EXTH-62. COMBINATORIAL THERAPY TARGETING TRANSCIPTION AND IMMUNO-METABOLISM IN RECURRENT GLIOBLASTOMA

Abstract Recurrent glioblastomas (GBM) are notoriously difficult to treat, and in spite of aggressive chemo- and radiation therapy they inevitably recur in almost all GBM patients within 14 months following initial diagnosis. To interrogate pathways driving therapeutic resistance, we compared matched primary and recurrent IDH wild type glioblastoma samples from a cohort of patients, using RNA-Seq. Our analyses showed that pathways involved with tumor immune and metabolic reprogramming were up-regulated in recurrent GBMs compared to untreated, primary samples. Based on these findings, we tested the anti-tumor efficacy of over 20 rationally selected targeted therapeutic agents alone and in combination in a high-throughput drug screen assay designed to measure long term cell viability of primary tumorspheres from patients who underwent surgery for recurrent GBM. Top performing drug combinations from these screens were validated in vivo using patient-derived intracranial mouse models of glioma. Our data demonstrated that the combination of the transcriptional/metabolic inhibitor TG02 and the dual PI3K/mTOR inhibitor GDC-0084 significantly prolonged in vivo survival of tumor bearing mice, performing better than either drug alone. Notably, both inhibitors are undergoing human clinical trials as single agents with positive initial safety profiles and superior blood brain barrier penetrance. RNA Seq and functional assays of tumor samples from treated mice showed that only the combination treatment (TG02 + GDC-0084) significantly inhibited expression of immunomodulatory cytokines driving tumor progression, including those identified by us to be overexpressed in recurrent human GBM samples. Overall, these data suggest that combining TG02 and GDC-0084 is an effective treatment for recurrent GBMs. Furthermore, our results support the use of a translational research platform consisting of a personalized pharmaco-genomics testing, using patient-derived tumor samples towards designing more effective treatment for recurrent GBM patients.

Download Full-text

CD70 as an actionable immunotherapeutic target in recurrent glioblastoma and its microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-003289 ◽

2022 ◽

Vol 10 (1) ◽

pp. e003289

Author(s):

Mathieu Seyfrid ◽

William Thomas Maich ◽

Vaseem Muhammad Shaikh ◽

Nazanin Tatari ◽

Deepak Upreti ◽

...

Keyword(s):

Standard Of Care ◽

Recurrent Glioblastoma ◽

Preclinical Model ◽

Dismal Prognosis ◽

Car T ◽

Cell Assays ◽

Human Gbm ◽

Recurrent Gbm

PurposeGlioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of cancer stem cells (CSCs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here, we identify CD70 as a potential therapeutic target for recurrent GBM CSCs.Experimental designIn the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We use CD70 knockdown studies, subsequent RNAseq pathway analysis, and in vivo xenotransplantation to validate CD70’s role in GBM. Next, we developed and tested an anti-CD70 chimeric antigen receptor (CAR)-T therapy, which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples.ResultsCD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations, notably putative M1 macrophages and CD4 T cells.ConclusionCD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable polytherapeutic avenue to co-target both GBM and its TIME.

Download Full-text

MODL-16. ABEMACICLIB, A SELECTIVE CDK4/6 INHIBITOR, RESTRICTS GROWTH OF PEDIATRIC GLIAL-LINEAGE TUMORS IN VITRO AND IN VIVO

Neuro-Oncology ◽

10.1093/neuonc/noaa222.590 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii414-iii414

Author(s):

Muh-Lii Liang ◽

Tsung-Han Hsieh ◽

Tai-Tong Wong

Keyword(s):

Dna Repair ◽

Therapeutic Strategy ◽

Target Genes ◽

Rna Seq ◽

Downstream Target ◽

Rb Phosphorylation ◽

Glial Lineage

Abstract BACKGROUND Glial-lineage tumors constitute a heterogeneous group of neoplasms, comprising gliomas, oligodendrogliomas, and ependymomas, which account for 40%–50% of all pediatric central nervous system tumors. Advances in modern neuro-oncological therapeutics are aimed at improving neoadjuvant chemotherapy and deferring radiotherapy because radiation exposure may cause long-term side effects on the developing brain in young children. Despite aggressive treatment, more than half the high-grade gliomas (pHGGs) and one-third of ependymomas exhibit recurrence within 2 years of initial treatment. METHODS By using integrated bioinformatics and through experimental validation, we found that at least one gene among CCND1, CDK4, and CDK6 was overexpressed in pHGGs and ependymomas. RESULTS The use of abemaciclib, a highly selective CDK4/6 inhibitor, effectively inhibited cell proliferation and reduced the expression of cell cycle–related and DNA repair–related gene expression, which was determined through RNA-seq analysis. The efficiency of abemaciclib was validated in vitro in pHGGs and ependymoma cells and in vivo by using subcutaneously implanted ependymoma cells from patient-derived xenograft (PDX) in mouse models. Abemaciclib demonstrated the suppression of RB phosphorylation, downstream target genes of E2F, G2M checkpoint, and DNA repair, resulting in tumor suppression. CONCLUSION Abemaciclib showed encouraging results in preclinical pediatric glial-lineage tumors models and represented a potential therapeutic strategy for treating challenging tumors in children.

Download Full-text

TMOD-08. DEVELOPING DERIVATIVE GBM PDX, IN VIVO, FROM TREATMENT NAÏVE SOURCES

Neuro-Oncology ◽

10.1093/neuonc/noab196.869 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi217-vi217

Author(s):

David James ◽

Craig Horbinski ◽

Roger Stupp ◽

Atique Ahmed

Keyword(s):

Mutagenic Effect ◽

Recurrent Glioblastoma ◽

Response To Treatment ◽

Mismatch Repair Gene ◽

Repair Gene ◽

Msh6 Mutation ◽

Treatment Naïve ◽

Post Therapy ◽

Recurrent Gbm

Abstract PURPOSE Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients. METHODS Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants. RESULTS Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant. CONCLUSIONS The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.

Download Full-text

Cycle initiation and colony formation in culture by murine marrow cells with long-term reconstituting potential in vivo

Blood ◽

10.1182/blood.v88.11.4149.4149 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4149-4158 ◽

Cited By ~ 3

Author(s):

M Trevisan ◽

XQ Yan ◽

NN Iscove

Keyword(s):

Colony Formation ◽

Interleukin 1 ◽

Methyl Cellulose ◽

Culture Conditions ◽

Liquid Suspension ◽

Almost All ◽

Marrow Cells

Abstract This investigation was directed at separating long-term reconstituting (LTR) stem cells in normal murine marrow from hematopoietic precursors detectable in short-term assays in vitro and in vivo, and then at determining whether purified LTR cells could themselves form colonies in culture. To do so, it was first necessary to identify culture conditions that would induce their growth while preserving their long- term reconstituting capacity. Marrow was cultured with various cytokines in liquid suspension for 4 days, after which the surviving LTR activity was quantitated in a competitive in vivo assay. Activity was preserved near input levels with combined murine c-kit ligand (KL), interleukin-1 (IL-1), IL-6, and IL-11. When the cultures also included tritiated or unlabeled thymidine, LTR potential was eliminated, indicating that essentially all LTR cells were induced into cell cycle with these cytokines. To purify them, marrow was sorted on the basis of Ly6A expression and Rhodamine 123 retention. The Ly6AhiRh123ls fraction contained 85% of total recovered LTR activity but only 1% of the recovered cells measured by multilineage colony formation in spleens or in vitro. This fraction was cultured in methyl cellulose with KL, IL-1, IL-6, and IL-11 for 4 to 6 days, after which colonies were isolated and injected into mice. High levels of permanent reconstitution were achievable in sublethally irradiated W41/W41 mice after the injection of a single reconstituting unit, and limiting dilution analysis estimated the frequency of multilineage LTR at 1 in 11,200 unpurified adult marrow cells. In either lethally irradiated normal or sublethally irradiated W41/W41 mice, 1-year lymphomyeloid reconstitutions were obtained from 1 in 65 to 84 colonies of 2 to 16 dispersed cells, but not from larger colonies or those with clumped cells. The results establish that resting marrow LTR cells can be separated from almost all of the more advanced clonogenic cells that are still pluripotential, can be induced to cycle in culture by defined cytokines with preservation of their reconstituting potential, and can be manipulated and assayed efficiently at single-cell and colony levels.

Download Full-text

SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY

The Journal of Cell Biology ◽

10.1083/jcb.61.3.789 ◽

1974 ◽

Vol 61 (3) ◽

pp. 789-807 ◽

Cited By ~ 49

Author(s):

Gert Kreibich ◽

David D. Sabatini

Keyword(s):

Serum Proteins ◽

Specific Activity ◽

Membrane Vesicles ◽

Rat Serum ◽

Coomassie Blue ◽

Specific Subset ◽

Low Levels ◽

Almost All

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER). More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons. The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation. A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content. Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER. Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and cross-reacted with antiserum against rat serum. Almost all microsomal glycoproteins were at least partly released with the microsomal content. Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [3H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER. On the other hand, after labeling for 30 min with [3H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes. This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae. The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [3H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue, with a main radioactive peak corresponding to cytochrome P 450. After the long-term labeling procedure most content proteins had low levels of radioactivity; this was especially true of serum proteins which were highly labeled after 30 min.

Download Full-text

Phase II trial of the phosphatidyinositol-3 kinase (PI3K) inhibitor BKM120 in recurrent glioblastoma (GBM).

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.2015 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 2015-2015 ◽

Cited By ~ 2

Author(s):

Patrick Y. Wen ◽

W. K. Alfred Yung ◽

Ingo K. Mellinghoff ◽

Kathleen Lamborn ◽

Shakti Ramkissoon ◽

...

Keyword(s):

Phase Ii ◽

Pi3k Pathway ◽

Homozygous Deletion ◽

Recurrent Glioblastoma ◽

Pi3k Inhibitor ◽

Eligibility Criteria ◽

Drug Concentrations ◽

Recurrent Gbm

2015 Background: The PI3K pathway is activated in most GBMs and represents a potential therapeutic target. BKM120 is an oral, pan-Class I PI3K inhibitor that enters the brain at therapeutic concentrations demonstrated to inhibit PI3K pathway, and potently inhibits the growth of U87 GBM tumors and human glioma tumor spheres in vitro and in vivo. Methods: The Ivy Foundation Early Phase Clinical Trials Consortium is conducting a phase II study of BKM120 in recurrent GBM patients with activation of the PI3K pathway (mutation, homozygous deletion or loss of IHC of PTEN, PIK3CA or PIK3RI mutations, or detectable pAKT). Additional eligibility criteria included radiologic progression, 1st or 2nd relapse, > 18 yrs, KPS > 60, adequate bone marrow and organ function, controlled blood glucose, and no enzyme-inducing antiepileptic drugs. Patients received BKM120 100mg daily. The study consisted of 2 parts conducted concurrently. Part 1 involved up to 15 patients who received BKM120 daily for 8-12 days prior to surgery for recurrent disease. Patients underwent FDG PET, pharmacokinetic (PK) studies, and tumor was obtained for drug concentrations and pharmacodynamic effects. Part 2 consisted of up to 50 patients with unresectable GBM treated with BKM120. The primary endpoint for Part 2 was 6-month progression-free survival (p0 =15%; p1= 32%). Results: To date 7 patients have been enrolled into Part 1, 33 into part 2. There were 5 women and 35 men. Median age was 54 yrs (29-68). Treatment was fairly well-tolerated. Major grades 3/4 toxicities were asymptomatic lipase elevation (5), fatigue (3), hyperglycemia (3), rash (3) elevated AST (1), and depression (1). Analysis of tumor from Part 1 showed reduction of pAkt by IHC. Genotyping of tumor specimens is ongoing. To date 33 patients had positive pAkt, 21 had PTEN loss by IHC. Of the first 19 patients who underwent whole exome sequencing, 3 had PIK3Ca mutations and 6 had PTEN mutations. Conclusions: BKM120 is generally well tolerated in patients with recurrent GBM and achieves adequate tumor concentration to inhibit pAkt. Updated PK and efficacy data and correlation of the latter with tumor genotype will be presented. Clinical trial information: NCT01339052.

Download Full-text

Cycle initiation and colony formation in culture by murine marrow cells with long-term reconstituting potential in vivo

Blood ◽

10.1182/blood.v88.11.4149.bloodjournal88114149 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4149-4158 ◽

Cited By ~ 50

Author(s):

M Trevisan ◽

XQ Yan ◽

NN Iscove

Keyword(s):

Colony Formation ◽

Interleukin 1 ◽

Methyl Cellulose ◽

Culture Conditions ◽

Liquid Suspension ◽

Almost All ◽

Marrow Cells

This investigation was directed at separating long-term reconstituting (LTR) stem cells in normal murine marrow from hematopoietic precursors detectable in short-term assays in vitro and in vivo, and then at determining whether purified LTR cells could themselves form colonies in culture. To do so, it was first necessary to identify culture conditions that would induce their growth while preserving their long- term reconstituting capacity. Marrow was cultured with various cytokines in liquid suspension for 4 days, after which the surviving LTR activity was quantitated in a competitive in vivo assay. Activity was preserved near input levels with combined murine c-kit ligand (KL), interleukin-1 (IL-1), IL-6, and IL-11. When the cultures also included tritiated or unlabeled thymidine, LTR potential was eliminated, indicating that essentially all LTR cells were induced into cell cycle with these cytokines. To purify them, marrow was sorted on the basis of Ly6A expression and Rhodamine 123 retention. The Ly6AhiRh123ls fraction contained 85% of total recovered LTR activity but only 1% of the recovered cells measured by multilineage colony formation in spleens or in vitro. This fraction was cultured in methyl cellulose with KL, IL-1, IL-6, and IL-11 for 4 to 6 days, after which colonies were isolated and injected into mice. High levels of permanent reconstitution were achievable in sublethally irradiated W41/W41 mice after the injection of a single reconstituting unit, and limiting dilution analysis estimated the frequency of multilineage LTR at 1 in 11,200 unpurified adult marrow cells. In either lethally irradiated normal or sublethally irradiated W41/W41 mice, 1-year lymphomyeloid reconstitutions were obtained from 1 in 65 to 84 colonies of 2 to 16 dispersed cells, but not from larger colonies or those with clumped cells. The results establish that resting marrow LTR cells can be separated from almost all of the more advanced clonogenic cells that are still pluripotential, can be induced to cycle in culture by defined cytokines with preservation of their reconstituting potential, and can be manipulated and assayed efficiently at single-cell and colony levels.

Download Full-text

Deep conservation of histone variants in Thermococcales archaea

10.1101/2021.09.07.455978 ◽

2021 ◽

Author(s):

Kathryn M Stevens ◽

Antoine Hocher ◽

Tobias Warnecke

Keyword(s):

Histone Variants ◽

Prima Facie ◽

Transcriptional Responses ◽

Functional Roles ◽

Histone Fold ◽

Dna Accessibility ◽

Almost All

AbstractHistones are ubiquitous in eukaryotes where they assemble into nucleosomes, binding and wrapping DNA to form chromatin. One process to modify chromatin and regulate DNA accessibility is the replacement of histones in the nucleosome with paralogous variants. Histones are also present in archaea but whether and how histone variants contribute to the generation of different physiologically relevant chromatin states in these organisms remains largely unknown. Conservation of paralogs with distinct properties can provide prima facie evidence for defined functional roles. We recently revealed deep conservation of histone paralogs with different properties in the Methanobacteriales, but little is known experimentally about these histones. In contrast, the two histones of the model archaeon Thermococcus kodakarensis, HTkA and HTkB, have been examined in some depth, both in vitro and in vivo. HTkA and HTkB exhibit distinct DNA-binding behaviours and elicit unique transcriptional responses when deleted. Here, we consider the evolution of HTkA/B and their orthologs across the order Thermococcales. We find histones with signature HTkA- and HTkB-like properties to be present in almost all Thermococcales genomes. Phylogenetic analysis indicates the presence of one HTkA- and one HTkB-like histone in the ancestor of Thermococcales and long-term maintenance of these two paralogs throughout Thermococcales diversification. Our results support the notion that archaea and eukaryotes have convergently evolved histone variants that carry out distinct adaptive functions. Intriguingly, we also detect more highly diverged histone-fold proteins, related to those found in some bacteria, in several Thermococcales genomes. The functions of these bacteria-type histones remain entirely unknown, but structural modelling suggests that they can form heterodimers with HTkA/B-like histones.

Download Full-text

P14.125 Retrospective analysis of long-term response to bevacizumab in combination with irinotecan for recurrent glioblastoma: identification of prognostic factors

Neuro-Oncology ◽

10.1093/neuonc/noz126.360 ◽

2019 ◽

Vol 21 (Supplement_3) ◽

pp. iii98-iii98

Author(s):

C Besson ◽

M Morisse ◽

H Brut ◽

W Waissi ◽

G Noel ◽

...

Keyword(s):

Overall Survival ◽

Prognostic Factors ◽

Retrospective Analysis ◽

Performance Status ◽

Relative Proportion ◽

Univariate Analysis ◽

Recurrent Glioblastoma ◽

Term Response ◽

Recurrent Gbm

Abstract BACKGROUND In absence of standard treatment for recurrent glioblastoma (rGBM), numerous prospective and retrospective studies have evaluated the off-label combination of bevacizumab (BEV) with irinotecan (IRI) in patients with rGBM. We report here our single center experience with this combination and we investigated prognostic factors for long-term response. MATERIAL AND METHODS We performed a retrospective analysis of consecutive patients treated initially by Stupp protocol and with BEV-IRI for a rGBM between 2007 and 2017. Times to progression and overall survival, as well as toxicities, were investigated and analysed. Patients without progression at least 12 month after the first administration of BEV-IRI were considered as long-term responders. The primary end-point was overall survival post-BEV-IRI (OS-BEV-IRI). RESULTS One-hundred eleven patients were eligible for the analysis. Median age at the diagnosis was 57 years and the value of WHO Performance Status (PS) at the recurrence was 0 to 1 for 67,5% of patients. Kaplan-Meier median progression-free survival (PFS-BEV-IRI) and overall survival (OS-BEV-IRI) at recurrence estimates (calculated from start of BEV-IRI) were 6.51 and 10.41 months, respectively. The median OS (calculated from diagnosis) was 22,4 months. Twenty-Three patients (20,7%) were long-term responders to BEV-IRI regimen. This subgroup was not significantly different than the short-term responders according to age or PS distribution, but the relative proportion of biopsy in comparison to other surgery modalities was significantly increased in long-term responders (p<0,0001). Univariate analysis showed that PS 0–1 (p=0,007), biopsy (p=0,0022) are significantly associated with a better prognosis, but not age. Eighty three patients (75%) had toxicities, mainly grade 1 and 2 (92%), such as hypertension, proteinuria, haemorrhage, thrombosis, nausea, diarrhoea, fatigue or neutropenia. Most of the grade 3 and grade 4 toxicities were related to BEV treatment. Adverse events were significantly more frequent in long-term responders (p=0,0096). CONCLUSION BEV-IRI Combination is well tolerated and may offer some clinical benefits in recurrent GBM patients, more particularly if only biopsy was performed instead of surgery. Our results strengthened the role of these agents for the treatment of recurrent GBM.

Download Full-text

Lactate Metabolism Regulates Tumour Growth and Progression in Glioblastoma

10.21203/rs.3.rs-965025/v1 ◽

2021 ◽

Author(s):

Lucia Longhitano ◽

Nunzio Vicario ◽

Daniele Tibullo ◽

Cesarina Giallongo ◽

Giuseppe Broggi ◽

...

Keyword(s):

Cell Proliferation ◽

Metabolic Reprogramming ◽

In Vivo Model ◽

Glycolytic Metabolism ◽

Mesenchymal Transition ◽

Signalling Molecule ◽

Extracellular Lactate ◽

Human Gbm

Abstract Background. Tumor microenvironment (TME) plays a pivotal role in establishing malignancy and it is associated with high glycolytic metabolism and increased lactate production accumulating in TME through monocarboxylate transporters (MCTs). Several lines of evidence suggest that lactate also serves as a signalling molecule through its receptor HCAR1thus functioning as a paracrine and autocrine signalling molecule in TME. The aim of the present study was to investigate the role of lactate in glioblastoma (GBM) progression and metabolic reprogramming in an in vitro and in vivo model.Methods. Cell proliferation, migration and clonogenicity assay were performed in vitro on three different human GBM cell lines. Protein expression of MCT1, MCT4 and pharmacological lactate receptor (GPR81) were evaluated both in vitro and in a zebrafish GBM in vivo model. These results were further validated in patient-derived GBM biopsies.Results. Our results showed that lactate significantly increased cell proliferation, migration and colony formation capacity of GBM cells, both in vitro and in vivo. We also showed that lactate increased MCT1 and HCAR1 expression. Moreover, lactate modulated epithelial-mesenchymal transition protein markers E-Cadherin and β-Catenin. Interestingly, lactate induced mitochondrial mass and OXPHOS gene suggesting an improved mitochondrial fitness. Similar effects were observed after treatment with 3,5-Dihydroxybenzoic acid, a known agonist of GPR81. Consistently, GBM zebrafish model exhibited an altered metabolism and increased expression of MCT1 and HCAR1 leading to high levels of extracellular lactate and thus supporting tumor cell proliferation. Our data from human GBM biopsies also showed that in high proliferative GBM biopsies, Ki67 positive cells expressed significantly higher levels of MCT1 compared to low proliferative GBM cells.Conclusions. Our data suggest that lactate favours proliferation of neighbourhood cells by cooperating with their glycolytic metabolism, sensing and removing extracellular lactate. In particular, lactate and its transporter and receptor play a major role in GBM proliferation and migration thus representing a potential target to develop new strategies to counteract tumor progression and recurrencies.

Download Full-text