Abstract 3347: Establishment of four novel HPV-16 positive cell lines of invasive cervical carcinoma of Indian origin and characterization of CD133 positive cancer stem cells
Authors: Javed Shifa , Sharma Bal Krishan , Sharma Sanjeev Kumar , Sood Swati , Bagga Rashmi , Bhattacharyya Shalmoli , Srinivasan Radhika 
Published: July 2016

Abstract
Background and Aim: Cancer of the cervix is the 2nd most common cancer in Indian women. Cell lines are essential in vitro models in cancer research. Till date, available cell lines in cervical cancer were highly passaged, developed from Caucasian, Black or Asian origin; hence, establishment of a low-passage cervical cancer derived cell line was the first aim of this study. Cancer stem cells (CSCs) are proposed to be responsible for metastasis and therapy resistance. Previously, we reported CD133 to be a putative marker for CSC, and they were further characterized.
Materials and Methods: 25 women were recruited after informed consent and approval from the Institute Ethics committee. A small portion of the biopsy was collected in DMEM/F12 media and subjected to primary culture. Successful propagation was possible in 4 cases. Their morphology, epithelial nature and HPV status were evaluated. Variable Number Tandem Repeat (VNTR) assay, cell cycle and kinetic studies were performed. Tissue expression of CD133 and CD49f stem cell markers was evaluated by direct immunofluorescence on frozen tumor samples. Adherent cells were sorted into CD133+ vs CD133- tumor bulk from all 4 cell lines by FACS. These two populations were evaluated for differences in tumorosphere formation, chemosensitivity to cisplatinum, expression of stemness (SOX2, OCT4, NANOG) and EMT (SNAIL, SLUG, TWIST, VIMENTIN and E-CADHERIN) markers at the transcript level.
Observations: Four cell lines designated RSBS-9, RSBS-14, RSBS-23 derived from non-keratinizing squamous cell carcinoma and RSBS-43 derived from adenocarcinoma cervix were established and passaged up to 50 times. The epithelial nature of cell lines was confirmed by cytokeratin and epithelial membrane antigen positivity. VNTR assay confirmed derivation of cell lines from respective parental tissue sample. All 4 cell lines were HPV-16positive. The cell doubling time was approximately 48 hours. Immunofluorescence on tissue samples revealed diffuse positivity for CD49f indicating that it may not represent a CSC marker as previously reported whereas CD133 staining showed scattered positive cells. No particular location of CD133+ CSC was observed. Comparison of CD133+ with CD133- bulk population cells revealed increased tumorosphere formation; however, no significant difference in the chemosensitivity to cisplatinum was seen. Real time quantitation of transcripts of stemness and EMT markers revealed CD133+ cells showing upregulation of EMT markers VIMENTIN, SNAIL, SLUG in the RSBS-23 cell line and of VIMENTIN and TWIST in RSBS-9 cell line whereas there was no significant difference in the RSBS-14 and RSBS-43 cell lines for any of the markers studied.
Conclusion: Low passage cell lines are useful model systems for understanding the biology of cervical cancer. CD133+ Cancer Stem cells exhibit some EMT markers in 2 of the 4 cell lines established.
Citation Format: Shifa Javed, Bal Krishan Sharma, Sanjeev Kumar Sharma, Swati Sood, Rashmi Bagga, Shalmoli Bhattacharyya, Radhika Srinivasan. Establishment of four novel HPV-16 positive cell lines of invasive cervical carcinoma of Indian origin and characterization of CD133 positive cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3347.