Bhas 42 cell transformation assay (CTA) has been used to estimate the carcinogenic potential of chemicals by exposing Bhas 42 cells to carcinogenic stimuli to form colonies, referred to as transformed foci, on the confluent monolayer. Transformed foci are classified and quantified by trained experts using morphological criteria.
Although the assay has been certified by international validation studies and issued as a guidance document by OECD, this classification process is laborious, time-consuming, and subjective.
We propose using deep neural network to classify foci more rapidly and objectively.
To obtain datasets, Bhas 42 CTA was conducted with a potent tumor promotor, 12-O-tetradecanoylphorbol-13-acetate, and focus images were classified by experts (1405 images in total).
The labeled focus images were augmented with random image processing and used to train a convolutional neural network (CNN). The trained CNN exhibited an area under the curve score of 0.95 on a test dataset significantly outperforming conventional classifiers by beginners of focus judgment.
The generalization performance of unknown chemicals was assessed by applying CNN to other tumor promotors exhibiting an area under the curve score of 0.87.
The CNN-based approach could support the assay for carcinogenicity as a fundamental tool in focus scoring.
In Vitro Osteoinductivity Assay of Hydroxylapatite Scaffolds, Obtained with Biomorphic Transformation Processes, Assessed Using Human Adipose Stem Cell Cultures
- In this study, the in vitro biocompatibility and osteoinductive ability of a recently developed biomorphic hydroxylapatite ceramic scaffold (B-HA) derived from transformation of wood structures were analyzed using human adipose stem cells (hASCs).
- Cell viability and metabolic activity were evaluated in hASCs, parental cells and in recombinant genetically engineered hASC-eGFP cells expressing the green fluorescence protein. B-HA osteoinductivity properties, such as differentially expressed genes (DEG) involved in the skeletal development pathway, osteocalcin (OCN) protein expression and mineral matrix deposition in hASCs, were evaluated.
- In vitro induction of osteoblastic genes, such as Alkaline phosphatase (ALPL), Bone gamma-carboxyglutamate (gla) protein (BGLAP), SMAD family member 3 (SMAD3), Sp7 transcription factor (SP7) and Transforming growth factor, beta 3 (TGFB3), and Tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11)/Receptor activator of NF-κB (RANK) ligand (RANKL), involved in osteoclast differentiation, was undertaken in cells grown on B-HA.
- Chondrogenic transcription factor SRY (sex determining region Y)-box 9 (SOX9), tested up-regulated in hASCs grown on the B-HA scaffold.
- Gene expression enhancement in the skeletal development pathway was detected in hASCs using B-HA compared to sintered hydroxylapatite (S-HA).
- OCN protein expression and calcium deposition were increased in hASCs grown on B-HA in comparison with the control.
- This study demonstrates the biocompatibility of the novel biomorphic B-HA scaffold and its potential use in osteogenic differentiation for hASCs. Our data highlight the relevance of B-HA for bone regeneration purposes.
Establishment of a Transformation Coupled in vitro End Joining Assay to Estimate Radiosensitivity in Tumor Cells.
Here, we present a modified in vitro end-joining (EJ) assay to quantify EJ capacity, accuracy as well as pathway switch to alternative end-joining (Alt-EJ) or single-strand annealing (SSA).
A novel transformation assay was established to specifically measure circular repair products, which correlate with classical EJ efficiency. The EJ assay was validated using EJ-deficient mammalian cell lines (Ku80, DNA-PKcs, LigIV, or XRCC4 mutants).
A pathway switch to Alt-EJ and SSA was seen exclusively in Ku-deficient cells. Circular EJ product formation correlated with cell survival and DSB repair capacity after X-irradiation.
Investigation of 14 HNSCC cell lines revealed differences in the total EJ capacity but a broader variation in the amount of circular repair products. Sequencing of repair junctions in HNSCC cells demonstrated a predominance of high-fidelity EJ and an avoidance of both Alt-EJ and SSA. A significant correlation was observed between the amount of circular repair products, repair of IR-induced DSB and radiosensitivity.
Collectively, these data indicate that the presented in vitro-EJ-assay can not only estimate the repair capacity of cancer cells to enable the stratification into radiosensitive or radioresistant, but can also identify repair pathway deregulation such as a switch to Alt-EJ or SSA, which enables tumor targeting.
Comparison of In Vitro Cell Transformation Assay Using Murine Fibroblasts and Human Keratinocytes.
The in vitro cell transformation assays (CTA) were performed using BALB/3T3 murine fibroblasts and HaCaT human keratinocytes in order to evaluate concordance between both in vitro CTAs and carcinogenicity with compounds differing in their genotoxic and carcinogenic potential.
Six test articles were evaluated, two each from three classes of compounds: genotoxic carcinogens (2-amino-5-nitrophenol and 4-nitroquinoline-N-oxide), genotoxic noncarcinogens (8-hydroxyquinoline and benzyl alcohol), and nongenotoxic carcinogens (methyl carbamate and N-nitrosodiphenylamine). Any foci of size ≥ 2 mm regardless of invasiveness and piling was scored as positive in CTA with BALB/3T3.
As expected, four carcinogens regardless of their genotoxicity had positive outcomes in two-stage CTA using BALB/3T3 cells.
However, of the two genotoxic noncarcinogens, benzyl alcohol was positive CTA finding. We concluded that, of the 6 chemicals tested, the sensitivity for BALB/3T3 system was reasonably high, being 100%. The respective specificity for BALB/3T3 assay was 50%.
We also investigated the correlation between results of BALB/3T3 assay and results from HaCaT assay in order to develop a reliable human cell transformation assay.
However, evaluation of staining at later time points beyond the confluency stage did not yield further assessable data because most of HaCaT cells were detached after 2~3 days of confluency.
CytoSelect 96-well Cell Transformation Assay |
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| CBA-130 | Cell Biolabs | 96 assays | 866.4 EUR |
CytoSelect 96-well Cell Transformation Assay |
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| CBA-130-5 | Cell Biolabs | 5 x 96 assays | 3463.2 EUR |
CytoSelect 384-well Cell Transformation Assay, Fluorometric |
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| CBA-145 | Cell Biolabs | 384 assays | 1208.4 EUR |
CytoSelect 384-well Cell Transformation Assay, Fluorometric |
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| CBA-145-5 | Cell Biolabs | 5 x 384 assays | 4681.2 EUR |
CytoSelect Cell Transformation Assay (Cell Recovery Compatible), Colorimetric |
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| CBA-135 | Cell Biolabs | 96 assays | 985.2 EUR |
CytoSelect Cell Transformation Assay (Cell Recovery Compatible), Colorimetric |
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| CBA-135-5 | Cell Biolabs | 5 x 96 assays | 4027.2 EUR |
CytoSelect Cell Transformation Assay (Cell Recovery Compatible), Fluorometric |
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| CBA-140 | Cell Biolabs | 96 assays | 1027.2 EUR |
CytoSelect Cell Transformation Assay (Cell Recovery Compatible), Fluorometric |
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| CBA-140-5 | Cell Biolabs | 5 x 96 assays | 4179.6 EUR |
CytoSelect Cell Transformation Assay (Cell Recovery Compatible), Colorimetric, Trial Size |
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| CBA-135-T | Cell Biolabs | 24 assays | 518.4 EUR |
CytoSelect 96-Well Cell Transformation Assay (Cell Recovery Compatible, Fluorometric), Trial Size |
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| CBA-140-T | Cell Biolabs | 24 assays | 547.2 EUR |
Cell Transformation Assay Kit (Colorimetric) |
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| K921-100 | Biovision | each | 744 EUR |
Cell Transformation Assay Kit (Fluorometric) |
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| K922-100 | Biovision | each | 718.8 EUR |
Thus, after test article treatment, HaCaT cells were split before massive cell death began.
In this modified protocol for this HaCaT system, growing attached colonies were counted instead of transformed foci 3 weeks since last subculture. Compared to BALB/3T3 assay, HaCaT assay showed moderate low sensitivity and high specificity.
Despite these differences in specificity and sensitivity, both cell systems did exhibit same good concordance between in vitro CTA and rodent carcinogenicity findings (overall 83% concordant results).
At present the major weakness of these in vitro CTA is lack of validation for regulatory acceptance and use. Thus, more controlled studies will be needed in order to be better able to assess and quantitatively estimate in vitro CTA data.