igg2a be0089  (Bio X Cell)

Journal: bioRxiv

Article Title: Mechanoimmunological Control of Metastatic Site Selection

doi: 10.1101/2025.05.10.653256

Figure Lengend Snippet: Luc + B16F10 cells were injected i.v. into IgG control and NK depleted (NK dep. ) recipient mice, which were then monitored for metastatic colonization of the lungs and bone. (A) Schematic diagram of the experimental approach. (B) Representative IVIS images of tumor-bearing IgG control and NK dep. mice, with metastatic burden in the lungs and femurs indicated by black and yellow arrowheads. (C) Quantification of relative femoral colonization, expressed as a ratio of IVIS signal in the legs to the total IVIS signal. Error bars denote SEM. Sample size is indicated above each bar. P-value calculated by unpaired Student’s t-test. (D) Survival and paralysis-free survival of tumor-bearing control and NK dep. mice. (E) Representative H&E image of B16F10 bone metastasis in the spine of an NK dep. mouse. Tumor cells indicated by black and yellow arrowheads. Scale bar = 500 μm. (F-H) C57BL/6 mice bearing stiff and soft subcutaneous hydrogel implants were treated with IgG control or NK cell depleting antibodies and then i.v. injected with Luc + B16F10 cells. B16F10 colonization of the implants was assessed after 2 weeks. (F) Schematic diagram of the experimental approach. (G) Representative images of hydrogel implants three weeks after implantation, with black and yellow arrowheads denoting vascularization. Scale bars = 2.5 mm. (H) Implant colonization by B16F10 cells in IgG control (left) and NK dep. (right) mice, measured by luciferase luminescence after hydrogel lysis. P-value calculated by paired Mann-Whitney test. All results are representative of at least two independent experiments.

Article Snippet: CD8⁺ T cell depletion was achieved by administering 250 μg of InVivoMab anti-mouse CD8α antibody (clone 53-6.7, BioXCell, BE0004-1) or IgG2a control (BioXCell, BE0089) by i.p. injection 2 days and 1 day before tumor delivery, followed by weekly injections, as previously described ( ).

Techniques: Injection, Control, Luciferase, Lysis, MANN-WHITNEY

Journal: bioRxiv

Article Title: Mechanoimmunological Control of Metastatic Site Selection

doi: 10.1101/2025.05.10.653256

Figure Lengend Snippet: (A) Flow cytometric validation of NK cell depletion by anti-asialoGM1 antibody treatment. NK1.1 staining of blood samples from representative IgG control and NK depleted (NK dep. ) mice are shown. (B) Mean fold change in B16F10 colonization in NK dep. mice relative to IgG controls, determined for total metastasis and femoral metastasis (legs). Paired values were derived from 3 independent experiments. (C) Flow cytometric validation of CD8 + T cell depletion by anti-CD8 antibody treatment. CD8 staining of blood samples from representative IgG control and CD8 depleted (CD8 dep. ) mice are shown. (D-E) Luc + B16F10 cells were injected i.v. into IgG control and CD8 dep. recipient mice, which were then monitored for metastatic colonization of the lungs and bone. (D) Representative IVIS images of tumor-bearing IgG control and CD8 dep. mice, with metastatic burden in the lungs and femurs indicated by black and yellow arrowheads. (E) Quantification of relative femoral colonization, expressed as a ratio of IVIS signal in the legs to the total IVIS signal. Error bars denote SEM. Sample size is indicated above each bar. P-value calculated by unpaired Student’s t-test.

Article Snippet: CD8⁺ T cell depletion was achieved by administering 250 μg of InVivoMab anti-mouse CD8α antibody (clone 53-6.7, BioXCell, BE0004-1) or IgG2a control (BioXCell, BE0089) by i.p. injection 2 days and 1 day before tumor delivery, followed by weekly injections, as previously described ( ).

Techniques: Biomarker Discovery, Staining, Control, Derivative Assay, Injection

Journal: bioRxiv

Article Title: Mechanoimmunological Control of Metastatic Site Selection

doi: 10.1101/2025.05.10.653256

Figure Lengend Snippet: Luc + EMT6 cells were injected i.v. into IgG control and CD8 dep. recipient mice, which were then monitored for metastatic colonization of the lungs and bone. (A) Representative IVIS images of tumor-bearing IgG control and CD8 dep. mice, with metastatic burden in the lungs and femurs indicated by black and yellow arrowheads. (B) Quantification of relative femoral colonization, expressed as a ratio of IVIS signal in the legs to the total IVIS signal. Error bars denote standard error of the mean (SEM). Sample size is indicated above each bar. (C) Mean fold change in EMT6 colonization in CD8 dep. mice relative to wild type controls, determined for total metastasis and femoral metastasis (legs). Paired values were derived from 3 independent experiments.

Article Snippet: CD8⁺ T cell depletion was achieved by administering 250 μg of InVivoMab anti-mouse CD8α antibody (clone 53-6.7, BioXCell, BE0004-1) or IgG2a control (BioXCell, BE0089) by i.p. injection 2 days and 1 day before tumor delivery, followed by weekly injections, as previously described ( ).

Techniques: Injection, Control, Derivative Assay

Journal: bioRxiv

Article Title: Mechanoimmunological Control of Metastatic Site Selection

doi: 10.1101/2025.05.10.653256

Figure Lengend Snippet: GFP + B16F10 cells were i.v. injected either into wild type or Prf1 -/- mice or into IgG control treated or NK cell depleted (NK dep. ) mice. After 2 weeks, MTCs from the resulting metastases in the lungs of wild type and IgG control mice and the lungs and bones of Prf1 -/- mice and NK dep. mice were extracted and subjected to SMR and scRNA-seq. (A) Schematic diagram of the experimental approach. (B-C) SMR of B16F10 MTCs isolated from the indicated organs of wild type and Prf1 -/- mice (B) or from IgG control and NK dep. mice (C). Violins encompass the entire data distribution, with dashed lines denoting the median and dotted lines indicating the upper and lower quartiles. Samples sizes (n=) are displayed below each violin. P-values calculated by one-way ANOVA. (D) UMAP visualization of scRNA-seq data from the indicated lung and bone metastases. Cells are clustered based on transcriptional similarity and are color-coded by Seurat cluster identity. Cluster 7 has been boxed in each graph. (E) Pie charts showing the proportion of cluster 7 cells in each MTC sample. (F) Violin plot showing Opn expression levels across Seurat clusters in the indicated lung and bone metastases. Each violin represents the distribution of Opn expression within a cluster, with width indicating more cells. (G) qRT-PCR analysis of Opn expression levels in MTCs extracted from the indicated organs in IgG-treated versus NK dep. tumor-bearing mice. Error bars denote SEM. P-values calculated by one-sample Wilcoxon test. All results are representative of at least two independent experiments.

Article Snippet: CD8⁺ T cell depletion was achieved by administering 250 μg of InVivoMab anti-mouse CD8α antibody (clone 53-6.7, BioXCell, BE0004-1) or IgG2a control (BioXCell, BE0089) by i.p. injection 2 days and 1 day before tumor delivery, followed by weekly injections, as previously described ( ).

Techniques: Injection, Control, Isolation, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Mechanoimmunological Control of Metastatic Site Selection

doi: 10.1101/2025.05.10.653256

Figure Lengend Snippet: (A-C) Luc + NT or Opn-KO1 B16F10 cells were injected i.v. into wild type and Prf1 -/- mice, which were then monitored for metastatic colonization of the lungs and bone. (A) Schematic diagram of the experimental approach. (B) Representative IVIS images of tumor-bearing wild type and Prf1 -/- mice injected with the indicated B16F10 lines. (C) Quantification of relative femoral colonization, expressed as a ratio of IVIS signal in the legs to the total IVIS signal. (D-F) Luc + NT or Opn-KO1 B16F10 cells were injected i.v. into IgG control and NK depleted (NK dep. ) recipient mice, which were then monitored for metastatic colonization of the lungs and bone. (D) Schematic diagram of the experimental approach. (E) Representative IVIS images of tumor-bearing IgG control and NK dep. mice injected with the indicated B16F10 lines. (F) Quantification of relative femoral colonization, expressed as a ratio of IVIS signal in the legs to the total IVIS signal. In B and E, metastatic burden in the lungs and femurs is denoted by black and yellow arrowheads. In C and F, error bars denote SEM, sample size is indicated above each bar, and P-values were calculated by one-way ANOVA. (G-I) NT and Opn-KO2 B16F10 cells were subjected to comparative bulk RNA-seq. (G) Heat map showing downregulation of selected ECM, adhesion, and cytoskeleton-related genes in Opn KO2 cells. (H) Gene Set Enrichment Analysis (GSEA) showing downregulation of genes related to focal adhesions (left) and ECM (right). NES = normalized enrichment score. (I-J) GFP + NT or Opn-KO B16F10 cells were injected i.v. into wild type and Prf1 -/- mice, and after 2 weeks, MTCs from the resulting lung metastases were extracted and subjected to SMR. (I) Schematic diagram of the experimental approach. (J) SMR of the indicated MTCs extracted from the indicated tumor-bearing mice. Violins encompass the entire data distribution, with dashed lines denoting the median and dotted lines indicating the upper and lower quartiles. Samples sizes (n=) are displayed below each violin. P-values calculated by one-way ANOVA. All results are representative of at least two independent experiments.

Article Snippet: CD8⁺ T cell depletion was achieved by administering 250 μg of InVivoMab anti-mouse CD8α antibody (clone 53-6.7, BioXCell, BE0004-1) or IgG2a control (BioXCell, BE0089) by i.p. injection 2 days and 1 day before tumor delivery, followed by weekly injections, as previously described ( ).

Techniques: Injection, Control, RNA Sequencing

Journal: bioRxiv

Article Title: Mechanoimmunological Control of Metastatic Site Selection

doi: 10.1101/2025.05.10.653256

Figure Lengend Snippet: (A-C) Luc + NT or Opn-KO2 B16F10 cells were injected i.v. into wild type and Prf1 -/- mice, which were then monitored for metastatic colonization of the lungs and bone. (A) Representative IVIS images of tumor-bearing wild type and Prf1 -/- mice injected with the indicated B16F10 lines, with metastatic burden in the lungs and femurs indicated by black and yellow arrowheads. (B) Quantification of relative femoral colonization, expressed as a ratio of IVIS signal in the legs to the total IVIS signal. (C) Representative H&E sections highlighting the absence of femoral colonization in Prf1 -/- mice injected with Opn-KO2 B16F10 cells. Lung metastases and B16-NT metastases in Prf1 -/- bone are indicated by black and yellow arrowheads. Scale bars = 200 μm. (D-F) Luc + NT or Opn-KO2 B16F10 cells were injected i.v. into IgG control and NK dep. recipient mice, which were then monitored for metastatic colonization of the lungs and bone. (D) Representative IVIS images of tumor-bearing IgG control and NK dep mice injected with the indicated B16F10 lines, with metastatic burden in the lungs and femurs indicated by black and yellow arrowheads. (E) Quantification of relative femoral colonization, expressed as a ratio of IVIS signal in the legs to the total IVIS signal. (F) Representative H&E sections highlighting the absence of femoral colonization in NK dep. mice injected with Opn-KO2 B16F10 cells. Lung metastases and B16-NT metastases in NK dep. bone are indicated by black and yellow arrowheads. Scale bars = 200 μm. In B and E, error bars denote SEM, sample size is indicated above each bar, and P-values were calculated by one-way ANOVA.

Article Snippet: CD8⁺ T cell depletion was achieved by administering 250 μg of InVivoMab anti-mouse CD8α antibody (clone 53-6.7, BioXCell, BE0004-1) or IgG2a control (BioXCell, BE0089) by i.p. injection 2 days and 1 day before tumor delivery, followed by weekly injections, as previously described ( ).

Techniques: Injection, Control