Journal: Journal of translational medicine

Article Title: RBM15 recruits myeloid-derived suppressor cells via the m6A-IGF2BP3/CBR3-AS1/miR-409-3p/CXCL1 axis, facilitating radioresistance in non-small-cell lung cancer.

doi: 10.1186/s12967-025-06205-y

Figure Lengend Snippet: Fig. 4 CBR3-AS1 recruits MDSCs through the miR-409-3p/CXCL1/CXCR2 axis. A Relative mRNA expression levels of CXCL1 in H520 cells transfected with a control empty vector (vecCtrl), miR-409-3p mimic plasmid (miR-409-3p), control shRNA (shCtrl), or specific interfering hairpin RNAs targeting miR-409-3p (shmiR-409-3p). The cells were exposed to radiation after 24 h of transfection. B Relative protein expression levels of CXCL1 in H520 cells. β-actin was used as the reference for normalization. C Relative luciferase activity of CXCL1 under miR-409-3p overexpression in the wild-type and mutant CXCL1 groups treated with radiation. D, E Relative mRNA and protein expression levels of CXCL1 in H520 cells transfected with shCtrl, shCBR3-AS1, or shmiR-409-3p. F Migration ability of MDSCs toward the conditioned medium of H520 cells transfected with CXCL1, shCXCL1, or treated with a CXCR2 inhibitor (SB265610, 10 mM) and treated with radiation. G, H Levels of interferon (IFN)-γ secreted by CD8+ and CD4.+ T cells co-cultured with H520 cells transfected with CXCL1, shCXCL1 or treated with a CXCR2 inhibitor (SB265610, 10 mM). IFN-γ expression was reported as the mean fluorescence intensity. The significance between two groups was analyzed using Student’s t-test. Data are presented as mean ± SD from three independent experiments. Statistical significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Membranes were probed with primary antibodies: RBM15 (1:500, Abclonal, USA, A4936), IGF2BP3 (1:1000, Abclonal, A4444), CXCL1 (1:500, Abclonal, USA, A5802), CXCR2 (1:1000, Proteintech, Wuhan, China, 20634-1-AP), or β-actin (1:500, Abclonal, USA, WL01372).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, shRNA, Luciferase, Activity Assay, Over Expression, Mutagenesis, Migration, Cell Culture, Fluorescence

Journal: Mucosal Immunology

Article Title: Butyrate regulates neutrophil homeostasis and impairs early antimicrobial activity in the lung

doi: 10.1016/j.mucimm.2023.05.005

Figure Lengend Snippet: Effect of butyrate on acute Pseudomonas aeruginosa immunity. (A) Quantification of the total number of cells in BALF 18 hours post-inoculation with 1 × 10 6 CFU of P. aeruginosa strain PAO1 in Ctrl and But mice. (B) Frequency and quantification of cell types in cytospins of BALF from control and butyrate-treated mice 18 hours after PAO1 infection; (C) Quantification of P. aeruginosa CFU in BALF 18 hours post-challenge. (D, E) Cytokine and chemokine production in BALF 18 hours following infection. (F) MFI of CXCR2 on lung AM and IM from Ctrl and But mice. (G) MFI of CXCR2 on Ctrl and But lung macrophages US or stimulated for 2 hours with LPS in vitro . (H) transmigration of Ctrl and But neutrophils toward a CXCL2 or CCL2 chemokine gradient after 3 hours. Results are a mean of two independent experiments. Values are expressed as mean ± standard error of mean; n = 6–14. Statistical significance was determined with one-way analysis of variance in (A–E, G, H) and Student’s t test (unpaired, two-tailed) in (F). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. AM = alveolar macrophages; BALF = bronchoalveolar lavage fluid; But = butyrate-treated; CFU = colony-forming units; Ctrl = control; IFN = interferon; IL = interleukin; IM = interstitial macrophages; LPS = lipopolysaccharide; MFI = mean fluorescence intensity; PAO1 = P. aeruginosa strain 1; TNF = tumor necrosis factor; US = unstimulated.

Article Snippet: To differentiate progenitor and neutrophil populations in the BM, cells were stained with antibodies to lineage cocktail, CD115 (BD Biosciences, clone 2B8), SiglecF (BD Biosciences, clone 2B8), CD117 BUV117 (BD Biosciences, clone 2B8), Sca1 APCFire (Biolegend, clone D7), FcgR biot (Miltenyi, clone 93), CD34 BV421 (Biolegend, clone SA376A4), CD11b BV510 (BD Biosciences, clone M1/70), Ly6G Pecy5 (eBioscience, clone 1A8-Ly6g), CXCR2 Pecy7 (Miltenyi, clone REA942) and streptavidin BUV395 (BD Biosciences).

Techniques: Control, Infection, In Vitro, Transmigration Assay, Two Tailed Test, Fluorescence

Journal: Advanced Science

Article Title: Interior Decorative VOCs Elevate T Cell‐Mediated Obstructive Lung Disease Risks via Osteogenesis‐Driven Lymphoid‐Biased Hematopoiesis

doi: 10.1002/advs.202512663

Figure Lengend Snippet: VOCs exposure enhances T cell maturation and their recruitment to lung tissues. Mice were exposed to CON or VOCs for 8‐week using the whole‐body inhalation exposure model. A) Representative images of H&E‐stained thymic sections. Black dashed lines indicate the cortical thickness of thymus. Scale bar: 100 µm. Immunohistochemical analysis for B) CD3 and C) CD8 in thymic sections, and the percentage of positive area. Yellow arrows indicate the positive expression. Scale bar: 40 µm. D) Representative flow cytometric dot plots and quantitative analysis of E) CD4 + T and F) CD8 + T cells in the PB. G) Immunostaining for the colocalization of CXCR3 (red) and CD8 (purple) in lung sections, and the corresponding quantitative analysis. Yellow arrows indicate CXCR3 colocalization with CD8. Scale bar: 20 µm. H) Immunostaining for the colocalization of CCR5 (green) and CD8 (purple) in lung sections, and the corresponding quantitative analysis. Yellow arrows indicate CCR5 colocalization with CD8. Scale bar: 20 µm. I) Quantitative analysis of CXCL9, CXCL10, and CXCL11 in the PB by ELISA assay. Protein expressions of CXCL10 in J) BALF and K) LI. L) A schematic representation summarizing the T cell maturation and recruitment to lung tissue. Statistical analysis was performed using two‐tailed unpaired t ‐test (A–C and E–K). * p < 0.05 and ** p < 0.01. Data are expressed as mean ± SEM ( n ≥ 3).

Article Snippet: Similarly, mouse lung sections were incubated with primary antibodies against CXCR3 (Bosterbio, China), CCR5 (Servicebio, China), and CD8 (Servicebio, China) at 4 °C overnight, followed by the application of secondary antibodies.

Techniques: Staining, Immunohistochemical staining, Expressing, Immunostaining, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: (A) Flow cytometry analysis of CXCR3 protein expression on a VGP melanoma cell line, C9. Cells were incubated with serum at 5% CO 2 . (B) Graph of CXCR3 protein expression on BOWES cells from 8 separate flow cytometry experiments. Cells were cultured either in normal culture conditions (far left bar), in serum-free media at 8% CO 2 for 24, 48, or 72 hrs, or in serum-free media at 8% CO 2 for 48 hrs followed by 48 hrs in normal culture conditions (far right bar). T-tests were performed on the averages for each time point, comparing treatment cells with the 0hr time point (NS = P>0.05, *** = P≤ 0.001). (C-D) Flow cytometry analysis of CXCR3 protein expression on an RGP cell line, BOWES. Cells were incubated (C) with serum at 5% CO 2 or (D) without serum at 8% CO 2 for 72 hrs. All flow cytometry plots and graphs are representative data from at least 3 separate experiments. Red lines = unstained cells, blue lines = cells stained with CXCR3.

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Expressing, Incubation, Cell Culture, Staining

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: Gene expression of stressed BOWES cells (cultured in serum-free media at 8% CO 2 ) relative to those cultured under normal serum-containing conditions (10% FBS media at 5% CO 2 ).

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: Gene Expression, Cell Culture

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: (A) BOWES cells were cultured for 48 hrs under stressful conditions (serum-free media at 8% CO 2 ) in the presence of ligand (100ng/ml CXCL9 and CXCL10), with the addition of DMSO, 0.2μM, or 1μM AMG487. IL-8 expression was measured with RT-PCR, fold change was calculated relative to cells treated 1μM AMG487. (B) Flow cytometry analysis of representative data from 3 separate sorts. CXCR3 Low and CXCR3 High cells were sorted based on the gates shown. Red line = unstained cells, blue line = cells stained with CXCR3. (C) CXCR3 and (D) IL-8 RT-PCR results from unsorted cells grown with serum in 5% CO 2 or without serum in 8% CO 2 (stressed), and sorted stressed cells (CXCR3 Low and CXCR3 High ). Fold change was calculated relative to unsorted stressed cells. T-tests were performed on the relative fold changes, pooled data from 3 separate experiments (* = P≤ 0.05, ** = P≤0.01, ***P≤ 0.001).

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: BOWES cells stably transfected with CXCR3 or an empty vector (PCMV6) were grown in serum-containing media. (A) Flow cytometry and (B) RT-PCR were used to measure CXCR3 expression. (A) BOWES WT (red line), BOWES PCMV6 clone #1(blue line), and BOWES CXCR3 clones #1 (orange line), #2 (light green), and #3 (dark green line). (B) RT-PCR was normalized to CXCR3 clone #2. (C) BOWES PCMV6 and CXCR3 cells were grown in serum-free media for 2 hrs, then in the presence of CXCL9 and CXCL10 for 2, 5, 10, 20, or 30 minutes. Total protein was isolated and probed for pERK and total ERK via immunoblot analysis. (D) BOWES PCMV6 and CXCR3 cells were plated on a membrane above serum-free media with CXCL9/10 (+ligand) or without (-ligand), and total migrated cells per field (40X objective) were quantified after 6hrs. Cells were also plated on the membrane in the presence or absence of AMG487. T-tests were performed on the BOWES CXCR3 migrated cells ± ligand in the presence and absence of AMG487 (NS = P>0.05, **** = P≤ 0.0001). (E) BOWES PCMV6 and CXCR3 cells were grown in serum-containing media for 48 hrs in the presence of ligand, with 0, 0.2, or 1 μM AMG487. Expression of IL-8 was measured via RT-PCR, fold change was calculated relative to BOWES PCMV6 0 μM. T-tests were performed on the relative fold changes, representative data from 3 separate experiments (**** = P≤ 0.0001).

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Expressing, Clone Assay, Isolation, Western Blot, Membrane

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: WM35 IL-8 signaling is CXCR3-signaling independent. (A) BOWES WT cells were grown in serum-free media for 48 hrs in the presence of ligand, with 0, 0.2, or 1 μM AMG487 and with DMSO (black bars) or 3 μM U0126 (gray bars). Expression of IL-8 was measured via RT-PCR, fold change was calculated relative to cells treated with 0 μM U0126 and 1 μM AMG487. (B-C) Flow cytometry analysis of CXCR3 protein expression on the BRAF V600E RGP cell line, WM35. Cells were incubated (B) with 5% serum at 5% CO 2 or (C) without serum at 8% CO 2 for 72 hrs. Red lines = unstained cells, blue lines = cells stained with CXCR3. (D) BOWES WT (black bars) and WM35 cells (gray bars) were grown in serum-free media for 48 hrs in the presence of ligand, with 0, 0.2, or 1 μM AMG487. Expression of IL-8 was measured via RT-PCR, fold change was calculated relative to BOWES cells treated with 0 μM AMG487. (E) WM35 cells were grown in serum-free media for 48 hrs in the presence of ligand, with 0, 0.2, or 1 μM AMG487 and either DMSO (black bars), 3 μM U0126 (striped bars), or 3 μM PLX4032 (gray bars). Expression of IL-8 was measured via RT-PCR, fold change was calculated relative to WM35 cells treated with 0 μM AMG487 and 3 μM U0126. All T-tests were performed on the relative fold changes, all data is representative from at least 3 separate experiments (NS = P>0.5, * = P≤ 0.05, ** = P≤0.01, **** = P≤ 0.0001).

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Incubation, Staining

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: Tumor growth in vivo .

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: In Vivo, Injection

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: BOWES PCMV6 and CXCR3 cells were injected interdermally into NSG mice (5 x 10 5 cells per injection). Tumors were measured weekly with calipers and tumor volume (mm 3 ) was calculated. Mean tumor volume and SEM were calculated from mice that had measurable tumors. (A) The graph shows mean tumor volume (with SEM) over the 6 week period. Linear regression analysis was performed on the two slopes (P = 0.006). (B) Tumor tissue from injected mice was resected at the time of harvest and RNA was isolated. IL-8 expression was measured in BOWES PCMV6 and CXCR3 tumor tissue via RT-PCR (**** = P≤ 0.0001). Representative individual tumors are presented in graph.

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: Injection, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Acta Pharmaceutica Sinica. B

Article Title: ACAT1 deficiency in myeloid cells promotes glioblastoma progression by enhancing the accumulation of myeloid-derived suppressor cells

doi: 10.1016/j.apsb.2023.09.005

Figure Lengend Snippet: CXCL1 is a key factor to promote MDSCs accumulation after ACAT1 depletion. (A) The protein levels of mouse CXCL1 in ectopic G422 tumors of LyzM-cre ( n = 8) and Acat1- CKO ( n = 8) mice at 14 days post-tumor cell inoculation. ∗∗∗ P = 0.000014. (B) Representative images of CXCL1-stained tumor sections (four images per mouse) of LyzM-cre and Acat1- CKO mice. (C) Serum CXCL1 level of tumor-free ( n = 3) mice and mice with ectopic ( n = 8) or orthotopic ( n = 5) G422 glioma model as described in Fig. 2 . ∗∗∗ P = 0.00076, ∗∗∗ P = 7.4E-6, ∗∗ P = 0.0080. (D) The indicated leukocyte ratio in the blood of normal LyzM-cre ( n = 5) and Acat1- CKO ( n = 5) mice without treatment. ∗∗ P = 0.00172, ∗∗ P = 0.00469, ∗ P = 0.0282, ∗∗∗ P = 0.000832. (E) Flow cytometry analysis (left) and percentage (right) of MDSCs (CD11b + /Gr1 + ) in the bone marrow cells cultured in complete RPMI medium containing GM-CSF and IL-6 from LyzM-cre ( n = 3) and Acat1- CKO ( n = 3) mice on the fourth day induced by CXCL1 of indicated concentration. ∗∗∗ P = 0.00097, ∗∗∗ P = 1.85E-5, ∗∗∗ P = 3.67E-7, ∗∗∗ P = 9.53E-7.

Article Snippet: To examine the effect of M φ -secreted cytokine on MDSC differentiation, the culture supernatants of normal BMDM and Acat1 knockout ( Acat1 −/− ) BMDM were collected and added to complete 1640 medium containing GM-CSF (100 ng/ml) and IL-6 (100 ng/mL) in a certain proportion, the C–X–C motif chemokine receptor 2 (CXCR2) inhibitor SB225002 (MCE) was added to selected cultures to a final concentration of 2 μmol/L to eliminate the effect of CXCL1 on the generation of MDSCs.

Techniques: Staining, Flow Cytometry, Cell Culture, Concentration Assay

Journal: Advanced Science

Article Title: The KDM6B/SLC10A2 Axis Suppresses MDSCs Recruitment via ERK/AP‐1 Signaling in Colorectal Cancer

doi: 10.1002/advs.202514086

Figure Lengend Snippet: KDM6B deficiency promotes MDSCs‐mediated immunosuppression via the AP‐1/CXCL–CXCR2 axis. A) Immunohistochemical (IHC) staining of Gr1 and CXCR2 in colon tissues from Villin Cre ; KDM6B fl/fl mice and KDM6B fl/fl littermate controls after 10 weeks of AOM/DSS treatment. Quantification of positive pixels per field. B) qPCR analysis of the expression of MDSC markers (S100A8, S100A9, Arg1, TGF‐β, and VEGF‐a) in the intestinal epithelium of Villin Cre ; KDM6B fl/fl mice and KDM6B fl/fl mice. C,D) MC38 murine colon cancer cells stably overexpressing KDM6B (OE‐KDM6B) or empty vector control (MCS) were subcutaneously injected into C57BL/6 mice (n = 5 per group). (C) Immunohistochemical (IHC) staining of Gr1 and CXCR2 in subcutaneous tumors derived from MC38‐MCS and MC38‐OE‐KDM6B cells at the endpoint (14 days post‐injection). Representative images are shown. Scale bars: 200 µm. D) qPCR of the expression of MDSCs markers (S100A8, S100A9, Arg1, TGF‐β, and VEGF‐a) in subcutaneous tumors derived from MC38‐MCS and MC38‐OE‐KDM6B cells at the endpoint. E) Transwell migration assays were performed to evaluate the ability of tumor‐conditioned media (TCM) from MC38 cells, including the MC38‐vector (MCS), MC38‐OE‐KDM6B (OE‐KDM6B), MC38‐shNC (NC), and MC38‐shKDM6B (sh‐KDM6B) groups, to recruit MDSCs. Representative images are shown. Each group had three biological replicates. F) Quantification of migrated MDSCs. G,H) qPCR of the expression of MDSC markers (S100A8, S100A9, Arg1, TGF‐β, and VEGF‐a) after TCM treatment. I,J) CXCR2 blockade experiments: (I) A CXCR2 inhibitor (SB265610, 10 µM) was added into tumor‐conditioned media (TCM) from MC38‐NC cells or MC38‐sh‐KDM6B cells, and Transwell assays were performed to evaluate the migration capacity of MDSCs. Representative images are shown. Each group had three biological replicates. (J) The statistical results of the migration cell count. K–M) In vivo CXCR2 inhibition: (K) Subcutaneous tumors were generated in C57BL/6 mice by injection of MC38 cells with stable KDM6B knockdown (sh‐KDM6B) or negative control (NC). Mice were treated daily with the CXCR2 inhibitor SB265610 (2 mg kg −1 day −1 , i.p.) or vehicle control for 15 days (n = 5). (L) Tumor growth curves. (M) Endpoint tumor weights. (N) Flow cytometry of tumor‐infiltrating PMN‐MDSCs (CD11b + Ly6G + Ly6C − ), CD4 + T cells (CD3 + CD4 + ) and CD8 + T cells (CD3 + CD8 + ). (O) Flow cytometry of peripheral blood from the four experimental groups (NC, NC+CXCR2i, sh‐KDM6B, and sh‐KDM6B+CXCR2i) was performed at the endpoint (Day 15). Gating strategy for PMN‐MDSCs (CD11b⁺Ly6G⁺Ly6C − ), CD4 + T cells (CD3 + CD4 + ) and CD8 + T cells (CD3 + CD8 + ). (P) Quantification of MDSCs and T cells in tumors and peripheral blood. (Q) Immunohistochemical (IHC) analysis of CXCR2 and Gr1⁺ cell infiltration in subcutaneous tumors. The sample size for each group was n = 5. The middle line in the box plots represents the median, whereas the whiskers denote the minimum‐to‐maximum range of the data distribution. The error bars show the means ± SEM. Statistical significance was determined by an unpaired, two‐tailed Student's t test. * p < 0.05, ** p < 0.01.

Article Snippet: The CXCR2 antibody SB‐225002 (MCE; Cat# HY‐16711) or phosphate‐buffered saline was administered via intraperitoneal injection at a dose of 2 mg kg −1 daily.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Stable Transfection, Plasmid Preparation, Control, Injection, Derivative Assay, Migration, Cell Characterization, In Vivo, Inhibition, Generated, Knockdown, Negative Control, Flow Cytometry, Two Tailed Test

Journal: Advanced Science

Article Title: The KDM6B/SLC10A2 Axis Suppresses MDSCs Recruitment via ERK/AP‐1 Signaling in Colorectal Cancer

doi: 10.1002/advs.202514086

Figure Lengend Snippet: SLC10A2 reverses KDM6B deficiency‐induced AP1/CXCL‐MDSC immunosuppression. A) Western blot analysis of SLC10A2 knockdown efficiency in MC38 cells using three independent shRNAs (sh1, sh2, and sh3). β‐actin served as a loading control. B) ELISA quantification of CXCL (1, 2, 3, and 5) secretion in conditioned media from MC38‐negative control (NC), SLC10A2‐sh1 and SLC10A2‐sh3 cells. C) Western blot confirmation of SLC10A2 overexpression in MC38 cells. β‐actin was used as a loading control. D) ELISA analysis of CXCL (1, 2, 3, and 5) secretion in SLC10A2‐overexpressing MC38 cells. E) Transwell migration assay showing MDSC recruitment by conditioned media from MC38‐negative control (NC), SLC10A2‐sh1 and SLC10A2‐sh3 cells. The right panel shows the statistical results. Each group had three biological replicates. F) Comparison of MDSC migration across four groups of MC38 cells: control, OE‐SLC10A2, sh‐KDM6B, and sh‐KDM6B+OE‐SLC10A2. The right panel shows the statistical results. Each group had three biological replicates. G) qPCR analysis of the expression of MDSC markers (S100A8, S100A9, Arg1, TGF‐β, and VEGF‐a) following stimulation with tumor supernatant. H) Chemokine secretion profiles across the four treatment groups determined by ELISA. I) Western blot analysis of KDM6B, SLC10A2, p‐FOS, c‐FOS, p‐JUN, c‐JUN and β‐actin expression. J) MC38 cells were subcutaneously injected into C57BL/6 mice to establish syngeneic transplant tumors. The experimental groups were Ctrl, sh‐KDM6B, sh‐KDM6B+OE‐SLC10A2, and OE‐SLC10A2. The mice (n = 5 per group) were sacrificed on Day 15 post‐injection, and the tumors were harvested for further analysis. K) Tumor growth kinetics (left) and endpoint weights (right) for the four treatment groups. L) Flow cytometry analysis of peripheral blood samples collected from the four experimental groups. Representative gating strategies illustrating the identification of PMN‐MDSCs (CD11b⁺Ly6G⁺Ly6C − ) and T‐cell subsets (CD3 + CD4 + T cells and CD3 + CD8 + T cells) are shown. Gating strategy for PMN‐MDSCs (CD11b⁺Ly6G⁺Ly6C − ), CD4 + T cells (CD3 + CD4 + ) and CD8 + T cells (CD3 + CD8 + ). M) Statistical analysis of peripheral blood immune cell frequencies. N–O) Flow cytometry analysis of tumor‐infiltrating PMN‐MDSCs (CD11b⁺Ly6G⁺Ly6C − ) and T‐cell subsets (CD3 + CD4 + T cells and CD3 + CD8 + T cells) (N). Quantitative analysis is shown (O). P) IHC analysis of CXCR2 and Gr1 expression in subcutaneous tumors derived from the MC38 cell lines Ctrl, sh‐KDM6B, sh‐KDM6B+OE‐SLC10A2, and OE‐SLC10A2. Representative images from each group (n = 5 biological replicates) were shown. The sample size for the (J–O) groups was 5 mice. The data are presented as the means ± SEM and were statistically analyzed by two‐tailed Student's t test or one‐way ANOVA. * p < 0.05, ** p < 0.01.

Article Snippet: The CXCR2 antibody SB‐225002 (MCE; Cat# HY‐16711) or phosphate‐buffered saline was administered via intraperitoneal injection at a dose of 2 mg kg −1 daily.

Techniques: Western Blot, Knockdown, Control, Enzyme-linked Immunosorbent Assay, Negative Control, Over Expression, Transwell Migration Assay, Comparison, Migration, Expressing, Injection, Flow Cytometry, Derivative Assay, Two Tailed Test

Journal: Science translational medicine

Article Title: Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer.

doi: 10.1126/scitranslmed.aax0428

Figure Lengend Snippet: Fig. 1. CXCR2 is a surface marker for NE cells in human PCa and is associated with disease progression. (A) Representative hematoxylin and eosin (H&E) staining (left) and IHC for NE marker CHGA (right) of human primary PCa. Nuclei (DAPI staining) are shown in blue. (B) Representative immunofluorescence images of CXCR2 (red) and NE marker CHGA (green) staining in human primary PCa. White arrowheads point to CXCR2+ CHGA+ NE tumor cells. (C) Immunofluorescence of CXCR2 (red) and luminal marker KRT8 (green) in human primary prostate adenocarcinoma. White arrowheads indicate CXCR2+ NE cells (red), which are negative for KRT8 (green). (D and E) Representative images (D) and quantification (E) of CXCR2 IHC staining on TMAs. Logistic regression analysis was performed using nonparametric Mann-Whitney U test; lines represent median and interquartile range. (F) Analysis of CXCR2 expres- sion among primary and metastatic PCa tumors from the dataset of Taylor et al. (11). Logistic regression analysis was performed using t test; lines represent means ± SD.

Article Snippet: CXCR2 cDNA plasmid was synthesized by OriGene (SC321915).

Techniques: Marker, Biomarker Discovery, Staining, Immunofluorescence, Immunohistochemistry, MANN-WHITNEY

Journal: Science translational medicine

Article Title: Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer.

doi: 10.1126/scitranslmed.aax0428

Figure Lengend Snippet: Fig. 2. Distinct signaling was observed between CXCR2+ NE and CXCR2− luminal cells isolated from human primary PCa. (A) Experimental scheme for gene expression and cellular function analysis of human PCa CXCR2+ NE and CXCR2− luminal populations. (B) Volcano plot of differentially expressed genes between CXCR2+ NE and CXCR2− luminal cells isolated from fresh primary human PCa tissue. (C) Heat map of genes from sorted benign basal cells (ITGA6+), NE tumor cells (CXCR2+), and luminal-type tumor cells (CXCR2−). (D and E) Tumor organoids generated by CXCR2+ NE cells (right) and CXCR2− luminal cells (left). Representative images (D) and quantification (E) of three independent patients’ samples are shown. 3D, three-dimensional. (F) Signaling pathways associated with CXCR2 expression in PCa. Gene expression of 459 primary PCa was obtained from TCGA prostate adenocarcinoma database (TCGA-PRAD). Ingenuity pathway analysis was performed using 682 genes that were correlated with CXCR2 expression (P < 0.05, Pearson’s correlation). (G and H) The enrichment scores of NE gene sets (G) and prostate adenocarcinoma gene sets (H) in CXCR2+ NE cells isolated from primary human PCa tissue (18). FDR, false discovery rate; NES, normalized enrichment score. (I) Heat map of gene expression among CXCR2+ NE and CXCR2− luminal tumor cells from fresh primary human PCa tissue, SCNC/neuroendocrine prostate cancer (NEPC), and prostate adenocarcinoma (18).

Article Snippet: CXCR2 cDNA plasmid was synthesized by OriGene (SC321915).

Techniques: Isolation, Gene Expression, Cell Function Assay, Generated, Protein-Protein interactions, Expressing

Journal: Science translational medicine

Article Title: Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer.

doi: 10.1126/scitranslmed.aax0428

Figure Lengend Snippet: Fig. 3. CXCR2-mediated phenotypic switch drives therapy resistance in PCa. (A) Flow cytometric quantification of CXCR2+ cells in parental LNCaP cell line (left) and LNCaP cells cultured in charcoal- stripped (androgen-deprived) medium for 2 weeks (right). Representative images of three independent experiments are shown. (B) Flow cytometric quantification of CXCR2+ cells in parental C4-2B cell line (left) and C4-2B cells treated with enzalutamide (20 M) for 2 weeks (right). Representative images of three independent experiments are shown. (C and D) Parental LNCaP cells and LNCaP cells overexpressing CXCR2 (LNCaP- CXCR2) were treated without (left) or with (right) enzalutamide (ENZA) for 2 weeks. Representative image (C) and quantification (D) of colony formation assay are shown. cDNA, complementary DNA. (E and F) CXCR2 expression induces a lineage switch from the luminal phenotype (AR+, KLK3+, and CHGA−) to the NE phenotype (AR−, KLK3−, and CHGA+) in an in vivo xenograft model. Representative images (E) and quantifica- tion (F) are shown. (G) Heat map of selected differentially expressed genes between LNCaP cells and LNCaP-CXCR2 cells. (H) Enrichment for binding sites for luminal-defining transcription factors in LNCaP cells measured by ChIP-seq analyses. The red dot represents the query signature of 1000 up- and down-regulated genes between LNCaP and LNCaP-CXCR2 cells. The blue dot represents one of a total of 100,000 randomly sampled gene lists of equal size to the query signature. The rank of the query gene list divided by the total number of resample instances was then used as the P value for the probability of enrichment by chance. (I) mRNA ex- pression of luminal markers in LNCaP cells with/without CXCR2 overexpression or C4-2B cells with/without CRISPR-Cas9 knock- out of CXCR2. (J) Growth curves of C4-2B/ MDVR-sgCtrl and C4-2B/MDVR-sgCXCR2 (CRISPR-Cas9 knockout of CXCR2) with or without enzalutamide (20 M) treatment. (K) The enrichment score of NE gene sets (18) in LNCaP-CXCR2 cell. (L) A model demon- strating how CXCR2 drives NE phenotype in PCa cells and renders CXCR2+ NE cells resis- tant to hormonal therapy [androgen depri- vation therapy or enzalutamide treatment (ADT/Enza)]. Logistic regression analysis was performed using nonparametric Mann-Whitney U test; lines represent median and inter- quartile range. ns, nonsignificant; *P < 0.05.

Article Snippet: CXCR2 cDNA plasmid was synthesized by OriGene (SC321915).

Techniques: Cell Culture, Colony Assay, Expressing, In Vivo, Binding Assay, ChIP-sequencing, Over Expression, CRISPR, Knock-Out, MANN-WHITNEY

Journal: Science translational medicine

Article Title: Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer.

doi: 10.1126/scitranslmed.aax0428

Figure Lengend Snippet: Fig. 4. CXCR2 expression in NE cells drives the secretion of proangiogenic factors and promotes the formation of premetastatic niche in the tumor environment. (A and B) Representa- tive images of immunofluorescence for CXCR2 (red) and vascular endothelial cell marker PECAM1 (green) in TMAs of human primary PCa are shown in (A). Nuclei (DAPI staining) are shown in blue. Spearman correlation between CXCR2 expression and blood vessel density in a panel of 77 PCa cases is shown in (B). (C and D) Representative IHC images (C) and quantification (D) of PECAM1 expression in LNCaP and LNCaP-CXCR2 xenograft tumors. (E to H) Representa- tive images (E) and quantification (F) of human cytokine antibody arrays of LNCaP or LNCaP-CXCR2. Representative images (G) and quantification (H) of human cyto- kine antibody arrays of C4-2B/MDVR- sgCtrl or C4-2B/MDVR-sgCXCR2. FBS, fetal bovine serum. (I to N) Representa- tive images (I) and quantification (J) of transwell Matrigel invasion assay of LNCaP and LNCaP-CXCR2 cells. The data were normalized to average in- vasion. Representative images (K) and quantification (L) of transwell Matrigel invasion assay of C4-2B/MDVR-sgCtrl and C4-2B/MDVR-sgCXCR2 cells. The data were normalized to average in- vasion. Representative images (M) and quantification (N) of spheroid invasion of C4-2B/MDVR-sgCtrl and C4-2B/ MDVR-sgCXCR2 cells at days 0 and 10, with leading edges indicated by the red arrows. Invasion distance normal- ized to day 0 distance for each sphere. (O) A model of CXCR2+ NE cells re- modeling the tumor microenviron- ment. MMP, matrix metalloproteinase. VEGF, vascular endothelial growth factor. Logistic regression analysis was performed using nonparametric Mann- Whitney U test; lines represent median and interquartile range. *P < 0.05.

Article Snippet: CXCR2 cDNA plasmid was synthesized by OriGene (SC321915).

Techniques: Expressing, Immunofluorescence, Marker, Staining, Invasion Assay, MANN-WHITNEY

Journal: Science translational medicine

Article Title: Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer.

doi: 10.1126/scitranslmed.aax0428

Figure Lengend Snippet: Fig. 5. Advanced and therapy-resistant PCa is sensitive to CXCR2 inhibition. (A to C) Tumorigenesis of C4-2B/MDVR cells with/without CRISPR-Cas9 knockout of CXCR2. Representative images (A), quantification of colony-forming efficiency (B), and colony size (C) of C4-2B/MDVR cells with/without the CXCR2 gene deleted. Cells were cul- tured for 2 weeks. (D to F) Effect of na- varixin on enzalutamide-resistant PCa C4-2B/ MDVR cells’ growth in vivo. Images (D), weights (E), and volumes (F) of C4-2B/MDVR tumors in mice treated with navarixin (70 mg/kg) or vehicle control. (G) Representative images of IHC staining for AR and prostate-specific antigen in control and navarixin-treated C4-2B/MDVR xenografts. (H to J) Repre- sentative images (H) and quantification (I) of immunostaining for terminal deoxy- nucleotidyl transferase–mediated deoxy- uridine triphosphate nick end labeling (TUNEL) in tumors from mice treated with vehicle or CXCR2 inhibitor navarixin for 3 weeks. Representative images (J) of BCL2 associated X, apoptosis regulator (BAX) immunostaining of xenograft tumors in mice treated with/without navarixin for 3 weeks. (K and L) Representative images (K) and quantification (L) of blood vessel marker CD31 in mice treated with vehicle or navarixin (70 mg/kg) for 3 weeks. (M) Quantification of C4-2B tumor burden in mice treated with vehicle, enzalutamide, navarixin, or both enzalutamide and navarixin. (N and O) Quantification (N) and representative images (O) of immunostain- ing for TUNEL in mice treated with vehicle, enzalutamide, navarixin, or both enzalut- amide and navarixin. ANOVA, analysis of variance. Logistic regression analysis was performed using nonparametric Mann- Whitney U test; lines represent median and interquartile range.

Article Snippet: CXCR2 cDNA plasmid was synthesized by OriGene (SC321915).

Techniques: Inhibition, CRISPR, Knock-Out, In Vivo, Control, Immunohistochemistry, Immunostaining, End Labeling, TUNEL Assay, Marker, MANN-WHITNEY

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-CD182 conjugated to 147Sm (clone 5E8/CXCR2) , Fluidigm , Cat# 3147010B.

Techniques: Ubiquitin Proteomics, Recombinant, Multiplex Assay, Reverse Transcription, SYBR Green Assay, In Situ, shRNA, Software