ccl5 levels  (Proteintech)

Journal: bioRxiv

Article Title: Adaptive anti-tumor immunity is orchestrated by a population of CCL5-producing tissue-resident NK cells

doi: 10.1101/2021.05.27.445981

Figure Lengend Snippet: a-b: −log10(p value) of chemokines and their receptors which are correlating with NK signature of skin tumor biopsies from melanoma patients responding to intra-tumoral treatment with ImmunoPulse IL-12 are shown. c: Normalized counts of CCL5 expression of patients with no-response (NR: PD) versus response (R: SD + PR) before and after intra-tumoral treatment with ImmunoPulse IL-12 determined by NanoString. d: NK cells were depleted using anti-AsialoGM1 antibody (i.p) every 3-4 days. On day 7 post EMT6-HER2 inoculation (tumor size 30–70 mm 3 ), tumors were isolated and lysed. CCL5 expression was determined by ELISA and normalized to total protein measured by BCA. n= 6 per condition. 2-tailed Student’s t test was used. e: Mice were treated with AdV5-IL12 or AdV5-control on day 7, 9 and 11 post EMT6-HER2 inoculation. On day 12 tumors were isolated and CCL5 expression (MFI) was analyzed on NK cells. n= 6 mice per condition. f: EMT6-HER2-engrafted mice were treated with AdV5-IL12 following the indicated schedule. Starting one day prior tumor inoculation or one day prior adenoviral therapy, CCL5 was neutralized using antibodies every 3-4 days. Kaplan-Meier survival curves are shown. g-i: Mice were treated with AdV5-IL12 or AdV5-control on day 7, 9 and 11 post EMT6-HER2 inoculation. On day 12 tumors were isolated and CCL5, GzmB, CXCR3 and CCR5 expression was analyzed on NK cell subsets. Percentage of CCL5 positive NK cells, as well as MFI of GzmB, CXCR3 and CCR5 were quantified after AdV5-IL12 treatment. Paired 2-tailed Student’s t test was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bar values represent SD or SEM (tumor growth curves). For comparisons between three or more groups, one-way ANOVA with multiple comparisons was used. For survival analysis, p values were computed using the Log Rank test. Two-way ANOVA was used to compare tumor growth curves. See also Supplementary Fig. S4.

Article Snippet: The supernatant was collected after 6 days of co-culture to assess IFNγ and CCL5 levels using a Human IFNγ ELISA Set (BD OptEIA, 555142) and ELISA MAX™ Deluxe Set Human CCL5 (Biolegend, 440804), respectively, according to the manufacturers’ instructions.

Techniques: Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Control

Journal: bioRxiv

Article Title: Adaptive anti-tumor immunity is orchestrated by a population of CCL5-producing tissue-resident NK cells

doi: 10.1101/2021.05.27.445981

Figure Lengend Snippet: a: Amount of tumor infiltration NK cells per g tumor was determined in untreated EMT6-HER2 and B16-HER2 tumors. b: Amount of trNK cells per g tumor was determined in untreated EMT6-HER2 and B16-HER2 tumors. c: Intra-tumoral CCL5 concentration was determined by ELISA and normalized to total protein in EMT6-HER2 and B16-HER2 tumor lysates. d: Wildtype (WT) mice were engrafted with 1 mio EMT6-HER2 (i.m.) or 0.5 mio B16-HER2 (s.c.). Mice were treated with AdV5-IL12 on day 7, 9, 11 and 14 or day 11, 13, 15 and 18 (tumor size 30–70 mm 3 ), respectively. NK cells were depleted using anti-AsialoGM1 and anti-NK1.1 antibody every 4-5 days starting one day prior adenoviral therapy. Tumor volume on day 25 post tumor inoculation is shown. e: Mice were treated with AdV5-IL12 and AdV5-CCL5 on day 11, 13, 15 and 18 (tumor size 30–70 mm 3 ) after B16-HER2 inoculation as indicated: Tumor growth and Kaplan-Meier survival curves are shown (n > 15 mice per condition). f: Mice were engrafted with 1 mio EMT6-HER2 (i.m.) or 0.5 Mio B16-HER2 (s.c.). Starting from day 7 or 11 (tumor size 30–70 mm 3 ), mice were treated with 1.5×10 8 PFU of HER2-targeted and shielded adenoviral vectors (p.t.) encoding for IL-12 on day 7/11, 9/13 and 11/15. On day 12/16 post inoculation, tumors were isolated, embedded in OCT and analyzed by multiparameter immuno-fluorescence microscopy. Visualization of odds ratios and p values for changes in cell-cell type interactions between EMT6-HER2 versus B16-HER2 focusing on interaction including CD8 T cells and NK cells. g-k: Mice were engrafted with 0.5 mio B16-HER2 (s.c.). Mice were treated with AdV5-IL12 and/or AdV5-CCL5 on day 11, 13 and 15 (tumor size 30–70 mm 3 ). On day 16 post inoculation, tumors were isolated and single cell suspensions were analyzed by flow cytometry or embedded in OCT and analyzed by multiparameter immuno-fluorescence microscopy. g-h: Number of cDC1s (CD11c+, F4/80-, Ly-6G-, MHCII+, CD103+, CD11b low ) and PD-L1 and CD80 expressing cDC1s, respectively. i: Interaction count per mm of CD8 T cells in close proximity to DCs. Representative IF pictures are showing AdV5-IL12 + AdV5-CCL5 treated tumors (MHCII: red, CD11c: green, CD8: blue). White arrows are showing CD8 T cells (CD45+, CD8+) neighboring DCs (CD45+, CD11c+, F4/80-). j: Ratio of PD-1, CD25 and CD69 expression on the CD8 T cell cluster in close (<50 μm) or distant (>50 μm) proximity to DC cluster. k: Proportion of granzyme B+ of CD8 T cells (CD3+, CD4-, NKp46-CD19-). l: Batf3 knockout mice (lacking cDC1s) were engrafted with 0.5 mio B16-HER2 (s.c.). Mice were treated with AdV5-IL12 and/or AdV5-CCL5. Tumor growth and Kaplan-Meier survival curves are shown. #x002A;p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bar values represent SEM. For survival analysis, p values were computed using the Log Rank test. Two-way ANOVA was used to compare tumor growth curves. See also Figure Supplementary Fig. S5.

Article Snippet: The supernatant was collected after 6 days of co-culture to assess IFNγ and CCL5 levels using a Human IFNγ ELISA Set (BD OptEIA, 555142) and ELISA MAX™ Deluxe Set Human CCL5 (Biolegend, 440804), respectively, according to the manufacturers’ instructions.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation, Fluorescence, Microscopy, Flow Cytometry, Expressing, Knock-Out

Journal: bioRxiv

Article Title: Adaptive anti-tumor immunity is orchestrated by a population of CCL5-producing tissue-resident NK cells

doi: 10.1101/2021.05.27.445981

Figure Lengend Snippet: a-e: Tumor digests of NSCLC patients were co-cultured with OVCAR-3 cells which were transduced with HER2-targeted AdV5 encoding human IL-12 or control virus. b: Quantification of OVCAR-3 viability normalized to untreated co-cultures after 96 h. c: IFNγ expression was determined in supernatants after 5d. d: Cell count of IFNγ+ CD8 T cells (CD3+, CD56-) and NK cells (CD56+, CD3-) per well after 96 h. e: Cell count of CCL5+ NK cells (CD56+, CD3-) after 96 h and CCL5 expression in supernatants after 5d. f-g: HER2+ ovarian cancer samples were dissected into tumor fragments and cultivated embedded in matrigel. Tumor fragments were treated with HER2-targeted AdV5 encoding human IL-12 for 48 h (8-12 fragments per condition). g: CCL5 concentration in supernatant was analyzed by ELISA. h: UMAP projection of scRNASeq data of tumor-infiltrating NK cells of NSCLC patients is shown. i: CCL5 expression in NK1 and NK2 subpopulations was quantified. j: CD56 bright CD16 low and trNK cell signature scores of NK1 and NK2 were determined. k-m: Tumor digests of NSCLC patients were co-cultured with OVCAR-3 cells which were transduced with HER2-targeted AdV5 encoding human IL-12. k: NK cell subsets were defined by CD16 and CD49a expression. l: CCL5 producing NK subsets were visualized after AdV5-IL12 treatment compared to untreated. m: Percentage of CCL5-positive NK cell subsets were quantified. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bar values represent SD. Paired 2-tailed Student’s t test was used. For comparisons between three or more groups, one-way ANOVA with multiple comparisons was used. See also Figure Supplementary Fig. S6 and S7.

Article Snippet: The supernatant was collected after 6 days of co-culture to assess IFNγ and CCL5 levels using a Human IFNγ ELISA Set (BD OptEIA, 555142) and ELISA MAX™ Deluxe Set Human CCL5 (Biolegend, 440804), respectively, according to the manufacturers’ instructions.

Techniques: Cell Culture, Transduction, Control, Virus, Expressing, Cell Counting, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Adaptive anti-tumor immunity is orchestrated by a population of CCL5-producing tissue-resident NK cells

doi: 10.1101/2021.05.27.445981

Figure Lengend Snippet: a-d: CCL5 expression in tumor biopsies of melanoma patients (n= 42) was analyzed pre and on nivolumab treatment . b: Normalized expression of CCL5 pre and on nivolumab treatment between patients with no response (NR) or response (R = SD+PR+CR) was quantified. c: Correlation between CCL5 expression and NK2 signature score or d: cDC1 signature score are shown. e-g: Cancer samples were dissected into tumor fragments and cultivated embedded in Matrigel. Tumor fragments were treated with nivolumab for 48 h (6-8 fragments per condition). IFNγ and CCL5 were determined in the supernatant. n = 6 tumor samples. h-k: Tumor digests of NSCLC patients treated with nivolumab were co-cultured with OVCAR-3 cells for 48h. i: OVCAR3 viability was normalized to untreated control group. j: IFNγ and k: CCL5 were determined in the supernatant. Percentage of CCL5 positive NK cells was quantified after nivolumab treatment. l: NK2/NK1 ratio of patients with no-response (NR: PD) versus response (R: SD + PR) before and after with ICI. m: WT mice were engrafted with 0.5 mio B16-HER2 (s.c.). Mice were treated with AdV5-CCL5 (p.t.) and/or anti-PD-1 (i.p.) antibodies on day 11, 13, 15 and 18 (tumor size 30–70 mm 3 ) as indicated. Tumor growth curves are shown. n= 6 mice per condition. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bar values represent SD or SEM (tumor growth curves). Paired 2-tailed Student’s t test was used. For comparisons between three or more groups, one-way ANOVA with multiple comparisons was used. Two-way ANOVA was used to compare tumor growth curves.

Article Snippet: The supernatant was collected after 6 days of co-culture to assess IFNγ and CCL5 levels using a Human IFNγ ELISA Set (BD OptEIA, 555142) and ELISA MAX™ Deluxe Set Human CCL5 (Biolegend, 440804), respectively, according to the manufacturers’ instructions.

Techniques: Expressing, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Association between an Increased Serum CCL5 Level and Pathophysiology of Degenerative Joint Disease in the Temporomandibular Joint in Females

doi: 10.3390/ijms24032775

Figure Lengend Snippet: Measurement of CCL5 and other markers related to bone metabolism and inflammation in the serum or urine obtained from all subjects. ( a ) Serum CCL5 levels are significantly higher in the group of all DJD-TMJ subjects (n = 17) than in control groups (n = 17). ( b ) Serum or urine markers related to bone metabolism and inflammation were compared between the control group (n = 17) and the group of all DJD-TMJ subjects (n = 17). t -test, two-tailed. * Indicates a significant difference at p < 0.05.

Article Snippet: CCL5 levels in serum were measured by an enzyme-linked linked immunosorbent assay (ELISA; Human CCL5 ELISA Kit, KE00093; Proteintech, IL, USA).

Techniques: Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Association between an Increased Serum CCL5 Level and Pathophysiology of Degenerative Joint Disease in the Temporomandibular Joint in Females

doi: 10.3390/ijms24032775

Figure Lengend Snippet: List of laboratory results for all DJD-TMJ and control subjects. Highlighted in yellow shows patients with systemic diseases.

Article Snippet: CCL5 levels in serum were measured by an enzyme-linked linked immunosorbent assay (ELISA; Human CCL5 ELISA Kit, KE00093; Proteintech, IL, USA).

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Association between an Increased Serum CCL5 Level and Pathophysiology of Degenerative Joint Disease in the Temporomandibular Joint in Females

doi: 10.3390/ijms24032775

Figure Lengend Snippet: Measurement of CCL5 and other markers in the serum or urine obtained from subjects at ≤42 years old (Younger subjects). ( a ) Serum CCL5 levels are significantly higher in the group of DJD-TMJ subjects ≤42 years old (n = 10) than in the age-matched control groups (n = 11). ( b ) Serum or urine markers related to bone metabolism and inflammation were compared between the corresponding control groups (n = 11) and the group of DJD-TMJ subjects ≤ 42 years old (n = 10). t -test, two-tailed. * Indicates a significant difference at p < 0.05.

Article Snippet: CCL5 levels in serum were measured by an enzyme-linked linked immunosorbent assay (ELISA; Human CCL5 ELISA Kit, KE00093; Proteintech, IL, USA).

Techniques: Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Association between an Increased Serum CCL5 Level and Pathophysiology of Degenerative Joint Disease in the Temporomandibular Joint in Females

doi: 10.3390/ijms24032775

Figure Lengend Snippet: Measurement of CCL5 and other markers in the serum or urine obtained from subjects >42 years old (Older subjects). ( a ) Serum CCL5 levels are significantly higher in the group of DJD-TMJ subjects > 42 years old (n = 7) than in the age-matched control groups (n = 6). ( b ) Serum or urine markers related to bone metabolism and inflammation were compared between the corresponding control groups (n = 7) and the group of DJD-TMJ subjects >42 years old (n = 6). t -test, two-tailed. * indicates a significant difference at p < 0.05.

Article Snippet: CCL5 levels in serum were measured by an enzyme-linked linked immunosorbent assay (ELISA; Human CCL5 ELISA Kit, KE00093; Proteintech, IL, USA).

Techniques: Control, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Association between an Increased Serum CCL5 Level and Pathophysiology of Degenerative Joint Disease in the Temporomandibular Joint in Females

doi: 10.3390/ijms24032775

Figure Lengend Snippet: List of laboratory results for DJD-TMJ and control subjects at ≤42 years old (Younger subjects). Highlighted in yellow shows patients with systemic diseases.

Article Snippet: CCL5 levels in serum were measured by an enzyme-linked linked immunosorbent assay (ELISA; Human CCL5 ELISA Kit, KE00093; Proteintech, IL, USA).

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Association between an Increased Serum CCL5 Level and Pathophysiology of Degenerative Joint Disease in the Temporomandibular Joint in Females

doi: 10.3390/ijms24032775

Figure Lengend Snippet: The list of laboratory results for DJD-TMJ and control subjects at >42 years old (Older subjects). Highlighted in yellow shows patients with systemic diseases.

Article Snippet: CCL5 levels in serum were measured by an enzyme-linked linked immunosorbent assay (ELISA; Human CCL5 ELISA Kit, KE00093; Proteintech, IL, USA).

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Association between an Increased Serum CCL5 Level and Pathophysiology of Degenerative Joint Disease in the Temporomandibular Joint in Females

doi: 10.3390/ijms24032775

Figure Lengend Snippet: ( a ) A principal component analysis of all control and DJD-TMJ subjects and ( b ) the measurement of serum CCL5 levels in an ovariectomized (OVX) rat model. ( a ) The principal component analysis showed that CCL5, bone metabolism markers, and TNF-α were independent. ( b ) Serum CCL5 levels in sham-operated, OVX and OVX-TPTD-treated rats did not differ significantly. Dunnett’s test.

Article Snippet: CCL5 levels in serum were measured by an enzyme-linked linked immunosorbent assay (ELISA; Human CCL5 ELISA Kit, KE00093; Proteintech, IL, USA).

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Association between an Increased Serum CCL5 Level and Pathophysiology of Degenerative Joint Disease in the Temporomandibular Joint in Females

doi: 10.3390/ijms24032775

Figure Lengend Snippet: ROC curve analyses of CCL5. ROC curve analyses of CCL5 in ( a ) all DJD-TMJ patients (n = 17), ( b ) the group of all DJD-TMJ subjects ≤ 42 years old (n = 11), ( c ) the group of all DJD-TMJ subjects > 42 years old (n = 7) are shown.

Article Snippet: CCL5 levels in serum were measured by an enzyme-linked linked immunosorbent assay (ELISA; Human CCL5 ELISA Kit, KE00093; Proteintech, IL, USA).

Techniques:

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Tumor bud-derived CCL5 recruits fibroblasts and promotes colorectal cancer progression via CCR5-SLC25A24 signaling.

doi: 10.1186/s13046-022-02300-w

Figure Lengend Snippet: Fig. 3 CCL5 recruits fibroblasts through the CCR5 receptor. a Relative expression of CCR1, CCR3, CCR4, CCR5, CD44, GPR75 mRNA in CCD-18Co/ siCtrl and CCD-18Co/siRNA. Data, mean ± SD; n = 3. b Immunoblots for CCR1, CCR3, CCR4, CCR5, CD44, GPR75 protein expression in CCD-18Co/ siCtrl and CCD-18Co/siRNA. c Model diagram of recruitment assay of 40 ng/ml CCL5 to CCR1, CCR3, CCR4, CCR5, CD44, or GPR75 knockdown fibroblasts. d Recruitment assay showing the recruitment ability of 40 ng/ml CCL5 to CCD-18Co/siCtrl and CCD-18Co/siRNA. Scale bar, 100 μm. Quantification of cell numbers of recruited CCD-18Co cells is shown in the upper panel. Data, mean ± SD; n = 5. e Model diagram of recruitment assay of 40 ng/ml CCL5 to fibroblasts without or with CCR1 inhibitor (BX471) or CCR5 inhibitor (Maraviroc). f Recruitment assay showing the recruitment ability of 40 ng/ml CCL5 to CCD-18Co without or with BX471 or Maraviroc. Scale bar, 100 μm. Quantification of cell numbers of recruited CCD-18Co cells is shown in the left panel. Data, mean ± SD; n = 5. ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Student’s t test (a, d, f)

Article Snippet: CCL5 supernatant levels in the CM samples from FHC and human CRC tumor cells were measured with ELISA using a commercially available kit (CUSABIO, CSBE17375h), as described by the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Knockdown, Two Tailed Test

Journal: Journal of Oncology

Article Title: Palbociclib Enhances Migration and Invasion of Cancer Cells via Senescence-Associated Secretory Phenotype-Related CCL5 in Non-Small-Cell Lung Cancer

doi: 10.1155/2022/2260625

Figure Lengend Snippet: Palbociclib induces SASP-related CCL5 in NSCLC cells. (a) The β -galactosidase staining in NSCLC cells induced by palbociclib. Left: images of β -Gal-stained H1650 and H226 cells with/without palbociclib. Scale bars represent 100 μ m. Right: the bar charts display the calculated numbers of β -Gal positive cells. (b) Protein expression levels of the genes involved in cell senescence after treatment with 2 μ M palbociclib. Left: gel images of western blot for genes' expression, with GAPDH as a loading control. Right: the bar charts display the relative expression of target genes on protein level. P16, P21, p-RB, and corresponding GAPDH were tested in the same experiment. And β -Gal, STING, and corresponding GAPDH were tested in another experiment. Western blot result of every target protein cropped from the same gel in the same experiment and the proteins with similar size came from different gels in the same experiment. (c) CCL5 mRNA expression levels in NSCLC cells treated with palbociclib of gradient concentration (left) and secreted CCL5 in the supernatants of NSCLC cell with 2 μ M palbociclib (right). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001.

Article Snippet: Thus, the secreted CCL5 levels in the media of palbociclib-treated cells and the control cells were assessed by enzyme-linked immunosorbent assay (ELISA) using CCL5 ELISA kit (EK0494, Boster Biological Technology, CA, USA) according to the manual instruction.

Techniques: Staining, Expressing, Western Blot, Control, Concentration Assay

Journal: Journal of Oncology

Article Title: Palbociclib Enhances Migration and Invasion of Cancer Cells via Senescence-Associated Secretory Phenotype-Related CCL5 in Non-Small-Cell Lung Cancer

doi: 10.1155/2022/2260625

Figure Lengend Snippet: Blocking SASP-related CCL5 inhibits the migration ability induced by Palbociclib. (a) The effect of antagonists of CCR5 TAK-779 on the migration ability of NSCLC cells treated with 2 μ M Palbociclib. Upper: Images of migrated cell with Palbociclib or TAK-779, bar: 100 μ m. Lower: The results were analyzed using unpaired t -test. (b) Diagram showing how Palbociclib contributes to the migration and invasion of NSCLC via SASP-related CCL5.

Article Snippet: Thus, the secreted CCL5 levels in the media of palbociclib-treated cells and the control cells were assessed by enzyme-linked immunosorbent assay (ELISA) using CCL5 ELISA kit (EK0494, Boster Biological Technology, CA, USA) according to the manual instruction.

Techniques: Blocking Assay, Migration