Journal: Cancer Research

Article Title: Chemotherapy-Treated Breast Cancer Cells Activate the WNT Signaling Pathway to Enter a Diapause-Like Early Persister State

doi: 10.1158/0008-5472.CAN-24-4165

Figure Lengend Snippet: Distinct chemotherapy treatments converge on robust WNT/β-catenin pathway activation during early persister cell enrichment. A, Venn diagram of commonly upregulated (UP; 1,381) genes between DOC (2,633) and CAR (2,525) vs. UNT. B, Gene ontology (GO) processes from one-tailed gene set enrichment analysis (GSEA) of shared upregulated genes ( A ), ranked by positive normalized enrichment score. Red line indicates significance [−log(NOM P value) = 1.3]; highlighted terms relate to WNT/β-catenin signaling regulation. C, Heatmap of 10 WNT/β-catenin signaling genes in the MDA-MB-231 cell line treated with DOC or CAR for 72 hours, presented as Z-scores (selected from WNT/β-catenin signaling hallmark, GSEA). Data for A–C were obtained from bulk mRNA-seq of the MDA-MB-231 cell line treated with chemotherapy for 72 hours. D–F, Western blots of active (nonphosphorylated) β-catenin in MDA-MB-231 ( D ), MDA-MB-468 ( E ), and PDC-BRC-101 ( F ) cells treated with chemotherapy for 12 to 72 hours. G–I, Quantification of blots in D–F , displayed as FC to UNT; signals normalized to β-actin. Mean ± SEM. Multiple t tests, n = 3. J, Immunofluorescence of active β-catenin in MDA-MB-231 cells treated with chemotherapy for 72 hours. Scale bar, 50 μm. Images show zoomed-in regions of interest. K–M, Quantification of nuclear active β-catenin (normalized to DAPI, find nuclei – method M) in TNBC cell lines treated with chemotherapy for 12 to 72 hours. Violin plots, all points shown. Multiple t tests, Holm–Sidak correction, n = 3. N and O, Flow cytometry of %CD24 Low /CD44 High cells in MDA-MB-468 ( N ) and PDC-BRC-101 ( O ) cell lines treated with chemotherapy for 48 or 96 hours. Box-and-whisker plots. Multiple t tests, Holm–Sidak correction, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: RNA sequencing (RNA-seq) libraries were prepared using 750 ng of total RNA using the KAPA Stranded mRNA-Seq Kit (Roche, 8098123702) according to the manufacturer’s specifications.

Techniques: Activation Assay, One-tailed Test, Western Blot, Immunofluorescence, Flow Cytometry, Whisker Assay

Journal: Cancer Research

Article Title: Chemotherapy-Treated Breast Cancer Cells Activate the WNT Signaling Pathway to Enter a Diapause-Like Early Persister State

doi: 10.1158/0008-5472.CAN-24-4165

Figure Lengend Snippet: Parental and early chemotherapy-treated WNT High persister cells display diapause-like cell properties. A–C, Flow cytometry of %WNT High (GFP + ) cells (bars, right y -axis) and %viable (DAPI − ) cells (dashed line, left y -axis) in TNBC-TGP cell lines treated with DOC or CAR for 24 hours, 72 hours, 6 days, or 9 days. One-way ANOVA, Holm–Sidak correction, n = 4. D, Experimental setup for transcriptional comparison of sorted WNT High vs. WNT Low MDA-MB-231-dTGP cells treated with chemotherapy for 72 hours (bulk mRNA-seq). E, Forest plots showing association between gene signatures and WNT status in sorted WNT High vs. WNT Low cells from DOC, CAR, and UNT samples. Gene signatures include in-house signatures and hallmark gene sets (MSigDB), analyzed using GSVA. Quartile regression used to observe the median change in rescaled gene signatures after accounting for batch effects. Signatures having a nonzero positive estimate indicate increased activity in WNT High cells. F and G, Forest plots for MYC hallmark (upregulated) and diapause-DTP (1; downregulated) signatures vs. WNT status, analyzed as in E . H, Binary pattern analysis between transcriptional DTP cells (2) and WNT status of sorted WNT High /WNT Low cells obtained from samples treated with CAR or DOC and UNT samples. Enrichment status score of −1 (light blue) indicates that a given hallmark/process is downregulated in DTPs or sorted WNT High cells, whereas a score of 1 (red) indicates that a given hallmark/process is upregulated in DTPs or sorted WNT High cells. Data for E–H were obtained from bulk mRNA-seq of sorted WNT High /WNT Low of MDA-MB-TGP cells treated with DOC or CAR for 72 hours. I, Absorbance values of cellular metabolic activity indicating cell number in sorted MDA-MB-231-TGP and MDA-MB-468-TGP cell lines 1 week after sorting (initial treatment was 72 hours of chemotherapy). Multiple t tests, Holm–Sidak correction, n = 3. J, Flow cytometry of apoptotic (% Annexin V + ) cells and corresponding WNT status (%WNT High cells) in MDA-MB-231-TGP and PDC-BRC-101-TGP cell lines treated with chemotherapy for after 96 hours. Multiple t tests, Holm–Sidak correction, n = 5. Unless specified otherwise, all data are presented as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. CI, confidence interval. D, Created in BioRender. Lluis Vinas, F. (2025) https://BioRender.com/yen63o9 .

Article Snippet: RNA sequencing (RNA-seq) libraries were prepared using 750 ng of total RNA using the KAPA Stranded mRNA-Seq Kit (Roche, 8098123702) according to the manufacturer’s specifications.

Techniques: Flow Cytometry, Comparison, Activity Assay