Evaluation of Interaction Between NK Cells and Colorectal Carcinoma Cells for Development of NK Cell-Based Immunotherapy in Patients with Refractory Disease
Authors: Todisco Elisabetta , Turin Ilaria , Ferulli Federica , Tanzi Matteo , Brugnatelli Silvia , Delfanti Sara , Maestri Marcello , Donadon Matteo , Cimino Matteo , Torzilli Guido , Santoro Armando , Pedrazzoli Paolo , Montagna Daniela 
Published: October 2019

Abstract
Background
The failure of conventional treatment and target therapies to significantly improve outcomes in metastatic colorectal cancer (mCRC) has prompted the development of immune-based therapies, including natural killer (NK) cells-based strategies. NK cells can kill target cells directly, as well as mediate antibody (Ab)-dependent cellular cytotoxicity (ADCC) via the membrane receptor FcgRIII, which binds to the Fc portion of IgG Ab. This feature makes them of particular importance in the immunotherapy of mCRC patients harboring the KRAS mutation that cannot benefit from administration of anti-epidermal growth factor receptor (EGFR) drugs. In this study we evaluated the capacity of patients derived NK cells, either resting or after cytokine activation, to lyse autologous mCRC cells. mCRC cells were analyzed for expression of ligands for adhesion and triggering NK receptors involved in their recognition and killing. We also evaluated whether KRAS mutated mCRC cells were susceptible to anti-EGFR-induced ADCC mediated by NK cells.
Patients and methods. After obtaining informed signed consent, 25 mCRC patients have been enrolled to date. Tumor cells were disaggregated by GentleMACS Dissociator (Miltenyi Biotec,Germany), in vitro expanded and analyzed for the expression of ligands for NK triggering receptors. Ligands expression was evaluated by cytofluorimetric analysis and gene expression by qPCR on mCRC cultured cells and by immunohistochemistry in sections of samples embedded in paraffin. Resting and IL-2- or IL-15-activated NK cells, were analyzed for expression of triggering and inhibitory receptors and for their ability to kill autologous mCRC cells alone or after incubation with anti-EGFR monoclonal antibodies (mAbs) in a 51Cr release cytotoxicity assay.
Results. Tumor cells were successfully expanded from 21 of 25 samples. Experiments performed in 10 patients showed the inability of patients resting NK cells to lyse mCRC cells (< 10% at effector:target ratio (E:T) of 20:1. Cytokine overnight (ON) activation resulted in an increased NK cytotoxic activity (IL-2: mean 28%; range:10-71; and IL-15: mean 40%; range 16-76 at E:T ratio of 20:1). Additional days of NK cell activation were able to further enhance their lytic capability. In resting NK cells the mean surface expression of activating receptors DNAM-1 and NKG2D was 79%, (range 75-91) and 39 % (range 29-58), respectively. The latter was up-regulated by ON cytokine activation (IL-2: mean 53%, range 48-70; IL-15: mean 70%, range 63-87). Among activating natural cytotoxicity receptors (NCRs), NKp46 is highly expressed both on resting and activated cells (>90%), while low surface expression of NKp30 and NKp44 was documented on resting NK cells (<10%). Their expression could be slightly up-regulated in particular after IL-15 ON activation (mean: 13%, range 9-30). Molecular analysis demonstrated a sizeable gene expression of NK ligands PVR, Nectin-2 and MICA/B on mCRC cells. Cytofluorimetric analysis showed that PVR and Nectin-2 are expressed in a vast majority of cultured mCRC cells (mean 65%, range 60-76; and mean 78%, range 70-98), respectively, while MICA/B were less expressed (mean 18%, range 15-25). The incubation of mCRC cells with anti-EGFR mAbs increased their susceptibility to NK-mediated lysis irrespective of KRAS status of tumor cells. The average rate of increase was greater using resting NK cells as effectors. The ongoing experiments comparing mCRC ligands expression evaluated on cultured tumor cells versus paraffin embedded sections will clarify whether results obtained in vitrocould be translated to the clinical setting.
Conclusions.
The evidence that ex vivo activated autologous NK cells are able to lyse patients tumor cells can offer new therapeutic options to a cohort of mCRC patients with a poor prognosis.

Disclosures
No relevant conflicts of interest to declare.