Impact of combination immunochemotherapies on progression of 4NQO-induced murine oral squamous cell carcinoma

Abstract

Advanced oral squamous cell carcinomas (OSCC) have limited therapeutic options. Although immune therapies are emerging as a potentially effective alternative or adjunct to chemotherapies, the therapeutic efficacy of combination immune chemotherapies has yet to be determined. Using a 4-nitroquinolone-N-oxide (4NQO) orthotopic model of OSCC in immunocompetent mice, we evaluated the therapeutic efficacy of single- and combined-agent treatment with a poly-epitope tumor peptide vaccine, cisplatin and/or an A_2AR inhibitor, ZM241385. The monotherapies or their combinations resulted in a partial inhibition of tumor growth and, in some cases, a significant but transient upregulation of systemic anti-tumor CD8⁺ T cell responses. These responses eroded in the face of expanding immunoregulatory cell populations at later stages of tumor progression. Our findings support the need for the further development of combinatorial therapeutic approaches that could more effectively silence dominant immune inhibitory pathways operating in OSCC and provide novel, more beneficial treatment options for this tumor.

Keywords: 4NQO model; A2AR inhibition; Carcinogenesis; Chemotherapy; Oral squamous cell carcinoma; Vaccination.

Fig. 1

Histopathology of 4NQO-induced OSCC. OSCC tumor-bearing mice were killed in week 24 and tumors harvested. In a H&E staining of a representative tongue tumor. In b immunofluorescence staining for Ki67 (green: proliferating cells), DAPI (blue: cell nuclei) and CD31 (red: tumor vasculature) in a representative tongue tumor harvested on day 23. The panel insert indicates the locations of tumors on the tongue

Fig. 2

Treatment of OSCC tumor-bearing mice with a poly-peptide vaccine results in the induction of specific CD8⁺ T cells but fails to impact disease progression. Mice bearing 16 week pre-malignant lesions were treated s.c. with a poly-epitope vaccine on weeks 18, 19, 20, 22 and 24. In a the average body weights of the vaccine-treated mice did not change vs. those of untreated control mice from week 19 onward (p > 0.05). In b, total tumor volumes (tongue and esophageal tumors) were equal in the vaccinated vs. control mice (p > 0.05). In c, d IFN-γ ELISPOT data are shown for splenic CD8⁺ T cells isolated from control (c) or vaccinated (d) mice after stimulation with EL4 thymoma cells presenting individual vaccine peptides or anti-CD3/anti-CD28 (positive control). The presented data are from one of the two independent experiments; *p < 0.05, **p < 0.01, ****p < 0.0001. In e OSCC tumors from vaccinated vs. control untreated mice were H&E stained [upper panels; tumor is marked by dotted boundary] Lower panels show results of fluorescence imaging of immune cell infiltrates using directly labeled antibody probes for CD4, CD8 and FoxP3. Quantitation of the e fluorescence images is reported in Supplemental Table 1

Fig. 3

Modest, but superior impact of treating OSCC tumor-bearing mice with cisplatin vs. poly-peptide vaccine monotherapy. In a, the average body weight of mice treated with cisplatin did not change relative to that in untreated mice, and it was not statistically different vs. mice treated with the vaccine (p > 0.05). Treatment with cisplatin but not vaccine was associated with significantly reduced total tumor volume (b) and the tongue tumor volume (c), but not the esophageal tumor volume (d) versus control untreated mice. NS not significant; *p > 0.05

Fig. 4

Modest but superior impact of monotherapy with high- vs. low-dose A_2AR antagonist ZM241385 on OSCC tumor growth. OSCC-bearing mice received therapy consisting of low- (0.2 μg/mouse; a) or high- (0.4 μg/mouse; e) dose A_2AR antagonist did not change the average body weight vs. untreated control mice (A_2AR low: NS; A_2AR high: NS). In b–d, f–h only therapy with high-dose A_2AR antagonist lead to significant inhibition of the total tumor volume of both tongue and esophageal tumors; *p < 0.05, **p < 0.01

Fig. 5

Minimal therapeutic impact for combination treatment using poly-peptide vaccination plus high-dose A_2AR antagonist (ATG). In a, changes over time in average body weights are compared for mice receiving the combined therapy vs. untreated controls (p < 0.01 for week 26 only; indicated by an asterisk; NS for other time points). In b–d while the tumor volumes appear to be smaller in mice receiving the combined therapy vs. control untreated mice, these differences failed to reach significance (NS for all)