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

Abstract

Summary 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-', the vascularised core became necrotic and haemorrhagic, while the outer avascular zone remained viable. Tumour Materials and methodsHistological studies EMT6 multicellular tumour spheroids were grown in spinner flask culture in a-MEM + 10% fetal calf serum, and between 15 and 25 spheroids, varying in size from 0.5 to 1.2 mm in diameter, were injected into the peritoneal cavities of anaesthetised Balb/C mice through a 161 gauge needle, as described previously (Zwi et al., 1989). Seven days later the mice in the treatment group (n = 7) were injected with FAA 0.8 mmol kg-' (kindly supplied by Dr K.D. Paull, National Cancer Institute) in 5% w/v sodium bicarbonate by the intravenous (i.v.) or intraperitoneal (i.p.) route, and were killed after a further 18 h. Untreated spheroid-bearing mice (n = 2) were killed after the same interval. The peritoneal cavities of the mice were opened, the free spheroids were collected, and those spheroids which had become attached to host structures were excised with a cuff of adjacent tissue. The spheroids were fixed in either 4% neutral formaldehyde or 2.5% phosphate buffered glutaraldehyde (pH 7.4). The formalin-fixed tissue was embedded in paraffin, and 5 gm sections were stained with haematoxylin and eosin (H&E). The glutaraldehyde-fixed spheroids were embedded in epoxy resin and 2 gim sections stained with toluidine blue. Multiple sections were examined from each spheroid.

Enhancement of the anti-tumour effects of the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) by combination with 5-hydroxytryptamine and bioreductive drugs

“…In support of the latter view, growth inhibition of murine colon tumours (but not antivascular effects) is compromised by T-cell depletion (Pratesi et al, 1990;Bibby et al, 1991) although similar studies by Ching et al (1992) showed little loss of activity of either FAA or DMXAA against colon 38 tumours in nude or thymectomized mice. Further, nonvascularized tumour tissue is relatively insensitive to FAA-induced killing in mice (Finlay et al, 1988;Zwi et al, 1989Zwi et al, , 1990, and a correlation has been demonstrated between blood flow inhibition by FAA and growth delay using a range of non-immunogenic mouse tumours (Hill et al, 1989). In the present study the effect of 5-HT on the anti-tumour effects of DMXAA was qualitatively similar for all three end points examined (blood flow inhibition at 4 h, necrosis at 12 h and growth delay to 3 x treatment size), although the magnitude of the dose-modifying effects (>3, 2.3 and 2.0 respectively) were not identical.…”

Necrosis in non-tumour tissues caused by flavone acetic acid and 5,6-dimethyl xanthenone acetic acid

“…The former type appeared to have accumulated over time, and the necrotic material furthest from the viable tissues showed the greatest degree of nuclear breakdown. Drug-induced necrososis was more uniform and less advanced, as described previously in spheroids (Zwi et al, 1990).…”

“…The functional vessels of tumour and non-tumour tissues in untreated mice were demonstrated by fluorescence microscopy on frozen sections, as described previously (Zwi et al, 1990). Briefly, a single injection (0.0 Iml g' body weight) containing Hoechst 33342 (H33342) 3.25mM and 10-nonyl acridine orange (NAO) 2 mM in 5% w/v D-glucose and 4% dimethylsulphoxide, was given five minutes before killing and the tumours and normal tissues were excised, frozen and sectioned.…”

“…EMT6 spheroids were treated with FAA i.v. or i.p., as described previously (Zwi et al, 1990). Tumour and nontumour tissues were excised, fixed in 4% formaldehyde, embedded in paraffin, stained with haematoxylin and eosin and examined by light microscopy.…”

Tumour vasculature - a potential therapeutic target

“…IL-2 has also been linked with endothelial damage, possibly by the stimulation of endogenous lymphokine-activated killer (LAK) cells (Kotasek et al, 1988). 1989;Zwi et al, 1990) and has been suggested as the prototype anti-vascular agent (Denekamp, 1990 (Murray et al, 1987). The accumulating evidence of a vascular effect in different forms of cancer therapy, and the realisation that some agents may operate by an exclusively anti-vascular action, has prompted the suggestion that novel chemicals be tested for anti-vascular effects over and above routine screening for potential tumoricidal properties (Denekamp, 1990 (Denekamp, 1993).…”