b16 f10 luc2  (ATCC)

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A 1014 cells were injected in C57BL/6 female mice. Treatment of C57BL/6 mice with aCD40 (30 μg/mouse every 3 days, intraperitoneal) started at Day 10 post tumor cell inoculation. Analysis of CD40 levels by flow cytometry of CD45 + CD19 + B cells prepared from tumor samples at day 22 post-treatment ( n = 5 per group) using a one-sided Mann–Whitney U test. B Splenocytes were isolated from C57BL/6 mice by magnetic separation, serum-free starved for 4hrs, and stimulated with or without 12.5 μg/ml aCD40. After 4 days of culture, splenic B cells were isolated by magnetic separation and stained for flow cytometric analysis ( n = 6 per group). C , D Tumor volume of 1014 melanoma in C57BL/6 female mice. Treatment with 200 μg/mouse αPD1 (every 3 days, intraperitoneal), 100 μg/mouse αCTLA4 (every 3 days, intraperitoneal), 30 μg/mouse agonist CD40 (aCD40, every 3 days, intraperitoneal), or IgG control antibody, starts at day 10 post tumor cell inoculation. E Frequency of CD19 + CD11b + regulatory B cells in 1014 melanoma tumors or tumor draining lymph node (TDLN) after 22 days of aCD40 treatment (every 3 days, intraperitoneal) TDLN IgG, n = 5; TDLN aCD40, n = 5; Tumor IgG, n = 4; Tumor aCD40, n = 5. F Flow cytometric analysis of CD45 + CD19 + B cells in the tumor microenvironment of 1014 melanoma after 3 weeks treatment of aCD40. Data were replicates from one experiment ( n = 5 mice per group). G C57BL/6 mice were intravenously injected with formalin-fixed 2 × 10 6 metastatic B16F10-Luc2 cells via the tail vein. Splenic CD8 + T and total B cells were isolated by magnetic separation after 2 days of tumor antigen priming. Total B cells were stimulated with 12.5 μg/ml aCD40 for 2 days and CD11b + B cells were isolated by magnetic separation. CD8 + cytotoxic T cell killing assay were performed via co-culturing CD11b + B cells, CD8 + T cells and B16F10-Luc2 cells in the conditioned RPMI complete medium containing 0.5 μg/ml of IL-7, 30U/ml of IL-2, and CD3/CD28 Dynabeads at a bead-to-cell ratio 2:1. Dead cells were removed and the luciferase activity of remaining live tumor cells was quantified to calculate % inhibition of CTL killing capacity. ( n = 3 independent experiments). Created in BioRender. Yan, C. (2026) https://BioRender.com/83jrya2 . Exact p values are provided as a Source Data file. H Splenic B cells were stimulated with 12.5 μg/ml aCD40 for 4 days. The CD11b - and CD11b + B cells were isolated by magnetic separation and restimulated with 12.5 μg/ml aCD40 for 30 min. Whole-cell extracts were harvested and immunoblotted. β-actin was used as a loading control for densitometry quantification. n = 3 independent experiments. One-way analysis of variance (ANOVA) with post hoc test. Source data are provided as a Source Data file.

Article Snippet: Three mouse melanoma tumor models were used in the study, including BRAF wt NRAS mut 1014, BRAF wt NRAS wt B16F10 (Cat#: CCL-6475, ATCC, RRID:CVCL_0159), and B16F10-Luc2 cells (Cat#: CRL-6475-Luc2, ATCC, RRID:CVCL_A4CJ).

Techniques: Injection, Flow Cytometry, MANN-WHITNEY, Isolation, Staining, Control, Luciferase, Activity Assay, Inhibition

Journal: Nature Communications

Article Title: RAS/MEK/PI3K pathway inhibition augments response to CD40 agonism by targeting CD11b + Bregs thereby overcoming melanoma PD1-resistance

doi: 10.1038/s41467-025-67315-1

Figure Lengend Snippet: A , B Tumor growth, created in BioRender. Yan, C. (2026) https://BioRender.com/83jrya2 , exact p values are provided as a Source Data file, and ( C ) Mouse body weight of B16F10 NRAS wt BRAF wt melanoma in C57BL/6 female mice, n = 8 mice per group. Treatment with 200 μg/mouse αPD1 (lead-in 2 doses, every 3 days, intraperitoneal), followed by 30 μg/mouse aCD40 (every 3 days, intraperitoneal), with or without 300 mg/kg rigosertib (RGS, 5 days a week, oral gavage) and 1 mg/kg trametinib (T, 5 days a week, oral gavage), starts at day 8 post tumor cell inoculation. D Tumor samples were collected from each treatment group at day 20 of treatment and immune profiled by FACS analysis. No αPD1-prime groups, Veh + IgG, n = 6, RGS + T + IgG, n = 8, Veh + aCD40, n = 8, RGS + T + aCD40, n = 7 tumors; with αPD1-prime groups, Veh + IgG, n = 5, RGS + T + IgG, n = 5, Veh + aCD40, n = 8, RGS + T + aCD40, n = 8 tumors. Exact p values are provided as a Source Data file. E The baseline frequency of CD11b + B cells and CD8 + Tc cells in melanoma tumors at 1-month post tumor cell inoculation (1014 tumors, n = 5; B16F10 tumors, n = 3). Exact p values are provided as a Source Data file. F The lung colonization model was established by intravenously injecting 0.5 × 10 6 metastatic B16F10-Luc2 cells via the tail vein. Splenic B cells were treated with 12.5 μg/ml aCD40 for 2 days then CD11b + B cells were isolated by magnetic separation for adoptive transfer. Starting at day 2 post tumor cell inoculation, ~2 × 10 6 CD11b + B cells were intravenously injected via the tail vein (once a week) in 200 μl HBSS buffer. Treatment of 200 μg/mouse αPD1 (every 3 days, intraperitoneal) starts at day 2 post tumor cell inoculation ( n = 5 mice per group). Twenty-one days after tumor implantation, tumor lung was weighed and subtracted from tumor-free lungs in mice. Created in BioRender. Yan, C. (2026) https://BioRender.com/83jrya2 . Exact p values are provided as a Source Data file. G Flow cytometry, exact p values are provided as a Source Data file, and ( H , I ) Immunohistochemistry (IHC) staining of B16F10 melanoma lung metastatic tumors after 21 days of treatment. The percentage of positive red IHC staining cells of Ki67 and CD8 was quantified from 20 fields of each FFPE slide sample per group ( n = 5 tumors per group). Exact p values are provided as a Source Data file. Tc, CD8 + CD3 + cytotoxic T cells. Th, CD4 + CD3 + T helper cells.

Article Snippet: Three mouse melanoma tumor models were used in the study, including BRAF wt NRAS mut 1014, BRAF wt NRAS wt B16F10 (Cat#: CCL-6475, ATCC, RRID:CVCL_0159), and B16F10-Luc2 cells (Cat#: CRL-6475-Luc2, ATCC, RRID:CVCL_A4CJ).

Techniques: Isolation, Adoptive Transfer Assay, Injection, Tumor Implantation, Flow Cytometry, Immunohistochemistry