Cytokine Induced Killer cells kill chemo-surviving melanoma cancer stem cells

PURPOSE: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSCs) was explored, as CSCs are considered responsible for chemo-resistance and relapses. EXPERIMENTAL DESIGN: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MCs) with a lentiviral-vector encoding eGFP under expression control by stemness gene promoter oct4. Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP+mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro. First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed vivo in two distinct immunodeficient murine models. RESULTS: We visualized eGFP+mCSCs (14±2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP-positive MCs (¼2) compared with the eGFP-negative counterpart (¼870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n=11, 8/11 autologous), with specific lysis ranging from 95% (E/T 40:1) to 20% (E/T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from 3 distinct melanoma demonstrated significant tumor responses involving CHT-spared eGFP+mCSCs (p=0.001). Sequential CHT-immunotherapy treatment retained antitumor activity (n=12, p=0.001) reducing mCSC rates (p=0.01). CONCLUSIONS: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving chemotherapy or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided.