Background Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and metastatic cancer. Despite the potential of cancer immunotherapy, PDAC is one of the least responsive solid tumors. In our previous studies, we have shown that NK cells and macrophages contribute to FcγR-dependent tumor control through antibody-dependent cellular cytotoxicity or phagocytosis (ADCC or ADCP), resulting in improved survival when combined with radiation (RT) and tumor-specific antibodies (mAbs). Based on these findings, we hypothesize that cell therapy with activated NK and macrophages could enhance the anti-tumor response when combined with mAbs and RT in PDAC.
Methods TCRαβ depletion of PBMCs from healthy donors resulted in the generation of a cell product (CP) that is enriched in NK/Macrophages. These cells were then stimulated overnight with IL-2 and zoledronic acid (ZA) (IL2/ZA). Parameters for the potency were assessed by Flow cytometry. Cytokine analysis was performed using cell culture supernatant. We tested the cytotoxic potency of the CP in vitro using PANC1, MiaPaCa, T3M4, and Capan2 human pancreatic cancer cell lines, measuring cell death through the Incucyte®. Furthermore, we assessed the therapeutic efficacy of the combination therapy in vivo using immunodeficient mice (NOD-Rag1null IL2rgnull, NOD rag gamma, NOD-RG) bearing human pancreatic tumors.
Results Flow cytometric analysis confirmed successful TCRαβ depletion from PBMCs in the CP with increased granzyme B and perforin expression on NK cells compared to unstimulated ones (P<0.0004). The IL2/ZA stimulated cells also exhibited increased release of IFN-γ and TNF-α, as assessed by cytokine assays on the cell culture supernatant (No stimulation vs IL2/ZA Stimulation; P<0.0001). When co-cultured with spheroids of four different pancreatic cancer cell lines expressing EGFR and HER2, the stimulated CP combined with tumor-specific mAbs resulted in significant cell lysis, while mAbs alone were ineffective (P<0.05.). Incorporating radiation into the CP+mAb treatment protocol enhanced the killing efficiency, as where radiation alone did not inhibit spheroid growth. The whole CP showed superior spheroid lysis compared to individual cell fractions of γδ T cells, NK cells, or macrophages alone. Human pancreatic tumors in immunodeficient mice showed tumor regression treated with 4 Gy radiation+mAb+cell therapy with no sign of toxicity (P<0.001). Moreover, in vivo tracking of the CP showed accumulation in the tumors that received radiation and correlated with their MHC class I polypeptide-related sequence A (MICA) expression.
Conclusions TCRαβ-depleted NK/macrophages-enriched IL-2/ZA-stimulated cell therapy exhibits enhanced killing potential against human pancreatic cancer cells when used in combination with tumor-specific monoclonal antibodies (mAbs) and radiation therapy.
Ethics Approval Animal studies were performed after approval by the University of Wisconsin-Madison Institutional Animal Care and Use Committee (IACUC).
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