Journal: Molecular Cancer Therapeutics

Article Title: Characterization of the CCL21-mediated melanoma-specific immune responses andin situmelanoma eradication

doi: 10.1158/1535-7163.mct-06-0709

Figure Lengend Snippet: Figure 1. CCL21 expression and secretion in B16F0 melanoma cells. A, CCL21 expression in pCMV-CCL21–transfected and pCUB-CCL21 – transfected Melan-a mouse melanocytes (lanes 1 and 3) and B16F0 mouse melanoma cells (lanes 2 and 4). B, expression and secretion of CCL21 in established CCL21-positive B16F0 cell lines. Lane 1, B16F0; lane 2, B16F0-CMV-CCL21; lane 3, B16F0-UB-CCL21; lane 4, culture medium from B16F0- CMV-CCL21; lane 5, culture medium from B16F0-UB-CCL21. Arrows on the left, point to apparent molecular weight (kDa). Arrows between the panels, point to CCL21 and actin (loading control). C, ELISA-based quantitation of CCL21 secretion in pooled and clonal B16F0 cells. Bottom, cell types. The assay was done in triplicates. Columns, mean of three independent experiments; bars, SD.

Article Snippet: For the quantitative ELISA assay, B16F0, B16F0-CMV-CCL21, and B16F0UB-CCL21 cells from pools and individual clones were seeded in 100 AL of culture medium onto 96-well plates (5 105 per well) and kept in culture for 48 h. Medium was then collected, purified, and subjected to the mouse CCL21 ELISA assay (R&D Systems) as advised by the manufacturer.

Techniques: Expressing, Transfection, Molecular Weight, Control, Enzyme-linked Immunosorbent Assay, Quantitation Assay

Journal: Molecular Cancer Therapeutics

Article Title: Characterization of the CCL21-mediated melanoma-specific immune responses andin situmelanoma eradication

doi: 10.1158/1535-7163.mct-06-0709

Figure Lengend Snippet: Figure 2. Development of the WT and CCL21-positive tumors. A, development of the WT and CCL21-positive tumors in C57BL6 and B6.CB17-Prkdcscid/SzJ mice. Area of the tumors was calculated by multiplication of the length of the longest axis to the shortest. Points, mean; bars, SD. 5 and n, B16F0 and B16F0-UB-Mock tumors in C57BL6 mice, respectively; o, B16F0-UB-CCL21 tumors in SCID mice; y, B16F0-CMV-CCL21 tumors in C57BL6 mice; E, B16F0-UB-CCL21 tumors in C57BL6 mice. B, photographs of the representative WT and CCL21-positive melanomas developed in C57BL6 mice 14 and 21 d after tumor inoculation. Top, tumor type. Tumor size and weight shown on the photo- graphs of the excised tumors.

Article Snippet: For the quantitative ELISA assay, B16F0, B16F0-CMV-CCL21, and B16F0UB-CCL21 cells from pools and individual clones were seeded in 100 AL of culture medium onto 96-well plates (5 105 per well) and kept in culture for 48 h. Medium was then collected, purified, and subjected to the mouse CCL21 ELISA assay (R&D Systems) as advised by the manufacturer.

Techniques:

Journal: Molecular Cancer Therapeutics

Article Title: Characterization of the CCL21-mediated melanoma-specific immune responses andin situmelanoma eradication

doi: 10.1158/1535-7163.mct-06-0709

Figure Lengend Snippet: Figure 3. Tumor infiltration and cytotoxic immune response. A, photographs of representative cryosections of tumors stained with antibodies specific to immune cell markers as indicated above the photographs (green or red). Blue, cell nuclei were stained with 4¶,6-diamidino-2-phenylindole. Left, types of tumors. B, FACS analysis of tumor infiltration 7 and 14 d after tumor inoculation. Percentage of tumor-infiltrating cells was calculated from the total number of the counted cells (1 105) and cells counted positive for the expression of the specific markers. Columns, mean of five independent counts; bars, SD. Black columns, B16F0-infiltrating cells; white columns, B16F0-UB-CCL21–infiltrating cells. C, cytotoxicity of splenocytes (E:T ratio of 1:50) isolated from tumor-rejecting (TR), B16F0-inoculated (B16F0), and naive mice against various target cells (indicated in the panel) as determined by Annexin-based cytotoxicity assay. Columns, mean of five independent experiments; bars, SD. D, cytotoxicity of splenocytes (n and .) and CD8+ T cells (5 and o) isolated from tumor-rejecting mice against B16F0 (n and 5) and YAC-1 (. and o) target cells at different E:T ratios. Points, mean of three independent experiments; bars, SD.

Article Snippet: For the quantitative ELISA assay, B16F0, B16F0-CMV-CCL21, and B16F0UB-CCL21 cells from pools and individual clones were seeded in 100 AL of culture medium onto 96-well plates (5 105 per well) and kept in culture for 48 h. Medium was then collected, purified, and subjected to the mouse CCL21 ELISA assay (R&D Systems) as advised by the manufacturer.

Techniques: Staining, Expressing, Isolation, Cytotoxicity Assay

Journal: Molecular Cancer Therapeutics

Article Title: Characterization of the CCL21-mediated melanoma-specific immune responses andin situmelanoma eradication

doi: 10.1158/1535-7163.mct-06-0709

Figure Lengend Snippet: Figure 4. Analysis of melanoma-specific antibody production. A, relative reactivity of the sera against B16F0 cells. Sera were collected from mice inoculated with WT (m1‘– m4’) or B16F0-UB-CCL21 melanomas (m1–m10). B, photographs of representative immunostainings of B16F0 melanoma cells with serum-derived antibodies. Top, source of sera or antibody. C, relative reactivity of the sera against Melan-a mouse melanocytes. Sera were collected from mice inoculated with WT or B16F0-UB-CCL21 melanomas. D, photographs of representative immunostainings of Melan-a cells with serum-derived antibodies. Top, source of sera or antibody. Data were collected from three independent measurements and presented as mean F SD.

Article Snippet: For the quantitative ELISA assay, B16F0, B16F0-CMV-CCL21, and B16F0UB-CCL21 cells from pools and individual clones were seeded in 100 AL of culture medium onto 96-well plates (5 105 per well) and kept in culture for 48 h. Medium was then collected, purified, and subjected to the mouse CCL21 ELISA assay (R&D Systems) as advised by the manufacturer.

Techniques: Derivative Assay

Journal: Molecular Cancer Therapeutics

Article Title: Characterization of the CCL21-mediated melanoma-specific immune responses andin situmelanoma eradication

doi: 10.1158/1535-7163.mct-06-0709

Figure Lengend Snippet: Figure 5. Challenge, adoptive transfer, and rejection of distant melanoma. A, photograph of mouse skin 40 d after inoculation of primary B16F0-UB- CCL21 melanoma. B, photograph of mouse skin 3 d after inoculation of the secondary B16F0 melanoma. C, photograph of primary (P) and secondary (S) melanoma sites 10 d after challenging inoculation. D, WT B16F0 melanoma outgrowth in naive mice that received control splenocytes (n) or splenocytes ( w) and TCM cells (D) from tumor-rejecting mice. Points, mean; bars, SD. Arrow, points to the time of adoptive transfer. E, development of preestablished WT B16F0 melanomas (n and .) after inoculation of B16F-UB-Mock (5) and B16F0-UB-CCL21 (o) tumors at distant sites. Arrow, points to a time of B16F-UB-Mock and B16F0-UB-CCL21 tumor inoculation. Same shapes (n and 5 or . and o) represent the development of B16F0 and B16F-UB-Mock or B16F0-UB-CCL21 in the same animals. Points, mean; bars, SD.

Article Snippet: For the quantitative ELISA assay, B16F0, B16F0-CMV-CCL21, and B16F0UB-CCL21 cells from pools and individual clones were seeded in 100 AL of culture medium onto 96-well plates (5 105 per well) and kept in culture for 48 h. Medium was then collected, purified, and subjected to the mouse CCL21 ELISA assay (R&D Systems) as advised by the manufacturer.

Techniques: Adoptive Transfer Assay, Control

Journal: Nature Cardiovascular Research

Article Title: The thrombin receptor PAR1 orchestrates changes in lymphatic endothelial cell junction morphology to augment lymphatic drainage during lung injury

doi: 10.1038/s44161-025-00681-7

Figure Lengend Snippet: ( a-d ) Tracing assay for VEGFR3Cre ERT2 using Lox-stop-lox ; TdTomato ; VEGFR3Cre ERT2 mice. ( a ) Merged image of Lox-stop-lox ; TdTomato ; VEGFR3Cre ERT2 mice after treatment with tamoxifen stained for anti-RFP (red, b ) and LEC marker anti-CCL21 (green, c ) demonstrating colocalization of TdTomato (red) and LECs (green) without significant TdTomato ( Cre reporter) expression outside of LECs. ( d ) Merged image of Lox-stop-lox ; TdTomato ; VEGFR3Cre ERT2 mice after treatment with tamoxifen stained for anti-RFP (red) and anti-VEGFR3 (LEC marker) also demonstrating colocalization of TdTomato (red) and LECs (green) without significant TdTomato expression outside of LECs. ( e ) Lung lymphatic endothelial cells (LECs) were isolated by flow cytometry for qPCR to assess knock-down efficiency of F2r (PAR1) in PAR1 iLECKO mice (n = 6) compared to PAR1 fl/fl mice (n = 3) with Gapd (GAPDH) as the reference housekeeping gene. Unpaired 2-tailed t- test; **, p = 0.0093. ( f ) BAL cell counts in room air PAR1 fl/fl (n = 4) and PAR1 iLEC KO (n = 4) mice compared to PAR1 fl/fl mice (n = 8) and PAR1 iLEC KO mice (n = 18) after 84 hours of hyperoxia exposure. One-way ANOVA; *, p = 0.0124; **, p = 0.0022; * p = 0.0332 from left to right. ( g, h ) Representative PCLS images demonstrating accumulation of CD45R + leukocytes (blue) in the peri-interstitial alveolar spaces adjacent to lymphatic collectors ( Prox1-EGFP , green) in PAR1 iLEC KO mice after hyperoxia-induced lung injury. BAL count and imaging data representative of at least 2 independent experiments. All error bars represent SEM. All scale bars = 200 µm.

Article Snippet: Slides from paraffin-embedded sections were stained with hematoxylin and eosin or immunostained as described previously , with the following antibodies: VEGFR3 (1:50 dilution, catalogue number AF743, R&D Systems); CCL21 (1:50 dilution, catalogue number AF457, R&D Systems); or RFP (1:100 dilution, catalogue number 600401379S, Rockland).

Techniques: Staining, Marker, Expressing, Isolation, Flow Cytometry, Knockdown, Imaging

Journal: Cancer Research Communications

Article Title: Chemokine Analysis in Patients with Metastatic Uveal Melanoma Suggests a Role for CCL21 Signaling in Combined Epigenetic Therapy and Checkpoint Immunotherapy

doi: 10.1158/2767-9764.crc-22-0490

Figure Lengend Snippet: FIGURE 5 Kaplan–Meier analysis showing. PFS (A) and OS (B), respectively using pretreatment plasma CCL21 (pg/mL) measurements based on their median values. C, Correlation between CCL21 (pg/mL) and CD3+CCR7+CD45RA+ (% counts) matched patient samples (n = 23). D, Flow cytometry–based comparison between short- and long-term survivors for CD3+CCR7+CD45RA+ % (T naïve and T stem cell memory) analysis using

Article Snippet: IHC was performed with an autostainer (Autostainer Link 48, Dako) using primary antibodies SOX10 (E6B6I, Cell Signaling Technology), TK1 (PA5-29686, Thermo Fisher Scientific), CCL21 (NBP2-37928, Novus), CCR7 (EPR23192-57, Abcam), and CD20 (Dako Clinical grade) antibodies.

Techniques: Clinical Proteomics, Flow Cytometry, Comparison

Journal: Cancer Research Communications

Article Title: Chemokine Analysis in Patients with Metastatic Uveal Melanoma Suggests a Role for CCL21 Signaling in Combined Epigenetic Therapy and Checkpoint Immunotherapy

doi: 10.1158/2767-9764.crc-22-0490

Figure Lengend Snippet: FIGURE 6 IHC magenta showing low mag of CD20 varied staining of TLS-like (borderline-tertiary lymphoid structures) followed by high magnification images of CD20, CD3, CCL21 within serial sections for Pt 2-027 (A), 3-010 (B). Also see Supplementary Fig. S5. Low and high

Article Snippet: IHC was performed with an autostainer (Autostainer Link 48, Dako) using primary antibodies SOX10 (E6B6I, Cell Signaling Technology), TK1 (PA5-29686, Thermo Fisher Scientific), CCL21 (NBP2-37928, Novus), CCR7 (EPR23192-57, Abcam), and CD20 (Dako Clinical grade) antibodies.

Techniques: Staining

Journal: Cell

Article Title: Inflammation switches the chemoattractant requirements for naive lymphocyte entry into lymph nodes

doi: 10.1016/j.cell.2024.11.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Goat Anti-Mouse Ccl21 / 6ckine Polyclonal antibody, Biotin Conjugated , R and D Systems , Cat# BAF457; RRID:AB_2072082.

Techniques: Control, Virus, Recombinant, Adjuvant, Reverse Transcription, RNAscope, SYBR Green Assay, Plasmid Preparation, Software