Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells.

Stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the "side population" (SP). In this study, we used flow cytometry and Hoechst 33342 dye efflux assay to isolate and characterize SP cells from six human lung cancer cell lines (H460, H23, HTB-58, A549, H441, and H2170). Nonobese diabetic/severe combined immunodeficiency xenograft experiments showed that SP cells were enriched in tumor-initiating capability compared with non-SP cells. Matrigel invasion assay showed that SP cells also have higher potential for invasiveness. Further characterization of this SP phenotype revealed several stem cell properties. We found evidence for repopulating ability by SP to regenerate a population resembling the original population. SP displayed elevated expression of ABCG2 as well as other ATP-binding cassette transporters and showed resistance to multiple chemotherapeutic drugs. Human telomerase reverse transcriptase expression was higher in the SP, suggesting that this fraction may represent a reservoir with unlimited proliferative potential for generating cancer cells. mRNA levels of minichromosome maintenance (MCM) 7, a member of the MCM family of proteins critical to the DNA replication complex, were lower in SP cells, suggesting that a majority of the SP fraction was in the G(0) quiescent state. Sixteen clinical lung cancer samples also displayed a smaller but persistent SP population. These findings indicate that SP is an enriched source of lung tumor-initiating cells with stem cell properties and may be an important target for effective therapy and a useful tool to investigate the tumorigenic process.

Polyfunctional T-cell responses are disrupted by the ovarian cancer ascites environment and only partially restored by clinically relevant cytokines.

BACKGROUND: Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment. METHODOLOGY/PRINCIPAL FINDINGS: Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1ß, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited. CONCLUSIONS/SIGNIFICANCE: The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.