b16f10 gfp cells  (Addgene inc)

Journal: Nature Communications

Article Title: CD133 + PD-L1 + cancer cells confer resistance to adoptively transferred engineered macrophage-based therapy in melanoma

doi: 10.1038/s41467-025-55876-0

Figure Lengend Snippet: a Schematic illustration of the preparation of NPs-MΦ and the mechanism of CuS NPs-induced M1 polarization. b Three treatment cycles of the adoptive macrophage transfer therapy with Un-MΦ (Un-MΦ 3 ) or NPs-MΦ (NPs-MΦ 3 ) in the B16F10 bearing C57BL/6 mice. Upper, the treatment regimens. Lower, individual tumor growth curves and the corresponding Kaplan-Meier survival curves, log-rank analysis ( n = 10 mice). Control, mice without treatment. c Heatmaps of the cluster of genes associated with the ECM, the key immunosuppressive molecules or the CD8 + T effector cells in the B16F10 tumor of C57BL/6 mice before the treatment at day 8, or after 1 cycle-treatment (NPs-MΦ 1 ) at days 16 or 35 ( n = 3 mice). d, e Images of haematoxylin and eosin (H&E) staining and force maps of the ECM stiffness (d), and the corresponding quantification of relative modulus measured using bio-atomic force microscopy (BioAFM) indentation (e). Tumors were collected from the NPs-MΦ 1 -treated mice at day 35. Mice before the treatment at day 8 or without treatment at day 16 were used as controls. One-way ANOVA with Tukey’s post-test ( n = 65536 biologically independent recorded points). f Expression of α-SMA and collagen IV in the tumor tissues following different treatments. Image shown is representative of n = 3 independent replicates of experiments with similar results. g Quantification of TGF-β in the tumor of mice by enzyme-linked immunosorbent assay (ELISA). One-way ANOVA with Tukey’s post-test ( n = 6 mice). h Fluorescence images of the injected DiD-labeled NPs-MΦ (red) and the immuno-stained collagen IV (green) in tumor sections. Samples were collected at 0 h or 24 h post-administration. The DiD-labeled NPs-MΦ were i.t. injected in the tumor of mice following NPs-MΦ 1 treatment at day 35 (NPs-MΦ 1 , day 35), or the tumors of mice without NPs-MΦ 1 treatment at days 8 (Without NPs-MΦ 1 , day 8) or 16 (Without NPs-MΦ 1 , day 16). The image shown is representative of n = 3 independent replicates of experiments with similar results. Data are expressed as mean ± s.e.m. Source data are provided as a Source Data file.

Article Snippet: B16F10 cells stably expressing green fluorescent protein (B16-GFP) and A375 cells stably expressing green fluorescent protein (A375-GFP) were generated through lentiviral transfection of B16F10 cells and A375 cells by Sunbio Medical Biotech Co. Ltd. (Shanghai, China), respectively.

Techniques: Control, Staining, Microscopy, Expressing, Enzyme-linked Immunosorbent Assay, Fluorescence, Injection, Labeling

Journal: Nature Communications

Article Title: CD133 + PD-L1 + cancer cells confer resistance to adoptively transferred engineered macrophage-based therapy in melanoma

doi: 10.1038/s41467-025-55876-0

Figure Lengend Snippet: a Expression of PD-L1 in B16F10 cancer cells or NIH3T3 fibroblast cells in the co-culture with or without TGF-β. The expression of PD-L1 was normalized with the corresponding untreated B16F10 or NIH3T3 cells cultured alone. Two-way ANOVA with Sidak’s post-test ( n = 4 biologically independent samples). b Expression of PD-L1 in the cancer cells and CAFs of mice following different treatments, normalized with the group of untreated day 8. Untreated day 16, the mice without treatment at day 16 reached a similar tumor size in volume ( ~ 500 mm 3 ) to that of mice following the NPs-MΦ 1 treatment at day 35. One-way ANOVA with Tukey’s post-test ( n = 6 biologically independent samples for untreated day 8, n = 5 biologically independent samples for untreated day 16 or NPs-MΦ 1 day 35). c Identification of CD133 + PD-L1 + from the B16-GFP cancer cells of mice receiving the adoptive NPs-MΦ therapy. Tumors were collected from the NPs-MΦ 1 -treated mice at day 35. One-way ANOVA with Tukey’s post-test ( n = 5 biologically independent mice). Right, representative dot plots of the flow cytometric analysis. d Flow cytometric analysis of TGF-β, IL-10 or VEGF-α production in GFP + CD133 - PD-L1 - , GFP + CD133 + PD-L1 - , GFP + CD133 - PD-L1 + and GFP + CD133 + PD-L1 + cancer cells of mice following the NPs-MΦ 1 treatment at day 35, respectively. The expression of TGF-β, IL-10, or VEGF-α were normalized with the corresponding GFP + CD133 + PD-L1 - cancer cells. One-way ANOVA with Tukey’s post-test ( n = 5 biologically independent samples for GFP + CD133 - PD-L1 - cancer cells, n = 4 biologically independent samples for GFP + CD133 + PD-L1 - , GFP + CD133 - PD-L1 + or GFP + CD133 + PD-L1 + cancer cells). e Volcano plot indicating differentially expressed genes relevant to the TGF-β pathway. The x- axis indicates the log 2 -fold change for GFP + CD133 + PD-L1 + versus GFP + CD133 - PD-L1 - cancer cells. The y- axis indicates the −1 × log 10 of the Q value. Statistical significance was calculated using the two-sided Wald test for DESeq2 data. Source data are provided as a Source Data file.

Article Snippet: B16F10 cells stably expressing green fluorescent protein (B16-GFP) and A375 cells stably expressing green fluorescent protein (A375-GFP) were generated through lentiviral transfection of B16F10 cells and A375 cells by Sunbio Medical Biotech Co. Ltd. (Shanghai, China), respectively.

Techniques: Expressing, Co-Culture Assay, Cell Culture

Journal: Nature Communications

Article Title: CD133 + PD-L1 + cancer cells confer resistance to adoptively transferred engineered macrophage-based therapy in melanoma

doi: 10.1038/s41467-025-55876-0

Figure Lengend Snippet: a Combinatorial αPD-L1 and the adoptive macrophage transfer therapy (NPs-MΦ 1 ) for B16F10 melanoma-bearing mice. Upper, the treatment regimens. Lower, individual tumor growth curves and the corresponding Kaplan-Meier survival curves, log-rank analysis ( n = 8 mice for Control, αPD-L1 or αPD-L1 + NPs-MΦ 1 groups, and n = 10 mice for NPs-MΦ 1 group). b Expression of PD-L1 in the B16F10 cells, Un-MΦ or NPs-MΦ in vitro, relative to isotype control. One-way ANOVA with Tukey’s post-test ( n = 3 biologically independent samples). c Uptake and degradation of αPD-L1 by Un-MΦ, NPs-MΦ, or the tumor cells. The amount of αPD-L1 within the cells after a 1-h incubation with αPD-L1 was measured by ELISA, and designated as ‘0 h’. After replacement with the fresh medium and incubation for another 4 h, the total amount of αPD-L1, designated as ‘4 h’, in the red box was calculated as the sum of the intracellular and exocytotic measurements listed on the right. Two-way ANOVA with Sidak’s post-test ( n = 5 biologically independent samples). d Schematic illustration of the PD-L1-mediated internalization and degradation of αPD-L1 by NPs-MΦ. e Combinatorial TGF-β inhibitor and adoptive macrophage transfer therapy (NPs-MΦ 1 ) for B16F10 melanoma-bearing mice. Upper, the treatment regimens and the corresponding Kaplan-Meier survival curves. Lower, individual tumor growth curves, log-rank analysis ( n = 10 mice). f The changes of immune cell populations in tumors after combinatorial TGF-β inhibitor and the adoptive NPs-MΦ 1 therapy in the B16F10 mouse model at day 22. CD11b + Gr-1 + MDSC subsets (CD11b + Gr-1 high granulocytic (G-MDSC) and CD11b + Gr-1 int monocytic (M-MDSC)), T reg cells (CD4 + CD25 + Foxp3 + ) as well as granzyme B-positive CD8 + T cells (CD8 + GranB + ) and IFN-γ-positive CD8 + T cells (CD8 + IFN-γ + ) in the tumor were analyzed. One-way ANOVA with Tukey’s post-test ( n = 5 mice). Data are expressed as mean ± s.e.m. Source data are provided as a Source Data file.

Article Snippet: B16F10 cells stably expressing green fluorescent protein (B16-GFP) and A375 cells stably expressing green fluorescent protein (A375-GFP) were generated through lentiviral transfection of B16F10 cells and A375 cells by Sunbio Medical Biotech Co. Ltd. (Shanghai, China), respectively.

Techniques: Control, Expressing, In Vitro, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: CD133 + PD-L1 + cancer cells confer resistance to adoptively transferred engineered macrophage-based therapy in melanoma

doi: 10.1038/s41467-025-55876-0

Figure Lengend Snippet: a Quantification of TGF-β, IL-10, and VEGF-α within the tumor tissues of mice by ELISA, respectively. Unpaired two-tailed t -test ( n = 10 mice). b Elevated surface CALR expression in the CD133 + PD-L1 + cancer cells under hyperthermia in vitro. Unpaired two-tailed t -test ( n = 6 biologically independent samples). c Enhanced phagocytosis of the hyperthermia-treated CD133 + PD-L1 + cancer cells by NPs-MΦ. Unpaired two-tailed t -test ( n = 5 biologically independent samples). d Combinatorial hyperthermia and adoptive transfer of Un-MΦ or NPs-MΦ in the B16F10-bearing mice. Upper, treatment regimens. Lower, the individual tumor growth curves and Kaplan-Meier survival curves, log-rank analysis ( n = 10 mice). e Hyperthermia attenuated TGF-β production in CD133 + PD-L1 + cancer cells and tumor stiffness in the relapsed B16F10 melanoma following the NPs-MΦ 1 treatment. Left, the changes in the number and the intracellular TGF-β level in GFP + CD133 + PD-L1 + cancer cells. The TGF-β level was normalized with that in the GFP + CD133 + PD-L1 + cancer cells without hyperthermia. Unpaired two-tailed t -test ( n = 6 mice). Right, micrographs of H&E staining and immunofluorescence staining of α-SMA and collagens. The image shown is representative of n = 3 independent replicates of experiments with similar results. f Combinatorial hyperthermia and adoptive transfer of Un-hMΦ or NPs-hMΦ in human A375 bearing B-NDG mice. Upper, treatment regimens. Lower, individual tumor growth curves and Kaplan-Meier survival curves, log-rank analysis ( n = 6 mice). g Hyperthermia attenuated TGF-β production in PD-L1 + cancer cells and tumor stiffness in relapsed A375 melanoma model following the NPs-hMΦ transfer. Left, the changes in the number and the intracellular TGF-β level in GFP + CD133 + PD-L1 + cancer cells. The TGF-β level was normalized with that in the GFP + CD133 + PD-L1 + cancer cells without hyperthermia. Unpaired two-tailed t -test ( n = 5 mice). Right, micrographs of H&E staining and immunofluorescence staining of α-SMA and collagens. The image shown is representative of n = 3 independent replicates of experiments with similar results. Data are expressed as mean ± s.e.m. Source data are provided as a Source Data file.

Article Snippet: B16F10 cells stably expressing green fluorescent protein (B16-GFP) and A375 cells stably expressing green fluorescent protein (A375-GFP) were generated through lentiviral transfection of B16F10 cells and A375 cells by Sunbio Medical Biotech Co. Ltd. (Shanghai, China), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Expressing, In Vitro, Adoptive Transfer Assay, Staining, Immunofluorescence