Journal: Advanced Science

Article Title: Therapy‐Induced ECM Remodeling Creates a Transient Immune Barrier in Residual Melanoma

doi: 10.1002/advs.202508451

Figure Lengend Snippet: Targeting ECM deposition enhances anti‐tumor immunity. A) Flow cytometry analysis of tumor‐infiltrating CD8+ T cells gated on live/CD45+ cells in BRAF/MEKi‐treated mice injected intraperitoneally with PBS, DHB, or BAPN. Left, representative flow cytometry plots. Numbers on the plots represent the percentage of cells within each gate. Right, quantification of tumor‐infiltrating CD8+ T cells in each group. n = 10, 13, and 12 tumors in the PBS, DHB, and BAPN groups, respectively. Data presented as mean with SEM. Statistics calculated using one‐way ANOVA with Dunnett's multiple comparisons test. B) Representative immunofluorescence images of CD45 (green), CD8a (red), and nuclear DAPI (blue) in the indicated BRAF/MEKi‐treated tumor groups. Scale bar, 100 µm. C) Scheme illustrating CD8+ T cell depletion in YUMM1.7‐bearing mice treated with BRAF/MEKi + DHB. BRAF/MEKi were administered by daily oral gavage when tumors reached ≈700mm 3 in size. DHB was injected intraperitoneally daily from day 10. CD8+ T cells were depleted by IP injection of anti‐CD8b antibodies. An IgG1 isotype control antibody was used in control animals. Tumors were collected when reaching the initial volume prior to BRAF/MEKi treatment. D) Flow cytometry analysis of circulating CD8+ T cells gated on live/CD45+ cells in BRAF/MEKi + DHB‐treated mice injected intraperitoneally with anti‐CD8b antibodies or isotype control. Left, representative flow cytometry plots. Numbers on the plots represent the percentage of cells within each gate. Right, quantification of circulating CD8+ T cells. n = 3 mice in both groups. Data presented as mean with SEM. Statistics were calculated using a two‐tailed unpaired t ‐test. E) Flow cytometry analysis of tumor‐infiltrating CD8+ T cells gated on live/CD45+ cells in BRAF/MEKi + DHB‐treated mice injected intraperitoneally with anti‐CD8b antibodies or isotype control. Left, representative flow cytometry plots. Numbers on the plots represent the percentage of cells within each gate. Right, quantification of tumor‐infiltrating CD8+ T cells. n = 5 and 6 tumors in anti‐CD8b and isotype control respectively. Data presented as mean with SEM. Statistics were calculated using a two‐tailed unpaired t ‐test. F) Kaplan–Meier curve for BRAF/MEKi + DHB‐treated mice bearing YUMM1.7 receiving anti‐CD8b antibodies or isotype control. The “percentage without full resistance” refers to the proportion of tumors in each cohort that have not yet reached full resistance. Statistics were calculated using the Log‐rank (Mantel‐Cox) test. G) Days for BRAF/MEKi + DHB‐treated mice with anti‐CD8b antibodies or isotype control injection to develop full resistance (tumors reached the initial size before BRAF/MEKi treatment, i.e., ≈700mm 3 in size). n = 5 tumors in both groups. Data presented as mean with SEM. Statistics were calculated using a two‐tailed unpaired t ‐test. Figure was created with BioRender.com.

Article Snippet: Control mice were injected with an equivalent amount of IgG1 isotype control (BioXcell, catalog no. BE0088).

Techniques: Flow Cytometry, Injection, Immunofluorescence, Control, Two Tailed Test