Jul 02, 2026
Additional methods to Duso, Messuti et al “NF1 modulates microtubule repair and sensitivity to antibody-drug conjugates” Nature Cancer 2026
- luca mazzarella1
- 1IEO
- mazzalab
Protocol Citation: luca mazzarella 2026. Additional methods to Duso, Messuti et al “NF1 modulates microtubule repair and sensitivity to antibody-drug conjugates” Nature Cancer 2026. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2x4z4v1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 30, 2026
Last Modified: July 02, 2026
Protocol Integer ID: 320091
Abstract
Antibody-Drug
Conjugates (ADCs) have transformed cancer therapy, but the determinants of
their differential activity among patients remain poorly understood, especially
in breast cancer, where several ADCs share the same target but differ in
payloads. NF1 is a tumor suppressor best characterized as a RAS pathway
inhibitor. Integrating patient data, in vitro/in vivo models, and
biochemistry, we show that NF1 loss sensitizes cancer cells to T-DM1 (the first
approved breast cancer ADC) through a novel, RAS-independent role in
microtubule dynamics and repair. NF1 functions as a bona fide
Microtubule-Associated Protein (MAP) enhancing intratubular repair; its loss
induces mitotic defects and aneuploidy. Clinically, NF1 loss correlates with
increased T-DM1 sensitivity in patients across Europe and the USA, defining NF1
as an ADC payload-associated predictive biomarker.
Materials
**Genetic engineering**
- pSpCas9(BB) 2A-GFP (PX458) plasmid421
- QIAquick Gel Extraction Kit (QIAGEN)
- Lipofectamine 2000
- Gene Knockout Kit v2 (Synthego)
**NF1 mammalian expression**
- pLVX-puro vector
- Calcium Chloride
- Polybrene (Sigma-Aldrich)
**RasG12V overexpression**
- pDONR223_KRAS_p.G12V (Addgene #81665)
- pLenti CMV hygro dest (Addgene #17454)
- Hygromycin B (InvivoGen)
- Active Ras Detection Kit (#8821, Cell Signalling Technology)
**Luciferase and H2B-GFP/mCherry expressing cells**
- Tet-Off-H2B-GFP lentiviral vector422
- H2B-mCherry retroviral vector423
- pLenti CMV Puro LUC (w168-1) (Addgene #17477)
- Polybrene (Sigma-Aldrich)
- Puromycin (Vinci-Biochem)
**Drug screen**
- PHERAstar FSX Microplate Reader
- GraphPad Prism 9
**Colony staining**
- Crystal violet solution (Sigma V5265)
- ColonyArea ImageJ plugin250
**Beta-galactosidase assay**
- β-Galactosidase staining kit (#9860, Cell Signaling Technology)
- Invitrogen EVOS XL Core 36microscope
- ImageJ
**Mass photometry**
- TwoMP instrument (Refeyn)
**Microtubule co-sedimentation**
- Tubulin (Cytoskeleton Inc)
- General Tubulin Buffer
- NuMA (gift from Marina Mapelli)
**GTPase activity**
- GTPase-Glo™ assay (Promega)
- GloMax®-Multi+ Detection System
**Turbidity assay**
- Tubulin Polymerization Buffer (80 mM PIPES pH 6.9, 2mM MgCl2, 0.5mM EGTA, 1mM GTP, 10% glycerol)
- Infinite 200 PRO-Tecan plate reader
**In vivo experiments**
- D-Luciferin (XenoLight, Perkin Elmer)
- Living Image v4.7.3 software (Perkin Elmer Inc)
**Cell imaging**
- Bond III IHC autostainer (Leica Biosystems)
- Bond Polymer Refine Detection Kit (DS9800 - Leica Biosystems)
- Aperio ScanScope XT instrument
**Co-localization immunofluorescence**
- Donkey Serum
- Anti-Neurofibromin (MilliporeSigma)
- NF1-A 376G3, #MABN2557
- Anti-alpha-Tubulin (Abcam, Ab18251)
- Anti-mouse Alexa Fluor-555
- Anti-rabbit Alexa Fluor-488
- DAPI (Sigma, D9542)
- CrestOptics confocal spinning disk X-light V3
**Proximity Ligation Assay**
- Duolink® flowPLA Detection Kit – Orange (Sigma-Aldrich, #92102)
**Chromosome alignment**
- RO3306 (Sigma-Aldrich)
- MG-132 (Tocris)
- Sodium Azide in PBS
**T-DM1 Internalisation**
- T-DM1 pulse (1.5 µg/mL)
- Triton X-100
- Sheep polyclonal anti-Human IgG-Cy5 (AC2115, Sigma)
- FITC-phalloidin (P5282, Sigma)
**HER2 expression**
- HER2 Alexa Fluor-488 conjugate (#FAB1129G-025, Biotechne)
- FlowJo 10
**Live imaging of chromosomal and microtubule dynamics**
- SiR-tubulin probe (#SC002, Spirochrome)
- Nikon Eclipse Ti microscope
**Microtubule tip tracking by EB3-GFP**
- LentiBrite™ EB3-GFP (Sigma, Cat. No. 17-10208)
- Yokogawa spinning disk field scanning confocal system CSU-W1 (Nikon Europe B.V., Strombaan 14, 1181 VX Amstelveen, The Netherlands)
- Photometrics Prime BSI camera
**Sequencing**
- DNeasy Blood 6 Tissue Kit (Qiagen)
- Qubit dsDNA Broad Range quantification assay kit (Thermo Fisher Scientific)
- Bioanalyzer 2100
- High Sensitivity DNA Kit (Agilent Technologies)
- Twist Comprehensive Exome Panel probes
- NovaSeq 6000 platform (Illumina)
- TruSeq low sample protocol (Illumina)
- STAR aligner version 2.7.1a
- DRAGEN Bio-IT Platform v4.0
- htseq-count
Turbidity assay
3 mg/mL Tubulin was mixed on ice in a 96 well plate with 0.6 µM NF1 and/or indicated drugs in a final volume of 100µL in Tubulin Polymerization Buffer (80 mM PIPES pH 6.9, 2mM MgCl2, 0.5mM EGTA, 1mM GTP, 10% glycerol). The polymerization reaction was started by the increase in temperature from 4°C to 37°C upon transfer of the reaction to pre-warmed microtiter spectrophotometer. Tubulin assembly was monitored for one hour at 340 nm in kinetic mode of 61 cycles for each sample and the readings were recorded at an interval of 1 min (Infinite 200 PRO-Tecan plate reader).
In vivo experiments
This protocol requires prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Bioluminescence imaging (BLI) acquisition and analysis. The images were acquired using PerkinElmer’s IVIS Lumina Series III instrument wavelengths (600- 800 nm). Scans were taken with an integrated CCD camera (Andor, Belfast, UK) supercooled down to −80 °C, with a 25 mm focal length lens (Navitar, Rochester, NY). The camera pointed straight down and was focused 10 mm above the imaging membrane. The F-number was kept at f/0.95 throughout the study. Prior to imaging, 200 µL of D-Luciferin (10 mg/mL, XenoLight, Perkin Elmer) were injected intraperitoneally and mice were anesthetised in induction chambers with 1-4% isoflurane. With animals in the supine position, BLI images were acquired after 10 min after luciferin injection. An exposure time of 2s and binning of 4 was used at the beginning of the study, and imaging parameters were updated as the tumors became brighter throughout the study, in order to maximize sensitivity of bioluminescence and avoid pixel saturation. Tumor burden was represented as total flux (photons/s), which is average radiance (flux per unit area and unit solid angle) integrated over the region of interest, using the Living Image v4.7.3 in vivo software package (Perkin Elmer Inc).
Cell imaging
Immunohistochemistry. Formalin fixed and paraffine embedded (FFPE) tumour sections of 3µm thickness were cut and left for an overnight incubation at 37°C before staining. All immunohistochemistry (IHC) were performed using Bond III IHC autostainer (Leica Biosystems). For a full automated IHC staining, the antibodies rabbit monoclonal anti-Caspase 3 Activated (#9661 - Cell Signaling, final dilution of 1:250), rabbit anti-Cleaved Caspase 7 (#9491 - Cell Signaling, final dilution 1:100), rabbit monoclonal anti-cleaved Parp (#5625 - Cell Signaling, final dilution 1:50), and mouse monoclonal antibody anti-BCL-2 (#ab692 - Abcam, final dilution 1:100) were unmasked with EDTA Ph9 (Bond Epitope Retrieval Sol2 Leica AR9640). All the antibodies were diluted with Bond Primary Antibody Diluent (AR9352 - Leica Biosystems), and BOND IHC Polymer Detection Kit (DS9800 - Leica Biosystems) was used to detect antibodies with DAB chromogen and counterstain tissues with haematoxylin. H 26E staining was performed with automated Leica Biosystems ST 5020 instrument, using Kit Infinity 2000 Test (3801698-Leica Biosystems) and pictures of stained sections were acquired with the Aperio ScanScope XT instrument.
Co-localization immunofluorescence of NF1 and microtubules. Cells were seeded (1 × 10^5^ in 12-well plates) on coverslips coated with 0.5% gelatin and allowed to attach overnight. Cells were synchronised and fixed as previously described. Fixed cells were then blocked with 5% Donkey Serum in IF buffer for 1 hour and subsequently incubated with primary antibodies mouse anti-Neurofibromin (MilliporeSigma, NF1-A 376G3, #MABN2557) diluted 1:100 and rabbit anti-alpha-Tubulin (Abcam, Ab18251) diluted 1:200 in IF buffer for 5 hours at RT. Primary antibodies were detected using anti-mouse Alexa Fluor-555 and anti-rabbit Alexa Fluor-488 secondary antibodies (Life Technologies). DAPI (Sigma, D9542) was used for nucleic staining. Images were acquired using the CrestOptics confocal spinning disk X-light V3.
Proximity Ligation Assay
Cells were synchronised and fixed as previously described. Fixed cells were then blocked with 5% Donkey Serum in IF buffer for 1 hour and subsequently incubated with mouse anti-Neurofibromin (MilliporeSigma, NF1-A 376G3, #MABN2557, 1:100 and rabbit anti-alpha-Tubulin (Abcam, Ab18251, 1:200 for 2.5 hours at RT. PLA staining was performed with Duolink® flowPLA Detection Kit – Orange (Sigma-Aldrich, #92102).
Chromosome alignment
Cells were seeded in late G2 with RO3306 (Sigma-Aldrich) at 9 µM for 39h (BT-474) and 20h (HCC1954) at 37°C. Cells were then washed three times with warm medium to release from G2 arrest and put back at 37°C. After 1 hour, cells were treated with MG-132 (Tocris) 10 µM for 90 min at 37°C. Finally, cells were fixed with cold methanol at −20°C for 3 min followed by rehydration in PBS. Fixed cells were then blocked with 5% Donkey Serum in IF buffer (1x TBS + 0,1% Triton X-100 + 2% BSA + 0,1% Sodium Azide in PBS) for 1 hour.
T-DM1 Internalisation
Cells were seeded on 0.5% gelatin-coated coverslips (7.5 × 10^5^ each) and allowed to attach overnight. After a 15 min T-DM1 pulse (1.5 µg/mL), the drug was washed off and cells were either immediately fixed with PFA 4% for 15 min RT or received fresh warm media and incubated at 37°C for 24 h, with or without chloroquine (CQ, 5 µM).
Cells were then fixed (permeabilised) with Triton X-100 0.5% in PBS and a sheep polyclonal anti-Human IgG-Cy5 (AC2115, Sigma) was used for the detection of T-DM1; a FITC-phalloidin (P5282, Sigma) was used for actin and nuclear counterstaining was done with DAPI. Images were acquired with an SP8 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) and a 63x/1.4NA oil immersion objective lens. Multichannel, Z-stack images were acquired with a voxel size of 72x72x200 nm3 by 3 different PMT detectors.
HER2 expression
Cells were blocked with 5% BSA in PBS for 30 minutes and then incubated with primary antibody anti-HER2 Alexa Fluor-488 conjugate (#FAB1129G-025, Biotechne) diluted in 1% BSA in PBS for 1 hour at 4°C in agitation. Flow cytometry was performed on BD FACS Celesta and all data were analysed using FlowJo 10.
Live imaging of chromosomal and microtubule dynamics
BT-474^^WT-H2B-GFP and BT-474^^KO-H2B-mCherry cells were seeded (4 × 10^4^ cells) in µ-Slide 8-well glass bottom chambers (80827, Ibidi). 24 hours later, growing cells were stained for 1.5h before acquisition with the SiR-tubulin probe (#SC002, Spirochrome; λabs 652 nm/λfl 674 nm) at 1 µM. Time-lapse microscopy was performed using an inverted microscope (Nikon Eclipse Ti) with a 20X/0.75NA objective. The microscope was equipped with an incubation chamber maintained at 37°C in an atmosphere of 5% CO2 and acquisitions were made every 4 min. A total of 28 cells were analysed for BT-474^^WT-H2B-GFP and 38 for BT-474^^KO-H2B-mCherry^^.
Microtubule tip tracking by EB3-GFP
To improve image quality, each timelapse movie was denoised with the built-in function Denoise.ai of Nis-Elements AR software (v. 5.42.01). For image analysis, cells were first segmented using Cellpose on GFP fluorescence of the first frame of each field of view, considering that cells were not migrating during the total duration of the acquisition (1 min). Per each field of view, the label image resulting from the segmentation was merged to the stack with EB3 fluorescence, in order to establish the cellular hierarchy of EB3 movements. Following segmentation, a customized script that execute TrackMate software (v. 7.10.2) in Fiji (v. 2.9.0/1.53t) was employed to detect and track EB3 tips throughout the time series: Laplacian of Gaussian detector was used to identify the EB3 spots, Kalman tracker was used to create tracks, and filters on Track duration ( 3e 4 seconds) were used to discard uninformative tracks. The label images from the Cellpose segmentation were integrated into the TrackMate analysis to identify the parent cell of each EB3 track, allowing us to associate specific EB3 dynamics with individual cells. EB3 dynamics was quantified by the Track Mean Speed parameter. Cell surface was calculated by Cellpose.
To calculate dynamic parameters, we selected tracks with confinement ratio 3e0.8 and track displacement 3e1 µm. Catastrophe events were calculated as events/µm^2^/min by dividing the number of tracks per cell by its area. Rescue events were calculated as events/µm^2^/min and were defined as events starting ≤ 1 µm from the ending coordinate of the previous event and ≤ 5 seconds after the previous event, divided by the cell area.
Cell cycle and ploidy
Cells were pelleted at 1200rpm for 5 minutes and fixed with 90% cold Ethanol for 30 minutes on ice, washed once with 1% BSA in PBS and stained with Propidium Iodide (PI) 2,5 ug/ml + RNase 0,25 mg/ml overnight at 4°C. Flow cytometry was performed on BD FACS Celesta and cell cycle gates were adjusted as needed to encompass the G0/G1, S, and G2/M populations. In some experiments, BrdU was pulsed for 1 h on treated cells with T-DM1 or vehicle for 36 h. Cells were fixed and stained according to manufacturer’s instructions (FITC BrdU Flow Kit, BD Biosciences).
Sequencing
DNA
Genomic DNA extraction was performed using the DNeasy Blood 6 Tissue Kit (Qiagen), in accordance with the manufacturer’s instructions. DNA concentrations were carried out using Qubit dsDNA Broad Range quantification assay kit (Thermo Fisher Scientific). For WES, Target regions were captured with Twist Comprehensive Exome Panel probes followed by PCR amplification and purification of the enriched library. Quantification of enriched libraries was performed with Qubit dsDNA High Sensitivity quantification assay kit (Thermo Fisher Scientific) and library size distribution was measured with Bioanalyzer 2100 and High Sensitivity DNA Kit (Agilent Technologies). Final DNA libraries sequencing was performed in Illumina NovaSeq 6000 platform using the NovaSeq 6000 S1 Reagent Kit 300 cycles (2 x 150 paired-end reads) (Illumina). Sequencing data were mapped against hg38 genome and analysed with Illumina DRAGEN Bio-IT Platform v4.0 using proprietary pipelines for variant calling. The resulting VCF with detected variants files were annotated and classified with the GATK-Funcotator.
RNA
mRNA-seq libraries were prepared according to the TruSeq low sample protocol (Illumina, San Diego, CA, USA), starting with 1 µg of total RNA per sample. RNA-seq libraries were pair-end sequenced on an Illumina NovaSeq 6000 sequencing platform. RNA-seq data were mapped using the STAR aligner version 2.7.1a against the human genome (hg19). Read counts were calculated with htseq-count with the standard UCSC reference GFF
Differential expression analysis was done with DESeq2 R package version 1.36.0. Genes of interest were selected using a false discovery rate (FDR) cut-off of 1x10^^-4^. Functional enrichment analysis war performed using EnrichR R package version 3.1 on differentially expressed genes with log2foldchange 3c -1 in NF1 knock-out T-DM1 treated vs NF1 knock-out and log2foldchange 3e -1 and log2foldchange 3c 1 in wild-type T-DM1 treated vs wild-type. Queries were performed over four different molecular signatures: Gene Ontology Biological Process, Gene Ontology Molecular Function, Gene Ontology Cellular Component (https://www.gsea-msigdb.org/gsea/msigdb/download_file.jsp?filePath=/msigdb/release/2022.1.Hs/c5.go.v2022.1.Hs.symbols.gmt) and Hallmark (https://www.gsea-msigdb.org/gsea/msigdb/download_file.jsp?filePath=/msigdb/release/2022.1.Hs/h.all.v2022.1.Hs.symbols.gmt). Enriched terms were selected using a standard FDR cut-off of 1x10^^-2^.
Protocol references
10.1073/pnas.0506580102, 10.1093/bioinformatics/btr260, 10.1016/j.cels.2015.12.004, https://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/genes/hg19.refGene.gtf.gz, https://www.gsea-msigdb.org/gsea/msigdb/download_file.jsp?filePath=/msigdb/release/2022.1.Hs/c5.go.v2022.1.Hs.symbols.gmt, https://www.gsea-msigdb.org/gsea/msigdb/download_file.jsp?filePath=/msigdb/release/2022.1.Hs/h.all.v2022.1.Hs.symbols.gmt