Growth studies of subcutaneous rat tumours: comparison of 31P-NMR spectroscopy, acid extracts and histology

Abstract

Summary 31P-NMR surface coil spectra of three subcutaneously implanted rat tumours (Morris hepatoma 7777, GH3 prolactinoma, Walker carcinosarcoma) and an N-methyl-N-nitrosourea induced rat mammary adenocarcinoma at different stages of growth were obtained and compared with histological sections taken immediately after NMR acquisitions. Metabolite ratios (phosphocreatine (PCr)/Pnucleoside triphosphate (PNTP), PCr/Pi, PNTP/Pi) calculated from the NMR spectra were compared with ratios obtained from acid extracts of tumours of similar size. Measurements of creatine and ADP were also made. Three of the tumours showed positive correlations between increasing tumour size and decreasing metabolite ratios measured both by NMR and in extracts, whereas the Walker carcinosarcoma showed no correlation between size and any parameters measured. Phosphorus metabolite ratios, measured in extracts of skin overlying the tumours, indicated a fall in high energy phosphate when there was histological evidence of skin invasion by the tumour. Surface coil 31P-NMR spectra of subcutaneously grown or induced tumours in the rat represent a slowly changing steady state as the tumour increases in size. We conclude that increasing numbers of hypoxic tumour cells, rather than large areas of necrotic tissue, contribute largely to the NMR spectrum.3'P-NMR spectroscopy has been used to monitor growth and response to therapy in several experimental tumour lines (Ng et al., 1982; Steen et al., 1988;Wehrle et al., 1987;Rodrigues et al., 1988;Tozer et al., 1989). Tumour response to various types of chemotherapy and X-irradiation therapy often involves the reversal of the changes seen during untreated progression; the treated tumour appears more highly energised (i.e. has increased phosphocreatine/pnucleoside triphosphate (PCr/PNTP), PCr/Pi or PNTP/Pi; see Methods) than before treatment.The changes in the 3'P-NMR spectra that occur as animal tumours grow could be due to several causes. Most tumours described in the literature have shown a fall in high energy phosphates with increasing age and size which is usually attributed to increasing hypoxic or necrotic fractions (Ng et al., 1982;Rofstad et al., 1988a). Other factors, such as host cell invasion, oedema, haemorrhage and cyst formation, will also affect the spectrum.Our recent studies (Stubbs et al., 1988a(Stubbs et al., , 1989 on the contribution of rat skin to surface coil spectra of subcutaneous tumours suggest that this, too, could be important in studies on animals.If the tumour invades and destroys the skin (and particularly the panniculus carnosus muscle which forms a major part of rodent skin) the skin contribution to the 3'P-NMR spectrum will decrease with increasing tumour size. In order to elucidate these processes we have embarked on a series of studies of tumour growth.Here we report the results of NMR studies of a variety of subcutaneously implanted rat tumours in vivo, at different time points in their growth, where we have compared the NMR spectra with histological sections of the t...

An assessment of 31P MRS as a method of measuring pH in rat tumours

“…However there are many reports of mouse, rat and human turn our^^^'.^^ which have neutral to alkaline pH values, as well as the ones reported here in which there appears to be no correlation with tumour growth or size (see also Ref. 15). …”

An assessment of artefacts in localized and non‐localized 31P MRS studies of phosphate metabolites and pH in rat tumours

“…Subsequent PCr and NTP measurements were made on acid extracts of the freezeclamped tissue either by enzymatic analysis (for PCr) or by HPLC (for NTP. the sum of ATP+GTP, the predominant NTPs in these tumours) as described by Stubbs et al 23 …”

The effect of blood flow modification on intra- and extracellular pH measured by 31P magnetic resonance spectroscopy in murine tumours

“…Further studies, in which pHe will be measured on the same tumour by both 31p MRS-3-APP and microelectrodes are planned to begin shortly and we hope these studies will resolve the differences between the two methods. In this study, ISIS localisation of the tumours was chosen to ensure that the intense PCr signal from the body wall did not contribute to the tumour spectra An additional caveat is that the MR spectrum includes all the nucleotide phosphates under the peaks which accounts for the conventional label of NTP, although the major proportion of this peak is ATP (Stubbs et al, 1989). However, the extracts provide data on ATP since HPLC separates ATP from other trinucleotides.…”

“…Because the chemical shift artefact causes more distortion to the P-NTP peak than the y-NTP, the latter has been used to calculate the NTP/Pi ratios. The contribution from free NDP is considered negligible and likely to be in the micromolar range (Stubbs et al, 1989). pH measurements pHi was measured from the difference in chemical shift between the Pi resonance and that of x-NTP at -7.57 p.p.m.…”