Journal: Frontiers in Oncology

Article Title: Tumor-associated MerTK promotes a pro-inflammatory microenvironment and enhances immune checkpoint inhibitor response in triple-negative breast cancer

doi: 10.3389/fonc.2025.1579214

Figure Lengend Snippet: Inducing MerTK expression impairs tumor growth and promotes anti-tumor immune infiltration. (A) Vector and MerTK-overexpressing EMT6 cells were treated for 18 hours with vehicle or doxycycline (dox, 1ug/mL) prior to collecting whole-cell lysate. Lysates were immunoblotted for MerTK and GAPDH as a loading control. (B) Vector and MerTK-overexpressing EMT6 cells were inoculated onto the flanks of BALB/c mice. Tumor-bearing mice were treated with vehicle or doxycycline via chow (about 200mg/kg/day) starting at day 10 post-tumor inoculation, and tumor volume was measured twice weekly. Mean values and SEMs are shown (n=4–8 tumors per group). (C) Tumors were harvested and stained using IHC. Representative images are shown at 20x magnification. (D) Tumor immune infiltrate by IHC was quantified using FIJI V2.14.0/1.54f. Mean values and SEMs are shown (n=3–6 tumors per group). *P<0.05; ** P< 0.01; ***P<0.001; ns, not significant.

Article Snippet: Membranes were incubated overnight at 4C with the following primary antibodies: MerTK (1:1000, ab184086, Abcam, Cambridge, United Kingdom), MerTK (1:1000, CS4319, Cell Signaling Technologies, Danvers, MA, USA), PD-L1 (1:1000, 17952-1-AP, Proteintech, Rosemont, IL, USA), GAPDH (1:5000, CS2118, Cell Signaling Technologies).

Techniques: Expressing, Plasmid Preparation, Control, Staining

Journal: Frontiers in Oncology

Article Title: Tumor-associated MerTK promotes a pro-inflammatory microenvironment and enhances immune checkpoint inhibitor response in triple-negative breast cancer

doi: 10.3389/fonc.2025.1579214

Figure Lengend Snippet: Tumors overexpressing MerTK are sensitive to immune checkpoint inhibition. A) 4T1 tumors overexpressing MerTK were inoculated onto the flanks of BALB/c mice. Tumor-bearing mice were treated with either IgG, αPD-L1 (15mg/kg), or αCTLA-4 (5mg/kg) therapeutic antibody three to four times, three days apart. Mean values and SEMs are shown (n=14–18 tumors per group). (B) Spaghetti plots for tumors presented in (A) Red lines indicate subjects whose tumors regressed without recurrence. (C, D) Immune profiling of 4–10 tumors in each group that were harvested three days after the third dose of antibody. (C) Tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. Mean values and SEMs are shown (8–10 tumors per group). (D) Heatmap of significantly altered genes involved in anti-tumor immunity for IgG control, PD-L1, and CTLA-4 treatment groups, clustered by upregulated (green) or downregulated (purple). Red and blue represent upregulation and downregulation, respectively. (E) Tumors were analyzed for gene expression by NanoString nCounter. Mean values and SEMs are shown (4 tumors per group). * P <0.05; ** P < 0.01; *** P <0.001; ns, not significant.

Article Snippet: Membranes were incubated overnight at 4C with the following primary antibodies: MerTK (1:1000, ab184086, Abcam, Cambridge, United Kingdom), MerTK (1:1000, CS4319, Cell Signaling Technologies, Danvers, MA, USA), PD-L1 (1:1000, 17952-1-AP, Proteintech, Rosemont, IL, USA), GAPDH (1:5000, CS2118, Cell Signaling Technologies).

Techniques: Inhibition, Flow Cytometry, Control, Gene Expression

Journal: Frontiers in Oncology

Article Title: Tumor-associated MerTK promotes a pro-inflammatory microenvironment and enhances immune checkpoint inhibitor response in triple-negative breast cancer

doi: 10.3389/fonc.2025.1579214

Figure Lengend Snippet: MerTK-negative tumors are resistant to immune checkpoint inhibition. (A) 4T1 vector and MerTK-overexpressing tumors were inoculated onto the flanks of BALB/c mice. Tumor-bearing mice were treated with IgG, αPD-L1 (15mg/kg), or αCTLA-4 (5mg/kg) therapeutic antibody four times 3–4 days apart. Mean values and SEMs are shown (n=12–16 tumors). (B) Spaghetti plots for each treatment group. Red lines indicate subjects whose tumors regressed without recurrence. (C) Cytotoxic granules (Granzyme B and Perforin), anti-tumor cytokines (IFN-γ and TNFα), and the apoptotic marker (CHOP/GADD153) were detected in tumor sections using IHC. Quantification was performed using FIJI V2.14.0/1.54f. Mean values and SEMs are shown (n=4–9 tumors per group). * P <0.05; ** P < 0.01; *** P <0.001; ns, not significant.

Article Snippet: Membranes were incubated overnight at 4C with the following primary antibodies: MerTK (1:1000, ab184086, Abcam, Cambridge, United Kingdom), MerTK (1:1000, CS4319, Cell Signaling Technologies, Danvers, MA, USA), PD-L1 (1:1000, 17952-1-AP, Proteintech, Rosemont, IL, USA), GAPDH (1:5000, CS2118, Cell Signaling Technologies).

Techniques: Inhibition, Plasmid Preparation, Marker

Journal: Frontiers in Oncology

Article Title: Tumor-associated MerTK promotes a pro-inflammatory microenvironment and enhances immune checkpoint inhibitor response in triple-negative breast cancer

doi: 10.3389/fonc.2025.1579214

Figure Lengend Snippet: Tumor-bound MerTK is not coupled to PD-L1 expression. (A) Whole cell lysate was collected from 4T1 cells overexpressing MerTK and the vector control and was immunoblotted for MerTK, PD-L1, and GAPDH, which was used as a loading control. (B) PD-L1 expression was analyzed in 4T1 cells overexpressing MerTK and vector control by flow cytometry. Mean values and SEMs are shown (n=3 per group). (C) PD-L1 RNA expression was evaluated in 4T1 cells overexpressing MerTK and vector control by NanoString nCounter. Mean Values and SEMs are shown (n=4 per group). (D) Correlation analysis of MerTK and PD-L1 mRNA expression in human TNBC patients. Data was obtained from the cBioportal (n=258 TNBC patients). (E) Correlation analysis of tumor-bound MerTK and stromal PD-L1 staining by mIF (n=101 TNBC patients). (F) Representative images of tumor-bound MerTK low, medium, and high-expressing patients. DAPI (blue), MerTK (cyan), and PD-L1 (magenta) staining are depicted for both stromal (green) and tumor (red) compartments. ns, not significant.

Article Snippet: Membranes were incubated overnight at 4C with the following primary antibodies: MerTK (1:1000, ab184086, Abcam, Cambridge, United Kingdom), MerTK (1:1000, CS4319, Cell Signaling Technologies, Danvers, MA, USA), PD-L1 (1:1000, 17952-1-AP, Proteintech, Rosemont, IL, USA), GAPDH (1:5000, CS2118, Cell Signaling Technologies).

Techniques: Expressing, Plasmid Preparation, Control, Flow Cytometry, RNA Expression, Staining