Transient perfusion in human melanoma xenografts.

Studies of transplantable rodent tumours have suggested that malignant tissue might experience transient perfusion at the microvascular level. The purpose of the work reported here was to investigate whether transient perfusion can be demonstrated in xenografted human tumours. Tumours of four melanoma lines (A-07, D-12, R-18, U-25), grown orthotopically in Balb/c nu/nu mice, were included in the study. Transient perfusion was studied by using the double-fluorescent staining technique. Hoechst 33342 and DiOC7(3) were either administered simultaneously or Hoechst 33342 was administered 20 min before DiOC7(3). Detection of transient perfusion by this method requires that vessels are non-functional for at least 5 min owing to the distribution half-lives of the dyes in the blood. Usable combinations of dye concentrations were found by varying the concentrations of Hoechst 33342 and DiOC7(3) systematically. The level of perfusion mismatch following simultaneous administration of the dyes ranged from approximately 1.5% for U-25 tumours to approximately 3.0% for R-18 tumours at these combinations. Moreover, the fraction of vessels stained only with Hoechst 33342 and the fraction of vessels stained only with DiOC7(3) were not significantly different whether the dyes were administered simultaneously or sequentially. Transient perfusion could not be demonstrated in any of the tumour lines. Thus, the fraction of vessels stained only with Hoechst 33342 and the fraction of vessels stained only with DiOC7(3) were not significantly higher after sequential than after simultaneous administration of the dyes. Moreover, the vessels stained only with Hoechst 33342 and the vessels stained only with DiOC7(3) were randomly distributed within the tumours whether the dyes were administered simultaneously or sequentially. Consequently, acute hypoxia caused by transient perfusion is probably a less pronounced phenomenon in malignant tissue than previous studies of rodent tumours have suggested.     

Quantification of tumour vasculature and hypoxia by immunohistochemical staining and HbO2 saturation measurements

Despite the possibility that tumour hypoxia may limit radiotherapeutic response, the underlying mechanisms remain poorly understood. A new methodology has been developed in which information from several sophisticated techniques is combined and analysed at a microregional level. First, tumour oxygen availability is spatially defined by measuring intravascular blood oxygen saturations (HbO2) cryospectrophotometrically in frozen tumour blocks. Second, hypoxic development is quantified in adjacent sections using immunohistochemical detection of a fluorescently conjugated monoclonal antibody (ELK3-51) to a nitroheterocyclic hypoxia marker (EF5), thereby providing information relating to both the oxygen consumption rates and the effective oxygen diffusion distances. Third, a combination of fluorescent (Hoechst 33342 or DiOC7(3)) and immunohistological (PECAM-1/CD31) stains is used to define the anatomical vascular densities and the fraction of blood vessels containing flow. Using a computer-interfaced microscope stage, image analysis software and a 3-CCD colour video camera, multiple images are digitized, combined to form a photo-montage and revisited after each of the three staining protocols. By applying image registration techniques, the spatial distribution of HbO2 saturations is matched to corresponding hypoxic marker intensities in adjacent sections. This permits vascular configuration to be related to oxygen availability and allows the hypoxic marker intensities to be quantitated in situ.     

Effect of vascular marker Hoechst 33342 on tumour perfusion and cardiovascular function in the mouse

The fluorescent stain Hoechst 33342 (H33342) has been employed extensively as an in vivo marker of functional tumour vasculature. We have found that H33342 causes a transient, dose-dependent decrease in tumour red blood cell (RBC) flow in SCCVII tumours as measured using laser Doppler flowmetry. After intravenous bolus injection of 15 mg kg-1 to anaesthetised mice, blood flow in subcutaneous back tumours declined to 19 +/- 11% of pretreatment values, returning to normal in less than 7 min. The effect was less pronounced in mice bearing foot tumours in which flow decreased to 52 +/- 14% of pretreatment values in unanaesthetised mice and to 50 +/- 15% in anaesthetised animals. RBC flow in foot tumours remained significantly depressed for only 2-3 min. A dose of 5 mg kg-1 was not significantly vasoactive in back tumours. H33342 also caused a transient 20 +/- 6 mmHg decline in mouse arterial blood pressure. Blood pH and haematocrit, and tumour cell oxygen consumption were unchanged by H33342. H33342-induced flow changes did not affect results obtained using an in vivo double staining protocol provided that the interval between stain injections was greater than 5 min. Due to its transient effects on tumour perfusion, the stain caused radiobiological tumour hypoxia if injected immediately prior to X-irradiation. Injection 20 min before irradiation had no influence on tumour radiation response. We conclude that the transient nature of H33342-induced perturbations in mouse cardiovascular physiology and tumour blood flow must always be considered but do not preclude the use of the stain as a vascular marker to detect spontaneous tumour blood flow fluctuations or acute hypoxia.     

Comparison of fluorescence intensity of Hoechst 33342-stained EMT6 tumour cells and tumour-infiltrating host cells

Hoechst 33342 is a fluorescent dye used for cell selection from tumours based upon intratumour location. When the dye is administered i.v. to tumour-bearing animals, cellular fluorescence is directly related to the proximity of cells to blood vessels. The present study compared inherent Hoechst fluorescence between in vitro-stained EMT6/Ro (mouse mammary sarcoma) cells and host cells, to determine if these populations have different staining characteristics that may influence cell selection procedures. Tumour cell fluorescence exceeded host cell staining by 8-fold when pure cell populations (EMT6/Ro monolayer cells, mouse spleen and peritoneal cells) were compared, and 3-fold for tumour cell-enriched and host cell-enriched populations from solid tumours. Inherent uptake of HO 33342 appeared to be correlated with cell volume. These differences in inherent dye uptake between host and tumour cells were found to be minor in comparison to the fluorescence gradient between the 10% brightest and 10% dimmest (78-fold) cell populations from in vivo-stained tumours.     

Intermittent blood flow in the KHT sarcoma--flow cytometry studies using Hoechst 33342

The administration of the fluorescent DNA stain, Hoechst 33342, to mice bearing the KHT sarcoma, combined with flow cytometry, can be used to select cells according to their proximity to functional vasculature. Different protocols of administration of Hoechst 33342 were used in order to differentiate between the presence of temporary and chronically hypoxic cells. The results show a large difference in radiosensitivity between cells close to, and distant from, functional vasculature. However, this pattern of radiosensitivity is observed only when the staining period with Hoechst 33342 is short and coincides with the period of irradiation. When the radiation treatment is temporally divorced from the staining period then the radiosensitivity and staining intensity are not related. This result can be interpreted as indicating that hypoxic cells exist within this tumour as a result of fluctuations in tumour blood flow.     

Validation of the fluorescent dye Hoechst 33342 as a vascular space marker in tumours

The DNA-binding fluorescent dye Hoechst 33342 (H33342) has been used in a series of investigations of the vascular parameters of two murine tumours. This dye has been shown, to have a short half-life in the circulation (T1/2 less than 2 min), but is stably bound for at least 2 h after it enters cells. It can be used in morphometric studies on frozen sections to determine the effective vascular volume, the capillary fraction and the size distribution of blood vessels in each tumour. These latter two parameters cannot be deduced from the less labour intensive techniques using radioactive isotopes. The effective vascular volume perfused in 1 min by H33342 was compared with the volume perfused in 30 min with 51Cr labelled erythrocytes. Similar volumes were estimated with the two techniques in a murine carcinoma and in a sarcoma. Both techniques showed that the vascular volume decreased in larger tumours. The H33342 analysis of vessel size showed the decrease in capillary vessels in the carcinomas was even greater, falling from 70% in small tumours to 20% in larger tumours. The deteriorating vascular network in larger tumours is associated with an increasing fraction of necrotic tissue. Experiments in which the isotopes and dye were co-injected suggest that at 40 mgkg-1 the dye may rapidly lead to a partial shutdown of the tumour vascular bed. This is less marked with 20 mg kg-1. In spite of this effect there is in general a close correlation between the volumes perfused by labelled red blood cells and the fluorescent dye.     

The use of fluorescent probes to identify regions of transient perfusion in murine tumors

Sequential intravenous injection of two fluorescent stains, Hoechst 33342 and DiOC7(3), can be used to quantify transient perfusion in experimental tumors. Regions of unmatched staining, indicative of intermittent perfusion, occur when vessels open or close in the 20 minute interval between administration of the dyes. In the murine SCCVII carcinoma, 8.9 +/- 2.4% (SD) of vessels in 0.5 g subcutaneous tumors had labelling of adjacent cells with only one stain, suggesting complete vessel closure lasting at least 5 minutes. Regions of intermittent perfusion were not homogeneously distributed throughout the tumor and larger tumors exhibited more mismatch than smaller tumors. Transient perfusion was observed in both subcutaneous and intramuscular tumor implants and was not significantly affected by restraint of the animal or by ketamine/diazepam anesthesia.     

Detection of multidrug resistance and quantification of responses of human tumour cells to cytotoxic agents using flow cytometric spectral shift analysis of Hoechst 33342-DNA fluorescence

Summary We describe the application of a flow cytometric technique for assessing the radiation or drug sensitivity characteristics of human tumour cells. The technique makes use of the phenomenon that a red shift occurs in the fluorescence emission spectrum of a DNA-specific dye (Hoechst 33342) as an increasing number of dye molecules bind to nuclear DNA. Intact, viable cells undergo a time-dependent spectral shift that can be distinguished from the rapid shift observed in cells with damaged membranes by the use of multiparametric flow cytometry. The responses of various human cell lines were compared, namely, those of normal and ataxia-telangiectasia (A-T) lymphoblastoid lines, a small-cell lung carcinoma line and its (in vitro) derived multidrug-resistant variants. A close correlation was found between dye toxicity and the degree of DNA binding of Hoechst 33342 independent of cellular DNA content, with lymphoblastoid and multidrug-resistant small-cell lung cancer cells showing enhanced and restricted dye-binding rates, respectively. VP16-and radiation-induced cell kill was found to result in a quantifiable increase in the fraction of cells undergoing a rapid spectral shift and was capable of detecting the increased radiation sensitivity of A-T-derived cells. Spectral shift analysis provides a rapid method for assessing the responses of tumour cells to cytotoxic agents and for determining the general ability of cells to protect cellular DNA from a model DNA-binding agent (Hoechst 33342) that participates in the multidrug resistance phenotype.     

Vascular perfusion and hypoxic areas in RIF-1 tumours after photodynamic therapy.

The influence of photodynamic therapy (PDT) on vascular perfusion and the development of hypoxia was investigated in the murine RIF-1 tumour. Image analysis was used to quantify changes in perfusion and hypoxia at 5 min after interstitial Photofrin-mediated PDT. The fluorescent stain Hoechst 33342 was used as an in vivo marker of functional vascular perfusion and the antibody anti-collagen type IV as a marker of the tumour vasculature. The percentage of total tumour vasculature that was perfused decreased to less than 30% of control values after PDT. For the lower light doses this decrease was more pronounced in the centre of the tumour. The observed reduction in vascular perfusion showed a good linear correlation (r = 0.98) with previously published tumour perfusion data obtained with the 86Rb extraction technique. The image analysis technique provides extra information concerning the localisation of (non)-perfused vessels. To detect hypoxic tumour areas in vivo, an immunohistochemical method was used employing NITP [7-(4''-(2-nitroimidazol-1-yl)-butyl)-theophylline]. A large increase in hypoxic areas was found for PDT-treated tumours. More than half the total tumour area was hypoxic after PDT, compared with < 4% for control tumours. Our studies illustrate the potential of image analysis systems for monitoring the functional consequences of PDT-mediated vascular damage early after treatment. This provides direct confirmation that the perfusion changes lead to tissue hypoxia, which has implications for the combined treatment of PDT with bioreductive drugs.     

Pharmacokinetics, binding and distribution of Hoechst 33342 in spheroids and murine tumours

The fluorescent stain Hoechst 33342, when injected i.v. into mice, has an LD50 of 300 micrograms g-1. The stain exits rapidly from the blood, with a half-life of 110 sec following an injection of 10 micrograms g-1, but remains bound within target cells, redistributing with a half-life longer than 2 h. This results in a gradient of drug binding outward from capillaries which can be used to estimate regional perfusion via fluorescence microscopy of frozen tissue sections. For tumour tissues that can be dispersed into single cell suspensions, intracellular Hoeschst 33342 can be quantified by flow cytometry, and cell populations can be selected on the basis of their fluorescence (distance from the vasculature) using a fluorescence-activated cell sorter. Our results in tumours and in spheroids indicate that the rate of stain uptake by different cell subpopulations in situ is much more dependent on stain delivery than on selective uptake. Retention of the stain in spheroids is sufficiently stable to allow cell sorting several hours post-injection. Hoechst 33342 thus appears to have considerable potential as an agent for quantifying tissue perfusion, and for allowing selection of tumour cell subpopulations to assess response to radiation and drugs.     

Local hypoxia is produced at sites of intratumour injection

Intratumour injection, commonly used for gene or drug delivery but also associated with needle biopsy or insertion of invasive measuring devices, may damage tumour microvessels. To examine this possibility, SCCVII tumours grown subcutaneously in C3H mice were injected with a 26 gauge needle containing 0.1 ml of the fluorescent dye Hoechst 33342 to label cells lining the track of the needle. Hoechst-labelled cells sorted from these tumours were more sensitive to killing by hypoxic cell cytotoxins (tirapazamine, RSU-1069) and less sensitive to damage by ionizing radiation. Hoechst-labelled cells also bound the hypoxia marker pimonidazole when given by i.p. injection. Intratumour injection transiently increased hypoxia from 18 to 70% in the tumour cells adjacent to the track of the needle. The half-time for return to pre-treatment oxygenation was about 30 min; oxygenation of tumour cells along the track had recovered by 20 h after intratumour injection. This effect could have significant implications for intratumour injection of drugs, cytokines or vectors that are affected by the oxygenation status of the tumour cells as well as potential effects on biodistribution via local microvasculature.     

The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells.

The human ATP-binding cassette superfamily G (White) member 2 (ABCG2) gene and its murine homologue breast cancer resistance protein 1 (Bcrp1) are recently described ATP-binding cassette transporters associated with drug resistance in tumor cell lines, including the MCF-7 cell line, selected for its resistance to mitoxantrone (MCF-7/MitoR). Infection of MCF-7 cells with the retroviral vector containing ABCG2 cDNA (G1-ABCG2) resulted in cells (MCF-7/ABCG2) that were resistant to mitoxantrone at levels similar to those observed in MCF-7/MitoR cells. Previous studies have shown that pluripotent hematopoietic stem cells overexpress the multidrug-resistant transport (MDR1) gene and efflux rhodamine, a substrate for the MDR1 transporter. Other studies have identified a primitive hematopoietic stem cell population, or side population (SP) cells, which are identified by their efflux of the fluorescent dye, Hoechst 33342. In an attempt to identify the transport genes responsible for this phenotype, we examined the uptake of Hoechst 33342 into MCF-7, MCF-7/MitoR, and MCF-7 cells infected with a retroviral vector expressing the ABCG2 gene (MCF-7/ABCG2). MCF-7/MitoR cells as well as MCF-7/ABCG2 cells demonstrated lower levels of Hoechst 33342 uptake compared with the parental MCF-7 cells. We also examined the level of the mouse Bcrp1 RNA in SP cells and non-SP cells isolated from mouse hematopoietic cells. Mouse SP cells expressed relatively high levels of Bcrp1 mRNA relative to non-SP cells. These results suggest that Hoechst 33342 is a substrate for the ABCG2 transporter and that ABCG2/Bcrp1 expression may serve as a marker for hematopoietic stem cells in hematopoietic cells.     

Cellular levels of photosensitisers in tumours: the role of proximity to the blood supply.

Flow cytometry using the tumour perfusion probe Hoechst 33342 was employed to examine the distribution of photosensitisers in tumour cells located at different distances from the blood supply. Two tumour models, the SCCVII squamous cell carcinoma and FsaR fibrosarcoma growing in C3H/HeN mice, were used in the experiments. Among the photosensitisers tested, only BPD (benzoporphyrin derivative monoacid) exhibited uniform distribution in tumour cells irrespective of their distance from the vasculature. In this respect, 5-aminolaevulinic acid (i.e. its metabolite protoporphyrin IX), di- and tetrasulphonated aluminium phthalocyanines (A1PcS2 and AlPcS4), di- and tetrasulphonated tetraphenylporphines (TPPS2 and TPPS4), Photofrin and bacteriochlorophyll-a (i.e. its metabolite bacteriopheophytin-a) followed BPD in decreasing order in their efficacy of accumulation in tumour cells remote from the blood supply. This photosensitiser property appeared not to depend on tumour type, tumour size, route of photosensitiser administration, time after the administration, photosensitiser lipophilicity or on the presence of host cell infiltrate in the tumour. Following treatment with photodynamic therapy (PDT) in vivo, tumour cells were sorted based on their blood vessel proximity and their survival was determined by colony formation assay. The data demonstrate that the direct killing of tumour cells by Photofrin- and A1PcS2-based PDT decreases with increasing distance of the cells from the blood supply.     

The use of vascularised spheroids to investigate the action of flavone acetic acid on tumour blood vessels

EMT6 multicellular spheroids were introduced into the peritoneal cavities of mice and allowed to become vascularised, resulting in solid spherical tumours. The necrotic cores of the initially avascular spheroids were replaced by vascularised tumour tissue but the outer zones of the spheroids failed to become vascularised. The presence of both vascular and avascular components in each spheroid allowed the role of the vasculature in the antitumour action of flavone acetic acid (FAA) to be determined. Eighteen hours after treatment with FAA 0.8 mmol kg-1, the vascularised core became necrotic and haemorrhagic, while the outer avascular zone remained viable. Tumour cells which were infiltrating superficial sub-mesothelial fat did not become necrotic despite the presence of numerous thrombi in associated vessels. Injection of two fluorescent vascular markers, the first (Hoechst 33342) together with FAA, and the second (10-nonyl acridine orange) 4 h later, demonstrated that there is a marked loss of blood flow in the spheroids. These results provide further evidence that FAA kills blood vessel-dependent tumour cells by interrupting the tumour blood supply.     

Leukemic blast and natural killer cell P-glycoprotein function and inhibition in a clinical trial of zosuquidar infusion in acute myeloid leukemia

A bioassay was developed to assess P-glycoprotein (P-gp) function of peripheral blood natural killer (NK) cells and AML blasts during zosuquidar infusion. Cells were incubated with the fluorescent dye DiOC(2)(3) in the presence and absence of zosuquidar, and dye accumulation measured by flow cytometry. The assay performance was assessed using NK cells and the P-gp-positive K562/R7 cell line, and then utilized to determine the function of P-gp and its inhibition by zosuquidar in AML blasts and NK cells from patients enrolled in a Phase I trial. The assay of zosuquidar-inhibitable accumulation of DiOC(2) is robust and reproducible.     

The effect of hydralazine on blood flow and misonidazole toxicity in human tumour xenografts

The effect of post-irradiation hypoxia induced by 5 or 30 mg/kg hydralazine has been studied in three human tumour xenografts (two rectocolic adenocarcinomas and one melanoma) treated with two doses of misonidazole similar to those used in patients (0.1 and 0.2 mg/g). Only a small sensitization was detected using an in vitro colony assay. These results are in marked contrast to the results obtained with rodent tumours. This difference between human tumour xenografts and rodent tumours might be explained by differences in the reduction of tumour blood flow after hydralazine administration (5 and/or 10 mg/kg). Using the laser Doppler technique, the tumour blood flow reduction was 33% and 25% of the control for NA11 and HRT18 tumours, respectively. In contrast, hydralazine induced a 60-70% reduction in blood flow in the murine SCCVII tumour. Using the fluorescent marker Hoechst 33342, the reduction in perfusion was again more pronounced in the murine tumour as compared to the Na11 and HRT18 xenografts. The differences between human tumour xenografts and rodent tumours are not linked to the mouse strain used (nude versus C3H) nor to a tumour bed effect.     

Tumor vascular shutdown following photodynamic therapy based on polyhematoporphyrin or 5-aminolevulinic Acid

The effects of photodynamic therapy (PDT) based on polyhaematoporphyrin (PHP) or 5-aminolaevulinic acid (ALA) on the tumour vascular perfusion (TVP) of a rat fibrosarcoma were examined using an (RbCl)-Rb-86 extraction technique. Both forms of therapy induced large and highly significant reductions in TVP, countering the previous suggestion that ALA-PDT has no vascular effects. Ultrasensitive digital imaging fluorescence microscopy indicated that ALA-induced PpIX was distributed relatively evenly throughout the tumour. Use of Hoechst 33342 as a vascular marker revealed that PpIX was also present in cells lining the tumour microvascular spaces, contrary to previous findings.     

荧光染料Hoechst 33342与细胞凋亡

荧光染料Hoechest33342在计数细胞周期及细胞凋亡研究方面得到广泛的应用。研究发现,Hoeehst33342可诱导如BC3H—1细胞、HL-60细胞、HT-144黑色素瘤细胞、肝细胞癌细胞等多种细胞凋亡。Hoeehest33342对拓扑异构酶I活性的抑制、对TATA盒结合蛋白的抑制、细胞内E2F-1蛋白表达的增加和线粒体功能不良可能是其诱导细胞凋亡的主要机制。     

Thymidine analogues to assess microperfusion in human tumors

PURPOSE: To validate the use of the thymidine analogues as local perfusion markers in human tumors (no labeling indicates no perfusion) by comparison with the well-characterized perfusion marker Hoechst 33342. METHODS AND MATERIALS: Human tumor xenografts from gliomas and head-and-neck cancers were injected with iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) and the fluorescent dye Hoechst 33342. In frozen sections, each blood vessel was scored for the presence of IdUrd/BrdUrd labeling and Hoechst in surrounding cells. The percentage of analogue-negative vessels was compared with the fraction of Hoechst-negative vessels. Collocalization of the two markers was also scored. RESULTS: We found considerable intertumor variation in the fraction of perfused vessels, measured by analogue labeling, both in the human tumor xenografts and in a series of tumor biopsies from head-and-neck cancer patients. There was a significant correlation between the Hoechst-negative and IdUrd/BrdUrd-negative vessels in the xenografts (r = 85, p = 0.0004), despite some mismatches on a per-vessel basis. CONCLUSIONS: Thymidine analogues can be successfully used to rank tumors according to their fraction of perfused vessels. Whether this fraction correlates with the extent of acute hypoxia needs further confirmation.     

The effects of melphalan and misonidazole on the vasculature of a murine sarcoma

A method for estimating both structural and functional vascular volumes in murine sarcomas is described. Intact vessels were demonstrated by the presence of laminin, a basement membrane-associated antigen, using an immunofluorescent technique, and functional vessels in the same sample by prior injection with the DNA binding dye Hoechst 33342. No significant vascular effects were seen after melphalan but a very pronounced decrease in both functional and structural vascular volume was seen after MISO. Combined chemotherapy of a murine sarcoma with melphalan and MISO induced a rapid decrease in the functional vascular volume, and there was a resumption of blood flow prior to measurable regrowth. The fully regrown tumour retained the vascular characteristics of untreated tumours of similar size.