Journal: PLoS Genetics

Article Title: Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence

doi: 10.1371/journal.pgen.1010271

Figure Lengend Snippet: ( A-C ) 1×10 5 6DT1 cells were implanted into the fourth left mammary fat pad of Clic4 wildtype (WT), n = 15, heterozygous (HET), n = 29, and knockout (KO), n = 22, FVB female mice. Primary tumors were resected and weighed (g = grams) ( A ), and superficial lung metastases quantified ( B ) after 28 days. ( C ) Immunohistochemistry for CLIC4 expression in 6DT1 primary tumors and superficial lung metastases from individual mice at 28 days post-implantation. ( D-F ) Primary tumor weight (g = grams) ( D ), number of lung metastases ( E ), and CLIC4 immunohistochemistry ( F) 28 days after implanting 2.5×10 5 E0771 cells into Clic4 wildtype (WT), n = 22, heterozygous (HET), n = 12, and knockout (KO), n = 25, C57BL/6 female mice. Values represent the mean ± s.d. P values were obtained after ANOVA followed by unpaired t test. Scale bar, 4 mm.

Article Snippet: The murine breast cancer cell line 6DT1 (FVB background) was grown as previously described [ ] and E0771 (C57BL/6 background) cells were purchased from CH3 BioSystems LLC and grown according to the manufacturer’s recommendations (Amherst, NY, No. 940001).

Techniques: Knock-Out, Immunohistochemistry, Expressing

Journal: PLoS Genetics

Article Title: Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence

doi: 10.1371/journal.pgen.1010271

Figure Lengend Snippet: Fourteen days after implantation of 1×10 5 6DT1 cells into the fourth left mammary fat pad of Clic4 wildtype (WT), n = 8, and knockout (KO), n = 8, FVB females, 1×10 5 GFP-labeled 6DT1 cells were injected via tail vein. ( A ) GFP immunostaining detects individual tail vein-injected GFP-labeled 6DT1 cells (red arrows) in lungs after 48 hours. Scale bar, 50 μm. ( B-C ) Primary tumor weight (g = grams) ( B ) and number of superficial lung metastases ( C ) 14 days post-tail vein injection. Values are means ± s.d., P values were determined by unpaired t test. ( D ) GFP-positive metastases, mixed GFP-positive and -negative metastases (black arrows), or residual single cells (red arrows) in lungs 14 days after tail vein injection of GFP-labeled 6DT1 cells. Scale bars, 5 mm (top) and 50 μm (bottom). Insert in bottom right panel, micro metastasis.

Article Snippet: The murine breast cancer cell line 6DT1 (FVB background) was grown as previously described [ ] and E0771 (C57BL/6 background) cells were purchased from CH3 BioSystems LLC and grown according to the manufacturer’s recommendations (Amherst, NY, No. 940001).

Techniques: Knock-Out, Labeling, Injection, Immunostaining

Journal: PLoS Genetics

Article Title: Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence

doi: 10.1371/journal.pgen.1010271

Figure Lengend Snippet: ( A ) Deletion of Clic4 in 6DT1 tumor cells (Parental) by CRISPR/Cas9 was confirmed by immunoblot. Two independent Clic4 wildtype (sgNT) and Clic4 knockout (sg Clic4 ) clones were selected. Densitometry-based quantification is shown below each protein band, normalized to the loading control (HSP90). NT = non-targeting. ( B ) 1×10 5 sgNT or sg Clic4 6DT1 cells were implanted into the fourth left mammary fat pad of Clic4 wildtype FVB females. Primary tumor weight (g = grams) and number of superficial lung metastases were evaluated 28 days post-implantation of the clonal sublines sgNT-1 (n = 15), sgNT-2 (n = 20), sg Clic4 -1 (n = 15), and sg Clic4 -2 (n = 20). Parental cell-derived tumors (n = 18; gray background) are shown for reference. P values were obtained using one-way ANOVA with comparisons between all clonal lines and the Sidak correction for multiple comparisons. ( C-D ) Representative immunohistochemistry showing the tissue distribution of CLIC4 expression in primary tumor and lung metastases from tumor-bearing mice at 28 days post-implantation of 1×10 5 sgNT or sg Clic4 6DT1 cells. Scale bar, 2 mm ( C ) and 30 μm ( D ). Yellow arrows, tumor stromal compartment. White arrows, tumor epithelial compartment.

Article Snippet: The murine breast cancer cell line 6DT1 (FVB background) was grown as previously described [ ] and E0771 (C57BL/6 background) cells were purchased from CH3 BioSystems LLC and grown according to the manufacturer’s recommendations (Amherst, NY, No. 940001).

Techniques: CRISPR, Western Blot, Knock-Out, Clone Assay, Control, Derivative Assay, Immunohistochemistry, Expressing

Journal: PLoS Genetics

Article Title: Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence

doi: 10.1371/journal.pgen.1010271

Figure Lengend Snippet: ( A ) Proteome Profiler Array heat map of 19 significantly differentially expressed cytokines and chemokines, from 111 tested, present in plasma from healthy control (0 days) (n = 3) or 6DT1 tumor-bearing Clic4 wildtype (WT) or knockout (KO) mice at 14 days (n = 3) or 28 days (n = 5) after mammary fat pad implantation. Raw values were obtained by determining the pixel intensity for each analyte followed by normalization to the corresponding internal control for each animal. These values were log2-transformed, and the heat map represents the mean log2 fold-change of each group from the WT healthy control group. P values were determined by unpaired t test comparing the raw values of WT and KO at each time point; *P value < 0.05. ( B-C ) Luminex Array heat map of 35 significantly differentially expressed proteins, from 51 tested, present in the primary tumors and lungs of Clic4 wildtype (WT) or knockout (KO) mice at 0 days (lung only, n = 5), 14 days (n = 8), or 28 days (n = 5) after 6DT1 tumor cell implantation in the mammary fat pad. ( B ) Tumor protein expression values shown are log2 fold-change of KO from WT at 14 or 28 days. P values were determined by unpaired t test comparing the raw values of WT and KO at each time point; * P value < 0.05. ( C ) Lung protein expression values shown are log2 fold-change of WT or KO at 14 or 28 days post-implantation from their respective genotype-matched baseline (0 days) values. P values were determined by unpaired t test comparing the raw values of WT or KO at 14 or 28 days to their respective baseline values; *P value < 0.05. ( D-E ) Quantification of selectively stained infiltrating inflammatory cells in primary tumor and lung tissues from 6DT1 tumor-bearing Clic4 wildtype (WT) and knockout (KO) mice by IHC. Values represent the mean ± s.d. of the total number of positive cells found in tissue sections from 3 animals analyzed by Image Scope. F4/80 was used to detect macrophages, Ly6G/GR1 for neutrophils, and CD4 and CD8 for mature T cell subsets in primary tumors ( D ) and lung tissues ( E ). P values were obtained after unpaired t test between genotypes at each time point.

Article Snippet: The murine breast cancer cell line 6DT1 (FVB background) was grown as previously described [ ] and E0771 (C57BL/6 background) cells were purchased from CH3 BioSystems LLC and grown according to the manufacturer’s recommendations (Amherst, NY, No. 940001).

Techniques: Clinical Proteomics, Control, Knock-Out, Transformation Assay, Luminex, Expressing, Staining

Journal: PLoS Genetics

Article Title: Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence

doi: 10.1371/journal.pgen.1010271

Figure Lengend Snippet: ( A ) Significant (FDR <0.25) Hallmark pathways identify transcriptomic changes that occur in the lungs of 6DT1 tumor-bearing Clic4 wildtype (WT) or knockout (KO) mice relative to the lungs of genotype-matched healthy control mice 14 days after implantation. Gene set enrichment analysis (GSEA) normalized enrichment scores (NES) are shown. Group 1, similar regulation between WT and KO; Group 2, significant for WT only; Group 3, significant for KO only; Group 4, opposite regulation between WT and KO. ( B ) Gene set enrichment analysis (GSEA) to identify significant enrichment (FDR <0.25) in Hallmark pathways based on genes differentially expressed in KO vs. WT lungs at 14 days after primary tumor implantation. ( C ) Enrichment plots for the “TNFA signaling via NFKB” Hallmark pathway for the three comparisons shown in panels A and B. The ten most significant leading edge genes are listed to the right of each plot.

Article Snippet: The murine breast cancer cell line 6DT1 (FVB background) was grown as previously described [ ] and E0771 (C57BL/6 background) cells were purchased from CH3 BioSystems LLC and grown according to the manufacturer’s recommendations (Amherst, NY, No. 940001).

Techniques: Knock-Out, Control, Tumor Implantation