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: bioRxiv

Article Title: Towards active vaccination against tumour endothelial marker Robo4

doi: 10.1101/2025.08.28.672833

Figure Lengend Snippet: A) Immunofluorescent staining mouse mature placenta (placental labyrinth part), adult mouse spleen, LLC1 and 4T1 tumour tissue. MECA32 stains endothelial cells. Scale bar: 50μm. B) Robo4 expression levels in LLC1 cell line, LLC1 tumours, 4T1 cell line, 4T1 tumours, placenta, spleen tissue from adult wild type mice measured by qRT-PCR. LLC1 and 4T1 cell line were used as comparators. Each dot represents one animal. Two-tailed Mann-Whitney U test. *, p < 0.05.

Article Snippet: Female Balb/c mice were s.c. implanted with 2×10 4 4T1 cells (Cytion, Germany; 300300) on 4 th mammary fat pad.

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

Journal: bioRxiv

Article Title: Towards active vaccination against tumour endothelial marker Robo4

doi: 10.1101/2025.08.28.672833

Figure Lengend Snippet: A) Experiment protocol. Mice were primed with TTc in alum, 4 weeks later boost with R4- TTc alum or TTc in alum. 4T1 cells were s.c. injected into 4 th mammary fat pad one day after the second boost with R4-TTc alum or TTc in alum. B) Robo4-specific IgG1 antibody after the R4-TTc injection. Dash line: detection level. Two- way ANOVA with mixed-effects analysis (Tukey multiple comparison). ****: p < 0.0001. C) Tumour growth. Two-way ANOVA with mixed-effects analysis. ****: p < 0.0001. D) Tumour weight at d28 after 4T1 cells transplantation. Two-tailed Mann-Witney U test. E) Correlation between the Robo4-specific IgG1 at d-1 (when tumour cell transplantation) and final tumour weight. Two-tailed Compute Pearson correlation coefficients. B-E:Each dot represents one animal, merged two independent experiments. A) Representative immunofluorescent images of tumours stained with MECA32, CD4, CD8, B220, CD11C, and DAPI. Scale bar: 50µm. G ) MECA32 + vessel density. Two-tailed Mann- Whitney U test. H ) Immune cell infiltration. Cells quantified within a 100 μm radius surrounding tumour-associated blood vessels (MECA32⁺). Each dot represents one animal. Two-tailed Mann-Whitney U test.

Article Snippet: Female Balb/c mice were s.c. implanted with 2×10 4 4T1 cells (Cytion, Germany; 300300) on 4 th mammary fat pad.

Techniques: Injection, Comparison, Transplantation Assay, Two Tailed Test, Staining, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: Obesity and triple‐negative‐breast‐cancer: Is apelin a new key target?

doi: 10.1111/jcmm.15639

Figure Lengend Snippet: Infusion of obesity‐related levels of apelin favours TNBC brain metastatization. A, Ex vivo bioluminescent signals from 4T1 Luc‐GFP in lungs of PBS or apelin‐infused (0.1 µmol/kg/d) Balb/c nude mice. B, Quantification of bioluminescent signals of lung metastases by intensity. C, Ex vivo bioluminescent signals from 4T1 Luc‐GFP in brains of PBS or Apelin‐infused Balb/c nude mice. D, Quantification of metastatic brains in PBS or Apelin‐infused mice. Number of mice per group for (A, B): Control: 9, Apelin: 9, and for (C, D): Control: 9, Apelin: 10. Data were analysed using two‐way ANOVA followed by Bonferroni post hoc test for (B). Data were analysed using Fisher's exact test for (D)

Article Snippet: The 4T1 Luc‐GFP cell line was obtained from Perkin Elmer (4T1 Red FLuc‐GFP, PerkinElmer, Waltham, MA, USA) and grows only in immunodeficient mice.

Techniques: Ex Vivo

Journal: British Journal of Cancer

Article Title: NGR-TNF, a novel vascular-targeting agent, does not induce cytokine recruitment of proangiogenic bone marrow-derived cells

doi: 10.1038/bjc.2013.347

Figure Lengend Snippet: NGR-mTNF does not increase the levels of circulating tumour-promoting BMDCs in a mouse model of breast cancer. Levels of circulating vCEPs, vCECs, TEMs and CD11b + Gr1 + MDSCs in 4T1 tumour-bearing mice ( n =5) were evaluated by FACS analysis 4 or 24 h or 1 week after the first treatment with the indicated drugs.

Article Snippet: 4T1 cells (7 × 10 3 ) were s.c. injected into syngeneic BALB/c mice (Charles River, Calco, Lecco, Italy).

Techniques:

Journal: Cancer cell

Article Title: Fasting mimicking diet reduces HO-1 to promote T cell-mediated tumor cytotoxicity

doi: 10.1016/j.ccell.2016.06.005

Figure Lengend Snippet: (A-D) 12-week old female BALB/c mice were grafted with breast cancer (4T1) cells and subject to (A) multiple cycles of FMD or STS alone or to (C, D) a combination of FMD and the chemotherapy drugs (C) doxorubicin (DXR) or (D) cyclophosphamide (CP). (B) Circulating IGF-1 levels at the end of STS or FMD in (A) were measured. (E) 12-week old female C57BL/6 mice were grafted with melanoma (B16) cells and subject to multiple cycles of FMD alone or in combination with DXR. Tumor volume at multiple time-points (on the left) and immediately prior to euthanasia (on the right) are reported for each set of treatments. The animals receiving chemotherapy were injected at the end of each FMD cycle (shaded area). [(A, B) n=10, (C) n=13, (D) n=10, and (E) n=15]. Data represented as mean ± SEM. One-way ANOVA (Tukey post-analysis test) was performed. p-values <0.05, 0.01 and 0.001 are indicated as *, **, and ***, respectively. See also Figure S1.

Article Snippet: Cancer cell lines and tumor cell injection MCF7 human breast adenocarcinoma (gift from Amy Lee – University of Southern California), 4T1- luc murine breast cancer (SibTech, Inc.) and B16 murine melanoma (gift from Noah Craft, UCLA) cell lines were cultured in high glucose (4.5g/L) Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (Thermo Fisher Scientific Inc.) at 37°C and 5% CO 2 .

Techniques: Injection

Journal: Cancer cell

Article Title: Fasting mimicking diet reduces HO-1 to promote T cell-mediated tumor cytotoxicity

doi: 10.1016/j.ccell.2016.06.005

Figure Lengend Snippet: (A) Bone marrow collected from BALB/c mice undergoing FMD/DXR treatments (see Figure S3A) was collected at the end of the experiment and analyzed with FACS (n=6) to assess the amount of Common Lymphoid Progenitors (CLP). (B) Breast cancer (4T1) tumor tissues collected at the end of the experiment (see Figure S3A) were analyzed using immunohistochemistry to assess CD3+/CD8+ TILs (n=8) (quantification on the right). (C, E) CD3+/CD8+ and (D, E) CD3+/CD4+/CD25+were also assessed by FACS analysis (n=7) in tumor tissue collected from a separate, equivalent experiment (see Figure 1C). (F) Melanoma (B16) tissue collected (see Figure 1E) was also processed to assess the levels of TILs (quantification on the right). Bar scale for DAPI, CD3 and CD8 is 75 μm. Bar scale for CD3+/CD8+ is 12.5 μm Data represented as mean ± SEM. The significance of the differences between experimental groups was determined by using one-way ANOVA (Tukey post-analysis test). p-values <0.05, 0.01 and 0.001 are indicated as *, **, and ***, respectively. See also Figure S2.

Article Snippet: Cancer cell lines and tumor cell injection MCF7 human breast adenocarcinoma (gift from Amy Lee – University of Southern California), 4T1- luc murine breast cancer (SibTech, Inc.) and B16 murine melanoma (gift from Noah Craft, UCLA) cell lines were cultured in high glucose (4.5g/L) Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (Thermo Fisher Scientific Inc.) at 37°C and 5% CO 2 .

Techniques: Immunohistochemistry

Journal: Cancer cell

Article Title: Fasting mimicking diet reduces HO-1 to promote T cell-mediated tumor cytotoxicity

doi: 10.1016/j.ccell.2016.06.005

Figure Lengend Snippet: (A) The effect of FMD on breast tumor growth (4T1) in immunodeficient BALB/c nude mice (n=15) (see Figure S4A-E); (B) The survival of wild type (WT) and nude mice injected with high dose of DXR and undergoing ad lib or FMD regimen. (C-H) 4T1 breast tumor-bearing mice were treated with DXR, FMD+DXR alone or in combination with [.alpha]CD8 monoclonal antibody and (C-E) circulating levels of (C, E) CD3+/CD8+ and (D, E) CD3+/CD4+/CD25+ were determined by FACS (n=7). (F) Tumor volumes of FMD+DXR and FMD+DXR+αCD8 were measured at multiple time points, and (G) TILs were also assessed in tumor samples collected from the same animals. (H) Circulating lymphocytes from DXR, FMD+DXR, DXR+αCD8, and FMD+DXR+αCD8 these animals were collected and cultured ex vivo with 4T1 cells for 24 hours and viability was assessed by MTT reduction. (I) Mice were immunized by subcutaneous inoculation (in the left flank) with 4T1 breast cancer cells preconditioned in vitro with either ad lib- (2 g/L glucose+10% FBS) or STS-medium (0.5 g/L glucose+1% FBS) with or without doxorubicin (5 μM). 7 days after the immunization, the same animals were inoculated with naïve 4T1 cells in the right flank (n=10). (J, K) Tumor progression for the immunization (left) and the naïve (right) sides are reported. Data represented as mean ± SEM. One-way ANOVA (Tukey post-analysis test) and Log-rank (Mantel-Cox) test (survival) was performed. Comparisons between groups were performed with Student's t test. p-values <0.05, 0.01 and 0.001 are indicated as *, **, and ***, respectively. See also Figure S3.

Article Snippet: Cancer cell lines and tumor cell injection MCF7 human breast adenocarcinoma (gift from Amy Lee – University of Southern California), 4T1- luc murine breast cancer (SibTech, Inc.) and B16 murine melanoma (gift from Noah Craft, UCLA) cell lines were cultured in high glucose (4.5g/L) Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (Thermo Fisher Scientific Inc.) at 37°C and 5% CO 2 .

Techniques: Injection, Cell Culture, Ex Vivo, In Vitro

Journal: Cancer cell

Article Title: Fasting mimicking diet reduces HO-1 to promote T cell-mediated tumor cytotoxicity

doi: 10.1016/j.ccell.2016.06.005

Figure Lengend Snippet: (A) HO-1 expression levels of grafted 4T1 tumor and normal (liver and skeletal muscle) tissues collected from ad lib fed and mice undergoing STS or FMD regimen were analyzed with qRT-PCR. (B) HO-1 levels in 4T1 cells following 48 hours of in vitro STS were measured by Western blotting (n=3) (Blot was captured with Bio-Rad ChemiDoc, and unedited, representative bands are shown. (C, E, G) Viability of 4T1 cells was determined by MTT reduction following (C) DXR and hemin (10 M), (E) CP and hemin (10 M), and (G) CP and ZnPP (20 M). (D, F) Viability of 4T1 cell stably over-expressing HO-1 (pHO-1) or empty vector (pEV) was determined by MTT following (D) DXR and (F) CP under control and STS conditions. Data represented as mean ± SEM. The significance of the differences between experimental groups was determined by using one-way ANOVA (Tukey post-analysis test). Comparisons between groups were performed with Student's t test. p-values <0.05, 0.01 and 0.001 are indicated as *, **, and ***, respectively. See also Figure S5.

Article Snippet: Cancer cell lines and tumor cell injection MCF7 human breast adenocarcinoma (gift from Amy Lee – University of Southern California), 4T1- luc murine breast cancer (SibTech, Inc.) and B16 murine melanoma (gift from Noah Craft, UCLA) cell lines were cultured in high glucose (4.5g/L) Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (Thermo Fisher Scientific Inc.) at 37°C and 5% CO 2 .

Techniques: Expressing, Quantitative RT-PCR, In Vitro, Western Blot, Stable Transfection, Plasmid Preparation, Control

Journal: Cancer cell

Article Title: Fasting mimicking diet reduces HO-1 to promote T cell-mediated tumor cytotoxicity

doi: 10.1016/j.ccell.2016.06.005

Figure Lengend Snippet: (A-C) 4T1 tumor-bearing mice were treated with (A) ZnPP (40 mg/kg/day; IP; n=7), (B) STS and hemin (30 mg/kg/day; IP; n=7), and (C) FMD and hemin and DXR. (D) Mice bearing 4T1 tumors stably over-expressing HO-1 (pHO-1), or empty vector (pEV) were treated with DXR under ad lib or FMD regimens (n=10-15) (see Figure 1C). Data represented as mean ± SEM. One-way ANOVA (Tukey post-analysis test) was performed. p-values <0.05, 0.01 and 0.001 are indicated as *, **, and ***, respectively. See also Figure S6.

Article Snippet: Cancer cell lines and tumor cell injection MCF7 human breast adenocarcinoma (gift from Amy Lee – University of Southern California), 4T1- luc murine breast cancer (SibTech, Inc.) and B16 murine melanoma (gift from Noah Craft, UCLA) cell lines were cultured in high glucose (4.5g/L) Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (Thermo Fisher Scientific Inc.) at 37°C and 5% CO 2 .

Techniques: Stable Transfection, Expressing, Plasmid Preparation

Journal: Cancer cell

Article Title: Fasting mimicking diet reduces HO-1 to promote T cell-mediated tumor cytotoxicity

doi: 10.1016/j.ccell.2016.06.005

Figure Lengend Snippet: Mice bearing 4T1 or 4T1-pHO-1 breast tumors under FMD were treated with DXR and either hemin (30 mg/kg/day; IP) or CD8+ specific neutralizing monoclonal antibodies (αCD8; clone YTS 169.4) (n=13-15) (see Figures 3F and ​and5D).5D). (A) Tumor volumes were measured at multiple time-points (left) and immediately prior to euthanasia (right). (B-D) CD3+/CD8+ (CTL) and CD3+/CD4+/CD25+ (Treg)lymphocytes from the tumor bed was analyzed by FACS. Data represented as mean ± SEM. One-way ANOVA (Tukey post-analysis test) was performed. p-values <0.05, 0.01 and 0.001 are indicated as *, **, and ***, respectively.

Article Snippet: Cancer cell lines and tumor cell injection MCF7 human breast adenocarcinoma (gift from Amy Lee – University of Southern California), 4T1- luc murine breast cancer (SibTech, Inc.) and B16 murine melanoma (gift from Noah Craft, UCLA) cell lines were cultured in high glucose (4.5g/L) Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (Thermo Fisher Scientific Inc.) at 37°C and 5% CO 2 .

Techniques: Bioprocessing

Journal: iScience

Article Title: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

doi: 10.1016/j.isci.2022.104827

Figure Lengend Snippet:

Article Snippet: 4T1 cells were injected orthotopically in the fourth mammary gland of female BALB/c mice (The Jackson Laboratory) at a concentration of 2.5 x10 5 cells.

Techniques: Virus, Plasmid Preparation, Derivative Assay, Recombinant, In Situ, Staining, RNA Sequencing, ChIP-sequencing, shRNA, Software

Journal: Journal of Biomedical Optics

Article Title: Second-harmonic generation scattering directionality predicts tumor cell motility in collagen gels

doi: 10.1117/1.JBO.20.5.051024

Figure Lengend Snippet: The relationship between metastasis to LN and SHG F/B in the 4T1 murine mammary adenocarcinoma model matches human data. (a) Diagram of the mouse torso revealing the location of the mammary fat pad where tumor cells were injected and the draining ...

Article Snippet: 2.3. tdTomato Transfected 4T1 Cell Culture Once the 4T1 cell line was chosen, it was cultured and eventually seeded on collagen gels : The tdTomato transfected 4T1 mouse mammary adenocarcinoma cell line (Caliper Life Sciences, Hopkinton, MA) was frozen after three initial passages.

Techniques: Injection