Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. VSports app下载.

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. V体育官网.

. 2014 Jan;184(1):271-81.
doi: 10.1016/j.ajpath.2013.09.017.

Expression of the homeobox gene HOXA9 in ovarian cancer induces peritoneal macrophages to acquire an M2 tumor-promoting phenotype

Song Yi Ko  1 , Andras Ladanyi  2 , Ernst Lengyel  2 , Honami Naora  3
Affiliations

"V体育官网" Expression of the homeobox gene HOXA9 in ovarian cancer induces peritoneal macrophages to acquire an M2 tumor-promoting phenotype

Song Yi Ko (VSports) et al. Am J Pathol. 2014 Jan.

"V体育ios版" Abstract

Tumor-associated macrophages (TAMs) exhibit an M2 macrophage phenotype that suppresses anti-tumor immune responses and often correlates with poor outcomes in patients with cancer. Patients with ovarian cancer frequently present with peritoneal carcinomatosis, but the mechanisms that induce naïve peritoneal macrophages into TAMs are poorly understood. In this study, we found an increased abundance of TAMs in mouse i. p. xenograft models of ovarian cancer that expressed HOXA9, a homeobox gene that is associated with poor prognosis in patients with ovarian cancer VSports手机版. HOXA9 expression in ovarian cancer cells stimulated chemotaxis of peritoneal macrophages and induced macrophages to acquire TAM-like features. These features included induction of the M2 markers, CD163 and CD206, and the immunosuppressive cytokines, IL-10 and chemokine ligand 17, and down-regulation of the immunostimulatory cytokine, IL-12. HOXA9-mediated induction of TAMs was primarily due to the combinatorial effects of HOXA9-induced, tumor-derived transforming growth factor-β2 and chemokine ligand 2 levels. High HOXA9 expression in clinical specimens of ovarian cancer was strongly associated with increased abundance of TAMs and intratumoral T-regulatory cells and decreased abundance of CD8(+) tumor-infiltrating lymphocytes. Levels of immunosuppressive cytokines were also elevated in ascites fluid of patients with tumors that highly expressed HOXA9. HOXA9 may, therefore, stimulate ovarian cancer progression by promoting an immunosuppressive microenvironment via paracrine effects on peritoneal macrophages. .

PubMed Disclaimer

Figures

Figure 1
Figure 1
HOXA9 expression is associated with abundance of M2 macrophages in i.p. xenograft models of ovarian cancer. A–C: Mouse i.p. xenograft models were established from vector-control and HOX-overexpressing MOSEC lines. Mice were sacrificed at 2 months after tumor cell inoculation. A: Representative examples of staining of F4/80+ macrophages in omental implants. Scale bar = 50 μm. B: Average numbers of F4/80+ cells in omental implants were calculated by scoring five random 200× microscopic fields of stained tumor tissue sections of each mouse (n = 5 mice per group). ∗∗∗P < 0.001. C: Ascitic cells of vector-control and +HOXA9 MOSEC xenograft models were stained with Abs to F4/80 and CD206 and evaluated by flow cytometric analysis. Shown are representative examples of the proportions of CD206+ cells within the gated population of F4/80+ macrophages. D: Mouse i.p. xenograft models were established from +HOXA9 control (empty vector and nontargeting) and HOXA9-knockdown (shA9-A and shA9-B) SKOV3ip lines. CD206 expression was evaluated by flow cytometric analysis in ascitic macrophages of control and HOXA9-knockdown groups of mice that were sacrificed at 25 days after tumor cell inoculation. Other HOXA9-knockdown groups of mice were analyzed at 45 days after tumor cell inoculation.
Figure 2
Figure 2
HOXA9 expression in ovarian cancer cells stimulates peritoneal macrophages to acquire M2 features. Primary cultures of naïve mouse peritoneal macrophages were stimulated with nonconditioned (No. cond.) medium and with medium conditioned by equivalent numbers of cells of +HOXA9 control (empty vector and nontargeting) and HOXA9-knockdown (shA9-A and shA9-B) SKOV3ip lines. At 5 days thereafter, macrophages were evaluated for expression of M2 markers, cytokines, and chemokines. A: Flow cytometric analysis of CD206 expression. B: RT-qPCR analysis of expression of Mrc1 (encoding CD206) and Cd163. C: RT-qPCR analysis of Il10, Ccl17, and Il12a expression. ∗P < 0.005, ∗∗P < 0.001.
Figure 3
Figure 3
Stimulatory effects of HOXA9 on macrophages are only partially mediated by HOXA9-induced, tumor-derived TGF-β2 levels. A: Primary cultures of naïve mouse peritoneal macrophages were stimulated with medium conditioned by control (nontargeting), TGF-β2–knockdown (shTGF-β2), and HOXA9-knockdown (shA9-B) SKOV3ip lines and an HOXA9-knockdown line that stably expressed TGF-β2 (shA9-B + TGF-β2). Macrophages were also stimulated with recombinant TGF-β2 (rTGF-β2) at the same concentration as that released by control SKOV3ip cells (240 pg/mL). At 5 days thereafter, macrophages were evaluated for CD206 expression by flow cytometric analysis. B: Chemotaxis of primary mouse peritoneal macrophages toward cells of the indicated SKOV3ip lines was assayed at 20 hours after seeding in Transwell chambers. Macrophages were also stimulated with rTGF-β2, as described for A. Shown are average results of three independent experiments. C and D: Mouse i.p. xenograft models were established from vector-control and +HOXA9 MOSEC lines and a +HOXA9 MOSEC line in which TGF-β2 was knocked down (+HOXA9 + shTGF-β2), and were evaluated at 2 months after tumor cell inoculation. C: Average numbers of F4/80+ cells in omental implants were calculated by scoring five random 200× microscopic fields of stained tumor tissue sections of each mouse (n = 5 mice per group). D: Representative examples of staining of F4/80+ macrophages in omental implants. ∗P < 0.0005, ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bar = 50 μm.
Figure 4
Figure 4
HOXA9 induces CCL2 expression in ovarian cancer cells and correlates with levels of CCL2, TGF-β2, and M2 cytokines in patient ascites. A: Relative CCL2 mRNA levels in SKOV3ip lines. B: Detection of binding of endogenous HOXA9 in SKOV3ip cells to the CCL2 promoter by ChIP. Negative controls included IP using cells expressing HOXA9 shRNA (shA9-B) and IgG. GAPDH was amplified as an irrelevant gene control. C: CCL2 levels in media conditioned (cond.) by SKOV3ip lines. Shown are average results of ELISAs using three independent sets of each type of SKOV3ip-conditioned medium. D: CCL2 levels in ascites fluid of mouse i.p. xenograft models established from vector-control and HOX-expressing MOSEC lines (n = 5 mice per group). E: Relative HOXA9 mRNA levels in ovarian tumor tissues and levels of CCL2, TGF-β2, IL-10, and CCL17 in matching specimens of ascites fluid from the same patients (n = 14 cases). Correlations were determined by Spearman test. All cases were postmenopausal patients with FIGO stage III/IV, high-grade serous ovarian carcinoma. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0005.
Figure 5
Figure 5
Combination of TGF-β2 and CCL2 recapitulates the stimulatory effects of HOXA9 on macrophages. A: Chemotaxis of primary mouse peritoneal macrophages toward +HOXA9 control (nontargeting) and HOXA9-knockdown (shA9-B) SKOV3ip cells. Where indicated, recombinant TGF-β2 (rTGF-β2) and CCL2 were added to achieve final concentrations of these factors that were the same as those secreted by nontargeting cells [ie, 240 pg/mL of TGF-β214 and 200 pg/mL of CCL2 (refer to Figure 4C)]. ∗P < 0.0005, ∗∗P < 0.005, and ∗∗∗P < 0.01. B: Primary cultures of naïve mouse peritoneal macrophages were stimulated with medium conditioned by SKOV3ip lines. Where indicated, conditioned medium was reconstituted with TGF-β2 and CCL2, as described for A. At 5 days thereafter, macrophages were evaluated for CD206 expression by flow cytometric analysis.
Figure 6
Figure 6
High HOXA9 expression in human ovarian cancer specimens is associated with increased abundance of M2 macrophages and Treg cells and decreased abundance of CD8+ TILs. A: IHC analysis of omental tumors from patients with FIGO stage III/IV, high-grade serous ovarian carcinoma. Cases with no or weak HOXA9 staining were scored as HOXA9-Low (n = 9). Cases with strong HOXA9 staining were scored as HOXA9-High (n = 16). Shown are representative examples of weak and strong HOXA9 staining in omental tumors of two different cases. Also shown are specimens of the same cases stained with Abs to CD68, CD163, FOXP3, and CD8. Scale bar = 50 μm. B: Numbers of tumor-infiltrating CD68+, CD163+, FOXP3+, and CD8+ cells were counted in five random 200× microscopic fields of stained tissue sections of each case. Average numbers of CD68+, FOXP3+, and CD8+ cells are shown for cases grouped by HOXA9 expression, with each symbol representing an individual case. The percentage of macrophages that were CD163+ was calculated from the numbers of CD163+ and CD68+ cells per field for a given case. The statistical significance of differences between groups of cases was determined by U-test. ∗P < 0.05, ∗∗P < 0.01, †P < 0.005, and ‡P < 0.002.
See this image and copyright information in PMC

References

    1. Tlsty T.D., Coussens L.M. Tumor stroma and regulation of cancer development. Annu Rev Pathol. 2006;1:119–150. - PubMed
    1. Kalluri R., Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer. 2006;6:392–401. - PubMed
    1. Mantovani A., Sozzani S., Locati M., Allavena P., Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 2002;23:549–555. - PubMed
    1. Solinas G., Germano G., Mantovani A., Allavena P. Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation. J Leukoc Biol. 2009;86:1065–1073. - PubMed
    1. Pollard J.W. Tumor-educated macrophages promote tumor progression and metastasis. Nat Rev Cancer. 2004;4:71–78. - "VSports" PubMed

"VSports手机版" MeSH terms

Substances (V体育ios版)