Pre-clinical assessment of the therapeutic efficacy of a nanovaccine in a mouse model of pancreatic cancer
by Daniele Ferrari
Date of Examination:2024-05-08
Date of issue:2024-08-09
Advisor:Prof. Dr. Frauke Alves
Referee:Prof. Dr. Frauke Alves
Referee:Prof. Dr. Philipp Ströbel
Referee:Prof. Dr. Lutz Walter
Referee:Prof. Dr. Luis A. Pardo
Referee:Prof. Dr. Susann Boretius
Persistent Address:
http://dx.doi.org/10.53846/goediss-10648
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Abstract
English
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest diseases with increasing incidence in developed countries. Due to the late diagnosis of PDAC, available treatments do not significantly improve patient survival. Immunotherapy-based strategies have shown encouraging results in many cancers, but have been disappointing in PDAC. This is because PDAC is characterized by an immunosuppressive microenvironment that supports tumor growth and consists of a physical barrier that prevents immune cell infiltration. In this study, we aimed to evaluate the therapeutic efficacy of a peptide-based nanovaccine targeting mesothelin (MSLN) for the treatment of PDAC using an orthotopic mouse model. In addition, since bioluminescence is an imaging technique widely used to preclinically monitor tumor development and is commonly applied in immunotherapy mouse studies for PDAC treatment, we investigated the anti-tumor immunity induced by the expression of the enzyme luciferase in the PDAC cells. The nanovaccine used in this study consisted of a synthetic long peptide of MSLN, a tumor-associated antigen that is overexpressed in PDAC and minimally expressed in healthy tissue. The peptide was conjugated to an AZDye™ 568 NHS Ester fluorophore to allow in vitro and ex vivo imaging. It was encapsulated in poly(lactic-co-glycolic acid)- chitosan nanoparticles (NPs) together with two adjuvants - polyinosinic: polycytidylic acid (Poly I: C) and Resiquimod (R848), both toll-like receptor agonists - to enhance immune cell activation. The nanovaccine was successfully taken up by bone marrow-derived dendritic cells in vitro and detected in vivo in the lymph nodes (LNs) 24 h after immunization of C57BL/6 mice. We evaluated the in vitro immunogenicity of the nanovaccine by re-stimulating splenocytes and cells isolated from the LNs of immunized mice with MSLN peptide or nanovaccine. Thereby we measured an increase in interferon-γ (IFN-γ) secretion in the supernatant of splenocytes by Enzyme-Linked Immunosorbent Assay and in CD8+ T cells by flow cytometry, in nanovaccine re-stimulated cells compared to NP control re-stimulated cells. We further found an increase in humoral response, characterized by MSLN-specific IgM and IgG antibodies in the serum of vaccinated mice compared to NP control mice. To evaluate the anti-tumor efficacy of the MSLN nanovaccine, we tested three vaccination schedules: (i) a prophylactic regimen involving vaccination before tumor induction; (ii) an early and (iii) a late therapeutic regimen involving vaccination after tumor cell inoculation. For the PDAC model generation, mice were orthotopically implanted with KrasG12D; Trp53R172H; Pdx-1Cre (KPC) cells in the head of the pancreas, and tumor growth was monitored by ultrasound imaging. MSLN nanovaccination resulted in the inhibition of tumor progression and metastasis and induced higher CD8+ T cell infiltration into the tumor as assessed by flow cytometry and immunofluorescence in response to the prophylactic and early therapeutic schedules, but not in the late vaccination approach. In addition, vaccination induced increased CD8+ T cell exhaustion in the therapeutic schedule. Our data support the potential of an MSLN-based nanovaccine as an immunotherapy for PDAC and suggest an innovative route of vaccine delivery using NPs. To evaluate the impact of luciferase expression in KPC tumor growth, mice were orthotopically implanted with either KPC or luciferase-transfected KPC cells (KPC-Luc). Bioluminescence imaging (BLI) was performed twice a week until we observed a drastic signal decrease, by the second week. Implantation of KPC-Luc cells resulted in tumor development and growth accompanied by BLI signals that increased until day 9. However, these signals were almost absent by day 12. At this time point, KPC-Luc tumors were significantly smaller than KPC tumors. Increased levels of IFN-γ were found intracellularly in CD8+ T cells and supernatants of splenocytes from KPC-Luc mice compared to splenocytes from KPC tumor-bearing mice after stimulation with luciferase peptide, suggesting a strong immunogenic response against the KPC-Luc tumors. When mice were euthanized before the BLI signal decreased, both groups of mice had similar tumor volumes on day 9 after tumor cell implantation. Peripheral blood and splenocytes from KPC-Luc tumor-bearing mice on day 9 showed no difference in the number of cytotoxic T cells compared to KPC tumor-bearing mice. In contrast, a significantly higher number of NK cells was found in both blood and splenocytes of KPC-Luc compared to KPC tumor mice on day 13. Furthermore, KPC-Luc tumors showed a higher infiltration of CD8+ T cells compared to KPC tumors. Our results confirm earlier reports in mouse models of other tumor entities that the expression of reporters such as luciferase by tumor cells affects tumor growth by inducing an increased immunogenic response. Such an immunogenic effect of luciferase reporters should be taken into account in syngeneic PDAC mouse models, especially when evaluating the efficacy of immunotherapies.
Keywords: PDAC; Immunotherapy; Nanovaccine
Schlagwörter: PDAC; Immunotherapy; Nanovaccine
