CHMP2A regulates tumor sensitivity to natural killer cell-mediated cytotoxicity

Natural killer (NK) cells are known to mediate killing of various cancer types, but tumor cells can develop resistance mechanisms to escape NK cell-mediated killing. Here, we use a “two cell type” whole genome CRISPR-Cas9 screening system to discover key regulators of tumor sensitivity and resistance to NK cell-mediated cytotoxicity in human glioblastoma stem cells (GSC). We identify CHMP2A as a regulator of GSC resistance to NK cell-mediated cytotoxicity and we confirm these findings in a head and neck squamous cells carcinoma (HNSCC) model. We show that deletion of CHMP2A activates NF-κB in tumor cells to mediate increased chemokine secretion that promotes NK cell migration towards tumor cells. In the HNSCC model we demonstrate that CHMP2A mediates tumor resistance to NK cells via secretion of extracellular vesicles (EVs) that express MICA/B and TRAIL. These secreted ligands induce apoptosis of NK cells to inhibit their antitumor activity. To confirm these in vitro studies, we demonstrate that deletion of CHMP2A in CAL27 HNSCC cells leads to increased NK cell-mediated killing in a xenograft immunodeficient mouse model. These findings illustrate a mechanism of tumor immune escape through EVs secretion and identify inhibition of CHMP2A and related targets as opportunities to improve NK cell-mediated immunotherapy.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Sample sizes were chosen based on preliminary data of tumor growth in this mouse model. Sample size was determined to be adequate based on the consistency of measurable differences within and between groups. The number of mice is rationalized following the "resource equation method" described in Festing et al., 2002 (Festing MF, Altman DG. Guidelines for the design and statistical analysis of experiments using laboratory animals. ILAR journal. 2002;43(4):244-58).
No data have been excluded from the analysis Every experiment was replicated at least three with near-identical results.
Based on the tumor volumes on the first day of treatment, tumor bearing mice were randomly assigned to treatment groups such that each treatment group had the same average tumor volume of 50 mm cube. Experiments other than mice experiments were not randomized because cells were collected from the same flask and no variation in cell growth or phenotype between samples is possible.
The data presented did not require the use of blinding. Data reported for mouse experiments were not subjective but rather based on quantitative analyses.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Instrument Software CHMP2A Antibody, rabbit policlonal, Proteintech, cat. n. 10477-1-AP, lot.n.00046622 GAPDH Loading Control Monoclonal Antibody (GA1R), Invitrogen, cat. n. MA5-15738, lot.n.WC318870 CD9, clone (D8O1A) Rabbit mAb, Cell Signaling, cat. n. 13174S R&D: It is hereby certified that the above product has been tested for proper performance and function under our established Quality Control testing criteria. Biolegend: This product lot has passed BioLegend's QC testing and is certified for use. For details on QC testing view our page at biolegend.com/en-us/quality-control. BD Biosciences: This Product Complies with all BDB release criteria.BD Biosciences, Life Science Research Reagents, is a registered facility with the British Standards Institute (BSI) for ISO 9001:2008. It is authorized by our Quality Assurance program to be released for sale Glioblastoma stem cell lines 387, 1517, CW468 and D456 were provided by Jeremy Rich. Head and neck squamous cells carinoma cell lines Cal27, Detroit568 and HNSCC17B were provided by J. Silvio Gutkind. K562 were purchased from ATCC None of these cell lines has been authenticated in our lab All the cell lines were frequently tested for mycoplasma contamination. No presence of mycoplasma contamination was detected according to PCR test.

No wild animals have been used in this study
No field-collected samples have been used in this study All mice were housed, treated, and handled in accordance with the guidelines set forth by the University of California, San Diego Institutional Animal Care and Use Committee and the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.
Cell lines were trypsinized and centrifuged at 1500 rpm for 5 min while NK cells resuspended in media and centrifuged at 1500 rpm for 5 minutes. Cells were resuspended in DPBS + 2% FCS (flow buffer) and stained with trypan blue, counted using an inverted microscope and 1 x 10^5 cells were dispensed per sample. Cells were incubated on ice in the dark for 20 minutes in flow buffer containing the antibodies of interest. Stained cells were centrifuged at 1500 rpm for 5 min and washed flow buffer two times. Finally, cells were resuspended in 300 "l of flow buffer containing SYTOX Blue Dead Cell Stain (Life Technologies) diluted by a factor of 1,000 and analyzed by FACS.