Chemoattractant Signaling between Tumor Cells and Macrophages Regulates Cancer Cell Migration, Metastasis and Neovascularization

Tumor-associated macrophages are known to influence cancer progression by modulation of immune function, angiogenesis, and cell metastasis, however, little is known about the chemokine signaling networks that regulate this process. Utilizing CT26 colon cancer cells and RAW 264.7 macrophages as a model cellular system, we demonstrate that treatment of CT26 cells with RAW 264.7 conditioned medium induces cell migration, invasion and metastasis. Inflammatory gene microarray analysis indicated CT26-stimulated RAW 264.7 macrophages upregulate SDF-1α and VEGF, and that these cytokines contribute to CT26 migration in vitro. RAW 264.7 macrophages also showed a robust chemotactic response towards CT26-derived chemokines. In particular, microarray analysis and functional testing revealed CSF-1 as the major chemoattractant for RAW 264.7 macrophages. Interestingly, in the chick CAM model of cancer progression, RAW 264.7 macrophages localized specifically to the tumor periphery where they were found to increase CT26 tumor growth, microvascular density, vascular disruption, and lung metastasis, suggesting these cells home to actively invading areas of the tumor, but not the hypoxic core of the tumor mass. In support of these findings, hypoxic conditions down regulated CSF-1 production in several tumor cell lines and decreased RAW 264.7 macrophage migration in vitro. Together our findings suggest a model where normoxic tumor cells release CSF-1 to recruit macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis

Increasing O-GlcNAcylation level on organ culture of soleus modulates the calcium activation parameters of muscle fibers.

O-N-acetylglucosaminylation is a reversible post-translational modification which presents a dynamic and highly regulated interplay with phosphorylation. New insights suggest that O-GlcNAcylation might be involved in striated muscle physiology, in particular in contractile properties such as the calcium activation parameters. By the inhibition of O-GlcNAcase, we investigated the effect of the increase of soleus O-GlcNAcylation level on the contractile properties by establishing T/pCa relationships. We increased the O-GlcNAcylation level on soleus biopsies performing an organ culture of soleus treated or not with PUGNAc or Thiamet-G, two O-GlcNAcase inhibitors. The enhancement of O-GlcNAcylation pattern was associated with an increase of calcium affinity on slow soleus skinned fibers. Analysis of the glycoproteins pattern showed that this effect is solely due to O-GlcNAcylation of proteins extracted from skinned biopsies. We also characterized the O-GlcNAcylated contractile proteins using a proteomic approach, and identified among others troponin T and I as being O-GlcNAc modified. We quantified the variation of O-GlcNAc level on all these identified proteins, and showed that several regulatory contractile proteins, predominantly fast isoforms, presented a drastic increase in their O-GlcNAc level. Since the only slow isoform of contractile protein presenting an increase of O-GlcNAc level was MLC2, the effect of enhanced O-GlcNAcylation pattern on calcium activation parameters could involve the O-GlcNAcylation of sMLC2, without excluding that an unidentified O-GlcNAc proteins, such as TnC, could be potentially involved in this mechanism. All these data strongly linked O-GlcNAcylation to the modulation of contractile activity of skeletal muscle

Activation threshold, pCa₅₀ and Hill parameter values of skinned fibers isolated from untreated and Thiamet-G-treated soleus.

*<p>p<0.05 untreated <i>versus</i> Thiamet-G-treated fibers.</p

Effect of the increase of O-GlcNAc level on calcium activation parameters of skinned fibers isolated from soleus.

<p>(A) T/pCa curves were representative of 13 fibers from untreated skinned biopsies (•) and 26 fibers from PUGNAc-treated skinned biopsies (○). (B) T/pCa curves were representative of 7 fibers from untreated skinned biopsies (•) and 7 fibers from Thiamet G-treated skinned biopsies (○). Data were presented as mean ± SEM. Curves were fitted with the Hill parameters. (C) Proteins from untreated (-) or PUGNAc-treated (+) skinned fibers included in T/pCa relationship analysis in A were separated on 10–20% linear gradient gel electrophoresis and analysed on CTD110.6 western blot (below) or actin (above). Three fibers were pooled per lane. (D) Proteins from untreated (-) or Thiamet G-treated (+) skinned fibers included in T/pCa relationship analysis in B were analysed by western blot using the CTD110.6 antibody (below) or actin (above). Total densitometry analysis of the western blots was performed and normalized to the control signal for PUGNAC- and Thiamet-G-treated fibers respectively; resulting histograms were presented next to the representative blot image. * indicates significant differences between untreated and treated biopsies (p<0.05).</p

Identification of O-GlcNAc bearing proteins purified from soleus skinned biopsies.

<p>(A) corresponds to a silver stained SDS-PAGE of O-GlcNAc proteins purified by RL-2 immunoprecipitation before (a) or after (b) an hexosaminidase treatment; (c) corresponds to immunoprecipitation protocol realized without proteins sample, and (d) to 25 µg of contractile proteins extract. O-GlcNAc proteins were purified by RL-2 immuno-precipitation followed by an electrophoretic separation on 7.5% (B) or 10–20% (C) SDS-PAGE. Gels were colloïdal blue stained. Each well-resolved bands were submitted to a mass spectrometry-based identification. Numbers on the two panels indicates the different identified proteins; note that the corresponding identifications are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048218#pone-0048218-t003" target="_blank">table 3</a>.</p

Glycopattern analysis of proteins extracted from skinned biopsies comparing with contractile proteins and whole proteins extracts.

<p>Soleus contractile proteins (lanes 1 and 2), proteins extracted from skinned biopsies (lanes 3 and 4) and whole proteins extracted from soleus (lanes 5 and 6) were separated on 12.5% SDS-PAGE and transferred onto nitrocellulose sheet for western blot analysis. Lanes 2, 4 and 6 corresponds to PNGaseF-treated proteins. (A) corresponds to Ponceau staining, (B) to AP-revelation for MAA binding on glycoproteins, (C) to AP-revelation of SNA binding on glycoproteins. Fet (fetuin) and Tf (transferin) correspond to positive glycoprotein markers.</p

Characterization of O-GlcNAcylation on regulatory proteins of thin filament.

<p>O-GlcNAc contractile proteins extracted from soleus were enriched using RL-2 immunoprecipitation protocol. The separation of immunoprecipitated proteins on 10–20% SDS-PAGE was followed by a western blot analysis using antibodies directed against troponin I (A), and slow and fast troponin T (respectively B and C); each isoform was indicated close to the blot. Lane 1 corresponds to whole contractile protein extract from soleus muscle (25 µg of contractile proteins); lane 2 to RL-2 immunoprecipitation protocol; lane 3 to immunoprecipitation experiment performed on hexosaminidase-treated proteins, <i>i.e</i>. on deglycosylated contractile proteins. Only the region of the protein of interest is shown.</p