Interleukin-8 mediates resistance to antiangiogenic agent sunitinib in renal cell carcinoma

Abstract

The broad spectrum kinase inhibitor sunitinib is a first-line therapy for advanced clear cell renal cell carcinoma (ccRCC), a deadly form of kidney cancer. Unfortunately, most patients develop sunitinib resistance and progressive disease after about 1 year of treatment. In this study, we evaluated the mechanisms of resistance to sunitinib to identify the potential tactics to overcome it. Xenograft models were generated that mimicked clinical resistance to sunitinib. Higher microvessel density was found in sunitinib-resistant tumors, indicating that an escape from antiangiogenesis occurred. Notably, escape coincided with increased secretion of interleukin-8 (IL-8) from tumors into the plasma, and coadministration of an IL-8 neutralizing antibody resensitized tumors to sunitinib treatment. In patients who were refractory to sunitinib treatment, IL-8 expression was elevated in ccRCC tumors, supporting the concept that IL-8 levels might predict clinical response to sunitinib. Our results reveal IL-8 as an important contributor to sunitinib resistance in ccRCC and a candidate therapeutic target to reverse acquired or intrinsic resistance to sunitinib in this malignancy.

Figures

Figure 1

Phenotypic resistance of ccRCC xenografts treated with sunitinib under an intermittent dosing regimen. A, A-498 ccRCC xenografts were treated with 40 mg/kg of sunitinib with a 3-wk-on and 4-wk-off dosing strategy, which mimicked the clinical regimen given to patients (4-wk-on and 2-wk-off) with modification. All A-498 xenograft mice (11 total) responded during the first round of sunitinib treatment and 4 mice developed resistance during the second round of sunitinib treatment (see text for definition of phenotypic resistance). Tumor growth ratio was determined by dividing the tumor volume measured at an indicated time by tumor volume at the start of sunitinib treatment. Tumor growth ratios for each group are presented as mean ± SD (*, P < 0.05). B, SN12C ccRCC xenografts were treated with 80 mg/kg of sunitinib with a 4-wk-on and 4-wk-off dosing strategy. Two mice (out of 10 total) showed phenotypic resistance during the first round of sunitinib treatment and two more mice showed resistance during the second round of sunitinib treatment.

Figure 2

Escape from antiangiogenesis and elevated plasma levels of IL-8 were found in sunitinib-resistant mice treated under an intermittent dosing regimen. A, increased MVD was found in sunitinib-resistant SN12C and A-498 xenograft tumors by the end of the treatment. Tumor sections from mice in Fig. 1 were stained for CD34, a vascular endothelial cell marker, and MVD was quantified using software as indicated in Materials and Methods. Arrows, blood vessels. Bar, 0.20 mm. B, reactivation of tumor angiogenesis was accompanied by a significant increase of IL-8 release in the plasma of resistant SN12C and A-498 xenograft mice, as analyzed by ELISA assay. The data points shown here represent the corresponding time points in Fig. 1. Bars, SD (*, P < 0.05 versus control; **, P < 0.01 versus control; #, P < 0.05 versus sensitive).

Figure 3

Phenotypic resistance of ccRCC xenografts and elevated plasma levels of IL-8 in sunitinib-resistant mice treated under a continuous dosing regimen. A, 786-O ccRCC xenograft tumors were treated with sunitinib at 40 mg/kg continuously for 34 d (days 33–67). Fifteen out of 18 mice (83%) developed resistance by day 67 (**, P < 0.01). B, plasma levels of human IL-8 were higher in resistant 786-O xenograft–bearing mice compared with sensitive mice. Plasma IL-8 levels were determined by ELISA and are normalized to tumor volume (*, P < 0.05 versus sensitive).

Figure 4

Neutralization of IL-8 activity resensitized ccRCC xenografts to sunitinib treatment under an intermittent dosing regimen. A, A-498 xenograft tumors were treated with 40 mg/kg of sunitinib daily with a 3-wk-on and 3-wk-off schedule. Mice that developed phenotypic resistance (the same animals as depicted in Supplemental Fig. S3A) were then randomly divided into three groups. One group of sunitinib-resistant mice was given sunitinib plus IL-8 neutralizing antibody (n = 4), one group was given sunitinib plus control IgG (n = 4), and the third group received IL-8 antibody alone (n = 5). IL-8 neutralizing antibody treatment started on day 98 and stopped on day 112. Tumor growth ratio was determined by dividing the tumor volume measured at an indicated time by tumor volume at the start of IL-8 antibody treatment (day 98) and presented as mean ± SD. Sunitinib plus IL-8 antibody treatment inhibited tumor growth compared with sunitinib treatment plus control IgG or IL-8 antibody treatment alone. B, bar graph of growth ratios of sunitinib-resistant tumors at 98 d (black columns, before IL-8 antibody treatment) and 112 d (white columns, after IL-8 antibody treatment). The means of tumor growth ratios for each treatment group are plotted. Bars, SD (*, P < 0.05; **, P < 0.01).

Figure 5

Neutralization of IL-8 activity resensitized ccRCC xenografts to sunitinib treatment under a continuous dosing regimen. A, attenuation of sunitinib resistance in the 786-O xenograft model. Starting from day 68, the same sunitinib-resistant animals depicted in Fig. 3A were divided into two groups: one group received sunitinib plus IL-8 neutralizing antibody (SU + IL-8 Ab; n = 7), and the other group received sunitinib plus control IgG (SU + IgG; n = 8). Tumor growth ratio was determined by dividing the tumor volume measured at an indicated time by tumor volume at the start of IL-8 antibody treatment and presented as mean ± SD. B, bar graph of tumor growth ratios plotted before (day 67) and after (day 85) IL-8 antibody treatment. C, the neutralizing activity of IL-8 antibody was confirmed by the detection of plasma levels of IL-8 using ELISA. High plasma levels of IL-8 were detected in sunitinib-resistant mice (SU + IL8 Ab and SU + IgG) compared with sensitive mice (SU-sen) on day 68. By day 76 and day 85, mice treated with neutralizing IL-8 antibody showed reduced levels of plasma IL-8; in contrast, IL-8 plasma levels remained high in the “sunitinib + IgG” group (*, P < 0.05; **, P < 0.01).

Figure 6

IL-8 expression is increased in human ccRCC tumors with intrinsic resistance to sunitinib treatment. ccRCC tumor samples were collected from patients prior to sunitinib treatment. Patient response to sunitinib treatment was evaluated by Response Evaluation Criteria in Solid Tumors guidelines. Nine patients showed no response to subsequent sunitinib treatment (intrinsic resistance), whereas 11 patients responded to sunitinib. IL-8 expression in ccRCC tumors was evaluated by immunohistochemistry. A, examples of immunohistochemical scoring for IL-8 expression. The cytoplasmic and membranous staining was scored as 0 (negative, left), 1 (weakly positive, middle), and 2 (strongly positive, right). In addition, the percentage of cells with each staining grade was recorded. B, representative IL-8 staining of tumor from a patient who was refractory to sunitinib treatment. Strong IL-8 expression is seen in the primary tumor. C, IL-8 staining from a patient who responded to sunitinib treatment. Only focally weak IL-8 expression is seen in the primary tumor (magnification, ×200). Table summarizes IL-8 staining scores from sunitinib-responsive and -nonresponsive patients.