Evaluation of the ecacy of potential antineoplastic drugs on tumour metastasis by a computer-assisted image analysis A. Lentini a , F. Autuori a , P. Mattioli a , M. Caraglia b , A. Abbruzzese b , S. Beninati a, * a Department of Biology, University of Rome `Tor Vergata' Via della Ricerca Scienti®ca, 00133 Rome, Italy b Department of Biochemistry and Biophysics, University of Naples, Via Costantinopoli, 80132 Naples, Italy Received 15 November 1999; received in revised form 15 March 2000; accepted 23 March 2000 Abstract Computerised image analysis, performed on histological sections of (C57BL6/N) mouse lungs that had been intravenously (i.v.) injected with B16-F10 melanoma cells was used to develop a novel method to quantify the ecacy of potential antineoplastic drugs. This procedure allowed the evaluation of the rate of inhibition of growth and the anti-invasive capability of new molecules, thus resulting in more accurate data than that obtained from common macroscopical counting of surface metastatic foci. Several mor- phological parameters can be measured by this method: the percentage of tissue area occupied by metastases, which accounts for tumour implantation into the organ; the growth index, related to the size of the metastases, and the invasion index, related to the frequency of foci. These morphometric data were found to be correlated to the levels of lung hydroxyproline and transglutaminase activity, well known markers of tumour invasion and cell dierentiation, respectively. The main objective of this computerised procedure was to evaluate how the tumour cell is aected in the host by the drug under investigation. The use of the method is exempli®ed by an analysis of the antitumour activity of some methylxanthines. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Melanoma; Metastasis; Image analysis; Tumour growth; Antineoplastic drugs 1. Introduction The metastatic spread of cancer cells to dierent organs represents the major cause of death in cancer patients. Despite the advances recently achieved in the treatment of tumours, the management of malignancies still remains a dicult task for the oncologist [1,2]. Indeed, one of the aims of most cancer researchers is the development of a suitable technical model which could allow both the evaluation of the metastatic potential of tumour cells, and the antineoplastic eect of new molecules. Computer-assisted image analysis is currently used for the quanti®cation of results for a wide range of biologi- cal experimental procedures [3±7]. In the present study, this technique coupled with a valid experimental metas- tasis model, like lung colony formations following intravenous (i.v.) injection of B16-F10 melanoma cells into syngeneic mice, has been shown to be a simple and reliable tool for a more accurate analysis of lung colo- nies formed by tumour cells. Moreover, the main advantage of this method is the possibility of evaluating how the antineoplastic activity of new drugs is exerted in vivo, discriminating between inhibition of tumour growth and anti-invasive properties. This information may be very useful to investigate whether the eects of the antineoplastic molecule could be quanti®ed and manipulated selectively, in the hope of establishing more targeted anticancer therapeutical interventions. We have previously shown that a set of morphometric parameters can be measured on histological sections of mouse target organs that have been colonised by B16- F10 melanoma cells [8]. In this report, we describe a method which facilitates the interpretation of these parameters for the objective and quantitative in vivo evaluation of invasion and tumour growth for pharma- cological studies. 2. Materials and methods 2.1. Materials Theophylline, caeine and theobromine were purchased from Sigma (St Louis, MO, USA). [ 3 H]-putrescine 2 0959-8049/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0959-8049(00)00147-7 European Journal of Cancer 36 (2000) 1572±1577 www.ejconline.com * Corresponding author. Tel.: +39-6-72594350; fax: +39-6-2023500. E-mail address: beninati@bio.uniroma2.it (S. Beninati).