S. Ananthasekar1, S. N. Banerjee2, S. Collawn3 1University of Alabama School of Medicine,Birmingham, AL, USA 2University Of Alabama at Birmingham,Department Of Biochemistry And Molecular Genetics,Birmingham, AL, USA 3University Of Alabama at Birmingham,Divison Of Plastic Surgery, Department Of Surgery,Birmingham, AL, USA
Introduction: With over one million new cases of nonmelanoma squamous cell carcinoma (SCC) annually and no guidelines for early detection of skin cancer, there is an urgent need of in vitro modeling of these cancers to accelerate the understanding of SCC. CD44, an epithelial cell surface marker, is a trans-membrane glycoprotein involved in cell-cell interaction, cell adhesion, and migration in normal epidermis. However, its function is altered during tumor progression. There is uncertainty on how CD44 overexpression regulates cancer cell proliferation and metastasis in vivo. In order to determine the exact impact of CD44 in skin cancer, it is first necessary to evaluate the maintenance of the tumor phenotype in an in vitro model.
Methods: Excised tumors were grown in 3D organotypic raft cultures (raft tumor) and harvested at 4, 6, or 8 weeks of growth. Formalin-fixed paraffin embedded original and raft tumors were cut into 4-micron thin sections for analysis. Histopathology using hematoxylin and eosin (H&E) was done on both the original and the raft tumors. Next, immunofluorescent histochemistry was done using a mouse monoclonal antibody to CD44 (Abcam) which was detected with an Alexa Fluor 488 conjugated anti-mouse antibody (Thermo Fisher). Slides were observed using a fluorescent microscope (BX 63). Images were captured with DP73 Olympus camera and CellSens software at 10X and 20X magnifications.
Results: (1) H&E images of the original tumor and raft tumor revealed similar invasion of the tumor into the dermis with classic characteristics of SCC such as keratin pearls, nests of tumor cells, and basal cell dysplasia. (2) CD44 is expressed in areas classified as SCC based on histopathology in both original and raft tumors.
Conclusion: SCC phenotype is maintained in vitro since both CD44 immunofluorescent histochemistry and H&E staining show similar characteristics in the in vivo and in vitro tumors. In vitro SCC models have the potential to not only study the role of CD44 in tumor growth and progression but also, cellular morphology and proliferation rate, upregulated pathways, and oncogenic factors released by the tumor.