Alpha-galactosylceramide (KRN7000) suppression of chemical- and oncogene-dependent carcinogenesis

Abstract

Recent studies have revealed significant efficacy of the marine sponge glycolipid, alpha-galactosylceramide (alpha-GalCer), in treatment of experimental metastatic cancers, infections, and autoimmune diseases. However, the capacity of alpha-GalCer to prevent tumor development had never, to our knowledge, been evaluated in mouse models of chemical- and oncogene-dependent carcinogenesis. In this study, we demonstrate that long-term administration of soluble alpha-GalCer, spanning the time of tumor initiation, inhibits primary tumor formation in three different models: methylcholanthrene-induced sarcomas, mammary carcinomas in Her-2/neu transgenic mice, and spontaneous sarcomas in p53-/- mice. Weekly treatment of mice with alpha-GalCer maintained lymphoid tissue natural killer cell and T cell activation and elevated serum IFN-gamma and IL-4 concentrations. Consistent with the antimetastatic activity of alpha-GalCer, prevention of methylcholanthrene-induced sarcoma was IFN-gammaand tumor necrosis factor-related apoptosis-inducing ligand dependent, but not perforin-dependent. Taken together, our results demonstrate that NK1.1+alphabetaTCR+ cell-based immune therapy can inhibit primary tumorigenesis.

Fig. 1.

α-GalCer-mediated control of MCA-induced fibrosarcoma. (a) Groups of 20 B6 WT mice were injected s.c. in the hind flank with 400 μg of MCA diluted in 0.1 ml of corn oil. Mice received 2 μg of α-GalCer i.p. as follows: (i) weekly from day –28 to day 0 (where day 0 = day of MCA inoculation); (ii) weekly from day 0 to day 28; (iii) weekly from day 0 to day 70; (iv) weekly from day 28 to day 98. Mice were observed weekly for tumor development over the course of 60–200 days. Tumors >4 mm in diameter and demonstrating progressive growth over 2 weeks were counted as positive. (b) Representative individual fibrosarcomas from the group of 20 B6 WT mice receiving 400 μgof MCA and vehicle (Left) were compared for growth with a similar group of B6 WT mice inoculated with 400 μg of MCA and receiving α-GalCer treatment (2μg of α-GalCer weekly; Right), commencing in each mouse when sarcoma was palpated for the second successive week. Tumor size was measured weekly with a caliper square as the product of two diameters, and results were recorded as the tumor size (cm²). The lines of best fit of each group demonstrate growth rate, are represented by the solid lines, and are not significantly different (P = 0.1649, Mann–Whitney U test). (c) Groups of 15 B6 WT mice were injected s.c. in the hind flank with 400 μg of MCA diluted in 0.1 ml of corn oil and received 0.02–2.0 μg of α-GalCer i.p. (as indicated) weekly from day 0 to day 70.

Fig. 2.

α-GalCer-mediated activation, cytokine production, and expansion of lymphocyte subsets. Groups of three B6 WT mice received either vehicle or 2 μg of α-GalCer i.p. on day 0 (α-GalCer ×1) or weekly from day 0 to day 28 (α-GalCer ×5). (a) Liver MNC were collected 7 days after each α-GalCer or vehicle injection, and the surface phenotype of the MNC was characterized by flow cytometry as indicated, NK1.1 vs. CD3 vs. CD69. (b) The percentage of CD8⁺ T cells and NK cells in the liver MNC were plotted. Cell numbers (×10⁵) were as follows: CD8⁺ T cells; vehicle, 2.5 ± 0.3; α-GalCer × 1, 18.7 ± 1.5; α-GalCer × 5, 13.5 ± 1.1; CD3^– NK1.1⁺ cells; vehicle, 1.2 ± 0.2; α-GalCer × 1, 16.1 ± 1.2; α-GalCer × 5, 4.8 ± 0.7; CD3⁺ NK1.1⁺ cells; vehicle, 4.1 ± 0.7; α-GalCer × 1, 15.4 ± 1.2; and α-GalCer × 5, 4.8 ± 0.7. (c) Sera were collected before (0 h), or4hor24h after a single (×1) or multiple (×5) i.p. injections of 2 μgof α-GalCer or vehicle. IFN-γ and IL-4 levels in the serum were evaluated by using ELISA, and statistical differences were determined by comparing levels with the 0-h time point for each treatment using a Mann–Whitney U test (*, P < 0.05). Levels of IFN-γ and IL-4 were below detection (<50 pg/ml) in vehicle-treated mice.

Fig. 3.

α-GalCer-mediated control of MCA-induced fibrosarcoma by innate immunity and IFN-γ activity. Groups of 15–20 B6 WT, B6 IFN-γ^–/–, B6 TRAIL^–/–,B6 pfp^–/–,orB6pfp^–/– IFN-γ^–/– mice were injected s.c. in the hind flank with 400 μg of MCA diluted in 0.1 ml of corn oil. Mice received either vehicle or 2 μg of α-GalCer i.p. weekly from day 0 to day 70. All mice were observed weekly for tumor development over the course of 60–200 days. Tumors >4 mm in diameter and demonstrating progressive growth over 2 weeks were counted as positive.

Fig. 4.

Appearance of the first tumor (a) and tumor multiplicity (b) in CBf1-neuT mice treated with α-GalCer and IL-12. Mice received 2 μg of α-GalCer (n = 13) or vehicle only (n = 20) i.p. at weeks 5, 9, 13, 17, 21, 25, and 29. Alternatively, a group of mice (n = 8) received 100 ng in 0.2 ml of IL-12/day for 5 days at weeks 5, 9, 13, 17, 21, 25, and 29. Progressively growing masses (>1-mm mean diameter) were regarded as tumors. Differences in tumor incidence at 25 weeks were statistically different between vehicle-treated and IL-12 or α-GalCer-treated mice, as evaluated by a Fisher's exact test (P < 0.0004). Tumor multiplicity was statistically different between vehicle-treated and IL-12- or α-GalCer-treated mice at week 25 and beyond, as demonstrated by using a Mann–Whitney U test (P < 0.004).

Fig. 5.

α-GalCer protects mice from spontaneous sarcoma. The appearance of tumors was recorded in groups of 34–35 B6 p53^–/– mice receiving either vehicle or 2 μgof α-GalCer i.p. weekly from 4 weeks of age. Mice were evaluated on a weekly basis. When mice were moribund, tumor type (open squares, thymic lymphoma; filled triangles, disseminated lymphoma; open circles, sarcoma; filled diamonds, other tumors) was recorded against the age (in days) at the time of death/autopsy. The mean age of p53^–/– mice developing spontaneous sarcoma is depicted by the cross bars, and the statistically later incidences of sarcomas in the α-GalCer-treated group were noted (192.6 ± 6.1 days), compared with vehicle-treated mice (147.1 ± 8.3 days; P = 0.0005, Mann–Whitney U test).