Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) neutralization counteracts T cell immune evasion in breast cancer

Abstract

Background: Nicotinamide phosphoribosyltransferase (NAMPT) is a key intracellular enzyme that participates in nicotinamide adenine dinucleotide (NAD) homeostasis as well as a released cytokine (eNAMPT) that is elevated in inflammatory conditions and in cancer. In patients with breast cancer, circulating eNAMPT is elevated and its plasma levels correlate with prognosis and staging. In light of this, we investigated the contribution of eNAMPT in triple negative mammary carcinoma progression by investigating the effect of its neutralization via a specific neutralizing monoclonal antibody (C269).

Methods: We used female BALB/c mice injected with 4T1 clone 5 cells and female C57BL6 injected with EO771 cells, evaluating tumoral size, spleen weight and number of metastases. We injected two times a week the anti-eNAMPT neutralizing antibody and we sacrificed the mice after 28 days. Harvested tumors were analyzed by histopathology, flow cytometry, western blot, immunohistochemistry, immunofluorescence and RNA sequencing to define tumor characteristics (isolating tumor infiltrating lymphocytes and tumoral cells) and to investigate the molecular mechanisms behind the observed phenotype. Moreover, we dissected the functional relationship between T cells and tumoral cells using three-dimensional (3D) co-cultures.

Results: The neutralization of eNAMPT with C269 led to decreased tumor size and reduced number of lung metastases. RNA sequencing and functional assays showed that eNAMPT controlled T-cell response via the programmed death-ligand 1/programmed cell death protein 1 (PD-L1/PD-1) axis and its neutralization led to a restoration of antitumoral immune responses. In particular, eNAMPT neutralization was able to activate CD8⁺IFNγ⁺GrzB⁺ T cells, reducing the immunosuppressive phenotype of T regulatory cells.

Conclusions: These studies indicate for the first time eNAMPT as a novel immunotherapeutic target for triple negative breast cancer.

Keywords: breast neoplasms; cytokines; immunotherapy; lymphocyte activation.

Figure 1

Neutralization of eNAMPT reduces 4T1 tumor growth and metastases. (A) Scheme of the treatment for data presented in panels B–G. 4T1 cells were established intramammary in female BALB/c mice. Control IgG1 (2.5 mg/kg i.p., two times a week) or C269 (2.5 mg/kg i.p., two times a week) were injected intraperitoneally. Data on untreated 4T1-bearing mice is present in online supplemental figure 1. No differences were found compared with IgG1-treated. (B) Serum eNAMPT levels in healthy, IgG1 or C269-treated 4T1-bearing mice. (C) Representative image of 4T1 tumor masses. (D) Tumor volume throughout the course of the experiment. (E) Tumor weight at sacrifice. (F) Spleen weight at sacrifice and in mice of the same age and weight. (G) Lung metastasis determined by the 6-thioguanine clonogenicity assay. Mean±SEM of eight independent experiments. (H) Scheme of the treatment for experiments presented in panels I–M. Control IgG1 (2.5 mg/kg i.p., two times a week) or C269 (2.5 mg/kg i.p., two times a week) were injected intraperitoneally when the tumors were palpable (around day 15). (I) Tumor growth determined by volume throughout the course of the experiment. (J) Tumor weight at sacrifice. (K) Spleen weight at sacrifice. (L) Serum eNAMPT levels at sacrifice. (M) Lung metastasis of 4T1 tumor cells determined by the 6-thioguanine clonogenicity assay. (N) Number of lung metastasis nodules from mice that received tail vein injection of 4T1 in presence or absence of C269. Mean±SEM of three (panels H–N) independent experiments. P value: *p<0.05; **p<0.01; ***p<0.001,****p<0.0001. eNAMPT, extracellular nicotinamide phosphoribosyltransferase; i.p., intraperitoneal.

Figure 2

Transcriptomic analysis of 4T1 cells from tumor explants. (A) Representative scheme of cells isolated from 4T1 tumor masses and subjected to RNAseq analysis. (B) Volcano plot of the differentially expressed genes in 4T1clone5-GFP cells (n=5 replicates/condition); FDR≤0.05. (C) Heat map and histogram depicting the upregulated genes by C269 treatment and relative GO analysis. (D) Patterns of transcription factor motif enrichment within the promoters of the C269-treated 4T1 cell-upregulated genes. (E–G) Heat map and histogram representation depicting the downregulated genes by the C269 treatment and relative GO analysis. (H) Patterns of transcription factor motif enrichment within the promoters of the C269-treated 4T1 cell-downregulated genes. FDR, false discovery rate; GO, gene ontology; RNAseq, RNA sequencing.

Figure 3

Neutralization of eNAMPT affects the PD-L1 axis in the tumor cells. (A–B) Cd274 and PD-L1 expression by qPCR and FACS of 4T1 cells in response to eNAMPT (500 ng/mL) or eNAMPT and C269 (10 µg/mL). IFNγ (100 ng/mL) was used as a positive control. Mean±SEM of three independent experiments. (C–D) Representative western blot and (E–F) densitometry of PD-L1 and ICOS-L in total cell lysates of 4T1 treated or not with rNAMPT for 24 hours. Mean±SEM of three independent experiments. (G–H) Representative western blot and (I–J) densitometry of PD-L1 and ICOS-L in homogenates from tumors of 4T1-bearing mice. Mean±SEM of 12 samples from two independent experiments. eNAMPT, extracellular nicotinamide phosphoribosyltransferase; FACS, fluorescence activated cell sorting; ICOS, inducible T-cell costimulator; IFN, interferon; PD-L1, programmed death-ligand 1; qPCR, quantitative PCR; rNAMPT, recombinant murine NAMPT; SSC, side scatter.

Figure 4

Neutralization of eNAMPT affects the PD-1/PD-L1 axis in the tumor microenvironment. (A–B) Representative western blot and (C–D) densitometry of PD-1 and ICOS in homogenates from tumors of 4T1-bearing mice. Mean±SEM of 12 samples from two independent experiments. (E–H) Quantification of: (E) T effector cells, T regulatory cells (gated on CD4⁺ cells) and CD8+cells; (F) PD-1⁺ T regulatory cells (gated on CD4⁺CD25⁺FoxP3⁺); (G) PD-1⁺CD8⁺ T cells (gated on CD8⁺ cells); (H) IFN⁺CD8⁺ and GrzB⁺CD8⁺ T cells (gated on CD8⁺ cells) from tumor of 4T1-bearing mice. Mean±SEM of five independent experiments. (I) IHC staining of CD8⁺ T cells in 4T1 tumor sections of 4T1-bearing mice. (J) Representative immunofluorescence analysis of GrzB⁺ in CD8⁺ T cells. (K) Suppression assay via [³H]-thymidine incorporation from spleen and draining lymph nodes. Mean±SEM of three independent experiments. P value: *p<0.05; **p<0.01; ***p<0.001,****p<0.0001. (L–O) 4T1 cells were established intramammary in female nude BALB/c mice. Control IgG1 (2.5 mg/kg i.p., two times a week) or C269 (2.5 mg/kg i.p., two times a week) were injected intraperitoneally. (L) Representative image of 4T1 tumor masses. (M) Tumor volume throughout the course of the experiment. (N) Tumor weight at sacrifice. (O) Spleen weight at sacrifice and in mice of the same age and weight. Mean±SEM of one experiment. DAPI, 4’,6-diamidino-2-phenylindole; eNAMPT, extracellular nicotinamide phosphoribosyltransferase; GrzB, granzyme B; ICOS-L, inducible costimulator-ligand; IFN, interferon; IHC, immunohistochemistry; i.p., intraperitoneal; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; Teff, T effector cells; Treg, regulatory T cell.

Figure 5

C269 shapes the immunophenotype of T-infiltrating cells. (A) Representative scheme of cells isolated from 4T1 tumor masses and subjected to RNAseq analysis. (B) Heat map and histogram depicting the downregulated genes by C269 treatment of CD4⁺ cells and relative GO analysis. (C) Heat map and histogram depicting the upregulated genes by C269 treatment of CD8⁺ cells and relative GO analysis. (D) Pdcd1 (PD-1) expression by qPCR of T cells in response to eNAMPT (500 ng/mL). Mean±SEM of three independent experiments. (E–F) Representative western blot and densitometry of PD-1 in total cell lysates of CD4⁺ and CD8⁺ T cells treated or untreated with rNAMPT for 24 hours and (G) quantification of PD-1 expression on T cells, pre-treated with eNAMPT for 24 hours and neutralized with C269 for other 24 hours. Mean±SEM of three independent experiments. P value: **p<0.01; ***p<0.001. eNAMPT, extracellular nicotinamide phosphoribosyltransferase; GO, gene ontology; PD-1, programmed cell death protein 1; qPCR, quantitative PCR; RNAseq, RNA sequencing; rNAMPT, recombinant murine NAMPT; SSC, side scatter.

Figure 6

eNAMPT promotes immunosuppressive phenotype of tumor infiltrating lymphocytes within 4T1 spheroids. (A) Experimental scheme for experiments depicted in the figure. (B) Representative immunofluorescence images and (C) ROI quantification of 4T1-GFP cells (green) and lymphocytes (red) in the spheroid area. N=6, Mean±SEM of two independent experiments. (D) Flow cytometry analyses of spheroid T cell infiltration. N=6. Mean±SEM of two independent experiments. (E) MFI of PD-1 in the IN and OUT compartments. Mean±SEM of two independent experiments. (F) Representative immunofluorescence images and (G) ROI quantification of 4T1-GFP cells (green) and lymphocytes (red) in the spheroid area Mean±SEM of three independent experiments. (H) Flow cytometry analyses of spheroid T-cell infiltration. N=6. Mean±SEM of three independent experiments. (I) MFI of PD-1 in the IN and OUT compartment of cocultured spheroids. Mean±SEM of three independent experiments. (J) MFI of IFNγ in the IN and OUT compartment of cocultured spheroids. Mean±SEM of three independent experiments. P value: *p<0.05; **p<0.01; ***p<0.001,****p<0.0001. eNAMPT, extracellular nicotinamide phosphoribosyltransferase; IFN, interferon; MFI, median fluorescence intensity; PD-1, programmed cell death protein 1, ROI, region on interest.