Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Inhibition of cell viability and colony formation, and induction of apoptosis by riluzole. ( A , B ), Pancreatic cancer cell lines (AsPC-1, PANC-1, and MIA PaCa-2), pancreatic cancer stem cells (Pan CSCs), and human normal pancreatic ductal epithelial (HPNE) cells were seeded in 96-well plates, treated with or without riluzole (0–40 μM) for 72 h and cell viability was measured by CellTiter-Glo Luminescent Cell Viability Assay (Promega). ( C ), Pancreatic cancer cell lines (AsPC-1, PANC-1, MIA PaCa-2 and Hs 766 T) and Pan CSCs were seeded in dishes and treated with riluzole (0–40 μM). Number of colonies formed at 21 days were counted as described in Material and Methods. ( D ), Pancreatic cancer cell lines (AsPC-1, PANC-1, MIA PaCa-2 and Hs 766 T) and CSCs were seeded and treated with riluzole (0–40 µM) for 72 h. Apoptosis was measured by TUNEL assay. Data represent mean ± SD (n = 4). *, #, and % = significantly different from control and each other; p < 0.05.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: Inhibition, Cell Viability Assay, TUNEL Assay, Control

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Riluzole regulates Wnt-β-catenin and mitochondrial pathways by RNA-Seq. Pancreatic cancer cells (MIA PaCa-2) were treated with Riluzole for 36 h. RNA was isolated. RNA-Seq was performed as described in Materials and methods. ( A ), Heat Map. Differential expression of genes in MIA PaCa-2 cells treated with or without riluzole. ( B ), Data analysis identified modulation of Wnt-β-catenin and mitochondrial pathways by riluzole in pancreatic cancer cells.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: RNA Sequencing, Isolation, Quantitative Proteomics

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Inhibition of β-catenin/TCF-LEF1 pathway by riluzole. ( A – F ), Pancreatic cancer MIA PaCa-2 cells were treated with riluzole (0–40 µM) for 36 h. RNA was extracted and the expression of β-catenin, Wnt3a, Wnt5a, WSP, TCF and LEF was measured by q-RT-PCR. Data represent mean ± SD (n = 4). *, and # = significantly different from control and each other; p < 0.05. ( G ), MIA PaCa-2 cells were stably transduced with TCF/LEF1-responsive GFP/firefly luciferase viral particles (pGreen Fire1-TCF/LEF1 with EF1, System Biosciences). After transduction, cells were treated with riluzole (0–40 µM) for 36 h. TCF/LEF1 reporter activity was measured by luciferase assay as we described . Data represent mean ± SD (n = 4). *, # and % = significantly different from control and each other; p < 0.05. ( H ), MIA PaCa-2 cells were treated with riluzole (0–40 µM) for 36 h. Cells were harvested and cytoplasmic and nuclear fractions were prepared. The expression of β-catenin in the cytoplasmic and nuclear fractions was measured by the Western blot analysis. β-Actin and Lamin B1 were used as loading controls in cytoplasmic and nuclear fractions, respectively. ( I–N ), Pancreatic cancer AsPC-1 cells were treated with riluzole (0–40 µM) for 36 h. RNA was extracted and the expression of β-catenin, Wnt3a, Wnt5a, WSP, TCF and LEF was measured by q-RT-PCR. Data represent mean ± SD (n = 4). *, # and % = significantly different from control and each other; p < 0.05. ( O ), AsPC-1 cells were stably transduced with TCF/LEF1-responsive GFP/firefly luciferase viral particles (pGreen Fire1-TCF/LEF1 with EF1, System Biosciences). After transduction, cells were treated with riluzole (0–40 µM) for 36 h. TCF/LEF1 reporter activity was measured by luciferase assay as we described . Data represent mean ± SD (n = 4). *, # and % = significantly different from control and each other; p < 0.05. ( P ), AsPC-1 cells were treated with riluzole (0–40 µM) for 36 h. Cells were harvested and cytoplasmic and nuclear fractions were prepared. The expression of β-catenin in the cytoplasmic and nuclear fractions was measured by the Western blot analysis. β-Actin and Lamin B1 were used as loading controls in cytoplasmic and nuclear fractions, respectively.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Stable Transfection, Transduction, Luciferase, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Inhibition of TCF-LEF1 target genes and induction of growth arrest at G2/M stage by riluzole. ( A ), Pancreatic cancer MIA PaCa-2 and AsPC-1 cells were treated with riluzole (0–40 µM) for 36 h. RNA was isolated and the expression of cyclin D1 was measured by q-RT-PCR. Data represent mean ± SD (n = 4). *, # and % = significantly different from control and each other; p < 0.05. ( B ), Cell cycle analyses. MIA PaCa-2 and AsPC-1 cells were treated with riluzole (0–40 µM) for 48 h. Cells were harvested, fixed, stained with propidium iodide (PI) and analysed with flow cytometer. *, # and % = significantly different from control and each other; p < 0.05. ( C ), MIA PaCa-2 cells were treated with riluzole (0–40 µM) for 36 h. RNA was isolated and the expression of cMyc, Axin1, GSK3β, PDK1, and MCT1 was measured by q-RT-PCR. Data represent mean ± SD (n = 4). *, # and % = significantly different from control and each other; p < 0.05. ( D ), AsPC-1 cells were treated with riluzole (0–40 µM) for 36 h. RNA was isolated and the expression of cMyc, Axin1, GSK3β, PDK1, and MCT1 was measured by q-RT-PCR. Data represent mean ± SD (n = 4). *, # and % = significantly different from control and each other; p < 0.05.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: Inhibition, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Riluzole inhibits Glut2 expression, glucose uptake, LDHA-A expression, and NAD + level. ( A ), Pancreatic cancer (PANC-1, Hs 766 T, MIA PaCa-2 and AsPC-1) cells and Pan CSCs were treated with riluzole (0–10 µM) for 36 h. RNA was extracted and the expression of Glut-2 was measured by q-RT-PCR. Data represent mean ± SD (n = 4). * = significantly different from control; p < 0.05. ( B ), Glucose uptake. Pancreatic cancer cells and CSCs were labelled with 2.5 µg/ml of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBD Glucose) for 30 min and treated with riluzole for 24 h. Cells were washed and resuspended in PBS. Fluorescence was measured (excitation 544 nm and emission at 590 nm). Data represent mean ± SD (n = 4). * = significantly different from control; P < 0.05. ( C ), Pancreatic cancer ( PANC-1, Hs 766 T, MIA PaCa-2 and AsPC-1) cells, and Pan CSCs were treated with riluzole (0–10 µM) for 36 h. RNA was isolated and the expression of LDH-A was measured by q-RT-PCR. Data represent mean ± SD (n = 4). * = significantly different from control; p < 0.05. ( D ), Pancreatic cancer ( PANC-1, Hs 766 T, MIA PaCa-2 and AsPC-1) cells were treated with riluzole (0–10 µM) for 1 h and total cellular NAD + concentration was measured at 450 nm by a NAD/NADH assay kit (Cayman). Data represent mean ± SD (n = 4). * = significantly different from control; p < 0.05.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Fluorescence, Isolation, Concentration Assay, Nad NADH Assay

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Requirement of glutamine, inhibition of glutamate efflux and GSH level, and upregulation of reactive oxygen species (ROS) in PDAC. ( A ), PANC1 cells were grown in the absence or presence of glutaminase (GLS) inhibitor (BPTES or DON, 5 mM) with glutamine (2 mM) in the medium. After 48 h, apoptosis was measured by TUNEL assay. Data represent mean ± SD (n = 4). * = significantly different from control, p < 0.05. ( B ), PANC-1 cells were grown in the presence (2 mM) or absence of glutamine for 48 h. Apoptosis was measured by TUNEL assay. Data represent mean ± SD (n = 4). * = significantly different from control, p < 0.05. ( C ), PANC-1, Hs 766 T, MIA PaCa-2, AsPC-1, and Pan CSCs were treated with riluzole (0–10 µM) for 24 h. Glutamate release was measured by the Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit with a fluorometer using excitation at 540 nm and emission at 590 nm (Invitrogen). Data represent mean ± SD (n = 4). * = significantly different from control, p < 0.05. ( D ), Pancreatic cancer PANC-1, Hs 7ssT, MIA PaCa-2, AsPC-1 cells and CSCs were treated with riluzole (0–10 µM) for 24 h. Intracellular total GSH was detected by measuring the product of glutathionylated DTNB at 405 nm (GSH assay kit, Cayman Chemical). Data represent mean ± SD (n = 4). * = significantly different from control, P < 0.05. ( E and F ), AsPC-1 and PANC-1 cells were pre-treated with NAC (3 mM) for 2 h, followed by treatment with riluzole (0–10 µM) for 24 h. Cells were labelled with 2’,7’-dichlorofluorescein diacetate (DCFDA / H2DCFDA) and ROS production was measured for various time points (0- 360 min) with a fluorometer using excitation at 495 nm and emission at 529 nm (Cellular Reactive Oxygen Species Detection Assay Kit, Abcam). Data represent mean ± SD (n = 4). *, @, #, $. %, &, and ** = significantly different from control; p < 0.05. ( G ), AsPC-1 and Pan CSCs were pre-treated with NAC (3 mM) for 2 h, followed by treatment with riluzole (0–10 µM) for 24 h. Cells were labelled with 2’,7’-dichlorofluorescein diacetate (DCFDA / H2DCFDA) and ROS production was measured at 120 min with a fluorometer using excitation at 495 nm and emission at 529 nm. Data represent mean ± SD (n = 4). * = significantly different from control and each other; p < 0.05.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: Inhibition, TUNEL Assay, Control, GSH Assay, Detection Assay

Journal: Scientific Reports

Article Title: Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

doi: 10.1038/s41598-022-13472-y

Figure Lengend Snippet: Riluzole inhibits pancreatic cancer growth in KPC (Pdx1-Cre, LSL-Trp53 R172H , and LSL-Kras G12D ) transgenic mice. KPC mice (about 6 weeks old) were injected ip with riluzole (20 mg/kg, Monday through Friday) for 12 weeks. At the end of the treatment, mice were sacrificed. ( A ) Pancreas Weight. Mice were euthanized and pancreas weight was taken from Cre mice (normal, untreated), KPC mice (control, untreated), and KPC mice (treated with riluzole 20 mg/kg). Data represent mean ± SD (n = 7). * and # = significantly different from control group; p < 0.05. ( B ), Histological examination of the pancreas was performed by H&E staining. Numbers of PanINs and PDAC were quantified. Data represent mean ± SD (n = 7). *, #, %, and & = significantly different from control group; p < 0.05. X = not detected.

Article Snippet: Human pancreatic cancer stem cells (CSCs, CD24 + /CD44 + /CD133 +) were isolated from primary tumors and grown in stem cell culture medium as per the supplier’s instructions (obtained from Celprogen, Torrance, CA).

Techniques: Transgenic Assay, Injection, Control, Staining

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Sesn3 deficiency promotes carcinogen-induced hepatocellular carcinoma via regulation of the hedgehog pathway

doi: 10.1016/j.bbadis.2019.07.011

Figure Lengend Snippet: Representative immunofluorescence analysis of Acta2 (α-SMA), Col1, and Col3 (A). Analysis of fluorescence intensity for Acta2, Col1, and Col3 (B). Hepatic mRNA levels of Pdgfrb, Col1a1, and Acta2 (C). Immunoblot analysis of hepatic Acta2 and Pdgfrb (D). Representative immunofluorescence analysis of hepatic AFP, CK-19, Arg-1, Cd133, and Cd44 (E). Analysis of fluorescence intensity of hepatic AFP, CK-19, Argl, Cd133, and Cd44 (F). Data are expressed as mean ± SEM (n = 4 to 6 each group). #p < 0.05 and ##p < 0.01 for KO vs. WT. Immunofluorescence image analyses were performed using a fluorescence microscope (ZEISS, x200 magnification).

Article Snippet: Immunofluorescence (IF) analysis was performed for detecting general HCC markers including Arginase-1 (Arg1, rabbit mAb, Cat. No. 93668S, Cell Signaling Technology), alpha-fetoprotein (AFP, mouse mAb, sc-8399, Santa Cruz Biotechnology), and cytokeratin 19 (CK-19, rat mAb, TROMA-III, DSHB), ECM molecules such as Collagen 1 and 3 (Col1, rabbit anti-Collagen I antibody, #ab21286; Col3, rabbit anti-Collagen III antibody, #ab7778, Abcam), and alpha-smooth muscle actin (α-SMA, rabbit anti-αSMA antibody, #ab5694, Abcam), and cancer stem cell markers like CD44 (mouse anti-CD44 antibody, #sc-7297, Santa Cruz Biotechnology) and CD 133 (rabbit anti-CD133 antibody, #NBP244250, Novus Biologicals).

Techniques: Immunofluorescence, Fluorescence, Western Blot, Microscopy

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Sesn3 deficiency promotes carcinogen-induced hepatocellular carcinoma via regulation of the hedgehog pathway

doi: 10.1016/j.bbadis.2019.07.011

Figure Lengend Snippet: Differentially expressed genes (DEGs) from the RNA-seq analysis of WT and Sens3-KO livers were presented by the volcano plot (A). A selected list of DEGs were presented in the table (B). Top 10 biological processes were over-represented in the significantly upregulated (C) and downregulated (D) genes. Hepatic Cd44 and Cd133 mRNA analysis by real-time qPCR (E). Immunoblot analysis of cancer signaling-related proteins in the liver of WT and Sesn3 KO mice (F). Co-IP analysis of Sesn3 and Gli2 interaction in HEK 293T cells (G). Data are expressed as mean ± SEM (n=4 for the immunoblot analysis, n=10 for ELISA, n=6 for qPCR analysis, respectively). #p < 0.05 and ##p < 0.01 for KO vs. WT.

Article Snippet: Immunofluorescence (IF) analysis was performed for detecting general HCC markers including Arginase-1 (Arg1, rabbit mAb, Cat. No. 93668S, Cell Signaling Technology), alpha-fetoprotein (AFP, mouse mAb, sc-8399, Santa Cruz Biotechnology), and cytokeratin 19 (CK-19, rat mAb, TROMA-III, DSHB), ECM molecules such as Collagen 1 and 3 (Col1, rabbit anti-Collagen I antibody, #ab21286; Col3, rabbit anti-Collagen III antibody, #ab7778, Abcam), and alpha-smooth muscle actin (α-SMA, rabbit anti-αSMA antibody, #ab5694, Abcam), and cancer stem cell markers like CD44 (mouse anti-CD44 antibody, #sc-7297, Santa Cruz Biotechnology) and CD 133 (rabbit anti-CD133 antibody, #NBP244250, Novus Biologicals).

Techniques: RNA Sequencing, Western Blot, Co-Immunoprecipitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Sesn3 deficiency promotes carcinogen-induced hepatocellular carcinoma via regulation of the hedgehog pathway

doi: 10.1016/j.bbadis.2019.07.011

Figure Lengend Snippet: Representative immunofluorescent images of Sesn3 and Gli2 after vector, Flag-Sesn3, and HA-Gli2 plasmid DNAs were transfected to Huh7 cells in the absence or presence of 100 nM SAG. Sesn3 and Gli2 were detected using anti-Sesn3 or anti-HA antibody followed by Alexa 594 and Alexa 488 secondary antibody, respectively (A, B). Vector or Flag-Sesn3 plus HA-Gli2 were transfected to Huh7 cells in the presence of 100 nM SAG. Sesn3, Gli2, and CD44 were detected using anti-Flag, anti-HA, and anti-CD44 antibody followed by Alexa 594, Alex 488, and Cy5 secondary antibody, respectively (C). Fluorescence images were captured using a Zeiss fluorescence microscope at x640 magnification. Real-time PCR analysis of endogenous GLI2 and CD44 mRNAs in vector or Sesn3 transfected Huh7 cells treated with vehicle or SAG (100 nM) for 24 hours (D, E). Data are expressed as mean ± SEM (n = 3). ###p < 0.001 for vehicle vs. SAG for vector or Sesn3 transfection, and ***p < 0.001 for vector vs. Sesn3 transfection after the SAG treatment.

Article Snippet: Immunofluorescence (IF) analysis was performed for detecting general HCC markers including Arginase-1 (Arg1, rabbit mAb, Cat. No. 93668S, Cell Signaling Technology), alpha-fetoprotein (AFP, mouse mAb, sc-8399, Santa Cruz Biotechnology), and cytokeratin 19 (CK-19, rat mAb, TROMA-III, DSHB), ECM molecules such as Collagen 1 and 3 (Col1, rabbit anti-Collagen I antibody, #ab21286; Col3, rabbit anti-Collagen III antibody, #ab7778, Abcam), and alpha-smooth muscle actin (α-SMA, rabbit anti-αSMA antibody, #ab5694, Abcam), and cancer stem cell markers like CD44 (mouse anti-CD44 antibody, #sc-7297, Santa Cruz Biotechnology) and CD 133 (rabbit anti-CD133 antibody, #NBP244250, Novus Biologicals).

Techniques: Plasmid Preparation, Transfection, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: Chronic High-Salt Diet Activates Tumor-Initiating Stem Cells Leading to Breast Cancer Proliferation

doi: 10.3390/cells13110912

Figure Lengend Snippet: Enumeration of TISC frequency in various murine passages following dietary salt modification. The frequency of CD44+CD24−TISCs cells in the isolated tumor cells among RS (grey) and HS (red) diet cohorts was enumerated by flow cytometry. ( A , B ) Representative flow cytometry plot of TISCs from passage 1 and passage 4 of Py230-C57Bl/6J HS diet cohort. ( C , D ) Changes in TISC frequency with each passage in Py230-C57Bl/6J ( C ) and 4T1-BALB/cJ ( D ) murine tumor models. ( E – N ) The mRNA expression of TISC markers Cadherin 1 ( E , J ), Snail2 ( F , K ), Aldh1A1 ( G , L ), Sox2 ( H , M ), and ITGA6 ( I , N ) in four passages of Py230-C57Bl/6J and 4T1-BALB/cJ tumor models (respectively). Data analyzed by one-way ANOVA for multiple comparisons and presented as mean ± SEM; n = 8 (biological replicates) per cohort; (*) p -value < 0.05.

Article Snippet: The TISCs were isolated from murine tumors by CD24 − CD44 + breast tumor stem cells using immunomagnetic beads as per the manufacturer’s specifications and reagents (catalog#MAGH111, R&D Systems, Minneapolis, MN, USA).

Techniques: Modification, Isolation, Flow Cytometry, Expressing

Journal: Cells

Article Title: Chronic High-Salt Diet Activates Tumor-Initiating Stem Cells Leading to Breast Cancer Proliferation

doi: 10.3390/cells13110912

Figure Lengend Snippet: TGFβ signaling induced the expression of immune-inhibitory CD80 on TISCs. ( A ) Representative flow cytometry histogram of TGFβR2 expression in passages 1 (blue), 2 (pink), 3 (olive green), and 4 (purple) from the HS diet cohort of the Py230-C57Bl/6J tumor model. ( B ) Comparison of the relative expression of TGFβR2 in each of the four passages on CD4+CD24-TISCs isolated from RS (grey) and HS (red) diet cohorts of the Py230-C57Bl/6J tumor model. ( C ) Representative flow cytometry histogram of pSMAD2/3 intracellular expression in each of the four passages from the HS diet cohort of the Py230-C57Bl/6J tumor model. ( D ) Comparison of the relative expression of pSMAD2/3 in each of the four passages on CD4+CD24-TISCs isolated from the RS and HS diet cohorts of the Py230-C57Bl/6J tumor model. ( E ) Representative flow cytometry histogram of CD80 surface expression in each of the four passages from the HS diet cohort of the Py230-C57Bl/6J tumor model. ( F ) Comparison of the relative expression of CD80 in each of the four passages on CD4+CD24-TISCs isolated from the RS and HS diet cohorts of the Py230-C57Bl/6J tumor model. ( G ) Representative flow cytometry plot to determine the TGFβR2/CD80 double-positive TISCs in passage 4 of the HS diet cohort from the Py230-C57Bl/6J tumor model. ( H ) Comparison of the relative expression of TGFβR2/CD80 double-positive TISCs in each of the four passages on CD4+CD24-TISCs isolated from the RS and HS diet cohorts of the Py230-C57Bl/6J tumor model. ( I ) Comparison of the relative expression of CD80 following TGFβ (80 ng/mL) stimulation for 5 days on CD4+CD24-TISCs isolated from each of the four passages of the RS and HS diet cohorts in the Py230-C57Bl/6J tumor model. ( J – M ) Comparison of the relative expression of TGFβR2 ( J ), pSMAd2/3 ( K ), CD80 ( L ), and TGFβR2/CD80 double-positive cells ( M ) in each of the four passages on CD4+CD24-TISCs isolated from the RS (grey) and HS (red) diet cohorts of the 4T1-BALB/cJ tumor model. ( N ) Comparison of the relative expression of CD80 following TGFβ (80 ng/mL) treatment for 5 days on CD4+CD24-TISCs isolated from each of the four passages of the RS and HS diet cohorts of the 4T1-BALB/cJ tumor model. Data analyzed by one-way ANOVA for multiple comparisons and presented as mean ± SEM; n = 8 (biological replicates) per cohort; (*) p -value < 0.05.

Article Snippet: The TISCs were isolated from murine tumors by CD24 − CD44 + breast tumor stem cells using immunomagnetic beads as per the manufacturer’s specifications and reagents (catalog#MAGH111, R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Flow Cytometry, Comparison, Isolation