Periodic Reporting for period 1 - PERSIST-SEQ (Building a reproducible single-cell experimental workflow to capture tumour drug persistence)
Période du rapport: 2021-07-01 au 2022-06-30
PERSIST-SEQ’s mission is to develop a standardised approach to single-cell sequencing workflows for the study of cells pre-treatment, at minimal residual disease (‘drug tolerant persister cells’) and at relapse. In so doing, we will create a connected community of researchers and industry parties skilled in the generation and interpretation of single- cell RNA-seq data. PERSIST-SEQ’s gold standard operating procedures and centralised European data infrastructure are envisioned to be sustained and built upon in an open access model, reducing duplication of effort, promoting collaboration across disciplines, and ensuring efficient adoption of state-of-the-art single cell technologies.
O1. Design and evaluate a reproducible single-cell sequencing workflow that can robustly extract and disentangle genetic, epigenetic and expression signatures that determine inherent and acquired resistance at the single-cell level, in the context of tumour cell populations of any level of diversity and the tumour microenvironment. This workflow will include the following steps: (1) Experimental design (2) Sampling, coding, storage and logistics, (3) Single-cell extraction and barcoding, (4) Single-cell & spatial sequencing, (5) Data storage, analysis, integration and interpretation and (6) Guidance on follow-up experiments.
O2. Incorporate beyond-state-of-the-art single-cell sequencing approaches and where necessary refine and improve them to handle complex biological samples (see Box 1 above for details).
O3. Develop novel PDO and/or PDX-based approaches to study the biology of plasticity in drug tolerant persister cells (DTPs).
O4. Select and analyse at the single cell level, tumour samples from patients affected by solid tumours (e.g. CRC) at baseline, on response and at progression to specific treatments.
O5. Devise blueprints (SOPs, guidelines, technical requirements, and quality assurance measures) of the developed sequencing workflow and experimental approaches to enable their reproducibility across academic and industrial research settings.
O6. Expand existing data infrastructures to provide wide access to single-cell sequencing data (Open Data) and ensure FAIRness of results, in line with parallel initiatives such as the Human Cell Atlas and COSMIC.
O7. Identify and replicate the molecular signatures of DTPs in clinical samples, cell lines, and 3D models (PDOs and PDXs).
O1. Design and evaluate a reproducible single-cell sequencing workflow that can robustly extract and disentangle genetic, epigenetic and expression signatures that determine inherent and acquired resistance at the single-cell level, in the context of tumour cell populations of any level of diversity and the tumour microenvironment. This workflow will include the following steps: (1) Experimental design (2) Sampling, coding, storage and logistics, (3) Single-cell extraction and barcoding, (4) Single-cell & spatial sequencing, (5) Data storage, analysis, integration and interpretation and (6) Guidance on follow-up experiments.
O2. Incorporate beyond-state-of-the-art single-cell sequencing approaches and where necessary refine and improve them to handle complex biological samples (see Box 1 above for details).
O3. Develop novel PDO and/or PDX-based approaches to study the biology of plasticity in drug tolerant persister cells (DTPs).
O4. Select and analyse at the single cell level, tumour samples from patients affected by solid tumours (e.g. CRC) at baseline, on response and at progression to specific treatments.
O5. Devise blueprints (SOPs, guidelines, technical requirements, and quality assurance measures) of the developed sequencing workflow and experimental approaches to enable their reproducibility across academic and industrial research settings.
O6. Expand existing data infrastructures to provide wide access to single-cell sequencing data (Open Data) and ensure FAIRness of results, in line with parallel initiatives such as the Human Cell Atlas and COSMIC.
O7. Identify and replicate the molecular signatures of DTPs in clinical samples, cell lines, and 3D models (PDOs and PDXs).
WP1
• Annual general meeting -first meeting with all of the PERSIST-SEQ partners is scheduled for 21 September 2022 and to be hosted by AstraZeneca in Cambridge, UK
• Lygature has created a PERSIST-SEQ SharePoint for document storage, project updates and to facilitate communication across all of the Efpia and non-Efpia partners (https://lygatureprojectplaza.sharepoint.com/sites/PERSIST-SEQ).
• A dedicated website has been created for PERSIST-SEQ (https://persist-seq.org/) to allow external communication, news and announcements of important discoveries/impact and publications.
• Established an ethics review panel to determine the criteria for projects to ensure that they are compliant with EU regulations on ethics of human material samples.
• a Management Team has been established and meets monthly to coordinate and prioritise activity in PERSIST-SEQ – decisions and updates are communicated directly to the lead investigators of all partner organisations.
• Created a bi-monthly newsletter of key updates that will also be sent to the lab members of each program lead.
• Each work package has 2 leads (Efpia and non-Efpia) and meet on a regular basis to plan and prioritise activity based on the tasks and deliverables of each work package - work packages 3 and 4 (Standardisation and benchmarking of Standard Operating Procedures; Single-cell acquisition from Models of Tumour Plasticity) and 7 (Analytical methods, integration and interpretation of single-cell datasets) are the most active at present focused as they are on the creation of SOPs for generating single cells for sequencing and the downstream analyses of that data.
WP2
• The Management team has created a Study Technical Plan that describes the proposed samples for submission for single-cell sequencing and that must be completed by each partner and approved by the team before the samples can be submitted. This Study Technical Plan includes experimental design information, whether any ethical constraints on data sharing, suitable QC metrics generated and analysis plans.
WP3, WP4, WP5
• The work performed by this group has led to the creation of the first SOPs for dissociating cancer cell lines or 3D organoids and cryopreservation prior to shipping for single-cell sequencing at Single Cell Discoveries, Netherlands. These SOPs have been uploaded to the PERSIST-SEQ SharePoint for all partners to access and will be used for the first 2 studies submitted.
• A tracking excel file have been created, and shared on the SharePoint, to capture all relevant metadata for any samples submitted including drug treatment, tumour type etc.
• A number of pilot studies are underway to test different ways to cryopreserve single cells as well as best methods to dissociate more difficult tissue samples such as patient-derived xenografts and clinical samples – based on these data we will define for the consortium preferred SOPs for single cell collection and make available via the PERSIST-SEQ SharePoint site
WP6
• At present we have limited activity in this WP given the focus initially on establishing robust single cell sequencing protocols using the 10X Genomics platform – we anticipate that this WP will be most relevant for clinical sample studies or in vivo mouse models.
WP7
• Established a monthly WP7 meeting that includes the lead Bioinformatician for each Efpia and non-Efpia partner to identify and prioritise single cell analysis algorithms that should be incorporated into the analysis pipeline that will be used to analyse all samples and is web-accessible to the bioinformaticians of each partner.
• Decision to use established Galaxy workflow created to support the Human Cell Atlas at the European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus and to part fund a position in the EBI lab of Irene Papatheodorou (Gene Expression Team Leader, EMBL-EBI) – using an established platform as the foundation for PERSIST-SEQ (rather than building as new) will allow us to start analyses quickly. The Galaxy workflow is ideal to add additional modules for data analysis as we identify these from publications/conferences as part of the WP7 monthly meetings.
• Annual general meeting -first meeting with all of the PERSIST-SEQ partners is scheduled for 21 September 2022 and to be hosted by AstraZeneca in Cambridge, UK
• Lygature has created a PERSIST-SEQ SharePoint for document storage, project updates and to facilitate communication across all of the Efpia and non-Efpia partners (https://lygatureprojectplaza.sharepoint.com/sites/PERSIST-SEQ).
• A dedicated website has been created for PERSIST-SEQ (https://persist-seq.org/) to allow external communication, news and announcements of important discoveries/impact and publications.
• Established an ethics review panel to determine the criteria for projects to ensure that they are compliant with EU regulations on ethics of human material samples.
• a Management Team has been established and meets monthly to coordinate and prioritise activity in PERSIST-SEQ – decisions and updates are communicated directly to the lead investigators of all partner organisations.
• Created a bi-monthly newsletter of key updates that will also be sent to the lab members of each program lead.
• Each work package has 2 leads (Efpia and non-Efpia) and meet on a regular basis to plan and prioritise activity based on the tasks and deliverables of each work package - work packages 3 and 4 (Standardisation and benchmarking of Standard Operating Procedures; Single-cell acquisition from Models of Tumour Plasticity) and 7 (Analytical methods, integration and interpretation of single-cell datasets) are the most active at present focused as they are on the creation of SOPs for generating single cells for sequencing and the downstream analyses of that data.
WP2
• The Management team has created a Study Technical Plan that describes the proposed samples for submission for single-cell sequencing and that must be completed by each partner and approved by the team before the samples can be submitted. This Study Technical Plan includes experimental design information, whether any ethical constraints on data sharing, suitable QC metrics generated and analysis plans.
WP3, WP4, WP5
• The work performed by this group has led to the creation of the first SOPs for dissociating cancer cell lines or 3D organoids and cryopreservation prior to shipping for single-cell sequencing at Single Cell Discoveries, Netherlands. These SOPs have been uploaded to the PERSIST-SEQ SharePoint for all partners to access and will be used for the first 2 studies submitted.
• A tracking excel file have been created, and shared on the SharePoint, to capture all relevant metadata for any samples submitted including drug treatment, tumour type etc.
• A number of pilot studies are underway to test different ways to cryopreserve single cells as well as best methods to dissociate more difficult tissue samples such as patient-derived xenografts and clinical samples – based on these data we will define for the consortium preferred SOPs for single cell collection and make available via the PERSIST-SEQ SharePoint site
WP6
• At present we have limited activity in this WP given the focus initially on establishing robust single cell sequencing protocols using the 10X Genomics platform – we anticipate that this WP will be most relevant for clinical sample studies or in vivo mouse models.
WP7
• Established a monthly WP7 meeting that includes the lead Bioinformatician for each Efpia and non-Efpia partner to identify and prioritise single cell analysis algorithms that should be incorporated into the analysis pipeline that will be used to analyse all samples and is web-accessible to the bioinformaticians of each partner.
• Decision to use established Galaxy workflow created to support the Human Cell Atlas at the European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus and to part fund a position in the EBI lab of Irene Papatheodorou (Gene Expression Team Leader, EMBL-EBI) – using an established platform as the foundation for PERSIST-SEQ (rather than building as new) will allow us to start analyses quickly. The Galaxy workflow is ideal to add additional modules for data analysis as we identify these from publications/conferences as part of the WP7 monthly meetings.
The project will deliver (a) a state-of-the-art single cell analysis pipeline in a cloud-based environment that is specific to the analysis of cancer models and (b) insights into the biology of cancer persister cells made from analysis of single cells from a range of cancer models and drug treatments using this analysis workflow. The project will generate among the largest such single-cell data from cancer models and we will constantly publish such data as we make new discoveries in this field. Both of these outputs will have impacts in the field of cancer research (providing new cancer targets and identification of the pathways that persister cells use to avoid cell death) and in the socio-economic arena (new drug targets will accelerate drug discovery programs in this area and ultimately lead to more effective treatments for cancer patients).