Journal: bioRxiv

Article Title: Local, but not circulating, complement C3 governs immune checkpoint blockade efficacy

doi: 10.1101/2025.05.12.653608

Figure Lengend Snippet: (A) MC-38 cells were transplanted subcutaneously into 6-week-old female control ( C3 fl/fl and Meflin -Cre + ; C3 WT/WT ) or Meflin-lineage C3 KO (mKO; Meflin -Cre + ; C3 fl/fl ) mice (day 0), followed by intraperitoneal administration of αPD-1 mAb or isotype IgG. Tumor measurements were performed blind to the genotypes. (B) Individual tumor size trajectories (grey lines) with linear approximations (dotted lines: isotype IgG, solid lines: αPD-1, cyan: control, magenta: mKO) on a log2 scale over 19 days. (C) Fixed effects of days post-inoculation ( time ), αPD-1 ( P ), mKO genotype ( M ), and their interactions on tumor growth rate, estimated using a generalized linear mixed model, and complete response (CR) odds ratio for αPD-1-treated control versus mKO tumors. Estimates and odds ratio are shown with 95% confidence intervals in square brackets. (D) Survival analysis of control and mKO mice treated with the indicated antibodies. Log-rank test P values are shown. (E) UMAP visualization of 12,528 cells from control and mKO tumor samples (n = 2 mice/group). Cells were clustered and colored according to the cell type. (F) scCODA analysis showing significant relative changes (FDR < 0.05) in cell composition between mKO and control tumors. (G) Cell-cell interaction network across cell types (left) and PageRank centrality scores showing M1-like and M2-like TAMs as network hubs (scores > 0.1) (right). (H) Flow cytometric quantification of iNOS + M1-like and MRC1 + M2-like TAMs within CD45 + cells isolated from control and mKO tumors (gating strategy and representative plots in ). (I) Total myeloid cells were stained for CD11b by IHC, followed by quantification of the number of CD11b + cells (n = 4 mice, mean of 4 high-power fields [HPFs]/mouse). All analyses were performed on tumors resected on day 9 (E to I).

Article Snippet: C3 inhibitor (AMY-101, CS-0119743, ChemScene) was administered subcutaneously near the tumor site on days 0, 1, 4, 7, 10, and 13 at a dose of 4 μg/gBW , CD11b depletion antibody (clone M1/70, 101272, BioLegend, RRID: AB_2561479) was administered intraperitoneally to mice on days 2, 4, 7, 10, and 13 at a dose of 10 μg/gBW , and CD11b partial agonist (ADH-503 or Leukadherin-1, S8306, Selleck) was administered intraperitoneally to mice every day from day 2 to 13 at a dose of 2 μg/gBW ( ).

Techniques: Control, Isolation, Staining

Journal: bioRxiv

Article Title: Local, but not circulating, complement C3 governs immune checkpoint blockade efficacy

doi: 10.1101/2025.05.12.653608

Figure Lengend Snippet: (A) Directional migration of mouse peritoneal CD11b + cells in response to serum-free conditioned media from mouse control ( C3 fl/fl ) and mKO (Meflin -Cre + ; C3 fl/fl ) embryonic fibroblasts (MEFs) using transwell assays (n = 2 wells, 5 fields/well). Arrowheads indicate transmigrated cells after 3 hours. (B) Directional migration of THP-1 cells toward HepG2 conditioned medium with anti-C3 or isotype control antibody (1 μM) in the lower chamber, with quantification (n = 3 wells, 5 fields/well). (C) Directional migration of THP-1 cells toward siRNA-transfected HepG2 conditioned medium. The effect of adding purified iC3b (2 μg/mL) to the lower chamber was also examined (n = 3 wells, 5 fields/well). (D) Effects of purified human iC3b (0–10 μg/mL) on THP-1 cell migration in medium containing 1% C3-depleted serum. (E) Directional migration of primary human CD14 + monocytes in response to iC3b in medium containing 1% C3-depleted serum (n = 2 donors, 2 wells/donor, 5 fields/well).

Article Snippet: C3 inhibitor (AMY-101, CS-0119743, ChemScene) was administered subcutaneously near the tumor site on days 0, 1, 4, 7, 10, and 13 at a dose of 4 μg/gBW , CD11b depletion antibody (clone M1/70, 101272, BioLegend, RRID: AB_2561479) was administered intraperitoneally to mice on days 2, 4, 7, 10, and 13 at a dose of 10 μg/gBW , and CD11b partial agonist (ADH-503 or Leukadherin-1, S8306, Selleck) was administered intraperitoneally to mice every day from day 2 to 13 at a dose of 2 μg/gBW ( ).

Techniques: Migration, Control, Transfection, Purification

Journal: bioRxiv

Article Title: Local, but not circulating, complement C3 governs immune checkpoint blockade efficacy

doi: 10.1101/2025.05.12.653608

Figure Lengend Snippet: (A) Flow cytometric analysis of THP-1 cells for CD11b expression using BV421-conjugated antibodies. (B) Detection of C3 in siRNA-transfected HepG2 cell supernatants by ELISA (left) and immunoblotting (right). Recombinant human C3b protein (10 ng) served as a positive control. The α-chain of C3/C3b (115 kDa) and the α-chain fragment of iC3b (68 kDa) were detected by IB using an anti-C3 antibody (upper right). Ponceau S staining indicated an equal loading of total proteins (lower right). (C) Characterization of shRNA-mediated CD11b ( ITGAM ) knockdown in THP-1 cells. Cells were transduced with either control (sh_scramble) or CD11b-targeting (sh_ ITGAM ) shRNAs, with sh_ITGAM-transduced cells further enriched via MACS using anti-CD11b microbeads. CD11b expression in both control and knockdown cells was assessed by flow cytometry.

Article Snippet: C3 inhibitor (AMY-101, CS-0119743, ChemScene) was administered subcutaneously near the tumor site on days 0, 1, 4, 7, 10, and 13 at a dose of 4 μg/gBW , CD11b depletion antibody (clone M1/70, 101272, BioLegend, RRID: AB_2561479) was administered intraperitoneally to mice on days 2, 4, 7, 10, and 13 at a dose of 10 μg/gBW , and CD11b partial agonist (ADH-503 or Leukadherin-1, S8306, Selleck) was administered intraperitoneally to mice every day from day 2 to 13 at a dose of 2 μg/gBW ( ).

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant, Positive Control, Staining, shRNA, Knockdown, Transduction, Control, Flow Cytometry

Journal: bioRxiv

Article Title: Local, but not circulating, complement C3 governs immune checkpoint blockade efficacy

doi: 10.1101/2025.05.12.653608

Figure Lengend Snippet: (A) Directional migration assay comparing THP-1 cells transduced with control (sh_scramble) or CD11b-targeting (sh_ ITGAM ) shRNAs in response to iC3b in medium containing 1% C3-depleted serum (n = 2 wells, 4 fields/well). Arrowheads indicate transmigrated cells after 3 hours. (B) Effect of selective Syk inhibitor BAY 61-3606 (1 μM) on THP-1 cell migration in response to iC3b in medium containing 1% C3-depleted serum (n = 3 wells, 6 fields/well). (C) Trajectory analysis of THP-1 cells with or without BAY 61-3606 pretreatment in medium containing 1% C3-depleted serum, with or without iC3b (n = 32 cells/group, tracked for 2 hours). (D) Adhesion assay of THP-1 cells cultured on plastic dishes with or without BAY 61-3606 pretreatment, then incubated for 1 hour with or without iC3b in medium containing 1% C3-depleted serum. Images were captured before and after gentle PBS washing to remove non-adherent cells, with quantification of detached cells (n = 2 wells, 3 low-power fields [LPFs]/well). Insets show higher magnification of the boxed areas. Dotted lines indicate regions where cells detached after PBS washing.

Article Snippet: C3 inhibitor (AMY-101, CS-0119743, ChemScene) was administered subcutaneously near the tumor site on days 0, 1, 4, 7, 10, and 13 at a dose of 4 μg/gBW , CD11b depletion antibody (clone M1/70, 101272, BioLegend, RRID: AB_2561479) was administered intraperitoneally to mice on days 2, 4, 7, 10, and 13 at a dose of 10 μg/gBW , and CD11b partial agonist (ADH-503 or Leukadherin-1, S8306, Selleck) was administered intraperitoneally to mice every day from day 2 to 13 at a dose of 2 μg/gBW ( ).

Techniques: Migration, Transduction, Control, Cell Adhesion Assay, Cell Culture, Incubation, Gentle

Journal: bioRxiv

Article Title: Local, but not circulating, complement C3 governs immune checkpoint blockade efficacy

doi: 10.1101/2025.05.12.653608

Figure Lengend Snippet: (A) Tissue sections from MC-38 tumors developed in WT and Meflin KO mice were stained for CD11b using IHC (left), followed by quantification (right) (n = 4 mice, mean of 4 HPFs/mouse). Representative IHC images are shown. CD11b + cells are indicated using arrowheads. (B) Tissue sections from mT5 tumors developed in WT and Meflin KO mice were stained for C3 using smISH (left), followed by quantification (right). Representative IHC images are shown. Boxed areas are magnified in the lower panels. Arrowheads indicate C3 + cells. T denotes tumor. (C) Graphical abstract. The data shown in the present study suggested that local, not circulating, complement C3 governs immune checkpoint blockade efficacy. C3 + cancer-associated fibroblasts (CAFs) inhibit monocyte infiltration into the tumor microenvironment (TME) via the iC3b-CR3 signaling, reducing immunosuppressive M2-like tumor-associated macrophage (TAM) accumulation and enhancing ICB efficacy. In contrast, TME enriched with C3 - CAFs shows increased monocyte infiltration, promoting ICB resistance. Notably, hepatocyte-derived circulating C3 does not influence ICB therapy outcomes, highlighting the distinct role of locally produced C3 in cancer immunotherapy.

Article Snippet: C3 inhibitor (AMY-101, CS-0119743, ChemScene) was administered subcutaneously near the tumor site on days 0, 1, 4, 7, 10, and 13 at a dose of 4 μg/gBW , CD11b depletion antibody (clone M1/70, 101272, BioLegend, RRID: AB_2561479) was administered intraperitoneally to mice on days 2, 4, 7, 10, and 13 at a dose of 10 μg/gBW , and CD11b partial agonist (ADH-503 or Leukadherin-1, S8306, Selleck) was administered intraperitoneally to mice every day from day 2 to 13 at a dose of 2 μg/gBW ( ).

Techniques: Staining, Derivative Assay, Produced

Journal: bioRxiv

Article Title: Local, but not circulating, complement C3 governs immune checkpoint blockade efficacy

doi: 10.1101/2025.05.12.653608

Figure Lengend Snippet: (A) MC-38 cells (1.0 × 10 cells) were transplanted subcutaneously into 6-week-old female Meflin KO mice (day 0), followed by intraperitoneal αPD-1 mAb or isotype IgG treatment in combinations with CD11b depletion antibody (M1/70: 10 μg/gBW, magenta arrowheads), CD11b partial agonist (ADH-503: 2 μg/gBW, cyan arrowheads), or control (PBS) at the indicated time points. (B) Individual tumor size trajectories (grey lines) with linear approximations (dotted lines: isotype IgG, solid lines: αPD-1, black for PBS, magenta for M1/70, cyan for ADH-503) on a log2 scale over 19 days. (C) Fixed effects of days post-inoculation ( time ), αPD-1 ( P ), M1/70 treatment ( M ), ADH-503 treatment ( A ), and their interactions on tumor growth rate estimated using a generalized linear mixed model. Estimates are shown with 95% confidence intervals in square brackets. (D) Survival analysis of Meflin KO mice treated with the indicated antibodies and agents. Log-rank test P values are shown. (E) Tissue sections from MC-38 tumors developed in Meflin KO mice treated with the indicated agents were stained for CD11b using IHC, followed by quantification of the number of CD11b + cells (n = 4 mice, mean of 4 HPFs/mouse).

Article Snippet: C3 inhibitor (AMY-101, CS-0119743, ChemScene) was administered subcutaneously near the tumor site on days 0, 1, 4, 7, 10, and 13 at a dose of 4 μg/gBW , CD11b depletion antibody (clone M1/70, 101272, BioLegend, RRID: AB_2561479) was administered intraperitoneally to mice on days 2, 4, 7, 10, and 13 at a dose of 10 μg/gBW , and CD11b partial agonist (ADH-503 or Leukadherin-1, S8306, Selleck) was administered intraperitoneally to mice every day from day 2 to 13 at a dose of 2 μg/gBW ( ).

Techniques: Control, Staining

Journal: Frontiers in Immunology

Article Title: Mechanosensitivity of macrophage polarization: comparing small molecule leukadherin-1 to substrate stiffness

doi: 10.3389/fimmu.2025.1420325

Figure Lengend Snippet: Summary of experimental systems and plan. (A) Schematic depiction of in vitro cell culture model to test mechanotransduction by incubation of murine BMDMs on collagen-coated silica gels with corresponding stiffness (quantified as kilopascal, kPa) (B) Brightfield images from BMDMs cultured on gels of 0.2 kPa (soft) or 64 kPa (stiff). Scale bar = 10 µm. (C) Area of BMDMs measured from microscopic images shown in (B) . Each symbol represents area of one cell, line at median, p-value determined by Mann-Whitney, data from three independent experiments. (D) Flow cytometric analysis of CD11b expression on BMDMs cultured on gels of indicated stiffness and primed with LPS, primed with LPS and activated with ATP, or left unstimulated. Each symbol represents value from one sample, bar at median, p-values determined by two-way ANOVA indicated significant effect of mechanosensation (MS) and cytokine stimulation (CS), with follow-up pairwise comparisons (***p< 0.001, ****p< 0.0001). Data from 3 independent experiments. (E) Molecular structure of CD11b activator LA-1 . (F) Schematic showing hypothesized activation of integrins on stiff substrate to induce mechanotransduction, compared to LA-1 ligation and activation of CD11b. (G) Summary of experimental plan to test if exposure to LA-1 and incubation on stiffer substrates similarly regulate BMDM activation and polarization.

Article Snippet: For engaging CD11b integrin receptor, specific activator Leukadherin-1 (LA-1 (ADH-503 (GB1275)) from Selleck Chemicals (Cat.#SO525) was administrated in culture 30 min before inflammasome activation (during the last 30 min of LPS-mediated priming.

Techniques: In Vitro, Cell Culture, Incubation, MANN-WHITNEY, Expressing, Activation Assay, Ligation

Journal: Frontiers in Immunology

Article Title: Mechanosensitivity of macrophage polarization: comparing small molecule leukadherin-1 to substrate stiffness

doi: 10.3389/fimmu.2025.1420325

Figure Lengend Snippet: Stiffness of collagen-coated silica gels, but not LA1 treatment, regulates BMDM differentiation. (A) Bone-marrow cells from WT mice were incubated on collagen-coated silica gels or TC plastic in M-CSF (L929-cells supernatant) for three or seven days. Cells were analyzed by flow cytometry for Ly6C and F4/80 to identify populations of monocytes (Ly6C high F4/80 low ) and BMDMs (Ly6C low F4/80 high ). Percentage of BMDMs indicated in each flow plot. (B) Percentage of total cells that were identified as BMDMs. Each symbol represents value from one of three independent experiments, line at median, Kruskal-Wallis with follow-up pairwise comparison used to determine p-values, “P” = plastic. (C) Monocytes were labeled with CellTrace-BV421 at start of incubation. After 3 days in culture, dilution of CellTrace-BV421 was analyzed by flow cytometry. Cells were defined as monocytes or macrophages as shown. (D) Bone marrow cells from WT mice were labeled with CellTrace-BV421, then incubated with or without LA1 (5 μg/ml) for 3 or 7 days. Flow cytometric analysis was used to determine surface expression of CD11b, Ly6C, and F4/80 and dilution of CellTrace-BV421. Monocytes and BMDMs were determined as in (A) . Each symbol shows value for one sample, with each experiment performed with technical triplicates. Line at median. Results of three independent experiments are shown. (E) Dilution of CellTrace-BV421 dilution from cells in (D) . Representative samples from one of three independent experiments shown.

Article Snippet: For engaging CD11b integrin receptor, specific activator Leukadherin-1 (LA-1 (ADH-503 (GB1275)) from Selleck Chemicals (Cat.#SO525) was administrated in culture 30 min before inflammasome activation (during the last 30 min of LPS-mediated priming.

Techniques: Incubation, Flow Cytometry, Comparison, Labeling, Expressing

Journal: Frontiers in Immunology

Article Title: Mechanosensitivity of macrophage polarization: comparing small molecule leukadherin-1 to substrate stiffness

doi: 10.3389/fimmu.2025.1420325

Figure Lengend Snippet: Macrophage surface markers are differentially regulated by integration of mechanical and cytokine cues. BMDMs were incubated on collagen-coated silica gels (open circles, light grey bars: 0.2 kPa; closed circles, dark grey bars: 64 kPa) with LPS+IFNγ, IL-4+IL-13, or without specific cytokine stimulation (none) for 24 h. Expression of indicated surface markers were analyzed by flow cytometry and quantified as median fluorescence intensity (MFI) of all BMDMs gated (CD11b pos F4/80 pos ). Each symbol represents value from one sample; bar shows median value; 95% confidence interval shown. Data were combined from three independent experiments, enabled by consistent fluorophore staining and identical cytometer settings for independent acquisitions. Two-way ANOVA was used to determine if substrate stiffness (MS, mechanosensation) and/or cytokine stimulation (CS) significantly altered marker expression, and to determine if there was a significant interaction of mechanosensation and cytokine stimulation on MFI. No specific pairwise comparisons were tested.

Article Snippet: For engaging CD11b integrin receptor, specific activator Leukadherin-1 (LA-1 (ADH-503 (GB1275)) from Selleck Chemicals (Cat.#SO525) was administrated in culture 30 min before inflammasome activation (during the last 30 min of LPS-mediated priming.

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence, Staining, Cytometry, Marker

Journal: Frontiers in Immunology

Article Title: Mechanosensitivity of macrophage polarization: comparing small molecule leukadherin-1 to substrate stiffness

doi: 10.3389/fimmu.2025.1420325

Figure Lengend Snippet: Macrophage surface markers are differentially regulated by LA1 treatment and cytokine cues. BMDMs were incubated with (closed circles) or without (gray circles) LA1 (5 µg/ml) and with LPS+IFNγ, IL-4+IL-13, or without specific cytokine stimulation (none) for 24 h. Expression of indicated surface markers were analyzed by flow cytometry and quantified as median fluorescence intensity (MFI) of all BMDMs gated (CD11b pos F4/80 pos ). Each symbol represents value from one sample; bar shows median value. Two-way ANOVA was used to determine if LA1 exposure (LA1) and/or cytokine stimulation (CS) significantly altered marker expression, and to determine if there was a significant interaction of LA1 exposure and cytokine stimulation on MFI. No specific pairwise comparisons were tested. Results from one representative experiment of two or three independent experiments shown; variability across independent acquisitions precluded combining data from multiple experiments. Results from other experiments are shown in Supplementary Figure 2 .

Article Snippet: For engaging CD11b integrin receptor, specific activator Leukadherin-1 (LA-1 (ADH-503 (GB1275)) from Selleck Chemicals (Cat.#SO525) was administrated in culture 30 min before inflammasome activation (during the last 30 min of LPS-mediated priming.

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence, Marker

Journal: Frontiers in Immunology

Article Title: Mechanosensitivity of macrophage polarization: comparing small molecule leukadherin-1 to substrate stiffness

doi: 10.3389/fimmu.2025.1420325

Figure Lengend Snippet: Interrogation of candidate tyrosine kinases downstream of LA1-mediated CD11b activation. (A) Immunoblot of lysates derived from BMDMs incubated with or without LA1 during final 30 min of LPS priming (4 h) and activation with nigericin (30 min). Densitometry of probed proteins shown below corresponding bands. (B–E) Quantification of (B) phospho-JNK1, (C) phospho-p65 (NF-κB) (D) phospho-ERK1/2, and (E) phospho-p38 normalized to total substrate (JNK1, p65, ERK1/2, or p38, respectively), then actin. Each symbol represents value from one of the 3 independent experiments, line at median, p-values determined using Mann-Whitney. Whole immunoblots with molecular weight markers shown in Supplementary Figure 7 .

Article Snippet: For engaging CD11b integrin receptor, specific activator Leukadherin-1 (LA-1 (ADH-503 (GB1275)) from Selleck Chemicals (Cat.#SO525) was administrated in culture 30 min before inflammasome activation (during the last 30 min of LPS-mediated priming.

Techniques: Activation Assay, Western Blot, Derivative Assay, Incubation, MANN-WHITNEY, Molecular Weight