3D bioprinting and the revolution in experimental cancer model systems-A review of developing new models and experiences with in vitro 3D bioprinted breast cancer tissue-mimetic structures.

Growing evidence propagates those alternative technologies (relevant human cell-based-e.g., organ-on-chips or biofabricated models-or artificial intelligence-combined technologies) that could help in vitro test and predict human response and toxicity in medical research more accurately. In vitro disease model developments have great efforts to create and serve the need of reducing and replacing animal experiments and establishing human cell-based in vitro test systems for research use, innovations, and drug tests. We need human cell-based test systems for disease models and experimental cancer research; therefore, in vitro three-dimensional (3D) models have a renaissance, and the rediscovery and development of these technologies are growing ever faster. This recent paper summarises the early history of cell biology/cellular pathology, cell-, tissue culturing, and cancer research models. In addition, we highlight the results of the increasing use of 3D model systems and the 3D bioprinted/biofabricated model developments. Moreover, we present our newly established 3D bioprinted luminal B type breast cancer model system, and the advantages of in vitro 3D models, especially the bioprinted ones. Based on our results and the reviewed developments of in vitro breast cancer models, the heterogeneity and the real in vivo situation of cancer tissues can be represented better by using 3D bioprinted, biofabricated models. However, standardising the 3D bioprinting methods is necessary for future applications in different high-throughput drug tests and patient-derived tumour models. Applying these standardised new models can lead to the point that cancer drug developments will be more successful, efficient, and consequently cost-effective in the near future.

Metabolic Adaptation as Potential Target in Papillary Renal Cell Carcinomas Based on Their In Situ Metabolic Characteristics.

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.

Characterisation of 3D Bioprinted Human Breast Cancer Model for In Vitro Drug and Metabolic Targeting.

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.

Survival of HT29 Cancer Cells Is Affected by IGF1R Inhibition via Modulation of Self-DNA-Triggered TLR9 Signaling and the Autophagy Response.

Purpose: In HT29 colon cancer cells, a close interplay between self-DNA-induced TLR9 signaling and autophagy response was found, with remarkable effects on cell survival and differentiation. IGF1R activation drives the development and malignant progression of colorectal cancer. IGF1R inhibition displays a controversial effect on autophagy. The interrelated roles of IGF1R inhibition and TLR9/autophagy signaling in HT29 cancer cells have not yet been clarified. In our study, we aimed to investigate the complex interplay of IGF1R inhibition and TLR9/autophagy signaling in HT29 cells. Methods: HT29 cells were incubated with tumor-originated self-DNA with or without inhibitors of IGF1R (picropodophyllin), autophagy (chloroquine), and TLR9 (ODN2088), respectively. Cell proliferation and metabolic activity measurements, direct cell counting, NanoString and Taqman gene expression analyses, immunocytochemistry, WES Simple Western blot, and transmission electron microscopy investigations were performed. Results: The concomitant use of tumor-derived self-DNA and IGF1R inhibitors displays anti-proliferative potential, which can be reversed by parallel TLR9 signaling inhibition. The distinct effects of picropodophyllin, ODN2088, and chloroquine per se or in combination on HT29 cell proliferation and autophagy suggest that either the IGF1R-associated or non-associated autophagy machinery is "Janus-faced" regarding its actions on cell proliferation. Autophagy, induced by different combinations of self-DNA and inhibitors is not sufficient to rescue HT29 cells from death but results in the survival of some CD133-positive stem-like HT29 cells. Conclusion: The creation of new types of combined IGF1R, autophagy, and/or TLR9 signaling inhibitors would play a significant role in the development of more personalized anti-tumor therapies for colorectal cancer.

Survival of HT29 cancer cells is influenced by hepatocyte growth factor receptor inhibition through modulation of self-DNA-triggered TLR9-dependent autophagy response.

HGFR activation drives the malignant progression of colorectal cancer, and its inhibition displays anti-autophagic activity. The interrelated role of HGFR inhibition and TLR9/autophagy signaling in HT29 cancer cells subjected to modified self-DNA treatments has not been clarified. We analyzed this complex interplay with cell metabolism and proliferation measurements, TLR9, HGFR and autophagy inhibitory assays and WES Simple Western blot-based autophagy flux measurements, gene expression analyses, immunocytochemistry, and transmission electron microscopy. The overexpression of MyD88 and caspase-3 was associated with enhanced HT29 cell proliferation, suggesting that incubation with self-DNAs could suppress the apoptosis-induced compensatory cell proliferation. HGFR inhibition blocked the proliferation-reducing effect of genomic and hypermethylated, but not that of fragmented DNA. Lowest cell proliferation was achieved with the concomitant use of genomic DNA, HGFR inhibitor, and chloroquine, when the proliferation stimulating effect of STAT3 overexpression could be outweighed by the inhibitory effect of LC3B, indicating the putative involvement of HGFR-mTOR-ULK1 molecular cascade in HGFR inhibitor-mediated autophagy. The most intense cell proliferation was caused by the co-administration of hypermethylated DNA, TLR9 and HGFR inhibitors, when decreased expression of both canonical and non-canonical HGFR signaling pathways and autophagy-related genes was present. The observed ultrastructural changes also support the context-dependent role of HGFR inhibition and autophagy on cell survival and proliferation. Further investigation of the influence of the studied signaling pathways and cellular processes can provide a basis for novel, individualized anti-cancer therapies.

Rapamycin Plus Doxycycline Combination Affects Growth Arrest and Selective Autophagy-Dependent Cell Death in Breast Cancer Cells.

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 WesTM, 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.

The role of metabolic ecosystem in cancer progression - metabolic plasticity and mTOR hyperactivity in tumor tissues.

Despite advancements in cancer management, tumor relapse and metastasis are associated with poor outcomes in many cancers. Over the past decade, oncogene-driven carcinogenesis, dysregulated cellular signaling networks, dynamic changes in the tissue microenvironment, epithelial-mesenchymal transitions, protein expression within regulatory pathways, and their part in tumor progression are described in several studies. However, the complexity of metabolic enzyme expression is considerably under evaluated. Alterations in cellular metabolism determine the individual phenotype and behavior of cells, which is a well-recognized hallmark of cancer progression, especially in the adaptation mechanisms underlying therapy resistance. In metabolic symbiosis, cells compete, communicate, and even feed each other, supervised by tumor cells. Metabolic reprogramming forms a unique fingerprint for each tumor tissue, depending on the cellular content and genetic, epigenetic, and microenvironmental alterations of the developing cancer. Based on its sensing and effector functions, the mechanistic target of rapamycin (mTOR) kinase is considered the master regulator of metabolic adaptation. Moreover, mTOR kinase hyperactivity is associated with poor prognosis in various tumor types. In situ metabolic phenotyping in recent studies highlights the importance of metabolic plasticity, mTOR hyperactivity, and their role in tumor progression. In this review, we update recent developments in metabolic phenotyping of the cancer ecosystem, metabolic symbiosis, and plasticity which could provide new research directions in tumor biology. In addition, we suggest pathomorphological and analytical studies relating to metabolic alterations, mTOR activity, and their associations which are necessary to improve understanding of tumor heterogeneity and expand the therapeutic management of cancer.

In Situ Metabolic Characterisation of Breast Cancer and Its Potential Impact on Therapy.

In spite of tremendous developments in breast cancer treatment, the relatively high incidence of relapsing cases indicates a great need to find new therapeutic strategies in recurrent, metastatic and advanced cases. The bioenergetic needs of growing tumours at the primary site or in metastases-accumulating genomic alterations and further heterogeneity-are supported by metabolic rewiring, an important hallmark of cancer. Adaptation mechanisms as well as altered anabolic and catabolic processes balance according to available nutrients, energy, oxygen demand and overgrowth or therapeutic resistance. Mammalian target of rapamycin (mTOR) hyperactivity may contribute to this metabolic plasticity and progression in breast carcinomas. We set out to assess the metabolic complexity in breast cancer cell lines and primary breast cancer cases. Cellular metabolism and mTOR-related protein expression were characterised in ten cell lines, along with their sensitivity to specific mTOR and other metabolic inhibitors. Selected immunohistochemical reactions were performed on ~100 surgically removed breast cancer specimens. The obtained protein expression scores were correlated with survival and other clinicopathological data. Metabolic and mTOR inhibitor mono-treatments had moderate antiproliferative effects in the studied cell lines in a subtype-independent manner, revealing their high adaptive capacity and survival/growth potential. Immunohistochemical analysis of p-S6, Rictor, lactate dehydrogenase A, glutaminase, fatty acid synthase and carnitine palmitoyltransferase 1A in human samples identified high mTOR activity and potential metabolic plasticity as negative prognostic factors for breast cancer patients, even in subtypes generally considered as low-risk. According to our results, breast cancer is characterised by considerable metabolic diversity, which can be targeted by combining antimetabolic treatments and recent therapies. Alterations in these pathways may provide novel targets for future drug development in breast cancer. We also propose a set of immunostainings for scoring metabolic heterogeneity in individual cases in order to select patients who may benefit from more accurate follow-up and specific therapies.

Inhibition of Metabolic Shift can Decrease Therapy Resistance in Human High-Grade Glioma Cells.

The high-grade brain malignancy, glioblastoma multiforme (GBM), is one of the most aggressive tumours in central nervous system. The developing resistance against recent therapies and the recurrence rate of GBMs are extremely high. In spite several new ongoing trials, GBM therapies could not significantly increase the survival rate of the patients as significantly. The presence of inter- and intra-tumoral heterogeneity of GBMs arise the problem to find both the pre-existing potential resistant clones and the cellular processes which promote the adaptation mechanisms such as multidrug resistance, stem cell-ness or metabolic alterations, etc. In our work, the in situ metabolic heterogeneity of high-grade human glioblastoma cases were analysed by immunohistochemistry using tissue-microarray. The potential importance of the detected metabolic heterogeneity was tested in three glioma cell lines (grade III-IV) using protein expression analyses (Western blot and WES Simple) and therapeutic drug (temozolomide), metabolic inhibitor treatments (including glutaminase inhibitor) to compare the effects of rapamycin (RAPA) and glutaminase inhibitor combinations in vitro (Alamar Blue and SRB tests). The importance of individual differences and metabolic alterations were observed in mono-therapeutic failures, especially the enhanced Rictor expressions after different mono-treatments in correlation to lower sensitivity (temozolomide, doxycycline, etomoxir, BPTES). RAPA combinations with other metabolic inhibitors were the best strategies except for RAPA+glutaminase inhibitor. These observations underline the importance of multi-targeting metabolic pathways. Finally, our data suggest that the detected metabolic heterogeneity (the high mTORC2 complex activity, enhanced expression of Rictor, p-Akt, p-S6, CPT1A, and LDHA enzymes in glioma cases) and the microenvironmental or treatment induced metabolic shift can be potential targets in combination therapy. Therefore, it should be considered to map tissue heterogeneity and alterations with several cellular metabolism markers in biopsy materials after applying recently available or new treatments.

Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells.

BACKGROUND: Glioma is the most common highly aggressive, primary adult brain tumour. Clinical data show that therapeutic approaches cannot reach the expectations in patients, thus gliomas are mainly incurable diseases. Tumour cells can adapt rapidly to alterations during therapeutic treatments related to their metabolic rewiring and profound heterogeneity in tissue environment. Renewed interests aim to develop effective treatments targeting angiogenesis, kinase activity and/or cellular metabolism. mTOR (mammalian target of rapamycin), whose hyper-activation is characteristic for many tumours, promotes metabolic alterations, macromolecule biosynthesis, cellular growth and survival. Unfortunately, mTOR inhibitors with their lower toxicity have not resulted in appreciable survival benefit. Analysing mTOR inhibitor sensitivity, other metabolism targeting treatments and their combinations could help to find potential agents and biomarkers for therapeutic development in glioma patients. METHODS: In vitro proliferation assays, protein expression and metabolite concentration analyses were used to study the effects of mTOR inhibitors, other metabolic treatments and their combinations in glioma cell lines. Furthermore, mTOR activity and cellular metabolism related protein expression patterns were also investigated by immunohistochemistry in human biopsies. Temozolomide and/or rapamycin treatments altered the expressions of enzymes related to lipid synthesis, glycolysis and mitochondrial functions as consequences of metabolic adaptation; therefore, other anti-metabolic drugs (chloroquine, etomoxir, doxycycline) were combined in vitro. RESULTS: Our results suggest that co-targeting metabolic pathways had tumour cell dependent additive/synergistic effects related to mTOR and metabolic protein expression patterns cell line dependently. Drug combinations, especially rapamycin + doxycycline may have promising anti-tumour effect in gliomas. Additionally, our immunohistochemistry results suggest that metabolic and mTOR activity alterations are not related to the recent glioma classification, and these protein expression profiles show individual differences in patients' materials. CONCLUSIONS: Based on these, combinations of different new/old drugs targeting cellular metabolism could be promising to inhibit high adaptation capacity of tumour cells depending on their metabolic shifts. Relating to this, such a development of current therapy needs to find special biomarkers to characterise metabolic heterogeneity of gliomas.