Cell selection from a murine tumour using the fluorescent probe Hoechst 33342

Dj, Chaplin; Durand, RE; Olive, Peggy L.

doi:10.1038/bjc.1985.79

Cited by 109 publications

(44 citation statements)

References 8 publications

(11 reference statements)

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“…Chaplin et al (1985Chaplin et al ( , 1986Chaplin et al ( , 1987 Loeffler et al, 1987). The sorted populations that result should be derived from different locations within the tumour relative to the blood supply at the time of staining.…”

mentioning

confidence: 99%

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

Minchinton

Durand²,

Dj³

1990

Br J Cancer

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mentioning

confidence: 99%

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

Minchinton

Durand²,

Dj³

1990

Br J Cancer

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“…Both panels of Figure 1 show cellular radiosensitivity as a function of Hoechst staining intensity; in spheroids, the symmetry of the model allows an easy calculation of the depth from which the cells were recovered (Durand, 1983), while in the tumour system, the data are simply presented as fraction number, where fraction I represented the 10% of the cells which were most intensely stained (thus nearest the functional blood supply, e.g. Chaplin et al, 1985Chaplin et al, ,1986a (Figure 2) indicated that the less well oxygenated cells were more sensitive to this nitrosourea, in agreement with previous results obtained with nitrosoureas in other spheroid systems (Deen et al, 1980;Kwok & Twentyman, 1985). Quite reproducible responses were found in the multiple experiments shown (note that in the case of spheroids, different starting sizes led to slightly different estimates of cell depth among experiments).…”

Section: Resultsmentioning

confidence: 99%

“…Cells were recognized by the forward scatter signal; the resulting signals from the Hoechst stain and the 900 scatter signals were processed through matched logarithmic amplifiers, and the ratio of these signals used to generate a 'stain concentration' profile which was integrated to define 10 windows of equal cell numbers each (Durand, 1983). A stained but untreated tumour or spheroid cell population was used to define the plating efficiency of each sorted fraction; as in previous studies (Durand, 1982(Durand, , 1983Chaplin et al, 1985Chaplin et al, , 1986a, no systematic variation was observed.…”

Section: Methodsmentioning

confidence: 99%

“…In both systems, cells were selectively recovered from known positions (Durand, 1982(Durand, , 1983Chaplin et al, 1985Chaplin et al, , 1986a using cell sorting techniques, permitting us to study drug interactions in specific cell subpopulations of known oxygenation status, and known radiosensitivities. Virtually identical conclusions were forthcoming from the two systems, despite the fact that they were utilized differently: spheroids allow control of many external variables, permitting mechanistic studies, whereas murine tumours are best used to address treatment efficacy in situ.…”

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confidence: 99%

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Chemosensitization by misonidazole in CCNU-treated spheroids and tumours

Durand

Dj²

1987

Br J Cancer

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Summary Misonidazole has been demonstrated to enhance the cytotoxicity of several common antineoplastic drugs in vitro and in vivo, and its mechanism of action as a chemosensitizer, though still unknown, is thought to be dependent upon hypoxia. We have used fluorescence-activated cell sorting to evaluate chemopotentiation by misonidazole as a function of cell position in V79 spheroids and KHT tumours. CCNU toxicity was enhanced in all cell subpopulations of both tumours and spheroids, with greater consistency than might be predicted on the basis of the known variations in oxygen tension. Further, both misonidazole and CCNU as single agents were preferentially toxic in the less well oxygenated regions of each system, arguing that differential toxicity canno t be implicated in the chemopotentiation observed. In fact, increased treatment toxicity did not necessarily lead to increased chemopotentiation, nor was potentiation directly related to the metabolism/binding of the misonidazole. Chemopotentiation in multicell systems thus appears to be a complex, multi-factorial process.Hypoxic cells develop spontaneously during the growth of many solid tumours, and are known to be resistant to ionizing radiation and implicated in resistance to several antineoplastic drugs. Clearly, their presence in human tumours may limit the effectiveness of cancer therapy. Development of hypoxic cell radiosensitizers thus has a clear rationale; the observations that many of these sensitizers were preferentially toxic to hypoxic cells (Olive & McCalla 1975; Hall & Roizin-Towle 1975;Moore et al., 1976) suggested that they may be complementary to many conventional cancer chemotherapeutic agents, and could thus be used effectively in combination treatments. Indeed, in a number of instances an interaction between the sensitizer and the chemotherapeutic agent was observed (Kelly et al., 1979;Clement et al., 1980;Rose et al., 1980, and reviewed by Siemann, 1984).Many factors have been implicated in the phenomenon of chemosensitization (see Brown 1982, and Siemann 1984 for reviews), including differential toxicity (Kelly et al., 1979), direct interactions between the sensitizer (or its toxic metabolites, which are generally produced under hypoxic conditions) and the chemotherapeutic agent itself (Taylor et al., 1982), alterations of drug pharmacology or delivery (Urtasun et al., 1982;Workman et al., 1983;Lee & Workman 1986), inhibition of repair processes by chemosensitizer treatment (Taylor et al., 1982;Mulcahy 1986), and alterations of cellular drug sensitivity, by, for example, the sensitizer selecting for surviving cells more vulnerable to the chemotherapeutic agent due to cell cycle status or other factors . Perhaps the most consistent observation, however, has been the apparent requirement of hypoxia both in vitro (Brown, 1982;Mulcahy, 1984), and in vivo Wheeler et al., 1984).In this report, we present data evaluating the cytotoxic effects of misonidazole, MISO, and N-(2-chloro-ethyl)-N'-cyclohexyl-N-nitrosourea, CCNU, singly and in combi...

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“…This dye penetrates tumour tissue producing a gradient of fluorescence (visible in the u.v. range) that identifies the position of cells in situ relative to any functional vasculature (Chaplin et al, 1985). Mice were given 0.1 ml intravenous injections of Hoechst 33342 20 minutes prior to tumour excision.…”

Section: Immune Cell Determinationmentioning

confidence: 99%

Proportion of infiltrating IgG-binding immune cells predict for tumour hypoxia

Collingridge

Hill

Dj³

2001

Br J Cancer

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Summary Macrophages can account for up to 50% of tumour mass and secrete many angiogenic factors. Furthermore, tumour hypoxia is thought to play a major role in the activation of macrophages and the regulation of angiogenesis. In this paper, we demonstrate a strong correlation between hypoxia and the recruitment of immune cells binding to IgG in 8 experimental tumours. We provide evidence that IgG binding immune cells in 3 tumour lines are predominately composed of macrophages. Reduced oxygenation may act as a stimulus for recruitment of immune cells to the tumour mass, and the detection of either IgG-positive host cells or macrophages may offer an alternative method for monitoring tumour hypoxia.

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Cell selection from a murine tumour using the fluorescent probe Hoechst 33342

Abstract: Images Figure 1

Cited by 109 publications

References 8 publications

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

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

Chemosensitization by misonidazole in CCNU-treated spheroids and tumours

Proportion of infiltrating IgG-binding immune cells predict for tumour hypoxia

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