Journal: bioRxiv

Article Title: Immune disease dialogue of chemokine-based cell communications as revealed by single-cell RNA sequencing meta-analysis

doi: 10.1101/2024.07.17.603936

Figure Lengend Snippet: a) Pan CD8 T cells migrated towards CXCL2 (at all dilutions for donor 1, and at 1 ng/ml and 10 ng/ml for donor 2), b) pan monocytes did not migrate towards RARRES2, c) pan monocytes migrated towards CCL8 (at 0.1 ng/ml, 1 ng/ml and 100 ng/ml for donor 1, and at 0.1 ng/ml and 100 ng/ml for donor 2).* p < 0.05. ** p < 0.01, *** p < 0.0001 compared to the medium control condition per donor.

Article Snippet: For the monocyte experiments, two positive controls were used; 5% (v/v) cobra venom activated human complement serum (CAS; Complement Technology Inc, cat. NC1769554), as well as CC motif chemokine ligand 8 (CCL8; R&D Systems, cat. 281-CP-010).

Techniques: Control

Journal: bioRxiv

Article Title: Chemokine profiling of melanoma-macrophage crosstalk identifies CCL8 and CCL15 as prognostic factors in cutaneous melanoma

doi: 10.1101/2023.10.04.560856

Figure Lengend Snippet: M1 and M2 macrophages were co-cultured with BLM melanoma cells for transcriptomic (24 h) and secretomic (72 h) analyses. (A) Gene set enrichment analyses (GSEA) comparing 24 hours exposed vs un-exposed isolated cells, as indicated (n=3 donors). Normalized Enrichment Score (NES) values from the main upregulated hallmark_pathwaysfor all groups,are shown. GOMF and KEGG gene sets were additionally studied for cytokine/ chemokine inflammatory pathways. False Discovery Rates are represented (FDR q value <0.01). (B) Representative scheme highlighting the coincident most upregulated secreted chemokines (fold >4 to both unex-posed macrophages and melanoma cells). (C) Secretomic analysis (38 chemokines) of 72 hours co-cultured supernatants comparing M1+ BLM or M2+BLM with unexposed macrophages (x-axis) or BLM (y-axis) cells. Fold logarithmic average changes are shown (n=4 donors). (D) CCL8 and CCL15 secretion assessed at 72 h macrophages ±BLM conditioned media. Mean ±standard deviation (SD) values are shown (n= 5 do-nors). (E) CCL8 and CCL15 mRNA expression levels in mutually conditioned isolated cells. Mean ±SD values relative to TBP mRNA are shown (n= 4). Significant differences to the respective untreated controls, are shown (*p< 0.05, unpaired t-test).

Article Snippet: Serum-deprived media ±recombinant human CCL8 or CCL15 (R&D Systems, Minnneapolis, MN) and ±5 μg/ml neutralizing antibodies for CCR1, CCR3 and CCR5 (supplementary material, Table S1) were added on top of the gels, and spheroids were allowed to invade for 72 hours.

Techniques: Cell Culture, Isolation, Standard Deviation, Expressing

Journal: bioRxiv

Article Title: Chemokine profiling of melanoma-macrophage crosstalk identifies CCL8 and CCL15 as prognostic factors in cutaneous melanoma

doi: 10.1101/2023.10.04.560856

Figure Lengend Snippet: (A) Serum deprivation survival assays of BLM, A375 and Skmel-103 melanoma cell lines in response to increasing concentra-tions of CCL8 and CCL15 chemokines (ng/ml), and to FCS 1%, as positive control. Mean ±SD optical density (OD) values, are shown (n= 4). (B) Proliferation response of melanoma cell lines to 200 ng/ml exogenous CCL8 and CCL15, and to FCS 1% as positive control. Mean ±SD percentages of BrdU+ nuclei, are shown (n= 3). (C) BLM spheroids embedded in 3D-collagen and allowed to invade for 72 h in the presence of increasing concentrations of exogenous CCL8 and CCL15, as indicated (ng/ml). Functional chemotactic axes were identified by using neutralizing antibodies against CCR1, CCR3, CCR5 and their corresponding control isotypes (5 μg/ml). Mean ±SD fold-invasion values, are shown (n= 3-6). Images show representative collagen-embedded spheroids showing basal and CCL15 stimulated invasion. Scale bar, 150 μm. (D) Immunoblots showing CCR1, CCR3 and CCR5 expression in whole-lysates of three melanoma cell lines and isolated CD14+ monocytes. (E) Serum deprivation survival and 3D-collagen invasion assays of monocytes in response to 100 ng/ml exogenous chemokines, including FCS 1%, as positive control (n= 5 donors). Significant differences to the respective untreated controls, are shown (*p< 0.05, paired t-test).

Article Snippet: Serum-deprived media ±recombinant human CCL8 or CCL15 (R&D Systems, Minnneapolis, MN) and ±5 μg/ml neutralizing antibodies for CCR1, CCR3 and CCR5 (supplementary material, Table S1) were added on top of the gels, and spheroids were allowed to invade for 72 hours.

Techniques: Positive Control, Functional Assay, Control, Western Blot, Expressing, Isolation

Journal: bioRxiv

Article Title: Chemokine profiling of melanoma-macrophage crosstalk identifies CCL8 and CCL15 as prognostic factors in cutaneous melanoma

doi: 10.1101/2023.10.04.560856

Figure Lengend Snippet: TAM (A) and tumor cell (B) average MFI of CCL15, CCL8, CCR1, CCR3 and CCR5 in non-metastasizing and metastasizing primary tumors.Mann-Whitney statistical analysis was used to compare non-metastasizing vs metastasizing melanomas; p-values are shown.(C) FFPE human melanoma samples stained for CD68 (TAM marker, red) and CCL15, CCL8, CCR1, CCR3 or CCR5 (green). Scale bar, 50 μm.

Article Snippet: Serum-deprived media ±recombinant human CCL8 or CCL15 (R&D Systems, Minnneapolis, MN) and ±5 μg/ml neutralizing antibodies for CCR1, CCR3 and CCR5 (supplementary material, Table S1) were added on top of the gels, and spheroids were allowed to invade for 72 hours.

Techniques: MANN-WHITNEY, Staining, Marker

Journal: bioRxiv

Article Title: Chemokine profiling of melanoma-macrophage crosstalk identifies CCL8 and CCL15 as prognostic factors in cutaneous melanoma

doi: 10.1101/2023.10.04.560856

Figure Lengend Snippet: (A)Disease-free and overall survival 10-year Kaplan–Meier curves. Youden’s index was used to choose a cutoff point to classify the 67 primary melanomas as ‘low’ or ‘high’: TC CCL8 (MFI, 80 a.u.), TC CCL15 (98a.u.),TAM CCL15 (69 a.u.) and TAM CCR3 (78 a.u.). Log-rank p values, are shown. (B) Summarizing model, created with BioRender.com .

Article Snippet: Serum-deprived media ±recombinant human CCL8 or CCL15 (R&D Systems, Minnneapolis, MN) and ±5 μg/ml neutralizing antibodies for CCR1, CCR3 and CCR5 (supplementary material, Table S1) were added on top of the gels, and spheroids were allowed to invade for 72 hours.

Techniques: