Journal: Cancer Cell

Article Title: Epithelial NOTCH Signaling Rewires the Tumor Microenvironment of Colorectal Cancer to Drive Poor-Prognosis Subtypes and Metastasis

doi: 10.1016/j.ccell.2019.08.003

Figure Lengend Snippet: Epithelial NOTCH1 Controls Neutrophil Recruitment to Drive Metastasis (A) Heatmap showing standardized infiltration-scores (calculated with MCPcounter) in GEMM tumors; AP, n = 3; APN, n = 3; KP, n = 3; KPN, n = 9. (B) Dot-plots showing standardized infiltration scores of neutrophils (calculated with MCPcounter); replicates as in (A). (C) Blood neutrophil count at endpoint of indicated genotype (n ≥ 6). (D) Representative Ly6G IHC. Scale bars, 100 μm. (E) Neutrophil infiltration-score in human adenoma. (F) Quantification of Ly6G + and S100A9 + cells per field of view (FOV); AP, n = 6; APN, n ≥ 5; KP, n ≥ 4; KPN, n ≥ 5. (G) Representative ISH of Cxcl5 expression. Scale bars, 100 μm. (H) Quantification of Cxcl5 + and Cxcr2 + cells; AP, n = 8; APN, n = 6; KP, n = 6; KPN, n ≥ 6. (I) Incidence of metastases at endpoint for KPN mice treated with: vehicle, n = 11; CXCR2sm, n = 10; 2A3, n = 10; 1A8, n = 9; analyzed by chi-square test, two-tailed. (J) Blood neutrophil count after 1 week of indicated treatments: vehicle, n = 5; CXCR2sm, n = 7; 2A3, n = 5; 1A8, n = 5; analyzed by Mann-Whitney U test, one-tailed. (K) Quantification of IHC on primary tumors of KPN mice after 1 week of indicated treatments: vehicle, n ≥ 4; CXCR2sm, n = 7; 2A3, n = 5; 1A8, n = 5. Error bars in (B), (C), (E), (F), (H), (J), and (K) represent mean ± SEM. Data in (B), (C), (E), (F), (H), and (K) analyzed by Mann-Whitney U test, two-tailed. See also and and and .

Article Snippet: Alk5 inhibitor (Alk5i) ( ) (AstraZeneca, AZ12601011) was administered at 50 mg/kg and CXCR2 small molecule (AstraZeneca, AZD5069) at 100 mg/kg, both in 0.5% Hydroxypropyl Methylcellulose (HPMC) and 0.1% Tween-80 twice daily by oral gavage.

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, One-tailed Test

Journal: Cancer Cell

Article Title: Epithelial NOTCH Signaling Rewires the Tumor Microenvironment of Colorectal Cancer to Drive Poor-Prognosis Subtypes and Metastasis

doi: 10.1016/j.ccell.2019.08.003

Figure Lengend Snippet: TGF-β or CXCR2 Inhibition Attenuates KPN Metastasis via T Cell Activation (A) Cartoon illustrating organoid isograft transplantation in the spleen. (B) Quantification of macroscopic liver metastases 4 weeks post-transplantation; KPN, n = 4; AKPT, n = 5. (C) Quantification of neutrophils in liver metastases by flow cytometry as in (B). (D) Representative contour plots of the analysis performed in (C). (E) Representative images for ISH analysis of Tgfb2 expression or IHC for Ly6G on liver metastases (n ≥ 3). Scale bars, 100 μm. (F) Schematic representation of the treatment regimen after organoid transplantation. (G) Number and burden of macroscopic liver metastases 4 weeks post-KPN organoid transplantation; vehicle, n = 5; Alk5i, n = 5. (H and I) Quantification of flow cytometry analysis for neutrophils in blood (H) or liver metastases 4 weeks post-KPN organoid transplantation (I); vehicle, n = 5; Alk5i, n ≥ 4. (J) Quantification of flow cytometry analysis for T cell subsets in liver metastases 4 weeks post-KPN organoid transplantation; vehicle, n = 5; Alk5i, n = 4. Error bars in (B), (C), (G), (H), (I), and (J) represent mean ± SEM. Data in (C), (G), (I), and (J) analyzed by Mann-Whitney U test, two-tailed. See also Figure S7 .

Article Snippet: Alk5 inhibitor (Alk5i) ( ) (AstraZeneca, AZ12601011) was administered at 50 mg/kg and CXCR2 small molecule (AstraZeneca, AZD5069) at 100 mg/kg, both in 0.5% Hydroxypropyl Methylcellulose (HPMC) and 0.1% Tween-80 twice daily by oral gavage.

Techniques: Inhibition, Activation Assay, Transplantation Assay, Flow Cytometry, Expressing, MANN-WHITNEY, Two Tailed Test

Journal: Cancer Cell

Article Title: Epithelial NOTCH Signaling Rewires the Tumor Microenvironment of Colorectal Cancer to Drive Poor-Prognosis Subtypes and Metastasis

doi: 10.1016/j.ccell.2019.08.003

Figure Lengend Snippet:

Article Snippet: Alk5 inhibitor (Alk5i) ( ) (AstraZeneca, AZ12601011) was administered at 50 mg/kg and CXCR2 small molecule (AstraZeneca, AZD5069) at 100 mg/kg, both in 0.5% Hydroxypropyl Methylcellulose (HPMC) and 0.1% Tween-80 twice daily by oral gavage.

Techniques: Purification, Produced, Recombinant, Staining, Chromatin Immunoprecipitation, Sequencing, Software, Real-time Polymerase Chain Reaction

Journal: Hepatology (Baltimore, Md.)

Article Title: C‐X‐C motif chemokine ligand 1 induced by Hedgehog signaling promotes mouse extrahepatic bile duct repair after acute injury

doi: 10.1002/hep.32492

Figure Lengend Snippet: CXCR2 inhibition reduces epithelial cell proliferation after acute injury. WT mice were treated with SB225002 or Veh (A), and EHBDs were examined 24 h after BDL (B–E). Neutrophils were marked with Ly6G (red) using immunofluorescence (B), enumerated, expressed as a percentage of all stromal cells, and compared with Veh‐treated controls (C). Proliferating epithelial cells were marked with EdU (green) (D), enumerated, expressed as a percentage of all epithelial cells in the CK19 + cell compartment, and compared with Veh‐treated controls (E). Asterisks mark EHBD lumen, arrows mark epithelial cells, and arrowheads mark Ly6G + cells. The data are presented as the mean ± SEM. ** p < 0.01, **** p < 0.0001. n = 4–6 mice/group. Unpaired Student t test. Scale bars, 100 μm (B) and 50 μm (D)

Article Snippet: SB225002 was used as a selective small molecule CXCR2 antagonist (B8200, ApeXBio).

Techniques: Inhibition, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Associated Microglia/Macrophages as a Predictor for Survival in Glioblastoma and Temozolomide-Induced Changes in CXCR2 Signaling with New Resistance Overcoming Strategy by Combination Therapy

doi: 10.3390/ijms222011180

Figure Lengend Snippet: The expression of proangiogenic molecules is distinctly changed in recurrent GBM tumors. Representative images of FFPE tissue sections of primary and recurrent tumors stained for ( a ) VEGF (red), ( c ) CXCL2 (green), ( e ) IL8 (green), ( g ) blood vessels (CD31; red), ( i ) CXCR2 (green), and cell nuclei (DAPI; blue); scale bars: 100 µm. ( b,d ) Graphs show calculation of the stained area of VEGF ( b ) and CXCL2 ( d ) as a percentage in relation to the total analyzed tumor area ( n = 38). ( f ) The percentage of patients expressing IL8 in primary and recurrent tumors was assessed ( n = 38). ( h , j ) Graphs depict vessels/mm 2 and the percentage of CXCR2 + vessels ( n = 28–30). All primary tumors are depicted in black and recurrent tumors in grey. Box plots depicting mean (shown as “+”), median, 1st and 3rd quartile (shown as the box), and min to max (shown as the whiskers). * p < 0.05. Paired Student’s t -test; box plots depicting mean (shown as “+”) and median ± standard deviation.

Article Snippet: In short, a mini-osmotic pump (model 2002/2001; ALZET, DURECT Corporation, Cupertino, CA, USA) was prepared and filled with the small molecule CXCR2 Antagonist SB225002 (Tocris, Bristol, UK) according to the manufacturer’s protocol prior to implantation on day 14.

Techniques: Expressing, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Associated Microglia/Macrophages as a Predictor for Survival in Glioblastoma and Temozolomide-Induced Changes in CXCR2 Signaling with New Resistance Overcoming Strategy by Combination Therapy

doi: 10.3390/ijms222011180

Figure Lengend Snippet: Influence of TMZ on TAM infiltration, tumor vascularization, and expression of proangiogenic molecules in matched primary and recurrent GBMs. ( a , b ) Kaplan–Meier curves for ( a ) progression-free (PFS) and ( b ) overall survival (OS) of the subgroup analysis comparing the standard with the not standard group. ( c – m ) Graphs show the calculation of the stained area of TAMs (IBA1) ( c – e ), proangiogenic molecules VEGF ( f ), CXCL2 ( g ), IL8 ( h ) tumor vessels (CD31) ( i – k ), and CXCR2 + vessels ( l , m ) on FFPE tissue sections of primary and recurrent tumors of all patients. ( c ) Graph depicts the amount of TAMs in primary and recurrent tumors of both groups, which were significantly reduced in recurrent tumors (standard: mean difference: 246.7; 95% confidence interval −400.5 to −92.87; not standard: mean difference: −192.2; 95% confidence interval −370.0 to −14.40). ( d,e ) Graphs depict CXCL2 + TAMs in primary and recurrent tumors of both groups. ( f – h ) Graphs show calculation of the stained area of VEGF ( f ) and CXCL2 ( g ) ( n = 38; standard: n = 18; not standard: n = 20). ( h ) The number of IL8 + patients in primary and recurrent tumors was calculated ( n = 38; standard: n = 18; not standard: n = 20). ( i – k ) Graphs depict vessels/mm 2 ( i ), the vessel area/vessel number ( j ), and vessel area/mm 2 ( k ) ( n = 30; standard: n = 15; not standard: n = 15). ( l , m ). The area of CXCR2 + vessels/CXCR2 + vessel number in ( l ) and the percentage of CXCR2 + vessels in relation to all vessels ( m ) was calculated ( n = 28; standard: n = 13; not standard: n = 15). * p < 0.05, ** p < 0.01, *** p < 0.001. ( c – m ) paired Student’s t -test; box plots depicting mean (shown as “+”), median, 1st and 3rd quartile (shown as the box) and min to max (shown as the whiskers).

Article Snippet: In short, a mini-osmotic pump (model 2002/2001; ALZET, DURECT Corporation, Cupertino, CA, USA) was prepared and filled with the small molecule CXCR2 Antagonist SB225002 (Tocris, Bristol, UK) according to the manufacturer’s protocol prior to implantation on day 14.

Techniques: Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Associated Microglia/Macrophages as a Predictor for Survival in Glioblastoma and Temozolomide-Induced Changes in CXCR2 Signaling with New Resistance Overcoming Strategy by Combination Therapy

doi: 10.3390/ijms222011180

Figure Lengend Snippet: Post treatment MRIs of tumor volume and therapy-induced effects on proliferation and apoptosis. ( a ) Representative T1w MRI images after contrast agent administration are showing a therapy-induced reduction of tumor volume after the treatment with TMZ and TMZ + SB. ( b ) Quantitative tumor volumetry showed a significantly reduced tumor volume by 75% in the combination group (TMZ + SB) whereas TMZ alone reduced tumor volume by 61% ( n = 7–8; * p = 0.0224; ** p = 0.0062). ( c ) Representative immunofluorescence staining of each group showing a reduction of proliferation in the TMZ and TMZ + SB group while apoptosis seemed to be unaffected (Ki67: green, TUNEL: red, scale bar = 100 µm). ( d ) The detailed analysis of proliferation showed significant impairment in both treatment groups. The proliferation was reduced by 37% in the combination group and 25% in the TMZ group ( n = 4; * p = 0.0205; ** p = 0.002; TMZ vs. TMZ + SB, p = 0.4416). ( e ) However, apoptosis remained unaltered ( n = 4; Control vs. TMZ, p = >0.9999; Control vs. TMZ + SB, p = 0.1616; TMZ vs. TMZ + SB, p = 0.1204). ( f ) If the ratio of apoptosis and proliferation was calculated, a significant shift towards apoptosis could be detected only in the combination group of TMZ + SB225002 ( n = 4; * p = 0.0388; ** p = 0.0014). ( g , h ) Apoptosis-regulating molecules Bax (proapoptotic) and Bcl2 (antiapoptotic) were unchanged in qRT-PCR analysis ( n = 3–4). ( d – f ) Graphs depict individual values with additional mean ± standard deviation; bar graphs ( g , h ) showing mean ± standard deviation. One-way ANOVA with Bonferroni correction was performed in all analyses.

Article Snippet: In short, a mini-osmotic pump (model 2002/2001; ALZET, DURECT Corporation, Cupertino, CA, USA) was prepared and filled with the small molecule CXCR2 Antagonist SB225002 (Tocris, Bristol, UK) according to the manufacturer’s protocol prior to implantation on day 14.

Techniques: Immunofluorescence, Staining, TUNEL Assay, Control, Quantitative RT-PCR, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Tumor-Associated Microglia/Macrophages as a Predictor for Survival in Glioblastoma and Temozolomide-Induced Changes in CXCR2 Signaling with New Resistance Overcoming Strategy by Combination Therapy

doi: 10.3390/ijms222011180

Figure Lengend Snippet: Upregulation of CXCL2/CXCR2 signaling in tumor tissue and the influence of therapy on CXCR2 signaling and alternative pathways. ( a ) Gene expression of Cxcr2 was significantly upregulated (7-fold) in tumor tissue of GL261 mouse GBMs compared to contralateral brain parenchyma ( n = 4; p = 0.0434). ( b ) Additionally, Cxcl2 was upregulated 5-fold but did not a significant level in this study ( n = 4; p = 0.0977). ( c ) The investigation of Cxcr2 showed a therapy-induced decrease of gene expression under TMZ and TMZ + SB treatment ( n = 3–4, Control vs. TMZ, p = 0.0693; Control vs. TMZ + SB p = 0.1637) but without reaching statistical significance. ( d ) However, Cxcl2 remained completely unchanged ( n = 3–4). ( e – h ) Representative immunofluorescence staining of CXCR2 and CXCL2 on the protein level showed no differences or group-specific distribution patterns amongst all groups (CXCR2: green, CXCL2: red). The measurement of the mean intensity of immunofluorescence images of both molecules showed no differences of protein expression. All images were captured with the same exposure time under comparable conditions ( n = 4). ( i – k ) qRT-PCR analysis of the classic angiogenic pathway with Vegf and its receptors Vegfr1 and Vegfr2 showed no compensatory upregulation during therapy with TMZ or TMZ + SB ( n = 3–4). ( l ) Gene expression of Cxcr1 , which has similar downstream effects as Cxcr2 , was also not altered significantly ( n = 3–4). ( a – d , i – l ) Bar graphs showing mean ± standard deviation; ( f , h ) graphs depict individual values with additional mean ± standard deviation; one-way ANOVA with Bonferroni correction was performed in all analyses.

Article Snippet: In short, a mini-osmotic pump (model 2002/2001; ALZET, DURECT Corporation, Cupertino, CA, USA) was prepared and filled with the small molecule CXCR2 Antagonist SB225002 (Tocris, Bristol, UK) according to the manufacturer’s protocol prior to implantation on day 14.

Techniques: Gene Expression, Control, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Standard Deviation