Mannan-BAM, TLR ligands, and anti-CD40 immunotherapy in established murine pancreatic adenocarcinoma: understanding therapeutic potentials and limitations

Abstract

Pancreatic adenocarcinoma is one of the leading causes of cancer-related deaths, and its therapy remains a challenge. Our proposed therapeutic approach is based on the intratumoral injections of mannan-BAM, toll-like receptor ligands, and anti-CD40 antibody (thus termed MBTA therapy), and has shown promising results in the elimination of subcutaneous murine melanoma, pheochromocytoma, colon carcinoma, and smaller pancreatic adenocarcinoma (Panc02). Here, we tested the short- and long-term effects of MBTA therapy in established subcutaneous Panc02 tumors two times larger than in previous study and bilateral Panc02 models as well as the roles of CD4⁺ and CD8⁺ T lymphocytes in this therapy. The MBTA therapy resulted in eradication of 67% of Panc02 tumors with the development of long-term memory as evidenced by the rejection of Panc02 cells after subcutaneous and intracranial transplantations. The initial Panc02 tumor elimination is not dependent on the presence of CD4⁺ T lymphocytes, although these cells seem to be important in long-term survival and resistance against tumor retransplantation. The resistance was revealed to be antigen-specific due to its inability to reject B16-F10 melanoma cells. In the bilateral Panc02 model, MBTA therapy manifested a lower therapeutic response. Despite numerous combinations of MBTA therapy with other therapeutic approaches, our results show that only simultaneous application of MBTA therapy into both tumors has potential for the treatment of the bilateral Panc02 model.

Keywords: Cancer immunotherapy; Checkpoint inhibitors; Mannan; Metastases; Pancreatic adenocarcinoma; TLR ligands.

Fig. 1

MBTA therapy in established Panc02 tumors. a C57BL/6 mice were subcutaneously injected with Panc02 cells in the right flank. After 16 days, mice were randomized into 4 groups (n = 6/group): the group treated with anti-CD40 antibody; the group treated with MBT therapy; the group treated with MBTA therapy; the group treated with PBS. Therapy was given intratumorally on days 0, 1, 2, 8, 9, 10, 16, 17, 18, 24, 25, and 26. The tumor volume was measured with a caliper. b The tumor volume growth is presented as a growth curve (*p < 0.05, ***p < 0.005). c The survival analysis is presented as a Kaplan–Meier curve (*p < 0.05)

Fig. 2

MBTA therapy of Panc02 tumors and the role of CD4⁺ T lymphocytes. a The CD4^−/− and CD8^−/− mice were subcutaneously injected with Panc02 cells in the right flank. After 12 days, both CD4^−/− and CD8^−/− mice were randomized into 4 groups (i) the CD4^−/− mice treated with MBTA therapy (n = 3); (ii) CD4^−/− mice treated with PBS (n = 3) (iii) the CD8^−/− mice treated with MBTA therapy (n = 6); (iv) the CD8^−/− mice treated with PBS (n = 5). Therapy was given intratumorally on days 0, 1, 2, 8, 9, 10, 16, 17, 18, 24, 25, and 26. The tumor volume was measured with a caliper. b The tumor volume growth is presented as a growth curve (*p < 0.05, ****p < 0.001). c The survival analysis is presented as a Kaplan–Meier curve (**p < 0.01)

Fig. 3

MBTA therapy of the bilateral Panc02 model. a C57BL/6 mice were subcutaneously injected with Panc02 cells in both right and left flanks. After 12 days, mice were randomized into 2 groups (n = 6/group): (i) the group treated with MBTA therapy; (ii) the group treated with PBS. Therapy was given intratumorally into the right tumor on days 0, 1, 2, 8, 9, 10, 16, 17, 18, 24, 25, and 26. The tumor volume of both tumors was measured with a caliper. b The tumor volume growth of the right MBTA-treated and (c) the left parallel (non-treated) tumors is presented as a growth curve (*****p < 0.0005). d The survival analysis is presented as a Kaplan–Meier curve

Fig. 4

Simultaneous MBTA therapy in the right and left parallel Panc02 tumors and irradiation of the left parallel tumor. a C57BL/6 mice were subcutaneously injected with Panc02 cells in both right and left flanks. After 12 days, mice were randomized into 5 groups (n = 6/group): the group treated with MBTA therapy (right and left tumor); the group treated with MBTA therapy (right tumor) and PBS (left tumor); the group treated with MBTA therapy (right tumor) and radiotherapy (RT) (left tumor); the group treated with PBS (right tumor) and RT (left tumor); the group treated with PBS (both tumors). Therapy was given intratumorally into the right or left parallel tumor on days 0, 1, 2, 8, 9, 10, 16, 17, 18, 24, 25, and 26. The left parallel tumors were irradiated on days 0, 14, and 28. The tumor volume of both tumors was measured with a caliper. b The tumor volume growth of the right MBTA-treated and (c) the left parallel-treated tumors is presented as a growth curve (*p < 0.05, **p < 0.01, ***p < 0.005, *****p < 0.0005). d The survival analysis is presented as a Kaplan–Meier curve (*p < 0.05, **p < 0.01)