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. 2022 Feb 24;12(1):3149.
doi: 10.1038/s41598-022-06427-w.

Oncological transformation in vitro of hepatic progenitor cell lines isolated from adult mice

Rocío Olivera-Salazar  1 , Mariano García-Arranz  2   3 , Aránzazu Sánchez  4 , Susana Olmedillas-López  2 , Luz Vega-Clemente  2 , Luis Javier Serrano  2 , Blanca Herrera  4 , Damián García-Olmo  2   3   5
Affiliations

Oncological transformation in vitro of hepatic progenitor cell lines isolated from adult mice

Rocío Olivera-Salazar et al. Sci Rep. .

Abstract

Colorectal cancer cells can transfer the oncogene KRAS to distant cells, predisposing them to malignant transformation (Genometastasis Theory). This process could contribute to liver metastasis; besides, hepatic progenitor cells (HPCs) have been found to be involved in liver malignant neoplasms. The objective of this study is to determine if mouse HPCs-Oval cells (OCs)-are susceptible to incorporate Kras GAT (G12D) mutation from mouse colorectal cancer cell line CT26.WT and if OCs with the incorporated mutation behave like malignant cells. To achieve this, three lines of OCs in different conditions were exposed to CT26.WT cells through transwell co-culture for a week. The presence of KrasG12D and capacity to form tumors were analyzed in treated samples by droplet digital PCR and colony-forming assays, respectively. The results showed that the KrasG12D mutation was detected in hepatic culture conditions of undifferentiated OCs and these cells were capable of forming tumors in vitro. Therefore, OCs are susceptible to malignant transformation by horizontal transfer of DNA with KrasG12D mutation in an undifferentiated condition associated with the liver microenvironment. This study contributes to a new step in the understanding of the colorectal metastatic process.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Percentage expression of surface and liver markers for three oval cell lines (OCs-1, OCs-2 and OCs-3) for characterization. Values are expressed as mean percentages with standard deviation (SD), one-way ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, α = 0.05.
Figure 2
Figure 2
Differentiation towards hepatic, osteogenic and adipogenic lineages of three oval cell lines (OCs-1, OCs-2 and OCs-3). (A–C) Controls of OCs-1, OCs-2 and OCs-3, (D–F) PAS staining for liver differentiation, (G–I) Alizarin Red S staining for osteogenic differentiation and (J–L) Oil Red staining for adipogenic differentiation. Objective 63 ×.
Figure 3
Figure 3
Droplet digital PCR (ddPCR) for the detection of mouse KrasG12D in control OCs-1 and treated OCs-1 with CT26.WT cells to confirm the malignant transformation. Black points (droplets without Kras amplification), green points (droplets containing KrasWT molecules), blue points (droplets containing amplified KrasG12D) and orange points (droplets containing both KrasWT and KrasG12D molecules).
Figure 4
Figure 4
Changes observed in KrasG12D OCs-1 with respect to KrasWT OCs-1. Three replicates of KrasWT OCs-1 and KrasG12D OCs-1, Student’s t-test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, α = 0.05. (A) Mean of expression percentages and SD of hepatic and surface markers. (B) Variation of gene expression of mouse EMT markers (N-Cadherin, Snail, Vimentin, E-Cadherin and Twist) between 2 and 4 weeks of culture (2 W and 4 W respectively) analyzed by RT-qPCR normalized to Gapdh housekeeping gene. (C) Morphology changes observed at optical microscopy (O.M) at 48 h and 4 weeks of culture. Objective 40 ×.
Figure 5
Figure 5
Alamar Blue assay for the proliferation of KrasWT OCs-1 and KrasG12D OCs-1. Mean and SD of three replicates of KrasWT OCs-1 and KrasG12D OCs-1. Relative fluorescence units (RFU). Student’s t-test *** p-value = 0.0003, (α = 0.05).
Figure 6
Figure 6
Noble Agar assay for KrasWT OCs-1 (A–C) versus KrasG12D OCs-1 (D–F). Photos by Loupe, Objective 16 × (A and D) and optical microscope (O.M), objectives 10 × (B and E) and 40 × (C and F).
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