Fraction Of Hypoxic Cells Research Articles

Tumor growth and vascular redistribution contributes to the dosimetric preferential effect of microbeam radiotherapy: a Monte Carlo study

The radiobiological mechanisms behind the favorable response of tissues to microbeam radiation therapy (MRT) are not fully described yet. Among other factors, the differential action to tumor and normal tissue vasculature is considered to contribute to MRT efficacy. This computational study evaluates the relevance of tumor growth stage and associated vascular redistribution to this effect. A multiscale approach was employed with two simulation softwares: TOPAS and CompuCell3D. Segmentation images of the angioarchitecture of a non-bearing tumor mouse brain were used. The tumor vasculature at different tumor growth stages was obtained by simulating the tumor proliferation and spatial vascular redistribution. The radiation-induced damage to vascular cells and consequent change in oxygen perfusion were simulated for normal and tumor tissues. The multiscale model showed that oxygen perfusion to tissues and vessels decreased as a function of the tumor proliferation stage, and with the decrease in uniformity of the vasculature spatial distribution in the tumor tissue. This led to an increase in the fraction of hypoxic (up to 60%) and necrotic (10%) tumor cells at advanced tumor stages, whereas normal tissues remained normoxic. These results showed that tumor stage and spatial vascular distribution contribute to the preferential effect of MRT in tumors.

Immunotherapy-based neoadjuvant therapy followed by concurrent chemoradiotherapy in locally advanced and advanced cervical cancer: A real-world study.

e17509 Background: Locally advanced and advanced cervical cancer (CC), characterized by extensive local invasion and potential distant metastasis, present substantial clinical challenges. Although concurrent chemoradiotherapy (CCRT) is the standard treatment for locally advanced CC, a considerable proportion (23%-34%) of treated pts experienced recurrence or subsequent metastasis. Recently, neoadjuvant therapy (NAT) has been proposed to increase the radiosensitivity and decreases the fraction of hypoxic cells. Particularly, immunotherapy (IO)-based NAT has shown high activity in treating CC. Moreover, IO plus CRT has shown promise in enhancing both local control and systemic response. Hence, we analyzed the efficacy and safety of IO-based NAT followed by CCRT for locally advanced and advanced CC in a real-world setting. Methods: This study retrospectively analyzed data from pts with locally advanced and advanced CC who underwent IO-based NAT followed by CCRT at Shengjing Hospital of China Medical University from January 2022 to January 2024. Outcome measures included complete response (CR), partial response (PR), objective response rate (ORR), progression free survival (PFS), and safety. Tumor responses after neoadjuvant or CCRT therapy were evaluated using RECIST v1.1 and PFS was analyzed with the Kaplan-Meir method. Safety was assessed by adverse events (AEs) referring to CTCAEs v 5.0. Results: A total of 20 pts (median age 57 years, range, 39-72) were included in this analysis, with 15% at stage IIIB, 60% at IIIC, 10% at IVA, and 15% at IVB. Eight (40%) pts received 1 cycle of neoadjuvant IO, 8 (40%) received 1-4 cycles of neoadjuvant IO + chemotherapy, and 4 (20%) received 2 cycles of neoadjuvant IO + bevacizumab (Bev) + chemotherapy. After NAT, all 20 pts received CCRT. In the overall population, ORR after NAT was 55% (11/20), with 5% CR and 50% PR. Regarding different IO-based NAT regimens, objective responses were achieved in 1 of 8 (12.5%) pts receiving neoadjuvant IO, 6 of 8 (75%) pts receiving neoadjuvant IO + chemotherapy, and 4 of 4 (100%) pts receiving neoadjuvant IO + Bev + chemotherapy. After CCRT, 6 (30%) pts had a CR and 7 (35%) pts had a PR, resulting in an ORR of 65.0%. At a median follow-up of 14.9 months, the median PFS was not reached, with a 12-month and 24-month PFS rates of 68% and 56%, respectively. The most common treatment-related AEs during NAT were white blood cell count decreased (50%) and neutrophil count decreased. Conclusions: IO-based NAT followed by CCRT were effective and well-tolerated for pts with locally advanced and advanced CC. This treatment modalities may provide a potential therapeutic strategy for locally advanced and advanced CC.

Hypoxia induces a glycolytic complex in intestinal epithelial cells independent of HIF-1-driven glycolytic gene expression

The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic adenosine triphosphate (ATP) production, an event with consequences for cellular bioenergetics and cell fate. This response is regulated at the transcriptional level by the hypoxia-inducible factor-1(HIF-1)-dependent transcriptional upregulation of glycolytic enzymes (GEs) and glucose transporters. However, this transcriptional upregulation alone is unlikely to account fully for the levels of glycolytic ATP produced during hypoxia. Here, we investigated additional mechanisms regulating glycolysis in hypoxia. We observed that intestinal epithelial cells treated with inhibitors of transcription or translation and human platelets (which lack nuclei and the capacity for canonical transcriptional activity) maintained the capacity for hypoxia-induced glycolysis, a finding which suggests the involvement of a nontranscriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. In our investigations into potential nontranscriptional mechanisms for glycolytic induction, we identified a hypoxia-sensitive formation of complexes comprising GEs and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of such glycolytic complexes occurs independent of HIF-1-driven transcription. Finally, we provide evidence for the presence of HIF-1α in cytosolic fractions of hypoxic cells which physically interacts with the glucose transporter GLUT1 and the GEs in a hypoxia-sensitive manner. In conclusion, we provide insights into the nontranscriptional regulation of hypoxia-induced glycolysis in intestinal epithelial cells.

Bcl-2/Bcl-xL inhibitor ABT-263 overcomes hypoxia-driven radioresistence and improves radiotherapy

Hypoxia, a characteristic of most human solid tumors, is a major obstacle to successful radiotherapy. While moderate acute hypoxia increases cell survival, chronic cycling hypoxia triggers adaptation processes, leading to the clonal selection of hypoxia-tolerant, apoptosis-resistant cancer cells. Our results demonstrate that exposure to acute and adaptation to chronic cycling hypoxia alters the balance of Bcl-2 family proteins in favor of anti-apoptotic family members, thereby elevating the apoptotic threshold and attenuating the success of radiotherapy. Of note, inhibition of Bcl-2 and Bcl-xL by BH3-mimetic ABT-263 enhanced the sensitivity of HCT116 colon cancer and NCI-H460 lung cancer cells to the cytotoxic action of ionizing radiation. Importantly, we observed this effect not only in normoxia, but also in severe hypoxia to a similar or even higher extent. ABT-263 furthermore enhanced the response of xenograft tumors of control and hypoxia-selected NCI-H460 cells to radiotherapy, thereby confirming the beneficial effect of combined treatment in vivo. Targeting the Bcl-2 rheostat with ABT-263, therefore, is a particularly promising approach to overcome radioresistance of cancer cells exposed to acute or chronic hypoxia with intermittent reoxygenation. Moreover, we found intrinsic as well as ABT-263- and irradiation-induced regulation of Bcl-2 family members to determine therapy sensitivity. In this context, we identified Mcl-1 as a resistance factor that interfered with apoptosis induction by ABT-263, ionizing radiation, and combinatorial treatment. Collectively, our findings provide novel insights into the molecular determinants of hypoxia-mediated resistance to apoptosis and radiotherapy and a rationale for future therapies of hypoxic and hypoxia-selected tumor cell fractions.

Hypoxia-activated pro-drugs of the KDAC inhibitor vorinostat (SAHA)

Hypoxia (lower than normal oxygen) is a characteristic of most solid tumours that results in poor cancer patient prognosis. The difference in cellular environment between normoxia (21% oxygen) or physoxia (4–7.5% oxygen) and hypoxia (<2.0% oxygen) causes increased resistance to radio- and chemotherapy, but also provides the opportunity to selectively release hypoxia-activated pro-drugs. This approach potentially allows targeting of chemotherapies, including lysine deacetylase (KDAC) inhibitors, to the hypoxic fraction of cells. Here, we report initial work on the development of KDAC inhibitors that are selectively released in hypoxic conditions. We have shown that the addition of a 4-nitrobenzyl (NB) or 1-methyl-2-nitroimidazole (NI) bioreductive group onto the hydroxamic acid moiety of SAHA, giving NB-SAHA and NI-SAHA, abolishes KDAC inhibition activity. Both NB-SAHA and NI-SAHA undergo enzyme-mediated bioreduction, in a hypoxia-dependent manner, to release SAHA selectively in <0.1% oxygen. This work provides an important foundation for further investigations into the targeted release of KDAC inhibitors in hypoxic tumours.

Molecular and biological rationale of hyperthermia as radio- and chemosensitizer.

Mild hyperthermia, local heating of the tumour up to temperatures <43°C, has been clinically applied for almost four decades and has been proven to substantially enhance the effectiveness of both radiotherapy and chemotherapy in treatment of primary and recurrent tumours. Clinical results and mechanisms of action are discussed in this review, including the molecular and biological rationale of hyperthermia as radio- and chemosensitizer as established in in vitro and in vivo experiments. Proven mechanisms include inhibition of different DNA repair processes, (in)direct reduction of the hypoxic tumour cell fraction, enhanced drug uptake, increased perfusion and oxygen levels. All mechanisms show different dose effect relationships and different optimal scheduling with radiotherapy and chemotherapy. Therefore, obtaining the ideal multi-modality treatment still requires elucidation of more detailed data on dose, sequence, duration, and possible synergisms between modalities. A multidisciplinary approach with different modalities including hyperthermia might further increase anti-tumour effects and diminish normal tissue damage.

WE‐AB‐202‐11: Radiobiological Modeling of Tumor Response During Radiotherapy Based On Pre‐Treatment Dynamic PET Imaging Data

Purpose:To evaluate the ability of a multiscale radiobiological model of tumor response to predict mid‐treatment hypoxia images, based on pretreatment imaging of perfusion and hypoxia with [18‐F]FMISO dynamic PET and glucose metabolism with [18‐F]FDG PET.Methods:A mechanistic tumor control probability (TCP) radiobiological model describing the interplay between tumor cell proliferation and hypoxia (Jeong et al., PMB 2013) was extended to account for intra‐tumor nutrient heterogeneity, dynamic cell migration due to nutrient gradients, and stromal cells. This extended model was tested on 10 head and neck cancer patients treated with chemoradiotherapy, randomly drawn from a larger MSKCC protocol involving baseline and mid‐therapy dynamic PET scans. For each voxel, initial fractions of proliferative and hypoxic tumor cells were obtained by finding an approximate solution to a system of linear equations relating cell fractions to voxel‐level FDG uptake, perfusion (FMISO K1) and hypoxia (FMISO k3). The TCP model then predicted their evolution over time up until the mid treatment scan. Finally, the linear model was reapplied to predict each lesion's median hypoxia level (k3[med,sim]) which in turn was compared to the FMISO k3[med] measured at mid‐therapy.Results:The average k3[med] of the tumors in pre‐treatment scans was 0.0035 min−1, with an inter‐tumor standard deviation of σ[pre]=0.0034 min−1. The initial simulated k3[med,sim] of each tumor agreed with the corresponding measurements within 0.1σ[pre]. In 7 out of 10 lesions, the mid‐treatment k3[med,sim] prediction agreed with the data within 0.3σ[pre]. The remaining cases corresponded to the most extreme relative changes in k3[med].Conclusion:This work presents a method to personalize the prediction of a TCP model using pre‐treatment kinetic imaging data, and validates the modeling of radiotherapy response by predicting changes in median hypoxia values during treatment. Variations from predicted response may be a useful biomarker, which should be further explored.Partially supported by NIH grant #1 R01 CA157770‐01A1 and a grant from Varian Corporation.

Hypoxia-mediated autophagic flux inhibits silver nanoparticle-triggered apoptosis in human lung cancer cells.

Solid tumors are frequently associated with resistance to chemotherapy because the fraction of hypoxic tumor cells is substantial. To understand the underlying mechanism of hypoxia on silver nanoparticle (AgNPs)-induced apoptosis, the expression of hypoxia-inducible factor (HIF)-1α, a hallmark of hypoxia, was measured in the presence and absence of AgNPs. The results showed that HIF-1α expression was upregulated after AgNPs treatment under both hypoxic and normoxic conditions. Cell viability assays showed that AgNPs promoted cell death in cancer cells but not in non-cancer cells, as cancer cells are slightly more acidic than normal cells. However, reactive oxygen species generation induced by AgNPs in lung cancer cells caused high susceptibility to oxidative stress, whereas pre-exposure to hypoxia blocked AgNPs-induced oxidative stress. Notably, HIF-1α inhibited AgNPs-induced mitochondria-mediated apoptosis by regulating autophagic flux through the regulation of ATG5, LC3-II, and p62. Further, cell viability after treatment of cancer cells with AgNPs under hypoxic conditions was lower in HIF-1α siRNA-transfected cells than in control siRNA-transfected cells, indicating that HIF-1α knockdown enhances hypoxia induced decrease in cell viability. Our results suggest that hypoxia-mediated autophagy may be a mechanism for the resistance of AgNPs-induced apoptosis and that strategies targeting HIF-1α may be used for cancer therapy.

Clinical evaluation of X-ray voxel Monte Carlo versus pencil beam-based dose calculation in stereotactic body radiotherapy of lung cancer under normal and deep inspiration breath hold.

The purpose of this study is to evaluate the differences between dose distributions calculated with the pencil beam (PB) and X-ray voxel Monte Carlo (MC) algorithms for patients with lung cancer using intensity-modulated radiotherapy (IMRT) or HybridArc techniques. The 2 algorithms were compared in terms of dose-volume histograms, under normal and deep inspiration breath hold, and in terms of the tumor control probability (TCP). The dependence of the differences in tumor volume and location was investigated. Dosimetric validation was performed using Gafchromic EBT3 (International Specialty Products, ISP, Wayne, NJ). Forty-five Computed Tomography (CT) data sets were used for this study; 40 Gy at 8 Gy/fraction was prescribed with 5 noncoplanar 6-MV IMRT beams or 3 to 4 dynamic conformal arcs with 3 to 5 IMRT beams distributed per arc. The plans were first calculated with PB and then recalculated with MC. The difference between the mean tumor doses was approximately 10% ± 4%; these differences were even larger under deep inspiration breath hold. Differences between the mean tumor dose correlated with tumor volume and path length of the beams. The TCP values changed from 99.87% ± 0.24% to 96.78% ± 4.81% for both PB- and MC-calculated plans (P = .009). When a fraction of hypoxic cells was considered, the mean TCP values changed from 76.01% ± 5.83% to 34.78% ± 18.06% for the differently calculated plans (P < .0001). When the plans were renormalized to the same mean dose at the tumor, the mean TCP for oxic cells was 99.05% ± 1.59% and for hypoxic cells was 60.20% ± 9.53%. This study confirms that the MC algorithm adequately accounts for inhomogeneities. The inclusion of the MC in the process of IMRT optimization could represent a further step in the complex problem of determining the optimal treatment plan.

Dihydroartemisinin is a Hypoxia-Active Anti-Cancer Drug in Colorectal Carcinoma Cells.

Tumor hypoxia is one main biological factor that drives resistance to chemotherapy and radiotherapy. To develop a novel strategy for overcoming hypoxia-induced therapy resistance, we examined the anti-neoplastic activity of the reactive oxygen donor dihydroartemisinin (DHA) in human colon cancer cell lines in normoxia and severe hypoxia. In addition, we analyzed the involvement of the intrinsic apoptosis pathway for DHA-mediated cytotoxicity in HCT116 cells in short-term and long-term in vitro assays. When applied at lower concentrations (≤25 μM), DHA induced apoptosis in Colo205, HCT15, and HCT116 cells, whereas necrotic cell death was increased when cells were treated with higher DHA concentrations (50 μM). However, no preference for DHA-induced apoptosis or necrosis could be detected between the treatment under normoxic or hypoxic conditions. Moreover, DHA potently reduced clonogenic survival of HCT116 cells in normoxia and hypoxia. Treatment of HCT116 cells with 25 μM DHA resulted in activation of Bax under normoxic and hypoxic conditions. Interestingly, cytochrome c release from the mitochondria and caspase-activation were observed only under normoxic conditions, whereas, under hypoxic conditions DHA induced a caspase-independent apoptosis-like cell death. However, under both conditions, generation of reactive oxygen species was an important mediator of DHA-induced toxicity. Further molecular analysis suggests that DHA-mediated cell death involves different sets of pro-apoptotic Bcl-2 family members. The pronounced cytotoxic activity of DHA in severe hypoxia as well as normoxia offers new perspectives for targeting the hypoxic tumor cell fraction to improve treatment outcome for cancer patients.

TU‐B‐137‐01: Radiobiological Principles of Fractionated Radiotherapy and the Potential for Hypofractionation

Firstly, we will review why we fractionate at all, looking first at physical reasons then at the radiobiological aspects. Mechanisms by which radiation kills cells and ways cell damage can be repaired will be reviewed. Using the linear‐quadratic model we will show the effect of radiobiological parameters which we can control for radiotherapy including dose/fraction, number of fractions, time between fractions, overall treatment time, and dose rate. We will show that there is a “Window of Opportunity” at low doses/fraction within which we can control cancers without exceeding normal tissue tolerance. We will then show how the use of highly conformal radiotherapy widens this “Window of Opportunity” so that we can use higher doses/fraction and hence hypofractionation. Finally, we will review some potential caveats that might reduce the effectiveness of hypofractionation and, possibly, make it inappropriate for some cancers. These are the presence of significant fractions of hypoxic cancer cells, the possibility that low‐dose hyper‐radiosensitivity in normal surrounding tissue cells might reduce the effectiveness of conformal therapies, and the lengthening of treatment times when using high doses/fraction thus allowing intra‐fraction repair of cancer cells.Learning Objectives1. To understand why we fractionate and how the linear‐quadratic model can be used to demonstrate the effect of radiobiological parameters which we can control for radiotherapy.2. To understand why hypofractionation might be possible with highly conformal radiotherapy.3. To understand the circumstances when hypofractionation might not be appropriate.

Abstract 4053: Hypoxic trans-activation of sodium dependent bicarbonate co-transporters regulates pH in 3D spheroids and promotes growth.

Abstract Background: Carbonic anhydrase IX (CAIX) is strongly induced by hypoxia. CAIX extracellularly hydrates CO2 to HCO3− and H+, and we previously showed CAIX regulates pH in 3D spheroids. CAIX expression correlates with poor prognosis in most tumour types. We recently showed CAIX regulates tumour growth, necrosis and that knockdown enhances Bevacizumab treatment, highlighting the role of pH regulation in the hypoxic tumour milieu. We hypothesised that transport of HCO3−, which had been produced by CAIX extracellularly could facilitate quenching of intracellular H+. We identified increased expression of SLC4A4 (which encodes a bicarbonate transporter), in response to Bevacizumab, a treatment which increases the hypoxic fraction of cells, in vivo. Method: We investigated a panel of cells lines (4 colon cancers, 2 glioblastomas, 1 breast cancer and 1 head and neck cancer) for changes in RNA expression of bicarbonate transporters in response to hypoxia (0.1% O2, 72 hours), and the role of HIF1α and HIF2α in key identified changes. We used a specific Na+ dependent bicarbonate transport inhibitor (SO859) to examine the effect of inhibition on pH regulation and growth in spheroids. Further to this we used shRNA knockdown of SLC4A4 and SLC4A9 in cell lines with increased hypoxic expression to further investigate their role. Results: Hypoxia increased the RNA expression of one or more bicarbonate transporter in 6/8 cancer cell lines. The largest increases in expression change were for SLC4A4 in Ls174T and SCC25 (79.4 fold, p&amp;lt;0.01 n=3: 2.4 fold, p&amp;lt;0.05, n=3) and SLC4A9 in U87 and Ls174T (10.5 fold, p&amp;lt;0.01 n=3; 19 fold, p&amp;lt;0.01 n=3). SLC4A4 expression was regulated by HIF1α in Ls174T and SCC25 (p&amp;lt;0.05, n=3). HIF1α bound directly to the HRE in the promoter of SLC4A4 in SCC25. SLC4A9 expression increase was regulated by HIF2α (80%) and HIF1α (29%) in Ls174T. SO859 treatment acidified the intracellular pH of cells in U87 spheroids. The most significant differences in pH were seen at the spheroid core (p&amp;lt;0.001, n=20) and periphery (p&amp;lt;0.001, n=20). U87 spheroids have HIF1α stabilisation and CAIX expression throughout, as detected by immunohistochemistry. Longer SO859 treatment of 24 hours resulted in a reduced effect on pH, suggesting a compensation mechanism. SO859 treatment reduced the spheroid growth rate of 4/4 cell lines tested, including MDA-MB-468 a cell line that showed no hypoxic increase in bicarbonate transport expression. In U87 spheroids, CAIX knockdown further reduced the reduction in growth rate seen with SO859. SLC4A4 and SLC4A9 knockdown reduced growth rate in Ls174T spheroids and SLC4A9 knockdown reduced spheroid growth rate in U87. Conclusion: This work highlights the value of developing small molecules or antibodies, which inhibit bicarbonate transport to clinically deregulate pH regulation in the hypoxic microenvironment of tumours. Citation Format: Alan McIntyre, Alzbeta Hulikova, Ioanna Ledaki, Peter Seden, Helen Turley, Dylan Jones, Angela Russell, Pawel Swietach, Adrian L. Harris. Hypoxic trans-activation of sodium dependent bicarbonate co-transporters regulates pH in 3D spheroids and promotes growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4053. doi:10.1158/1538-7445.AM2013-4053

Эффективность фракционированного воздействия γ-излучения и быстрых нейтронов на саркому М-1

The effectiveness of fractionated exposure to gamma- and neutron radiation in their separate and combined use on the growth and functional morphology of mutant p53 sarcoma M-1 in rats was studied. Investigation techniques included immunostaining of PCNA and mutant p53 expressing cells, determination of mitotic activity and apoptotic death of tumor cells, as well as computer analysis of microscopic images. The antitumor efficacy of different types of radiation is shown to be determined by different levels of apoptosis induction, reduced proliferation and cellularity. Neutron radiation of the impulse generator has a marked damaging effect on the vasculature and the development of tumor necrosis. Fractionated irradiation at equal daily doses led to the decrease in the relative effectiveness of radio-inactivation of tumor cells. After 9 fractions of irradiation, the calculated value of the RBE of fast neutrons normalized to the input dose of 1 Gy by the coefficient of tumor growth inhibition, a reduced proliferative activity of PCNA and induced apoptosis of tumor cells was 3.4, 3.7 and 3.1, respectively. In the mode of daily superfractionation with splitting the dose in two fractions, the effectiveness of the combined exposure corresponded to the additive effect of gamma- and neutron radiation with a tendency toward synergism. There are reasons to believe that high resistance of sarcoma M-1 to the ionizing radiation impact is due not only to a fraction of hypoxic cells, but also the mutant status of p53 gene.

Vascular abnormalities associated with acute hypoxia in human melanoma xenografts

Background and purposeThe fraction of hypoxic cells has been shown to differ substantially among human tumors of the same histological type. In this study, a window chamber model was used to identify possible mechanisms leading to the development of highly different hypoxic fractions in A-07 and R-18 human melanoma xenografts. Materials and methodsChronic and acute hypoxia was assessed in intradermal tumors using an immunohistochemical and a radiobiological assay. Functional and morphological parameters of the vascular networks of tumors growing in dorsal window chambers were assessed with intravital microscopy. ResultsR-18 tumors showed significantly higher hypoxic fractions than A-07 tumors, and the difference was mostly due to acute hypoxia. Compared to A-07 tumors, R-18 tumors showed low vascular densities, low vessel diameters, long vessel segments, low blood flow velocities, frequent fluctuations in blood flow, and a high fraction of narrow vessels with absent or very low and varying flux of red blood cells. ConclusionThe high fraction of acute hypoxia in R-18 tumors was a consequence of frequent fluctuations in blood flow and red blood cell flux combined with low vascular density. The fluctuations were most likely caused by high geometric resistance to blood flow in the tumor microvasculature.

A modified hypoxia-based TCP model to investigate the clinical outcome of stereotactic hypofractionated regimes for early stage non-small-cell lung cancer (NSCLC)

Stereotactic body radiotherapy (SBRT) has been applied to lung tumors at different stages and sizes with good local tumor control (LC) rates. The linear quadratic model (LQM), in its basic formulation, does not seem to be appropriate to describe the response to radiotherapy for clinical trials, based on a few fractions. Thus, the main aim of this work was to develop a model, which takes into account the hypoxic cells and their reoxygenation. A parameter named B has been introduced in a modified tumor control probability (TCP) from LQM and linear-quadratic-linear model (LQLM), and represents the fraction of hypoxic cells that survive and become oxygenated after each irradiation. Based on published trials evaluating LC at 3 yr (LC3), values of B were obtained by maximum likelihood minimization between predicted TCP and clinical LC3. Two oxygen enhancement ratio (OER) parameter sets (1 and 2) from literature have been adopted to calculate the B-factors. Initial hypoxic cell fractions (η(h)) from 0.05 to 0.50 were assumed. Log-likelihood (L) and Akaike information criterion (AIC) were determined in an independent clinical validation dataset. The B-values of modified TCPs spanned the whole interval from 0 to 1, depending on the fractionation scheme (number of fractions and dose/fraction), showing a maximum (close to 1) at doses/fraction of 8-12 Gy. The B-values calculated using the OER parameter set 1 exhibited a smoother falloff than set 2. An analytical expression was derived to describe the B-value's dependence on the fractionation scheme. The R(2)-adjusted values varied from 0.63 to 0.67 for LQ models and OER set 1 and from 0.75 to 0.78 for LQ model and OER set 2. Lower values of R(2)-adjusted were found for LQLM and both OER sets. L and AIC, calculated using a fraction of η(h) = 0.15 and the B-value from the authors analytical expression were higher than for other η(h)-values, irrespective of model or OER set. The authors model allows to predict the clinical outcome associated with SBRT treatment, taking into account both direct killing and indirect vasculature or stromal damage.

Imaging biomarkers to monitor response to the hypoxia-activated prodrug TH-302 in the MiaPaCa2 flank xenograft model

TH-302, a hypoxia-activated anticancer prodrug, was evaluated for antitumor activity and changes in dynamic contrast-enhanced (DCE) and diffusion-weighted (DW) magnetic resonance imaging (MRI) in a mouse model of pancreatic cancer. TH-302 monotherapy resulted in a significant delay in tumor growth compared to vehicle-treated controls. TH-302 treatment was also associated with a significant decrease in the volume transfer constant (Ktrans) compared to vehicle-treated controls 1 day following the first dose measured using DCE-MRI. This early decrease in Ktrans following the first dose as measured is consistent with selective killing of the hypoxic fraction of cells which are associated with enhanced expression of hypoxia inducible transcription factor-1 alpha that regulates expression of permeability and perfusion factors including vascular endothelial growth factor-A. No changes were observed in DW-MRI following treatment with TH-302, which may indicate that this technique is not sensitive enough to detect changes in small hypoxic fractions of the tumor targeted by TH-302. These results suggest that changes in tumor permeability and/or perfusion may be an early imaging biomarker for response to TH-302 therapy.

Elusive Goal of Targeting Tumor Hypoxia for Therapeutic Gain

In the article that accompanies this editorial, Janssens et al report the outcome results of a phase III trial of accelerated radiotherapy with carbogen and nicotinamide (ARCON) versus accelerated radiotherapy alone in the treatment of 345 patients with stages T2 to T4, any nodal stage, M0 laryngeal cancer. Despite the sound biologic rationale for accelerated radiotherapy plus carbogen and nicotinamide (ARCON) and the promising results from a large preceding nonrandomized study, no evidence of a therapeutic gain was seen in the definitive phase III trial. Specifically, no significant differences were observed in laryngeal preservation rates, disease-free survival, overall survival, treatment-related toxicity, or functional outcomes. The authors did note an improved regional nodal complete response rate in the ARCON arm as assessed clinically, but this did not translate into any improvement in disease-free survival after “salvage” neck dissection. Unfortunately, both the article and the protocol are unclear as to whether a neck dissection was stipulated for patients who failed to achieve a regional complete response by a certain time after treatment. If this was the case, the required neck dissection would normally be considered part of the definitive treatment package, and patients undergoing surgery would not be scored as having sustained a regional failure. In a translational research substudy involving 79 patients, the investigators estimated the hypoxic fraction of tumor cells in biopsies of the primary tumor by using pimonidazole staining. They reported that regional (but not local) control was significantly improved by treatment with ARCON in patients with more hypoxic primary tumors (defined as 2.6% pimonidazole staining) but not in patients with better oxygenated tumors. However, this conclusion is based on small numbers of neck failures and appears to take no account of the original neck node classification of these patients. It can therefore be considered no more than a hypothesisgenerating observation. Although these findings are consistent with the notion that any benefit from a hypoxia-targeting therapy will be restricted to the population with demonstrable hypoxia, an important caveat is that they did not actually assess hypoxia in the nodes. The hypoxia status of the primary and nodes has been reported to be discordant in studies that used hypoxic positron emission tomography (PET) imaging. ARCON is the latest in the growing list of interventions targeting hypoxia that have failed to realize their predicted potential in the treatment of unselected populations of patients with head and neck cancers. Strategies thathavepreviouslybeensubjectedtorandomizedclinical trials includehyperbaricoxygen, hypoxiccell radiosensitizers(misonidazole, etanidazole, and nimorazole), fast neutron therapy, concurrent chemotherapy with mitomycin C, and the hypoxic cell cytotoxin tirapazamine. Although a recent meta-analysis of trials of hypoxic modification did show a small but significant survival benefit, only nimorazole has entered clinical practice, and then only in Denmark, in combination with radiation without chemotherapy. It is an axiom of rational targeted therapy that the intervention can be successful only if the target is present and cure limiting. In the case of therapies targeting hypoxia, this requires not only that tumors harbor hypoxic clonogenic cells before treatment, but also that effective reoxygenation does not occur during treatment. Another important consideration as to whether hypoxia is cure limiting, at least for oropharyngeal tumors, is a tumor’s human papillomavirus (HPV) status. Recent data suggest that HPV-associated oropharyngeal cancers have a similarly high incidence of hypoxia compared with HPVnegative head and neck cancers but nonetheless retain their favorable prognosis. Furthermore, there are data from exploratory analyses of three randomized trials of hypoxia-targeting therapies that suggest that any benefit from these therapies may be restricted to patients with HPV-negative tumors. Several strategies have been used to detect tumor hypoxia before treatment, albeit with inconsistent correlations with outcome. These include oxygen electrode measurements, histochemical markers, plasma markers, functional imaging, and gene expression profiling. Use of such tests may enlarge the patient population potentially able to benefit from hypoxiatargeted therapy. Retrospective analyses of the Danish Head and Neck Cancer Study (DAHANCA) 5 trial have suggested that some of these tests may be predictive of benefit from nimorazole, and we have previously reported that patients with PETimageable hypoxia may benefit from tirapazamine. Nevertheless, such approaches have not been incorporated into phase III trials, at least in part because of the lack of a validated, reproducible test of tumor hypoxia that has been demonstrated to be suitable for use in multi-institutional clinical trials. The efficiency of reoxygenation during treatment is more difficult to measure and interpret. There are limited data from three small [18F]-misonidazole PET imaging studies about the persistence of hypoxia during treatment and correlation with outcome. It is noteworthy that in the Trans Tasman Radiation Oncology Group JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 30 NUMBER 15 MAY 2

Recombinant Human Endostatin Normalizes Tumor Vasculature and Enhances Radiation Response in Xenografted Human Nasopharyngeal Carcinoma Models

BackgroundHypoxic tumor cells can reduce the efficacy of radiation. Antiangiogenic therapy may transiently “normalize” the tumor vasculature to make it more efficient for oxygen delivery. The aim of this study is to investigate whether the recombinant human endostatin (endostar) can create a “vascular normalization window” to alleviate hypoxia and enhance the inhibitory effects of radiation therapy in human nasopharyngeal carcinoma (NPC) in mice.Methodology/Principal FindingsTransient changes in morphology of tumor vasculature and hypoxic tumor cell fraction in response to endostar were detected in mice bearing CNE-2 and 5–8F human NPC xenografts. Various treatment schedules were tested to assess the influence of endostar on the effect of radiation therapy. Several important factors relevant to the angiogenesis were identified through immunohistochemical staining. During endostar treatment, tumor vascularity decreased, while the basement membrane and pericyte coverage associated with endothelial cells increased, which supported the idea of vessel normalization. Hypoxic tumor cell fraction also decreased after the treatment. The transient modulation of tumor physiology caused by endostar improved the effect of radiation treatment compared with other treatment schedules. The expressions of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14 decreased, while the level of pigment epithelium-derived factor (PEDF) increased.ConclusionsEndostar normalized tumor vasculature, which alleviated hypoxia and significantly sensitized the function of radiation in anti-tumor in human NPC. The results provide an important experimental basis for combining endostar with radiation therapy in human NPC.

Dynamic contrast‐enhanced‐MRI of tumor hypoxia

Patients with highly hypoxic primary tumors show increased frequency of locoregional treatment failure and poor survival rates and may benefit from particularly aggressive treatment. The potential of gadolinium diethylene-triamine penta-acetic acid-based dynamic contrast-enhanced-MRI in assessing tumor hypoxia was investigated in this preclinical study. Xenografted tumors of eight human melanoma lines were subjected to dynamic contrast-enhanced-MRI and measurement of the fraction of radiobiologically hypoxic cells and the fraction of pimonidazole-positive hypoxic cells. Tumor images of K(trans) (the volume transfer constant of gadolinium diethylene-triamine penta-acetic acid) and v(e) (the fractional distribution volume of gadolinium diethylene-triamine penta-acetic acid) were produced by pharmacokinetic analysis of the dynamic contrast-enhanced-MRI data, and K(trans) and v(e) frequency distributions of the non-necrotic tumor tissue were established and related to the extent of hypoxia. Tumors showing high K(trans) values and high v(e) values had low fractions of hypoxic cells, whereas tumors showing both low K(trans) values and low v(e) values had high hypoxic fractions. K(trans) differentiated better between tumors with low and high hypoxic fractions than did v(e). This study supports the current attempts to establish dynamic contrast-enhanced-MRI as a method for assessing the extent of hypoxia in human tumors, and it provides guidelines for the clinical development of valid assays.

Microtubule-targeting-compound PBOX-15 radiosensitizes cancer cells in vitro

BackgroundWe proposed to investigate the radiosensitizing properties of PBOX-15, a novel microtubule-disrupting agent, in a panel of cancer cell lines. ResultsPBOX-15 treatment was associated with significant cell kill and increased radiosensitivity in all three cell lines tested. The number of surviving cells in response to the combined treatment was significantly less than PBOX -15 alone in 22Rv1 cells. In these cells, radiosensitisation correlated with induction of G2/M cell cycle arrest by PBOX-15. The compound sustained its activity and increased HIF-1Α expression under hypoxic conditions. PBOX-15 prevented onset of hypoxia-induced radioresistance in hypoxic prostate cells and reduced the surviving fraction of irradiated hypoxic cells to levels similar to those achieved under aerobic conditions.MethodsClonogenic assays were used to determine sensitivity of a panel of cancer cell lines (22Rv1, A549, U87) to PBOX-15 alone or in combination with a single 2Gy dose fraction. Induction of cell cycle arrest and apoptosis was investigated in 22Rv1 prostate cancer cells. The cytotoxic properties of the compound under hypoxic conditions were correlated with Hypoxia Inducible Factor 1 alpha (HIF-1Α) gene and protein expression levels and its radiosensitisation potential was investigated in hypoxic 22Rv1 using clonogenic assays. ConclusionsThis preliminary data identifies the potential of PBOX-15 as a novel radiosensitising agent for the management of solid tumours and eradication of hypoxic cells.