A novel polyamine blockade therapy activates an anti-tumor immune response

Abstract

Most tumors maintain elevated levels of polyamines to support their growth and survival. This study explores the anti-tumor effect of polyamine starvation via both inhibiting polyamine biosynthesis and blocking the upregulated import of polyamines into the tumor. We demonstrate that polyamine blockade therapy (PBT) co-treatment with both DFMO and a novel polyamine transport inhibitor, Trimer PTI, significantly inhibits tumor growth more than treatment with DFMO or the Trimer PTI alone. The anti-tumor effect of PBT was lost in mice where CD4⁺ and CD8⁺ T cells were antibody depleted, implying that PBT stimulates an anti-tumor immune effect that is T-cell dependent. The PBT anti-tumor effect was accompanied by an increase in granzyme B⁺, IFN-γ⁺ CD8⁺ T-cells and a decrease in immunosuppressive tumor infiltrating cells including Gr-1⁺CD11b⁺ myeloid derived suppressor cells (MDSCs), CD4⁺CD25⁺ Tregs, and CD206⁺F4/80⁺ M2 macrophages. Stimulation with tumor-specific peptides elicited elevated antigen-specific IFN-γ secretion in splenocytes from PBT-treated mice, indicating that PBT treatment stimulates the activation of T-cells in a tumor-specific manner. These data show that combined treatment with both DFMO and the Trimer PTI not only deprives polyamine-addicted tumor cells of polyamines, but also relieves polyamine-mediated immunosuppression in the tumor microenvironment, thus allowing the activation of tumoricidal T-cells.

Keywords: difluoromethylornithine; immunomodulation; polyamines; transport inhibitor; tumor microenvironment.

Figure 1. Structure of DFMO and Trimer PTI

Polyamine blockade therapy consists of combined treatment with α-difluoromethylornithine (DFMO, an ODC inhibitor), and the Trimer PTI (N1, N1′, N1″-(benzene-1, 3, 5-triyltris(methylene))tris(N4-(4-(methylamino)butyl)butane- 1, 4-diamine), an inhibitor of the polyamine transport system [29].

Figure 2. B16F10-sTAC tumor growth inhibition with DFMO and Trimer PTI

(A) Mice were subcutaneously injected with 5×10⁵ B16F10-sTAC melanoma cells. When tumors were 50-100 mm³ in size, treatment was initiated with either saline, 0.25% DFMO (w/v) in the drinking water, Trimer PTI (i.p., 3 mg/kg, once a day) or both DFMO and Trimer PTI. Graph shows B16F10-sTAC tumor growth under different treatments (mean tumor volume ± SEM). (B) Spleen weight was determined upon sacrifice (mean ± SEM). (C) Upon sacrifice, tumors were excised and weighed (mean ± SEM). (D) Polyamine levels were determined in tumors by HPLC and normalized to DNA levels in the tissue extracts (nmol/mg DNA). (E) Tumor and non-tumor bearing skin tissues were excised from PBT-treated mice, flash frozen, finely pulverized in liquid nitrogen with mortar and pestle, homogenized in a solution of 33% water, 66% methanol and 1% acetic acid, and then centrifuged at 5000 rpm for 10 min at 25°C. Trimer PTI levels were determined in tumor and skin supernatants by mass spectrometry and normalized to tissue protein concentration (nmol/mg protein). n = 5-10 mice per group; ^* = p ≤ 0.05 and # = p ≤ 0.01 compared to vehicle-treated mice.

Figure 3. DFMO and Trimer PTI co-treatment increases cytotoxic T-cell activity and promotes macrophage infiltration of the tumor

(A) The frequency of IFN-γ producing T-cells was measured by the ELISpot assay as IFN-γ spot forming units (SFU) per million spleen cells. (B) The number of F4/80⁺ cells per million B16F10-sTAC melanoma cells from either vehicle treated mice or mice co-treated with DFMO and Trimer PTI. Representative images of B16F10-sTAC tumor sections from mice treated with vehicle (C) or DFMO and Trimer PTI (D) and stained for F4/80⁺ macrophages. n = 5-10 mice per group; ^* = p ≤ 0.05 and # = p ≤ 0.01 compared to vehicle treated mice.

Figure 4. DFMO and Trimer PTI co-treatment increases levels of pro-inflammatory cytokines in B16F10-sTAC tumors

Upon sacrifice, tumors were excised, flash frozen in liquid nitrogen and then homogenized to produce tumor lysates which were assayed for levels of TNF-α, IL-10, IFN-γ, IL-6, MCP-1 and VEGF by Cytokine Bead Array or ELISA. n = 5-10 mice per group; ^* = p ≤ 0.05 and # = p ≤ 0.01 compared to indicated group.

Figure 5. Depletion of CD4⁺ and CD8⁺ T-cells reverses PBT inhibition of tumor growth

(A) Mice were subcutaneously injected with 5×10⁵ CT26.CL25 colon carcinoma cells. When tumors were 50-100 mm³ in size, treatment was initiated with either saline or 0.25% DFMO (w/v) in the drinking water plus Trimer PTI (i.p. 3 mg/kg, once a day). Mice in the anti-CD4/CD8 groups were i.p. injected with 75 μg of anti-CD4 and anti-CD8 antibodies every three days starting 3 days prior to the initiation of treatment with a total of four doses. Graph shows CT26.CL25 tumor growth under different treatments (mean tumor volume ± SEM). Upon sacrifice, tumors (B) and spleens (C) were excised and weighed (mean ± SEM). n = 5-10 mice per group; ^* = p ≤ 0.05 and # = p ≤ 0.01 compared to indicated group.

Figure 6. DFMO and Trimer PTI co-treatment reduces the immunosuppressive and pro-tumorigenic cells populations

Upon sacrifice, tumors were excised from CT26.CL25-tumor bearing mice and processed for analysis by flow cytometry. CD45⁺ tumor cells were analyzed for the percentage of the indicated cell subpopulations. n = 5-10 mice per group; ^* = p ≤ 0.05 and # = p ≤ 0.01 compared to indicated group.

Figure 7. Co-treatment with DFMO and Trimer PTI increases the cytotoxic T-cell activity in the tumor

Upon sacrifice, tumors were excised from CT26.CL25-tumor bearing mice and infiltrating leukocytes were isolated by discontinuous Percoll gradients. CD8⁺ T-cells were analyzed for the percentage of IFN-γ⁺ (A and B) and granzyme B⁺ (A and C) cells by flow cytometry. (D) The frequency of IFN-γ producing T-cells was measured by the ELISpot assay as IFN-γ spot forming units (SFU) per 5 x10⁵ tumor infiltrating leukocytes. n = 5-10 mice per group; ^* = p ≤ 0.05 and # = p ≤ 0.01 compared to indicated group.

Figure 8. Co-treatment with DFMO and Trimer PTI decreases the immunosuppressive activity of MDSCs

(A) One week prior to sacrifice, MDSCs were isolated from the spleens of CT26.CL25 tumor-bearing mice that were treated either with vehicle or Co-treatment with DFMO and Trimer PTI decreases. Isolated MDSCs (5.2 × 10⁶ per mouse) were then adoptively transferred into separate groups of recipient tumor-bearing mice in the PBT-treated group. (B) Upon sacrifice, splenocytes from the recipient mice were used to measure the frequency of IFN-γ producing T-cells by ELISpot assay following challenge with tumor-expressing β-galactosidase peptide. n = 5-10 mice per group; ^* = p ≤ 0.05 and # = p ≤ 0.01 compared to indicated group.