Stem cell proliferation patterns as an alternative for in vivo prediction and discrimination of carcinogenic compounds

An-Sofie Stevens,Maxime Willems,Tom Artois,Jan-Pieter Ploem,Ellen Winckelmans,Michelle Plusquin,Karen Smeets

doi:10.1038/srep45616

An-Sofie Stevens, Maxime Willems + Show 5 more

Open Access

https://doi.org/10.1038/srep45616

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Abstract

One of the major challenges in the development of alternative carcinogenicity assays is the prediction of non-genotoxic carcinogens. The variety of non-genotoxic cancer pathways complicates the search for reliable parameters expressing their carcinogenicity. As non-genotoxic and genotoxic carcinogens have different cancer risks, the objective of this study was to develop a concept for an in vivo test, based on flatworm stem cell dynamics, to detect and classify carcinogenic compounds. Our methodology entails an exposure to carcinogenic compounds during the animal’s regeneration process, which revealed differences in proliferative responses between non-genotoxic and genotoxic carcinogens during the initial stages of the regeneration process. A proof of concept was obtained after an extensive study of proliferation dynamics of a genotoxic and a non-genotoxic compound. A pilot validation with a limited set of compounds showed that the proposed concept not only enabled a simple prediction of genotoxic and non-genotoxic carcinogens, but also had the power to discriminate between both. We further optimized this discrimination by combining stem cell proliferation responses with a phenotypic screening and by using specific knockdowns. In the future, more compounds will be tested to further validate and prove this concept.

Highlights

The present alternative in vitro and short term in vivo assays identify the majority of genotoxic carcinogens improvements in the predictive capacity are still needed to diminish false-positive and -negative results10. 75–95% of non-carcinogens give irrelevant positive results in at least one test of the core battery genotoxicity tests (i.e. Ames-test, mouse lymphoma cell test and in vitro chromosome aberration test), which requires expensive and time-consuming follow-up in vitro and in vivo testing[10]
Non-genotoxic carcinogens often slip through the detection net and become wrongly classified as non-carcinogens
We artificially created a proliferative environment by inducing the regeneration process in the animals. In this proliferation-promoting setting, we exposed the organisms to genotoxic and non-genotoxic carcinogens and monitored in vivo stem cell responses and organismal phenotypes

Summary

Introduction

The present alternative in vitro and short term in vivo assays identify the majority of genotoxic carcinogens improvements in the predictive capacity are still needed to diminish false-positive and -negative results10. 75–95% of non-carcinogens give irrelevant positive results in at least one test of the core battery genotoxicity tests (i.e. Ames-test, mouse lymphoma cell test and in vitro chromosome aberration test), which requires expensive and time-consuming follow-up in vitro and in vivo testing[10]. (3) The initiation and promotion stages of chemically-induced carcinogenesis are described in flatworms and underlying cancer and regeneration-related genes and signaling pathways were identified e.g. PTEN, FOXO, caspases, cyclins, (PI3K)-Akt pathway, RAS pathway, p53 pathway, MAPK pathway[19,20,21,22,23,24] (cancer and regeneration share underlying molecular events[18]). We exploited these flatworm characteristics to develop a concept for an in vivo assay to predict and classify carcinogenic compounds based on stem cell responses in the flatworm Schmidtea mediterranea. Possibilities to further subdivide each carcinogenic group according to mechanisms of action (MOA) were tested by comparing carcinogen-induced cell proliferation patterns in cancer-related gene knockdowns

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