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Fig.2.E showing Flow cytometry/Cell sorting from the publication: Concomitant activation of GLI1 and Notch1 contributes to racial disparity of human triple negative breast cancer progression.

Fig.1.A showing Flow cytometry/Cell sorting in a Homo sapiens (Human) sample from the publication: Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in ductal carcinoma in situ.

Fig.2.A showing Flow cytometry/Cell sorting in a Homo sapiens (Human) sample from the publication: Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in ductal carcinoma in situ.

Fig.1.A showing Flow cytometry/Cell sorting in a Homo sapiens (Human) sample from the publication: Wnts enhance neurotrophin-induced neuronal differentiation in adult bone-marrow-derived mesenchymal stem cells via canonical and noncanonical signaling pathways.

Fig.2.F showing Western Blotting from the publication: Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

Fig.2.G showing Western Blotting from the publication: Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

Fig.4.A showing Western Blotting from the publication: Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

Fig.5.F showing Immunocytochemistry-immunofluorescence from the publication: Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

Fig.6.A showing Immunocytochemistry-immunofluorescence from the publication: Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

Fig.6.B showing Immunocytochemistry-immunofluorescence from the publication: Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

Fig.6.D showing Immunocytochemistry-immunofluorescence from the publication: Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

11 images found

Xanthohumol modulate autophagy and ER stress to counteract stemness and enhance cisplatin efficacy in head and neck squamous cell carcinoma.

In Scientific Reports on 16 April 2025 by Del Vecchio, V., Sanchez-Pajares, I. R., et al.

Natural compounds have been increasingly investigated for their efficient anti-cancer activity. Xanthohumol (XN), a flavonoid derived from hops, has shown promise in preclinical studies for various cancers due to its unique biological properties. This study investigates the effects of XN and a cost-effective hop extract (HOP EX) on head and neck squamous cell carcinoma (HNSCC), focusing on their potential to modulate cancer stemness and enhance the efficacy of Cisplatin chemotherapy. Using a combination of flow cytometry, qPCR, and cellular assays, we assessed the impact of XN and HOP EX on cell viability, stemness, and chemoresistance in HNSCC cell lines. Further, we explored the underlying mechanisms by examining the induction of apoptosis, ER stress, and autophagy activation. Our findings demonstrate that both XN and HOP EX significantly decrease cell viability and stemness in HNSCC cells and enhance the cytotoxic effects of Cisplatin, suggesting a synergistic interaction. Mechanistically, we identified that the induction of ER stress and subsequent activation of the unfolded protein response (UPR) promote autophagy, leading to increased apoptosis. By modulating key cellular pathways such as ER stress and autophagy, these natural compounds could be developed into supportive treatments for HNSCC.
© 2025. The Author(s).

BMSC-derived exosomes promote osteoporosis alleviation via M2 macrophage polarization.

In Molecular Medicine on 19 November 2024 by Zhang, Y., Bai, J., et al.

Osteoporosis is characterized by reduced bone mass due to imbalanced bone metabolism. Exosomes derived from bone mesenchymal stem cells (BMSCs) have been shown to play roles in various diseases. This study aimed to clarify the regulatory function and molecular mechanism of BMSCs-derived exosomes in osteogenic differentiation and their potential therapeutic effects on osteoporosis. Exosomes were extracted from BMSCs. Bone marrow-derived macrophages (BMDMs) were cultured and internalized with BMSCs-derived exosomes. Real-time quantitative PCR was used to detect the expression of macrophage surface markers and tripartite motif (TRIM) family genes. BMDMs were co-cultured with human osteoblasts to assess osteogenic differentiation. Western blot was performed to analyze the ubiquitination of triggering receptor expressed on myeloid cell 1 (TREM1) mediated by TRIM25. An ovariectomized mice model was established to evaluate the role of TRIM25 and exosomes in osteoporosis. Exosomes were successfully isolated from BMSCs. BMSCs-derived exosomes upregulated TRIM25 expression, promoting M2 macrophage polarization and osteogenic differentiation. TRIM25 facilitated the ubiquitination and degradation of TREM1. Overexpression of TREM1 reversed the enhanced M2 macrophage polarization and osteogenic differentiation caused by TRIM25 overexpression. TRIM25 enhanced the protective effect of BMSCs-derived exosomes against bone loss in mice. These findings suggested that BMSCs-derived exosomes promoted osteogenic differentiation by regulating M2 macrophage polarization through TRIM25-mediated ubiquitination and degradation of TREM1. This mechanism might provide a novel approach for treating osteoporosis.
© 2024. The Author(s).

NSDHL contributes to breast cancer stem-like cell maintenance and tumor-initiating capacity through TGF-β/Smad signaling pathway in MCF-7 tumor spheroid.

In BMC Cancer on 8 November 2024 by Yoon, S. H., Lee, S., et al.

NAD(P)-dependent steroid dehydrogenase-like protein (NSDHL), which is involved in breast tumor growth and metastasis, has been implicated in the maintenance of cancer stem cells. However, its role in regulating breast cancer stem-like cells (BCSCs) remains unclear. We have previously reported the clinical significance of NSDHL in patients with estrogen receptor-positive (ER +) breast cancer. This study aimed to elucidate the molecular mechanisms by which NSDHL regulates the capacity of BCSCs in the ER + human breast cancer cell line, MCF-7.
NSDHL knockdown suppressed tumor spheroid formation in MCF-7 human breast cancer cells grown on ultralow-attachment plates. RNA sequencing revealed that NSDHL knockdown induced widespread transcriptional changes in the MCF-7 spheroids. TGF-β signaling pathway was the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (fold change ≥ 2, P ≤ 0.05) identified in NSDHL-knockdown MCF-7 spheroids compared with the control. In orthotopic tumor models injected with NSDHL-knockdown MCF-7 spheroids, tumor initiation and growth were strongly suppressed compared with those in the control.
BCSC populations with CD44+/CD24- and CD49f+/EpCAM + phenotypes and high ALDH activity were decreased in NSDHL-knockdown MCF-7 spheroids and xenograft tumors relative to controls, along with decreased secretion of TGF-β1 and 3, phosphorylation of Smad2/3, and expression of SOX2. In RNA-sequencing data from The (TCGA) database, a positive correlation between the expression of NSDHL and SOX2 was found in luminal-type breast cancer specimens (n = 998). Our findings revealed that NSDHL plays an important role in maintaining the BCSC population and tumor-initiating capacity of ER-positive MCF-7 spheroids, suggesting that NSDHL is an attractive therapeutic target for eliminating BCSCs, thus preventing breast cancer initiation and progression.
Our findings suggest that NSDHL regulates the BCSC/tumor-initiating cell population in MCF-7 spheroids and xenograft tumors.
© 2024. The Author(s).

Preconditioning of Human Umbilical Cord Mesenchymal Stem Cells with a Histone Deacetylase Inhibitor: Valproic Acid.

In Balkan Medical Journal on 6 September 2024 by Isildar, B., Ozkan, S., et al.

Mesenchymal stem cells (MSCs) play a key role in regenerative medicine due to their capacity to differentiate into multiple cell lines, regulate the immune system, and exert paracrine effects. The therapeutic impact of MSCs is primarily mediated through their secretome. The secretory and therapeutic potential of MSCs can be improved through preconditioning, which entails the application of hypoxic environments, 3-dimensional cell cultures, and pharmacological agents. Valproic acid (VPA) is a histone deacetylase inhibitor that is employed in medical practice for treating epilepsy and bipolar disorder. Hence, preconditioning MSCs with VPA is expected to induce histone acetylation, enhance gene expression, and beneficially modify the cells' secretomes.
To assess the effectiveness of VPA in enhancing and regulating the therapeutic potential of cells as well as its impact on MSC secretome profiles and ultrastructural morphologies.
Expiremental study.
Human umbilical cord MSCs were preconditioned with 2 mM VPA for 24 and 48 hours; untreated MSCs served as controls. The secretome secreted by the cells was assessed for its total protein content. Subsequently, interferon-gamma (IFN-γ), interleukin-17 (IL-17), IL-10, vascular endothelial growth factor, nerve growth factor (NGF), glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor (BDNF) levels in the secretome were analyzed using the ELISA method. The ultrastructural properties of the cells were studied under transmission electron microscopy.
Ultrastructural examinations revealed that the chromatin content of VPA-treated cells was reduced. VPA-preconditioned cells exhibited a higher density of rough endoplasmic reticulum, autophagic vesicles, and myelin figures on cytoplasmic structure analysis, which was indicative of increased secretion. Protein secretion was elevated in those cells, with notable increases in NGF and BDNF levels. Furthermore, the cytoskeletal rearrangement and elevated autophagic activity observed in the 48-hour preconditioned cells could indicate the initiation of neuronal differentiation. IL-10, IL-17, and IFN-γ were not detected in the secretome.
This study indicate that preconditioning with VPA enhances MSC activity and subsequently modifies the secretome content.

Human post-implantation blastocyst-like characteristics of Muse cells isolated from human umbilical cord.

In Cellular and Molecular Life Sciences : CMLS on 11 July 2024 by Kushida, Y., Oguma, Y., et al.

Muse cells, identified as cells positive for the pluripotent surface marker SSEA-3, are pluripotent-like endogenous stem cells located in the bone marrow (BM), peripheral blood, and organ connective tissues. The detailed characteristics of SSEA-3(+) cells in extraembryonic tissue, however, are unknown. Here, we demonstrated that similar to human-adult tissue-Muse cells collected from the BM, adipose tissue, and dermis as SSEA-3(+), human-umbilical cord (UC)-SSEA-3(+) cells express pluripotency markers, differentiate into triploblastic-lineage cells at a single cell level, migrate to damaged tissue, and exhibit low telomerase activity and non-tumorigenicity. Notably, ~ 20% of human-UC-SSEA-3(+) cells were negative for X-inactive specific transcript (XIST), a naïve pluripotent stem cell characteristic, whereas all human adult tissue-Muse cells are XIST-positive. Single-cell RNA sequencing revealed that the gene expression profile of human-UC-SSEA-3(+) cells was more similar to that of human post-implantation blastocysts than human-adult tissue-Muse cells. The DNA methylation level showed the same trend, and notably, the methylation levels in genes particularly related to differentiation were lower in human-UC-SSEA-3(+) cells than in human-adult tissue-Muse cells. Furthermore, human-UC-SSEA-3(+) cells newly express markers specific to extraembryonic-, germline-, and hematopoietic-lineages after differentiation induction in vitro whereas human-adult tissue-Muse cells respond only partially to the induction. Among various stem/progenitor cells in living bodies, those that exhibit properties similar to post-implantation blastocysts in a naïve state have not yet been found in humans. Easily accessible human-UC-SSEA-3(+) cells may be a valuable tool for studying early-stage human development and human reproductive medicine.
© 2024. The Author(s).

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Concomitant activation of GLI1 and Notch1 contributes to racial disparity of human triple negative breast cancer progression.

In Elife on 10 December 2021 by Siddharth, S., Parida, S., et al.

Fig.2.E

African American (AA)-triple negative breast cancer (TNBC) cells possess higher stem cell enriched population than White American (WA)-TNBC cells.(A) Gene set enrichment analysis (GSEA) of hallmark epithelial-mesenchymal transition and Tavazoie metastasis pathway in cancer between AA-TNBC samples and WA-TNBC samples from GSE46581 dataset (Gene Expression Omnibus). (B) Differential expression statistics comparing AA-TNBC cells (MDA-MB-468, HCC1806, HCC1569) with WA-TNBC cells (Hs578t, BT549, BT20, HCC1937, MDA-MB-231) were calculated and results are shown in a heatmap (p ≤ 0.05). Genes are marked on the side. (C, D) Representative images of mammospheres formed by HCC1806, HCC1569, MDA-MB-468, HCC1937, BT549, and Hs578t cells. Bar graphs show quantification of solid mammospheres. Data represents n = 4 independent experiments. *p ≤ 0.05. (E) Flow cytometry analysis of AA-TNBC and WA-TNBC cells using CD44 and CD49f staining. (F) Immunoblot analysis of NANOG, OCT4, SOX2, KLF4, and c-MYC in HCC1806, HCC1569, Hs578t, and BT549 cells. Expression of ACTB served as the loading control. (G) Immunofluorescence analysis of cMyc, OCT4, NANOG, and SOX2 in MDA-MB-468, HCC1569, HCC1806, HCC1937, BT549, and Hs578t cells. Nuclei are stained with DAPI. Scale bar, 20 µm. (H) Normalized mRNA expression of CD44 (*p = 0.102), KLF4 (*p = 0.010), MYC (*p = 0.002), and NANOG (*p = 0.023) in WA- and AA-TNBC patients from TCGA and Affymatrix datasets.Figure 2—source data 1.Western blot data for Figure 2F.Western blot data for Figure 2F. less

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Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in ductal carcinoma in situ.

In Nat Commun on 13 September 2019 by Ding, L., Su, Y., et al.

Fig.1.A

Heterogeneity of the CD10+ cell population. a FACS analysis of CD10+ cells according to expression of CD44 in normal breast tissues of nulliparous (NP) and parous (P) control women and BRCA1 and BRCA2 mutation carriers. b Quantification of percentage of CD10+CD44− and CD10+CD44+ in total epithelial cells (n = 6/group). p-values indicate statistical significance of difference in total CD10+ cells between groups by t-test. c viSNE maps from CyTOF analysis of normal breast tissues colored for expression of SMA, CD10, CD44, and CD49f (control n = 6, BRCA1n = 6, BRCA2n = 7). Color scale indicates minimum and maximum values of expression. d Pathways enriched in genes differentially expressed between CD10+CD44− and CD10+CD44+ cells. Color scale corresponds to −log(p-value) of significance of enrichment, calculated by MetaCore Enrichment Analysis test. e 3D Principal component analysis plots of CD10+ gene expression data from the indicated samples. f Transcription factors differentially expressed between CD10+CD44− and CD10+CD44+ cells. Red highlight indicates genes selected for further analyses. g Multicolor immunofluorescence analysis of p63, TCF7, and CD10 expression in normal breast tissues. Images are a montage of nine fields captured from one area of the tissue. Scale bar 50 μm. h Relative quantification of CD10+ myoepithelial cells positive for p63 or TCF7 or both proteins. p-value indicates the significant association of the p63/TCF7 positive/negative status of CD10+ myoepithelial cells with condition (Control, BRCA1, or BRCA2), as assessed by Pearson’s chi-squared test among averages of estimated cell counts across replicates (total population size was conservatively estimated to 100 cells). Source data are provided as a Source Data file less

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Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in ductal carcinoma in situ.

In Nat Commun on 13 September 2019 by Ding, L., Su, Y., et al.

Fig.2.A

CD10, p63, and TCF7 expression in DCIS and in invasive breast tumors. a FACS analysis of CD10 and CD44 expression in DCIS and in normal breast tissue. b Relative quantification of CD10+CD44− and CD10+CD44+ in total epithelial cells. p-value indicates the significant association of the CD44 positive/negative status of CD10+ cells with condition (Normal, DCIS), as assessed by Pearson’s chi-squared test among averages of estimated cell counts across replicates (total population size was conservatively estimated to 100 cells). c 3D principal component analysis plot of gene expression data. d Pathways enriched in genes differentially expressed between CD10+ cells in DCIS and in normal breast. Color scale corresponds to −log(p-value) of significance of enrichment, calculated by MetaCore Enrichment Analysis test. e Multicolor immunofluorescence analysis of p63, TCF7, and CD10 expression in low grade (LG) and high grade (HG) DCIS and DCIS-IDC. Left panels are a montage of nine fields captured from one area of the tissue, while right panels are high magnification of selected areas. Scale bar 50 μm. f Relative quantification of CD10+ cells positive for p63 or TCF7 or both proteins in low (LG) and high (HG) grade pure DCIS and DCIS adjacent to IDC (DCIS-IDC). p-value indicates the significant association of the p63/TCF7 positive/negative status of CD10+ cells with condition (LG-DCIS, HG-DCIS, and DCIS-IDC), as assessed by Pearson’s chi-squared test among averages of estimated cell counts across replicates (total population size was conservatively estimated to 100 cells). g Multicolor immunofluorescence analysis of PanCK, p63, and TCF7 in luminal, basal-like, and metaplastic invasive breast tumors. Images are a montage of nine fields captured from one area of the tissue. Scale bar 50 μm. h Scoring the expression and co-localization of p63 and TCF7 in different subtypes of metaplastic breast tumors. Source data are provided as a Source Data file less

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Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

In PLoS One on 11 December 2013 by Kim, K. J., Godarova, A., et al.

Fig.2.F

Loss of Rb expression promotes metastatic behavior in human breast cancer cell lines.(A) Phase contrast images and quantification of mammosphere-forming potency of MCF7ras and T47D cells with Rb knockdown. Arrows indicate protrusions formed by invading cells and cell clusters. Scale bar, 50 μm; unequal variance Student's t-test, * p<0.05, ** p<0.01, *** p<0.001. (B) Morphology of primary tumors and lymphovascular (LV) or mammary fat pad (MFP) invasion. MCF7ras cells stably expressing shRNA against Rb or scrambled sequence were injected into the MFP of NOD/SCID mice. Primary tumors and other tissues were harvested after 8 weeks and H&E stained. Bar graph depicts primary tumor weight in each group expressed as mean ± SD where n is the number of animals in each group. Arrows indicate sites of lymphovascular invasion in Rb knockdown experiment. Scale bar, 50 μm (left panel) and 25 μm (middle panel). (C) Fluorescent and H&E stained images of metastatic tumor growth in lungs. Metastases containing EGFP-expressing Rb knockdown or control cells originated from orthotopic site. Images from n number of animals were quantified using ImageJ software (NIH). Data are presented as mean ± SD. Scale bar, 5 mm or 100 μm; equal variance Student's t-test, ** p<0.01, *** p<0.001. (D) Scheme showing isolation of circulating cancer cells (CCC) from mice bearing primary tumors. CCC were co-separated with the mononuclear fraction of blood and adhered to Poly-L-lysine-coated plates. (E) Fluorescent images and quantification of isolated EGFP-expressing circulating cancer cells and cell clusters. Scale bar, 200 μm; unequal variance Student's t-test, * p<0.05, ** p<0.01. (F) Western blot analysis of cell lysates from in vitro cultures of MCF7ras cells expressing Rb or control shRNA. β-tubulin staining was used as a loading control. (G) Western blot analysis of lysates from primary tumors. Total Akt was used as a loading control. less

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Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

In PLoS One on 11 December 2013 by Kim, K. J., Godarova, A., et al.

Fig.2.G

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Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

In PLoS One on 11 December 2013 by Kim, K. J., Godarova, A., et al.

Fig.5.F

CD44 expression is essential for lymphovascular invasion and lung metastases initiated by Rb knockdown.(A) Weight of orthotopic primary tumors initiated by MCF7ras cells with double knockdown of Rb and CD44 eight weeks after mammary fat pad injection. Rb shRNAs and one of the control shRNAs were expressed in a vector conferring puromycin resistance. CD44 shRNAs and the second control shRNA were expressed in an EGFP vector. Data are presented as mean ± SD, n is the number of mice in each group. Equal variance Student's t-test, * p<0.05, *** p<0.001. (B) Representative phase contrast and fluorescent images of tumor-attached EGFP-positive cancer cells/clusters invading mammary fat pad or adjacent capillaries. Analysis was performed on the whole animal post mortem. Scale bar, 2 mm. (C) Representative high magnification phase contrast and fluorescent images of Rb-1 shRNA from Figure 5B with visible MFP and lymphovascular invasion. Scale bar, 500 μm. (D) H&E staining of primary tumors from mice injected with Rb and CD44 double knockdown cells. Arrows indicate areas of lymphovascular invasion. Scale bar, 200 μm. (E) Quantification of collective cell invasion into host mammary fat pad and extratumoral capillaries. Mammary fat pad invasion was quantified as incidence of EGFP-positive cell clusters in the area adjacent to tumor and not within a capillary (as judged by presence of erythrocytes in the phase contrast image). Cell clusters considered to be within a capillary were counted as lymphovascular invasion. Number of animals in each group is indicated as n. Unequal variance Student's t-test, * p<0.05, ** p<0.01, *** p<0.001. (F) Fluorescent images and quantification of lung metastases' numbers initiated by primary tumors 8 weeks after implantation of MCF7ras cells with double knockdown of Rb and CD44 into mammary fat pad. Number of metastases were quantified by ImageJ and normalized to the weight of primary tumor; number of animals in each group is indicated by n. Scale bar on fluorescent images, 3 mm. Unequal variance Student's t-test, * p<0.05, ** p<0.01, *** p<0.001. less

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Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

In PLoS One on 11 December 2013 by Kim, K. J., Godarova, A., et al.

Fig.6.A

Regulation of incidence of circulating single/clustered cancer cells (CCCS/CCCC) by Rb and CD44.(A) Fluorescent images and quantification of circulating live cancer cells and cell clusters co-isolated with mononuclear fraction from blood of animals bearing tumors. The tumors were induced by orthotopic implantation of MCF7ras cells expressing shRNAs against Rb, CD44, and their combination, along with respective controls. EGFP is expressed in cytoplasm and its presence indicates both cancer cell identity and presumably intact membrane. Number of animals in each group is depicted on the graph. Scale bar, 10 μm; unequal variance Student's t-test, ** p<0.01, *** p<0.001. (B) Fluorescent images and quantification of total circulating cancer cells (both alive and dead) detected with a human-specific antibody to a nuclear antigen. Data are presented as mean ± SEM. Scale bar, 10 μm; unequal variance Student's t-test, ** p<0.01, *** p<0.001. (C) Number of CCCS and CCCC in the total number of cancer cells detected with a human-specific antibody to a nuclear antigen. Data are presented as mean ± SEM; unequal variance Student's t-test, ** p<0.01, *** p<0.001. (D) Fluorescent images and quantification of CD44 positive cancer cells detected with a human-specific anti-CD44 antibody. Data are presented as mean ± SEM. Scale bar, 10 μm; unequal variance Student's t-test, ** p<0.01, *** p<0.001. less

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Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

In PLoS One on 11 December 2013 by Kim, K. J., Godarova, A., et al.