Journal: Advanced Science

Article Title: CDK5 Inhibition Abrogates TNBC Stem‐Cell Property and Enhances Anti‐PD‐1 Therapy

doi: 10.1002/advs.202001417

Figure Lengend Snippet: CDK5 blockade inhibits metastasis and stemness in the TNBC mouse model. A) The growth of orthotopic 4T1 tumors in mice with different treatments as indicated ( n = 6 for each group). B) Left, representative images of tumor tissues isolated from mice described in A). Right, mean tumor weights are shown. C) Representative bioluminescence images of 4T1‐bearing mice with indicated treatments. D) Left, representative pictures of hematoxylin and eosin‐stained (H&E) sections of lung tissues. Right, quantification of pulmonary metastatic nodules. E) Representative flow cytometric analysis of CD44 + CD24 −/low BCSC population in 4T1 tumors. F) Immunoblot analysis of CSCs marker, ALDH1, expression in tumor tissues with or without Rosc treatment. β ‐Actin was analyzed as a loading control. The ALDH1 expression level was quantified using ImageJ. G) Representative IHC images for ALDH1 expression in 4T1 tumor tissues with indicated treatments. The ratios of ALDH1‐positive cells in each field were quantified. H) Gene set enrichment analysis of alterations in BCSC signature genes after CDK5 inhibition. I) Immunoblot analysis of CDK5 and CDK5R1 expression in each subtype of 4T1 cells as indicated. J) Colony formation analysis of each subtype of 4T1 cells. K) Mammosphere formation in 4T1 cells treated as indicated. Left, a representative image of mammospheres. Right, quantification of mammospheres, counted using a microscope with size ≥ 50 µm. Scale bars = 50 µm. L) Representative flow cytometric analysis of CD44 high CD24 −/low BCSC population in mammospheres. M) mRNA level of BCSC‐related signature genes (Aldh1, Fgfr1, Notch1, Oct4, and Sox1). Data represent mean ± SEM. The experiments were repeated at least twice to observe concordant statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The 4T1 (GDC0294) murine mammary cell lines were obtained from China Center for Type Culture Collection (Wuhan, China), and maintained in RPMI 1640 media supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (Gibco).

Techniques: Isolation, Staining, Western Blot, Marker, Expressing, Control, Inhibition, Microscopy

Journal: Advanced Science

Article Title: CDK5 Inhibition Abrogates TNBC Stem‐Cell Property and Enhances Anti‐PD‐1 Therapy

doi: 10.1002/advs.202001417

Figure Lengend Snippet: CDK5 controls CD44v isoform switching promoting TNBC stemness. A) Sashimi plot showing a large number of reads spanning CD44 exons 5 and 14 junctions in Rosc‐treated cells. B) RT‐PCR detection of CD44 variant fragment expression. C) Representative immunofluorescence microscopy images for the analysis of CD44v expression (red) in 4T1‐bearing mice with/without Rosc treatment. Nuclei were counterstained with DAPI. Scale bars, 50 and 20 µm for the magnified field. D) Flow cytometric analysis of CD44v expression in 4T1 tumors. E) Mammosphere formation assay detecting self‐renewal capacity. The genetic recovery experiment was conducted by overexpressing CD44s/CD44v in CDK5‐KO cells as indicated. F) Immunoblotting analysis of the activation of CD44v downstream EGFR signaling pathway. Data represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The 4T1 (GDC0294) murine mammary cell lines were obtained from China Center for Type Culture Collection (Wuhan, China), and maintained in RPMI 1640 media supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (Gibco).

Techniques: Reverse Transcription Polymerase Chain Reaction, Variant Assay, Expressing, Immunofluorescence, Microscopy, Tube Formation Assay, Western Blot, Activation Assay

Journal: Advanced Science

Article Title: CDK5 Inhibition Abrogates TNBC Stem‐Cell Property and Enhances Anti‐PD‐1 Therapy

doi: 10.1002/advs.202001417

Figure Lengend Snippet: PPAR γ phosphorylation is necessary for CDK5‐induced CD44v class switching. A) Representative IHC images for CDK5 and pho‐PPAR γ expression in tissue microarrays, including normal breast and tumor tissues and negative and positive lymph nodes. Typical staining in the box region was magnified and shown in the adjacent columns. Scale bars, 20 × 10 −6 m for (5×) and 50 × 10 −6 m for (magnified). B) Correlation of CDK5 expression and phosphorylation level of PPAR γ ( n = 70, Pearson's correlation coefficient R and p ‐value are shown). C) Histoscores of pho‐PPAR γ IHC staining. Normal breast tissue ( n = 11); stage 1 ( n = 26) and stage 2 ( n = 44). D,E) Immunoblotting analyzing the expression of phosphorylation of PPAR γ at the Ser273 site in 4T1 cells after CDK5 interruption. D) Rosc treatment; E) CDK5 knockout. The levels of pho‐PPAR γ were semi‐quantified according to the gray value calculated using ImageJ (bottom panel). F) CD44v‐targeted RT‐PCR detection. Examination of CD44v expression using G) immunoblotting and H) flow cytometry in 4T1 tumors after SR‐1664 treatment. I) Mammosphere formation assay (MFA) detecting the self‐renewal capacity of transgenic 4T1 cells as indicated. J) qPCR detection of stemness‐related gene expression. Data represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The 4T1 (GDC0294) murine mammary cell lines were obtained from China Center for Type Culture Collection (Wuhan, China), and maintained in RPMI 1640 media supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (Gibco).

Techniques: Phospho-proteomics, Expressing, Staining, Immunohistochemistry, Western Blot, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Tube Formation Assay, Transgenic Assay, Gene Expression

Journal: Advanced Science

Article Title: CDK5 Inhibition Abrogates TNBC Stem‐Cell Property and Enhances Anti‐PD‐1 Therapy

doi: 10.1002/advs.202001417

Figure Lengend Snippet: Interruption of the CDK5/pho‐PPAR γ axis induces fate alteration of TNBC stem cells. A) 4T1 cells were overexpressed with PPAR γ ‐S273A and PPAR γ ‐S273D. Cycloheximide treatment revealed the half‐life of PPAR γ with a different mutation. B) The transgenic cells described in (A) were pre‐incubated with MG132 (10 × 10 −6 m ) for 2 h, then the cell lysates were subjected to ubiquitination detection. C) Immunoblotting analysis of ESPR1 expression in transgenic 4T1 cells as indicated (top) or after incubation with Rosc in different concentrations (bottom). D) RT‐qPCR detection of the expression of ESRP1 at the mRNA level. E) Co‐immunoprecipitation (co‐IP) assay analyzing the interaction between PPAR γ and ESRP1 (top). The interaction was further confirmed using reverse co‐IP (bottom). F) Representative fluorescence confocal microscopy images analyzing the co‐localization (yellow) between PPAR γ (red) and ESRP1 (green) in different treatments as indicated. Nuclei are stained with DAPI (blue). Scale bars, 50 µm. G,H) In 4T1 cells with different treatments as indicated, cycloheximide treatment examined the half‐life of ESRP1. G) Immunoblotting detected the residual of ESRP1 at the protein level and H) consequently quantified it by normalizing the intensity of the actin band. I) Transient overexpression of ESRP1 in CDK5‐KO and PPAR γ ‐S273A cells. MFA detected self‐renewal capacity. J) CD44v‐targeted RT‐PCR detection examined CD44v generation. K) Immunoblotting detected CD44v expression in the indicated cells after ESRP1 transient overexpression. Data represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The 4T1 (GDC0294) murine mammary cell lines were obtained from China Center for Type Culture Collection (Wuhan, China), and maintained in RPMI 1640 media supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (Gibco).

Techniques: Mutagenesis, Transgenic Assay, Incubation, Ubiquitin Proteomics, Western Blot, Expressing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Fluorescence, Confocal Microscopy, Staining, Over Expression, Reverse Transcription Polymerase Chain Reaction

Journal: Advanced Science

Article Title: CDK5 Inhibition Abrogates TNBC Stem‐Cell Property and Enhances Anti‐PD‐1 Therapy

doi: 10.1002/advs.202001417

Figure Lengend Snippet: Pharmacological inhibition of the stemness transformation of tumor cells enhances anti‐PD‐1 therapeutic efficacy. A) Therapy regimen (top panel) and mean tumor volume of orthotopic 4T1‐bearing mice treated with vehicle or Rosc (2 mg kg −1 ), combinational treatment with isotype and anti‐PD‐1 antibodies (100 mg per mouse) for every five days ( n = 8, 1 dead in the control group, bottom panel). B) Representative photographs of 4T1 tumor tissues isolated at the time of termination of the experiment (scale bars, 1 cm). Tumor weights are presented in the right panel. C) Representative photographs of lung metastasis are shown in the left panel, and quantification of metastatic sites is shown in the right panel. D) Kaplan–Meier survival analysis for the overall survival of mice. E) Representative IHC images of Ki‐67 and ALDH1 in 4T1 tumors with indicated treatments. Apoptosis was detected using the TUNEL assay. Scale bars, 50 µm. F) Quantification of CD3+ T‐cell, G, left) CTL, and G, right) Treg populations and H) the ratio of CTLs/Tregs in 4T1 tumors at days 14 and 28 post tumor graft ( n = 6) using flow cytometry. I) Representative flow cytometry images and quantification of IFN‐ γ expression in CD8 + TILs ( n = 6). J,K) Examination of the therapeutic effects of the combined treatment with anti‐PD‐1 and SR‐1664 in 4T1 tumors. J) mean tumor volumes were recorded at the indicated times. K) The overall survival of each group was analyzed using the Kaplan–Meier survival analysis. Data represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The 4T1 (GDC0294) murine mammary cell lines were obtained from China Center for Type Culture Collection (Wuhan, China), and maintained in RPMI 1640 media supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (Gibco).

Techniques: Inhibition, Transformation Assay, Drug discovery, Control, Isolation, TUNEL Assay, Flow Cytometry, Expressing

Journal: Scientific Reports

Article Title: In vitro and in vivo anti-cancer effects of hibernating common carp ( Cyprinus carpio ) plasma on metastatic triple-negative breast cancer

doi: 10.1038/s41598-022-06368-4

Figure Lengend Snippet: Effects of hibernating plasma on 4T1 breast cancer and L929 normal cell viability according to MTT assay. Comparison of different concentrations (1, 2, 4, 8, 16, and 32 mg/ml) of hibernating and non-hibernating plasma effects on the viability of ( A ) 4T1 breast cancer and ( B ) L929 normal fibroblast cells after 24 h incubation. ( C ) Comparison of anti-proliferative effect of different concentrations (1, 2, 4, 8, 16, and 32 mg/ml) of hibernating plasma on 4T1 cancer cells after different incubation times (24, 48, and 72 h). (ns: non-significant. *: P < 0.05).

Article Snippet: The murine triple-negative mammary cancer (4T1) and normal murine fibroblast (L929) cell lines were obtained from the Pasteur Institute (Tehran, Iran).

Techniques: Clinical Proteomics, MTT Assay, Comparison, Incubation

Journal: Scientific Reports

Article Title: In vitro and in vivo anti-cancer effects of hibernating common carp ( Cyprinus carpio ) plasma on metastatic triple-negative breast cancer

doi: 10.1038/s41598-022-06368-4

Figure Lengend Snippet: Effects of hibernating plasma on the morphology of 4T1 breast cancer and L929 normal cells. ( A ) 4T1 and ( B ) L929 cells photographs were obtained using a digital light microscope before (0 h) and 24 h after treatment with non-hibernating plasma (16 mg/ml) and hibernating plasma (16 mg/ml). The control cells were incubated with standard culture media. Arrow: rounded cell. Arrowhead: cell with irregular shape. Star: cell debris. Two-head arrow: cell with elongated cellular extensions.

Article Snippet: The murine triple-negative mammary cancer (4T1) and normal murine fibroblast (L929) cell lines were obtained from the Pasteur Institute (Tehran, Iran).

Techniques: Clinical Proteomics, Light Microscopy, Control, Incubation