EMT-derived alterations in glutamine metabolism sensitize mesenchymal breast cells to mTOR inhibition

Author's accepted version (postprint).This is an Accepted Manuscript of an article published by American Association for Cancer Research in Molecular Cancer Research on 04/06/2021.Available online: https://mcr.aacrjournals.org/content/19/9/1546acceptedVersio

Metabolic profiling of colorectal cancer organoids: A comparison between high-resolution magic angle spinning magnetic resonance spectroscopy and solution nuclear magnetic resonance spectroscopy of polar extracts

publishedVersio

Biodistribution of Poly(alkyl cyanoacrylate) Nanoparticles in Mice and Effect on Tumor Infiltration of Macrophages into a Patient-Derived Breast Cancer Xenograft

We have investigated the biodistribution and tumor macrophage infiltration after intravenous injection of the poly(alkyl cyanoacrylate) nanoparticles (NPs): PEBCA (poly(2-ethyl-butyl cyanoacrylate), PBCA (poly(n-butyl cyanoacrylate), and POCA (poly(octyl cyanoacrylate), in mice. These NPs are structurally similar, have similar PEGylation, and have previously been shown to give large variations in cellular responses in vitro. The PEBCA NPs had the highest uptake both in the patient-derived breast cancer xenograft MAS98.12 and in lymph nodes, and therefore, they are the most promising of these NPs for delivery of cancer drugs. High-resolution magic angle spinning magnetic resonance (HR MAS MR) spectroscopy did not reveal any differences in the metabolic profiles of tumors following injection of the NPs, but the PEBCA NPs resulted in higher tumor infiltration of the anti-tumorigenic M1 macrophages than obtained with the two other NPs. The PEBCA NPs also increased the ratio of M1/M2 (anti-tumorigenic/pro-tumorigenic) macrophages in the tumors, suggesting that these NPs might be used both as a vehicle for drug delivery and to modulate the immune response in favor of enhanced therapeutic effects

Characterization of breast cancer using MR metabolomics and gene expression analysis

Brystkreft er den vanligste kreftformen blant kvinner i Norge. Prognose og overlevelse avhenger av type kreft, tumorstørrelse, lymfeknutemetastaser og reseptorstatus (østrogen, progesteron og HER2). Basert på genekspresjonanalyser av tumorvev kan brystkreft deles inn i fem grupper; luminal A, luminal B, basal-like, HER2 positive og normal-like. Prognosene til pasientene i de ulike gruppene varierer der pasienter med basal-like brystkreft har den dårligste prognosen, mens pasienter med luminal A brystkreft har de beste prognosene. Metabolismen i kreftceller og normale celler er svært forskjellig. Et kjennetegn ved kreftceller er endret glykolytisk aktivitet. Kreftcellene kan forbruke glukose og omdanne dette til laktat til tross for at det er rikelig med oksygen til stede. Denne effekten er ofte omtalt som Warburgeffekten. En annen karakteristisk egenskap ved kreftceller er endringer i kolinmetabolismen. High resolution magic angle spinning MR spektroskopi (HR-MAS MRS) er en metode som er egnet for å studere biokjemiske forbindelser, kalt metabolitter, i vev. Glykolyseog kolinmetabolitter i kreftvev kan derfor studeres med denne teknikken. Proton (1H) MRS gir et spekter med informasjon om hvilke metabolitter som finnes i vev. 13C MRS er velegnet til å studere metabolsk omsetning i celler, dyr eller mennesker. Ved administrering av 13C-merkede metabolitter, kan man derfor kartlegge metabolske reaksjonsveier. Siden MRS er en kvantitativ metode kan den brukes til å beregne metabolittkonsentrasjoner i vev. Ved bruk av multivariate dataanalyser kan flere metabolitter i HR-MAS MR-spektrene analyseres samtidig. Denne metoden er derfor egnet til å studere metabolske forskjeller mellom ulike brystkreftgrupper. Siden vevet er intakt etter HR-MAS MRS kan det brukes til andre analyser etterpå, som for eksempel histopatologi eller genekspresjonsanalyser. Genekspresjonsanalyse er en egnet metode for å kartlegge hele eller deler av genomet. Med denne metoden kan man undersøke de genetiske forandringene som oppstår i kreftceller. Denne doktorgraden består av tre studier. Målet med det første studiet var å kartlegge prognostiske faktorer i brystkreftvev ved bruk HR-MAS MRS og multivariate dataanalyser. Tre ulike typer multivariate metoder ble benyttet for å undersøke om HRMAS MR spektrene inneholder informasjon som kan brukes til å prediktere østrogenreseptorstatus, progesteronreseptorstatus og lymfeknutestatus. Resultatene viste at det finnes metabolske forskjeller mellom tumorer som har positiv og negativ hormonreseptorstatus. I det andre studiet ble 13C HR-MAS MRS og genekspresjonsanalyser brukt til å kartlegge den glykolytiske aktiviteten i to ulike brystkreft musemodeller som representerer luminal-like og basal-like brystkreft. 13C-merket glukose ble injisert i de to modellene og tumorvev samlet 10 eller 15 minutter etter injeksjon. HR-MAS MRSanalysene av tumorvevet viste at glukose/laktat (Glc/Lac) og glukose/alanin (Glc/Ala)- ratioene var større i de raskt voksende basal-like svulstene sammenlignet med den luminal-like modellen. De fleste glykolytiske genene var dessuten oppregulert i den luminal-like modellen. Disse resultatene indikerer at den luminal-like modellen har større glykolytisk aktivitet enn den basal-like modellen, og at tumorvekst ikke nødvendigvis er en avgjørende faktor for glykolytisk aktivitet. Hensikten med det tredje studiet var å beskrive den metabolske profilen til et større utvalg av brystkreft musemodeller som representerer både luminal A, luminal B, basallike og HER2 positiv brystkreft. Resultatene viste at luminal B-svulstene hadde en større fosfokolin/glyserofosfokoline (PCho/GPC)-ratio enn de fleste basal-like svulstene. I tillegg var kolin, PCho og GPC korrelert til andre gener i kolinmetabolismen i luminal B- svulstene enn i de basal-like svulstene. Dette kan bety at reguleringen av kolinmetabolismen er ulik i de to undergruppene av brystkreft. Det var i tillegg god overensstemmelse mellom både metabolitt- og genekspresjonsprofiler mellom xenograftprøvene og brystkreftprøver fra pasienter i de to undergruppene. Resultatene fra studiet viser at dette panelet av xenograftmodeller er representativt for brystkreft hos mennesker, og betyr at modellene kan brukes til å identifisere nye behandlingsregimer ved bruk av HR-MAS MRS og genekspresjonsanalyser. Studiene beskrevet i denne avhandlingen har vist at HR-MAS MRS og genekspresjonsanalyser reflekterer ulike karakteristikker i brystkreft og at disse metodene derfor kan brukes til å utvikle prognostiske verktøy for brystkreftpasienter

Ionizing radiation abrogates the pro-tumorigenic capacity of cancer-associated fibroblasts co-implanted in xenografts

Cancer-associated fibroblasts (CAFs) are abundantly present in solid tumors and affect tumorigenesis and therapeutic responses. In the context of clinical radiotherapy, the impact of irradiated CAFs to treatment outcomes is largely unexplored. Aiming at improving radiotherapy efficacy, we have here explored the effect of radiation on the inherent pro-tumorigenic capacity of CAFs in animals. Ionizing radiation was delivered to cultured CAFs as single-high or fractionated doses. Tumor development was compared in mice receiving A549 lung tumor cells admixed with irradiated or control CAFs. Biological mechanisms behind tumor growth regulation were investigated by quantitative histology and immunohistochemistry. Viability assessments confirmed that irradiated CAFs are fully functional prior to implantation. However, the enhanced tumorigenic effect observed in tumors co-implanted with control CAFs was abrogated in tumors established with irradiated CAFs. Experiments to ascertain fate of implanted fibroblasts showed that exogenously administered CAFs reside at the implantation site for few days, suggesting that tumor growth regulation from admixed CAFs take place during initial tumor formation. Our work demonstrate that irradiated CAFs lose their pro-tumorigenic potential in vivo, affecting angiogenesis and tumor engraftment. This finding propose a previously unknown advantageous effect induced by radiotherapy, adding to the direct cytotoxic effects on transformed epithelial cells

Glutamine to proline conversion is associated with response to glutaminase inhibition in breast cancer

Introduction Glutaminase inhibitors target cancer cells by blocking the conversion of glutamine to glutamate, thereby potentially interfering with anaplerosis and synthesis of amino acids and glutathione. The drug CB-839 has shown promising effects in preclinical experiments and is currently undergoing clinical trials in several human malignancies, including triple-negative breast cancer (TNBC). However, response to glutaminase inhibitors is variable and there is a need for identification of predictive response biomarkers. The aim of this study was to determine how glutamine is utilized in two patient-derived xenograft (PDX) models of breast cancer representing luminal-like/ER+ (MAS98.06) and basal-like/triple-negative (MAS98.12) breast cancer and to explore the metabolic effects of CB-839 treatment. Experimental MAS98.06 and MAS98.12 PDX mice received CB-839 (200 mg/kg) or drug vehicle two times daily p.o. for up to 28 days (n = 5 per group), and the effect on tumor growth was evaluated. Expression of 60 genes and seven glutaminolysis key enzymes were determined using gene expression microarray analysis and immunohistochemistry (IHC), respectively, in untreated tumors. Uptake and conversion of glutamine were determined in the PDX models using HR MAS MRS after i.v. infusion of [5-13C] glutamine when the models had received CB-839 (200 mg/kg) or vehicle for 2 days (n = 5 per group). Results Tumor growth measurements showed that CB-839 significantly inhibited tumor growth in MAS98.06 tumors, but not in MAS98.12 tumors. Gene expression and IHC analysis indicated a higher proline synthesis from glutamine in untreated MAS98.06 tumors. This was confirmed by HR MAS MRS of untreated tumors demonstrating that MAS98.06 used glutamine to produce proline, glutamate, and alanine, and MAS98.12 to produce glutamate and lactate. In both models, treatment with CB-839 resulted in accumulation of glutamine. In addition, CB-839 caused depletion of alanine, proline, and glutamate ([1-13C] glutamate) in the MAS98.06 model. Conclusion Our findings indicate that TNBCs may not be universally sensitive to glutaminase inhibitors. The major difference in the metabolic fate of glutamine between responding MAS98.06 xenografts and non-responding MAS98.12 xenografts is the utilization of glutamine for production of proline. We therefore suggest that addiction to proline synthesis from glutamine is associated with response to CB-839 in breast cancer

EMT-derived alterations in glutamine metabolism sensitize mesenchymal breast cells to mTOR inhibition

Epithelial-to-mesenchymal transition (EMT) is a fundamental developmental process with strong implications in cancer progression. Understanding the metabolic alterations associated with EMT may open new avenues of treatment and prevention. Here we used 13C carbon analogs of glucose and glutamine to examine differences in their utilization within central carbon and lipid metabolism following EMT in breast epithelial cell lines. We found that there are inherent differences in metabolic profiles before and after EMT. We observed EMT-dependent re-routing of the TCA-cycle, characterized by increased mitochondrial IDH2-mediated reductive carboxylation of glutamine to lipid biosynthesis with a concomitant lowering of glycolytic rates and glutamine-dependent glutathione (GSH) generation. Using weighted correlation network analysis, we identified cancer drugs whose efficacy against the NCI-60 Human Tumor Cell Line panel is significantly associated with GSH abundance and confirmed these in vitro. We report that EMT-linked alterations in GSH synthesis modulate the sensitivity of breast epithelial cells to mTOR inhibitors

Glutamine to proline conversion is associated with response to glutaminase inhibition in breast cancer

Introduction Glutaminase inhibitors target cancer cells by blocking the conversion of glutamine to glutamate, thereby potentially interfering with anaplerosis and synthesis of amino acids and glutathione. The drug CB-839 has shown promising effects in preclinical experiments and is currently undergoing clinical trials in several human malignancies, including triple-negative breast cancer (TNBC). However, response to glutaminase inhibitors is variable and there is a need for identification of predictive response biomarkers. The aim of this study was to determine how glutamine is utilized in two patient-derived xenograft (PDX) models of breast cancer representing luminal-like/ER+ (MAS98.06) and basal-like/triple-negative (MAS98.12) breast cancer and to explore the metabolic effects of CB-839 treatment. Experimental MAS98.06 and MAS98.12 PDX mice received CB-839 (200 mg/kg) or drug vehicle two times daily p.o. for up to 28 days (n = 5 per group), and the effect on tumor growth was evaluated. Expression of 60 genes and seven glutaminolysis key enzymes were determined using gene expression microarray analysis and immunohistochemistry (IHC), respectively, in untreated tumors. Uptake and conversion of glutamine were determined in the PDX models using HR MAS MRS after i.v. infusion of [5-13C] glutamine when the models had received CB-839 (200 mg/kg) or vehicle for 2 days (n = 5 per group). Results Tumor growth measurements showed that CB-839 significantly inhibited tumor growth in MAS98.06 tumors, but not in MAS98.12 tumors. Gene expression and IHC analysis indicated a higher proline synthesis from glutamine in untreated MAS98.06 tumors. This was confirmed by HR MAS MRS of untreated tumors demonstrating that MAS98.06 used glutamine to produce proline, glutamate, and alanine, and MAS98.12 to produce glutamate and lactate. In both models, treatment with CB-839 resulted in accumulation of glutamine. In addition, CB-839 caused depletion of alanine, proline, and glutamate ([1-13C] glutamate) in the MAS98.06 model. Conclusion Our findings indicate that TNBCs may not be universally sensitive to glutaminase inhibitors. The major difference in the metabolic fate of glutamine between responding MAS98.06 xenografts and non-responding MAS98.12 xenografts is the utilization of glutamine for production of proline. We therefore suggest that addiction to proline synthesis from glutamine is associated with response to CB-839 in breast cancer. Graphical abstract The effect of glutaminase inhibition in two breast cancer patient-derived xenograft (PDX) models. 13C HR MAS MRS analysis of tumor tissue from CB-839-treated and untreated models receiving 13C-labeled glutamine ([5-13C] Gln) shows that the glutaminase inhibitor CB-839 is causing an accumulation of glutamine (arrow up) in two PDX models representing luminal-like breast cancer (MAS98.06) and basal-like breast cancer (MAS98.12). In MAS98.06 tumors, CB-839 is in addition causing depletion of proline ([5-13C] Pro), alanine ([1-13C] Ala), and glutamate ([1-13C] Glu), which could explain why CB-839 causes tumor growth inhibition in MAS98.06 tumors, but not in MAS98.12 tumors

Interplay of choline metabolites and genes in patient-derived breast cancer xenografts

Introduction Dysregulated choline metabolism is a well-known feature of breast cancer, but the underlying mechanisms are not fully understood. In this study, the metabolomic and transcriptomic characteristics of a large panel of human breast cancer xenograft models were mapped, with focus on choline metabolism. Methods Tumor specimens from 34 patient-derived xenograft models were collected and divided in two. One part was examined using high-resolution magic angle spinning (HR-MAS) MR spectroscopy while another part was analyzed using gene expression microarrays. Expression data of genes encoding proteins in the choline metabolism pathway were analyzed and correlated to the levels of choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) using Pearson’s correlation analysis. For comparison purposes, metabolic and gene expression data were collected from human breast tumors belonging to corresponding molecular subgroups. Results Most of the xenograft models were classified as basal-like (N = 19) or luminal B (N = 7). These two subgroups showed significantly different choline metabolic and gene expression profiles. The luminal B xenografts were characterized by a high PCho/GPC ratio while the basal-like xenografts were characterized by highly variable PCho/GPC ratio. Also, Cho, PCho and GPC levels were correlated to expression of several genes encoding proteins in the choline metabolism pathway, including choline kinase alpha (CHKA) and glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). These characteristics were similar to those found in human tumor samples. Conclusion The higher PCho/GPC ratio found in luminal B compared with most basal-like breast cancer xenograft models and human tissue samples do not correspond to results observed from in vitro studies. It is likely that microenvironmental factors play a role in the in vivo regulation of choline metabolism. Cho, PCho and GPC were correlated to different choline pathway-encoding genes in luminal B compared with basal-like xenografts, suggesting that regulation of choline metabolism may vary between different breast cancer subgroups. The concordance between the metabolic and gene expression profiles from xenograft models with breast cancer tissue samples from patients indicates that these xenografts are representative models of human breast cancer and represent relevant models to study tumor metabolism in vivo