Heparanase is required for activation and function of macrophages Lilach Gutter-Kapona, Dror Alishekevitzb, Yuval Shakedb, Jin-Ping Lic, Ami Aronheimd, Neta Ilana, and Israel Vlodavskya,1 aCancer and Vascular Biology Research Center, Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel; bDepartment of Cell Biology and Cancer Science, Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel; cDepartment of Medical Biochemistry and Microbiology, University of Uppsala, SE-751 05 Uppsala, Sweden; and dDepartment of Molecular Genetics, the Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel Edited by Joseph Schlessinger, Yale University School of Medicine, New Haven, CT, and approved October 17, 2016 (received for review July 13, 2016) The emerging role of heparanase in tumor initiation, growth, The carcinoma microenvironment includes nontransformed metastasis, and chemoresistance is well recognized and is encouraging epithelial cells, fibroblasts, endothelial cells, and infiltrated immune the development of heparanase inhibitors as anticancer drugs. Unlike cells. Endothelial cells lining blood and lymph vessels are major the function of heparanase in cancer cells, very little attention has component of the tumor microenvironment, and antiangiogenesis been given to heparanase contributed by cells composing the tumor therapy, targeting vascular endothelial growth factor (VEGF) or microenvironment. Here we used a genetic approach and examined its receptor (VEGFR2), is implemented clinically (15). In addi- the behavior and function of macrophages isolated from wild-type tion, recent research has revealed the critical roles of inflam- (WT) and heparanase-knockout (Hpa-KO) mice. Hpa-KO macrophages matory responses in different stages of tumor development and express lower levels of cytokines (e.g., TNFα,IL1-β) and exhibit lower metastasis (16). motility and phagocytic capacities. Intriguingly, inoculation of control The most plentiful immune cells within the tumor microenviron- monocytes together with Lewis lung carcinoma (LLC) cells into Hpa-KO ment are tumor-associated macrophages (TAMs) (16, 17). Func- mice resulted in nearly complete inhibition of tumor growth. In tionally, two distinct states have been described for macrophages: striking contrast, inoculating LLC cells together with monocytes M1 (or classically activated) and M2 (or type II, alternatively acti- isolated from Hpa-KO mice did not affect tumor growth, indicating vated). The M1 phenotype is proinflammatory and characterized by that heparanase is critically required for activation and function of the release of inflammatory cytokines (e.g., IL-1β,TNFα), reactive macrophages. Mechanistically, we describe a linear cascade by nitrogen and oxygen intermediates, and microbicidal/tumoricidal ac- which heparanase activates Erk, p38, and JNK signaling in macro- tivity. In contrast, M2 macrophages are polarized by anti-inflammatory phages, leading to increased c-Fos levels and induction of cytokine molecules (e.g., IL-4, IL-13) and support angiogenesis, tissue expression in a manner that apparently does not require heparanase remodeling, and repair (18, 19). Thus, macrophages are thought enzymatic activity. These results identify heparanase as a key medi- to play a dual role in tumor growth, initiating an immune re- ator of macrophage activation and function in tumorigenesis and sponse against transformed cells on the one hand and promoting cross-talk with the tumor microenvironment. tumor growth and angiogenesis on the other hand (20–23). Here we used a genetic approach to examine the behavior heparanase | macrophage | tumor growth | cytokine expression | and function of macrophages isolated from wild-type (WT) and knockout mice heparanase-knockout (Hpa-KO) mice (24). Hpa-KO macro- phages express lower levels of cytokines (e.g,, TNFα,IL-1β) eparanase is an endo-β-glucuronidase that cleaves heparan previously shown to be induced by the addition of heparanase or1its overexpression (25), and appear less motile. Inoculating control Hsulfate (HS) side chains presumably at sites of low sulfation. + Traditionally, heparanase activity was correlated with the meta- monocytes (CD11b ) together with Lewis lung carcinoma (LLC) static potential of tumor-derivedcells,attributedtoenhanced cells into Hpa-KO mice resulted in nearly complete inhibition of cell dissemination as a consequence of HS cleavage and remodeling of the extracellular matrix (ECM) barrier (1, 2). Significance Intensive research efforts over the last decade have revealed that heparanase expression is up-regulated in an increasing The tumor microenvironment is now considered to play a major number of human carcinomas and hematologic malignancies. role in cancer growth and metastasis. Heparanase is the only In many cases, heparanase induction correlates with increased enzyme in mammals capable of cleaving heparan sulfate, an ac- tumor metastasis, vascular density, and shorter postoperative tivity that is highly implicated in tumor growth, metastasis, and survival of cancer patients (3–6), providing strong clinical inflammation. Here we provide evidence that heparanase is crit- support for the protumorigenic function of the enzyme and ically required for the activation and function of macrophages, an encouraging the development of heparanase inhibitors as anti- important constituent of the tumor microenvironment. Mecha- cancer drugs (3, 7, 8). More recent studies have provided com- nistically, we describe a linear cascade by which heparanase ac- pelling evidence associating heparanase level with all stages of tivates Erk, p38, and JNK signalinginmacrophages,leadingto tumor formation, including tumor initiation, growth, metastasis, increased c-Fos levels and induction of cytokine expression in a and chemoresistance (9–14). manner that apparently does not require heparanase enzymatic Although heparanase up-regulation by tumor cells is well activity. These results identify heparanase as a key mediator of documented, the protumorigenic contribution of heparanase pro- macrophage activation and function in tumorigenesis and cross- vided by cells composing the tumor microenvironment has not talk with the tumor microenvironment. been sufficiently explored. We recently reported that heparanase- neutralizing polyclonal and monoclonal antibodies attenuated the Author contributions: N.I. and I.V. designed research; L.G.-K., D.A., Y.S., and N.I. per- formed research; J.-P.L. and A.A. contributed new reagents/analytic tools; L.G.-K., D.A., growth of myeloma and lymphoma cells within bones (14). No- Y.S., J.-P.L., A.A., N.I., and I.V. analyzed data; and N.I. and I.V. wrote the paper. tably, the neutralizing antibodies also attenuated the growth of The authors declare no conflict of interest. Raji lymphoma cells, which do not express heparanase owing to This article is a PNAS Direct Submission. methylation of the gene, implying that neutralization of heparanase 1To whom correspondence should be addressed. Email: [email protected]. contributed by the tumor microenvironment is sufficient to restrain This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tumor growth (14). 1073/pnas.1611380113/-/DCSupplemental. E7808–E7817 | PNAS | Published online November 14, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1611380113 Downloaded by guest on September 28, 2021 tumor growth. In striking contrast, inoculating LLC cells to- Results PNAS PLUS gether with monocytes isolated from Hpa-KO mice did not affect Reduced Cytokine Expression by Hpa-KO Macrophages. We have tumor growth, suggesting that heparanase is required for the previously shown that the exogenous addition or overexpression proper activation and function of macrophages. of heparanase activates macrophages and stimulates cytokine Fig. 1. Reduced cytokine expression and motility of heparanase-deficient macrophages. (A) Heparanase activity. Cell exudates were collected from the peritoneum of control (WT) and Hpa-KO mice at 3 d after thioglycolate administration. (Left) After washing, cells (2 × 106) were lysed by three freeze/thaw cycles, and cell lysates were applied on sulfate-labeled ECM dishes. Determination of heparanase activity was carried out as described in Materials and Methods.(Right) Heparanase activity was evaluated similarly in freshly isolated WT macrophages (Mac) and an equal number (2 × 106) of LLC cells. (B) Cytokine expression. Cell exudates were collected from the peritoneum of control (WT) and Hpa-KO mice at 3 d after thioglycolate administration. Cells were plated on tissue culture dishes, and nonadherent cells were removed after 24 h. Total RNA was extracted from the adherent macrophages, and corresponding cDNAs were subjected to quantitative real-time PCR analyses using a set of primers specific for the indicated cytokines. Cytokine expression in Hpa-KO macrophages is shown graphically in relation to the level in control macrophages set arbitrarily to a value of 1. Note that the expression level of most cytokines is reduced in Hpa-KO macrophages. *P < 0.04. (C) WT C57BL/6 mice were administrated with the indicated anti–heparanase-neutralizing antibody (250 μg/mouse) or control rabbit IgG (Control) 30 min before the administration of thioglycolate. Cell exudate was collected 3 d later, and cytokine expression was evaluated as above. Note the reduced cytokine expression by peritoneal macrophages following treatment with the heparanase- neutralizing antibodies. *P < 0.02. (D) Heparanase small-molecule