202 In vitro and in vivo combination therapy of Low Molecular Weight Heparins, chemotherapy and immunotherapy, induce antitumor activity in Pancreatic Cancer

BackgroundPDAC is recognized as a highly thrombogenic tumor; thus, low-molecular-weight heparin (LMWH) is routinely used for PDAC patients. Based on the combinatorial therapy approach to treating highly malignant and refractory cancers such as PDAC, we hypothesized that LMWHs could augment the effectiveness of immune checkpoint inhibitors and induce an efficient antitumoral activity.1MethodsBxPC-3, PANC-1, and MIA-PaCa2 were incubated alone or in combination with Tinzaparin (T) and/or Nab-Paclitaxel (A) and/or Gemcitabine (G) and/or Nivolumab (NI), Pembrolizumab (PE) and/or Ipilimumab (IPI). The effect of these regimes on various signaling pathways controlling proliferation and apoptosis was identified in vitro through Western blot. Cell viability was measured with MTT assay. NOD/SCID mice will be used to generate xenografts with the PANC-1 cell line. Human peripheral blood mononuclear cells (PBMCs) from healthy donors will be injected to give mice a human-like immune system.2ResultsIn a triple combinatorial scheme, NI/PE+IPI+T, the protein levels of VEGFR2 were decreased (0.1 to 0.7 folds) in a dose-dependent way in mtKRAS PC cell lines (PANC1 and MIAPACA2). The number of PANC-1 cells was decreased around 40% in a triple combinatorial scheme of T+IPI+(NI or PE) after 48 hours. The triple combination of Gemcitabine + Nab-paclitaxel + Tinzaparin leads to a decrease in tumor size relative to control by 51% and relative to Nab-P + G by 15%. The combination of chemotherapy, immunotherapy, and Tinzaparin leads to a reduction in tumor size compared to control by up to 60%. Tinzaparin contributes an additional 20% Preliminary data show that the quadruple therapeutic regimen increases the percentage of CD8+ cells from 5% to 27% and decreases Tregs’ percentage from 9.5% to 4% (in TILs).ConclusionsIn vitro experiments show a decrease in the cell viability of PC cell lines and a reduction in the protein levels of VEGFR2 in mtKRAS cell lines. In vivo experiments with NOD/SCID mice and humanized NOD/SCID mice show a significant reduction in tumor volume in the combination therapy regimens with Tinzaparin. Possible mechanisms for these effects include an increase in CD8+ cells, a decrease in Tregs cells, a reduction in VEGFR-2 expression, and an increase in cancer cell apoptosis. This synergistic strategy can create new avenues for the treatment of patients with pancreatic cancer, achieving a better clinical outcome and greater survival.ReferencesBokas A, Papakotoulas P, Sarantis P, Papadimitropoulou A, Papavassiliou AG, Karamouzis MV. Mechanisms of the Antitumor Activity of Low Molecular Weight Heparins in Pancreatic Adenocarcinomas. Cancers (Basel). 2020;12(2):432. Published 2020 Feb 13. doi:10.3390/cancers12020432.Chen Q, Wang J, Liu WN, Zhao Y. Cancer Immunotherapies and Humanized Mouse Drug Testing Platforms. Transl Oncol. 2019;12(7):987–995. doi:10.1016/j.tranon.2019.04.020.

Low molecular weight heparin (LMWH) enhances immunotherapy (I) activity in pancreatic cancer cells.

50 Background: The aim is to investigate the effect of LMWH in combination with chemotherapy (C) and I in PC. Methods: BxPC-3, PANC-1, MIA PACA-2 cell lines were exposed in Tinzaparin (T) (0.5, 0.7, 0.9 μΜ) and/or 1 μΜ Nab-Paclitaxel (A) and/or 1 μΜ Gemcitabine (G) and/or 1 μΜ Nivolumab (NI), 1 μΜ, Pembrolizumab (PE) and 1 μΜ ipilimumab (IPI). Protein levels of VEGFR2, p-ERK1/2, p-AKT detected by Western blotting. Cells viability was measured through MTT assay. Results: Increasing protein levels of VEGFR2 was observed in all PC cell lines exposed to a constant dose of T and/or A,G. NI and PE +/- with IPI increased VEGFR2. In NI/PE+IPI+T scheme VEGFR2 levels were decreased (0.1-0.7 folds) in a dose dependent way in mtKRAS cells (PANC1, MIAPACA2). C (G or A) + I decreased VEGFR2 protein levels in mtKRAS cells PANC1 (0.1-0.4 folds), MIAPACA2 (0.1-0.6 folds) in double scheme. T+G/A+NI/PE+IPI scheme increased VEGFR2 in all PC cells. PANC-1 cells were decreased 40% in 0,7T+IPI+(NI or PE) after 48hours. The effect of these schemes on signaling pathways (MEK/EKR, AKT/mTOR) and apoptosis was identified through pERK1/2, pAKT, PARP, cl. caspase-3. Conclusions: These results identify the different effect of I alone or in combination with T and C in PC cells bearing mutant or wild type KRAS. Double or triple combination reduced VEGFR2 protein levels in mtKRAS and not in wtKRAS PC cells. The 0,7T+IPI+(NI or PE) combination decreases cell viability of PC cells through apoptosis. Testing of the combinatorial scheme T+chemo+IPI+(NI or PE) with PANC-1 PC in scid mice is ongoing and will be presented. NC:not changed, -:<20% protein vs control, +:>20% protein vs control, ++:>40% protein vs control. [Table: see text]