Differential impact of sex on regulation of skeletal muscle mitochondrial function and protein homeostasis by hypoxia-inducible factor-1α in normoxia.

Hypoxia-inducible factor (HIF)-1α is continuously synthesized and degraded in normoxia. During hypoxia, HIF1α stabilization restricts cellular/mitochondrial oxygen utilization. Cellular stressors can stabilize HIF1α even during normoxia. However, less is known about HIF1α function(s) and sex-specific effects during normoxia in the basal state. Since skeletal muscle is the largest protein store in mammals and protein homeostasis has high energy demands, we determined HIF1α function at baseline during normoxia in skeletal muscle. Untargeted multiomics data analyses were followed by experimental validation in differentiated murine myotubes with loss/gain of function and skeletal muscle from mice without/with post-natal muscle-specific Hif1a deletion (Hif1amsd). Mitochondrial oxygen consumption studies using substrate, uncoupler, inhibitor, titration protocols; targeted metabolite quantification by gas chromatography-mass spectrometry; and post-mitotic senescence markers using biochemical assays were performed. Multiomics analyses showed enrichment in mitochondrial and cell cycle regulatory pathways in Hif1a deleted cells/tissue. Experimentally, mitochondrial oxidative functions and ATP content were higher with less mitochondrial free radical generation with Hif1a deletion. Deletion of Hif1a also resulted in higher concentrations of TCA cycle intermediates and HIF2α proteins in myotubes. Overall responses to Hif1amsd were similar in male and female mice, but changes in complex II function, maximum respiration, Sirt3 and HIF1ß protein expression and muscle fibre diameter were sex-dependent. Adaptive responses to hypoxia are mediated by stabilization of constantly synthesized HIF1α. Despite rapid degradation, the presence of HIF1α during normoxia contributes to lower mitochondrial oxidative efficiency and greater post-mitotic senescence in skeletal muscle. In vivo responses to HIF1α in skeletal muscle were differentially impacted by sex. KEY POINTS: Hypoxia-inducible factor -1α (HIF1α), a critical transcription factor, undergoes continuous synthesis and proteolysis, enabling rapid adaptive responses to hypoxia by reducing mitochondrial oxygen consumption. In mammals, skeletal muscle is the largest protein store which is determined by a balance between protein synthesis and breakdown and is sensitive to mitochondrial oxidative function. To investigate the functional consequences of transient HIF1α expression during normoxia in the basal state, myotubes and skeletal muscle from male and female mice with HIF1α knockout were studied using complementary multiomics, biochemical and metabolite assays. HIF1α knockout altered the electron transport chain, mitochondrial oxidative function, signalling molecules for protein homeostasis, and post-mitotic senescence markers, some of which were differentially impacted by sex. The cost of rapid adaptive responses mediated by HIF1α is lower mitochondrial oxidative efficiency and post-mitotic senescence during normoxia.

Lonidamine Induced Selective Acidification and De-Energization of Prostate Cancer Xenografts: Enhanced Tumor Response to Radiation Therapy.

Prostate cancer is a multi-focal disease that can be treated using surgery, radiation, androgen deprivation, and chemotherapy, depending on its presentation. Standard dose-escalated radiation therapy (RT) in the range of 70-80 Gray (GY) is a standard treatment option for prostate cancer. It could be used at different phases of the disease (e.g., as the only primary treatment when the cancer is confined to the prostate gland, combined with other therapies, or as an adjuvant treatment after surgery). Unfortunately, RT for prostate cancer is associated with gastro-intestinal and genitourinary toxicity. We have previously reported that the metabolic modulator lonidamine (LND) produces cancer sensitization through tumor acidification and de-energization in diverse neoplasms. We hypothesized that LND could allow lower RT doses by producing the same effect in prostate cancer, thus reducing the detrimental side effects associated with RT. Using the Seahorse XFe96 and YSI 2300 Stat Plus analyzers, we corroborated the expected LND-induced intracellular acidification and de-energization of isolated human prostate cancer cells using the PC3 cell line. These results were substantiated by non-invasive 31P magnetic resonance spectroscopy (MRS), studying PC3 prostate cancer xenografts treated with LND (100 mg/kg, i.p.). In addition, we found that LND significantly increased tumor lactate levels in the xenografts using 1H MRS non-invasively. Subsequently, LND was combined with radiation therapy in a growth delay experiment, where we found that 150 µM LND followed by 4 GY RT produced a significant growth delay in PC3 prostate cancer xenografts, compared to either control, LND, or RT alone. We conclude that the metabolic modulator LND radio-sensitizes experimental prostate cancer models, allowing the use of lower radiation doses and diminishing the potential side effects of RT. These results suggest the possible clinical translation of LND as a radio-sensitizer in patients with prostate cancer.

Radiation-Chemical Oxygen Depletion Depends on Chemical Environment and Dose Rate: Implications for the FLASH Effect.

PURPOSE: FLASH (dose rates >40 Gy/s) radiation therapy protects normal tissues from radiation damage, compared with conventional radiation therapy (∼Gy/m). Radiation-chemical oxygen depletion (ROD) occurs when oxygen reacts with radiation-induced free radicals, so a possible mechanism for FLASH involves radioprotection by the decreased oxygen as ROD occurs. High ROD rates would favor this mechanism, but prior studies have reported low ROD values (∼0.35 µM/Gy) in chemical environments such as water and protein/nutrient solutions. We proposed that intracellular ROD might be much larger, possibly promoted by its strongly reducing chemical environment. METHODS AND MATERIALS: ROD was measured, using precision polarographic sensors, from ∼100 µM to zero in solutions containing intracellular reducing agents ± glycerol (1M), to simulate intracellular reducing and hydroxyl-radical-scavenging capacity. Cs irradiators and a research proton beamline allowed dose rates from 0.0085 to 100 Gy/s. RESULTS: Reducing agents significantly altered ROD values. Most greatly increased ROD but some (eg, ascorbate) actually decreased ROD and additionally imposed an oxygen dependence of ROD at low oxygen concentrations. The highest values of ROD were found at low dose rates, but these montonically decreased with increasing dose rate. CONCLUSIONS: ROD was greatly augmented by some intracellular reducing agents but others (eg, ascorbate) effectively reversed this effect. Ascorbate had its greatest effect at low oxygen concentrations. ROD decreased with increasing dose rate in most cases.

A phenomenological model of proton FLASH oxygen depletion effects depending on tissue vasculature and oxygen supply.

Introduction: Radiation-induced oxygen depletion in tissue is assumed as a contributor to the FLASH sparing effects. In this study, we simulated the heterogeneous oxygen depletion in the tissue surrounding the vessels and calculated the proton FLASH effective-dose-modifying factor (FEDMF), which could be used for biology-based treatment planning. Methods: The dose and dose-weighted linear energy transfer (LET) of a small animal proton irradiator was simulated with Monte Carlo simulation. We deployed a parabolic partial differential equation to account for the generalized radiation oxygen depletion, tissue oxygen diffusion, and metabolic processes to investigate oxygen distribution in 1D, 2D, and 3D solution space. Dose and dose rates, particle LET, vasculature spacing, and blood oxygen supplies were considered. Using a similar framework for the hypoxic reduction factor (HRF) developed previously, the FEDMF was derived as the ratio of the cumulative normoxic-equivalent dose (CNED) between CONV and UHDR deliveries. Results: Dynamic equilibrium between oxygen diffusion and tissue metabolism can result in tissue hypoxia. The hypoxic region displayed enhanced radio-resistance and resulted in lower CNED under UHDR deliveries. In 1D solution, comparing 15 Gy proton dose delivered at CONV 0.5 and UHDR 125 Gy/s, 61.5% of the tissue exhibited ≥20% FEDMF at 175 µm vasculature spacing and 18.9 µM boundary condition. This percentage reduced to 34.5% and 0% for 8 and 2 Gy deliveries, respectively. Similar trends were observed in the 3D solution space. The FLASH versus CONV differential effect remained at larger vasculature spacings. A higher FLASH dose rate showed an increased region with ≥20% FEDMF. A higher LET near the proton Bragg peak region did not appear to alter the FLASH effect. Conclusion: We developed 1D, 2D, and 3D oxygen depletion simulation process to obtain the dynamic HRF and derive the proton FEDMF related to the dose delivery parameters and the local tissue vasculature information. The phenomenological model can be used to simulate or predict FLASH effects based on tissue vasculature and oxygen concentration data obtained from other experiments.

Oxygen Monitoring in Model Solutions and In Vivo in Mice During Proton Irradiation at Conventional and FLASH Dose Rates.

FLASH is a high-dose-rate form of radiation therapy that has the reported ability, compared with conventional dose rates, to spare normal tissues while being equipotent in tumor control, thereby increasing the therapeutic ratio. The mechanism underlying this normal tissue sparing effect is currently unknown, however one possibility is radiochemical oxygen depletion (ROD) during dose delivery in tissue at FLASH dose rates. In order to investigate this possibility, we used the phosphorescence quenching method to measure oxygen partial pressure before, during and after proton radiation delivery in model solutions and in normal muscle and sarcoma tumors in mice, at both conventional (Conv) (∼0.5 Gy/s) and FLASH (∼100 Gy/s) dose rates. Radiation dosimetry was determined by Advanced Markus Chamber and EBT-XL film. For solutions contained in sealed glass vials, phosphorescent probe Oxyphor PtG4 (1 µM) was dissolved in a buffer (10 mM HEPES) containing glycerol (1 M), glucose (5 mM) and glutathione (5 mM), designed to mimic the reducing and free radical-scavenging nature of the intracellular environment. In vivo oxygen measurements were performed 24 h after injection of PtG4 into the interstitial space of either normal thigh muscle or subcutaneous sarcoma tumors in mice. The "g-value" for ROD is reported in mmHg/Gy, which represents a slight modification of the more standard chemical definition (µM/Gy). In solutions, proton irradiation at conventional dose rates resulted in a g-value for ROD of up to 0.55 mmHg/Gy, consistent with earlier studies using X or gamma rays. At FLASH dose rates, the g-value for ROD was ∼25% lower, 0.37 mmHg/Gy. pO2 levels were stable after each dose delivery. For normal muscle in vivo, oxygen depletion during irradiation was counterbalanced by resupply from the vasculature. This process was fast enough to maintain tissue pO2 virtually unchanged at Conv dose rates. However, during FLASH irradiation there was a stepwise decrease in pO2 (g-value ∼0.28 mmHg/Gy), followed by a rebound to the initial level after ∼8 s. The g-values were smaller and recovery times longer in tumor tissue when compared to muscle and may be related to the lower initial endogenous pO2 levels in the former. Considering that the FLASH effect is seen in vivo even at doses as low as 10 Gy, it is difficult to reconcile the amount of protection seen by oxygen depletion alone. However, the phosphorescence probe in our experiments was confined to the extracellular space, and it remains possible that intracellular oxygen depletion was greater than observed herein. In cell-mimicking solutions the oxygen depletion g-vales were indeed significantly higher than observed in vivo.

Ultrafast Tracking of Oxygen Dynamics During Proton FLASH.

PURPOSE: Radiation therapy delivered at ultrafast dose rates, known as FLASH RT, has been shown to provide a therapeutic advantage compared with conventional radiation therapy by selectively protecting normal tissues. Radiochemical depletion of oxygen has been proposed to underpin the FLASH effect; however, experimental validation of this hypothesis has been lacking, in part owing to the inability to measure oxygenation at rates compatible with FLASH. METHODS AND MATERIALS: We present a new variant of the phosphorescence quenching method for tracking oxygen dynamics with rates reaching up to ∼3.3 kHz. Using soluble Oxyphor probes we were able to resolve, both in vitro and in vivo, oxygen dynamics during the time of delivery of proton FLASH. RESULTS: In vitro in solutions containing bovine serum albumin the O2 depletion g values (moles/L of O2 depleted per radiation dose, eg, µM/Gy) are higher for conventional irradiation (by ∼13% at 75 µM [O2]) than for FLASH, and in the low-oxygen region (<25 µM [O2]) they decrease with oxygen concentration. In vivo, depletion of oxygen by a single FLASH is insufficient to achieve severe hypoxia in initially well-oxygenated tissue, and the g values measured appear to correlate with baseline oxygen levels. CONCLUSIONS: The developed method should be instrumental in radiobiological studies, such as studies aimed at unraveling the mechanism of the FLASH effect. The FLASH effect could in part originate from the difference in the oxygen dependencies of the oxygen consumption g values for conventional versus FLASH RT.

Effects of Ultra-high doserate FLASH Irradiation on the Tumor Microenvironment in Lewis Lung Carcinoma: Role of Myosin Light Chain.

PURPOSE: To investigate whether the vascular collapse in tumors by conventional dose rate (CONV) irradiation (IR) would also occur by the ultra-high dose rate FLASH IR. METHODS AND MATERIALS: Lewis lung carcinoma (LLC) cells were subcutaneously implanted in mice. This was followed by CONV or FLASH IR at 15 Gy. Tumors were harvested at 6 or 48 hours after IR and stained for CD31, phosphorylated myosin light chain (p-MLC), γH2AX (a surrogate marker for DNA double strand break), intracellular reactive oxygen species (ROS), or immune cells such as myeloid and CD8α T cells. Cell lines were irradiated with CONV IR for Western blot analyses. ML-7 was intraperitoneally administered daily to LLC-bearing mice for 7 days before 15 Gy CONV IR. Tumors were similarly harvested and analyzed. RESULTS: By immunostaining, we observed that CONV IR at 6 hours resulted in constricted vessel morphology, increased expression of p-MLC, and much higher numbers of γH2AX-positive cells in tumors, which were not observed with FLASH IR. Mechanistically, MLC activation by ROS is unlikely, because FLASH IR produced significantly more ROS than CONV IR in tumors. In vitro studies demonstrated that ML-7, an inhibitor of MLC kinase, abrogated IR-induced γH2AX formation and disappearance kinetics. Lastly, we observed that CONV IR when combined with ML-7 produced some effects similar to FLASH IR, including reduction in the vasculature collapse, fewer γH2AX-positive cells, and increased immune cell influx to the tumors. CONCLUSIONS: FLASH IR produced novel changes in the tumor microenvironment that were not observed with CONV IR. We believe that MLC activation in tumors may be responsible for some of the microenvironmental changes differentially regulated between CONV and FLASH IR.

Intratumoral Hypoxia Reduces IFN-γ-Mediated Immunity and MHC Class I Induction in a Preclinical Tumor Model.

Tumor hypoxia occurs because of an increased demand for oxygen by the rapidly growing tumor cells, together with reduction in the oxygen supply due to malformed and nonfunctional tumor vasculature. The effects of tumor hypoxia on radiotherapy (RT) are well known; however, recent findings suggest it may also suppress immunotherapy, although the mechanisms governing this observation remain undetermined. Our laboratory and others have shown that IFN-γ conditions the tumor milieu and is important for the efficacy of RT. Thus, we hypothesized that hypoxia could inhibit IFN-γ-mediated antitumor responses, resulting in decreased RT efficacy. This inhibition could involve the production and/or the cellular response to IFN-γ. To test this, we used murine tumor cell lines B16F0 and Colon38. We observed that hypoxia inhibited upregulation of IFN-γ-dependent MHC class I expression by tumor cells along with the gene expression of IFN-γ-dependent chemokines CXCL9 and CXCL10, essential for immune cell infiltration. Furthermore, CD8+ T cells, an important source of IFN-γ, which mediate effector antitumor responses, had reduced ability to proliferate and generate IFN-γ under hypoxic conditions in vitro. Interestingly, reoxygenation restored the cytokine-producing capability of these cells. Studies performed in vivo using a mouse tumor model and the hypoxia marker EF5 demonstrated that RT could reverse the hypoxia within treated tumors. This study has identified a unique mechanism of hypoxia-induced immune suppression involving the downregulation of IFN-γ production and cellular responsiveness to this essential cytokine. These results suggest that therapies that target and reduce tumor hypoxia can potentially boost antitumor immune responses.

A Two-Component Assay for Hypoxia Incorporating Long-Term Nitroreduction and Short-Term DNA-Damage Allows Differentiation of the Three Hypoxia Sub-types.

Hypoxia in tumors has many well-characterized effects that are known to prevent optimal cancer treatment. Despite the existence of a large number of assays that have supported hypoxia as an important diagnostic, there is no routine clinical assay in use, and anti-hypoxia therapies have often not included parallel hypoxia measurements. Even with a functioning hypoxia assay, it is difficult to match the oxygen dependence of treatment resistance to that of the assay, and this mismatch can vary substantially from assay to assay and even from tumor to tumor [e.g., caused by endogenous variations in non-protein sulfhydryls (NPSH)]. An underlying concern is the current inability to measure the three types of hypoxia; in particular, cycling hypoxia can affect all aspects of detection and treatment strategy. Here we present data that help validate a new two-component hypoxia assay recently suggested by our laboratory. This assay incorporates the long-term bioreduction of the 2-nitroimidazole, EF5, and the short-term production of γ-H2AX (e.g., time of ionizing radiation exposure). The former can be calibrated to provide the average tissue pO2 over the EF5 exposure time while the latter provides the combined sum of microenvironmental radiation response modifiers (e.g., oxygen and NPSH) at the time of irradiation. Importantly, formation of γ-H2AX is not dependent on blood flow, while EF5 binding is only minimally so, due to the rapid and extensive diffusion characteristics of lipophilic compounds. While both individual assays have their limitations, which are addressed in this article, their combination can dissect the type of hypoxia present. In particular, a mismatch between the two assays can directly detect cycling hypoxia in a therapeutically relevant manner. Preliminary use of this two-component assay in small PC3 tumors showed essentially no binding of EF5. Similarly, there were no tumor regions (for uniform irradiation with 12 Gy) with the low levels of γ-H2AX expected for a condition of cycling hypoxia. Thus, both assays were consistent with an essentially aerobic, radiation-responsive tumor. In a larger PC3 tumor, all regions of high EF5 binding had low levels of γ-H2AX.

Novel Double-Hit Model of Radiation and Hyperoxia-Induced Oxidative Cell Damage Relevant to Space Travel.

Spaceflight occasionally requires multiple extravehicular activities (EVA) that potentially subject astronauts to repeated changes in ambient oxygen superimposed on those of space radiation exposure. We thus developed a novel in vitro model system to test lung cell damage following repeated exposure to radiation and hyperoxia. Non-tumorigenic murine alveolar type II epithelial cells (C10) were exposed to >95% O2 for 8 h only (O2), 0.25 Gy ionizing γ-radiation (IR) only, or a double-hit combination of both challenges (O2 + IR) followed by 16 h of normoxia (ambient air containing 21% O2 and 5% CO2) (1 cycle = 24 h, 2 cycles = 48 h). Cell survival, DNA damage, apoptosis, and indicators of oxidative stress were evaluated after 1 and 2 cycles of exposure. We observed a significant (p < 0.05) decrease in cell survival across all challenge conditions along with an increase in DNA damage, determined by Comet analysis and H2AX phosphorylation, and apoptosis, determined by Annexin-V staining, relative to cells unexposed to hyperoxia or radiation. DNA damage (GADD45α and cleaved-PARP), apoptotic (cleaved caspase-3 and BAX), and antioxidant (HO-1 and Nqo1) proteins were increased following radiation and hyperoxia exposure after 1 and 2 cycles of exposure. Importantly, exposure to combination challenge O2 + IR exacerbated cell death and DNA damage compared to individual exposures O2 or IR alone. Additionally levels of cell cycle proteins phospho-p53 and p21 were significantly increased, while levels of CDK1 and Cyclin B1 were decreased at both time points for all exposure groups. Similarly, proteins involved in cell cycle arrest was more profoundly changed with the combination challenges as compared to each stressor alone. These results correlate with a significant 4- to 6-fold increase in the ratio of cells in G2/G1 after 2 cycles of exposure to hyperoxic conditions. We have characterized a novel in vitro model of double-hit, low-level radiation and hyperoxia exposure that leads to oxidative lung cell injury, DNA damage, apoptosis, and cell cycle arrest.

Letter to the Editor Re: Ogawa, Y. Cancers 2016, 8, 28.

We read with interest the recently published paper by Dr. Ogawa "Paradigm Shift in Radiation Biology/Radiation Oncology-Exploitation of the H2O2 Effect" for Radiotherapy Using Low-LET (Linear Energy Transfer) Radiation such as X-rays and High-Energy Electrons".[...].

Initial evidence that blood-borne microvesicles are biomarkers for recurrence and survival in newly diagnosed glioblastoma patients.

The purpose of this pilot study was to determine whether blood-borne microvesicles from newly diagnosed glioblastoma patients could be used as biomarkers. We collected 2.8 mL blood from 16 post-operative patients at the time that they were being simulated for chemoradiation therapy (radiation with concurrent temozolomide). Two additional samples were collected during chemoradiation therapy and a final sample was collected at the end of chemoradiation therapy. Patients continued with the therapy suggested by their physicians, based on tumor conference consensus and were followed for recurrence and overall survival. Microvesicles were isolated using serial centrifugation and stained for surface markers (Annexin V for phosphotidyl serine, CD41 for platelets, anti-EGFR for tumor cells, and CD235 for red blood cells). Flow cytometry analysis was performed. Our findings provide initial evidence that increases in Annexin V positive microvesicle levels during chemoradiation therapy are associated with earlier recurrence and shorter overall survival in newly diagnosed glioblastoma patients. The effect is dramatic, with over a four-fold increase in the hazard ratio for an individual at the 75th versus the 25th percentile. Moreover the pattern of Annexin V positive microvesicles remain significant after adjustment for confounding clinical variables that have previously been shown to be prognostic for recurrence and survival. Inclusion of neutrophil levels at the start of chemoradiation therapy in the model yielded the largest attenuation of the observed association. Further studies will be needed to verify and further investigate the association between these two entities.

Improved Methods to Generate Spheroid Cultures from Tumor Cells, Tumor Cells & Fibroblasts or Tumor-Fragments: Microenvironment, Microvesicles and MiRNA.

Diagnostic and prognostic indicators are key components to achieve the goal of personalized cancer therapy. Two distinct approaches to this goal include predicting response by genetic analysis and direct testing of possible therapies using cultures derived from biopsy specimens. Optimally, the latter method requires a rapid assessment, but growing xenograft tumors or developing patient-derived cell lines can involve a great deal of time and expense. Furthermore, tumor cells have much different responses when grown in 2D versus 3D tissue environments. Using a modification of existing methods, we show that it is possible to make tumor-fragment (TF) spheroids in only 2-3 days. TF spheroids appear to closely model characteristics of the original tumor and may be used to assess critical therapy-modulating features of the microenvironment such as hypoxia. A similar method allows the reproducible development of spheroids from mixed tumor cells and fibroblasts (mixed-cell spheroids). Prior literature reports have shown highly variable development and properties of mixed-cell spheroids and this has hampered the detailed study of how individual tumor-cell components interact. In this study, we illustrate this approach and describe similarities and differences using two tumor models (U87 glioma and SQ20B squamous-cell carcinoma) with supporting data from additional cell lines. We show that U87 and SQ20B spheroids predict a key microenvironmental factor in tumors (hypoxia) and that SQ20B cells and spheroids generate similar numbers of microvesicles. We also present pilot data for miRNA expression under conditions of cells, tumors, and TF spheroids.

Optimizing hypoxia detection and treatment strategies.

Clinical studies using Eppendorf needle sensors have invariably documented the resistance of hypoxic human tumors to therapy. These studies first documented the need for individual patient measurement of hypoxia, as hypoxia varied from tumor to tumor. Furthermore, hypoxia in sarcomas and cervical cancer leads to distant metastasis or local or regional spread, respectively. For various reasons, the field has moved away from direct needle sensor oxygen measurements to indirect assays (hypoxia-inducible factor-related changes and bioreductive metabolism) and the latter can be imaged noninvasively. Many of hypoxia's detrimental therapeutic effects are reversible in mice but little treatment improvement in hypoxic human tumors has been seen. The question is why? What factors cause human tumors to be refractory to antihypoxia strategies? We suggest the primary cause to be the complexity of hypoxia formation and its characteristics. Three basic types of hypoxia exist, encompassing various diffusional (distance from perfused vessel), temporal (on or off cycling), and perfusional (blood flow efficiency) limitations. Surprisingly, there is no current information on their relative prevalence in human tumors and even animal models. This is important because different hypoxia subtypes are predicted to require different diagnostic and therapeutic approaches, but the implications of this remain unknown. Even more challenging, no agreement exists for the best way to measure hypoxia. Some results even suggest that hypoxia is unlikely to be targetable therapeutically. In this review, the authors revisit various critical aspects of this field that are sometimes forgotten or misrepresented in the recent literature. As most current noninvasive imaging studies involve PET-isotope-labeled 2-nitroimidazoles, we emphasize key findings made in our studies using 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) and F-18-labeled EF5. These show the importance of differentiating hypoxia subtypes, optimizing drug pharmacology, ensuring drug and isotope stability, identifying key biochemical and physiological variables in tumors, and suggesting therapeutic strategies that are most likely to succeed.

Single-cell measurement of the uptake, intratumoral distribution and cell cycle effects of cisplatin using mass cytometry.

Although of fundamental importance to the treatment of cancer patients, the quantitative study of drug distribution and action in vivo at the single cell level is challenging. We used the recently-developed technique of mass cytometry to measure cisplatin uptake into individual tumor cells (Pt atoms/cell), combined with measurement of the rate of IdU incorporation into DNA (I(127) atoms/cell/min) and tumor hypoxia identified by the 2-nitroimidazole EF5 in cisplatin-treated BxPC-3 and ME-180 xenografts. Pt levels of 10(5) to 10(6) atoms/cell were obtained following a single cisplatin treatment using clinically relevant doses. Cisplatin caused cell cycle arrest in a dose- and time-dependent manner that paralleled effects in vitro, and it readily penetrated into hypoxic tumor regions. Similar levels of Pt/cell were found in xenografts treated with oxaliplatin. Mass cytometry offers the unique capability to study the cellular uptake and anticancer effects of platinum-containing drugs at the single cell level in animal models, and it has the potential for application to samples obtained from cancer patients during treatment.

Microvesicles as a Biomarker for Tumor Progression versus Treatment Effect in Radiation/Temozolomide-Treated Glioblastoma Patients.

UNLABELLED: The standard of care for glioblastoma (GB) is surgery followed by concurrent radiation therapy (RT) and temozolomide (TMZ) and then adjuvant TMZ. This regime is associated with increased survival but also increased occurrence of equivocal imaging findings, e.g., tumor progression (TP) versus treatment effect (TE), which is also referred to as pseudoprogression (PsP). Equivocal findings make decisions regarding further treatment difficult and often delayed. Because none of the current imaging assays have proven sensitive and specific for differentiation of TP versus TE/PsP, we investigated whether blood-derived microvesicles (MVs) would be a relevant assay. METHODS: 2.8 ml of citrated blood was collected from patients with GB at the time of their RT simulation, at the end of chemoradiation therapy (CRT), and multiple times following treatment. MVs were collected following multiple centrifugations (300g, 2500g, and 15,000g). The pellet from the final spin was analyzed using flow cytometry. A diameter of approximately 300 nm or greater and Pacific Blue-labeled Annexin V positivity were used to identify the MVs reported herein. RESULTS: We analyzed 19 blood samples from 11 patients with GB. MV counts in the patients with stable disease or TE/PsP were significantly lower than patients who developed TP (P = .014). CONCLUSION: These preliminary data suggest that blood analysis for MVs from GB patients receiving CRT may be useful to distinguish TE/PsP from TP. MVs may add clarity to standard imaging for decision making in patients with equivocal imaging findings.