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. 2019 Apr;7(2):127-138.
doi: 10.1093/gastro/goy017. Epub 2018 Jun 8.

Bone marrow-derived CXCR4-overexpressing MSCs display increased homing to intestine and ameliorate colitis-associated tumorigenesis in mice

Xiao-Bin Zheng  1   2 , Xiao-Wen He  1   2 , Long-Juan Zhang  3 , Hua-Bo Qin  1   2 , Xu-Tao Lin  1   2   4 , Xuan-Hui Liu  1   2 , Chi Zhou  1   2 , Hua-Shan Liu  1   2 , Tuo Hu  1   2 , Hai-Chun Cheng  5 , Xiao-Sheng He  1   2 , Xian-Rui Wu  1   2 , Yu-Feng Chen  1   2 , Jia Ke  1   2 , Xiao-Jian Wu  1   2 , Ping Lan  1   2
Affiliations

Bone marrow-derived CXCR4-overexpressing MSCs display increased homing to intestine and ameliorate colitis-associated tumorigenesis in mice

Xiao-Bin Zheng et al. Gastroenterol Rep (Oxf). 2019 Apr.

Abstract

Background and objective: Increasing interest has developed in the therapeutic potential of bone marrow-derived mesenchymal stem cells (MSCs) for the treatment of inflammatory bowel disease (IBD) and IBD-induced cancer. However, whether MSCs have the ability to suppress or promote tumor development remains controversial. The stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis is well known to play a critical role in the homing of MSCs. In this study, we aimed to evaluate the role of CXCR4-overexpressing MSCs on the tumorigenesis of IBD.

Methods: MSCs were transduced with lentiviral vector carrying either CXCR4 or green fluorescent protein (GFP). Chemotaxis and invasion assays were used to detect CXCR4 expression. A mouse model of colitis-associated tumorigenesis was established using azoxymethane and dextran sulfate sodium (DSS). The mice were divided into three groups and then injected with phosphate buffer saline (PBS), MSC-GFP or MSC-CXCR4.

Results: Compared with the mice injected with MSC-GFP, the mice injected with MSC-CXCR4 showed relieved weight loss, longer colons, lower tumor numbers and decreased tumor load; expression of pro-inflammatory cytokines decreased, and signal transducer and activator of transcription 3 (STAT3) phosphorylation level in colon tissue was down-regulated.

Conclusion: CXCR4-overexpressing MSCs exhibited effective anti-tumor function, which may be associated with enhanced homing to inflamed intestinal tissues.

Keywords: CXCR4; Inflammatory bowel disease; mesenchymal stem cells; mice; tumorigenesis.

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Figures

Figure 1.
Figure 1.
Flow cytometry analysis of C57BL/6 bone marrow MSCs. Fluorescent immunostaining results show that cultivated cells express CD29, CD44 and CD105, but do not express CD11b, CD31 or CD45. MSCs, mesenchymal stem cells.
Figure 2.
Figure 2.
Transfection efficiency in MSCs and the capability of multi-potential differentiation. The results of RT-PCR (A) and Western blot analysis (B) show that CXCR4 mRNA and protein expressions are higher in MSCs infected with pWSLV-07-EF1α-CXCR4-Puro-GFP than in MSCs infected with pWSLV-07-EF1α-Puro-GFP. The results of inductive experiment (C) indicate that MSC, MSC-GFP and MSC-CXCR4 present similar capability of multi-potential differentiation. Cells exhibit basic features as fibroblast-like morphology (a, b, c). MSC, MSC-GFP and MSC-CXCR4 are all able to differentiate into adipocytes (d, e, f) and osteocytes (g, h, i) (magnification,  ×200). MSCs, mesenchymal stem cells; RT-PCR, real-time polymerase chain reaction; CXCR4, C-X-C chemokine receptor type 4; GFP, green fluorescent protein.
Figure 3.
Figure 3.
Regulation of CXCR4 expression affects the proliferation, chemotaxis and invasiveness toward SDF-1 of MSCs. (A) RTCA assay results show no differences in proliferation rates of MSC-GFP and MSC-CXCR4. The results of scratch assay (B) and invasion assay (C) indicate that CXCR4 overexpression enhanced the chemotaxis potential and invasiveness toward SDF-1 of MSCs (magnification, ×200). Quantitative results of the scratch assay (D) and the invasion assay (E) indicate that the wound-closure rate of MSC-CXCR4 is significantly higher than that of MSC-GFP and more cells pass through the membrane in the MSC-CXCR4 group than in the MSC-GFP group. MSCs, mesenchymal stem cells; CXCR4, C-X-C chemokine receptor type 4; SDF-1, stromal cell-derived factor 1; RTCA, real-time cell analysis; GFP, green fluorescent protein.
Figure 4.
Figure 4.
MSC-CXCR4 plays a more effective role in attenuating AOM/DSS-induced colitis-associated tumorigenesis. (A) Schematic overview of MSCs administration during colitis-associated tumorigenesis induced by AOM/DSS. PBS, MSC-GFP or MSC-CXCR4 was administered by injection via the tail vein on Days 4, 14 and 24. Mice were sacrificed on Day 84. (B) Changes in the body weight of mice show that the body-weight loss was significantly relieved in the MSCs-CXCR4 treated group. (C, D) DSS treatment shortened the length of the colon, and the following MSC treatment reduced the extent of colon shortening. The colons isolated from the mice are longer in the MSC-CXCR4 group than in the MSC-GFP group. (E–H) The tumor number is less and tumor load (measured as the sum of all tumor diameters in a given mouse) is smaller in the MSC-CXCR4 group than in the MSC-GFP group. However, the tumor size shows no significant difference among three groups. Arrows in (E) indicate tumors. Values are expressed as mean ± standard error mean (n = 6 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001. MSCs, mesenchymal stem cells; CXCR4, C-X-C chemokine receptor type 4; AOM, azoxymethane; DSS, dextran sulfate sodium; GFP, green fluorescent protein; ns, no significant difference.
Figure 5.
Figure 5.
The role of the SDF-1/CXCR4 pathway in the homing of MSCs to inflamed intestine in vivo. (A) The results of RT-PCR and Western blot show that SDF-1 mRNA and protein levels were up-regulated in the intestinal tissue of mice after the treatment of AOM/DSS. (B, C) Homing of MSCs to inflamed intestine was measured by RT-PCR, Western blot and immunohistochemistry, and the results indicate that levels of GFP and CXCR4 were both up-regulated in the MSC-CXCR4 group compared with the MSC-GFP group. Recipients treated with MSC-GFP were used as control. *P < 0.05 vs control. (D) RT-PCR results show that GFP expression was detected in the intestine as well as the renal, liver, spleen and lung tissues. SDF-1, stromal cell-derived factor 1; CXCR4, C-X-C chemokine receptor type 4; MSCs, mesenchymal stem cells; RT-PCR, real-time polymerase chain reaction; GFP, green fluorescent protein.
Figure 6.
Figure 6.
MSC-CXCR4 reduced histological damage of the colon tissue more effectively. (A) Representative HE staining of tumor tissues from PBS-, MSC-GFP- and MSC-CXCR4-treated mice. All tumors are characterized as adenomas with high-grade dysplasia. Representative HE staining (B) and histological score (C) of inflammatory tissue from the mice treated with PBS, MSC-GFP and MSC-CXCR4 show that histological scores of colon tissues from MSCs-treated mice were significantly decreased compared with those of colon tissues from PBS-treated mice. The infusion of MSCs-CXCR4 exhibited enhanced therapeutic effects compared with the administration of MSC-GFP. Values are expressed as the mean ± standard error mean (n = 4 mice per group). *P < 0.05. MSCs, mesenchymal stem cells; CXCR4, C-X-C chemokine receptor type 4; HE, hematoxylin and eosin; GFP, green fluorescent protein.
Figure 7.
Figure 7.
MSC-CXCR4 more effectively reduced pro-inflammatory cytokines and STAT3 phosphorylation in colon tissue. (A) Real-time quantitative polymerase chain reaction results show mRNA expressions of inflammatory cytokines were down-regulated in colon tissues isolated from MSCs-treated mice, which could be more obviously observed in mice from the MSC-CXCR4 group than in mice from the MSC-GFP group. Values are expressed as the mean ± standard error mean (n = 4 mice per group). *P < 0.05, **P < 0.01. Both Western blot (B) and immunohistochemistry (C) results show that the level of STAT3 phosphorylation in colon tissues decreased after the treatment of MSC and the overexpression of CXCR4 further down-regulated the level of STAT3 phosphorylation. Arrows indicate positively stained areas. MSCs, mesenchymal stem cells; CXCR4, C-X-C chemokine receptor type 4; TNF-α, tumor necrosis factor α; IL-1β, interleukin 1β; IL-6, interleukin 6; IL-8, interleukin 8; GFP, green fluorescent protein; STAT3, signal transducer and activator of transcription 3.
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