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

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.

Figure 1. Time profile of stem cell proliferation responses following carcinogenic exposures.

Mitotic divisions after 1 day (day = d), 3 days, 1 week (week = w) and 2 weeks exposure to high and low concentrations of a genotoxic carcinogen (MMS), non-genotoxic carcinogen (CsA) and non-carcinogen (Dmann). The mitotic divisions after 6 weeks exposure to MMS and CsA are also represented. The number of mitotic cells was normalized against the total body area of the worms and expressed relative to the corresponding MMS, CsA and Dmann control group per time point, respectively culture medium, culture medium with 0.05% DMSO (vehicle control) and culture medium. Average cell proliferation values at each time point are connected by lines, individual measurements are also represented (≥3 per time point). At each time point, concentrations were chosen according to the criteria described in the text and classified accordingly. Significant effects are indicated in the table: ***p < 0.01; *p < 0.1. The mean and standard errors (se) of MMS control groups are: 172.0 ± 21.9 cells/mm² (1d); 368.6 ± 37.4 cells/mm² (3d); 347.5 ± 8.9 cells/mm² (1 w); 83.8 ± 26.0 cells/mm² (2 w); 80.3 ± 9.7 cells/mm² (6 w). The mean and se of CsA control groups are: 344.1 ± 42.5 cells/mm² (1d); 328.3 ± 46.6 cells/mm² (3d); 288.8 ± 17.7 cells/mm² (1 w); 180.9 ± 15.8 cells/mm² (2 w); 74.9 ± 11.6 cells/mm² (6 w). The mean and se of Dmann control groups are: 352.5 ± 42.2 cells/mm² (1d); 317.7 ± 26.9 cells/mm² (3d); 260.6 ± 44.0 cells/mm² (1 w); 135.9 ± 15.6 cells/mm² (2 w). High concentrations of MMS (≤1 w: ≥200 μM; 2 w: ≥50 μM; 6 w: ≥20 μM), low concentrations of MMS (≤3d: ≤100 μM; 1 w: ≤50 μM; 2 w: ≤20 μM; 6 w: ≤1 μM), high concentrations of CsA (≤2 w: ≥0.5 μM; 6 w: ≥0.25 μM), low concentrations of CsA (≤2 w: ≤0.25 μM; 6 w: ≤0.125 μM), high concentrations of Dmann (≤3d: ≥27.4 mM; ≥1 w: ≥16.5 mM), low concentrations of Dmann (≤3d: ≤16.5 mM; ≥1 w: ≤5.5 mM). MMS methyl methane sulphonate, CsA cyclosporine A, Dmann d-mannitol

Figure 2. Comparison of stem cell proliferation responses after 1 and 3 days of carcinogenic exposure.

Mitotic divisions after 1 and 3 days (day = d) exposure to genotoxic carcinogens (MMS, 4NQO), non-genotoxic carcinogens (CsA, S-PB) and non-carcinogens (Dmann). The number of mitotic cells was normalized against the total body area of the worms and expressed relative to the corresponding MMS, 4NQO, CsA, S-PB and Dmann control group per time point, which was culture medium for all compounds except for CsA, where culture medium with 0.05% DMSO (vehicle control) was used. Cell proliferation values of each concentration are connected by lines and are the average and standard error (se) of minimum 3 biological repeats. Significant effects are indicated in the table: ***p < 0.01; **p < 0.05; *p < 0. 1. The mean and se of MMS control groups are: 172.0 ± 21.9 cells/mm² (1d) and 368.6 ± 37.4 cells/mm² (3d). The average and se of 4NQO control groups are: 293.5 ± 21.6 cells/mm² (1d) and 452.7 ± 30.3 cells/mm² (3d). The average and se of CsA control groups are: 344.1 ± 42.5 cells/mm² (1d) and 328.3 ± 46.6 cells/mm² (3d). The average and se of S-PB control groups are: 289.6 ± 41.1 cells/mm² (1d) and 424.5 ± 10.9 cells/mm² (3d). The average and se of Dmann control groups are: 352.5 ± 42.2 cells/mm² (1d) and 317.7 ± 26.9 cells/mm² (3d). MMS methyl methane sulphonate, 4NQO 4-nitroquinoline-1-oxide, CsA cyclosporine A, S-PB sodium phenobarbital, Dmann d-mannitol

Figure 3. Stem cell activity responses at the appearance of phenotypic effects.

Stem cell proliferation responses at the appearance of phenotypic effects (cat 2) following genotoxic (MMS and 4NQO) and non-genotoxic (S-PB, CPZ and MPH) carcinogen exposures. The number of mitotic cells was normalized against the total body area of the worms and expressed relative to the corresponding non-exposed group. The values indicated in the graphs are the mean and se of min. 3 biological repeats, except for S-PB and CPZ because of severe lysis of the animals. Significant effects as compared to the corresponding control group are indicated with stars: ***p < 0.01. Exposure times were 3 days (MMS), 1 week (4NQO) and 2 weeks (S-PB, CPZ and MPH). The mean and se of groups exposed to culture medium are: 227.7 ± 26.7 cells/mm² (3 days); 212.0 ± 4.7 cells/mm² (1 w); 130.9 ± 10.2 cells/mm² (2 w). MMS methyl methane sulphonate, 4NQO 4-nitroquinoline-1-oxide, S-PB sodium phenobarbital, CPZ chlorpromazine hydrochloride, MPH methapyrilene hydrochloride

Figure 4. Workflow to predict and discriminate genotoxic (GTX), non-genotoxic (NGTX) and non-carcinogens (NC) based on in vivo proliferation responses of adult stem cells and phenotypic effects in the regenerating flatworm S. mediterranea.

Step 1 of the workflow: measurement of the amount of dividing cells in a regenerating worm after 1 and 3 days of exposure (mitotic minimum and maximum during regeneration). GTX carcinogens: the amount of cells in mitosis is lower at 3 days as compared to 1 day of exposure (expressed relative to the non-exposed group) or is lower than 1. NGTX carcinogens: the amount of cells in mitosis is higher at 3 days of exposure as compared to 1 day of exposure (expressed relative to the non-exposed group) or is higher than 1. Non-carcinogens: the amount of cells in mitosis is the same at both days (expressed relative to the non-exposed group). Step 2 of the workflow: measurement of the amount of dividing cells at the moment of phenotypic appearance. A strong drop in cell proliferation is noticed when phenotype appears during gtx exposure. Step 3 of the workflow: when carcinogens are detected, a third step using knock donws can be included to increase sensitivity or to specify the mode of action of the compound.