Realidad Virtual en IES Francesc Borja i Moll

Cells were passaged by tipping off the medium, washing once with MEM, adding 10 ml o f 0.5% typsin, and re-incubating for a further 5 minutes. After re-incubation, the enzyme was neutralised with an equal quantity o f MEM^, and the suspension centrifuged at 1000 rpm for 5 minutes. The supernatant was decanted off and the pellet resuspended in 10 ml MEM^. The cells were counted using a Coulter counter and re-seeded at a density o f 1.8 x 10^ cells/flask.

4.1.1.7 Calculation of surviving fraction

The colony forming efficiency (CFE), or plating efficiency, was calculated by counting the number o f colonies to develop on each petri dish and dividing this by the number o f cells originally seeded. The CFE was averaged for at least 4 replicate petri dishes. The number o f clonogens per 100 mg o f treated tumour was calculated by multiplying the average CFE, by the tumour cell yield.

The surviving fraction (SF) was determined by dividing the number o f clonogens per 100 mg treated tumour by the number o f clonogens per 100 mg o f control tumour. A standard error o f the mean was determined based upon the multiple repeats o f each treatment group.

4.1.1.8 Calculation of hypoxic fraction

The SF’s o f the aerobic and hypoxic treatments were plotted on a semi-log scale depicting radiation dose versus SF. The best linear fit for the hypoxic data was determined and superimposed on the aerobic data. The hypoxic fraction was obtained from the vertical displacement o f the aerobic curve from the hypoxic curve according to the following equation (Moulder and Rockwell, 1984);

log (hypoxic fraction) = log (SFaerobic) - log (SFhypoxic)

To obtain an error on the hypoxic fraction, the ratios o f the four dose points were averaged and the standard error o f the mean calculated.

This calculation is based upon the assumption that tumours contain two populations o f cells; an aerobic component with maximal radiosensitivity, and a anoxic component with full radioresistance.

4.1.2 Relationship between hypoxic fraction, labelling of hypoxic cells and pOz

4.1.2.1 Experimental design

This study was designed to obtain an assessment for the hypoxic fraction o f 30 individual SaF tumours by three techniques; cell labelling, pOz and paired survival curve assay. The protocol included the following sequence o f events: (1) each tumour was irradiated for the paired survival curve assay, (2) tumours were labelled, in situ, with a hypoxic cell marker, 2-nitroimidazole-theophylline (NITP), (3) tumours were assessed for pOz, and (4) tumours were excised and processed to quantify the radiobiologically hypoxic fraction and the NITP labelling index.

Radiobiological hypoxia was determined at a single dose, based upon the assumption that a single pair o f hypoxic and oxic cell survivals would be representative o f a linear fit displacement applied through four different doses. Control groups included 9 randomised untreated SaF tumours and 9 anoxic SaF tumours for SF calculations, 9 untreated SaF tumours for NITP analyses, and a further 20 SaF tumours for oxygenation studies (see below). The following sections are laid out in accordance with the running order o f the study.

4.1.2.2 Tumour irradiation

All animals were irradiated, as previously described in 4.1.1.1, at a single dose o f 20 Gy. Anoxia was induced 10 minutes prior to irradiation by cervical dislocation.

4.1.2.3 NITP formulation and dosing

A formulation suitable for oral dosing was used. 10 g o f a p-cyclodextrin derivative, molecusol, was dissolved in 7.2 ml o f double distilled water at 37°C. 100.8 g o f NITP was added and the solution returned to the water bath. Dissolution o f the NITP generally took upto 48 hours.

Doses were given to each mouse, dependent upon weight, by orally feeding 0.022 ml/gram o f mouse. This was equivalent to approximately 0.91 |imol.

4.1.2.4 pOz assessment

All oxygen measurements were performed using the Eppendorf pOi histograph on unanaesthetised animals as outlined previously (2.1.2). Control groups, each containing 5 animals, included; untreated, subcutis, 20 minutes post-irradiation, and 120 minutes post- NTTP administration.

4.1.2.5 Tumour excision

120 minutes after NTTP administration, animals were sacrificed and the tumours aseptically excised, digested and a known number o f cells plated in petri dishes with a feeder layer. The procedure for this clonogenic assay has already been described in section 4.1.1.2. Remaining cells were centrifuged at 1000 rpm, resuspended in 1 ml phosphate buffered saline (PBS), and added to 9 ml o f ice cold 70% ethanol. Fixed cells were then stored at 4°C until required for immunohistochemical staining.

4.1.2.6 The hypoxic probe: 2-nitroimidazole theophylline

In vivo, 2-nitroimidazole is metabolised by nitroreductases under low oxygen concentrations. Cellular binding is nuclear, and probably to the DNA. The theophylline side chain facilitates labelling with specific antibodies which allow identification and thus quantification o f the degree o f cellular hypoxia. NITP, at a

concentration o f 100 jiM, has a ‘k’ value, in vitro, o f 0.3% (2.3 mmHg) for SaF cells (L.Webster, PhD Thesis, pg. 125, University o f London, 1994) and V79 Chinese hamster cells (Hodgkiss et al, 1991). This describes the oxygen concentration which achieves half maximal binding o f the anti-theophylline antibody.

4.1.2.7 Immunohistochemical staining of theophylline adducts

Labelling o f the theophylline adducts was carried out via a two step antibody protocol compatible with flow cytometry. FACScan resolution was improved by preparing nuclei suspensions to eliminate cellular debris. All samples, including the 9 untreated control tumours, were processed according to the following protocol:

1. Using a haemocytometer count cells in ethanol. Aliquot 2 x 10^ into centrifuge tubes.