b16f10 ova tumor bearing mice  (ATCC)

Journal: Nature Communications

Article Title: Lymphatic-derived oxysterols promote anti-tumor immunity and response to immunotherapy in melanoma

doi: 10.1038/s41467-025-55969-w

Figure Lengend Snippet: A B16F10-OVA VEGF-C tumors were inoculated in C57BL/6 mice. LECs were sorted by flow cytometry (CD45 neg CD31 + GP38 + ) from tumors, tumor-draining LNs (TdLN) and non-draining LNs (NdLN) after 11 days. Ch25h mRNA levels (RPKM) provided by RNA sequencing. n = 5 mice/group. Data were presented as mean values ± SD. B Correlation between Ch25h expression and lymphatic vessel (LV) signature (LV signature 1: pdpn, vegfc, lyve1 , and LV signature 2: prox1, flt4, lyve1, pdpn, vegfc ) in SKCM (Skin cutaneous melanoma) patients (TPM transcript per million). SKCM patient overall survival (OS) of high and low Ch25h expression. Data extracted from TCGA. C Predictive activity (OS) of Ch25h high and Ch25h low-expressors SKCM patients treated with anti-PD-1 and anti-CTLA-4, from an integrated dataset of multiple clinical trial studies. Significance was determined by log-rank analysis. D – F B16F10-OVA VEGF-C tumor cells were injected into Ch25h-GFP and WT mice. D , E Ch25h expression by tumor infiltrated cells on day 14. (DC dendritic cells, CAF cancer-associated fibroblasts, BEC blood endothelial cells). Results are representative of two independent experiments, with n = 4 mice/ group. F Ch25h expression by LECs in indicated organs at different time points. Results are pooled from two independent experiments, with n = 2–3 mice/group each. Two-way ANOVA, **** P < 0.0001. G , H B16F10-OVA VEGF-C tumor cells were injected in LEC ΔCh25h and LEC WT mice. G Ch25h expression by LECs and BECs in tumors on day 11. Results are representative of two independent experiments, with n = 5 mice/group. D – G Ch25h expression is represented as MFI Ch25h-GFP – MFI-WT mice. H 25-HC levels in tumor interstitial fluid measured by liquid chromatography–mass spectrometry. Results are pooled from two experiments, with n = 4–13 mice /group. G , H Two-tailed unpaired t -test. * P < 0.05; **** P < 0.0001. I VEGF-C expression in human melanoma cell lines by Q-PCR. Histograms depict technical triplicates from one experiment. E – I Data were presented as mean values ± SD. J , K Human melanoma VEGF-C high T362C and VEGF-C low T618A cells were injected in NSG (WT), NSG-LEC ΔCh25h , and NSG-LEC WT mice. Ch25h expression (MFI Ch25h-GFP-MFI-WT mice) by LECs from tumors and skin was assessed by flow cytometry. Data were presented as mean values ± SD. Results are representative of two independent experiments. L Ch25h mRNA and PDPN staining on human melanoma sections ( n = 3 patients). Representative images are shown for patients 1 (Pt1) and 2 (Pt2). Scale bar, 20 and 5 µm (zoomed).

Article Snippet: B16F10-OVA VEGFC tumor-bearing mice were injected subcutaneously at days 4 and 8 post tumor inoculation with OVA protein (50 µg/mice; Invivogen) and CpG-B 1826 (30 µg/mice; Invivogen).

Techniques: Flow Cytometry, RNA Sequencing, Expressing, Activity Assay, Injection, Liquid Chromatography, Mass Spectrometry, Two Tailed Test, Staining

Journal: Nature Communications

Article Title: Lymphatic-derived oxysterols promote anti-tumor immunity and response to immunotherapy in melanoma

doi: 10.1038/s41467-025-55969-w

Figure Lengend Snippet: A – D B16F10-OVA VEGF-C cells were injected in LEC ΔCh25h and LEC WT mice ( A , C , D ), in NSG-LEC ΔCh25h and NSG-LEC WT mice, or in Rag2 KO -LEC ΔCh25h and Rag2 KO -LEC WT mice ( B ). A , B Tumor growth was followed and normalized to the size of tumors in LEC WT , NSG-LEC WT , and Rag2 KO -LEC WT mice, respectively, at day 6 for tumor growth, two-way ANOVA, * P < 0.05. Data were presented as mean values ± SEM. Tumor cell proliferation (Ki67 + ) was evaluated by flow cytometry on day 11. Data were presented as mean values ± SD. Results are pooled from two independent experiments ( n = 4–8 mice per group each) and normalized to WT ( A ), and Ch25h expression (MFI Ch25h-GFP – MFI-WT mice) was measured by flow cytometry at day 12 ( B ). C , D Tumors were harvested on day 11. C CD4 + and CD8 + T cells were analyzed for their expression of indicated markers. Representative flow cytometry dot plots and histograms provide the frequency of positive cells among T cell subsets. Data are presented as mean values ± SD. Results are pooled from two independent experiments ( n = 4–8 mice/ group each) and normalized to WT. D Myeloid cells (CD11b + CD68 + ) were separated into monocytes (Ly6C + F4/80 - ) and macrophages (Ly6C - F4/80 + ) and were analyzed for their expression of indicated markers. Representative flow cytometry dot plots provide the frequency of positive cells among indicated cells. Data were presented as mean values ± SD. Results are representative of two independent experiments, n = 5–7 mice/group each. A – D For FACS analysis, two-tailed unpaired t -test. * P < 0.05; ** P < 0.01; *** P < 0.0001.

Article Snippet: B16F10-OVA VEGFC tumor-bearing mice were injected subcutaneously at days 4 and 8 post tumor inoculation with OVA protein (50 µg/mice; Invivogen) and CpG-B 1826 (30 µg/mice; Invivogen).

Techniques: Injection, Flow Cytometry, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Lymphatic-derived oxysterols promote anti-tumor immunity and response to immunotherapy in melanoma

doi: 10.1038/s41467-025-55969-w

Figure Lengend Snippet: A – E LEC ΔCh25h and LEC WT mice were inoculated with B16F10-OVA VEGF-C (B16-OVA-VC) cells and vaccinated with OVA protein and CpG-B at day 5 ( A ) or at day 4 and day 8 ( B – E ). A Ch25h expression (GFP MFI) was measured by flow cytometry on day 11. n = 5 mice/ group. B Tumor growth was followed and compared to unvaccinated B16F10-OVA VEGF-C tumor-bearing WT mice. Representative of four independent experiments, n = 5–6 mice/group each. C – E Tumor were harvested on day 24 and analyzed by flow cytometry. C CD45 + /tumor cell ratio and tumor cell proliferation (Ki67 + ) and CD45 + cell frequency in living cells. D Myeloid cells (CD11b + CD68 + ) were separated into monocytes (Ly6C + F4/80 - ) and macrophages (Ly6C - F4/80 + ) and were analyzed for their expression of indicated markers. E CD4 + and CD8 + T cells were analyzed for their expression of indicated markers. C – E Representative of two independent experiments, n = 5–8 mice/group each. Data were presented as mean values ± SD ( F , G ) LEC ΔCh25h and LEC WT mice were inoculated with B16F10-OVA VEGF-C cells and adoptively transferred 8 days later with OT-1 effector T cells. Tumor growth was followed and normalized to the size of WT at day 6 ( F ), and OT-1 cells were analyzed in tumors 2 days after transfer for indicated markers ( G ). Data were pooled from two independent experiments, n = 2–5 mice/group each, and are presented as mean values ± SEM ( F ) or as mean values ± SD ( G ). B , F Two-way ANOVA, * P < 0. 05. ( A , C , D , E , G ) Two-tailed unpaired t -test. * P < 0.05; ** P < 0.01; *** P < 0.0001.

Article Snippet: B16F10-OVA VEGFC tumor-bearing mice were injected subcutaneously at days 4 and 8 post tumor inoculation with OVA protein (50 µg/mice; Invivogen) and CpG-B 1826 (30 µg/mice; Invivogen).

Techniques: Expressing, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Lymphatic-derived oxysterols promote anti-tumor immunity and response to immunotherapy in melanoma

doi: 10.1038/s41467-025-55969-w

Figure Lengend Snippet: A LEC ΔCh25h and LEC WT mice were inoculated with B16F10-OVA VEGF-C cells and tumors were harvested at day 11. Montages of maximum projected 3D confocal images of representative sections ( n = 3 mice/group) immunostained for lymphatic vessels (Lyve-1, green), T cells (CD8, red), myeloid cells (CD68, white), and nuclei (blue). Images were obtained using a 10x objective, including a 3x relative magnification. Selected regions of interest are indicated by dashed squares and denoted magnified areas are shown in images beside. Scale bars, 1 mm, 100 μm (zoomed). B BMDMs were incubated with B16F10-OVA VEGF-C tumor-conditioned medium (TCM) and treated or not with 25-HC for 48 h. BMDM phenotype was analyzed by flow cytometry. Data were representative of three independent experiments with n = 3–6 replicates each. Data were presented as mean values ± SD . C BMDM were treated or not with IFN-γ or IL-4 and further treated or not with 25-HC for 48 h. BMDM phenotype was analyzed by flow cytometry. Data were representative of two independent experiments with n = 4–8 replicates each. Data were presented as mean values ± SD . D TCM-exposed BMDM were treated or not with 25-HC for 48 h, washed, and incubated with OT-1 effector cells (5:1 ratio) for 48 h. T-cell phenotype was analyzed by flow cytometry. Data were representative of two independent experiments with n = 3–5 replicates each. Data were presented as mean values ± SD. B Two-tailed unpaired t -test. C Two-way ANOVA, D One-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: B16F10-OVA VEGFC tumor-bearing mice were injected subcutaneously at days 4 and 8 post tumor inoculation with OVA protein (50 µg/mice; Invivogen) and CpG-B 1826 (30 µg/mice; Invivogen).

Techniques: Incubation, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Lymphatic-derived oxysterols promote anti-tumor immunity and response to immunotherapy in melanoma

doi: 10.1038/s41467-025-55969-w

Figure Lengend Snippet: A LEC ΔCh25h and LEC WT mice were inoculated with B16F10-OVA VEGF-C cells and vaccinated with OVA protein and CpG-B at day 4 and day 8. Mice were additionally injected with anti-CSF1R (1 mg / mouse at day 10, followed by 500 µg/mouse every 3 days or PBS), as represented in the scheme. Tumor growth was followed. Two-way ANOVA, ** P < 0.01. Data were representative of two independent experiments with n = 5–7 mice/group each. B NSG-LEC ΔCh25h and NSG-LEC WT mice were inoculated with B16F10-OVA VEGF-C cells mixed or not with BMDM. Tumor-bearing mice were further adoptively transferred with OT-1 effectors on day 8, as represented in the scheme. Tumor growth was followed and normalized to the size individually from day 6. Two-way ANOVA, ** P < 0.01. Data were pooled from two independent experiments with n = 5–8 mice/group each. A , B Data were presented as mean values ± SEM.

Article Snippet: B16F10-OVA VEGFC tumor-bearing mice were injected subcutaneously at days 4 and 8 post tumor inoculation with OVA protein (50 µg/mice; Invivogen) and CpG-B 1826 (30 µg/mice; Invivogen).

Techniques: Injection

Journal: Nature Communications

Article Title: Lymphatic-derived oxysterols promote anti-tumor immunity and response to immunotherapy in melanoma

doi: 10.1038/s41467-025-55969-w

Figure Lengend Snippet: A – C LEC ΔCh25h and LEC WT mice were inoculated with B16F10-OVA VEGF-C cells and vaccinated with OVA protein and CpG-B on day 4 and day 8. Tumors were harvested on day 24, and monocytes and macrophages were sorted by flow cytometry (see Fig. for gating strategy). RNA sequencing was performed (Illumina). A Top 10 Hallmark pathways expressed in monocytes and macrophages in tumors from LEC ΔCh25h and LEC WT mice. B Heatmap showing expression levels of genes implicated in “anti-tumor” or “pro-tumor” immune signatures in monocytes and macrophages and in tumors from LEC ΔCh25h and LEC WT mice. C GSEA enrichment of M1 and M2 signatures in monocytes and macrophages from tumor in LEC ΔCh25h and LEC WT mice using gene set from ref. . D B16F10-OVA VEGF-C tumor-bearing C57BL/6 mice were treated with 25-HC every 2 days, starting at day 4. Tumor growth was followed. Two-way ANOVA, * P < 0.05. Data were a pool of three independent experiments with n = 4–6 mice/group each. On day 11, tumors were harvested, and the CD68 + CD11b + myeloid cell (macrophage, F4/80 + Ly6C - and monocytes, F4/80 - Ly6C + ) phenotype was assessed by flow cytometry. Ratio iNOS + /CD206 + cells are provided. Two-tailed unpaired t -test, *** p < 0.001. E Correlation between Ch25h expression and M1/M2 signatures in normal skin and in SKCM patients. Data were extracted from the TCGA database.

Article Snippet: B16F10-OVA VEGFC tumor-bearing mice were injected subcutaneously at days 4 and 8 post tumor inoculation with OVA protein (50 µg/mice; Invivogen) and CpG-B 1826 (30 µg/mice; Invivogen).

Techniques: Flow Cytometry, RNA Sequencing, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Lymphatic-derived oxysterols promote anti-tumor immunity and response to immunotherapy in melanoma

doi: 10.1038/s41467-025-55969-w

Figure Lengend Snippet: A BMDM were incubated with B16F10-OVA VEGF-C tumor-conditioned medium (TCM), treated or not with 25-HC for 48 h, and analyzed by Q-PCR for PPAR-γ mRNA levels. Data were representative of two experiments with n = 2–3 replicates each. Data were presented as mean values ± SD. Two-tailed unpaired t -test. * P < 0.05. B BMDMs were incubated with tumor-conditioned medium (TCM), treated or not with 25-HC, Troglitazone, or the combination of 25-HC and Troglitazone for 48 h. BMDM phenotype was analyzed by flow cytometry. Data were representative of two independent experiments with n = 3 replicates each. Data were presented as mean values ± SD. One-way ANOVA. *** P < 0.001; **** P < 0.0001. C TCM-exposed BMDM were treated or not with 25-HC, Troglitazone, or the combination of 25-HC and Troglitazone for 48 h, washed, and incubated with OT-1 effector cells for 48 h. T-cell phenotype was analyzed by flow cytometry. Data were representative of two independent experiments with n = 6 replicates each. Data were presented as mean values ± SD. One-way ANOVA. * P < 0.05; *** P < 0.001; *** P < 0.0001. D LEC ΔCh25h and LEC WT mice were inoculated with B16F10-OVA VEGF-C cells mixed with either WT or PPAR-γ deficient (PPAR-γ KO ) BMDM and vaccinated with OVA protein and CpG-B on day 4 and day 8. Tumor growth was followed. Data are presented as mean values ± SEM. Two-way ANOVA, ** P < 0.01. Data were representative of two independent experiments with n = 4–5 mice/group each.

Article Snippet: B16F10-OVA VEGFC tumor-bearing mice were injected subcutaneously at days 4 and 8 post tumor inoculation with OVA protein (50 µg/mice; Invivogen) and CpG-B 1826 (30 µg/mice; Invivogen).

Techniques: Incubation, Two Tailed Test, Flow Cytometry