Systemic high-dose dexamethasone treatment may modulate the efficacy of intratumoral viral oncolytic immunotherapy in glioblastoma models

Abstract

Background: Intratumoral viral oncolytic immunotherapy is a promising new approach for the treatment of a variety of solid cancers. CAN-2409 is a replication-deficient adenovirus that delivers herpes simplex virus thymidine kinase to cancer cells, resulting in local conversion of ganciclovir or valacyclovir into a toxic metabolite. This leads to highly immunogenic cell death, followed by a local immune response against a variety of cancer neoantigens and, next, a systemic immune response against the injected tumor and uninjected distant metastases. CAN-2409 treatment has shown promising results in clinical studies in glioblastoma (GBM). Patients with GBM are usually given the corticosteroid dexamethasone to manage edema. Previous work has suggested that concurrent dexamethasone therapy may have a negative effect in patients treated with immune checkpoint inhibitors in patients with GBM. However, the effects of dexamethasone on the efficacy of CAN-2409 treatment have not been explored.

Methods: In vitro experiments included cell viability and neurosphere T-cell killing assays. Effects of dexamethasone on CAN-2409 in vivo were examined using a syngeneic murine GBM model; survival was assessed according to Kaplan-Meier; analyses of tumor-infiltrating lymphocytes were performed with mass cytometry (CyTOF - cytometry by time-of-flight). Data were analyzed using a general linear model, with one-way analysis of variance followed by Dunnett's multiple comparison test, Kruskal-Wallis test, Dunn's multiple comparison test or statistical significance analysis of microarrays.

Results: In a mouse model of GBM, we found that high doses of dexamethasone combined with CAN-2409 led to significantly reduced median survival (29.0 days) compared with CAN-2409 treatment alone (39.5 days). CyTOF analyses of tumor-infiltrating immune cells demonstrated potent immune stimulation induced by CAN-2409 treatment. These effects were diminished when high-dose dexamethasone was used. Functional immune cell characterization suggested increased immune cell exhaustion and tumor promoting profiles after dexamethasone treatment.

Conclusion: Our data suggest that concurrent high-dose dexamethasone treatment may impair the efficacy of oncolytic viral immunotherapy of GBM, supporting the notion that dexamethasone use should be balanced between symptom control and impact on the therapeutic outcome.

Keywords: brain neoplasms; oncolytic virotherapy; translational medical research; tumor microenvironment.

Figure 1

Dexamethasone interferes with CAN-2409/GCV cytotoxic and immunogenic effect. (A) Mechanism of CAN-2409/GCV. After intratumoral injection of CAN-2409, HSV-TK is expressed in tumor cells and metabolizes the administered prodrug GCV, inducing DNA damage. This results in (1) apoptotic and necrotic tumor cell death and hence leads to (2) immune cell activation. (B, C) Dexamethasone reduces the immunogenic effect of CAN-2409/GCV. For T-cell killing assays, G9_pCDH (B, C) and U87_GFP (D, E) were cocultured with either activated CD8⁺ T cells, CAN-2409/GCV, dexamethasone 1 µM/10 µM or the combination as indicated and imaged daily for 6 days. Sphere area was normalized to sphere size on day 0. Images on day 5 show a smaller sphere size between CAN-2409/GCV and combination of both ±CD8⁺ T cells. Addition of CD8⁺ T cells is associated with reduced sphere size and fluorescence intensity (×4 magnification, scale bar=500 µm). GCV, ganciclovir.

Figure 2

Dexamethasone blocks responses to CAN-2409 in vivo. (A) Experimental set-up in vivo. A total of 100 000 GL261fluc cells were injected intracranially in the right hemisphere; 9 days after tumor implantation, CAN-2409 was injected at the same location, followed 24 hours later by daily GCV (group AdV-tk +GCV) and/or dexamethasone (group combination) treatment for a total of 7 days. Each cohort n=6. (B) Kaplan-Meier survival curves. While treatment with CAN-2409 (39.5 days) and dexamethasone (33.5 days) alone led to a significant longer median survival compared with control (25 days) (CAN-2409 vs control, log-rank test: p=0.0022; dexamethasone vs control, p=0.0386), the combination resulted in significant reduction of the CAN-2409-effect (combination vs CAN-2409, log-rank test: p=0.0184) and was not statistically different compared with control (combination vs control, log-rank test: ns). Curves were right-censored on day 86. One outlier animal from the control group was excluded from the study because it did not display any evidence of lasting successful tumor formation by immunostaining (online supplemental figure S3) and was therefore excluded from the analysis. (C) MRI. Representative T2 -weighed magnetic resonance images 22 and 29 days after tumor implantation from animals of the survival study (B). All animals (n=2/group) displayed tumor formation on day 22. On day 29, five animals had reached endpoint and therefore images are blank. Among the surviving animals on day 29, the tumor volume in the CAN-2409-treated animal was less, whereas the tumor in the dexamethasone and the combination-treated animal had increased. (D) Tumor volumetry. Tumor volume per animal of days 22 and 29. Animals that had reached endpoint on day 29 are marked with (+). GCV, ganciclovir; ns, not significant.

Figure 3

Altered CAN-2409-induced tumor microenvironment after DEX. (A) tSNE plots of all samples and single therapies. tSNE plots displaying immune cell subset identification overlay plots and density dot plots of CyTOF-analyzed murine tumor infiltrating leukocytes. (B) Therapy-related alteration of leukocyte populations. Percentages of total cell populations are displayed. CAN-2409 increases the percentages of CD8⁺, CD4⁺, B-cell, Treg and DPT cell populations, whereas combinatorial treatment is associated with a reduction of these populations and an increase in NK cells, neutrophils and macrophages. Testing with GLM showed significant reduction of CD8⁺ (p=0.016), CD4⁺ (p=0.02) T cells, B cells (p=0.01), and DPT cells (p=0.02) as well as an increase of neutrophils (p=0.02) after DEX treatment compared with CAN-2409, and a significant reduction of Tregs after DEX treatment compared with control and CAN-2409 (p=0.01 resp. p=0.016) (bars show mean±SD). t-SNE, t-distributed stchastic neighbor embedding; DEX, dexamethasone; DNT, double negative T; DPT, double positive T; GLM, general linear model; NK, natural killer; tSNE, t-distributed stochastic neighbor embedding.

Figure 4

Treatment with dexamethasone ±CAN-2409 affects T-cell profiles. (A) Overlay of leukocyte clusters on tSNE plot with focus on T cells. Cluster of interest for CD8⁺ T cells are pC07, 11, and 20, and for CD4⁺ T cells pC04 and 14. (B) Cluster distribution within CD8⁺ and CD4⁺ T cells in relation to therapy. CD8⁺ and CD4⁺ T cells consist of indicated clusters. Predominant cluster throughout all treatment groups is pC07 for CD8⁺ and pC04 for CD4⁺ T cells. CAN-2409 leads to increased cell abundance among all clusters, whereas dexamethasone and the combination therapy induces the opposite effect. This effect is statistically significant in pC07 (p=0.0084) (bars show mean±SD). (C, D) Expression of significantly altered markers on CD8⁺ and CD4⁺ T cells. Graphs display the median expression of CD44 and PD-L1 without or after treatment with dexamethasone, CAN-2409 and the combination on CD8⁺ (C) and the median expression of CD44, I-A/I-E, CD39, PD-L1, CD152 and Ly6C on CD4⁺ T cells (D). Combination treatment is associated with increased expression of CD44, PD-L1, I-A/I-E, CD39 and Ly6C compared with CAN-2409, with CD44 (p adj.=0.045) and PD-L1 (p adj.=0.045) being significantly upregulated in the combination treatment for CD8⁺ cells and CD44 being significantly upregulated in CD4⁺ cells (p adj.=0.028). PD-L1, programmed death-ligand 1.

Figure 5

Dexamethasone ±CAN-2409 induces a protumorigenic macrophage profile. (A) Overlay of leukocyte clusters on tSNE plot with focus on macrophages. Cluster of interest for macrophages are pC01, 05, 06, 09, 12, 13, 15, 16, 19, 24, and 28. (B) Cluster distribution of the macrophage population in relation to therapy. Compared with CAN-2409, dexamethasone and combination treatment correlate with an increased macrophage population, being statistically significant in cluster pC01 (p=0.0024) (bars show mean±SD). (C) Expression of significantly altered markers on macrophages. Treatment with dexamethasone and the combination leads to an increase of CD44, CD196 and CD172a compared with CAN-2409, with CD172a being statistically significant in the contrast control versus combination (adj. p=0.034).

Figure 6

Increased neutrophil count and activation profile after treatment with dexamethasone ±CAN-2409. (A) Overlay of leukocyte clusters on tSNE plot with focus on neutrophils. Cluster of interest for neutrophils are pC02, pC17, and pC29. (B) Cluster distribution of the neutrophil population in relation to therapy. Dexamethasone and combination treatment is associated with an increase in the neutrophil cell population in all clusters (bars show mean±SD). (C) Expression of significantly altered markers on macrophages. Combinatorial treatment with CAN-2409 and dexamethasone is associated with increased median expression of CD44, Ly6G, CD172a and CD68 with CD44 (adj. p=0.004) being significantly upregulated in combination versus CAN_2409 and Ly6G (adj. p=0.015) being significantly upregulated in the combination group compared with control.