Abstract 2290: Effective immunotherapy with cytokine-induced killer cells against autologous melanoma cancer stem cells
Authors: Gammaitoni Loretta , Giraudo Lidia , Macagno Marco , Cattaneo Giulia , Leuci Valeria , Sassi Francesco , Zaccagna Alessandro , Pisacane Alberto , Coha Valentina , Carnevale-Schianca Fabrizio , Aglietta Massimo , Sangiolo Dario 
Published: July 2016

Abstract
Purpose of the study was to explore the activity of cytokine-induced killer (CIK) cells against autologous chemo-surviving melanoma cancer stem cells (mCSCs) in vivo.
Elimination of mCSCs is an ambitious goal as considered responsible for chemo-resistance and relapses.
CIK cells are ex vivo expanded T-NK lymphocytes with MHC-independent antitumor activity. We provided proof of concept that CIK cells can kill mCSCs in vitro (Gammaitoni et al. Clin Cancer Res 2013) and demonstration of their activity in vivo, within an autologous model, would be relevant in clinical perspective.
Experimental procedures and results.
In vivo activity of CIK cells against autologous mCSCs was explored in NOD/SCID mice bearing tumors generated by subcutaneous implantation of primary melanoma cells or alternatively surgically resected tumor samples (patient-derived xenografts, PDX).
mCSCs were visualized by a lentiviral CSC-detector encoding eGFP under control of stem-gene promoter OCT4 (Gammaitoni et al. Clin Cancer Res 2013). Melanoma cells were engineered with the CSC-detector before implantation or alternatively, in PDX, after removal of residual tumors at the end of experimental treatments.
In vivo chemotherapy (CHT) consisted of intravenous (i.v.) Fotemustine (600μg days 1;15). Immunotherapy consisted of i.v. CIK cells (1×107) every 5 days. Antitumor activity was evaluated assessing tumor proliferative index by Ki67 expression in residual tumors. Activity against mCSCs was evaluated assessing the rate of residual GFP+-mCSCs in residual tumors after treatments.
In vivo infusion of CIK cells for 2 weeks determined significant tumor response (n = 6, p &lt; 0.0001) that fully involved autologous eGFP+-mCSCs. On the contrary, CHT spared mCSCs that resulted significantly increased in residual tumors compared with mice treated with CIK cells (p = 0.001) or untreated controls (p = 0.001). CIK cells infused after a previous CHT treatment (n = 6) retained significant antitumor activity (p = 0.001) and were capable to revert the CHT-induced enrichment of eGFP+-mCSCs, compared to mice treated with CHT alone (p = 0.01).
Similarly, in the PDX model, the infusion (n = 4) of autologous CIK cells exerted significant antitumor activity that involved eGFP+mCSCs (p = 0.009), confirmed by the absence of their increment in residual tumors (p = 0.3).
In vitro CHT treatment of melanoma from 8 different patients significantly enriched eGFP+ mCSCs (2.2 ±0.2 fold, p = 0.0018) compared to untreated controls. Melanoma treated with CHT, enriched in eGFP+mCSCs, were efficiently killed by autologous CIK cells with mean killing values ranging from 94±2% (40:1 E/T) to 21%±4 (1:3 E/T).
Conclusions.
Our findings provide first demonstration that immunotherapy with CIK cells is active against autologous mCSCs, surviving chemotherapy, in vitro and in vivo. We provide an experimental platform to study mCSCs and rationale to design clinical studies with CIK cells against melanoma.
Citation Format: Loretta Gammaitoni, Lidia Giraudo, Marco Macagno, Giulia Cattaneo, Valeria Leuci, Francesco Sassi, Alessandro Zaccagna, Alberto Pisacane, Valentina Coha, Fabrizio Carnevale-Schianca, Massimo Aglietta, Dario Sangiolo. Effective immunotherapy with cytokine-induced killer cells against autologous melanoma cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2290.