Radiation Biology

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SU_43_2437 - Novel strategy to develop orthotopic prostate tumor using androgen dependent LNCaP transduced with miR133b

Sunday, October 21
1:15 PM - 2:45 PM
Location: Innovation Hub, Exhibit Hall 3

Novel strategy to develop orthotopic prostate tumor using androgen dependent LNCaP transduced with miR133b
S. Samanta1, M. Creed2, A. Anvari3, J. Mahmood4, T. Kingsbury5, and A. Sawant6; 1University of Maryland, Dept. of Radiation Oncology, Baltimore, MD, 2University of Maryland, Baltimore, Baltimore, MD, 3University of Maryland, Dept of Radiation Oncology, Baltimore, MD, 4University of Maryland Department of Radiation Oncology, Baltimore, MD, 5University of Maryland, Dept of Physiology, Baltimore, MD, 6Department of Radiation Oncology, University of Maryland, School of Medicine, Baltimore, MD

Purpose/Objective(s): Most xenograft preclinical models of prostate cancer (PCa) are developed using androgen-insensitive cells (e.g., PC3), because they are faster growing. However, androgen-insensitive cells represent only a small fraction (<5%) of all PCa cases. Local or locally advanced PCa patients (over 90% of prostate cancer cases) exhibit androgen sensitivity. Unfortunately, androgen sensitive PCa cell lines exhibit relatively much slower growth and are therefore difficult to implement in vivo. In this work, we present a novel technique to significantly enhance the proliferation of human-derived, androgen-sensitive LNCaP cells by targeting and increasing the expression of micro RNA (miR) 133b. Our objective, beyond the current scope, is to use these proliferative cells to develop clinically translatable orthotopic prostate cancer models in immunocompetent small animals (mice, rats).

Materials/Methods: LNCaP cells grown in RPMI media with 10%FBS dihydrotestosterone (DHT) were transduced with Lentivirus expressing miR133b along with Green Fluorescent protein(GFP) and other necessary package components. After 72 hours, the transduction efficiency was quantified via flow cytometry to measure GFP expression. Cells expressing more than 90% GFP were selected and re-cultured for further characterization. Quantification of miR133b expression was performed via qPCR (Taqman Assay). Transduction stability was quantified via GFP competition assay by performing weekly flow cytometry. Cell proliferation rate for the LNCap cells expressing miR133b was compared to the parental cells using MTS assay at 10 and 12 days.

Results: LNCap cells transduced with miR133b at a titer of 6-8ul/ml showed 90% GFP positivity compared to the control(WCC52). qPCR measurements showed increased expression of miR133b in LNCap transduced cells (relative expression of 2,300 at a titer of 8ul/ml) compared to WCC52. LNCap cells expressing miR133b showed increased proliferation rate by 21% and 27% compared to the WCC52 at 10 days and 12 days, respectively, when measured using MTS assay at 490 nm. GFP competition assay using flow cytometry done over 2 weeks confirmed stable transduction.

Conclusion: We successfully developed LNCap cells expressing higher miR133b, that show increased proliferation. These early results suggest the possible role of miR133b in aggressive androgen dependent prostate cancer. Future work involves using this modified LNCap cell line to develop a clinically relevant, androgen-dependent orthotopic prostate tumor model in immune competent mice and rats.

Author Disclosure: S. Samanta: None. M. Creed: None. A. Anvari: None. T. Kingsbury: None.

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