The combination of immunotherapy and a glutamine metabolism inhibitor represents an effective therapeutic strategy for advanced and metastatic murine pancreatic adenocarcinoma

Abstract

Despite constant advances in cancer research, the treatment of pancreatic adenocarcinoma remains extremely challenging. The intratumoral immunotherapy approach that was developed by our research group and was based on a combination of mannan-BAM, TLR ligands, and anti-CD40 antibody (MBTA) showed promising therapeutic effects in various murine tumor models, including a pancreatic adenocarcinoma model (Panc02). However, the efficacy of MBTA therapy in the Panc02 model was negatively correlated with tumor size at the time of therapy initiation. Here, we aimed to further improve the outcome of MBTA therapy in the Panc02 model using the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). The combination of intratumoral MBTA therapy and intraperitoneal administration of DON resulted in the complete elimination of advanced Panc02 subcutaneous tumors (140.8 ± 46.8 mm³) in 50% of treated animals and was followed by development of long-term immune memory. In the bilateral Panc02 subcutaneous tumor model, we observed a significant reduction in tumor growth in both tumors as well as prolonged survival of treated animals. The appropriate timing and method of administration of DON were also addressed to maximize its therapeutic effects and minimize its side effects. In summary, our findings demonstrate that the intraperitoneal application of DON significantly improves the efficacy of intratumoral MBTA therapy in both advanced and bilateral Panc02 subcutaneous tumor murine models.

Keywords: Cancer; Glutamine antagonist; Immunotherapy; Intratumoral; Pancreatic adenocarcinoma.

Fig. 1. Synergistic effect of the combination of intratumoral MBTA therapy and glutamine antagonist (DON) in murine advanced and bilateral Panc02 models.

(A) C57BL/6 mice were subcutaneously (s.c.) inoculated with Panc02 cells into the right flank. After 20 days, mice were randomized into three groups (n = 6/group): group treated with PBS i.t.; group treated with MBTA i.t.; group treated with MBTA i.t. and DON i.p. MBTA therapy (PBS in controls) was administered through pulse regimens on days 0, 1, 2, 8, 9, 10, 16, 17, 18, 24, 25, and 26. DON was administered on days 0, 7, 14, 21, and 28. (B) The growth of the injected tumors is presented as a growth curve (** p < 0.01). (C) The growth of tumors only for MBTA and MBTA/DON groups. (D) The survival curve analysis (* p < 0.05, ** p < 0.01). (E) C57BL/6 mice were s.c. inoculated with Panc02 cells to both the right and left flanks. After 14 days, mice were randomized into four groups (n = 6/group): group treated with PBS i.t. and PBS i.p.; group treated with PBS i.t. and DON i.p.; group treated with MBTA i.t. and PBS i.p.; group treated with MBTA i.t. and DON i.p.. MBTA (PBS in the control group) was administered intratumorally (i.t.) into the right-treated tumors through a pulse regimen on days 0, 1, 2, 8, 9, 10, 16, 17, 18, 24, 25, and 26. DON (PBS in the control group) was administered i.p. on days 5, 13, 21, 29, 37. (F) The growth of the injected tumor is presented as a growth curve (** p < 0.01). (G) The growth of the non-injected (distant) tumor is presented as a growth curve. (H) The survival curve analysis (* p < 0.05).

Fig. 2. Combination of intratumoral (i.t.) MBTA therapy and intraperitoneal (i.p.) administration of glutamine antagonist (DON) in two time regimens; subcutaneous administration of DON in different dosages; and comparison of i.t. and i.p. routes of DON administration in the bilateral Panc02 model.

(A) C57BL/6 mice were s.c. inoculated with Panc02 cells in both right and left flanks. After 12 days, mice were randomized into four groups (n = 6/group): group treated with PBS applied in the right tumor (black); group treated with MBTA applied in the right tumor (blue); group treated with MBTA applied in the right tumor, and DON applied i.p. (13 doses, red); group treated with MBTA applied in the right tumor, and DON applied i.p. (5 doses, green). MBTA (PBS in the control group) was administered through a pulse regimen on days 0, 1, 2, 8, 9, 10, 16, 17, 18, 24, 25, and 26. DON was administered i.p. as indicated. (B, C) The tumor growth of MBTA treated and non-treated (distant) tumors is presented as a growth curve (* p < 0.05, **** p < 0.001, ***** p < 0.0005). (D) The survival curve analysis. (E) C57BL/6 mice were s.c. inoculated with Panc02 cells to both the right and left flanks. After 12 days, mice were randomized into four groups (n = 6/group): Group treated with PBS applied in the right tumor (black); group treated with MBTA applied in the right tumor (blue); group treated with MBTA applied in the right tumor, and low doses of DON applied s.c. (red); group treated with MBTA applied in the right tumor and high doses of DON applied s.c. (green). MBTA (PBS in the control group) was administered through a pulse regimen as shown in Fig.2A. DON was administered s.c. as indicated. (F, G) The growth (in mm³) in MBTA treated and non-treated (distant) tumors is presented as a growth curve (* p < 0.05, ***** p < 0.0005). (H) The survival curve analysis (* p < 0.05). (I) C57BL/6 mice were s.c. inoculated with Panc02 cells in both right and left flanks. After 12 days, mice were randomized into four groups (n = 6/group): Group treated with PBS applied in the right tumor (black); group treated with MBTA applied in the right tumor (blue); group treated with MBTA applied in the right tumor, and DON applied in left tumor (green); group treated with MBTA applied in the right tumor, and DON applied i.p. (red). MBTA (PBS in the control group) was applied in the pulse regime as in Fig. 2A. DON was applied i.t. or i.p. (J, K) The growth (in mm³) of both right and left tumors is presented as a growth curve (** p < 0.01, Kruskal-Wallis test). (L) The survival curve analysis (* p < 0.05).

Fig. 3. Combination of MBTA immunotherapy and glutamine metabolism inhibition by DON in bilateral Panc02 model: evaluation of side effects.

(A) C57BL/6 mice were subcutaneously inoculated with Panc02 cells to both the right and left flanks. After 12 days, mice were randomized into three groups (n = 6/group): Group treated with PBS applied in the right tumor (black); group treated with MBTA applied in the right tumor (blue); group treated with MBTA applied in the right tumor, and DON applied i.p. (red). MBTA (PBS in the control group) was applied in the pulse regime as in Fig. 2A. DON was applied i.p. as indicated. (B, C) The growth of MBTA treated and non-treated (distant) tumors is presented as a growth curve (* p < 0.05). (D) The survival analysis (* p < 0.05). (E, F, G) Evaluation of food and water consumption and weight changes in mice during the experiment.