Fast and sensitive dynamic oxygen-enhanced MRI with a cycling gas challenge and independent component analysis.

PURPOSE: There is a critical need for non-invasive imaging biomarkers of tumor oxygenation to assist in patient stratification and development of hypoxia targeting therapies. Using a cycling gas challenge and independent component analysis (ICA), we sought to improve the sensitivity and speed of existing oxygen enhanced MRI (OE-MRI) techniques to detect changes in oxygenation with dynamically acquired T1 W signal intensity images (dOE-MRI). METHODS: Mice were implanted with SCCVII, HCT-116, BT-474, or SKOV3 tumors in the dorsal subcutaneous region and imaged at 7T. T1 W images were acquired during a respiratory challenge with alternating 2-minute periods of air and 100% oxygen for three cycles. Data were analyzed with ICA and oxygenation maps were generated and compared to corresponding histology sections stained for hypoxia (pimonidazole) and blood vessels (CD31). RESULTS: Cycling air-oxygen-air gas challenges were well tolerated and ICA permitted extraction of the oxygen-enhancing component in all imaged tumors from four different models. Comparison with synthetic response functions showed that dOE-MRI does not require any a-priori knowledge of the physiological response. The fraction of O2 -negative dOE-MRI voxels that correlate inversely with the ICA gas-cycling component correspond well with the histological hypoxic fraction in SCCVII tumors (r = 0.91, p = 0.0016) but did not correlate in HCT-116 tumors (r = 0.13, p = 0.81). CONCLUSIONS: Using ICA and adding a cycling gas challenge extends the sensitivity of OE-MRI and allows the oxygenation status of tumors to be assessed in as little as six minutes. These findings support further development of OE-MRI as a biomarker of tumor oxygenation.

The differential effects of metronomic gemcitabine and antiangiogenic treatment in patient-derived xenografts of pancreatic cancer: treatment effects on metabolism, vascular function, cell proliferation, and tumor growth.

BACKGROUND: Metronomic chemotherapy has shown promising activity against solid tumors and is believed to act in an antiangiogenic manner. The current study describes and quantifies the therapeutic efficacy, and mode of activity, of metronomic gemcitabine and a dedicated antiangiogenic agent (DC101) in patient-derived xenografts of pancreatic cancer. METHODS: Two primary human pancreatic cancer xenograft lines were dosed metronomically with gemcitabine or DC101 weekly. Changes in tumor growth, vascular function, and metabolism over time were measured with magnetic resonance imaging, positron emission tomography, and immunofluorescence microscopy to determine the anti-tumor effects of the respective treatments. RESULTS: Tumors treated with metronomic gemcitabine were 10-fold smaller than those in the control and DC101 groups. Metronomic gemcitabine, but not DC101, reduced the tumors' avidity for glucose, proliferation, and apoptosis. Metronomic gemcitabine-treated tumors had higher perfusion rates and uniformly distributed blood flow within the tumor, whereas perfusion rates in DC101-treated tumors were lower and confined to the periphery. DC101 treatment reduced the tumor's vascular density, but did not change their function. In contrast, metronomic gemcitabine increased vessel density, improved tumor perfusion transiently, and decreased hypoxia. CONCLUSION: The aggregate data suggest that metronomic gemcitabine treatment affects both tumor vasculature and tumor cells continuously, and the overall effect is to significantly slow tumor growth. The observed increase in tumor perfusion induced by metronomic gemcitabine may be used as a therapeutic window for the administration of a second drug or radiation therapy. Non-invasive imaging could be used to detect early changes in tumor physiology before reductions in tumor volume were evident.

Drug penetration in solid tumours.

To be most effective anticancer drugs must penetrate tissue efficiently, reaching all the cancer cells that comprise the target population in a concentration sufficient to exert a therapeutic effect. Most research into the resistance of cancers to chemotherapy has concentrated on molecular mechanisms of resistance, whereas the role of limited drug distribution within tumours has been neglected. We summarize the evidence that indicates that the distribution of many anticancer drugs in tumour tissue is incomplete, and we suggest strategies that might be used either to improve drug penetration through tumour tissue or to select compounds based on their abilities to penetrate tissue, thereby increasing the therapeutic index.

Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling.

Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1(+)CD31(dim)vascular endothelial (VE)-cadherin(-)CD45(-) precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1(+) precursor cell, but not the VE-cadherin(+) endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1(+) precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1(+) local precursor to VSMC in a spatiotemporal fashion across all vascular beds.

DNA-PK inhibitor AZD7648 is a more portent radiosensitizer than PARP inhibitor Olaparib in BRCA1/2 deficient tumors.

The effectiveness of radiotherapy depends on the sensitivities of 'normal' and cancer cells to the administered radiation dose. Increasing the radiosensitivity of cancers by inhibiting DNA damage repair is a goal of much current research, however success depends on avoiding concomitant sensitization of normal tissues inevitably irradiated during therapy. In this study we investigated the mechanisms of radiosensitization for DNA-PK and PARP inhibitors by examining the impacts on proliferating vs quiescent cell populations. Experiments were performed in BRCA1/2null and wild-type parental cancer models in vitro and in vivo. Overall AZD7648 has greater radiosensitizing activity relative to Olaparib, with BRCA2-deficient models showing the greatest sensitivity. However, DNA-PK inhibitor AZD7648 also produced greater toxicity in all irradiated mice. While both DNA-PK and PARP inhibition sensitizes wild type tumor cells to radiation, in BRCA1/2 deficient cells PARP inhibition by Olaparib had limited radiosensitization capacity. Quiescent cells are more radioresistant than proliferating cells, and these were also effectively sensitized by AZD7648 while Olaparib was unable to increase radiation-induced cell kill, even in BRCA1/2null cells. These findings underscore the distinct mechanisms of radiosensitization for DNA-PK and PARP inhibitors. While DNA-PK inhibitors are able to target both proliferating and non-proliferating tumor cells for greater overall anti-cancer benefit, their application is limited by exacerbation of normal tissue toxicities. Conversely, PARP inhibitors exhibit selective activity for proliferating cells, providing a mechanism for targeting activity to cancers, but due to poor activity in non-proliferating cells they have an overall reduced impact on tumor growth control. This study highlights the importance of creating a therapeutic ratio with DNA damage repair inhibition radiation sensitizing strategies.

Radiation and Chemo-Sensitizing Effects of DNA-PK Inhibitors Are Proportional in Tumors and Normal Tissues.

Inhibitors of DNA-dependent protein kinase (PRKDC; DNA-PK) sensitize cancers to radiotherapy and DNA-damaging chemotherapies, with candidates in clinical trials. However, the degree to which DNA-PK inhibitors also sensitize normal tissues remains poorly characterized. In this study, we compare tumor growth control and normal tissue sensitization following DNA-PK inhibitors in combination with radiation and etoposide. FaDu tumor xenografts implanted in mice were treated with 10 to 15 Gy irradiation ± 3 to 100 mg/kg AZD7648. A dose-dependent increase in time to tumor volume doubling following AZD7648 was proportional to an increase in toxicity scores of the overlying skin. Similar effects were seen in the intestinal jejunum, tongue, and FaDu tumor xenografts of mice assessed for proliferation rates at 3.5 days after treatment with etoposide or 5 Gy whole body irradiation ± DNA-PK inhibitors AZD7648 or peposertib (M3814). Additional organs were examined for sensitivity to DNA-PK inhibitor activity in ATM-deficient mice, where DNA-PK activity is indicated by surrogate marker γH2AX. Inhibition was observed in the heart, brain, pancreas, thymus, tongue, and salivary glands of ATM-deficient mice treated with the DNA-PK inhibitors relative to radiation alone. Similar reductions are also seen in ATM-deficient FaDu tumor xenografts where both pDNA-PK and γH2AX staining could be performed. DNA-PK inhibitor-mediated sensitization to radiation and DNA-damaging chemotherapy are not only limited to tumor tissues, but also extends to normal tissues sustaining DNA damage. These data are useful for interpretation of the sensitizing effects of DNA damage repair inhibitors, where a therapeutic index showing greater cell-killing effects on cancer cells is crucial for optimal clinical translation.

Detection of FLASH-radiotherapy tissue sparing in a 3D-spheroid model using DNA damage response markers.

PURPOSE: The oxygen depletion hypothesis has been proposed as a rationale to explain the observed phenomenon of FLASH-radiotherapy (FLASH-RT) sparing normal tissues while simultaneously maintaining tumor control. In this study we examined the distribution of DNA Damage Response (DDR) markers in irradiated 3D multicellular spheroids to explore the relationship between FLASH-RT protection and radiolytic-oxygen-consumption (ROC) in tissues. METHODS: Studies were performed using a Varian Truebeam linear accelerator delivering 10 MeV electrons with an average dose rate above 50 Gy/s. Irradiations were carried out on 3D spheroids maintained under a range of O2 and temperature conditions to control O2 consumption and create gradients representative of in vivo tissues. RESULTS: Staining for pDNA-PK (Ser2056) produced a linear radiation dose response whereas γH2AX (Ser139) showed saturation with increasing dose. Using the pDNA-PK staining, radiation response was then characterised for FLASH compared to standard-dose-rates as a function of depth into the spheroids. At 4 °C, chosen to minimize the development of metabolic oxygen gradients within the tissues, FLASH protection could be observed at all distances under oxygen conditions of 0.3-1 % O2. Whereas at 37 °C a FLASH-protective effect was limited to the outer cell layers of tissues, an effect only observed at 3 % O2. Modelling of changes in the pDNA-PK-based oxygen enhancement ratio (OER) yielded a tissue ROC g0-value estimate of 0.73 ± 0.25 µM/Gy with a km of 5.4 µM at FLASH dose rates. CONCLUSIONS: DNA damage response markers are sensitive to the effects of transient oxygen depletion during FLASH radiotherapy. Findings support the rationale that well-oxygenated tissues would benefit more from FLASH-dose-rate protection relative to poorly-oxygenated tissues.

DNA-PK inhibition extends the therapeutic effects of Top2 poisoning to non-proliferating cells, increasing activity at a cost.

Type II topoisomerase (Top2) poisoning therapy is used to treat a broad range of cancers via induction of double strand breaks (DSBs) in cells undergoing replication and transcription. Preventing the repair of DSBs via inhibition of DNA-PK, an inhibitor of non-homologous end-joining (NHEJ), increases cell kill with Top2 poisons and has led to the initiation of several clinical trials. To elucidate the cellular mechanisms leading to synergistic activity of dual DNA-PK/Top2 inhibition we looked at their effects in cycling versus non-cycling cells, in 3D spheroids and in xenograft models. Combined DNA-PK/Top2 inhibition was found to not only increase the cell kill in proliferating cells, the cell population that is typically most vulnerable to Top2 poisoning, but also in non-proliferative but transcriptionally active cells. This effect was observed in both cancer and normal tissue models, killing more cells than high concentrations of etoposide alone. The combination treatment delayed tumor growth in mice compared to Top2 poisoning alone, but also led to increased toxicity. These findings demonstrate sensitization of Top2ß-expressing, non-cycling cells to Top2 poisoning by DNA-PK inhibition. Expansion of the target cell population of Top2 poison treatment to include non-proliferating cells via combination with DNA damage repair inhibitors has implications for efficacy and toxicity of these combinations, including for inhibitors of DNA-PK currently in clinical trial.

The combination of gefitinib and RAD001 inhibits growth of HER2 overexpressing breast cancer cells and tumors irrespective of trastuzumab sensitivity.

BACKGROUND: HER2-positive breast cancers exhibit high rates of innate and acquired resistance to trastuzumab (TZ), a HER2-directed antibody used as a first line treatment for this disease. TZ resistance may in part be mediated by frequent co-expression of EGFR and by sustained activation of the mammalian target of rapamycin (mTOR) pathway. Here, we assessed feasibility of combining the EGFR inhibitor gefitinib and the mTOR inhibitor everolimus (RAD001) for treating HER2 overexpressing breast cancers with different sensitivity to TZ. METHODS: The gefitinib and RAD001 combination was broadly evaluated in TZ sensitive (SKBR3 and MCF7-HER2) and TZ resistant (JIMT-1) breast cancer models. The effects on cell growth were measured in cell based assays using the fixed molar ratio design and the median effect principle. In vivo studies were performed in Rag2M mice bearing established tumors. Analysis of cell cycle, changes in targeted signaling pathways and tumor characteristics were conducted to assess gefitinib and RAD001 interactions. RESULTS: The gefitinib and RAD001 combination inhibited cell growth in vitro in a synergistic fashion as defined by the Chou and Talalay median effect principle and increased tumor xenograft growth delay. The improvement in therapeutic efficacy by the combination was associated in vitro with cell line dependent increases in cytotoxicity and cytostasis while treatment in vivo promoted cytostasis. The most striking and consistent therapeutic effect of the combination was increased inhibition of the mTOR pathway (in vitro and in vivo) and EGFR signaling in vivo relative to the single drugs. CONCLUSIONS: The gefitinib and RAD001 combination provides effective control over growth of HER2 overexpressing cells and tumors irrespective of the TZ sensitivity status.

Vascularization predicts overall survival and risk of transformation in follicular lymphoma.

Follicular lymphoma patients display heterogeneous overall survival and variable risk of transformation. Recent studies have highlighted the role of the microenvironment. The contribution of microvessel density to follicular lymphoma survival remains controversial. We used a quantitative tumor mapping approach to determine whether the degree of vascularization correlated with outcome in a uniformly treated cohort. Whole-tissue sections of diagnostic biopsies from 84 cases were stained for CD34 and tumor-to-vessel-distance that encompassed 90% of the tumor (TVD(90)) was determined using image analysis. Twenty-one cases with lower TVD(90) showed inferior overall survival (P=0.0001) and high risk of transformation (P=0.01). These cases significantly correlated with increased Lymphoma-Associated Macrophages (χ(2)=0.025). In multivariate analysis macrophages content, IPI and TVD(90) were independent predictors of overall survival (P=0.05, P=0.001 and P=0.01, respectively) and IPI and TVD(90) predicted risk of transformation (P=0.008 and P=0.08, respectively). Increased angiogenesis is an independent marker of inferior survival and may promote transformation.

Direct visualization of heterogeneous extravascular distribution of trastuzumab in human epidermal growth factor receptor type 2 overexpressing xenografts.

PURPOSE: The high molecular weight and binding affinity of trastuzumab, a monoclonal antibody in use for treatment of breast cancers overexpressing human epidermal growth factor receptor type 2 (HER2), in combination with microenvironmental factors, may limit its distribution and efficacy. We assessed and mapped the distribution of systemically given, unlabeled trastuzumab at micrometer resolution in tumor xenografts using immunohistochemistry. EXPERIMENTAL DESIGN: Mice bearing MDA-435/LCC6(HER2) xenografts were given single doses of 4 or 20 mg/kg unlabeled trastuzumab with tumor harvest at various time points thereafter; bound trastuzumab was imaged directly in tumor cryosections using fluorescently tagged antihuman secondary antibodies. Combinations of additional markers, including HER2, 5-bromo-2-deoxyuridine, CD31, DioC(7)(3), desmin, and collagen IV were also mapped on the same tumor sections. RESULTS: Distribution of trastuzumab in MDA-435/LCC6(HER2) tumors is found to be heterogeneous, with tumor margins saturating more thoroughly in doses and times analyzed. Considerable intervessel heterogeneity is also seen. For example, in unsaturated tissues, there remain perfused vessels without any trastuzumab in addition to vessels with a few layers of positively stained perivascular cells, in addition to vessels with bound drug up to 150 microm away. This heterogeneity is independent of HER2 expression, microvessel density, and perfusion. A slightly greater proportion of vessels were associated with pericytes in sections with greater trastuzumab saturation, but this would not adequately account for observed heterogeneous trastuzumab distribution. CONCLUSIONS: Complete penetration of trastuzumab in tumor tissue was not seen in our study, leaving the possibility that inadequate distribution may represent a mechanism for resistance to trastuzumab.

Irinophore C, a novel nanoformulation of irinotecan, alters tumor vascular function and enhances the distribution of 5-fluorouracil and doxorubicin.

PURPOSE: To examine the antitumor effects of Irinophore C, a nanopharmaceutical formulation of irinotecan, on the tissue morphology and function of tumor vasculature in HT-29 human colorectal tumors. EXPERIMENTAL DESIGN: Fluorescence microscopy was used to map and quantify changes in tissue density, tumor vasculature, hypoxia, and the distribution of Hoechst 33342, a perfusion marker, and the anticancer drug, doxorubicin. Noninvasive magnetic resonance imaging was used to quantify Ktrans, the volume transfer constant of a solute between the blood vessels and extracellular tissue compartment of the tumor, as a measure of vascular function. Following treatment with Irinophore C, 19F magnetic resonance spectroscopy was used to monitor the delivery of 5-fluorouracil (5-FU) to the tumor tissue, whereas scintigraphy was used to quantify the presence of bound [14C]5-FU. RESULTS: Irinophore C decreased cell density (P = 8.42 x 10(-5)), the overall number of endothelial cells in the entire section (P = 0.014), tumor hypoxia (P = 5.32 x 10(-9)), and K(trans) (P = 0.050). However, treatment increased the ratio of endothelial cells to cell density (P = 0.00024) and the accumulation of Hoechst 33342 (P = 0.022), doxorubicin (P = 0.243 x 10(-5)), and 5-FU (P = 0.0002) in the tumor. Vascular endothelial growth factor and interleukin-8, two proangiogenic factors, were down-regulated, whereas the antiangiogenic factor TIMP-1 was up-regulated in Irinophore C-treated tumors. CONCLUSIONS: Irinophore C treatment improves the vascular function of the tumor, thereby reducing tumor hypoxia and increasing the delivery and accumulation of a second drug. Reducing hypoxia would enhance radiotherapy, whereas improving delivery of a second drug to the tumor should result in higher cell kill.

Suppression of VEGF secretion and changes in glioblastoma multiforme microenvironment by inhibition of integrin-linked kinase (ILK).

Integrin-linked kinase (ILK) was assesed as a therapeutic target in glioblastoma xenograft models through multiple endpoints including treatment related changes in the tumor microenvironment. Glioblastoma cell lines were tested in vitro for sensitivity toward the small-molecule inhibitors QLT0254 and QLT0267. Cell viability, cell cycle, and apoptosis were evaluated using MTT assay, flow cytometry, caspase activation, and DAPI staining. Western blotting and ELISA were used for protein analysis (ILK, PKB/Akt, VEGF, and HIF-1alpha). In vivo assessment of growth rate, cell proliferation, BrdUrd, blood vessel mass (CD31 labeling), vessel perfusion (Hoechst 33342), and hypoxia (EF-5) was done using U87MG glioblastoma xenografts in RAG2-M mice treated orally with QLT0267 (200 mg/kg q.d.). ILK inhibition in vitro with QLT0254 and QLT0267 resulted in decreased levels of phospho-PKB/Akt (Ser473), secreted VEGF, G2-M block, and apoptosis induction. Mice treated with QLT0267 exhibited significant delays in tumor growth (treated 213 mm3 versus control 549 mm3). In situ analysis of U87MG tumor cell proliferation from QLT0267-treated mice was significantly lower relative to untreated mice. Importantly, VEGF and HIF-1alpha expression decreased in QLT0267-treated tumors as did the percentage of blood vessel mass and numbers of Hoechst 33342 perfused tumor vessels compared with control tumors (35% versus 83%). ILK inhibition with novel small-molecule inhibitors leads to treatment-associated delays in tumor growth, decreased tumor angiogenesis, and functionality of tumor vasculature. The therapeutic effects of a selected ILK inhibitor (QLT0267) should be determined in the clinic in cancers that exhibit dysregulated ILK, such as PTEN-null glioblastomas.

Targeting Hypoxia-Induced Carbonic Anhydrase IX Enhances Immune-Checkpoint Blockade Locally and Systemically.

Treatment strategies involving immune-checkpoint blockade (ICB) have significantly improved survival for a subset of patients across a broad spectrum of advanced solid cancers. Despite this, considerable room for improving response rates remains. The tumor microenvironment (TME) is a hurdle to immune function, as the altered metabolism-related acidic microenvironment of solid tumors decreases immune activity. Here, we determined that expression of the hypoxia-induced, cell-surface pH regulatory enzyme carbonic anhydrase IX (CAIX) is associated with worse overall survival in a cohort of 449 patients with melanoma. We found that targeting CAIX with the small-molecule SLC-0111 reduced glycolytic metabolism of tumor cells and extracellular acidification, resulting in increased immune cell killing. SLC-0111 treatment in combination with immune-checkpoint inhibitors led to the sensitization of tumors to ICB, which led to an enhanced Th1 response, decreased tumor growth, and reduced metastasis. We identified that increased expression of CA9 is associated with a reduced Th1 response in metastatic melanoma and basal-like breast cancer TCGA cohorts. These data suggest that targeting CAIX in the TME in combination with ICB is a potential therapeutic strategy for enhancing response and survival in patients with hypoxic solid malignancies.

Endothelial Sash1 Is Required for Lung Maturation through Nitric Oxide Signaling.

The sterile alpha motif (SAM) and SRC homology 3 (SH3) domain containing protein 1 (Sash1) acts as a scaffold in TLR4 signaling. We generated Sash1-/- mice, which die in the perinatal period due to respiratory distress. Constitutive or endothelial-restricted Sash1 loss leads to a delay in maturation of alveolar epithelial cells causing reduced surfactant-associated protein synthesis. We show that Sash1 interacts with ß-arrestin 1 downstream of the TLR4 pathway to activate Akt and endothelial nitric oxide synthase (eNOS) in microvascular endothelial cells. Generation of nitric oxide downstream of Sash1 in endothelial cells affects alveolar epithelial cells in a cGMP-dependent manner, inducing maturation of alveolar type 1 and 2 cells. Thus, we identify a critical cell nonautonomous function for Sash1 in embryonic development in which endothelial Sash1 regulates alveolar epithelial cell maturation and promotes pulmonary surfactant production through nitric oxide signaling. Lung immaturity is a major cause of respiratory distress and mortality in preterm infants, and these findings identify the endothelium as a potential target for therapy.

Limited tissue penetration of taxanes: a mechanism for resistance in solid tumors.

PURPOSE: Limited drug penetration in solid tumors is a potential mechanism of resistance for many anticancer drugs. Taxanes represent a class of drugs that are currently undergoing a new round of development, but with little known of their ability to penetrate and distribute relative to blood vessels within solid tumors. EXPERIMENTAL DESIGN: We assessed the tissue penetration of paclitaxel and docetaxel in HCT-116 tumor xenografts and in multilayered cell culture (MCC), a three-dimensional cell culture model of the tumor extravascular compartment. In xenografts, taxanes were mapped relative to blood vessels to obtain drug profiles as a function of distance from vasculature. For MCC, cultures were exposed to stirred drug reservoirs and taxanes measured as a function of depth into tissue. RESULTS: Both taxanes exhibited limited penetration, with little drug reaching further than 100 microm into the tissue. Of the two, paclitaxel exhibited up to 2-fold greater penetration than docetaxel. Mapping tumor cell proliferation following treatment allowed the consequences of limited drug penetration to be assessed. In tumor xenografts where reduced drug exposure to cells far from vasculature is one of several factors influencing response to treatment, up to a 75% reduction in S-phase cells was achieved in cells nearest the vessels, but only 50% reduction was observed in the tissue 150 microm away. In MCC-based data, where the influence of reduced cell proliferation with depth into tissue was circumvented, a 5-fold (paclitaxel) and 10-fold (docetaxel) increase in reservoir drug concentration was required to produce a response in cells 150 microm into the tissue equivalent to that seen in cells directly exposed to the drug. CONCLUSION: These results indicate that limited distribution is an important mechanism of tumor resistance to taxanes.

The impact of complement activation on tumor oxygenation during photodynamic therapy.

The response to photodynamic therapy (PDT) mediated by photosensitizer Photofrin was examined with Lewis lung carcinomas growing in either complement-proficient C57BL/6 (B6) or complement-deficient complement C3 knockout (C3KO) mice. The results reveal that Photofrin-PDT was more effective in attaining cures of tumors in C3KO than in B6 hosts. Colony-forming ability of cells from tumors excised immediately after Photofrin-PDT confirmed that the direct cell killing effect was more pronounced in C3KO than in B6 hosts. In contrast, PDT mediated by photosensitizer benzoporphyrin derivative (BPD) produced higher cure rates of tumors in B6 hosts than those in C3KO hosts. Determination of tumor C3 levels by ELISA showed that Photofrin-PDT induced markedly more pronounced complement activation than BPD-PDT. Measurements of tumor oxygen tension immediately after PDT by Eppendorf pO2 histograph showed that Photofrin-PDT induced a marked decline in the oxygenation of tumors growing in B6 mice that was much less pronounced in C3KO hosts. With BPD-PDT the oxygen tensions in tumors in B6 and C3KO hosts decreased to a similar extent. This study indicates that complement activation in PDT-treated tumors that varies with different photosensitizers is an important determinant of tumor oxygen limitation effects directly associated with photodynamic action.

Tirapazamine causes vascular dysfunction in HCT-116 tumour xenografts.

BACKGROUND AND PURPOSE: Tirapazamine is a hypoxic cytotoxin currently undergoing Phase II/III clinical evaluation in combination with radiation and chemotherapeutics for the treatment of non-hematological cancers. Tissue penetration studies using multicellular models have suggested that tirapazamine exposure may be limited to cells close to blood vessels. However, animal studies show tirapazamine enhances the anti-tumour activity of radiation and chemotherapy and clinical studies with tirapazamine, so far, are promising. To investigate this apparent paradox we examined the microregional effects of tirapazamine in vivo by mapping drug effects with respect to the position of blood vessels in tumour cryosections. PATIENTS AND METHODS: Tirapazamine was administered i.p. to mice bearing HCT-116 tumours, which were excised at various times after treatment. Images of multiple-stained cryosections were overlaid to provide microregional information on the relative position of proliferating cells, hypoxia, perfusion and vasculature. RESULTS: We observed extensive and permanent vascular dysfunction in a large proportion of tumours from mice treated with tirapazamine. In the affected tumours, blood flow ceased in the centrally located tumour vessels, leaving a rim of functional vessels around the periphery of the tumour. This vascular dysfunction commenced within 24 h after tirapazamine administration and the areas affected appeared to be replaced by necrosis over the following 24-48 h. CONCLUSIONS: Because the majority of hypoxic cells are located in the center of tumours we propose that the activity of tirapazamine in vivo may be related to its effects on tumour vasculature and that its activity against hypoxic cells located distal to functional blood vessels may not be as important as previously believed.

Tumor distribution of bromodeoxyuridine-labeled cells is strongly dose dependent.

Bromodeoxyuridine (BrdUrd) is used extensively to measure the fraction of proliferating cells in tumors. Unlike endogenous markers of proliferation such as proliferating cell nuclear antigen (PCNA) and Ki-67, BrdUrd is exogenously administered and reaches the tumor via vasculature where it must then diffuse throughout the tissue to label S-phase cells. In this study, we examine the dose dependence of BrdUrd on the tumor distribution of labeled cells in histological sections. Analysis of the distribution of labeled cells in SiHa tumor xenografts showed that a dose between 400 and 1000 mg/kg was required to label cells 150 micro m from blood vessels, approaching the border of necrosis. Lower doses resulted in only the cells close to blood vessels being labeled. Interestingly, cells residing furthest from blood vessels still labeled albeit at half the level of cells situated proximal to the tumor vasculature. Results were compared with the penetration of BrdUrd seen in vitro using multilayered cell culture (MCC), a three-dimensional tissue culture model of solid tumors. Using MCC, an exposure of 100 micro M BrdUrd for 1 h was required for labeling of S-phase cells 150 micro m into the tissue, whereas cells adjacent to the edge of the tissue could be adequately labeled with just 5 micro M BrdUrd for 1 h. The area under the curve for a 100 mg/kg BrdUrd dose in mice was found to be approximately 30 micro M x h.

Microregional effects of gemcitabine in HCT-116 xenografts.

To examine the tumor microregional effects after gemcitabine administration to mice, we mapped the location of proliferating and hypoxic cells relative to vasculature in human colon cancer xenografts. The S-phase marker bromodeoxyuridine was used as a surrogate of drug effect and administered 2 hours before tumor excision, whereas vessel position and perfusion were assessed via staining for CD31 and intravenous injection of carbocyanine, respectively. Hypoxia was detected using pimonidazole. Images of the four markers were overlaid to reveal the spatial relationship between proliferation, vasculature, and hypoxia and to examine the microregional effects. Within 1 day after administration of 240 mg/kg of gemcitabine, proliferation throughout the tumor was completely inhibited. Over time, a reemergence of dividing cells occurred in relation to the distance from vasculature. Microregional analysis revealed that cells located distal to vasculature commenced cycling sooner than cells located proximal to vasculature. A similar trend was seen after multiple doses of gemcitabine (40 mg/kg on days 1, 4, 7, and 10). The possibility that the effect of gemcitabine could be attributed to changes in oxygenation was discounted after examining the vessel perfusion and patterns of hypoxia. The effect of gemcitabine was examined in multilayered cell culture, and at doses <30 micromol/L, a gradient in proliferation between the exposed and unexposed sides was observed. We show a differential effect on cell proliferation in relation to vasculature and conclude that cells distal to blood vessels are less affected by gemcitabine probably because of limited penetration.