Generation of Pancreatic Organoid-Derived Isografts.

This protocol is a procedure for generating orthotopic isografts using mouse pancreatic cancer organoids. These isografts can be used to track the evolution of pancreatic ductal adenocarcinoma (PDA) from a preinvasive lesion to a metastatic disease and therefore represent a suitable model for identification of determinants of PDA progression. For complete details on the use and execution of this protocol, please refer to Boj et al. (2015) and Filippini et al. (2019).

CD4+ and CD8a+ PET imaging predicts response to novel PD-1 checkpoint inhibitor: studies of Sym021 in syngeneic mouse cancer models.

Predicting the outcome of immunotherapy is essential for efficient treatment. The recent clinical success of immunotherapy is increasingly changing the paradigm of cancer treatment. Accordingly, the development of immune-based agents is accelerating and the number of agents in the global immuno-oncology pipeline has grown 60-70% over the past year. However, despite remarkable clinical efficacy in some patients, only few achieve a lasting clinical response. Treatment failure can be attributed to poorly immunogenic tumors that do not attract tumor infiltrating lymphocytes (TILs). Therefore, we developed positron emission tomography (PET) radiotracers for non-invasive detection of CD4+ and CD8a+ TILs in syngeneic mouse tumor models for preclinical studies. Methods: Seven syngeneic mouse tumor models (B16F10, P815, CT26, MC38, Renca, 4T1, Sa1N) were quantified for CD4+ and CD8a+ TILs using flow cytometry and immunohistochemistry (IHC), as well as for tumor growth response to Sym021, a humanized PD-1 antibody cross-reactive with mouse PD-1. Radiotracers were generated from F(ab)'2 fragments of rat-anti-mouse CD4 and CD8a antibodies conjugated to the p-SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator and radiolabeled with Zirconium-89 (89Zr-DFO-CD4/89Zr-DFO-CD8a). Tracers were optimized for in vivo PET/CT imaging in CT26 tumor-bearing mice and specificity was evaluated by depletion studies and isotype control imaging. 89Zr-DFO-CD4 and 89Zr-DFO-CD8a PET/CT imaging was conducted in the panel of syngeneic mouse models prior to immunotherapy with Sym021. Results: Syngeneic tumor models were characterized as "hot" or "cold" according to number of TILs determined by flow cytometry and IHC. 89Zr-DFO-CD4 and 89Zr-DFO-CD8a were successfully generated with a radiochemical purity >99% and immunoreactivity >85%. The optimal imaging time-point was 24 hours post-injection of ~1 MBq tracer with 30 µg non-labeled co-dose. Reduced tumor and spleen uptake of 89Zr-DFO-CD8a was observed in CD8a+ depleted mice and the uptake was comparable with that of isotype control (89Zr-DFO-IgG2b) confirming specificity. PET imaging in syngeneic tumor models revealed a varying maximum tumor-to-heart ratio of 89Zr-DFO-CD4 and 89Zr-DFO-CD8a across tumor types and in-between subjects that correlated with individual response to Sym021 at day 10 relative to start of therapy (p=0.0002 and p=0.0354, respectively). The maximum 89Zr-DFO-CD4 tumor-to-heart ratio could be used to stratify mice according to Sym021 therapy response and overall survival was improved in mice with a 89Zr-DFO-CD4 ratio >9 (p=0.0018). Conclusion: We developed 89Zr-DFO-CD4 and 89Zr-DFO-CD8a PET radiotracers for specific detection and whole-body assessment of CD4+ and CD8a+ status. These radiotracers can be used to phenotype preclinical syngeneic mouse tumor models and to predict response to an immune checkpoint inhibitor. We foresee development of such non-invasive in vivo biomarkers for prediction and evaluation of clinical efficacy of immunotherapeutic agents, such as Sym021.

Cyanidin-3-O-glucoside enhanced the function of syngeneic mouse islets transplanted under the kidney capsule or into the portal vein.

BACKGROUND: It was previously shown that islets are susceptible to oxidative stress due to their inherent low antioxidant capacity. Therefore, in this study, we determined whether treatment of mouse islets with an antioxidant cyanidin-3-O-glucoside (C3G) could enhance their function after transplantation under the kidney capsule or into the portal vein. METHODS: B6 mouse islets were treated with various concentrations of C3G, their viability, and the expression of antioxidant (HO-1) and antiapoptotic (Bcl-2 and Survivin) genes were determined. The C3G-treated (1.0 µM) or untreated B6 mouse islets (100, 200, and 400 IEQ) were transplanted under the kidney capsule or into the portal vein of diabetic B6 mice, and their blood glucose levels were monitored for more than 100 days after transplantation. RESULTS: The C3G-treated islets showed higher cell viability compared to untreated control and the expression of HO-1; Bcl-2 and Survivin genes were enhanced in a concentration-dependent manner. All mice that were transplanted with C3G-treated islets achieved normoglycemia faster than recipients of untreated islets. Mice that received 400, 200, or 100 treated islets transplanted under the kidney capsule achieved normoglycemia, whereas only mice that were transplanted with 400 and 200 treated islets into the portal vein achieved normoglycemia. The mean blood glucose levels of mice that received C3G-treated islets were lower compared to those observed in mouse recipients of untreated islets transplanted under the kidney capsule or into the portal vein. CONCLUSIONS: Our results show that C3G could enhance the viability of mouse islets and improve their function after transplantation.