Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Plac1 expression and lentivirus-mediated reduction of Plac1 in EO771 cells. ( a ) EO771 mouse mammary tumor cells expressed high levels of Plac1 in comparison to mouse placenta. ( b ) EO771 cells were transduced with lentiviruses expressing crambled RNA (Scr) or four Plac1 shRNAs designated sh81, sh187, sh300 and sh490; sh490 inhibited RNA expression >98%, and these cells were designated EO771/shPlac1. ( c ) EO771/Scr and EO771/shPlac1 cells were grown as monolayers, and the number of viable cells were quantified by sulforhodamine B staining. Shown is the mean ± S.D. of triplicate analysis of three samples. The growth of EO771/shPlac1 cells differed significantly ( P < 0.001) from EO771/Scr cells by the two-sided Student’s t test. ( d ) qRT-PCR analysis of immune cell-related gene expression downregulated in EO771/shPlac1 cells. Shown is the mean ± S.D. of triplicate analysis of three samples. Significant differences between EO771/Scr and EO771/shPlac1 cells were obtained for CD274 ( P < 0.01), Plac1 ( P < 0.01), Cxcl1 ( P < 0.001), Ccl5 ( P < 0.001) and Lif ( P < 0.001) using the two-tailed Student’s t test; values for Ccl7 were not significantly different ( P > 0.05). ( e ) Heatmap of gene expression as determined by Affymetrix microarray analysis of EO771/Scr (Ctl) and EO771/shPlac1 (sh) cells. Shown are immune cell-related transcripts (Table ) representing ≥3.0-fold change in expression.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Expressing, Comparison, Transduction, RNA Expression, Staining, Quantitative RT-PCR, Gene Expression, Two Tailed Test, Microarray

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Growth of EO771/Scr and EO771/shPlac1 cells in syngeneic and SCID mice. ( a ) Syngeneic C57BL/6 mice or ( b ) SCID mice at five weeks of age, were inoculated in the mammary gland with 1 × 10 6 cells. Tumor size was measured by calipers in two dimensions. Tumor growth for EO771/Scr and EO771/shPlac1 cells in syngeneic mice differed significantly (P = 0.040) by the unpaired Student’s t test. There was no significant difference (P > 0.05) in tumor growth between the two cell lines in SCID mice. Shown is the mean ± SD, N = 5 per group. ( c ) H&E staining and Plac1 IHC in isografts of EO771/Scr and EO771/shPlac1 cells. Magnification 400X.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Staining

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Growth of EO771 cells in syngeneic mice following treatment with a Cxcr 2 antagonist. ( a ) Syngeneic 57BL/6 mice were inoculated in the mammary gland with 1 × 10 6 at five weeks of age, and injected i.p. daily with vehicle (blue) or 2 mg/kg (red) or 20 mg/kg (green) SB225002 beginning 11 days after cell inoculation. SB225002 completely suppressed tumor growth after 14 days. Differences between vehicle- and 2 mg/kg SB225002-treated mice were not significantly different (P = 0.145); differences between vehicle- and 20 mg/kg SB225002-treated mice were significantly different (P = 0.005) by the unpaired two-tailed Student’s t test. Shown is the mean ± SD, N = 5 per group. ( b ) Immune gene expression in tumors 17 days after treatment with 20 mg/kg SB225002. Shown is the relative expression in control and SB225002-treated mice in comparison to their changes in EO771/shPlac1 cells (Table ). ( c ) FACS analysis of immune cell tumor infiltrates in isografts after treatment with vehicle or SB225002 as in ( b ). SB225002 treatment reduced the percentage of immune cell tumor infiltrates of CD11b + /Gr-1 + myeloid-derived suppressor cells ( MDSC ) and Foxp3 + /CD25 + T cells ( Treg ), and increased the percentages of CD8 + /CD4 + T cells ( T) , CD3 + /NK1.1 + NK cells ( NK ) and F4/80 + /CD80/86 + macrophages ( Mϕ ) and CD11c + /CD80/86 + dendritic cells ( DC ). Numbers in parentheses ( ) represent the percentages of each cell population. ( d ) Bar graph represents the mean±SD of the percent distribution of immune cell tumor infiltrates as in ( c ); P values were determined by the unpaired two-tailed Student’s t test, N = 4 per group. ( e ) CD8 + T cell infiltration determined by IHC in tumor isografts from vehicle-treated ( EO771/Ctl ) and SB225002-treated ( EO771/SB ) mice. Infiltration of CD8 + T cells increased after treatment with 20 mg/kg SB225002. Magnification 600X. ( f ) Macrophage ( F4/80 ) and Treg cell ( Foxp3 ) infiltration, Plac1 expression and apoptosis by cleaved caspase-3 expression ( Caspase ) in tumor isografts from vehicle-treated ( EO771/Ctl ) and SB225002-treated ( EO771/SB ) mice. Infiltration of macrophages and Treg cells were reduced and apoptosis was increased after treatment with 20 mg/kg SB225002. Magnification 400X

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Injection, Two Tailed Test, Gene Expression, Expressing, Control, Comparison, Derivative Assay

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Lentivirus-mediated reduction of Cxcl1 in EO771 cells. ( a ) EO771 cells were transduced with lentiviruses expressing scrambled RNA (Scr) or three Cxcl1 shRNAs designated sh118, sh174, sh218; sh174 inhibited RNA expression >99% (EO771/shCxcl1). ( b ) EO771/Scr and EO771/shCxcl1 cells were grown as monolayers, and the number of viable cells were determined by sulforhodamine B staining. Shown is the mean ± S.D. of triplicate analysis from three samples, which were significantly different ( P < 0.001) by the two-tailed Student’s t test. ( c ) Growth of EO771/Scr and EO771/shCxcl1 cells in syngeneic mice. Mice at five weeks of age were inoculated in the mammary gland with 1x10 6 cells, and tumor size was measured by calipers in two dimensions. Differences in tumor growth between EO771/Scr and EO771/shCxcl1 cells were significantly different (P = 0.006) by the unpaired two-tailed Student’s t test; N = 5. ( d ) qRT-PCR analysis of genes downregulated in EO771/shCxcl1 cells. Shown is the mean ± SD of triplicate analysis of 3 samples. Significant differences between EO771/Scr and EO771/shCxcl1 cells were obtained for Plau ( P < 0.02), C3 ( P < 0.01), Ly6a ( P < 0.01), Ccl7 ( P < 0.001) and Il23a ( P < 0.01) by the two-sided Student’s t test; differences for CD68 were not significantly different ( P > 0.05).

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Transduction, Expressing, RNA Expression, Staining, Two Tailed Test, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Expression of immune-related genes in E0771/shCxcl1 cells. Shown are ≥3-fold changes in gene expression with a raw score ≥300 in EO771/shCxcl1 or E0771/Scr cells.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Expressing, Gene Expression

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Gene expression common to EO771/shPlac1 and EO771/shCxcl1 cells. Shown is the ratio between EO771/shPlac1 or EO771/shCxcl1 cells to EO771/Scr control cells for genes with ≥3.0-fold changes in expression and a raw score ≥300.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Gene Expression, Control, Expressing

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Cxcl1 rescue of EO771/sh490 cells. ( a ) EO771/Scr and EO771/sh490 cells expressing eGFP were transduced with a lentivirus expressing Cxcl1 and mCherry, and selected for 35 days in 3.5 mg/ml G418. The merged photo shows cells co-expressing eGFP and mCherry (yellow). Magnification 200X. ( b ) qRT-PCR for Plac1 and Cxcl1 in EO771/Scr, EO771/sh490 and EO771/sh490/Cxcl1 cells. Shown is the mean ± S.D. of triplicate determinations.( c ) EO771/sh490/Cxcl1 cells were grown in 96-well plates at an initial density of 5,000 cells per well in media supplemented with 3.5 mg/ml G418. Cell density was determined by sulforhodamine B staining. Shown is the mean ± SD of triplicate determinations. ( d ) Syngeneic C57BL/6 mice were inoculated in the mammary gland with 1 × 10 6 at five weeks of age. There was a significant difference in the growth EO771/sh490 cells (P = 0.021) and EO771/sh490/Cxcl1 cells (P = 0.034) vs. EO771/Scr cells by the unpaired two-tailed Student’s t test. Shown is the mean ± SD, N=6 per group.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Expressing, Transduction, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Mechanical stimulations can inhibit local and remote tumor progression by downregulating WISP1

doi: 10.1096/fj.202000713RR

Figure Lengend Snippet: Reduction in bone loss by knee loading. The single and double asterisks indicate p < 0.05 and p < 0.01, respectively. (A) Schematic illustration of intra-tibia injection of EO771 mammary tumor cells and the timeline. (B) Representative μCT images of the proximal tibia. (C) Comparison of BV/TV, BMD, Tb.n, and Tb.s in the proximal tibia between the placebo and loaded groups. (D) Representative μCT images of the distal femur. (E) Comparison of BV/TV, BMD, Tb.n, and Tb.s in the distal femur between the placebo and loaded groups. (F) HE staining of the proximal tibia in the placebo and loaded groups, and the relative tumor area. The dotted area (green) indicates the tumor-grown region. (G) Reduction in the protein levels of MMP9, Runx2, TGFβ, and Snail in bone marrow-derived cells in the loaded group.

Article Snippet: Eight-week-old C57BL/6 female mice (8 per group; Envigo RMS, Inc., Indianapolis, IN, USA) received subcutaneous injections of EO771 cells (3.0 × 10 5 cells in 50 μl PBS), to the mammary fat pad on day 0 ( 25 ).

Techniques: Injection, Comparison, Staining, Derivative Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Mechanical stimulations can inhibit local and remote tumor progression by downregulating WISP1

doi: 10.1096/fj.202000713RR

Figure Lengend Snippet: Load-driven reduction in the mammary tumor. The single and double asterisks indicate p < 0.05 and p < 0.01, respectively. (A) Schematic illustration of the injection of EO771 mammary tumor cells to the mammary fat pad. (B) Mammary tumors 2 weeks after tumor inoculation in the placebo and loaded group. (C&D) Decrease in the tumor weight and size in the loaded group.

Article Snippet: Eight-week-old C57BL/6 female mice (8 per group; Envigo RMS, Inc., Indianapolis, IN, USA) received subcutaneous injections of EO771 cells (3.0 × 10 5 cells in 50 μl PBS), to the mammary fat pad on day 0 ( 25 ).

Techniques: Injection

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Mechanical stimulations can inhibit local and remote tumor progression by downregulating WISP1

doi: 10.1096/fj.202000713RR

Figure Lengend Snippet: Differential effects of pre- and post-loading urine samples on the expression of WISP1, MMP9, Runx2, and TGFβ in EO771 cells. Of note, pre-S & post-S = pre- & post-step aerobics, and pre-L & post-L = pre- and post-loading. The bar chart is based on 10 pairs of human samples and 10 pairs of mouse samples. (A&B) Downregulation of MMP9, RUNX2, TGFβ, and WISP1 in response to human post-step aerobics urine samples. (C&D) Downregulation of MMP9, RUNX2, TGFβ, and WISP1 in response to murine post-knee loading urine samples.

Article Snippet: Eight-week-old C57BL/6 female mice (8 per group; Envigo RMS, Inc., Indianapolis, IN, USA) received subcutaneous injections of EO771 cells (3.0 × 10 5 cells in 50 μl PBS), to the mammary fat pad on day 0 ( 25 ).

Techniques: Expressing

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Mechanical stimulations can inhibit local and remote tumor progression by downregulating WISP1

doi: 10.1096/fj.202000713RR

Figure Lengend Snippet: Effects of TGFβ and calcitriol in the urine. Of note, CN = control, pl = placebo, load = knee loading, and VD = vitamin D3 (calcitriol). The single and double asterisks indicate p < 0.05 and p < 0.01, respectively. (A) Cholesterol levels in the serum and urine in the normal, placebo, and loaded groups. (B) TGFβ levels in the serum and urine before and after knee loading. (C) Effects of TGFβ on the expression of MMP9, Runx2, and Snail. (D) Calcitriol levels in the serum and urine before and after knee loading. (E) Effects of calcitriol on the expression of MMP9, Runx2, and Snail. (F-H) Calcitriol-driven reduction in EdU-based proliferation, Transwell invasion, and cellular motility of EO771 cells.

Article Snippet: Eight-week-old C57BL/6 female mice (8 per group; Envigo RMS, Inc., Indianapolis, IN, USA) received subcutaneous injections of EO771 cells (3.0 × 10 5 cells in 50 μl PBS), to the mammary fat pad on day 0 ( 25 ).

Techniques: Control, Expressing

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Mechanical stimulations can inhibit local and remote tumor progression by downregulating WISP1

doi: 10.1096/fj.202000713RR

Figure Lengend Snippet: Load-driven downregulation of WISP1 in the post-loading murine urine. Of note, CN = control, and VD = vitamin D3 (calcitriol). (A) Expression of 111 cytokines and chemokines in the mouse XL cytokine array. (B-D) WISP1-driven stimulation of EdU-based proliferation, Transwell invasion, and cellular motility of EO771 cells. (E) Elevation of MMP9, Runx2, TGFβ, and Snail by WISP1 in EO771 cells. (F) Size changes in fragmented breast cancer tumor tissue by calcitriol and WISP1.

Article Snippet: Eight-week-old C57BL/6 female mice (8 per group; Envigo RMS, Inc., Indianapolis, IN, USA) received subcutaneous injections of EO771 cells (3.0 × 10 5 cells in 50 μl PBS), to the mammary fat pad on day 0 ( 25 ).

Techniques: Control, Expressing