Well-differentiated gastroenteropancreatic neuroendocrine neoplasms are rare tumors with a slow proliferation. They are virtually resistant to many DNA-damaging therapeutic approaches, such as chemo- and external beam therapy, which might be overcome by DNA damage inhibition induced by proteasome inhibitors such as bortezomib. In this study, we assessed several combined treatment modalities in vitro and in vivo. By cell-based functional analyses, in a 3D in ovo and an orthotopic mouse model, we demonstrated sensitizing effects of bortezomib combined with cisplatin, radiation and peptide receptor radionuclide therapy (PRRT). By gene expression profiling and western blot, we explored the underlying mechanisms, which resulted in an impaired DNA damage repair. Therapy-induced DNA damage triggered extrinsic proapoptotic signaling as well as the induction of cell cycle arrest, leading to a decreased vital tumor volume and altered tissue composition shown by magnetic resonance imaging and F-18-FDG-PET in vivo, however with no significant additional benefit related to PRRT alone. We demonstrated that bortezomib has short-term sensitizing effects when combined with DNA damaging therapy by interfering with DNA repair in vitro and in ovo. Nevertheless, due to high tumor heterogeneity after PRRT in long-term observations, we were not able to prove a therapeutic advantage of bortezomib-combined PRRT in an in vivo mouse model.

This study identified and confirmed angiotensin II (ATII) as a strong activator of signaling in neuroendocrine neoplasm (NEN) cells. Expression analyses of the ATII receptor type 1 (AGTR1) revealed an upregulation of mRNA levels (RT-qPCR) and radioligand binding (autoradiography) in small-intestinal (n = 71) NEN tissues compared to controls (n = 25). NEN cells with high AGTR1 expression exhibited concentration-dependent calcium mobilization and chromogranin A secretion upon stimulation with ATII, blocked by AGTR1 antagonism and Gαq inhibition. ATII also stimulated serotonin secretion from BON cells. AGTR1 ligand saralasin was coupled to a near-infrared fluorescent (NIRF) dye and tested for its biodistribution in a nude mouse model bearing AGTR1-positive BON and negative QGP-1 xenograft tumors. NIRF imaging showed significantly higher uptake in BON tumors. This proof of concept establishes AGTR1 as a novel target in NEN, paving the way for translational chelator-based probes for diagnostic PET imaging and radioligand therapy.

Patient-derived 3D cell culture models (PD3D®) developed as a powerful tool for disease modelling, biomarker and drug discovery. Currently, they are gaining increasing significance in the field of personalized oncology, as they recapitulate the histopathology of the original tumor tissue and preserve its genetic markup. PD3D® can be used to model intratumoral heterogeneity and for medium and high throughput drug screens. Using a reverse clinical engineering approach, PD3D® models allow identification of chemoresistance/sensitivity signatures (i.e., biomarkers) and can be applied in personalized oncology to identify treatment for an individual patient. We successfully established PD3D® models from more than 300 tumor tissue samples, ranging from more prevalent cancers like colorectal, breast and pancreas carcinoma, to rare tumor entities including various sarcoma types and thymoma. PD3D® models from different tumor entities differ in morphology and culture media requirements. When treating PD3D® from the same tumor entity with standard of care drugs, we found that their response differed, as does clinical response of patients. Furthermore, we successfully used PD3D® models to identify a biomarker for predicting chemosensitivity towards a targeted drug. For application of PD3D® in truly personalized oncology, we developed a protocol that allows us to generate a PD3D® culture and perform a drug sensitivity assay for an individual patient within a therapy-relevant timeframe. Using this protocol, we identified a combination therapy for a pretreated, metastasized appendix carcinoma within 29 days, that resulted in stable disease of the patient. In conclusion, PD3D® models can be derived from various cancer entities and used to analyze drug response in cohorts of models for drug development or identification of signatures related to drug resistance or sensitivity. Furthermore, PD3D® models can be used to predict a patient tumor’s drug response in a personalized manner, supporting the oncologist to identify the best treatment option for the patient. Citation Format: Alina Pflaume, Samantha Exner, Katja Herrera-Glomm, Jürgen Loskutov, Ulrike Pfohl, Manuela Regenbrecht, Sushmitha Sankarasubramanian, Lena Wedeken, Sabine Finkler, Larissa Ruhe, Quirin Graf Adelmann, Christoph Reinhard, Philipp Stroebel, Christian R. Regenbrecht. PD3D^®models: New age in cancer research and clinical diagnostics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6223.

Peptide receptor radioligand therapy (PRRT) has evolved as an important second-line treatment option in the management of inoperable and metastatic neuroendocrine neoplasms (NEN). Though high radiation doses can be delivered to the tumors, complete remission is still rare. Radiosensitization prior to PRRT is therefore considered to be a promising strategy to improve the treatment effect. In this study, effect and mechanism of mTOR inhibitors were investigated in a comprehensive panel of five NEN cell lines (BON, QGP-1, LCC-18, H727, UMC-11), employing assays for cellular proliferation, clonogenic survival, cell cycle modification and signaling. mTOR inhibition lead to growth arrest with a biphasic concentration-response pattern: a partial response at approximately 1 nM and full response at micromolar concentrations (8-48 μM). All cell lines demonstrated elevated p70S6K phosphorylation yet also increased phosphorylation of counterregulatory Akt. The pulmonary NEN cell line UMC-11 showed the lowest induction of phospho-Akt and strongest growth arrest by mTOR inhibitors. Radiation sensitivity of the cells (50% reduction versus control) was found to range between 4 and 8 Gy. Further, mTOR inhibition was employed together with irradiation to evaluate radiosensitizing effects of this combination treatment. mTOR inhibition was found to radiosensitize all five NEN cells in an additive manner with a moderate overall effect. The radiation-induced G2/M arrest was diminished under combination treatment, leading to an increased G1 arrest. Further investigation involving a suitable animal model as well as radioligand application such as ¹⁷⁷ Lu-DOTATATE or ¹⁷⁷ Lu-DOTATOC will have to demonstrate the full potential of this strategy for radiosensitization in NEN.