Journal: JCI Insight

Article Title: Bioactive extracellular vesicles from a subset of endothelial progenitor cells rescue retinal ischemia and neurodegeneration

doi: 10.1172/jci.insight.155928

Figure Lengend Snippet: ( A – D ) ECFC characterization and CD44 sorting and KD. ( A ) Representative images of confluent ECFC colonies taken at 5× (top) and 10× (bottom) original magnification. Scale bar: 500 μm (top), 200 μm (bottom). ( B ) Immunophenotypic characterization of ECFCs. Representative flow cytometry histograms of ECFCs demonstrated positive expression of CD13, CD31, CD105, and HLA-ABC and negative expression of hematopoietic markers CD14 and CD45, mesenchymal stem cell marker CD90, as well as HLA-DR (right-shifted, black-filled curves in comparison with gray-filled curves representing the appropriate isotype controls; n = 3 replicates). ( C ) Representative gating strategy to sort CD44 hi and CD44 lo ECFCs using FACS. ( D ) Flow cytometric analysis of CD44 in ECFCs-shCD44 (black-filled curves) and ECFCs-scrRNA (gray-filled curves) following lentivirus-mediated transduction of ECFCs with shCD44. ( E – H ) EV isolation protocol, yield, morphology, and immunophenotype. ( E ) Schematic of UF-SEC-UF protocol. ( F ) UF-SEC-UF obtained a significantly higher EV yield than differential UC. Two-tailed Student’s t test; n = 4 EV isolations. Error bars represent SEM. ( G ) TEM of EV samples isolated via differential UC (left) or UF-SEC-UF (right). Differential UC samples demonstrated aggregation of macromolecules (red arrows) and EVs (yellow arrow). UF-SEC-UF produced EV samples devoid of contaminating aggregates. Scale bars: 0.2 μm. ( H ) Representative magnetic bead–assisted flow cytometry histograms of EVs hi and EVs lo . Both populations positively expressed tetraspanins CD9, CD63, and CD81, as well as endothelial marker CD31 (right-shifted, black filled curves compared with gray-filled curves of negative control samples; n = 3 replicates). * P < 0.05. MWCO, molecular weight cutoff.

Article Snippet: CD44 expression was knocked down in ECFCs using green fluorescent protein (GFP) lentiviral shRNA clones to CD44 (CMV-Neo, GenTarget Inc., clones TRCN0000308110 and TRCN0000296190) to generate ECFCs-shCD44.

Techniques: Flow Cytometry, Expressing, Marker, Comparison, Transduction, Isolation, Two Tailed Test, Produced, Negative Control, Molecular Weight

Journal: JCI Insight

Article Title: Bioactive extracellular vesicles from a subset of endothelial progenitor cells rescue retinal ischemia and neurodegeneration

doi: 10.1172/jci.insight.155928

Figure Lengend Snippet: ( A and B ) CD44 hi ECFCs rescued OIR mice. ( A ) Representative images and ( B ) quantification of NV (red) and VO (yellow) of retinal flat mounts from OIR mice. One-way ANOVA with Tukey’s; n = 7 retinas for CD44 hi ECFCs, n = 7 retinas for CD44 lo ECFCs, n = 5 retinas for HUVECs. ( C and D ) EVs hi rescued OIR mice. ( C ) Representative images and ( D ) quantification of retinal flat mounts. Inserts in A and C depict the original unquantified images; scale bars: 1 mm. Additional controls included EVs lo , HUVEC EVs, nonconditioned ECFC and HUVEC media subjected to UF-SEC-UF (XFM UF-SEC-UF and M200 UF-SEC-UF, respectively), and EVs hi sample depleted of vesicles via overnight UC (EVs hi depleted). Data in D are represented as a box-and-whisker plot where the top and bottom of the box represent mean of the upper and lower quartiles, horizontal line within the box represents the mean, and bars outside the box represent the min and max data points. One-way ANOVA with Tukey’s; n = 100 retinas for EVs hi , n = 102 retinas for EVs lo , n = 10 retinas for HUVEC EVs, n = 11 retinas for XFM UF-SEC-UF, n = 11 retinas for M200 UF-SEC-UF, n = 12 retinas for EVs hi depleted. ( E ) Pharmacologic exosome inhibition of CD44 hi ECFCs via GW4869 (20 μM) in DMSO (+GW4869, n = 12 retinas) attenuated the effects of CD44 hi ECFCs compared with cells incubated with DMSO alone (-GW4869, n = 12 retinas). Two-tailed Student’s t test. ( F and G ) ECFCs-shCD44 and their EVs failed to rescue OIR mice. Quantification of NV and VO in mice injected with ( F ) ECFCs-scrRNA ( n = 10 retinas) versus ECFCs-shCD44 ( n = 11 retinas) and ( G ) EVs from ECFCs-scrRNA ( n = 10 retinas) versus EVs from ECFCs-shCD44 ( n = 11 retinas). Two-tailed Student’s t test. ( H ) Dose-response curve of OIR mice injected with EVs hi . Mice were treated with a starting dose of 1.25 × 10 6 particles/0.5 μL/eye and serial 10-fold dilutions. Kruskal-Wallis test with Dunn’s multiple-comparison test; n = 6–9 retinas per group. Error bars represent SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: CD44 expression was knocked down in ECFCs using green fluorescent protein (GFP) lentiviral shRNA clones to CD44 (CMV-Neo, GenTarget Inc., clones TRCN0000308110 and TRCN0000296190) to generate ECFCs-shCD44.

Techniques: Whisker Assay, Inhibition, Incubation, Two Tailed Test, Injection, Comparison

Journal: JCI Insight

Article Title: Bioactive extracellular vesicles from a subset of endothelial progenitor cells rescue retinal ischemia and neurodegeneration

doi: 10.1172/jci.insight.155928

Figure Lengend Snippet: DICER1 KD attenuated rescue effects of CD44 hi ECFCs in A and B and their EVs in C and D in OIR mice. ( A ) Representative images and ( B ) quantification of NV and VO in retinal flat mounts from OIR mice demonstrated that CD44 hi ECFCs-shDICER1 failed to rescue NV relative to mice treated with scrRNA-transfected control cells (CD44 hi ECFCs-scrRNA). ( C ) Representative images and ( D ) quantification of retinal flat mounts demonstrated that EVs from CD44 hi ECFCs-shDICER1 failed to rescue both NV and VO relative to mice treated with EVs from CD44 hi ECFCs-scrRNA EVs. Two-tailed Student’s t test; n = 10 retinas for CD44 hi ECFCs-scrRNA, n = 9 retinas for CD44 hi ECFCs-shDICER1, n = 28 retinas for CD44 hi ECFCs-scrRNA EVs, n = 18 retinas for CD44 hi ECFCs-DICER1 EVs. Error bars represent SEM. * P < 0.05, **** P < 0.0001.

Article Snippet: CD44 expression was knocked down in ECFCs using green fluorescent protein (GFP) lentiviral shRNA clones to CD44 (CMV-Neo, GenTarget Inc., clones TRCN0000308110 and TRCN0000296190) to generate ECFCs-shCD44.

Techniques: Transfection, Control, Two Tailed Test

Journal: JCI Insight

Article Title: Bioactive extracellular vesicles from a subset of endothelial progenitor cells rescue retinal ischemia and neurodegeneration

doi: 10.1172/jci.insight.155928

Figure Lengend Snippet: ( A ) Heatmap of differentially expressed ( q < 0.05) miRs on small RNA sequencing of EVs hi (H, n = 2) and EVs lo (L, n = 3). ( B ) Injection of miR mimics upregulated in EVs hi in A rescued NV (miR-7-5p, miR-26a-3p miR-30a-5p, miR-216a-3p, miR-381-3p, miR-503-5p) and VO (miR-30a-5p, miR-216a-3p, miR-503-5p) compared to scrmiR-injected controls. n = 12–16 retinas for miR mimics, n = 72 retinas for scrmiR. ( C ) Combinatorial injection of miR mimics rescued OIR mice dose-dependently. “Candidate miRs” miR-7-5p, miR-23a-3p, miR-216a-3p, and miR-503-5p were upregulated on small RNA sequencing in A , validated on RT-qPCR, and functional in rescuing OIR mice in B . Combination injection of the 2 most effective miR mimics (miR-216a-3p and miR-503-5p) in B and, separately, all candidate miR mimics rescued OIR mice. n = 9–14 retinas for miR-216a-3p and miR-503-5p, n = 11–16 retinas for all candidate miRs, n = 24 retinas for scrmiR. ( D ) EVs from CD44 hi ECFCs with KD expression of individual or combinatorial miRs no longer rescued OIR mice. Multiple lines of ECFCs were generated with KD expression of both miR-216a-3p and miR-503-5p, all candidate miRs together, and each candidate miR individually. EVs from miR-23a-3p KD CD44 hi ECFCs failed to rescue VO; EVs from miR-503-5p KD CD44 hi ECFCs failed to rescue NV; and EVs from CD44 hi ECFCs with KD expression of all candidate miRs failed to rescue both NV and VO in OIR mice. n = 11–20 retinas for individual miR KD, n = 7 retinas for miR-216a-3p and miR-503-5p KD, n = 12 for all candidate miR KD, n = 18 for scrmiR KD, n = 20 for PBS. One-way ANOVA with Tukey’s analysis. Error bars represent SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: CD44 expression was knocked down in ECFCs using green fluorescent protein (GFP) lentiviral shRNA clones to CD44 (CMV-Neo, GenTarget Inc., clones TRCN0000308110 and TRCN0000296190) to generate ECFCs-shCD44.

Techniques: RNA Sequencing Assay, Injection, Quantitative RT-PCR, Functional Assay, Expressing, Generated

Journal: Oncotarget

Article Title: CD44 drives aggressiveness and chemoresistance of a metastatic human osteosarcoma xenograft model

doi: 10.18632/oncotarget.23125

Figure Lengend Snippet: (A) Gains/losses within chromosome 11 in hyper-metastatic U-2 OS/M1 as compared to parental U-2 OS cells were detected by indirect aCGH. The left panel displays entire chromosome 11 with a major gain (peak to the right side) of the region 11p11.12 to 11p14.1 including the CD44 gene locus (arrow). The right panel zooms into the CD44 gene locus on 11p13 with the genes located in the respective region depicted as bars. (B) Log2 gene dose ratios of U-2 OS-derived DNA as compared to non-malignant, diploid reference DNA as well as of U-2 OS/M1 and U-2 OS/M2 as compared to the parental U-2 OS DNA are depicted for the five CD44 oligonucleotides on the microarray. (C) Whole genome gene expression analysis: normalized intensity values for CD44 of the three indicated cell models are shown. The data are evaluated with the GeneSpring software. One-way ANOVA with Bonferroni’s post hoc test; * p < 0.05; ** p < 0.01.

Article Snippet: For transient gene silencing cells were seeded (3 × 10 5 per well) in six well plates and incubated for 24 hours before transient transfection using Lipofectamin 2000 (Invitrogen, Carlsbad, CA) with a final concentration of 2 μg shRNA for CD44 (shCD44-2 pRRL; Addgene, Cambridge, MA) or 2 μg empty vector control (shLuc pRRL; Addgene) as described [ ].

Techniques: Derivative Assay, Microarray, Gene Expression, Software

Journal: Oncotarget

Article Title: CD44 drives aggressiveness and chemoresistance of a metastatic human osteosarcoma xenograft model

doi: 10.18632/oncotarget.23125

Figure Lengend Snippet: (A) CD44 mRNA expression was determined by real-time PCR with the indicated primer sets. Two experiments were performed in triplicates. (B-D) Protein expression levels were determined by Western blot analysis in the indicated cell lines either untransfected (control) or transfected with a shRNA targeting CD44 mRNA (shRNA CD44 ) or a shLuc vector control. One out of three experiments is shown representatively. (C, D) Numbers depict densitometric quantification of the Western blots (ImageJ Software) for the indicted genes and data are in all cases given as expression levels relative to the untransfected control of each OS cell line. Values are means of three experiments. One-way ANOVA with Bonferroni’s post hoc test; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: For transient gene silencing cells were seeded (3 × 10 5 per well) in six well plates and incubated for 24 hours before transient transfection using Lipofectamin 2000 (Invitrogen, Carlsbad, CA) with a final concentration of 2 μg shRNA for CD44 (shCD44-2 pRRL; Addgene, Cambridge, MA) or 2 μg empty vector control (shLuc pRRL; Addgene) as described [ ].

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Transfection, shRNA, Plasmid Preparation, Software

Journal: Oncotarget

Article Title: CD44 drives aggressiveness and chemoresistance of a metastatic human osteosarcoma xenograft model

doi: 10.18632/oncotarget.23125

Figure Lengend Snippet: (A) Transwell migration assays were performed for 48 hours under standard culture conditions. Cell migration to the lower side of the membrane was determined by densitometric quantification of crystal violet staining. Three experiments were performed in duplicates. (B) Invasion assays were performed with matrigel covered filters for 48 hours and evaluated as under (A). Three experiments in duplicates were performed. (C) Clonogenic assays were performed under standard culture conditions with and without 5 μg anti-CD44 antibody for 7 days. Densitometric quantification of the crystal violet stained cell clones are shown. Two experiments were performed in duplicates. (D) Clonogenic assays were performed under standard culture conditions with and without 10 nM doxorubicin for 7 days. Means of three experiments in duplicates are depicted. One-way ANOVA with Bonferroni’s post hoc test; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: For transient gene silencing cells were seeded (3 × 10 5 per well) in six well plates and incubated for 24 hours before transient transfection using Lipofectamin 2000 (Invitrogen, Carlsbad, CA) with a final concentration of 2 μg shRNA for CD44 (shCD44-2 pRRL; Addgene, Cambridge, MA) or 2 μg empty vector control (shLuc pRRL; Addgene) as described [ ].

Techniques: Migration, Membrane, Staining, Clone Assay

Journal: Oncotarget

Article Title: CD44 drives aggressiveness and chemoresistance of a metastatic human osteosarcoma xenograft model

doi: 10.18632/oncotarget.23125

Figure Lengend Snippet: Anticancer activity of doxorubicin against OS cell models and impact of a CD44 knockdown

Article Snippet: For transient gene silencing cells were seeded (3 × 10 5 per well) in six well plates and incubated for 24 hours before transient transfection using Lipofectamin 2000 (Invitrogen, Carlsbad, CA) with a final concentration of 2 μg shRNA for CD44 (shCD44-2 pRRL; Addgene, Cambridge, MA) or 2 μg empty vector control (shLuc pRRL; Addgene) as described [ ].

Techniques: Activity Assay, Knockdown, shRNA

Journal: Oncotarget

Article Title: CD44 drives aggressiveness and chemoresistance of a metastatic human osteosarcoma xenograft model

doi: 10.18632/oncotarget.23125

Figure Lengend Snippet: Tissue sections of subcutaneous OS xenografts (A) and tail vein injection-induced lung metastases (B) were analysed immunohistochemically. Sections were stained for CD44. (C) Quantification of the proportion of positively stained areas for CD44 analyzed by Definiens software. Values are given as percentage positively-stained areas for CD44 in comparison to the total tissue in metastases divided by subcutaneous tumors (ratio). Students t-test; * p < 0.05.

Article Snippet: For transient gene silencing cells were seeded (3 × 10 5 per well) in six well plates and incubated for 24 hours before transient transfection using Lipofectamin 2000 (Invitrogen, Carlsbad, CA) with a final concentration of 2 μg shRNA for CD44 (shCD44-2 pRRL; Addgene, Cambridge, MA) or 2 μg empty vector control (shLuc pRRL; Addgene) as described [ ].

Techniques: Injection, Staining, Software, Comparison

Journal: Cancer Research

Article Title: CD44v8-10 Is a Cancer-Specific Marker for Gastric Cancer Stem Cells

doi: 10.1158/0008-5472.can-13-2309

Figure Lengend Snippet: Figure 1. EpCAM-expressing cells in gastric cancer tissues are tumor epithelial cells containing subpopulations of CD44þ and CD133þ cells. Flow cytometric analyses of surface marker expression in cells isolated from 28 paired gastric tumors and adjacent nontumor tissue. A, EpCAM levels in 28 gastric tumors compared with adjacent nontumor tissue. MFI was calculated using FlowJo software (Supplementary Fig. S1B). Horizontal bars, the mean value. Patient clinical information is included in Supplementary Table S1. B, proportion of CD44þ and CD133þ cells in gastric cancer compared with adjacent nontumor tissue. Representative flow cytometry data plots from 3 patients with gastric cancer (GC16, GC59, and GC71) are shown. Proportion (%) of CD44þ or CD133þ cells within the EpCAMþ

Article Snippet: For CD44 "rescue" experiments, TMK1 cells were cotransfected with 6 mg each of CD44 shRNA plasmids (Origene) and either CD44s, CD44v8-10, or pcDNA3 vector as shRNA-resistant rescue constructs.

Techniques: Expressing, Marker, Isolation, Software, Cytometry

Journal: Cancer Research

Article Title: CD44v8-10 Is a Cancer-Specific Marker for Gastric Cancer Stem Cells

doi: 10.1158/0008-5472.can-13-2309

Figure Lengend Snippet: Figure 2. Gastric CSCs are enriched in EpCAMþCD44þ cell fractions. A, gastric cancer xenograft lines from three clinical biopsies of well-differentiated (GC16), moderately differentiated (GC21), and poorly differentiated (GC38) gastric cancer. Top, H&E staining of primary gastric cancer biopsies. Bottom, H&E staining of respective xenogeneic patient-derived tumors. Scale bars, 200 mm. B, proportion of CD44þ cells in respective xenograft lines. C, summary of limiting dilution assays estimating the frequency of CSCs in unsorted, EpCAMþCD44þ, and EpCAMþCD44 cell fractions from GC16, GC21, and GC38 xenografts. Differences between groups were tested for statistical significance using x2 test (degrees of freedom ¼ 1; 95% confidence intervals). , statistically significant P values. Detailed data for limiting dilution analysis and estimation of gastric CSC frequency are shown in Table 1. D, flow cytometric analysis of EpCAMþCD44þ population in primary and secondary xenograft tumors from serial transplantation experiments generated from sorted EpCAMþCD44þ cells. E, H&E staining showing histology and cellular composition of tumors initiated by EpCAMþCD44þ cells from first and second xenograft transplants. Scale bars, 100 mm.

Article Snippet: For CD44 "rescue" experiments, TMK1 cells were cotransfected with 6 mg each of CD44 shRNA plasmids (Origene) and either CD44s, CD44v8-10, or pcDNA3 vector as shRNA-resistant rescue constructs.

Techniques: Staining, Derivative Assay, Transplantation Assay, Generated

Journal: Cancer Research

Article Title: CD44v8-10 Is a Cancer-Specific Marker for Gastric Cancer Stem Cells

doi: 10.1158/0008-5472.can-13-2309

Figure Lengend Snippet: Figure 3. CD44v8-10 is the major CD44 variant found on cells that express surface CD44. A, PCR of cDNA from xenograft lines GC16 (lane 1), GC21 (lane 2), and GC38 (lane 3). MW, molecular weight marker. Diagram of human CD44 gene structure showing alternatively spliced variant exons v1-10 in colored boxes and constant exons in gray boxes. CD44v8-10 comprises all constant exons and variant exons 8–10 while CD44s contains only constant exons. PCR primers are located in constant exons 5 and 16/17. B, qRT-PCR of CD44v8-10 expression in xenograft tumors of GC16, GC21, and GC38 normalized to 18S rRNA internal control. Error bars, mean SD. C, flow cytometric analyses of surface expression of CD44v8-10 in EpCAMþCD45 and CD45þ cells isolated from tumor and adjacent normal tissues of patient GC101. Proportion (%) of CD44v8-10– positive (CD44vþ) and CD44v8- 10–negative cells within the EpCAMþCD45 or CD45þ

Article Snippet: For CD44 "rescue" experiments, TMK1 cells were cotransfected with 6 mg each of CD44 shRNA plasmids (Origene) and either CD44s, CD44v8-10, or pcDNA3 vector as shRNA-resistant rescue constructs.

Techniques: Variant Assay, Molecular Weight, Marker, Quantitative RT-PCR, Expressing, Control, Isolation

Journal: Cancer Research

Article Title: CD44v8-10 Is a Cancer-Specific Marker for Gastric Cancer Stem Cells

doi: 10.1158/0008-5472.can-13-2309

Figure Lengend Snippet: Figure 5. CD44v8-10 is upregulated in clinical gastric cancer and plays a functional role in tumor initiation. A, immunoblot of primary gastric cancer cells GC38-adh stably expressing CD44s and CD44v8-10. Cell proliferation assay was performed over 5 days. Data points represent mean SD of five replicates. Difference in slopes indicates cell proliferation rate. Results are representative of two independent experiments. B, xenograft tumor formation potential of fractionated GC38-adh cells overexpressing CD44s and CD44v8-10. Chi-squared tests were performed for statistical significance (degrees of freedom ¼ 1). , statistically significant P values. Data from two independent experiments are represented, with cell doses and number of mice used reflected in Supplementary Table S3A. C, top, CD44v8-10 expression in TMK1 cells analyzed by flow cytometry. Bottom, post-sort analyses of CD44-silenced TMK1 cells rescued with indicated shRNA-refractory genes. Percentage purity of sorted cells is indicated. These cells were used in xenograft tumor formation assay. D, xenograft tumor formation in NSG mice by CD44-silenced TMK1 cells harboring the indicated shRNA-refractory CD44 variants. Xenograft tumor formation potential for the indicated cell population was determined by limiting dilution assay. Differences between groups were tested for statistical significance using x2 test (degrees of freedom ¼ 1; 95% confidence intervals). , statistically significant P values. Detailed data for limiting dilution analysis are presented in Supplementary Table S3B.

Article Snippet: For CD44 "rescue" experiments, TMK1 cells were cotransfected with 6 mg each of CD44 shRNA plasmids (Origene) and either CD44s, CD44v8-10, or pcDNA3 vector as shRNA-resistant rescue constructs.

Techniques: Functional Assay, Western Blot, Stable Transfection, Expressing, Proliferation Assay, Cytometry, shRNA, Tube Formation Assay, Limiting Dilution Assay

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A Representative images of cell clusters in the vasculature of human primary breast tumor. The distributions of CD206 (brown), CD44 (brown), CK (brown), and CD31 (red, indicating vessels) in tissue serial sections from luminal-like BrCa ( n = 15) were determined by immunohistochemical staining. Nuclei are stained with hematoxylin. B Immunohistochemical staining of CD206, CD44, and CD31 was performed in serial tissue sections of human BrCa. Tumors with and without microemboli from a tissue microarray (85 human breast lesions) were analyzed. Using Image-Pro Plus software, the expression intensity of each molecule was evaluated by integral optical density and compared with and without microemboli. Data were analyzed with the Mann-Whitney test. * p < 0.05, *** p < 0.001. C The expression of CD44 and CD206 in the invasion front versus non-invasive front of serial sections from human BrCa tissues was analyzed. Data represents the mean ± SEM. Mann-Whitney test. D A positive association between high frequency of CD206 + -macrophage and CD44 high -cancer cells in BrCa.

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Immunohistochemical staining, Staining, Microarray, Software, Expressing, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A Adaptive CD44 expression of luminal-like BrCa cells (MCF7 and T47D) upon THP-1-derived M2-like macrophages stimulation in a non-contact transwell system was assessed by immunoblotting analysis. GAPDH was used as control. The band intensities were analyzed by densitometry analysis. * p < 0.05. B Representative confocal images of CD44 expression in disseminated cell clusters on top-3D matrix membrane during collective detachment. DAPI was used to stain the nuclei. An acquired CD44 high state upon TAMs stimulation in primary BrCa cell clusters (purified from MMTV-PyMT tumor) was observed by immunofluorescence analysis. C Co-immunoprecipitation experiment from whole-cell extracts demonstrating the interaction between CD44 and Ezrin after co-cultured with THP-1-derived M2-like macrophages in a non-contact transwell system. The band intensities were analyzed by densitometry analysis. * p < 0.05. D The influence of CD44 knockdown on collective invasion of cell clusters in 3D matrix membrane. The sh-Control and sh-CD44 primary BrCa cell clusters, premixed with or without TAMs, were embedded in 3D system and recorded by time-lapse microscopy. TAMs were pre-stained with Vybrant CM-DiI (red). E Representative confocal images of localization of pEzrin, pMyosin, β-catenin and ZO-1 at the invasive and disseminating clusters. The sh-Control and sh-CD44 of primary BrCa cell clusters, premixed with or without TAMs, were cultured in top-3D basement. F Knockdown of CD44 inhibited TAMs-induced formation of mammosphere. The mammosphere-forming capacity of primary BrCa cells purified from MMTV-PyMT tumors was detected. Primary BrCa cells were directly sorted into wells of ultra-low attachment 24-well plate containing mammosphere growth medium, and then co-cultured with or without TAMs for 12 days in a non-contact transwell system. G Representative intravital images of subcutaneous xenografts derived from MCF7/sh-Control/GFP + and MCF7/sh-CD44/GFP + cells in vivo, which were injected with or without THP-1-derived M2-like macrophages, showing cohesive-invading MCF7/GFP + cells approaching around vascular vessel. Tomato lectin (DyLight649, Cat. L32472, Thermo Fisher) was injected via tail vein to label vascular structures. Scale bars, 100 μm. See Supplementary Video – . H Representative multiphoton confocal images of thick tumor tissues showed collective dissemination in subcutaneous xenografts. Tumor tissues were cut into 10- to 12-µm thick for paraffin sections, and the blood vessels were labeled by CD31 (Red channel) using immunohistochemical staining. Scale bars, 100 μm. See Supplementary Video – .

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Expressing, Derivative Assay, Western Blot, Control, Membrane, Staining, Purification, Immunofluorescence, Immunoprecipitation, Cell Culture, Knockdown, Time-lapse Microscopy, In Vivo, Injection, Labeling, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A The mRNA levels of a panel of cytokines in TAMs versus PBMCs isolated from five primary breast tumors (MMTV-PyMT) were detected by qRT-PCR. B The intracellular CCL8 and IL-10 in TAMs versus PBMCs isolated from normal mouse or tumor-bearing mouse (MMTV-PyMT tumor) were determined by ELISA. n = 5/group. Bars correspond to mean ± SD. * p < 0.05, ** p < 0.01, ns, nonsignificant. C The secreted CCL8 and IL-10 in TAMs versus primary BrCa cells were determined by ELISA. Cells were isolated from five primary breast tumors (MMTV-PyMT) and cultured separately. Bars correspond to mean ± SD. ** p < 0.01, ns, nonsignificant. D Fluorescence in situ hybridization analysis of human BrCa tissue sections revealed that CCL8 mRNA is found in CD206 + TAMs but not in cancer cells. The expression of CD206 was detected using immunochemical staining. Inset representing a CD206 + macrophage-expressing CCL8 mRNA. Scale bars, 100 μm ( n = 3). E Blocking of CCL8 inhibited the collective migration induced by THP-1-derived M2-like macrophages. In vitro scratch assay of untreated MCF7 or treated with THP-1-derived M2-like macrophages, THP-1-derived M2-like macrophages plus normal IgG, or THP-1-derived M2-like macrophages plus CCL8 antibody for the indicated period of time. Red line, cell culture margins ( n = 4). F Blocking of CCL8 inhibited the re-distribution of invadopodia molecules in collective migration induced by THP-1-derived M2-like macrophages. Representative confocal images showed the re-distribution of Cortactin (red) and TKS5 (green) at the migrating front of clusters after CCL8 blocking for 3 days. MCF7 cells co-cultured with THP-1-derived M2-like macrophages in a non-contact transwell system, followed by treatment with normal IgG or CCL8 antibody. Scale bars, 20 μm. G Knocking down of CCL8 in THP-1-derived M2-like macrophages inhibited the increase of Vinculin at the migrating front of clusters. Wound healing assay was used to observe the changes of Vinculin expression at the migrating front. M2-like macrophages derived from THP-1 were transfected with siRNAs (si-Control or si-CCL8) and then co-cultured with MCF7 cells in a non-contact transwell system for 3 days. The expression of Vinculin was determined by immunofluorescence assay. Scale bars, 10 μm. H Blocking of CCL8 inhibited the onset of xenografts induced by THP-1-derived M2-like macrophages in vivo. MCF7 cells were orthotopically implanted into mammary pad of nu/nu mice, followed by intraperitoneal administration of a neutralizing antibody for CCL8. I HE staining of MCF7 xenografts in normal IgG or CCL8 antibody-treated mice. J CD44 (brown) expression of MCF7 xenografts in normal IgG- or CCL8 antibody-treated mice. K CD31 (red) expression of MCF7 xenografts in normal IgG- or CCL8 antibody-treated mice.

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Fluorescence, In Situ Hybridization, Expressing, Staining, Blocking Assay, Migration, Derivative Assay, In Vitro, Wound Healing Assay, Transfection, Control, Immunofluorescence, In Vivo

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A The influence of CCL8 on the expression of CD44 in MCF7 cells was evaluated by western blot. The band intensities were analyzed by densitometry analysis. * p < 0.05. B Blocking of CCL8 inhibited the acquisition of CD44 high state in MCF7 cells induced by M2-like macrophages, which was obtained from human monocytic cell line THP-1 stimulated by PMA/m-CSF/IL-4/IL-10/IL-13. The band intensities were analyzed by densitometry analysis. ** p < 0.01, ns: no significance. C Scheme of identifying differential CD44-interacting peptides upon THP-1-derived M2-like macrophages/CCL8 stimulation. The intersection showed the CD44-interacting proteins, which were analyzed by co-immunoprecipitation-based mass spectrometry assay. MCF7 and T47D cells were stimulated by THP-1-derived M2-like macrophages or CCL8 for 3 days, respectively. The proteins recruited to CD44 were precipitated by CD44 antibody, and then subject to mass spectrometry assay. D Functional associations of the regulatory networks of MDM2/p53-correlated genes from analysis of STRING data are presented. E The effects of CCL8 on the activation of MDM2 in MCF7 cells were evaluated by immunoblotting assay. The band intensities were analyzed by densitometry analysis. * p < 0.05. F Blocking of CCL8 inhibited the activation of MDM2 in MCF7 cells induced by THP1-derived M2-like macrophages. The band intensities were analyzed by densitometry analysis. * p < 0.05, ** p < 0.01, ns: no significance. G Repression of p53 expression by M2-like macrophages in human BrCa cells was detected by immunoblotting assay. The band intensities were analyzed by densitometry analysis. * p < 0.05. H Knocking down of MDM2 in primary BrCa cells inhibited the up-regulation of CD44 induced by TAMs. The band intensities were analyzed by densitometry analysis. *** p < 0.001, ns: no significance. I The influence of MDM2 inhibitor on collective dissemination of cell clusters induced by TAMs in 2D culture system. Before co-cultured with TAMs on 2D culture system, primary BrCa cell clusters were obtained from an ultra-low attachment culture system. TAMs were pre-stained with Vybrant CM-DiI (red).

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Blocking Assay, Derivative Assay, Immunoprecipitation, Mass Spectrometry, Functional Assay, Activation Assay, Cell Culture, Staining

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A Activation of Ezrin and p38 after MCF7 cells co-cultured with THP-1-derived M2-like macrophages or CCL8 by a non-contact transwell system was determined by immunoblotting assay. The band intensities were analyzed by densitometry analysis. * p < 0.05, *** p < 0.001. B Knocking down of CD44 inhibited the activation of Ezrin and p38 induced by THP-1-derived M2-like macrophages or CCL8. The band intensities were analyzed by densitometry analysis. *** p < 0.001, ns: no significance. C Knocking down of Ezrin in primary BrCa cells inhibited collective dissemination induced by TAMs in 2D culture system. The primary BrCa cell clusters were transfected by si-Ezrin. TAMs were pre-stained with Vybrant CM-DiI (red). D The p38 inhibitor attenuated collective dissemination of primary BrCa cell clusters induced by TAMs. TAMs were prestained with Vybrant CM-DiI (red). E Knocking down of Ezrin inhibited phosphorylation of Cortactin and p38 triggered by CCL8. The band intensities were analyzed by densitometry analysis. *** p < 0.001, ns: no significance. F Knocking down of p38 repressed the phosphorylation of Cortactin induced by CCL8. The band intensities were analyzed by densitometry analysis. *** p < 0.001, ns: no significance. G Scheme summarizing the proposed mechanism by which MDM2-p53-p38 signaling pathway mediated the acquisition of CD44 high state, which leading to cohesive detachment.

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Activation Assay, Cell Culture, Derivative Assay, Western Blot, Transfection, Staining, Phospho-proteomics

Journal: Proteomics. Clinical applications

Article Title: A proteomic investigation of isogenic radiation resistant prostate cancer cell lines.

doi: 10.1002/prca.202100037

Figure Lengend Snippet: FIGURE 4 Proteins identified to be enriched in radiation resistance. A. Pathway enrichment analysis performed using g:Profiler on the (i) significantly downregulated and (ii) significantly upregulated proteins in Figure S3 Large clusters of similar pathways are outlined as internal cell components (purple), cell adhesion (yellow), cytoskeleton (pink), transport (green), extracellular (blue), blood related (red) and oxygen related (orange). B. Heatmap showing the 72 radiation resistant proteins which had a significant change in expression (represented as circles in (Figure S3 . Hard clustering was applied, with an optimum km = 2 identified. CD44, a protein of interest, has been manually highlighted. C. Log2 normalized iBAQ values for CD44 in DU145-PAR (green), DU145-CF (blue), and DU145-HF (red). Statistics were performed using Student’s t-test. p values < 0.05 was considered statistically significant

Article Snippet: After 16 h, siRNA control or CD44 siRNA (Origene Inc., USA) were transiently transfected into cells using SiTran Clinical relevance Radiation therapy remains a definitive treatment modality for prostate cancer with curative intent.

Techniques: Expressing

Journal: Proteomics. Clinical applications

Article Title: A proteomic investigation of isogenic radiation resistant prostate cancer cell lines.

doi: 10.1002/prca.202100037

Figure Lengend Snippet: FIGURE 5 Expression of CD44 is linked to radiation resistance. A. Western blot showing the expression of CD44 in whole cell lysates from DU145-PAR, DU145-CF, DU145-HF, PC3-PAR, and PC3-CF with β-actin as a loading control. B. Flow cytometry analysis of surface expression of CD44 on DU145-PAR, DU145-CF, and DU145-HF (N = 3). Geometric mean of FITC-CD44 fluorescence intensity is normalised to isotype control A representative replicate of CD44 fluorescence intensity compared to isotype control is shown. Data are expressed as mean ± standard error of the mean. Statistical analyses were performed using Student’s t-test. p value < 0.05 was considered to be statistically significant. (C) Flow cytometry analysis of surface expression of CD44 on PC3-PAR and PC3-CF (N = 3). Geometric mean of FITC-CD44 fluorescence intensity is normalised to isotype control. A representative replicate of CD44 fluorescence intensity compared to isotype control is shown. Data are expressed as mean ± standard error of the mean. Statistical analyses were performed using Student’s t-test. p value < 0.05 was considered to be statistically significant. D. DU145 PAR (green), DU145 CF (blue), and DU145 HF (red) treated with 0 Gy or 6 Gy irradiation in combination with 0 μg/mL or 10 μg/mL anti-CD44 monoclonal antibody (mAb) (N = 3). Clonogenic survival of each treatment is normalized to 0 Gy + 0 μg/mL mAb. Data was expressed as mean ± standard error of the mean. Statistical analyses were performed using Student’s t-test. p value < 0.05 was considered to be statistically significant. E. DU145-PAR (green), DU145-CF (blue), and DU145-HF (red) treated with 0 Gy or 6 Gy irradiation in combination with CD44 siRNA or control siRNA (n = 3). Clonogenic survival of each treatment is normalized to 0 Gy + control siRNA. Data was expressed as mean ± standard error of the mean. Statistical analyses were performed using Student’s t-test. p value < 0.05 was considered to be statistically significant. F. Western blot showing knockdown of CD44 in whole cell lysates from DU145-PAR, DU145-CF, and DU145 with β-actin as a loading control.

Article Snippet: After 16 h, siRNA control or CD44 siRNA (Origene Inc., USA) were transiently transfected into cells using SiTran Clinical relevance Radiation therapy remains a definitive treatment modality for prostate cancer with curative intent.

Techniques: Expressing, Western Blot, Control, Flow Cytometry, Fluorescence, Irradiation, Knockdown

Journal: Molecular Cancer Therapeutics

Article Title: MTI-101 (Cyclized HYD1) Binds a CD44 Containing Complex and Induces Necrotic Cell Death in Multiple Myeloma

doi: 10.1158/1535-7163.mct-13-0310

Figure Lengend Snippet: Figure 3. Reduction in CD44 expression induces cell death in U266 and NCI-H929, as determined through cleaved caspase-3 activation. A and C, CD44 expression determined by FACS analysis in U266 and NCI-H929 multiple myeloma cells. B and D, reduction of CD44 surface expression in U266 and NCI-H929 multiple myeloma cells results in the activation of cleaved caspase-3 (NS, nonsilencing). Experiments were run in triplicate and repeated 3 times. Statistical significance was determined using Student t test, , P 0.05.

Article Snippet: Briefly, 293FT cells were grown to 90% confluence in 100-mmPetri dishes and then transfectedwith the pBabe-puro CD44s retroviral vector (Addgene plasmid 19127) synthesized by the Weinberg lab or a scrambled retroviral CD44 shRNA (Origene) using the pVPack system (Clontech).

Techniques: Expressing, Activation Assay

Journal: Molecular Cancer Therapeutics

Article Title: MTI-101 (Cyclized HYD1) Binds a CD44 Containing Complex and Induces Necrotic Cell Death in Multiple Myeloma

doi: 10.1158/1535-7163.mct-13-0310

Figure Lengend Snippet: Figure 4. MTI-101 treatment induces transient phospho-Erk1/2 and CD44-associated Pyk2 complex in NCI-H929 and U266 multiple myeloma cells. A and B, CD44 immunoprecipation was performed after MTI-101 treatment in NCI-H929 and U266 cells and probed for Pyk2 and CD44. GAPDH levels in the total lysaste were used as a loading control. C and D, NCI-H929 and U266 cells were treated with 5 and 10 mmol/L MTI-101, respectively, for various time points before whole cell lysis in RIPA buffer and probed for pErk (T202/Y204) and total Erk. Experiments were repeated 3 times and shown is a representative Western blot.

Article Snippet: Briefly, 293FT cells were grown to 90% confluence in 100-mmPetri dishes and then transfectedwith the pBabe-puro CD44s retroviral vector (Addgene plasmid 19127) synthesized by the Weinberg lab or a scrambled retroviral CD44 shRNA (Origene) using the pVPack system (Clontech).

Techniques: Control, Lysis, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: MTI-101 (Cyclized HYD1) Binds a CD44 Containing Complex and Induces Necrotic Cell Death in Multiple Myeloma

doi: 10.1158/1535-7163.mct-13-0310

Figure Lengend Snippet: Figure 6. MTI-101 has activity as a single agent in vivo and MTI-101 necrotic cell death is Drp1 independent in NCI-H929 cell line. A, NCI-H929 cells were incubated with siRNA targeting DRP1 for 72 hours before treatment with 5 mmol/L MTI-101 for 6 hours, and cell death was determined by FACS analysis. NS, nonsilencing. B, in the SCID-Hu model, tumor was allowed to engraft for 28 days and mice were randomized into control and treatment group. On day 28, represented as day 0 on the graph, mice (N ¼ 10 for each treatment) were treated intraperitoneally with 8 mg/kg of MTI-101 or PBS daily for 21 days. Tumor burden was assessed weekly by measuring k levels in the sera. MTI-101 treatment demonstrated a significant reduction in tumor burden (P < 0.05, ANOVA). C, in the 5TGM1 mouse myeloma model, tumor was allowed to engraft for 10 days and mice (N ¼ 10 for each treatment) were treated intraperitoneally at day 10 with agents (MTI-101, bortezomib, or PBS) 3 times a week for 3 weeks. MTI-101 treatment resulted in a significant increase in survival compared with control mice (P < 0.05, Mantel–Cox test). Mice were monitored daily for survival with all remaining mice euthanized at day 100 after treatment. D, proposed model of MTI-101 mechanism of action. MTI-101 binds to a CD44 and a4-integrin containing complex, resulting in the recruitment of Pyk2. This complex leads to a pro-survival signal mediated through Erk1/2 and a necrotic cell signal through Rip1/Rip3, and Drp1. There is also a Rip1/Rip3/Drp1 independent pathway that ultimately leads to necrosis. Statistical significance was determined using ANOVA, P < 0.05. Knockdown and inhibitor experiments were run in triplicate and repeated 3 times. Statistical significance was determined using Student t test, #, P > 0.05.

Article Snippet: Briefly, 293FT cells were grown to 90% confluence in 100-mmPetri dishes and then transfectedwith the pBabe-puro CD44s retroviral vector (Addgene plasmid 19127) synthesized by the Weinberg lab or a scrambled retroviral CD44 shRNA (Origene) using the pVPack system (Clontech).

Techniques: Activity Assay, In Vivo, Incubation, Control, Knockdown