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Biomedical subjects

R E Durand

Publications and source records attributed to R E Durand.

At least 19 recordsLinked to original sources
PubMed2006

Comparison between pimonidazole binding, oxygen electrode measurements, and expression of endogenous hypoxia markers in cancer of the uterine cervix.

BACKGROUND: Although tumor hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is no consensus as to the method best suited for routine measurement. Binding of the chemical hypoxia marker, pimonidazole, and expression of the endogenous hypoxia markers HIF-1alpha and CAIX were compared for their ability to detect hypoxia in tumor biopsies from 67 patients with advanced carcinoma of the cervix. METHODS: Two biopsies were taken one day after administration of pimonidazole and were analyzed for pimonidazole binding using flow cytometry or immunohistochemistry. CAIX and HIF-1alpha expression and degree of colocalization were measured in sequential antibody-stained sections. Patient subsets were examined for tumor oxygen tension using an Eppendorf electrode, S phase DNA content, or change in HIF-1alpha expression over the course of treatment. RESULTS: Approximately 6% of the tumor area stained positive for pimonidazole, HIF-1alpha, or CAIX. The CAIX positive fraction correlated with the pimonidazole positive fraction (r = 0.60). Weaker but significant correlations were observed between pimonidazole and HIF-1alpha (r = 0.31) and CAIX and HIF-1alpha (r = 0.41). Taking the extent of marker colocalization into consideration increased the confidence that all markers were identifying hypoxic regions. Over 65% of stained areas showed a high degree of colocalization with the other markers. Oxygen microelectrode measurements and S phase fraction were not correlated with the hypoxic fraction measured using the three hypoxia markers. HIF-1alpha levels tended to decrease with time after the start of therapy. CONCLUSIONS: Endogenous hypoxia marker binding shows reasonable agreement, in extent and location, with binding of pimonidazole. CAIX staining pattern is a better match to the pimonidazole staining pattern than is HIF-1alpha, and high CAIX expression in the absence (or low levels) of HIF-1alpha may indicate a different biology.

Antigens, Neoplasm↗
PubMed2001

Drug-induced alterations in tumour perfusion yield increases in tumour cell radiosensitivity.

The perfusion of human tumour xenografts was manipulated by administration of diltiazem and pentoxifylline, and the extent that observed changes in tumour perfusion altered tumour radiosensitivity was determined. 2 tumour systems having intrinsically different types of hypoxia were studied. The responses of SiHa tumours, which have essentially no transient hypoxia, were compared to the responses of WiDr tumours, which contain chronically and transiently hypoxic cells. We found that relatively modest increases in net tumour perfusion increased tumour cell radiosensitivity in WiDr tumours to a greater extent than in SiHa tumours. Moreover, redistribution of blood flow within WiDr tumours was observed on a micro-regional level that was largely independent of changes in net tumour perfusion. Through fluorescence-activated cell sorting coupled with an in vivo-in vitro cloning assay, increases in the radiosensitivity of WiDr tumour cells at intermediate levels of oxygenation were observed, consistent with the expectation that a redistribution of tumour blood flow had increased oxygen delivery to transiently hypoxic tumour cells. Our data therefore suggest that drug-induced changes in tumour micro-perfusion can alter the radiosensitivity of transiently hypoxic tumour cells, and that increasing the radiosensitivity of tumour cells at intermediate levels of oxygenation is therapeutically relevant.

Animals↗
PubMed2001

Intermittent blood flow in solid tumours--an under-appreciated source of 'drug resistance'.

As the search for improved anti-cancer drugs continues, new paradigms concerning the reasons for clinical failures in common human solid tumours are also evolving. Classical drug resistance is now perhaps less often invoked to explain lack of treatment efficacy than are newer concepts, including 'contact resistance', 'tumour heterogeneity', 'regrowth resistance', and 'physiological barriers' to drug delivery. This commentary will explore the resistance of solid tumours to chemotherapy from yet another, largely ignored perspective: that of tumour-specific fluctuations in blood flow. Transient decreases in blood flow have significant implications for delivery of chemotherapeutic agents, cellular responsiveness to those agents, and the regrowth potential of the surviving tumour cells.

Antineoplastic Agents↗
PubMed2001

Non-constant tumour blood flow--implications for therapy.

In the past few years, 'perfusion-limited' hypoxia caused by intermittent decreases in tumour blood flow has received increasing attention. Little effort, however, has gone into characterizing the nature, magnitude or duration of these changes, or their functional significance other than as modifiers of radiotherapy. We have therefore undertaken multiple, quantitative analyses of tumour blood flow in human tumour xenograft systems, and rigorously examined the ramifications of transient blood flow changes. Tumour blood flow in these experimental tumours is much less constant than has previously been assumed, and not only impacts on response to radiotherapy and chemotherapy, but also on the more fundamental processes of tumour growth and repopulation. Notably, responses entirely consistent with the laboratory results have been seen in our initial studies of human tumours sequentially biopsied during treatment.

Adenocarcinoma↗
PubMed2001

Clinical relevance of intermittent tumour blood flow.

One of the goals of translational cancer research is to understand basic 'phenomena' so that tumour response to therapy can be improved. One such phenomenon is intermittent tumour blood flow. The impact of the transient hypoxia that results from decreased tumour blood flow is now beginning to be appreciated in preclinical systems, and also receiving some attention in clinical practise. Thus in this article we review the nature and frequency of microregional blood flow changes in preclinical and clinical tumours and examine the impact of those changes on response to both radiotherapy and chemotherapy. Additionally, the implications of non-constant blood flow for both the growth of the unperturbed tumour and the regrowth of surviving tumour clonogens during and after therapy are examined.

Antineoplastic Agents↗
PubMed2000

Effects of mitomycin C on the oxygenation and radiosensitivity of murine and human tumours in mice.

BACKGROUND AND PURPOSE: Mitomycin C was one of the first chemotherapeutic agents to be shown to have preferential cytotoxicity toward hypoxic cells in vitro. Consequently, it has been used clinically with radiotherapy, and has stimulated considerable interest for analogue development. More recent studies also suggested a possible role for the drug in enhancing tumour blood flow; we therefore undertook a comprehensive examination of mitomycin C as a potential radiosensitizer in murine and human tumours growing in mice. MATERIALS AND METHODS: Two dissimilar human tumour xenograft systems, SiHa and WiDr cells, were used as was the murine SCCVII line. Effects of mitomycin C treatment on the regional and microregional blood flow in these tumours was evaluated, and cell sorting based on dye perfusion techniques was used to study the cytotoxicity of mitomycin C as a single agent or in combination with radiation in the xenograft systems. RESULTS: Contrary to our expectations, no preferential killing of less-well oxygenated tumour cells in situ was observed, nor were any consistent effects on tumour blood flow found. The inclusion of mitomycin C with radiation did, however, produce a modest increase in cell killing in the hypoxic subpopulations of the xenograft system with the largest hypoxic fraction. CONCLUSIONS: Our results indicate that combined treatment with mitomycin C and radiation cannot be rationalized on the expectation of either complementary cytotoxicity of the modalities, or of drug-induced improvement in tumour oxygenation.

Animals↗
PubMed2000

Comparison between the comet assay and pimonidazole binding for measuring tumour hypoxia.

Pimonidazole is finding increasing use in histochemical analyses of hypoxia in tumours. Whether it can identify every hypoxic cell in a tumour, and whether the usual subjective criteria used to define 'positive' cells are optimal, are less certain. Therefore, our aim was to develop an objective flow cytometry procedure for quantifying pimonidazole binding in tumours, and to validate this method by using a more direct indicator of radiobiologic hypoxia, the comet assay. SCCVII tumours in C3H mice were analysed for pimonidazole binding using flow cytometry and an iterative curve-fitting procedure, and the results were compared to the comet assay for the same cell suspensions. On average, cells defined as anoxic by flow analysis (n = 43 tumours) bound 10.8 +/- 0.95 times more antibody than aerobic cells. In samples containing known mixtures of aerobic and anoxic cells, hypoxic fractions as low as 0.5% could easily be detected. To assess the flow cytometry assay under a wider range of tumour oxygen contents, mice were injected with hydralazine to reduce tumour blood flow, or allowed to breathe various gas mixtures during the 90 min exposure to pimonidazole. Hypoxic fraction estimated by the pimonidazole binding method agreed well with the hypoxic fraction measured using the comet assay in SCCVII tumours (r2 = 0.87, slope = 0.98), with similar results in human U87 glioma cells and SiHa cervical carcinoma xenografts. We therefore conclude that this objective analysis of pimonidazole labelling by flow cytometry gives a convenient and accurate estimate of radiobiological hypoxia. Preliminary analyses of biopsies from 3 patients given 0.5 g m-2 pimonidazole also suggest the suitability of this approach for human tumours.

Animals↗
PubMed1999

Repopulation characteristics and cell kinetic parameters resulting from multi-fraction irradiation of xenograft tumors in SCID mice.

PURPOSE: Cell kinetics and repopulation rates during multifraction irradiation have previously been measured in SiHa human cervical carcinoma cells grown as spheroids. The current study applied similar techniques to SiHa tumor xenografts with the ultimate goal of assessing the clinical prognostic value of in situ cell kinetics. METHODS AND MATERIALS: SiHa (human squamous cell cervical tumor) cells were inoculated subcutaneously in the flank or back of SCID mice. When tumors reached a size of 200-300 mg, they received 25 Gy in 10 fractions over 5 days. Tumor regrowth and cell kinetics parameters were followed during treatment, and for 10 days after completion by measuring tumor volume and analyzing cellular BrdUrd and IdUrd incorporation with flow cytometry. RESULTS: Tumor volume was of limited use in assessing response to irradiation. The fraction of proliferating cells increased early during irradiation as did the labeling index; potential doubling time (Tpot) decreased during treatment and returned to the pre-irradiation value after treatment. Cell cycle time remained relatively constant throughout the experiments. CONCLUSION: These results confirm the feasibility of evaluating cell cycle kinetics and repopulation parameters in a murine tumor model undergoing a fractionated course of irradiation. Repopulation of clonogenic tumor cells occurred more rapidly than predicted by pretreatment measurements, primarily due to an increased growth fraction and consequent decrease in Tpot.

Animals↗
PubMed1999

Cell kinetics and repopulation parameters of irradiated xenograft tumours in SCID mice: comparison of two dose-fractionation regimens.

The extent and mechanism(s) of repopulation were assessed in SiHa (human cervical squamous cell carcinoma) xenografts in SCID mice for two fractionated irradiation regimens. Mice in one arm of the study received 50 Gy in 20 fractions over 23 days with a 14 day split between 10 fraction, 5 day courses. The other tumours were treated with 50 Gy in 20 fractions over 10 consecutive days. Cell kinetics and tumour regrowth parameters were monitored during and after treatment by measuring tumour volume and analysing cellular DNA content and proliferation parameters with flow cytometry. Repopulation occurred rapidly, beginning during irradiation and largely attributable to an increased growth fraction and decreased potential doubling time, apparently triggered by increased cell loss. Cell cycle time, in contrast, remained relatively constant throughout. Extrapolation of these results to humans suggests that treatment times should be minimised whenever possible, since regrowth rates exceeded those predicted from pretreatment Tpot measurements.

Animals↗
PubMed1999

The comet assay in clinical practice.

The comet assay is a single-cell gel electrophoresis technique that measures DNA damage in individual cells. Since radiation produces 3-4 times more DNA damage in well-oxygenated cells compared with hypoxic cells, this assay can quantify the fraction of radiation-resistant hypoxic cells found in many solid tumours. This paper summarizes our results with 73 accessible metastatic tumours irradiated with palliative intent. Hypoxic fractions ranged from 0.0 to 0.67 with a mean of 0.15; 62% of these advanced tumours showed a hypoxic fraction > 0.05. Comparisons between two sequential aspirates in 33 tumours gave a slope of 0.92 (r2 = 0.88), suggesting that a single aspirate is generally representative of the tumour. A limitation, however, is that the hypoxic fraction could not be measured in clinical samples given a conventional dose of 2 Gy.

Animals↗
PubMed1998

The lifetime of hypoxic human tumor cells.

PURPOSE: For hypoxic and anoxic cells in solid tumors to be a therapeutic problem, they must live long enough to be therapeutically relevant, or else be rapidly recruited into the proliferating compartment during therapy. We have, therefore, estimated lifetime and recruitment rate of hypoxic human tumor cells in multicell spheroids in vitro, or in xenografted tumors in SCID mice. MATERIALS AND METHODS: Cell turnover was followed by flow cytometry techniques, using antibodies directed at incorporated halogenated pyrimidines. The disappearance of labeled cells was quantified, and verified to be cell loss rather than label dilution. Repopulation was studied in SiHa tumor xenografts during twice-daily 2.5-Gy radiation exposures. RESULTS: The longevity of hypoxic human tumor cells in spheroids or xenografts exceeded that of rodent cell lines, and cell turnover was slower in xenografts than under static growth as spheroids. Human tumor cells remained viable in the hypoxic regions of xenografts for 4-10 days, compared to 3-5 days in spheroids, and 1-3 days for most rodent cells in spheroids. Repopulation was observed within the first few radiation treatments for the SiHa xenografts and, with accumulated doses of more than 10 Gy, virtually all recovered cells had progressed through at least one S-phase. CONCLUSION: Our results suggest an important difference in the ability of human vs. rodent tumor cells to withstand hypoxia, and raise questions concerning the increased longevity seen in vivo relative to the steady-state spheroid system.

Animals↗
PubMed1998

Identification of nonproliferating but viable hypoxic tumor cells in vivo.

We have used the combination of pimonidazole labeling of hypoxic cells, bromodeoxyuridine labeling of proliferating cells, and cell sorting based on Hoechst 33342 perfusion to directly study hypoxia and proliferation in human tumor xenografts and transplantable murine tumors in vivo. Hypoxia was largely confined to cells in regions with the least perfusion, although in tumors exhibiting transient blood flow, hypoxic cells were not as highly localized. Similarly, proliferation and hypoxia were mutually exclusive except in areas of a tumor subjected to transient changes in perfusion. By determining the clonogenic potential, pimonidazole labeling intensity, and radiosensitivity of sorted tumor cell subpopulations, we have provided direct evidence that pimonidazole identifies hypoxic tumor cells of therapeutic relevance in vivo. Given that pimonidazole exhibits few diffusion or delivery problems and no apparent cytotoxicity, it appears to be a versatile and useful label for hypoxic cells in solid tumors.

Animals↗
PubMed1998

Cell kinetics and repopulation mechanisms during multifraction irradiation of spheroids.

BACKGROUND AND PURPOSE: The objective of the present study was to evaluate the predictive potential of cell kinetic parameters and repopulation rates determined by flow cytometry during multifraction irradiation of spheroids, a system in which the fate of all cells can be determined with high precision. Ultimately, similar analytical techniques should provide a reproducible and prognostically significant clinical predictive assay. MATERIALS AND METHODS: Multicellular spheroids of Chinese hamster V79 lung cells were irradiated with 2.5 Gy of 250 kVp X-rays twice daily to a total dose of 25 Gy. Repopulation parameters and cell kinetic parameters were followed throughout the irradiation period and for 5 days after completion of exposure. RESULTS: (1) Regrowth (RG) took place early during multifraction irradiation. (2) Potential doubling time (Tpot) decreased steadily from the early part of treatment, remaining of short duration until the spheroids almost attained the pre-treatment number of clonogenic cells. (3) Accelerated repopulation was mainly due to a decreased cell loss factor (phi) and increased growth fraction (GF), although a modest decrease in cell cycle time (tc) was suggested. (4) Phi decreased during exponential RG. (5) Other parameters such as observed doubling time (td) and labelling index (LI) paralleled these findings. CONCLUSIONS: Clonogen repopulation that began early in the irradiation scheme and accelerated rapidly is not consistent with the prevailing view that accelerated repopulation begins several weeks into clinical protocols. Also, pre-treatment Tpot did not adequately estimate the repopulation speed in the spheroids. Equivalent studies in animal tumour systems, and then in the clinic, are consequently indicated and of some urgency.

Animals↗
PubMed1998

Use of the comet assay for assessment of drug resistance and its modulation in vivo.

Drug resistance is generally considered to be a major impediment to successful cancer chemotherapy, yet it is generally not possible to predict the degree or timing of the emergence of tumour resistance in most chemotherapy protocols. Recent developments with the single-cell gel electrophoresis or 'comet' assay for DNA damage at the single-cell level suggest that this technique might provide a method for identifying and potentially monitoring tumour cell responsiveness to many anti-cancer agents in situ. In principle, this assay could be applied to any accessible tumour being treated with chemotherapeutic agents that cause overt DNA damage. We have investigated that supposition using several rodent and human tumour cell lines exhibiting a spectrum of resistance to the DNA strand-breaking drug, etoposide. By assessing cells grown as monolayers, spheroids and xenografted tumours in immunodeficient mice, we found that the comet assay can provide not only an index of sensitivity to etoposide, but, additionally, can demonstrate the efficacy (or lack thereof) of multidrug resistance (MDR) reversing agents for cells in vitro, and tumours in vivo.

Animals↗
PubMed1997

Tumor repopulation during radiotheraphy: quantitation in two xenografted human tumors.

PURPOSE: "Accelerated repopulation" has generated considerable recent interest. Our purpose in this study was to determine whether flow cytometry measurements like those used for classical Tpot determinations could be used to quantify the rate of repopulation, and the time of its initiation in irradiated human tumor xenografts. METHODS AND MATERIALS: Two human tumor cell lines (SiHa, a squamous cell cervix carcinoma, and WiDr, an adenocarcinoma of the colon) were grown as subcutaneous xenografts in SCID mice. Tpot was measured in a conventional manner using flow cytometry, for control tumors and for tumors exposed to five fractions of 4 Gy twice daily over a 2-day interval. For the irradiated tumors, Tpot measurements were conducted 48 h following the final exposure. RESULTS: Active proliferation even after irradiation was observed in both the radiosensitive SiHa and more radioresistant WiDr tumors, and the estimated repopulation rate was at least as fast as would have been predicted by the pre-treatment Tpot estimates. CONCLUSIONS: Our data clearly indicate tumor cell proliferation after only a few fractions of radiation exposure in these human tumor xenografts. Additionally, the data suggest that pretreatment Tpot values may underestimate the actual regrowth rate.

Adenocarcinoma↗