On-chip photodynamic therapy - monitoring cell metabolism using electrochemical microsensors.

We introduce a new system which combines metabolic monitoring using electrochemical microsensors with photodynamic therapy on-chip for the first time. Oxygen consumption of T-47D breast cancer cells was measured during therapy with protoporphyrin IX. We determined the efficacy of the therapy and revealed its recovery effects, which underlines the high relevance of continuous monitoring.

Sensor Access to the Cellular Microenvironment Using the Sensing Cell Culture Flask.

The Sensing Cell Culture Flask (SCCF) is a cell culture monitoring system accessing the cellular microenvironment in 2D cell culture using electrochemical microsensors. The system is based on microfabricated sensor chips embedded in standard cell culture flasks. Ideally, the sensor chips could be equipped with any electrochemical sensor. Its transparency allows optical inspection of the cells during measurement. The surface of the sensor chip is in-plane with the flask surface allowing undisturbed cell growth on the sensor chip. A custom developed rack system allows easy usage of multiple flasks in parallel within an incubator. The presented data demonstrates the application of the SCCF with brain tumor (T98G) and breast cancer (T-47D) cells. Amperometric oxygen sensors were used to monitor cellular respiration with different incubation conditions. Cellular acidification was accessed with potentiometric pH sensors using electrodeposited iridium oxide films. The system itself provides the foundation for electrochemical monitoring systems in 3D cell culture.

Low-Dose-Rate Irradiation for 1 Hour Induces Protection Against Lethal Radiation Doses but Does Not Affect Life Span of DBA/2 Mice.

Prior findings showed that serum from DBA/2 mice that had been given whole-body irradiation for 1 hour at a low dose rate (LDR) of 30 cGy/h induced protection against radiation in reporter cells by a mechanism depending on transforming growth factor ß3 and inducible nitric oxide synthase activity. In the present study, the effect of the 1 hour of LDR irradiation on the response of the preirradiated mice to a subsequent lethal dose and on the life span is examined. These DBA/2 mice were prime irradiated for 1 hour at 30 cGy/h. Two experiments with 9 and 9.5 Gy challenge doses given 6 weeks after priming showed increased survival in primed mice compared to unprimed mice followed up to 225 and 81 days after challenge irradiation, respectively. There was no overall significant difference in life span between primed and unprimed mice when no challenge irradiation was given. The males seemed to have a slight increase in lifespan after priming while the opposite was seen for the females.

Hypoxia Strongly Affects Mitochondrial Ribosomal Proteins and Translocases, as Shown by Quantitative Proteomics of HeLa Cells.

Hypoxia is an important and common characteristic of many human tumors. It is a challenge clinically due to the correlation with poor prognosis and resistance to radiation and chemotherapy. Understanding the biochemical response to hypoxia would facilitate the development of novel therapeutics for cancer treatment. Here, we investigate alterations in gene expression in response to hypoxia by quantitative proteome analysis using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with LCMS/MS. Human HeLa cells were kept either in a hypoxic environment or under normoxic conditions. 125 proteins were found to be regulated, with maximum alteration of 18-fold. In particular, three clusters of differentially regulated proteins were identified, showing significant upregulation of glycolysis and downregulation of mitochondrial ribosomal proteins and translocases. This interaction is likely orchestrated by HIF-1. We also investigated the effect of hypoxia on the cell cycle, which shows accumulation in G1 and a prolonged S phase under these conditions. Implications. This work not only improves our understanding of the response to hypoxia, but also reveals proteins important for malignant progression, which may be targeted in future therapies.

Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium.

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

Alterations of monocarboxylate transporter densities during hypoxia in brain and breast tumour cells.

BACKGROUND: Tumour cells are characterized by aerobic glycolysis, which provides biomass for tumour proliferation and leads to extracellular acidification through efflux of lactate via monocarboxylate transporters (MCTs). Deficient and spasm-prone tumour vasculature causes variable hypoxia, which favours tumour cell survival and metastases. Brain metastases frequently occur in patients with advanced breast cancer.Effective treatment strategies are therefore needed against brain metastasis from breast carcinoma. MATERIAL AND METHODS: In order to identify differences in the capacity for lactate exchange, human T-47D breast cancer cells and human glioblastoma T98G cells were grown under 4 % or 20 % oxygen conditions and examined for MCT1, MCT2 and MCT4 expression on plasma membranes by quantitative post embedding immunogold electron microscopy. Whereas previous studies on MCT expression in tumours have recorded mRNA and protein levels in cell extracts, we examined concentrations of the proteins in the microvillous plasma membrane protrusions specialized for transmembrane transport. RESULTS: In normoxia, both tumour cell types highly expressed the low affinity transporter MCT4, which is thought to mainly mediate monocarboxylate efflux, while for high affinity transport the breast tumour cells preferentially expressed MCT1 and the brain tumour cells resembled brain neurons in expressing MCT2, rather than MCT1. The expressions of MCT1 and MCT4 were upregulated in hypoxic conditions in both breast and brain tumour cells. The expression of MCT2 also increased in hypoxic breast cancer cells, but decreased in hypoxic brain tumour cells. Quantitative immunoblots showed similar hypoxia induced changes in the protein levels. CONCLUSION: The differential expression and regulation of MCTs in the surface membranes of hypoxic and normoxic tumour cells of different types provide a foundation for innovation in tumour therapy through the selective targeting of MCTs. Selective inhibition of various MCTs could be an efficient way to quench an important energy source in both original breast tumour and metastatic cancer tissue in the brain.

Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies.

Cancer cells in hypoxic areas of solid tumors are to a large extent protected against the action of radiation as well as many chemotherapeutic drugs. There are, however, two different aspects of the problem caused by tumor hypoxia when cancer therapy is concerned: One is due to the chemical reactions that molecular oxygen enters into therapeutically targeted cells. This results in a direct chemical protection against therapy by the hypoxic microenvironment, which has little to do with cellular biological regulatory processes. This part of the protective effect of hypoxia has been known for more than half a century and has been studied extensively. However, in recent years there has been more focus on the other aspect of hypoxia, namely the effect of this microenvironmental condition on selecting cells with certain genetic prerequisites that are negative with respect to patient prognosis. There are adaptive mechanisms, where hypoxia induces regulatory cascades in cells resulting in a changed metabolism or changes in extracellular signaling. These processes may lead to changes in cellular intrinsic sensitivity to treatment irrespective of oxygenation and, furthermore, may also have consequences for tissue organization. Thus, the adaptive mechanisms induced by hypoxia itself may have a selective effect on cells, with a fine-tuned protection against damage and stress of many kinds. It therefore could be that the adaptive mechanisms may take advantage of for new tumor labeling/imaging and treatment strategies. One of the Achilles' heels of hypoxia research has always been the exact measurements of tissue oxygenation as well as the control of oxygenation in biological tumor models. Thus, development of technology that can ease this control is vital in order to study mechanisms and perform drug development under relevant conditions. An integrated EU Framework project 2004-2009, termed EUROXY, demonstrates several pathways involved in transcription and translation control of the hypoxic cell phenotype and evidence of cross-talk with responses to pH and redox changes. The carbonic anhydrase isoenzyme CA IX was selected for further studies due to its expression on the surface of many types of hypoxic tumors. The effort has led to marketable culture flasks with sensors and incubation equipment, and the synthesis of new drug candidates against new molecular targets. New labeling/imaging methods for cancer diagnosing and imaging of hypoxic cancer tissue are now being tested in xenograft models and are also in early clinical testing, while new potential anti-cancer drugs are undergoing tests using xenografted tumor cancers. The present article describes the above results in individual consortium partner presentations.

Radiobiological responses for two cell lines following continuous low dose-rate (CLDR) and pulsed dose rate (PDR) brachytherapy.

The iso-effective irradiation of continuous low-dose-rate (CLDR) irradiation was compared with that of various schedules of pulsed dose rate (PDR) irradiation for cells of two established human lines, T-47D and NHIK 3025. Complete single-dose response curves were obtained for determination of parameters alpha and beta by fitting of the linear quadratic formula. Sublethal damage repair constants micro and T(1/2) were determined by split-dose recovery experiments. On basis of the acquired parameters of each cell type the relative effectiveness of the two regimens of irradiation (CLDR and PDR) was calculated by use of Fowler's radiobiological model for iso-effect irradiation for repeated fractions of dose delivered at medium dose rates. For both cell types the predicted and observed relative effectiveness was compared at low and high iso-effect levels. The results indicate that the effect of PDR irradiation predicted by Fowlers model is equal to that of CLDR irradiation for both small and large doses with T-47D cells. With NHIK 3025 cells PDR irradiation induces a larger effect than predicted by the model for small doses, while it induces the predicted effect for high doses. The underlying cause of this difference is unclear, but cell-cycle parameters, like G2-accumulation is tested and found to be the same for the two cell lines.

The role of p27 in controlling the oxygen-dependent checkpoint of mammalian cells in late G1.

We have studied hypoxia-induced cell cycle arrest in human cells where the retinoblastoma tumour suppressor protein (pRb) is either functional (T-47D cells) or abrogated by expression of the HPV18 E7 oncoprotein (NHIK 3025 cells). Cells of both types are arrested in a restriction point in late G1, here denoted as the oxygen-dependent restriction point in late G1. This arrest seems to occur under extreme hypoxia in all types of mammalian cells so far tested. During an 18-h exposure to extreme hypoxia, the p27 protein level increased in G1-phase in both cells lines investigated and was followed by a binding between p27 and CDK2. This was observed both in the pRb-positive T-47D cells and in the pRb-negative NHIK 3025 cells. We, therefore, believe that p27 and not pRb is the mediator of this oxygen-dependent checkpoint in late G1. Our results also suggest that p27 regulates the restart of cell cycle progression of these arrested cells after reoxygenation.

Pericellular oxygen depletion during ordinary tissue culturing, measured with oxygen microsensors.

Recent research has found important differences in oxygen tension in proximity to certain mammalian cells when grown in culture. Oxygen has a low diffusion rate through cell culture media, thus, as a result of normal respiration, a decrease in oxygen tension develops close to the cells. Therefore, for the purpose of standardization and optimization, it is important to monitor pericellular oxygen tension and cell oxygen consumption. Here, we describe an integrated oxygen microsensor and recording system that allows measurement of oxygen concentration profiles in vertical transects through a 1.6-mm deep, stagnant, medium layer covering a cell culture. The measurement set-up reveals that, when confluent, a conventional culture of adherent cells, although exposed to the constant oxygen tension of ambient air, may experience pericellular oxygen tensions below the level required to sustain full oxidative metabolism. Depletions reported are even more prominent and potentially aggravating when the cell culture is incubated at reduced oxygen tensions (down to around 4% oxygen). Our results demonstrate that, if the pericellular oxygen tension is not measured, it is impossible to relate in vitro culture results (for example, gene expression to the oxygen tension experienced by the cell), as this concentration may deviate very substantially from the oxygen concentration recorded in the gas phase.

pH effects on the cellular uptake of four photosensitizing drugs evaluated for use in photodynamic therapy of cancer.

The difference in extracellular pH in malignant as compared to normal healthy tissues has been proposed to contribute to selective uptake of photosensitizers in tumors. Hematoporphyrin IX (HpIX), disulfonated meso-tetraphenylporphine (TPPS(2a)), meso-tetra(3-hydroxyphenyl)porphine (mTHPP) and meso-tetra(3-hydroxyphenyl)chlorin (mTHPC) were chosen to examine the pH dependence of their cellular drug uptake. The study was performed in the pH range 6.5-8.0 and showed that significantly higher amounts of the drug are taken up by T-47D cells at low pH values only in the case of HpIX. The pH value of the incubation medium did not influence the cellular uptake of mTHPP, mTHPC and TPPS(2a) significantly. The present work indicates that tumor selectivity of dyes, which get more lipophilic with decreasing pH value, may be related to the low extracellular pH value.

Cell cycle inhibitors and outcome after radiotherapy in bladder cancer patients.

The aim of this study was to correlate the expression of cell cycle inhibitors with outcome of patients with muscle-invasive bladder cancer treated with preoperative radiotherapy (46 Gy/4-5 weeks or 20 Gy/1 week) and cystectomy. Patients with pT3b (n = 42) or pT0 (n = 17) were included in the study. Expression of p16INK4a and p27KIP1 was assessed immunohistochemically in pre-radiotherapy biopsies and cystectomy specimens. Previously reported results of p21CP1 expression were also included. No difference in pretreatment protein expression was found between patients with pT0 and pT3b. Expression of p21CIP1 and p27KIP1 was lower in cystectomy specimens than in pretreatment biopsies. None of the proteins showed significant impact on survival when analysed separately. However, patients with tumours showing > 50% expression of p16INK4a, p21CIP1, or p27KIP1 displayed poorer cancer-specific survival rates compared with the remaining patients (p = 0.025). This effect was more pronounced in patients receiving 46 Gy than in those receiving 20 Gy. In conclusion, low expression of cell cycle inhibitors is related to favourable survival after pre-cystectomy radiotherapy.

Role of ribonucleotide reductase in regulation of cell cycle progression during and after exposure to moderate hypoxia.

Earlier studies have shown that the retinoblastoma protein (pRb) is involved in cell-cycle regulation under conditions of moderate hypoxia, which is the term we use to denote oxygen concentrations just above the lower level giving full respiration, ie. 1300 ppm O2. We have studied the cell cycle regulatory influence of varying levels of ribonucleotide reductase under moderate hypoxia in human cancer cells with either functional (T47D and T47DHU-res) or non-functional pRb due to expression of HPV18 E7 (HeLa S3). In this study we adapted a cell-line to hydroxyurea (T47DHU-res) resulting in an increased level of ribonucleotide reductase tyrosyl-radical that is not cell-cycle dependent. Under moderate hypoxic stress, pRb becomes dephosphorylated and rebound in the cell nucleus in all phases of the cell cycle, in contrast to its function under aerobic conditions where it binds only during early G1. We have shown in this paper that, upon reoxygenation, dephosphorylation and binding to the nucleus of pRb is reversible in T47D cells, although phosphorylation is delayed by more than 12 hours. As a result of the dephosphorylation of pRb, T47D cells are between 12 and 24 hours slower in reinitiating their S-phase progression than HeLa S3 cells (not containing functional pRb) following reoxygenation. However, with an increased level of ribonucleotide reductase tyrosyl-radical in the T47DHU-res cells, the S-phase progression after reoxygenation is reinitiated earlier, although the pRb status of these cells is the same as in T47D cells.