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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.

Activation of the mTOR pathway has been observed in osteosarcoma, however the inhibition of mammalian target of rapamycin (mTOR) complex 1 has had limited results in osteosarcoma treatment. Certain metabolic pathways can be altered by mTOR activation, which can affect survival. Our aim was to characterize the mTOR profile and certain metabolic alterations in pediatric osteosarcoma to determine the interactions between the mTOR pathway and metabolic pathways. We performed immunohistochemistry on 28 samples to analyze the expression of mTOR complexes such as phospho-mTOR (pmTOR), phosphorylated ribosomal S6 (pS6), and rapamycin-insensitive companion of mTOR (rictor). To characterize metabolic pathway markers, we investigated the expression of phosphofructokinase (PFK), lactate dehydrogenase-A (LDHA), β-F1-ATPase (ATPB), glucose-6-phosphate dehydrogenase (G6PDH), glutaminase (GLS), fatty acid synthetase (FASN), and carnitin-O-palmitoyltransferase-1 (CPT1A). In total, 61% of the cases showed low mTOR activity, but higher pmTOR expression was associated with poor histological response to chemotherapy and osteoblastic subtype. Rictor expression was higher in metastatic disease and older age at the time of diagnosis. Our findings suggest the importance of the Warburg-effect, pentose-phosphate pathway, glutamine demand, and fatty-acid beta oxidation in osteosarcoma cells. mTOR activation is linked to several metabolic pathways. We suggest performing a detailed investigation of the mTOR profile before considering mTORC1 inhibitor therapy. Our findings highlight that targeting certain metabolic pathways could be an alternative therapeutic approach.

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.

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.

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&mdash, accumulating genomic alterations and further heterogeneity&mdash, 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.

mTOR activation has been observed in rhabdomyosarcoma (RMS), however, mTOR complex (mTORC) 1 inhibition has had limited success thus far. mTOR activation alters the metabolic pathways, which is linked to survival and metastasis. These pathways have not been thoroughly analyzed in RMSs. We performed immunohistochemistry on 65 samples to analyze the expression of mTOR complexes (pmTOR, pS6, Rictor), and several metabolic enzymes (phosphofructokinase, lactate dehydrogenase-A, &beta, F1-ATPase, glucose-6-phosphate dehydrogenase, glutaminase). RICTOR amplification, as a potential mechanism of Rictor overexpression, was analyzed by FISH and digital droplet PCR. In total, 64% of the studied primary samples showed mTOR activity with an mTORC2 dominance (82%). Chemotherapy did not cause any relevant change in mTOR activity. Elevated mTOR activity was associated with a worse prognosis in relapsed cases. RICTOR amplification was not confirmed in any of the cases. Our findings suggest the importance of the Warburg effect and the pentose-phosphate pathway beside a glutamine demand in RMS cells. The expression pattern of the studied mTOR markers can explain the inefficacy of mTORC1 inhibitor therapy. Therefore, we suggest performing a detailed investigation of the mTOR profile before administering mTORC1 inhibitor therapy. Furthermore, our findings highlight that targeting the metabolic plasticity could be an alternative therapeutic approach.

Pheochromocytoma/paragangliomas (Pheo/PGL) are rare endocrine cancers with strong genetic background. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) predispose patients to malignant disease with limited therapeutic options and poor prognosis. Using a host of cellular and molecular biology techniques in 2D and 3D cell culture formats we show that SDH inhibition had cell line specific biological and biochemical consequences. Based on our studies performed on PC12 (rat chromaffin cell line), Hela (human cervix epithelial cell line), and H295R (human adrenocortical cell line) cells, we demonstrated that chromaffin cells were not affected negatively by the inhibition of SDH either by siRNA directed against SDHB or treatment with SDH inhibitors (itaconate and atpenin A5). Cell viability and intracellular metabolite measurements pointed to the cell line specific consequences of SDH impairment and to the importance of glutamate metabolism in chromaffin cells. A significant increase in glutaminase-1 (GLS-1) expression after SDH impairment was observed in PC12 cells. GLS-1 inhibitor BPTES was capable of significantly decreasing proliferation of SDH impaired PC12 cells. Glutaminase-1 and SDHB expressions were tested in 35 Pheo/PGL tumor tissues. Expression of GLS1 was higher in the SDHB low expressed group compared to SDHB high expressed tumors. Our data suggest that the SDH-associated malignant potential of Pheo/PGL is strongly dependent on GLS-1 expression and glutaminases may be novel targets for therapy.

With the discovery of rapamycin 45 years ago, studies in the mechanistic target of rapamycin (mTOR) field started 2 decades before the identification of the mTOR kinase. Over the years, studies revealed that the mTOR signaling is a master regulator of homeostasis and integrates a variety of environmental signals to regulate cell growth, proliferation, and metabolism. Deregulation of mTOR signaling, particularly hyperactivation, frequently occurs in human tumors. Recent advances in molecular profiling have identified mutations or amplification of certain genes coding proteins involved in the mTOR pathway (eg, PIK3CA, PTEN, STK11, and RICTOR) as the most common reasons contributing to mTOR hyperactivation. These genetic alterations of the mTOR pathway are frequently observed in lung neoplasms and may serve as a target for personalized therapy. mTOR inhibitor monotherapy has met limited clinical success so far; however, rational drug combinations are promising to improve efficacy and overcome acquired resistance. A better understanding of mTOR signaling may have the potential to help translation of mTOR pathway inhibitors into the clinical setting.

Loss of liver kinase B (LKB1, encoded by STK11) has been observed in about one-third of lung adenocarcinomas (ADCs) and can cause the inactivation of its major target AMP-activated protein kinase (AMPK) and subsequently, activation of the mammalian target of rapamycin (mTOR) pathway. In addition to its inhibitory effects on tumor growth and proliferation, AMPK may affect acetyl-CoA homeostasis and induces histone acetylation through regulating the activity and nuclear translocation of acyl-CoA synthetase short-chain family member 2 (ACSS2) and ATP citrate lyase (ACLY). Nuclear and/or cytoplasmic expression of LKB1/AMPK/mTOR pathway markers (LKB1, p-AMPKα, p-S6), enzymes involved in acetate and lipid metabolism (p-ACC, ACSS2, ACLY), acetyl-histone H3 (ac-H3), and immune regulatory proteins (PD-L1, COX-2) was assessed in lung ADCs (N=100) by immunohistochemistry. The expression of mRNAs coding for proteins involved in antitumor immunity was also analyzed in STK11-mutant and wild-type cases with using TCGA data. Associations with the clinicopathological parameters as well as the antiproliferative effects of mTOR and metabolic inhibitors (rapamycin, metformin, phenformin, and ACSS2 inhibitor) and a histone deacetylase (HDAC) inhibitor (valproic acid) were also studied in ADC cell lines harboring KRAS and/or STK11 mutations. In cases with low or no LKB1 expression, we observed significantly lower expression of p-AMPKα, p-ACC, ac-H3, ACSS2, and ACLY (P < .01). PD-L1 expression was also slightly decreased in these samples. The expression of most of the studied mRNAs, particularly those involved in innate immunity, was also lower in STK11 mutant cases. In our in vitro studies, rapamycin had a significant antiproliferative effect on KRAS- and/or STK11-mutant cell lines with high mTOR activity. The ACSS2 inhibitor and valproic acid were not effective as monotherapies, however, valproic acid significantly increased the antitumoral effects of rapamycin in STK11-mutant cells similarly to phenformin. Our results suggest that histone acetylation levels are lower in ADCs with LKB1 loss, perhaps by lower expression and decreased nuclear translocation of ACSS2 and ACLY. HDAC inhibitors may have the benefit to maintain histone acetylation in STK11-mutant ADCs, therefore, restoring the expression of tumor suppressors or proteins involved in antitumor immunity, which are silenced in cancer cells. Simultaneous inhibition of mTOR and HDACs may exhibit significant antitumor properties, especially in cancer cells with STK11 mutation and high mTOR activity. Supported by the Hungarian Respiratory Foundation, National Bionics Program of National Research, Development and Innovation Fund of Hungary (ED_17-1-2017-0009), and grants of the Hungarian National Research, Development and Innovation Office (NKFI-FK-128404). Citation Format: Ildiko Krencz, Daniel Sztankovics, Titanilla Danko, Gabor Petovari, Eniko Vetlenyi, Regina Raffay, Judit Moldvay, Judit Papay, Anna Sebestyen. LKB1 loss-associated changes in histone acetylation are potential therapeutic targets in lung adenocarcinoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-046.

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.

Background Bioenergetic characterisation of malignant tissues revealed that different tumour cells can catabolise multiple substrates as salvage pathways, in response to metabolic stress. Altered metabolism in gliomas has received a lot of attention, especially in relation to IDH mutations, and the associated oncometabolite D-2-hydroxyglutarate (2-HG) that impact on metabolism, epigenetics and redox status. Astrocytomas and oligodendrogliomas, collectively called diffuse gliomas, are derived from astrocytes and oligodendrocytes that are in metabolic symbiosis with neurons; astrocytes can catabolise neuron-derived glutamate and gamma-aminobutyric acid (GABA) for supporting and regulating neuronal functions. Methods Metabolic characteristics of human glioma cell models – including mitochondrial function, glycolytic pathway and energy substrate oxidation – in relation to IDH mutation status and after 2-HG incubation were studied to understand the Janus-faced role of IDH1 mutations in the progression of gliomas/astrocytomas. The metabolic and bioenergetic features were identified in glioma cells using wild-type and genetically engineered IDH1-mutant glioblastoma cell lines by metabolic analyses with Seahorse, protein expression studies and liquid chromatography-mass spectrometry. Results U251 glioma cells were characterised by high levels of glutamine, glutamate and GABA oxidation. Succinic semialdehyde dehydrogenase (SSADH) expression was correlated to GABA oxidation. GABA addition to glioma cells increased proliferation rates. Expression of mutated IDH1 and treatment with 2-HG reduced glutamine and GABA oxidation, diminished the pro-proliferative effect of GABA in SSADH expressing cells. SSADH protein overexpression was found in almost all studied human cases with no significant association between SSADH expression and clinicopathological parameters (e.g. IDH mutation). Conclusions Our findings demonstrate that SSADH expression may participate in the oxidation and/or consumption of GABA in gliomas, furthermore, GABA oxidation capacity may contribute to proliferation and worse prognosis of gliomas. Moreover, IDH mutation and 2-HG production inhibit GABA oxidation in glioma cells. Based on these data, GABA oxidation and SSADH activity could be additional therapeutic targets in gliomas/glioblastomas. Electronic supplementary material The online version of this article (10.1186/s13046-018-0946-5) contains supplementary material, which is available to authorized users.

Background Multiple studies concluded that oncometabolites (e.g. D-2-hydroxyglutarate (2-HG) related to mutant isocitrate dehydrogenase 1/2 (IDH1/2) and lactate) have tumour promoting potential. Regulatory mechanisms implicated in the maintenance of oncometabolite production have great interest. mTOR (mammalian target of rapamycin) orchestrates different pathways, influences cellular growth and metabolism. Considering hyperactivation of mTOR in several malignancies, the question has been addressed whether mTOR operates through controlling of oncometabolite accumulation in metabolic reprogramming. Methods HT-1080 cells – carrying originally endogenous IDH1 mutation – were used in vitro and in vivo. Anti-tumour effects of rapamycin were studied using different assays. The main sources and productions of the oncometabolites (2-HG and lactate) were analysed by 13C-labeled substrates. Alterations at protein and metabolite levels were followed by Western blot, flow cytometry, immunohistochemistry and liquid chromatography mass spectrometry using rapamycin, PP242 and different glutaminase inhibitors, as well. Results Rapamycin (mTORC1 inhibitor) inhibited proliferation, migration and altered the metabolic activity of IDH1 mutant HT-1080 cells. Rapamycin reduced the level of 2-HG sourced mainly from glutamine and glucose derived lactate which correlated to the decreased incorporation of 13C atoms from 13C-substrates. Additionally, decreased expressions of lactate dehydrogenase A and glutaminase were also observed both in vitro and in vivo. Conclusions Considering the role of lactate and 2-HG in regulatory network and in metabolic symbiosis it could be assumed that mTOR inhibitors have additional effects besides their anti-proliferative effects in tumours with glycolytic phenotype, especially in case of IDH1 mutation (e.g. acute myeloid leukemias, gliomas, chondrosarcomas). Based on our new results, we suggest targeting mTOR activity depending on the metabolic and besides molecular genetic phenotype of tumours to increase the success of therapies. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0544-y) contains supplementary material, which is available to authorized users.

Introduction The mTOR (mammalian target of rapamycin) is a receptor tyrosine kinase which plays an important role in cell growth regulation, proliferation and tumour cell survival in cancer cells. Differences of mTOR activity were described in tumours. mTOR dependent and independent metabolic activity of tumour cells could be essential in survival and therapy resistance of breast cancer. Changes of mTOR and metabolic activity of breast cancer could correlate to the patients’ treatment outcome. Material and methods In our work, the correlations between the metabolic phenotype and mTOR activity of breast cancer cell lines were studied. Furthermore, the expressions of mTOR activity related proteins and several metabolic transporter, enzymes were studied by Western blot, ICC and IHC using breast cancer cell lines and human tissue samples. The metabolic phenotypes were also analysed using LC-MS. Moreover, the effect of mTOR, metabolic inhibitors and chemotherapeutics were also studied in vitro. Results and discussions Subtypes independent differences in mTORC1/C2 complex and metabolic activities were detected in breast cancer cell lines. Certain cell lines were characterised with high glucose uptake, high mTORC1 complex activity and high lactate production, while other cell lines showed balanced mTORC1 and mTORC2 activity, well-functioning oxidative phosphorylation capacity and higher levels of TCA metabolites which were related to intact mitochondrial functions. The metabolic phenotype and mTORC1/C2 activity correlated to the detected mTOR inhibitors, metabolic inhibitors and chemotherapeutic sensitivity of breast cancer cell lines in vitro. The metabolic and mTOR activity differences can be observed in human tissue samples independently of breast carcinoma cell subtypes, as well. Conclusion Differences of metabolic and mTOR related protein levels may explain the different therapeutic sensitivity. We suggest evaluating the metabolic phenotype and mTOR activity in breast cancer subtypes, which may help to introduce therapeutic drug combinations and predict therapy resistance. Supported by STIA-KF-17(SA), Bolyai-590/2015(SA), UNKP-17-3(HZ)−17-3(KI)−17-2(DT) and EFOP-3.6.3-VEKOP-16-2017-00009.

Introduction Kidney transplantation improves survival and life quality for patients with end-stage renal disease (ESRD), however, immunosuppression leads to increased risk of cancer, including renal cell carcinoma (RCC). The PI3K/Akt/mTOR pathway has a fundamental role in renal physiology and its inhibitors are used as immunosuppressive as well as antitumour agents. Recent studies suggest that immunosuppressive regimen, especially calcineurin inhibitors, may affect mTOR pathway activation. The aim of our study was to analyse the expression of mTORC1 and mTORC2-related proteins in RCCs developed in kidneys of transplant recipients and healthy individuals as controls. Material and methods Expression of p-mTOR (catalytic subunit of mTORC1 and mTORC2), p-S6 (mTORC1-related protein) and Rictor (mTORC2-related protein) was assessed in RCC tissues obtained from 46 kidney transplant recipients (15 clear cell RCCs, 31 papillary RCCs) and 46 healthy individuals (28 clear cell RCCs, 18 papillary RCCs) using IHC on tissue microarrays. Tissues from patients with ESRD (n=10) and normal kidney (n=3) were also studied. Results and discussions In normal kidney, expression of p-S6 and p-mTOR was weak in tubular epithelial cells. In contrast, expression of Rictor was high in the epithelium of both proximal and distal tubules. In ESRD, expression of p-mTOR, p-S6, and Rictor appeared to be higher in transplant recipients (n=6) compared to those who have not received immunosuppression (n=4). Expression of p-mTOR and Rictor was significantly higher in RCCs of diseased native kidneys of renal transplant recipients compared to RCCs developed in healthy individuals (p 0.01 and p 0.001, respectively) providing information about mTORC2 activation in post-transplant RCCs. According to previous observations, expression of p-S6 was significantly higher in clear cell RCCs as compared to papillary RCCs in both post-transplant and non-transplant patients (p 0.01 and p 0.05, respectively). Conclusion Our results highlighted the role of mTORC2 in the pathobiology of post-transplant RCCs. There are insufficient evidences for the protective role of mTORC1 inhibitors as immunosuppressive agents against post-transplant malignancies. Use of dual mTORC1/C2 inhibitors in the antitumour and immunosuppressive therapy needs further investigation and may represent a promising opportunity in the treatment or prevention of post-transplant RCCs. Supported by the UNKP-17–3, Bolyai Found of Hungarian Academy Sciences, and Semmelweis University Innovation Found STIA-KF-17.

Introduction IDH mutations are expressed in 80% of low grade gliomas and secondary glioblastomas with a favourable prognosis comparing to non IDH mutant gliomas [According to WHO (2016)] classification, IDH1 mutation is an important marker to classify gliomas. Our main interest is to study the role of certain metabolic alterations in glioma models, especially mitochondrial functions, glycolytic activity, IDH1 mutations and its regulation. Material and methods U87 MG, U373 MG and U251 MG and U251 MG IDH1 R132H (kind gift of Dr. W. Leenders) overexpressing cells were used in vitro . Cellular oxygen consumption, glycolytic activity, energy substrates oxidation were measured by Seahorse technique. Furthermore, the amount and the activity of several proteins related mTOR (mammalian target of rapamycin) and other metabolic pathways were studied. The proliferation and apoptosis rates of in vitro cell cultures were also studied. We analysed extra- and intracellular metabolite levels (citrate, succinate, fumarate, alfa-ketoglutarate, malate, 2-hydroxyglutarate – D-2-HG, glutamate) using LC-MS measurements. Addressing the question what are the sources of D-2-HG in IDH1 mutant U251 MG cells, we fed the cells with 13 C-labelled energy substrates. Results and discussions The glycolytic activity and oxygen consumption rates of the four cell lines show differences. The wild type U251 MG cell line has higher glycolytic activity and lower basal respiration than its IDH1 mutant variant glioma cell line pair. Addition of D-2-HG (72 hour) to wild type U251 MG cells increased the basal respiration and decreased the glycolytic activity of U251 MG cells. Using various bioenergetic substrates, it has been shown that U251 MG cells can oxidise glutamine, glutamate and malate at significantly higher level than IDH1 mutant U251 MG cells. This may be due to the alternative use of glutamine, we found that glutamine is the main source of D-2-HG production in these IDH1 mutant glioma cells. Furthermore, differences were found in the expression pattern of proteins which regulate energy metabolism in the studied glioma cells. Conclusion Based on the results of the examined glioma cells, IDH1 mutant cells showed a lower glycolytic capacity, a higher respiration and altered glutamine metabolism, which could be therapeutic targets in the future. Supported by UNKP-17–2, UNKP-17–3, Richter Centenarium Foundation, Bolyai-590/2015 and STIA-KF-17.