Characterization of Renal Cell Carcinoma Heterotypic 3D Co-Cultures with Immune Cell Subsets.

Two-dimensional cell culture-based platforms are easy and reproducible, however, they do not resemble the heterotypic cell-cell interactions or the complex tumor microenvironment. These parameters influence the treatment response and the cancer cell fate. Platforms to study the efficacy of anti-cancer treatments and their impact on the tumor microenvironment are currently being developed. In this study, we established robust, reproducible, and easy-to-use short-term spheroid cultures to mimic clear cell renal cell carcinoma (ccRCC). These 3D co-cultures included human endothelial cells, fibroblasts, immune cell subsets, and ccRCC cell lines, both parental and sunitinib-resistant. During spheroid formation, cells induce the production and secretion of the extracellular matrix. We monitored immune cell infiltration, surface protein expression, and the response to a treatment showing that the immune cells infiltrated the spheroid co-cultures within 6 h. Treatment with an optimized drug combination or the small molecule-based targeted drug sunitinib increased immune cell infiltration significantly. Assessing the therapeutic potential of this drug combination in this platform, we revealed that the expression of PD-L1 increased in 3D co-cultures. The cost- and time-effective establishment of our 3D co-culture model and its application as a pre-clinical drug screening platform can facilitate the treatment validation and clinical translation.

Diclofenac Potentiates Sorafenib-Based Treatments of Hepatocellular Carcinoma by Enhancing Oxidative Stress.

Sorafenib is the first developed systemic treatment for advanced forms of hepatocellular carcinoma, which constitutes the most frequent form of primary liver cancers and is a major global health burden. Although statistically significant, the positive effect of sorafenib on median survival remains modest, highlighting the need to develop novel therapeutic approaches. In this report, we introduce diclofenac, a nonsteroidal anti-inflammatory drug, as a potent catalyzer of sorafenib anticancer efficacy. Treatment of three different hepatocellular cancer cells (Huh-7, HepG2, and PLC-PRF-5) with sorafenib (5 µM, 24 h) and diclofenac (100 µM, 24 h) significantly increased cancer cell death compared to sorafenib or diclofenac alone. Anti-oxidant compounds, including N-acetyl-cysteine and ascorbic acid, reversed the deleterious effects of diclofenac/sorafenib co-therapy, suggesting that the generation of toxic levels of oxidative stress was responsible for cell death. Accordingly, whereas diclofenac increased production of mitochondrial oxygen reactive species, sorafenib decreased concentrations of glutathione. We further show that tumor burden was significantly diminished in mice bearing tumor xenografts following sorafenib/diclofenac co-therapy when compared to sorafenib or diclofenac alone. Taken together, these results highlight the anticancer benefits of sorafenib/diclofenac co-therapy in hepatocellular carcinoma. They further indicate that combining sorafenib with compounds that increase oxidative stress represents a valuable treatment strategy in hepatocellular carcinoma.

Rebound pathway overactivation by cancer cells following discontinuation of PI3K or mTOR inhibition promotes cancer cell growth.

Whilst effects of anti-cancer drugs have been thoroughly explored, little is known about the repercussion of drug cessation. However, this has important clinical relevance since several clinical protocols such as intermittent drug scheduling lead to frequent drug discontinuation. In this study, we have thus investigated the consequences of withdrawal of agents that target the PI3K/AKT/mTOR signaling pathway in cancer cells. We report that washout of kinase inhibitors of mTOR or PI3K inhibitors led to a rapid and sustainable overactivation of AKT. Consequently, proliferation of tumor cells was significantly higher following drug washout in cancer cells that were pre-treated with mTOR or PI3K inhibitors compared to untreated cells. This effect was prevented by the addition of an AKT inhibitor following drug washout. Rebound AKT overactivation induced by mTOR or PI3K inhibitors discontinuation was mediated by IGF-1R, as demonstrated by its prevention in the presence of an IGF-1R inhibitor and by increased IGF-1R phosphorylation in treated cells versus control cells. Taken together, our results show that discontinuation of PI3K or mTOR inhibitors results in AKT overactivation that promotes tumor growth. They further highlight the benefit of adding an AKT inhibitor following cessation of PI3K or mTOR inhibitors.

Genetic manipulation of Vibrio cholerae by combining natural transformation with FLP recombination.

Even though Vibrio cholerae is a well-known human pathogen, it is also a normal member of aquatic habitats. Within this environment it often forms biofilms on the chitin-containing exoskeleton of crustaceans and their molts. Chitin not only serves as nutrient source but also induces a developmental program called natural competence. Naturally competent bacteria take up free DNA and integrate it into their genome by homologous recombination, thereby becoming naturally transformed. In this study, we made use of the knowledge on the environmental lifestyle of V. cholerae to genetically manipulate its genome. We achieved this by combining the methods of chitin-induced natural transformation and Flp recombination. Using this approach, we disrupted several genes by insertion of FRT-site-flanked antibiotic-resistance cassettes. The cassettes were subsequently excised by induction of the Flp recombinase, which acts on the FRT sites. This method represents a simplified and faster alternative to standard gene deletion techniques, which often depend on bacterial conjugation and the availability of suicide vectors.