In this study, we have found that both HL cell lines and primary HL tumor cells contain a minor side-population of tumor cells that have increased resistance to gemcitabine, but that also express tumor associated antigens which render them susceptible to killing by tumor-specific CTLs following demethylation with decitabine. While this primary drug resistant SP subset may therefore contribute to resurgent disease, a combination of chemotherapy and targeted immunotherapy may be able to reduce tumor burden and decrease the risk of relapse.
An SP subset of cells has been identified in a variety of hematologic malignancies and solid tumors [9
], but had not yet been reported in HL. We detected SP cells in two HL cell lines, HLDM2 and L428, which conferred primary drug resistance to the cell lines compared to those lacking SP cells. Importantly, we also detected SP cells in primary HL biopsy samples that possessed an HRS phenotype, including increased expression of CD30, CD3 (T cell rosettes [28
]) and lack of CD20 (). Because not all SP cells were CD30+, it is likely that SP cells found in these biopsies include normal B and T lymphocytes in addition to malignant cells, and that definitive identification of HRS cells with SP properties will need to be confirmed by clonal analyses, However, these results are suggestive that a malignant chemoresistant cell type exists that could contribute to relapse following disease treatment and may be targetable by antigen-specific CTL.
In other malignancies, SP cell resistance to lipophilic anti-neoplastic drugs, such as doxorubicin and mitoxantrone, can be attributed to the expression of the multi-drug transporter proteins including ABCG2 and MDR1 [5
]. More recent studies, however, have indicated that malignant SP cells may be more generally resistant to cytotoxic drugs, such as gemcitabine, independent of their excretion by transporter proteins [8
] and may also resist radiotherapy [38
], This may help explain the resistance of refractory HL to these commonly used treatment modalities [39
]. Because HL SP cells have lower apoptosis rates following exposure to gemcitabine compared to NSP cells, and can proliferate even in the presence of the drug, such chemotherapy exposure can simply enrich for drug resistant SP cells ( and ). Although we do not know the mechanisms for this transporter-protein independent resistance in HL-SP cells study of SP cells in pancreatic [41
] and gastrointestinal derived cell lines [8
], show the cells express CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6), which modulates Akt-associated anti-apoptotic activity, which would provide gemcitabine resistance [42
]. Additionally, Bleau and colleagues found that PI3K/Akt regulates ABCG2 expression in SP cells in malignant gliomas, suggesting that malignant SP cells reside in a distinct biological state that renders them insensitive to radiotherapy and a variety of chemotherapy agents [43
The potential benefits of CTL for chemoresistant hematological malignancy is supported by the long-term cures obtained following adoptive transfer of donor leukocyte infusions (DLI) to relapsed patients after allogeneic stem cell transplant [44
]. Predictably safe and effective immunotherapy, however, requires identification of specific target antigens that reside within the chemoresistant population. We have now shown that HL SP cells from cell lines have enhanced expression of TAAs, including MAGEA4, SSX2, NY-ESO-1 and survivin compared to NSP cells, although such expression was only seen at consistently high levels following decitabine demethylation (). Due to technical difficulties in culturing and isolating sufficient numbers of viable SP cells from primary samples following demethylation, we were unable to determine whether this effect is also relevant to HRS cells in vivo. However, other studies have shown that HL cells have increased CpG methylation [46
], and global hypomethylation is commonly seen in leukemias [47
], so that demethylating treatments may be a generally applicable tool for enhancing TAA expression in lymphoma and leukemia if the malignant cells are to be made susceptible to immunotherapies [48
]. Since multiple TAA are upregulated within chemoresistant SP cells, it should be possible to target several antigens simultaneously, thereby reducing the risk of tumor immune escape [55
In summary, we have identified a distinct SP subset in HL cell lines and primary tumor biopsies that are resistant to chemotherapy, but can be eradicated with TAA-specific CTL following decitabine demethylation. Combination therapeutic strategies that use conventional chemotherapy to debulk tumor burden, followed by novel drugs such as histone deacetylation (HDAC) inhibitors and T cell immunotherapy may eliminate residual chemoresistant tumor cells and help prevent disease relapse.