Macrophage-Derived Neuropilin-2 Exhibits Novel Tumor-Promoting Functions

Abstract

Tumor-associated macrophages (TAM) are causally associated with tumorigenesis as well as regulation of antitumor immune responses and have emerged as potential immunotherapeutic targets. Recent evidence suggests TAM phagocytose apoptotic tumor cells within the tumor microenvironment through efferocytosis in an immunologically silent manner, thus maintaining an immunosuppressed microenvironment. The signal transduction pathways coupling efferocytosis and immunosuppression are not well known. Neuropilin-2 (NRP2) is a member of the membrane-associated neuropilin family and has been reported in different immune cells but is poorly characterized. In this study, we show that NRP2 is expressed during macrophage differentiation, is induced by tumor cells, and regulates phagocytosis in macrophages. Furthermore, NRP2 in TAM promoted efferocytosis and facilitated tumor growth. Deletion of NRP2 from TAM impaired the clearance of apoptotic tumor cells and increased secondary necrosis within tumors. This resulted in a break in the immune tolerance and reinitiated antitumor immune responses, characterized by robust infiltration of CD8⁺ T and natural killer cells. This result suggests NRP2 may act as a molecular mediator that connects efferocytosis and immune suppression. Deletion of NRP2 in TAM downregulated several immunosuppressive and tumor-promoting genes and upregulated immunostimulatory genes in the myeloid compartment. Taken together, our study demonstrates that TAM-derived NRP2 plays a crucial role in tumor promotion through efferocytosis, opening the enticing option for the development of effective immunotherapy targeting TAM.Significance: Neuropilin-2 in macrophages promotes tumor growth by regulating efferocytosis of apoptotic tumor cells and orchestrating immune suppression.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/19/5600/F1.large.jpg Cancer Res; 78(19); 5600-17. ©2018 AACR.

Figure 1.. Expression of NRP2 in macrophages.

(A, B) NRP2 expression in freshly isolated human monocytes and bone marrow cells from C57BL/6 mice differentiated to macrophages with M-CSF. (C) Representative confocal image showing the presence of NRP2 expressing CD68⁺ TAMs in human PDAC tissue. The first panel shows only NRP2 positive cells (red). The second panel shows CD68⁺ macrophages (green). The third panel represents merged image showing NRP2⁺CD68⁺ macrophage in the tumor. Scale bar, 20μm. Inset shows magnified image of part of the tissue. Dapi was used to stain the nucleus. (D) Graphical representation showing relative abundance of NRP2⁺ macrophages in a cohort of treatment naïve PDAC tissues derived from RAPP. Table shows details of patient tissues procured. (E, F) Immunoblot analyses showing NRP2 expression in human and mouse macrophages differentiated with pancreatic cancer cell line-derived Conditioned medium (CM) from Panc-1 or UNKC-6141 cells respectively in vitro.

Figure 2.. NRP2 regulates phagosome maturation without affecting uptake of phagocytic cargo.

(A) BMDM from NRP2^fl/flCSF1R-iCre mice were assessed for phagosome maturation and degradation of internalized E.coli bioparticles at time = 0min, 15min, 45min, 90min, 120min and 240min. Scale bars, 10 μm. Magnified images within the inset show the phagosomes containing E.coli particles. (B) Phagosome maturation and degradation of bioparticles was assessed as corrected total cell fluorescence using Image J software, at times indicated at the graph abscissa and represented in bar graphs as mean ± SEM. (C,F,I,L) Western Blots showing knock out or deletion of NRP2 for each experiment. (C) shows NRP1 protein level following NRP2 knock out in macrophages. (D) M-CSF treated BMDM from NRP2^fl/flCSF1R-iCre mice were analyzed for phagosome maturation and degradation of internalized zymosan particlesat time= 0hr, 6hr, 10hr, 14hr and 18hr. Scale bars, 10μm. Magnified images within the inset show the phagosomes containing zymosan particles. (E) Phagosome maturation and degradation of zymosan particles was scored as corrected total cell fluorescence using Image J software at times indicated and represented in bar graph as mean ± SEM. (G) Phagocytosis assay for assessing the ability of human macrophages to uptake E.coli bioparticles (green) and phagosome maturation (red) following NRP2 knockdown. The first column shows phagosome maturation (red) in the scr (upper) and siNRP2 treated (bottom) cells. The second column indicates the uptake efficiency (green) in the scr (upper) and siNRP2 treated (bottom) cells. The third column represents merged images showing the role of NRP2 on uptake and phagosome maturation. Scale bars, 20μm. Single cell magnified within the boxed region shows green or red E.coli particle. (H) Uptake efficiency was measured as green cellular fluorescence whereas the intensity of red fluorescence indicated phagosome maturation. Results were represented graphically, values as mean ± SEM. Dapi was used for staining the nucleus. (J) Phagocytosis assay to show the effect of NRP1 depletion on cargo uptake (green) as well as phagosome maturation (red) in human macrophages. Insets show magnified image of cell containing bacteria. Scale bars 10μm. (K) Phagosome maturation and uptake efficiency in the presence and absence of NRP1 were quantified as in (H), and represented graphically as mean ± SEM. (L) Western Blot showing NRP1 knockdown.

Figure 3.. Effect of NRP2 on early and late phagosomes.

Knockdown or deletion of NRP2 inhibits maturation of early to late phagosomes in macrophages. (A) Immunostaining of early and late phagosomal maturation markers in human macrophages following NRP2 knockdown. The upper panel represents early phagosome marker Rab5 (green). Scale bars 10μm. The lower panel shows representative the late phagosomal marker Rab7 (green). Scale bars 20μm. Magnified images of individual cell are shown in the inset for each condition. (B) Immunostaining data for Rab5 and Rab7 were quantified as cellular fluorescence using Image J software and represented graphically. The upper and lower panels show graphical representation of Rab5 and Rab7 respectively, in the presence and absence of NRP2. (D) Immunostaining of early and late phagosomal maturation markers in mouse BMDM following NRP2 deletion. The upper and lower panels represent early phagosome marker Rab5 (red) and the late phagosomal marker Rab7 (red) respectively. Scale bars 10μm. The insets are magnified image of individual cell for each condition. (E) Representative bar graphs showing quantification of Rab5 and Rab7 using Image J software. The upper and lower panels represent changes in Rab5 and Rab7 respectively, following NRP2 deletion. All values are shown as mean ± SEM. Dapi was used for staining the nucleus. (C,F) Western Blot showing total cellular Rab5 and Rab7 in whole cell lysates from human and mouse macrophages following knockdown or knockout of NRP2 respectively.

Figure 4.. NRP2 regulates efferocytosis of apoptotic cells by macrophages in vitro.

Efferocytosis assay to assess the effect of NRP2 deletion on the ability of macrophages to degrade the apoptotic cells. (A,D,G) BMDM from NRP2^fl/flCSF1R-iCre mice were treated with either M-CSF (A) or UNKC-6141 CM (D,G) and then assessed for degradation of internalized apoptotic Jurkat cells (A,D) or UKC-6141 cells (G) at time points= 0hr, 2hr, 6hr, and 8 or 12hr. Scale bars, 10μm (A,D), 20μm (G). Insets show single macrophage containing apoptotic cells. (B,E,H) Corrected total cellular fluorescence (red) was analyzed at the time points indicated using Image J software as a measure for efferosome maturation and degradation of the apoptotic cargo. Results are represented graphically as mean ± SEM. (C,F,I) Immunoblot to show knock out of NRP2 for A,D,G. Dapi was used for staining the nucleus.

Figure 5.. NRP2 in macrophages affects tumor growth

(A) Schematic diagram for subcutaneous tumor progression model. 2×10⁶ or 500,000 UNKC-6141 cells were subcutaneously implanted into NRP2^fl/flCSF1R-iCre mice. Tumor progression was monitored for the indicated time periods. (B) Graph showing relative tumor volume for control and test tumors (2×10⁶ cells implanted). (C) Scatter plot representation of the final volume of the harvested tumors (2×10⁶ cells implanted). (D,F) Immunoblot analysis showing efficient knock out of NRP2 from macrophages for experiment B and E. (E, G) Graphical representation of final volume of harvested tumors (n=3 or 5, 500,000 cells implanted). (H) RT-PCR showing efficient knock out of NRP2 from CD11b⁺ myeloid cells isolated from test tumors for G.

Figure 6.. NRP2 in TAMs regulates the efferocytosis of apoptotic tumor cells and immune responses.

(A) Representative image showing TUNEL⁺ cells in control and test tumors. (B) The number of necrotic foci relative to tumor volume shown graphically. (C) Representative images showing deposition of MSU crystals in control and test tumors. Scale bar, 500 μm. (D) Scatter plot comparing the formation of MSU crystals in control versus test tumors. (E) Representative image showing role of NRP2 in the migration of macrophages (F4/80⁺, red) to the tumor. Scale bar, 20μm. Inset shows magnified image of a single F4/80⁺ macrophage. (F) the number of F4/80⁺ cells per field were counted using Image J software and represented graphically. (G) Representative immunohistochemistry image showing CD8⁺ T cell infiltration in control and test tumors. Image Scale, 20× magnification. (H) Number of CD8⁺ T cells per field was counted using Image J software, and represented graphically. (I) Representative confocal microscopy image for CD69⁺ T cell infiltration (green) in the control and test tumors. Scale bar 20μm. (J) Number of CD69⁺ T cells per field was counted using Image J software and represented as a scatter plot. (K) Representative confocal microscopy image for NK1.1⁺ cells (green) in control and test tumors. Scale bar 20μm. (L) Number of NK1.1⁺ cells per field was counted using Image J software, and represented graphically. Dapi was used to stain the nucleus. All values are mean± SEM.

Figure 7.. Transcriptome analysis from CD11b⁺ myeloid cells by next generation RNA-Seq.

(A) Schematic diagram showing experimental design. (B) Representative canonical pathways from Ingenuity Pathway Analysis are shown with gene counts and -log(p-value). (C) Representative functional annotation clusters from DAVID database are shown. (D) Representative list of genes related to macrophage polarization whose expressions were significantly altered in the CD11b⁺ myeloid population, following NRP2 deletion in macrophages in test tumors. (E) RT-PCR analysis in separate biological replicates (pooled, n=3 in either control or test) showing altered expression of genes in CD11b⁺ cells following NRP2 deletion in macrophages in test tumors.