Journal: Stem Cell Research & Therapy

Article Title: Identification of CD141 + vasculogenic precursor cells from human bone marrow and their endothelial engagement in the arteriogenesis by co-transplantation with mesenchymal stem cells

doi: 10.1186/s13287-024-03994-9

Figure Lengend Snippet: CD141 is a critical marker that distinguishes BM-VPCs from BM-MSCs. A List of markers with an expression difference of > 50% between BM-VPCs and BM-MSCs screened among 376 surface markers. B Differential expression of CD141 and CD282 in 15–20 donors assessed using flow cytometry analysis (*** p < 0.001, Student's t-test, two-tailed). C–F BM-MNCs were sorted using magnetic bead-conjugated CD141 antibody. The selected MNCs were plated in EGMPL. C Colonies that appeared from CD141 + sorted BM-MNCs on days 5, 7, and 9. Scale bar = 500 μm. D Marker expression profile of ex vivo-cultured cells from CD141 + sorted colonies at passages 1 to 4 analyzed using flow cytometry. E, F CD141 + sorted BM-MNCs were cultured in EGMPL at passage 0; from passage 1 to 3 and then, the cells were divided and cultured in EGMPL or StemMACS. E Morphology of replated CD141 + sorted cells in EGMPL and StemMACS at passages 1 to 3, and their tube-forming ability in MEMα + 0.2%hPL at passage 2. Scale bar = 500 μm. F Marker expression profile of CD141 + sorted cells cultured in StemMACS

Article Snippet: StemMACS is an abbreviation for StemMACS MSC expansion medium XF, human (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Marker, Expressing, Quantitative Proteomics, Flow Cytometry, Two Tailed Test, Ex Vivo, Cell Culture

Journal: Heliyon

Article Title: Inhibition of CTLA-4 accelerates atherosclerosis in hyperlipidemic mice by modulating the Th1/Th2 balance via the NF-κB signaling pathway

doi: 10.1016/j.heliyon.2024.e37278

Figure Lengend Snippet: The anti-CTLA-4 antibody promotes CD4 + T-cell infiltration and T-cell differentiation into Th1 cells in the aortic root. (A) GO enrichment analysis showing the significantly enriched biological processes in hyperlipidemic mice administered the anti-IgG antibody or anti-CTLA-4 antibody. n = 3 per group. (B) GSEA of RNA-seq data showing the enrichment of “regulation of Th1-type immune response” and “Th1 cell differentiation” in hyperlipidemic mice administered the anti-IgG antibody or anti-CTLA-4 antibody. NES: normalized enrichment score; FDR: adjusted p value. (C) Representative immunofluorescence staining of CD4 (red), α-SMA (green), and DAPI (blue) in mice in the indicated groups. The square represents the colocalization of CD4 with the smooth muscle marker α-SMA. The scale bars are 100 μm and 20 μm. (D) Quantitative analysis of CD4 fluorescence intensity in aortic roots in the indicated groups. The data are presented as the means ± SEM (n = 6). (E) Flow cytometric analysis of the distributions of CD4 + IFN-γ + T cells and CD4 + IL-4 + T cells in peripheral blood in the indicated groups. (F) Relative quantification of the percentages of CD4 + IFN-γ + T cells and CD4 + IL-4 + T cells among the sorted CD4 + T cells. The data are presented as the means ± SEM (n = 5). (G) The Th1/Th2 ratio was estimated by determining the ratio of CD4 + IFN-γ + IL-4 − cells to CD4 + IFN-γ − IL-4 + cells. The data are presented as the means ± SEM (n = 5). (H) Quantitative results were obtained by real-time PCR showing the relative mRNA expression of IFN-γ and IL-4 in the indicated groups. The data are presented as the means ± SEM (n = 6). *P < 0.05 vs . the anti-IgG group. (I – J) Quantitative analyses demonstrating the number of CD4 + IFN-γ + T cells and CD4 + IL-4 + T cells in aortic roots from each indicated experimental group. The data are presented as the means ± SEM (n = 4). *P < 0.05 vs . the anti-IgG group.

Article Snippet: One hundred microliters of mouse peripheral blood were collected in isotonic RPMI 1640 complete culture medium (R8758, Sigma-Aldrich) and stimulated with 2 μL of leukocyte activation cocktail (550583, BD Biosciences) in 5 % CO 2 at 37 °C for 4 h. Then, the cells were treated with anti-mouse CD3 (APC-Cy7-CD3, 1:200, 557596, BD Biosciences) and anti-mouse CD4 (BV510-CD4, 1:200, 563106, BD Biosciences) for 30 min and lysed in red blood cell lysis solution (130-094-183, Miltenyi Biotec) for 10 min. After being treated with fixation/permeabilization concentrate at room temperature for 1 h, total T lymphocytes were further stained with anti-mouse IFN-γ (FITC-IFN-γ, 1:100, 554411, BD Biosciences) and anti-mouse IL-4 (PE-IL-4, 1:100, 554435, BD Biosciences) for 30 min. Isotype controls were used for compensation and to confirm antibody specificity.

Techniques: Cell Differentiation, RNA Sequencing, Immunofluorescence, Staining, Marker, Fluorescence, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Expressing

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 2. Cardiomyocyte-specific knockout of ETV1 slows atrial and His-Purkinje system conduction. Etv1flox/

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Knock-Out

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 3. Cardiomyocyte deletion of ETV1 resulted in decreased expression of fast conduction genes in atrial and His-Purkinje system (HPS) myocytes. (A) Quantitative RT-PCR of fast conduction gene RNA levels (normalized to Gapdh) comparing 10–12-week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6- Cre) FACS-purified ventricular, atrial, and Purkinje myocytes. Relative Nkx2–5, Gja5, and Scn5a expression displayed versus control, Etv1 WT (n = 4). (B) Immunoblot assessment of Etv1 WT and Etv1 cKO atrial tissue lysates detecting NKX2–5, Cx40, NaV1.5, and Vinculin (loading control). (C) Protein level densitometric quantification (normalized to vinculin), displayed relative to Etv1 WT (n = 5). (D) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO atria/ventricular sections. (E) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO HPS sections. Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). LA, left atria; LV, left ventricle. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test. Scale bars: 50 um.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Purification, Control, Western Blot, Immunofluorescence

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 5. ETV1 regulates the diversity of sodium channel biophysical properties between ventricular, atrial, and Purkinje myocytes. Whole-cell patch clamp data from dissociated cardiomyocytes (ventricular, right atrial, Purkinje myocytes) using 10–12 week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6-Cre) mice in a Cntn2-EGFP background (n = 4). (A) Comparison of sodium current–voltage (I–V) relationship. Maximum conductance was calculated to assess significant differences among experimental groups. (B) Voltage dependence of steady-state activation. Voltage at half activation (V0.5, activation) was calculated to assess significant differences among experimental groups. (C) Voltage dependence of steady-state inactivation. Voltage at half inactivation (V0.5, inactivation) was calculated to assess significant differences among experimental groups. (D) Time course of recovery from inactivation. Tau of recovery (τrecovery) was calculated to assess significant differences among experimental groups. Number of cells analyzed per cell type (ventricle, right atria, Purkinje) included in each graph legend. Patch clamp protocol diagrams are included for each endpoint. Data represent mean ± SEM. *P < 0.05, 1-way ANOVA.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Patch Clamp, Comparison, Activation Assay

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 6. ETV1-transduced neonatal rat ventricular myocytes (NRVMs) upregulates a His-Purkinje system gene signature. (A) Volcano plot of relative transcript expression from NRVMs transduced with either Ad-Etv1- EGFP or Ad-EGFP. RNA-sequencing (RNA-seq) comparison revealed a total of 9,236 differentially expressed genes (normalized counts ≥ 5, padj < 0.05). All significantly different genes (padj < 0.05) are labeled blue (downregulated) or red (enriched) and all nonsignificantly different transcripts labeled in gray. Of these there were 4,696 upregulated and 4,540 downregulated genes in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs. (B) Functional clustering of upregulated genes in Ad-Etv1 transduced NRVMs highlighted significantly enriched ETV1-dependent cellular processes (top 20 non-redundant categories are shown). Pathways are color coded to represent genes clustered into functional classes for heat maps in C. (C) Comparative RNA-seq between 21-day-old (P21) wild-type mouse FACS-purified Purkinje cell (PC)/ventricular myocytes (VM) and Ad-Etv1-EGFP/Ad-EGFP transduced NRVMs. Heat map representation of 88 genes differentially expressed in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs (n = 3) plotted adjacent to average fold change expression in PCs and VMs. Genes clustered into functional groups demonstrate that ETV1 regulates a PC transcriptome in neonatal cardiomyocytes.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Transduction, RNA Sequencing, Comparison, Labeling, Functional Assay, Purification

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 8. Activation of ETV1 in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) leads to increased expression of rapid conduction genes and sodium current. (A) Schematic representation of hiPSC-CM generation and maturation (day 0–21), transduction of Ad-Etv1-EGFP or Ad-EGFP (day 24), and timepoint for experimentation (day 38–40). (B) Quantitative RT-PCR analysis of Etv1, NKX2–5, GJA5, SCN5A, and MYL2 in hiPSC-CM transduced with either Ad-Etv1-EGFP or Ad-EGFP (n = 4). (C) Whole-cell patch clamp was performed on Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Sodium current–voltage (I–V) relationship comparison. (D) hiPSC-CM NaV peak conductance (gNaV-peak). gNaV-peak following −120 mV to −35 mV depolarization step was measured for Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Activation Assay, Derivative Assay, Expressing, Transduction, Quantitative RT-PCR, Patch Clamp, Comparison

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) Representative flow cytometry graphs of CD11b and GFP in peripheral blood of KDRGFP mice bearing gliomas. Astrocytic tumors were generated by RCAS/TVA-mediated overexpression of PDGF as described in Methods. Low-grade gliomas were observed by week 5 and high grade by weeks 7–8. Controls (CTL) were mice bearing an intact Kdr locus without GFP knockin. (B) Quantification of CD11b+KDRGFP+ cell frequency in peripheral blood of RCAS/TVA tumor mice at low-grade and high-grade stages. ***P < 0.001, Student’s t test. LEU, Leukocytes. (C) Further characterization of murine CD11b+KDRGFP+ cells in peripheral blood by Ly6C and Ly6G staining. (D) Quantification of Ly6G+ and Ly6C+ frequency out of total CD11b+ cells in the CD11b+KDRGFP+ and CD11b+KDRGFP– populations in peripheral blood at high-grade stage. **P < 0.01, for Ly6Chi cells, Student’s t test. (E) CFU assays were performed on Lin–KDRGFP+ hematopoietic cells from BM, and macrophage (M) colonies and macrophage/granulocyte (GM) colonies, but not granulocyte (G) colonies, were observed. Quantification of various colony types formed from lineage-negative KDRGFP– or KDRGFP+ cells. ***P < 0.001, in granulocytes, Student’s t test. (F) Gene expression (quantitative RT-PCR) of various markers, including Kdr in in vitro–cultured BM Lin– cells exposed to GL261-conditioned medium at different time points. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA. Data are shown as mean ± SD.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Flow Cytometry, Generated, Over Expression, Knock-In, Staining, Gene Expression, Quantitative RT-PCR, In Vitro, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: Lethal dose–irradiated C57BL/6 mice were transplanted with Ubc-GFP Rosa26-CreERT2Kdrfl/fl and Rosa26-CreERT2KDRfl/+ BM cells, and GL261 tumors were implanted after BM engraftment. (A) Peripheral white blood cells were analyzed on side scatter (SSC) and GFP by flow cytometry. The GFP+ and GFP– populations were gated for further analysis. CD11b versus GFP (B), Ly6C versus Ly6G (C), and CD3 versus B220 (D) are shown on both GFP+ and GFP– populations. (E) BM cells were also analyzed on side scatter and GFP. (F) Lineage-negative cells were gated for analysis of HSCs and HPCs by c-Kit versus Sca-1 (G). (H) HPCs (Lin–c-Kit+Sca-1–) were further characterized by FcγR versus CD34 within both GFP+ and GFP– populations. The experiment was conducted 5 times.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Irradiation, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) The unsupervised principal component analysis of significantly altered genes in Lin–c-Kit+Sca-1– from naive mice, Lin–c-Kit+Sca-1– KDR-GFP+ cells, and Lin–c-Kit+Sca-1–KDR-GFP– cells from BM of tumor-bearing mice. (B) Lin–c-Kit+Sca-1– cells from naive mice (black), Lin–c-Kit+Sca-1– KDR-GFP+ cells (red), and Lin–c-Kit+Sca-1–KDR-GFP– cells (green) from BM of tumor-bearing mice, based on similarity of gene profiles. PC1, principal component 1. (C) The candidate genes (P < 0.05, >1.5-fold change; Lin–c-Kit+Sca-1– KDR-GFP+ versus Lin–c-Kit+Sca-1–KDR-GFP–) were divided according to the subset with the highest expression and analyzed for categories with significant enrichment (P < 0.05) of categories in GO biologic processes using DAVID tools. Similar categories were grouped accordingly. The presence of association between functions and genes was color highlighted (black [negative] versus green [positive]). (D) Expression of Id2 in various lineages of hematopoietic cells. ***P < 0.001, 1-way ANOVA. (E) Expression of ID2 in HPCs (CD45–CD34+) from patients with low-grade or high-grade gliomas. ***P < 0.001, 1-way ANOVA. n = 21. (F) Expression of ID2 in CD11b+ blood cells from patients with low-grade or high-grade gliomas. ***P < 0.001, 1-way ANOVA. n = 20. Data are shown as mean ± SD.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) Chimeric C57BL/6 mice transplanted with Id2–/– BM cells (Id2+/+ BM cells as control) were implanted with luciferase-labeled GL261 tumors intracranially. Tamoxifen was applied at day 3 after implantation. The tumors were monitored by bioluminescence. Representative images were taken at day 14. The color bar on the right represents photon intensity. The experiments had 2 replicates. The tumor growth curve is based on bioluminescence. n = 10. **P < 0.01, 1-way ANOVA. (B) Flow cytometry analysis of peripheral blood cells on CD11b, Ly6C, and Ly6G in Id2+/+ BMT and Id2–/– BMT groups. Quantification of CD11b+ cell frequency out of total white blood cells and of Ly6Chi cell frequency out of CD11b+ cells. **P < 0.01, 1-way ANOVA, for each group. n = 7. (C) Tubule formations of HCMEC/D3 (GFP) cocultured with lineage-negative Id2+/+, Id2–/–, Id2–/– scrambled sequence (SC) control, or Id2–/– KDR overexpression (OE) HPCs pretreated with TGF-β/GM-CSF. Quantifications of tubule lengths in the indicated group. ***P < 0.0001, 1-way ANOVA. n = 6. (D) Growth factor–reduced Matrigel plugs of lineage-negative Id2+/+, Id2–/–, Id2–/– SC CTL, or Id2–/– KDR overexpression HPCs pretreated with TGF-β/GM-CSF. Blood vessels (red) were perfused with rhodamine-dextran. Lower panels show magnified views to highlight vascular permeability. Quantifications of based blood vessels density (BVD) in each group. **P < 0.01, 1-way ANOVA. n = 6. Data are represented as mean ± SD. Scale bars: 50 μm (D, upper panels); 20 μm (C, D, lower panels).

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Control, Luciferase, Labeling, Flow Cytometry, Sequencing, Over Expression, Permeability

Journal: Cancer Cell

Article Title: Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER + Breast Cancer

doi: 10.1016/j.ccell.2020.08.016

Figure Lengend Snippet: Persistent FOXM1 Expression in Breast Cancer PDXs That Have Acquired Resistance to PI3Kα Inhibition (A) Relapse of an HCI-011 PDX after treatment with GDC-0032 (HCI-011R). (B) Mean tumor volumes (cm 3 ) ± SEM of HCI011R xenografts receiving drug vehicle (n = 5) or GDC-0032 (n = 8). p values were defined using two-sided Wald t tests. (C) Immunoblots of lysates from (B) and from an HCI-011 tumor. (D) Mean tumor volumes (cm 3 ) ± SEM of HCI011R xenografts treated with drug vehicle (n = 2), drug vehicle plus tamoxifen (n = 3), and tamoxifen plus GDC-0032 (n = 5). p values were defined using two-sided Wald t tests. (E) Immunoblots of lysates from (D). Mean ± standard deviation (n = 2 to 4). p values were calculated using two-sided Welch's t tests. (F) Gene expression (Log2) enrichment (heatmap: red, positive; white, neutral; blue, negative) of FOXM1 gene targets in HCI-011 (n = 4) and in HCI-011R xenografts (n = 8). The top row shows false discovery rate (FDR)-adjusted p values. (G) Gene expression (Log2) enrichment of genes mediating resistance to BYL-719 in HCI-011 (n = 4 for vehicle and drug) and in HCI-011R (n = 5 vehicle, n = 8 GDC-0032). FDR-adjusted p values of drug-resistant versus drug-sensitive differential expression are shown in the top row. (H) Mean tumor volume (cm 3 ) ± SEM of drug-resistant T47DR tumors after long-term treatment with GDC-0032. p values were defined using two-sided Wald t tests. (I) Immunoblot of tumors lysates from (H). See also Figure S5 .

Article Snippet: PDTCs from HCI-001, HCI-011, HCI-011R tumors and cells from T47DR tumors were obtained by dissociating them using a kit (human 130-095-929, Miltenyi Biotec), as described previously ( ).

Techniques: Expressing, Inhibition, Western Blot, Standard Deviation, Gene Expression, Quantitative Proteomics

Journal: Cancer Cell

Article Title: Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER + Breast Cancer

doi: 10.1016/j.ccell.2020.08.016

Figure Lengend Snippet: Persistent FOXM1 Expression Contributes to Drug Resistance and Can Be Explained by Cytoplasmic Localization of FOXO3a (A) Representative confocal microscopy images of T47D PTEN wt (Ctrl) and PTEN KO cells drug-treated for 72 h. FOXO-3a staining (green), DAPI staining (blue). (B) Relative number of red fluorescent MCF7 and T47D cells (mStrawberry-positive cells, co-expressing FOXM1C) in mixtures with parental controls following treatment for 120 h. p values were calculated using two-sided Welch's t tests. (C) Viability of HCI-011R PDTCs treated with the indicated drugs for 120 h. Left: viability of HCI-011R PDTCs treated for 120 h. Mean ± standard deviation (five technical replicates, n = 3 experiments). Right: mean ± standard deviation (five technical replicates, n = 4 experiments). p values were calculated using two-sided Welch's t tests on the averages of replicates. (D) Viability of cells dissociated from a T47DR tumor treated with the indicated drugs for 120 h. Left: mean ± standard deviation (five technical replicates, n = 4 experiments). Right: mean ± standard deviation (five technical replicates, n = 3 experiments). p values were calculated using two-sided Welch's t tests on the averages of replicates. (E) Viability of T47D PTEN KO cells expressing FOXM1 targeting sequences (shFOXM1 [sequence 1 or 2]) or a non-targeting control sequence (shCtrl) treated for 120 h. Mean ± standard deviation (n = 5, five technical replicates). p values were calculated using two-sided Welch's t tests on the averages of replicates. (F) Viability of HCI-011R PDTCs expressing doxycycline-inducible shRNA sequences targeting FOXM1 (sequence 1) or a control shRNA and treated for 120 h. Mean ± standard deviation (three technical replicates). p values were calculated using two-sided Welch's t tests on the averages of the replicates. (G) LDH activity in the cells used in (E) and treated for 96 h. Figure shows the mean ± standard deviation (n = 3 experiments). p values were calculated using two-sided Welch's t tests on the averages of replicates. (H) Immunoblots of lysates from cells used in (G). (I) Mean tumor volume (cm 3 ) ± SEM of T47D PTEN KO xenografts expressing doxycycline-inducible shFOXM1 (sequence 1) or shCtrl, following prolonged GDC-0032 treatment. One cohort received standard food (shCtrl n = 3 and shFOXM1 n = 3) and the other cohort food plus doxycycline (shCtrl n = 3 and shFOXM1 n = 4; 0.2 g/kg food pellet Harlan D.98186). p values were defined using two-sided Wald t tests. (J) Immunoblot of lysates from tumors in (I). p values were calculated using two-sided Welch's t tests. See also Figure S6 .

Article Snippet: PDTCs from HCI-001, HCI-011, HCI-011R tumors and cells from T47DR tumors were obtained by dissociating them using a kit (human 130-095-929, Miltenyi Biotec), as described previously ( ).

Techniques: Expressing, Confocal Microscopy, Staining, Standard Deviation, Sequencing, Control, shRNA, Activity Assay, Western Blot

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 1. Schematic representation of experimental protocol and workflow of this present work. The top panel is the schematic illustration of the SNA substrates inserts into commercial dishes for CD34+HSPCs enrichment. Below which is the schematic representation of SMNP delivery CBE- and sgRNA-plasmids into commercial dishes with SNA substrates inserts. Then, after obtaining umbilical cord blood cells, SNA substrate and SMNP delivery mediated CBE base editing (SNA⋅SMNP⋅CBE)— enable efficiently and precisely modify BCL11A promoter to achieve C-T conversion and HBG elevation in CD34+HSPCs. The edited human CD34+HSPC was successively transplanted into SCID mouse by intraosseous injection to detect the CD34 engraftment.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Injection

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 3. supramolecular nanoparticle (SMNP) enables delivery of CBE and sgRNA plasmids to modify BCL11A to achieve HBG over expression in vitro. (A), Schematic representation of base-editing experiments in human umbilical cord blood derived CD34+HSPCs enriched by SNA substrate CBE-, sgRNA-(with a red fluorescent label (mCherry) expressing plasmid were co-delivered by SMNP in human CD34+HSPCs enriched by SNA substrate. (B), Representative images of mCherry + cells at 2 h post-transfection following SMNP delivery CBE-, sgRNA-plasmids. (C), Representative flow cytometry results of edited CD34 positive cells ratio after SMNP delivery CBE- and sgRNA- plasmids. (D), Editing efficiency of targeted BCL11A PCR products was detected by Sanger sequencing, with C-T conversion value of 32.7 % base edits at position C1, and 9.5 % at position C2.(E), Real-time PCR analysis of mCherry (left panel), BCL11A (middle panel) and HBG (right panel) expression in CD34+HSPCs subjected with/without SMNP delivering co-encapsulated CBE- and sgRNA targeting BCL11A- plasmids (edited CD34+HSPCs/ naïve CD34+HSPCs). CD34+HSPCs delivered by SMNP co-encapsulated CBE- and sgRNA targeting BCL11A- plasmids was designated as edited group, and unedited group. Results are normalized to GAPDH and shown as mean±SEM. (n=3 biological replicates). Statistical tests compare edited samples and unedited control group (**P＜ 0.01). (F), Western blots were used to detect HBG expression at protein level in edited CD34+HSPCs and naïve CD34+HSPCs. Results are representative of three biological replicates.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Over Expression, In Vitro, Derivative Assay, Expressing, Plasmid Preparation, Transfection, Flow Cytometry, Sequencing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 4. Comparison of our novel system and conventional strategy in the aspect of editing efficiency and CD34+HSPCs property. (A), Editing efficiency of targeted BCL11A PCR products from CD34+HSPCs subjected to different strategies. The top panel (SNA⋅SMNP group) is CD34+HSPCs was enriched by our SNA substrate and transfected with CBE- and sgRNA targeting BCL11A- plasmids by SMNP delivery (with C-T conversion value of 32.7 % base edits at position C1, and 9.5 % at position C2). The middle panel (IMS⋅EP group) is CD34+HSPCs was isolated by conventional immunomagnetic bead method (IMS) and transfected CBE- and sgRNA targeting BCL11A- plasmids with electroporation (EP) (with C-T conversion value of 33.7 % base edits at position C1, and 10.2 % at position C2). The bottom panel (control group) was CD34+HSPCs without edited treatment. (B), Transcriptome analysis was performed in edited CD34+HSPCs with different strategies, and the control naïve CD34+HSPCs. Venn diagram of differential expression genes among the three groups: SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs and naïve CD34+HSPCs (left panel). Gene cluster analysis of transcriptome results among the three groups: SNA⋅SMNP treated CD34+HSPCs, IMS⋅EP treated CD34+HSPCs and naïve CD34+ (right panel). (C), CD34 mRNA expression in three groups SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs and naïve CD34+HSPCs. Results shown as mean±SEM (n=3 biological replicates). (D) mRNA expression of hematopoietic stem cell marker (CD133, CD90) and cell differentiation markers (GATA-1, SPI1, CD235a and CD33) in four groups SNA⋅SMNP CD34+HSPCs, IMS⋅EP CD34+HSPCs, naïve CD34+HSPCs, and PMBC. CD133 and CD90 were shown in left panel, while differ entiation markers including GATA-1, SPI1, CD235a and CD33 were presented in right panel. Results shown as mean±SEM (n=3 biological replicates).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Comparison, Transfection, Isolation, Electroporation, Control, Quantitative Proteomics, Expressing, Marker, Cell Differentiation

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 5. Tracks of editing feasibility of SNA to maintain CD34 property by Intraosseous injection of transplanted edited CD34+HSPCs into SCID mouse. (A), Gene edited human CD34+HSPCs were transplanted into SCD mouse with two different injection strategies: intraosseous injection into the bone marrow of the mouse tibia (left panel) and tail vein injection (right panel). (B), Representative immunofluorescence images (scale bar=8μm) of CD34 in the bone marrow (left panel) and peripheral blood of mice at 14 weeks of intraosseous injection (top) and tail vein injection (below) strategy, respectively. (C), Representative flow cytometry results of CD45+ proportion in the bone marrow (left panel) and peripheral blood of transplanted mice with intraosseous injection (labeled with Red) and tail vein injection (labeled with green) strategy, respectively. Detection time was set at 10-, 12-, 14-week after transplantation. (D), Real-time PCR analysis of CD34 mRNA expression in the bone marrow (left panel) and peripheral blood of mice with intraosseous injection (labeled with Red) and tail vein injection (labeled with green) strategy, respectively. Detection time was set at 10-, 12-, 14-week after transplantation. Results shown as mean±SEM (n=3 biological replicates, *P<0.05, **P<0.01).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Injection, Immunofluorescence, Flow Cytometry, Labeling, Transplantation Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 6. Detection of BCL11A gene modification and HBG/HBB expression after transplantation of edited human CD34+HSPCs. (A), Representative Sanger sequencing of BCL11A from bone marrow (left panel) and peripheral blood (right panel) of transplanted mice, evidenced from C-T conversions. (B), (C), Real-time PCR was used to detect human HBG and HBB expression in the bone marrow (B) and peripheral blood (C) of SCID mice at 10, 12, 14weeks after transplantation of edited human CD34+HSPCs.

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Modification, Expressing, Transplantation Assay, Sequencing, Real-time Polymerase Chain Reaction

Journal: Nano Today

Article Title: SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

doi: 10.1016/j.nantod.2024.102558

Figure Lengend Snippet: Fig. 7. Comparison of our novel strategy and conventional strategy in maintain CD34 property in vivo. (A), Schematic illustration of protocol. The top panel is the process of conventional strategy IMS⋅EP⋅CBE3⋅intravenous (tail vein) injection, while the below panel is the process of our novel strategy SNA⋅SMNP⋅CBE3 ⋅intraosseous injection. After transplantation edited human CD34+HSPCs with different treatment at 14 weeks, SCID mice from the two different groups were euthanatized, mononuclear cells were resorted and enriched from the bone marrow of mice in both groups. Then, transcriptome analysis was performed to detect and compare the gene expression profiles in each group with naïve CD34 controls. (B), Representative flow cytometry results of CD45+ proportion in the bone marrow of SNA⋅SMNP⋅CBE3⋅intraosseous injection mice and IMS⋅EP⋅CBE3⋅intravenous (tail vein) injection counterparts. (C), Real-time PCR analysis of human HBG expression in the bone marrow (left panel) and peripheral blood (right panel) of SNA⋅SMNP⋅CBE3⋅intraosseous injection mice and IMS⋅EP⋅CBE3⋅ intravenous (tail vein) injection counterparts. (D), Transcriptome analysis was performed in edited CD34+HSPCs with different strategies, and the control naïve CD34+HSPCs. Venn diagram of differential expression genes among the three groups: IMS⋅EP⋅CBE3⋅intravenous, SNA⋅SMNP⋅CBE3⋅intraosseous injection and naïve CD34+HSPCs (left panel). Gene cluster analysis of transcriptome results among the three groups (right panel). (E), Representative CD34 expression by Real-time PCR in the three groups: SNA⋅SMNP⋅CBE3⋅intraosseous group, IMS⋅EP⋅CBE3⋅tail vein injection counterparts, as well as naïve CD34+HSPCs. Results shown as mean±SEM (n=3 biological replicates, **P<0.01).

Article Snippet: Method 2: Immno-magnetic isolation (IMS) For CD34 microbead magnetic-activated cell sorting isolation, a cell separation kit (CD34 MicroBead Kit, Miltenyi Biotec, 130–046–702) was used, as per the guided instruction.

Techniques: Comparison, In Vivo, Injection, Transplantation Assay, Gene Expression, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing, Control, Quantitative Proteomics

Journal: Advanced Science

Article Title: Chronic Stress‐Induced and Tumor Derived SP1 + Exosomes Polarizing IL‐1β + Neutrophils to Increase Lung Metastasis of Breast Cancer

doi: 10.1002/advs.202310266

Figure Lengend Snippet: Chronic stress strengthens neutrophil self‐recruitment via CXCL2. Representative images of flow cytometry and quantification of A) PB‐infiltrating and B) lung‐infiltrating neutrophils in 4‐week tumor‐bearing mice from the control group ( n = 6), chronic stress group ( n = 6), and chronic stress and propranolol co‐treated group ( n = 6). C) Representative lung immunofluorescence staining of mice injected with DiI + vivo S‐exo. Red indicates DiI + exosomes, blue indicates DAPI, and green indicates MPO. Scale bar: 10 µm (bottom) and 40 µm (up). D) Representative images of flow cytometry and quantification of lung‐infiltrating neutrophils 4 h after injection with DiI + vivo C‐exo ( n = 5) and vivo S‐exo ( n = 5). E) Representative image of DiI‐labeled neutrophils absorbing DiO‐labeled TDEs after 4 h of co‐culture. 488 nm, TDE; 405 nm, Hoechst; 555 nm, neutrophils. The arrows indicate neutrophils that have taken up DiO‐labeled exosomes. F) Representative images of Bouin's and H&E staining for lungs from the 4‐week tumor‐bearing mice. Quantification of the number of lung metastatic nodules and burden of lung metastases ( n = 5 mice/group). G,H) Transwell migration assay to detect the neutrophil recruitment ability of the conditioned medium ( n = 4). I) qRT‐PCR analysis of chemokine expression in naïve neutrophils treated alone ( n = 3), with vivo C‐exo ( n = 3), and vivo S‐exo ( n = 3), respectively for 4 h. J) ELISA analysis of CXCL2 concentrations in the supernatant of naïve neutrophils treated alone ( n = 5), with vivo C‐exo ( n = 5), and vivo S‐exo ( n = 5), respectively for 4 h. K) qRT‐PCR analysis of Cxcl2 expression in naïve neutrophils treated with vivo C‐exo ( n = 3), vivo S‐exo ( n = 3), and vivo S+P‐exo ( n = 3) for 4 h. L) ELISA analysis of CXCL2 concentrations in the lung supernatant of 4‐week tumor‐bearing mice in the control group ( n = 7), chronic stress group ( n = 7), and chronic stress and propranolol co‐treated group ( n = 7). M,N) Transwell migration assay to detect the neutrophil recruitment ability of N+vivo S‐exo CM ( n = 6) and N+vitro ISO‐exo CM ( n = 6) with or without the addition of anti‐CXCL2 antibody. The data are shown as mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p < 0.0001.

Article Snippet: Neutrophils were isolated using a mouse neutrophil isolation kit (Miltenyi) according to the manufacturer's instructions.

Techniques: Flow Cytometry, Control, Immunofluorescence, Staining, Injection, Labeling, Co-Culture Assay, Transwell Migration Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Chronic Stress‐Induced and Tumor Derived SP1 + Exosomes Polarizing IL‐1β + Neutrophils to Increase Lung Metastasis of Breast Cancer

doi: 10.1002/advs.202310266

Figure Lengend Snippet: TDEs under chronic stress polarize IL‐1β + neutrophils through the TLR4‐NFκβ pathway. A) qRT‐PCR analysis of Il‐1β expression in naïve neutrophils treated alone ( n = 3), with vivo C‐exo ( n = 3), and vivo S‐exo ( n = 3), respectively for 4 h. B) qRT‐PCR analysis of Il‐1β expression of naïve neutrophils treated alone ( n = 3), with vitro C‐exo ( n = 3), and vitro ISO‐exo ( n = 3), respectively for 4 h. C) WB analysis of IL‐1β ( n = 11) and NFκβ pp65 ( n = 5) expression in naïve neutrophils treated alone, with vivo C‐exo, and vivo S‐exo, respectively for 4 h. D) WB analysis of IL‐1β ( n = 6) and NFκβ pp65 ( n = 3) expression in naïve neutrophils treated alone, with vitro C‐exo, and vitro ISO‐exo, respectively for 4 h. E) ELISA analysis of IL‐1β concentrations in the supernatant of naïve neutrophils treated alone ( n = 6), with vivo C‐exo ( n = 6), vivo S‐exo ( n = 6), and ISO ( n = 6), respectively for 4 h. F) IL‐1β + neutrophils quantification by flow cytometry of naïve neutrophils treated alone ( n = 6), with vivo C‐exo ( n = 6), vivo S‐exo ( n = 6), and vivo S+P‐exo ( n = 6), respectively for 4 h. G) Flow cytometry quantification of lung‐infiltrating IL‐1β + neutrophils in 4‐week tumor‐bearing mice in the control group ( n = 8), chronic stress group ( n = 8), and chronic stress & propranolol co‐treated group ( n = 8). H) ELISA analysis of IL‐1β concentrations in the lung supernatant of 4‐week tumor‐bearing mice in the control group ( n = 24), chronic stress group ( n = 24), and chronic stress & propranolol co‐treated group ( n = 24). I) qRT‐PCR analysis of Nfκβ1 , Tlr2 , and Tlr4 expression of naïve neutrophils treated alone ( n = 3), with vivo C‐exo ( n = 3), and vivo S‐exo ( n = 3), respectively for 4 h. J) qRT‐PCR analysis of Nfκβ1 , Tlr2 , and Tlr4 expression of naïve neutrophils treated alone ( n = 3), with vitro C‐exo ( n = 3), and vitro ISO‐exo ( n = 3), respectively for 4 h. K) Flow cytometry quantification of lung‐infiltrating TLR4 + neutrophils in 4‐week tumor‐bearing mice of the control group ( n = 8) and chronic stress group ( n = 8). L) Quantification of IL‐1β + neutrophils by flow cytometry in naïve neutrophils treated alone ( n = 5), with vivo C‐exo ( n = 5), vivo S‐exo ( n = 5), vivo S‐exo and TLR2 inhibitor ( n = 5), vivo S‐exo and TLR4 inhibitor ( n = 5), respectively for 4 h. M) Quantification of IL‐1β + neutrophils by flow cytometry in naïve neutrophils treated alone, with vivo C‐exo ( n = 6), vitro C‐exo ( n = 6), vivo C‐exo and a TLR4 agonist ( n = 6), vitro C‐exo and a TLR4 agonist ( n = 5), vivo C‐exo and a TLR4 agonist and a NFκβ inhibitor ( n = 6), and vitro C‐exo and a TLR4 agonist and a NFκβ inhibitor ( n = 6), respectively for 4 h. The data are shown as mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p < 0.0001.

Article Snippet: Neutrophils were isolated using a mouse neutrophil isolation kit (Miltenyi) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control

Journal: Advanced Science

Article Title: Chronic Stress‐Induced and Tumor Derived SP1 + Exosomes Polarizing IL‐1β + Neutrophils to Increase Lung Metastasis of Breast Cancer

doi: 10.1002/advs.202310266

Figure Lengend Snippet: Exosomal SP1 activates the neutrophil TLR4‐NFκβ‐IL‐1β pathway, leading to lung metastasis. A) WB analysis of SP1 expression in vivo C‐exo ( n = 3) and vivo S‐exo ( n = 3). B) WB analysis of SP1 expression in BM‐PMNs treated alone ( n = 6), with vivo C‐exo ( n = 6), or with vivo S‐exo ( n = 6) for 4 hours. C) WB analysis of SP1 expression of BM‐PMNs treated alone ( n = 5), with vitro C‐exo ( n = 5), and vitro ISO‐exo ( n = 5), respectively for 4 h. D) Flow cytometry quantification of IL‐1β + neutrophils in BM‐PMNs treated with vivo C‐exo ( n = 6), vivo S‐exo ( n = 6), vivo S‐exo and the 500 n m SP1 inhibitor mithramycin A ( n = 6) for 4 h. E) Flow cytometry quantification of IL‐1β + neutrophils of BM‐PMNs treated alone ( n = 3), with 500 n m mithramycin A ( n = 3), vitro C‐exo ( n = 3), vitro ISO‐exo ( n = 3), and vitro ISO‐exo and 500 n m mithramycin A ( n = 3), respectively for 4h. F) Representative H&E staining and quantification of lung metastases in 4‐week tumor‐bearing mice of the chronic stress + PBS injection group ( n = 5) and the chronic stress + 1 mg kg −1 mithramycin A injection group ( n = 5). Scale bar: 1 mm. G,H) Flow cytometry quantification of lung‐infiltrating neutrophils and lung‐infiltrating IL‐1β + neutrophils in 4‐week tumor‐bearing mice from the chronic stress + PBS injection group ( n = 5) and the chronic stress + 1 mg kg −1 mithramycin A injection group ( n = 5). I) Diagram illustrating the injection of SP1 control exosomes (4T1‐shCTRL C‐exo) and SP1 knockdown exosomes (4T1‐shSP1#2 C‐exo) into mouse via the tail vein. Exosomes were injected every 3 days into BALB/C mice that had received a subcutaneous injection of 10 5 4T1 tumor cells 7 days earlier. J) Representative images of Bouin's and H&E staining for lungs from the 4‐week tumor‐bearing mice injected with 4T1‐shCTRL C‐exo ( n = 5) or 4T1‐shSP1#2 C‐exo ( n = 5). Quantification of the number of lung metastatic nodules and burden of lung metastases are shown. K) Flow cytometry quantification of lung‐infiltrating neutrophils and lung‐infiltrating IL‐1β + neutrophils in 4‐week tumor‐bearing mice injected with 4T1‐shCTRL C‐exo ( n = 5) or 4T1‐shSP1#2 C‐exo ( n = 5). The data are shown as mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p < 0.0001.

Article Snippet: Neutrophils were isolated using a mouse neutrophil isolation kit (Miltenyi) according to the manufacturer's instructions.

Techniques: Expressing, In Vivo, Flow Cytometry, Staining, Injection, Control, Knockdown

Journal: Advanced Science

Article Title: Chronic Stress‐Induced and Tumor Derived SP1 + Exosomes Polarizing IL‐1β + Neutrophils to Increase Lung Metastasis of Breast Cancer

doi: 10.1002/advs.202310266

Figure Lengend Snippet: Chronic stress significantly promotes the secretion of TDEs and alters the contents of exosomes by stimulating the adrenergic β receptor. Chronic stress strengthens neutrophil self‐recruitment through the CXCL2 autocrine. Upon exposure to chronic stress, the level of SP1 in TDEs increases, which promotes neutrophil secretion of IL‐1β through the TLR4‐NFκβ pathway, thereby remodeling the lung microenvironment and contributing to the promotion of lung metastasis of breast cancer.

Article Snippet: Neutrophils were isolated using a mouse neutrophil isolation kit (Miltenyi) according to the manufacturer's instructions.

Techniques:

Journal: Research

Article Title: Gene Signature-Based Drug Screening Reveals Ponatinib Enhances Immunotherapy Efficacy in Triple-Negative Breast Cancer by Reversing MDSC-Mediated Immunosuppressive Tumor Microenvironment

doi: 10.34133/research.0915

Figure Lengend Snippet: Ponatinib inhibits the infiltration of immunosuppressive MDSCs into TNBC TME by repressing CXCL1 and CXCL2 expression. (A) Growth of 4T1 tumors in BALB/c nude mice treated with ponatinib or vehicle (control, n = 5; ponatinib, n = 8). Tumor volume kinetics were monitored by vernier calipers (left). Statistical significance was determined by 2-way ANOVA. Terminal tumor weight quantification is shown (middle), with statistical significance determined by unpaired 2-tailed Student’s t tests. *** *P < 0.0001. The tumor image (right) shows 4T1 tumors from the indicated groups. (B) Growth of 4T1 tumors in NSG mice treated with ponatinib or vehicle ( n = 6 mice per group). Tumor volume kinetics monitored by vernier calipers (left). Statistical significance was determined by 2-way ANOVA. Terminal tumor weight quantification is shown (middle), with statistical significance determined by unpaired 2-tailed Student’s t tests. ns (not significant), P > 0.05. The tumor image (right) shows 4T1 tumors from the indicated groups. (C) RT-qPCR analysis of Cxcl1 and Cxcl2 mRNA levels in 4T1 tumors from BALB/c WT mice (left) or nude mice (right) receiving ponatinib or control treatments as described in Figs. H and A. Heatmap representing the relative expression of the indicated chemokine genes normalized to Gapdh . Expression scaled from high (red) to low (blue). Each square represents individual tumors from a single mouse (gray squares indicate tumors from mice sacrificed due to the requirement of animal ethics). (D) MDSC identification using publicly available scRNA-seq data from breast cancer patients. Cells were colored by cell types defined in a breast cancer scRNA-seq dataset (left) and by MDSC types predicted using the scPred package (right). MDSCs were identified in a dataset of 29 breast cancer patients receiving ICB therapy (EGAD00001006608) by training on another well-defined breast cancer MDSC dataset ( GSE139125 ) . (E) Percentage of different cell types in the breast cancer TME, grouped by high or low expression of CXCL1 and CXCL2 in scRNA-seq data from breast cancer patients . P values were determined using the chi-square test. (F) Percentage of MDSCs (relative to all myeloid cells) in breast cancer patients with high or low expression of CXCL1 and CXCL2 , calculated using the breast cancer scRNA-seq dataset . (G) Migration of MDSCs toward CM from 4T1 cells treated with ponatinib or DMSO, evaluated using in vitro Transwell migration assays. (H) Migration of MDSCs toward CM from 4T1 cells treated with either DMSO or ponatinib and supplemented with or without recombinant mouse CXCL1 and CXCL2. (I) Transwell migration assays for MDSC migration toward CM from MC38 cells treated with either ponatinib or DMSO. Data are presented as means ± SEM. Statistical significance was assessed using unpaired 2-tailed Student’s t tests. * *P < 0.01, ** *P < 0.001, and *** *P < 0.0001.

Article Snippet: MDSCs were isolated from the spleens of 4T1 tumor-bearing mice using the Mouse MDSC Isolation Kit (Miltenyi Biotec, catalog no. 130-094-538).

Techniques: Expressing, Control, Quantitative RT-PCR, Migration, In Vitro, Recombinant

Journal: Research

Article Title: Gene Signature-Based Drug Screening Reveals Ponatinib Enhances Immunotherapy Efficacy in Triple-Negative Breast Cancer by Reversing MDSC-Mediated Immunosuppressive Tumor Microenvironment

doi: 10.34133/research.0915

Figure Lengend Snippet: Ponatinib reduces the infiltration of protumor-related immune cells and enhances the infiltration of antitumor-related immune cells into the TME in vivo. (A) Flow cytometry analysis of MDSCs (CD45 + CD11b + F4/80 − Gr-1 + ) in 4T1 tumors of BALB/c WT mice treated with ponatinib or vehicle. (B) Flow cytometry analysis of PMN-MDSCs (CD45 + CD11b + Ly-6G + Ly-6C − ) and M-MDSCs (CD45 + CD11b + Ly-6G − Ly-6C + ) in 4T1 tumors of BALB/c WT mice treated with ponatinib or vehicle. (C) Flow cytometry analysis of MDSC in 4T1 tumors from BALB/c nude mice treated with ponatinib or vehicle. Data are presented as means ± SEM. Statistical significance was determined using unpaired 2-tailed Student’s t tests. **P < 0.01. (D) A mixture of 4T1 cells and MDSCs (isolated from tumor-bearing mice) was inoculated into the fat pad of BALB/c WT mice ( n = 10). The growth of the tumor was monitored, and ponatinib (30 mg/kg) or vehicle was administered 4 days a week when the tumor volume reached 100 mm 3 . Data represent mean ± SEM. Two-way ANOVA was used for statistical significance. ns, P > 0.05, ****P < 0.0001. (E) Representative images of 4T1 tumors from the indicated groups in (D). (F) Flow cytometry analysis of CD3 + T cells (CD45 + CD3e + ) in 4T1 tumors of BALB/c WT mice treated with ponatinib or vehicle. (G) Flow cytometry analysis of CD4 + T cells (CD45 + CD3e + CD8 − CD4 + ) and CD8 + T cells (CD45 + CD3e + CD8 + CD4 − ) in 4T1 tumors of BALB/c WT mice treated with ponatinib or vehicle. (H) Flow cytometry analysis of NK cells (CD45 + CD49b + ) in 4T1 tumors from BALB/c nude mice treated with ponatinib or vehicle. (I) Flow cytometry data analysis of NK cells (CD45 + Nkp46 + ) in spleens from 4T1 tumor-bearing BALB/c WT mice treated with ponatinib or vehicle. Data are presented as means ± SEM. Statistical significance was determined using unpaired 2-tailed Student’s t tests. **P < 0.01, ***P < 0.001.

Article Snippet: MDSCs were isolated from the spleens of 4T1 tumor-bearing mice using the Mouse MDSC Isolation Kit (Miltenyi Biotec, catalog no. 130-094-538).

Techniques: In Vivo, Flow Cytometry, Isolation

Journal: Research

Article Title: Gene Signature-Based Drug Screening Reveals Ponatinib Enhances Immunotherapy Efficacy in Triple-Negative Breast Cancer by Reversing MDSC-Mediated Immunosuppressive Tumor Microenvironment

doi: 10.34133/research.0915

Figure Lengend Snippet: Combination treatment with ponatinib enhances the therapeutic efficacy of anti-PD-L1 against TNBC. (A) Expression levels of CXCL1 or CXCL2 and therapeutic responses assessed from publicly available scRNA-seq data for 29 breast cancer patients who received ICB therapy . Cells were colored based on ICB response results (top) and gene expression levels of CXCL1 and CXCL2 (bottom). Es, patients with clonotype expansion ( n = 9); NEs, patients with limited or no clonotype expansion ( n = 20). (B) Receiver operating characteristic (ROC) curves depicting the predictive value of CXCL1 and CXCL2 expression levels for anti-PD-1 therapy response in breast cancer patients. (C) CXCL1 and CXCL2 expression in tumor cells, comparing Es with NEs, calculated using the breast cancer scRNA-seq dataset . P values were determined using the Mann–Whitney test. (D) Expression of known MDSC-related genes [ , , , , ] in MDSCs determined in this study, comparing Es with NEs. MDSCs were identified in the breast cancer scRNA-seq dataset (EGAD00001006608) , through learning from another breast cancer scRNA-seq dataset with MDSC well-defined ( GSE139125 ) , using the cell type prediction method of scPred package . P values were determined using the Mann–Whitney test. (E) Expression of known MDSC-related genes [ , , , , ] in MDSCs, comparing pretreatment with on-treatment conditions. Pre, biopsy collected before anti-PD-1 treatment; On, biopsy collected during subsequent surgery. P values were determined using the Mann–Whitney test. (F) Expression of known MDSC-related genes in the breast TME, comparing Es with NEs from the breast cancer scRNA-seq dataset . P values were determined using the Mann–Whitney test. (G) Expression of known MDSC-related genes in tumor cells, comparing Es with NEs, calculated using the breast cancer scRNA-seq dataset . P values were determined using the Mann–Whitney test. (H) Expression of known MDSC-related genes in MDSCs, comparing across 3 different breast cancer types. P values were determined using the Mann–Whitney test. (I) Expression of known MDSC-related genes in the TME, comparing across 3 different breast cancer types, was calculated using the breast cancer scRNA-seq dataset . P values were determined using the Mann–Whitney test. (J) Schematic diagram of the combination treatment protocol for 4T1-bearing mice; ponatinib (30 mg/kg) or vehicle was administered 4 days per week, and PD-L1 monoclonal antibody (200 μg per injection, twice a week), either alone or in combination, starting when the tumor volume reached 100 mm 3 ( n = 12 to 14 mice per group). (K and L) Growth of 4T1 tumors in BALB/c WT mice treated with the indicated conditions from (J). Tumor volume kinetics were monitored by vernier calipers. Data represent mean ± SEM of n mice. Two-way ANOVA determined statistical significance. * *P < 0.01, ** *P < 0.001, and *** *P < 0.0001. (M) Survival curves for the indicated groups. Statistical significance was determined using the log-rank (Mantel–Cox) test. ns, P > 0.05, * *P < 0.01, and ** *P < 0.001.

Article Snippet: MDSCs were isolated from the spleens of 4T1 tumor-bearing mice using the Mouse MDSC Isolation Kit (Miltenyi Biotec, catalog no. 130-094-538).

Techniques: Drug discovery, Expressing, Gene Expression, MANN-WHITNEY, Injection

Journal: Frontiers in Immunology

Article Title: Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

doi: 10.3389/fimmu.2017.01163

Figure Lengend Snippet: Experimental setup and quality controls for phosphoproteomics in primary human T cells. (A) Conventional CD4 + CD25 – T cells (Tcons) were cocultured either with allogeneic Tcons or regulatory T cells (Tregs), and cocultures were stimulated for 5 min with cross-linked anti-CD3/anti-CD28 antibodies. Stimulation was stopped on ice. Tstim (blue) and Tsup (red) were obtained after separation of T cell receptor (TCR)-stimulated Tcon:Tcon or Tcon:Treg cocultures, respectively. Unstimulated Tcons (Trest; gray) from the same donor were processed in parallel. Proteins were digested, peptides dimethyl-labeled and mixed, before phosphopeptides were enriched and measured by mass spectrometry (MS). Relative abundance of phosphopeptides was quantified by calculating the intensity ratios between the different samples as indicated. (B) An aliquot of cells used for phosphoproteomics was stimulated for 3 h before coculture separation, and suppression of cytokine mRNA was measured in re-isolated responder Tcons [Trest, Tstim, and Tsup as in panel (A) ]. As additional control, responder Tcons were stimulated without allogeneic Tcons (control Tstim). IL2 and IFNG mRNA were measured by quantitative RT-PCR, normalized to GAPDH mRNA. Results are presented as fold change compared to Trest (set to 1). The upper panel shows a representative donor (mean ± SD of technical PCR duplicates). Percentage suppression of respective cytokines in Tsup as compared to Tstim was calculated and is summarized for the three phosphoproteomics donors (lower panel). T cells were processed in three independent experiments (one experiment/donor) and phosphopeptide enrichment was performed in two independent experiments. (C) The number of unique phosphopeptides detected in each donor was determined, and the overlap is depicted as Venn diagram.

Article Snippet: We first isolated CD25 high cells with CD25-specific MACS beads (2 μl/10 7 cells, Miltenyi Biotec; cat. no. 130-092-983) as described previously ( ).

Techniques: Phospho-proteomics, Labeling, Mass Spectrometry, Isolation, Control, Quantitative RT-PCR