Monocyte-Derived Fibrocyte Differentiation Breast Cancer Cells

Galectin-3 Binding Protein Secreted by Breast Cancer Cells Inhibits Monocyte-Derived Fibrocyte Differentiation

This information is current as Michael J. V. White, David Roife and Richard H. Gomer of September 25, 2021. J Immunol 2015; 195:1858-1867; Prepublished online 1 July 2015; doi: 10.4049/jimmunol.1500365 http://www.jimmunol.org/content/195/4/1858 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Galectin-3 Binding Protein Secreted by Breast Cancer Cells Inhibits Monocyte-Derived Fibrocyte Differentiation

Michael J. V. White,* David Roife,† and Richard H. Gomer*

To metastasize, tumor cells often need to migrate through a layer of collagen-containing scar tissue which encapsulates the tumor. A key component of scar tissue and fibrosing diseases is the monocyte-derived fibrocyte, a collagen-secreting profibrotic cell. To test the hypothesis that invasive tumor cells may block the formation of the fibrous sheath, we determined whether tumor cells secrete factors that inhibit monocyte-derived fibrocyte differentiation. We found that the human metastatic breast cancer cell line MDA-MB-231 secretes activity that inhibits human monocyte-derived fibrocyte differentiation, whereas less aggressive breast cancer cell lines secrete less of this activity. Purification indicated that Galectin-3 binding protein (LGALS3BP) is the active factor. Recombinant LGALS3BP inhibits monocyte-derived fibrocyte differentiation, and immunodepletion of LGALS3BP from MDA-

MB 231 conditioned media removes the monocyte-derived fibrocyte differentiation-inhibiting activity. LGALS3BP inhibits the dif- Downloaded from ferentiation of monocyte-derived fibrocytes from wild-type mouse spleen cells, but not from SIGN-R12/2 mouse spleen cells, suggesting that CD209/SIGN-R1 is required for the LGALS3BP effect. Galectin-3 and galectin-1, binding partners of LGALS3BP, potentiate monocyte-derived fibrocyte differentiation. In breast cancer biopsies, increased levels of tumor cell-associated LGALS3BP were observed in regions of the tumor that were invading the surrounding stroma. These findings suggest LGALS3BP and galectin-3 as new targets to treat metastatic cancer and fibrosing diseases. The Journal of Immunology, 2015, 195: 1858–1867. http://www.jimmunol.org/ key component of scar tissue is the fibrocyte, a CD34+, vate nearby fibroblasts to proliferate and secrete collagen (3, 17– CD45+,collagen+ cell found in healing wounds and fi- 20). Increased monocyte-derived fibrocyte differentiation correlates A brotic lesions. Monocytes are recruited to wounds or with increased fibrosis in animal models (21, 22). Elevated circu- fibrotic lesions by chemokines (1, 2), and in response to wound lating fibrocyte counts also associate with poor prognosis in human signals such as tryptase released from mast cells, or thrombin diseases (23). activated during blood clotting, differentiate into monocyte- In response to a foreign object or inflammatory environment, the derived fibrocytes (3–5). The term “fibrocyte” has been used to immune system can initiate a desmoplastic response in which refer to multiple cell types, including cells of the ear (6), CD34+, monocytes differentiate into monocyte-derived fibrocytes to form + + CD45 ,collagen circulating cells (4, 7), and spindle-shaped a sheath of fibrotic tissue around the foreign object (24–27). In by guest on September 25, 2021 CD34+,CD45+,collagen+ cells that differentiate in a tissue or in response to some tumors, the immune system also initiates a cell culture (4, 8). Spindle-shaped CD34+,CD45+,collagen+ fibro- desmoplastic response, attempting to contain the tumor (9, 28). This cytes are found in scar tissue (9, 10), and it appears likely that desmoplastic sheath is a dynamic, responsive tissue that adjusts circulating CD34+,CD45+,collagen+ PBMC are either precursors to to changing conditions in the tumor microenvironment (29, 30). the fibrocytes in scar tissue (11, 12) or are fibrocytes that have left To metastasize through this desmoplastic tissue, cancer cells the scar tissue. must find a way to remove scar tissue or to prevent scar tissue from Although circulating fibrocytes can be directly purified (12–14), in forming (29–34). As cancer progresses toward metastasis and this study, we cultured PBMC or monocytes to obtain monocyte- a more mesenchymal phenotype, it interacts with the immune derived fibrocytes (15). Monocytes isolated from PBMC differen- system in different ways. Some tumors attempt to evade the im- tiate in vitro in a defined medium into monocyte-derived fibrocytes mune system, and others act to suppress the immune system (16). Monocyte-derived fibrocytes express collagen and other ex- (35–39). tracellular matrix proteins, secrete proangiogenic factors, and acti- The MDA-MB 231 (231) cell line was isolated from metastases of a breast cancer patient (40). 231 cells behave aggressively in culture and murine models, displaying a metastatic phenotype that *Department of Biology, Texas A&M University, College Station, TX 77843; and †Department of Surgical Oncology, University of Texas M.D. Anderson Cancer suggests that these cells retain the protein expression profile which Center, Houston, TX 77030 allowed them to metastasize through the basement membrane of Received for publication February 18, 2015. Accepted for publication June 2, 2015. the original patient (41). This work was supported in part by National Institutes of Health Grants Galectin-3 binding protein (LGALS3BP), previously called T32CA009599 (to D.R.) and HL118507 (to R.H.G.). Mac-2 binding protein and tumor-associated Ag 90K, is a heavily Address correspondence and reprint requests to Dr. Richard H. Gomer, Department glycosylated 90-kDa protein (42). LGALS3BP binds to galectins of Biology, Texas A&M University, 301 Old Main Drive, ILSB MS 3474, College 1, 3, and 7, fibronectin, and collagen IV, V, and VI (42–44). Station, TX 77843-3474. E-mail address: [email protected] LGALS3BP is a member of the scavenger receptor cysteine-rich The online version of this article contains supplemental material. domain (SRCR) family of proteins (45). LGALS3BP is ubiq- Abbreviations used in this article: 231, MDA-MB 231; 435, MDA-MB-435; CHO, Chinese hamster ovary; CM, conditioned medium; DC-SIGN, dendritic cell–specific uitously expressed in bodily secretions, including milk, tears, intercellular adhesion molecule 3-grabbing nonintegrin; LGALS3BP, Galectin-3 semen, and serum, usually 10 mg/ml (46). In patients with aggres- binding protein; PFM, protein-free medium; SFM, serum-free medium; SRCR, scav- sive hormone-regulated cancers, including breast cancer, serum enger receptor cysteine-rich. LGALS3BP concentration can be an order of magnitude higher Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 than in normal serum (47–49). In breast milk, LGALS3BP www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500365 The Journal of Immunology 1859 concentration can rise and fall over the same range (∼10–100 mg/ml) (Liebniz Institute: German Collection of Microogranisms and Cell Culture, depending on the length of time after the pregnancy (46). LGALS3BP Braunschwieg, Germany), and U-937 (83), HL-60 (84), THP-1 (85), and is produced mostly by epithelial cells in glands (breast and tear ducts) HEK-293 (86) cells were from the American Type Culture Collection (Manassas, VA). The MDA-MB 435 cell line has previously been classed and cancer cells (especially breast cancer cells) (50). as a breast cancer cell line but is currently classed as a melanoma cell line Higher levels of serum LGALS3BP correlate with worse out- (66, 87). Each tumor cell line was tested for mycoplasma contamination comes in breast cancer patients (48, 49, 51, 52), whereas higher using a PCR detection kit (MDBioproducts, St. Paul, MN) following the levels of LGALS3BP’s binding partner galectin-3 correlate with manufacturer’s instructions, and all work was done with cell lines containing undetectable levels of mycoplasma. better outcomes for breast cancer patients (53). LGALS3BP pro- Tumor cell lines were grown in DMEM supplemented with 10% FCS motes angiogenesis by increasing vascular endothelial growth (Genesee Scientific, San Diego, CA) in 75-cm2 flasks (BD Biosciences, factor signaling and directly signaling endothelial cells (43, 54). Franklin Lakes, NJ) until 70% confluent. Adhered cells were washed three Mouse knockouts of LGALS3BP show higher circulating levels of times with PBS and were then incubated with 10 ml PFM. After 24 or 48 h, 3 TNF-a, IL-12, and IFN-g, suggesting a role of LGALS3BP in the CM was collected and clarified by centrifugation at 300 g for 10 min. CM from 231 cells was further clarified by centrifugation at regulating the immune system (55). 1,000 3 g for 10 min, followed by clarification at 200,000 3 g for 1 h. The Galectin-3 is a ∼30-kDa protein expressed nearly ubiquitously supernatant was then concentrated with a 100-kDa centrifugal filter in human tissues and can be secreted from cells, associated with (EMD Millipore, Billerica, MD), and buffer was exchanged with 20 mM membrane-bound carbohydrates, or located in the cytoplasm (53, sodium phosphate (pH 7.2). Proteins were visualized by silver stain on 4–20% SDS-PAGE gels (Bio-Rad, Hercules, CA), and protein concen- 56–59). Galectin-3 is a biomarker of fibrosing diseases such tration was assessed by absorbance at 280 nm (Synergy MX, Bio-Tek, as heart disease and pulmonary fibrosis (60, 61). As the disease Winooski, VT). severity increases, serum galectin-3 concentrations increase. Downloaded from Protein purification and identification Galectin-3 is widely expressed by immune system cells and promotes the differentiation of monocytes into macrophages A total of 300 ml 231 CM was clarified by ultracentrifugation, concentrated, (62). Galectin-3 interacts with a number of intercellular and and buffer-exchanged as described above and resuspended in 1 ml. This was loaded on a 5-ml MonoQ anion exchange column on an AKTA chroma- intracellular receptors and ligands and is theorized to have roles tography system (GE Healthcare, Piscataway, NJ). The column was washed in inflammation, host response to a virus, and wound healing with 6 ml 20 mM NaPO4 (pH 7.4) buffer (first six fractions), and bound

(57, 62, 63). proteins were then eluted with a 24-ml gradient of 0–0.5 M NaCl in http://www.jimmunol.org/ In this paper, we show that 231 cells secrete LGALS3BP, which 20 mM NaPO4 (pH 7.4), collecting 0.5-ml fractions. Serial doubling in turn inhibits monocyte-derived fibrocyte differentiation, and that dilutions of fractions were then mixed with PBMC, and their effect on monocyte-derived fibrocyte differentiation was measured as described conversely galectin-3 promotes monocyte-derived fibrocyte dif- previously (15). Trypsin digestion of samples, purification of peptides with ferentiation. LGALS3BP and galectin-3 are new modulators of Zip tips (EMD Millipore), and mass spectrometry were performed by the fibrosis in the tumor microenvironment. In addition, the effects of University of Utah Mass Spectrometry Core Facility, and peptides with LGALS3BP and galectin-3 on monocyte-derived fibrocytes show MASCOT scores . 40 and mass errors , 3 ppm were used for protein identification. these proteins are active signaling molecules in cancer and fibrosis, respectively, and not passive biomarkers. Flow cytometry by guest on September 25, 2021 PBMC were placed on an ultralow attachment plate (Corning, Corning, NY) Materials and Methods and incubated with 231 CM at the indicated concentrations for the indicated PBMC isolation and culture time. These cells were then removed from the plate using ice-cold 5 mM EDTA in PBS (Rockland, Limerick, PA) with gentle pipetting. PBMC were Human blood was collected from adult volunteers who gave written consent collected by centrifugation at 300 3 g for 10 min, resuspended in 100 ml and with specific approval from the Texas A&M University human subjects ice-cold PBS, and analyzed for viability using propidium iodide (Sigma- Institutional Review Board. PBMC were isolated and cultured as described Aldrich) and forward/side scatter via flow cytometry (Accuri-BD, Franklin previously (64, 65). Protein-free medium (PFM) was Fibrolife basal me- Lakes, NJ) as described previously (88, 89). dium (Lifeline, Walkersville, MD) supplemented with 10 mM HEPES (Sigma-Aldrich, St. Louis, MO), 13 nonessential amino acids (Sigma- Immunodepletion of CM Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 2 mM glutamine (Lonza, Basel, Switzerland), 100 U/ml penicillin, and 100 mg/ml strepto- For immunodepletion, rabbit polyclonal anti–galectin-3 binding protein mycin (Lonza). Serum-free medium (SFM) was PFM further supplemented (BIOSS, Woburn, MA), mouse monoclonal anti–galectin-3 (BioLegend), with 10 mg/ml recombinant human insulin (Sigma-Aldrich), 5 mg/ml mouse IgG isotype control (Jackson ImmunoResearch Laboratories, West recombinant human transferrin (Sigma-Aldrich), and 550 mg/ml filter- Grove, PA), and rabbit polyclonal anti-protein S (Sigma-Aldrich) Abs were sterilized human albumin (65). PBMC were cultured in SFM with the in- bound to protein G–coated Dynabeads (Invitrogen, Carlsbad, CA) beads dicated concentrations of conditioned medium (CM), recombinant human following the manufacturer’s instructions. Beads complexed with Abs galectin-1 and galectin-3 (PeproTech, Rocky Hill, NJ), or recombinant hu- were mixed 1:10 with CM at 37˚C for 2 h. Beads were then removed from man galectin-3 binding protein (R&D Systems, Minneapolis, MN) for 5 d, the CM following the manufacturer’s instructions. after which PBMC were stained, and monocyte-derived fibrocytes were Sequencing 231 LGALS3BP identified by morphology as 40- to 100-mm-long, spindle-shaped cells tapered at both ends with oval nuclei (65). The same phenotypic criteria Total RNAwas isolated from 231 cells using a kit (Omega Biotek, Norcross, were used to identify the 25- to 60-mm-long mouse monocyte-derived GA), and cDNA was generated using a kit (Thermo Scientific, Waltham, fibrocytes. Adhered cells and macrophages were counted as described MA). LGALS3BP was amplified using Phusion polymerase (New England previously (65). Human monocytes were purified, tested for purity, and Biolabs, Ipswich, MA) with primers 59-AACTCGAGGTCCACACCTGAGTT- cultured as described previously (8, 65). For immunohistochemistry, GG-39 and 59-AAACTCCTAGTCCACACCTGAGG-39 that encompassed all PBMC were fixed and stained for CD209 (BioLegend, San Diego, CA) as known or predicted transcript variants (90) and resulted in a single band on described previously (8). a DNA gel. Amplified LGALS3BP was ligated into pCMV and sequenced at Lonestar Labs (Houston, TX), using the primers listed above and internal Tumor cell lines and CM primers (59-CGCCCTGGGCTTCTGTGG-39 and 59-GGTCTATCAGTCCA- 231 (40), 435 (66), and DCIS.com (67) cells were gifts from Dr. W. Porter, GACG-39). Texas A&M University (College Station, TX). HT-29 (68), SW480 (69), Isolation of mouse spleen cells DKOB8 (70), and HCT (71) cells were gifts from Dr. R. Chapkin (Texas A&M University, College Station, TX). MCF-7 (72), ADR-RES (73), SIGN-R12/2 spleens were developed by A. McKenzie (Medical Research OVCAR-8 (74, 75), SNU 398 (76), HEP-G2 (77), SW 1088 (78), U87 MG Council, Cambridge, U.K.) (91) and were gifts from Dr. J. Ravetch at (79), and PANC-1 (80) cells were gifts from Dr. D. Wallis (Texas A&M Rockefeller University (New York, NY). Mouse spleen cells were isolated University). Mono mac-1 (81) and Mono mac-6 (82) were from the DSMZ and cultured as described previously (92). 1860 LGALS3BP INHIBITS FIBROCYTE DIFFERENTIATION

Staining of biopsies derived fibrocyte differentiation. In our culture conditions, Deidentified slides of formalin-fixed paraffin-embedded biopsies or surgical monocyte-derived fibrocytes express canonical fibrocyte markers specimens from patients with confirmed infiltrative ductal carcinoma of the (8). Human PBMC were incubated in the presence or absence of breast were provided by Dr. K. Hunt at the University of Texas M.D. tumor cell line CM, and after 5 d, monocyte-derived fibrocytes Anderson Cancer Center. Patients signed informed consent prior to the were counted. In the absence of CM, we observed 81–1374 initiation of treatment. The M.D. Anderson Institutional Review Board monocyte-derived fibrocytes per 105 PBMCs from the different approved the use of all patient-derived tissues and data. Slides were 5 deparaffinized in xylene and rehydrated through graded ethanols. Ags were donors, similar to what we have observed previously (65). Per 10 retrieved by incubating sections with Ag Unmasking Solution H-3300 PBMC originally added to each well, 17,617 6 5,720 (mean 6 (Vector Laboratories, Burlingame, CA) in a steamer for 20 min. Slides SD, n = 38) remained adhered to the plate after fixation. Of these, were then permeabilized by incubating with 0.2% Triton X-100 in PBS for 638 6 278 had a monocyte-derived fibrocyte morphology, and 45 min at room temperature. Slides were blocked by incubating with 1% 6 BSA in PBS for 1 h at room temperature. Slides were then incubated with 6062 2208 had a phenotype similar to macrophages cultured in primary Ab diluted in 0.1% BSA/PBS overnight at 4˚C. Primary Abs were serum and MCSF (8). Because of this variability, monocyte- collagen I (rabbit pAb, 1:500, Abcam number ab34710), CD45RO (mouse derived fibrocyte numbers were thus normalized to CM-free mAb, 1:100, BioLegend number 304202), LGALS3BP (Rabbit pAb, controls. CM from 231 (40) and 435 cells (66) inhibited 1:200, GeneTex number GTX116497), and galectin-3 (Rat mAb, 1:200, monocyte-derived fibrocyte differentiation in a concentration- BioLegend number 125401). Secondary Ab in 0.1% BSA/PBS was then added for 1 h at room temperature. Secondary Abs were donkey anti- dependent manner (Fig. 1A, Supplemental Fig. 1A), and this ef- mouse DyLight 488, donkey anti-rabbit Red X, and goat anti-rat 488 fect was observed for PBMC from all donors tested. The 435 CM (1:500, Jackson ImmunoResearch Laboratories). Sections were DAPI did not affect the number of adherent cells after removing weakly stained for 10 min and mounted with Dako Fluorescent mounting medium adhering cells, and then fixing and staining, whereas 231 CM Downloaded from (DakoCytomation, Carpinteria, CA). Images were captured with an Olympus BX51 microscope and Olympus DP72 camera (Olympus, Tokyo, slightly inhibited adherent cell number only at 12.5% CM, which Japan) and CellSens software (Center Valley, PA). is well above the IC50 for monocyte-derived fibrocyte inhibition (Fig. 1B, Supplemental Fig. 1B). PBMC exposed to 231 CM or Statistics 435 CM for 5 d did not have significantly increased cell death as Statistics were performed using Prism (Graphpad Software, San Diego, assessed by propidium iodide staining (Fig. 1C, Supplemental CA). Differences were assessed by two-tailed t tests or two-tailed Mann– Fig. 1C) or decreased total cell number (this includes the http://www.jimmunol.org/ Whitney tests. Significance was defined as p , 0.05. weakly adherent cells removed before fixing and staining) as assessed by removing all cells from the assay well and counting by Results flow cytometry (Fig. 1D, Supplemental Fig. 1D). Some concen- 231 and 435 cells secrete factors that inhibit monocyte-derived trations of 231 CM increased total cell numbers (Fig. 1D). This fibrocyte differentiation may due to factors in the CM that promote cell survival and/or cell To determine whether factors secreted from tumors might promote proliferation. or inhibit monocyte-derived fibrocyte differentiation, we examined Monocyte-derived fibrocytes differentiate from monocytes (3, the effect of CM from a variety of human tumor cell lines on human 16, 17). Cells in the PBMC population include T cells, B cells, NK monocytes cultured in SFM and measured the resulting monocyte- cells, and other cells in addition to monocytes (15). To determine by guest on September 25, 2021

FIGURE 1. 231 CM inhibits monocyte-derived fibrocyte differentiation. (A) PBMC were cultured in SFM in the presence of the indicated concentrations of 231 CM for 5 d. Monocyte-derived fibrocyte counts were normalized for each donor to the SFM control. (B) Counts of total adherent PBMC per five fields of view at the indicated concentrations of 231 CM. Total propidium iodide–positive PBMC (C) and total PBMC (D) after 5 d at the indicated concentrations of 231 CM, measured by flow cytometry. (E) Total number of monocyte-derived fibrocytes from monocytes cultured at the indicated concentrations of 231 CM for 5 d. Monocytes were 16 6 9% (mean 6 SEM, n =3)ofthePBMCand926 5% in the purified fraction. (F) Total number of macrophages from PBMC cultured at the indicated concentrations of 231 CM for 5 d. Values are mean 6 SEM. The absence of error bars indicates that the error was smaller than the plot symbol. (A, B, and F) n = 38, (C–E) n =3.*p , 0.05, **p , 0.01, ***p , 0.001 compared with the control (t test). The Journal of Immunology 1861 whether the CM effect on monocyte-derived fibrocyte differenti- cient 0.73 6 0.20). When added to monocytes, the 231 and ation is a direct effect on monocytes or is mediated by the other 435 IC50swere1.26 0.2% (n = 5, Hill coefficient 1.08 6 0.15) cells in a PBMC population, 231 or 435 CM was added to and 1.9 6 0.7% (n = 3, Hill coefficient 1.70 6 0.60), respec- purified human monocytes. 231 CM and 435 CM inhibited tively. The difference in IC50s for 231 CM between PBMCs and monocyte-derived fibrocyte differentiation from purified monocytes monocytes was significant with p , 0.05 (t test); the difference (Fig. 1E, Supplemental Fig. 1E). For the inhibition of monocyte- for 435 CM was not significant. Because purifying monocytes derived fibrocyte differentiation from PBMC, the IC50 of 231 CM and thus removing other cells from the PBMC population was 0.33 6 0.05% (mean 6 SEM, n = 37, Hill coefficient 1.06 6 modestly increased the 231 CM IC50, these data suggest that 0.13) and that of 435 CM was 0.45 6 0.17% (n = 7, Hill coeffi- either the monocyte purification procedure modestly reduced the ability of monocytes to respond to the factor(s) in CM that inhibits monocyte-derived fibrocyte differentiation or else the presence of the other cells in the PBMC population somewhat potentiates the ability of monocytes to respond to the factor(s). In either case, the data indicate that monocytes can respond direct to the factor(s). In addition to differentiating into monocyte-derived fibrocytes, monocytes can also differentiate into macrophages. To determine whether the CMs that inhibit monocyte-derived fibrocyte differ-

entiation also affect macrophage differentiation from monocytes, Downloaded from we counted the total number of adhered macrophages, as assessed by morphology. 231 CM caused a decrease in macrophage differentiation at 12.5% CM but did not affect the number of macrophages at lower concentrations, which inhibited monocyte- derived ﬁbrocyte differentiation, and 435 did not signiﬁcantly

inhibit macrophage differentiation (Fig. 1F, Supplemental Fig. 1F). http://www.jimmunol.org/ Taken together, the data indicate that factors in 231 and 435 CM affect monocytes to strongly inhibit monocyte-derived ﬁbrocyte differentiation, while having no effect, or a relatively modest effect, on cell death, total cell numbers, numbers of macrophages, and the numbers of adherent cells. Some but not all human cancer cell lines also secrete a monocyte-derived ﬁbrocyte inhibitory activity

To determine whether other tumor types might secrete factors that by guest on September 25, 2021 inhibit monocyte-derived fibrocyte differentiation, we exposed PBMC to CM from cancer cell lines derived from human breast, skin, colon, liver, pancreas, brain, ovary, and leukocyte tissue. We defined units of activity as the inverse of the CM’s IC50 for monocyte-derived fibrocyte inhibition. Of the cell lines tested, CM from OVCAR-8, U-87 MG, 231, 435, MCF-7, and DCIS.com significantly inhibited monocyte-derived fibrocyte differentiation (Fig. 2). OVCAR-8 is an ovarian cancer cell line derived from a metastatic site (74, 75). U87-mg is derived from a glioblastoma (79). 231 is derived from a breast cancer metastasis (40). 435 has an uncertain origin: originally the cell line was listed as a breast cancer line by the American Type Culture Collection (66). Cur- FIGURE 2. The effect of CM from cancer cell lines on monocyte- rently, the cell line is listed as a melanoma cell line (87). MCF-7 is derived fibrocyte differentiation. (A) CM from 25 different cancer cell lines show different levels of potentiation and inhibition for monocyte-derived fibrocyte differentiation. Activity units are the dilution of CM that inhibited monocyte-derived fibrocyte differentiation to 50% of the control value. Higher activity units indicate more potent inhibition of monocyte- derived fibrocyte differentiation. n = 3 for Mono mac-1, Mono mac-6, U937, HL-60, THP-1, DK0B8, HT-29, HCT-15, HCT (P21+/P53+), HCT (P212/P53+), HCT (P21+/P532), HCT (P212/P532), HEK-293, K562; n = 6 for ADR-RES, OVCAR-8, SNU-398, HEP-G2, U-87 MG, PANC-1, SW-1088, SW480, n = 7 for DCIS.com, n = 10 for MCF-7 and 435, and n = 38 for 231. A, cancers derived from breast tissue; B, skin; C, leukocyte; D, colon; E, embryonic kidney; F, ovarian; G, liver; H, brain; and I, pancreas. The absence of error bars indicates that the error was smaller than the plot symbol. #Inconsistent inhibition of monocyte-derived fibrocyte differentiation (variability of response of PBMC from different FIGURE 3. 231 CM’s monocyte-derived fibrocyte inhibitory activity is donors). *p , 0.05, **p , 0.01, ***p , 0.001 compared with the control .100 kDa. The indicated fractions were assessed for their ability to inhibit (t test). (B) CM from SW480 cells potentiated monocyte-derived fibrocyte monocyte-derived fibrocyte differentiation. The activity units were normal- differentiation. Values are mean 6 SEM, n =6.*p , 0.05, **p , 0.01, ized to the value for the CM. Values are mean 6 SEM, n =24.#Fibrocyte ***p , 0.001 compared with the control (t test). potentiation. 1862 LGALS3BP INHIBITS FIBROCYTE DIFFERENTIATION a breast cancer cell line derived from a pleural effusion (72). in fraction 27 (Fig. 4B). Tryptic fragments of proteins in fraction DCIS.com is derived from a normal breast tissue cell line 27 were analyzed by mass spectrometry. In decreasing order of the (MCF10A) passaged through a mouse and forms a nonmetastatic number of identified peptides, the identified proteins were human ductal carcinoma in situ when injected into mice (67). Each cell galectin-3 binding protein, desmoplakin, plakoglobin, desmoglein line whose CM inhibited monocyte-derived fibrocyte differentia- type 1, pentraxin-3, adult intestinal phosphatase, and dermcidin. tion was isolated from hormone-secreting tissues (breast and However, after purifying peptides with a Ziptip, the only identified ovarian), with the exception of the U-87 MG and 435 cell lines. peptides corresponded to LGALS3BP (GI:5031863). No colon, liver, pancreatic, or leukemia CM significantly inhibited Immunodepletion of LGALS3BP from CM removes most of the monocyte-derived fibrocyte differentiation (Fig. 2A), and 0.4– monocyte-derived fibrocyte inhibitory activity 10% SW480 colon cancer CM modestly potentiated monocyte- derived fibrocyte differentiation (Fig. 2B). To determine whether the LGALS3BP detected in CM affects monocyte-derived fibrocyte differentiation, we immunodepleted The 231 monocyte–derived fibrocyte inhibitor is a protein and LGALS3BP from 231 and 435 CM. Immunodepletion with a can be concentrated and purified control Ab had little effect on the ability of CM to inhibit To determine whether the monocyte-derived fibrocyte inhibition monocyte-derived fibrocyte differentiation (Fig. 5, Supplemental activity in 231 CM is due to a protein, we exposed conditioned Fig. 3). Immunodepletion of LGALS3BP from 231 CM (Fig. 5) media to trypsin, heat, and freeze-thaw cycles. All three treatments increased the IC50 by 8.6 6 1.3-fold (mean 6 SEM, n =7,p , 0. strongly decreased the ability of 231 CM to inhibit monocyte- 001, t test). Similarly, immunodepletion of LGALS3BP from derived fibrocyte differentiation (Supplemental Fig. 2). The 435 CM (Supplemental Fig. 3) increased the IC50 by 21 6 11-fold Downloaded from 231 CM retained ∼80% activity after clarification by ultracentri- (mean 6 SEM, n =7,p , 0.001, t test). These results suggest that fugation and was largely retained by a 100-kDa filter (Fig. 3A). LGALS3BP is a significant component of the 231 and 435 CM The components of 231 CM that passed through a 100-kDa filter monocyte–derived fibrocyte inhibitory activity. potentiated monocyte-derived fibrocyte differentiation (Supplemental Recombinant LGALS3BP inhibits monocyte-derived fibrocyte Fig. 3A). This potentiating activity was retained by a 10-kDa filter differentiation

(Supplemental Fig. 3B). Taken together, these results suggest that the http://www.jimmunol.org/ 231 CM activity, which inhibits monocyte-derived fibrocyte differ- To test the hypothesis that LGALS3BP inhibits monocyte-derived entiation, is a protein. fibrocyte differentiation, we incubated PBMC with recombinant To purify the 231 CM monocyte-derived fibrocyte differentiation human LGALS3BP. LGALS3BP significantly inhibited monocyte- 6 inhibitor, the 100-kDa retentate was fractionated by anion ex- derived fibrocyte differentiation with an IC50 of 0.22 0.05 mg/ml change chromatography, and fractions were assayed for activity (Hill coefficient 6.5 6 2.2) (Fig. 6) but, unlike 231 CM (Fig. 6A), (Fig. 4A). There was little activity in the flow-through, some ac- did not completely inhibit monocyte-derived fibrocyte differenti- tivity throughout the elution, and a large peak of activity in ation. Taken together with the immunodepletion assays, the data fractions 27 and 28, corresponding to a NaCl concentration of 375 indicate that LGALS3BP does inhibit monocyte-derived fibrocyte mM. The IC50 of both of these fractions occurred at a ∼4,096-fold differentiation. by guest on September 25, 2021 6 6 dilution, with a Hill coefficient of 4.6 1.7 (mean SEM, n = 6). The 231 LGALS3BP mRNA encodes a canonical LGALS3BP Silver-stained gels showed prominent bands at ∼85 and ∼39 kDa LGALS3BP has an experimentally verified transcript variant and several predicted transcript variants (90). To determine whether 231 cells secreted a truncated or alternatively spliced variant of LGALS3BP, mRNA was isolated from 231 and converted to cDNA. Using primers that encompass all known or predicted

FIGURE 4. Anion exchange chromatography of the partially purified factor. (A) A 100-kDa concentrated 231 CM, produced as in Fig. 3, was fractionated on an anion exchange column. Fractions were assayed as in FIGURE 5. Immunodepletion of LGALS3BP decreases 231 CM’s Fig. 1, and the resulting monocyte-derived fibrocyte inhibition was mea- monocyte-derived fibrocyte inhibitory activity. 231 CM was immunode- sured by activity units, as in Fig. 2. (B) SDS-PAGE gel of the fractions, pleted with anti-LGALS3BP or isotype control Abs. Values are mean 6 silver stained. SEM, n =7. The Journal of Immunology 1863

as a single band, the recombinant LGALS3BP from Chinese hamster ovary (CHO) cells has several bands. R&D Systems is the only manufacturer of recombinant LGALS3BP produced in eukaryotic cells. 231 and 435 CM inhibit monocyte-derived fibrocyte differentiation using a dendritic cell–specific intercellular adhesion molecule 3-grabbing nonintegrin –dependent mechanism The C-type lectin receptor dendritic cell–specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)/CD209 is expressed on monocytes and is upregulated as monocytes differentiate into dendritic cells (94). IgG is capable of inducing a pro- FIGURE 6. Recombinant LGALS3BP inhibits monocyte-derived fibro- or anti-inflammatory macrophage phenotype by interacting with cyte differentiation. Recombinant LGALS3BP was added to PBMC, and monocytes (95). IgG that is glycosylated with N-linked sialic-acid monocyte-derived fibrocyte differentiation was assessed as in Fig. 1. Values glycans binds DC-SIGN and causes macrophages to secrete 6 , are mean SEM, n =8.***p 0.001 compared with the control (t test). IL-10, reducing inflammation (95, 96). Both glycosylated IgG and LGALS3BP bind human DC-SIGN (44, 95). To determine whether the DC-SIGN receptor might mediate the effect of LGALS3BP on transcript variants (90), LGALS3BP cDNA was amplified via PCR monocyte-derived fibrocyte differentiation, we added 231 CM and Downloaded from and sequenced. The sequence encoded the four LGALS3BP 435 CM to PBMC from wild-type and CD2092/2 (SIGN-R12/2) peptides detected by mass spectrometry and was identical to hu- mice. 231 CM and 435 CM inhibited monocyte-derived fibrocyte man LGALS3BP from oral squamous carcinoma cells (93) and is differentiation from wild-type C57BL/6 mouse spleen cells but identical to the sequence of the recombinant full-length human potentiated monocyte-derived fibrocyte differentiation from CD209 LGALS3BP (R&D datasheet). knockout mouse spleen cells (Fig. 8) in a similar manner to 231 CM

LGALS3BP produced in cancer cells has a higher mass than components that passed through a 100-kDa filter (Supplemental http://www.jimmunol.org/ recombinant LGALS3BP Fig. 3). Recombinant LGALS3BP, 231 CM, and 435 CM each upregulated CD209 expression on PBMC and decreased the number To determine the concentration of LGALS3BP in CM, Western of fibronectin-positive monocyte-derived fibrocytes (Supplemental blots of CM and known amounts of recombinant LGALS3BP were Fig. 4). stained for LGALS3BP. 231, 435, and OVCAR-8 CMs showed high concentrations of LGALS3BP compared with MCF-7, DCIS. com, and U87-mg CMs (Fig. 7). 435, 231, and OVCAR-8 cells accumulated ∼1 mg/ml LGALS3BP in their CMs. U-87 MG CM had very little LGALS3BP, suggesting that glioma cells may in- by guest on September 25, 2021 hibit monocyte-derived fibrocyte differentiation by other means. LGALS3BP has a predicted mass of ∼60 kDa, but LGALS3BP isolated from the serum of cancer patients has a mass of ∼90 kDa (50) and the LGALS3BP we observed in cancer cell CM has a mass of ∼85 kDa. Although LGALS3BP in cancer CM appears

FIGURE 8. CD209 (SIGN-R1) is needed for the effect of 231 and 435 CM on mouse monocyte–derived ﬁbrocyte differentiation. Cells were isolated from mouse spleens and incubated with the indicated concentrations of FIGURE 7. High concentrations of LGALS3BP are present in 321 and CM. Spleen cells from wild-type C57BL/6 (A) and SIGN-R12/2 knockout 435 CM. Western blot of equal volumes of the indicated concentrations (in (B) mice were incubated with the indicated concentrations of 231 or nanograms per milliliter) of recombinant LGALS3BP and CM from the 435 CM. After 5 d, monocyte-derived ﬁbrocytes were counted. Values indicated cell types was stained with anti-LGALS3BP Abs. Blot image is are mean 6 SEM, n =3;*p , 0.05 and ***p , 0.001comparedwiththe representative of three separate experiments. control (t test). 1864 LGALS3BP INHIBITS FIBROCYTE DIFFERENTIATION

Galectin-3 and galectin-1 potentiate monocyte-derived procollagen, collagen-I, galectin-3, and LGALS3BP. For all bi- fibrocyte differentiation opsies tested, the tumor cells strongly expressed LGALS3BP and Galectin-1 and galectin-3 are binding partners for LGALS3BP procollagen. The staining intensity of LGALS3BP increased at the (50, 97). To determine whether galectin-1 and -3 affect monocyte- interface of tumor cells and stroma, particularly where tumor cells derived fibrocyte differentiation, we incubated PBMC with were invading through layers of collagen-rich stroma (Fig. 9). recombinant human galectin-1 and -3 (98, 99). Galectin-1 and -3 Monocyte-derived fibrocytes at the tumor edge were procollagen significantly potentiated monocyte-derived fibrocyte differen- and collagen-1 positive, whereas the scar tissue was negative for tiation (Supplemental Fig. 4C). To determine how a mixture of procollagen and positive for collagen-1. “X” indicates the tumor, galectin-3 and LGALS3BP would influence monocyte-derived the arrow indicates tumor infiltration into scar tissue, and “*” fibrocyte differentiation, recombinant galectin-3 and LGALS3BP indicates areas with monocyte-derived fibrocytes. Galectin-3 were coincubated with PBMC. Concentrations of galectin-3 that colocalized with the monocyte-derived fibrocyte markers CD45RO potentiated monocyte-derived fibrocyte differentiation continued and collagen-I (Fig. 9). These data suggest that in some breast to potentiate monocyte-derived fibrocyte differentiation when tumors LGALS3BP expression is increased at the interface be- mixed with concentrations of LGALS3BP that inhibited monocyte- tween the tumor and desmoplastic tissue and that monocyte- derived fibrocyte differentiation (Fig. 6, Supplemental Fig. 4D). derived fibrocytes are reduced when LGALS3BP expression is Galectin-3 did not potentiate monocyte-derived fibrocyte differen- increased. Intriguingly, galectin-3, which we observed to potentiation when mixed with a 3-fold higher quantity of LGALS3BP tiate monocyte-derived fibrocyte differentiation, colocalized with (Supplemental Fig. 4D). This suggests that the monocyte-derived monocyte-derived fibrocytes (Fig. 9). fibrocyte-potentiating effect of galectin-3 competes with the Downloaded from monocyte-derived fibrocyte-inhibiting effect of LGALS3BP. Discussion In this paper, we show that several human tumor cell lines secrete Increased LGALS3BP expression at the interface between activity that inhibits the differentiation of human monocytes into breast cancer and scar tissue monocyte-derived fibrocytes. For a metastatic breast cell line and To determine how tumor cells, LGALS3BP, galectin-3, and a metastatic melanoma cell line, the majority of the activity is monocyte-derived fibrocytes interact in human tumors, we stained LGALS3BP. LGALS3BP produced by cancer cells appears to act http://www.jimmunol.org/ sections of human infiltrative ductal carcinomas for CD45RO, through the CD209 receptor to inhibit monocyte-derived fibrocyte by guest on September 25, 2021

FIGURE 9. Breast cancer tumor sections visualized by immunofluorescence show increased LGALS3BP at the edge of tumors. Human infiltrative ductal carcinoma tumor specimens were sectioned and stained for CD45RO, collagen-1, procollagen LGALS3BP, and galectin-3. Images are H&E stain of a representative biopsy section (A and B), immunofluorescence for collagen (red) and CD45RO (green) (C), and immunofluorescence for LGALS3BP (red) and galectin-3 (green) (D), and immunofluorescence for procollagen (red) and CD45 (green) (E and F). Yellow indicates colocalization. The boxes in (A) and (E) indicate areas of magnification for (B)–(E) and (F), respectively. The image in (F) extends slightly below the white box in (E). X indicates the tumor, the arrow indicates tumor infiltration into scar tissue, and * indicates areas with monocyte-derived fibrocytes. Scale bar, 200 mm(A); scale bars, 20 mm(B–F). The Journal of Immunology 1865 differentiation. LGALS3BP’s binding partner, galectin-3, is up- Galectin-3 is expressed by monocytes and macrophages (122). regulated in fibrotic tissue surrounding breast cancer tumors and Serum galectin-3 is upregulated in heart disease and other fibrosing promotes monocyte-derived fibrocyte differentiation at physio- diseases (123), and inhibitors of galectin-3 are currently in clinical logical concentrations. In breast cancer biopsies, LGALS3BP is trials for the treatment of fibrosing diseases (124, 125). Although concentrated at the edge of the tumor in regions with fewer serum galectin-3 is 100–900 ng/ml (98), we observed potentiation monocyte-derived fibrocytes. Taken together, this suggests that of monocyte-derived fibrocyte differentiation at 2.5–5 mg/ml. LGALS3BP may inhibit monocyte-derived fibrocyte differentia- Similar concentrations of galectin-3 are necessary to induce tion to facilitate metastasis in breast cancer and melanoma. changes in other cells (99), suggesting that either the effects of LGALS3BP (Mac2-BP and Ag 90K) is a secreted member of the galectin-3 we and others observed in tissue culture are not physi- SRCR family of proteins (50). LGALS3BP binds to galectin-1 ological or that extracellular concentrations of galectin-3 in some and -3 (100), both collagen and fibronectin (101), and increases tissues may be much higher than in the serum. If galectin-3 levels in cell adhesion (97). Mouse knockouts of LGALS3BP show higher a tissue are high enough to potentiate monocyte-derived fibrocyte circulating levels of TNF-a, IL-12, and IFN-g, suggesting multi- differentiation, this would suggest that high levels of galectin-3 ple roles in regulating the immune system (102). could potentiate fibrosis in part by potentiating monocyte-derived MCF-7 and DCIS.com cells, which are derived from non- fibrocyte differentiation. Because we observed that galectin-1 also metastatic breast cancers (67, 72), accumulate relatively low ex- potentiates monocyte-derived fibrocyte differentiattion, high levels tracellular levels of LGALS3BP, whereas 231 cells, which are of galectin-1 may similarly potentiate fibrosis. derived from a metastatic breast cancer (40), accumulate high Taken together, this work elucidates a signal and receptor used extracellular levels of LGALS3BP (103–109). Patients with met- by metastatic tumor cells to block a response of the innate immune Downloaded from astatic breast cancer tend to have abnormally high serum levels of system. An intriguing possibility is that blocking LGALS3BP may LGALS3BP (50, 52). 435 accumulates high concentrations of decrease the ability of tumor cells to inhibit the desmoplastic re- extracellular LGALS3BP compared with other cancer cell lines sponse and metastasize. Conversely, LGALS3BP might be useful (110), and patients with metastatic melanoma have higher serum to decrease monocyte-derived fibrocyte differentiation and thus LGALS3BP than patients with benign skin cancer (111). In decrease fibrosis. Because galectin-3 and galectin-1 potentiate patients with breast cancer (112, 113), ovarian cancer (114–117), monocyte-derived fibrocyte differentiation, these proteins might be http://www.jimmunol.org/ or melanoma (118–120), serum LGALS3BP concentrations in- useful to inhibit metastasis, although with the danger that they crease during the progression to metastasis (49, 121). An intriguing might promote fibrosis. possibility is that LGALS3BP may play a role in metastasis by inhibiting monocyte-derived fibrocyte differentiation. The Hill coefficient of recombinant LGALS3BP for monocyte- Acknowledgments derived fibrocyte inhibition is 6.5, but 231 and 435 CM have Hill We thank Drs. Kelly Hunt and Cansu Karakas at the University of Texas M.D. Anderson Cancer Center for providing human breast cancer patient coefficients close to 1. Fractions of 231 CM containing LGALS3BP slides and aiding in interpretation of the immunofluorescent staining. We have a Hill coefficient of 4.6. Both 231 CM and 435 CM contain also thank Darrell Pilling and Nehemiah Cox for advice. We thank the vol- by guest on September 25, 2021 a factor (or factors) that potentiate monocyte-derived fibrocyte unteers who donated blood and the phlebotomy staff at the Texas A&M differentiation (Supplemental Fig. 3), suggesting that perhaps Beutel Student Health Center. LGALS3BP competes with these factors to produce a Hill coefficient of ∼1. Recombinant LGALS3BP, 231 CM, and 435 CM all Disclosures increase CD209 staining on monocytes, suggesting that LGALS3BP The authors have no financial conflicts of interest. may cooperatively bind to monocytes by increasing CD209 expression. LGALS3BP secreted from 231 cells has a higher apparent mass References than recombinant LGALS3BP produced in CHO cells, despite the 1. Hamilton, J. A. 2008. Colony-stimulating factors in inflammation and auto- immunity. Nat. Rev. Immunol. 8: 533–544. two having an identical primary structure. Because LGALS3BP is 2. Murray, P. J., and T. A. Wynn. 2011. Protective and pathogenic functions of glycosylated (44), this suggests that the LGALS3BPs from the two macrophage subsets. Nat. Rev. Immunol. 11: 723–737. cell lines have different glycosylations and/or other posttransla- 3. Abe, R., S. C. Donnelly, T. Peng, R. Bucala, and C. N. Metz. 2001. Peripheral ∼ blood fibrocytes: differentiation pathway and migration to wound sites. J. tional modifications. 231 and 435 secrete 1 mg/ml LGALS3BP Immunol. 166: 7556–7562. into CM, and 231 and 435 CM have an IC50 for monocyte- 4. Bucala, R., L. A. Spiegel, J. Chesney, M. Hogan, and A. Cerami. 1994. Cir- derived fibrocyte differentiation of ∼0.3% (Fig. 1, Supplemental culating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol. Med. 1: 71–81. Fig. 1). Taken together, these results indicate that the IC50 for 5. White, M. J., E. D. Galvis-Carvajal, and R. H. Gomer. 2015. A brief exposure LGALS3BP should be ∼0.3% 3 1 mg/ml = ∼3 ng/ml. Recombinant to tryptase or thrombin potentiates fibrocyte differentiation in the presence of LGALS3BP’s IC for monocyte-derived fibrocyte inhibition serum or serum amyloid p. J. Immunol. 194: 142‑150. 50 6. Suko, T., I. Ichimiya, K. Yoshida, M. Suzuki, and G. Mogi. 2000. Classification was 300 ng/ml. This then suggests that glycosylation and/or and culture of spiral ligament fibrocytes from mice. Hear. Res. 140: 137–144. posttranslational modification of LGALS3BP may affect its 7. Strieter, R. M., E. C. Keeley, M. A. Hughes, M. D. Burdick, and B. Mehrad. ability to inhibit monocyte-derived fibrocyte differentiation. In 2009. The role of circulating mesenchymal progenitor cells (fibrocytes) in the pathogenesis of pulmonary fibrosis. J. Leukoc. Biol. 86: 1111–1118. agreement with this, LGALS3BP produced by cancer cells has 8. Pilling, D., T. Fan, D. Huang, B. Kaul, and R. H. Gomer. 2009. Identification of at least a 4-fold higher affinity for CD209 (DC-SIGN) receptors markers that distinguish monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts. PLoS One 4: e7475. than LGALS3BP produced by noncancer cells (44). The CD209 9. Bellini, A., and S. Mattoli. 2007. The role of the fibrocyte, a bone marrow- receptor, which we found to be necessary for the ability of derived mesenchymal progenitor, in reactive and reparative fibroses. Lab. In- LGALS3BP to inhibit monocyte-derived fibrocyte differentia- vest. 87: 858–870. 10. Paget, J. 1853. Lectures on Surgical Pathology Delivered at the Royal College tion, is activated by posttranslational glycosylations (95). A of Surgeons of England. Wilson and Ogilvy, London. reasonable possibility is thus that the posttranslational glyco- 11. Mattoli, S., A. Bellini, and M. Schmidt. 2009. The role of a human hemato- sylations of LGALS3BP produced in CHO and 231 cells have poietic mesenchymal progenitor in wound healing and fibrotic diseases and implications for therapy. Curr. Stem Cell Res. Ther. 4: 266–280. different affinities for CD209, resulting in the observed dif- 12. Bellini, A., M. A. Marini, L. Bianchetti, M. Barczyk, M. Schmidt, and ferences in IC50s. S. Mattoli. 2012. Interleukin (IL)-4, IL-13, and IL-17A differentially affect the 1866 LGALS3BP INHIBITS FIBROCYTE DIFFERENTIATION

profibrotic and proinflammatory functions of fibrocytes from asthmatic patients. 43. Ullrich, A., I. Sures, M. D’Egidio, B. Jallal, T. J. Powell, R. Herbst, A. Dreps, Mucosal Immunol. 5: 140–149. M. Azam, M. Rubinstein, C. Natoli, et al. 1994. The secreted tumor-associated 13. Isgro`, M., L. Bianchetti, M. A. Marini, A. Bellini, M. Schmidt, and S. Mattoli. antigen 90K is a potent immune stimulator. J. Biol. Chem. 269: 18401–18407. 2013. The C-C motif chemokine ligands CCL5, CCL11, and CCL24 induce the 44. Nonaka, M., B. Y. Ma, H. Imaeda, K. Kawabe, N. Kawasaki, K. Hodohara, migration of circulating fibrocytes from patients with severe asthma. Mucosal N. Kawasaki, A. Andoh, Y. Fujiyama, and T. Kawasaki. 2011. Dendritic cell- Immunol. 6: 718–727. specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) 14. Bianchetti, L., M. A. Marini, M. Isgro`, A. Bellini, M. Schmidt, and S. Mattoli. recognizes a novel ligand, Mac-2-binding protein, characteristically expressed 2012. IL-33 promotes the migration and proliferation of circulating fibrocytes on human colorectal carcinomas. J. Biol. Chem. 286: 22403–22413. from patients with allergen-exacerbated asthma. Biochem. Biophys. Res. 45. Resnick, D., A. Pearson, and M. Krieger. 1994. The SRCR superfamily: Commun. 426: 116–121. a family reminiscent of the Ig superfamily. Trends Biochem. Sci. 19: 5–8. 15. Pilling, D., C. D. Buckley, M. Salmon, and R. H. Gomer. 2003. Inhibition of 46. D’Ostilio, N., G. Sabatino, C. Natoli, A. Ullrich, and S. Iacobelli. 1996. 90K fibrocyte differentiation by serum amyloid P. J. Immunol. 171: 5537–5546. (Mac-2 BP) in human milk. Clin. Exp. Immunol. 104: 543–546. 16. Pilling, D., V. Vakil, and R. H. Gomer. 2009. Improved serum-free culture 47. Iacobelli, S., E. Arno`, A. D’Orazio, and G. Coletti. 1986. Detection of antigens conditions for the differentiation of human and murine fibrocytes. J. Immunol. recognized by a novel monoclonal antibody in tissue and serum from patients Methods 351: 62–70. with breast cancer. Cancer Res. 46: 3005–3010. 17. Reilkoff, R. A., R. Bucala, and E. L. Herzog. 2011. Fibrocytes: emerging ef- 48. Wang, Y., X. Ao, H. Vuong, M. Konanur, F. R. Miller, S. Goodison, and fector cells in chronic inflammation. Nat. Rev. Immunol. 11: 427–435. D. M. Lubman. 2008. Membrane glycoproteins associated with breast tumor cell 18. Moeller, A., S. E. Gilpin, K. Ask, G. Cox, D. Cook, J. Gauldie, P. J. Margetts, progression identified by a lectin affinity approach. J. Proteome Res. 7: 4313–4325. L. Farkas, J. Dobranowski, C. Boylan, et al. 2009. Circulating fibrocytes are an 49. Mbeunkui, F., B. J. Metge, L. A. Shevde, and L. K. Pannell. 2007. Identification indicator of poor prognosis in idiopathic pulmonary fibrosis. Am. J. Respir. Crit. of differentially secreted biomarkers using LC-MS/MS in isogenic cell lines Care Med. 179: 588–594. representing a progression of breast cancer. J. Proteome Res. 6: 2993–3002. 19. Quan, T. E., S. E. Cowper, and R. Bucala. 2006. The role of circulating 50. Grassadonia, A., N. Tinari, I. Iurisci, E. Piccolo, A. Cumashi, P. Innominato, fibrocytes in fibrosis. Curr. Rheumatol. Rep. 8: 145–150. M. D’Egidio, C. Natoli, M. Piantelli, and S. Iacobelli. 2004. 90K (Mac-2 BP) 20. Strieter, R. M., E. C. Keeley, M. D. Burdick, and B. Mehrad. 2009. The role of and galectins in tumor progression and metastasis. Glycoconj. J. 19: 551–556. circulating mesenchymal progenitor cells, fibrocytes, in promoting pulmonary 51. Tinari, N., R. Lattanzio, P. Querzoli, C. Natoli, A. Grassadonia, S. Alberti, fibrosis. Trans. Am. Clin. Climatol. Assoc. 120: 49–59. M. Hubalek, D. Reimer, I. Nenci, P. Bruzzi, et al. 2009. High expression of 90K Downloaded from 21. Pilling, D., D. Roife, M. Wang, S. D. Ronkainen, J. R. Crawford, E. L. Travis, (Mac-2 BP) is associated with poor survival in node-negative breast cancer and R. H. Gomer. 2007. Reduction of bleomycin-induced pulmonary fibrosis by patients not receiving adjuvant systemic therapies. Int. J. Cancer 124: 333‑338. serum amyloid P. J. Immunol. 179: 4035–4044. 52. Iacobelli, S., P. Sismondi, M. Giai, M. D’Egidio, N. Tinari, C. Amatetti, P. Di 22. Naik-Mathuria, B., D. Pilling, J. R. Crawford, A. N. Gay, C. W. Smith, Stefano, and C. Natoli. 1994. Prognostic value of a novel circulating serum 90K R. H. Gomer, and O. O. Olutoye. 2008. Serum amyloid P inhibits dermal antigen in breast cancer. Br. J. Cancer 69: 172–176. wound healing. Wound Repair Regen. 16: 266–273. 53. Hughes, R. C. 1999. Secretion of the galectin family of mammalian 23. Herzog, E. L., and R. Bucala. 2010. Fibrocytes in health and disease. Exp. carbohydrate-binding proteins. Biochim. Biophys. Acta 1473: 172–185.

Hematol. 38: 548–556. 54. Piccolo, E., N. Tinari, D. Semeraro, S. Traini, I. Fichera, A. Cumashi, R. La http://www.jimmunol.org/ 24. Andrade, Z. A. 2009. Schistosomiasis and liver fibrosis. Parasite Immunol. 31: Sorda, F. Spinella, A. Bagnato, R. Lattanzio, et al. 2013. LGALS3BP, lectin 656–663. galactoside-binding soluble 3 binding protein, induces vascular endothelial 25. Boros, D. L. 1989. Immunopathology of Schistosoma mansoni infection. Clin. growth factor in human breast cancer cells and promotes angiogenesis. J. Mol. Microbiol. Rev. 2: 250–269. Med. 91: 83–94. 26. Wahl, S. M., M. Frazier-Jessen, W. W. Jin, J. B. Kopp, A. Sher, and 55. Trahey, M., and I. L. Weissman. 1999. Cyclophilin C-associated protein: A. W. Cheever. 1997. Cytokine regulation of schistosome-induced granuloma a normal secreted glycoprotein that down-modulates endotoxin and proin- and fibrosis. Kidney Int. 51: 1370–1375. flammatory responses in vivo. Proc. Natl. Acad. Sci. USA 96: 3006–3011. 27. Wyler, D. J., S. M. Wahl, and L. M. Wahl. 1978. Hepatic fibrosis in schisto- 56. Moutsatsos, I. K., M. Wade, M. Schindler, and J. L. Wang. 1987. Endogenous somiasis: egg granulomas secrete fibroblast stimulating factor in vitro. Science lectins from cultured cells: nuclear localization of carbohydrate-binding protein 202: 438–440. 35 in proliferating 3T3 fibroblasts. Proc. Natl. Acad. Sci. USA 84: 6452–6456. 28. Schedin, P., and A. Elias. 2004. Multistep tumorigenesis and the microenvi- 57. Hughes, R. C. 1997. The galectin family of mammalian carbohydrate-binding ronment. Breast Cancer Res. 6: 93–101. molecules. Biochem. Soc. Trans. 25: 1194–1198. by guest on September 25, 2021 29. Kim, B. G., H. J. An, S. Kang, Y. P. Choi, M. Q. Gao, H. Park, and N. H. Cho. 58. Xu, X. C., A. K. el-Naggar, and R. Lotan. 1995. Differential expression of 2011. Laminin-332-rich tumor microenvironment for tumor invasion in the galectin-1 and galectin-3 in thyroid tumors: potential diagnostic implications. interface zone of breast cancer. Am. J. Pathol. 178: 373–381. Am. J. Pathol. 147: 815–822. 30. Coulson-Thomas, V. J., Y. M. Coulson-Thomas, T. F. Gesteira, C. A. de Paula, 59. Coli, A., G. Bigotti, F. Zucchetti, F. Negro, and G. Massi. 2002. Galectin-3, A. M. Mader, J. Waisberg, M. A. Pinhal, A. Friedl, L. Toma, and H. B. Nader. a marker of well-differentiated thyroid carcinoma, is expressed in thyroid 2011. Colorectal cancer desmoplastic reaction up-regulates collagen synthesis nodules with cytological atypia. Histopathology 40: 80–87. and restricts cancer cell invasion. Cell Tissue Res. 346: 223–236. 60. Gruson, D., and G. Ko. 2012. Galectins testing: new promises for the diagnosis 31. Cichon, M. A., A. C. Degnim, D. W. Visscher, and D. C. Radisky. 2010. Mi- and risk stratification of chronic diseases? Clin. Biochem. 45: 719–726. croenvironmental influences that drive progression from benign breast disease 61. McCullough, P. A., A. Olobatoke, and T. E. Vanhecke. 2011. Galectin-3: to invasive breast cancer. J. Mammary Gland Biol. Neoplasia 15: 389–397. a novel blood test for the evaluation and management of patients with heart 32. Shao, Z. M., M. Nguyen, and S. H. Barsky. 2000. Human breast carcinoma failure. Rev. Cardiovasc. Med. 12: 200–210. desmoplasia is PDGF initiated. Oncogene 19: 4337–4345. 62. Sundblad, V., D. O. Croci, and G. A. Rabinovich. 2011. Regulated expression 33. Walker, R. A. 2001. The complexities of breast cancer desmoplasia. Breast of galectin-3, a multifunctional glycan-binding protein, in haematopoietic and Cancer Res. 3: 143–145. non-haematopoietic tissues. Histol. Histopathol. 26: 247–265. 34. Coulson-Thomas, V. J., Y. M. Coulson-Thomas, T. F. Gesteira, C. A. de Paula, 63. Krzeslak, A., and A. Lipinska. 2004. Galectin-3 as a multifunctional protein. A. M. Mader, J. Waisberg, M. A. Pinhal, A. Friedl, L. Toma, and H. B. Nader. Cell. Mol. Biol. Lett. 9: 305–328. 2011. Colorectal cancer desmoplastic reaction up-regulates collagen synthesis 64. Cox, N., D. Pilling, and R. H. Gomer. 2012. NaCl potentiates human fibrocyte and restricts cancer cell invasion. Cell Tissue Res. 346: 223–236. differentiation. PLoS One 7: e45674. 35. Finn, O. J. 2012. Immuno-oncology: understanding the function and dysfunc- 65. White, M. J., M. Glenn, and R. H. Gomer. 2013. Trypsin potentiates human tion of the immune system in cancer. Ann. Oncol. 23(Suppl. 8): viii6‑viii9. fibrocyte differentiation. PLoS One 8: e70795. 36. Gajewski, T. F., H. Schreiber, and Y. X. Fu. 2013. Innate and adaptive immune 66. Cailleau, R., M. Olive´, and Q. V. Cruciger. 1978. Long-term human breast cells in the tumor microenvironment. Nat. Immunol. 14: 1014–1022. carcinoma cell lines of metastatic origin: preliminary characterization. In Vitro 37. Solito, S., L. Pinton, V. Damuzzo, and S. Mandruzzato. 2012. Highlights on 14: 911–915. molecular mechanisms of MDSC-mediated immune suppression: paving the 67. Miller, F. R., S. J. Santner, L. Tait, and P. J. Dawson. 2000. MCF10DCIS.com way for new working hypotheses. Immunol. Invest. 41: 722–737. xenograft model of human comedo ductal carcinoma in situ. J. Natl. Cancer 38. Biragyn, A., and D. L. Longo. 2012. Neoplastic “Black Ops”: cancer’s sub- Inst. 92: 1185–1186. versive tactics in overcoming host defenses. Semin. Cancer Biol. 22: 50–59. 68. von Kleist, S., E. Chany, P. Burtin, M. King, and J. Fogh. 1975. Immunohis- 39. Lakshmi Narendra, B., K. Eshvendar Reddy, S. Shantikumar, and tology of the antigenic pattern of a continuous cell line from a human colon S. Ramakrishna. 2013. Immune system: a double-edged sword in cancer. tumor. J. Natl. Cancer Inst. 55: 555–560. Inflamm. Res. 62: 823‑834. 69. Leibovitz, A., J. C. Stinson, W. B. McCombs, III, C. E. McCoy, K. C. Mazur, 40. Cailleau, R., R. Young, M. Olive´, and W. J. Reeves, Jr. 1974. Breast tumor cell and N. D. Mabry. 1976. Classification of human colorectal adenocarcinoma cell lines from pleural effusions. J. Natl. Cancer Inst. 53: 661–674. lines. Cancer Res. 36: 4562–4569. 41. Abdelkarim, M., N. Vintonenko, A. Starzec, A. Robles, J. Aubert, M. L. Martin, 70. Shirasawa, S., M. Furuse, N. Yokoyama, and T. Sasazuki. 1993. Altered growth S. Mourah, M. P. Podgorniak, S. Rodrigues-Ferreira, C. Nahmias, et al. 2011. of human colon cancer cell lines disrupted at activated Ki-ras. Science 260: 85– Invading basement membrane matrix is sufficient for MDA-MB-231 breast 88. cancer cells to develop a stable in vivo metastatic phenotype. PLoS One 6: 71. Dexter, D. L., J. A. Barbosa, and P. Calabresi. 1979. N,N-Dimethylformamide- e23334. induced alteration of cell culture characteristics and loss of tumorigenicity in 42. Koths, K., E. Taylor, R. Halenbeck, C. Casipit, and A. Wang. 1993. Cloning and cultured human colon carcinoma cells. Cancer Res. 39: 1020–1025. characterization of a human Mac-2-binding protein, a new member of the su- 72. Soule, H. D., J. Vazguez, A. Long, S. Albert, and M. Brennan. 1973. A human perfamily defined by the macrophage scavenger receptor cysteine-rich domain. cell line from a pleural effusion derived from a breast carcinoma. J. Natl. J. Biol. Chem. 268: 14245–14249. Cancer Inst. 51: 1409–1416. The Journal of Immunology 1867

73. Scudiero, D. A., A. Monks, and E. A. Sausville. 1998. Cell line designation 101. Sasaki, T., C. Brakebusch, J. Engel, and R. Timpl. 1998. Mac-2 binding protein change: multidrug-resistant cell line in the NCI anticancer screen. J. Natl. is a cell-adhesive protein of the extracellular matrix which self-assembles into Cancer Inst. 90: 862. ring-like structures and binds b1 integrins, collagens and fibronectin. EMBO J. 74. Hamilton, T. C., R. C. Young, W. M. McKoy, K. R. Grotzinger, J. A. Green, 17: 1606–1613. E. W. Chu, J. Whang-Peng, A. M. Rogan, W. R. Green, and R. F. Ozols. 1983. 102. Blake, J. A., C. J. Bult, J. T. Eppig, J. A. Kadin, and J. E. Richardson, Mouse Characterization of a human ovarian carcinoma cell line (NIH:OVCAR-3) with Genome Database Group. 2014. The Mouse Genome Database: integration of androgen and estrogen receptors. Cancer Res. 43: 5379–5389. and access to knowledge about the laboratory mouse. Nucleic Acids Res. 42: 75. Hamilton, T. C., R. C. Young, and R. F. Ozols. 1984. Experimental model D810–D817. systems of ovarian cancer: applications to the design and evaluation of new 103. Whelan, S. A., J. He, M. Lu, P. Souda, R. E. Saxton, K. F. Faull, treatment approaches. Semin. Oncol. 11: 285–298. J. P. Whitelegge, and H. R. Chang. 2012. Mass spectrometry (LC-MS/MS) 76. Park, J. G., J. H. Lee, M. S. Kang, K. J. Park, Y. M. Jeon, H. J. Lee, H. S. Kwon, identified proteomic biosignatures of breast cancer in proximal fluid. J. Pro- H. S. Park, K. S. Yeo, K. U. Lee, et al. 1995. Characterization of cell lines teome Res. 11: 5034–5045. established from human hepatocellular carcinoma. Int. J. Cancer 62: 276–282. 104. Lawlor, K., A. Nazarian, L. Lacomis, P. Tempst, and J. Villanueva. 2009. 77. Aden, D. P., A. Fogel, S. Plotkin, I. Damjanov, and B. B. Knowles. 1979. Pathway-based biomarker search by high-throughput proteomics profiling of Controlled synthesis of HBsAg in a differentiated human liver carcinoma- secretomes. J. Proteome Res. 8: 1489–1503. derived cell line. Nature 282: 615–616. 105. Kulasingam, V., and E. P. Diamandis. 2008. Tissue culture-based breast cancer 78. Fogh, J., J. M. Fogh, and T. Orfeo. 1977. One hundred and twenty-seven cul- biomarker discovery platform. Int. J. Cancer 123: 2007–2012. tured human tumor cell lines producing tumors in nude mice. J. Natl. Cancer 106. Pavlou, M. P., and E. P. Diamandis. 2010. The cancer cell secretome: a good Inst. 59: 221–226. source for discovering biomarkers? J. Proteomics 73: 1896–1906. ´ 79. Ponten, J., and E. H. Macintyre. 1968. Long term culture of normal and neo- 107. Colzani, M., P. Waridel, J. Laurent, E. Faes, C. Ruegg,€ and M. Quadroni. 2009. plastic human glia. Acta Pathol. Microbiol. Scand. 74: 465–486. Metabolic labeling and protein linearization technology allow the study of 80. Lieber, M., J. Mazzetta, W. Nelson-Rees, M. Kaplan, and G. Todaro. 1975. proteins secreted by cultured cells in serum-containing media. J. Proteome Res. Establishment of a continuous tumor-cell line (panc-1) from a human carci- 8: 4779–4788. noma of the exocrine pancreas. Int. J. Cancer 15: 741–747. 108. Palazzolo, G., N. N. Albanese, G. DI Cara, D. Gygax, M. L. Vittorelli, and 81. Steube, K. G., D. Teepe, C. Meyer, M. Zaborski, and H. G. Drexler. 1997. A model system in haematology and immunology: the human monocytic cell line I. Pucci-Minafra. 2012. Proteomic analysis of exosome-like vesicles derived Downloaded from MONO-MAC-1. Leuk. Res. 21: 327–335. from breast cancer cells. Anticancer Res. 32: 847–860. 82. Ziegler-Heitbrock, H. W., E. Thiel, A. Futterer,€ V. Herzog, A. Wirtz, and 109. Zhang, Y., X. Tang, L. Yao, K. Chen, W. Jia, X. Hu, and L. X. Xu. 2012. G. Riethmuller.€ 1988. Establishment of a human cell line (Mono Mac 6) with Lectin capture strategy for effective analysis of cell secretome. Proteomics 12: characteristics of mature monocytes. Int. J. Cancer 41: 456–461. 32–36. 83. Sundstro¨m, C., and K. Nilsson. 1976. Establishment and characterization of 110. Ross, D. T., U. Scherf, M. B. Eisen, C. M. Perou, C. Rees, P. Spellman, V. Iyer, a human histiocytic lymphoma cell line (U-937). Int. J. Cancer 17: 565–577. S. S. Jeffrey, M. Van de Rijn, M. Waltham, et al. 2000. Systematic variation in 84. Collins, S. J., R. C. Gallo, and R. E. Gallagher. 1977. Continuous growth and gene expression patterns in human cancer cell lines. Nat. Genet. 24: 227–235. 111. Cesinaro, A. M., C. Natoli, A. Grassadonia, N. Tinari, S. Iacobelli, and

differentiation of human myeloid leukaemic cells in suspension culture. Nature http://www.jimmunol.org/ 270: 347–349. G. P. Trentini. 2002. Expression of the 90K tumor-associated protein in benign 85. Tsuchiya, S., M. Yamabe, Y. Yamaguchi, Y. Kobayashi, T. Konno, and K. Tada. and malignant melanocytic lesions. J. Invest. Dermatol. 119: 187–190. 1980. Establishment and characterization of a human acute monocytic leuke- 112. He, J., S. A. Whelan, M. Lu, D. Shen, D. U. Chung, R. E. Saxton, K. F. Faull, mia cell line (THP-1). Int. J. Cancer 26: 171–176. J. P. Whitelegge, and H. R. Chang. 2011. Proteomic-based biosignatures in 86. Graham, F. L., J. Smiley, W. C. Russell, and R. Nairn. 1977. Characteristics of breast cancer classiﬁcation and prediction of therapeutic response. Int. J. a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Proteomics 2011: 896476. Virol. 36: 59–74. 113. Kostianets, O., S. Antoniuk, V. Filonenko, and R. Kiyamova. 2012. Immuno- 87. Rae, J. M., C. J. Creighton, J. M. Meck, B. R. Haddad, and M. D. Johnson. histochemical analysis of medullary breast carcinoma autoantigens in different 2007. MDA-MB-435 cells are derived from M14 melanoma cells—a loss for histological types of breast carcinomas. Diagn. Pathol. 7: 161. breast cancer, but a boon for melanoma research. Breast Cancer Res. Treat. 114. Lee, J. H., X. Zhang, B. K. Shin, E. S. Lee, and I. Kim. 2009. Mac-2 binding 104: 13–19. protein and galectin-3 expression in mucinous tumours of the ovary: an

88. Buckley, C. D., D. Pilling, N. V. Henriquez, G. Parsonage, K. Threlfall, annealing control primer system and immunohistochemical study. Pathology by guest on September 25, 2021 D. Scheel-Toellner, D. L. Simmons, A. N. Akbar, J. M. Lord, and M. Salmon. 41: 229–233. 1999. RGD peptides induce apoptosis by direct caspase-3 activation. Nature 115. Iacobelli, S., I. Bucci, M. D’Egidio, C. Giuliani, C. Natoli, N. Tinari, 397: 534–539. M. Rubistein, and J. Schlessinger. 1993. Purification and characterization of 89. Salmon, M., D. Scheel-Toellner, A. P. Huissoon, D. Pilling, N. Shamsadeen, a 90 kDa protein released from human tumors and tumor cell lines. FEBS Lett. H. Hyde, A. D. D’Angeac, P. A. Bacon, P. Emery, and A. N. Akbar. 1997. 319: 59–65. Inhibition of T cell apoptosis in the rheumatoid synovium. J. Clin. Invest. 99: 116. Scambia, G., P. B. Panici, G. Baiocchi, L. Perrone, S. Iacobelli, and 439–446. S. Mancuso. 1988. Measurement of a monoclonal-antibody-defined antigen 90. UniProt Consortium. 2014. Activities at the Universal Protein Resource (Uni- (90K) in the sera of patients with ovarian cancer. Anticancer Res. 8: 761–764. Prot). Nucleic Acids Res. 42: D191–D198. 117. Gortzak-Uzan, L., A. Ignatchenko, A. I. Evangelou, M. Agochiya, K. A. Brown, 91. Lanoue, A., M. R. Clatworthy, P. Smith, S. Green, M. J. Townsend, H. E. Jolin, P. St. Onge, I. Kireeva, G. Schmitt-Ulms, T. J. Brown, J. Murphy, et al. 2008. A K. G. Smith, P. G. Fallon, and A. N. McKenzie. 2004. SIGN-R1 contributes to proteome resource of ovarian cancer ascites: integrated proteomic and bio- protection against lethal pneumococcal infection in mice. J. Exp. Med. 200: informatic analyses to identify putative biomarkers. J. Proteome Res. 7: 339– 1383–1393. 351. 92. Crawford, J. R., D. Pilling, and R. H. Gomer. 2010. Improved serum-free culture 118. Inohara, H., and A. Raz. 1994. Identification of human melanoma cellular and conditions for spleen-derived murine fibrocytes. J. Immunol. Methods 363: 9–20. secreted ligands for galectin-3. Biochem. Biophys. Res. Commun. 201: 1366– 93. Endo, H., T. Muramatsu, M. Furuta, N. Uzawa, A. Pimkhaokham, T. Amagasa, 1375. J. Inazawa, and K. Kozaki. 2013. Potential of tumor-suppressive miR-596 119. Traini, S., E. Piccolo, N. Tinari, C. Rossi, R. La Sorda, F. Spinella, A. Bagnato, targeting LGALS3BP as a therapeutic agent in oral cancer. Carcinogenesis R. Lattanzio, M. D’Egidio, A. Di Risio, et al. 2014. Inhibition of tumor growth 34: 560–569. and angiogenesis by SP-2, an anti-lectin, galactoside-binding soluble 3 binding 94. Bullwinkel, J., A. Ludemann, J. Debarry, and P. B. Singh. 2011. Epigenotype protein (LGALS3BP) antibody. Mol. Cancer Ther. 13: 916–925. switching at the CD14 and CD209 genes during differentiation of human 120. Morandi, F., M. V. Corrias, I. Levreri, P. Scaruffi, L. Raffaghello, B. Carlini, monocytes to dendritic cells. Epigenetics 6: 45‑51. 95. Anthony, R. M., F. Wermeling, M. C. Karlsson, and J. V. Ravetch. 2008. P. Bocca, I. Prigione, S. Stigliani, L. Amoroso, et al. 2011. Serum levels of Identification of a receptor required for the anti-inflammatory activity of IVIG. cytoplasmic melanoma-associated antigen at diagnosis may predict clinical Proc. Natl. Acad. Sci. USA 105: 19571–19578. relapse in neuroblastoma patients. Cancer Immunol. Immunother. 60: 1485– 96. Anthony, R. M., and J. V. Ravetch. 2010. A novel role for the IgG Fc glycan: 1495. the anti-inflammatory activity of sialylated IgG Fcs. J. Clin. Immunol. 30 121. Chen, S. T., T. L. Pan, H. F. Juan, T. Y. Chen, Y. S. Lin, and C. M. Huang. 2008. (Suppl. 1): S9–S14. Breast tumor microenvironment: proteomics highlights the treatments targeting 97. Inohara, H., S. Akahani, K. Koths, and A. Raz. 1996. Interactions between secretome. J. Proteome Res. 7: 1379–1387. galectin-3 and Mac-2-binding protein mediate cell-cell adhesion. Cancer Res. 122. Liu, F. T., D. K. Hsu, R. I. Zuberi, I. Kuwabara, E. Y. Chi, and W. R. Henderson, 56: 4530–4534. Jr. 1995. Expression and function of galectin-3, a b-galactoside‑binding lectin, in 98. Iurisci, I., N. Tinari, C. Natoli, D. Angelucci, E. Cianchetti, and S. Iacobelli. human monocytes and macrophages. Am. J. Pathol. 147: 1016–1028. 2000. Concentrations of galectin-3 in the sera of normal controls and cancer 123. Grandin, E. W., P. Jarolim, S. A. Murphy, L. Ritterova, C. P. Cannon, patients. Clin. Cancer Res. 6: 1389‑1393. E. Braunwald, and D. A. Morrow. 2012. Galectin-3 and the development of 99. Kohatsu, L., D. K. Hsu, A. G. Jegalian, F. T. Liu, and L. G. Baum. 2006. heart failure after acute coronary syndrome: pilot experience from PROVE Galectin-3 induces death of Candida species expressing specific b-1,2‑linked IT-TIMI 22. Clin. Chem. 58: 267–273. mannans. J. Immunol. 177: 4718–4726. 124. Traber, P. G., and E. Zomer. 2013. Therapy of experimental NASH and fibrosis 100. Iurisci, I., A. Cumashi, A. A. Sherman, Y. E. Tsvetkov, N. Tinari, E. Piccolo, with galectin inhibitors. PLoS One 8: e83481. M. D’Egidio, V. Adamo, C. Natoli, G. A. Rabinovich, et al. 2009. Synthetic 125. Traber, P. G., H. Chou, E. Zomer, F. Hong, A. Klyosov, M. I. Fiel, and inhibitors of galectin-1 and -3 selectively modulate homotypic cell aggregation S. L. Friedman. 2013. Regression of fibrosis and reversal of cirrhosis in rats by and tumor cell apoptosis. Anticancer Res. 29: 403–410. galectin inhibitors in thioacetamide-induced liver disease. PLoS One 8: e75361.