Journal: Advanced Science

Article Title: PRMT5 Inhibitor Synergizes with Chemotherapy to Induce Resembling Mismatch Repair Deficiency and Enhance Anti‐TIGIT Therapy in Microsatellite‐Stable Colorectal Cancer

doi: 10.1002/advs.202500271

Figure Lengend Snippet: PRMT5 as a DDR‐related epigenetic gene is upregulated in MSS CRC. A) Schematic illustration of the DDR‐related epigenetic gene screening from MSS CRC. The schematic illustration was created using Biorender. B) Uniform manifold approximation and projection (UMAP) plots of single cells identified by scRNA‐seq, color‐coded by major cell types. C) epiUMAP plot representing malignant and non‐malignant cells detected by inferCNV. D) Venn diagram illustrating the intersection of highly expressed epigenetic genes, DDR‐related gene sets in malignant cells, and a malignancy‐promoting gene dataset. E–G) Correlation between PRMT5 levels and the therapeutic effect of DNA‐damaging drugs: CPT‐11 (E), oxaliplatin (F), and 5‐fluorouracil (G), analyzed using ROC plotter ( https://rocplot.org/ ). H,I) Kaplan–Meier survival curves showing OS (H) and PFS (I) for CRC patients with low versus high PRMT5 expression, from the Kaplan–Meier plotter ( https://www.kmplot.com/analysis/ ). J) Western blot analysis of PRMT5 levels in MSS CRC tissues and NATs ( n = 9). K,L) Representative images (K) and quantitative proportions (L) from IHC staining showing PRMT5 expression in MSS CRC tissues paired with corresponding NATs ( n = 31). The p ‐value was calculated using a two‐tailed Student's t ‐test. Scale bar = 50 µm. Error bars show the mean ± SD. ns, p > 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: When the tumors reached ≈200 mm 3 , mice were treated with DMSO (1%, Solarbio, Beijing, China), PRMT5i (40 mg kg −1 , Selleck Chemicals, Houston, TX, USA), CPT‐11 (40 mg kg −1 , Medchemexpress, Shanghai, China), 5‐FU (40 mg kg −1 , Medchemexpress, Shanghai, China), Oxaliplatin (2.5 mg kg −1 , Medchemexpress, Shanghai, China), αPD‐1 (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), αTIGIT (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), or αCD8 (200 μg mouse −1 , Selleck Chemicals, Houston, TX, USA) by intraperitoneal injection every three days.

Techniques: Expressing, Western Blot, Immunohistochemistry, Two Tailed Test

Journal: Advanced Science

Article Title: PRMT5 Inhibitor Synergizes with Chemotherapy to Induce Resembling Mismatch Repair Deficiency and Enhance Anti‐TIGIT Therapy in Microsatellite‐Stable Colorectal Cancer

doi: 10.1002/advs.202500271

Figure Lengend Snippet: PRMT5 inhibition enhances CPT‐11 sensitivity in vitro. A) qRT‐PCR (top) and Western blot (bottom) analysis of PRMT5 levels in normal human colon mucosal epithelial cells and CRC cell lines. The p‐values were calculated using one‐way ANOVA. B,C) PRMT5 levels were assessed using Western blot in PRMT5 knockdown (B), PRMT5 overexpression (C), and paired control CRC cells. D, E) PRMT5 knockdown (D), PRMT5 overexpression (E), and paired control CRC cells were exposed to increasing concentrations of CPT‐11 for 48 h, and IC50 values were determined using the CCK‐8 assay. The p ‐values were calculated using one‐way ANOVA. F–I) Flow cytometry analysis of apoptosis in PRMT5 knockdown (F,G), PRMT5 overexpression (H,I), and paired control SW480 cells treated with DMSO or CPT‐11 (50 µ m ) for 48 h. The p ‐values were calculated using one‐way ANOVA. J–M) Western blot detection of cleaved caspase‐3 and γ‐H2AX levels in PRMT5 knockdown (J,K), PRMT5 overexpression (L,M), and paired control CRC cells treated with DMSO or CPT‐11 (50 µ m ). N–P) Representative fluorescence images showing cellular localization and expression of γ‐H2AX (N), dsDNA (O), and Picogreen (P) in PRMT5 knockdown and paired control CRC cells, with or without CPT‐11 (50 µ m ) treatment. Scale bar = 5 µm. Error bars show the mean ± SD. ns, p > 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: When the tumors reached ≈200 mm 3 , mice were treated with DMSO (1%, Solarbio, Beijing, China), PRMT5i (40 mg kg −1 , Selleck Chemicals, Houston, TX, USA), CPT‐11 (40 mg kg −1 , Medchemexpress, Shanghai, China), 5‐FU (40 mg kg −1 , Medchemexpress, Shanghai, China), Oxaliplatin (2.5 mg kg −1 , Medchemexpress, Shanghai, China), αPD‐1 (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), αTIGIT (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), or αCD8 (200 μg mouse −1 , Selleck Chemicals, Houston, TX, USA) by intraperitoneal injection every three days.

Techniques: Inhibition, In Vitro, Quantitative RT-PCR, Western Blot, Knockdown, Over Expression, Control, CCK-8 Assay, Flow Cytometry, Fluorescence, Expressing

Journal: Advanced Science

Article Title: PRMT5 Inhibitor Synergizes with Chemotherapy to Induce Resembling Mismatch Repair Deficiency and Enhance Anti‐TIGIT Therapy in Microsatellite‐Stable Colorectal Cancer

doi: 10.1002/advs.202500271

Figure Lengend Snippet: The combination of PRMT5 inhibitor and CPT‐11 impairs tumor growth in MSS mice. A) Schematic timeline for the treatment of mice carrying subcutaneous syngeneic tumors. B–E) Representative tumor images (B), tumor growth curves (C), body weight (D), and relative tumor weight (E) of mice treated with DMSO, PRMT5i (40 mg kg −1 ), CPT‐11 (40 mg kg −1 ), or PRMT5i + CPT‐11 ( n = 5). The p ‐values were calculated using two‐way ANOVA (C) and one‐way ANOVA (E). F) Schematic overview of the AOM/DSS model. G–K) Representative mini‐endoscopy images (G), tumor images (H), H&E staining (I), tumor numbers (J), and tumor load (K) in AOM/DSS‐induced tumor‐bearing mice treated with DMSO, PRMT5i (40 mg kg −1 ), CPT‐11 (40 mg kg −1 ), or PRMT5i + CPT‐11 (n = 5). The p ‐values were calculated using one‐way ANOVA. Scale bar (upper part) = 2 mm, Scale bar (lower part) = 50 µm. L) H&E, ki‐67, and TUNEL staining of tumor tissues across different groups. Scale bar = 50 µm. M) Representative fluorescence images of cleaved caspase‐3, γ‐H2AX, and ki‐67 in the indicated tumor tissues. Scale bar = 50 µm. Error bars show the mean ± SD. ns, p > 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: When the tumors reached ≈200 mm 3 , mice were treated with DMSO (1%, Solarbio, Beijing, China), PRMT5i (40 mg kg −1 , Selleck Chemicals, Houston, TX, USA), CPT‐11 (40 mg kg −1 , Medchemexpress, Shanghai, China), 5‐FU (40 mg kg −1 , Medchemexpress, Shanghai, China), Oxaliplatin (2.5 mg kg −1 , Medchemexpress, Shanghai, China), αPD‐1 (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), αTIGIT (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), or αCD8 (200 μg mouse −1 , Selleck Chemicals, Houston, TX, USA) by intraperitoneal injection every three days.

Techniques: Staining, TUNEL Assay, Fluorescence

Journal: Advanced Science

Article Title: PRMT5 Inhibitor Synergizes with Chemotherapy to Induce Resembling Mismatch Repair Deficiency and Enhance Anti‐TIGIT Therapy in Microsatellite‐Stable Colorectal Cancer

doi: 10.1002/advs.202500271

Figure Lengend Snippet: The dual‐drug treatment facilitates the PMS2‐related transformation of dMMR‐like CRC. A,B) Enrichment analysis of DNA damage repair (A) and mismatch repair (B) signaling pathways in SW620 cells from CPT‐11 versus PRMT5 inhition + CPT‐11 groups, conducted using GSEA ( n = 3). NES, normalized enrichment score; FDR, false discovery rate. C,D) qRT‐PCR analysis of the expression of 13 candidate DDR genes in control and PRMT5‐silenced SW480 (C) and SW620 (D) cells following CPT‐11 treatment. The p ‐values were calculated using a two‐tailed Student's t ‐test. E,F) Western blot analysis of PMS2 protein levels in PRMT5 knockdown (E), PRMT5 overexpression (F), and paired control SW480 and SW620 cells treated with CPT‐11. G,H) Luciferase reporter assay showing the effect of PRMT5 on the transcriptional activity of the PMS2 promoter in CPT‐11‐treated SW480 (G) and SW620 (H) cells. The p ‐values were calculated using a two‐tailed Student's t ‐test. I) Schematic illustration of putative PRMT5‐binding sites on the PMS2 promoter region and the design of corresponding primers. J,K) ChIP‐qPCR analysis showing the enrichment of PRMT5, H3R2me2s, or IgG at the PMS2 promoter. The p ‐values were calculated using two‐way ANOVA. L, M) IC50 values of SW480 (L) and SW620 (M) cells assessed using the CCK‐8 assay following 48 h treatment with increasing concentrations of CPT‐11 across different groups. The p ‐values were calculated using one‐way ANOVA. N) IHC staining showing PMS2 protein levels in mouse tumor tissues from the indicated groups. Scale bar = 50 µm. O) ESTIMATE immune score estimation in mice tumors treated with DMSO versus PRMT5i combined with CPT‐11, based on RNA‐seq data ( n = 3). The p ‐values were calculated using a two‐tailed Student's t ‐test. P) mMCPCounter analysis showing CD8 + T cell infiltration in the indicated treatment groups ( n = 3). The p ‐values were calculated using a two‐tailed Student's t ‐test. Q) DEPTH2 scores for mice samples from the indicated treatment groups ( n = 3). The p ‐values were calculated using a two‐tailed Student's t ‐test. R) Heatmap showing the relative expression of MMR‐related genes in the indicated treatment groups ( n = 3). S) MSI status as determined by PreMSIm in the indicated treatment groups ( n = 3). MSI‐low (MSI‐L) represents pMMR, and MSI‐high (MSI‐H) represents dMMR. Error bars show the mean ± SD. ns, p > 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: When the tumors reached ≈200 mm 3 , mice were treated with DMSO (1%, Solarbio, Beijing, China), PRMT5i (40 mg kg −1 , Selleck Chemicals, Houston, TX, USA), CPT‐11 (40 mg kg −1 , Medchemexpress, Shanghai, China), 5‐FU (40 mg kg −1 , Medchemexpress, Shanghai, China), Oxaliplatin (2.5 mg kg −1 , Medchemexpress, Shanghai, China), αPD‐1 (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), αTIGIT (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), or αCD8 (200 μg mouse −1 , Selleck Chemicals, Houston, TX, USA) by intraperitoneal injection every three days.

Techniques: Transformation Assay, Protein-Protein interactions, Quantitative RT-PCR, Expressing, Control, Two Tailed Test, Western Blot, Knockdown, Over Expression, Luciferase, Reporter Assay, Activity Assay, Binding Assay, ChIP-qPCR, CCK-8 Assay, Immunohistochemistry, RNA Sequencing

Journal: Advanced Science

Article Title: PRMT5 Inhibitor Synergizes with Chemotherapy to Induce Resembling Mismatch Repair Deficiency and Enhance Anti‐TIGIT Therapy in Microsatellite‐Stable Colorectal Cancer

doi: 10.1002/advs.202500271

Figure Lengend Snippet: Combinational of PRMT5 silencing and CPT‐11 activates cGAS/STING pathway via restrain PMS2 expression. A,B) Western blot analysis showing the levels of p‐TBK1, TBK1, p‐IRF3, IRF3, p‐STING, and STING in SW480 (A) and CT26 (B) cells treated with control, PRMT5 silencing, CPT‐11, and PRMT5 silencing combined with CPT‐11. C–F) qRT‐PCR analysis of IFN‐β (C), ISG15 (D), CCL5 (E), and CXCL10 (F) levels in CT26 cells across all groups. The p ‐values were calculated using one‐way ANOVA. G) Representative flow cytometry images showing CD80 + and CD86 + expression on CD11c + DCs in the indicated groups ( n = 3). H,I) Expression levels of CD80 and CD86 (H), and I‐A/I‐E (I) on CD11c + DCs across all groups ( n = 3). The p‐ values were calculated using one‐way ANOVA. J,K) Expression levels of IFNγ (J) and GZMB (K) on CD8 + T cells in the indicated groups ( n = 3). The p ‐values were calculated using one‐way ANOVA. L,M) Western blot analysis showing the effect of PRMT5 silencing combined with CPT‐11 on PMS2 overexpression‐induced cGAS‐STING pathway inactivation in SW480 (L) and CT26 (M) cells. Error bars show the mean ± SD. ns, p > 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: When the tumors reached ≈200 mm 3 , mice were treated with DMSO (1%, Solarbio, Beijing, China), PRMT5i (40 mg kg −1 , Selleck Chemicals, Houston, TX, USA), CPT‐11 (40 mg kg −1 , Medchemexpress, Shanghai, China), 5‐FU (40 mg kg −1 , Medchemexpress, Shanghai, China), Oxaliplatin (2.5 mg kg −1 , Medchemexpress, Shanghai, China), αPD‐1 (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), αTIGIT (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), or αCD8 (200 μg mouse −1 , Selleck Chemicals, Houston, TX, USA) by intraperitoneal injection every three days.

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Flow Cytometry, Over Expression

Journal: Advanced Science

Article Title: PRMT5 Inhibitor Synergizes with Chemotherapy to Induce Resembling Mismatch Repair Deficiency and Enhance Anti‐TIGIT Therapy in Microsatellite‐Stable Colorectal Cancer

doi: 10.1002/advs.202500271

Figure Lengend Snippet: The dual‐drug combination therapy elevates the level of TIGIT in MSS CRC. A) Workflow illustrating the isolation of CD45 + live cells from a single‐cell suspension for further analysis. B–D) Representative flow cytometry images of CD80 + and CD86 + expression (B), and expression levels of CD80 and CD86 (C), and I‐A/I‐E (D) on CD11c + DCs isolated from tumor‐bearing mice treated with DMSO, PRMT5i, CPT‐11, or PRMT5i + CPT‐11 ( n = 5). The p ‐values were calculated using one‐way ANOVA. E–G) Representative flow cytometry images (F) and quantitative analysis (E,G) of CD4 + and CD8 + on CD3 + T cells in the indicated mice ( n = 5). The p ‐values were calculated using one‐way ANOVA. H–K) Representative flow cytometry images (J,K) and quantitative analysis (H,I) of IFNγ + and GZMB + on CD8 + CD3 + T cells in the indicated tumor tissues ( n = 5). The p‐ values were calculated using one‐way ANOVA. L–O) Representative fluorescence images (L) and quantitative analysis (M–O) of CD11c, CD8, and CD4 in the indicated mouse tissues. Fluorescence intensity normalized to control. The p ‐values were calculated using one‐way ANOVA. Scale bar = 50 µm. P) levels of TIGIT on CD8 + T cells isolated from the spleens of mice in the indicated groups ( n = 3). The p ‐values were calculated using one‐way ANOVA. Q,R) Representative flow cytometry images (R) and quantitative analysis (Q) of TIGIT on CD8 + T cells isolated from the tumor tissues of the indicated mice ( n = 5). The p ‐values were calculated using one‐way ANOVA. S,T) Representative fluorescence images (T) and quantitative analysis (S) of TIGIT levels on CD8 + cells in the indicated mouse tissues. Fluorescence intensity normalized to control. The p ‐values were calculated using one‐way ANOVA. Scale bar = 50 µm. Error bars show the mean ± SD. ns, p > 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: When the tumors reached ≈200 mm 3 , mice were treated with DMSO (1%, Solarbio, Beijing, China), PRMT5i (40 mg kg −1 , Selleck Chemicals, Houston, TX, USA), CPT‐11 (40 mg kg −1 , Medchemexpress, Shanghai, China), 5‐FU (40 mg kg −1 , Medchemexpress, Shanghai, China), Oxaliplatin (2.5 mg kg −1 , Medchemexpress, Shanghai, China), αPD‐1 (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), αTIGIT (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), or αCD8 (200 μg mouse −1 , Selleck Chemicals, Houston, TX, USA) by intraperitoneal injection every three days.

Techniques: Isolation, Suspension, Flow Cytometry, Expressing, Fluorescence, Control

Journal: Advanced Science

Article Title: PRMT5 Inhibitor Synergizes with Chemotherapy to Induce Resembling Mismatch Repair Deficiency and Enhance Anti‐TIGIT Therapy in Microsatellite‐Stable Colorectal Cancer

doi: 10.1002/advs.202500271

Figure Lengend Snippet: The dual‐drug combination therapy induced a dMMR‐like response to anti‐TIGIT therapy. A) Timeline schematic showing the treatment of subcutaneous syngeneic tumor mice and AOM/DSS‐induced tumor‐bearing mice. B–E) Representative tumor images (B), tumor growth curves (C), body weight (D), and relative tumor weight (E) of subcutaneous syngeneic tumor mice treated with DMSO, PRMT5i (40 mg kg −1 ) + CPT‐11 (40 mg kg −1 ), αTIGIT (100 µg per mouse), or PRMT5i + CPT‐11 + αTIGIT ( n = 6). The p ‐values were calculated using two‐way ANOVA (C) and one‐way ANOVA (E). F–J) Representative mini‐endoscopy images (F), tumor images (G), H&E staining (H), tumor numbers (I), and tumor load (J) of AOM/DSS‐induced tumor‐bearing mice treated with the indicated therapies ( n = 6). Scale bar (left part) = 2 mm, Scale bar (right part) = 50 µm. The p ‐values were calculated using one‐way ANOVA. K–M) Representative images (K) and quantitative analysis (L,M) of ki‐67 and TUNEL staining in tumor tissues from the indicated groups. The p ‐values were calculated using one‐way ANOVA. Scale bar = 50 µm. N–Q) Levels of ALT (N), AST (O), CRE (P), and BUN (Q) in blood from AOM/DSS‐induced mice in the indicated groups. The p‐ values were calculated using one‐way ANOVA. R) Representative images of organ indexes, including heart, liver, spleen, lungs, and kidneys, in AOM/DSS‐induced mice. Scale bar = 50 µm. S,T) Representative fluorescence images (S) and quantitative analysis (T) of CD8 + cells in the indicated mouse tissues. The p ‐values were calculated using one‐way ANOVA. Scale bar = 50 µm. Error bars show the mean ± SD. ns, p > 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: When the tumors reached ≈200 mm 3 , mice were treated with DMSO (1%, Solarbio, Beijing, China), PRMT5i (40 mg kg −1 , Selleck Chemicals, Houston, TX, USA), CPT‐11 (40 mg kg −1 , Medchemexpress, Shanghai, China), 5‐FU (40 mg kg −1 , Medchemexpress, Shanghai, China), Oxaliplatin (2.5 mg kg −1 , Medchemexpress, Shanghai, China), αPD‐1 (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), αTIGIT (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), or αCD8 (200 μg mouse −1 , Selleck Chemicals, Houston, TX, USA) by intraperitoneal injection every three days.

Techniques: Staining, TUNEL Assay, Fluorescence

Journal: Advanced Science

Article Title: PRMT5 Inhibitor Synergizes with Chemotherapy to Induce Resembling Mismatch Repair Deficiency and Enhance Anti‐TIGIT Therapy in Microsatellite‐Stable Colorectal Cancer

doi: 10.1002/advs.202500271

Figure Lengend Snippet: Schematic illustrating the potential mechanism by which the combination of PRMT5i and CPT‐11 elicited a dMMR‐like state and enhanced αTIGIT therapy in MSS CRC. The schematic illustration was created using Biorender.

Article Snippet: When the tumors reached ≈200 mm 3 , mice were treated with DMSO (1%, Solarbio, Beijing, China), PRMT5i (40 mg kg −1 , Selleck Chemicals, Houston, TX, USA), CPT‐11 (40 mg kg −1 , Medchemexpress, Shanghai, China), 5‐FU (40 mg kg −1 , Medchemexpress, Shanghai, China), Oxaliplatin (2.5 mg kg −1 , Medchemexpress, Shanghai, China), αPD‐1 (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), αTIGIT (100 μg mouse −1 , Selleck Chemicals, Houston, TX, USA), or αCD8 (200 μg mouse −1 , Selleck Chemicals, Houston, TX, USA) by intraperitoneal injection every three days.

Techniques: