Cancer Res Treat.  2020 Apr;52(2):622-633. 10.4143/crt.2019.593.

CXCL-13 Regulates Resistance to 5-Fluorouracil in Colorectal Cancer

• Zhang G
• Luo X
• Zhang W
• Chen E
• Xu J
• Wang F
• Cao G
• Ju Z
• Jin D
• Huang X
• Zhou W
• Song Z

• KMID: 2500346
• DOI: http://doi.org/10.4143/crt.2019.593

Abstract

Purpose
5-Fluorouracil (5-Fu) is used as a conventional chemotherapy drug in chemotherapy for patients with advanced colorectal cancer, but many patients still suffer from treatment failure due to 5-Fu resistance. Emerging observations revealed the important role of chemokine (C-X-C motif) ligand 13 (CXCL-13) in tumor microenvironment and its relationship with prognosis in patients with colorectal cancer. This study is designed to reveal the important role of CXCL-13 in causing colorectal cancer resistance to 5-Fu.
Materials and Methods
CXCL-13 levels of patient's serum or cell culture supernatants were measured separately by enzyme-linked immunosorbent assay. In cell assays, cell viability is detected by Cell Counting Kit-8. Therefore, the recombinant human CXCL-13 was used to simulate its high expression in cells while its antibody and siRNA were used to reduce CXCL-13 expression in cells.
Results
In this study, we demonstrated that CXCL-13 is associated with 5-Fu resistance by culture medium exchange experiments and cytokine arrays of colorectal cancer resistant and nonresistant cells. Clinical studies showed that CXCL-13 is highly expressed in the serum of 5-Fu–resistant patients. High levels of serum CXCL-13 also predict a worse clinical outcome. The addition of recombinant CXCL-13 cytokine resulted in 5-Fu resistance, while its antibody overcame 5-Fu resistance, and knockdown of CXCL-13 expression by siRNA also reduced 5-Fu resistance, which can be saved by added recombination CXCL-13.
Conclusion
These results not only identify a CXCL-13 mediated 5-Fu resistance mechanism but also provide a novel target for 5-Fu–resistant colorectal cancer in prevention and treatment strategies.

Keyword

CXCL13; 5-Fluorouracil; Colorectal neoplasms; Drug resistance; Chemokines

Figure

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  Fig. 1. Chemokine (C-X-C motif) ligand 13 (CXCL-13) is elevated in 5-fluorouracil (5-Fu) resistant (5-FuR) colorectal cancer cells. (A, B) Viability of DLD-1 and DLD-1 5-FuR cells, HCT116, and HCT116 5-FuR cells treated with different concentrations of 5-Fu for 3 days. (C, D) Viability of DLD-1 cells or HCT116 cells treated with different concentrations of 5-Fu for 3 days after pretreatment with DLD-1 5-FuR or HCT116 5-FuR cells conditioned medium (CM) for 2 days. (E) Semi-quantitative of cytokine arrays analysis of expression levels of different cytokines measured in the CM of paired DLD-1 and DLD-1 5-FuR cells. (F, G) Box-plot showing the CXCL-13 expression level in colorectal cancer parental cells (DLD-1, HCT116) and 5-FuR cells (DLD-1 5-FuR, HCT116 5-FuR). Data from three independent experiments were tested in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test.

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  Fig. 2. Chemokine (C-X-C motif) ligand 13 (CXCL-13) is elevated in 5-fluorouracil (5-Fu) resistant colorectal cancer patient and predicts clinical outcomes. (A) Comparison of the levels of CXCL-13 protein were measured by enzyme-linked immunosorbent assay in 5-Fu–sensitive (n=9) and –resistant (n=9) colorectal cancer patients, patients are shown in Table 1. Sen, sensitive cases; Res, resistant cases. (B) 6-Year overall survival (OS) Kaplan–Meier survival curves grouped by serum CXCL-13 expression levels (118 pg/mL) of 64 colorectal cancer patients, as patient information is shown in Table 2. (C) Six-year disease-free survival (DFS) Kaplan–Meier survival curves grouped by serum CXCL-13 expression levels (118 pg/mL) of 64 colorectal cancer patients, as patient information is shown in Table 2. (A) *p < 0.05, **p < 0.01 by unpaired Student’s t test. (B, C) *p < 0.05 by log-rank (Mantel-Cox), hazard ratios (HRs) and 95% confidence interval (CI) are shown.

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  Fig. 3. Chemokine (C-X-C motif) ligand 13 (CXCL-13) promotes colorectal cancer cells resistance to 5-fluorouracil (5-Fu) through autocrine mechanism. (A, B) Viability of DLD-1 cells or HCT116 cells treated with different concentrations of 5-Fu for 3 days after pretreatment with 10 ng/mL of CXCL-13 for 6 hours. (C, D) Viability of DLD-1 5-Fu resistant (5-FuR) cells or HCT116 5-FuR cells treated with different concentrations of 5-Fu for 3 days after pretreatment with a control IgG or a CXCL-13 neutralizing antibody (5 μg/mL) for 6 hours. Data from three independent experiments were tested in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test or one-way ANOVA.

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  Fig. 4. Knockdown of chemokine (C-X-C motif) ligand 13 (CXCL-13) overcomes 5-fluorouracil (5-Fu) resistance (5-FuR) in colorectal cancer cells. (A) Box-plot showing the CXCL-13 expression level in DLD-1 5-FuR cells or HCT116 5-FuR cells, DLD-1 5-FuR or HCT116 5-FuR siCXCL-13 cells (30 pg/mL for siCXCL-13). (B, C) Viability of DLD-1 5-FuR cells or HCT116 5-FuR cells, DLD-1 5-FuR or HCT116 5-FuR siCXCL-13 cells (30 pg/mL for siCXCL-13) and recombinant CXCL-13 (10 ng/mL) reversed the effect of CXCL-13 siRNA on the sensitivity of DLD-1 5-FuR cells or HCT116 5-FuR cells treated with different concentrations of 5-Fu for 3 days after pretreatment with CXCL-13 siRNA. (D) The 5-Fu concentration of 50% inhibition of cell growth (IC50) of six cells in B and C above. Data from three independent experiments were tested in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test or one-way ANOVA.

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  Fig. 5. 5-Fluorouracil (5-Fu) treatment induces chemokine (C-X-C motif) ligand 13 (CXCL-13) secretion in vivo. (A) A simple schematic of the experimental process. (B) Line chart showing the serum CXCL-13 expression level in mice bearing patient-derived tumor xenograft (PDX) tumors and treated with vehicle or 5-Fu (30 mg/kg/twice a week for 4 weeks) as in A. (C) Representative H&E, immunohistochemistry (IHC) images of subcutaneous tumors formed by PDX tumors treated as in A. (D) Quantification of IHC staining for CXCL-13 of subcutaneous tumors formed by PDX tumors treated as in A. ELISA, enzyme-linked immunosorbent assay. Data are represented as mean±standard deviation. n=3 mice/group while n=6 tumors/group. **p < 0.01 by Student’s t test or two-way ANOVA.

Reference

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