Fig 1.
4T1- and LLC-conditioned macrophages exhibit increased pro-inflammatory cytokine production in response to LPS.
(A) C57BL/6 or (B) BALB/c BMDMs were cultured in DMEM alone or in the presence of culture medium conditioned by 4T1 mammary carcinoma, E0771 mammary carcinoma, ID8 ovarian carcinoma, or LLC cells for 24 h. Medium conditioned by HC11 normal mouse epithelial cells was used as a negative control. BMDMs were then washed with PBS and stimulated with 100 ng/ml LPS for 4 h, then culture supernatants were collected and protein levels of cytokines TNFα, IL-6, CCL2, and IL-10 were determined by ELISA. Results are shown as the mean (± SEM) of at least 5 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 2.
Macrophages exposed to 4T1-conditioned culture medium demonstrate increased CCL2 production and promote macrophage recruitment.
(A) Gene expression of monocyte/macrophage chemokines by BMDMs treated for 24 h with 4T1- or HC11-conditioned medium (CM). Expression levels are displayed as the fold change relative to gene expression of BMDMs treated with DMEM alone. (B) CCL2 protein production by BMDMs (Mϕ) treated for 24h with 4T1- or HC11-conditioned medium as measured by ELISA. (C) Macrophage chemotaxis towards the culture supernatants of 4T1- or HC11-conditioned BMDMs (Mϕ). (D) Macrophage chemotaxis towards macrophage culture supernatants pre-treated with 10 μg/ml of a CCL2-neutralizing or an isotype Ab for 1h. Results are shown as the mean (± SEM) of at least 3 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; * p < 0.05.
Fig 3.
Increased sensitivity of 4T1-conditioned macrophages to LPS is transient.
BMDMs were treated with 4T1- or HC11-conditioned medium (CM) for the indicated times. Following treatment, BMDMs were then washed with PBS and stimulated with 100 ng/ml LPS for 4 h. Culture supernatants were collected and protein levels of cytokines CCL2, TNFα, and IL-6 were determined by ELISA. Results are the mean (± SEM) of 3 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; * p < 0.05.
Fig 4.
4T1-conditioned macrophages show increased inflammatory cytokine production in response to bacterial agonists.
BMDMs were treated with 4T1- or HC11-conditioned medium (CM) for 24 h. BMDMs were then washed with PBS and stimulated for 24 h with CpG ODN (5 μg/ml), flagellin (5 μg/ml), or peptidoglycan (10 μg/ml). Culture supernatants were collected and protein levels of cytokines TNFα, IL-6, and CCL2 were determined by ELISA. Results are shown as the mean (± SEM) of at least 5 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 5.
4T1-conditioned macrophages demonstrate increased nitrite production in response to LPS.
BMDMs were treated with 4T1- or HC11-conditioned medium for 24 h. (A) To assess nitrite production, BMDMs were then washed with PBS followed by stimulation with LPS (100 ng/ml), flagellin (5 μg/ml), peptidoglycan (10 μg/ml), or CpG ODN (5 μg/ml) for another 24 h. Culture supernatants were collected and the nitrite production was assessed by the addition of Griess reagent for 15 min. Absorbance (570 nm) of the culture supernatants was then measured by a spectrophotometer. (B) To measure the production of reactive oxygen species, tumor-conditioned BMDMs were stimulated with bacterial TLR agonists for 24 h in the presence of Amplex Red reagent and horseradish peroxidase. Absorbance (450 nm) of the cultures was then measured by a spectrophotometer. Results are shown as the mean (± SEM) of at least 4 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; ** p < 0.01, *** p < 0.001.
Fig 6.
4T1-conditioned macrophages exhibit increased phagocytosis of E. coli bioparticles.
BMDMs were treated for 24 h with 4T1- or HC11-conditioned medium (CM). BMDMs were then washed with PBS and incubated for 2 h with E. coli bioparticles, which fluoresce only under acidic conditions present in endosomes. E. coli phagocytosis was determined by measuring BMDM fluorescence by flow cytometry. As a negative control, BMDMs were pre-treated for 1 h with actin polymerization inhibitor cytochalasin D (10 μM) prior to the addition of E. coli bioparticles. Phagocytic activity was normalized to the uptake of E. coli bioparticles by DMDMs treated with DMEM alone. Results are shown as the mean (± SEM) of at least 4 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; * p < 0.05.
Fig 7.
Increased F4/80 expression by 4T1-conditioned macrophages.
BMDMs were treated for 24 h with 4T1- or HC11-conditioned medium (CM). BMDMs were stained with fluorochrome-labeled antibodies against macrophage markers F4/80, CD11b, CD80, and MHC II, as well as receptors CD40 and CD206. Cell fluorescence was measured via flow cytometry. Protein surface expression of tumor-conditioned BMDMs is expressed as the geometric mean fluorescence intensity. Results are the mean (± SEM) of at least 4 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; ** p < 0.01.
Fig 8.
The increased sensitivity of 4T1-conditioned macrophages to LPS is dependent on actin polymerization.
(A) BMDMs were pre-treated for 1 h with the indicated concentrations of AG490, BAY 11–7082, cytochalasin D, GW2580, JNK inhibitor II, nocodazole, PD184352, SB203580, wortmannin, or the DMSO vehicle prior to a 24 h treatment with 4T1- or HC11-conditioned medium (CM). (B) BMDMs were transfected with IRF-1 or control siRNA for 24 h prior to a 24 h treatment with 4T1- or HC11-CM. BMDMs were then washed with PBS and stimulated with 100 ng/ml LPS for 4 h. Culture supernatants were collected and assessed for the levels of IL-6 by ELISA. Results are shown as the mean (± SEM) IL-6 levels of at least 4 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; * p < 0.05.
Fig 9.
4T1- and HC11-conditioned media contain extracellular vesicles.
Conditioned culture media were absorbed onto carbon-coated grids, negatively stained with 1% uranyl acetate, and visualized by electron microscopy. The displayed images are representative images of (A) fresh medium, (B) 4T1-conditioned medium, (C) HC11-conditioned medium, (D) sonicated 4T1-conditioned medium, and (E) sonicated HC11-conditioned medium.
Fig 10.
Disruption of extracellular vesicle integrity diminishes 4T1-mediated priming of macrophage inflammatory responses.
BMDMs were treated for 24 h with sonicated 4T1- or HC11-conditioned medium (CM). BMDMs were then washed with PBS and stimulated with 100 ng/ml LPS for 4 h. Culture supernatants were collected and assessed for the levels of cytokines by ELISA. Results are shown as the mean (± SEM) cytokine levels of at least 3 independent experiments. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 11.
Peritoneal macrophages from 4T1 tumor-bearing mice demonstrate enhanced sensitivity to LPS.
Peritoneal macrophages from control (n = 4) or 4T1-bearing mice (n = 6) were stimulated with 100 ng/ml LPS for 4 h. Culture supernatants were collected and assessed for the levels of cytokines by ELISA. Statistical analyses were done by ANOVA, followed by Tukey’s multiple comparison tests; * p < 0.05.
Table 1.
Primer sequences for real-time PCR.