Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 1 CXCL17 is upregulated in lung metastatic breast cancer cells. a Scheme of the animal model. 4T1 cells (500,000 cells per fat pad) were implanted into the mammary fat pads and allowed to spontaneously metastasize to the lung for 24 days. Whole primary tumor in mammary or tumor nodules in the lungs (n = 3), livers (n = 4), and intestine (n = 4) and lymph nodes (tumor nodules = 7) were subjected to gene profiling. b Specific gene profile of breast cancer metastasized to lung. c The upregulation of CXCL17 protein in lung metastatic 4T1 cells. 4T1 cells were implanted into mice in an orthotic model. The expression of CXCL17 protein of 4T1 cells isolated from primary sites (mammary) or lung tissue (six pairs of 4T1 cell isolated from in mammary and lung) was assessed by ELISA after 48-h incubation. d In the left panel, the box plot generated from Kao Huang Breast GSE20685 dataset of SurvExpress showed the amount of CXCL17 expression in each group. The right panel showed the Kaplan-Meier time to metastasis curve. e The results were from Van De Vijver Nature 2002 dataset of SurvExpress. The left and right panels showed CXCL17 expression and the Kaplan-Meier time to metastasis curve respectively. The group was divided according to the “Maximize Risk Groups” in SurvExpress website. f Kaplan-Meier distant metastasis-free survival via KM plotter database. High and low CXCL17 expression groups were divided according to “Auto select best cutoff” in the KM plotter website. Each value is the mean ± SD of three determinations; *p < 0.05

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Animal Model, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Incubation, Generated

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 2 CXCL17 increased lung metastasis in vivo. a CXCL17 increased the lung metastasis of 4T1 cells in an orthotropic model. The total number of tumor nodule per whole lung lobes was counted and averaged among the animals of each group. b The H&E staining of tumor sections. BALB/c mice were treated with PBS or recombinant mouse CXCL17 protein by intra-tracheal administration for 14 days (1 μg/mouse, 2 times/week, n = 6 per group). 4T1 were implanted into the fat pads of mice. Tumor nodules of 4T1 in the primary site and lungs were collected after 24 days of injections. c CXCL17 increased lung metastasis in human breast MDA-MB-231. Nude mice were treated with PBS or recombinant CXCL17 protein by intra-tracheal administration for 14 days (1 μg/mouse, 2 times/week, n = 6 per group). MDA-MB-231 cells were implanted into mice by tail vein injection. The MDA- MB-231 tumor nodules in the lungs of mice were collected after 90 days of injections. d The H&E staining of tumor sections. e Knockdown of CXCL17 decreased the spontaneous metastatic ability of L4T1 cells. f The H&E staining of tumor sections. CXCL17-knockdown-L4T1 were implanted into the fat pads of mice (n = 6 per group). Tumor nodules in the lungs were collected after 24 days of injections. Representative lung tissue sections were stained with H&E and photographed at × 100 magnification. Each value is the mean ± SEM; Mann-Whitney U test was performed, *p < 0.05. T tumor

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: In Vivo, Staining, Recombinant, Injection, Knockdown, MANN-WHITNEY

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 3 CXCL17 increases the recruitment of MDSCs in metastatic lungs of mice. The effect of CXCL17 in the recruitment of CD11b+Gr-1+ MDSCs (a), CD11b+Gr-1−MDSCs (b), and CD11b+F4/80+ macrophages (c) in the lungs of mice. BALB/c mice were treated with PBS or recombinant mouse CXCL17 protein by intra-tracheal administration for 14 days (1 μg/mouse, 2 times/week, n = 6 per group). Various immune cells were isolated from the lungs of mice by antibody conjugated magnetic beads. Each value is the mean ± SEM; *p < 0.05. CXCL17 increased the migration (d) and transendothelial migration (e) of CD11b+Gr-1+ MDSCs in vitro. GPR35 inhibitor decreased the migration (f) and transendothelial migration (g) of CD11b+Gr-1+ MDSCs induced by CXCL17. CD11b+Gr-1+ MDSCs were isolated from the lungs of normal mice (n = 3). PKH26-labeled CD11b+Gr-1+ MDSCs cells were seeded onto inserts (1 × 105 cells in 3-μm pore insert for migration analysis). For transendothelial migration analysis, C166 cells were seeded in 3-μm pore collagen-coated inserts for confluent monolayer, and PKH26-labeled CD11b+Gr-1+ MDSCs cells (1 × 105/insert) were seeded onto C166 confluent monolayer inserts, and the migration of cancer cells was assessed by fluorescence microscope. CXCL17 (1 ng/ml) were added in bottom well as chemoattractant. For blocking experiment, GPR35 inhibitor (CID2745687, 2 μM) was added in the inserts. Results are representative of at least three independent experiments, and each value is the mean ± SD of three determinations. *Significant difference between the two test groups (p < 0.05)

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Recombinant, Isolation, Magnetic Beads, Migration, In Vitro, Labeling, Fluorescence, Microscopy, Blocking Assay

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 4 CXCL17 increases angiogenesis in lung metastatic niche by recruiting CD11b+Gr-1+ MDSCs. a CXCL17 increased CD31+ cells in the lungs of mice. Digital images of tissues were captured and analyzed with ImageJ software to calculate the percentage of positive cells (high positive + positive + low positive cells). b Conditioned medium (CM) (50%) of CD11b+Gr-1+ MDSCs isolated from the lungs of CXCL17-treated mice (n = 5) increased tube formation of C166 cells. The effect of CXCL17 in the PDGF-AA (c), PDGF-BB (d), VEGF-A (e), and EGF basic (f) secretions of CD11b+Gr-1+ MDSCs in vivo. BALB/c mice were treated with PBS or recombinant mouse CXCL17 protein by intra-tracheal administration for 14 days (1 μg/mouse, 2 times/ week, n = 6 per group). The lungs of these mice were harvested and constrained by CD31 antibody and H&E. Alternatively, CD11b+Gr-1+ MDSCs were isolated from lungs of PBS or CXCL17 treated mice (n = 6 per group) by antibody conjugated magnetic beads, and CM was collected after culturing for 24 h. The expression of various angiogenic factors was assessed by Luminex Assays. g CXCL17 increased the expression of PDGF-BB in CD11b+Gr- 1+ MDSCs isolated from the lungs of normal mice. CD11b+Gr-1+ MDSCs were isolated from the lungs of normal mice (n = 5) by antibody conjugated magnetic beads and treated with rmCXCL17 (10 ng/ml) for 24 h. The expression of PDGF-BB was assessed by Luminex Assays. h Inhibition of PDGFR-β by specific inhibitor (DMPQ-2HCl, 5 μM) prevents CD11b+Gr-1+ MDSC-mediated C166 tube formation. Results are representative of at least three independent experiments in vitro studies, and each value is the mean ± SD of three determinations. *Significant difference between the two test groups (p < 0.05)

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Software, Isolation, In Vivo, Recombinant, Magnetic Beads, Expressing, Luminex, Inhibition, In Vitro

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 5 CD11b+Gr-1+ myeloid cells in lungs metastatic niche promote cancer cell colonization. a CXCL17 increased cancer cell extravasation into the lungs of mice. Athymic nude mice were treated with recombinant CXCL17 protein (1 μg/mouse, 2 times/week, n = 6 per group) for 14 days; MDA-MB-231-RFP-Luc cells were injected into mice by tail vein. The lungs of these mice were harvested after 48-h injection, then examined by a confocal microscope. b Scheme of CD11b+Gr-1+ depletion in the animal model. Depletion of CD11b+Gr-1+ cells decreased CXCL17-mediated cancer extravasation (c) and tumor nodules formation (d) in the lungs of mice. e The H&E staining of tumor sections of lungs. For MDSC depletion studies, mice (n = 6 per group) were treated with isotype or anti-Gr-1 antibodies (Bio X Cell) at 100 μg/mouse intraperitoneal injections every 4th day, and 4T1 cells were injected via tail vein into the mice (n = 6 per group). The control group received intraperitoneal injection of purified rat immunoglobulins. PKH26-labeled 4T1 cells were injected into mice by tail vein for indicated times (24 days for lung metastasis and 48 h for extravasation). The lungs of these mice were harvested and examined by a confocal microscope. Each value is the mean ± SEM; *p < 0.05. White arrows indicate cancer cells

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Recombinant, Injection, Microscopy, Animal Model, Staining, Control, Purification, Labeling

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 6 CD11b+Gr-1+ MDSCs increased breast cancer extravasation and survival via PDGF-BB. CM of CD11b+Gr-1+ MDSC isolated from the lungs of CXCL17-treated or 4T1-bearing mice (n = 5) increased transendothelial migration (a) and colony formation (b) of 4T1 cells. rmPDGF enhanced 4T1 cell transendothelial migration (c) and colony formation (d). Blockade of PDGFR-β prevented transendothelial migration (e) and colony formation (f) of 4T1 cells induced by CM of CD11b+Gr-1+ MDSCs. CD11b+Gr-1+ MDSC isolation and the collection of their CMs have been described in the legend of Fig. 2. C166 cells were seeded 8-μm pore collagen-coated inserts for confluent monolayer, and PKH26-labeled 4T1 cells (1 × 105/insert) were seeded onto C166 confluent monolayer inserts, and the CM of CD11b+Gr-1+ MDSCs (50%) or rmPDGF-BB protein (20 ng/ml) were placed in the bottom well as chemoattractant. The migration of cancer cells was assessed by a fluorescence microscope. For colony formation analysis, 4T1 cells were treated with different CMs (50%) or PDGF-BB (20 ng/ml), and the colonies counted after staining. g PDGFR inhibitor imatinib decreased lung metastasis in mice (n = 6 per group). h H&E staining of lung sections. Representative lung tissue sections were stained with H&E and photographed at × 100 magnification. Results are representative of at least three independent experiments and each value is the mean ± SD of three determinations. *Significant difference between the two test groups (p < 0.05)

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Isolation, Migration, Labeling, Fluorescence, Microscopy, Staining

Journal: Breast cancer research : BCR

Article Title: CXCL17-derived CD11b + Gr-1 + myeloid-derived suppressor cells contribute to lung metastasis of breast cancer through platelet-derived growth factor-BB.

doi: 10.1186/s13058-019-1114-3

Figure Lengend Snippet: Fig. 7 A novel mechanism underlying the contribution of primary cancer to lung metastatic niche formation in breast cancer. Primary cancer- secreted CXCL17 increases the accumulation of CD11b+Gr-1+ MDSCs in the lungs, which produce PDGF-BB, resulting in enhanced angiogenesis in the lung tissue before cancer cells’ arrival. In addition, CXCL17 also drives CD11b+Gr-1+ MDSCs to exhibit supportive activity for cancer extravasation and survival by PDGF-BB production under cancer cell arrival

Article Snippet: CXCL17 levels were assessed by human or mouse CXCL17 ELISA kits (Cusabio Biotech, Wuhan, China).

Techniques: Activity Assay