Lighting up PNET: Creating murine models with a novel bioluminescent cell line.

Abstract

e16352 Background: Pancreatic neuroendocrine tumors (PNETs) are rare malignancies characterized by dysregulation of complex molecular signaling pathways. Despite advancements in their biology, treatment options for advanced PNETs remain limited to surgery. This highlights an urgent need for novel therapies, which require developing cost-effective and biologically relevant in vivo models, a current gap in the field. Methods: Murine enteroendocrine tumor-derived STC-1 cells were transduced with a lentivirus encoding GFP and firefly luciferase (F-Luc), selected for puromycin resistance, and purified using fluorescence-activated cell sorting (FACS) to ensure stable GFP and F-Luc expression. These cells were implanted into immunocompromised mice via subcutaneous (SQ), renal capsule (RC), or orthotopic pancreatic (OP) injection. Tumor growth was monitored by SPECTRAL bioluminescence live imaging, while histological and immunohistochemical (IHC) analyses were performed to confirm PNET characteristics. Results: Histological analysis verified tumor formation in all three xenograft models. The SQ model showed rapid growth and produced larger tumors (43+/-25.6 mm) but lacked key PNET characteristics, as evidenced by H&E and IHC staining. The RC model supported moderate tumor growth (68+/-17.3 mm) but lacked expression of specific PNET-specific markers. The OP model, though technically challenging, demonstrated robust tumor growth (108+/-16.3 mm) within 5 weeks and closely resembled well-differentiated human PNETs, as confirmed by H&E morphology, IHC, and co-immunofluorescence staining for PNET markers with GFP. Conclusions: We systematically compared the SQ, RC, and OP PNET xenograft models. The SQ and RC models are technically simple but fail to replicate human PNET characteristics fully. Despite being more technically challenging, the OP model accurately recapitulates human PNET features, making it ideal for studying the molecular mechanism underlying PNET initiation, progression, and metastasis. This is the first report of an orthotopic injected STC1-PNET xenograft model. The STC-1/OP xenograft model is a cost-effective and efficient system for studying the molecular mechanisms underlying PNET. It also serves as a valuable platform for therapeutic drug testing, given its strong resemblance to human PNETs. Comparison of in vivo bioluminescent PNET methods: IHC, tumor size. Number of STC1 cells being Injected (M) Surgical time per injection (min) Average tumor size 6 weeks of implantation (mm) PNET development after 6 weeks of implantation (H&E) Chromogranin A IHC staining Synaptophysin IHC staining Subcutaneous 4 10 61.7 Negative Negative Negative Renal capsule 0.5 20 68.7 Equivocal Positive Negative Orthotopic pancreas 0.5 15 118.3 Well-differentiated Grade 1 PNET Positive Positive