Data_Sheet_1_Breast Cancer-Derived Exosomes Alter Macrophage Polarization via gp130/STAT3 Signaling.doc

<p>Tumor-derived exosomes are being recognized as essential mediators of intercellular communication between cancer and immune cells. It is well established that bone marrow-derived macrophages (BMDMs) take up tumor-derived exosomes. However, the functional impact of these exosomes on macrophage phenotypes is controversial and not well studied. Here, we show that breast cancer-derived exosomes alter the phenotype of macrophages through the interleukin-6 (IL-6) receptor beta (glycoprotein 130, gp130)-STAT3 signaling pathway. Addition of breast cancer-derived exosomes to macrophages results in the activation of the IL-6 response pathway, including phosphorylation of the key downstream transcription factor STAT3. Exosomal gp130, which is highly enriched in cancer exosomes, triggers the secretion of IL-6 from BMDMs. Moreover, the exposure of BMDMs to cancer-derived exosomes triggers changes from a conventional toward a polarized phenotype often observed in tumor-associated macrophages. All of these effects can be inhibited through the addition of a gp130 inhibitor to cancer-derived exosomes or by blocking BMDMs exosome uptake. Collectively, this work demonstrates that breast cancer-derived exosomes are capable of inducing IL-6 secretion and a pro-survival phenotype in macrophages, partially via gp130/STAT3 signaling.</p

Glycolipids from <i>T. rangeli</i> and <i>T. cruzi</i> suppress NOS expression.

<p><i>Rhodnius</i> were injected with either Tr GIPL or Tc GIPL and three days later salivary glands were dissected, homogenized and NOS expression evaluated by Western blotting. A. Western blotting against NOS in salivary glands obtained from control and insects injected with Tr GIPLs. B. Western blotting against NOS in salivary glands obtained from control and insects injected with Tc GIPLs.</p

Glycolipid-mediated suppression of NO synthesis occurs through the manipulation of intracellular phosphorylation-dephosphorylation circuits.

<p>Intracellular circuits of protein-phosphorylation and dephosphorylation were evaluated through different assays. A. Salivary glands obtained from either control or <i>T. rangeli</i>-infected insects were dissected three days after the injection, homogeneized and phosphorylated in the presence of ³²P-ATP followed by SDS-gel electrophoresis and autoradiograph. B. A similar experiment was conducted with salivary glands isolated from insects injected with Tr GIPL or Tc GIPL. C. Following a blood meal on rabbit ear salivary glands were dissected at different points in time. Total protein phosphatase activity was followed during the refilling cycle of salivary glands using pNPP as substrate. Data is the mean ± S.E. of three different experiments. D. Insects were injected with Tr GIPL and evaluated for protein phosphatase activity in the presence and in the absence of SO. The fraction of enzyme activity inhibited by SO in control and Tr GIPL-injected insects is shown. Data is the mean ± S.E. of three different experiments.</p

<i>T. rangeli</i> infection downregulates NOS synthesis.

<p><i>Rhodnius</i> were infected with <i>T. rangeli</i> and three days later salivary glands were dissected and incubated in the presence of the NO fluorescent probe DAF-FM. A, C are contrast-phase imaging of B and D, respectively. A. Control salivary gland. B. DAF-FM fluorescent image of a control salivary gland shown on A. C. Infected salivary gland. D. DAF-FM fluorescence image of an infected salivary gland shown on C.</p

Chemical composition of GIPL purified from <i>T</i>. <i>rangeli</i> (Tr GIPL).

a<p>Determined by GC as trimethylsilyl derivatives of methylglycosides.</p>b<p>Determined by GC and GC-MS as fatty acid methyl esters (FAMEs).</p>c<p>Determined by GC and GC-MS after N-acetylation and trimethylsilylation.</p

Infection with <i>T. rangeli</i> reduces the NOS activity and the levels of NOS protein in the salivary glands of <i>R. prolixus</i>.

<p>A. <i>Rhodnius</i> were dissected 7 days after control injection of water or <i>T. rangeli</i> and assayed for NADPH-diaphorase activity. Results from three experiments were evaluated statistically using the Student t test (* p<0.05). B. Salivary gland extracts from control or <i>T. rangeli-</i>injected insects were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with primary antibody anti-NOS and then with an anti-rabbit antibody conjugated to alkaline phosphatase. This experiment was performed three times. Tr, <i>Trypanosoma rangeli</i> cells evalutated for NOS blotting. N, salivary glands from non-injected insects. C, control salivary glands from insects injected with water. I, Salivary glands from <i>T. rangeli</i>-injected insects. C. NADPH-diaphorase activity was measured in salivary gland extracts of salivary glands three days after injection with 100 ng of glycolipids from either <i>T. rangeli</i> (Tr GIPL), <i>P. serpens (</i>Ps GIPL<i>)</i> or <i>T. cruzi</i> eGPI-mucin (Tc Mucin). The experiment was performed three times and analyzed by ANOVA (* p<0.05).</p

NADPH-diaphorase activity of NOS in <i>Rhodnius prolixus</i> salivary glands after a blood meal and the expression of NOS.

<p>A. Salivary glands were dissected in different days after blood feeding and evaluated for NOS NAPDH-diaphorase activity. Salivary glands were assayed in 10 mM Tris-HCl pH 8,0, 0,05 M NaCl, 0,1%, Triton X-100, 1 mM CaCl₂, 5 µM FAD, 1 mM NADPH and 0,5 mg/mL MTT. MTT reduction was followed at 540 nm for 30 min at 37°C. Also samples were obtained and NOS content evaluated by Western blotting. Each point is the average and SE of 05 different experiments. B. Immunoblotting using an anti-NOS antibody. Blottings were developed with the use of a secondary antibody conjugated to alkaline phosphatase in the presence of the substrate Western Blue. Molecular mass markers are indicated at the left. C. Upper panel<b>,</b> total RNA from the salivary glands at different days after feeding was isolated and cDNA was synthesized. Samples were then analyzed by semi-quantitative PCR with temperatures of 55, 72 and 94°C for 27 cycles with primers for NOS. Lower panel, analysis of 18 S RNA levels. In this case reaction occurred for 25 cycles. The products of reactions shown on panels C were separated on agarose gel 1.4% stained with ethidium bromide and photographed under ultraviolet light. Molecular mass standards are indicated at the left.</p

Tc GIPL-mediated suppression of NO synthesis is mediated through the inhibition of a protein tyrosine phosphatase.

<p><i>Rhodnius</i> were injected with either water, Tc GIPLs or sodium orthovanadate (SO) and three days later analyzed for NO synthesis by DAF-FM fluorescence. Also samples were collected for the evaluation of NOS mRNA levels by RT-PCR. A. DAF-FM fluorescence image of control insects. B. DAF-FM fluorescence image form salivary glands dissected from GIPL-injected insects. C. DAF-FM fluorescence image from salivary glands dissected from SO-injected insects. D. RT-PCR analysis of NOS mRNA from control, Tc GIPL- and SO-injected insects. Data is the mean ± S.E. of three different experiments. The experiment was performed three times and analyzed by NOVA (p>0.05) which indicated that there is no statistically significant difference among groups.</p

Tr GIPLs suppress NO synthesis.

<p><i>Rhodnius</i> were injected either with 100 ng of Tr GIPL or water and three days later salivary glands were dissected and incubated with DAF-FM. A. Image of control salivary glands. B. DAF-FM fluorescent image of a control salivary gland. C. Image of salivary glands isolated from insects injected with Tr GIPL. D. DAF-FM fluorescence image of salivary glands isolated from insects injected with Tr GIPL.</p

Tc GIPL does not affect regular blood feeding, anti-clotting and apyrase activity.

<p><i>Rhodnius</i> injected or not with Tc GIPLs were evaluated for their ability to feed on blood. Parallel controls in each panel were obtained in insects inject with GIPL solvent. Three days after the injection insects were either allowed to feed on a rabbit ear or their salivary glands were dissected and evaluated for anti-hemostatic activities. A. Weight gain after blood feeding. B. Apyrase activity. C. aPTT activity. Data is the mean ± S.E. of three different experiments.</p