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.

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.

Elevated Microsatellite Alterations at Selected Tetranucleotides (EMAST) in Colorectal Cancer is Associated with an Elderly, Frail Phenotype and Improved Recurrence-Free Survival.

BACKGROUND: Elevated microsatellite alterations at selected tetranucleotides (EMAST) is a poorly investigated form of microsatellite instability (MSI) in colorectal cancer (CRC). OBJECTIVE: The aim of this study was to investigate the clinicopathological features of EMAST in CRC and its relation to outcome. METHODS: A population-based, consecutive cohort of surgically treated stage I-III CRC patients investigated for high-frequency MSI (MSI-H) and EMAST. Clinicopathological differences were reported as odds ratios (OR) and survival was presented as hazard ratios (HR) with 95% confidence intervals (CIs). RESULTS: Of 161 patients included, 25% were aged > 79 years. There was a large overlap in the prevalence of EMAST (31.7%) and MSI-H (27.3%) [82.4% of EMAST were also MSI-H]. EMAST had the highest prevalence in the proximal colon (OR 15.9, 95% CI 5.6-45.1; p < 0.001) and in women (OR 4.1, 95% CI 1.9-8.6; p < 0.001), and were poorly differentiated (OR 5.0, 95% CI 2.3-10.7; p < 0.001). Compared with EMAST-negative patients, EMAST-positive patients were older (median age 77 vs. 69 years; p < 0.001), leaner (median weight 67.5 vs. 77 kg; p = 0.001), had significantly higher rates of hypoalbuminemia (24% vs. 6%; OR 2.3, 95% CI 1.5-3.6; p = 0.002) and anemia (45% vs. 20%; OR 3.3, 95% CI 1.6-6.8; p = 0.001), and had elevated preoperative C-reactive protein (CRP) levels (51% vs. 34%; OR 1.9, 95% CI 1.0-3.9; p = 0.046). Improved recurrence-free survival was found in both MSI-H and EMAST subtypes. In multivariable analysis, node status (pN +), together with elevated CRP and MSI-positive, were the strongest prognostic factors for recurrence-free survival. CONCLUSIONS: EMAST in CRC is associated with an older, leaner, and frailer phenotype with a lower risk of recurrence. The relevance of, and putative mechanisms to, EMAST warrants further investigation.

Assessment of clinically related outcomes and biomarker analysis for translational integration in colorectal cancer (ACROBATICC): study protocol for a population-based, consecutive cohort of surgically treated colorectal cancers and resected colorectal liver metastasis.

BACKGROUND: More accurate predictive and prognostic biomarkers for patients with colorectal cancer (CRC) primaries or colorectal liver metastasis (CLM) are needed. Outside clinical trials, the translational integration of emerging pathways and novel techniques should facilitate exploration of biomarkers for improved staging and prognosis. METHODS: An observational study exploring predictive and prognostic biomarkers in a population-based, consecutive cohort of surgically treated colorectal cancers and resected colorectal liver metastases. Long-term outcomes will be cancer-specific survival, recurrence-free survival and overall survival at 5 years from diagnosis. Beyond routine clinicopathological and anthropometric characteristics and laboratory and biochemistry results, the project allows for additional blood samples and fresh-frozen tumour and normal tissue for investigation of circulating tumour cells (CTCs) and novel biomarkers (e.g. immune cells, microRNAs etc.). Tumour specimens will be investigated by immunohistochemistry in full slides. Extracted DNA/RNA will be analysed for genomic markers using specific PCR techniques and next-generation sequencing (NGS) panels. Flow cytometry will be used to characterise biomarkers in blood. Collaboration is open and welcomed, with particular interest in mutual opportunities for validation studies. STATUS AND PERSPECTIVES: The project is ongoing and recruiting at an expected rate of 120-150 patients per year, since January 2013. A project on circulating tumour cells (CTCs) has commenced, with analysis being prepared. Investigating molecular classes beyond the TNM staging is under way, including characteristics of microsatellite instability (MSI) and elevated microsatellite alterations in selected tetranucleotides (EMAST). Hot spot panels for known mutations in CRC are being investigated using NGS. Immune-cell characteristics are being performed by IHC and flow cytometry in tumour and peripheral blood samples. The project has ethical approval (REK Helse Vest, #2012/742), is financially supported with a Ph.D.-Grant (EMAST project; Folke Hermansen Cancer Fund) and a CTC-project (Norwegian Research Council; O. Nordgård). The ACROBATICC clinical and molecular biobank repository will serve as a long-term source for novel exploratory analysis and invite collaborators for mutual validation of promising biomarker results. The project aims to generate results that can help better discern prognostic groups in stage II/III cancers; explore prognostic and predictive biomarkers, and help detail the biology of colorectal liver metastasis for better patient selection and tailored treatment. The project is registered at http://www.ClinicalTrials.gov NCT01762813.

Accuracy of TNM staging in colorectal cancer: a review of current culprits, the modern role of morphology and stepping-stones for improvements in the molecular era.

Colorectal cancer (CRC) is the third most common cancer worldwide. Survival is largely stage-dependant, guided by the tumor-node-metastases (TNM) system for TNM assessment. Histopathological evaluation, including assessment of lymph node status, is important for correct TNM staging. However, recent updates in the TNM system have resulted in controversy. A continued debate on definitions resulting in potential up- and downstaging of patients, which may obscure survival data, has led the investigators to investigate other or alternative staging tools. Consequently, additional prognostic factors have been searched for using the regular light microscopy. Among the factors evaluated by histopathology include the evaluation of tumor budding and stromal environment, angiogenesis, as well as involvement of the immune system (including the 'Immunoscore'). We review the current role of histopathology, controversies in TNM-staging and suggested alternatives to better predict outcome for CRC patients in the era of genomic medicine.

Comparing in vitro cytotoxic drug sensitivity in colon and pancreatic cancer using 2D and 3D cell models: Contrasting viability and growth inhibition in clinically relevant dose and repeated drug cycles.

BACKGROUND: In vitro drug screening that is more translatable to the in vivo tumor environment can reduce both time and cost of cancer drug development. Here we address some of the shortcomings in screening and show how treatment with 5-fluorouracil (5-FU) in 2D and 3D culture models of colorectal cancer (CRC) and pancreatic ductal adenocarcinomas (PDAC) give different responses regarding growth inhibition. METHODS: The sensitivity of the cell lines at clinically relevant 5-FU concentrations was monitored over 4 days of treatment in both 2D and 3D cultures for CRC (SW948 and HCT116) and PDAC (Panc-1 and MIA-Pa-Ca-2) cell lines. The 3D cultures were maintained beyond this point to enable a second treatment cycle at Day 14, following the timeline of a standard clinical 5-FU regimen. RESULTS: Evaluation after one cycle did not reveal significant growth inhibition in any of the CRC or PDAC 2D models. By the end of the second cycle of treatment the CRC spheroids reached 50% inhibition at clinically achievable concentrations in the 3D model, but not in the 2D model. The PDAC models were not sensitive to clinical doses even after two cycles. High content viability metrics point to even lower response in the resistant PDAC models. CONCLUSION: This study reveals the limitations of testing drugs in 2D cancer models and short exposure in 3D models, and the importance of using appropriate growth inhibition analysis. We found that screening with longer exposure and several cycles of treatment in 3D models suggests a more reliable way to assess drug sensitivity.

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.

Molecular pathways and cellular metabolism in colorectal cancer.

Colorectal cancer (CRC) is, for sporadic forms, most strongly related to lifestyle factors. The epidemic of obesity and physical inactivity has great impact on disease patterns. Likewise, an altered metabolism has consequences at the cellular and molecular level with implications for cancer initiation and growth. Understanding the genetic hallmarks of cancers has improved over the years and now also includes cancer metabolic reprogramming. The initiation of cancer through genetic instability, including chromosomal instability, microsatellite instability and epigenetic silencing through the CpG island methylator phenotype follows pathways with distinct clinical, pathological, and genetic characteristics. These can potentially be used for molecular classification and comprehensive tumor profiling for improved diagnostics, prognosis and treatment in CRC. For one, epidermal growth factor receptor-directed treatment now considerably prolongs survival in metastatic disease, but defining the true responders from non-responders has emerged as complex. Further, the use of both non-steroidal anti-inflammatory drugs including cyclooxygenase-2 inhibitors is associated with a decreased incidence of adenoma and reduced mortality rate of CRC. This review gives a brief yet updated overview of the current understanding of CRC as a genetic and molecular disease with potential for clinical pathways of prevention, improved prediction and better prognosis in the future.

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.

Graphene-based phenformin carriers for cancer cell treatment: a comparative study between oxidized and pegylated pristine graphene in human cells and zebrafish.

Graphene is an attractive choice for the development of an effective drug carrier in cancer treatment due to its high adsorption area and pH-responsive drug affinity. In combination with the highly potent metabolic drug phenformin, increased doses could be efficiently delivered to cancer cells. This study compares the use of graphene oxide (GO) and polyethylene glycol stabilized (PEGylated) pristine graphene nanosheets (PGNSs) for drug delivery applications with phenformin. The cytotoxicity and mitotoxicity of the graphene-based systems were assessed in human cells and zebrafish larvae. Targeted drug release from GO and PGNSs was evaluated at different pH levels known to arise in proliferating tumor microenvironments. PGNSs were less cytotoxic and mitotoxic than GO, and showed an increased release of phenformin at lower pH in cells, compared to GO. In addition, the systemic phenformin effect was mitigated in zebrafish larvae when bound to GO and PGNSs compared to free phenformin, as measured by flavin metabolic lifetime imaging. These results pave the way for improved phenformin-based cancer therapy using graphene nano-sheets, where PGNSs were superior to GO.

Improved pH-Responsive Release of Phenformin from Low-Defect Graphene Compared to Graphene Oxide.

Graphene-based drug carriers provide a promising addition to current cancer drug delivery options. Increased accessibility of high-quality graphene made by plasma-enhanced chemical vapor deposition (PE-CVD) makes it an attractive material to revisit in comparison to the widely studied graphene oxide (GO) in drug delivery. Here, we show the potential of repurposing the metabolic drug phenformin for cancer treatment in terms of stability, binding, and pH-responsive release. Using covalent attachment of poly(ethylene glycol) (PEG) onto pristine (PE-CVD) graphene, we show that PEG stabilized graphene nanosheets (PGNS) are stable in aqueous solutions and exhibit higher binding affinity toward phenformin than GO. Moreover, we experimentally demonstrate an improved drug release from PGNS than GO at pH levels lower than physiological conditions, yet comparable to that found in tumor microenvironments.

Metformin treatment response is dependent on glucose growth conditions and metabolic phenotype in colorectal cancer cells.

Cancer cells exhibit altered metabolism, a phenomenon described a century ago by Otto Warburg. However, metabolic drug targeting is considered an underutilized and poorly understood area of cancer therapy. Metformin, a metabolic drug commonly used to treat type 2 diabetes, has been associated with lower cancer incidence, although studies are inconclusive concerning effectiveness of the drug in treatment or cancer prevention. The aim of this study was to determine how glucose concentration influences cancer cells' response to metformin, highlighting why metformin studies are inconsistent. We used two colorectal cancer cell lines with different growth rates and clinically achievable metformin concentrations. We found that fast growing SW948 are more glycolytic in terms of metabolism, while the slower growing SW1116 are reliant on mitochondrial respiration. Both cell lines show inhibitory growth after metformin treatment under physiological glucose conditions, but not in high glucose conditions. Furthermore, SW1116 converges with SW948 at a more glycolytic phenotype after metformin treatment. This metabolic shift is supported by changed GLUT1 expression. Thus, cells having different metabolic phenotypes, show a clear differential response to metformin treatment based on glucose concentration. This demonstrates the importance of growth conditions for experiments or clinical studies involving metabolic drugs such as metformin.

Cellular stress induced by resazurin leads to autophagy and cell death via production of reactive oxygen species and mitochondrial impairment.

Mitochondrial bioenergetics and reactive oxygen species (ROS) often play important roles in cellular stress mechanisms. In this study we investigated how these factors are involved in the stress response triggered by resazurin (Alamar Blue) in cultured cancer cells. Resazurin is a redox reactive compound widely used as reporter agent in assays of cell biology (e.g. cell viability and metabolic activity) due to its colorimetric and fluorimetric properties. In order to investigate resazurin-induced stress mechanisms we employed cells affording different metabolic and regulatory phenotypes. In HL-60 and Jurkat leukemia cells resazurin caused mitochondrial disintegration, respiratory dysfunction, reduced proliferation, and cell death. These effects were preceded by a burst of ROS, especially in HL-60 cells which were also more sensitive and contained autophagic vesicles. Studies in Rho(0) cells (devoid of mitochondrial DNA) indicated that the stress response does not depend on the rates of mitochondrial respiration. The anti-proliferative effect of resazurin was confirmed in native acute myelogenous leukemia (AML) blasts. In conclusion, the data suggest that resazurin triggers cellular ROS production and thereby initiates a stress response leading to mitochondrial dysfunction, reduced proliferation, autophagy, and cell degradation. The ability of cells to tolerate this type of stress may be important in toxicity and chemoresistance.

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.

BACKGROUND: An organic extract of the recreational herb khat (Catha edulis Forsk.) triggers cell death in various leukemia cell lines in vitro. 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. RESULTS: 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-FLIPL cleavage and procaspase-8 activation. CONCLUSION: 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.

Elevated Microsatellite Alterations at Selected Tetranucleotides (EMAST) Is Not Attributed to MSH3 Loss in Stage I-III Colon cancer: An Automated, Digitalized Assessment by Immunohistochemistry of Whole Slides and Hot Spots.

INTRODUCTION: EMAST is a poorly understood form of microsatellite instability (MSI) in colorectal cancer (CRC) for which loss of MSH3 has been proposed as the underlying mechanism, based on experimental studies. We aimed to evaluate whether MSH3 loss is associated with EMAST in CRC. METHODS: A consecutive cohort of patients with stage I-III CRC. Digital image analysis using heatmap-derived hot spots investigated MSH3 expression by immunohistochemistry. Fragment analysis of multiplex PCR was used to assess MSI and EMAST, and results cross-examined with MSH3 protein expression. RESULTS: Of 152 patients, EMAST was found in 50 (33%) and exclusively in the colon. Most EMAST-positive cancers had instability at all 5 markers, and EMAST overlapped with MSI-H in 42/50 cases (84%). The most frequently altered tetranucleotide markers were D8S321 (38.2% of tumors) and D20S82 (34.4%). Subjective evaluation of MSH3 expression by IHC in tumor found ≤10% negative tumor cells in all samples, most being ≤5% negative. Digital analysis improved the detection but showed a similar spread of MSH3 loss (range 0.1-15.7%, mean 2.2%). Hotspot MSH3 negativity ranged between 0.1 to 95.0%, (mean 8.6%) with significant correlation with the whole slide analysis (Spearman's rho=0.677 P<.001). Loss of MSH3 expression did not correlate with EMAST. CONCLUSIONS: In a well-defined cohort of patients with CRC, loss of MSH3 was not associated with EMAST. Further investigation into the mechanisms leading to EMAST in CRC is needed.

Aging, Metabolism, and Cancer Development: from Peto's Paradox to the Warburg Effect.

Medical advances made over the last century have increased our lifespan, but age-related diseases are a fundamental health burden worldwide. Aging is therefore a major risk factor for cardiovascular disease, cancer, diabetes, obesity, and neurodegenerative diseases, all increasing in prevalence. However, huge inter-individual variations in aging and disease risk exist, which cannot be explained by chronological age, but rather physiological age decline initiated even at young age due to lifestyle. At the heart of this lies the metabolic system and how this is regulated in each individual. Metabolic turnover of food to energy leads to accumulation of co-factors, byproducts, and certain proteins, which all influence gene expression through epigenetic regulation. How these epigenetic markers accumulate over time is now being investigated as the possible link between aging and many diseases, such as cancer. The relationship between metabolism and cancer was described as early as the late 1950s by Dr. Otto Warburg, before the identification of DNA and much earlier than our knowledge of epigenetics. However, when the stepwise gene mutation theory of cancer was presented, Warburg's theories garnered little attention. Only in the last decade, with epigenetic discoveries, have Warburg's data on the metabolic shift in cancers been brought back to life. The stepwise gene mutation theory fails to explain why large animals with more cells, do not have a greater cancer incidence than humans, known as Peto's paradox. The resurgence of research into the Warburg effect has given us insight to what may explain Peto's paradox. In this review, we discuss these connections and how age-related changes in metabolism are tightly linked to cancer development, which is further affected by lifestyle choices modulating the risk of aging and cancer through epigenetic control.

Correlation of Blood T-Cells to Intratumoural Density and Location of CD3+ and CD8+ T-Cells in Colorectal Cancer.

AIM: To test the feasibility of conducting parallel analyses of circulating T-cells in blood and intratumoural T-cells in colorectal cancer. A pre-operative 'liquid biopsy' to determine immune status would facilitate clinical decision-making. MATERIALS AND METHODS: A total of 18 patients with stage I-III colorectal cancer (CRC) were included. Blood was analyzed for T-cell type (CD3+, CD4+ and CD8+) and count using flow cytometry. Intratumoural T-cells were stained using immunohistochemistry and quantified by digital pathology. Tumour location was defined as invasive front (IF) or tumour center (TC). RESULTS: The number of CD3+ and CD4+ T-cells in pre-surgical blood samples correlated with the number of CD3+ T-cells found in the IF (Spearman ϱ=0.558, p<0.05 and 0.598, p<0.01 respectively) and CD3+ in the TC (ϱ=0.496, p<0.05, and ϱ=0.637, p<0.01, respectively). A strong correlation was found between CD4+ cells in blood and CD8+ T-cells found in the TC and IF (ϱ=0.602 and ϱ=0.591, p<0.01). CONCLUSION: There is a correlation between blood CD3+ and CD4+ T-cells and the T-cells found at the TC and IF.

The angiogenic switch leads to a metabolic shift in human glioblastoma.

Background: Invasion and angiogenesis are major hallmarks of glioblastoma (GBM) growth. While invasive tumor cells grow adjacent to blood vessels in normal brain tissue, tumor cells within neovascularized regions exhibit hypoxic stress and promote angiogenesis. The distinct microenvironments likely differentially affect metabolic processes within the tumor cells. Methods: In the present study, we analyzed gene expression and metabolic changes in a human GBM xenograft model that displayed invasive and angiogenic phenotypes. In addition, we used glioma patient biopsies to confirm the results from the xenograft model. Results: We demonstrate that the angiogenic switch in our xenograft model is linked to a proneural-to-mesenchymal transition that is associated with upregulation of the transcription factors BHLHE40, CEBPB, and STAT3. Metabolic analyses revealed that angiogenic xenografts employed higher rates of glycolysis compared with invasive xenografts. Likewise, patient biopsies exhibited higher expression of the glycolytic enzyme lactate dehydrogenase A and glucose transporter 1 in hypoxic areas compared with the invasive edge and lower-grade tumors. Analysis of the mitochondrial respiratory chain showed reduction of complex I in angiogenic xenografts and hypoxic regions of GBM samples compared with invasive xenografts, nonhypoxic GBM regions, and lower-grade tumors. In vitro hypoxia experiments additionally revealed metabolic adaptation of invasive tumor cells, which increased lactate production under long-term hypoxia. Conclusions: The use of glycolysis versus mitochondrial respiration for energy production within human GBM tumors is highly dependent on the specific microenvironment. The metabolic adaptability of GBM cells highlights the difficulty of targeting one specific metabolic pathway for effective therapeutic intervention.