Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Munc13-4 deficiency in tumor cells inhibits tumor growth in an immunity-dependent way (A) Representative immunohistochemical images showing Munc13-4 expression in breast cancer, thyroid cancer, cholangiocarcinoma, gastrointestinal stromal tumors, pancreatic cancer, and hepatocellular carcinoma tissues, along with their corresponding adjacent normal tissues, assessed using a multi-organ carcinoma tissue array. Scale bar, 500 μm. (B–D) Tumor growth in BALB/c mice inoculated with wild-type (WT), control or Munc13-4 knockout (KO) 4T1 cells (n = 9). (B) Schematic of experimental design. (C) Tumor growth curves following mammary gland inoculation. (D) Percentage change in tumor volume, normalized to WT group. (E–G) Tumor growth in BALB/Nude mice inoculated with WT, control or Munc13-4 KO 4T1 cells (n = 8). (E) Schematic of experimental design. (F) Tumor growth curves following mammary gland inoculation. (G) Percentage change in tumor volume, normalized to WT group. (H–J) Tumor growth in NOD/SCID mice inoculated with WT, control or Munc13-4 KO 4T1 cells (n = 6). (H) Schematic of experimental design. (I) Tumor growth curves following mammary gland inoculation. (J) Percentage change in tumor volume, normalized to WT group. Data are presented as means ± SEM, p -values were calculated by one-way ANOVA with multiple comparisons (C, F and I), ns, not significant. See also Figure S1 and S2.

Article Snippet: The lysates were then centrifuged at 4°C for 10 minutes at 12,000 × g. The supernatant was incubated overnight at 4°C with Munc13-4 Antibody (C-12) (Santa Cruz, sc-271301, 1:100) or HGS Polyclonal antibody (Proteintech, 10390-1-AP, 1:500), in conjunction with Protein A/G magnetic beads (Biolinkedin, L- 1004), on a rotator.

Techniques: Immunohistochemical staining, Expressing, Control, Knock-Out

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Munc13-4 deficiency in tumor cells enhances T cell infiltration and activation (A–C) Flow cytometric quantification of the percentage of CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells among total cells in the tumors (A) , spleens (B) , and draining lymph nodes (C) of BALB/c mice (n = 5), 21 days after mammary gland injection with 3 × 10 5 control or Munc13-4 KO 4T1 cells per mouse. (D–F) Quantification of the percentage of granzyme B + (GzmB + ) (D) , Ki67 + (E) and IFNγ + (F) cells among CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells within tumors from orthotopic mouse models of breast cancer generated by control or Munc13-4 KO 4T1 cells (n = 5). (G–I) Quantification of the percentage of granzyme B + (G) , Ki67 + (H) and IFNγ + (I) cells among CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells within spleens from orthotopic mouse models of breast cancer generated by control or Munc13-4 KO 4T1 cells (n = 5). (J–L) Quantification of the percentage of granzyme B + (J) , Ki67 + (K) and IFNγ + (L) cells among CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells within the draining lymph nodes from orthotopic mouse models of breast cancer generated by control or Munc13-4 KO 4T1 cells (n = 5). Box plots show all data points, all p -values were calculated by Multiple t tests. See also Figure S3.

Article Snippet: The lysates were then centrifuged at 4°C for 10 minutes at 12,000 × g. The supernatant was incubated overnight at 4°C with Munc13-4 Antibody (C-12) (Santa Cruz, sc-271301, 1:100) or HGS Polyclonal antibody (Proteintech, 10390-1-AP, 1:500), in conjunction with Protein A/G magnetic beads (Biolinkedin, L- 1004), on a rotator.

Techniques: Activation Assay, Injection, Control, Generated

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Facilitating PD-L1 secretion by Munc13-4 suppresses anti-Tumor efficacy of T cells (A) Western blot analysis of total PD-L1 level in control and Munc13-4 KO SUM159 or 4T1 cells (n = 3). (B) Representative TEM images of EVs secreted by control and Munc13-4 KO SUM159 or 4T1 cells. Scale bar, 50 nm. (C) Quantification of exosomes secreted by equal numbers of control and Munc13-4 KO SUM159 (left) or 4T1 (right) cells through NTA (n = 3). (D–F) Analysis of EVs by optiprep TM density gradient centrifugation. (D) Schematic of experimental design. Western blot analysis of PD-L1, Alix, CD63 and CD81 in EVs secreted by equal numbers of control and Munc13-4 KO SUM159 (E) or 4T1 (F) cell, collected from factions 1–6 (F1–6) (n = 3). (G and H) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and Munc13-4 KO SUM159 cells (G) and corresponding quantification of blot band intensities (H) (n = 3). (I and J) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and Munc13-4 KO 4T1 cells (I) and corresponding quantification of blot band intensities (J) (n = 3). (K and L) Assessment of cytotoxicity elicited by activated mouse spleen lymphocytes against control and Munc13-4 KO 4T1 cells. (K) Schematic of experimental design. (L) Quantification of killing efficiency against control and Munc13-4 KO 4T1 cells (n = 3). (M and N) Evaluation of the relationship between decreased oncogenicity and impaired PD-L1 secretion in Munc13-4-deficient 4T1 cells. (M) Schematic of experimental design. (N) Tumor growth curves following mammary gland inoculation of control or Munc13-4 KO 4T1 cells, with subsequent injection of PBS or the indicated exosomes (n = 6). Data are presented as means ± SEM (C, H, J and N), p -values were calculated by unpaired t test (C), two-way ANOVA (H and J), paired t test (L) and one-way ANOVA with multiple comparisons (N). See also Figure S4 and S5.

Article Snippet: The lysates were then centrifuged at 4°C for 10 minutes at 12,000 × g. The supernatant was incubated overnight at 4°C with Munc13-4 Antibody (C-12) (Santa Cruz, sc-271301, 1:100) or HGS Polyclonal antibody (Proteintech, 10390-1-AP, 1:500), in conjunction with Protein A/G magnetic beads (Biolinkedin, L- 1004), on a rotator.

Techniques: Western Blot, Control, Gradient Centrifugation, Injection

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Munc13-4 facilitates MVB docking and fusion with the plasma membrane (A) Cryo-EM structure of the Munc13-4–Rab27a complex (upper panel) and detailed interface view (lower panel). Munc13-4 is represented in orange and Rab27a is shown in light green with the GppNHp nucleotide (red) bound. Residues on Rab27a (F46, W73, F88) and Munc13-4 (V660, K661, N739, T740), which are implicated in stabilizing the complex, are highlighted in darker colors. (B and C) GST pull-down assays examining the effects of VKAA and NTGG mutations in Munc13- 4 (B) , and F46S, W73S, F88S mutations in Rab27a (C) , on the formation of the Munc13-4–Rab27a complex (n = 3). (D and E) TIRF microscopy analysis of the effects of mutations in Munc13-4 and Rab27a on MVB mobility. Quantification of the mean diffusion coefficient ( D ), an index of MVB mobility, in SUM159 cells with the indicated mutations in Munc13-4 (D) or Rab27a (E) (n ≥ 962 for each group from triplicate experiments). (F) Quantification of exosomes secreted by equal numbers of indicated SUM159 cells though NTA (n = 3). (G and H) FRET-based detection of the role of Munc13-4 in SNARE complex assembly. (G) Illustration of FRET assay used to detect SNARE complex assembly. VAMP-7 SNARE motif (V7 SNARE) labeled with donor dye BODIPY FL, SNAP-23 (SN-23) labeled with acceptor dye 5- TAMRA, and syntaxin-4 (with its transmembrane domain deleted, termed as Syx4 ΔTM) together form a SNARE complex, leading to FRET between V7 SNARE and SN-23. (H) Representative graph of time-dependent SNARE complex assembly measured by the development of FRET between the 5-TAMRA labeled SN-23 and the BODIPY FL labeled V7 SNARE (n = 3). (I–K) FRET-based detection of the role of Munc13-4 in liposome fusion. (I) Illustration of the liposome fusion experiment. Syntaxin-4 (Syx-4) was incorporated into DiD-labeled liposomes and VAMP-7 was incorporated into DiI-labeled liposomes. Munc13-4 accelerates liposome fusion mediated by SNARE complex, leading to FRET between two liposome populations. (J) Time- dependent liposome fusion measured from the development of FRET between the DiD-labeled liposomes and the DiI-labeled liposomes. (K) Quantification of the FRET efficiency at the end of the detection (n = 3). Box plots show 10–90% percentile range of all data, with outliers represented as individual dots (D and E), data are represented as means ± SEM (F, J and K), p -values were calculated by Kruskal- Wallis test (D and E) and one-way ANOVA with multiple comparisons (F and K). See also Figure S6, S7 and S8.

Article Snippet: The lysates were then centrifuged at 4°C for 10 minutes at 12,000 × g. The supernatant was incubated overnight at 4°C with Munc13-4 Antibody (C-12) (Santa Cruz, sc-271301, 1:100) or HGS Polyclonal antibody (Proteintech, 10390-1-AP, 1:500), in conjunction with Protein A/G magnetic beads (Biolinkedin, L- 1004), on a rotator.

Techniques: Membrane, Cryo-EM Sample Prep, Microscopy, Diffusion-based Assay, Labeling, Liposomes

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Exosomal sorting of PD-L1 by Munc13-4 and HRS (A) Representative confocal microscopy images of control and Munc13-4 KO SUM159 cells co- expressing GFP-tagged PD-L1 with Orange-tagged CD63 or Orange-tagged LAMP1. Scale bar, 20 μm. (B) Quantification of Pearson’s correlation coefficient between PD-L1 and CD63, as well as between PD-L1 and LAMP1, in control and Munc13-4 KO SUM159 cells (n = 30 from triplicate experiments). (C) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and HRS KO SUM159 cells (n = 2). (D) Western blot analysis of total HRS amount in control and Munc13-4 KO SUM159 cells (n = 3). (E) Co-IP and immunoblotting (IB) analysis in control and Munc13-4 KO SUM159 cells transfected with indicated constructs to investigate the effect of Munc13-4 knockout on HRS–PD-L1 interaction (n = 3). (F and G) PLA to assess the effect of Munc13-4 knockout on the endogenous interaction between HRS and PD-L1. (F) Representative confocal microscopy images of control and Munc13-4 KO SUM159 cells in the PLA. Scale bar, 10 μm. (G) Quantification of puncta (n = 207 for control group and 175 for Munc13-4 KO group, from triplicate experiments). (H) Co-IP and IB analysis in Munc13-4 KO SUM159 cells transfected with indicated constructs to demonstrate that expression of Munc13-4 restored HRS–PD-L1 interaction (n = 3). (I) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs to test the interaction of Munc13-4 with HRS and STAM (n = 3). (J) Representative confocal microscopy image of SUM159 cells subjected to PLA, detecting the endogenous interaction between Munc13-4 and HRS (n = 3). Scale bar, 10 μm. (K) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs to explore the interaction between Munc13-4 and PD-L1 (n = 3). (L) Representative confocal microscopy image of SUM159 cells subjected to PLA, showing the endogenous interaction between Munc13-4 and PD-L1 (n = 3). Scale bar, 10 μm. (M and N) Direct binding between Munc13-4 and PD-L1 determined by in vitro liposome co- flotation assay. (M) Schematic of experimental design. (N) Western blot analysis of Munc13-4 and PD-L1 in top three fractions and bottom fraction of the mixture. (O) Co-IP and IB analysis in control and HRS KO SUM159 cells transfected with indicated constructs to examine the effect of HRS knockout on Munc13-4–PD-L1 interaction (n = 3). Box plots show all data points (B and G), p -values were calculated by two-way ANOVA (B) and Mann-Whitney U test (G).

Article Snippet: The lysates were then centrifuged at 4°C for 10 minutes at 12,000 × g. The supernatant was incubated overnight at 4°C with Munc13-4 Antibody (C-12) (Santa Cruz, sc-271301, 1:100) or HGS Polyclonal antibody (Proteintech, 10390-1-AP, 1:500), in conjunction with Protein A/G magnetic beads (Biolinkedin, L- 1004), on a rotator.

Techniques: Confocal Microscopy, Control, Expressing, Western Blot, Co-Immunoprecipitation Assay, Transfection, Construct, Knock-Out, Binding Assay, In Vitro, MANN-WHITNEY

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: IFN γ -induced modifications of Munc13-4 and HRS exert opposing effects on PD-L1 sorting (A and B) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and CBP KO SUM159 cells under IFNγ treatment (A) and corresponding quantification of blot band intensities (B) (n = 3). (C and D) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and HDAC3 KO SUM159 cells under IFNγ treatment (C) and corresponding quantification of blot band intensities (D) (n = 3). (E and F) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and NEDD4L knockdown (KD) SUM159 cells under IFNγ treatment (E) and corresponding quantification of blot band intensities (F) (n = 3). (G and H) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by SUM159 cells co-treated with IFNγ and PR-619 or DMSO (G) and corresponding quantification of blot band intensities (H) (n = 3). (I and J) PLA to assess the effects of Munc13-4 mutations on the interaction between endogenous HRS and PD-L1 in SUM159 cells. Representative confocal images of indicated SUM159 cells in the PLA (I) and quantification of PLA puncta (J) (n = 181 for control group, 252 for KO group, 174 for KO + WT group, 240 for KO + KKQQ group and 185 for KO + KKRR group from triplicate experiments). Scale bar, 10 μm. (K and L) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by indicated SUM159 cells (K) and corresponding quantification of blot band intensities (L) (n = 3). (M) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs to investigate the effect of HRS ubiquitylation on its interaction with PD-L1 (n = 3). (N) IP and IB analysis in control and Munc13-4 KO SUM159 cells, with or without IFNγ treatment, to explore the ubiquitylation of HRS (n = 3). Data are represented as means ± SEM (B, D, F, H and L), box plot shows 5–95% percentile range of all data, with outliers represented as individual dots (J), p -values were calculated by two-way ANOVA (B, D, F, H and L) and Kruskal-Wallis test (J). See also Figure S9–S11.

Article Snippet: The lysates were then centrifuged at 4°C for 10 minutes at 12,000 × g. The supernatant was incubated overnight at 4°C with Munc13-4 Antibody (C-12) (Santa Cruz, sc-271301, 1:100) or HGS Polyclonal antibody (Proteintech, 10390-1-AP, 1:500), in conjunction with Protein A/G magnetic beads (Biolinkedin, L- 1004), on a rotator.

Techniques: Western Blot, Control, Knockdown, Co-Immunoprecipitation Assay, Transfection, Construct

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: A peptide disrupting PD-L1 – Munc13-4 interaction inhibits tumor growth (A) Prediction of the interaction between PD-L1 (residues 253–290) and Munc13-4 (residues 1049–1080). Top insets show five ensembles generated by AlphaFold multimer, where N- and C-terminus of the proteins are indicated. PD-L1 and Munc13-4 are colored in blue and red, respectively. Residues that show potential contacts with each other (within 4 Å) are shown in sticks. Bottom inset displays the statistics of the residues in PD-L1 that show contacts with Munc13-4. The absolute and averaged contact numbers for each ensemble are illustrated by stacked histograms and line-scatter plot. The potential Munc13-4–interacting sequence of PD-L1 is shaded in magenta. (B) Diagram of the sequences for P-pep and S-pep. P-pep comprises a cell-penetrating peptide (CPP) fused to the human PD-L1 256–273 motif, whereas S-pep consists of a CPP linked to a scrambled sequence containing the same amino acid composition as the human PD-L1 256–273 motif. (C) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs and incubated with P-pep or S-pep to examine the effect of P-pep on Munc13-4–PD-L1 interaction (n = 3). (D) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs and incubated with P-pep or S-pep to assess the effect of P-pep on the interactions of HRS with PD-L1 and Munc13-4 (n = 3). (E) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on the same number of exosomes secreted from equal number of SUM159 cells treated with S-pep or P-pep. (F and G) Assessment of in vivo anti-tumor efficacy of P-pep. (F) Schematic of experimental design. (G) Tumor growth curves of orthotopic mouse models of breast cancer treated with P-pep or S-pep (n = 9). (H and I) Flow cytometric quantification of the percentage of CD45 + CD3 + CD4 + (H) and CD45 + CD3 + CD8 + (I) T cells among total cells in tumors (n = 5). (J and L) Representative contour plots depicting CD45 + CD3 + CD4 + (J) and CD45 + CD3 + CD8 + (L) T cell populations within tumors, showing the expression of granzyme B. (K and M) Quantification of the percentage of granzyme B + cells among CD45 + CD3 + CD4 + (K) and CD45 + CD3 + CD8 + (M) T cells within tumors (n = 5). Data are represented as means ± SEM (G), box plots show all data points (H, I, K and M), p -values were all calculated by unpaired t test. See also Figure S12.

Article Snippet: The lysates were then centrifuged at 4°C for 10 minutes at 12,000 × g. The supernatant was incubated overnight at 4°C with Munc13-4 Antibody (C-12) (Santa Cruz, sc-271301, 1:100) or HGS Polyclonal antibody (Proteintech, 10390-1-AP, 1:500), in conjunction with Protein A/G magnetic beads (Biolinkedin, L- 1004), on a rotator.

Techniques: Generated, Sequencing, Co-Immunoprecipitation Assay, Transfection, Construct, Incubation, Western Blot, In Vivo, Expressing

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Mechanistic model of Munc13-4-mediated tumor immune evasion through the regulation of PD-L1 sorting and secretion via exosomes

Article Snippet: The lysates were then centrifuged at 4°C for 10 minutes at 12,000 × g. The supernatant was incubated overnight at 4°C with Munc13-4 Antibody (C-12) (Santa Cruz, sc-271301, 1:100) or HGS Polyclonal antibody (Proteintech, 10390-1-AP, 1:500), in conjunction with Protein A/G magnetic beads (Biolinkedin, L- 1004), on a rotator.

Techniques:

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Munc13-4 deficiency in tumor cells inhibits tumor growth in an immunity-dependent way (A) Representative immunohistochemical images showing Munc13-4 expression in breast cancer, thyroid cancer, cholangiocarcinoma, gastrointestinal stromal tumors, pancreatic cancer, and hepatocellular carcinoma tissues, along with their corresponding adjacent normal tissues, assessed using a multi-organ carcinoma tissue array. Scale bar, 500 μm. (B–D) Tumor growth in BALB/c mice inoculated with wild-type (WT), control or Munc13-4 knockout (KO) 4T1 cells (n = 9). (B) Schematic of experimental design. (C) Tumor growth curves following mammary gland inoculation. (D) Percentage change in tumor volume, normalized to WT group. (E–G) Tumor growth in BALB/Nude mice inoculated with WT, control or Munc13-4 KO 4T1 cells (n = 8). (E) Schematic of experimental design. (F) Tumor growth curves following mammary gland inoculation. (G) Percentage change in tumor volume, normalized to WT group. (H–J) Tumor growth in NOD/SCID mice inoculated with WT, control or Munc13-4 KO 4T1 cells (n = 6). (H) Schematic of experimental design. (I) Tumor growth curves following mammary gland inoculation. (J) Percentage change in tumor volume, normalized to WT group. Data are presented as means ± SEM, p -values were calculated by one-way ANOVA with multiple comparisons (C, F and I), ns, not significant. See also Figure S1 and S2.

Article Snippet: To determine the interaction between Munc13-4 and PD-L1, Munc13-4 Antibody (C-2) (Santa Cruz, sc-271300, 1:200) and PD-L1/CD274 (C-terminal) Polyclonal antibody (Proteintech, 28076-1-AP, 1:200) were used.

Techniques: Immunohistochemical staining, Expressing, Control, Knock-Out

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Munc13-4 deficiency in tumor cells enhances T cell infiltration and activation (A–C) Flow cytometric quantification of the percentage of CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells among total cells in the tumors (A) , spleens (B) , and draining lymph nodes (C) of BALB/c mice (n = 5), 21 days after mammary gland injection with 3 × 10 5 control or Munc13-4 KO 4T1 cells per mouse. (D–F) Quantification of the percentage of granzyme B + (GzmB + ) (D) , Ki67 + (E) and IFNγ + (F) cells among CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells within tumors from orthotopic mouse models of breast cancer generated by control or Munc13-4 KO 4T1 cells (n = 5). (G–I) Quantification of the percentage of granzyme B + (G) , Ki67 + (H) and IFNγ + (I) cells among CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells within spleens from orthotopic mouse models of breast cancer generated by control or Munc13-4 KO 4T1 cells (n = 5). (J–L) Quantification of the percentage of granzyme B + (J) , Ki67 + (K) and IFNγ + (L) cells among CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells within the draining lymph nodes from orthotopic mouse models of breast cancer generated by control or Munc13-4 KO 4T1 cells (n = 5). Box plots show all data points, all p -values were calculated by Multiple t tests. See also Figure S3.

Article Snippet: To determine the interaction between Munc13-4 and PD-L1, Munc13-4 Antibody (C-2) (Santa Cruz, sc-271300, 1:200) and PD-L1/CD274 (C-terminal) Polyclonal antibody (Proteintech, 28076-1-AP, 1:200) were used.

Techniques: Activation Assay, Injection, Control, Generated

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Facilitating PD-L1 secretion by Munc13-4 suppresses anti-Tumor efficacy of T cells (A) Western blot analysis of total PD-L1 level in control and Munc13-4 KO SUM159 or 4T1 cells (n = 3). (B) Representative TEM images of EVs secreted by control and Munc13-4 KO SUM159 or 4T1 cells. Scale bar, 50 nm. (C) Quantification of exosomes secreted by equal numbers of control and Munc13-4 KO SUM159 (left) or 4T1 (right) cells through NTA (n = 3). (D–F) Analysis of EVs by optiprep TM density gradient centrifugation. (D) Schematic of experimental design. Western blot analysis of PD-L1, Alix, CD63 and CD81 in EVs secreted by equal numbers of control and Munc13-4 KO SUM159 (E) or 4T1 (F) cell, collected from factions 1–6 (F1–6) (n = 3). (G and H) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and Munc13-4 KO SUM159 cells (G) and corresponding quantification of blot band intensities (H) (n = 3). (I and J) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and Munc13-4 KO 4T1 cells (I) and corresponding quantification of blot band intensities (J) (n = 3). (K and L) Assessment of cytotoxicity elicited by activated mouse spleen lymphocytes against control and Munc13-4 KO 4T1 cells. (K) Schematic of experimental design. (L) Quantification of killing efficiency against control and Munc13-4 KO 4T1 cells (n = 3). (M and N) Evaluation of the relationship between decreased oncogenicity and impaired PD-L1 secretion in Munc13-4-deficient 4T1 cells. (M) Schematic of experimental design. (N) Tumor growth curves following mammary gland inoculation of control or Munc13-4 KO 4T1 cells, with subsequent injection of PBS or the indicated exosomes (n = 6). Data are presented as means ± SEM (C, H, J and N), p -values were calculated by unpaired t test (C), two-way ANOVA (H and J), paired t test (L) and one-way ANOVA with multiple comparisons (N). See also Figure S4 and S5.

Article Snippet: To determine the interaction between Munc13-4 and PD-L1, Munc13-4 Antibody (C-2) (Santa Cruz, sc-271300, 1:200) and PD-L1/CD274 (C-terminal) Polyclonal antibody (Proteintech, 28076-1-AP, 1:200) were used.

Techniques: Western Blot, Control, Gradient Centrifugation, Injection

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Munc13-4 facilitates MVB docking and fusion with the plasma membrane (A) Cryo-EM structure of the Munc13-4–Rab27a complex (upper panel) and detailed interface view (lower panel). Munc13-4 is represented in orange and Rab27a is shown in light green with the GppNHp nucleotide (red) bound. Residues on Rab27a (F46, W73, F88) and Munc13-4 (V660, K661, N739, T740), which are implicated in stabilizing the complex, are highlighted in darker colors. (B and C) GST pull-down assays examining the effects of VKAA and NTGG mutations in Munc13- 4 (B) , and F46S, W73S, F88S mutations in Rab27a (C) , on the formation of the Munc13-4–Rab27a complex (n = 3). (D and E) TIRF microscopy analysis of the effects of mutations in Munc13-4 and Rab27a on MVB mobility. Quantification of the mean diffusion coefficient ( D ), an index of MVB mobility, in SUM159 cells with the indicated mutations in Munc13-4 (D) or Rab27a (E) (n ≥ 962 for each group from triplicate experiments). (F) Quantification of exosomes secreted by equal numbers of indicated SUM159 cells though NTA (n = 3). (G and H) FRET-based detection of the role of Munc13-4 in SNARE complex assembly. (G) Illustration of FRET assay used to detect SNARE complex assembly. VAMP-7 SNARE motif (V7 SNARE) labeled with donor dye BODIPY FL, SNAP-23 (SN-23) labeled with acceptor dye 5- TAMRA, and syntaxin-4 (with its transmembrane domain deleted, termed as Syx4 ΔTM) together form a SNARE complex, leading to FRET between V7 SNARE and SN-23. (H) Representative graph of time-dependent SNARE complex assembly measured by the development of FRET between the 5-TAMRA labeled SN-23 and the BODIPY FL labeled V7 SNARE (n = 3). (I–K) FRET-based detection of the role of Munc13-4 in liposome fusion. (I) Illustration of the liposome fusion experiment. Syntaxin-4 (Syx-4) was incorporated into DiD-labeled liposomes and VAMP-7 was incorporated into DiI-labeled liposomes. Munc13-4 accelerates liposome fusion mediated by SNARE complex, leading to FRET between two liposome populations. (J) Time- dependent liposome fusion measured from the development of FRET between the DiD-labeled liposomes and the DiI-labeled liposomes. (K) Quantification of the FRET efficiency at the end of the detection (n = 3). Box plots show 10–90% percentile range of all data, with outliers represented as individual dots (D and E), data are represented as means ± SEM (F, J and K), p -values were calculated by Kruskal- Wallis test (D and E) and one-way ANOVA with multiple comparisons (F and K). See also Figure S6, S7 and S8.

Article Snippet: To determine the interaction between Munc13-4 and PD-L1, Munc13-4 Antibody (C-2) (Santa Cruz, sc-271300, 1:200) and PD-L1/CD274 (C-terminal) Polyclonal antibody (Proteintech, 28076-1-AP, 1:200) were used.

Techniques: Membrane, Cryo-EM Sample Prep, Microscopy, Diffusion-based Assay, Labeling, Liposomes

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Exosomal sorting of PD-L1 by Munc13-4 and HRS (A) Representative confocal microscopy images of control and Munc13-4 KO SUM159 cells co- expressing GFP-tagged PD-L1 with Orange-tagged CD63 or Orange-tagged LAMP1. Scale bar, 20 μm. (B) Quantification of Pearson’s correlation coefficient between PD-L1 and CD63, as well as between PD-L1 and LAMP1, in control and Munc13-4 KO SUM159 cells (n = 30 from triplicate experiments). (C) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and HRS KO SUM159 cells (n = 2). (D) Western blot analysis of total HRS amount in control and Munc13-4 KO SUM159 cells (n = 3). (E) Co-IP and immunoblotting (IB) analysis in control and Munc13-4 KO SUM159 cells transfected with indicated constructs to investigate the effect of Munc13-4 knockout on HRS–PD-L1 interaction (n = 3). (F and G) PLA to assess the effect of Munc13-4 knockout on the endogenous interaction between HRS and PD-L1. (F) Representative confocal microscopy images of control and Munc13-4 KO SUM159 cells in the PLA. Scale bar, 10 μm. (G) Quantification of puncta (n = 207 for control group and 175 for Munc13-4 KO group, from triplicate experiments). (H) Co-IP and IB analysis in Munc13-4 KO SUM159 cells transfected with indicated constructs to demonstrate that expression of Munc13-4 restored HRS–PD-L1 interaction (n = 3). (I) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs to test the interaction of Munc13-4 with HRS and STAM (n = 3). (J) Representative confocal microscopy image of SUM159 cells subjected to PLA, detecting the endogenous interaction between Munc13-4 and HRS (n = 3). Scale bar, 10 μm. (K) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs to explore the interaction between Munc13-4 and PD-L1 (n = 3). (L) Representative confocal microscopy image of SUM159 cells subjected to PLA, showing the endogenous interaction between Munc13-4 and PD-L1 (n = 3). Scale bar, 10 μm. (M and N) Direct binding between Munc13-4 and PD-L1 determined by in vitro liposome co- flotation assay. (M) Schematic of experimental design. (N) Western blot analysis of Munc13-4 and PD-L1 in top three fractions and bottom fraction of the mixture. (O) Co-IP and IB analysis in control and HRS KO SUM159 cells transfected with indicated constructs to examine the effect of HRS knockout on Munc13-4–PD-L1 interaction (n = 3). Box plots show all data points (B and G), p -values were calculated by two-way ANOVA (B) and Mann-Whitney U test (G).

Article Snippet: To determine the interaction between Munc13-4 and PD-L1, Munc13-4 Antibody (C-2) (Santa Cruz, sc-271300, 1:200) and PD-L1/CD274 (C-terminal) Polyclonal antibody (Proteintech, 28076-1-AP, 1:200) were used.

Techniques: Confocal Microscopy, Control, Expressing, Western Blot, Co-Immunoprecipitation Assay, Transfection, Construct, Knock-Out, Binding Assay, In Vitro, MANN-WHITNEY

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: IFN γ -induced modifications of Munc13-4 and HRS exert opposing effects on PD-L1 sorting (A and B) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and CBP KO SUM159 cells under IFNγ treatment (A) and corresponding quantification of blot band intensities (B) (n = 3). (C and D) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and HDAC3 KO SUM159 cells under IFNγ treatment (C) and corresponding quantification of blot band intensities (D) (n = 3). (E and F) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and NEDD4L knockdown (KD) SUM159 cells under IFNγ treatment (E) and corresponding quantification of blot band intensities (F) (n = 3). (G and H) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by SUM159 cells co-treated with IFNγ and PR-619 or DMSO (G) and corresponding quantification of blot band intensities (H) (n = 3). (I and J) PLA to assess the effects of Munc13-4 mutations on the interaction between endogenous HRS and PD-L1 in SUM159 cells. Representative confocal images of indicated SUM159 cells in the PLA (I) and quantification of PLA puncta (J) (n = 181 for control group, 252 for KO group, 174 for KO + WT group, 240 for KO + KKQQ group and 185 for KO + KKRR group from triplicate experiments). Scale bar, 10 μm. (K and L) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by indicated SUM159 cells (K) and corresponding quantification of blot band intensities (L) (n = 3). (M) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs to investigate the effect of HRS ubiquitylation on its interaction with PD-L1 (n = 3). (N) IP and IB analysis in control and Munc13-4 KO SUM159 cells, with or without IFNγ treatment, to explore the ubiquitylation of HRS (n = 3). Data are represented as means ± SEM (B, D, F, H and L), box plot shows 5–95% percentile range of all data, with outliers represented as individual dots (J), p -values were calculated by two-way ANOVA (B, D, F, H and L) and Kruskal-Wallis test (J). See also Figure S9–S11.

Article Snippet: To determine the interaction between Munc13-4 and PD-L1, Munc13-4 Antibody (C-2) (Santa Cruz, sc-271300, 1:200) and PD-L1/CD274 (C-terminal) Polyclonal antibody (Proteintech, 28076-1-AP, 1:200) were used.

Techniques: Western Blot, Control, Knockdown, Co-Immunoprecipitation Assay, Transfection, Construct

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: A peptide disrupting PD-L1 – Munc13-4 interaction inhibits tumor growth (A) Prediction of the interaction between PD-L1 (residues 253–290) and Munc13-4 (residues 1049–1080). Top insets show five ensembles generated by AlphaFold multimer, where N- and C-terminus of the proteins are indicated. PD-L1 and Munc13-4 are colored in blue and red, respectively. Residues that show potential contacts with each other (within 4 Å) are shown in sticks. Bottom inset displays the statistics of the residues in PD-L1 that show contacts with Munc13-4. The absolute and averaged contact numbers for each ensemble are illustrated by stacked histograms and line-scatter plot. The potential Munc13-4–interacting sequence of PD-L1 is shaded in magenta. (B) Diagram of the sequences for P-pep and S-pep. P-pep comprises a cell-penetrating peptide (CPP) fused to the human PD-L1 256–273 motif, whereas S-pep consists of a CPP linked to a scrambled sequence containing the same amino acid composition as the human PD-L1 256–273 motif. (C) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs and incubated with P-pep or S-pep to examine the effect of P-pep on Munc13-4–PD-L1 interaction (n = 3). (D) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs and incubated with P-pep or S-pep to assess the effect of P-pep on the interactions of HRS with PD-L1 and Munc13-4 (n = 3). (E) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on the same number of exosomes secreted from equal number of SUM159 cells treated with S-pep or P-pep. (F and G) Assessment of in vivo anti-tumor efficacy of P-pep. (F) Schematic of experimental design. (G) Tumor growth curves of orthotopic mouse models of breast cancer treated with P-pep or S-pep (n = 9). (H and I) Flow cytometric quantification of the percentage of CD45 + CD3 + CD4 + (H) and CD45 + CD3 + CD8 + (I) T cells among total cells in tumors (n = 5). (J and L) Representative contour plots depicting CD45 + CD3 + CD4 + (J) and CD45 + CD3 + CD8 + (L) T cell populations within tumors, showing the expression of granzyme B. (K and M) Quantification of the percentage of granzyme B + cells among CD45 + CD3 + CD4 + (K) and CD45 + CD3 + CD8 + (M) T cells within tumors (n = 5). Data are represented as means ± SEM (G), box plots show all data points (H, I, K and M), p -values were all calculated by unpaired t test. See also Figure S12.

Article Snippet: To determine the interaction between Munc13-4 and PD-L1, Munc13-4 Antibody (C-2) (Santa Cruz, sc-271300, 1:200) and PD-L1/CD274 (C-terminal) Polyclonal antibody (Proteintech, 28076-1-AP, 1:200) were used.

Techniques: Generated, Sequencing, Co-Immunoprecipitation Assay, Transfection, Construct, Incubation, Western Blot, In Vivo, Expressing

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Mechanistic model of Munc13-4-mediated tumor immune evasion through the regulation of PD-L1 sorting and secretion via exosomes

Article Snippet: To determine the interaction between Munc13-4 and PD-L1, Munc13-4 Antibody (C-2) (Santa Cruz, sc-271300, 1:200) and PD-L1/CD274 (C-terminal) Polyclonal antibody (Proteintech, 28076-1-AP, 1:200) were used.

Techniques:

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Munc13-4 deficiency in tumor cells inhibits tumor growth in an immunity-dependent way (A) Representative immunohistochemical images showing Munc13-4 expression in breast cancer, thyroid cancer, cholangiocarcinoma, gastrointestinal stromal tumors, pancreatic cancer, and hepatocellular carcinoma tissues, along with their corresponding adjacent normal tissues, assessed using a multi-organ carcinoma tissue array. Scale bar, 500 μm. (B–D) Tumor growth in BALB/c mice inoculated with wild-type (WT), control or Munc13-4 knockout (KO) 4T1 cells (n = 9). (B) Schematic of experimental design. (C) Tumor growth curves following mammary gland inoculation. (D) Percentage change in tumor volume, normalized to WT group. (E–G) Tumor growth in BALB/Nude mice inoculated with WT, control or Munc13-4 KO 4T1 cells (n = 8). (E) Schematic of experimental design. (F) Tumor growth curves following mammary gland inoculation. (G) Percentage change in tumor volume, normalized to WT group. (H–J) Tumor growth in NOD/SCID mice inoculated with WT, control or Munc13-4 KO 4T1 cells (n = 6). (H) Schematic of experimental design. (I) Tumor growth curves following mammary gland inoculation. (J) Percentage change in tumor volume, normalized to WT group. Data are presented as means ± SEM, p -values were calculated by one-way ANOVA with multiple comparisons (C, F and I), ns, not significant. See also Figure S1 and S2.

Article Snippet: The diluted Munc13-4 primary antibody working solution (1:50, Santa Cruz, sc-271300) was applied, and the slides were incubated overnight at 4°C.

Techniques: Immunohistochemical staining, Expressing, Control, Knock-Out

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Munc13-4 deficiency in tumor cells enhances T cell infiltration and activation (A–C) Flow cytometric quantification of the percentage of CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells among total cells in the tumors (A) , spleens (B) , and draining lymph nodes (C) of BALB/c mice (n = 5), 21 days after mammary gland injection with 3 × 10 5 control or Munc13-4 KO 4T1 cells per mouse. (D–F) Quantification of the percentage of granzyme B + (GzmB + ) (D) , Ki67 + (E) and IFNγ + (F) cells among CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells within tumors from orthotopic mouse models of breast cancer generated by control or Munc13-4 KO 4T1 cells (n = 5). (G–I) Quantification of the percentage of granzyme B + (G) , Ki67 + (H) and IFNγ + (I) cells among CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells within spleens from orthotopic mouse models of breast cancer generated by control or Munc13-4 KO 4T1 cells (n = 5). (J–L) Quantification of the percentage of granzyme B + (J) , Ki67 + (K) and IFNγ + (L) cells among CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells within the draining lymph nodes from orthotopic mouse models of breast cancer generated by control or Munc13-4 KO 4T1 cells (n = 5). Box plots show all data points, all p -values were calculated by Multiple t tests. See also Figure S3.

Article Snippet: The diluted Munc13-4 primary antibody working solution (1:50, Santa Cruz, sc-271300) was applied, and the slides were incubated overnight at 4°C.

Techniques: Activation Assay, Injection, Control, Generated

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Facilitating PD-L1 secretion by Munc13-4 suppresses anti-Tumor efficacy of T cells (A) Western blot analysis of total PD-L1 level in control and Munc13-4 KO SUM159 or 4T1 cells (n = 3). (B) Representative TEM images of EVs secreted by control and Munc13-4 KO SUM159 or 4T1 cells. Scale bar, 50 nm. (C) Quantification of exosomes secreted by equal numbers of control and Munc13-4 KO SUM159 (left) or 4T1 (right) cells through NTA (n = 3). (D–F) Analysis of EVs by optiprep TM density gradient centrifugation. (D) Schematic of experimental design. Western blot analysis of PD-L1, Alix, CD63 and CD81 in EVs secreted by equal numbers of control and Munc13-4 KO SUM159 (E) or 4T1 (F) cell, collected from factions 1–6 (F1–6) (n = 3). (G and H) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and Munc13-4 KO SUM159 cells (G) and corresponding quantification of blot band intensities (H) (n = 3). (I and J) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and Munc13-4 KO 4T1 cells (I) and corresponding quantification of blot band intensities (J) (n = 3). (K and L) Assessment of cytotoxicity elicited by activated mouse spleen lymphocytes against control and Munc13-4 KO 4T1 cells. (K) Schematic of experimental design. (L) Quantification of killing efficiency against control and Munc13-4 KO 4T1 cells (n = 3). (M and N) Evaluation of the relationship between decreased oncogenicity and impaired PD-L1 secretion in Munc13-4-deficient 4T1 cells. (M) Schematic of experimental design. (N) Tumor growth curves following mammary gland inoculation of control or Munc13-4 KO 4T1 cells, with subsequent injection of PBS or the indicated exosomes (n = 6). Data are presented as means ± SEM (C, H, J and N), p -values were calculated by unpaired t test (C), two-way ANOVA (H and J), paired t test (L) and one-way ANOVA with multiple comparisons (N). See also Figure S4 and S5.

Article Snippet: The diluted Munc13-4 primary antibody working solution (1:50, Santa Cruz, sc-271300) was applied, and the slides were incubated overnight at 4°C.

Techniques: Western Blot, Control, Gradient Centrifugation, Injection

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Munc13-4 facilitates MVB docking and fusion with the plasma membrane (A) Cryo-EM structure of the Munc13-4–Rab27a complex (upper panel) and detailed interface view (lower panel). Munc13-4 is represented in orange and Rab27a is shown in light green with the GppNHp nucleotide (red) bound. Residues on Rab27a (F46, W73, F88) and Munc13-4 (V660, K661, N739, T740), which are implicated in stabilizing the complex, are highlighted in darker colors. (B and C) GST pull-down assays examining the effects of VKAA and NTGG mutations in Munc13- 4 (B) , and F46S, W73S, F88S mutations in Rab27a (C) , on the formation of the Munc13-4–Rab27a complex (n = 3). (D and E) TIRF microscopy analysis of the effects of mutations in Munc13-4 and Rab27a on MVB mobility. Quantification of the mean diffusion coefficient ( D ), an index of MVB mobility, in SUM159 cells with the indicated mutations in Munc13-4 (D) or Rab27a (E) (n ≥ 962 for each group from triplicate experiments). (F) Quantification of exosomes secreted by equal numbers of indicated SUM159 cells though NTA (n = 3). (G and H) FRET-based detection of the role of Munc13-4 in SNARE complex assembly. (G) Illustration of FRET assay used to detect SNARE complex assembly. VAMP-7 SNARE motif (V7 SNARE) labeled with donor dye BODIPY FL, SNAP-23 (SN-23) labeled with acceptor dye 5- TAMRA, and syntaxin-4 (with its transmembrane domain deleted, termed as Syx4 ΔTM) together form a SNARE complex, leading to FRET between V7 SNARE and SN-23. (H) Representative graph of time-dependent SNARE complex assembly measured by the development of FRET between the 5-TAMRA labeled SN-23 and the BODIPY FL labeled V7 SNARE (n = 3). (I–K) FRET-based detection of the role of Munc13-4 in liposome fusion. (I) Illustration of the liposome fusion experiment. Syntaxin-4 (Syx-4) was incorporated into DiD-labeled liposomes and VAMP-7 was incorporated into DiI-labeled liposomes. Munc13-4 accelerates liposome fusion mediated by SNARE complex, leading to FRET between two liposome populations. (J) Time- dependent liposome fusion measured from the development of FRET between the DiD-labeled liposomes and the DiI-labeled liposomes. (K) Quantification of the FRET efficiency at the end of the detection (n = 3). Box plots show 10–90% percentile range of all data, with outliers represented as individual dots (D and E), data are represented as means ± SEM (F, J and K), p -values were calculated by Kruskal- Wallis test (D and E) and one-way ANOVA with multiple comparisons (F and K). See also Figure S6, S7 and S8.

Article Snippet: The diluted Munc13-4 primary antibody working solution (1:50, Santa Cruz, sc-271300) was applied, and the slides were incubated overnight at 4°C.

Techniques: Membrane, Cryo-EM Sample Prep, Microscopy, Diffusion-based Assay, Labeling, Liposomes

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Exosomal sorting of PD-L1 by Munc13-4 and HRS (A) Representative confocal microscopy images of control and Munc13-4 KO SUM159 cells co- expressing GFP-tagged PD-L1 with Orange-tagged CD63 or Orange-tagged LAMP1. Scale bar, 20 μm. (B) Quantification of Pearson’s correlation coefficient between PD-L1 and CD63, as well as between PD-L1 and LAMP1, in control and Munc13-4 KO SUM159 cells (n = 30 from triplicate experiments). (C) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and HRS KO SUM159 cells (n = 2). (D) Western blot analysis of total HRS amount in control and Munc13-4 KO SUM159 cells (n = 3). (E) Co-IP and immunoblotting (IB) analysis in control and Munc13-4 KO SUM159 cells transfected with indicated constructs to investigate the effect of Munc13-4 knockout on HRS–PD-L1 interaction (n = 3). (F and G) PLA to assess the effect of Munc13-4 knockout on the endogenous interaction between HRS and PD-L1. (F) Representative confocal microscopy images of control and Munc13-4 KO SUM159 cells in the PLA. Scale bar, 10 μm. (G) Quantification of puncta (n = 207 for control group and 175 for Munc13-4 KO group, from triplicate experiments). (H) Co-IP and IB analysis in Munc13-4 KO SUM159 cells transfected with indicated constructs to demonstrate that expression of Munc13-4 restored HRS–PD-L1 interaction (n = 3). (I) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs to test the interaction of Munc13-4 with HRS and STAM (n = 3). (J) Representative confocal microscopy image of SUM159 cells subjected to PLA, detecting the endogenous interaction between Munc13-4 and HRS (n = 3). Scale bar, 10 μm. (K) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs to explore the interaction between Munc13-4 and PD-L1 (n = 3). (L) Representative confocal microscopy image of SUM159 cells subjected to PLA, showing the endogenous interaction between Munc13-4 and PD-L1 (n = 3). Scale bar, 10 μm. (M and N) Direct binding between Munc13-4 and PD-L1 determined by in vitro liposome co- flotation assay. (M) Schematic of experimental design. (N) Western blot analysis of Munc13-4 and PD-L1 in top three fractions and bottom fraction of the mixture. (O) Co-IP and IB analysis in control and HRS KO SUM159 cells transfected with indicated constructs to examine the effect of HRS knockout on Munc13-4–PD-L1 interaction (n = 3). Box plots show all data points (B and G), p -values were calculated by two-way ANOVA (B) and Mann-Whitney U test (G).

Article Snippet: The diluted Munc13-4 primary antibody working solution (1:50, Santa Cruz, sc-271300) was applied, and the slides were incubated overnight at 4°C.

Techniques: Confocal Microscopy, Control, Expressing, Western Blot, Co-Immunoprecipitation Assay, Transfection, Construct, Knock-Out, Binding Assay, In Vitro, MANN-WHITNEY

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: IFN γ -induced modifications of Munc13-4 and HRS exert opposing effects on PD-L1 sorting (A and B) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and CBP KO SUM159 cells under IFNγ treatment (A) and corresponding quantification of blot band intensities (B) (n = 3). (C and D) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and HDAC3 KO SUM159 cells under IFNγ treatment (C) and corresponding quantification of blot band intensities (D) (n = 3). (E and F) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by control and NEDD4L knockdown (KD) SUM159 cells under IFNγ treatment (E) and corresponding quantification of blot band intensities (F) (n = 3). (G and H) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by SUM159 cells co-treated with IFNγ and PR-619 or DMSO (G) and corresponding quantification of blot band intensities (H) (n = 3). (I and J) PLA to assess the effects of Munc13-4 mutations on the interaction between endogenous HRS and PD-L1 in SUM159 cells. Representative confocal images of indicated SUM159 cells in the PLA (I) and quantification of PLA puncta (J) (n = 181 for control group, 252 for KO group, 174 for KO + WT group, 240 for KO + KKQQ group and 185 for KO + KKRR group from triplicate experiments). Scale bar, 10 μm. (K and L) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on equal numbers of exosomes secreted by indicated SUM159 cells (K) and corresponding quantification of blot band intensities (L) (n = 3). (M) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs to investigate the effect of HRS ubiquitylation on its interaction with PD-L1 (n = 3). (N) IP and IB analysis in control and Munc13-4 KO SUM159 cells, with or without IFNγ treatment, to explore the ubiquitylation of HRS (n = 3). Data are represented as means ± SEM (B, D, F, H and L), box plot shows 5–95% percentile range of all data, with outliers represented as individual dots (J), p -values were calculated by two-way ANOVA (B, D, F, H and L) and Kruskal-Wallis test (J). See also Figure S9–S11.

Article Snippet: The diluted Munc13-4 primary antibody working solution (1:50, Santa Cruz, sc-271300) was applied, and the slides were incubated overnight at 4°C.

Techniques: Western Blot, Control, Knockdown, Co-Immunoprecipitation Assay, Transfection, Construct

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: A peptide disrupting PD-L1 – Munc13-4 interaction inhibits tumor growth (A) Prediction of the interaction between PD-L1 (residues 253–290) and Munc13-4 (residues 1049–1080). Top insets show five ensembles generated by AlphaFold multimer, where N- and C-terminus of the proteins are indicated. PD-L1 and Munc13-4 are colored in blue and red, respectively. Residues that show potential contacts with each other (within 4 Å) are shown in sticks. Bottom inset displays the statistics of the residues in PD-L1 that show contacts with Munc13-4. The absolute and averaged contact numbers for each ensemble are illustrated by stacked histograms and line-scatter plot. The potential Munc13-4–interacting sequence of PD-L1 is shaded in magenta. (B) Diagram of the sequences for P-pep and S-pep. P-pep comprises a cell-penetrating peptide (CPP) fused to the human PD-L1 256–273 motif, whereas S-pep consists of a CPP linked to a scrambled sequence containing the same amino acid composition as the human PD-L1 256–273 motif. (C) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs and incubated with P-pep or S-pep to examine the effect of P-pep on Munc13-4–PD-L1 interaction (n = 3). (D) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs and incubated with P-pep or S-pep to assess the effect of P-pep on the interactions of HRS with PD-L1 and Munc13-4 (n = 3). (E) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on the same number of exosomes secreted from equal number of SUM159 cells treated with S-pep or P-pep. (F and G) Assessment of in vivo anti-tumor efficacy of P-pep. (F) Schematic of experimental design. (G) Tumor growth curves of orthotopic mouse models of breast cancer treated with P-pep or S-pep (n = 9). (H and I) Flow cytometric quantification of the percentage of CD45 + CD3 + CD4 + (H) and CD45 + CD3 + CD8 + (I) T cells among total cells in tumors (n = 5). (J and L) Representative contour plots depicting CD45 + CD3 + CD4 + (J) and CD45 + CD3 + CD8 + (L) T cell populations within tumors, showing the expression of granzyme B. (K and M) Quantification of the percentage of granzyme B + cells among CD45 + CD3 + CD4 + (K) and CD45 + CD3 + CD8 + (M) T cells within tumors (n = 5). Data are represented as means ± SEM (G), box plots show all data points (H, I, K and M), p -values were all calculated by unpaired t test. See also Figure S12.

Article Snippet: The diluted Munc13-4 primary antibody working solution (1:50, Santa Cruz, sc-271300) was applied, and the slides were incubated overnight at 4°C.

Techniques: Generated, Sequencing, Co-Immunoprecipitation Assay, Transfection, Construct, Incubation, Western Blot, In Vivo, Expressing

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Mechanistic model of Munc13-4-mediated tumor immune evasion through the regulation of PD-L1 sorting and secretion via exosomes

Article Snippet: The diluted Munc13-4 primary antibody working solution (1:50, Santa Cruz, sc-271300) was applied, and the slides were incubated overnight at 4°C.

Techniques: