Colorectal cancer-infiltrating NK cell landscape analysis unravels tissue-resident PD-1 + NK cells in microsatellite instability tumors

Valentina Obino¹Chiara Giordano¹Simona Carlomagno²Chiara Setti¹Marco Greppi¹Matteo Bozzo³Silvia Pesce^1,4Elisa Ferretti^1,5Simona Candiani^3,4Letizia Muccio¹Enrico Ciferri⁶TANIA BUTTIRON WEBBER⁷Agnese Solari¹Fulvia Ortolani²Laura Paleari⁸Matteo Clavarezza⁷Andrea Barberis⁶Marco Filauro⁶Nicoletta Provinciali^1,7Mariangela Rutigliani⁹Emanuela Marcenaro^1,4*Andrea DeCensi^10,7Mariella Della Chiesa^1,4*Simona Sivori^1,4*

• ¹Department of Experimental Medicine (DIMES), University of Genoa, Genoa, Italy
• ²Department of Medicine (DMED), University of Udine, Udine, Italy
• ³Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy
• ⁴Direzione Scientifica, San Martino Hospital (IRCCS), Genova, Liguria, Italy
• ⁵U.O. Medicina di Laboratorio, San Martino Hospital (IRCCS), Genova, Liguria, Italy
• ⁶Department of Abdominal Surgery – General and Hepatobiliopancreatic Surgery Unit, Ente Ospedaliero Ospedali Galliera, Genoa, Italy
• ⁷Division of Medical Oncology, Ente Ospedaliero Ospedali Galliera, Genoa, Italy
• ⁸ALiSa, Liguria Health Authority, Genoa, Italy
• ⁹Department of Laboratory and Service – Histological and Anatomical Pathology Unit, Ente Ospedaliero Ospedali Galliera, Genoa, Italy
• ¹⁰Queen Mary University of London, London, United Kingdom

Background Natural killer (NK) cells are innate lymphocytes endowed with potent cytotoxic activity. The presence of tumor-associated NK cells has been correlated with better prognosis in several solid tumors including colorectal cancer (CRC). This malignant disease is the second cause of cancer death worldwide and is in urgent need for novel approaches to improve current immunotherapies. Since CRC microenvironment can induce NK cell dysfunction and hinder cancer control, understanding tumorassociated NK cell features is mandatory to fully unlock their immunotherapeutic potential.Our study aims at elucidating the molecular and functional characteristics of tumor-associated NK cells in CRC focusing on the expression of immune checkpoints that critically regulate NK cell function. We performed an in-depth cytofluorimetric analysis of tumor-associated NK cells obtained by tissue dissociation of samples derived from 80 CRC patients comparing tumor with matched tumorfree tissue and peripheral blood, stratifying patients by tumor stage or MSI/MSS condition. Tumor tissue was also analysed by immunohistochemistry.Results NK cells expressing immune checkpoints (i.e., KIR, NKG2A and TIM-3) were significantly enriched in tumor compared to tumor-free tissue, and an increase in PD-1 + NK cells was observed in tumors compared to peripheral blood and tumor-free tissue, indicating TME-induced modulation. Notably, tumor-associated PD-1 + NK cells characterized MSI rather than MSS CRC. In addition, tumor-associated NK cells also expressed tissue residency markers (CD103 and/or CD49a) and displayed a distinct profile also including the PD-1 + NK cell subset in MSI CRC, possibly representing NK cells recruited from circulation, retained in tumors, and reconfigured by TME signals. Importantly, tissue resident NK cells adequately expressed activating NK receptors and cytotoxic molecules.