ABSTRACT
The limited recapitulation of critical cancer features in 2D cultures causes poor translatability of preclinical results from in vitro assays to in vivo tumor models. This contributes to slow drug development with a low success rate. 3D cultures better recapitulate the tumor microenvironment, enabling more accurate predictions when screening drug candidates and improving the development of chemotherapeutics. Platinum (Pt) (IV) compounds are promising prodrugs designed to reduce the severe systemic toxicity of widely used Food and Drug Administration (FDA)-approved Pt(II) drugs such as cisplatin. Here, this work presents spatiotemporal evaluations in 3D colorectal cancer (CRC) spheroids of mitochondria-targeting Pt(IV) complexes. CRC spheroids provide a greater pathophysiological recapitulation of in vivo tumors than 2D cultures by a marked upregulation of the ABCG2 chemoresistance marker expression. Furthermore, new 3D-staining protocols are introduced to evaluate the real-time decrease in mitochondria membrane potential (ΔΨ) in CRC spheroids, and a Pt-sensing dye to quantify the Pt mitochondrial accumulation. Finally, this work demonstrates a correlation between in vitro results and the efficacy of the compounds in vivo. Overall, the CRC spheroids represent a fast and cost-effective model to assess the behavior of Pt compounds in vitro and predict their translational potential in CRC treatment.
ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies nowadays. The available chemo- and immunotherapies are often ineffective in treating PDAC due to its immunosuppressive and highly desmoplastic tumor immune microenvironment (TIME), which is hardly reproduced in the existing preclinical models. The PDAC TIME results from a peculiar spatial organization between different cell types. For this reason, developing new human models recapitulating the tissue organization and cell heterogeneity of PDAC is highly desirable. We developed human 3D heterocellular tumor spheroids of PDAC formed by cancer cells, endothelial cells, pancreatic stellate cells (PSC), and monocytes. As a control, we formed spheroids using immortalized epithelial pancreatic ductal cells (non-cancerous spheroids) with cellular heterogeneity similar to the tumor spheroids. Normal spheroids containing endothelial cells formed a complex 3D endothelial network significantly compromised in tumor spheroids. Monocyte/macrophages within the 4-culture tumor spheroids were characterized by a higher expression of CD163, CD206, PD-L1, and CD40 than those in the non-cancerous spheroids suggesting their differentiation towards an immunosuppressive phenotype. The heterocellular tumor spheroids presented a hypoxic core populated with PSC and monocytes/macrophages. The 4-culture tumor spheroids were characterized by spatial proximity of PSC and monocytes to the endothelial cells and a cytokine signature with increased concentrations of CXCL10, CCL2, and IL-6, which have been observed in PDAC patients and associated with poor survival. Further, 4-culture tumor spheroids decreased the concentrations of T-cell chemoattracting cytokines, i.e., CCL4, CCL5, and CXCL9, when compared with the non-cancerous spheroids, revealing a critical immunosuppressive feature of the different types of cells forming the tumor spheroids. Our results showed that the 4-culture tumor spheroids better resembled some critical features of patients' PDAC TIME than monoculture tumor spheroids. Using the proposed human 3D spheroid model for therapy testing at the preclinical stage may reveal pitfalls of chemo- and immuno-therapies to help the development of better anti-tumor therapies.
ABSTRACT
Giant clams harbor three genera of symbiotic dinoflagellates (Symbiodinium, Cladocopium, and Durusdinium) as extracellular symbionts (zooxanthellae). While symbiotic dinoflagellates can synthesize amino acids to benefit the host, they are nitrogen-deficient. Hence, the host must supply them with nitrogen including urea, which can be degraded to ammonia and carbon dioxide by urease (URE). Here, we report three complete coding cDNA sequences of URE, one for each genus of dinoflagellate, obtained from the colorful outer mantle of the giant clam, Tridacna squamosa. The outer mantle had higher transcript level of Tridacna squamosa zooxanthellae URE (TSZURE) than the whitish inner mantle, foot muscle, hepatopancreas, and ctenidium. TSZURE was immunolocalized strongly and atypically in the plastid, moderately in the cytoplasm, and weakly in the cell wall and plasma membrane of symbiotic dinoflagellates. In the outer mantle, illumination upregulated the protein abundance of TSZURE, which could enhance urea degradation in photosynthesizing dinoflagellates. The urea-nitrogen released could then augment synthesis of amino acids to be shared with the host for its general needs. Illumination also enhanced gene and protein expression levels of TSZURE/TSZURE in the inner mantle and foot muscle, which contain only small quantities of symbiotic dinoflagellate, have no iridocyte, and lack direct exposure to light. With low phototrophic potential, dinoflagellates in the inner mantle and foot muscle might need to absorb carbohydrates in order to assimilate the urea-nitrogen into amino acids. Amino acids donated by dinoflagellates to the inner mantle and the foot muscle could be used especially for synthesis of organic matrix needed for light-enhanced shell formation and muscle protein, respectively.
