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CXCL10 silencing abrogates aged ZnO NPs–induced accumulation of pro‐metastatic stromal populations. (A) Schematic of coculture model: NIH‐3T3 or Raw264.7 cells with CXCL10 knockdown were stimulated with PBS, fresh or aged NPs (7.5 µg/mL), co‐cultured with 4T1 cells for 72 h. (B) qPCR analysis confirming CXCL10 knockdown in Raw264.7 cells ( n = 3). (C‐E) CXCL10 knockdown in Raw264.7 macrophages attenuates ZnO NP‐enhanced M2 polarization, as shown by reduced Arg1, Mrc1 expression (C) and CD206 + cell frequency (D, E) ( n = 3). (F) qPCR analysis confirming CXCL10 knockdown in NIH‐3T3 cells ( n = 3). (G, H) Flow cytometric analysis (G) and quantification (H) of the percentage of CD140a‐positive cells among CXCL10‐knockdown NIH‐3T3 cells in the co‐culture system ( n = 3). (I) qPCR analysis of CAF‐related genes (FN1, FSP1, α‐SMA, CD140a) in CXCL10‐knockdown NIH‐3T3 cells from the co‐culture model ( n = 3). (J) Schematic illustration of primary MDSCs preparation: primary mouse bone marrow‐derived macrophages were differentiated into MDSCs by supplement of GM‐CSF (40 ng/mL) and IL‐6 (40 ng/mL). MDSCs with CXCL10 knockdown were stimulated with PBS, fresh ZnO NPs, or aged ZnO NPs (7.5 µg/mL), then co‐cultured with 4T1 cells for 72 h. Cells were collected for further experiments. (K) qPCR analysis of relative CXCL10 mRNA expression in MDSCs with CXCL10 knockdown ( n = 3). (L, M) Flow cytometric analysis (L) and quantification (M) of the proportion of granulocytic CD11b + Ly6G + Ly6C low PMN‐MDSCs in the co‐culture system after CXCL10 knockdown ( n = 3). All data were represented as the mean ± SD. P values were calculated by one‐way ANOVA followed by Tukey's test. * p <0.05; ** p <0.01.
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CXCL10 silencing abrogates aged ZnO NPs–induced accumulation of pro‐metastatic stromal populations. (A) Schematic of coculture model: NIH‐3T3 or Raw264.7 cells with CXCL10 knockdown were stimulated with PBS, fresh or aged NPs (7.5 µg/mL), co‐cultured with 4T1 cells for 72 h. (B) qPCR analysis confirming CXCL10 knockdown in Raw264.7 cells ( n = 3). (C‐E) CXCL10 knockdown in Raw264.7 macrophages attenuates ZnO NP‐enhanced M2 polarization, as shown by reduced Arg1, Mrc1 expression (C) and CD206 + cell frequency (D, E) ( n = 3). (F) qPCR analysis confirming CXCL10 knockdown in NIH‐3T3 cells ( n = 3). (G, H) Flow cytometric analysis (G) and quantification (H) of the percentage of CD140a‐positive cells among CXCL10‐knockdown NIH‐3T3 cells in the co‐culture system ( n = 3). (I) qPCR analysis of CAF‐related genes (FN1, FSP1, α‐SMA, CD140a) in CXCL10‐knockdown NIH‐3T3 cells from the co‐culture model ( n = 3). (J) Schematic illustration of primary MDSCs preparation: primary mouse bone marrow‐derived macrophages were differentiated into MDSCs by supplement of GM‐CSF (40 ng/mL) and IL‐6 (40 ng/mL). MDSCs with CXCL10 knockdown were stimulated with PBS, fresh ZnO NPs, or aged ZnO NPs (7.5 µg/mL), then co‐cultured with 4T1 cells for 72 h. Cells were collected for further experiments. (K) qPCR analysis of relative CXCL10 mRNA expression in MDSCs with CXCL10 knockdown ( n = 3). (L, M) Flow cytometric analysis (L) and quantification (M) of the proportion of granulocytic CD11b + Ly6G + Ly6C low PMN‐MDSCs in the co‐culture system after CXCL10 knockdown ( n = 3). All data were represented as the mean ± SD. P values were calculated by one‐way ANOVA followed by Tukey's test. * p <0.05; ** p <0.01.
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CXCL10 silencing abrogates aged ZnO NPs–induced accumulation of pro‐metastatic stromal populations. (A) Schematic of coculture model: NIH‐3T3 or Raw264.7 cells with CXCL10 knockdown were stimulated with PBS, fresh or aged NPs (7.5 µg/mL), co‐cultured with 4T1 cells for 72 h. (B) qPCR analysis confirming CXCL10 knockdown in Raw264.7 cells ( n = 3). (C‐E) CXCL10 knockdown in Raw264.7 macrophages attenuates ZnO NP‐enhanced M2 polarization, as shown by reduced Arg1, Mrc1 expression (C) and CD206 + cell frequency (D, E) ( n = 3). (F) qPCR analysis confirming CXCL10 knockdown in NIH‐3T3 cells ( n = 3). (G, H) Flow cytometric analysis (G) and quantification (H) of the percentage of CD140a‐positive cells among CXCL10‐knockdown NIH‐3T3 cells in the co‐culture system ( n = 3). (I) qPCR analysis of CAF‐related genes (FN1, FSP1, α‐SMA, CD140a) in CXCL10‐knockdown NIH‐3T3 cells from the co‐culture model ( n = 3). (J) Schematic illustration of primary MDSCs preparation: primary mouse bone marrow‐derived macrophages were differentiated into MDSCs by supplement of GM‐CSF (40 ng/mL) and IL‐6 (40 ng/mL). MDSCs with CXCL10 knockdown were stimulated with PBS, fresh ZnO NPs, or aged ZnO NPs (7.5 µg/mL), then co‐cultured with 4T1 cells for 72 h. Cells were collected for further experiments. (K) qPCR analysis of relative CXCL10 mRNA expression in MDSCs with CXCL10 knockdown ( n = 3). (L, M) Flow cytometric analysis (L) and quantification (M) of the proportion of granulocytic CD11b + Ly6G + Ly6C low PMN‐MDSCs in the co‐culture system after CXCL10 knockdown ( n = 3). All data were represented as the mean ± SD. P values were calculated by one‐way ANOVA followed by Tukey's test. * p <0.05; ** p <0.01.
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CXCL10 silencing abrogates aged ZnO NPs–induced accumulation of pro‐metastatic stromal populations. (A) Schematic of coculture model: NIH‐3T3 or Raw264.7 cells with CXCL10 knockdown were stimulated with PBS, fresh or aged NPs (7.5 µg/mL), co‐cultured with 4T1 cells for 72 h. (B) qPCR analysis confirming CXCL10 knockdown in Raw264.7 cells ( n = 3). (C‐E) CXCL10 knockdown in Raw264.7 macrophages attenuates ZnO NP‐enhanced M2 polarization, as shown by reduced Arg1, Mrc1 expression (C) and CD206 + cell frequency (D, E) ( n = 3). (F) qPCR analysis confirming CXCL10 knockdown in NIH‐3T3 cells ( n = 3). (G, H) Flow cytometric analysis (G) and quantification (H) of the percentage of CD140a‐positive cells among CXCL10‐knockdown NIH‐3T3 cells in the co‐culture system ( n = 3). (I) qPCR analysis of CAF‐related genes (FN1, FSP1, α‐SMA, CD140a) in CXCL10‐knockdown NIH‐3T3 cells from the co‐culture model ( n = 3). (J) Schematic illustration of primary MDSCs preparation: primary mouse bone marrow‐derived macrophages were differentiated into MDSCs by supplement of GM‐CSF (40 ng/mL) and IL‐6 (40 ng/mL). MDSCs with CXCL10 knockdown were stimulated with PBS, fresh ZnO NPs, or aged ZnO NPs (7.5 µg/mL), then co‐cultured with 4T1 cells for 72 h. Cells were collected for further experiments. (K) qPCR analysis of relative CXCL10 mRNA expression in MDSCs with CXCL10 knockdown ( n = 3). (L, M) Flow cytometric analysis (L) and quantification (M) of the proportion of granulocytic CD11b + Ly6G + Ly6C low PMN‐MDSCs in the co‐culture system after CXCL10 knockdown ( n = 3). All data were represented as the mean ± SD. P values were calculated by one‐way ANOVA followed by Tukey's test. * p <0.05; ** p <0.01.
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CXCL10 silencing abrogates aged ZnO NPs–induced accumulation of pro‐metastatic stromal populations. (A) Schematic of coculture model: NIH‐3T3 or Raw264.7 cells with CXCL10 knockdown were stimulated with PBS, fresh or aged NPs (7.5 µg/mL), co‐cultured with 4T1 cells for 72 h. (B) qPCR analysis confirming CXCL10 knockdown in Raw264.7 cells ( n = 3). (C‐E) CXCL10 knockdown in Raw264.7 macrophages attenuates ZnO NP‐enhanced M2 polarization, as shown by reduced Arg1, Mrc1 expression (C) and CD206 + cell frequency (D, E) ( n = 3). (F) qPCR analysis confirming CXCL10 knockdown in NIH‐3T3 cells ( n = 3). (G, H) Flow cytometric analysis (G) and quantification (H) of the percentage of CD140a‐positive cells among CXCL10‐knockdown NIH‐3T3 cells in the co‐culture system ( n = 3). (I) qPCR analysis of CAF‐related genes (FN1, FSP1, α‐SMA, CD140a) in CXCL10‐knockdown NIH‐3T3 cells from the co‐culture model ( n = 3). (J) Schematic illustration of primary MDSCs preparation: primary mouse bone marrow‐derived macrophages were differentiated into MDSCs by supplement of GM‐CSF (40 ng/mL) and IL‐6 (40 ng/mL). MDSCs with CXCL10 knockdown were stimulated with PBS, fresh ZnO NPs, or aged ZnO NPs (7.5 µg/mL), then co‐cultured with 4T1 cells for 72 h. Cells were collected for further experiments. (K) qPCR analysis of relative CXCL10 mRNA expression in MDSCs with CXCL10 knockdown ( n = 3). (L, M) Flow cytometric analysis (L) and quantification (M) of the proportion of granulocytic CD11b + Ly6G + Ly6C low PMN‐MDSCs in the co‐culture system after CXCL10 knockdown ( n = 3). All data were represented as the mean ± SD. P values were calculated by one‐way ANOVA followed by Tukey's test. * p <0.05; ** p <0.01.
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CXCL10 silencing abrogates aged ZnO NPs–induced accumulation of pro‐metastatic stromal populations. (A) Schematic of coculture model: NIH‐3T3 or Raw264.7 cells with CXCL10 knockdown were stimulated with PBS, fresh or aged NPs (7.5 µg/mL), co‐cultured with 4T1 cells for 72 h. (B) qPCR analysis confirming CXCL10 knockdown in Raw264.7 cells ( n = 3). (C‐E) CXCL10 knockdown in Raw264.7 macrophages attenuates ZnO NP‐enhanced M2 polarization, as shown by reduced Arg1, Mrc1 expression (C) and CD206 + cell frequency (D, E) ( n = 3). (F) qPCR analysis confirming CXCL10 knockdown in NIH‐3T3 cells ( n = 3). (G, H) Flow cytometric analysis (G) and quantification (H) of the percentage of CD140a‐positive cells among CXCL10‐knockdown NIH‐3T3 cells in the co‐culture system ( n = 3). (I) qPCR analysis of CAF‐related genes (FN1, FSP1, α‐SMA, CD140a) in CXCL10‐knockdown NIH‐3T3 cells from the co‐culture model ( n = 3). (J) Schematic illustration of primary MDSCs preparation: primary mouse bone marrow‐derived macrophages were differentiated into MDSCs by supplement of GM‐CSF (40 ng/mL) and IL‐6 (40 ng/mL). MDSCs with CXCL10 knockdown were stimulated with PBS, fresh ZnO NPs, or aged ZnO NPs (7.5 µg/mL), then co‐cultured with 4T1 cells for 72 h. Cells were collected for further experiments. (K) qPCR analysis of relative CXCL10 mRNA expression in MDSCs with CXCL10 knockdown ( n = 3). (L, M) Flow cytometric analysis (L) and quantification (M) of the proportion of granulocytic CD11b + Ly6G + Ly6C low PMN‐MDSCs in the co‐culture system after CXCL10 knockdown ( n = 3). All data were represented as the mean ± SD. P values were calculated by one‐way ANOVA followed by Tukey's test. * p <0.05; ** p <0.01.
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CXCL10 silencing abrogates aged ZnO NPs–induced accumulation of pro‐metastatic stromal populations. (A) Schematic of coculture model: NIH‐3T3 or Raw264.7 cells with CXCL10 knockdown were stimulated with PBS, fresh or aged NPs (7.5 µg/mL), co‐cultured with 4T1 cells for 72 h. (B) qPCR analysis confirming CXCL10 knockdown in Raw264.7 cells ( n = 3). (C‐E) CXCL10 knockdown in Raw264.7 macrophages attenuates ZnO NP‐enhanced M2 polarization, as shown by reduced Arg1, Mrc1 expression (C) and CD206 + cell frequency (D, E) ( n = 3). (F) qPCR analysis confirming CXCL10 knockdown in NIH‐3T3 cells ( n = 3). (G, H) Flow cytometric analysis (G) and quantification (H) of the percentage of CD140a‐positive cells among CXCL10‐knockdown NIH‐3T3 cells in the co‐culture system ( n = 3). (I) qPCR analysis of CAF‐related genes (FN1, FSP1, α‐SMA, CD140a) in CXCL10‐knockdown NIH‐3T3 cells from the co‐culture model ( n = 3). (J) Schematic illustration of primary MDSCs preparation: primary mouse bone marrow‐derived macrophages were differentiated into MDSCs by supplement of GM‐CSF (40 ng/mL) and IL‐6 (40 ng/mL). MDSCs with CXCL10 knockdown were stimulated with PBS, fresh ZnO NPs, or aged ZnO NPs (7.5 µg/mL), then co‐cultured with 4T1 cells for 72 h. Cells were collected for further experiments. (K) qPCR analysis of relative CXCL10 mRNA expression in MDSCs with CXCL10 knockdown ( n = 3). (L, M) Flow cytometric analysis (L) and quantification (M) of the proportion of granulocytic CD11b + Ly6G + Ly6C low PMN‐MDSCs in the co‐culture system after CXCL10 knockdown ( n = 3). All data were represented as the mean ± SD. P values were calculated by one‐way ANOVA followed by Tukey's test. * p <0.05; ** p <0.01.
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Image Search Results


CXCL10 silencing abrogates aged ZnO NPs–induced accumulation of pro‐metastatic stromal populations. (A) Schematic of coculture model: NIH‐3T3 or Raw264.7 cells with CXCL10 knockdown were stimulated with PBS, fresh or aged NPs (7.5 µg/mL), co‐cultured with 4T1 cells for 72 h. (B) qPCR analysis confirming CXCL10 knockdown in Raw264.7 cells ( n = 3). (C‐E) CXCL10 knockdown in Raw264.7 macrophages attenuates ZnO NP‐enhanced M2 polarization, as shown by reduced Arg1, Mrc1 expression (C) and CD206 + cell frequency (D, E) ( n = 3). (F) qPCR analysis confirming CXCL10 knockdown in NIH‐3T3 cells ( n = 3). (G, H) Flow cytometric analysis (G) and quantification (H) of the percentage of CD140a‐positive cells among CXCL10‐knockdown NIH‐3T3 cells in the co‐culture system ( n = 3). (I) qPCR analysis of CAF‐related genes (FN1, FSP1, α‐SMA, CD140a) in CXCL10‐knockdown NIH‐3T3 cells from the co‐culture model ( n = 3). (J) Schematic illustration of primary MDSCs preparation: primary mouse bone marrow‐derived macrophages were differentiated into MDSCs by supplement of GM‐CSF (40 ng/mL) and IL‐6 (40 ng/mL). MDSCs with CXCL10 knockdown were stimulated with PBS, fresh ZnO NPs, or aged ZnO NPs (7.5 µg/mL), then co‐cultured with 4T1 cells for 72 h. Cells were collected for further experiments. (K) qPCR analysis of relative CXCL10 mRNA expression in MDSCs with CXCL10 knockdown ( n = 3). (L, M) Flow cytometric analysis (L) and quantification (M) of the proportion of granulocytic CD11b + Ly6G + Ly6C low PMN‐MDSCs in the co‐culture system after CXCL10 knockdown ( n = 3). All data were represented as the mean ± SD. P values were calculated by one‐way ANOVA followed by Tukey's test. * p <0.05; ** p <0.01.

Journal: Advanced Science

Article Title: Aged ZnO Nanoparticles Promote Breast Cancer Lung Metastasis by Remodeling the Pulmonary Microenvironment Through the CXCL10/Wnt/β‐Catenin Axis

doi: 10.1002/advs.78249

Figure Lengend Snippet: CXCL10 silencing abrogates aged ZnO NPs–induced accumulation of pro‐metastatic stromal populations. (A) Schematic of coculture model: NIH‐3T3 or Raw264.7 cells with CXCL10 knockdown were stimulated with PBS, fresh or aged NPs (7.5 µg/mL), co‐cultured with 4T1 cells for 72 h. (B) qPCR analysis confirming CXCL10 knockdown in Raw264.7 cells ( n = 3). (C‐E) CXCL10 knockdown in Raw264.7 macrophages attenuates ZnO NP‐enhanced M2 polarization, as shown by reduced Arg1, Mrc1 expression (C) and CD206 + cell frequency (D, E) ( n = 3). (F) qPCR analysis confirming CXCL10 knockdown in NIH‐3T3 cells ( n = 3). (G, H) Flow cytometric analysis (G) and quantification (H) of the percentage of CD140a‐positive cells among CXCL10‐knockdown NIH‐3T3 cells in the co‐culture system ( n = 3). (I) qPCR analysis of CAF‐related genes (FN1, FSP1, α‐SMA, CD140a) in CXCL10‐knockdown NIH‐3T3 cells from the co‐culture model ( n = 3). (J) Schematic illustration of primary MDSCs preparation: primary mouse bone marrow‐derived macrophages were differentiated into MDSCs by supplement of GM‐CSF (40 ng/mL) and IL‐6 (40 ng/mL). MDSCs with CXCL10 knockdown were stimulated with PBS, fresh ZnO NPs, or aged ZnO NPs (7.5 µg/mL), then co‐cultured with 4T1 cells for 72 h. Cells were collected for further experiments. (K) qPCR analysis of relative CXCL10 mRNA expression in MDSCs with CXCL10 knockdown ( n = 3). (L, M) Flow cytometric analysis (L) and quantification (M) of the proportion of granulocytic CD11b + Ly6G + Ly6C low PMN‐MDSCs in the co‐culture system after CXCL10 knockdown ( n = 3). All data were represented as the mean ± SD. P values were calculated by one‐way ANOVA followed by Tukey's test. * p <0.05; ** p <0.01.

Article Snippet: Sections were then incubated overnight at 4°C with the following primary antibodies: F4/80 (Servicebio, GB11027; 1:750), Ki‐67 (Abcam, ab15580; 1:200), FSP‐1 (Bioss, bs‐1015R; 1:250), FN‐1 (BD Pharmingen, 610077; 1:250), CD206 (Affinity, DF7564; 1:250), αSMA (Affinity, AF1032; 1:500), bFGF (Proteintech, 14726‐1‐AP; 1:200), COX‐2 (Cell Signaling Technology, 12282S; 1:100).

Techniques: Knockdown, Cell Culture, Analysis, Expressing, Co-culture Assay, Derivative Assay