CHMP2A regulates broad immune cell-mediated antitumor activity in an immunocompetent in vivo head and neck squamous cell carcinoma model

Abstract

Background: Natural killer (NK) cells are key effector cells of antitumor immunity. However, tumors can acquire resistance programs to escape NK cell-mediated immunosurveillance. Identifying mechanisms that mediate this resistance enables us to define approaches to improve immune-mediate antitumor activity. In previous studies from our group, a genome-wide CRISPR-Cas9 screen identified Charged Multivesicular Body Protein 2A (CHMP2A) as a novel mechanism that mediates tumor intrinsic resistance to NK cell activity.

Methods: Here, we use an immunocompetent mouse model to demonstrate that CHMP2A serves as a targetable regulator of not only NK cell-mediated immunity but also other immune cell populations. Using the recently characterized murine 4MOSC model system, a syngeneic, tobacco-signature murine head and neck squamous cell carcinoma model, we deleted mCHMP2A using CRISPR/Cas9-mediated knock-out (KO), following orthotopic transplantation into immunocompetent hosts.

Results: We found that mCHMP2A KO in 4MOSC1 cells leads to more potent NK-mediated tumor cell killing in vitro in these tumor cells. Moreover, following orthotopic transplantation, KO of mCHMP2A in 4MOSC1 cells, but not the more immune-resistant 4MOSC2 cells enables both T cells and NK cells to better mediate antitumor activity compared with wild type (WT) tumors. However, there was no difference in tumor development between WT and mCHMP2A KO 4MOSC1 or 4MOSC2 tumors when implanted in immunodeficient mice. Mechanistically, we find that mCHMP2A KO 4MOSC1 tumors transplanted into the immunocompetent mice had significantly increased CD4⁺T cells, CD8⁺T cells. NK cell, as well as fewer myeloid-derived suppressor cells (MDSC).

Conclusions: Together, these studies demonstrate that CHMP2A is a targetable inhibitor of cellular antitumor immunity.

Keywords: Immunity, Innate; Immunotherapy; Immunotherapy, Adoptive; Killer Cells, Natural; Myeloid-Derived Suppressor Cells.

Figure 1

Deletion of mCHMP2A leads to increased NK cell cytotoxicity in vitro against 4MOSC1 cells, but not in 4MOSC2 tumor cells. (A) In vitro 4-hour cytotoxicity assay against 4MOSC1 WT and mCHMP2A KO cells. Mouse primary NK cells were used as effectors. Statistical analysis was performed by two-way ANOVA (10:1 p=0.0019; 5:1 p=0.0374; 2.5:1 p=0.0197 and 0.625:1 ns); and (B) cytotoxicity against 4MOSC2 WT and mCHMP2A KO cells. Statistical analysis was performed by two-way ANOVA (all E:T ratio ns). Data shown in (A, B), representative of n=2 independent experiments. ANOVA, analysis of variance; E:T, effector to target; KO, knock-out; NK, natural killer; WT, wild type.

Figure 2

NK cell-mediated antitumor immunosurveillance is active in 4MOSC1 but not 4MOSC2 tumors. (A) C57BL/6 mice were implanted with 1×10⁶ of 4MOSC 1 WT and 4MOSC 1 mCHMP2A KO cells into the tongue. Growth kinetic curves of 4MOSC1 tumor-bearing mice are shown as average tumor volumes (n=5 mice per group; two sited Student’s t-test). (B) C57Bl/6 mice were implanted with 1×10⁶ of 4MOSC2 WT and 4MOSC2 mCHMP2A KO cells into the tongue (n=5 mice per group, p=0.942). (C) Average tumor volumes on day 16 depicted (Individual dots represent volume of each tumor, n=5 mice per group). KO, knock-out; NK, natural killer; WT, wild type.

Figure 3

4MOSC1 and 4MOSC2 tumor growth in immunodeficient NSG mice. (A) NSG mice were implanted with 1×10⁶ of 4MOSC 1 WT and 4MOSC 1 mCHMP2A KO cells into the tongue. Shown is the average volume of each tumor (n=5 mice per group; two-sided Student’s t-test, p=0.868). (B) NSG mice were implanted with 1×10⁶ of 4MOSC2 WT and 4MOSC2 mCHMP2A KO cells into the tongue. Shown is the average volume of each tumor (n=5 mice per group p=0.930). KO, knock-out; WT, wild type.

Figure 4

Infiltration of immune cells in 4MOSC tumor. C57BL/6 mice were implanted with 1×10⁶ of (A–D) 4MOSC1 (n=5 mice for WT n=4 mice for mCHMP2A KO, one mouse injected with mCHMP2A KO failed to show tumor engraftment). (E–H) 4MOSC2 WT or mCHMP2A KO cells into the tongue analyzed by flow cytometry of the tumors at day 11, showing the infiltration of (A, E) NK cells (B, F) CD4⁺ T cells (C, G) CD8⁺ T cells (D, H) CD11b⁺ MDSCs in tumors. KO, knock-out; MDSCs, myeloid-derived suppressor cells; NK, natural killer; WT, wild type.

Figure 5

Difference in immune cell infiltration between 4MOSC WT and KO tumors. (A) Representative immunofluorescent staining of histological tissue sections from B6 mouse tongues with 4MOSC1 WT, 4MOSC1 KO, 4MOSC2 WT, and 4MOSC2 KO tumors on day 10 post injection. Images of stained tumors to show expression of CD3 (Red) NK1.1 (green) DAPI (blue). Immunostaining of CD3 and NK1.1 highlights an increase of CD45+CD3+ T cell and CD45+NK1.1+ NK cell recruitment in 4MOSC1 KO tumor compared with 4MOSC1 WT tumors. (B) Representative tumor tissue sections stained for CD3 by immunohistochemistry show increase in CD3+T cell infiltration in 4MOSC1 KO compared with 4MOSC1 WT but no significant difference in 4MOSC2 WT and 4MOSC2 KO tumors. (C) Immunohistochemistry staining for NK1.1 to show increase in NK cell infiltration in 4MOSC1 KO than in 4MOSC1 WT. No significant change in NK infiltration observed in 4MOSC2 WT and 4MOSC2 KO tumors. KO, knock-out; NK, natural killer; WT, wild type.

Figure 6

Depletion of immune cells reduces antitumor effect of mCHMP2A deletion on 4MOSC1 cells. (A) C57BL/6 mice were implanted with 1×10⁶ of 4MOSC 1 mCHMP2A KO or 4MOSC1 WT cells into the tongue. Mice were treated with IP with 10 mg/kg of (B) anti-CD8 (C) anti-NK1.1 or (D) anti-CD4 (n = 7 per group). Individual growth curve of the 4MOSC1 mCHMP2A tumor-bearing mice are shown. KO, knock-out; NK, natural killer; WT, wild type.

Figure 7

EVs secreted by 4MOSC cell lines express mouse NKG2D ligands. (A) 4MOSC1 and 4MOSC2 WT and mCHMP2A KO derived EVs were collected and analyzed by flow cytometry for mouse NKG2D ligands mRae, mULBP, mH60 (blue histogram). Red histogram represents isotype. EVs, extracellular vehicles; KO, knock-out.