Non-hypoxic Regions Research Articles

64Cu-ATSM/64Cu-Cl2 and their relationship to hypoxia in glioblastoma: a preclinical study

BackgroundDiacetyl-bis(N4-methylthiosemicarbazone), labeled with 64Cu (64Cu-ATSM) has been suggested as a promising tracer for imaging hypoxia. However, various controversial studies highlighted potential pitfalls that may disable its use as a selective hypoxic marker. They also highlighted that the results may be tumor location dependent. Here, we first analyzed uptake of Cu-ATSM and its less lipophilic counterpart Cu-Cl2 in the tumor over time in an orthotopic glioblastoma model. An in vitro study was also conducted to investigate the hypoxia-dependent copper uptake in tumor cells. We then further performed a comprehensive ex vivo study to compare 64Cu uptake to hypoxic markers, specific cellular reactions, and also transporter expression.MethodsμPET was performed 14 days (18F-FMISO), 15 days (64Cu-ATSM and 64Cu-Cl2), and 16 days (64Cu-ATSM and 64Cu-Cl2) after C6 cell inoculation. Thereafter, the brains were withdrawn for further autoradiography and immunohistochemistry. C6 cells were also grown in hypoxic workstation to analyze cellular uptake of Cu complexes in different oxygen levels.ResultsIn vivo results showed that Cu-ASTM and Cu-Cl2 accumulated in hypoxic areas of the tumors. Cu-ATSM also stained, to a lesser extent, non-hypoxic regions, such as regions of astrogliosis, with high expression of copper transporters and in particular DMT-1 and CTR1, and also characterized by the expression of elevated astrogliosis. In vitro results show that 64Cu-ATSM showed an increase in the uptake only in severe hypoxia at 0.5 and 0.2% of oxygen while for 64Cu-Cl2, the cell retention was significantly increased at 5% and 1% of oxygen with no significant rise at lower oxygen percentages.ConclusionIn the present study, we show that Cu-complexes undoubtedly accumulate in hypoxic areas of the tumors. This uptake may be the reflection of a direct dependency to a redox metabolism and also a reflection of hypoxic-induced overexpression of transporters. We also show that Cu-ATSM also stained non-hypoxic regions such as astrogliosis.

Effect of HNSCC Radiochemotherapy on Imaging Biomarker T2* MRI and its Relation to FMISO-PET Derived Hypoxia

Previous studies using hypoxia PET have shown significant reduction of tumor hypoxia (reoxygenation) during primary radiochemotherapy (RCT) of HNSCC and a significant correlation between reoxygenation on hypoxia PET and local control. Assessment of tumor hypoxia using MRI would offer independence from hypoxia PET tracer availability. The apparent MRI transverse relaxation time T2* has been proposed as an imaging biomarker and as a potential surrogate for hypoxia PET. The aim of this study was to assess the effect of primary RCT on T2* at an early (week 2) and late (week 5) time point during RCT and to analyse the relation between T2* and established hypoxia PET tracer 18F-misonidazole PET/CT (FMISO PET) and standard 18F-FDG PET/CT (FDG PET). In 10 T2-4N+ HNSCC patients, FDG PET was obtained at baseline and repeat FMISO PET and 3 Tesla MRI T2* in weeks 0, 2 and 5. MRI gross tumor volumes for tumor and lymph nodes (GTV-T, GTV-LN) were contoured and FMISO PET derived hypoxic tumor/lymph node subvolumes (HSV-T, HSV-LN) and complementary non-hypoxic subvolumes (nonHSV-T, nonHSV-LN) were generated using a threshold level of 1.4 times the mean SUV FMISO within muscle. Volumes were contoured for all time points (week 0, 2 and 5) individually and mean values for T2* and SUVmean FDG PET were obtained. Within GTVs (T, LN), r2 (FMISO to T2*) was calculated for [FMISO SUVmax GTV/SUVmean muscle] to [T2* mean GTV] and r2 (FMISO to FDG) was calculated for [FMISO SUVmax GTV/SUVmean muscle] to [FDG SUVmax GTV/SUVmean muscle]. From week 0 to 5 GTV-T and GTV-LN decreased by -56% and -63%, respectively and HSV-T and HSV-LN nearly completely resolved (n = 10). Mean T2* signal showed no significant change for GTV-T or GTV-LN. Within HSV-T mean T2* values were smaller compared to nonHSV-T: 15.0+/-4.6 vs. 18.3+/-2.9 (p = 0.051), whereas FDG SUVmean was significantly higher within hypoxic as compared to non-hypoxic regions: HSV-T 12.1+/-5.5 vs. nonHSV-T 6.1+/-2.6 and HSV-LN 10.2+/-3.9 vs. nonHSV-LN 4.7+/-1.9 (week 0, p≤0.026 and p≤0.011). Correlation between FMISO PET and FDG PET was higher than between FMSIO PET and T2*: r2 for GTV-T (FMISO/FDG)=0.81, r2 for GTV-T (FMISO/T2*)=0.32. Marked reduction of tumor hypoxia between week 0, 2 and 5 found on FMISO PET was not accompanied by a significant T2*change within GTVs over time. At baseline, smaller T2* values and significantly larger FDG SUVmean were found within HSV-T as compared to nonHSV-T. These results suggest a relation between tumor oxygenation status and T2* at baseline, however with the correlation coefficient r2 FMISO/T2* being lower than r2 FMISO/FDG. Overall, T2* quantitation was feasible and the findings on T2* imaging warrant further investigations but do not indicate a simple surrogate role for T2* as hypoxia imaging marker due to lack of correlation to established hypoxia marker FMISO PET. Our findings on reduction of FMISO uptake during RCT were in line with previous reports.

Intensity-Modulated Proton Therapy with Dose Painting based on Hypoxia Imaging for Nasopharyngeal Cancer

In precision medicine, reducing recurrence rates and toxicities is necessary for nasopharyngeal cancer (NPC) patients. Hypoxia is a well-known risk factor for radioresistance. Radiation dose escalation (DE) to hypoxic regions is one strategy, especially for locally advanced cases in which tumor control may be unsuccessful. Radiation dose reduction is also a valuable approach. A clinical trial of dose de-escalation (DDE) for oropharyngeal cancer based on hypoxia status was recently reported. DDE may also be applicable for NPC patients with good prognostic characteristics. The purpose of this study was to investigate the feasibility of hypoxia-based dose painting for intensity-modulated proton therapy (IMPT) and intensity-modulated X-ray therapy (IMXT) with constraints on doses to organs at risk (OARs) and normal tissue complication probabilities (NTCPs) in DE and DDE scenarios. DE and DDE treatment plans for IMPT and IMXT were generated for ten NPC patients who underwent 18F-fluoromisonidazole (FMISO)-PET before definitive radiotherapy. In the DE scenario, 70 Gy was prescribed to the tumor in a standard manner, with DE to 84 Gy to FMISO-PET-positive (hypoxic) regions. In the DDE scenario, 70 Gy was prescribed to the hypoxic region, with DDE to 60 Gy to non-hypoxic regions. Prescribed doses to high- and low-risk areas were 63 and 56 Gy, respectively, in the DE scenario and 54 and 48 Gy, respectively, in the DDE scenario. The constraints for doses to OARs were a parotid gland mean dose (Dmean) <26 Gy and an oral cavity Dmean <39 Gy. The criterion for NTCP was probability ≤50% for all five toxicities: salivary flow reduction, xerostomia, dysphagia, tube feeding dependence and dysgeusia. The determined doses to hypoxic and non-hypoxic target volumes were prescribed in all IMPT and IMXT plans in the DE and DDE scenarios. The numbers of patients who satisfied the parotid gland, oral cavity and NTCP criteria were 7, 10 and 10, respectively, for IMPT and 0, 1 and 0, respectively, for IMXT in the DE scenario and 10, 10 and 10, respectively, for IMPT and 1, 2 and 5, respectively, for IMXT in the DDE scenario. Dmean for the contralateral parotid gland was 25.2±3.0 for IMPT and 36.3±3.4 for IMXT (p=0.0003) in the DE scenario and 21.4±2.6 for IMPT and 30.9±3.0 for IMXT (p=0.0003) in the DDE scenario. Dmean for the oral cavity was 28.8±7.3 for IMPT and 49.3±4.9 for IMXT (p=0.0003) in the DE scenario and 24.5±6.2 for IMPT and 41.3±4.3 for IMXT (p=0.0003) in the DDE scenario. For all toxicities, NTCPs were lower for IMPT than for IMXT for all patients. ΔNTCP, calculated as NTCPIMXT-NTCPIMPT, was more than 10% for 3 or more toxicities for 9 patients in the DE scenario and 8 patients in the DDE scenario. Compared with IMXT, IMPT satisfied parotid, oral cavity and NTCP criteria in more patients and reduced doses to OARs and NTCPs in both the DE and DDE scenarios. IMPT could be more useful than IMXT when conducting hypoxia-based dose painting while maintaining low toxicities.

Abstract 2433: The hypoxic tumor microenvironment in vivo selects the tumor cells with cancer stem cell characteristics and increased resistance against genotoxic stresses

Abstract Tumor hypoxia has been established as an independent prognostic factor associated with poor patient survival and resistance to conventional therapies. Emerging evidence suggests that hypoxia has the potential to maintain or enhance the stem cell phenotype and confers chemo- or radio-resistance to tumor cells. However, it remains to be determined whether cell fate and acquisition of therapy resistance is regulated in vivo by the hypoxic tumor microenvironment (TME). In this study, we have developed a hypoxia-sensing human breast cancer cell xenograft model using the hypoxia-response element (HRE)-driven enhanced green fluorescence protein (EGFP) as a hypoxia reporter. We have found that the hypoxic tumor cells isolated from xenografts contain increased subpopulations of tumor cells showing the cancer stem cell (CSC) characteristics. Upon re-implantation, the CSC characteristics of the hypoxic tumor cells continue to be further enriched during in vivo growth, whereas secondary xenografts derived from the non-hypoxic tumor cells remain similar to the primary xenografts. Consistent with this finding, the phenotypes exhibited by the hypoxic tumor cells are stably different from those of the non-hypoxic tumor cells isolated from the same tumor mass even when they are maintained under the same ambient culture conditions. Mechanistically, we find that the PI3K/AKT pathway is strongly potentiated in the hypoxic tumor cells and is required to maintain the CSC phenotype. Cell fate differences between hypoxic and non-hypoxic tumor cells are found only in tumor cells isolated from the hypoxic TME in vivo, and are not seen in tumor cells treated by hypoxia in vitro alone. In addition, we have found that naturally hypoxic tumor cells in a breast cancer xenograft model are moderately radio- and chemo-resistant compared to their neighboring non-hypoxic tumor cells in the same tumor. These naturally hypoxic tumor cells are proficient at repairing DNA damages and resist apoptosis induced by genotoxic stresses. The ex vivo hypoxic tumor cells are capable of proficient activation of the DNA damage-sensing pathways, especially the ATM pathway. These previously unrecognized observations suggest that the hypoxic TME may promote malignant progression by coordinating induction, selection and preferential maintenance of the CSC phenotype in tumor cells. This presentation will review our results which strongly suggest that the hypoxic TME may induce adaptive responses, either by clonal evolution or selection, such that hypoxic tumor cells in vivo acquire new functional traits that confer resistance to genotoxic stresses and enhance survival. It can be postulated that previously hypoxic tumor cells may continue to exhibit their aggressive phenotypes long after they have migrated into non-hypoxic regions or after their native niche become normoxic. Citation Format: Hoon Kim, Qun Lin, Zhong Yun. The hypoxic tumor microenvironment in vivo selects the tumor cells with cancer stem cell characteristics and increased resistance against genotoxic stresses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2433.

HIF1-alpha expressing cells induce a hypoxic-like response in neighbouring cancer cells

BackgroundHypoxia stimulates metastasis in cancer and is linked to poor patient prognosis. In tumours, oxygen levels vary and hypoxic regions exist within a generally well-oxygenated tumour. However, whilst the heterogeneous environment is known to contribute to metastatic progression, little is known about the mechanism by which heterogeneic hypoxia contributes to cancer progression. This is largely because existing experimental models do not recapitulate the heterogeneous nature of hypoxia.The primary effector of the hypoxic response is the transcription factor Hypoxia inducible factor 1-alpha (HIF1-alpha). HIF1-alpha is stabilised in response to low oxygen levels in the cellular environment and its expression is seen in hypoxic regions throughout the tumour.MethodsWe have developed a model system in which HIF1-alpha can be induced within a sub-population of cancer cells, thus enabling us to mimic the effects of heterogeneic HIF1-alpha expression.ResultsWe show that induction of HIF1-alpha not only recapitulates elements of the hypoxic response in the induced cells but also results in significant changes in proliferation, gene expression and mammosphere formation within the HIF1-alpha negative population.ConclusionsThese findings suggest that the HIF1-alpha expressing cells found within hypoxic regions are likely to contribute to the subsequent progression of a tumour by modifying the behaviour of cells in the non-hypoxic regions of the local micro-environment.

Abstract 2001: Preclinical investigation of a 15 gene hypoxia profile in different prostate cancer cell lines

Abstract Purpose: Hypoxia is known to reduce the response to radiotherapy and has been found to be a common feature in prostate cancer. Identifying patients with hypoxic tumors will allow us to select those patients that require specific hypoxic modifiers. Previously our department has developed a gene profile consisting of 15 genes, which demonstrated prognostic and predictive impact for hypoxic modification in head and neck squamous cell carcinomas (HNSCC). In the current study we investigated the 15 gene profile in vitro in different prostate cancer cell lines, and subsequently in vivo in the PC3 xenograft model. Methods: For the in vitro experiments the prostate cancer cell lines investigated were PC-3, LNCaP and DU-145. Cell lines were cultured under normoxic (21% O2) or varying hypoxic conditions (0%, 0.5%, 1.0%, 2.0%, and 5.0% O2) for 24 hours, totRNA was extracted and gene expression levels measured by qPCR. Individual reference genes were selected (PSMC4, TBP, NDFIP1) and applied in the normalization of the relative expression levels, together with the reference genes previously used in the HNSCC study. For in vivo experiments, the PC3 cell line was inoculated on the flank of female NMRI nu/nu mice. Two hypoxia-sensitive tracers (18F-FAZA and Pimonidazole) were administered in order to determine hypoxic and non-hypoxic regions on excised tumor sections. These regions were isolated by laser-assisted microdissection, after which totRNA was extracted and gene expression levels measured by qPCR. Results: In the in vitro experiments, all three prostate cancer cell lines had 14 of the 15 genes induced by hypoxia, when comparing the anoxic cells (0% O2) to the normoxic.cells (21% O2). The only discrepancy was ALDOA, which was not upregulated in the hypoxic cells. In the PC3 and DU-145 cell lines genes were investigated at additional hypoxic levels (0.5%, 1.0%, 2.0%, and 5.0% O2), giving a more intricate expression of the 15 genes. In vivo experiments are still ongoing but preliminary results show a hypoxia induced upregulation in 10 out of the 15 genes, of which 4 were significantly upregulated (ADM, ANKRD37, FAM162A, and LOX). Conclusion: We investigated the 15 gene hypoxic profile in different prostate cancer cell lines, both in vitro and in vivo. A hypoxia dependent induction of genes was observed in both types of experiments, however with a differential gene expression with regard to hypoxia level. From the performed experiments, and looking only at oxygen dependency, it appears that the gene profile could be suitable for prostate cancers as well as HNSCC. Funding sources: Danish Cancer Society ; The Danish Council for Independent Research: Medical Sciences ; EC FP7 project METOXIA (project no. 222741) ; CIRRO: The Lundbeck Foundation Center for Interventional Research in Radiation Oncology and The Danish Council for Strategic Research. Citation Format: Thomas R. Wittenborn, Brita S. Sorensen, Morten Busk, Mathilde B. Thomsen, Steffen Nielsen, Michael R. Horsman. Preclinical investigation of a 15 gene hypoxia profile in different prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2001. doi:10.1158/1538-7445.AM2017-2001

Quantitative evaluation of oxygen metabolism in the intratumoral hypoxia: 18F-fluoromisonidazole and 15O-labelled gases inhalation PET

BackgroundIntratumoral hypoxia is one of the resistant factors in radiotherapy and chemotherapy for cancer. Although it is detected by 18F-fluoromisonidazole (FMISO) PET, the relationship between intratumoral hypoxia and oxygen metabolism has not been studied. The purpose of this study was to evaluate the intratumoral perfusion and oxygen metabolism in hypoxic regions using the rat xenograft model. Ten male Fischer rats with C6 glioma (body weight = 220 ± 15 g) were investigated with 18F-FMISO PET and steady-state inhalation method of 15O-labelled gases PET. The tumoral blood flow (TBF), tumoral metabolic rate of oxygen (TMRO2), oxygen extraction fraction (OEF), and tumoral blood volume (TBV) were measured under artificial ventilation with 15O–CO2, 15O–O2, and 15O–CO gases. Multiple volumes of interest (1-mm diameter sphere) were placed on the co-registered 18F-FMISO (3 h post injection) and functional 15O-labelled gases PET images. The TBF, TMRO2, OEF, and TBV values were compared among the three groups classified by the 18F-FMISO uptake as follows: group Low (L), less than 1.0; group Medium (M), between 1.0 and 2.0; and group High (H), more than 2.0 in the 18F-FMISO standardized uptake value (SUV).ResultsThere were moderate negative correlations between 18F-FMISO SUV and TBF (r = −0.56 and p < 0.01), and weak negative correlations between 18F-FMISO SUV and TMRO2 (r = −0.38 and p < 0.01) and 18F-FMISO SUV and TBV (r = −0.38 and p < 0.01). Quantitative values were as follows: TBF, (L) 55 ± 30, (M) 32 ± 17, and (H) 30 ± 15 mL/100 mL/min; OEF, (L) 33 ± 14, (M) 36 ± 17, and (H) 41 ± 16%; TMRO2, (L) 2.8 ± 1.3, (M) 1.9 ± 1.0, and (H) 2.1 ± 1.1 mL/100 mL/min; and TBV, (L) 5.7 ± 2.1, (M) 4.3 ± 1.9, and (H) 3.9 ± 1.2 mL/100 mL, respectively. Intratumoral hypoxic regions (M and H) showed significantly lower TBF, TMRO2, and TBV values than non-hypoxic regions (L). OEF showed significant increase in the severe hypoxic region compared to non-hypoxic and mild hypoxic regions.ConclusionsThis study demonstrated that intratumoral hypoxic regions showed decreased blood flow with increased oxygen extraction, suggesting the need for a treatment strategy to normalize the blood flow for oxygen-avid active tumor cells in hypoxic regions.

Targeting carbonic anhydrase IX improves the anti-cancer efficacy of mTOR inhibitors.

The inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) by chemical inhibitors, such as rapamycin, has demonstrated anti-cancer activity in preclinical and clinical trials. Their efficacy is, however, limited and tumors eventually relapse through resistance formation. In this study, using two different cancer mouse models, we identify tumor hypoxia as a novel mechanism of resistance of cancer cells against mTORC1 inhibitors. Indeed, we show that the activity of mTORC1 is mainly restricted to the non-hypoxic tumor compartment, as evidenced by a mutually exclusive staining pattern of the mTORC1 activity marker pS6 and the hypoxia marker pimonidazole. Consequently, whereas rapamycin reduces cancer cell proliferation in non-hypoxic regions, it has no effect in hypoxic areas, suggesting that cancer cells proliferate independently of mTORC1 under hypoxia. Targeting the hypoxic tumor compartment by knockdown of carbonic anhydrase IX (CAIX) using short hairpin RNA or by chemical inhibition of CAIX with acetazolamide potentiates the anti-cancer activity of rapamycin. Taken together, these data emphasize that hypoxia impairs the anti-cancer efficacy of rapalogs. Therapeutic strategies targeting the hypoxic tumor compartment, such as the inhibition of CAIX, potentiate the efficacy of rapamycin and warrant further clinical evaluation.

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.

Abstract 3949: In vivo imaging of tumor hypoxia by a new near-infrared fluorescent carbonic anhydrase IX-targeted agent

Abstract Carbonic anhydrase IX (CA IX), a cell surface enzyme involved in tumor cell acidification, is induced during tumor hypoxia (lack of oxygen) in many different types of tumors. CA IX expression is correlated to tumor cell proliferation and metastasis, poor prognosis, and resistance to therapeutic intervention, making CA IX an important biomarker in the study of hypoxia, tumor cell proliferation, and therapy. Therefore, determination of CAIX expression and identification of hypoxic tumors in vivo are critical for cancer research and treatments. We have designed and synthesized a near-infrared (NIR) fluorescent agent (VM3219) for in vivo detection and quantification of a cell surface biomarker of hypoxia, CA IX based on a well-known CA IX inhibitor, acetazolamide. VM3219 was characterized extensively in HeLa cells and in mouse HeLa tumor xenografts. Preliminary chemical and biochemical characterization of VM3219 showed excellent CA IX selectivity and affinity. In vitro, VM3219 detected 4-7-fold up-regulation of CA IX in live hypoxic HeLa cells as visualized by fluorescence microscopy and quantified by flow cytometry. VM3219 fluorescence signals were blocked by 80% when cells were pre-incubated with unlabeled acetazolamide. Fluorescent signal of VM3219 was low in normoxic HeLa cells and was comparable to the signal that were detected by using a non-binding control agent (VM3182) incubated with normoxic or hypoxic cells. In vivo imaging by fluorescence molecular tomography (FMTTM) showed that approximately 5% of the injected dose (2 nmol VM3219) was retained in the tumor tissues after 24 h (100 -120 pmol), 10-20-fold more than measured with a non-binding control agent. Tumor fluorescence was blocked &amp;gt;65% by prophylactic acetazolamide treatment. Maintenance of tumor-bearing mice on low oxygen (8%), as compared to normally housed tumor bearing mice (20% oxygen), induced a &amp;gt;2X (∼250 pmol) increase in VM3219 tumor fluorescence signal suggesting that CAIX expression was up-regulated within tumors by low oxygen. In tissue sections, VM3219 signal was shown to be specifically co-localized in the tumor hypoxic regions with CA IX antibody and Hypoxyprobe (pimonidazole), and the signal was away from non-hypoxic regions as determined by Hoechst stain. In conclusion, VM3219 is a new non-invasive tumor hypoxia imaging agent that offers a tool for specific imaging of a biologically important hypoxia biomarker, CA IX. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3949. doi:1538-7445.AM2012-3949

Tumour-infiltrating CD11b+ myelomonocytes and response to fractionated irradiation of human squamous cell carcinoma (hSCC) xenografts

PurposeBone marrow derived CD11b+ myelomonocytes have been shown to be recruited by the tumour and to promote tumour regrowth after irradiation. Here we investigated in a panel of well characterised hSCC tumour models the number of tumour-infiltrating CD11b+ cells and the association with response to clinically relevant fractionated irradiation. MethodsSix hSCC tumour models (UT-SCC-5, -14, -15, XF354, FaDu, SAS) xenografted in nude mice were excised after injection of pimonidazole hypoxia marker before irradiation and after 5 and 10 fractions. In parallel, TCD50 (dose to cure 50% of the tumours) assays were performed to determine the response to 30 fractions within 6weeks. The TCD50 values have been previously published [1]. Double staining of CD11b and pimonidazole was performed using immunofluorescence. CD11b+ cells were counted in viable pimonidazole-negative areas (non-hypoxic) and pimonidazole-positive areas (hypoxic) of whole tumour cross-sections. ResultsThe median number of tumour-infiltrating CD11b+ cells either decreased or remained unchanged after 5 and 10 fractions in most of the tumour models. The density of CD11b+ cells in hypoxic areas was similar or lower than in non-hypoxic regions independently on treatment in majority of the tumour models. After 10 fractions the median CD11b+ cell density was significantly associated with the TCD50 values after 30 fractions. ConclusionThe data from our exploratory study suggest that tumour-infiltrating CD11b+ cells may contribute to local tumour control after fractionated irradiation, which supports to further study their prognostic value and to evaluate specific myelomonocyte targeting strategies to overcome radiation resistance.

Rethinking the Warburg Effect with Myc Micromanaging Glutamine Metabolism

The MYC oncogene, which is frequently deregulated in human cancers, encodes a master transcription factor c-Myc (herein termed Myc) that integrates cell proliferation with metabolism through its regulation of thousands of genes including microRNAs (miRNA). In addition to its known function in regulating the cell cycle and glucose metabolism, recent studies document a role for Myc in stimulating glutamine catabolism, in part through the repression of miRNAs miR-23a and miR-23b. These observations suggest an additional level of complexity in tumor metabolism, which includes the commensal metabolic relationship between hypoxic and nonhypoxic regions of tumors as well as the surrounding stroma. Thus, a reevaluation of cancer metabolism considering glutamine catabolism with a better understanding of the tumor histological complexity is needed before cancer metabolism can be effectively targeted in therapy.

Basic characterization of 64Cu-ATSM as a radiotherapy agent

64Cu-diacetyl-bis( N 4-methylthiosemicarbazone) ( 64Cu-ATSM) is a promising radiotherapy agent for the treatment of hypoxic tumors. In an attempt to elucidate the radiobiological basis of 64Cu-ATSM radiotherapy, we have investigated the cellular response patterns in vitro cell line models. Cells were incubated with 64Cu-ATSM, and the dose–response curves were obtained by performing a clonogenic survival assay. Radiation-induced damage in DNA was evaluated using the alkali comet assay and apoptotic cells were detected using Annexin V-FITC and propidium iodide staining methods. Washout rate and subcellular distribution of 64Cu in cells were investigated to further assess the effectiveness of 64Cu-ATSM therapy on a molecular basis. A direct comparison of subcellular localization of Cu-ATSM was made with the flow tracer analog Cu-pyruvladehyde-bis( N 4-methylthiosemicarbazone). In this study, 64Cu-ATSM was shown to reduce the clonogenic survival rate of tumor cells in a dose-dependent manner. Under hypoxic conditions, cells took up 64Cu-ATSM and radioactive 64Cu was highly accumulated in the cells. In the 64Cu-ATSM-treated cells, DNA damage by the radiation emitted from 64Cu was detected, and inhibition of cell proliferation and induction of apoptosis was observed at 24 and 36 h after the treatment. The typical features of postmitotic apoptosis induced by radiation were observed following 64Cu-ATSM treatment. The majority of the 64Cu taken up into the cells remained in the postmitochondrial supernatant (the cellular residue after removal of the nuclei and mitochondria), which indicates that the β − particle emitted from 64Cu may be as effective as the Auger electrons in 64Cu-ATSM therapy. These data allow us to postulate that 64Cu-ATSM will be able to attack the hypoxic tumor cells directly, as well as potentially affecting the peripheral nonhypoxic regions indirectly by the β − particle decay of 64Cu.

Differential effects of buthionine sulphoximine in hypoxic and non-hypoxic regions of human cervical carcinoma xenografts

Background and Purpose: Recently we reported increased glutathione (GSH) levels in hypoxic regions of ME 180 and SiHa cervical cancer xenografts. Since this association might act synergistically to protect from radiotherapy, we examined the differential effects of the GSH depleting agent buthionine suiphoximine (BSO) in relation to tumor oxygenation. Materials and methods: The nitroimidazole EF5 was used to label tumor hypoxia. GSH levels were determined in cryostat sections using a sensitive HPLC assay and in parallel sections using fluorescence image analysis. Using a dual-labeling method, GSH levels were determined selectively in hypoxic and non-hypoxic tumor regions. Results: GSH levels were higher in hypoxic than in non-hypoxic regions of cervical carcinoma xenografts. Treatment with BSO produced a more pronounced GSH depletion in regions of hypoxia, resulting in similar post-treatment levels in hypoxic and non-hypoxic areas. Conclusions: BSO effectively depletes GSH in hypoxic microregions of tumors. These findings suggest a potential role for BSO as an adjunct to radiotherapy in cervical cancer patients.

Estimation of oxygen distribution in RIF-1 tumors by diffusion model-based interpretation of pimonidazole hypoxia and eppendorf measurements.

Numerical simulations of oxygen diffusion from the capillaries in tumor tissue were used to predict the capillary oxygen supply within and near hypoxic regions of the RIF-1 tumor. A finite element method to simulate the oxygen distribution from a histology section is presented, along with a method to iteratively estimate capillary oxygen concentrations. Pathological structural data for these simulations came from sections of the tumor stained with hematoxylin and eosin and were used to define the capillary positions and shapes, while overlapping regions of low oxygen concentration were defined by the hypoxia marker pimonidazole. These simulations were used to calculate spatial maps of the oxygen concentration and were tested for their ability to reproduce Eppendorf pO(2) histograms from the same tumor line. This simulation study predicted that capillary oxygen concentrations ranged from zero to above 20 microM, with a dominant peak in the hypoxic regions showing 78% of capillaries with less than 1 microM oxygen concentration, compared to only 12% in the non-hypoxic regions. The results were not highly sensitive to the metabolic oxygen consumption rate, within the range of 2 to 16 microM/s. This numerical method for oxygen capillary simulation is readily adaptable to histology sections and provides a method to examine the heterogeneity of oxygen within the capillaries and throughout the tumor tissue section being examined.

Almitrine enhances in low dose the reactivity of pulmonary vessels to hypoxia

Almitrine bismesylate (Alm) has been shown to increase arterial oxygen tension in patients with chronic obstructive pulmonary disease. This effect is thought to be attributable to the enhancement of hypoxic pulmonary vasoconstruction (HPV). We evaluated the effect of various doses of Alm on HPV in terms of blood flow diversion associated with anoxic challenge to the left lower lobe (LLL) in dogs with the LLL and the rest of the lung separately ventilated. The stimilus-response curve shifted to the right with increasing rate of Alm infusion, suggesting that Alm enhances the reactivity of the pulmonary vessels to hypoxia. Low doses of Alm enhanced HPV, while higher doses attenuated it. This suggests that the degree of vasoconstriction in hypoxic and non-hypoxic regions depends on the dose of Alm. The same effects of Alm were also observed in peripheral chemoreceptor denervated dogs. It is supposed that the vasoconstriction induced by Alm may be attributable to a direct effect on pulmonary vessels rather than to a nervous reflex effect.