In preclinical cancer research, three-dimensional (3D) cell culture systems such as multicellular spheroids and organoids are becoming increasingly important. They provide valuable information before studies on animal models begin and, in some cases, are even suitable for reducing or replacing animal experiments. Furthermore, they recapitulate microtumors, metastases, and the tumor microenvironment much better than monolayer culture systems could. Three-dimensional models show higher structural complexity and diverse cell interactions while reflecting (patho)physiological phenomena such as oxygen and nutrient gradients in the course of their growth or development. These interactions and properties are of great importance for understanding the pathophysiological importance of stromal cells and the extracellular matrix for tumor progression, treatment response, or resistance mechanisms of solid tumors. Special emphasis is placed on co-cultivation with tumor-associated cells, which further increases the predictive value of 3D models, e.g., for drug development. The aim of this overview is to shed light on selected 3D models and their advantages and disadvantages, especially from the radiopharmacist’s point of view with focus on the suitability of 3D models for the radiopharmacological characterization of novel radiotracers and radiotherapeutics. Special attention is paid to pancreatic ductal adenocarcinoma (PDAC) as a predestined target for the development of new radionuclide-based theranostics.

Glioblastoma (GBM) is the most aggressive form of malignant gliomas, with the eicosanoid-synthesizing enzyme cyclooxygenase-2 (COX-2) playing a pivotal role in its progression via the COX-2/prostaglandin E2/4 axis. COX-2 upregulations in tumor cells induces a pro-inflammatory tumor microenvironment (TME), affecting the behavior of invading bone marrow-derived macrophages (Mϕ) and brain-resident microglia (MG) through unclear autocrine and paracrine mechanisms. Using CRISPR/Cas9 technology, we generated COX-2 knockout U87 glioblastoma cells. In spheroids and in vivo xenografts, this resulted in a significant inhibition of tumorigenic properties, while not observed in standard adherent monolayer culture. Here, the knockout induced a G1 cell cycle arrest in adherent cells, accompanied by increased ROS, mitochondrial activity, and cytochrome c-mediated apoptosis. In spheroids and xenograft models, COX-2 knockout led to notable growth delays and increased cell death, characterized by features of both apoptosis and autophagy. Interestingly, these effects were partially reversed in subcutaneous xenografts after co-culture with Mϕ, while co-culture with MG enhanced the growth-suppressive effects. In an orthotopic model, COX-2 knockout tumors displayed reduced proliferation (fewer Ki-67 positive cells), increased numbers of GFAP-positive astrocytes, and signs of membrane blebbing. These findings highlight the potential of COX-2 knockout and suppression as a therapeutic strategy in GBM, particularly when combined with suppression of infiltrating macrophages and stabilization of resident microglia populations to enhance anti-tumor effects.

Background/Objective Pancreatic stellate cells (PSCs) in pancreatic adenocarcinoma (PDAC) are producing extracellular matrix, which promotes the formation of a dense fibrotic microenvironment. This makes PDAC a highly heterogeneous tumor-stroma-driven entity, associated with reduced perfusion, limited oxygen supply, high interstitial fluid pressure, and limited bioavailability of therapeutic agents. Methods In this study, spheroid and tumor xenograft models of human PSCs and PanC-1 cells were characterized radiopharmacologically using a combined positron emission tomography (PET) radiotracer approach. [¹⁸F]FDG, [¹⁸F]FMISO, and [¹⁸F]FAPI-74 were employed to monitor metabolic activity, hypoxic metabolic state, and functional expression of fibroblast activation protein alpha (FAPα), a marker of activated PSCs. Results In vitro, PanC-1 and multi-cellular tumor spheroids demonstrated comparable glucose uptake and hypoxia, whereas FAPα expression was significantly higher in PSC spheroids. In vivo, glucose uptake as well as the transition to hypoxia were comparable in PanC-1 and multi-cellular xenograft models. In mice injected with PSCs, FAPα expression decreased over a period of four weeks post-injection, which was attributed to the successive death of PSCs. In contrast, FAPα expression increased in both PanC-1 and multi-cellular xenograft models over time due to invasion of mouse fibroblasts. Conclusion The presented models are suitable for subsequently characterizing stromal cell-induced metabolic changes in tumors using noninvasive molecular imaging techniques.

Pancreatic ductal adenocarcinoma (PDAC) is a highly heterogeneous cancer with a severe stromal reaction mediated by pancreatic stellate cells (PSCs), leading to increased resistance to chemotherapy and radiotherapy. Following a repurposing concept, this preclinical study investigates the potential of approved drugs, known to be modulators of cellular copper transport, in combination with cisplatin for therapeutic approaches in PDAC. Two major strategies were pursued: (i) inhibiting copper transporters ATP7A and B with tranilast (TR) and omeprazole (OM) to block the cellular copper and, potentially, also cisplatin efflux, and (ii) using the chelator elesclomol (ES) to elevate intracellular copper and cisplatin levels. Human cell lines PanC-1 (PDAC), HPaSteC (PSC), and their co-culture, as well as the hepatocellular carcinoma cell line HepG2 as a reference model, were used. In addition to an analysis of the expression of copper transport proteins, the dynamics of cellular copper uptake and transport were monitored using a [⁶⁴Cu]CuCl₂ radiotracer approach. In vitro, all drugs enhanced cellular copper uptake and/or reduced copper efflux. Moreover, all drugs contributed to the enhanced cellular anticancer activity of cisplatin, with ES being the most effective compound. The results suggest that the targeted modulation of copper transport mechanisms may offer novel adjuvant approaches for the treatment of PDAC.