Author Manuscript Published OnlineFirst on September 26, 2017; DOI: 10.1158/0008-5472.CAN-17-1099 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Modeling cytostatic and cytotoxic responses to new treatment regimens for ovarian cancer Francesca Falcetta1, Francesca Bizzaro2, Elisa D’Agostini2, Maria Rosa Bani2, Raffaella Giavazzi2* and Paolo Ubezio1* 1 Biophysics Unit, Laboratory of Anticancer Pharmacology and 2 Laboratory of Biology and Treatment of Metastasis, Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy. * These authors share last authorship Running title: Decoding tumor growth with different treatment schemes Keywords: cell proliferation, paclitaxel, cisplatin, bevacizumab, drug scheduling, mathematical modeling Financial support: This study was supported by grants from the Italian Association for Cancer Research (AIRC IG2016 n. 18853) and by Nerina and Mario Mattioli Foundation Correspondence to: Dr. Paolo Ubezio Istituto di Ricerche Farmacologiche "Mario Negri", Via La Masa 19, 20156 Milano, Italy Phone:+39-02-39014438; Fax:+39-02-3546277 E-mail: [email protected] 1 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 26, 2017; DOI: 10.1158/0008-5472.CAN-17-1099 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT The margin for optimizing polychemotherapy is wide, but a quantitative comparison of current and new protocols is rare even in preclinical settings. In silico reconstruction of the proliferation process and the main perturbations induced by treatment provides insight into the complexity of drug response and grounds for a more objective rationale to treatment schemes. We analysed a 12 treatment groups trial on an ovarian cancer xenograft, reproducing current therapeutic options for this cancer including one-, two- and three-drug schemes of cisplatin (DDP), bevacizumab (BEV) and paclitaxel (PTX) with conventional and two levels (“equi” and “high”) of dose-dense schedules. All individual tumor growth curves were decoded via separate measurements of cell death and other antiproliferative effects, gaining fresh insight in the differences between treatment options. Individual drug treatments were cytostatic, but only DDP and PTX were also cytotoxic. After treatment, regrowth stabilized with increased propensity to quiescence, particularly with BEV. More cells were killed by PTX dose-dense-equi than with PTX conventional, but with the addition of DDP cytotoxicity was similar and considerably less than expected from that of individual drugs. In the DDP/PTX dose-dense-high scheme both cell death and regrowth impairment were intensified enough to achieve complete remission and addition of BEV increased cell death in all schemes. The results support the option for dose-dense PTX chemotherapy with active single doses, showing the relative additional contribution of BEV, but also indicate negative drug interactions in concomitant DDP/PTX treatments, suggesting that sequential schedules could improve antitumor efficacy. 2 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 26, 2017; DOI: 10.1158/0008-5472.CAN-17-1099 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. INTRODUCTION Though new targeted drugs are under study for ovarian cancer, first-line options are still based on a combination of the cytotoxic drugs with proven activity: platinum compounds, carboplatin and cisplatin (DDP), and paclitaxel (PTX). Conventional treatments are based on repeated of 21-day cycles where carboplatin and PTX are both given once on day 1. A dose-dense schedule of PTX treatment has been proposed, splitting the PTX dose into three parts in each cycle (1). A debate is ongoing and clinical trials have demonstrated some superiority of the dose-dense scheme; some trials adopting a dose-dense scheme with total dose equivalent (2) others 50% higher (3) respect to the conventional one. However, clinical studies aimed at optimizing the schedule are rare, the actual choices often being empirically driven by practice. A further element of complexity was introduced with the inclusion of angiogenesis inhibitors such as bevacizumab (BEV) into the previous schemes, with the better timing of these drugs which is still controversial (4). While the addition of bevacizumab to chemotherapy showed benefits in term of progression-free survival (5,6), leading to its incorporation in the primary treatment of ovarian cancer, the advantage of each of the different schedules and doses of chemotherapy in combination with bevacizumab was recently argued (7,8). Clinical studies rely on late outcomes, with no direct measure of the antitumor activities of a treatment, e.g. the fraction of tumor cells killed. The different schemes have not been compared yet for such parameters of activity, in either clinical or preclinical studies. Although the difficulties of comparing several schemes/doses in a single study are probably insurmountable in the clinical setting, this is not impossible in preclinical studies. The present study demonstrates its feasibility, directly comparing single vs. double vs. triple treatments with DDP, PTX and BEV, conventional or dose-dense PTX, and two dose-dense levels of PTX, on the basis of different parameters of antitumor activity obtained from analysis of the growth curves of individual tumors. This required a reconsideration of the methods currently used to evaluate growth curves, like the popular 3 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 26, 2017; DOI: 10.1158/0008-5472.CAN-17-1099 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. T/C ratio at an arbitrary time, which catches only a small piece of the information conveyed by the curves and depends strongly on the growth rate of untreated tumors. A tumor growth curve during and after treatment reflects –not in a trivial way- the underlying drug- induced phenomena, like cell cycle arrest, cell kill and changes in the tumor microenvironment (including extracellular matrix, tumor vasculature and all kinds of host cells (9) ), which intertwine with drugs’ dose- and time- dependence to determine the growth curve. It is clear therefore that evaluation of the response at a single time point –as in most preclinical studies- only very partially exploits the available data and may be misleading. This is particularly true with targeted drugs, such as the angiogenesis inhibitors, which mainly show a cytostatic-long lasting, rather than a cytotoxic effect (10,11). Integration of data with mathematical modeling of the relevant processes in principle would allow to extract the full information conveyed by each growth curve and to frame the comparisons between treatment groups on a common and objective ground. Moving in that direction, in the present study we reproduced the growth curves of individual tumors, in a wide preclinical trial, in terms of a computer model rendering the proliferation process, exploiting methods previously developed in our laboratory (12–16) to extract a few parameters measuring separately the main drug effects. The approach provided a new insight into the response to single and combined treatments and allowed in-depth comparison of the different treatment options, contributing to the controversial issues on the use of dose-dense vs. conventional PTX treatment with the addition of an angiogenesis inhibitor. 4 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 26, 2017; DOI: 10.1158/0008-5472.CAN-17-1099 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. MATERIALS AND METHODS Experimental procedures Six- to eight-week-old female NCr-nu/nu mice were from Envigo Laboratories. Mice were maintained under specific-pathogen-free conditions, housed in isolated vented cages, and handled using aseptic procedures. Procedures involving animals and their care were conducted in conformity with institutional guidelines that comply with national (DLgs. 26,/2014) and international (EEC Council Directive 2010/63) laws and policies. Animals studies were approved by the Italian Ministry of Health (decree no. 84-2013). The human tumor xenograft MNHOC18, derived from a primary ovarian tumor in a patient with high-grade serous ovarian carcinoma, was recovered from frozen stocks and used within 5-6 mouse passages after establishment from the patient. This xenograft, transplanted subcutaneously in female NCr-nu/nu mice, was molecularly, biologically and pharmacologically characterized and found similar to the original patient tumor (17). MNHOC18 bearing mice were randomized at 100-300 mm3 tumor volume, six mice per group, in 12 treatment groups and one untreated group. PTX (Indena S.p.A.), dissolved in 50% Cremophor and 50% ethanol and diluted with saline, DDP (Sigma-Aldrich) and BEV (Avastin, Roche S.p.A.), dissolved in saline, were injected intravenously (i.v.) at different schedules and doses, as specified in Table