Spreading of Isolated Ptch Mutant Basal Cell Carcinoma Precursors Is Physiologically Suppressed and Counteracts Tumor Formation in Mice
Abstract
:1. Introduction
2. Results
2.1. Wildtype Ptch Progeny of CD4Cre-Targeted Cells Spread over the HF/Skin Complex with Increasing Mouse Age
2.2. The CD4Cre-Deleter Targets Keratin 5+ Epidermal Cells That Are the Origin of DMBA/TPA-Induced BCC in Ptchf/f CD4Cre Mice
2.3. Isolated Ptch Mutant Epidermal Cells do Not Spread Like Their Wildtype Ptch Counterparts
3. Discussion
4. Materials and Methods
4.1. Mice
4.2. Isolation of Keratinocytes, Epidermal Sheets and Individual Hair Follicles
4.3. Tissue Embedding and Sectioning
4.4. Primary Keratinocyte Culture
4.5. LacZ Staining, Antibody Staining, Flowcytometric Analyses and Microscopy
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
AEC | 3-amino-9-ethylcarbazole |
BCC | basal cell carcinoma |
cSCC | cutaneous squamous cell carcinoma |
CTL | cytotoxic T cells |
DK-SFM | defined keratinocyte-serum-free medium |
DMBA | 7,12-Dimethylbenz[a]anthracene |
DN | double-negative |
DP | double-positive |
EDAC | epithelial defense against cancer |
EGFP | enhanced GFP |
FITC | fluorescein isothiocyanate |
FSC | forward scatter |
GFP | green fluorescent protein |
H&E | hematoxylin and eosin |
HF | hair follicle |
Hh | hedgehog |
IFE | interfollicular epidermis |
IRS | inner root sheath |
K5 | Keratin 5 |
ORS | outer root sheath |
PBS | phosphate buffered saline |
PE | phycoerythrin |
PerCP | peridinin-chlorophyll-protein |
Ptch | Patched |
RFP | red fluorescent protein |
SG | sebaceous gland |
SP | single-positive |
SSC | side scatter |
TBS | tris buffered saline |
TCR | T cell receptor |
tdT | tdTomato |
TPA | 12-O-tetradecanoylphorbol-13-acetate |
Appendix A
Antigen | Antibody | Reactivity | Manufacturer (Clone) | Antigen Retrieval | Dilution | Fluorochrome-Labeled Secondary Antibody (Dilution, Manufacturer, Catalogue Code) |
---|---|---|---|---|---|---|
GFP | g anti-GFP | Novus Biologicals, Littleton, CO, USA (NB100-1770) | citric acid, pH6 | 1:200 | bov anti-gt-Alexa488 (1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; #805-545-150) | |
K5 | rb anti-K5 | m/h | Bio Legend, San Diego, CA, USA (Poly19055) | citric acid, pH6 | 1:1000 | d anti-rb-Alexa488 (1:200; Jackson ImmunoResearch; #711-545-152) d anti-rb-Cy3 (1:400; Jackson ImmunoResearch; #711-165-152) |
RFP * | g anti-RFP | My BioSource, San Diego, CA, USA (MBS448122) | boric acid or citric acid, pH6 | 1:200 | d anti-g-FITC (1:200; Jackson ImmunoResearch; #705-095-147) bovine anti-g-Alexa488 (1:1000, Jackson ImmunoResearch; #805-545-150) | |
RFP * | rb anti-RFP | Rockland Immunochemicals, Inc., Gilbertsville, PA, USA (600-401-379S) | boric acid or citric acid, pH6 | 1:500 | d anti-rb-Cy3 (1:400; Jackson ImmunoResearch; #711-165-152) g anti-rb-Cy3 (1:400; Jackson ImmunoResearch; #111-165-003) |
Antigen | Antibody | Reactivity | Clone/Catalogue Code |
---|---|---|---|
CD16/CD32 | r anti-CD16/CD32-PE_Cy7 | m | 2.4G2 |
CD3ε | ha anti-CD3e-PE-Cy7 | m | 145-2C11 |
CD4 | r anti-CD4-FITC | m | RM4-5 |
CD49f | r anti-CD49f-PerCP-Cy5.5 | m/h | GoH3 |
TCRβ | ha anti-TCRβ-PE-Cy7 | m | H57-597 |
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Brandes, N.; Mitkovska, S.H.; Botermann, D.S.; Maurer, W.; Müllen, A.; Scheile, H.; Zabel, S.; Frommhold, A.; Heß, I.; Hahn, H.; et al. Spreading of Isolated Ptch Mutant Basal Cell Carcinoma Precursors Is Physiologically Suppressed and Counteracts Tumor Formation in Mice. Int. J. Mol. Sci. 2020, 21, 9295. https://doi.org/10.3390/ijms21239295
Brandes N, Mitkovska SH, Botermann DS, Maurer W, Müllen A, Scheile H, Zabel S, Frommhold A, Heß I, Hahn H, et al. Spreading of Isolated Ptch Mutant Basal Cell Carcinoma Precursors Is Physiologically Suppressed and Counteracts Tumor Formation in Mice. International Journal of Molecular Sciences. 2020; 21(23):9295. https://doi.org/10.3390/ijms21239295
Chicago/Turabian StyleBrandes, Nadine, Slavica Hristomanova Mitkovska, Dominik Simon Botermann, Wiebke Maurer, Anna Müllen, Hanna Scheile, Sebastian Zabel, Anke Frommhold, Ina Heß, Heidi Hahn, and et al. 2020. "Spreading of Isolated Ptch Mutant Basal Cell Carcinoma Precursors Is Physiologically Suppressed and Counteracts Tumor Formation in Mice" International Journal of Molecular Sciences 21, no. 23: 9295. https://doi.org/10.3390/ijms21239295