The Role Of Cd44 In Modulating Radiosensitivity In Vitro

CD44 has been correlated with radiation sensitivity, locoregional recurrence, and distant metastasis in clinical trials. However, there is a paucity of preclinical evidence investigating CD44’s causative role in controlling radiosensitivity by manipulating CD44 expression. We hypothesized that modulating CD44 receptor signaling will alter radiosensitivity. Low passage human head and neck (H&N) squamous cell carcinoma (SCC) cell lines UT-8-SCC, UT-14-SCC, and UT-15-SCC were stably transduced via lentivirus or transiently transfected using lipofectamine 3000 with human CD44v6 or shRNA against total CD44 to either overexpress or knockdown CD44. The mRNA levels were quantified via qRT-PCR. H4C4 antibody against the CD44 receptor hyaluronan binding domain was used to abrogate CD44 signaling. Cell proliferation was compared using growth curves and MTT assays. Radiosensitivity was compared using MTT assays after photon irradiation dose of 0, 2, 4, 6, or 8 Gy. Statistical analyses were done using one-way or two-way ANOVA tests. Successful transfection or transduction was confirmed by GFP expression via both immunofluorescence and flow cytometry. CD44v6 overexpression was at least 70-fold above endogenous levels and vector controls as quantified via qRT-PCR. Stable knockdown of total CD44 via lentiviral shRNA was at least 60%. CD44 antibody against the hyaluronan binding domain staining was confirmed via immunofluorescence. CD44v6 expression levels affected primary UT H&N SCC cell line proliferation rates: UT-14-SCC doubling time 39.7 hours (vector), 85.3 hours (CD44 knockdown), and 35.1 hours (CD44v6 overexpression) (P<0.001). Antibody blocking of CD44 produced a dose dependent decrease in UT-14-SCC proliferation as seen in MTT growth curves (P<0.001). H4C4 antibody blocking of CD44 receptor also caused UT-14-SCC to become more radiosensitive as seen in MTT assays after irradiation (P=0.02). UT-14-SCC was rendered either more radioresistant with CD44v6 overexpression or radiosensitive with CD44 knockdown as assessed using MTT assays after irradiation (P<0.001). In an in vitro preclinical model of primary H&N SCC cell lines, CD44v6 overexpression was sufficient to increase radioresistance while blocking total CD44 signaling decreased radioresistance. Ongoing research will examine the effect of CD44 manipulation on radiosensitivity using clonogenic assays.