Gro Vatne Røsland

CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells

CD133 is a cell surface marker expressed on progenitors of haematopoietic and endothelial cell lineages. Moreover, several studies have identified CD133 as a marker of brain tumor‐initiating cells. In this study, human glioblastoma multiforme biopsies were engrafted intracerebrally into nude rats. The resulting tumors were serially passaged in vivo, and monitored by magnetic resonance imaging. CD133 expression was analyzed at various passages. Tumors initiated directly from the biopsies expressed little or no CD133, and showed no contrast enhancement suggesting an intact blood‐brain barrier. During passaging, the tumors gradually displayed more contrast enhancement, increased angiogenesis and a shorter survival. Real‐time qPCR and immunoblots showed that this was accompanied by increased CD133 expression. Primary biopsy spheroids and xenograft tumors were subsequently dissociated and flow sorted into CD133 negative and CD133 positive cell populations. Both populations incorporated BrdU in cell culture, and expressed the neural precursor marker nestin. Notably, CD133 negative cells derived from 6 different patients were tumorgenic when implanted into the rat brains. For 3 of these patients, analysis showed that the resulting tumors contained CD133 positive cells. In conclusion, we show that CD133 negative glioma cells are tumorgenic in nude rats, and that CD133 positive cells can be obtained from these tumors. Upon passaging of the tumors in vivo, CD133 expression is upregulated, coinciding with the onset of angiogenesis and a shorter survival. Thus, our findings do not suggest that CD133 expression is required for brain tumor initiation, but that it may be involved during brain tumor progression.

Long-term Cultures of Bone Marrow-Derived Human Mesenchymal Stem Cells Frequently Undergo Spontaneous Malignant Transformation

Human mesenchymal stem cells (hMSC) aid in tissue maintenance and repair by differentiating into specialized cell types. Due to this ability, hMSC are currently being evaluated for cell-based therapies of tissue injury and degenerative diseases. However, extensive expansion ex vivo is a prerequisite to obtain the cell numbers required for human cell-based therapy protocols. Recent studies indicate that hMSC may contribute to cancer development and progression either by acting as cancer-initiating cells or through interactions with stromal elements. If spontaneous transformation ex vivo occurs, this may jeopardize the use of hMSC as therapeutic tools. Whereas murine MSC readily undergo spontaneous transformation, there are conflicting reports about spontaneous transformation of hMSC. We have addressed this controversy in a two-center study by growing bone marrow-derived hMSC in long-term cultures (5-106 weeks). We report for the first time spontaneous malignant transformation to occur in 45.8% (11 of 24) of these cultures. In comparison with hMSC, the transformed mesenchymal cells (TMC) showed a significantly increased proliferation rate and altered morphology and phenotype. In contrast to hMSC, TMC grew well in soft agar assays and were unable to undergo complete differentiation. Importantly, TMC were highly tumorigenic, causing multiple fast-growing lung deposits when injected into immunodeficient mice. We conclude that spontaneous malignant transformation may represent a biohazard in long-term ex vivo expansion of hMSC. On the other hand, this spontaneous transformation process may represent a unique model for studying molecular pathways initiating malignant transformation of hMSC.

Spontaneous Malignant Transformation of Human Mesenchymal Stem Cells Reflects Cross-Contamination: Putting the Research Field on Track - Letter

Several groups, including ours, have published results showing spontaneous transformation of human mesenchymal stem cells (MSC). Recently, we reported in this journal spontaneous transformation of bone marrow-derived human MSC (hMSC), isolated and expanded independently in two laboratories ([1][1

EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis

Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth.

Metabolic profiling indicates impaired pyruvate dehydrogenase function in myalgic encephalopathy/chronic fatigue syndrome

Myalgic encephalopathy/chronic fatigue syndrome (ME/CFS) is a debilitating disease of unknown etiology, with hallmark symptoms including postexertional malaise and poor recovery. Metabolic dysfunction is a plausible contributing factor. We hypothesized that changes in serum amino acids may disclose specific defects in energy metabolism in ME/CFS. Analysis in 200 ME/CFS patients and 102 healthy individuals showed a specific reduction of amino acids that fuel oxidative metabolism via the TCA cycle, mainly in female ME/CFS patients. Serum 3-methylhistidine, a marker of endogenous protein catabolism, was significantly increased in male patients. The amino acid pattern suggested functional impairment of pyruvate dehydrogenase (PDH), supported by increased mRNA expression of the inhibitory PDH kinases 1, 2, and 4; sirtuin 4; and PPARδ in peripheral blood mononuclear cells from both sexes. Myoblasts grown in presence of serum from patients with severe ME/CFS showed metabolic adaptations, including increased mitochondrial respiration and excessive lactate secretion. The amino acid changes could not be explained by symptom severity, disease duration, age, BMI, or physical activity level among patients. These findings are in agreement with the clinical disease presentation of ME/CFS, with inadequate ATP generation by oxidative phosphorylation and excessive lactate generation upon exertion.

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.

Novel Points of Attack for Targeted Cancer Therapy

New molecular insight reveals novel points of attack for targeted cancer therapy. The recent advances in cancer genomics and novel insight into the complex biology of cancer make the promise of personalized, targeted cancer medicine closer than ever. The massive parallel sequencing endeavours performed by The Cancer Genome Atlas, the International Cancer Genome Consortium and by numerous individual investigators have provided a comprehensive genomic characterization of a wide range of cancers. The joint efforts enabled by the improved sequencing technology have demonstrated that individual cancers comprise mutational repertoires with only a few frequently recurrent driver genes. Thus, the identification of new drug targets and novel drugs have accelerated and renewed the hopes of personalized cancer therapy achieving clinical reality for a wider range of cancers. Together with cost-effective sequencing technology to perform comprehensive mutational profiling of each individual cancer, this provides the basis for a personalized cancer medicine revolution within the next few years. The aim of this MiniReview is to provide an overview of the history and evolution of targeted cancer therapy, exemplified by molecularly targeted drugs successfully implemented in the clinic. Furthermore, we aim to highlight novel molecular targets for therapeutic intervention, as well as the main present challenges including inter- and intratumor heterogeneity and cellular plasticity in addition to the importance of the tumor micro-environment. Many cancer patients already receive some form of tailored therapy, and recent evidence suggests that novel and highly innovative, targeted approaches are on their way into the clinic.

Spontaneous Transformation of Stem Cells In Vitro and the Issue of Cross-Contamination

We have read with interest the paper “Long-term cultured human neural stem cells undergo spontaneous transformation to tumor-initiating cells”, recently published by Wu et al. 1. In this study the authors show spontaneous transformation of human fetal striatum neural stem cells (hsNSCs) in culture, and that the transformed cells (T1) are characterized by stem cell-like features, the expression of neural stem cell markers, abnormal karyotype and an increased growth rate. In the text they refer to previous reports on spontaneous MSC transformation 2, 3. However, they fail to inform the readers that both these publications have later been retracted or corrected since both research groups detected that their transformed cells were cross-contaminated with cancer cells 4, 5. In the article by Wu and colleagues, they have characterized the T1 cells by DNA fingerprinting. Most interesting, the DNA fingerprint of the transformed cells (T1) did not match the “cell of origin”, and the authors explain this by genetic instability. However, we have compared the T1 fingerprint published by Wu et al., with public available cell line STR profiles, and find that the T1 STR profile published by Wu et al. is surprisingly similar to HeLa cells, Table ​Table1.1. The DNA fingerprinting profile of cancer cells compared to normal cells is characterized by large differences in peak height at one or more loci, indicating genetic instability, occasional additional alleles at a locus, indicating gene duplication events, and loss of heterozygosity (LOH), at one or more loci 6, 7. Genetic imbalance will in other words not generate a completely new fingerprinting profile. Table 1 STR fingerprinting profile of hsNSC, T1 and HeLa STR profiling is currently the recommended test for cell line authentication due to its high power of discrimination and the possibility to compare the numerical code obtained from various laboratories 7. Wu and colleagues analyzed their cells by using the PowerPlex 16 System Kit from Promega. The kit provides 15 STR markers as well as the gender determinator Amelogenin, and it has a matching probability of > 1 in 1.83×10e17 (www.promega.com). The same kit was recently used to determine the STR profile of HeLa cells 8, showing 97% identity between T1 and HeLa with only one LOH (Table ​(Table1).1). According to general recommendations, the profile of identical or closely related profiles should match at 80% or more of the alleles 6, and profiles with an identity level between 50 and 75% must be regarded with suspicion 7. The HeLa profiles listed in Table ​Table11 match T1 with 80-97% accuracy. A minor variation is seen at one locus when comparing the STR profile reported by ATCC and CLS (D13S317: 12,13.3 and 13,13.3) and Wu (D13S317: 12,14). According to the Promega Protocol for PowerPlex16, each allele at the D13S317 locus separates by 4 nucleotides, and it is therefore unclear if the allele at D13S317 13.3 is correct. There is at present several batches of HeLa cells available, and minor differences exists between them 6. A number of scientists have pointed at the problem of cross-contamination for decades, and it is now highly recommended to authenticate cells by DNA fingerprinting 7, 9. Several databases for checking the fingerprinted profiles are available, such as STR profile databases at ATCC (www.lgcstandards-atcc.org) and DSMZ (www2.dsmz.de). Also a list of 360 cross-contaminated cell lines is available to help researchers quality-check their work 10, and HeLa is still the most frequent cross-contaminating cell line 10. In conclusion, it is highly questionable if the article presented by Wu et al., actually describes a transforming event of hsNSCs.

Increased lymphatic vascular density is seen before colorectal cancers reach stage II and growth factor FGF-2 is downregulated in tumor tissue compared with normal mucosa.

Lymphangiogenesis is an important event in progression of colorectal cancer (CRC), and the estimated lymphatic vascular density (LVD) probably indicates facilitated lymphatic tumor cell invasion and metastasis. However, at what time point during tumor progression this process is triggered, is unclear. The aim of this study was twofold. Firstly, to examine LVD in paired samples of CRC tissue and normal mucosa with specific emphasis on possible difference in LVD between tumors stages II and III, and secondly, the expression of the lymphangiogenic growth factor fibroblast growth factor‐2 (FGF‐2). Eighteen patients were studied. Immunostaining for podoplanin was performed to highlight lymphatic vessels. FGF‐2 mRNA expression was determined by quantitative real‐time RT‐PCR, whereas protein expression was quantitatively assessed by densitometric analysis of Western blot signal intensity. The immunoblots were further validated by FGF‐2 immunostaining of histological sections. LVD was significantly increased in tumor tissue compared with the normal mucosa but no changes in LVD between stages II and III CRC was observed. FGF‐2 was found to be downregulated both at the mRNA and protein level in tumor tissues compared with normal mucosa. Lymphangiogenesis was triggered early in tumor development. An increased LVD was established before the tumor reached stage II. FGF‐2 was downregulated in tumor tissue. The importance of this finding remains unclear.

A new live-cell reporter strategy to simultaneously monitor mitochondrial biogenesis and morphology

Changes in mitochondrial amount and shape are intimately linked to maintenance of cell homeostasis via adaptation of vital functions. Here, we developed a new live-cell reporter strategy to simultaneously monitor mitochondrial biogenesis and morphology. This was achieved by making a genetic reporter construct where a master regulator of mitochondrial biogenesis, nuclear respiratory factor 1 (NRF-1), controls expression of mitochondria targeted green fluorescent protein (mitoGFP). HeLa cells with the reporter construct demonstrated inducible expression of mitoGFP upon activation of AMP-dependent protein kinase (AMPK) with AICAR. We established stable reporter cells where the mitoGFP reporter activity corresponded with mitochondrial biogenesis both in magnitude and kinetics, as confirmed by biochemical markers and confocal microscopy. Quantitative 3D image analysis confirmed accordant increase in mitochondrial biomass, in addition to filament/network promoting and protecting effects on mitochondrial morphology, after treatment with AICAR. The level of mitoGFP reversed upon removal of AICAR, in parallel with decrease in mtDNA. In summary, we here present a new GFP-based genetic reporter strategy to study mitochondrial regulation and dynamics in living cells. This combinatorial reporter concept can readily be transferred to other cell models and contexts to address specific physiological mechanisms.