The “Side-Population” of Human Lymphoma Cells Have Increased Chemo-Resistance, Stem-Cell Like Properties and Are Potential Targets for Immunotherapy
Authors: Shafer Jessica Ann , Leen Ann M. , Cruz Conrad Russell , Craddock John A , Heslop Helen E. , Rooney Cliona M. , Brenner Malcolm K , Bollard Catherine M. , Foster Aaron 
Published: April 2020

Abstract
We have previously demonstrated that hematological malignancies contain a distinct “side-population” (SP), which is characterized by the active transport of Hoechst fluorescent dye. The mechanisms responsible for Hoechst efflux also contribute to resistance of the SP to chemotherapy. This population has also been shown to be enriched for tumor initiating cells in several human cancer models. We hypothesize that targeting the cancer “SP” cells using immunotherapy may prevent relapse by eliminating the cell population that:
is least sensitive to chemotherapy and has tumor repopulating potential.
In this study we characterized SP cells in ten human lymphoma cell lines. Five of the 11 lymphoma cell lines tested (HDLM2, L428, Ramos, Toledo, Karpas) had a distinct SP ranging from 0.8–2% of total cells. We subsequently found SP cells with tumor associated markers in biopsy samples from patients with Hodgkin’s Lymphoma, T-cell Lymphoma, and Follicular lymphoma. Culture of the sorted cell lines show that SP but not the non-SP cells produced progeny that were both SP and non-SP cells. Gene expression analysis of the SP showed increased expression of ABCG2 and other ABC transporters that mediate both the transport of Hoechst dye and selected chemotherapeutic agents out of the cell. We evaluated whether lymphoma cell lines with an SP are resistant to the chemotherapeutic drug gemcitabine. The viability of lymphoma lines containing SP cells was significantly higher (mean 59.8%; range 37.4–87.8%%) than the viability of lines without an SP (mean 9.3% range 3.0%–12.2%) when cultured with 10nM gemcitabine for 3 days. The SP component of the lymphoma cells became enriched 10 fold when cultured with gemcitabine. Furthermore, when equal numbers of SP and non-SP cells were grown in separate fractions with gemcitabine, there was reduced viability of the non-SP (2,247 +/−294 mean counts per minute (cpm)) by thymidine assay. In contrast, the viability of the SP was preserved (13,200 +/− 7500 mean cpm). There was no difference in the viability between the untreated SP and Non SP, so that the gemcitabine rather than the Hoechst dye accounts for the differences in viability. Although lymphoma SP cells are resistant to chemotherapeutic agents, they also express higher levels of tumor associated antigens that are known targets for cytotoxic T cells. Hence RT-PCR and immunohistochemistry both show that SP cells are HLA positive and have increased expression of MAGE-4, SSX2, Survivin and NY-ESO transcripts and protein compared to non-SP cells from the same tumor. In IFNγ ELISPOT assays, we have shown that T cells secrete IFNγ in response to SP targets. Hence, lymphoma cell lines and primary lymphoma tissue contain chemotherapy-resistant SP cells which express tumor associated antigens and may be targeted by specific T cells.