Therapeutic Modification of Hypoxia.

Regions of reduced oxygenation (hypoxia) are a characteristic feature of virtually all animal and human solid tumours. Numerous preclinical studies, both in vitro and in vivo, have shown that decreasing oxygen concentration induces resistance to radiation. Importantly, hypoxia in human tumours is a negative indicator of radiotherapy outcome. Hypoxia also contributes to resistance to other cancer therapeutics, including immunotherapy, and increases malignant progression as well as cancer cell dissemination. Consequently, substantial effort has been made to detect hypoxia in human tumours and identify realistic approaches to overcome hypoxia and improve cancer therapy outcomes. Hypoxia-targeting strategies include improving oxygen availability, sensitising hypoxic cells to radiation, preferentially killing these cells, locating the hypoxic regions in tumours and increasing the radiation dose to those areas, or applying high energy transfer radiation, which is less affected by hypoxia. Despite numerous clinical studies with each of these hypoxia-modifying approaches, many of which improved both local tumour control and overall survival, hypoxic modification has not been established in routine clinical practice. Here we review the background and significance of hypoxia, how it can be imaged clinically and focus on the various hypoxia-modifying techniques that have undergone, or are currently in, clinical evaluation.

Realizing the Potential of Vascular Targeted Therapy: The Rationale for Combining Vascular Disrupting Agents and Anti-Angiogenic Agents to Treat Cancer.

Vascular targeted therapies (VTTs) are agents that target tumor vasculature and can be classified into two categories: those that inhibit angiogenesis and those that directly interfere with established tumor vasculature. Although both the anti-angiogenic agents (AAs) and the vascular disrupting agents (VDAs) target tumor vasculature, they differ in their mechanism of action and therapeutic application. Combining these two agents may realize the full potential of VTT and produce an effective therapeutic regimen. Here, we review AAs and VDAs (monotherapy and in combination with conventional therapies). We also discuss the rationale of combined VTT and its potential to treat cancer.

Evaluations of the renal cell carcinoma model Caki-1 using a silicon based microvascular casting technique.

The vascular development of orthotopically grown human renal cell carcinoma (Caki-1) was examined using a silicon based casting technique in nude mice. Images taken of these vascular casts showed Caki-1 tumors to be highly vascularized and invasive. The spread of the tumor within the cortex of the kidney revealed that Caki-1 recruits the kidney's own vasculature, destroying the functional glomeruli in the process. The loss of these glomeruli was further highlighted by the presence of enlarged glomeruli resulting from the development of super nephrons. Vessel size and density measurements were then made in this model. This was done using both computer-based and manual measurement methods. In the vessel size studies the computer-based method tended to overestimate the number of larger diameter vessels whereas the vessels density assessment showed good agreement between the two techniques. Nevertheless, both methods showed that Caki-1 tumors possessed a higher proportion of larger diameter vessels and a lower vessel density than normal kidney cortex. In summary, silicon based vascular casting proved to be a simple and effective tool for the study of tumor vasculature. In particular this technique could readily be used to examine the invasion of tumor into normal tissue. The computer-based technique for evaluating vessel number and vascular density was found to have merit in both normal and tumor tissues, particularly in the vascular density studies. However, in both settings this technique did tend to overestimate the number of larger diameter vessels.

Inhibition of renal cell carcinoma angiogenesis and growth by antisense oligonucleotides targeting vascular endothelial growth factor.

Angiogenesis is critical for growth and metastatic spread of solid tumours. It is tightly controlled by specific regulatory factors. Vascular endothelial growth factor has been implicated as the key factor in tumour angiogenesis. In the present studies we evaluated the effects of blocking vascular endothelial growth factor production by antisense phosphorothioate oligodeoxynucleotides on the growth and angiogenic activity of a pre-clinical model of renal cell carcinoma (Caki-1). In vitro studies showed that treating Caki-1 cells with antisense phosphorothioate oligodeoxynucleotides directed against vascular endothelial growth factor mRNA led to a reduction in expressed vascular endothelial growth factor levels sufficient to impair the proliferation and migration of co-cultured endothelial cells. The observed effects were antisense sequence specific, dose dependent, and could be achieved at a low, non-toxic concentration of phosphorothioate oligodeoxynucleotides. When vascular endothelial growth factor antisense treated Caki-1 cells were injected into nude mice and evaluated for their angiogenic potential, the number of vessels initiated were approximately half that induced by untreated Caki-1 cells. To test the anti-tumour efficacy of vascular endothelial growth factor antisense, phosphorothioate oligodeoxynucleotides were administrated to nude mice bearing macroscopic Caki-1 xenografts. The results showed that the systemic administration of two doses of vascular endothelial growth factor antisense phosphorothioate oligodeoxynucleotides given 1 and 4 days after the tumours reached a size of approximately 200 mm(3) significantly increased the time for tumours to grow to 1000 mm(3).

Improved tumor response by combining radiation and the vascular-damaging drug 5,6-dimethylxanthenone-4-acetic acid.

The interaction between 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and radiation was investigated in two different mouse tumor models and a normal mouse tissue. C3H mouse mammary carcinomas transplanted in the feet of CDF1 mice and KHT mouse sarcomas growing in the leg muscles of C3H/HeJ mice were used. DMXAA was dissolved in saline and injected intraperitoneally. Tumors were irradiated locally in nonanesthetized mice, and response was assessed using tumor growth for the C3H mammary carcinoma and in vivo/in vitro clonogenic cell survival for the KHT sarcoma. DMXAA alone had an antitumor effect in both tumor types, but only at doses above 15 mg/kg. DMXAA also enhanced radiation damage, and again there was a threshold dose. No enhancement was seen in the C3H mammary carcinoma at 10 mg/kg and below, while in the KHT sarcoma, doses above 15 mg/kg were necessary. This enhancement of radiation damage was also dependent on the sequence of and interval between the treatments with DMXAA and radiation. Combining radiation with DMXAA at the maximum tolerated dose (i.e., the highest dose that could be injected without causing any lethality) of either 20 mg/kg (CDF1 mice) or 17.5 mg/kg (C3H/HeJ mice) gave an additive response when the two agents were administered simultaneously. Even greater antitumor effects were achieved when DMXAA was administered 1-3 h after irradiation. However, when administration of DMXAA preceded irradiation, the effect was similar to that seen for radiation alone, suggesting that appropriate timing is essential to maximize the utility of this agent. When such conditions were met, DMXAA was found to increase the tumor response significantly in the absence of an enhancement of radiation damage in normal skin, thus giving rise to therapeutic gain.

Interaction between combretastatin A-4 disodium phosphate and radiation in murine tumors.

BACKGROUND AND PURPOSE: The ability of combretastatin A-4 disodium phosphate (CA4DP) to induce vascular damage and enhance the radiation response of murine tumors was investigated. MATERIALS AND METHODS: A C3H mouse mammary carcinoma transplanted in the foot of CDF1 mice and the KHT mouse sarcoma growing in the leg muscle of C3H/HeJ mice were used. CA4DP was dissolved in saline and injected intraperitoneally. Tumor blood perfusion was estimated using 86RbCl extraction and Hoechst 33342 fluorescent labelling. Necrotic fraction was determined from histological sections. Tumors were locally irradiated in non-anaesthetised mice and response assessed by local tumor control for the C3H mammary carcinoma and in vivo/in vitro clonogenic cell survival for the KHT sarcoma. RESULTS: CA4DP decreased tumor blood perfusion and increased necrosis in a dose-dependent fashion in the C3H mammary carcinoma, which was maximal at 250 mg/kg. The decrease in perfusion and induction of necrosis by CA4DP was more extensive in the KHT sarcoma. CA4DP enhanced radiation damage in both tumor types. In the KHT sarcoma this enhancement was independent of whether the drug was given before or after irradiating, whereas for C3H mammary carcinoma the enhancement was only significant when administered at the same time or after the radiation, with no enhancement seen if CA4DP was given before. These effects were drug-dose dependent. CA4DP did not enhance radiation damage in normal skin. CONCLUSIONS: CA4DP enhanced radiation damage in the two tumor models without enhancing normal tissue damage. These radiation effects were clearly consistent with the anti-vascular action of CA4DP.

Targeting tumor blood vessels: an adjuvant strategy for radiation therapy.

BACKGROUND AND PURPOSE: The neovascularization of tumor cells is a prerequisite if a clinically relevant tumor size is to be reached. A continuously expanding vessel network supplying nutritional requirements and removing waste products is essential for continued tumor development, growth and survival. RESULTS: In many tumors, the growing endothelium is unable to fully support the demands of the neoplastic cell population. As a consequence of the inadequacies of the resulting aberrant vasculature, microenvironmental conditions develop in tumors which are not only detrimental to the response of tumors to conventional anticancer treatments, but may lead to or predispose cells to genetic modifications resulting in more aggressive phenotypes and higher metastatic potential. Yet the utter dependence of the tumor on its induced vessel formation for growth, survival and spread has also created a great deal of enthusiasm for developing therapeutic approaches to specifically targeting the tumor microcirculation. CONCLUSIONS: The application of such strategies as adjuvants to conventional radiation treatments offers unique opportunities to develop more effective cancer therapies.

Altered fractionation in definitive irradiation of squamous cell carcinoma of the head and neck.

The likelihood of local control after radiation therapy may be improved by increasing total dose or decreasing overall time. The probability of late complications increases with dose per fraction. Altered fractionation techniques usually employ two or more fractions per day using a dose per fraction that is similar or less than that employed in conventional fractionation. Altered fractionation may be broadly classified as hyperfractionation or accelerated fractionation. Data suggest that altered fractionation schedules may improve local control (and to a lesser extent, survival) compared with conventional irradiation.

Targeting the tumor vasculature with combretastatin A-4 disodium phosphate: effects on radiation therapy.

PURPOSE: The aim of this study was to evaluate the antitumor efficacy of combretastatin A-4 disodium phosphate (combretastatin prodrug) in the rodent KHT sarcoma model either alone or in combination with radiation therapy. METHODS: KHT tumors were grown in C3H/HeJ mice. Combretastatin A-4 prodrug was injected intraperitoneally at doses ranging from 10 to 100 mg/kg. Tumors were irradiated in unanesthetized mice using a 137Cs source. Tumor response to combretastatin A-4 prodrug was assessed by histological evaluations as well as an in vivo to in vitro cell survival assay. RESULTS: Histological evaluation showed morphological damage of tumor cells within a few hours after drug treatment, followed by extensive central necrosis. Administering increasing doses of combretastatin A-4 prodrug to tumor-bearing mice resulted in a dose-dependent increase in cell killing irrespective of whether the tumors were irradiated or not. When combined with radiation, a 100 mg/kg dose of combretastatin A-4 prodrug reduced tumor cell survival 10-500-fold lower than that seen with radiation alone. Further, the shape of the cell survival curve observed following the combination therapy suggested that including combretastatin in the treatment had a major effect on the radiation-resistant hypoxic cell subpopulation associated with this tumor. CONCLUSION: The present results demonstrated that in the KHT sarcoma, combretastatin A-4 prodrug caused rapid vascular shutdown, a concentration-dependent direct cell killing, and effective enhancement of the antitumor effects of radiation therapy.

Developing VDEPT for DT-diaphorase (NQO1) using an AAV vector plasmid.

PURPOSE: One enzyme/prodrug combination that has the potential to be used in virally directed enzyme/prodrug therapy (VDEPT) is the obligate 2-electron reducing enzyme, DT-diaphorase (NQO1), with bioreductive agents such as EO9. The present studies were undertaken to determine if this enzyme, as well as the reporter molecule, green fluorescent protein (GFP), could be expressed from a single dicistronic unit under control of the CMV promoter in an adeno-associated virus (AAV) background. METHODS: The human ovarian tumor cell line, SAU, and the mouse sarcoma cell line, KHT/iv, were studied due to their low level of NQO1 expression. These cells were transfected with pTRUF3-NQO1 using a liposome-mediated protocol. RESULTS: The results indicate that this construct has the ability to increase the total protein level of NQO1 by 66-fold in SAU and 102-fold in KHT/iv after 24 h. Furthermore, the level of NQO1 activity in SAU increased from undetectable levels to approximately 200 nmol/min/mg, and the NQO1 activity in KHT/iv increased approximately 10-fold following transfection. Expression of the GFP reporter was readily detectable in both cell types using FACS analysis. CONCLUSIONS: Taken together, these results indicate that this proviral AAV vector plasmid will allow for the production of a recombinant AAV, which can coordinately express the enzyme NQO1 and the GFP reporter for use in vivo in VDEPT studies with various bioreductive agents which are substrates for NQO1.

Potentiation of cisplatin activity by the bioreductive agent tirapazamine.

PURPOSE: The chemosensitizing potential of the benzotriazine-N-oxide tirapazamine was determined in rodent mammary tumor cells grown as solid tumors. MATERIALS AND METHODS: C3H/HeJ mice bearing i.m. transplanted 16C mammary carcinomas were treated with varying doses of either cisplatin alone or cisplatin in combination with a 0.27 mmol/kg dose of tirapazamine. Tumor response to single agent or combination therapy was assessed using an in situ tumor growth delay assay. Normal tissue toxicity resulting from the tirapazamine, cisplatin, or tirapazamine plus cisplatin was determined by measuring bone marrow stem cell (CFU-GM) toxicities and blood urea nitrogen (BUN) levels. RESULTS: Tirapazamine itself had no measurable effect on the growth of this tumor. However, when administered from 3 h before to simultaneously with a single dose of cisplatin, the resultant tumor growth delay was significantly increased as compared to that seen with cisplatin alone. The administration of tirapazamine 3 h prior to a range of doses of cisplatin was found to result in a dose modifying factor (DMF) of approximately 1.7 in tumor response compared to cisplatin alone. Tirapazamine did demonstrate some hematologic toxicity on its own but it did not potentiate the toxicity of cisplatin when the two agents were administered in combination. BUN analysis showed that tirapazamine had little effect on BUN levels but did suppress the BUN values of mice treated with the combination of tirapazamine and 15 mg/kg cisplatin as compared to cisplatin alone. CONCLUSIONS: The present findings suggest that the addition of tirapazamine to cisplatin therapy may lead to a therapeutic benefit.

Measurement of proliferation activities in human tumor models: a comparison of flow cytometric methods.

Proliferating cells in tumors may be of considerable relevance in cancer therapy. Not only do such cells dictate the rate of tumor progression, but evidence exists that they may also play an important role in the diagnosis and prognosis of tumor regrowth. Consequently, the identification of this subset of cells in the overall neoplastic cell population is of considerable importance. The aim of the present investigations was to compare four flow cytometric methodologies commonly used to study cell proliferation. These included nuclear antigen Ki67 detection, acridine orange (AO) and bromodeoxyuridine (BrdUrd) staining, and percent S-phase determinations. Three human tumor cell lines (HEp3, A549, H226) were examined in various stages of growth. Further, a direct comparison was made of the proliferation activities of HEp3 cells grown in culture or as xenografts in nude mice. The results showed that of the techniques investigated, detection of the nuclear antigen Ki67 may be most useful for marking proliferating tumor cells and determining tumor growth fractions.

Radiobiological hypoxia in the KHT sarcoma: predictions using the Eppendorf histograph.

PURPOSE: To investigate whether electrode measurements of tumor oxygenation obtained under a range of different treatment conditions designed to alter the degree of tumor hypoxia could be correlated with estimates of radiobiological hypoxia measured under the same conditions. METHODS AND MATERIALS: Experiments were performed in restrained, nonanesthetized, female C3H/He mice, which had approximately 0.5 g KHT sarcomas growing intramuscularly in the hind limbs. The treatments used to modify tumor oxygenation status included breathing gas mixtures of varying oxygen content, altering tumor blood flow, and shifting the hemoglobin oxygen dissociation curve. Radiobiological hypoxic fraction was estimated using the paired survival curve assay, while electrode measurements of tumor oxygenation were obtained with an Eppendorf histograph. RESULTS: With the selected manipulations it was possible to vary the radiobiological hypoxic fraction in the tumors from approximately 1 to approximately 100% of the total viable cell population. Furthermore, these changes in radiation response were directly reflected in the changes in tumor oxygenation measurements made with the Eppendorf histograph. CONCLUSION: These findings suggest that in the KHT tumor model the Eppendorf electrode measurements could predict the response of the tumors to radiation as determined by the proportion of hypoxic cells.

Tumor cell heterogeneity: impact on mechanisms of therapeutic drug resistance.

PURPOSE: The aim of these studies was to determine whether chemotherapy-resistant tumor cell sublines derived from a single starting cell population with identical treatment protocols, have the same mechanism of resistance. METHODS AND MATERIALS: Twelve cyclophosphamide-resistant sublines were derived from KHT-iv murine sarcoma cells by repeated exposures to 2, 4, or 8 microg/ml doses of 4-hydroperoxycyclophosphamide (4-OOHCP). To investigate possible mechanisms of resistance, glutathione (GSH) levels, glutathione S-transferase (GST) activity, and aldehyde dehydrogenase (ALDH) activity were determined. In addition, studies with the GSH depletor buthionine sulfoximine (BSO) and the ALDH inhibitor diethylamino-benzaldehyde (DEAB) were undertaken. RESULTS: Resistant factors to 4-OOHCP, assessed at 10% clonogenic cell survival, ranged from 1.5-7.0 for the various cell lines. Crossresistance to melphalan and adriamycin also were commonly observed. Increased GSH levels, GST activity and ALDH activity were detected in the sublines but not all exhibited the same pattern of biochemical alterations. The response to GSH and ALDH inhibitors also varied among the sublines; the resistance being reversible in some cell lines but not others. CONCLUSION: The present results indicate that when resistant sublines are derived simultaneously from the same starting cell population, the observed mechanisms of resistance may not be the same in each of the variants. These findings support the hypothesis that preexisting cellular heterogeneity may affect mechanisms of acquired resistance.

Effects of ionizing radiation on the adhesive interaction of human tumor and endothelial cells in vitro.

A centrifugation assay was used to determine the effects of ionizing radiation on the adhesive interaction of A549 human lung adenocarcinoma tumor cells and human umbilical vein endothelial cells (HUVEC). The tumor cells were fluorescently labeled and divided into control (sham-irradiated) and irradiated groups. The irradiated groups were exposed to irradiation levels ranging from 5 to 20 Gy using a 137Cs source. A specified number of these A549 tumor cells were then delivered into each well of 96-well cell culture plates containing confluent monolayers of human umbilical cord vein endothelial cells (HUVEC), and were given time to adhere to the endothelial cells. The wells were then sealed and were exposed to an acceleration field varying from 1 to 42 g (0-500 rpm) for 10 min. Finally, the wells were drained, and the number of tumor cells adhering to the endothelial monolayer were counted using a fluorescent microscope system. Our results indicate that the irradiation of A549 tumor cells significantly increased their adhesive interaction with endothelial cells (number of adhering irradiated cells/number of adhering control cells = 1.0, 1.3, 1.9, 2.2 for 0, 5, 10, 20 Gy respectively). In contrast, when endothelial cells were irradiated, rather than tumor cells, adhesive interaction decreased with an increase in the radiation dose (irradiated/control = 1.0, 0.9, 0. 8, 0.5 for 0, 5, 10, 20 Gy respectively). Simultaneous irradiation of both the tumor cells and the endothelial cells did not alter their adhesive interaction significantly. These findings may have important implications for the metastatic ability of irradiated tumor cells.

Nicotinamide as a radiosensitizer in tumours and normal tissues: the importance of drug dose and timing.

BACKGROUND AND PURPOSE: Nicotinamide is a radiation sensitizer currently undergoing clinical testing. This was an experimental study to determine the importance of drug dose and time interval between drug administration and irradiation for radiosensitization. MATERIALS AND METHODS: Nicotinamide (50-500 mg/kg) was injected intraperitoneally into CDFI or C3H mice and drug plasma pharmacokinetics were determined by HPLC. Radiosensitization was measured in tumours and normal tissues after local irradiation. The tumours were a C3H mammary carcinoma, the KHT sarcoma and the SCCVII carcinoma. Tumour response was assessed using either growth delay (C3H) or clonogenic survival (KHT/SCCVII). Normal tissue toxicities evaluated included early responding skin (development of moist desquamation of the foot) and late responding bladder (reservoir function estimated by cystometry) and lung (ventilation rate measured by plethysmography). RESULTS: All nicotinamide peak plasma concentrations were seen within 30 min after injection. Irradiating tumours at peak times resulted in enhancement ratios (ERs) of 1.27 (C3H), 1.75 (KHT) and 1.45 (SCCVII) with high nicotinamide doses and 1.27 (C3H), 1.28 (KHT) and 1.36 (SCCVII) after giving clinically relevant doses (100-200 mg/kg). Lower ERs were observed when the time interval between drug injection and irradiation was increased beyond the peak time. Irradiating normal tissues at peak times after injecting 100-200 mg/kg nicotinamide gave ERs of 1.20 (skin), 0.90 (bladder) and 1.02 (lung). CONCLUSIONS: Clinically achievable doses of nicotinamide will enhance tumour radiation damage while having minimal effects in normal tissues, but for the best tumour effect radiation should be given at the time of peak plasma drug concentrations.

The in situ tumour response to combinations of cyclophosphamide and tirapazamine.

The potential of tirapazamine to enhance the in situ efficacy of the anti-cancer drug cyclophosphamide (CP) was evaluated in two rodent tumours (KHT sarcoma, 16C mammary carcinoma) and one human ovarian tumour xenograft (MLS) using end points of in vivo to in vitro cell survival or in situ tumour growth delay. For comparison, bone marrow toxicity under similar treatment conditions was determined using a CFU-GM stem cell survival assay. The results showed that a 0.27 mmol kg-1 dose of tirapazamine alone had little anti-tumour effect. However, when given prior to a range of CP doses, tirapazamine increased the efficacy of this chemotherapeutic agent in all three tumour models investigated. CFU-GM stem cell toxicity, assessed under similar treatment conditions, demonstrated that this dose of tirapazamine (1) led to some bone marrow toxicity on its own; and (2) increased the toxicity of CP beyond that seen with CP alone. The present findings demonstrate that the bioreductive agent tirapazamine can potentiate the in situ anti-tumour efficacy of the alkylating agent CP. However, in the preclinical models investigated, the enhanced anti-tumour effect did not translate into a therapeutic benefit because a similar increase in bone marrow toxicity also resulted from this treatment combination.

Direct relationship between radiobiological hypoxia in tumors and monoclonal antibody detection of EF5 cellular adducts.

While the potential importance of hypoxia in limiting the sensitivity of tumor cells to ionizing radiation has long been appreciated, methods for accurately quantifying the number of radiation-resistant hypoxic cells within tumors have been lacking. We have used the pentafluorinated derivative [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acet amide] of etanidazole (EF5), which binds selectively to hypoxic cells. The adducts formed between EF5 and cellular proteins in the hypoxic cells were detected using the specific monoclonal antibody (MAb), ELK3-51 conjugated to the flurochrome Cy3, and the number of hypoxic cells was quantified by flow cytometry. To verify the validity of this technique for the detection of hypoxic cells, mice bearing KHT sarcomas were treated with various agents to alter tumor oxygenation and hence vary the fraction of radiobiologically hypoxic tumor cells. The percentage of EF5 binding cells was then compared directly with the clonogenic survival of the tumor cells following radiation treatment under the various pretreatment conditions. The results showed that allowing the mice to breathe carbogen (5% CO2/95% O2) prior to irradiation reduced clonogenic cell survival approx. 6-fold and led to an absence of cells binding high levels of EF5. In contrast, pretreating the tumor-bearing animals with either hydralazine, which decreased tumor blood flow, or phenylhydrazine hydrochloride, which made the mice anemic, increased tumor cell survival following irradiation 2- to 4-fold, indicative of an increase in the fraction of hypoxic tumor cells. EF5 measurements made under identical conditions illustrated a shift in the cells in the tumor to high EF5 binding. Our results demonstrate that flow cytometric measurement by fluorescent MAb binding to EF5 adducts may relate directly to radiobiological hypoxia in KHT tumors measured by conventional methods.

Does neck stage predict local control after irradiation for head and neck cancer?

The impact of neck stage (N stage) on local control after treatment for head and neck cancer is controversial. This article reviews the pertinent literature. Based on this review, the authors conclude that although N stage may be inversely related to local control, the relationship is relatively weak. Hence, current data are insufficient to justify altering treatment of the primary lesion based solely on N stage.