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: Materials Today Bio

Article Title: Injectable versatile liquid-solid transformation implants alliance checkpoint blockade for magnetothermal dynamic-immunotherapy

doi: 10.1016/j.mtbio.2022.100442

Figure Lengend Snippet: Magnetothermal performance and therapeutic efficacy of F/A@P with various components in vitro. (a) Magnetothermal curves of different volumes and mass fractions of Fe 3 O 4 in F/A@P under AMF actuation and (b) the corresponding infrared thermal images. (c) Temperature curves of F/A@P over seven on-off cycles under AMF actuation. (d–e) Viability of 4T1 cells after different treatments assessed by CCK8 assay. (f) Live (green)/dead (red) fluorescence images of 4T1 cells after different treatments (scale bars: 50 ​μm). (g) FCM was used to determine the apoptosis level of 4T1 cells after various treatments. The data are shown as the mean ​± ​SD, n ​= ​3 per group; ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001.

Article Snippet: 4T1 breast tumor cells were purchased from Procell (Wuhan, China), cultured in complete DMEM and grown in an atmosphere composed of 5% carbon dioxide (CO 2 ) and 95% air at 37 °C.

Techniques: Drug discovery, In Vitro, CCK-8 Assay, Fluorescence

Journal: Materials Today Bio

Article Title: Injectable versatile liquid-solid transformation implants alliance checkpoint blockade for magnetothermal dynamic-immunotherapy

doi: 10.1016/j.mtbio.2022.100442

Figure Lengend Snippet: Oxygen-irrelevant free radical-generating ability of F/A@P and the mechanism of ER stress in vitro. (a) UV–vis spectrum of oxidized TMB in different groups after AMF actuation for 400 ​s (inset: digital photos of the TMB solutions). (b) UV–vis spectrum of oxidized TMB after treatment with F/A@P under different AMF actuation times (inset: digital photos of the TMB solutions). (c) Absorbance curves of oxidized TMB based on a) and b). (d) Typical DCF fluorescence images of ROS levels in 4T1 cells after various treatments (scale bars: 50 ​μm). (e) Intracellular ROS levels after different treatments analyzed by FCM and (f) the corresponding quantitative analyses of ROS levels. (g) Intracellular GSH levels after different treatments. (h) Representative immunoblot results of various protein expression levels from each group. (i) Corresponding quantitative analyses of the ratios of p-PERK/GAPDH, ATF4/GAPDH and CHOP/GAPDH based on h). (The data are shown as the mean ​± ​SD, n ​= ​3 per group; n.s. represents no significance, ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001 in comparison with the control group, ##p ​< ​0.01 in comparison with the ICG ​+ ​laser group).

Article Snippet: 4T1 breast tumor cells were purchased from Procell (Wuhan, China), cultured in complete DMEM and grown in an atmosphere composed of 5% carbon dioxide (CO 2 ) and 95% air at 37 °C.

Techniques: In Vitro, Fluorescence, Western Blot, Expressing, Comparison, Control

Journal: Materials Today Bio

Article Title: Injectable versatile liquid-solid transformation implants alliance checkpoint blockade for magnetothermal dynamic-immunotherapy

doi: 10.1016/j.mtbio.2022.100442

Figure Lengend Snippet: F/A@P mediates immunogenic cell death and DC maturation in vitro. (a) Schematic diagram of the coculture process of immature DCs with 4T1 cell residues in vitro. (b) Immunofluorescence images of HMGB1, CRT, HSP70, and HSP90 expression in 4T1 cells (scale bars: 25 ​μm). Nuclei: blue, DAPI-labeled; specific cell surface proteins: green, FITC-IgG secondary antibody-stained. (c) The expression of CD80 and CD86 on the surface of the DCs in each group was analyzed by FCM. (d) Corresponding quantitative analysis of the DC maturation rates and levels of the cytokines IL-6, IL-12 and TNF-α in the supernatants of the DC culture medium. (The data are shown as the mean ​± ​SD, n ​= ​3 per group; n.s. represents no significance, ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001 in comparison with the control group).

Article Snippet: 4T1 breast tumor cells were purchased from Procell (Wuhan, China), cultured in complete DMEM and grown in an atmosphere composed of 5% carbon dioxide (CO 2 ) and 95% air at 37 °C.

Techniques: In Vitro, Immunofluorescence, Expressing, Labeling, Staining, Comparison, Control

Journal: Materials Today Bio

Article Title: Injectable versatile liquid-solid transformation implants alliance checkpoint blockade for magnetothermal dynamic-immunotherapy

doi: 10.1016/j.mtbio.2022.100442

Figure Lengend Snippet: Antitumor effect of F/A@P-mediated MDT combined with CTLA4 blockade therapy in an orthotopic 4T1 bilateral tumor-bearing mouse model. (a) Schematic diagram of F/A@P-mediated MHT combined with CTLA4 blockade therapy to inhibit primary and distant tumors. (b) Digital photographs of the mice in the five groups at 0, 1, 7, 14 and 21 days after various treatments. (c) Primary tumor growth curves and the average primary tumor growth curve of the mice in each group (n ​= ​6). (d) Distant tumor growth curves and average distant growth curve in each group (n ​= ​6). (e) Primary tumor inhibition rates and (f) distant tumor inhibition rates of the mice in each group. (g) Body weights and (h) morbidity-free survival of the mice in each group. (i) Digital photographs and H&E staining images of representative pulmonary metastatic nodules (circles) from the mice in each group at the end of treatment (scale bars: 100 ​μm). (The data are shown as the mean ​± ​SD, n ​= ​6 per group; n.s. represents no significance, ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001 in comparison with the control group, #p ​< ​0.05, ##p ​< ​0.01, ###p ​< ​0.001 in comparison with the F/A@P ​+ ​AMF group).

Article Snippet: 4T1 breast tumor cells were purchased from Procell (Wuhan, China), cultured in complete DMEM and grown in an atmosphere composed of 5% carbon dioxide (CO 2 ) and 95% air at 37 °C.

Techniques: Inhibition, Staining, Comparison, Control

Journal: Materials Today Bio

Article Title: Injectable versatile liquid-solid transformation implants alliance checkpoint blockade for magnetothermal dynamic-immunotherapy

doi: 10.1016/j.mtbio.2022.100442

Figure Lengend Snippet: Immune responses to combination therapy in an orthotopic bilateral 4T1 tumor-bearing mouse model. (a) FCM diagrams and quantification analysis of DC maturation in drain LNs adjacent to the primary tumors. (b) FCM diagrams and quantification analysis of DC maturation in spleens. (c) FCM diagrams and (d) quantification analysis of CTLs and Tregs in spleens. (e) FCM diagrams and (f) quantification analysis of CTLs and Tregs in distant tumors. (g) FCM diagrams and (h) quantification analysis of T EM cells in the mouse spleens of the control group and F/A@P ​+ ​AMF ​+ ​anti-CTLA4 group. (i) Proportions of Teff cells in secondary tumors based on (e) and (f). (j) Teff:Treg and CTL:Treg ratios in the secondary tumors. (The data are shown as the mean ​± ​SD, n ​= ​3 per group; n.s. represents no significance, ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001 in comparison with the control group, #p ​< ​0.05, ##p ​< ​0.01, ###p ​< ​0.001 in comparison with the F/A@P ​+ ​AMF group).

Article Snippet: 4T1 breast tumor cells were purchased from Procell (Wuhan, China), cultured in complete DMEM and grown in an atmosphere composed of 5% carbon dioxide (CO 2 ) and 95% air at 37 °C.

Techniques: Control, Comparison