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. 2012 May 22;106(11):1833-41.
doi: 10.1038/bjc.2012.177.

Interleukin-8 and its receptor CXCR2 in the tumour microenvironment promote colon cancer growth, progression and metastasis

Y S Lee  1 , I Choi, Y Ning, N Y Kim, V Khatchadourian, D Yang, H K Chung, D Choi, M J LaBonte, R D Ladner, K C Nagulapalli Venkata, D O Rosenberg, N A Petasis, H-J Lenz, Y-K Hong
Affiliations

Interleukin-8 and its receptor CXCR2 in the tumour microenvironment promote colon cancer growth, progression and metastasis

Y S Lee et al. Br J Cancer. .

Abstract

Background: Colorectal cancer (CRC) is a leading cause of death in the United States. Increased level of interleukin-8 (IL-8) and CXCR2 on tumours and in the tumour microenvironment has been associated with CRC growth, progression and recurrence in patients. Here, we aimed to evaluate the effects of tissue microenvironment-encoded IL-8 and CXCR2 on colon cancer progression and metastasis.

Methods: A novel immunodeficient, skin-specific IL-8-expressing transgenic model was generated to evaluate colon cancer growth and metastasis. Syngeneic mouse colon cancer cells were grafted in CXCR2 knockout (KO) mice to study the contribution of CXCR2 in the microenvironment to cancer growth.

Results: Elevated levels of IL-8 in the serum and tumour microenvironment profoundly enhanced the growth of human and mouse colon cancer cells with increased peri-tumoural angiogenesis, and also promoted the extravasation of the cancer cells into the lung and liver. The tumour growth was inhibited in CXCR2 KO mice with significantly reduced tumour angiogenesis and increased tumour necrosis.

Conclusion: Increased expression of IL-8 in the tumour microenvironment enhanced colon cancer growth and metastasis. Moreover, the absence of its receptor CXCR2 in the tumour microenvironment prevented colon cancer cell growth. Together, our study demonstrates the critical roles of the tumour microenvironment-encoded IL-8/CXCR2 in colon cancer pathogenesis, validating the pathway as an important therapeutic target.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Proliferation of CT26 and HCT116 cancer cells was activated by the IL-8 and CXCR2 pathway. (A–D) IF images of CXCR2 on CT26 (A) and HCT116 (C) cancer cell lines. The same IF assays were performed without the primary antibody to confirm the specificity of the primary antibody for CXCR2 (B, D). Bar, 20 μm. (E, F) Proliferation assays were performed on CT26 (E) or HCT116 (F) cells in a low serum (1%) media containing vehicle (DMSO), hIL-8 (10 ng ml−1) or Gro-α (10 ng ml−1). (G, H) The CXCR2 antagonist, SCH-527123, efficiently blocked the IL-8-induced activation of cell proliferation. Proliferation assay was performed with vehicle (DMSO), or IL-8 (10 ng ml−1) in the presence of SCH-527123 (10 or 25 μM) on CT26 (G) and HCT116 (H) cells. SCH, SCH-527123. Data are expressed as mean±s.d.; *P<0.05; **P<0.005.
Figure 2
Figure 2
Generation and characterisation of a transgenic mouse model producing functional hIL-8 in the skin. (A) The upper image represents a transgene construct used to generate the K14-hIL-8 transgenic mice. K14 pro, Keratin-14 promoter; hGH ex/int, human growth hormone exons/introns; PA, poly A. The lower graph shows the serum level of hIL-8 protein in different founder mice determined by ELISA, which is consistent with the PCR-based genotyping results. (B, C) IHC analyses detecting hIL-8 in the skin of the WT control (B) vs K14-hIL-8 transgenic (C) mice. (D) DTH reactions were induced in the right ear of WT and K14-hIL-8 transgenic (TG) mice using Oxazolone. Left ears of both groups were treated with vehicle alone as a negative control to set the base line. Data are expressed as an average per cent thickness±s.d. (nine mice per group). Note more prominent swelling was detected in the ears of the TG mice than those of WT mice during the first week. After 16 days, the swelling was largely resolved in both groups. *P<0.05; **P<0.005.
Figure 3
Figure 3
Enhanced tumour growth in the K14-hIL-8/nu mouse with increased peri-tumoural angiogenesis (A) One million CT26 cells were subcutaneously injected into the left and right flank of the K14-hIL-8/nu (n=10) or WT nude mice (n=10) and tumour volume was measured every 2 days for 2 weeks. Tumour size was expressed as an average±s.d. Number/area (B) and size (C) of CD31-positive vessels found in the peri-tumour (n=6) and intra-tumour areas (n=6) were measured in both groups of tumour and displayed as an average±s.d. (D) One million HCT116 cells were subcutaneously injected into the left and right flank of the K14-hIL-8/nu (n=6) or WT nude mice (n=4), and tumour volume was measured every 2 days for 23 days. Number/area (E) and size (F) of CD31-positive vessels in the peri-tumoural (n=8) and intra-tumoural areas (n=6) were measured in both groups of tumour and displayed as an average±s.d. *P<0.05, **P<0.005.
Figure 4
Figure 4
Enhanced metastatic potential of human colon cancer cells in K14-hIL-8/nu mice (A) Bright field, red fluorescent and combined images of cultured HCT116LR cells ( × 100) (B) RFP signals from both WT nude and K14-hIL-8/nu mouse. Both mice were skinned to better detect the RFP signal. (C) Liver, lung, heart, kidney, spleen and subcutaneous lesions were harvested from K14-hIL-8/nu mice and their bioluminescent signals from firefly luciferase and fluorescent signals from RFP were simultaneously captured. (D) Bright field and red fluorescent images showing lung metastases in the WT nude and K14-hIL-8/nu mice. (E) Average number of metastatic lesions in the lung, liver and other organs (skin, heart, spleen and kidney) in the WT nude (n=5) and K14-hIL-8/nu (n=6) mice. Graph represents the average number of total metastases in the WT nude vs K14-hIL-8/nu mice. *P<0.05. The colour reproduction of this figure is available at the British Journal of Cancer online.
Figure 5
Figure 5
Expression of CXCR2 in the tumour microenvironment is required for optimal growth of colon cancer cells. (A) CT26 tumour growth in the WT and CXCR2 KO mice. One million CT26 cells were injected into the flanks of the WT (n=10) and CXCR2 KO (n=10) mice and tumour growth was measured every 2 days for 15 days. Number/area (B) and size (C) of CD31-positive vessels found in the peri-tumoural and intra-tumoural areas were measured in both groups and displayed as an average±s.d. (D) Necrotic lesions (red dotted line) in CT26 tumours of the WT and CXCR2 KO mice are shown (H&E staining). Bar, 200 μm. (E) Percentage of necrotic area in tumours in the WT vs CXCR2 KO mice. (F) IHC staining showing CD31-positive vessels in the peri-tumoural areas in the WT vs CXCR2 KO mice. (G) Expression of CXCR1 and CXCR2 was determined in the skin of the WT or three independent CXCR2 KO (KO1∼3) mice by semi-quantitative conventional RT–PCR. *P<0.05; **P<0.005. The colour reproduction of this figure is available at the British Journal of Cancer online.
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