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Cancer Research 68, 5639, July 15, 2008. doi: 10.1158/0008-5472.CAN-07-5804
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

A Special Linker between Macrophage and Hematopoietic Malignant Cells: Membrane Form of Macrophage Colony-Stimulating Factor

Lin Wang, Guo-Guang Zheng, Cui-Hua Ma, Yong-Min Lin, Hai-Yan Zhang, Yuan-Yuan Ma, Jing-Hui Chong and Ke-Fu Wu

State Key Laboratory for Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China

Requests for reprints: Guo-Guang Zheng, State Key Laboratory for Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 288 Nanjing Road, Tianjin 300020, P. R. China. Phone: 86-22-23909053; Fax: 86-22-23909032; E-mail: zhengggtjchn{at}yahoo.com.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The membrane form of macrophage colony–stimulating factor (mM-CSF) is an alternative splicing variant of this cytokine. Although its high expression was detected in hematopoietic malignancies, its physiologic and pathologic roles in hematopoietic system have not been established. In this report, stable transfectant clones expressing mM-CSF (Namalwa-M and Ramos-M) were obtained, which showed reduced proliferation potential in vitro. Moreover, the in vivo study showed that Namalwa-M and Ramos-M exhibited enhanced oncogenicity in tumor size in nude mice model, which could be inhibited by M-CSF monoclonal antibody. A remarkable increase in infiltrating macrophage and the vessel densities was found in tumor tissues formed by lymphoma cell lines that stably expressed mM-CSF, which suggested the involvement of macrophages in this process. The in vitro results using coculture system showed that macrophages could promote Namalwa-M and Ramos-M proliferation and activate extracellular signal-regulated kinase/mitogen-activated protein kinase signal pathway. In addition, the expression of murine origin vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor was elevated in Namalwa-M formed tumor tissues. These results suggested that mM-CSF should be a positive regulator in the development of hematopoietic malignancies by abnormally activating infiltrating macrophages, which in turn promote the malignant development. Thus, mM-CSF may be a critical linker between macrophages and malignant cells in the development of hematopoietic malignancies. [Cancer Res 2008;68(14):5639–47]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Macrophage colony–stimulating factor (M-CSF) is an important member in the cytokine network regulating hematopoiesis. It is a homodimeric glycoprotein that stimulates the proliferation, differentiation, and function of the monocyte/macrophage lineage (1). By alternative splicing from a single gene, M-CSF exists in at least three forms: secretory (sM-CSF), membrane-bound (mM-CSF), and extracellular matrix or proteoglycan (2). All three forms can bind the same receptor, M-CSFR, which is encoded by the c-fms gene (3). M-CSF can play important roles in innate immunity, cancer, and inflammatory diseases (4). The circulating secretory and proteoglycan isoforms can function at a distance. The membrane-bound isoform is involved in local regulation, whereas the secreted proteoglycan isoform might be preferentially localized to specific extracellular matrices (5).

Macrophages are important participants in many cellular functions, including immune defense and normal tissue development (5). Furthermore, macrophages represent major components of the tumor leukocytic infiltrate and can serve as both positive and negative mediators in tumor development by sophisticated mechanisms (6). Previous studies showed that increased circulating M-CSF was found in various malignancies, including breast cancer, ovarian cancer, endometrial carcinoma, and cervical cancer (7, 8). Furthermore, the high sM-CSF level was associated with poor prognosis in colorectal cancer, and coexpression of M-CSF and M-CSFR was associated with a more malignant phenotype and poor prognosis in gynecologic malignancies (9, 10). The abnormal high-serum M-CSF level was also reported in preleukemia, leukemia, and lymphoid malignancies (11). Recent studies revealed a role for sM-CSF in promoting tumor growth and tumor progression to metastasis, which was associated with macrophages (12, 13). M-CSF acts as one of tumor-derived chemoattractants and can recruit macrophages into tumor tissues. Then, it is possible that macrophages are educated to become tumor-associated macrophages (TAM) toward a tumor-promoting phenotype (14). TAM can secrete a wide range of growth factors and angiogenic factors to promote tumor development (15). On the contrary, it can also act as negative regulator. The sM-CSF can directly activate the antitumor activities by enhancing the antibody-dependent cytotoxicity of macrophages and through the activation of natural killer cells (16, 17). In addition, sM-CSF can also promote macrophages to secrete some factors, such as IL-1, tumor necrosis factor {alpha}, and IFN-{gamma}, to modulate tumor immunity (18, 19).

As the proteolytic cleavage sites used to release the secreted isoforms have been spliced out, mM-CSF is stably expressed at the cell surface where it is inefficiently cleaved to yield a soluble form of the cytokine (20). It can bind to M-CSFR and shares the common biological functions with sM-CSF due to their common NH2-terminal parts (1–149 amino acids; ref. 2). The early studies showed that mM-CSF was a functional molecule to stimulate macrophage colony formation (21). In vitro experiments showed that mM-CSF was biologically active as an extracted membrane protein, and it was active in situ for macrophage proliferation for myelopoiesis (22–25). Transgenic expression of mM-CSF could partly restore M-CSF function in M-CSF–deficient Csf1op/Csf1op mice, i.e., the growth retardation, failure of tooth eruption, and abnormal male and female reproductive functions could be corrected in transgenic mice (26). However, unlike sM-CSF, which can regulate cells nearby or at long distance through autocrine, paracrine, or endocrine mechanisms, the inefficient cleavage of mM-CSF limits its functional distance. It was proposed to function through juxtacrine mechanism and play adhesion molecule–like roles in its interaction with the M-CSFR between stromal cells and hematopoietic cells as well as between leukemia cells (21, 24). Nevertheless, the physiologic and pathologic significance of this isoform, especially in tumorigenesis and tumor progression, is not fully understood. It was reported that, in some solid tumors, mM-CSF might activate macrophages, which could mediate direct antitumor cytotoxicity or the presentation of tumor-associated antigens (TAA). Tumor expressing mM-CSF elicited efficient antitumoral activity of macrophages in glioblastoma, glioma, hepatocellular carcinoma, and breast cancer (27–30). However, distinctive antitumoral mechanisms were proposed in different cases. For example, antitumoral mechanism was via paraptosis that was associated with increased expression of three different heat shock proteins in glioma, whereas it was by CD8+ T-cell immunity in hepatocellular cancer model (28, 29). The knowledge of mM-CSF in hematopoietic malignancies is limited. Our previous reports showed that mM-CSF was highly expressed in human leukemia cell line J6-1, and the growth of the cells was promoted by the interaction of mM-CSF and M-CSFR through an autojuxtacrine mechanism (31). Furthermore, high expression of mM-CSF was detected in Hodgkin's lymphoma (HL) and myeloid leukemia (32). However, the detailed function and mechanism of this isoform in leukemogenesis are poorly understood.

In this report, we studied the role of mM-CSF by establishing lymphoma cell lines stably expressing mM-CSF. The mM-CSF acted as a positive regulator in the tumor progression in nude mice model. Elevated infiltrating macrophages and angiogenesis were found in tumors formed by mM-CSF–expressing cells, suggesting the involvement of macrophages in this process. In vitro evidence showed that macrophages, which might be abnormally activated by mM-CSF, in turn, directly stimulated the growth of mM-CSF–expressing cells through the activation of extracellular signal-regulated kinase (ERK)/ mitogen-activated protein kinase (MAPK) cascade.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell lines, cell culture, and antibodies. The human Burkitt's lymphoma cell lines (Namalwa and Ramos) were maintained in our laboratory. All cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; Life Technologies) and antibiotics in a humidified atmosphere of 5% CO2 at 37 °C. All culture supplies were screened and selected on the basis of being endotoxin free. Anti–M-CSF monoclonal antibody (McAb) was from R&D Systems. Anti–M-CSF McAb used in in vivo experiments was prepared as ascites of B5, a mouse McAb developed in our laboratory recognizing the N part of M-CSF. Abs against ERK (C-16), c-Jun-NH2-kinase (JNK; C-17), and P38 (N-20) were purchased from Santa Cruz Biotechnology, Inc. Antiphosphotyrosine McAb (4G10) was from Upstate. FITC anti-mouse F4/80 (BM8) and PE anti-mouse CD31 (390) polyclonal antibodies were purchased from Biolegend.

Semiquantitative reverse transcription-PCR and quantitative reverse transcription-PCR. Total RNA was extracted using Trizol reagent (Invitrogen), following the manufacturer's instructions. Complementary DNA (cDNA) was subsequently synthesized from 5 µg total RNA using M-MLV reverse transcriptase (Life Technologies).

Semiquantitative reverse transcription-PCR (RT-PCR) was performed using a Gene-Amp PCR System 2400 thermocycler (Perkin-Elmer). The primers used were as follows: M-CSF (human) forward, 5'-GCGCTTCAGAGATAACACC-3'; reverse, 5'-CCTCCGCCTCCACCTGTAGA-3'. M-CSFR (human): forward, 5'-ACACTAAGCTCGCAATCCC-3'; reverse, 5'-GTATCGAAGGGTGAGCTCAAA-3'. Neo gene: forward, 5'-GGTGGAGAGGCTATTCGGCT-3'; reverse, 5'-GATAGAAGGCGATGCGCTGC-3'. GAPDH: forward, 5'-TGAAGGTCGGAGTCAACGGATTTGG-3'; reverse, 5'-CATGTGGGCCATGAGGTCCACCAC-3'.

For quantitative RT-PCR measurement of murine basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF), day 30 nude mice tumor tissues were used. An aliquot of 0.1 to 0.2 grams tumor tissue was homogenized with 1 mL Trizol reagent, and total RNA was extracted. Real-time PCR was performed using an ABI-Prism 7500 Sequence Detector (Applied Biosystems). Typically, 5 ng of reverse-transcribed cDNA per sample was used in triplicate. The murine origin primers used (33) were as follows: bFGF forward, 5'-AGCGACCCACACGTCAAACTAC-3'; reverse, 5'-CAGCCGTCCATCTTCCTTCATA-3'; HGF forward, 5'-TGCCCTATTTCCCGTTGTGA-3'; reverse, 5'-CCATTTACAACCCGCAGTTGTT-3'; VEGF forward, 5'-TGCACCCACGACAGAAGG-3'; reverse, 5'-GCACACAGGACGGCTTGA-3'; GAPDH forward, 5'-CACTTGAAGGGTGGAGC-3'; reverse, 5'-GGGCTAAGCAGTTGGTG-3'.

Plasmid construction, transfection procedures, and selection of clones. pTARGET vector carrying mM-CSF (pTARGET-mM-CSF) was constructed by the following procedure. The cDNA fragment encoding human mM-CSF was obtained from human leukemia cell line J6-1 by RT-PCR. The recognition sequences of XhoI and KpnI were added to the two primers, respectively. The PCR product was digested and cloned into pTARGET vector containing a selective marker (neo, the neomycin phosphotransferase gene). The construct was verified by DNA sequencing and purified using a plasmid purification kit (Qbioscience). Then, cells were transfected with either pTARGET blank vector or pTARGET-mM-CSF using Lipofectamine 2000 reagent (Life Technologies). After 18 h, the medium was changed with fresh RPMI 1640 containing 1.4 mg/mL of G418 (Invitrogen Life Technologies) in 24-well plates. Limiting dilution and screening were performed for the establishment of cell clones stably expressing mM-CSF, which were verified by RT-PCR, Western blotting, and confocal microscopy. Clones stably transfected with pTARGET blank vector were also obtained by a similar method and verified by RT-PCR analysis of neo gene.

Immunoprecipitation and Western blotting. Cells were collected by centrifugation and lysed for 15 min on ice in cell lysis buffer containing 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L Na2EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium PPi, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, and 1 µg/mL leupeptin (Cell Signaling Technology). Then, the detergent-insoluble material was removed by centrifugation at 12,000 rpm for 15 min at 4 °C. Twenty microlitres of lysates with equal amount of proteins were collected at each vial and mixed with 2 x reducing buffer [40% w/v glycerol, 9.2% w/v SDS, 250 mmol/L Tris (pH 6.8), 20% w/v β-mercaptoethanol, and 0.04% bromophenol blue] for electrophoresis on 10% SDS-polyacrylamide gels before blotted to polyvinylidene difluoride (PVDF) membrane (Bio-Rad).

For IP studies, lysates containing 500 µg of proteins were immunoprecipitated overnight at 4 °C by incubation in cell lysis buffer with 2 µg/mL Ab (against ERK, c-Jun-NH2-kinase, and P38, respectively). Immunocomplexes were captured on protein A-agarose (Life Technologies) for 2 h at 4 °C. After washing thrice with PBS, pellets were resuspended in SDS-PAGE sample buffer, boiled for 5 min, subjected to 10% SDS-PAGE, and transferred onto PVDF membrane.

The membrane was first blocked with 5% nonfat milk in TBST [150 mmol/L NaCl, 25 mmol/L Tris, and 0.1% Tween 20 (pH 7.5)] overnight at 4°C. After incubations with primary Abs for 2 h at room temperature (RT) and horseradish peroxidase–conjugated secondary Abs for 2 h, visualization of specific proteins was carried out by an enhanced chemiluminescence method using ECL Western blotting detection reagents (Pierce Biotechnology, Inc.) according to the manufacturer's instructions. Membrane was washed with TBST thrice between every two steps.

Tumor growth in nude mice. Animal use was carried out according to the Animal Studies Committee–approved protocols. Female BALB/c nude mice, which were 5- to 6-wk-old, were purchased from Center for Experimental Animals, the Academy of Military Medical Sciences. The immunodeficient mice were housed in sterile microisolators. After irradicated by CS137 with 300 cGy, nude mice were injected s.c. on the dorsal side with 5 x 107 Namalwa cells or 5 x 106 Ramos cells in a volume of 200 µL. After 7 to 10 d, when the tumors were palpable, they were measured with metric calipers according to the length and width of the tumor. The tumor volume was then calculated by the equation: volume = length x width x width/2 (mm3; ref. 28). Data at each time point were compared using a Student's t test. A P value of <0.05 was considered significant. Mice were sacrificed on day 17 (for Ramos series cells) or day 30 (for Namalwa series cells).

For neutralizing experiments, Namalwa-M or Ramos-M cells were treated with or without anti–M-CSF diluted ascites, containing ~500 µg/mL B5 McAb, for 30 min before s.c. injected into female BALB/c nude mice (5 per group, 5 x 107 cells per mouse for Namalwa-M and 5 x 106 cells per mouse for Ramos-M). B5 McAb (50 µg/100 µL/mouse) was injected intratumorally on day 7 and 14 for Namalwa-M formed tumors and day 7, 10, and 13 for Ramos-M formed tumors. Mice were sacrificed on day 17 (for Ramos-M) or day 30 (for Namalwa-M).

Confocal microscopy. The formalin-fixed, paraffin-embedded tissue sections (8 µm) were deparaffinized and rehydrated in PBS. Antigen retrieval was performed by heating the sections in 0.01 mol/L citrate buffer in a microwave oven. Nonspecific binding was blocked by incubating the tissue sections with 10% normal goat serum (NGS; Sigma) in PBS for 20 min. Slides were then incubated with FITC-conjugated anti-mouse F4/80 antibody at a dilution of 1:100 at RT for 30 min.

Frozen tissue sections (5 µm) were fixed with cold acetone for 10 min at RT. Nonspecific binding was blocked by incubating the tissue sections with 10% NGS/PBS for 20 min. Then, sections were stained at RT for 30 min with phycoerythrin (PE)-conjugated anti-mouse CD31 at a dilution of 1:100.

All samples were washed thrice with PBS, and then nuclei were stained using 4,6 diamidino-2-phenylindole dye (Sigma). The specimens were mounted in 80% Glycerine/PBS solution and analyzed by confocal scanning microscopy (Leica TCS SP).

Macrophages/cell lines coculture experiment. Nude mice had been injected i.p. with 2 mL of 10% FBS in RPMI 1640 2 to 3 d previously. Murine peritoneal exudate cells (PEC) were obtained by injecting 4 mL of cold RPMI 1640 into the peritoneal cavity and withdrawing the cell suspension after gentle massage. The PECs were centrifuged at 1,200 rpm and resuspended at the desired concentration in RPMI 1640 with 2% FBS. The PECs suspension was dispensed into 96-well or 6-well plate and incubated for 2 h at 37°C. The culture medium was removed gently by aspiration and changed to fresh culture medium. The PECs used for experiments routinely contained 87% to 92% macrophages (34). Then, cell lines were implanted into these wells at a ratio of 1:1. For proliferative assay, these cells were cocultured for 7 d. For cell signal experiment, cells were cocultured for 48 h.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Establishment of lymphoma cell lines stably expressing membrane form of mM-CSF. To investigate the biological characteristics of mM-CSF, the Namalwa and Ramos cells, which do not express any endogenous M-CSF or M-CSFR (Fig. 1A ), had been transfected with either the blank vector or mM-CSF–expressing vector before underwent G418 selection. The stable transfectant clones were designated as Namalwa-B/Ramos-B and Namalwa-M/Ramos-M, respectively. Namalwa-M and Ramos-M were verified by RT-PCR (Fig. 1B) and Western blotting (Fig. 1C) methods. Namalwa-B and Ramos-B were verified by RT-PCR analysis of the neo gene expression (Fig. 1B). At lease three clones were obtained for each construct. The confocal microscopy results showed that mM-CSF was expressed not only on cell membrane but also in cytoplasm and nucleus (data not shown).


Figure 1
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  		Figure 1. Establishment of lymphoma cell lines stably expressing mM-CSF. A, the expressions of mM-CSF and M-CSFR in Namalwa and Ramos cells were analyzed by RT-PCR. Human leukemia cell line J6-1 was used as a positive control and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. B, Namalwa and Ramos cells were transfected with mM-CSF–expressing vector or blank vector. Stably transfected clones (Namalwa-M/Ramos-M and Namalwa-B/Ramos-B) were obtained by G418 selection followed by RT-PCR verification using primers for either mM-CSF or neo gene. The amount of cDNA was equalized by PCR amplification of GAPDH. C, Western blot analysis of mM-CSF expression at protein level in the above clones was performed as detailed in Materials and Methods. Actin was used as an internal control.

 
The oncogenicity of Namalwa-M and Ramos-M in vivo. The in vitro data suggested that the expression of mM-CSF resulted in quiescence phenotype (data not shown). Whether it had any role on the in vivo oncogenicity of lymphoma cell lines was of interest. To this object, BALB/c nude mice were s.c. implanted 5 x 107 cells/200 µL with Namalwa-M, Namalwa, or Namalwa-B. Palpable tumors could be detected after 7 days postinoculation. Then, the size of tumors was measured with metric calipers twice or thrice a week. At early stage, no difference in the tumor progression between Namalwa-M and its controls was observed. Then, the tumor progression of Namalwa-M was much faster than the two controls (Fig. 2B ). Nude mice were killed on day 30, the size (Fig. 2A) of tumors formed by Namalwa-M showed significant difference when compared with those formed by Namalwa or Namalwa-B. Different Namalwa-M clones were used in this experiment and similar results were obtained. Similar results were also obtained when Namalwa series cells were replaced by 5 x 106 cells/200 µL Ramos series cells (Fig. 2A and B). In this case, nude mice were killed on day 17 because they grew faster than Namalwa series.


Figure 2
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  		Figure 2. The oncogenicity of Namalwa-M and Ramos-M cells in vivo. Female BALB/c nude mice (5 per group) were injected s.c. on day 0 with 5 x 107 Namalwa cells or 5 x 106 Ramos cells, and the tumor size was measured on the date indicated. The size of each tumor from the groups was pooled. Points, mean; bars, SD. *, Student's t test P values are significantly different (P < 0.05) from the control mice. A, tumors formed by Namalwa, Namalwa-B, and Namalwa-M cells on day 30, and tumors formed by Ramos, Ramos-B, and Ramos-M cells on day 17, respectively. Different transfectant clones were used, and typical results are shown. B, tumor volumes. C, Namalwa-M or Ramos-M cells were treated with or without diluted ascites containing 500 µg/mL B5 McAb for 30 min before s.c. injected into female BALB/c nude mice (5 per group, 5 x 107 cells per mouse for Namalwa-M and 5 x 106 for Ramos-M). B5 McAb (50 µg/100 µL/mouse) was injected intratumorally on day 7 and 14 for Namalwa-M formed tumors and day 7, 10, and 13 for Ramos-formed tumors. On day 30 (for Namalwa-M) or day 17 (for Ramos-M), mice were sacrificed and tumors were analyzed.

 
The H&E staining results showed no significant different among tumor samples. Light microscopy analysis showed that no tumor capsule was detected. Necrosis could be observed in the middle of tumor tissues. Tumor cell infiltration to fat and muscle tissues was detected. Tumor cells undergoing mitosis were frequently observed. Tumor cells were closely packed with scant cytoplasm, prominent nuclear membrane, and large nuclei with prominent nucleoli.

To further verify the protumor effect of mM-CSF, B5 McAb was used at day 0 before inoculation and after palpable tumors could be detected. The administration of B5 decreased the tumor size in both Namalwa-M and Ramos-M–formed tumors (Fig. 2C). The weight of tumors formed by Namalwa-M on day 30 was 0.08 ± 0.15 grams in B5 McAb group versus 0.71 ± 0.49 grams in control group (P < 0.05). For Ramos-M on day 17, the weight was 4.00 ± 1.27 grams in B5 McAb group versus 5.12 ± 1.21 grams in control group. The results showed that treatment of M-CSF antibody could inhibit in vivo tumor growth in these models and prominent inhibitory effect could be observed in Namalwa-M formed tumors.

The above results suggested that there should be more complicated mechanisms linking the expression of mM-CSF and oncogenicity of lymphoma cells in vivo. Furthermore, the expression of mM-CSF can enhance the oncogenicity of lymphoma cells.

Macrophage infiltration and vessel density in tumor tissues. Previous studies have shown that sM-CSF can recruit macrophages into the tumor site and promote the aggressive development of tumors (12). Besides acting as adhesion molecule mediating intercellular contact (21, 24), mM-CSF might also be cleaved in the juxtamembrane position to release a soluble form (22). So we studied the distribution of macrophages in tumor samples examined using macrophage-specific marker anti-F4/80 antibody. Confocal microscopy analysis showed that a few macrophages were found in the Namalwa/Ramos or Namalwa-B/Ramos-B–formed tumor tissues, whereas a remarkable increase in macrophage infiltration occurred in both Namalwa-M and Ramos-M formed tumor tissues (Fig. 3A, top and B, top ). Similar results were obtained by using anti-mouse CD11b antibody (data not shown).


Figure 3
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  		Figure 3. Macrophage infiltration and vessel density in tumor tissues. Sections were obtained from nude mice tumor tissues. A, confocal images of macrophage infiltration in tumor tissues formed by Namalwa, Namalwa-B, and Namalwa-M cells on day 30 (top). Paraffin-embedded sections were stained with FITC conjugated anti-mouse F4/80 antibody. Bottom, confocal images of vessels in tumor tissues formed by above cells. Frozen sections were stained with PE-conjugated anti-mouse CD31 antibody. B, confocal images of macrophage infiltration in tumor tissues formed by Ramos, Ramos-B, and Ramos-M cells on day 17 (top). Bottom, confocal images of vessels in tumor tissues formed by Ramos, Ramos-B, and Ramos-M cells. C and D, quantitative analysis of the vessel density in tumors. Vessels were counted from five random high-power fields. Columns, means of vessel numbers; bars, SD. *, P < 0.05 by Student's t test.

 
High-level angiogenesis is associated with tumor progression (35). In addition, in human breast cancers, macrophages clustered in human breast cancer samples, which correlated with a high level of angiogenesis (36, 37). Then, we analyzed the vessel density in these tumor tissues using PE-CD31 antibody. The high density of vessels was found in the Namalwa-M and Ramos-M–formed tumor tissues but not in the control samples (Fig. 3A, bottom and B, bottom). A quantitative analysis, by counting the vessel number under confocal microscope, indicated a significant increase of vessel density in Namalwa-M (Fig. 3C; P = 0.002) or Ramos-M (Fig. 3D; P = 0.012) samples compared with their control samples.

These observations suggested that macrophages might be recruited into mM-CSF–expressing tumors and the infiltrating macrophages might play important roles, including promoting angiogenesis, in these models. These may be parts of the mechanisms explaining the in vivo high progression of mM-CSF expressing lymphoma cell lines.

Peritoneal cavity macrophage from nude mice promoted Namalwa-M and Ramos-M proliferation and enhanced the phosphorylation of ERK in vitro. To further specify whether macrophages play a critical role in prompting mM-CSF–expressing lymphoma cell proliferation, we cocultured nude mice abdominal cavity macrophages with those cell lines in vitro in 96-well plate at a ratio of 1:1 for 7 days. When Namalwa-B or Namalwa-M cells were removed by aspiration after gentle resuspending, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method analyzing macrophages that adhered to the well bottom showed no statistical difference between macrophage wells and coculture wells. Cell counting analyzing the resuspending cells showed a stimulatory effect of macrophage on Namalwa-M but not Namalwa-B cells (Namalwa-M, 5.2 ± 0.2 x 105/mL; Namalwa-M coculture, 9.2 ± 0.1 x 105/mL; Namalwa-B, 7.5 ± 0.2 x 105/mL; Namalwa-B coculture, 8.1 ± 0.3 x 105/mL). MTT assay further verified that macrophages promoted Namalwa-M (P = 0.037) and Ramos-M (P = 0.046) growth (Fig. 4A ). Moreover, this effect was blocked by the addition of anti–M-CSF McAb (Fig. 4B). These results showed that macrophages could promote Namalwa-M and Ramos-M cells proliferation in this model; furthermore, mM-CSF was indispensable in this process.


Figure 4
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  		Figure 4. Peritoneal cavity macrophage from nude mice promoted Namalwa-M and Ramos-M cells proliferation. Cells were cultured in triplicate in 96-well plate. Columns, means of MTT units (absorbance); bars, SD. A, peritoneal cavity macrophages from nude mice were obtained. Tumor cells (Namalwa-B, Namalwa-M, Ramos-B, and Ramos-M) were cultured alone or cocultured with macrophages at a ratio of 1:1 for 7 d. Then, tumor cell proliferation was measured by MTT assay. Blank control was set when the same number of macrophages were cultured alone. The MTT units (OD) of cocultured tumor cells were calculated as: ODcocultured cell line = ODcoculture – ODmacrophages. B, Namalwa-M cells or Ramos-M cells were cultured alone or cocultured with macrophages with or without 2.5 µg/mL M-CSF McAb. Columns, means; bars, SD. *, P < 0.05, statistical analysis was performed using Student's t test.

 
Next, we asked which signal pathways are involved in this process. To address this question, we first analyzed the pattern of protein phosphorylation in Namalwa-M and Namalwa-B cells after cocultured with macrophages for 48 hour. Figure 5A illustrated that the tyrosine phosphorylation of several proteins with molecular masses of ~27, 42, 55, and 85 to 120 kDa was increased, whereas with molecular masses of ~45 to 50 kDa, tyrosine phosphorylation was decreased in Namalwa-M cells cocultured with macrophages. However, these effects could not be observed in Namalwa-B (Fig. 5A) or Namalwa (data not shown) cells.


Figure 5
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  		Figure 5. Macrophages enhanced the phosphorylation of ERK in Namalwa-M and Ramos-M cells in vitro. Lane 1, Namalwa-B; lane 2, Namalwa-B cocultured with macrophages (Namalwa-B CO); lane 3, Namalwa-M; lane 4, Namalwa-M cocultured with macrophages (Namalwa-M CO). A, tumor cells were cocultured with macrophages at 1:1 ratio for 48 h. Then, the cells were lysed and lysates were subjected to electrophoresis and Western blotting. Tyrosine-phosphorylated proteins were detected using antiphosphotyrosine McAb (4G10). Actin was used as internal control. B, ERK, JNK, and p38 kinases were IP from cell lysates using specific Abs, and the phosphorylation level was detected by probing the membrane with 4G10. Membrane was probed with Ab recognizing native proteins as loading control. C, the relative phosphorylation intensities of ERK, JNK, and p38; columns, mean; bars, SD. D, Ramos-B or Ramos-M cells were cocultured with or without macrophages, ERK was IP from these cell lysates using above methods. The relative phosphorylation intensities of ERK; columns, mean; bars, SD. *, P < 0.05, statistical analysis was performed using Student's t test.

 
As MAPK cascades are important pathways mediating the cell growth promotion effects (38), we investigated the tyrosine phosphorylation level of three members (ERK, JNK, and P38) of MAPK family in Namalwa-M and Namalwa-B cells with or without macrophage stimulation (Fig. 5B and C). The phosphorylation of ERK, but not JNK or P38, in Namalwa-M cells was enhanced by coculturing with macrophages for 48 hours. However, it kept unaltered in Namalwa-B cells. Furthermore, these observations could also be detected in Ramos-M cells (Fig. 5D). Taken together, the proliferation of Namalwa-M and Ramos-M were enhanced by coculturing with macrophages through the activation of ERK/MAPK signal pathway.

The expression of angiogenic factors were up-regulated in Namalwa-M formed tumor tissues. Angiogenesis, which is stimulated by proangiogenic factors including VEGF, bFGF, HGF, etc., play a pivotal role in tumor progression. Recent work showed that macrophages played a key role in promoting the angiogenesis in tumor models (37). The remarkable increase in macrophage infiltration and the high-density vessel were observed in mM-CSF–expressing cell–formed tumor tissues. To further determine the proangiogenic mechanism, the expressions of VEGF, bFGF, and HGF were measured using mouse specific primers by real-time RT-PCR. The levels of the three factors in Namalwa-M formed tumor tissues were higher than those in control samples (Fig. 6 ). By contrast, when human specific primers for these factors were used to analyze whether the effect was also from malignant cells, unaltered or slightly decreased levels were observed. These results strongly suggested that the proangiogenic effect might be induced mainly by macrophages.


Figure 6
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  		Figure 6. The expression of murine angiogenic factors in tumor tissues. Samples were obtained from day 30 nude mice tumor tissues indicated. For each sample, 0.1 to 0.2 grams of tumor tissue were homogenized with 1 mL Trizol reagent, and total RNA was extracted. cDNA was subsequently synthesized from 5 µg total RNA using M-MLV reverse transcriptase. The expressions of bFGF, HGF, and VEGF were analyzed by real-time PCR using specific murine primers detailed in Materials and Methods. A, bFGF. B, HGF. C, VEGF.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
The abnormal expression of M-CSF was found in different tumors, and the role of mM-CSF in malignancies was of interest (7, 8). In glioblastoma, glioma, hepatocellular carcinoma, and breast cancer, tumor-expressed mM-CSF elicited efficient antitumoral activity of macrophages, which suggested a negatively regulatory role in the development of those malignancies (27–30). However, this cannot explain why it was highly expressed in some malignancies. The mM-CSF together with M-CSFR were highly expressed in some leukemia cell lines as well as samples from patients who suffered leukemia and HL (32). Hence, they were considered potential TAA for tumor immunotherapy, and DNA vaccines targeting mM-CSF and/or M-CSFR were developed (39). However, the roles of this isoform in the development of hematopoietic malignancies are still unknown. Thus, lymphoma cell lines stably expressing mM-CSF were established. The in vitro growth pattern of these cells suggested a quiescence and less adhesive phenotype, which raise the speculation that mM-CSF might promote some cancer stem cell–like features. Moreover, except early stage, the tumor development of mM-CSF–expressing cells was much faster than their controls in nude mice model. It was reported that local synthesis of mM-CSF was sufficient for the regulation of several tissue macrophage populations (26). A remarkable increase in macrophage infiltration was found in the Namalwa-M–formed tumor tissues on day 30 and the Ramos-M–formed tumor tissues on day 17. These results implied that macrophage infiltration should account for the in vivo development of mM-CSF–expressing cell–formed tumors, i.e., once infiltrating and abnormally activated macrophages are sufficient in the tumor microenvironment, growth-promoting effect could be observed. These results suggested that the infiltrating and abnormally active macrophages should play important roles on stimulating the growth of Namalwa-M cells or Ramos-M. This notion was further verify by in vitro coculture experiment. Furthermore, the phosphorylation of ERK was enhanced in Namalwa-M and Ramos-M cells upon coculturing with macrophage. Although in vivo conditions are much more complex than in vitro conditions, our in vitro results were in accordance with the in vivo early stage results. Hence, we suggested that these results could at least partly account for the action of the macrophages in vivo, especially in the early stage. Taken together, in this model, infiltrating and abnormally active macrophages enhanced the proliferation of Namalwa-M and Ramos-M through the activation of ERK/MAPK signal pathway.

Angiogenesis plays pivotal roles in tumor development (40). Here, we found the elevated angiogenesis in Namalwa-M and Ramos-M formed tumor tissues. Increasing evidences have emerged that macrophages play important roles not only in tumor cell survival, proliferation, invasion, and metastasis but also in stimulating angiogenesis by secreting a wide range of angiogenic factors (14, 15). In this article, the expression of mouse bFGF, HGF, and VEGF was higher in Namalwa-M tumor tissues, respectively, whereas no significant difference was found in that of human origin. These results suggested that the infiltrating macrophages contributed to the high-level angiogenesis in Namalwa-M tumor tissues. Furthermore, we attempted to use in vitro coculture system to further verify the in vivo results. However, the results were irregular (data not shown), which suggested that the system could not mimic in vivo conditions. Angiogenesis is strictly regulated by the microenvironment, which consists of complex conditions and multifold stimuli. The mechanisms macrophages use to promote angiogenesis in tumor are unclear (37). Our results suggested that mM-CSF itself could not directly stimulate macrophages to overexpress angiogenic factors. Nevertheless, the activation of macrophages by mM-CSF should be the first and essential step.

The development of mM-CSF–expressing lymphoma cells in nude mice can be outlined in the following steps: (a) Macrophage infiltration. The mM-CSF could be cleaved to release a soluble form with a t1/2 of ~11 hours compared with that of 20 minutes for secretion from the soluble form, and sM-CSF is a chemoattractant for monocyte/macrophage and can induce macrophages into tumor sites (14). Although our in vitro experiment using conditioned medium of mM-CSF–expressing cells failed to chemoattract macrophages, this proteolytic cleavage might be sufficient in vivo, to build up high concentrations of soluble M-CSF. The detailed mechanism is unclear, and other chemoattractants, which can be expressed by both malignant cells and already infiltrated macrophages, might contribute to the process. (b) Abnormal activation of macrophages. As M-CSFR is expressed in infiltrating macrophages (21), mM-CSF can act as an adhesion molecule to adhere the infiltrated macrophages in tumor sites. Moreover, it can act as ligand leading the activation of M-CSFR signaling. This signal together with other costimulating signals, which are elicited by other membrane and/or soluble molecules, result in the activation of infiltrating macrophages. Notably, the abundant existing mM-CSF in the tumor microenvironment causes a so-called "cytokine storm" (local mM-CSF storm; ref. 41) to infiltrating macrophages, causing high-level activation of M-CSFR signaling. Furthermore, the M-CSFR signal elicited by sM-CSF can be blocked by degradation after internalization, which is via clathrin-coated pits and vesicles and targeted to lysosomes (42, 43). Whereas, the membrane-integrated mM-CSF hinders the internalization and degradation process. Taken together, the persistent activation signals cause the abnormal activation of infiltrating macrophages, which might be taught to TAM (6). (c) Promoting tumor development. The abnormally activated macrophages, in turn, can express some membrane proteins, directly stimulating the growth of mM-CSF–expressing cells through juxtacrine mechanism, and/or secrete a panel of cytokines, function through paracrine mechanism. These stimuli cause the activation of ERK/MAPK pathway. Furthermore, at late stage, these macrophages secrete angiogenic factors to promote tumor angiogenesis.

The mM-CSF can act as both positive and negative regulators in tumor development by activating macrophages depending on cell types. Although expression of this isoform in some tumors resulted in the activation of antitumor effects of macrophages, the infiltrating macrophages were reported to correlate with tumor cell survival, proliferation, invasion, metastasis, and poor prognosis (14). Especially, in hematopoietic malignancies, high expression of mM-CSF was detected in HL and myeloid leukemia (32). Furthermore, stromal macrophages predominantly increased in malignant lymphoma represented the poor prognosis and short life span, whereas macrophages were rare or absent in most of the long-term survivors (44, 45). Thus, we presume that mM-CSF may be a double-edged sword in tumor development and should be a positive regulator in the development of hematopoietic malignancies via macrophages. How macrophages decide its fate is still unknown. However, macrophage subpopulations, which differ in immunoregulatory properties, were found in histiocytic lymphoma (46). A possible answer might be that, in different tumors, distinct macrophage subpopulations were recruited, adhered, and activated. Whether positive or negative effects can be observed depends on which subpopulation was predominantly activated in the tumor microenvironment.

Although several groups focused on mM-CSF, the mechanisms regulating mM-CSF expression are still unclear. Evidence has shown that virus takes part in the genesis and development of tumors. For example, the EBV has been linked to Hodgkin's disease, Burkitt's lymphoma, and nasopharyngeal cancer, as well as certain rare cancers in immunosupressed transplant patients (47). Our previous work showed that human herpes virus 6 promoted the expression of mM-CSF in leukemic cells from patient samples (48). Thus, mM-CSF might be a bridge linking herpes virus infection/activation and hematopoietic malignancies.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: National Natural Science Foundation of China (Grant No. 30470897) and Major State Basic Research Development Program (973 program) of China (Grant No. 2006CB910406).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Mei Zhang, Hong-Guang Ying, Yan-Han Li, Jie Gu, Qing Rao, Hai-Rong Jia, Xin Fu, and Rong-Xiang Zhao for their technical help.

Received 10/ 8/07. Revised 5/ 4/08. Accepted 5/14/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. Kawasaki ES, Ladner MB, Wang AM, et al. Molecular cloning of a complementary DNA encoding human macrophage-specific colony-stimulating factor (CSF-1). Science 1985;230:291–6.[Abstract/Free Full Text]
  2. Cerretti DP, Wignall J, Anderson D, et al. Human macrophage-colony stimulating factor: alternative RNA and protein processing from a single gene. Mol Immunol 1988;25:761–70.[CrossRef][Medline]
  3. Sherr CJ, Rettenmier CW, Sacca R, Roussel MF, Look AT, Stanley ER. The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1. Cell 1985;41:665–76.[CrossRef][Medline]
  4. Chitu V, Stanley ER. Colony-stimulating factor-1 in immunity and inflammation. Curr Opin Immunol 2006;18:39–48.[CrossRef][Medline]
  5. Pixley FJ, Stanley ER. CSF-1 regulation of the wandering macrophage: complexity in action. Trends Cell Biol 2004;14:628–38.[CrossRef][Medline]
  6. Pollard JW. Tumour-educated macrophages promote tumor progression and metastasis. Nat Rev Cancer 2004;4:71–8.[CrossRef][Medline]
  7. Kacinski BM. CSF-1 and its receptor in ovarian, endometrial and breast cancer. Ann Med 1995;27:79–85.[Medline]
  8. Kirma N, Hammes LS, Liu YG, et al. Elevated expression of the oncogene c-fms and its ligand, the macrophage colony-stimulating factor-1, in cervical cancer and the role of transforming growth factor-β1 in inducing c-fms expression. Cancer Res 2007;67:1918–26.[Abstract/Free Full Text]
  9. Mroczko B, Groblewska M, Wereszczyñska-Siemiatkowska U, et al. Serum macrophage-colony stimulating factor levels in colorectal cancer patients correlate with lymph node metastasis and poor prognosis. Clin Chim Acta 2007;380:208–12.[CrossRef][Medline]
  10. Baiocchi G, Kavanagh JJ, Talpaz M, Wharton JT, Gutterman JU, Kurzrock R. Expression of the macrophage colony-stimulating factor and its receptor in gynecologic malignancies. Cancer 1991;67:990–6.[CrossRef][Medline]
  11. Janowska-Wieczorek A, Belch AR, Jacobs A, et al. Increased circulating colony-stimulating factor-1 in patients with pre-leukemia, leukemia and lymphoid malignancies. Blood 1991;77:1796–803.[Abstract/Free Full Text]
  12. Lin EY, Nguyen AV, Russell RG, Pollard JW. Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy. J Exp Med 2001;193:727–40.[Abstract/Free Full Text]
  13. Wyckoff J, Wang W, Lin EY, et al. A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res 2004;64:7022–9.[Abstract/Free Full Text]
  14. Lewis CE, Pollard JW. Distinct role of macrophages in different tumor microenvironments. Cancer Res 2006;66:605–12.[Abstract/Free Full Text]
  15. Crowther M, Brown NJ, Bishop ET, Lewis CE. Microenvironmental influence on macrophage regulation of angiogenesis in wounds and malignant tumors. J Leukoc Biol 2001;70:478–90.[Abstract/Free Full Text]
  16. Yano S, Hanibuchi M, Nishioka Y, et al. Combined therapy with anti-P-glycoprotein antibody and macrophage colony-stimulating factor gene transduction for multiorgan metastases of multidrug-resistant human small cell lung cancer in NK cell-depleted SCID mice. Int J Cancer 1999;82:105–11.[CrossRef][Medline]
  17. Baldwin GC, Chung GY, Kaslander C, Esmail T, Reisfeld RA, Golde DW. Colony-stimulating factor enhancement of myeloid effector cell cytotoxicity towards neuroectodermal tumour cells. Br J Haematol 1993;83:545–53.[Medline]
  18. Moore RN, Oppenheim JJ, Farrar JJ, Carter CS, Jr., Waheed A, Shadduck RK. Production of lymphocyte-activating factor (Interleukin1) by macrophages activated with colony-stimulating factors. J Immunol 1980;125:1302–5.[Abstract]
  19. Warren MK, Ralph P. Macrophage growth factor CSF-1 stimulates human monocyte production of interferon, tumor necrosis factor, and colony stimulating activity. J Immunol 1986;137:2281–5.[Abstract]
  20. Heard JM, Roussel MF, Rettenmier CW, Sherr CJ. Synthesis, post-translational processing, and autocrine transforming activity of a carboxylterminal truncated form of colony stimulating factor-1. Oncogene Res 1987;1:423–40.[Medline]
  21. Uemura N, Ozawa K, Takahashi K, et al. Binding of membrane-anchored macrophage colony-stimulating factor (M-CSF) to its receptor mediates specific adhesion between stromal cells and M-CSF receptor-bearing hematopoietic cells. Blood 1993;82:2634–40.[Abstract/Free Full Text]
  22. Tuck DP, Cerretti DP, Hand A, Guha A, Sorba S, Dainiak N. Human macrophage colony-stimulating factor is expressed at and shed from the cell surface. Blood 1994;84:2182–8.[Abstract/Free Full Text]
  23. Stein J, Borzillo GV, Rettenmier CW. Direct stimulation of cells expressing receptors for macrophage colony-stimulating factor (CSF-1) by a plasma membrane-bound precursor of human CSF-1. Blood 1990;76:1308–14.[Abstract/Free Full Text]
  24. Zheng G, Rao Q, Wu K, He Z, Geng Y. Membrane-bound macrophage colony-stimulating factor and its receptor play adhesion molecule-like roles in leukemic cells. Leuk Res 2000;24:375–83.[CrossRef][Medline]
  25. Tsuboi I, Revol V, Blanchet JP, Mouchiroud G. Role of the membrane form of human colony stimulating factor-1 (CSF-1) in proliferation of multipotent hematopoietic FDCP-mix cells expressing human CSF-1 receptor. Leukemia 2000;14:1460–6.[CrossRef][Medline]
  26. Dai XM, Zong XH, Sylvestre V, Stanley ER. Incomplete restoration of colony-stimulating factor 1 (CSF-1) function in CSF-1-deficient Csf1op/Csf1op mice by transgenic expression of cell surface csf-1. Blood 2004;103:1114–23.[Abstract/Free Full Text]
  27. Graf MR, Jadus MR, Hiserodt JC, Wepsic HT, Granger GA. Development of systemic immunity to glioblastoma multiforme using tumor cells genetically engineered to express the membrane-associated isoform of macrophage colony-stimulating factor. J Immunol 1999;163:5544–51.[Abstract/Free Full Text]
  28. Jadus MR, Chen Y, Boldaji MT, et al. Human U251MG glioma cells expressing the membrane form of macrophage colony-stimulating factor (mM-CSF) are killed by human monocytes in vitro and are rejected within immunodeficient mice via paraptosis that is associated with increased expression of three different heat shock proteins. Cancer Gene Ther 2003;10:411–20.[CrossRef][Medline]
  29. Dan Q, Sanchez R, Delgado C, et al. Non-immunogenic murine hepatocellular carcinoma Hepa1–6 cells expressing the membrane form of macrophage colony stimulating factor are rejected in vivo and lead to CD8+ T-cell immunity against the parental tumor. Mol Ther 2001;4:427–37.[CrossRef][Medline]
  30. Williams CC, Trinh H, Tran TV, et al. Membrane macrophage colony-stimulating factor on MADB106 breast cancer cells does not activate cytotoxic macrophages but immunizes rats against breast cancer. Mol Ther 2001;3:216–24.[CrossRef][Medline]
  31. Wu KF, Rao Q, Zheng GG, et al. Enhancement of J6–1 human leukemic cell proliferation by membrane bound M-CSF through a cell-cell contact mechanism II: Role of an M-CSF receptor like membrane protein. Leuk Res 1998;22:55–60.[CrossRef][Medline]
  32. Zheng GG, Wu KF, Geng YQ, et al. Expression of membrane-associated macrophage colony-stimulating factor (mM-CSF) in Hodgkin's diseases and other hematologic malignancies. Leuk Lymphoma 1999;32:339–44.[Medline]
  33. Asano T, Kaneko E, Shinozaki S, et al. Hyperbaric oxygen induces basic fibroblast growth factor and hepatocyte growth factor expression, and enhances blood perfusion and muscle regeneration in mouse ischemic hind limbs. Circ J 2007;71:405–11.[CrossRef][Medline]
  34. Tominaga K, Saito S, Matsuura M, Funatogawa K, Matsumura H, Nakano M. Role of IFN-{gamma} on dissociation between nitric oxide and TNF/IL-6 production by murine peritoneal cells after restimulation with bacterial lipopolysaccharide. J Leukoc Biol 1999;66:974–80.[Abstract]
  35. Albini A, Tosetti F, Benelli R, Noonan DM. Tumor inflammatory angiogenesis and its chemoprevention. Cancer Res 2005;65:10637–41.[Abstract/Free Full Text]
  36. Leek RD, Lewis CE, Whitehouse R, Greenall M, Clarke J, Harris AL. Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res 1996;56:4625–9.[Abstract/Free Full Text]
  37. Lin EY, Li JF, Gnatovskiy L, et al. Macrophages regulate the angiogenic switch in a mouse model of breast cancer. Cancer Res 2006;66:11238–46.[Abstract/Free Full Text]
  38. Torii S, Yamamoto T, Tsuchiya Y, Nishida E. ERK map kinase in G cell cycle progression and cancer. Cancer Sci 2006;97:697–702.[CrossRef][Medline]
  39. Wang MH, Zheng GG, Wu KF, et al. Co-immunization with M-CSFR and mM-CSF DNA vaccines is better than M-CSFR-mM-CSF fusion DNA vaccine. Haematologica 2002;87:1087–94.[Abstract/Free Full Text]
  40. Folkman J. The role of angiogenesis in tumor growth. Semin Cancer Biol 1992;3:65–71.[Medline]
  41. Antin JH, Ferrara JL. Cytokine dysregulation and acute graft-versus-host disease. Blood 1992;80:2964–8.[Abstract/Free Full Text]
  42. Manger R, Najita L, Nichols EJ, Hakomori S, Rohrschneider L. Cell surface expression of the McDonough strain of feline sarcoma virus fms gene product (gp 140fms). Cell 1984;34:327–37.
  43. Guilbert LJ, Stanley ER. The interaction of 125I-colony stimulating factor-1 with bone marrow-derived macrophages. J Biol Chem 1986;261:4024–32.[Abstract/Free Full Text]
  44. Ree HJ, Crowley JP, Leone LA. Macrophage-histiocytes in malignant lymphoma, small lymphocytic type (well-differentiated lymphocytic lymphoma). Cancer 1985;56:1117–23.[Medline]
  45. Ree HJ, Kadin ME. Macrophage-histiocytes in Hodgkin's disease. The relation of peanut-agglutinin-binding macrophage-histiocytes to clinicopathologic presentation and course of disease. Cancer 1985;56:333–8.[Medline]
  46. Lavie G, Shoenfeld Y, Shaklai M, Aderka D, Pick AI, Pinkhas J. Homogeneous populations of macrophages from histiocytic lymphoma patients as a source for macrophage subpopulations which differ in immunoregulatory properties. Cancer 1982;50:69–77.[Medline]
  47. Iwakiri D, Samanta M, Takada K. Mechanisms of EBV-mediated oncogenesis. Uirusu 2006;56:201–8.[CrossRef][Medline]
  48. Yang W, Wu K, Song Y. Effect of HHV-6 on expression of M-CSF in patients with hematological disorders. Zhong hua Xue Ye Xue Za Zhi 1998;19:646–8.




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