Tumor Tissue Oxygenation Research Articles

Alleviating tumor hypoxia and immunosuppression via sononeoperfusion: A new Ally for potentiating anti-PD-L1 blockade of solid tumor

The hypoxic and immunosuppressive tumor microenvironment (TME) remains a major obstacle to impede cancer immunotherapy. Here, we found that sononeoperfusion—a new effect of tumor perfusion enhancement induced by low mechanical index ultrasound stimulated microbubble cavitation (USMC)—ameliorated tumor tissue oxygenation and induced tumor vascular normalization (TVN). This TVN might be associated with the down-regulation of hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) within tumors. Moreover, the sononeoperfusion effect reduced the accumulation of immunosuppressive cells, such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and M2-like tumor-associated macrophages (M2-TAMs), and decreased the production of immune inhibitory factors like transforming growth factor-β1 (TGF-β1), interleukin 10 (IL-10), chemoattractant chemokines CC-chemokine ligand 22 (CCL22), CCL28, adenosine and lactate within tumors. Notably, flow cytometry analysis revealed that sononeoperfusion not only increased the percentage of tumor infiltrating-CD8+ T cells, but also promoted the generation of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) by these cells. Furthermore, the improved immune TME by sononeoperfusion effect sensitized anti-PD-L1 treatment both in MC38 colon cancer and Lewis lung carcinoma mice, resulting in tumor regression and prolonged survival. Mechanically, the enhanced efficacy of combination therapy was mainly based on promoting the infiltration and function of CD8+ T cells within tumors. Together, sononeoperfusion could ameliorate hypoxia and immunosuppression in the TME, thereby potentiating anti-PD-L1 therapy for solid tumors. This novel method of USMC generating sononeoperfusion effect may provide a new therapeutic modality for facilitating cancer immunotherapy.

Sonodynamic-chemotherapy synergy with chlorin e6-based carrier-free nanoparticles for non-small cell lung cancer.

Sonodynamic therapy (SDT), an emerging cancer treatment with significant potential, offers the advantages of non-invasiveness and deep tissue penetrability. The method involves activating sonosensitizers with ultrasound to generate reactive oxygen species (ROS) capable of eradicating cancer cells, addressing the challenge faced by photodynamic therapy (PDT) where conventional light sources struggle to penetrate deep tissues, impacting treatment efficacy. This study addresses prevalent challenges in numerous nanodiagnostic and therapeutic agents, such as intricate synthesis, poor repeatability, low stability, and high cost, by introducing a streamlined one-step assembly method for nanoparticle preparation. Specifically, the sonosensitizer Chlorin e6 (Ce6) and the chemotherapy drug erlotinib are effortlessly combined and self-assembled under sonication, yielding carrier-free nanoparticles (EC-NPs) for non-small cell lung cancer (NSCLC) treatment. The resulting EC-NPs exhibit optimal drug loading capacity, a simplified preparation process, and robust stability both in vitro and in vivo, owing to their carrier-free characteristics. Under the synergistic treatment of sonodynamic therapy and chemotherapy, EC-NPs induce an excess of reactive oxygen in tumor tissue, prompting apoptosis of cancer cells and reducing their proliferative capacity. Both in vitro and in vivo experiments demonstrate superior therapeutic effects of EC-NPs under ultrasound conditions compared to free Ce6. In summary, our research findings highlight that the innovatively designed carrier-free sonosensitizer EC-NPs present a therapeutic option with commendable efficacy and minimal side effects.

Efficient type Ⅰ and type Ⅱ ROS generated aggregation-induced emission photosensitizer for mitochondria targeted photodynamic therapy

Fluorescence imaging-guided photodynamic therapy (PDT) is considered as an ideal alternative strategy to treat tumors. However, insufficient supply of oxygen in solid tumor tissue, aggregation-caused quenching (ACQ) of photosensitizers (PSs), poor targeting-specificity of cancer cells and short wavelengths of emission are representative obstacles for effective PDT. In this paper, three D-π-A structured PSs TSB-OH, TSBPy-OH, TSBPy-DNT with aggregation induced emission (AIE) properties and integrated type I and type II ROS generation were synthesized by modulating distinct acceptor units. Among them, TSBPy-OH is optimal with multiple advantages including near-infrared emission, good biocompatibility, remarkably intracellular ROS production ability, mitochondria targeting capability inducing cell apoptosis and enhancing PDT, and long-term tumor retention ability for imaging-guided photodynamic therapy. More importantly, efficient in vitro cancer cells damage, in vivo tumor ablation and ignorable damage in major organs during PDT are demonstrated, suggesting potential capability of TSBPy-OH in safe and effective fluorescence imaging-guided PDT. This successful example of AIE theranostics design would provide a reference for the modification of D-π-A structure organic AIEgens.

Sononeoperfusion effect by ultrasound and microbubble promotes nitric oxide release to alleviate hypoxia in a mouse MC38 tumor model

Tumor hypoperfusion not only impedes therapeutic drug delivery and accumulation, but also leads to a hypoxic and acidic tumor microenvironment, resulting in tumor proliferation, invasion, and therapeutic resistance. Sononeoperfusion effect refers to tumor perfusion enhancement using ultrasound and microbubbles. This study aimed to further investigate hypoxia alleviation by sononeoperfusion effect and explore the characteristics and mechanism of sononeoperfusion effect. To stimulate the sononeoperfusion effect, mice bearing MC38 colon cancers were included in this study and diagnostic ultrasound for therapy was set at a mechanical index (MI) of 0.1, 0.3, and 0.5, frequency of 3 MHz, pulse length of 5 cycles, and pulse repetition frequency of 2000 Hz. The results demonstrated that a single ultrasound and microbubble (USMB) treatment resulted in tumor perfusion enhancement at MI = 0.3, and nitric oxide (NO) concentration increased at MI = 0.3/0.5 (P < 0.05). However, there were no significant difference in the hypoxia-inducible factor-1α (HIF-1α) or D-lactate (D-LA) (P > 0.05) levels. Multiple sononeoperfusion effects were observed at MI = 0.3/0.5 (P < 0.05). For each treatment, USMB slightly but steadily improved the tumor tissue oxygen partial pressure (pO2) during and post treatment. It alleviated tumor hypoxia by decreasing HIF-1α, D-LA level and the hypoxic immunofluorescence intensity at MI = 0.3/0.5 (P < 0.05). The sononeoperfusion effect was not stimulated after eNOS inhibition. In conclusion, USMB with appropriate MI could lead to a sononeoperfusion effect via NO release, resulting in hypoxia amelioration. The tumors were not resistant to multiple sononeoperfusion effects. Repeated sononeoperfusion is a promising approach for relieving tumor hypoxia and resistance to therapy.

Radiosensitizing oxygenation changes in murine tumors treated with VEGF-ablation therapy are measurable using oxygen enhanced-MRI (OE-MRI)

PurposeThere is a significant need for a widely available, translatable, sensitive and non-invasive imaging biomarker for tumor hypoxia in radiation oncology. Treatment-induced changes in tumor tissue oxygenation can alter the sensitivity of cancer tissues to radiation, but the relative difficulty in monitoring the tumor microenvironment results in scarce clinical and research data. Oxygen-Enhanced MRI (OE-MRI) uses inhaled oxygen as a contrast agent to measure tissue oxygenation. Here we investigate the utility of dOE-MRI, a previously validated imaging approach employing a cycling gas challenge and independent component analysis (ICA), to detect VEGF-ablation treatment-induced changes in tumor oxygenation that result in radiosensitization. MethodsMurine squamous cell carcinoma (SCCVII) tumor-bearing mice were treated with 5 mg/kg anti-VEGF murine antibody B20 (B20-4.1.1, Genentech) 2–7 days prior to radiation treatment, tissue collection or MR imaging using a 7 T scanner. dOE-MRI scans were acquired for a total of three repeated cycles of air (2 min) and 100% oxygen (2 min) with responding voxels indicating tissue oxygenation. DCE-MRI scans were acquired using a high molecular weight (MW) contrast agent (Gd-DOTA based hyperbranched polygylcerol; HPG-GdF, 500 kDa) to obtain fractional plasma volume (fPV) and apparent permeability-surface area product (aPS) parameters derived from the MR concentration–time curves. Changes to the tumor microenvironment were evaluated histologically, with cryosections stained and imaged for hypoxia, DNA damage, vasculature and perfusion. Radiosensitizing effects of B20-mediated increases in oxygenation were evaluated by clonogenic survival assays and by staining for DNA damage marker γH2AX. ResultsTumors from mice treated with B20 exhibit changes to their vasculature that are consistent with a vascular normalization response, and result in a temporary period of reduced hypoxia. DCE-MRI using injectable contrast agent HPG-GDF measured decreased vessel permeability in treated tumors, while dOE-MRI using inhaled oxygen as a contrast agent showed greater tissue oxygenation. These treatment-induced changes to the tumor microenvironment result in significantly increased radiation sensitivity, illustrating the utility of dOE-MRI as a non-invasive biomarker of treatment response and tumor sensitivity during cancer interventions. ConclusionsVEGF-ablation therapy-mediated changes to tumor vascular function measurable using DCE-MRI techniques may be monitored using the less invasive approach of dOE-MRI, an effective biomarker of tissue oxygenation that can monitor treatment response and predict radiation sensitivity.

Normalizing tumor microenvironment with nanomedicine and metronomic therapy to improve immunotherapy

Nanomedicine offered hope for improving the treatment of cancer but the survival benefits of the clinically approved nanomedicines are modest in many cases when compared to conventional chemotherapy. Metronomic therapy, defined as the frequent, low dose administration of chemotherapeutics – is being tested in clinical trials as an alternative to the conventional maximum tolerated dose (MTD) chemotherapy schedule. Although metronomic chemotherapy has not been clinically approved yet, it has shown better survival than MTD in many preclinical studies. When beneficial, metronomic therapy seems to be associated with normalization of the tumor microenvironment including improvements in tumor perfusion, tissue oxygenation and drug delivery as well as activation of the immune system. Recent preclinical studies suggest that nanomedicines can cause similar changes in the tumor microenvironment. Here, by employing a mathematical framework, we show that both approaches can serve as normalization strategies to enhance treatment. Furthermore, employing murine breast and fibrosarcoma tumor models as well as ultrasound shear wave elastography and contrast-enhanced ultrasound, we provide evidence that the approved nanomedicine Doxil can induce normalization in a dose-dependent manner by improving tumor perfusion as a result of tissue softening. Finally, we show that pretreatment with a normalizing dose of Doxil can improve the efficacy of immune checkpoint inhibition.

Novel humanized monoclonal antibodies for targeting hypoxic human tumors via two distinct extracellular domains of carbonic anhydrase IX

BackgroundHypoxia in the tumor microenvironment (TME) is often the main factor in the cancer progression. Moreover, low levels of oxygen in tumor tissue may signal that the first- or second-line therapy will not be successful. This knowledge triggers the inevitable search for different kinds of treatment that will successfully cure aggressive tumors. Due to its exclusive expression on cancer cells, carbonic anhydrase IX belongs to the group of the most precise targets in hypoxic tumors. CA IX possesses several exceptional qualities that predetermine its crucial role in targeted therapy. Its expression on the cell membrane makes it an easily accessible target, while its absence in healthy corresponding tissues makes the treatment practically harmless. The presence of CA IX in solid tumors causes an acidic environment that may lead to the failure of standard therapy.MethodsParental mouse hybridomas (IV/18 and VII/20) were humanized to antibodies which were subsequently named CA9hu-1 and CA9hu-2. From each hybridoma, we obtained 25 clones. Each clone was tested for antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, affinity, extracellular pH measurement, multicellular aggregation analysis, and real-time monitoring of invasion with the xCELLigence system.ResultsBased on the results from in vivo experiments, we have selected mouse monoclonal antibodies VII/20 and IV/18. The first one is directed at the conformational epitope of the catalytic domain, internalizes after binding to the antigen, and halts tumor growth while blocking extracellular acidification. The second targets the sequential epitope of the proteo-glycan domain, does not internalize, and is able to block the attachment of cancer cells to the matrix preventing metastasis formation. In vitro experiments prove that humanized versions of the parental murine antibodies, CA9hu-1 and CA9hu-2, have preserved these characteristics. They can reverse the failure of standard therapy as a result of an acidic environment by modulating the TME, and both are able to induce an immune response and have high affinity, as well as ADCC and CDC activity.ConclusionCA9hu-1 and CA9hu-2 are the very first humanized antibodies against CA IX that are likely to become suitable therapies for hypoxic tumors. These antibodies can be applied in the treatment therapy of primary tumors and suppression of metastases formation.

Recent advances in in situ oxygen-generating and oxygen-replenishing strategies for hypoxic-enhanced photodynamic therapy.

Cancer is a leading cause of death worldwide, accounting for an estimated 10 million deaths by 2020. Over the decades, various strategies for tumor therapy have been developed and evaluated. Photodynamic therapy (PDT) has attracted increasing attention due to its unique characteristics, including low systemic toxicity and minimally invasive nature. Despite the excellent clinical promise of PDT, hypoxia is still the Achilles' heel associated with its oxygen-dependent nature related to increased tumor proliferation, angiogenesis, and distant metastases. Moreover, PDT-mediated oxygen consumption further exacerbates the hypoxia condition, which will eventually lead to the poor effect of drug treatment and resistance and irreversible tumor metastasis, even limiting its effective application in the treatment of hypoxic tumors. Hypoxia, with increased oxygen consumption, may occur in acute and chronic hypoxia conditions in developing tumors. Tumor cells farther away from the capillaries have much lower oxygen levels than cells in adjacent areas. However, it is difficult to change the tumor's deep hypoxia state through different ways to reduce the tumor tissue's oxygen consumption. Therefore, it will become more difficult to cure malignant tumors completely. In recent years, numerous investigations have focused on improving PDT therapy's efficacy by providing molecular oxygen directly or indirectly to tumor tissues. In this review, different molecular oxygen supplementation methods are summarized to alleviate tumor hypoxia from the innovative perspective of using supplemental oxygen. Besides, the existing problems, future prospects and potential challenges of this strategy are also discussed.

The Metabolic Features of Tumor-Associated Macrophages: Opportunities for Immunotherapy?

Besides transformed cells, the tumors are composed of various cell types that contribute to undesirable tumor progression. Tumor-associated macrophages (TAMs) are the most abundant innate immune cells in the tumor microenvironment (TME). Within the TME, TAMs exhibit high plasticity and undergo specific functional metabolic alterations according to the availability of tumor tissue oxygen and nutrients, thus further contributing to tumorigenesis and cancer progression. Here, we review the main functional TAM metabolic patterns influenced by TME, including glycolysis, amino acid, and fatty acid metabolism. Moreover, this review discusses antitumor immunotherapies that affect TAM functionality by inducing cell repolarizing and metabolic profiles towards an antitumoral phenotype. Also, new macrophage-based cell therapeutic technologies recently developed using chimeric antigen receptor bioengineering are exposed, which may overcome all solid tumor physical barriers impeding the current adoptive cell therapies and contribute to developing novel cancer immunotherapies.

A versatile gas-generator promoting drug release and oxygen replenishment for amplifying photodynamic-chemotherapy synergetic anti-tumor effects

Excellent efficiency of combinational therapy of chemotherapy and photodynamic therapy (PDT) highly depends on the amounts of drug and oxygen in tumor tissue. However, how to cleverly promote drug release accompanied with improving oxygen concentration remains a challenge. Herein, we proposed a gas-generator that realized a high drug loading and integrated facilitation of drug release with oxygen replenishment into a single and simple system, utilizing huge cavities and mesoporous channels of hollow mesoporous silica nanoparticles (HMSNs) for encapsulating oxygen (O2) saturated perfluoropentane (PFP) droplets, indocyanine green (ICG) and doxorubicin (DOX), biocompatible polydopamine (PDA) as the gatekeepers. Under irradiation of 808 nm laser, the thermal effect of PDA caused PFP droplets occur liquid-gas phase transition that triggered the burst release of DOX and O2, finally amplifying the synergetic effects of PDT and chemotherapy both in vitro and in vivo. The influence of PFP, GSH and laser on drug release kinetic was explored through mathematical models. Notably, the mechanism of gas-generator on accelerating drug release under irradiation based on doing volume work and enhancing diffusion coefficient was clarified by researching the relation between DOX release, PFP release and temperature change. Additionally, the way of replenishing O2 did not rely on intracellular components but timely offered abundant “fuels” for producing reactive oxygen species (ROS) when compared with traditional manners. This work provides a new research strategy for boosting drug release and opens an avenue for constructing multifunctional controlled delivery systems.

Synthesis and Characterisation of a Boron-Rich Symmetric Triazine Bearing a Hypoxia-Targeting Nitroimidazole Moiety

Boron Neutron Capture Therapy (BNCT) is a binary therapy that promises to be suitable in treating many non-curable cancers. To that, the discovery of new boron compounds able to accumulate selectively in the tumour tissue is still required. Hypoxia, a deficiency of oxygen in tumor tissue, is a great challenge in the conventional treatment of cancer, because hypoxic areas are resistant to conventional anticancer treatments. 2-Nitroimidazole derivatives are known to be hypoxia markers due to their enrichment by bioreduction in hypoxic cells. In the present work, 2-nitroimidazole was chosen as the starting point for the synthesis of a new boron-containing compound based on a 1,3,5-triazine skeleton. Two o-carborane moieties were inserted to achieve a high ratio of boron on the molecular weight, exploiting a short PEG spacer to enhance the polarity of the compound and outdistance the active part from the core. The compound showed no toxicity on normal human primary fibroblasts, while it showed noteworthy toxicity in multiple myeloma cells together with a consistent intracellular boron accumulation.

Quantitative In Vivo Monitoring of Hypoxia and Vascularization of Patient-Derived Murine Xenografts of Mantle Cell Lymphoma Using Photoacoustic and Ultrasound Imaging

Tumor oxygenation and vascularization are important parameters that determine the aggressiveness of the tumor and its resistance to cancer therapies. We introduce dual-modality ultrasound and photoacoustic imaging (US-PAI) for the direct, non-invasive real-time in vivo evaluation of oxygenation and vascularization of patient-derived xenografts (PDXs) of B-cell mantle cell lymphomas. The different optical properties of oxyhemoglobin and deoxyhemoglobin make it possible to determine oxygen saturation (sO2) in tissues using PAI. High-frequency color Doppler imaging enables the visualization of blood flow with high resolution. Tumor oxygenation and vascularization were studied in vivo during the growth of three different subcutaneously implanted patient-derived xenograft (PDX) lymphomas (VFN-M1, VFN-M2 and VFN-M5 R1). Similar values of sO2 (sO2 Vital), determined from US-PAI volumetric analysis, were obtained in small and large VFN-M1 tumors ranging from 37.9 ± 2.2 to 40.5 ± 6.0 sO2 Vital (%) and 37.5 ± 4.0 to 35.7 ± 4.6 sO2 Vital (%) for small and large VFN-M2 PDXs. In contrast, the higher sO2 Vital values ranging from 57.1 ± 4.8 to 40.8 ± 5.7 sO2 Vital (%) (small to large) of VFN-M5 R1 tumors corresponds with the higher aggressiveness of that PDX model. The different tumor percentage vascularization (assessed as micro-vessel areas) of VFN-M1, VFN-M2 and VFN-M5 R1 obtained by color Doppler (2.8 ± 0.1%, 3.8 ± 0.8% and 10.3 ± 2.7%) in large-stage tumors clearly corresponds with their diverse growth and aggressiveness. The data obtained by color Doppler were validated by histology. In conclusion, US-PAI rapidly and accurately provided relevant and reproducible information on tissue oxygenation in PDX tumors in real time without the need for a contrast agent.

Abnormal morphology biases hematocrit distribution in tumor vasculature and contributes to heterogeneity in tissue oxygenation

Oxygen heterogeneity in solid tumors is recognized as a limiting factor for therapeutic efficacy. This heterogeneity arises from the abnormal vascular structure of the tumor, but the precise mechanisms linking abnormal structure and compromised oxygen transport are only partially understood. In this paper, we investigate the role that red blood cell (RBC) transport plays in establishing oxygen heterogeneity in tumor tissue. We focus on heterogeneity driven by network effects, which are challenging to observe experimentally due to the reduced fields of view typically considered. Motivated by our findings of abnormal vascular patterns linked to deviations from current RBC transport theory, we calculated average vessel lengths [Formula: see text] and diameters [Formula: see text] from tumor allografts of three cancer cell lines and observed a substantial reduction in the ratio [Formula: see text] compared to physiological conditions. Mathematical modeling reveals that small values of the ratio λ (i.e., [Formula: see text]) can bias hematocrit distribution in tumor vascular networks and drive heterogeneous oxygenation of tumor tissue. Finally, we show an increase in the value of λ in tumor vascular networks following treatment with the antiangiogenic cancer agent DC101. Based on our findings, we propose λ as an effective way of monitoring the efficacy of antiangiogenic agents and as a proxy measure of perfusion and oxygenation in tumor tissue undergoing antiangiogenic treatment.

Hyperbaric oxygen increases glioma cell sensitivity to antitumor treatment with a novel isothiourea derivative invitro.

Glioblastoma (GBM) is the most common primary brain tumor. Tumor hypoxia is a pivotal factor responsible for the progression of this malignant glioma, and its resistance to radiation and chemotherapy. Thus, improved tumor tissue oxygenation may promote greater sensitivity to anticancer treatment. Protein kinase D1 (PKD1) protects cells from oxidative stress, and its abnormal activity serves an important role in multiple malignancies. The present study examined the effects of various oxygen conditions on the cytotoxic potential of the novel isothiourea derivate N,N′-dimethyl-S-(2,3,4,5,6-pentabromobenzyl)- isothiouronium bromide (ZKK-3) against the T98G GBM cell line. ZKK-3 was applied at concentrations of 10, 25 and 50 µM, and cells were maintained under conditions of normoxia, anoxia, hypoxia, hyperbaric oxygen (HBO), hypoxia/hypoxia and hypoxia/HBO. The proliferation and viability of neoplastic cells, and protein expression levels of hypoxia-inducible factor 1α (HIF-1α), PKD1, phosphorylated (p)PKD1 (Ser 916) and pPKD1 (Ser 744/748) kinases were evaluated. Oxygen deficiency, particularly regarding hypoxia, could diminish the cytotoxic effect of ZKK-3 at 25 and 50 µM and improve T98G cell survival compared with normoxia. HBO significantly reduced cell proliferation and increased T98G cell sensitivity to ZKK-3 when compared with normoxia. HIF-1α expression levels were increased under hypoxia compared with normoxia and decreased under HBO compared with hypoxia/hypoxia at 0, 10 and 50 µM ZKK-3, suggesting that HBO improved oxygenation of the cells. ZKK-3 exhibited inhibitory activity against pPKD1 (Ser 916) kinase; however, the examined oxygen conditions did not appear to significantly influence the expression of this phosphorylated form in cells treated with the tested compound. Regarding pPKD1 (Ser 744/748), a significant difference in expression was observed only for cells treated with 10 µM ZKK-3 and hypoxia/hypoxia compared with normoxia. However, there were significant differences in the expression levels of both phosphorylated forms of PKD1 under different oxygen conditions in the controls. In conclusion, the combination of isothiourea derivatives and hyperbaric oxygenation appears to be a promising therapeutic approach for malignant glioma treatment.

Dynamic influence of pentoxifylline on the oxygen status of Pliss’s lymph sarcoma in rat

Tumor oxygenation is one of the key factors influencing disease prognosis and the effectiveness of treatment. Assessment of tumor oxygenation levels facilitates the selection of optimum conditions for radiation therapy, and plays an important role in creating alternative regimes of irradiation. Treating tumors with agents capable of increasing tumor oxygenation in order to increase radiosensitivity is a promising avenue of enquiry. Diffuse optical spectroscopy (DOS) allows a noninvasive determination of tissue oxygen levels based on information about the local changes in optical parameters, and the visualization of metabolic processes in the region of interest. DOS allows reconstruction of the two-dimensional distribution of main tissue chromophores that characterize the processes of oxygen supply (oxygenated hemoglobin) and oxygen consumption (deoxygenated hemoglobin), as well as the blood oxygen saturation levels, which indirectly reflect the tissue oxygenation levels. In the present study, a hemorheologic drug, pentoxifylline, which can improve microcirculation in regions with circulatory disturbances, was used for modifying tumor tissue oxygenation. Pliss’s lymph sarcoma (PLS), which is characterized by rapid growth and early occurrence of necrotic areas, was chosen as a tumor model. Tumor oxygenation was monitored by DOS with parallel plane geometry. Pentoxifylline could improve tumor oxygenation by increasing the concentration of oxyhemoglobin. The increased blood oxygen saturation persisted from 30 to 120 min after drug administration. Normal healthy tissue (muscle) and tumor tissue responded differently to the drug. DOS can be used for testing new agents that influence tissue oxygen status and blood-filling rate.

Noninvasive measurement of tissue blood oxygenation with Cerenkov imaging during therapeutic radiation delivery.

Tumor tissue oxygenation significantly affects the outcome of radiotherapy. Real-time monitoring of tumor hypoxia is highly desirable for effective radiotherapy, and is the basis for improved treatment because hypoxic tumor cells are more resistant to radiation damage than fully oxygenated cells. We propose to use Cerenkov imaging to monitor tumor hypoxia by means of tissue blood oxygenation without the need for any exogenous contrast agent. Using a rodent hypoxia model, we demonstrate that Cerenkov imaging can be used as a noninvasive and noncontact method to measure tissue blood oxygenation level during radiation delivery. The data from Cerenkov imaging were validated using near infrared spectrometry methods. The results demonstrate the feasibility of using Cerenkov imaging to monitor tumor hypoxia during therapeutic radiation delivery.

The effect of imatinib therapy on tumour cycling hypoxia, tissue oxygenation and vascular reactivity

Background: Several biomedical imaging techniques have recently been developed to probe hypoxia in tumours, including oxygen-enhanced (OE) and blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI). These techniques have strong potential for measuring both chronic and transient (cycling) changes in hypoxia, and to assess response to vascular-targeting therapies in the clinic. Methods: In this study, we investigated the use of BOLD and OE-MRI to assess changes in cycling hypoxia, tissue oxygenation and vascular reactivity to hyperoxic gas challenges, in mouse models of colorectal therapy, following treatment with the PDGF-receptor inhibitor, imatinib mesylate (Glivec). Results: Whilst no changes were observed in imaging biomarkers of cycling hypoxia (from BOLD) or chronic hypoxia (from OE-MRI), the BOLD response to carbogen-breathing became significantly more positive in some tumour regions and more negative in other regions, thereby increasing overall heterogeneity. Conclusions: Imatinib did not affect the magnitude of cycling hypoxia or OE-MRI signal, but increased the heterogeneity of the spatial distribution of BOLD MRI changes in response to gas challenges.

P08.58 Impact of selected pentabromobenzylisothiourea (ZKK-3) combined with hyperbaric oxygen therapy on therapeutic response and oxygenation status of T98G glioblastoma cell line

Abstract Introduction: Glioblastoma (GBM), the primary brain tumour derived from astroglial cells, is the most frequently diagnosed intracranial malignancy in adults. Poor prognosis for GBM patients results from high invasiveness of neoplastic cells related with faulty angiogenesis and persistent hypoxia states. In such conditions glioma cells develop radio- and chemotherapy-resistant phenotype. It is postulated that application of hyperbaric oxygen (HBO) may improve therapeutic response via refinement of tumour tissue oxygenation. The more effective therapeutic strategies are also required. One of the novel compounds that exhibit anti-tumour properties are pentabromobenzylisothioureas. The aim of this study was to examine whether HBO may enhance anti-tumour efficacy of selected pentabromobenzylisothiourea when both agents were administered as combined ZKK-3/HBO therapy. Materials and Methods: Human glioblastoma T98G cell line was cultured in medium supplemented with novel isothiourea derivative - ZKK-3. The proliferation and viability of glioma cells cultured in hypoxia or hyperbaric oxygen conditions were examined. Cell proliferation was tested 24 hours after ZKK-3 addition using Nikon Inverted Microscope and Multisizer 3 Beckman Coulter. The viability assay was taken 24 and 48 hours post ZKK-3 supplementation by CellTiter 96®AQueous One Solution Cell Proliferation Assay (Promega). Hypoxia state of T98G cells was determined via the evaluation of HIF-1α protein expression using HIF-1A ELISA Kit (Thermo Scientific). Tests were performed on cells treated with ZKK-3 and cultured in various oxygen conditions: 1/ normoxia (24 hours), 2/ hypoxia (24 hours), 3/ HBO (2 ATA, 1 hour followed by 23 hours of normoxia), 4/ double hypoxia, 5/ hypoxia/HBO. Results: Proliferation of T98G cells treated with ZKK-3 was significantly decreased after hyperbaric oxygenation in comparison to hypoxia conditions. Administration of HBO resulted also in statistically significant diminution of glioma cells’ viability. Expression of HIF-1α in T98G cells strongly depended on oxygen conditions: in hypoxia and double hypoxia the level of protein was significantly elevated compared with normoxia, while after hyperbaric oxygenation no changes or even decrease of HIF-1α level was observed. Comparison of double hypoxia and hypoxia/HBO groups showed that HBO addition after ZKK-3 supplementation caused significant reduction of HIF-1α expression. Conclusions: Treatment of malignant glioma cells with selected isothiourea derivative is significantly more efficient when combined with hyperbaric oxygen administration. Beneficial effect of HBO on decreasing HIF-1α expression allows to consider hyperbaric oxygenation as the way to overcome tumour hypoxia.ACKNOWLEDGEMENT: The research was supported by the KNOW-MMRC project and Foundation for the Development of Diagnostic and Therapy.

TROSATIVE STRESS PARAMETERS IN COLON CANCER TUMOR, ADJACENT AND HEALTHY TISSUE

Colorectal cancer is one of the most frequent human malignant diseases and one of the most common causes of malignant diseases death. Oxidative and nitrosative stress have an important role in cancer initiation and propagation. That is why this study is focused on the determination of oxidative and nitrosative stress markers in tumor, adjacent and health tissue, which are important for the estimation of tumor proliferative and angiogenic potential. The study encompassed 50 patients who underwent surgery due to colorectal cancer. In the tissue samples from resected colon preparation (tumor, adjacent and healthy tissue, at least 10 cm distant from tumor), oxidative and nitrosative stress markers, malondialdehyde (MDA) and nitric oxide (NO) were determined. The obtained results prove the presence of oxidative stress in tumor tissue. Highly significantly (p<0.001) increased MDA concentrations in both tumor and adjacent tissue (12.43±9.39 and 11.57±5.56 nmol/mg proteins) compared to healthy one (7.25±5.52) reflect higher tumor aggressiveness and metastatic capacity. Higher NO concentrations in adjacent tissue (85.100±37.972 nmol/mg prot.) compared to the tumor one (58.608±22.789) point out high angiogenic potential of tumor surrounding tissue, which could have the clinical importance in the assessment of tumor invasiveness and the probability of local recurrence. In conclusion, the determination of the intensity of reactive oxygen and nitrogen species generation in tumor and adjacent colon tissue of patients with colorectal carcinoma could be useful in the estimation of the cancer invasive and metastatic capacity related to the prognosis of the disease and the choice of adjuvant therapy.

The Cure from Nature: The Extraordinary Anticancer Properties of Ascorbate (Vitamin C)

The anticancer properties of Vitamin C (ascorbic acid o sodium ascorbate) are known since at least four decades, However, being a cheap and natural product, Vitamin C is not patentable and therefore has never been developed as an anticancer molecule. Recent in vitro investigations have confirmed the extraordinary antitumor properties of high doses of Vitamin C (sodium ascorbate), particularly when administered by the intravenous route, and phase I/II randomized, controlled clinical trials have been started to verify its anticancer properties in vivo. Unfortunately, the controlled clinical trials performed so far, do not confirm the extraordinary results obtained with Vitamin C (sodium ascorbate) in vitro. However, this may depend on a number of different factors, such as the pharmaceutical preparation (Sodium ascorbate may be more suitable than buffered ascorbic acid), the schedule of administration (slow infusion better than rapid infusion), tumor tissue oxygenation (Cancer tissue oxygenation is lower that oxygenation of tumor cell lines, in vitro), etc., which deserve further in depth investigation. Even with these limitations, Vitamin C (sodium ascorbate) in high doses, administered by intravenous route, beyond being extremely effective in vitro, against a number of human tumor cell lines, is safe, has minimal contraindications, improves the quality of life of patients, and is highly selective for cancer cells. The Authors discuss these important aspects and suggest possible solutions to improve the in vivo anticancer effects of Vitamin C (sodium ascorbate).