Residual OXPHOS is required to drive primary and metastatic lung tumours in an orthotopic breast cancer model.

Background: Fast adaptation of glycolytic and mitochondrial energy pathways to changes in the tumour microenvironment is a hallmark of cancer. Purely glycolytic ρ0 tumour cells do not form primary tumours unless they acquire healthy mitochondria from their micro-environment. Here we explored the effects of severely compromised respiration on the metastatic capability of 4T1 mouse breast cancer cells. Methods: 4T1 cell lines with different levels of respiratory capacity were generated; the Seahorse extracellular flux analyser was used to evaluate oxygen consumption rates, fluorescent confocal microscopy to assess the number of SYBR gold-stained mitochondrial DNA nucleoids, and the presence of the ATP5B protein in the cytoplasm and fluorescent in situ nuclear hybridization was used to establish ploidy. MinION nanopore RNA sequence analysis was used to compare mitochondrial DNA transcription between cell lines. Orthotopic injection was used to determine the ability of cells to metastasize to the lungs of female Balb/c mice. Results: OXPHOS-deficient ATP5B-KO3.1 cells did not generate primary tumours. Severely OXPHOS compromised ρ0D5 cells generated both primary tumours and lung metastases. Cells generated from lung metastasis of both OXPHOS-competent and OXPHOS-compromised cells formed primary tumours but no metastases when re-injected into mice. OXPHOS-compromised cells significantly increased their mtDNA content, but this did not result in increased OXPHOS capacity, which was not due to decreased mtDNA transcription. Gene set enrichment analysis suggests that certain cells derived from lung metastases downregulate their epithelial-to-mesenchymal related pathways. Conclusion: In summary, OXPHOS is required for tumorigenesis in this orthotopic mouse breast cancer model but even very low levels of OXPHOS are sufficient to generate both primary tumours and lung metastases.

A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity.

PURPOSE: Cancer cells rapidly adjust their balance between glycolytic and mitochondrial ATP production in response to changes in their microenvironment and to treatments like radiation and chemotherapy. Reliable, simple, high throughput assays that measure the levels of mitochondrial energy metabolism in cells are useful determinants of treatment effects. Mitochondrial metabolism is routinely determined by measuring the rate of oxygen consumption (OCR). We have previously shown that indirect inhibition of plasma membrane electron transport (PMET) by the mitochondrial uncoupler, FCCP, may also be a reliable measure of mitochondrial energy metabolism. Here, we aimed to validate these earlier findings by exploring the relationship between stimulation of oxygen consumption by FCCP and inhibition of PMET. METHODS: We measured PMET by reduction of the cell impermeable tetrazolium salt WST-1/PMS. We characterised the effect of different growth conditions on the extent of PMET inhibition by FCCP. Next, we compared FCCP-mediated PMET inhibition with FCCP-mediated stimulation of OCR using the Seahorse XF96e flux analyser, in a panel of cancer cell lines. RESULTS: We found a strong inverse correlation between stimulation of OCR and PMET inhibition by FCCP. PMET and OCR were much more severely affected by FCCP in cells that rely on mitochondrial energy production than in cells with a more glycolytic phenotype. CONCLUSION: Indirect inhibition of PMET by FCCP is a reliable, simple and inexpensive high throughput assay to determine the level of mitochondrial energy metabolism in cancer cells.

Mitochondrial DNA Affects the Expression of Nuclear Genes Involved in Immune and Stress Responses in a Breast Cancer Model.

Tumor cells without mitochondrial (mt) DNA (ρ0 cells) are auxotrophic for uridine, and their growth is supported by pyruvate. While ATP synthesis in ρ0 cells relies on glycolysis, they fail to form tumors unless they acquire mitochondria from stromal cells. Mitochondrial acquisition restores respiration that is essential for de novo pyrimidine biosynthesis and for mitochondrial ATP production. The physiological processes that underpin intercellular mitochondrial transfer to tumor cells lacking mtDNA and the metabolic remodeling and restored tumorigenic properties of cells that acquire mitochondria are not well understood. Here, we investigated the changes in mitochondrial and nuclear gene expression that accompany mtDNA deletion and acquisition in metastatic murine 4T1 breast cancer cells. Loss of mitochondrial gene expression in 4T1ρ0 cells was restored in cells recovered from subcutaneous tumors that grew from 4T1ρ0 cells following acquisition of mtDNA from host cells. In contrast, the expression of most nuclear genes that encode respiratory complex subunits and mitochondrial ribosomal subunits was not greatly affected by loss of mtDNA, indicating ineffective mitochondria-to-nucleus communication systems for these nuclear genes. Further, analysis of nuclear genes whose expression was compromised in 4T1ρ0 cells showed that immune- and stress-related genes were the most highly differentially expressed, representing over 70% of those with greater than 16-fold higher expression in 4T1 compared with 4T1ρ0 cells. The monocyte recruiting chemokine, Ccl2, and Psmb8, a subunit of the immunoproteasome that generates MHCI-binding peptides, were the most highly differentially expressed. Early monocyte/macrophage recruitment into the tumor mass was compromised in 4T1ρ0 cells but recovered before mtDNA could be detected. Taken together, our results show that mitochondrial acquisition by tumor cells without mtDNA results in bioenergetic remodeling and re-expression of genes involved in immune function and stress adaptation.

Vaccines adjuvanted with an NKT cell agonist induce effective T-cell responses in models of CNS lymphoma.

Aim: The efficacy of anti-lymphoma vaccines that exploit the cellular adjuvant properties of activated natural killer T (NKT) cells were examined in mouse models of CNS lymphoma. Materials & methods: Vaccines were prepared by either loading the NKT cell agonist, α-galactosylceramide onto irradiated and heat-shocked B- and T-lymphoma cells, or chemically conjugating α-galactosylceramide to MHC-binding peptides from a lymphoma-associated antigen. Vaccine efficacy was analyzed in mice bearing intracranial tumors. Results: Both forms of vaccine proved to be effective in preventing lymphoma engraftment through activity of T cells that accessed the CNS. Established lymphoma was harder to treat with responses constrained by Tregs, but this could be overcome by depleting Tregs prior to vaccination. Conclusion: Simply designed NKT cell-activating vaccines enhance T-cell responses and have the potential to protect against CNS lymphoma development or prevent CNS relapse. To be effective against established CNS lymphoma, vaccines need to be combined with Treg suppression.

Perfluorocarbon emulsions radiosensitise brain tumors in carbogen breathing mice with orthotopic GL261 gliomas.

BACKGROUND: Tumour hypoxia limits the effectiveness of radiation therapy. Delivering normobaric or hyperbaric oxygen therapy elevates pO2 in both tumour and normal brain tissue. However, pO2 levels return to baseline within 15 minutes of stopping therapy. AIM: To investigate the effect of perfluorocarbon (PFC) emulsions on hypoxia in subcutaneous and intracranial mouse gliomas and their radiosensitising effect in orthotopic gliomas in mice breathing carbogen (95%O2 and 5%CO2). RESULTS: PFC emulsions completely abrogated hypoxia in both subcutaneous and intracranial GL261 models and conferred a significant survival advantage orthotopically (Mantel Cox: p = 0.048) in carbogen breathing mice injected intravenously (IV) with PFC emulsions before radiation versus mice receiving radiation alone. Carbogen alone decreased hypoxia levels substantially and conferred a smaller but not statistically significant survival advantage over and above radiation alone. CONCLUSION: IV injections of PFC emulsions followed by 1h carbogen breathing, radiosensitises GL261 intracranial tumors.

Iterative sorting reveals CD133+ and CD133- melanoma cells as phenotypically distinct populations.

BACKGROUND: The heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial. METHODS: We iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice. RESULTS: We demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment. CONCLUSION: We show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma.

Horizontal transfer of mitochondria between mammalian cells: beyond co-culture approaches.

Current dogma holds that genes are the property of individual mammalian cells and partition between daughter cells during cell division. However, and rather unexpectedly, recent research has demonstrated horizontal cell-to-cell transfer of mitochondria and mitochondrial DNA in several mammalian cell culture systems. Furthermore, unequivocal evidence that mitochondrial DNA transfer occurs in vivo has now been published. While these studies show horizontal transfer of mitochondrial DNA in pathological settings, it is also possible that intercellular mitochondrial transfer is a fundamental physiological process with a role in development and tissue homeostasis.

Pharmacological doses of daily ascorbate protect tumors from radiation damage after a single dose of radiation in an intracranial mouse glioma model.

Pharmacological ascorbate is currently used as an anti-cancer treatment, potentially in combination with radiation therapy, by integrative medicine practitioners. In the acidic, metal-rich tumor environment, ascorbate acts as a pro-oxidant, with a mode of action similar to that of ionizing radiation; both treatments kill cells predominantly by free radical-mediated DNA damage. The brain tumor, glioblastoma multiforme (GBM), is very resistant to radiation; radiosensitizing GBM cells will improve survival of GBM patients. Here, we demonstrate that a single fraction (6 Gy) of radiation combined with a 1 h exposure to ascorbate (5 mM) sensitized murine glioma GL261 cells to radiation in survival and colony-forming assays in vitro. In addition, we report the effect of a single fraction (4.5 Gy) of whole brain radiation combined with daily intraperitoneal injections of ascorbate (1 mg/kg) in an intracranial GL261 glioma mouse model. Tumor-bearing C57BL/6 mice were divided into four groups: one group received a single dose of 4.5 Gy to the brain 8 days after tumor implantation, a second group received daily intraperitoneal injections of ascorbate (day 8-45) after implantation, a third group received both treatments and a fourth control group received no treatment. While radiation delayed tumor progression, intraperitoneal ascorbate alone had no effect on tumor progression. Tumor progression was faster in tumor-bearing mice treated with radiation and daily ascorbate than in those treated with radiation alone. Histological analysis showed less necrosis in tumors treated with both radiation and ascorbate, consistent with a radio-protective effect of ascorbate in vivo. Discrepancies between our in vitro and in vivo results may be explained by differences in the tumor microenvironment, which determines whether ascorbate remains outside the cell, acting as a pro-oxidant, or whether it enters the cells and acts as an anti-oxidant.

Anti-leukemic activity of ubiquinone-based compounds targeting trans-plasma membrane electron transport.

Trans-plasma membrane electron transport (tPMET) is a ubiquinone-dependent cell survival pathway for maintaining intracellular redox homeostasis in rapidly dividing cells. To target this pathway, fifteen ubiquinone-based compounds were designed and synthesized to position at the plasma membrane and disrupt tPMET. We established that quaternary ammonium salt moieties carrying highly hindered, positive electronic charges located to the plasma membrane. A ten-carbon chain linked to these moieties was effective at positioning the redox-active ubiquinone-like function within the lipid bilayer to disrupt tPMET in human leukemic cells (IC50 9 ± 1 µM). TPMET inhibition alone was not sufficient to induce significant cell death, but positively charged compounds could also enter the cell and disrupt intracellular redox balance, distinct from their effects on mitochondrial electron transport. The synergistic effect of tPMET inhibition plus intracellular redox disruption gave strong antiproliferative activity (IC50 2 ± 0.2 µM). Positively charged ubiquinone-based compounds inhibit human leukemic cell growth.

Side population is not necessary or sufficient for a cancer stem cell phenotype in glioblastoma multiforme.

There is strong evidence for the existence of cancer stem cells (CSCs) in the aggressive brain tumor glioblastoma multiforme (GBM). These cells have stem-like self-renewal activity and increased tumor initiation capacity and are believed to be responsible for recurrence due to their resistance to therapy. Several techniques have been used to enrich for CSC, including growth in serum-free defined media to induce sphere formation, and isolation of a stem-like cell using exclusion of the fluorescent dye Hoechst 33342, the side population (SP). We show that sphere formation in GBM cell lines and primary GBM cells enriches for a CSC-like phenotype of increased self-renewal gene expression in vitro and increased tumor initiation in vivo. However, the SP was absent from all sphere cultures. Direct isolation of the SP from the GBM lines did not enrich for stem-like activity in vitro, and tumor-initiating activity was lower in sorted SP compared with non-SP and parental cells. Transient exposure to doxorubicin enhanced both CSC and SP frequency. However, doxorubicin treatment altered the cytometric profile and obscured the SP demonstrating the difficulty of identifying SP in cells under stress. Doxorubicin-exposed cells showed a transient increase in SP, but the doxorubicin-SP cells were still not enriched for a stem-like self-renewal phenotype. These data demonstrate that the GBM SP does not necessarily contribute to self-renewal or tumor initiation, key properties of a CSC, and we advise against using SP to enumerate or isolate CSC.

The anti-cancer, anti-inflammatory and tuberculostatic activities of a series of 6,7-substituted-5,8-quinolinequinones.

A variety of 6,7-substituted-5,8-quinolinequinones were synthesised and assessed for their anti-tumour and anti-inflammatory activities, and their ability to inhibit the growth of Mycobacterium bovis BCG. In particular, the introduction of a sulfur group at the 7-position of the quinolinequinone led to the discovery of two compounds, 6-methylamino-7-methylsulfanyl-5,8-quinolinequinone (10a) and 6-amino-7-methylsulfonyl-5,8-quinolinequinone (12), that exhibited selectivity for leukemic cells over T-cells, a highly desirable property for an anti-cancer drug. A number of anti-inflammatory (AI) compounds were also identified, with 6,7-bis-methylsulfanyl-5,8-quinolinequinone (18a) exhibiting the highest AI activity (0.11 microM), while 6,7-dichloro-5,8-quinolinequinone (7a), 6,7-dichloro-2-methyl-5,8-quinolinequinone (7b), and 6,7-bis-phenylsulfanyl-quinoline-5,8-diol (19) also exhibited good AI activity and specificity. Several quinolinequinone TB-drug candidates were identified. Of these, 6-amino-7-chloro-5,8-quinolinequinone (11) and 6-amino-7-methanesulfinyl-5,8-quinolinequinone (14), exhibited low MICs (1.56-3.13 microg/mL) for the 100% growth inhibition of M. Bovis BCG. Some general trends pertaining to the functional group substitution of the quinolinequinone core and biological activity were also identified.

Inhibition of trans-plasma membrane electron transport: a potential anti-leukemic strategy.

The recently demonstrated reliance of glycolytic cancer cells on trans-plasma membrane electron transport (tPMET) for survival raises the question of its suitability as a target for anticancer drug development. In this study, the effects of several new and known compounds on proliferation, tPMET activity and NAD(P)H intrinsic fluorescence in human myelogenous leukemic cell lines were investigated. The whole data confirm the importance of tPMET in leukemic cell survival and suggest this activity as a new potential anti-leukemic target.

Antitumor activity of bis-indole derivatives.

This paper reports the synthesis of compounds formed by two indole systems separated by a heterocycle (pyridine or piperazine). As a primary screening, the new compounds were submitted to the National Cancer Institute for evaluation of antitumor activity in the human cell line screen. The pyridine derivatives were far more active than the piperazine derivatives. For the study of the mechanism of action, the most active compounds were subjected to COMPARE analysis and to further biological tests including proteasome inhibition and inhibition of plasma membrane electron transport. The compound bearing the 5-methoxy-2-indolinone moiety was subjected to the first in vivo experiment (hollow fiber assay) and was active. It was therefore selected for the second in vivo experiment (human tumor xenograft in mice). In conclusion we demonstrated that this approach was successful, since some of the compounds described are much more active than the numerous, so far prepared and tested 3-indolylmethylene-2-indolinones.