Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Tumor-derived exosomes drive pre-metastatic niche formation in lung via modulating CCL1 + fibroblast and CCR8 + Treg cell interactions

doi: 10.1007/s00262-022-03196-3

Figure Lengend Snippet: LLC-exo upregulate CCL1 secretion by lung fibroblasts. A Lung fibroblasts were stimulated for 24 h with either LLC-exo or PBS and inflammatory cytokines in the culture supernatant were detected using mouse cytokine microarrays. B CCL1 protein concentrations in LLC-exo stimulated fibroblasts were analyzed by mouse CCL1 ELISA. C Ccl1 mRNA levels in LLC-exo treated lung fibroblasts were detected by qRT-PCR. D CCL1 expression in LLC-exo pre-treated lungs and co-localization with α-SMA+ lung fibroblasts were determined by immunofluorescence staining; scale bar, 100 μm. Data represent the mean ± SD (n = 5). **P < 0.01 (Student’s t-test)

Article Snippet: To detect CCL1 and CCR8 expression in the lung or fibroblasts, lung sections were stained overnight at 4 °C with the following primary antibodies: CCL1 (RD system, MAB8451-SP, 1:300), CCR8 (ABclonal, A4288, 1:200) and α-SMA (Abcam, ab5694, 1:200).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Tumor-derived exosomes drive pre-metastatic niche formation in lung via modulating CCL1 + fibroblast and CCR8 + Treg cell interactions

doi: 10.1007/s00262-022-03196-3

Figure Lengend Snippet: LLC-exo induces Treg differentiation via the CCL1-CCR8 axis. A MPF were stimulated with either PBS or LLC-exo for 24 h and the culture media were collected (PBS and LLC-exo MPF CM, respectively). Splenic T cells were co-cultured with the LLC-exo MPF CM in the presence and absence of ZK756326 (5 μg/ml, daily) for 4 days. B, C The number of CD4+Foxp3+ Tregs was analyzed by flow cytometry; data represent the mean ± SD (n = 5). D Schematic diagram of the in vivo LLC-exo MPF CM and ZK756326 treatment experiment. E, F The proportion of Tregs in the lungs of mice in each group was determined by flow cytometry; data represent the mean ± SD (n = 5). (G) Immunofluorescence staining of CD4+Foxp3+ Tregs in LLC-exo CM ± ZK756326 treated mouse lung: CD4 (red), Foxp3 (green). Nuclei were stained with DAPI. Scale bars, 100 µm. *P < 0.05, **P < 0.01 (one-way ANOVA)

Article Snippet: To detect CCL1 and CCR8 expression in the lung or fibroblasts, lung sections were stained overnight at 4 °C with the following primary antibodies: CCL1 (RD system, MAB8451-SP, 1:300), CCR8 (ABclonal, A4288, 1:200) and α-SMA (Abcam, ab5694, 1:200).

Techniques: Cell Culture, Flow Cytometry, In Vivo, Immunofluorescence, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Tumor-derived exosomes drive pre-metastatic niche formation in lung via modulating CCL1 + fibroblast and CCR8 + Treg cell interactions

doi: 10.1007/s00262-022-03196-3

Figure Lengend Snippet: Schematic diagram of the proposed mechanism by which TDEs drive tumor cell colonization of the lung via fibroblast CCL1 production and Treg CCR8 activation

Article Snippet: To detect CCL1 and CCR8 expression in the lung or fibroblasts, lung sections were stained overnight at 4 °C with the following primary antibodies: CCL1 (RD system, MAB8451-SP, 1:300), CCR8 (ABclonal, A4288, 1:200) and α-SMA (Abcam, ab5694, 1:200).

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Gut-derived memory γδ T17 cells exacerbate sepsis-induced acute lung injury in mice

doi: 10.1038/s41467-024-51209-9

Figure Lengend Snippet: a Heatmap showing the chemokine expression signature in sham-operated and CLP mice (n = 4). b The protein levels of CCL1 in the murine lung after combined prophylactic and therapeutic administration of S-KT or 0.9% saline (n = 6). c Transwell assay was used to assess the ability of CCL1 to chemotaxis sorted-γδ T cells from spleen mononuclear cells (n = 6). d CCR8 expression by intestinal γδ T cells in sham-operated and CLP mice was measured by FCM analysis (n = 6 for sham, 7 for CLP). Sorted-γδ T cells from spleen mononuclear cells in sham-operated or CLP mice were co-cultured with AMs. Expression of e IL-17A and f IFN-γ by γδ T cells was measured by FCM analysis (n = 3). g Heatmap of the top 29 up-regulated DEGs in the lung between the sham-operated mice and CLP mice (n = 3). h The protein levels of the components of the Wnt signaling pathway (Wnt5a, FZD5, β-catenin, and lymphoid enhancer-binding factor 1 (Lef1)) in murine lungs after combined prophylactic and therapeutic administration of S-KT or 0.9% saline (n = 6). i Average plots and heat maps showing the signals at the TSS in the ChIP-seq data sets. j Pie charts showing the distribution of all Lef1 peak locations. k Distal intergenic region-specific binding peaks between Lef1 and CCL1 gene. Data are shown as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns not significant. P values were calculated using one-way ANOVA followed by Tukey’s multiple-comparison test ( b , h ), and two-sided Student’s unpaired t-test ( c – f ). Source data and exact P values are provided as a Source Data file.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies against IL-17A (1:1000, Cat# sc-374218, Santa Cruz), IL-6 (1:1000, Cat# ab229381, Abcam), IL-1β (1:1000, Cat# A1112, ABclonal), Mucin-2 (MUC-2, 1:1000, Cat# 27675-1-AP, Proteintech), ZO-1 (1:1000, Cat# 21773-1-AP, Proteintech), Occludin (1:1500, Cat# 27260-1-AP, Proteintech), Wnt5a (1:1000, Cat# 619919, Zen Bio), FZD5 (1:1000, Cat# 222996, Zen Bio), β-catenin (1:2000, Cat# 51067-2-AP, Proteintech), Lef1 (1:500, Cat# 14972-1-AP, Proteintech), TCF1/7 (1:1000, Cat# A20835, ABclonal), anti-human CCL1 (1:1000, Cat# A10670, ABclonal), anti-mouse CCL1 (1:1000, Cat# MAB845, R&D Systems), GAPDH (1:2000, Cat# 60004-1-Ig, Proteintech) and β-actin (1:2000, Cat# 66009-1-Ig, Proteintech).

Techniques: Expressing, Saline, Transwell Assay, Chemotaxis Assay, Cell Culture, Binding Assay, ChIP-sequencing, Comparison

Journal: Nature Communications

Article Title: Gut-derived memory γδ T17 cells exacerbate sepsis-induced acute lung injury in mice

doi: 10.1038/s41467-024-51209-9

Figure Lengend Snippet: a The protein expression of β-catenin, Lef1, TCF1/7 and b CCL1 in the lung (n = 3 for Lef1, 6 for TCF1/7 and CCL1). c A diagram of MH-S cell line treated with iCRT14 or vehicle followed by stimulation with LPS or PBS for 24 h in vitro to analyze the protein level of CCL1. d The protein level of CCL1 in MH-S cells (n = 7). e Gating strategy and frequency for Kaede red + γδ T17 cells in the lung of Kaede-tg septic mice treated with CCL1- shRNA (Adv-shRNA (CCL1)) or adenovirus-negative control (Adv-NC) (n = 6). f Gating strategy and frequency of Kaede red + γδ T17 cells in the lung of Kaede-tg septic mice treated with anti-CCL1 mAb or control IgG (n = 6). g Gating strategy and quantification of γδ T cells in the small intestine (n = 8). h Representative FCM plots and absolute number of Kaede red + γδ T17 cells in the lung of Kaede-tg septic mice treated with vehicle or iCRT14 (n = 6). i , j Representative H&E sections of lung and histological injury scores (n = 7). 20×, Scale bar = 100 μm. 40×, Scale bar = 50 μm. k The levels of IL-17A (n = 10 for sham + vehicle, 7 for CLP + vehicle, 6 for CLP + iCRT14), IL-6 (n = 10 for sham + vehicle, 8 for CLP + vehicle and CLP + iCRT14) and total protein (n = 6) in the BALF. l The protein level of IL-17A and IL-6 in the lung (n = 6). Data are shown as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns not significant. P values were calculated using one-way ANOVA followed by Tukey’s multiple-comparison test ( a , b , d , k , l ), and two-sided Student’s unpaired t-test ( e – h , j ). Source data and exact P values are provided as a Source Data file.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies against IL-17A (1:1000, Cat# sc-374218, Santa Cruz), IL-6 (1:1000, Cat# ab229381, Abcam), IL-1β (1:1000, Cat# A1112, ABclonal), Mucin-2 (MUC-2, 1:1000, Cat# 27675-1-AP, Proteintech), ZO-1 (1:1000, Cat# 21773-1-AP, Proteintech), Occludin (1:1500, Cat# 27260-1-AP, Proteintech), Wnt5a (1:1000, Cat# 619919, Zen Bio), FZD5 (1:1000, Cat# 222996, Zen Bio), β-catenin (1:2000, Cat# 51067-2-AP, Proteintech), Lef1 (1:500, Cat# 14972-1-AP, Proteintech), TCF1/7 (1:1000, Cat# A20835, ABclonal), anti-human CCL1 (1:1000, Cat# A10670, ABclonal), anti-mouse CCL1 (1:1000, Cat# MAB845, R&D Systems), GAPDH (1:2000, Cat# 60004-1-Ig, Proteintech) and β-actin (1:2000, Cat# 66009-1-Ig, Proteintech).

Techniques: Expressing, In Vitro, shRNA, Negative Control, Control, Comparison

Journal: Journal of experimental & clinical cancer research : CR

Article Title: High and selective cytotoxicity of ex vivo expanded allogeneic human natural killer cells from peripheral blood against bladder cancer: implications for natural killer cell instillation after transurethral resection of bladder tumor.

doi: 10.1186/s13046-024-02955-7

Figure Lengend Snippet: Fig. 8 Receptors of the chemokines expressed on CD3+ T cells and T cell count migrating across the transwell after blocking CCL1/2/20 in the superna tant of coculture medium. (A) The expressions of the chemokine receptors on CD3+CD56− T cells were detected using flow cytometry (n = 3 for all mol ecules). The representative images are shown. (B) Summary data of the cumulative numbers of T cells determined using a fluorescence cell analyzer after being harvested from the lower chambers, which were added into blocking antibodies against CCL1 (0.25 µg/mL), CCL2 (40 ng/mL), or CCL20 (10 µg/ mL) (n = 3 for each). Data are shown as mean ± SD. Statistical significance was determined using an unpaired t-test. *p < 0.05. Abbreviations: CXCR: C-X-C chemokine receptor; CCR:C-C chemokine receptor; XCR:X-C chemokine receptor

Article Snippet: For blocking experiments, blocking antibodies against CCL1 (0.25 μg/mL; MAB272; R&D Systems), CCL2 (40 ng/mL; MAB679; R&D Systems), and CCL20 (10 μg/ mL; MAB360; R&D Systems) were added into the conditioned media in the lower chambers.

Techniques: Cell Counting, Blocking Assay, Flow Cytometry, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: ODN 2088 modulates the release of chemokines by SC astrocytes, in vitro. a Representative chemokine arrays used to detect chemokines in CM of vehicle- and ODN 2088-treated astrocytes. The chemokine arrays were independently repeated twice, showing similar results. Results from a representative experiment are shown. The dots enclosed in rectangular boxes show chemokines whose levels were decreased (> 5% difference) in the CM of ODN 2088-treated astrocytes compared to the CM of vehicle-treated astrocytes. The dots enclosed in the oval box show the chemokine whose levels were increased (> 5% difference) in the CM of ODN 2088-treated astrocytes compared to CM of vehicle-treated astrocytes. 1: CCL1; 2: CCL9/MIP-1γ; 3: CCL2/MCP-1; 4: CCL20/MIP-3α; 5: CX3CL1. b Densitometric quantification of the signal obtained in the chemokine array using the Image Lab software (Bio-Rad). c Quantification of CCL9 levels in CM obtained from ODN 2088- or vehicle-treated astrocytes [** p < 0.01, independent-sample t -test, two-tailed]. The experiment was independently repeated four times, and the mean of 4 experiments ( n = 4) is shown. d Quantification of CCL2 levels in CM obtained from ODN 2088- or vehicle-treated astrocytes [* p < 0.05, independent-sample t -test, two-tailed]. The experiment was independently repeated three times, and the mean of 3 experiments ( n = 3) is shown

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: In Vitro, Software, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: CCL1 released by ODN 2088-treated astrocytes mediates the chemotaxis of peritoneal macrophages. Quantification of F4/80 + cells that crossed to the lower surface of the membrane in response to CM derived from vehicle- or ODN 2088-treated TLR9 astrocytes in the absence or presence of CCL1 neutralizing antibody or IgG 2A isotype control [ F (5, 48) = 81.03, p < 0.0001 by one-way ANOVA, **** p < 0.0001 by Tukey’s post hoc test]. The results of three independent experiments ( n = 3) are shown. Data are presented as mean ± SEM

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: Chemotaxis Assay, Membrane, Derivative Assay, Control

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: Astrocyte-derived CCL2 and CCL9 but not CCL1 regulate macrophage polarization, in vitro. a Macrophage cultures were exposed to ODN 2088-treated astrocyte CM (ODN 2088-CM), in the absence or presence of CCL1 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [ p = 0.7228, independent-sample t -test, two-tailed]. b Macrophage cultures were exposed to vehicle-treated astrocyte CM, in the absence or presence of CCL2 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [** p < 0.01, independent-sample t -test, two-tailed]. c Macrophage cultures were exposed to vehicle-treated astrocyte CM, in the absence or presence of CCL9 neutralizing Ab. The graph shows the quantification of the F4/80 + /Arg-1 + cell number expressed as percentage of total F4/80 + cells in the macrophage cultures [*** p < 0.001, independent-sample t -test, two-tailed]. d Macrophage cultures were exposed to vehicle-treated astrocyte CM (Veh-CM) or ODN 2088-treated astrocyte CM (ODN 2088-CM) for 24 h, with or without (control) addition of rmCCL9 (20 pg/ml). The graph shows the quantification of the F4/80 + /Arg-1 + double-labeled cells expressed as percent of total F4/80 + cells in macrophage cultures [ F (2, 6) = 53.68, p < 0.0001 by one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 by Tukey’s post hoc test]. The experiments were independently repeated twice, yielding similar results. Results from a representative experiment are shown. Results obtained from additional biological repeats of these experiments can be found in Additional file D-G. Data are presented as mean ± SEM

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: Derivative Assay, In Vitro, Two Tailed Test, Control, Labeling

Journal: Journal of Neuroinflammation

Article Title: Astroglial TLR9 antagonism promotes chemotaxis and alternative activation of macrophages via modulation of astrocyte-derived signals: implications for spinal cord injury

doi: 10.1186/s12974-020-01748-x

Figure Lengend Snippet: A scheme summarizing the effects of ODN 2088-treated astrocytes on macrophages. TLR9 antagonism increases the release of CCL1 by astrocytes, which enhances macrophage chemotaxis. In contrast, CCL2 and CCL9 release are decreased in response to ODN 2088. This reduces the negative regulatory effect of CCL2 and CCL9 on M2 macrophage polarization and fosters the M2 phenotype

Article Snippet: In addition, CCL1 concentrations were independently measured by a second ELISA kit purchased from a different vendor (Rockland Immunochemicals; Limerick, PA, USA).

Techniques: Chemotaxis Assay

Journal: The Journal of Experimental Medicine

Article Title: Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses

doi: 10.1084/jem.20111627

Figure Lengend Snippet: Tumor cell chemotaxis to LECs is mediated by CCL1. (A) Migration of breast cancer and melanoma cells to LEC-CM in a Boyden chamber assay. MCF-10F and 184-B5, nontumorigenic human breast epithelial cell lines; MCF-7, MDA-MB-231, and MDA-MB-435, breast cancer cell lines; SK-MEL-28, SK-MEL-25, and MEL-501, melanomas. (B) Migration of MDA-MB-435 cells to LEC-CM in presence of pertussis toxin (PTx) or cholera toxin (CTx). (C) Effects of a monoclonal anti-CCL1 neutralizing antibody on MDA-MB-435 and SK-MEL-25 chemotaxis to LEC-CM. (D) Tumor cell migration to LEC-CM depleted of CCL1 with a polyclonal anti-CCL1 antibody. (E) Migration of tumor cells to 50 ng/ml rhCCL1, in comparison to LEC-CM. (F) Cell shape change of MDA-MB-435 cells stimulated with 50 ng/ml human CCL1. Cells are stained with rhodamine-labeled phalloidin (red) and Hoechst (nuclei; blue). Data are representative of three experiments. Migration (fold increase) is calculated as cell migration (total cell area) to LEC-CM/cell migration to basal medium. Error bars indicate SD, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bar, 10 µm.

Article Snippet: Recombinant human CCL1, mouse CCL1, and viral MCV-type II chemokine-like protein MC148 were purchased from R&D Systems.

Techniques: Chemotaxis Assay, Migration, Boyden Chamber Assay, Comparison, Staining, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses

doi: 10.1084/jem.20111627

Figure Lengend Snippet: Inflammatory cytokines increase CCL1 production by LECs and tumor cell migration to LEC-CM. (A) Real-time qPCR analysis for CCL1 mRNA upon treatment of LECs with 50 ng/ml TNF, 500 ng/ml LPS, or 50 ng/ml IL-1β for 3, 6, and 18 h. Error bars indicate mean ± SD, n = 3. (B) RT-PCR for CCL1 mRNA expression in cultured LECs. cDNA from PMA-stimulated Jurkat cells (50 ng/ml, 48 h) was used as a positive control. (C and D) Western blot (C) and ELISA (D) for CCL1 protein in conditioned medium from LECs treated with 100 ng/ml TNF, 500 ng/ml LPS, or 50 ng/ml IL-1β for 48 h, and CM from Jurkat cells treated with 50 ng/ml PMA for 48 h. Error bars indicate mean ± SD, n = 2. (E and F) Effects of CCL1 depletion on tumor cell (MDA-MB-435) migration to LEC-CM generated in presence of 50 ng/ml TNF (E) or 500 ng/ml LPS (F) for 48 h. Bars indicate mean ± SD, n = 3. Data are representative of three experiments. *, P < 0.05. (G–L) Immunostaining of mouse skin for either LYVE-1 or podoplanin (both green) and for CCL1 (red); boxed areas in J and L depict the overlay. Images are representative of at least six skin samples per group. Arrows, lymphatic vessels; ep, epidermis. Bars, 50 µm.

Article Snippet: Recombinant human CCL1, mouse CCL1, and viral MCV-type II chemokine-like protein MC148 were purchased from R&D Systems.

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Positive Control, Western Blot, Enzyme-linked Immunosorbent Assay, Generated, Immunostaining

Journal: The Journal of Experimental Medicine

Article Title: Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses

doi: 10.1084/jem.20111627

Figure Lengend Snippet: Blocking CCR8 inhibits lymph node metastasis. (A and B) Expression of CCR8 inhibitor MC148 in tumor cell lines as indicated. (A) RT-PCR of cultured tumor cells for hCCL1, MC148, and hCCR8 mRNA. cDNA from LECs served as a positive control for CCL1. β-Actin served as a loading control. (B) Western blot for hCCR8 protein in lysates of cultured cells, MC148 protein in tumor cell CM in vitro, and MC148 in tumor lysates in vivo. β-Actin served as a loading control. C, pcDNA vector control; M, MC148. (C) Tumor growth of pcDNA control and MC148-expressing MDAcl.6 or MDAcl.13 cells. Each data point represents mean tumor volume ± SEM, n = 10 mice per group. (D) Quantification of total vessels (CD31 + ) in tumors as indicated. Bars represent the mean vessel area ± SEM per 10 6 µm 2 total area; MDAcl.13, n = 4; SK-MEL-25, n = 3 tumors. (E and F) Efficiency of shRNA-mediated CCR8 knockdown in tumor cells. qPCR for human CCR8 (E) and Western blot analysis (F) in different tumor cell lines in vitro and in tumors in vivo, as indicated. (F) CCR8 knockdown in MDA-MB-435 cells was performed with shCCR8 sequence 1, shCCR8(1); in SK-MEL-25 with the sequence 2, shCCR8(2); and in MDA cl. 13 with shCCR8 sequences 1 and 2. Error bars indicate mean ± SD, n = 3. (G) Incidence of intranodal metastases upon CCR8 inhibition with the MC148 soluble antagonist or upon CCR8 knockdown with shRNA. Metastasis incidence is calculated as the number of LN with GFP + tumor cells, divided by the total number of LN examined. ND, not determined; ns, not significant; ***, P < 0.001.

Article Snippet: Recombinant human CCL1, mouse CCL1, and viral MCV-type II chemokine-like protein MC148 were purchased from R&D Systems.

Techniques: Blocking Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Positive Control, Control, Western Blot, In Vitro, In Vivo, Plasmid Preparation, shRNA, Knockdown, Sequencing, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses

doi: 10.1084/jem.20111627

Figure Lengend Snippet: CCR8 expression and function in tumor cell lines. (A) Expression of CCR8 mRNA (RT-PCR, in vitro) and protein (Western blot, in vitro and in vivo) by MDA-MB-435 and SK-MEL-25 cells. (B) FACS analysis was performed on cultured tumor cells and primary melanocytes. (C) Effects of CCR8 antagonist MC148 on tumor cell chemotaxis to LEC-CM. Error bars indicate mean ± SD, n = 3. *, P < 0.05. (D) Effects of CCR8 antagonist MC148 and pertussis toxin (PTx) on cytoskeletal rearrangements induced by rhCCL1 in MDA-MB-435 cells. Cells are stained with phalloidin (red) and Hoechst (nuclei, blue). Data are representative of three experiments. (E and F) Changes of cytosolic Ca 2+ concentration measured by FACS in Fluo-4–labeled cells upon addition of human (E) or mouse (F) CCL1 (12.5 or 25 nM as indicated). The black arrow indicates the time of CCL1 addition. MC148 was added at 25 nM. Data are expressed as percentile fluorescence intensity of Fluo-4 dye bound to Ca 2+ . Data are representative of at least two experiments. (G) Cell shape change of MDA-MB-435 cells stimulated with 50 ng/ml mouse CCL1 for the times indicated. Cells are stained with rhodamine-labeled phalloidin (red). Bars, 10 µm.

Article Snippet: Recombinant human CCL1, mouse CCL1, and viral MCV-type II chemokine-like protein MC148 were purchased from R&D Systems.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, In Vitro, Western Blot, In Vivo, Cell Culture, Chemotaxis Assay, Staining, Concentration Assay, Labeling, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses

doi: 10.1084/jem.20111627

Figure Lengend Snippet: Expression of CCL1 in the mouse skin, tumor, and lymph node lymphatic vessels. (A–O) Double immunostaining for either LYVE-1 or podoplanin (lymphatic vessels, red) and mouse CCL1 (green). (A–C) Mouse skin overlying the tumor. (D–F) MDAcl.13 tumor xenograft. (G–L) Capsule and the lymph node cortex of the naive (G–I) and the tumor-draining (J–L) lymph node. (M–O) Lymph node medullary sinuses staining negative for CCL1. Images are representative at least n = 5 samples per group. ep, epidermis. Bars, 100 µm.

Article Snippet: Recombinant human CCL1, mouse CCL1, and viral MCV-type II chemokine-like protein MC148 were purchased from R&D Systems.

Techniques: Expressing, Double Immunostaining, Staining

Journal: The Journal of Experimental Medicine

Article Title: Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses

doi: 10.1084/jem.20111627

Figure Lengend Snippet: CCL1 expression in human tissues. Immunohistochemical staining for podoplanin (lymphatic vessels) and for human CCL1 on serial sections of normal human skin (A and B), melanoma in the skin (C–F), and lymph nodes with melanoma metastases (G–J). (E and F) Higher magnification of the boxed area in C and D. (G–J) Serial sections of human lymph node with melanoma metastases stained with H&E (G) or immunostained for podoplanin (H) or CCL1 (I and J). (H) Podoplanin identifies lymphatic vessels of the SCS (arrows) and lymphatics associated with melanoma metastases in the cortex. (I) CCL1 staining (red, arrows) on a serial section. (J) Higher magnification of the boxed area in I. Images are representative of at least 10 skin samples, 14 primary tumors, and 8 lymph node samples. Arrows, lymphatic vessels; t, tumor; ep, epidermis; c, capsule. Bars: (A–F, H, and I) 100 µm; (G) 250 µm; (J) 25 µm.

Article Snippet: Recombinant human CCL1, mouse CCL1, and viral MCV-type II chemokine-like protein MC148 were purchased from R&D Systems.

Techniques: Expressing, Immunohistochemical staining, Staining