Targeted Knock-Down of CD44 Expression In DLBCL Cells with Retrovirus-Mediated shRNA Interference

Abstract 1997

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma, characterized by heterogeneity in its clinical, immunophenotypic, and genetic features. Gene-expression profiling studies have distinguished 3 molecular subtypes of DLBCL known as germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and type III. When treated with CHOP or other CHOP-like regimens, patients with GCB-DLBCL have a better survival than those with ABC-DLBCL. CD44 is a widely expressed type I transmembrane glycoprotein and functions as the major hyaluronan receptor on many cell types. In a number of solid tumors, CD44 plays an important role in tumor invasion and metastasis. In DLBCL, CD44 is expressed at significantly higher levels in ABC-DLBCL compared to GCB-DLBCL. In addition, co-expression of CD44 splice variants with the hyaluronan receptor predicts a very poor prognosis in patients treated with standard CHOP therapy. Despite these published observations, there is only limited understanding regarding the role of CD44 in the cellular behavior and gene expression control of DLBCL cells. To address this issue, we first profiled cell surface expression of CD44 in 4 DLBCL cell lines by flow cytometry. Two GCB-DLBCL lines (OCI-Ly1 and OCI-Ly8) and two ABC-DLBCL lines (SUDHL-2 and OCI-Ly3) were analyzed. The percentage of CD44 positive cells in SUDHL-2, OCI-Ly3, OCI-Ly1 and OCI-Ly8 cells was 74.57±5.03%, 44.01±3.70%, 0.12±0.05%, 0.14±0.03%, respectively. Next, we evaluated the invasion ability of 3 cell lines by the Matrigel Transwell assay. For the SUDHL-2, OCI-Ly1, and OCI-Ly8 cells, the percentage of invaded cells in 4 hours was 72.7±11.7%, 20.0±8.6%, and 30.3±7.5%, respectively. The ABC-DLBCL line, SUDHL-2, was significantly more invasive compared with OCI-Ly1 and OCI-Ly8 (P<0.01). No significant difference was found between the two GCB-DLBCL lines, OCI-Ly1 and OCI-Ly8 (P=0.238). To study the role of CD44 in cell invasion, we down-regulated the expression of CD44 with retrovirus -mediated short hairpin RNA (shRNA) interference. After stable integration of retroviral vectors, stable subclones were obtained by puromycin selection. Effect of the knock-down on CD44 expression in ABC-DLBCL cells (SUDHL-2) was confirmed by flow cytometry. For SUDHL-2, CD44 level in shRNA expressing cells was reduced to 51.72±0.49% (one week), 38.31±1.24% (two weeks), and 16.29±11.17% (three weeks), respectively. In conclusion, we have confirmed the differential expression pattern of CD44 in DLBCL subtypes using cell line models. Our data also indicates that the level of CD44 expression positively correlates with cell invasion capability, such that ABC-DLBCL cells are more invasive compared with GCB-DLBCL cells. This difference may potentially contribute to the unfavorable treatment outcome of ABC-DLBCL. Lastly, we demonstrate that shRNA-mediated knock-down can stably and substantially reduce the endogenous CD44 expression on the cell surface. This experimental system is expected to provide a useful tool for further investigation of CD44 as a regulator of cellular behavior and chemotherapy response in DLBCL.