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Plasticity in Tumor-Promoting Inflammation: Impairment of Macrophage Recruitment Evokes a Compensatory Neutrophil Response

Pahler, Jessica C., Tazzyman, Simon, Erez, Neta, Chen, Yung-Yi, Murdoch, Craig, Nozawa, Hiroaki, Lewis, Claire E., Hanahan, Douglas·2008·Neoplasia
DOI 10.1593/neo.07871
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Figure 1 CCL2 and CCR2 are upregulated during K14-HPV/E2 tumorigenesis. RNA was isolated from the cervix of three estrogentreated nontransgenic FVB/n, and K14-HPV mice with dysplasia (3 months) and real-time quantitative PCR used to determine (A) CCL2 and (B) CCR2 expression. Additionally, RNA was isolated from sorted fibroblasts (PDGFR α + ), myeloid cells (F4/80 + and/or CD68 + ), and the remaining unstained cells from a pool of 10 cervixes from 3-month-old mice and qRT-PCR used to measure CCL2 expression in these populations (C). CCL2 expression was 0.07 in the PDGFR α + population. Expression levels were normalized to those of the housekeeping gene GUS . Error bars show SEM.
Figure 1 CCL2 and CCR2 are upregulated during K14-HPV/E2 tumorigenesis. RNA was isolated from the cervix of three estrogentreated nontransgenic FVB/n, and K14-HPV mice with dysplasia (3 months) and real-time quantitative PCR used to determine (A) CCL2 and (B) CCR2 expression. Additionally, RNA was isolated from sorted fibroblasts (PDGFR α + ), myeloid cells (F4/80 + and/or CD68 + ), and the remaining unstained cells from a pool of 10 cervixes from 3-month-old mice and qRT-PCR used to measure CCL2 expression in these populations (C). CCL2 expression was 0.07 in the PDGFR α + population. Expression levels were normalized to those of the housekeeping gene GUS . Error bars show SEM.CC-BY-NC-ND · From the PDF
Figure 2 Absence of CCR2 delays time-to-tumor onset but does not affect tumor growth or incidence. Tumor incidence and burden were assessed using hematoxylin and eosin -stained serial sections using tissue collected from mice at 4 months, between 4.25 and 5 months, and more than 5 months of age. (A) Tumor volume (growth) was not significantly different between any genotype at end stage. (B) Tumor incidence was significantly delayed ( P < .001) at the earliest stages in CCR2-null K14-HPV/E2 mice. The n values for each group are displayed at the base of each bar.
Figure 2 Absence of CCR2 delays time-to-tumor onset but does not affect tumor growth or incidence. Tumor incidence and burden were assessed using hematoxylin and eosin -stained serial sections using tissue collected from mice at 4 months, between 4.25 and 5 months, and more than 5 months of age. (A) Tumor volume (growth) was not significantly different between any genotype at end stage. (B) Tumor incidence was significantly delayed ( P < .001) at the earliest stages in CCR2-null K14-HPV/E2 mice. The n values for each group are displayed at the base of each bar.CC-BY-NC-ND · From the PDF
Figure 3 Effect of CCR2 deficiency on tumor angiogenesis and MMP-9 expression. (A) Immunohistochemical analysis of Meca-32 (Rhodamine, red; endothelial cells) in tumor sections at 1-month intervals during tumorigenesis revealed no striking differences. E indicates the epithelium; S, stroma. Nuclei are shown in blue by staining with DAPI; original magnification, ×200. (B) Tissue sections were immunohistochemically stained with an antibody against MMP-9, and expression per field was quantified in the cervical stroma of 4-month-old K14-HPV/E2 and CCR2-null K14-HPV/E2 mice ( P = .03). (C) Tissues were collected from mice at the same age, and nonquantitative substrate zymography was performed to assess the presence of MMP activity.
Figure 3 Effect of CCR2 deficiency on tumor angiogenesis and MMP-9 expression. (A) Immunohistochemical analysis of Meca-32 (Rhodamine, red; endothelial cells) in tumor sections at 1-month intervals during tumorigenesis revealed no striking differences. E indicates the epithelium; S, stroma. Nuclei are shown in blue by staining with DAPI; original magnification, ×200. (B) Tissue sections were immunohistochemically stained with an antibody against MMP-9, and expression per field was quantified in the cervical stroma of 4-month-old K14-HPV/E2 and CCR2-null K14-HPV/E2 mice ( P = .03). (C) Tissues were collected from mice at the same age, and nonquantitative substrate zymography was performed to assess the presence of MMP activity.CC-BY-NC-ND · From the PDF
Figure 4 CCR2 inhibition blocks macrophage recruitment to the cervical stroma. (A) Immunohistochemical analysis of F4/80 and CD68 was performed, and staining was quantified per field in nontransgenic FVB/n and 4-month-old CCR2 wild-type, heterozygous, and null K14-HPV/E2 mice. In all cases, there are significantly fewer positively stained cells in the CCR2-null mice compared to wild-type K14-HPV/E2 mice. There is no significant difference in expression between nontransgenic and CCR2-deficient transgenic mice. (B) Images of MMP-9 (FITC, green) colocalization with the macrophage marker F4/80 (Rhodamine, red) in 4-month-old wild-type and CCR2null K14-HPV/E2 mice. Yellow indicates colocalization, and green arrows indicate MMP-9 + , F4/80 -cells. Original magnification, ×400. (C) % of all MMP-9 cells expressing F4/80.
Figure 4 CCR2 inhibition blocks macrophage recruitment to the cervical stroma. (A) Immunohistochemical analysis of F4/80 and CD68 was performed, and staining was quantified per field in nontransgenic FVB/n and 4-month-old CCR2 wild-type, heterozygous, and null K14-HPV/E2 mice. In all cases, there are significantly fewer positively stained cells in the CCR2-null mice compared to wild-type K14-HPV/E2 mice. There is no significant difference in expression between nontransgenic and CCR2-deficient transgenic mice. (B) Images of MMP-9 (FITC, green) colocalization with the macrophage marker F4/80 (Rhodamine, red) in 4-month-old wild-type and CCR2null K14-HPV/E2 mice. Yellow indicates colocalization, and green arrows indicate MMP-9 + , F4/80 -cells. Original magnification, ×400. (C) % of all MMP-9 cells expressing F4/80.CC-BY-NC-ND · From the PDF
Image 5
CC-BY-NC-ND · From the PDF
Image 6
CC-BY-NC-ND · From the PDF
Figure 5 Neutrophil expression colocalizes with MMP-9 in CCR2deficient mice. Immunohistochemistry was performed with MMP-9 and the neutrophil antibody clone 7/4 on tissues from 4-month-old mice. (A) Representative images of MMP-9 (Rhodamine, red) colocalization with the 7/4 clone (FITC, green) in wild-type and CCR2-null K14-HPV/E2 cervical tissues. (B) Hematoxylin and eosin staining (original magnifications: ×100 for large panel and ×1000 for inset) with the 7/4 clone shows that 7/4 is expressed on macrophages in K14-HPV/E2 mice and on neutrophils in the CCR2-null counterparts. Neutrophils can be clearly identified by morphology in the null mice but were rare in the cervical stroma of wild-type mice. 7/4 staining (FITC, green) of macrophages was further confirmed by colocalization with the macrophage marker CSF1R (Rhodamine, red) only in wild-type mice. Original magnification, ×400 for all fluorescent images. (C) Staining for both 7/4 single-positive and 7/4 and CSF1R double-positive populations was determined in both mouse genotypes ( P < .001).
Figure 5 Neutrophil expression colocalizes with MMP-9 in CCR2deficient mice. Immunohistochemistry was performed with MMP-9 and the neutrophil antibody clone 7/4 on tissues from 4-month-old mice. (A) Representative images of MMP-9 (Rhodamine, red) colocalization with the 7/4 clone (FITC, green) in wild-type and CCR2-null K14-HPV/E2 cervical tissues. (B) Hematoxylin and eosin staining (original magnifications: ×100 for large panel and ×1000 for inset) with the 7/4 clone shows that 7/4 is expressed on macrophages in K14-HPV/E2 mice and on neutrophils in the CCR2-null counterparts. Neutrophils can be clearly identified by morphology in the null mice but were rare in the cervical stroma of wild-type mice. 7/4 staining (FITC, green) of macrophages was further confirmed by colocalization with the macrophage marker CSF1R (Rhodamine, red) only in wild-type mice. Original magnification, ×400 for all fluorescent images. (C) Staining for both 7/4 single-positive and 7/4 and CSF1R double-positive populations was determined in both mouse genotypes ( P < .001).CC-BY-NC-ND · From the PDF
Figure 6 Colocalization of 7/4 + cells with macrophage and neutrophil markers in the cervix. Tissue sections from both wild-type and CCR2-deficient mice were stained with antibodies against CD11b, GR-1, CD68, and F4/80 (Rhodamine, red) and with the 7/4 clone (FITC, green) to further characterize these cells as having neutrophil markers in CCR2-null mice and macrophage markers in wild-type mice. Yellow arrows indicate coexpression. Original magnification, ×400.
Figure 6 Colocalization of 7/4 + cells with macrophage and neutrophil markers in the cervix. Tissue sections from both wild-type and CCR2-deficient mice were stained with antibodies against CD11b, GR-1, CD68, and F4/80 (Rhodamine, red) and with the 7/4 clone (FITC, green) to further characterize these cells as having neutrophil markers in CCR2-null mice and macrophage markers in wild-type mice. Yellow arrows indicate coexpression. Original magnification, ×400.CC-BY-NC-ND · From the PDF
Figure 7 Monocytes inhibit neutrophil migration and infiltration into human tumor spheroids in vitro . (A) Cell migration assays were carried out using control, monocyte-conditioned medium (CM), or tumor cell CM spiked with the neutrophil chemoattractant CXCL8 as described in Materials and Methods. CXCL8 promoted neutrophil migration that was significantly ablated in the presence of monocyte CM but not of tumor cell CM. (B) Human tumor (cervical; HeLa or lung; A549) spheroids were cocultured with either neutrophils alone for 5 hours or monocytes for 16 hours followed by neutrophils for 5 hours. Spheroids were then washed and enzymatically digested, and flow cytometry was used to quantify the number of infiltrating neutrophils. Significantly ( P < .001) fewer neutrophils infiltrated into spheroids when monocytes were already present in these structures than when they were absent. (C) Immunohistochemistry using an antimyeloperoxidase antibody (brown; arrows) illustrates the presence of neutrophils in tumor spheroids following coculture. This marker could detect both monocytes and neutrophils in HeLa spheroids when the latter were cocultured with each cell type alone. However, there was no (cumulative) increase in MPO + cell number when monocytes were allowed to infiltrate spheroids before their culture with neutrophils (data not shown). N indicates the necrotic core at the center of spheroids. Scale bar, 150 μ m.
Figure 7 Monocytes inhibit neutrophil migration and infiltration into human tumor spheroids in vitro . (A) Cell migration assays were carried out using control, monocyte-conditioned medium (CM), or tumor cell CM spiked with the neutrophil chemoattractant CXCL8 as described in Materials and Methods. CXCL8 promoted neutrophil migration that was significantly ablated in the presence of monocyte CM but not of tumor cell CM. (B) Human tumor (cervical; HeLa or lung; A549) spheroids were cocultured with either neutrophils alone for 5 hours or monocytes for 16 hours followed by neutrophils for 5 hours. Spheroids were then washed and enzymatically digested, and flow cytometry was used to quantify the number of infiltrating neutrophils. Significantly ( P < .001) fewer neutrophils infiltrated into spheroids when monocytes were already present in these structures than when they were absent. (C) Immunohistochemistry using an antimyeloperoxidase antibody (brown; arrows) illustrates the presence of neutrophils in tumor spheroids following coculture. This marker could detect both monocytes and neutrophils in HeLa spheroids when the latter were cocultured with each cell type alone. However, there was no (cumulative) increase in MPO + cell number when monocytes were allowed to infiltrate spheroids before their culture with neutrophils (data not shown). N indicates the necrotic core at the center of spheroids. Scale bar, 150 μ m.CC-BY-NC-ND · From the PDF
Figure W1. Neutrophils do not inhibit monocyte infiltration into human tumor spheroids in vitro . Human tumor (cervical; HeLa or lung; A549) spheroids were cocultured with either monocytes alone for 24 hours or neutrophils for 5 hours followed by monocytes for 24 hours. Flow cytometry of enzymatically dispersed spheroids showed that there were no significant differences ( P =.252) in the level of monocyte infiltration into spheroids when neutrophils were already present in these structures compared to when they were absent.
Figure W1. Neutrophils do not inhibit monocyte infiltration into human tumor spheroids in vitro . Human tumor (cervical; HeLa or lung; A549) spheroids were cocultured with either monocytes alone for 24 hours or neutrophils for 5 hours followed by monocytes for 24 hours. Flow cytometry of enzymatically dispersed spheroids showed that there were no significant differences ( P =.252) in the level of monocyte infiltration into spheroids when neutrophils were already present in these structures compared to when they were absent.CC-BY-NC-ND · From the PDF

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Cited by

Metadata

Authors
Pahler, J. C., Tazzyman, S., Erez, N., Chen, Y.-Y., Murdoch, C., Nozawa, H., Lewis, C. E., & Hanahan, D.
Venue
Neoplasia
Publisher
Elsevier BV
Published
2008-04
Volume
10
Issue
4
Pages
329-IN2
ISSN
1476-5586
DOI
10.1593/neo.07871
Cited by
184
References
3
Language
en
Licence

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