5).

2. Primary culture was performed to obtain CAFs and NAFs in this research. However, there was no demonstrations or photos to show the status of cells in order to verify the results. And the isolations and purifications of CAFs and NAFs were not mentioned either.

We added representative pictures of CAFs and NFs by Podoplanin staining in Fig 3A. The method for the primary culture of CAFs and NFs was added in the manuscript. CAFs and NFs were defined by αSMA staining, as previously reported. (on page 5 line 4-9) (on page 5 line 12-16)

1. Fuyuhiro Y, Yashiro M, Noda S, Matsuoka J, Hasegawa T, Kato Y, et al. Cancer-associated orthotopic myofibroblasts stimulates the motility of gastric carcinoma cells. Cancer Sci. 2012;103(4):797-805.

2. Fuyuhiro Y, Yashiro M, Noda S, Kashiwagi S, Matsuoka J, Doi Y, et al. Myofibroblasts are associated with the progression of scirrhous gastric carcinoma. Exp Ther Med. 2010;1(4):547-51.

3. Inconsistent data were found between abstract and main text (e.g. positive rate of THBS4 expression).

We corrected them. (on page 2 line 11)

4. For WB assay, the authors did not elucidate the sample size and necessary statistical analysis (t-test). A solitary result cannot clarify the real world.

We added statistical analysis of western blot of THBS4 in CAFs and NFs in Fig 3B. We added the comments in the results. (on page 11 line 9-12) (on page 11 line 17-18).

5. According to the clinical data, THBS4 in stroma has obvious correlations with tumor invasion and lymph node metastasis. Since the primary CAFs have been cultured successfully, the authors should perform gene function experiments (e.g. migration & invasion) subsequently and explore the potential mechanisms to explain the phenomena they observed.

We agree that additional experiments on function of THBS4 would be valuable, as reviewer commented. We plan to investigate THBS4 function in CAFs, and submit as a new paper of in vitro experiment in the near future. Then, we added these comments in the discussion, as follows. Muppala S et al. reported that THBS4 is upregulated in response to TGFβ and promotes the effect of TGFβ on angiogenesis, mediating cell proliferation and survival [3]. Forester et al. showed THBS4 transcription on CAFs was stimulated by incubation with conditioned medium derived from human diffuse gastric cancer lines [4]. It was speculated that TGF-β derived from cancer cells stimulated THBS4 expression on CAFs resulting in cancer proliferation and progression. We added these comments in the text. (on page 12 line 23-29)

3. Muppala S, Xiao R, Krukovets I, Verbovetsky D, Yendamuri R, Habib N, et al. Thrombospondin-4 mediates TGF-beta-induced angiogenesis. Oncogene. 2017;36(36):5189-98.

4. Forster S, Gretschel S, Jons T, Yashiro M, Kemmner W. THBS4, a novel stromal molecule of diffuse-type gastric adenocarcinomas, identified by transcriptome-wide expression profiling. Mod Pathol. 2011;24(10):1390-403.

6. Are there any references to support the IHC evaluating method of THBS4 expression? If yes, please cite the articles, otherwise the authors should state the reason why score 3 was the watershed.

We cited a reference which supports our IHC evaluating method of Thrombospondin4 (THBS4), as described below.

5. Huang T, Liu D, Wang Y, Li P, Sun L, Xiong H, et al. FGFR2 Promotes Gastric Cancer Progression by Inhibiting the Expression of Thrombospondin4 via PI3K-Akt-Mtor Pathway. Cell Physiol Biochem. 2018;50(4):1332-45.

7. Terms were not used properly in this article. NAF was not common in the description of normal fibroblast. And definitions of positive and high were confused.

We corrected NAF to NF. We also corrected “positive” to” high”. (on page 2 line 13) (on page 7 line 8-9) (on page 11 line 8)

Reviewer #3: Dear editors,

In this study, the authors mainly investigated the clinicopathological value of THBS4

In gastric cancer based on the retrospective cohort from their own center. The research methods were statistical analyses of clinical data, including chi-square test, Cox proportional hazards regression models, as well as survival curves. The endpoint parameter was overall survival (OS). The article was well written, and focused on practical clinical aspects. While some problems still existed and should be improved.

1. The exact correlation of α-SMA or CAFs and THBS4 should be explained more apparently both in Introduction and Discussion.

THBS4-high expression in stroma was significantly associated with higher αSMA expression. Forester et al. showed THBS4 is expressed on CAFs which are stimulated by diffuse gastric cancer cells. These findings suggest that CAFs with THBS4 expression might be activated by diffuse gastric cancer cells. Moreover, multivariate analysis for overall survival showed that not αSMA expression on CAFs but THBS4 expression on CAFs was an independent prognostic factor. CAFs with THBS4 expression might be associated with malignant potential of gastric cancer cells. We added these comments in the text. (Table 1) (Table 2) (on page 3 line 14-16) (on page 4 line 28-29) (on page 7 line 22) (on page 10 line 8) (on page 12 line 7-8) (on page 12 line 12-20)

Forster S, Gretschel S, Jons T, Yashiro M, Kemmner W. THBS4, a novel stromal molecule of diffuse-type gastric adenocarcinomas, identified by transcriptome-wide expression profiling. Mod Pathol. 2011;24(10):1390-403.

2. The qualities of all K-M curves were poor. We believe this problem should be focused since key results of this study were reflected herein. Some normative forms could be referenced, for instance, ANNALS OF ONCOLOGY.

In accordance with the Reviewer’s comment, we renew the Kaplan-Meier curve referencing ANNALS OF ONCOLOGY. Graph size and line style were changed and censors were added.

3. Information from Figure 3 was too simple to explain exact relation between the expression of THBS4 and α-SMA or CAFs.

We added the comments of Fig 3 more in detail, as follows. (on page 11 line 14-18)

Figure legends

Fig 3. The picture of CAFs and NFs stained with Podoplanin and the western blot analysis of THBS4. (A), Representative picture of Podoplanin staining. Podoplanin was mainly stained at the cytoplasm fibroblasts. The expression level of Podoplanin was higher in CAFs, in compared with that in NFs. (B). THBS4 expression. CAFs expressed THBS4, but NFs and cancer cells did not.

4. References should be updated by using articles in recent 5 years.

We updated references using recent articles.

Reviewer #4

Thank you very much for the careful review of the Reviewer #4. We correct several points according to the descriptions by the Reviewer #4, as follows.

I have some comments with respect to the methodology of CAFs and NAFs isolation for western blot analysis. The authors have not elaborated on the specific markers that were used to identify these from the samples.

We used the specific markers, Podoplanin, to identify CAFs and NFs in Figure 3A. We also added the comments in the materials and methods and the results. (on page 5 line 12-16)

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

22 Oct 2019

The Clinicopathological Significance of Thrombospondin-4 Expression in the Tumor Microenvironment of Gastric Cancer

PONE-D-19-19270R1

Dear Dr. Yashiro,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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Reviewer #1: All comments have been addressed

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Reviewer #3: All comments have been addressed

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

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Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors revised manuscript according to reviewers' comments.

It is acceptable to be published in PlosOne

Reviewer #2: The authors answered most of the questions I raised, and modified the mistakes. I am satisfied with the revised version.

Reviewer #3: (No Response)

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Reviewer #2: No

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29 Oct 2019

PONE-D-19-19270R1

The Clinicopathological Significance of Thrombospondin-4 Expression in the Tumor Microenvironment of Gastric Cancer

Dear Dr. Yashiro:

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