A Spontaneous Melanoma Mouse Model Applicable for a Longitudinal Chemotherapy and Immunotherapy Study

Abstract

Mouse models that reflect human disorders provide invaluable tools for the translation of basic science discoveries to clinical therapies. However, many of these in vivo therapeutic studies are short term and do not accurately mimic patient conditions. In this study, we used a fully immunocompetent, transgenic mouse model, TGS, in which the spontaneous development of metastatic melanoma is driven by the ectopic expression of a normal neuronal receptor, mGluR1, as a model to assess longitudinal treatment response (up to 8 months) with an inhibitor of glutamatergic signaling, troriluzole, which is a prodrug of riluzole, plus an antibody against PD-1, an immune checkpoint inhibitor. Our results reveal a sex-biased treatment response that led to improved survival in troriluzole and/or anti-PD-1-treated male mice that correlated with differential CD8⁺ T cells and CD11b⁺ myeloid cell populations in the tumor-stromal interface, supporting the notion that this model is a responsive and tractable system for evaluating therapeutic regimens for melanoma in an immunocompetent setting.

Figure 1:. Sex-bias responses were observed in mice treated with inhibitors against glutamatergic signaling and immune checkpoint.

(a) TGS mice at 80 days old. (b) Treatment groups: vehicle (DMSO + rat IgG, n=13 males, n=9 females), troriluzole (n=8 males, n=8 females), anti-PD-1 (n=8 males, n=8 females), and troriluzole + anti-PD-1 (n=11 males, n=9 females). (c) Data is presented as an average tumor burden ± SD. Growth rate was calculated as percent change between 18-weeks and the treatment groups respective 0-weeks. Statistical significance was determined using a two-way ANOVA test with Bonferroni post-hoc comparisons. *** (p-value: 0.001), comparisons between 0 and 18 weeks for vehicle (DMSO + rat IgG) treated male mice. #, comparisons between 0 and 18 weeks in female mice treated with troriluzole (p-value: 0.001), anti-PD-1 (p-value: 0.002), and troriluzole + anti-PD-1 (p-value: 4.56 E-7).

Figure 2:. Distribution of individual mouse tumor growth rates at 6 and 18 weeks from baseline.

(a) Male (top-6 weeks and bottom-18-weeks) and (b) female (top-6 weeks, bottom-18-weeks) mice were stratified into progressive disease (at least 20% increase), stable disease (neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease), and partial response (at least 30% decrease) using the RECIST criteria, a tumor centric criterion used evaluate response to treatment. Sample size is as described in Figure 1b.

Figure 3:. Riluzole and glutamate levels across treatment groups in male and female mice.

LC-MS/MS was performed with blood plasma samples collected at 0, 6, 12, and 18 weeks. Blood was collected at least 10 hours post-treatment. The same mouse samples were used in both assays for (a) riluzole and (b) glutamate levels and data are shown as the average levels ± SEM. To note, anti-PD-1 samples were analyzed at a different time than those of the other three groups. The samples used were vehicle (DMSO + rat IgG, n=9 males, n=8 females), troriluzole (n=8 males, n=6 females), anti-PD-1 (n=7 males, n=8 females) and troriluzole + anti-PD-1 (n=10 males, n=6 females). (c) Immunofluorescence (IF) staining was performed on excised tumor skin samples from 18 week treated mice. At least 2 male and 2 female mice were used except in troriluzole female mice where only 1 mouse was used, and each specimen had a variable number of tumors. Images were quantified using QuPath (v0.4.1) (Bankhead et al., 2017). Data is presented as average percent of glutamate stain/area μm² ± SEM. Statistical significance was determined using a two-way ANOVA test with Bonferroni post-hoc comparisons. * p < 0.05, ** p<0.01, *** p<0.001.

Figure 4:. CD8⁺ and CD11b⁺ cell populations within the tumor-stromal interface.

Immunohistochemical (IHC) staining was performed on excised tumor skin samples from 18 week treated mice. At least 2 male and 2 female mice were used, and each specimen had a variable number of tumors. Images were quantified using Aperio ImageScope (v12.4.3.5008). (a) Data is presented as average percent positivity ± SEM. Positivity is defined as the number of positive cells (weak positive + positive + strong positive)/total number of cells (positive + negative). (b) Representative images of stained slides (50 μm and 200 μm). Brown cells represent melanomas while antibody staining is in red. Statistical significance was determined using a two-way ANOVA test with Bonferroni post-hoc analysis. * p < 0.05, ** p<0.01, *** p<0.001.

Figure 5:. Improved overall survival observed in male mice after treatment removal.

After 18 weeks of treatment, a random subset of TGS mice were taken off treatment to determine their survival outcomes for another 18 weeks. The survival study groups were the same as the treatment ones: vehicle (DMSO + rat IgG, n=4 males, n=4 females), troriluzole (n=3 males, n=1 females), anti-PD-1 (n=3 males, n=1 females), and troriluzole + anti-PD-1 (n=3 males, n=4 females). The Log Rank (Mantel-Cox) statistical test was used to evaluate the significance of each treatment group compared to vehicle controls, only if there was sufficient samples size.

Figure 6:. Tumor analysis on TGS mice.

TGS heterozygote at 71 (left) and 186 days old (right). The same randomly chosen area was selected for quantification of pigmented lesions for the duration of the study. This area was selected, cropped, and converted to binary images to allow for quantification of lesions in that area.