Evaluation of the expression of bone marrow‐derived mesenchymal stem cells and cancer‐associated fibroblasts in the stroma of gastric cancer tissue

Abstract Aim Cancer‐associated fibroblasts (CAFs) generated by bone marrow‐derived mesenchymal stem cells (BM‐MSCs) play an important role in cancer progression. In this study, we investigated the relationships of BM‐MSCs and CAFs in resected gastric cancers with the clinicopathological factors of patients. Methods We analyzed 120 gastric cancer patients who underwent gastrectomy. Immunostaining was performed with an anti‐CD271 antibody (BM‐MSCs) and anti‐α‐smooth muscle actin (αSMA) antibody (CAFs). Staining intensity was used to divide patients into low and high expression groups. Observation sites in cancer tissues were invasive, central, and whole portions. Results Expression of αSMA was significantly related to depth of tumor invasion (T), lymph node metastasis (N), lymphatic invasion (ly), venous invasion (v), and stage. Expression of CD271 was significantly related to v, stage, stromal volume, and tumor infiltration pattern (INF). Overall survival (OS) of the high expression group was significantly lower than that of the low expression group for both αSMA and CD271. Multivariate analysis showed that N, αSMA (whole), and CD271 (invasive) were independent prognostic factors. Conclusions Cancer‐associated fibroblasts and BM‐MSCs are related to the progression, invasion, and prognosis of gastric cancer and may be therapeutic targets of gastric cancer.

Mesenchymal stem cells (MSCs) are somatic stem cells derived from mesodermal tissue (mesenchyma), which are present in all tissues.
Among tissue stem cells, although MSCs are considered to differentiate into various mesenchymal cell types such as bone, cartilage, muscle, fat, and nerves, MSCs originally exist in bone marrow, and MSCs that are isolated from bone marrow can reconstitute various mesenchymal tissues. 2 Changes in cancer cells themselves along with microenvironmental changes in the cancer surrounding the stroma are critically involved the development and metastasis of cancer. The peritumoral microenvironment includes various stromal cell types, cancer-associated fibroblasts (CAFs), endothelial cells, adipocytes, and immune cells. 3 Among these cell types, CAFs are related to tumor invasion and metastasis. 4,5 Furthermore, CAFs have recently been shown to be derived from  6 However, there are few reports on BM-MSCs in gastric cancer tissue.
In this study, the relationships of BM-MSCs and CAFs with the clinicopathological factors and prognosis of gastric cancer patients were examined immunohistochemically in resected gastric cancer tissues.

| Immunohistochemical staining
Immunohistochemical staining was performed using paraffin-embedded sections, including the deepest part of the tumor in each case, with an anti-CD271 (BM-MSC marker) antibody 8 and anti-α-smooth muscle actin (αSMA; CAF marker) antibody. 9 Several researchers previously isolated MSCs from bone marrow using an anti-CD271 antibody. 8,[10][11][12] Therefore, CD271 is considered a marker for BM-MSCs. CD271 is also known as affinity nerve growth factor receptor (LNGFR), nerve growth factor receptor (NGFR), and p75NTR (neurotrophin receptor), and belongs to the tumor necrosis factor superfamily. 13 Paraffin-embedded tissue samples were sectioned at 4 μm were used as primary antibodies. For CD271 staining, heat treatment was applied in Epitope Recovery Solution 2 (pH 9.0) for 30 minutes, followed by incubation with the anti-CD271 antibody for 15 minutes. For αSMA staining, the antibody was applied without antigen recovery for 15 minutes. The automated system used for detection was a refined polymer detection system (Leica Microsystems) with horseradish peroxidase-polymer as the secondary antibody, 3,3′-Diaminobenzidine was used as the chromogen. The negative control for both αSMA and CD271 was phosphate-buffered saline instead of primary antibody. The positive control for αSMA was the proper muscle layer of the stomach, and that for CD271 was melanoma tissue.

| Evaluation of αSMA and CD271 expression
To evaluate myofibroblasts as CAFs, αSMA was stained in myofi-

| Statistical analysis
Statistical analysis was performed using JMP (version 2018).
Statistical significance of analytical data was evaluated using the chisquared test or t-test. P < .05 was deemed as statistically significant.
The cumulative survival time was calculated using the Kaplan-Meier method and analyzed by the log-rank test. The stepwise method was used for multivariate analysis to calculate independent prognostic factors after prognostic variables were identified by univariate analysis. Spearman rank correlation coefficient analysis was used for comparisons of staining intensity between αSMA expression and CD271 expression in the invasive, central, and whole portions.

| Terms
Clinicopathological terms were used in compliance with the Japanese classification of gastric carcinoma 3rd English edition. central [P = .015]), no associations were found with age, sex, gross type, T factor, N factor, or ly ( Table 2).

| Correlations of αSMA and CD271 expression
Expression of αSMA and CD271 tended to be correlated at the invasive, central, and whole portions (P = .08, .07, and .08, respectively; Table 3).
By Spearman rank correlation coefficient analysis, there were correlation between αSMA expression and CD271 expression in the invasive portion (r = .3922, P < .0001), and whole portion (r = .3450,  Note: T1 tumor confined to the submucosa, T2 tumor invades the muscularis propria, T3 tumor invades the subserosa, T4 tumor invasion is contiguous to or exposed beyond the serosa or tumor invades adjacent structures, N0 no regional lymph node metastasis, N1 metastasis in one to two regional lymph nodes, N2 metastasis in three to six regional lymph nodes, N3 metastasis in seven or more regional lymph nodes, ly0 no lymphatic invasion, ly1 minimal lymphatic invasion, ly2 moderate lymphatic invasion, ly3 marked lymphatic invasion, Note: T1 tumor confined to the submucosa, T2 tumor invades the muscularis propria, T3 tumor invades the subserosa, T4 tumor invasion is contiguous to or exposed beyond the serosa or tumor invades adjacent structures, N0 no regional lymph node metastasis, N1 metastasis in one to two regional lymph nodes, N2 metastasis in three to six regional lymph nodes, N3 metastasis in seven or more regional lymph nodes, ly0 no lymphatic invasion, ly1 minimal lymphatic invasion, ly2 moderate lymphatic invasion, ly3 marked lymphatic invasion, P < .0001), and strong correlation in the central portion (r = .4225, P < .0001). Note: Groups A-D are αSMA Low and CD271 Low, αSMA Low and CD271 High, αSMA High and CD271 Low, and αSMA High and CD271 High, respectively (invasive: P = .08; central: P = .07; whole: P = .08).
Therefore, the presence of CAFs in the whole tumor and BM-MSCs in the invasive portion was considered to be involved in the prognosis of gastric cancer.

| D ISCUSS I ON
In this study, gastric cancer tissues were immunostained for As tumors grow larger, they need to obtain oxygen and nutrition from newly developed vessels. CAFs generated from BM-MSCs were recruited to the tumor after first accumulating in the peripheral blood. 15 Histologically, many BM-MSCs were observed around cancer cells in the invasive portion of the tumor, together with CAFs throughout the tumor stroma.

F I G U R E 4
Groups A-D are α-smooth muscle actin (αSMA) Low and CD271 Low, αSMA Low and CD271 High, αSMA High and CD271 Low, and αSMA High and CD271 High, respectively. Overall survival of both αSMA and CD271 high expression groups was significantly poorer than that of the both αSMA and CD271 low expression groups in terms of invasive, central, and whole portions (P = .0011, .016, and .0232, respectively)

Epithelial-mesenchymal transition was originally reported by
Hay et al as a phenomenon observed early in ontogeny. 16 The importance of EMT for tumor growth progression has been reported in recent years. Cytokines that induce EMT include Wnt and TGF-β produced by cancer cells. 17  with the autocrine action of Wnt-5a secreted from cancer cells. 28 In this study, we immunohistochemically examined BM-MSCs Note: T1 tumor confined to the submucosa, T2 tumor invades the muscularis propria, T3 tumor invades the subserosa, T4 tumor invasion is contiguous to or exposed beyond the serosa or tumor invades adjacent structures, N0 no regional lymph node metastasis, N1 metastasis in one to two regional lymph nodes, N2 metastasis in three to six regional lymph nodes, N3 metastasis in seven or more regional lymph nodes, v0 no venous invasion, v1 minimal venous invasion, v2 moderate venous invasion, v3 marked venous invasion, med sparse stroma, sci abundant stroma, int the quality of stroma is intermediate between med and sci, HR hazard ratio, CI confidence interval.

D I SCLOS U R E
Funding: This study was not funded.

I N FO R M E D CO N S E NT
Informed consent was obtained from all individual participants included in this study.