State-of-art: Colorectal cancer is the third most prevalent malignancy and one of the leading causes of cancer death worldwide. Aldehyde dehydrogenase 1A3 (ALDH1A3) belongs to the detoxifying enzyme responsible also for the oxidation of retinal to retinoic acid associated with the cell differentiation. ALDH1A3 play an important role in the tumour progression and it’s correlated with worse prognosis in patients and chemoresistance. Evidences indicate that the inhibition of ALDH1 activity sensitize cancer cells to various chemotherapeutics.

Model: Chemoresistant colorectal adenocarcinoma cell line HT-29/EGFP/FUR (prepared by the long-term maintenance of HT-29/EGFP cell line in 5-fluorouracil (5-FU)) was injected subcutaneously to SCID/bg mouse. Metastatic cells (FURiv-met) colonising the lung were isolated, selected and classified as a spontaneous model of metastasis. They are cross-resistant to 5-FU, cisplatin, oxaliplatin. While ALDH1A1 isoform was greatly upregulated in the parental HT-29/EGFP cells, ALDH1A3 isoform is highly upregulated in FURiv-met.

Aim: To analyse if genetic attenuation of ALDH1A3 can affect the functional traits of FURiv-met.

Methods: Lentiviral plasmid lentiCRISPRv2 (Addgene #52961) contained 2 expression cassettes for SpCas9 and sgRNA with the commercially cloned crRNA parts: GeneScript-ALDH1A3-2, ALDH1A3-4 and ALDH1A3-6. HEK293T packaging cell line were transfected with lentiCRISPRv2, psPAX2 (Addgene #12260) and pCMV-VSV-G (Addgene #8454) using CalPhos™ Mammalian Transfection Kit (Clontech Laboratories). FURiv-met were transduced by chosen lentivirus combination and selected by Puromycin. Dilution cloning was performed to generate single cell colonies. Prepared knockout clones were confirmed by QPCR and Western Blot and subjected to functional in vitro tests: the sensitivity to 5-FU and oxaliplatin using Cell viability assay (Promega) and Incucyte® cell migration and invasion assays (Essen) for all tested knockouts vs. sg controls. ALDH1A3 and ALDH1A1 expressions were detected by QPCR.

Results: We induced knockout of ALDH1A3 gene in FURiv-met by CRISPR/Cas9. Genetic editing led to more than 90% attenuation of ALDH1A3 mRNA expression and protein production in C4 and A2 knockouts.

Resistance to 5-fluorouracil decreased 6-times in C4 knockout (IC₅₀ was 6µg/ml for C4 KO vs. 37µg/ml for sg control cells). ALDH1A3 knockouts had lower migratory ability than ctrl and were not invasive in vitro. ALDH1A3 attenuation in FURiv-met led to the rise of ALDH1A1 isoform (3x in A2 KO vs. sg ctrl). ALDH1A3 attenuation resulted in ALDH1A1 isoform switch and the increased sensitivity to chemotherapy and the decline of migratory ability in vitro. That proved ALDH1A3 upregulation is associated with drug resistance and migratory ability in FURiv-met cells. More functional studies for characterization of clonogenic potential in vitro and tumorigenic and metastatic potential in vivo are planned.