Monolayer cultures, the less standard three-dimensional (3D) culturing systems, and xenografts are the main tools used in current basic and drug development studies of cancer research. The aim of biofabrication is to design and construct a more representative in vivo 3D environment, replacing two-dimensional (2D) cell cultures. Here, we aim to provide a complex comparative analysis of 2D and 3D spheroid culturing, and 3D bioprinted and xenografted breast cancer models. We established a protocol to produce alginate-based hydrogel bioink for 3D bioprinting and the long-term culturing of tumour cells in vitro. Cell proliferation and tumourigenicity were assessed with various tests. Additionally, the results of rapamycin, doxycycline and doxorubicin monotreatments and combinations were also compared. The sensitivity and protein expression profile of 3D bioprinted tissue-mimetic scaffolds showed the highest similarity to the less drug-sensitive xenograft models. Several metabolic protein expressions were examined, and the in situ tissue heterogeneity representing the characteristics of human breast cancers was also verified in 3D bioprinted and cultured tissue-mimetic structures. Our results provide additional steps in the direction of representing in vivo 3D situations in in vitro studies. Future use of these models could help to reduce the number of animal experiments and increase the success rate of clinical phase trials.

Metabolic alteration is characteristic during tumour growth and therapy; however, targeting metabolic rewiring could overcome therapy resistance. mTOR hyperactivity, autophagy and other metabolic processes, including mitochondrial functions, could be targeted in breast cancer progression. We investigated the growth inhibitory mechanism of rapamycin + doxycycline treatment in human breast cancer model systems. Cell cycle and cell viability, including apoptotic and necrotic cell death, were analysed using flow cytometry, caspase activity measurements and caspase-3 immunostainings. mTOR-, autophagy-, necroptosis-related proteins and treatment-induced morphological alterations were analysed by Wes^TM, Western blot, immunostainings and transmission electron microscopy. The rapamycin + doxycycline combination decreased tumour proliferation in about 2/3rd of the investigated cell lines. The continuous treatment reduced tumour growth significantly both in vivo and in vitro. The effect after short-term treatment was reversible; however, autophagic vacuoles and degrading mitochondria were detected simultaneously, and the presence of mitophagy was also observed after the long-term rapamycin + doxycycline combination treatment. The rapamycin + doxycycline combination did not cause apoptosis or necrosis/necroptosis, but the alterations in autophagy- and mitochondria-related protein levels (LC3-B-II/I, p62, MitoTracker, TOM20 and certain co-stainings) were correlated to autophagy induction and mitophagy, without mitochondria repopulation. Based on these results, we suggest considering inducing metabolic stress and targeting mTOR hyperactivity and mitochondrial functions in combined anti-cancer treatments.

Drug repositioning is a high-priority and feasible strategy in the field of oncology research, where the unmet medical needs are continuously unbalanced. Disulfiram is a potential non-chemotherapeutic, adjuvant anticancer agent. However, the clinical translation is limited by the drug's poor bioavailability. Therefore, the molecular encapsulation of disulfiram with cyclodextrins is evaluated to enhance the solubility and stability of the drug. The present work describes for the first time the complexation of disulfiram with randomly methylated-β-cyclodextrin. A parallel analytical andin vitrobiological comparison of disulfiram inclusion complexes with hydroxypropyl-β-cyclodextrin, randomly methylated-β-cyclodextrin and sulfobutylether-β-cyclodextrin is conducted. A significant drug solubility enhancement by about 1000-folds and fast dissolution in 1 min is demonstrated. Thein vitrodissolution-permeation studies and proliferation assays demonstrate the solubility-dependent efficacy of the drug. Throughout the different cancer cell lines' characteristics and disulfiram unspecific antitumoral activity, the inhibitory efficacy of the cyclodextrin encapsulated drug on melanoma (IC₅₀ about 100 nM) and on glioblastoma (IC₅₀ about 7000 nM) cell lines differ by a magnitude. This pre-formulation screening experiment serves as a proof of concept of using cyclodextrin encapsulation as a platform tool for further drug delivery development in repositioning areas.

Lung carcinoma is one of the most common cancer types for both men and women. Despite recent breakthroughs in targeted therapy and immunotherapy, it is characterized by a high metastatic rate, which can significantly affect quality of life and prognosis. Rictor (encoded by the RICTOR gene) is known as a scaffold protein for the multiprotein complex mTORC2. Among its diverse roles in regulating essential cellular functions, mTORC2 also facilitates epithelial–mesenchymal transition and metastasis formation. Amplification of the RICTOR gene and subsequent overexpression of the Rictor protein can result in the activation of mTORC2, which promotes cell survival and migration. Based on recent studies, RICTOR amplification or Rictor overexpression can serve as a marker for mTORC2 activation, which in turn provides a promising druggable target. Although selective inhibitors of Rictor and the Rictor-mTOR association are only in a preclinical phase, they seem to be potent novel approaches to reduce tumor cell migration and metastasis formation. Here, we summarize recent advances that support an important role for Rictor and mTORC2 as potential therapeutic targets in the treatment of lung cancer. This is a traditional (narrative) review based on Pubmed and Google Scholar searches for the following keywords: Rictor, RICTOR amplification, mTORC2, Rictor complexes, lung cancer, metastasis, progression, mTOR inhibitors.

Metabolic characteristics of kidney cancers have mainly been obtained from the most frequent clear cell renal cell carcinoma (CCRCC) studies. Moreover, the bioenergetic perturbances that affect metabolic adaptation possibilities of papillary renal cell carcinoma (PRCC) have not yet been detailed. Therefore, our study aimed to analyze the in situ metabolic features of PRCC vs. CCRCC tissues and compared the metabolic characteristics of PRCC, CCRCC, and normal tubular epithelial cell lines. The protein and mRNA expressions of the molecular elements in mammalian target of rapamycin (mTOR) and additional metabolic pathways were analyzed in human PRCC cases compared to CCRCC. The metabolic protein expression pattern, metabolite content, mTOR, and metabolic inhibitor sensitivity of renal carcinoma cell lines were also studied and compared with tubular epithelial cells, as “normal” control. We observed higher protein expressions of the “alternative bioenergetic pathway” elements, in correlation with the possible higher glutamine and acetate consumption in PRCC cells instead of higher glycolytic and mTOR activity in CCRCCs. Increased expression of certain metabolic pathway markers correlates with the detected differences in metabolite ratios, as well. The lower lactate/pyruvate, lactate/malate, and higher pyruvate/citrate intracellular metabolite ratios in PRCC compared to CCRCC cell lines suggest that ACHN (PRCC) have lower Warburg glycolytic capacity, less pronounced pyruvate to lactate producing activity and shifted OXPHOS phenotype. However, both studied renal carcinoma cell lines showed higher mTOR activity than tubular epithelial cells cultured in vitro, the metabolite ratio, the enzyme expression profiles, and the higher mitochondrial content also suggest increased importance of mitochondrial functions, including mitochondrial OXPHOS in PRCCs. Additionally, PRCC cells showed significant mTOR inhibitor sensitivity and the used metabolic inhibitors increased the effect of rapamycin in combined treatments. Our study revealed in situ metabolic differences in mTOR and metabolic protein expression patterns of human PRCC and CCRCC tissues as well as in cell lines. These underline the importance in the development of specific new treatment strategies, new mTOR inhibitors, and other anti-metabolic drug combinations in PRCC therapy.