Journal: Cancer Research

Article Title: Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

doi: 10.1158/0008-5472.can-10-0690

Figure Lengend Snippet: Figure 1. Retinal lesions and ASCs are caused by Notch3 signaling. A, virus is microinjected into the ventricles of an E10.5 mouse embryo. At the time of injection, the forebrain and optic cup are a continuous structure, allowing viral infection of the anlage giving rise to the retina and optic nerve. Viral particles are released into the amniotic sac near the injection site on needle withdrawal and infect the developing lens. B, E17.5 retina in a N3CLE-injected animal with a PLAP-positive clone (i). H&E staining showed that N3CLE-induced retinal lesions can span 1 (ii) or all 3 layers (iii) of the retina. Retinal lesions contained BIII- tubulin–positive (iv) and GS-positive (v) cells that were often double positive for nuclear Notch3 (N3). C, Pax6/Chx10 double-positive cells in retinal lesions (arrowheads). D, i, white cataractous lenses developed in N3CLE-injected animals. ii, the ALE (arrowhead) expanded into multiple epithelial layers (arrow) and contained mitotic bodies in the N3CLE-induced ASCs (inset). iii, areas of hyperproliferation were PLAP positive and contained Ki67-positive cells (DAPI counterstain). Original magnifications: 20 (Bi, Biv, and C), 40 (Bv and Dii), 200 (Bii, Biii, and Di).

Article Snippet: Primary antibodies used were a-Notch3 (rabbit, 1:1,000; Santa Cruz Biotechnology), a-Pax6 (mouse, 1:400, Millipore Corporation), a-Chx10 (sheep, 1:100, Exalpha Biologicals), a-GFAP (rabbit, 1:1,000; DAKO), a-Nestin (chicken, 1:250; Aves Labs, Inc.), and Hes5 (rabbit, Gaiano Laboratory, only recognizes overexpression).

Techniques: Virus, Injection, Infection, Staining

Journal: Cancer Research

Article Title: Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

doi: 10.1158/0008-5472.can-10-0690

Figure Lengend Snippet: Figure 2. Invasive choroidal tumors and retinal glial lesions arise following Notch3 activation. N3CLE-induced cellular choroidal tumors in the adult eye were PLAP positive (A), pigmented, and arose in the choroidal layer beneath an intact retinal pigment epithelium (Bi, arrow). Many choroidal tumors invaded outward through breaks in the sclera (Bi, arrowheads) and could diffusely infiltrate periocular tissues. Choroidal tumors contained nuclear N3 and Ki67-positive cells (Bii and Biii). C and D, N3CLE-injected animals contained PLAP-positive retinal glial lesions (C), GFAP-positive (Dii), and GS-positive (Diii). Original magnifications: 10 (A), 20 (Bi), 40 (Bii and Diii), 63 (Biii, Di, and Dii), 64 (Bii), 200 (C).

Article Snippet: Primary antibodies used were a-Notch3 (rabbit, 1:1,000; Santa Cruz Biotechnology), a-Pax6 (mouse, 1:400, Millipore Corporation), a-Chx10 (sheep, 1:100, Exalpha Biologicals), a-GFAP (rabbit, 1:1,000; DAKO), a-Nestin (chicken, 1:250; Aves Labs, Inc.), and Hes5 (rabbit, Gaiano Laboratory, only recognizes overexpression).

Techniques: Activation Assay, Injection

Journal: Cancer Research

Article Title: Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

doi: 10.1158/0008-5472.can-10-0690

Figure Lengend Snippet: Figure 3. N3CLE induced formation of glial tumors along the optic nerve. A, glial tumors (arrowheads) formed along the optic nerve (arrow) in N3CLE-injected animals and contained mitotic bodies (ii, inset). B, the optic nerve tumors expressed GFAP and Notch3 and invaded orbital tissues surrounding the optic nerve. Some glial tumors involved both the retina (arrowhead) and optic nerve (arrow). C, i, glial tumors contained Ki67-positive proliferating cells. Nestin-positive and Nestin/GFAP-positive cells within the lesions coexpressing Hes5 or Notch3, respectively (Cii and D). Original magnifications: 63 (C and D), 100 (Ai and Bi), 200 (Ai, Aii, and Bii), 400 (Aii, inset).

Article Snippet: Primary antibodies used were a-Notch3 (rabbit, 1:1,000; Santa Cruz Biotechnology), a-Pax6 (mouse, 1:400, Millipore Corporation), a-Chx10 (sheep, 1:100, Exalpha Biologicals), a-GFAP (rabbit, 1:1,000; DAKO), a-Nestin (chicken, 1:250; Aves Labs, Inc.), and Hes5 (rabbit, Gaiano Laboratory, only recognizes overexpression).

Techniques: Injection

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3 is overexpressed in multiple human tumors (A) Expression of NOTCH3 mRNA in primary human tumors from TCGA compared to normal tissues. Box and whiskers plots are drawn with individual points below 10 th and above 90 th percentiles. Median values are drawn as a line in the middle of the box. TPM, transcripts per kilobase million, ns, non-significant. (B) NOTCH3 mRNA fold change in primary human lung, breast, and ovarian tumors and xenografts compared to normal tissues (baseline, dashed line). Data represent mean (n = 2–4). (C) Quantitation of NOTCH3 ISH staining as a measure of percentage of area stained on breast (ER + , TNBC), lung (LUAD, LUSC), ovarian, and normal (breast, lung, and ovary) tissue sections.

Article Snippet: Notch3 probe targeting nucleotides 727-2210 of human NOTCH3 mRNA (NM_000435.2) , Advanced Cell Diagnostics , 558996.

Techniques: Expressing, Quantitation Assay, Staining

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Generation and characterization of therapeutic anti-NOTCH3 mAbs (A) NOTCH3-dependent report gene assay with NOTCH3-targeted and control mAbs. Data represent mean ± SEM from 3 biological replicates (n = 4 per replicate), ns, non-significant. (B) qRT-PCR of HES1 gene expression in treated cells. Data represent mean ± SD from 2 biological replicates (n = 3 per replicate), ns, non-significant. (C) NOTCH3 immunoblot from mAb-treated cells and xenografts. GAPDH is shown as a loading control. Schematic diagram of NOTCH3 cleavage events and protein fragments. M, mouse number. (D and E) Immunoblot using N- or C-terminal domain antibodies to detect NOTCH3 fragments after DLL4 activation in treated cells. β-actin is shown as a loading control. (F) Epitope mapping of anti-NOTCH3 mAbs using NRR3-NRR1 domain swap chimeric constructs. NRR3 domains are shown in black and NRR1 domains are shown in gray. Representative data represent mean (n = 2). (G) Binding interface of anti-N3(i) Fab on the NOTCH3-NRR domain as determined from the co-crystal structure is shown in red, LNR-A (green), LNR-B (beige), LNR-C (blue), HD1 (pink), and HD2 (magenta).

Article Snippet: Notch3 probe targeting nucleotides 727-2210 of human NOTCH3 mRNA (NM_000435.2) , Advanced Cell Diagnostics , 558996.

Techniques: Gene Assay, Control, Quantitative RT-PCR, Gene Expression, Western Blot, Activation Assay, Construct, Binding Assay

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Cell - membrane distribution of anti-NOTCH3 mAbs depends on signaling status (A) Confocal images of anti-NOTCH3 mAbs on fixed cells. Scale bar, 10 μm. (B) Single optical sections from live-cell confocal imaging of anti-NOTCH3 mAbs simultaneously bound to cells and imaged over time with anti-N3(i)-Alexa 488 (green) and anti-N3-DyLight647 (magenta). Scale bar, 10 μm. (C) z stack of maximum intensity projections from live-cell confocal imaging of anti-NOTCH3 mAbs over time. Scale bar, left = 8 μm, right = 10 μm. (D) Cross-section of cells bound by NOTCH3-targeted mAbs from images in Figure 2 C. Scale bar, 10 μm.

Article Snippet: Notch3 probe targeting nucleotides 727-2210 of human NOTCH3 mRNA (NM_000435.2) , Advanced Cell Diagnostics , 558996.

Techniques: Membrane, Imaging

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Anti-NOTCH3 mAbs have differential rates of endocytosis and lysosomal trafficking (A and B) Colocalization of mAbs with pHrodo Red dextran compartments calculated by PCC and MCC from live-cell confocal images. (A) Data represent mean ± SEM of 3 biological replicates (n = 3 fields imaged per replicate). (B) Data represent mean ± SEM of 2 biological replicates (n = 2 fields imaged per replicate), ns, non-significant. (C) Colocalization of mAbs with CT-β were calculated by PCC and MCC from live-cell confocal images. Data represent mean ± SEM of 2 biological replicates (n = 5 fields imaged per replicate). (D) Single optical sections of mAbs with CT-β from live-cell confocal imaging. Scale bar, 10 μm. (E) Colocalization of mAbs with CAV-1 or clathrin were calculated by MCC in fixed cells. Data represent mean ± SEM (n = 3 fields imaged). (F) Colocalization of mAbs with pHrodo Red dextran compartments were calculated by MCC from live-cell confocal images of MβCD-treated cells. Cross hairs were set at a value of 0.2 MCC to highlight more rapid colocalization of both mAbs with pHrodo Red dextran compartments. Data represent mean ± SEM of 2 biological replicates (n = 5 fields imaged per replicate).

Article Snippet: Notch3 probe targeting nucleotides 727-2210 of human NOTCH3 mRNA (NM_000435.2) , Advanced Cell Diagnostics , 558996.

Techniques: Imaging

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Anti-NOTCH3 mAbs transendocytose into DLL4 ligand cells (A and B) TEC of labeled mAbs (green) that were bound to U2OS-hN3 cells and then co-cultured with HEK-DLL4 or HEK-parental cells (red) from live-cell confocal imaging. (A) Single optical sections of a U2OS-hN3 cell and a migrating HEK-DLL4 cell (asterisk) before and after contact (arrows). Scale bar, 10 μm. (B) z stack of maximum intensity projections. Arrows, anti-NOTCH3 mAbs inside HEK-DLL4 cells. (C) Confocal images of maximum intensity projections acquired from indirect immunofluorescence of mAbs bound to U2OS-hN3 cells (magenta) and then co-cultured with HEK-DLL4 (green) from. Dashed white line demarcates anti-NOTCH3 mAbs inside HEK-DLL4 cells. Scale bar, 10 μm.

Article Snippet: Notch3 probe targeting nucleotides 727-2210 of human NOTCH3 mRNA (NM_000435.2) , Advanced Cell Diagnostics , 558996.

Techniques: Labeling, Cell Culture, Imaging, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3-targeted ADCs induce cytotoxicity in both receptor and ligand cells (A) General structure of NOTCH3-targeted ADCs that were generated with mAbs, a cleavable dipeptide-based linker and the Aur0101 payload (blue). (B) NOTCH3-ADC induction of caspase-3/7 activity. Data represent mean ± SEM of 3 biological replicates (n = 3 per replicate). (C) In vitro cytotoxicity of NOTCH3-ADCs after control ( Control:siRNA ) or siRNA knockdown of NOTCH3 mRNA ( N3:siRNA ). Data represent mean ± SEM of 2 biological replicates (n = 3 per replicate). (D) In vitro cytotoxicity of NOTCH3-ADCs using parental MDA-MB-468 cells under 2D and 3D culture conditions. Data represent mean ± SEM (n = 3). (E and F) TEC of NOTCH3-ADCs induces caspase activity in HEK-DLL4 cells. (E) z stack of maximum intensity projections from live-cell confocal imaging of anti-N3(i) ADC bound to U2OS-hN3 cells and co-cultured with HEK-DLL4 cells labeled with pHrodo Red dextran. Caspase compartments (magenta line), pHrodo Red dextran compartments (blue line) and the merged image. (F) Percentage of the caspase-positive compartments that were calculated after treatment with NOTCH3-ADCs. Data represent mean ± SEM for 3 biological replicates (n = 15 fields imaged per replicate), ns, non-significant.

Article Snippet: Notch3 probe targeting nucleotides 727-2210 of human NOTCH3 mRNA (NM_000435.2) , Advanced Cell Diagnostics , 558996.

Techniques: Generated, Activity Assay, In Vitro, Control, Knockdown, Imaging, Cell Culture, Labeling

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3-targeted ADCs induce prolonged tumor regressions (A) Tumor growth inhibition of HCC2429 CLXs treated with control and NOTCH3-targeted mAbs and ADCs. Data represent mean ± SEM (n = 7–8 per group). (B) Tumor growth inhibition of 37622 PDXs treated with NOTCH3-ADCs compared to cisplatin or control ADC. Data represent mean ± SEM (n = 9–10 per group). (C) Tumor growth inhibition of MDA-MB-468 CLXs treated with control and anti-N3 ADCs. Data represent mean ± SEM (n = 7–8 per group). (D) Tumor growth inhibition of OVCAR3 CLXs treated with control and anti-N3 ADCs compared with carboplatin alone, and carboplatin then anti-N3 ADC. Data represent mean ± SEM (n = 7–8 per group).

Article Snippet: Notch3 probe targeting nucleotides 727-2210 of human NOTCH3 mRNA (NM_000435.2) , Advanced Cell Diagnostics , 558996.

Techniques: Inhibition, Control

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet:

Article Snippet: Notch3 probe targeting nucleotides 727-2210 of human NOTCH3 mRNA (NM_000435.2) , Advanced Cell Diagnostics , 558996.

Techniques: Control, Polymer, Plasmid Preparation, Blocking Assay, Electron Microscopy, Recombinant, Adjuvant, Antibody Labeling, RNAscope, Sequencing, Software

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: The distribution of NOTCH3 cys variants in CADASIL cohorts and in population databases and the NVFOR per EGFr domain. ( A ) Distribution of NOTCH3 cys variants in CADASIL cohorts ( top ) and population databases ( bottom ) along the 34 EGFr domains. ( B ) Interval plot showing the NVFOR of each EGFr domain with error bars indicating 95% confidence intervals. Each EGFr domain is classified in an EGFr risk category, based on their respective NVFOR: HR-EGFr domains have NVFOR >5, MR-EGFr domains have an NVFOR between 0.20 and 5 and LR-EGFr domains have an NVFOR <0.20. If an EGFr domain included less than 10 NOTCH3 cys variant counts, then it was classified as an UR-EGFr domain. The dotted lines indicate NVFOR 0.2 and 5.0, which are the cut-off points used for the EGFr risk categories.

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques: Variant Assay

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: Distribution of high-risk, medium-risk and low-risk EGFr domains along the NOTCH3 protein and in CADASIL and population genotype–phenotype data sets. ( A ) Schematic representation of the transmembrane NOTCH3 protein. The NOTCH3 gene contains 33 exons, of which exons 2–24 encode the 34 EGFr domains located in the extracellular domain. Each EGFr domain is classified in an EGFr risk category based on their respective NVFOR. ( B ) Proportional bar charts showing the proportion of individuals with a NOTCH3 cys variant for each EGFr risk category in the CADASIL and population genotype–phenotype data sets. Most CADASIL patients harbour an HR-EGFr variant (66.1%), whereas the majority of individuals in the population genotype–phenotype data set harbour an LR-EGFr variant (85.7%).

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques: Variant Assay

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: Characteristics of individuals with a NOTCH3 cys variant in the CADASIL and population data sets

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques: Variant Assay

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: CADASIL patients with a NOTCH3 cys variant located in the newly identified HR-EGFr domains 8, 11 and 26 are clinically and radiologically as severe as patients with HR-EGFr 1–6 variants . Two scatterplots and three interval plots showing nWMHv ( A ), PSMD ( B ), nLV ( C ), risk of stroke ( D ) and disability ( E ) stratified per EGFr risk category in the CADASIL genotype–phenotype data set after correction for cardiovascular risk factors and sex. CADASIL patients with NOTCH3 cys variants located in the newly identified HR-EGFr domains 8, 11 and 26 had a significantly higher nWMHv ( P = 1.8 × 10 −8 ), PSMD ( P = 2.6 × 10 −8 ), nLV ( P = 0.006), risk of stroke ( P = 0.002) and disability ( P = 0.041) than CADASIL patients with MR-EGFr variants. There were no significant differences between patients with HR-EGFr 1–6 variants and those with variants in HR-EGFR domains 8, 11 and 26 in nWMHv ( P = 0.27), PSMD ( P = 0.087), nLV ( P = 0.87), disability ( P = 0.26) and risk of stroke ( P = 0.75). Error bars indicate 95% confidence interval of the group means. Statistical significance after correction for multiple testing: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques: Variant Assay

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: NOTCH3 cys variants in EGFr domains 1–5, 14 and 26 are most frequent in the CADASIL genotype–phenotype data set: associations with disease severity . Five interval plots showing the estimated marginal means, odds ratios or hazard ratios of SVD imaging markers, risk of stroke and disability for the EGFr domains most frequently harbouring NOTCH3 cys variants in the CADASIL genotype–phenotype data set, after correction for cardiovascular risk factors and sex. Only EGFr domains which had high NOTCH3 cys variant counts (≥10 individuals for SVD imaging markers and ≥20 individuals for clinical outcomes) were included in the analyses. Error bars indicate 95% confidence intervals of the group means. ( A – D ) Of all frequently mutated HR-EGFr domains, EGFr domains 2 and 3 were associated with the highest burden of nWMHv, PSMD, nLV and risk of stroke. There was an evident decrease in the SVD imaging marker loads and risk of stroke in EGFr domain 3 through EGFr domain 5. EGFr domain 26 was associated with as high a burden of SVD imaging markers and risk of stroke as the other HR-EGFr domains. EGFr domain 14, which was the only MR-EGFr domain with a high frequency of NOTCH3 cys variants in CADASIL patients, was associated with the lowest burden of SVD imaging markers and risk of stroke. ( E ) There were no significant differences in disability. Further details including statistically significant differences in the burden of SVD imaging markers and risk of stroke between EGFr domains are shown in .

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques: Imaging, Variant Assay, Marker

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: The risk of stroke between the NOTCH3 cys EGFr risk categories in the CADASIL and population genotype–phenotype data set. A survival curve ( A ) and interval plot ( B ), both corrected for cardiovascular risk factors and sex, showing that MR-EGFr and HR-EGFr patients in the CADASIL genotype–phenotype data set had a significant earlier onset of stroke than individuals from the same EGFr risk category in the population genotype–phenotype data set [MR-EGFr: median 72 versus >78 years ( P = 8.1 × 10 −6 ); HR-EGFr: median 57 versus 74 years ( P = 3.5 × 10 −5 )]. The risk of stroke of HR-EGFr individuals in the population data set and MR-EGFr patients from the CADASIL data set was similar ( P = 0.54).

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques:

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: Differences in NOTCH3 aggregation and signalling between EGFr risk categories. ( A – C ) NOTCH3 aggregation load, expressed as immunohistochemical NOTCH3 score, was determined in skin tissue samples of LR-EGFr ( n = 3), MR-EGFr ( n = 6) and HR-EGFr ( n = 12) patients, of which six had an HR-EGFr 1–6 NOTCH3 cys variant and six had a NOTCH3 cys variant located in the newly identified HR-EGFr domain 26. ( A ) As there were only tissue samples available of three LR-EGFr patients, these patients were excluded from the group comparisons. HR-EGFr 1–6 CADASIL patients had a significantly higher NOTCH3 score than MR-EGFr patients: mean NOTCH3 score 43.0 (95% CI: 30.3–55.6) versus 23.2 (95% CI: 10.5–35.8) ( P = 0.033). HR-EGFr 26 patients had a mean NOTCH3 score of 32.8 (95% CI: 20.1–45.5), which did not significantly differ from that of HR-EGFr 1–6 ( P = 0.24) and MR-EGFr ( P = 0.27) patients. The three LR-EGFr patients had a mean NOTCH3 score of 15.4 (95% CI: 0–32.6). ( B ) NVFOR was significantly associated with a NOTCH3 score ( P = 0.003), also after correction for age ( P = 0.006). ( C ) Representative images of NOTCH3-immunohistochemistry of skin biopsies of HR-EGFr 1–6, HR-EGFr 26, MR-EGFr and LR-EGFr patients, showing increasing granular NOTCH3 staining from low- to high-risk EGFr domains. Bar represents 20 µm. ( D ) NOTCH3 signalling activity was assessed in NIH 3T3 cells using a CBF1-responsive luciferase assay with and without Jagged1 stimulation. NOTCH3 signalling was expressed as fold increase of luciferase activity upon Jagged1 stimulation (= ‘luciferase activity with Jagged1 stimulation’/‘luciferase activity without Jagged1 stimulation’). Each dot represents an individual experiment. Signalling activity did not differ significantly between NOTCH3 cys variants located in the other HR-EGFr domains and the MR-EGFr and LR-EGFr domains (all P > 0.58). However, NOTCH3 cys variants in HR-EGFr and MR-EGFr showed a significantly lower NOTCH3 signalling activity compared to wild-type ( P = 0.008 and P = 0.02). As expected, NOTCH3 cys variants located in the HR-EGFr domain 11 had strongly impaired ligand-induced signalling activity, comparable to a deletion of the ligand binding domain ( P = 0.53). There was no significant difference in NOTCH3 signalling activity between HR-EGFr domain 26 and other HR-EGFr domains, MR-EGFr domains and LR-EGFr domains (all P > 0.47). ( E ) NVFOR was not associated with NOTCH3 signalling activity ( P = 0.88). Error bars indicate 95% confidence intervals of the group means. Statistical significance after correction for multiple testing: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques: Immunohistochemical staining, Variant Assay, Immunohistochemistry, Staining, Activity Assay, Luciferase, Ligand Binding Assay

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Schematic representation of Notch signaling. (1) Furin (S1 cleavage) cleaves the NOTCH3 precursor protein in the Golgi system, resulting in a non-covalently bound heterodimeric protein that is transported to the cell surface. (2) A mechanical traction force is applied to the NOTCH3 ECD when a Notch ligand binds to the EGF repeats 10-11, exposing the extracellular NRR near the cell membrane, which consists of LNR and the heterodimerization domain (in green). Subsequently, ADAM17 cleaves the C-terminal portion of the heterodimerization domain (S2-cleavage). (3) The NEXT, which is made up of a RAM domain, many ANK domains, a PEST domain, and a transmembrane domain, is cleaved by the γ-secretase (S3-cleavage) releasing the N3ICD. (4) The N3ICD binds to the CSL complex protein and together with the co-activator Mastermind-like (MAM) trigger downstream gene transcription in the nucleus. (5) The NOTCH3 ECD and ligand are normally endocytosed by the ligand expressing cell and is degraded in the lysosome. B) Schematic representation of NOTCH3 cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) mutations. NOTCH3 ECD contains 34 EGF repeat domains, each of which has 6 cysteine residues (WT). Mutations in CADASIL change the number of cysteines to an uneven number of cysteines (Mutant). These unpaired cysteines residues result in incorrect EGF repeat folding, irregular protein folding which leads to an enhanced NOTCH3 ECD multimerization. Distribution of the cysteine-altering mutations that cause CADASIL are shown. In the CADASIL mutant NOTCH3 ECD, the endocytosis is hampered, and NOTCH ECD remains outside of the VSMC and starts to accumulate and aggregate around the vessels. ADAM17, a disintegrin and metalloproteinase domain-containing protein 17; ANK, ankyrin repeats; EGF, epidermal growth factor; HD, heterodimerization domain; LNR, Lin-Notch repeats; PEST, proline (P), glutamic acid (E), serine (S) and threonine (T) degradation domain; RAM, Rbp-associated molecule domain; TM, transmembrane domain.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Membrane, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Schematic representation of NOTCH3 and NOTCH3 EGF 1-5 . NOTCH3 represents the full-length protein, and NOTCH3 EGF 1-5 represents the NOTCH3 protein with exon 1 to 5 fused with a myc-His-Tag at the C-terminus used for purification of the aggregated protein. B) Western blot of the NOTCH3 EGF 1-5 WT and R133C purified protein. The eluate fractions were visualized by western blot using an α-myc antibody. C) Western blot of NOTCH3 EGF 1-5 WT and R133C aggregated proteins. The incubated fractions of NOTCH3 EGF 1-5 WT and R133C were visualized on a western blot using an α-myc antibody. The purified proteins and the aggregates were verified after 1-5 days incubation by western blot using α-myc ab under non-reducing conditions.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Purification, Western Blot, Incubation

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Schematic and work plan of the subcutaneous active immunization on the TgN3R182C 150 mouse model. B) Antibody titre validation of serum from TgN3R182C 150 CADASIL mice immunized with NOTCH3 EGF 1-5 aggregates (vaccinated) and PBS (sham) at 4, 5 and 7 months old. A direct ELISA with NOTCH3 aggregate-coated plates and different dilutions of serum was performed.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Biomarker Discovery, Direct ELISA

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Representative images of TgN3R182C 150 , sham- and NOTCH3 EGF 1-5 - immunized mice at 7 months of age and TgN3R182C 150 at 18 months of age. Representative images show brain arteries of TgN3R182C 150 (7 and 18 months), sham and NOTCH3 EGF 1-5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an α-SMA antibody (green). B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF 1-5 - immunized, sham and non-vaccinated TgN3R182C 150 mice at 7 months of age. NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per vessel increases significantly in the TgN3R182C 150 mice at 18 months of age versus NOTCH3 EGF 1-5 - immunized, sham and non-vaccinated TgN3R182C 150 mice at 7 months of age. (*p < 0.05, **p < 0.01, ns= non-significant). Scale bar =20µm.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Staining

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: Quantitative real-time PCR analysis of the Notch downstream target genes NOTCH3, Hes1, Hey1 and Nrip2 on TgN3R182C150 mice at 5 and 12 months of age.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Representative images of TgN3R182C 150 , sham- and NOTCH3 EGF 1-5 - immunized mice at 3, 7 and 18 months of age. Representative images show brain arteries and capillaries of TgN3R182C 150 , sham and NOTCH3 EGF 1-5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an anti-perlecan antibody (green). B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3-ECD stained area and average size per vessel revealed a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non-vaccinated 3 months old TgN3R182C 150 (n=3) and 7 months old TgN3R182C 150 (n=6) mice and 18 months old TgN3R182C 150 (n=3). Quantification of NOTCH3-ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3-ECD stained area and average size per vessel revealed a significant decrease in NOTCH3-ECD deposition in brain arteries and capillaries between NOTCH3 EGF 1-5 - immunized (n=11), sham (n=9) and non-vaccinated TgN3R182C 150 (n=6) mice. (*p < 0.05, **p < 0.01, ****p < 0.0001, ns= non-significant). Scale bar =20µm.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Staining

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: Quantification of human NOTCH3 ECD protein present in the whole blood serum of sham, immunized and non-vaccinated TgN3R182C 150 mice (at 3 and 7 months old). A) NOTCH3 ECD was detected in the whole blood serum of the non-treated TgN3R182C 150 mice at three months of age and further increased at seven months of age. B) NOTCH3 ECD in the TgN3R182C 150 mice was significantly reduced in the vaccinated TgN3R182C 150 mice. Statistical analysis was performed using unpaired t test with Welch’s correction. P < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques:

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Immunostaining for smooth muscle actin (ASMA) revealed that there were no significant differences in the composition of the smooth muscle cell coating of vessels in the retinal vasculature in WT (C57Bl6/J) versus TgN3R182C 150 mice at 7 months of age. B) Immunostaining for smooth muscle actin (ASMA) shows no significant differences in the composition of the smooth muscle cell coating of vessels in the retinal vasculature in NOTCH3 EGF 1-5 - vaccinated versus sham-vaccinated TgN3R182C 150 mice. C) Immunostaining for smooth muscle actin (ASMA) shows an extensive loss of VSMC in the Notch3 -/- mice when compared to a WT (C57Bl6/J) at 3 months of age. Scale bar =50µm.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Immunostaining

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: A) Representative images of TgN3R182C 150 , sham- and NOTCH3 EGF1-5 - immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an antibody against microglia (Iba1, green). B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF1-5 - immunized (n=11), sham (n=9) and non-vaccinated TgN3R182C 150 (n=6) mice at 7 months of age. (ns= non-significant). Scale bar =20µm.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Staining

Journal: bioRxiv

Article Title: NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.1101/2022.07.11.499563

Figure Lengend Snippet: NIH3T3 cells were transfected with the control, wild type NOTCH3, or NOTCH3 R182C plasmids, as well as the β-gal and 12XCSL-luc reporter plasmids and cultured on immobilized jagged2 (Jag2) in the presence of DMSO or DAPT (n=3 and two technical replicates). Statistical analysis was performed using 2-way ANOVA followed by Tukey’s multiple comparisons tests. P < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, ns= non-significant). RLU, relative luminescence units.

Article Snippet: Briefly, high-affinity binding 96 well plates were coated with a NOTCH3 capture monoclonal antibody (MAB1559; R&D Systems) at 0.625 ng/μL in 100 μL of PBS and agitated overnight at 4 °C.

Techniques: Transfection, Control, Cell Culture