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Keywords:

  • CSF-1R;
  • GS-1101;
  • invasion: macrophage;
  • PI3K p110δ

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Colony stimulating factor-1 (CSF-1) stimulates mononuclear phagocytic cell survival, growth and differentiation into macrophages through activation and autophosphorylation of the CSF-1 receptor (CSF-1R). We have previously demonstrated that CSF-1-induced phosphorylation of Y721 (pY721) in the receptor kinase insert triggers its association with the p85 regulatory subunit of phosphoinositide 3′-kinase (PI3K). Binding of p85 PI3K to the CSF-1R pY721 motif activates the associated p110 PI3K catalytic subunit and stimulates spreading and motility in macrophages and enhancement of tumor cell invasion. Here we show that pY721-based signaling is necessary for CSF-1-stimulated PtdIns(3,4,5)P production. While primary bone marrow-derived macrophages and the immortalized bone marrow-derived macrophage cell line M−/−.WT express all three class IA PI3K isoforms, p110δ predominates in the cell line. Treatment with p110δ-specific inhibitors demonstrates that the hematopoietically enriched isoform, p110δ, mediates CSF-1-regulated spreading and invasion in macrophages. Thus GS-1101, a potent and selective p110δ inhibitor, may have therapeutic potential by targeting the infiltrative capacity of tumor-associated macrophages that is critical for their enhancement of tumor invasion and metastasis.


Abbreviations
BMMs

bone marrow-derived macrophages

CSF-1

colony-stimulating factor-1

CSF-1R

colony-stimulating factor-1 receptor

DMSO

dimethylsulfoxide

EGF

epidermal growth factor

PI3K

phosphoinositide 3′-kinase

PIP3

phosphatidylinositol 3,4,5-trisphosphate

TAM

tumor-associated macrophage

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Macrophages are fully differentiated cells of the mononuclear phagocytic lineage, and are important for normal development and homeostasis as well as immune surveillance [1, 2]. They also promote disease progression in conditions ranging from cancer to atherosclerosis and arthritis [3, 4]. Interstitial motility is an essential element of macrophage function, and is required for both their beneficial and deleterious activities [5]. Colony stimulating factor-1 (CSF-1), originally identified as a mononuclear phagocytic cell growth and differentiation factor [6], potently stimulates macrophage motility [5, 7]. Furthermore, high circulating levels of CSF-1 are found in advanced breast, endometrial and ovarian cancers [8-10], and breast carcinoma cells express high levels of CSF-1 at invasive fronts where large numbers of tumor-associated macrophages (TAMs) have infiltrated and gathered [11]. Indeed, a lack of CSF-1 in the mammary carcinoma-prone MMTV-PyMT mouse significantly inhibits tumor progression, invasion and pulmonary metastasis, while CSF-1 over-expression accelerates disease progression [12]. An important mechanism underlying the promotion of tumor invasion and metastasis by TAMs is establishment of a paracrine chemotactic loop between carcinoma cells and TAMs [13]. Carcinoma cells secrete CSF-1 and TAMs secrete epidermal growth factor (EGF) to induce co-migration of both cell types [14]. This multicellular migratory streaming, which has been imaged in vivo, is dependent upon CSF-1 signaling [14, 15].

The pleiotrophic effects of CSF-1 are mediated by the CSF-1 receptor (CSF-1R), a receptor tyrosine kinase of the PDGF-R/c-Kit family, via trans-autophosphorylation of a series of intracellular tyrosine residues [2]. We have developed a unique macrophage cell line system in which immortalized macrophages derived from the CSF-1R−/− mouse retrovirally express either wild-type (WT) or tyrosine mutant (e.g. Y721F) receptors [16]. We have recently used this model system to show that signaling triggered by the pY721 motif regulates CSF-1-induced motility in macrophages [17]. This motif is necessary and sufficient for association of the receptor with the p85 regulatory subunit of phosphoinositide 3′-kinase (PI3K), and subsequent PI3K activation leads to polarized production of PtdIns(3,4,5)P (PIP3) at the leading edge of the cell [17]. The p85 subunit is constitutively bound to one of three class IA catalytic p110 PI3K subunits, p110α, p110β and p110δ [18]. These class IA PI3K heterodimers signal downstream of receptor tyrosine kinases to mediate a variety of biological effects in various cell types [18, 19]. Macrophages express the ubiquitous isoforms, p110α and p110β, as well as the hematopoietically enriched p110δ isoform [20]. Although p110δ has been shown to be the main isoform recruited to the CSF-1R in primary macrophages, p110α associates with the receptor in the immortalized BAC1.2F5 macrophage cell line [20]. Nevertheless, p110δ has been demonstrated to regulate migration in both primary and immortalized macrophages [20].

Although the precise signaling pathways by which PI3K mediates CSF-1-induced macrophage motility are not yet clear, more than one downstream pathway is probably involved [17]. Targeting upstream PI3K activation to inhibit the motility of infiltrating TAMs is therefore more likely to be effective than targeting several possible downstream effectors. GS-1101, a potent and selective inhibitor of p110δ [21], is in phase III clinical trials as a targeted therapy for B-cell lymphoproliferative disorders, including chronic lymphocytic leukemia and indolent non-Hodgkin lymphomas [22, 23]. Here, we confirm the necessity of pY721-based signaling for CSF-1-induced PIP3 production in macrophages, and demonstrate that p110α and p110δ are the major class IA PI3K isoforms expressed in bone marrow-derived macrophages (BMMs) and the immortalized bone marrow-derived macrophage cell line M−/−.WT. We also show that specific inhibition of p110δ by GS-1101 abrogates CSF-1-induced PIP3 production to block macrophage spreading and infiltrative capacity.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

pY721 CSF-1R-based signaling is necessary for CSF-1-induced PIP3 production in macrophages

We and others have previously shown that phosphorylation of Y721 in the CSF-1R kinase insert mediates rapid and prolonged association of p85 PI3K with the receptor in macrophages in response to CSF-1 [17, 24]. The resultant activation of PI3K produces an approximately ninefold increase in total PIP3 levels (Fig. 1A, M−/−.WT cells). In contrast, macrophages expressing a mutant Y721F receptor (M−/−.Y721F) fail to show a CSF-1-induced increase in PIP3 levels, indicating that PI3K requires direct association with the receptor via the pY721 motif for p110 activation and subsequent PIP3 production (Fig. 1A).

image

Figure 1. CSF-1R pY721-based signaling is required for CSF-1-induced PIP3 production. (A) PIP3 levels in CSF-1-starved M−/−.WT and M−/−.Y721F cells stimulated with CSF-1 for 0 or 30 s were measured by PIP3 ELISA (= 4). Asterisks indicate a statistically significant difference compared with 0 s time point (***< 0.001). (B) Primary and M−/−.WT macrophages grown in the continuous presence of CSF-1 were lysed, and the lysates examined for expression of PI3K class IA isoforms by SDS/PAGE and immunoblotting. MCF10A mammary epithelial cell lysates were included for comparison, and GAPDH immunoblotting was performed as a loading control. A representative blot (= 4) is shown.

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Macrophages express all three class IA PI3K isoforms at variable levels in cell lines compared to primary macrophages

To identify the class IA PI3K isoforms activated by CSF-1 in macrophages, we initially examined their relative expression levels in BMM and M−/−.WT cells compared with MCF10A cells, which are a widely used breast epithelial cell line. MCF10A cells expressed the ubiquitous isoforms p110α and p110β, but not the hematopoietically enriched isoform p110δ (Fig. 1B). Compared to the epithelial cell line, M−/−.WT cells expressed very high levels of p110δ with very little p110α and p110β, while primary BMMs expressed relatively low levels of all three isoforms (Fig. 1B). Thus, while primary and immortalized macrophages express all three class IA PI3K isoforms, their expression of the ubiquitous isoforms is very low compared to MCF10A cells. Moreover, in contrast to a previous study, which demonstrated an increased role for p110α downstream of the CSF-1R in the BAC1.2F5 spleen-derived macrophage cell line compared to BMMs [20], expression of the hematopoietic isoform predominates in M−/−.WT cells, which are bone marrow-derived, indicating that isoform expression levels differ between macrophage cell lines as well as when compared to BMMs. Thus PI3K isoform signaling studies in macrophages should include reference BMMs where possible.

p110δ is the primary mediator of CSF-1-induced PI3K signaling in macrophages

Class IA PI3K isoforms exert distinct actions downstream of receptor tyrosine kinases that are not necessarily correlated with their relative expression levels. Studies with microinjected inhibitory antibodies against specific PI3K isoforms indicated that the CSF-1R signals through p110α to mitogenesis, but regulates actin cytoskeletal remodeling and motility through p110β and p110δ in BAC1.2F5 cells [25]. Subsequent studies with p110α or p110δ PI3K-deficient macrophages and specific small molecule inhibitors demonstrated that p110δ is the primary mediator of CSF-1-induced chemotaxis [20]. To more closely examine the role of p110δ in macrophage motility, GS-1101, a highly selective and potent p110δ small molecule inhibitor (IC50 = 2.5 nm) [21], and IC488743, a p110δ inhibitor with an IC50 of 13 nm [26] from Gilead Sciences (Seattle, WA, USA), were used in a series of signaling and single-cell assays in both BMMs and M−/−.WT macrophages.

CSF-1-induced PI3K activation in M−/−.WT macrophages results in Akt phosphorylation in the activation loop (T308) and the C-terminus (S473) that peaks at 2.5 min [17]. CSF-1-stimulated Akt phosphorylation was abrogated by pre-treatment with the pan-PI3K inhibitor, wortmannin (Fig. 2A). Akt phosphorylation was significantly inhibited by 1 μm of GS-1101 (pS473 53%, pT308 62%) and IC488743 (pS473 68%, pT308 72%), with almost complete inhibition at greater inhibitor concentrations (Fig. 2A). As p110 isoform levels may vary between macrophage cell lines and primary macrophages (Fig. 1B), the effect of GS-1101 was examined in BMMs, and the extent of p110δ inhibition was comparable to that seen in the macrophage cell line (Fig. 2B). PI3K inhibition did not affect CSF-1-induced tyrosine phosphorylation in macrophages (data not shown).

image

Figure 2. Inhibition of PI3K p110δ by GS-1101 abrogates PIP3 production and Akt activation in macrophages. (A) CSF-1-starved M−/−.WT cells were pre-treated with the indicated doses of GS-1101, IC488743 or wortmannin for 1 h prior to CSF-1 stimulation for 0 or 2.5 min. Akt activation was measured by densitometry of pS473 and pT308 Akt bands, and expressed as the percentage reduction in signal compared to DMSO-treated control cells, as indicated below the respective blots. (B) CSF-1-starved BMMs were pre-treated with the indicated doses of GS-1101 prior to CSF-1 stimulation for 0 or 2.5 min, and Akt activation measured by SDS/PAGE and immunoblotting as in figure 2A. (C) PIP3 levels in CSF-1-starved M−/−.WT cells pre-treated with GS-1101 (5 μm), IC488743 (5 μm) or DMSO (0.1%), then stimulated with CSF-1 for 0 or 30 s, were measured by PIP3 ELISA (= 3). Asterisks indicate a statistically significant difference compared with 0 s (***< 0.001).

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To confirm that inhibition of p110δ alone prevents Akt activation, a PIP3 ELISA of M−/−.WT cells in the presence or absence of the isoform-specific inhibitors was performed. Consistent with the phospho-Akt levels, blockade of p110δ alone by GS-1101 prevented a CSF-1-induced rise in PIP3 production similar to that observed with macrophages expressing the Y721F CSF-1R mutant (Fig. 2C). Taken together, these results indicate that p110δ is the primary PI3K isoform activating the PI3K/Akt pathway downstream of the CSF-1R in macrophages.

PI3K p110δ mediates CSF-1-induced spreading and invasion

CSF-1 stimulates rapid and circumferential spreading in macrophages prior to elongation and commencement of migration [5]. The spreading response, which increases the macrophage footprint area by approximately 40% at 5 min, is dependent upon pY721-based CSF-1R signaling [17]. To determine whether PI3K p110δ regulates CSF-1-stimulated spreading, we plated M−/−.WT cells on fibronectin-coated cover slips for 2 days prior to removing CSF-1 overnight. The cells were then pre-treated with GS-1101 (5 μm) or vehicle control (dimethylsulfoxide, DMSO) and stimulated with CSF-1 for 0 or 5 min before fixation and staining for F-actin. Stained cells were individually traced to determine mean footprint area before and after CSF-1 stimulation. DMSO is a widely used solvent for cell-based assays and is required to solubilize GS-1101 and IC488743. Although it is widely considered to be non-toxic to cells in culture at concentrations as high as 1%, macrophages in 0.1% DMSO failed to show the expected spreading response upon CSF-1 treatment (data not shown). To assess whether DMSO inhibits CSF-1-induced spreading, we exposed M−/−.WT cells to 0, 0.04 or 0.1% DMSO, and measured the mean footprint area 0 and 5 min after CSF-1 stimulation. The footprint of cells treated with 0.04% DMSO increased by 30% in response to CSF-1 (> 30 cells), which was not significantly different to control cells (42%), but there was a significant decrease in the spreading response of cells in 0.1% DMSO (16%, < 0.01) (Fig. 3A). To reduce the DMSO volume to a final concentration of 0.04%, the concentration of isoform-specific inhibitors had to be reduced accordingly. Thus, we compared CSF-1-induced Akt phosphorylation in cells pre-treated with 2 or 5 μm of either GS-1101 or IC488743 to confirm that reduced inhibitor concentrations significantly inhibited CSF-1-stimulated PI3K activation in M−/−.WT macrophages. Both inhibitors were as effective at 2 μm as at 5 μm (Fig. 3B).

image

Figure 3. GS-1101 inhibits macrophage spreading. (A) To examine the effect of DMSO on CSF-1-induced macrophage spreading, CSF-1-starved M−/−.WT cells were pre-treated with the indicated concentrations of DMSO, then stimulated with CSF-1 for 0 or 5 min, fixed and stained for F-actin. Cell outlines of at least 30 cells/sample were traced using imagej to determine footprint area, and the results of a representative experiment (= 3) are shown. Asterisks indicate statistically significant differences (*< 0.05, **< 0.01, ***< 0.001). (B) CSF-1-starved M−/−.WT cells were pre-treated with the indicated doses of GS-1101 or IC488743 prior to CSF-1 stimulation for 0 or 2.5 min, and Akt activation was measured by SDS/PAGE and immunoblotting as in Figure 3A. (C) CSF-1-starved M−/−.WT cells were pre-treated with 2 μm GS-1101 or 0.04% DMSO, then stimulated with CSF-1 for 0 or 5 min, fixed and stained for F-actin, and cell outlines were traced using imagej to determine footprint area. Asterisks indicate statistically significant differences (***< 0.001). (D) M−/−.WT macrophages treated with DMSO or GS-1101 before CSF-1 stimulation for the indicated times were fixed and stained for Factin. Scale bar = 20 μm.

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Thus, we examined the effect of inhibition of p110δ on the CSF-1-induced cell spreading response using GS-1101 at 2 μm. CSF-1-starved M−/−.WT cells were pre-treated with DMSO or GS-1101 prior to CSF-1 stimulation, and their footprint area was measured at 0 and 5 min. While control macrophages demonstrated the expected increase in footprint area (39%), spreading failed to occur following inhibition of p110δ (Fig. 3C). Representative images of M−/−.WT cells pre-treated with DMSO or GS-1101 then stimulated with CSF-1 are shown in Fig. 3D. Thus p110δ PI3K mediates CSF-1-stimulated spreading, underpinned by actin polymerization and adhesion structure formation [5, 17], in macrophages.

To determine whether p110δ inhibition similarly affects macrophage motility and infiltrative capacity, a Matrigel invasion assay was performed. Compared to control cells, M−/−.WT macrophages pre-treated with GS-1101 or IC488743 showed a 65–70% reduction in their ability to infiltrate through Matrigel in a modified Boyden chamber assay (Fig. 4A). To further examine the role of p110δ in the regulation of macrophage invasive capacity, a matrix degradation assay was performed. BMMs, which degrade matrix more efficiently than M−/−.WT cells (data not shown), were plated on Cy3-labeled gelatin in the presence or absence of GS-1101 for 24 h before fixation, F-actin staining and immunofluorescent microscopy. Inhibition of p110δ produced a significant reduction in both the number of macrophages that were actively degrading Cy3-labeled gelatin (Fig. 4B) and the area of digestion (Fig 4C). Importantly, inhibition of p110δ for 24 h did not affect cell proliferation, as the mean number of cells per field was equivalent in the DMSO- and GS-1101-treated samples (data not shown). Taken together, the results indicate a profound reduction in total matrix area degraded in the presence of GS-1101. Representative images for BMMs treated with either DMSO or GS-1101 illustrate the profound reduction in extracellular matrix digestion upon inhibition of p110δ (Fig. 4D). Thus PI3K signaling regulates the infiltrative and degradative capacity of macrophages, largely through the activity of p110δ.

image

Figure 4. GS-1101 inhibits macrophage invasion and matrix degradation. (A) CSF-1-starved M−/−.WT cells pre-treated with GS-1101 (5 μm), IC488743 (5 μm) or DMSO (0.1%) were added to Matrigel-coated inserts and their capacity to migrate towards CSF-1 in the bottom chamber over 16 h was measured (= 4). Asterisks indicate statistically significant differences compared with DMSO (***< 0.001). (B, C) For the matrix degradation assay, BMMs were seeded onto Cy3-labeled gelatin with GS-1101 (2 μm) or DMSO (0.04%) for 24 h prior to fixation and staining for F-actin. Five fields were captured per sample at 20× magnification, and areas of degraded matrix were matched to overlying F-actin-stained cells and traced in imagej to determine the percentage of BMMs actively degrading matrix (B) and the percentage area of matrix degraded per field (C). (D) Representative images of BMMs treated with DMSO or GS-1101 showing F-actin-stained BMMs (green) and Cy3-labeled gelatin (red). Scale bar = 20 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Most epithelially derived tumors contain large numbers of TAMs, which are the dominant innate immune cell in mammary cancers of humans and mice [3]. There is a strong correlation between poor prognosis and TAM numbers in many solid tumors, and high circulating levels of CSF-1 are found in advanced breast, endometrial and ovarian cancers [8-10, 27]. CSF-1 stimulates TAMs to secrete a variety of factors to promote tumor growth and progression via several mechanisms, including stimulation of angiogenesis and lymphangiogenesis [28-30] and suppression of the immune response [31]. TAMs also promote tumor invasion and metastasis by secreting EGF to establish an EGF/CSF-1 paracrine dialogue with CSF-1-secreting carcinoma cells [13, 14]. CSF-1 and EGF are both potent chemokines for their respective target cells [5, 32], and alternating tumor cells and TAMs may be seen migrating out of primary tumors along collagen fibers [15].

If the CSF-1/EGF chemotactic circuit between TAMs and tumor cells is an underlying driver of invasion and metastasis, inhibition of macrophage migration should break the circuit and prevent egress of carcinoma cells, thereby inhibiting tumor invasion and metastasis. To investigate this possibility, we initially used our novel cell line system [16] to identify the CSF-1R tyrosine residue(s) responsible for transducing the CSF-1 signal to motility in macrophages. Phosphorylation of a single receptor tyrosine, Y721, mediates association of PI3K with the receptor to trigger both macrophage migration and enhancement of tumor cell invasion in vitro [17]. Here, we have extended these findings to confirm that CSF-1-stimulated PIP3 production requires CSF-1R Y721, and show that the predominant PI3K isoform activated by association with the receptor is p110δ. To determine whether specific inhibition of this hematopoietically enriched PI3K isoform is sufficient to prevent macrophage migration, we examined the effects of GS-1101, a potent and highly selective p110δ inhibitor, on macrophage motility signaling. Confirming our initial findings, p110δ inhibition not only completely blocks CSF-1-induced PIP3 production, but GS-1101 also prevents macrophage spreading, invasive capacity and degradation of the extracellular matrix.

Thus, we have identified p110δ as a primary drug target to inhibit macrophage infiltration into disease sites, including tumors, and next we aim to examine whether GS-1101 prevents macrophage infiltration of mammary spheroids and stimulation of spheroid invasion into the surrounding matrix. GS-1101 is currently in phase III clinical trials for hematopoietic malignancies. GS-1101 has been shown to promote apoptosis in B-cell lines and primary cells from patients with various B-cell malignancies, including chronic lymphocytic leukemia, mantle cell lymphoma, multiple myeloma, and Hodgkin lymphoma [21, 22, 26, 33]. Several lines of evidence demonstrate that GS-1101 interferes with the cross-talk between malignant B cells and their microenvironment, suggesting that disruption of intrinsic and extrinsic survival signals may be a critical mechanism for the clinical activity of GS-1101 [22, 34]. We believe that the clinical usefulness of this drug may extend to the treatment of invasive solid tumors.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Cell culture

M−/−.WT and M−/−.Y721F macrophages were cultured in supplemented alpha modified Minimal Essential Medium (α + MEM) containing 10% newborn calf serum (Life Technologies, Grand Island, NY, USA), as described previously [16]. Primary BMMs were extracted from C57BL/6 mouse femurs and tibias, differentiated and maintained in α + MEM containing 10% fetal bovine serum as described previously [35]. All macrophages were grown in 120 ng·mL−1 human recombinant CSF-1 (a gift from Chiron Corporation, Emeryville, CA, USA) for at least 1 week when thawed from GM-CSF-maintained stocks. MCF10A cells (a gift from E. Thomas, School of Surgery, University of Western Australia) were maintained in HuMEC basal serum-free medium supplemented with the HuMEC supplement kit (Life Technologies).

Antibodies and other reagents

GS-1101 and IC488743 were supplied by Gilead Sciences. Wortmannin (W1628) was purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies used included anti-phosphotyrosine (4G10; Millipore, Billerica, MA, USA), anti-GAPDH (Abcam, Cambridge, MA, USA), anti-Akt (BD Transduction Laboratories, San Jose, CA, USA) and anti-PI3K p110δ (Y387) (GeneTex, Irvine, CA, USA). Cell Signaling Technology (Danvers, MA, USA) supplied anti-PI3K p110α (polyclonal), anti-PI3K p110β (C33D4), anti-PI3K p110γ (D55D5), anti-phospho-Akt Ser473 (193H12) and anti-phospho-Akt Thr308 (polyclonal). Horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling Technology, and Alexa Fluor 488-conjugated phalloidin and Prolong Gold anti-fade reagent with 4,6-diamidino-2-phenylindole were obtained from Molecular Probes/Life Technologies.

PIP3 ELISA

PIP3 production was detected using a PI3-Kinase Activity ELISA: Pico kit (Echelon Biosciences, Logan, UT, USA). Briefly, cells were starved of CSF-1 for 16 h, treated with 5 μm GS-1101, IC488743 or DMSO for 1 h, and stimulated with CSF-1 for 0 or 30 s. PIP3 was extracted by sequential centrifugation in methanol/chloroform/HCl buffer as per manufacturer's instructions, and measured using a PIP3 Mass ELISA kit (Echelon Biosciences) according to manufacturer's instructions.

Immunoblotting

Sub-confluent (approximately 70–80%) 100 mm dish cultures of cells were starved of CSF-1 for 16 h to up-regulate CSF-1R expression, and then incubated with 240 ng·mL−1 CSF-1 at 37 °C for the indicated times. Following incubation, cells were rinsed in ice-cold NaCl/Pi, scraped into 200 μL of lysis buffer (1% Nonidet P-40, 10 mm Tris/HCl, 50 mm NaCl, 30 mm Na4P2O7, 50 mm NaF, 500 μm Na3VO4, 5 μm ZnCl2, 1 mm benzamidine, 10 μg·mL−1 leupeptin and 10 μg·mL−1 aprotinin, pH 7.2) at 4 °C, vortexed and centrifuged at 13 000 g for 30 min. Protein (35–50 μg) was loaded for SDS/PAGE. SDS/PAGE and western blots were performed using a Criterion Bis-Tris XT gel electrophoresis system (Bio-Rad, Hercules, CA, USA). Blotted membranes were incubated with horseradish peroxidase substrate (Millipore), and the chemiluminescent signal was detected using an ImageQuant™LAS 4000 biomolecular imager (GE Healthcare, Piscataway, NJ, USA).

F-actin staining and footprint area quantification

Cells were seeded onto fibronectin-coated cover slips (BD Biosciences, San Jose, CA, USA) in six-well tissue culture dishes, and grown for a minimum of 2 days. When 60–70% confluent, cells were starved of CSF-1 for 16 h. Up-regulated cells were treated with either DMSO or GS-1101 (2 μm) for 1 h, stimulated with 120 ng·mL−1 CSF-1 for the indicated times, then fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100 and quenched with 0.1% glycine in Fix buffer as previously described [35]. Cover slips were blocked using 10% goat serum in 1% BSA/TBS with Alexa Fluor 488-conjugated phalloidin for F-actin staining. Cover slips were mounted on slides using Prolong Gold anti-fade reagent with 4,6-diamidino-2-phenylindole. Samples were imaged using an Olympus IX-81 inverted fluorescence microscope (Olympus, Tokyo, Japan), and images were analyzed by imagej (http://rsbweb.nih.gov/ij/) to calculate the footprint area from cell outline tracing [35].

Matrigel invasion assay

Cells were seeded at 1 × 105 cells/insert on Matrigel™ invasion chambers (BD Biosciences) in 24-well tissue culture dishes in the presence of 5 μm GS-1101, 5 μm IC488743 or 0.1% DMSO for 16 h in a CSF-1 gradient with no CSF-1 in the chamber insert and 120 ng·mL−1 CSF-1 below. Inserts were fixed in 4% paraformaldehyde, and cells remaining on the chamber were removed. The membranes were cut and mounted, then examined by phase contrast using an Olympus IX-81 microscope. Ten representative fields at 20× magnification were captured per sample to calculate the mean number of cells that had traversed through the Matrigel.

Matrix degradation assay

Glass-bottomed 35 mm dishes (MatTek, Ashland, MA, USA) were coated with a thin layer of Cy3-labeled gelatin using a QCM™ Gelatin Invadopodia Assay (Red) kit (Millipore) according to the manufacturer's instructions. BMMs were seeded at a density of 2 × 105 cells per dish in medium containing 10% fetal bovine serum, 120 ng·mL−1 CSF-1, 20 ng·mL−1 recombinant murine interleukin-4 (PeproTech, Rocky Hill, NJ, USA) and either 2 μm GS-1101 or DMSO for 24 h. Dishes were fixed using 4% paraformaldehyde, permeabilized using 0.25% Triton X-100, quenched with 0.1% glycine, blocked with 10% goat serum in 1% BSA/TBS with Alexa Fluor 488-conjugated phalloidin for F-actin staining, and mounted in 1% N-propyl gallate (Sigma-Aldrich, St Louis, MO, USA) in 1× NaCl/Pi. Samples were imaged using an Olympus IX-81 inverted fluorescence microscope, and representative images were analyzed by imagej for cell number, and the number and area of matrix degraded regions normalized to cell number for each field.

Significance testing

Student's t test was used for all comparisons.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This work was supported by the Australian National Health and Medical Research Foundation (F.J.P., 513817) and the Cancer Council of Western Australia (F.J.P., APP102957) and US National Institutes of Health grants CA26504 and PO1 CA100324 to E.R.S. The authors acknowledge the facilities, and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, a facility that is funded by the university and the state and commonwealth governments.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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