Journal: OncoImmunology

Article Title: Hyperdifferentiated murine melanoma cells promote adaptive anti-tumor immunity but activate the immune checkpoint system

doi: 10.1080/2162402x.2024.2437211

Figure Lengend Snippet: Figure 1. Characterization of melanoma cells surviving exposure to low dose staurosporine. (a) The percentage of viable cells 24 hours after exposure to staurosporine was measured by MTT assay. Data are expressed as the mean ± SD (n = 4). (b) The sensitivity of cell populations derived from 10 subclones isolated from parental B16F10 cells to 0.1 µg/ml staurosporine. (c) Morphological changes and senescence-associated β-galactosidase accumulation of surviving cells. Scale bars, 50 µm. (d) DNA polyploidy in surviving cells. (e) The cell cycle of surviving cells was evaluated using a FUCCI probe. Percentages indicate the fraction of G1 or S/G2/M cells for each day. (f) Representative images of mitosis slippage between days 2 and 3. Arrows indicate the timelapse images of the same cell. (g) Enlarged multinucleated cells

Article Snippet: To generate PD-L1 knockout B16F10 (B16F10-PD-L1 KO) cells, Pdcd-1L1 CRISPR/Cas9 Knockout Plasmid (Santa Cruz Biotechnology, Dallas, Tx, USA) was used.

Techniques: MTT Assay, Derivative Assay, Isolation

Journal: OncoImmunology

Article Title: Hyperdifferentiated murine melanoma cells promote adaptive anti-tumor immunity but activate the immune checkpoint system

doi: 10.1080/2162402x.2024.2437211

Figure Lengend Snippet: Figure 3. Hyperdifferentiated melanoma cells elicit antitumor immune responses. (a) Establishment of B16F10-OVA. Histograms show the cell surface expression of ova- derived epitope peptide-mhc complexes. (b) and (c) comparison of the expressions of cell surface ova-peptide-mhc complexes in control and staurosporine-exposed surviving B16F10-OVA cells. (d) and (e) an ELISpot assay was used to evaluate tumor-derived antigen-specific immune responses. Representative ELISpot images (d) and data quantification (e) are shown. Data are shown as the mean ± SD in the right panels (n = 9 per group). Statistical significance was determined by one-way ANOVA (****p < 0.0001, Dunnett’s test). (f) Establishment of a PD-L1 knockout B16F10 cell line. Histograms show the expression of PD-L1 on the cell surface of B16F10 cells 48 hours after culture in the presence of 100 U ifn-γ. (g) Measurement of the immunological elimination of cB16F10 or sB16F10 cells by CTLs derived from pmel-1 mice over time. The cell counts are shown relative to the initial time of measurement. (h) Altered susceptibility of sB16F10 cells to T cell mediated cytotoxicity in the presence or absence of PD-L1. Data are shown as the mean ± SD. Statistical significance was determined by Student’s t-test (***p < 0.001). (i) C57BL/6J mice were subcutaneously inoculated with cB16F10-ova or pB16F10-ova cells (1 × 105 cells/head) followed by the intraperitoneal administration of anti-PD-1 mAbs or isotype mAb as a control. The effect of immune checkpoint inhibition on the tumor growth (j) and survival time (k) of tumor-bearing mice. In (k), the data were analyzed by log-rank test (***p < 0.001). The images in (c) are from TogoTV (https://togotv.Dbcls.jp © 2016 DBCLS TogoTV, CC-BY-4.0 https://creativecommons.Org/licenses/by/4.0/deed.Ja).

Article Snippet: To generate PD-L1 knockout B16F10 (B16F10-PD-L1 KO) cells, Pdcd-1L1 CRISPR/Cas9 Knockout Plasmid (Santa Cruz Biotechnology, Dallas, Tx, USA) was used.

Techniques: Expressing, Derivative Assay, Comparison, Control, Enzyme-linked Immunospot, Knock-Out, Inhibition

Journal: Cancer research

Article Title: Tumor-localized secretion of soluble PD1 enhances oncolytic virotherapy

doi: 10.1158/0008-5472.CAN-16-1638

Figure Lengend Snippet: (A) B16/F10 cells were either mock infected or infected with MYXV at MOI=10. 24 hours post infection cells were harvested and the total level of PDL1 was analyzed using westernblot (band representing PDL1 is indicated by arrow). Expression of β Actin is shown as a loading control. Data shown is representative is three independent experiments. (B) B16/F10 cells were either mock infected or infected with MYXV at MOI=10. 24 hours post infection cells were harvested and the cell surface expression of PDL1 was analyzed using flowcytometry. Data shown is representative of two independent experiments. (C) C57/B6 mice were injected SQ with 4x105 B16/F10 cells. On days 7, 9, and 11 post injection mice were given IT injection of either saline (n=9) or 1x107 FFU of MYXV (n=9). On day 12, mice were euthanized and the percentage of either CD4+ or CD8+ splenocytes expressing PD1 was determined using flowcytometry. Data represents the summation of four independent experiments. Significance was determined using unpaired students T-Test. (D) C57/B6 mice were injected SQ with either 4x105 B16/F10-wt or B16/F10-PDL1−/− cells. On days 7, 9, and 11 post injection mice were given IT injection of either saline (B16/F10 n=5, B16/F10-PDL1−/− n=5) or 1x107 FFU of MYXV (B16/F10 n=5, B16/F10-PDL1−/− n=14). Animals were then monitored for tumor burden and euthanized when tumors reached 15mm in any direction. Data is representative of two independent experiments. (E) C57/B6 mice were injected SQ with 4x105 B16/F10 cells. On day 7 post injection, mice were randomly separated into one of four cohorts: saline + isotype (n=9), saline + αPD1 antibody (n=8), MYXV + isotype (n=5), MYXV + αPD1 antibody (n=7). Viral injections consisted of 1x107 FFU of MYXV and were given on days 7, 9, and 11. Antibody injections consisted of 100μg/injection and were given on days 7, 11, 14, and 18. Animals were then monitored for tumor burden and euthanized when tumors reached 15mm in any direction. Data is representative >four independent experiments. (F) Example of alopecia in mice on day 70 post-treatment and H&E stain from the belly skin of displayed animal. (G) Average alopecia score from MYXV + αPD1 antibody treated animals (n=7, shown in 1E) and B16/F10-PDL1−/− bearing animals treated with MYXV (n=14, shown in 1D). * denotes significance of p<0.05 for the indicated day and was determined using unpaired students T-Test.

Article Snippet: PDL1 deficient B16/F10 cells (B16/F10-PDL1 −/− ) were generated using the CRISPR/CAS9 system (Genscript, Piscataway, NJ, USA) per the manufacturer’s recommendations.

Techniques: Infection, Expressing, Control, Injection, Saline, Staining