Journal: Pharmaceutics

Article Title: Biosynthetic Nanobubble-Mediated CRISPR/Cas9 Gene Editing of Cdh2 Inhibits Breast Cancer Metastasis

doi: 10.3390/pharmaceutics14071382

Figure Lengend Snippet: Schematic diagram of ultrasound-mediated CRISPR/Cas9 gene editing of Cdh2 to inhibit tumor invasion and metastasis.

Article Snippet: 4T1 cells were purchased from ATCC (Manassas, VA, USA) and modified according to the needs of the experiment to stably express Cas9 protein and enhanced green fluorescent protein (EGFP), which was termed CRISPR Cas9 4T1-Cas9-hyg stable cell line (SL581; GeneCopoeia, Inc., Rockville, MD, USA).

Techniques: CRISPR

Journal: Pharmaceutics

Article Title: Biosynthetic Nanobubble-Mediated CRISPR/Cas9 Gene Editing of Cdh2 Inhibits Breast Cancer Metastasis

doi: 10.3390/pharmaceutics14071382

Figure Lengend Snippet: ( A ) Fluorescence images of Cas9- and EGFP-stably expressed 4T1 cells transfected with only pU6-sgRNA (EGFP)-mCherry (control), GPD, or GPD + US (GPD: the abbreviation of GVs-PEI-DNA). Scale bar = 200 μm; ( B ) determination of the transfection efficiency by flow cytometry through counting of the red-emitting cells; ( C ) quantitative analysis of the mCherry-expressed cells from ( B ) ( n = 3); ( D ) determination of the EGFP knockout efficiency ( n = 3). ns denotes p > 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: 4T1 cells were purchased from ATCC (Manassas, VA, USA) and modified according to the needs of the experiment to stably express Cas9 protein and enhanced green fluorescent protein (EGFP), which was termed CRISPR Cas9 4T1-Cas9-hyg stable cell line (SL581; GeneCopoeia, Inc., Rockville, MD, USA).

Techniques: Fluorescence, Stable Transfection, Transfection, Flow Cytometry, Knock-Out

Journal: Pharmaceutics

Article Title: Biosynthetic Nanobubble-Mediated CRISPR/Cas9 Gene Editing of Cdh2 Inhibits Breast Cancer Metastasis

doi: 10.3390/pharmaceutics14071382

Figure Lengend Snippet: ( A ) Fluorescence images of Cas9- and EGFP-stably expressed 4T1 cells transfected with only pU6-sgRNA (Cdh2)-mCherry (control), GPD, or GPD + US (GPD: the abbreviation of GVs–PEI–DNA). Scale bar = 100 μm; ( B ) determination of the transfection efficiency by flow cytometry through the counting of the red-emitting cells in EGFP-expressed cells; ( C ) quantitative analysis of the mCherry-expressed cells from ( B ) ( n = 3) *** p < 0.001. ( D ) Western blotting assay of the expression of N-cadherin in the wild-type (WT) and two Cdh2-KO cell lines. GAPDH was used as an internal marker.

Article Snippet: 4T1 cells were purchased from ATCC (Manassas, VA, USA) and modified according to the needs of the experiment to stably express Cas9 protein and enhanced green fluorescent protein (EGFP), which was termed CRISPR Cas9 4T1-Cas9-hyg stable cell line (SL581; GeneCopoeia, Inc., Rockville, MD, USA).

Techniques: Fluorescence, Stable Transfection, Transfection, Flow Cytometry, Western Blot, Expressing, Marker

Journal: Biomedical Optics Express

Article Title: Matching an immersion medium’s refractive index to a cell’s cytosol isolates organelle scattering

doi: 10.1364/BOE.461874

Figure Lengend Snippet: Amplitude (a,b) and phase (c,d) images of the same 4t1 cell immersed in cell media (a,c) with nm=1.33 and in RI-matching solution (b,d) of iodixanol mixed with media to achieve nm=1.37 .

Article Snippet: Cells from the 4T1 mouse mammary adenocarcinoma cell line (Caliper Life Sciences, Hopkinton, MA) were frozen after three initial passages.

Techniques:

Journal: Biomedical Optics Express

Article Title: Matching an immersion medium’s refractive index to a cell’s cytosol isolates organelle scattering

doi: 10.1364/BOE.461874

Figure Lengend Snippet: Angular scattering intensity of light scattered from the 4t1 cell shown in Fig. 5, immersed in (a) cell media and (b) RI-matching solution. (c) Azimuthally averaged 1D scattered intensity for the cell showing higher low-angle scattering for the cell in media. White dashed circles indicate 5/55 degrees.

Article Snippet: Cells from the 4T1 mouse mammary adenocarcinoma cell line (Caliper Life Sciences, Hopkinton, MA) were frozen after three initial passages.

Techniques:

Journal: Biomedical Optics Express

Article Title: Matching an immersion medium’s refractive index to a cell’s cytosol isolates organelle scattering

doi: 10.1364/BOE.461874

Figure Lengend Snippet: Distribution of α measurements on multiple cells.

Article Snippet: Cells from the 4T1 mouse mammary adenocarcinoma cell line (Caliper Life Sciences, Hopkinton, MA) were frozen after three initial passages.

Techniques:

Journal: Scientific Reports

Article Title: Fast and efficient isolation of murine circulating tumor cells using screencell technology for pre-clinical analyzes

doi: 10.1038/s41598-024-66032-x

Figure Lengend Snippet: Different volumes of WT mice blood collected either within ( A ) heparin tubes or ( B ) EDTA tubes were filtered with ScreenCell Cyto kits. Similar volumes of PyMT cancer mice blood were also processed with ScreenCell Cyto kits ( C ). To evaluate the correct morphology of cancer cells in EDTA tubes, MCF7, and 4T1 breast cancer cell lines were spiked into WT mice blood and processed with ScreenCell Cyto kits. The images were taken using 40X magnification ( D ). To evaluate the recovery rate of ScreenCell technology, either 0, 5, or 10 MCF7 cells were spiked into 100 µl of blood samples of healthy WT mice. Captured cancer cells were counted on Cyto ISs and recovery rate was calculated in percentage. Data are presented as mean ± SEM (n = 6) ( E ). All experiments were repeated at least 2 times.

Article Snippet: 4T1 mouse breast cancer cell line (passages 12–15) and MCF7 human breast cancer cell line (passages 7–10) were received from Inserm U1197 (Paul Brousse Hospital, Villejuif, France) and cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Life Technologies, USA) supplemented with 10% FBS (Pan biotech, Aidenbach, Germany) and 1% Penicillin/Streptomycin (P/S) (Thermofisher Scientific, Waltham, MA, USA).

Techniques:

Journal: Scientific Reports

Article Title: Fast and efficient isolation of murine circulating tumor cells using screencell technology for pre-clinical analyzes

doi: 10.1038/s41598-024-66032-x

Figure Lengend Snippet: This figure represents the 4T1 cancer cells isolated using ScreenCell Cyto kits either immediately after the blood draw (left image, 40X) or after 24 h of preservation at 4 °C in EDTA tubes (right image, 40X). This experiment was performed 4 times. T0: time zero.

Article Snippet: 4T1 mouse breast cancer cell line (passages 12–15) and MCF7 human breast cancer cell line (passages 7–10) were received from Inserm U1197 (Paul Brousse Hospital, Villejuif, France) and cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Life Technologies, USA) supplemented with 10% FBS (Pan biotech, Aidenbach, Germany) and 1% Penicillin/Streptomycin (P/S) (Thermofisher Scientific, Waltham, MA, USA).

Techniques: Isolation, Preserving

Journal: Scientific Reports

Article Title: Fast and efficient isolation of murine circulating tumor cells using screencell technology for pre-clinical analyzes

doi: 10.1038/s41598-024-66032-x

Figure Lengend Snippet: ScreenCell Cyto kits can isolate both single and cluster CTCs. ( A ) After spiking into WT mice blood, both single and cluster 4T1 cells were captured by ScreenCell Cyto kits. ( B ) Single and cluster CTCs were also efficiently captured from breast cancer mice blood. In all conditions, CTCs were distinguishable from normal epithelial cells according to their morphological features. These experiments were performed at least 3 times. All images are taken using 40X magnification, except for the CTC cluster (lower right) which is in 20X.

Article Snippet: 4T1 mouse breast cancer cell line (passages 12–15) and MCF7 human breast cancer cell line (passages 7–10) were received from Inserm U1197 (Paul Brousse Hospital, Villejuif, France) and cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Life Technologies, USA) supplemented with 10% FBS (Pan biotech, Aidenbach, Germany) and 1% Penicillin/Streptomycin (P/S) (Thermofisher Scientific, Waltham, MA, USA).

Techniques:

Journal: Scientific Reports

Article Title: Fast and efficient isolation of murine circulating tumor cells using screencell technology for pre-clinical analyzes

doi: 10.1038/s41598-024-66032-x

Figure Lengend Snippet: DNA concentration from WT mouse blood with or without spiked 4T1 cells isolated by ScreenCell MB device.

Article Snippet: 4T1 mouse breast cancer cell line (passages 12–15) and MCF7 human breast cancer cell line (passages 7–10) were received from Inserm U1197 (Paul Brousse Hospital, Villejuif, France) and cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Life Technologies, USA) supplemented with 10% FBS (Pan biotech, Aidenbach, Germany) and 1% Penicillin/Streptomycin (P/S) (Thermofisher Scientific, Waltham, MA, USA).

Techniques: Concentration Assay, Isolation

Journal: Scientific Reports

Article Title: Fast and efficient isolation of murine circulating tumor cells using screencell technology for pre-clinical analyzes

doi: 10.1038/s41598-024-66032-x

Figure Lengend Snippet: 4T1 cells were spiked into WT mice blood and filtered with ScreenCell MB kits. ISs were then directly inserted into 24 well plates and cultured for 10 days. The images of D0, D5, and D10 with RAL coloration are taken using 40X, and D10 without coloration is taken with 20X magnification. This experiment was performed 4 times. D: Day.

Article Snippet: 4T1 mouse breast cancer cell line (passages 12–15) and MCF7 human breast cancer cell line (passages 7–10) were received from Inserm U1197 (Paul Brousse Hospital, Villejuif, France) and cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Life Technologies, USA) supplemented with 10% FBS (Pan biotech, Aidenbach, Germany) and 1% Penicillin/Streptomycin (P/S) (Thermofisher Scientific, Waltham, MA, USA).

Techniques: Cell Culture

Journal: International Journal of Nanomedicine

Article Title: Improving the solubility and in vitro cytotoxicity (anticancer activity) of ferulic acid by loading it into cyclodextrin nanosponges

doi: 10.2147/IJN.S206350

Figure Lengend Snippet: MTT viability assay for ( A ) MCF7 and ( B ) 4T1 cell lines treated with different formulations of ferulic acid (FA)-loaded cyclodextrin nanosponges (NS) and various interval times at 570 nm.

Article Snippet: MCF7 (human breast cancer) and 4T1 (mouse breast cancer) cell lines were purchased from Pasteur Institute of Iran.

Techniques: MTT Viability Assay

Journal: International Journal of Nanomedicine

Article Title: Improving the solubility and in vitro cytotoxicity (anticancer activity) of ferulic acid by loading it into cyclodextrin nanosponges

doi: 10.2147/IJN.S206350

Figure Lengend Snippet: 4T1 cell lines treated with different formulations: control cells ( A ); ferulic acid, 250 µM ( B ); nanosponges, 250 µM ( C ); ferulic acid-loaded nanosponges, 250 µM ( D ). Microscopic magnification ×10 K, after 24 hrs of incubation.

Article Snippet: MCF7 (human breast cancer) and 4T1 (mouse breast cancer) cell lines were purchased from Pasteur Institute of Iran.

Techniques: Incubation

Journal: International Journal of Molecular Medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple-negative breast cancer metastasis via the inhibition of CXCR4 expression

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: The HER3 pathway is associated with the DMFS of patients with TNBC. (A) c-Met, EGFR, IGF1R, IR and HER3 protein phosphorylation levels were measured using Luminex ® 200. The experiments were performed in duplicate and repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with 4T1 cells. (B) The expression levels of p-HER3, HER3, p-Akt, Akt, p-mTOR, mTOR, p-ERK and ERK were detected using western blot analysis. The expression levels of β-actin were used as internal controls. Quantification of signals is presented as fold of changes relative to phosphorylated proteins vs. total proteins. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with 4T1 cells. (C) Kaplan-Meier Plotter analysis indicated that DMFS was associated with HER3 expression (low expression group, n=212; high expression group, n=212) among 424 TNBC cases (log-rank, P=0.0035). DMFS, distant metastasis-free survival; TNBC, triple-negative breast cancer; HER3, human epidermal growth factor receptor 3; EGFR, epidermal growth factor receptor; IGF1R, insulin-like growth factor 1 receptor; IR, insulin receptor; mTOR, mammalian target of rapamycin; p-phosphorylated.

Article Snippet: The mouse TNBC cell line, luciferase-expressing 4T1 (cat. no. JCRB1447), was obtained from the Japanese Collection of Research Bioresources (JCRB).

Techniques: Phospho-proteomics, Luminex, Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple-negative breast cancer metastasis via the inhibition of CXCR4 expression

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: Knockdown of HER3 decreases the migration, invasion and metastasis of metastatic TNBC cells by inhibiting Akt and mTOR. (A-C) The 4T1-L8 cells were transfected with HER siRNA (50 nM) or control siRNA. (A) The expression levels of HER3, p-Akt, Akt, p-mTOR and mTOR were detected using western blot analysis. The expression levels of β-actin were used as internal controls. Quantification of signals is presented as fold of changes relative to phosphorylated protein vs. total proteins or total proteins vs. β-actin. The experiments were repeated four times. Data are presented as the mean ± SD. * P<0.05, compared with control siRNA. (B) The number of cells stained with trypan blue counted on days 1, 2 and 3. The experiments were performed in triplicate and repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with control siRNA. (C) Cell migration and invasion analysis was performed using the cell culture insert. The cells passing through the cell culture insert were counted. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with control siRNA. Representative images of cells transfected with control siRNA and HER3 siRNA are shown. Scale bars, 100 µ m. (D) The 4T1-L8 cells transfected with HER siRNA (50 nM) or control siRNA were injected into BALB/c mice via the tail vain. Metastasis in the lungs was monitored using IVIS. After 8 days, the mice were sacrificed and the number of metastatic nodules in the lungs were counted. The results are expressed as the mean ± SD. * P<0.05, compared with control siRNA. TNBC, triple-negative breast cancer; HER3, human epidermal growth factor receptor 3; mTOR, mammalian target of rapamycin; p-phosphorylated.

Article Snippet: The mouse TNBC cell line, luciferase-expressing 4T1 (cat. no. JCRB1447), was obtained from the Japanese Collection of Research Bioresources (JCRB).

Techniques: Knockdown, Migration, Transfection, Control, Expressing, Western Blot, Staining, Cell Culture, Injection

Journal: International Journal of Molecular Medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple-negative breast cancer metastasis via the inhibition of CXCR4 expression

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: The HER3/Akt/mTOR pathway promotes the migration and invasion of metastatic triple-negative breast cancer cells by increasing CXCR4 expression. (A) The expression levels of CCR2, CCR7 and CXCR4 were detected using western blot analysis. The expression levels of β-actin were used as internal controls. Quantification of signals is presented as fold of changes relative to total proteins vs. β-actin. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with 4T1 cells. (B) Cell migration and invasion analysis was performed using the cell culture insert. The cells passing through the cell culture insert were counted. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with SDF-1(-). Representative images of SDF-1-untreated 4T1 or 4T1-L8 and SDF-1-treated 4T1 or 4T1-L8 cells are shown. Scale bars, 100 µ m. (C and D) 4T1-L8 cells were transfected with HER siRNA (50 nM) or control siRNA (Stealth™ RNAi Negative Control). (C) The expression levels of CXCR4 were detected using western blotting. The expression levels of β-actin were used as internal controls. Quantification of signals is presented as fold change or relative levels of CXCR4 vs. β-actin. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with control siRNA. (D) Cell migration and invasion analysis was performed using the cell culture insert. The cells passing through the cell culture insert were counted. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with control siRNA. Representative images of control siRNA and HER3 siRNA are shown. Scale bars, 100 µ m. HER3, human epidermal growth factor receptor 3; mTOR, mammalian target of rapamycin; CCR, chemokine receptor; CXCR4, C-X-C chemokine receptor type 4; SDF-1, stromal derived factor-1.

Article Snippet: The mouse TNBC cell line, luciferase-expressing 4T1 (cat. no. JCRB1447), was obtained from the Japanese Collection of Research Bioresources (JCRB).

Techniques: Migration, Expressing, Western Blot, Cell Culture, Transfection, Control, Negative Control, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple-negative breast cancer metastasis via the inhibition of CXCR4 expression

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: Everolimus decreases the expression of CXCR4 via mTOR inhibition. (A and B) 4T1-L8 cells were treated with everolimus (1, 5 and 10 µ M). (A) The expression levels of p-mTOR, mTOR and CXCR4 were detected using western blot analysis. The expression levels of β-actin were used as internal controls. Quantification of signals is presented as the fold change or relative levels of phosphorylated protein vs. total protein or total protein vs. β-actin. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with the control. (B) The number of cells stained with trypan blue counted on days 1, 2 and 3. The experiments were performed in triplicate and repeated three times. Data are presented as the mean ± SD. mTOR, mammalian target of rapamycin; CCR, chemokine receptor; CXCR4, C-X-C chemokine receptor type 4; p-phosphorylated.

Article Snippet: The mouse TNBC cell line, luciferase-expressing 4T1 (cat. no. JCRB1447), was obtained from the Japanese Collection of Research Bioresources (JCRB).

Techniques: Expressing, Inhibition, Western Blot, Control, Staining

Journal: International Journal of Molecular Medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple-negative breast cancer metastasis via the inhibition of CXCR4 expression

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: Everolimus inhibits the metastasis of triple-negative breast cancer cells in vivo . The 4T1-L8 cells were injected via the tail vein into BALB/c mice. The mice were then randomly divided into three groups, namely, the control, 5 mg/kg everolimus, and 10 mg/kg everolimus groups. Metastasis in the lungs was monitored using an in vivo imaging system. After 8 days, the mice were sacrificed, and the number of metastatic nodules in the lungs was counted. The results are expressed as the mean ± SD. * P<0.05, compared with the control.

Article Snippet: The mouse TNBC cell line, luciferase-expressing 4T1 (cat. no. JCRB1447), was obtained from the Japanese Collection of Research Bioresources (JCRB).

Techniques: In Vivo, Injection, Control, In Vivo Imaging

Journal: bioRxiv

Article Title: Low albumin status accompanies multi-layered immunosuppressive phenotypes in metastatic breast cancer patients

doi: 10.1101/2023.09.05.556440

Figure Lengend Snippet: a. Heatmap showing the Pearson correlation coefficients between the clinical characteristics (age and albumin levels) and the module eigengenes generated by WGCNA. In each column, the upper number indicates the Pearson correlation coefficient and the lower number in parentheses indicates a p -value calculated by WGCNA. See also Extended Data Fig. 2a, b for scale independence and mean connectivity of this analysis. b. Bar plot showing log 2 fold changes of module-3 genes between metastatic breast cancer patients (MBC) and healthy volunteers (HV). Genes showing negative correlations to albumin levels are shown in green. Genes showing positive correlations to albumin levels are shown in orange. Genes are ordered according to their correlation to albumin levels. The names of the top10 genes most strongly negatively correlating with albumin levels are indicated. c. The plasma albumin levels of sham-operated and 4T1 breast cancer-bearing mice measured 14 days after transplantation. Data are presented as the mean ± SEM. The p- value is shown (non-paired, two-tailed Student t -test). n = 11 for sham-operated mice, n = 11 for 4T1-bearing mice. d. Heatmap of module-3 genes showing the same direction of changes in the 4T1 breast cancer model compared to the human datasets. Genes are ordered according to their correlation to albumin levels in humans. The names of the top10 genes most strongly negatively correlating with albumin levels in humans are indicated. See also Extended Data Fig. 2c, d for the mouse data.

Article Snippet: 4T1 mouse breast cancer cell line was cultured and maintained in RPMI1640 (nacalai tesque) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Saint Louis, Missouri, USA), 1% penicillin/streptomycin (nacalai tesque) in a 5% CO 2 tissue culture incubator at 37°C.

Techniques: Generated, Clinical Proteomics, Transplantation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Low albumin status accompanies multi-layered immunosuppressive phenotypes in metastatic breast cancer patients

doi: 10.1101/2023.09.05.556440

Figure Lengend Snippet: a. Heatmap of select module-2 genes showing the same direction of changes in the 4T1 breast cancer model compared to the human datasets. Genes are ordered according to their correlation to albumin levels in humans. The names of the top10 genes most strongly positively correlating with albumin levels in humans are indicated. b. Correlation of plasma albumin levels and log 2 normalized scores of CD8A in PBMC. c. Correlation of plasma albumin levels and log 2 normalized scores of KLRG1 in PBMC. d. Correlation of plasma albumin levels and log 2 normalized scores of CRTAM in PBMC. e. UMAP plot for CD8A . f. UMAP plot for KLRG1 . g. UMAP plot for CRTAM h. Estimated abundance of CD8 + T cells in PBMC calculated by ImmuCell-AI and its correlation to plasma albumin levels. i. Estimated abundance of γδ T cells in PBMC calculated by ImmuCell-AI and its correlation to plasma albumin levels. See also Extended Data Fig. 4b for the data related to natural killer cells. See also Extended Data Fig. 4a, c, d for the mouse data for CD8 + T cells, γδ T cells, and natural killer cells, respectively. j. Correlation of plasma albumin levels and log 2 normalized scores of TRDV2 in PBMC. k. UMAP plot for TRDV2 . b-d, j. Scores are normalized to the average scores of five healthy volunteers. b-d, h, i, j. The Pearson correlation coefficients and p -values obtained by simple regression analysis (GraphPad Prism) are shown. e-g, k. The data are retrieved from previously published single-cell RNA-seq (scRNA-seq) datasets from PBMC of Japanese healthy volunteers ( n = 3 (females)) . Clusters corresponding to CD8 + T cells ( e-g ) and γδ T cells ( k ) are highlighted.

Article Snippet: 4T1 mouse breast cancer cell line was cultured and maintained in RPMI1640 (nacalai tesque) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Saint Louis, Missouri, USA), 1% penicillin/streptomycin (nacalai tesque) in a 5% CO 2 tissue culture incubator at 37°C.

Techniques: Clinical Proteomics, RNA Sequencing