Jagged2 targeting in lung cancer activates anti-tumor immunity via Notch-induced functional reprogramming of tumor-associated macrophages

Abstract

Signaling through Notch receptors intrinsically regulates tumor cell development and growth. Here, we studied the role of the Notch ligand Jagged2 on immune evasion in non-small cell lung cancer (NSCLC). Higher expression of JAG2 in NSCLC negatively correlated with survival. In NSCLC pre-clinical models, deletion of Jag2, but not Jag1, in cancer cells attenuated tumor growth and activated protective anti-tumor T cell responses. Jag2^-/- lung tumors exhibited higher frequencies of macrophages that expressed immunostimulatory mediators and triggered T cell-dependent anti-tumor immunity. Mechanistically, Jag2 ablation promoted Nr4a-mediated induction of Notch ligands DLL1/4 on cancer cells. DLL1/4-initiated Notch1/2 signaling in macrophages induced the expression of transcription factor IRF4 and macrophage immunostimulatory functionality. IRF4 expression was required for the anti-tumor effects of Jag2 deletion in lung tumors. Antibody targeting of Jagged2 inhibited tumor growth and activated IRF4-driven macrophage-mediated anti-tumor immunity. Thus, Jagged2 orchestrates immunosuppressive systems in NSCLC that can be overcome to incite macrophage-mediated anti-tumor immunity.

Keywords: Jagged; Notch ligands; immunosuppression in cancer; immunosuppressive myelopoiesis; macrophage reprogramming; tumor-associated macrophages.

Figure 1.. Jagged2 ablation directs anti-tumor T cell immunity in lung cancer.

(A) Probability of survival ± 95% CI in NSCLC patients (TCGA PanCancer Atlas lung adenocarcinoma (LUAD); Schabath LUAD; ORIEN NSCLC) stratified by JAG2 mRNA expression (TCGA: JAG2^Lo n = 79, JAG2^Hi n = 395; Schabath JAG2^Lo n = 55, JAG2^Hi n = 343; ORIEN JAG2^Lo n = 15, JAG2^Hi n = 99). HR: log rank hazard ratio. (B,C) From two NSCLC TMAs, (B) representative automated multispectral images showing Pan-cytokeratin (PCK; cyan), JAG2 (yellow), CD3 (green), and DAPI, and (C) the median ± IQR of intra-tumor CD3⁺ T cells in DAPI⁺ cells within all tumors (JAG2^Lo n = 91, JAG2^Hi n = 28) or primary tumors (JAG2^Lo n = 71; JAG2^Hi n = 37). See also Table S1 and Table S2. (D, E) (D) H&E staining and (E) median ± quartiles in lung tumor areas from mice orthotopically inj ected with Scramble or Jag2^−/−LLC (n = 6 mice/group). Triangles note total tumor regions. (F, G) (F) H&E staining and (G) median ± quartiles in lung tumor areas from mice orthotopically injected with Scramble or Jag2^−/− KPM^Kin (n = 5 mice/group). Triangles note representative tumor regions. (H) Tumor volume ± SEM in C57BL/6 mice bearing subcutaneous wildtype (n = 6), Scramble (n = 18) or Jag2^−/− (n = 19) LLC tumors. (I) Tumor volume ± SEM in mice bearing subcutaneous wildtype (n = 3), Scramble (n = 7), or Jag2^−/− (n = 15) KPM^Kin tumors. (J, K) (J) H&E staining and (K) median ± quartiles in lung tumor areas from KP (n = 14) or KPJ (n = 12) mice intratracheally challenged with Cre-expressing adenovirus. Triangles note representative tumor regions. (L) Tumor volume ± SEM of Scramble and Jag2^−/− tumors in C57BL/6 or Rag1^−/− mice (n = 5/group). (M) Tumor volume ± SEM in mice bearing Scramble or Jag2^−/− tumors treated with isotype (ISO; n = 10), αCD4 (n = 9;), or αCD8 (n = 9). (N, O) (N) Immunohistochemistry images and (O) percent per area ± SEM of CD8⁺ and CD4⁺ from Scramble (22 fields) and Jag2^−/− (20 fields) LLC tumors (n = 3/group). (P, Q) (P) Immunohistochemistry images and (Q) percent per area ± SEM of CD8⁺ and CD4⁺ from Scramble (22 fields) and Jag2^−/− (5 fields) KPM^Kin tumors. (R, S) Proportion ± SEM of intratumor (R) CD4⁺ or CD8⁺ T cells within CD45⁺ cells and (S) CD44⁺CD69⁺ or IFNγ⁺TNFα⁺ CD8⁺ T cells from Scramble (n = 4, 8) and Jag2^−/− (n = 4, 14) LLC tumors. Statistics were applied using log-rank Mantel-Cox test, one-way ANOVA, Student’s xt-test, or Mann-Whitney t-test *, p<0.05; **, p<0.01, ***, p<0.001. See also Figure S1.

Figure 2.. Jagged2 deletion in NSCLC cells reprograms intra-tumor immune repertoire.

(A) Uniform manifold approximation and projection (UMAP) embedding of CD45⁺ tumor-infiltrating leukocytes clusters from Scramble (n = 3) and Jag2^−/− (n = 2) LLC tumors. (B, C) T lymphocyte cluster (B) UMAP and (C) proportions and cell counts. (D) Heatmap displaying T Lymphocyte cluster genes, identified in (B, C), from Jag2^−/− relative to Scramble. (E-H) The macrophage and neutrophil myeloid cell clusters (E) UMAP, (F) heatmap, (G) proportions and cell counts, and (H) psuedotime trajectory analysis. See also Table S3. (I) Volcano plot comparing cluster 0 from Scramble-enriched macrophages to cluster 2 from Jag2^−/−-enriched macrophages. See also Figure S2.

Figure 3.. Jagged2 ablated tumors expand immunostimulatory macrophages.

(A) Immunohistochemistry images and quantification of F4/80 from mice bearing Scramble (18 fields) or Jag2^−/− (17 fields) LLC tumors (n = 3/group). (B) Flow cytometry percentages ± SEM of conventional DC1 (cDC1), conventional DC2 (cDC2), monocytic DC (moDC), monocytic (M-MDSC) or polymorphonuclear (PMN-MDSC) MDSC subsets, and macrophages (Mac) within CD45⁺ cells in Scramble and Jag2^−/− LLC tumors (n = 7, 14/group). (C) Flow cytometry percentages ± SEM of intratumor Ly6C^HiMHC-II⁺ and Ly6C^LoMHC-II⁺ subsets within CD45⁺ cells or macrophages from Scramble (n = 5) and Jag2^−/− (n = 6) LLC tumors. (D) G-CSF, M-CSF, and GM-CSF from Scramble or Jag2^−/− LLC tumor homogenates (n = 5/group). (E-H) Histogram and mean fluorescent intensity (MFI ± SEM) of (E) CSF1R, (F) IFNγ, (G) TNFα, and (H) MHC-II on macrophages from Scramble or Jag2^−/− LLC tumors (n = 6, 9/group). (I) Proportion ± SEM of MHC-II⁺ cells in CTV-labeled Ly6C⁺ cells from Scramble tumors transferred into established Scramble or Jag2^−/− tumors (n = 4) after 24 h. (J) eGFP MFI from tumoral total macrophages or Ly6C^HiMHC-II⁺ macrophages from eGFP⁺Scramble (n = 10) or eGFP⁺Jag2^−/− (n = 9) LLC tumors. (K, L) (K) Histograms and (L) MFI ± SEM of tumor injected cleaved DQ-OVA in macrophages from Scramble (n = 5) or Jag2^−/− (n = 4) LLC tumors. (M, N) F4/80⁺ cells from Scramble or Jag2^−/− LLC tumors (n = 4/group) were pulsed ex vivo with DQ-OVA and co-cultured with OT-II CD4⁺ T cells. (M) DQ-OVA MFI ± SEM and (N) OT-II CD4⁺ CD25 expression. (O, P) (O) Tumor volume ± SEM in C57BL/6 (n = 5) or Ccr2^−/− (n = 4) mice bearing Scramble or Jag2^−/− LLC tumors and (P) intratumoral proportions ± SEM of Ly6C^HiMHC-II⁺ or Ly6C^LoMHC-II⁺ macrophages in CD45⁺ cells. (Q, R) (Q) Tumor volume ± SEM in mice treated with isotype (ISO; n = 5) or aCSFIR (n = 5) bearing Scramble or Jag2^−/− tumors and I intratumoral proportions ± SEM of Ly6C^HiMHC-II⁺ or Ly6C^LoMHC-II⁺ macrophage subsets in total tumor-infiltrating CD45⁺ cells. (S) Tumor volume ± SEM in C57BL/6 (n = 10, 12/group) or Rag1^−/− mice (n = 4/group) mice receiving 1:1 co-transfer of LLC tumor cells with F4/80⁺ cells isolated from LLC Scramble or LLC Jag2^−/− tumors. Statistics were applied using one-way ANOVA or Student’s t-test, *, p<0.05; **, p<0.01, ***, p<0.001. See also Figure S3.

Figure 4.. Jagged2 null tumor cells provoke contact dependent expansion of macrophages via DLL1 and DLL4 induction.

(A,B) Fold change in the proportion ± SEM of BM-derived macrophages (BM-Mac) in CD45⁺ from BM-derived monocytes (BM-Mono) co-cultured with irradiated Scramble or Jag2^−/− LLC tumor cells at varying ratios (A) without or (B) with transwells (2 independent experiments each). (C) Mean fluorescent intensity (MFI) ± SEM of cleaved DQ-OVA from BM-Mac post-coculture with irradiated Scramble or Jag2^−/− LLC cells or BM-Mac cultured alone (2 experiments). (D) Fold change in the proportion ± SD of BM-Mac in CD45⁺ cells from BM-Mono cultures supplemented with TCS or TES from Scramble or Jag2^−/− LLC. (E, F) I Volcano plot indicating bulk RNAseq transcripts differentially expressed in Scramble LLC-eGFP versus Jag2^−/− LLC-eGFP from mice (n = 3 replicates/group) and (F) GSEA and enrichment plots for differentially expressed genes. See alsoTable S4 and Table S5. (G) MFI ± SEM of DLL1 and DLL4 on Scramble or Jag2^−/− LLC tumor cells (n = 4 mice/group). (H-K) (H) Tumor volume ± SEM from Scramble (n = 9), Jag2^−/−x (n = 10), Dll1^−/−Dll4^−/− (n = 9), and Jag2^−/−Dll1^−/−Dll4^−/− (n = 9) LLC tumors and (I) the intratumoral proportions ± SEM of total macrophages in tumor-infiltrating CD45⁺ cells. After intratumoral DQ-OVA injection, macrophages were measured for (J) DQ-OVA MFI ± SEM (n = 3/group) then isolated and co-cultured for (K) proliferation of OT-II CD4⁺ T cells. (L-N) (L) Representative automated multispectral images of a NSCLC TMA showing Pan-cytokeratin (PCK; cyan), JAG2 (red), CD3 (orange), CD8 (magenta), DLL1 (yellow), DLL4 (green), and DAPI and the median ± quartiles of intra-tumor CD8⁺ T cells in DAPI⁺ cells stratified by PCK⁺ region expression of (M) DLL1^LoJAG2^Hi (n = 16) or DLL1^HiJAG2^Lo (n = 48) or (N) DLL4^LoJAG2^Hi (n = 18) or DLL4^HiJAG2^Lo (n = 45). (O) Immunoblot of Nor1 and Nurr7 in CD45 Scramble or Jag2^−/− LLC tumor cells. (P) Relative fold change in DLL1 and DLL4 MFI ± SEM in Jag2^−/− tumors transduced with a non-targeting (control) siRNA, Nor1 siRNA and Nur77 siRNA alone or both, and treated with LPS and IFNγ or LLC TES (3 independent experiments). (Q) Fold change in the mean ± SEM relative luciferase units (RLU) of the Nr4a-reporter in Scramble or Jag2^−/− LLC treated with vehicle, LPS and IFNγ, or LLC TES (3 experiments). (R) ChIP of Dll1 and Dll4 promoter pulldown with IgG or Nor1 in Scramble or Jag2^−/− LLC cells. Mean of 2 biological replicates. Statistics were applied using one-way ANOVA, Student’s t-test, or Welch’s t-test *, p<0.05; **, p<0.01, ***, p<0.001. See also Figure S4

Figure 5.. Jagged2 deficient tumors direct Notch-mediated programming of macrophages.

(A) Relative fold change of Notch1 and Notch2 mRNA expression in tumor-infiltrating F4/80⁺ cells from Scramble (n = 9) and Jag2^−/− (n = 8 or 11) LLC tumors. (B, C) Histogram and mean fluorescent intensity quantification (MFI) ± SEM of Notch1 (B) and Notch2 (C) in macrophages from Scramble (n = 4, 5) or Jag2^−/− (n = 5) LLC tumors. (B) MFI ± SEM of Notch2 and Notch1 on Ly6C^HiMHC-II⁺ and Ly6C^LoMHC-II⁺ macrophages from Scramble ox Jag2^−/− LLC tumors (n = 5/group). (C) Notch2 and Notch1 MFI ± SEM on Ly6C^HiMHC-II⁺ and Ly6C^LoMHC-II⁺ macrophages from co-cultures with irradiated Scramble or Jag2^−/− LLC cells (2 experiments). (D) Immunoblot of cleaved Notch2 in tumor-infiltrating CD11b⁺ cells from Scramble or Jag2^−/− LLC tumors. Representative of 3 replicates from 2 distinct experiments. (E) Relative fold change ± SEM of Hes1 mRNA in tumor-infiltrating F4/80⁺ cells from Scramble (n = 7) and Jag2^−/− (n = 6) LLC tumors. (F) MFI ± SEM of cleaved Notch2 (NICD2) in macrophage subsets from mice bearing Scramble or Jag2^−/− LLC tumors (n = 9/group). (I, J) The (I) relative expression and (J) distribution of genes related to Notch signaling between Scramble-enriched macrophage cluster 0 and Jag2^−/−-enriched macrophage cluster 2. (K) Proportion of BM-Mac in CD45⁺ cells from NICD inhibitor pre-treated BM-Mono then co-cultured with irradiated Scramble or Jag2^−/− LLC cells. Mean ± SEM (2 experiments). (L-O) (L) LLC tumor volume ± SEM in RosaSoichy^NotchIC (n = 5) or Rosa^NotchICLysM^Cre/+− (n = 14) mice and intratumoral proportions ± SEM of (M) Ly6C^HiMHC-II⁺ and Ly6C^LoMHC-II⁺ macrophages in CD45⁺ cells, (N) CD4⁺ and CD8⁺ T cells in CD45⁺ cells, and (O) CD44⁺CD69⁺ CD4⁺ or CD8⁺ T cells in CD45⁺ cells. (P) Notch2 MFI on F4/80⁺ macrophages from Scramble (n = 4), Jag2^−/− (n = 3), Dll1^−/−Dll4^−/− (n = 4) or Jag2^−/−Dll1^−/−Dll4^−/− (n = 4) LLC tumors. Statistics were applied using one-way ANOVA or Student’s t-test, *, p<0.05; **, p<0.01, ***, p<0.001. See also Figure S5.

Figure 6.. Notch2 directs IRF4-dependent anti-tumor macrophage polarization.

(A, B) (A) Relative fold change of Irf4 mRNA expression and (B) immunoblot of IRF4 in F4/80⁺ cells from Scramble, Jag2^−/−, Dll1^−/−Dll4^−/−, and Jag2^−/−Dll1^−/−Dll4^−/− LLC tumors. (C) Mean fluorescent intensity (MFI) ± SEM of IRF4 in total macrophages or Ly6C^HiMHC-II⁺ or Ly6C^LoMHC-II⁺ subsets from Scramble, Jag2^−/−, Dll1^−/−Dll4^−/−; or Jag2^−/−Dll1^−/−Dll4^−/− LLC tumors (n = 4/group). (D) MFI ± SEM of IRF4 in total macrophages co-cultured with irradiated Scramble, Jag2^−/−, Dll1^−/−Dll4^−/−, or Jag2^−/−Dll1^−/−Dll4^−/− LLC tumors cells (2 experiments). (E) ChIP of Irf4 and Hes1 promoter pulldown with IgG, Notch1, or Notch2 antibodies in F4/80⁺ cells from Scramble or Jag2^−/− LLC tumors. Mean of 3 independent experiments with 3 biological replicates. (F) MFI ± SEM of IRF4 in BM-Mac from NICD inhibitor pre-treated BM-Mono then co-cultured with irradiated Scramble or Jag2^−/− LLC cells. Mean ± SEM (2 experiments). (G) MFI ± SEM of IRF4 in GFP⁺ (NICD⁺) macrophages from Rosa^NotchICLysM^Cre+/− mice and total macrophages from Rosa^NotchIC bearing wildtype LLC tumors (n = 5/group). (H, I) Spleen macrophages (sMac), (H) proportions and (I) cleaved DQ-OVA MFI ± SEM, derived from splenic monocytes (sMono) of Irf4^F/F or Irf4^F/FLysM^Cre+/− mice (n = 4/group) co-cultured with irradiated Scramble or Jag2^−/− LLC tumor cells. (J, K) (J) Tumor volume ± SEM of Scramble or Jag2^−/− LLC tumors implanted in Irf4^F/F (n = 18, 21) or Irf4^F/FLysM^F/F (n = 14, 18) mice and (K) intratumor proportions of Ly6C^LoMHC-II⁺ macrophages in total macrophages or tumor-infiltrating CD45⁺ cells. Statistics were applied using one-way ANOVA or Student’s t-test, *, p<0.05; **, p<0.01, ***, p<0.001. See also Figure S6.

Figure 7.. Therapeutic targeting of Jagged2 recapitulates anti-tumor effects of Jagged2 genetic ablation.

(A) LLC tumor volume ± SEM in mice injected with isotype (ISO) or αJag2 (n = 15/treatment). (B) LLC tumor volume ± SEM in Rag1^−/− mice injected with ISO or αJag2 (n = 5/treatment). (C, D) (C) Immunohistochemistry images and (D) percent ± SEM of F4/80, CD8, or CD4 in tumors from (A); ISO: 15 fields; αJag2: 10 fields. (E-G) Intratumoral proportions ± SEM of (E) CD4⁺ and CD8⁺ T cells (TIL) in CD45⁺ cells, (F) CD44⁺CD69⁺ CD4⁺ or CD8⁺ in TILs, and (G) Ly6C^HiMHC-II⁺ or Ly6C^LoMHC-II⁺ macrophages from (A). (H) Relative fold change of Nr4a1 and Csf1 mRNA in CD45⁻ tumor cells from mice treated (n = 3/treatment) as in (A). (I) MFI ± SEM of DLL 1 or DLL4 expression on LLC tumor cells from mice treated (n = 7, 9/treatment) as in (A). (J, K) (J) Relative fold change ± SEM of Irf4 mRNA and (K) immunoblot of IRF4 in intra-tumor F4/80⁺ cells from mice treated (n = 3/treatment) as in (A). (L) Representative histogram of tumor-injected cleaved DQ-OVA in total tumor-infiltrating macrophages from mice treated (n = 2/treatment) as in (A). (M) MFI ± SEM of cleaved Notch2 (NICD2) in macrophage subsets from mice treated (n = 6/treatment) as in (A). (N) LLC tumor volume ± SEM in C57BL/6 or Ccr2^−/− mice treated with ISO (n = 4, 5) or αJag2 (n = 5). (O) LLC tumor volume ± SEM in Irf4^F/F (n = 5, 6/treatment), LysM^Cre+/− (n = 5/treatment), or Irf4^F/FLysM^Cre+/− (n = 5, 6/treatment) mice treated with ISO or αJag2. Statistics were applied using one-way ANOVA or Student’s t-test, *, p<0.05; **, p<0.01, ***, p<0.001. See also Figure S7.