Cellular metabolism in colorectal carcinogenesis: Influence of lifestyle, gut microbiome and metabolic pathways

The interconnectivity between diet, gut microbiota and cell molecular responses is well known; however, only recently has technology allowed the identification of strains of microorganisms harbored in the gastrointestinal tract that may increase susceptibility to cancer. The colonic environment appears to play a role in the development of colon cancer, which is influenced by the human metabolic lifestyle and changes in the gut microbiome. Studying metabolic changes at the cellular level in cancer be useful for developing novel improved preventative measures, such as screening through metabolic breath-tests or treatment options that directly affect the metabolic pathways responsible for the carcinogenicity

Induction of mitochondrial biogenesis and respiration is associated with mTOR regulation in hepatocytes of rats treated with the pan-PPAR activator tetradecylthioacetic acid (TTA)

The hypolipidemic effect of peroxisome proliferator-activated receptor (PPAR) activators has been explained by increasing mitochondrial fatty acid oxidation, as observed in livers of rats treated with the pan-PPAR activator tetradecylthioacetic acid (TTA). PPAR-activation does, however, not fully explain the metabolic adaptations observed in hepatocytes after treatment with TTA. We therefore characterized the mitochondrial effects, and linked this to signalling by the metabolic sensor, the mammalian target of rapamycin (mTOR). In hepatocytes isolated from TTA-treated rats, the changes in cellular content and morphology were consistent with hypertrophy. This was associated with induction of multiple mitochondrial biomarkers, including mitochondrial DNA, citrate synthase and mRNAs of mitochondrial proteins. Transcription analysis further confirmed activation of PPARα-associated genes, in addition to genes related to mitochondrial biogenesis and function. Analysis of mitochondrial respiration revealed that the capacity of both electron transport and oxidative phosphorylation were increased. These effects coincided with activation of the stress related factor, ERK1/2, and mTOR. The protein level and phosphorylation of the downstream mTOR actors eIF4G and 4E-BP1 were induced. In summary, TTA increases mitochondrial respiration by inducing hypertrophy and mitochondrial biogenesis in rat hepatocytes, via adaptive regulation of PPARs as well as mTOR.acceptedVersio

Metabolic flux analysis of 3D spheroids reveals significant differences in glucose metabolism from matched 2D cultures of colorectal cancer and pancreatic ductal adenocarcinoma cell lines

Background Most in vitro cancer cell experiments have been performed using 2D models. However, 3D spheroid cultures are increasingly favored for being more representative of in vivo tumor conditions. To overcome the translational challenges with 2D cell cultures, 3D systems better model more complex cell-to-cell contact and nutrient levels present in a tumor, improving our understanding of cancer complexity. Despite this need, there are few reports on how 3D cultures differ metabolically from 2D cultures. Methods Well-described cell lines from colorectal cancer (HCT116 and SW948) and pancreatic ductal adenocarcinoma (Panc-1 and MIA-Pa-Ca-2) were used to investigate metabolism in 3D spheroid models. The metabolic variation under normal glucose conditions were investigated comparing 2D and 3D cultures by metabolic flux analysis and expression of key metabolic proteins. Results We find significant differences in glucose metabolism of 3D cultures compared to 2D cultures, both related to glycolysis and oxidative phosphorylation. Spheroids have higher ATP-linked respiration in standard nutrient conditions and higher non-aerobic ATP production in the absence of supplemented glucose. In addition, ATP-linked respiration is significantly inversely correlated with OCR/ECAR (p = 0.0096). Mitochondrial transport protein, TOMM20, expression decreases in all spheroid models compared to 2D, and monocarboxylate transporter (MCT) expression increases in 3 of the 4 spheroid models. Conclusions In this study of CRC and PDAC cell lines, we demonstrate that glucose metabolism in 3D spheroids differs significantly from 2D cultures, both in terms of glycolytic and oxidative phosphorylation metrics. The metabolic phenotype shift from 2D to 3D culture in one cell line is greater than the phenotypic differences between each cell line and tumor source. The results herein emphasize the need to use 3D cell models for investigating nutrient utilization and metabolic flux for a better understanding of tumor metabolism and potential metabolic therapeutic targets.publishedVersio

Prevalence of PD‑L1 expression is associated with EMAST, density of peritumoral T‑cells and recurrence‑free survival in operable non‑metastatic colorectal cancer

Introduction Microsatellite instability (MSI) predict response to anti-PD1 immunotherapy in colorectal cancer (CRC). CRCs with MSI have higher infiltration of immune cells related to a better survival. Elevated Microsatellite Alterations at Tetranucleotides (EMAST) is a form of MSI but its association with PD-L1 expression and immune-cell infiltration is not known. Methods A consecutive, observational cohort of patients undergoing surgery for CRC. EMAST and clinicopathological characteristics were investigated against PD-L1, as well as CD3 and CD8 expression in the invasive margin or tumour centre (Immunoscore). Difference in survival between groups was assessed by log rank test. Results A total of 149 stage I–III CRCs patients, with a median follow up of 60.1 months. Patients with PD-L1+ tumours (7%) were older (median 79 vs 71 years, p = 0.045) and had EMAST+ cancers (OR 10.7, 95% CI 2.2–51.4, p = 0.001). Recurrence-free survival was longer in cancers with PD-L1+ immune cells (HR 0.35, 95% CI 0.16–0.76, p = 0.008, independent of EMAST) and high Immunoscore (HR 0.10, 95% CI 0.01–0.72, p = 0.022). Patients expressing PD-L1 in immune cells had longer disease-specific survival (HR 0.28, 95% CI 0.10–0.77, p = 0.014). Conclusions Higher Immunoscore (CD3/CD8 cells) and expression of tumour PD-L1 is found in CRCs with EMAST. Lymphocytic infiltrate and peritumoral PD-L1 expression have prognostic value in CRC.publishedVersio

A template to quantify the location and density of CD3 + and CD8 + tumor-infiltrating lymphocytes in colon cancer by digital pathology on whole slides for an objective, standardized immune score assessment

Background In colon cancer, the location and density of tumor-infiltrating lymphocytes (TILs) can classify patients into low and high-risk groups for prognostication. While a commercially available ‘Immunoscore®’ exists, the incurred expenses and copyrights may prevent universal use. The aim of this study was to develop a robust and objective quantification method of TILs in colon cancer. Methods A consecutive, unselected series of specimens from patients with colon cancer were available for immunohistochemistry and assessment of TILs by automated digital pathology. CD3 + and CD8 + cells at the invasive margin and in tumor center were assessed on consecutive sections using automated digital pathology and image analysis software (Visiopharm®). An algorithm template for whole slide assessment, generated cell counts per square millimeters (cells/mm2), from which the immune score was calculated using distribution volumes. Furthermore, immune score was compared with clinical and histopathological characteristics to confirm its relevance. Results Based on the quantified TILs numbers by digital image analyses, patients were classified into low (n = 83, 69.7%), intermediate (n = 14, 11.8%) and high (n = 22, 18.5%) immune score groups. High immune score was associated with stage I–II tumors (p = 0.017) and a higher prevalence of microsatellite instable (MSI) tumors (p = 0.030). MSI tumors had a significantly higher numbers of CD3 + TILs in the invasive margin and CD8 + TILs in both tumor center and invasive margin, compared to microsatellite stable (MSS) tumors. Conclusion A digital template to quantify an easy-to-use immune score corresponds with clinicopathological features and MSI in colon cancer.publishedVersio

A template to quantify the location and density of CD3 + and CD8 + tumor-infiltrating lymphocytes in colon cancer by digital pathology on whole slides for an objective, standardized immune score assessment

Background: In colon cancer, the location and density of tumor-infiltrating lymphocytes (TILs) can classify patients into low and high-risk groups for prognostication. While a commercially available ‘Immunoscore®’ exists, the incurred expenses and copyrights may prevent universal use. The aim of this study was to develop a robust and objective quantification method of TILs in colon cancer. Methods: A consecutive, unselected series of specimens from patients with colon cancer were available for immunohistochemistry and assessment of TILs by automated digital pathology. CD3 + and CD8 + cells at the invasive margin and in tumor center were assessed on consecutive sections using automated digital pathology and image analysis software (Visiopharm®). An algorithm template for whole slide assessment, generated cell counts per square millimeters (cells/mm2), from which the immune score was calculated using distribution volumes. Furthermore, immune score was compared with clinical and histopathological characteristics to confirm its relevance. Results: Based on the quantified TILs numbers by digital image analyses, patients were classified into low (n = 83, 69.7%), intermediate (n = 14, 11.8%) and high (n = 22, 18.5%) immune score groups. High immune score was associated with stage I–II tumors (p = 0.017) and a higher prevalence of microsatellite instable (MSI) tumors (p = 0.030). MSI tumors had a significantly higher numbers of CD3 + TILs in the invasive margin and CD8 + TILs in both tumor center and invasive margin, compared to microsatellite stable (MSS) tumors. Conclusion: A digital template to quantify an easy-to-use immune score corresponds with clinicopathological features and MSI in colon cancer.publishedVersio

Camptothecin and khat (Catha edulis Forsk.) induced distinct cell death phenotypes involving modulation of c-FLIPL, Mcl-1, procaspase-8 and mitochondrial function in acute myeloid leukemia cell lines

<p>Abstract</p> <p>Background</p> <p>An organic extract of the recreational herb khat (<it>Catha edulis </it>Forsk.) triggers cell death in various leukemia cell lines <it>in vitro</it>. The chemotherapeutics camptothecin, a plant alkaloid topoisomerase I inhibitor, was tested side-by-side with khat in a panel of acute myeloid leukemia cell lines to elucidate mechanisms of toxicity.</p> <p>Results</p> <p>Khat had a profound effect on MOLM-13 cells inducing mitochondrial damage, chromatin margination and morphological features of autophagy. The effects of khat on mitochondrial ultrastructure in MOLM-13 correlated with strongly impaired routine respiration, an effect neither found in the khat-resistant MV-4-11 cells nor in camptothecin treated cells. Enforced expression of anti-apoptotic Bcl-2 protein provided protection against camptothecin-induced cell death and partly against khat toxicity. Khat-induced cell death in MOLM-13 cells included reduced levels of anti-apoptotic Mcl-1 protein, while both khat and camptothecin induced c-FLIP_Lcleavage and procaspase-8 activation.</p> <p>Conclusion</p> <p>Khat activated a distinct cell death pathway in sensitive leukemic cells as compared to camptothecin, involving mitochondrial damage and morphological features of autophagy. This suggests that khat should be further explored in the search for novel experimental therapeutics.</p

Cyclic production of biocompatible few-layer graphene ink with in-line shear-mixing for inkjet-printed electrodes and Li-ion energy storage

The scalable production of two-dimensional (2D) materials is needed to accelerate their adoption to industry. In this work, we present a low-cost in-line and enclosed process of exfoliation based on high-shear mixing to create aqueous dispersions of few-layer graphene, on a large scale with a Yw ~ 100% yield by weight and throughput of ϕ ~ 8.3 g h−1. The in-line process minimises basal plane defects compared to traditional beaker-based shear mixing which we attribute to a reduced Reynolds number, Re ~ 105. We demonstrate highly conductive graphene material with conductivities as high as σ ∼ 1.5 × 104 S m−1 leading to sheet-resistances as low as Rs ∼ 2.6 Ω □−1 (t ∼ 25 μm). The process is ideal for formulating non-toxic, biocompatible and highly concentrated (c ∼ 100 mg ml−1) inks. We utilise the graphene inks for inkjet printable conductive interconnects and lithium-ion battery anode composites that demonstrate a low-rate lithium storage capability of 370 mAh g−1, close to the theoretical capacity of graphite. Finally, we demonstrate the biocompatibility of the graphene inks with human colon cells and human umbilical vein endothelial cells at high c ∼ 1 mg ml−1 facilitating a route for the use of the graphene inks in applications that require biocompatibility at high c such as electronic textiles.publishedVersio

Å krysse fremfor å beskytte grenser. Om ergoterapeut-, fysioterapeut-, lege- og sykepleierstudenters deltakelse og læring i tverrprofesjonell praksis

Tema for avhandlingen er studenters møte med og deltakelse i tverrprofesjonell virksomhet i sine praksisstudier. Jeg utforsker hvordan ergoterapeut-, fysioterapeut-, lege- og sykepleierstudenter lærer ved å delta i tverrprofesjonell praksis. I helseprofesjonsutdanninger er praksisstudier obligatoriske og viktige i utvikling av handling, handlingsgrunnlag og profesjonalitet. Studenters kliniske studier ved sykehus omfatter læring i profesjonenes lokale praksiser. Avhandlingens empiriske utforskning om studenters deltakelse og læring ved praksisstedet gir derfor også innblikk i profesjonenes samhandling og praksis på mikronivå. I helse- og sykdomsfeltet er kunnskapsutviklingen stor og mangfoldig som følge av økende differensiering og spesialisering. Samtidig innebærer helsepolitiske føringer og forventninger fra pasienter og pårørende at profesjonelle samarbeider slik at deres innsats og kunnskaper samlet former enhetlige pasientforløp. Det er ikke gitt at profesjonelle samarbeider, og samarbeidsproblem blir gjerne knyttet til at profesjoner hegner om sine oppgave- og kunnskapsfelt. Dette står i kontrast til tverrprofesjonell praksis der felles oppgave blir løst ved å forbinde deltakernes bidrag. Profesjonenes oppgaver og kunnskapsbruk ved praksisstedet påvirker studenters oppfatning av hva som er relevant og meningsfylt å lære. Deltakelse og kunnskaper får betydning når de bidrar i forståelse av hva profesjonalitet er, og ikke bare for å kunne løse konkrete oppgaver. Hvordan utvikler og forstår studenter profesjonalitet, og hvordan inngår tverrprofesjonell praksis i deres bilde av fremtidig yrkesutøvelse? Hvordan er den konkrete samhandling mellom profesjonene som studenter møter i sykehus? Hva betyr deltakelse i lokale praksiser i studenters læring? Dette er sentrale spørsmål i avhandlingen. Det empiriske materiale som er analysert og tolket, er frembragt ved deltakende observasjon, intervjuer med ergoterapeut-, fysioterapeut-, lege- og sykepleierstudenter samt fokusgruppesamtale med veilederne. Praksisstedene er sykehusposter ved medisinsk seksjon i to sykehus. En post er studenttett, der studenter overtok mange av sin profesjons oppgaver. Det teoretiske tilfanget er hentet fra sosial læringsteori og profesjonsteori. Wengers (1998) sosiale læringsteori om praksisfellesskap er den sentrale analytiske rammen for å belyse hvordan lokal praksis tilrettela for studenters deltakelse og læring. Abbotts (1988) teori om profesjonssystemet gir en analytisk spenning mellom profesjonenes grenser forstått som skillelinjer og grenser forstått som forbindelsesflater (Wenger 1998). Det var forskjeller i postenes praksis i hvordan pasientens problem og tiltak ble vurdert og fastsatt. Forskjellene knyttes til aktørenes direkte samhandling og bruk av felles redskap. Begrepet grenseobjekt (Star & Griesemer 1989) bidrar i analysene til å tydeliggjøre hvordan elektronisk pasientjournal og felles skjema påvirket samhandling, praksis og studentenes læring. Avhandlingen løfter frem hvordan deltaker- og tingliggjorte forbindelser (Wenger 1998), inklusiv felles redskap, samspiller og former praksis. Deltakerforbindelser var avgjørende i utvikling av tverrprofesjonelle praksisfellesskap og i studentenes læring, de dominerer tverrprofesjonell praksis i Studenttett post. Betydningen av tingliggjorte forbindelser var tydelig i etablert tverrprofesjonell praksis og dominerer i det andre sykehuset. Ved Studenttett post deltok studentene i utvikling av felles interessefelt om pasienten. Ved å være deltakere i meningsforhandling og meningsdannelse kjeder studentene sammen aktivitet og kunnskaper mellom profesjonene. De danner tverrprofesjonelle deltakerforbindelser og praksisfellesskap. De utvikler tillit til hverandres kunnskap og handling. De engasjerer seg i hverandres praksis, forståelse og læring på tvers av profesjonsgrensene. Legestudentene engasjerer seg lite i postens praksis og skiller seg ut. Til forskjell var samhandlingen om felles interessefelt ved det andre sykehuset preget av effektivitet og innordning. Møtepraksis speiler profesjonenes daglige funksjonsdeling og tillit til hver profesjons ansvarlighet. Dette hemmer studentene i å oppdage og lære hvordan bidrag er forbundet over profesjonsgrensene. De får begrenset innblikk i postens virksomhet og praksis. Studentene tilhører profesjonens studentfellesskap og utvikler ikke tverrprofesjonelle relasjoner. Møtepraksis bidrar til å sosialisere studentene inn i profesjonens ansvarsfelt. I hverdagen er postens tverrprofesjonelle praksis lite synlig i direkte samhandling og fremstår som flerprofesjonell. Dette bildet endrer seg når bruk av elektronisk pasientjournal trekkes inn i analysene. Når de tverrprofesjonelle kompetanseområder trer frem i analysene er postens tverrprofesjonelle praksis tydelig. Praktiske kunnskaper og synteser (Grimen 2008) er sentrale perspektiv for å forstå tverrprofesjonell praksis. Praktiske synteser innebærer at kunnskapselement blir forbundet på bestemte måter ut i fra hva formålet er. Analysene løfter frem hvordan deltakerne former meningsfulle sammenhenger ved å forbinde praktiske og teoretiske kunnskaper over profesjonsgrensene. Tverrprofesjonelle kompetanseområder er vesentlige for lokal praksis, og for å belyse tverrprofesjonalitet i studenters læring. Det var forskjeller i praksisstedenes tverrprofesjonelle kompetanseområder. Ved Studenttett post var tverrprofesjonell praksis og kompetanseområder utviklet for å sikre forsvarlig pasientbehandling når studenter overtok, og samtidig skape et inkluderende læringsmiljø. Sentralt var å kunne utvikle enhetlige pasientforløp ved felles vurdering av problem og tiltak. Veiledning og kunnskaper som bidro til studenters mestring, hadde betydning i veiledernes tverrprofesjonelle praksis. Postens praksis og kompetansesystem åpnet for at studenter var nær fullverdige deltakere. Ved det andre sykehuset var formålet å gjøre pasienter mest mulig selvstendige. Dette tvang frem sammenkjeding av profesjonenes kunnskaper, fordi pasientens problem var delt opp og fordelt mellom profesjonene. Tre tverrprofesjonelle kompetanseområder blir eksemplifisert: Forflytning, kognitiv funksjon og svelgeproblem. Hvert av kompetanseområdene forbandt relevante kunnskaper om problemet. For hvert område var det laget et hefte. Alle profesjoner deltok i utvikling av tverrprofesjonelle kompetanseområder og praksis. I utviklingsarbeidet danner de tverrprofesjonelle praksisfellesskap, som muliggjør postens møtepraksis, bruk av skriftliggjøring og profesjonenes funksjonsdeling i hverdagen. Posten kompetansesystem og praksis gjør studentene til svært perifere deltakere. Studenters læring er ikke del av postens felles praksis, og som veiledere er ikke veilederne et tverrprofesjonelt praksisfellesskap. Flere former for deltakerbaner (Wenger 1998) får frem hvordan praksis i ulik grad åpner for studenters deltakelse og læring. Ved Studenttett post går studentenes deltakerbaner via tverrprofesjonelle praksisfellesskap inn i fremtidig profesjon. Analysene får også frem betydningen av å kunne følge deltakerbaner i profesjonens lokale praksisfellesskap. Det vil si lære å bruke profesjonens handlingsgrunnlag, dokumentasjonsmåte og kriterier for praksis. Deltakerbaner i tverrprofesjonelle praksisfellesskap hjalp studentene ved Studenttett post til å forme fremtidig profesjonalitet som går utover det å mestre konkrete oppgaver. De lærer å krysse profesjonsgrensene i utvikling av felles ansvarlighet, praksis og handlingsgrunnlag. Å være deltaker i tverrprofesjonelle praksisfellesskap innebærer å krysse profesjonsgrenser for å utvikle kunnskap og meningsfulle sammenhenger. Studentene ved det andre sykehuset har små muligheter til å følge deltakerbaner inn i tverrprofesjonell praksis. I deres profesjonalitet inngår i mindre grad å utvikle praksis og forståelse ved å forbinde flere profesjoners bidrag

Cellular responses to metabolic stress. A study on the role of mitochondria in the crosstalk between cell metabolism and survival signaling

Metabolic imbalance is associated with increased risk of several diseases, including cancer. Metabolism affects or is affected by virtually all other cellular processes, which is not surprising considering their conserved role throughout evolution. Mitochondria are important sensors of the intracellular metabolic environment and play a major role in bioenergetics and survival signaling in mammalian cells. Accordingly, these cell organelles have been implicated in the development of cancer, which includes changes in cellular metabolism and cell death signaling. Metabolic reprogramming in cancer cells increases cell growth and proliferation. This entails greater nutrient uptake which is metabolized to provide energy and intermediates for cell constituents. This reprogramming has generally been associated with increased therapeutic resistance, but may also prove to be utilized in metabolic targeted therapies. Major signaling networks that are involved in tumor growth and metabolic reprogramming are PI3K/Akt/mTOR signaling pathways often found mutated in cancer cells. Activation of this pathway results in high glucose dependency and aggressive tumors. On the other hand, the activation of a lowenergy- responsive AMP-activated protein kinase (AMPK) signaling pathway leads to growth inhibition. Both of these signaling pathways may regulate or be regulated by mitochondrial functions, which has been the basis of this Ph.D study. The aim of this work was to investigate interactions between cell metabolism and signalling that affect mitochondrial function under conditions of energetic and metabolic stress. In order to reflect different contexts of mitochondrial regulation, we studied these mechanisms in both metabolically restricted cancer cells, as well as metabolically flexible primary rat hepatocytes. We found that leukemia cells that have mutations in the PI3K/Akt/mTOR signaling pathway (Jurkat) were more susceptible to glucose deprivation and agents that challenge this metabolism (such as palmitic acid). Alternatively, leukemia cells that use more oxidative phosphorylation for their metabolism (HL-60) showed increased resistance to glucose deprivation, but higher susceptibility to agents interfering with their mitochondrial function (such as resazurin and AICAR). Metabolically flexible primary cells (hepatocytes) were found to adjust their metabolism in response to agents that induce increased metabolic stress (TTA). Interestingly, this involved the nutrient sensing mTOR signaling pathway, which may play a role in regulating cell size, whereas we found no indications of hyperplasia (no neoplastic growth of the liver). These studies support the growing understanding that metabolic characterization of cancer cells and its effects on and by mutations in cell signalling pathways, not only gives us a better understanding of tumour biology, but may also provide additional treatment targets and strategies in cancer therapy. It is thus a possibility that treatments targeted to metabolism cause cell stress and death in metabolically compromised cancer cells, while more benign reversible stress responses may occur in normal cells