Immunity, Hypoxia, and Metabolism-the Ménage à Trois of Cancer: Implications for Immunotherapy.

It is generally accepted that metabolism is able to shape the immune response. Only recently we are gaining awareness that the metabolic crosstalk between different tumor compartments strongly contributes to the harsh tumor microenvironment (TME) and ultimately impairs immune cell fitness and effector functions. The major aims of this review are to provide an overview on the immune system in cancer; to position oxygen shortage and metabolic competition as the ground of a restrictive TME and as important players in the anti-tumor immune response; to define how immunotherapies affect hypoxia/oxygen delivery and the metabolic landscape of the tumor; and vice versa, how oxygen and metabolites within the TME impinge on the success of immunotherapies. By analyzing preclinical and clinical endeavors, we will discuss how a metabolic characterization of the TME can identify novel targets and signatures that could be exploited in combination with standard immunotherapies and can help to predict the benefit of new and traditional immunotherapeutic drugs.

Targeting monocarboxylate transporters (MCTs) in cancer: How close are we to the clinics?

As a result of metabolic reprogramming, cancer cells display high rates of glycolysis, causing an excess production of lactate along with an increase in extracellular acidity. Proton-linked monocarboxylate transporters (MCTs) are crucial in the maintenance of this metabolic phenotype, by mediating the proton-coupled lactate flux across cell membranes, also contributing to cancer cell pH regulation. Among the proteins codified by the SLC16 gene family, MCT1 and MCT4 isoforms are the most explored in cancers, being overexpressed in many cancer types, from solid tumours to haematological malignancies. Similarly to what occurs in particular physiological settings, MCT1 and MCT4 are able to mediate lactate shuttles among cancer cells, and also between cancer and stromal cells in the tumour microenvironment. This form of metabolic cooperation is responsible for important cancer aggressiveness features, such as cell proliferation, survival, angiogenesis, migration, invasion, metastasis, immune tolerance and therapy resistance. The growing understanding of MCT functions and regulation is offering a new path to the design of novel inhibitors that can be foreseen in clinical practices. This review provides an overview of the role of MCT isoforms in cancer and summarizes the recent advances in their pharmacological targeting, highlighting the potential of new potent and selective MCT1 and/or MCT4 inhibitors in cancer therapeutics, and anticipating its inclusion in clinical practice.

Clinical and Prognostic Impact of the Warburg Effect in Esophageal Carcinoma: Monocarboxylate Transporters as Candidates for Therapeutic Targeting.

INTRODUCTION: Esophageal cancer (EC) seems to display increased glycolytic activity, but clinical studies on the expression/prognostic significance of glycometabolism-related proteins, as well as functional assays, are missing. METHODS: Expression of 10 glycolytic biomarkers was evaluated by immunohistochemistry in tissue sections from 95 patients. Two esophageal squamous cell carcinoma (ESCC) cell lines were used to assess the effect of monocarboxylate transporter (MCT) downregulation on cell viability and extracellular lactate/glucose accumulation. RESULTS: Expression of MCT1, MCT4, CD147, and GLUT1 was significantly associated with an ESCC histopathology, while a poor clinicopathological profile was seen in GLUT1- and LDHA-positive EC cases. In the ESCC group, MCT1 immunoreactivity is associated with high TNM stage and metastasis. The 3-year overall survival (OS) rate was significantly influenced by MCT4 and CAIX positivity and HKII negativity. Those biomarkers were considered independent prognostic factors of OS in multivariate analysis. Dual inhibition of MCT1/4 expression decreased cell viability and extracellular lactate accumulation in ESCC cells. CONCLUSION: Elevated glycolytic rates correlate with a poor clinicopathological profile in EC patients. MCT4 and CAIX positivity independently predict a worse prognosis. Due to the lack of information on treatment modalities, we could not further infer the role of these biomarkers in predicting response to therapy, which needs to be assessed in future studies. In addition, MCT1/4 targeting should be performed both "in vitro" and "in vivo" to further explore its impact on tumor growth and response to classical therapies. HKII expression and function, particularly in the tumor stroma, should be investigated.

Prognostic Value of Monocarboxylate Transporter 1 Overexpression in Cancer: A Systematic Review.

Energy production by cancer is driven by accelerated glycolysis, independently of oxygen levels, which results in increased lactate production. Lactate is shuttled to and from cancer cells via monocarboxylate transporters (MCTs). MCT1 works both as an importer and an extruder of lactate, being widely studied in recent years and generally associated with a cancer aggressiveness phenotype. The aim of this systematic review was to assess the prognostic value of MCT1 immunoexpression in different malignancies. Study collection was performed by searching nine different databases (PubMed, EMBASE, ScienceDirect, Scopus, Cochrane Library, Web of Science, OVID, TRIP and PsycINFO), using the keywords "cancer", "Monocarboxylate transporter 1", "SLC16A1" and "prognosis". Results showed that MCT1 is an indicator of poor prognosis and decreased survival for cancer patients in sixteen types of malignancies; associations between the transporter's overexpression and larger tumour sizes, higher disease stage/grade and metastasis occurrence were also frequently observed. Yet, MCT1 overexpression correlated with better outcomes in colorectal cancer, pancreatic ductal adenocarcinoma and non-small cell lung cancer patients. These results support the applicability of MCT1 as a biomarker of prognosis, although larger cohorts would be necessary to validate the overall role of MCT1 as an outcome predictor.

Phenolic Compounds Contribution to Portuguese Propolis Anti-Melanoma Activity.

Melanoma is the deadliest type of skin cancer, with about 61,000 deaths annually worldwide. Late diagnosis increases mortality rates due to melanoma's capacity to metastasise rapidly and patients' resistance to the available conventional therapies. Consequently, the interest in natural products as a strategy for drug discovery has been emerging. Propolis, a natural product produced by bees, has several biological properties, including anticancer effects. Propolis from Gerês is one of the most studied Portuguese propolis. Our group has previously demonstrated that an ethanol extract of Gerês propolis collected in 2018 (G18.EE) and its fractions (n-hexane, ethyl acetate, and n-butanol) decrease melanoma cell viability. Out of all the fractions, G18.EE-n-BuOH showed the highest potential as a melanoma pharmacological therapy. Thus, in this work, G18.EE-n-BuOH was fractioned into 17 subfractions whose effect was evaluated in A375 BRAF-mutated melanoma cells. The subfractions with the highest cytotoxic activity were analysed by UPLC-DAD-ESI/MSn in an attempt to understand which phenolic compounds could account for the anti-melanoma activity. The compounds identified are typical of the Gerês propolis, and some of them have already been linked with antitumor effectiveness. These results reaffirm that propolis compounds can be a source of new drugs and the isolation of compounds could allow its use in traditional medicine.

Non-Alcoholic Fatty Liver Disease-Related Hepatocellular Carcinoma: Immunohistochemical Assessment of Markers of Cancer Cell Metabolism.

INTRODUCTION: Hepatocellular carcinoma (HCC) has been associated to non-alcoholic fatty liver disease (NAFLD). We sought to investigate the immunoexpression of several glycolytic metabolism-associated markers in patients with HCC associated to NAFLD and associate these factors to their clinical-pathological characteristics. METHODS: We evaluated 35 HCC specimens from 21 patients diagnosed with non-alcoholic steatohepatitis (NASH) undergoing liver resection (12 patients), liver transplantation (8 patients), or both (1 patient). Histological features, clinical aspects, demographic and biochemical data, as well as the immunohistochemical reactivity for monocarboxylate transporters 1, 2, and 4; their chaperone CD147; carbonic anhydrase IX; and glucose transporter-1 (GLUT1) were assessed. RESULTS: Metabolic-associated cirrhosis was present in 12 of the 21 patients (8 child A and 4 child B scores). From 9 patients without cirrhosis, 3 presented NASH F3 and 6 NASH F2. Sixteen (76%) had diabetes mellitus, 17 (81%) arterial hypertension, and 19 (90%) body mass index above 25 kg/m2; 8 (38%) had dyslipidemia. From 35 nodules, steatosis was found in 26, ballooning in 31 nodules, 25 of them diagnosed as steatohepatitic subtype of HCC. MCT4 immunoexpression was associated with extensive intratumoral fibrosis, advanced clinical stages, and shorter overall survival. GLUT1 was noticeable in nodules with extensive intratumoral steatosis, higher intratumoral fibrosis, and advanced clinical stages. Immunohistochemical expression of the metabolic biomarkers MCT4 and GLUT1 was higher in patients with Barcelona-clinic liver cancer B or C. GLUT1 correlated with higher degree of steatosis, marked ballooning, intratumoral fibrosis, and higher parenchymal necroinflammatory activity. CONCLUSION: Our data indicate that the expression of the glycolytic phenotype of metabolic markers, especially GLUT1 and MCT4, correlates with a more severe course of HCC occurring in NASH patients.

Targeting Lysosomes in Colorectal Cancer: Exploring the Anticancer Activity of a New Benzo[a]phenoxazine Derivative.

Colorectal cancer (CRC) has been ranked as one of the cancer types with a higher incidence and one of the most mortal. There are limited therapies available for CRC, which urges the finding of intracellular targets and the discovery of new drugs for innovative therapeutic approaches. In addition to the limited number of effective anticancer agents approved for use in humans, CRC resistance and secondary effects stemming from classical chemotherapy remain a major clinical problem, reinforcing the need for the development of novel drugs. In the recent years, the phenoxazines derivatives, Nile Blue analogues, have been shown to possess anticancer activity, which has created interest in exploring the potential of these compounds as anticancer drugs. In this context, we have synthetized and evaluated the anticancer activity of different benzo[a]phenoxazine derivatives for CRC therapy. Our results revealed that one particular compound, BaP1, displayed promising anticancer activity against CRC cells. We found that BaP1 is selective for CRC cells and reduces cell proliferation, cell survival, and cell migration. We observed that the compound is associated with reactive oxygen species (ROS) generation, accumulates in the lysosomes, and leads to lysosomal membrane permeabilization, cytosolic acidification, and apoptotic cell death. In vivo results using a chicken embryo choriollantoic membrane (CAM) assay showed that BaP1 inhibits tumor growth, angiogenesis, and tumor proliferation. These observations highlight that BaP1 as a very interesting agent to disturb and counteract the important roles of lysosomes in cancer and suggests BaP1 as a promising candidate to be exploited as new anticancer lysosomal-targeted agent, which uses lysosome membrane permeabilization (LMP) as a therapeutic approach in CRC.

3-Bromopyruvate Suppresses the Malignant Phenotype of Vemurafenib-Resistant Melanoma Cells.

(1) BRAF mutations are associated with high mortality and are a substantial factor in therapeutic decisions. Therapies targeting BRAF-mutated tumors, such as vemurafenib (PLX), have significantly improved the overall survival of melanoma patients. However, patient relapse and low response rates remain challenging, even with contemporary therapeutic alternatives. Highly proliferative tumors often rely on glycolysis to sustain their aggressive phenotype. 3-bromopyruvate (3BP) is a promising glycolysis inhibitor reported to mitigate resistance in tumors. This study aimed to evaluate the potential of 3BP as an antineoplastic agent for PLX-resistant melanoma treatment. (2) The effect of 3BP alone or in combination with PLX on viability, proliferation, colony formation, cell death, migration, invasion, epithelial-mesenchymal marker and metabolic protein expression, extracellular glucose and lactate, and reactive species were evaluated in two PLX-resistant melanoma cell lines. (3) 3BP treatment, which was more effective as monotherapy than combined with PLX, disturbed the metabolic and epithelial-mesenchymal profile of PLX-resistant cells, impairing their proliferation, migration, and invasion and triggering cell death. (4) 3BP monotherapy is a potent metabolic-disrupting agent against PLX-resistant melanomas, supporting the suppression of the malignant phenotype in this type of neoplasia.

Portuguese Propolis Antitumoral Activity in Melanoma Involves ROS Production and Induction of Apoptosis.

Melanoma is the most aggressive and life-threatening skin cancer type. The melanoma genome is the most frequently mutated, with the BRAF mutation present in 40-60% of melanoma cases. BRAF-mutated melanomas are characterized by a higher aggressiveness and progression. Adjuvant targeted treatments, such as BRAF and MEK inhibitors, are added to surgical excision in BRAF-mutated metastatic melanomas to maximize treatment effectiveness. However, resistance remains the major therapeutic problem. Interest in natural products, like propolis, for therapeutic applications, has increased in the last years. Propolis healing proprieties offer great potential for the development of novel cancer drugs. As the activity of Portuguese propolis has never been studied in melanoma, we evaluated the antitumoral activity of propolis from Gerês (G18.EE) and its fractions (n-hexane, ethyl acetate (EtOAc), and n-butanol) in A375 and WM9 melanoma cell lines. Results from DPPH•/ABTS• radical scavenging assays indicated that the samples had relevant antioxidant activity, however, this was not confirmed in the cell models. G18.EE and its fractions decreased cell viability (SRB assay) and promoted ROS production (DHE/Mitotracker probes by flow cytometry), leading to activation of apoptotic signaling (expression of apoptosis markers). Our results suggest that the n-BuOH fraction has the potential to be explored in the pharmacological therapy of melanoma.

Selective Cytotoxicity of Portuguese Propolis Ethyl Acetate Fraction towards Renal Cancer Cells.

Renal cell carcinoma is the most lethal cancer of the urological system due to late diagnosis and treatment resistance. Propolis, a beehive product, is a valuable natural source of compounds with bioactivities and may be a beneficial addition to current anticancer treatments. A Portuguese propolis sample, its fractions (n-hexane, ethyl acetate, n-butanol and water) and three subfractions (P1-P3), were tested for their toxicity on A498, 786-O and Caki-2 renal cell carcinoma cell lines and the non-neoplastic HK2 kidney cells. The ethyl acetate fraction showed the strongest toxicity against A498 (IC50 = 0.162 µg mL-1) and 786-O (IC50 = 0.271 µg mL-1) cells. With similar toxicity against 786-O, P1 (IC50 = 3.8 µg mL-1) and P3 (IC50 = 3.1 µg mL-1) exhibited greater effect when combined (IC50 = 2.5 µg mL-1). Results support the potential of propolis and its constituents as promising coadjuvants in renal cell carcinoma treatment.

In Vivo Anticancer Activity of AZD3965: A Systematic Review.

Proliferating cancer cells have high energy demands, which is mainly obtained through glycolysis. The transmembrane trafficking of lactate, a major metabolite produced by glycolytic cancer cells, relies on monocarboxylate transporters (MCTs). MCT1 optimally imports lactate, although it can work bidirectionally, and its activity has been linked to cancer aggressiveness and poor outcomes. AZD3965, a specific MCT1 inhibitor, was tested both in vitro and in vivo, with encouraging results; a phase I clinical trial has already been undertaken. Thus, analysis of the experimental evidence using AZD3965 in different cancer types could give valuable information for its clinical use. This systematic review aimed to assess the in vivo anticancer activity of AZD3965 either alone (monotherapy) or with other interventions (combination therapy). Study search was performed in nine different databases using the keywords "AZD3965 in vivo" as search terms. The results show that AZD3965 successfully decreased tumor growth and promoted intracellular lactate accumulation, which confirmed its effectiveness, especially in combined therapy. These results support the setup of clinical trials, but other important findings, namely AZD3965 enhanced activity when given in combination with other therapies, or MCT4-induced treatment resistance, should be further considered in the clinical trial design to improve therapy response.

Unravelling the anticancer potential of functionalized chromeno[2,3-b]pyridines for breast cancer treatment.

A selection of new chromeno[2,3-b]pyridines was prepared from chromenylacrylonitriles and N-substituted piperazines, using a novel and efficient synthetic procedure. The compounds were tested for their anticancer activity using breast cancer cell lines MCF-7, Hs578t and MDA-MB-231 and the non-neoplastic cell line MCF-10A for toxicity evaluation. In general, compounds showed higher activity towards the luminal breast cancer subtype (MCF-7), competitive with the reference compound Doxorubicin. The in vivo toxicity assay using C. elegans demonstrated a safe profile for the most active compounds. Chromene 3f revealed a promising drug profile, inhibiting cell growth and proliferation, inducing cell cycle arrest in G2/M phase, apoptosis and microtubule destabilization. The new compounds presented exciting bioactive features and may be used as lead compounds in cancer related drug discovery.

Lactate and Lactate Transporters as Key Players in the Maintenance of the Warburg Effect.

Reprogramming of energy metabolism is a key hallmark of cancer. Most cancer cells display a glycolytic phenotype, with increased glucose consumption and glycolysis rates, and production of lactate as the end product, independently of oxygen concentrations. This phenomenon, known as "Warburg Effect", provides several survival advantages to cancer cells and modulates the metabolism and function of neighbour cells in the tumour microenvironment. However, due to the presence of metabolic heterogeneity within a tumour, cancer cells can also display an oxidative phenotype, and corruptible cells from the microenvironment become glycolytic, cooperating with oxidative cancer cells to boost tumour growth. This phenomenon is known as "Reverse Warburg Effect". In either way, lactate is a key mediator in the metabolic crosstalk between cancer cells and the microenvironment, and lactate transporters are expressed differentially by existing cell populations, to support this crosstalk.In this review, we will focus on lactate and on lactate transporters in distinct cells of the tumour microenvironment, aiming at a better understanding of their role in the acquisition and maintenance of the direct/reverse "Warburg effect" phenotype, which modulate cancer progression.

Microbes and Cancer: Friends or Faux?

Cancer is one of the most aggressive and deadly diseases in the world, representing the second leading cause of death. It is a multifactorial disease, in which genetic alterations play a key role, but several environmental factors also contribute to its development and progression. Infections induced by certain viruses, bacteria, fungi and parasites constitute risk factors for cancer, being chronic infection associated to the development of certain types of cancer. On the other hand, susceptibility to infectious diseases is higher in cancer patients. The state of the host immune system plays a crucial role in the susceptibility to both infection and cancer. Importantly, immunosuppressive cancer treatments increase the risk of infection, by decreasing the host defenses. Furthermore, alterations in the host microbiota is also a key factor in the susceptibility to develop cancer. More recently, the identification of a tumor microbiota, in which bacteria establish a symbiotic relationship with cancer cells, opened a new area of research. There is evidence demonstrating that the interaction between bacteria and cancer cells can modulate the anticancer drug response and toxicity. The present review focuses on the interaction between microbes and cancer, specifically aiming to: (1) review the main infectious agents associated with development of cancer and the role of microbiota in cancer susceptibility; (2) highlight the higher vulnerability of cancer patients to acquire infectious diseases; (3) document the relationship between cancer cells and tissue microbiota; (4) describe the role of intratumoral bacteria in the response and toxicity to cancer therapy.

Direct actions of adiponectin on changes in reproductive, metabolic, and anti-oxidative enzymes status in the testis of adult mice.

Obesity is a major health problem that is linked to decreased sperm count. It is hypothesized that an obesity-associated reduction in adiponectin secretion may be responsible for impairment of spermatogenesis. Therefore, the aim of the study was to evaluate the direct role of adiponectin in spermatogenesis and steroid synthesis in adult mice. This study showed that adiponectin receptors (AdipoR1 and AdipoR2) were localized in Leydig cells and seminiferous tubules in the testis of adult mice. The result of the in vitro study showed the direct action of adiponectin on spermatogenesis by stimulating cell proliferation (PCNA) and survival (Bcl2) and by suppressing cell apoptosis. Treatment of testis with adiponectin also enhanced transport of the energetic substrates glucose and lactate to protect cells from undergoing apoptosis. Adiponectin treatment further showed a significant reduction in oxidative stress and nitric oxide. Our findings suggest that adiponectin effectively facilitates cell survival and proliferation, as well as protects from apoptosis. Thus, adiponectin treatment may be responsible for enhancing sperm counts. Interestingly, this study showed the stimulatory effect of adiponectin in spermatogenesis but showed an inhibitory effect on testosterone and estradiol synthesis in the testes. Based on the present study, it is hypothesized that systemic adiponectin treatment may be a promising therapeutic strategy for the improvement of spermatogenesis and sperm count.

Value of pH regulators in the diagnosis, prognosis and treatment of cancer.

Altered metabolism, associated with acidification of the extracellular milieu, is one of the major features of cancer. As pH regulation is crucial for the maintenance of all biological functions, cancer cells rely on the activity of lactate exporters and proton transporters to regulate their intracellular pH. The major players in cancer pH regulation are proton pump ATPases, sodium-proton exchangers (NHEs), monocarboxylate transporters (MCTs), carbonic anhydrases (CAs) and anion exchangers (AEs), which have been shown to be upregulated in several human malignancies. Thanks to the activity of the proton pumps and transporters, tumours acidify their microenvironment, becoming more aggressive and resistant to therapy. Thus, targeting tumour pH may contribute to more effective anticancer strategies for controlling tumour progression and therapeutic resistance. In the present study, we review the role of the main pH regulators expressed in human cancer cells, including their diagnostic and prognostic value, as well as their usefulness as therapeutic targets.

Bioenergetic modulators hamper cancer cell viability and enhance response to chemotherapy.

Gliomas are characterized by a marked glycolytic metabolism with a consequent production of massive amounts of lactate, even in the presence of normal levels of oxygen, associated to increased invasion capacity and to higher resistance to conventional treatment. This work aimed to understand how the metabolic modulation can influence tumour aggressive features and its potential to be used as complementary therapy. We assessed the effect of bioenergetic modulators (BMs) targeting different metabolic pathways in glioma cell characteristics. The in vivo effect of BMs was evaluated using the chicken chorioallantoic membrane model. Additionally, the effect of pre-treatment with BMs in the response to the antitumour drug temozolomide (TMZ) was analysed in vitro. Cell treatment with the BMs induced a decrease in cell viability and in migratory/invasion abilities, as well as modifications in metabolic parameters (glucose, lactate and ATP) and increased the cytotoxicity of the conventional drug TMZ. Furthermore, all BMs decreased the tumour growth and the number of blood vessels in an in vivo model. Our results demonstrate that metabolic modulation has the potential to be used as therapy to decrease the aggressiveness of the tumours or to be combined with conventional drugs used in glioma treatment.

Monocarboxylate transporter 1 is a key player in glioma-endothelial cell crosstalk.

Glioblastoma (GBM) is one of the most glycolytic and angiogenic human tumors, characteristics that contribute to the poor prognosis associated with this type of tumor. A lactate shuttle has been described between tumor cells and endothelial cells (ECs), with the monocarboxylate transporters (MCTs) acting as important players in this tumor-EC communication. In this study, we aimed to understand how the tumor microenvironment modulates EC metabolism, and to characterize the role of MCTs in the glioma-brain EC crosstalk. Exposure of human brain microvascular ECs (HBMEC) to GBM cell-conditioned media increased the expression of MCT1, which corresponded to activation of oxidative metabolism and an increase in angiogenic capacity, as determined by increased proliferation, migration, and vessel assembly. Lactate depletion from the microenvironment or inhibition of lactate uptake in HBMEC induced an increase in lactate production and a decrease in proliferation, migration, and vessel assembly. Moreover, addition of lactate to HBMEC media promoted activation of AKT and AMPK pathways and increased expression in NFκB, HIF-1α, and the lactate receptor GPR81. Here, we demonstrate a role for MCT1 as a mediator of lactate signaling between glioma cells and brain ECs. Our results suggest that MCT1 can mediate EC metabolic reprograming, proliferation, and vessel sprouting in response to tumor signaling. Thus, targeting MCT1 in both tumor cells and brain EC may be a promising therapeutic strategy for the treatment of GBM.

Impact of mesenchymal stem cells' secretome on glioblastoma pathophysiology.

BACKGROUND: Glioblastoma (GBM) is a highly aggressive primary brain cancer, for which curative therapies are not available. An emerging therapeutic approach suggested to have potential to target malignant gliomas has been based on the use of multipotent mesenchymal stem cells (MSCs), either unmodified or engineered to deliver anticancer therapeutic agents, as these cells present an intrinsic capacity to migrate towards malignant tumors. Nevertheless, it is still controversial whether this innate tropism of MSCs towards the tumor area is associated with cancer promotion or suppression. Considering that one of the major mechanisms by which MSCs interact with and modulate tumor cells is via secreted factors, we studied how the secretome of MSCs modulates critical hallmark features of GBM cells. METHODS: The effect of conditioned media (CM) from human umbilical cord perivascular cells (HUCPVCs, a MSC population present in the Wharton's jelly of the umbilical cord) on GBM cell viability, migration, proliferation and sensitivity to temozolomide treatment of U251 and SNB-19 GBM cells was evaluated. The in vivo chicken chorioallantoic membrane (CAM) assay was used to evaluate the effect of HUCPVCs CM on tumor growth and angiogenesis. The secretome of HUCPVCs was characterized by proteomic analyses. RESULTS: We found that both tested GBM cell lines exposed to HUCPVCs CM presented significantly higher cellular viability, proliferation and migration. In contrast, resistance of GBM cells to temozolomide chemotherapy was not significantly affected by HUCPVCs CM. In the in vivo CAM assay, CM from HUCPVCs promoted U251 and SNB-19 tumor cells growth. Proteomic analysis to characterize the secretome of HUCPVCs identified several proteins involved in promotion of cell survival, proliferation and migration, revealing novel putative molecular mediators for the effects observed in GBM cells exposed to HUCPVCs CM. CONCLUSIONS: These findings provide novel insights to better understand the interplay between GBM cells and MSCs, raising awareness to potential safety issues regarding the use of MSCs as stem-cell based therapies for GBM.

Hotspot TERT promoter mutations are rare events in testicular germ cell tumors.

The abnormal activation of telomerase, codified by the telomerase reverse transcriptase (TERT) gene, is related to one of cancer hallmarks. Hotspot somatic mutations in the promoter region of TERT, specifically the c.-124:C>T and c.-146:C>T, were recently identified in a range of human cancers and have been associated with a more aggressive behavior. Testicular germ cell tumors frequently exhibit a good prognosis; however, the development of refractory disease is still a clinical challenge. In this study, we aim to evaluate for the first time the presence of the hotspot telomerase reverse transcriptase gene promoter mutations in testicular germ cell tumors. A series of 150 testicular germ cell tumor cases and four germ cell tumor cell lines were evaluated by PCR followed by direct Sanger sequencing and correlated with patient's clinical pathological features. Additionally, we genotyped the telomerase reverse transcriptase gene promoter single nucleotide polymorphism rs2853669 (T>C) located at -245 position. We observed the presence of the TERT promoter mutation in four patients, one exhibited the c.-124:C>T and three the c.-146:C>T. No association between TERT mutation status and clinicopathological features could be identified. The analysis of the rs2853669 showed that variant C was present in 22.8 % of the cases. In conclusion, we showed for the first time that TERT promoter mutations occur in a small subset (~3 %) of testicular germ cell tumors.