NK cells and innate lymphoid cells provide protection against pathogens and help to maintain homeostasis in distinct tissues, including the intestine. However, their deregulated function has been associated with intestinal pathology, comprised inflammatory disorders and CRC. Gene expression signature and heterogeneity of NK cells and ILCs subsets have been recently defined by distinct high-dimensional single cell RNA sequencing approaches. However, several molecular aspects of NK cells as well as their role in tumorigenesis is still debated. In particular, diversity of tumor infiltrated-NK cell subsets at protein level has not been clarified, yet.
In the proposed research plan, we will combine single cell sorting approach with high-throughput proteomic based mass-spectrometry analysis to deconvolute at single cell level the proteomic profile of tumor-infiltrating NK cells using mouse models of CRC. We aim to understand the plasticity of proteome in NK cells and to clarify the NK cell-mediated mechanisms of immunosurveillance and immunoevasion.
Accumulation of genetic alterations and the establishment of a pro-tumoral microenvironment characterize the CRC development and progression. Due to the complexity of this pathology, the characterization of novel cellular subsets offers the opportunity to clarify their role in immunosurveillance and CRC tumorigenesis. Among several types of infiltrating immune cells, heterogeneity of ILCs subset remains controversial. Despite the broadly demonstrated roles of NK cells in cancer immunosurveillance and their favorable use in cancer immunotherapy, complete studies analyzing phenotypes and functions of these cells in CRC are still missing. Initial results have led to the conclusion that a low level of CRC infiltrated NK cells correlate with poor patient survival and CRC relapse (Scanocchia et al, 2014). Conversely, functional analysis of circulating and tumor-infiltrating NK cells in CRC patients show alterations in the IFN-¿ production, receptor repertoire and in their cytotoxic function. As proof of this, decreased levels of NKG2D expression in NK cells, occurring along with an increase of NKG2D soluble ligands released by tumor cells, has been associated with NK cell tolerance in CRC (Gharangozloo et al, 2018). In addition, upregulated levels of the inhibitory receptor TIGIT was associated with dysfunctional phenotype of tumor-infiltrating NK cells favoring cancer growth (Zhang et al, 2018). Remarkably, the capacity of NK cells to acquire an ILC1-like phenotype both in physiological and pathological conditions shows a novel a mechanism of tumor immune evasion characterized by NK plasticity (Gao et al, 2017).
Only recent works, based on single-cell RNA sequencing (sc-RNAseq), allowed to better characterize the transcriptional programs of ILCs-NK cells in mice and humans, revealing blood- and tissue- specific signature able to define respectively NK cells and ILC1 subsets. Although this technology gave a rapid growth in knowledge for measuring the genomic properties of individual cells, all these projects lack of information at the protein level. In fact, to better understand the mechanism of cellular processes is necessary measure the effector molecules of the process, which are usually the proteins encoded by the mRNA.
Only a combined analysis of RNA and protein information from single cells could be useful to entirely understood a biological system and the role of NK cells in pathological conditions.
Here, we propose to perform a FACS-sorting approach coupled with high-dimensional LC-MS technology to isolate and characterize proteomic profile of single NK cells. Globally, our project will clarify the role of CRC to modulate NK cell function.