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HUMAN EPITHELIAL CELL LINE FOR 3-D MODELIZING OF CANCER AND TREATMENT THEREOF

20190242874 · 2019-08-08

    Inventors

    Cpc classification

    International classification

    Abstract

    A cellular model is described that targets dysregulation or inappropriate activation of the Sonic Hedgehog/Patched (SHH/PTCH) pathway. Also described is a screening method using this cellular model to screen for pharmacological compounds that can treat or prevent skin cancer, in particular, Basal Cell Carcinoma (BCC) lesions.

    Claims

    1. An immortalized cell line of human nevoid basal cell carcinoma syndrome keratinocytes (KNBCCS6 E6/E7), wherein the cell line has a heterozygous mutation in the PATCHED1 (PTCH 1) gene; and the cell line has stable expression overtime, even after a high rate from passages in tissue culture.

    2. The immortalized cell line of claim 1, wherein the cell line is immortalized by retroviral transduction of pLE6/E7SN.

    3. The immortalized cell line of claim 1, wherein the cell line is produced in the absence of serum or without using feeder cells.

    4. The immortalized cell line of claim 3, wherein the cell line expresses PTCH 1 mRNA.

    5. The immortalized cell line of claim 4, wherein the cell line expresses the other members of the pathway SHH/PTCH necessary to appropriately inhibit and activate the pathway.

    6. A process for obtaining the immortalized human keratinocytes of claim 1, the process comprising the following steps: a. isolating human primary keratinocytes from a healthy individual having Nevoid Basal Cell Carcinoma Syndrome (NBCCS), b. immortalizing human primary keratinocytes from NBCCS patients by retroviral transduction with pLE6/E7SN, and c. selecting a cell line expression with a medium of selection.

    7. A drug screening method, the method comprising screening for a drug with the immortalized cell line of claim 1.

    8. The drug screening method of claim 7, the method comprising the following steps of: a. bringing one sample of organotypic skin cultures reconstituted using the immortalized cell line of claim 1 into contact with one or more test compounds or with a mixture of compounds, b. measuring histology of a skin equivalent to assess development of epidermal invasion in the skin equivalent or other conservative method after stable labeling of the cell line using GFP, and c. selecting the compounds for which a modulation of epidermal invasion is measured in step b) when compared to the epidermal invasion in the absence of any test compounds or mixture of compounds.

    9. The drug screening method of claim 8, wherein the sample is analyzed in vitro or in vivo in real time without need of sample fixation and/or animal vector sacrifice.

    10. The drug screening method of claim 7, wherein the identified drug is an antitumor drug.

    11. An in vitro method for screening candidate compounds for preventive and/or curative treatment of cutaneous cancer, the method comprising the following steps of: a. bringing one sample of the immortalized cell line of claim 1 into contact with one or more test compounds or with a mixture of compounds, b. measuring invasive properties of the cell line, and c. selecting the compounds for which a modulation of epidermal invasion is measured in step b when compared to epidermal invasion in the absence of any test compounds or mixture of compounds.

    12. A drug identified by the drug screening method of claim 7.

    13. Organotypic skin cultures, reconstituted using the immortalized cell line of claim 1, overlaying a dermal equivalent containing autologous NBCCS6 primary keratinocytes.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0071] FIG. 1: Schematic map of the LE6E7 SN proviral construct. E6E7, sequence of the human papilloma virus 16 encoding the E6 and E7 transforming proteins.

    [0072] FIG. 2: westerm blot analysis of the expression of the P53 protein in the indicated cells. GAPDH is a control of loading attenting that similar amout of protein is present in each lane.

    [0073] FIG. 3: Organotypic skin reconstruction showing invasive properties of KNBCC6 E6/E7 cell line over a demal equivalent composed of autologous NBCCS6 primary fibroblasts

    [0074] FIG. 4: Organotypic skin reconstruction and effect of SHH/PTCH inhibitor

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