Ongoing chromosomal instability in cancer: real-time imaging and single cell genetics of missegregating chromosomes.

Projektinformationen

OCIC

ID Finanzhilfevereinbarung: 896393

DOI

10.3030/896393

Projekt abgeschlossen

EK-Unterschriftsdatum 15 April 2020

Startdatum 1 Januar 2021

Enddatum 31 Dezember 2022

Finanziert unter

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Gesamtkosten

€ 187 572,48

EU-Beitrag

€ 187 572,48

Koordiniert durch

UNIVERSITAIR MEDISCH CENTRUM UTRECHT
Netherlands

Periodic Reporting for period 1 - OCIC (Ongoing chromosomal instability in cancer: real-time imaging and single cell genetics of missegregating chromosomes.)

Berichtszeitraum: 2021-01-01 bis 2022-12-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

While many cancer treatments are improving, cancer remains a major cause of death and suffering, affecting both patients and their families. Increased life expectancy has further increased cancer incidence, putting a heavy burden on society,increasing pressure and healthcare and costs. The objectives of this project are to add fundamental knowledge on how genetic instability is related to ongoing tumor development.
DNA analysis of patient tumors show many different genomic alterations, ranging from point mutations, gene amplifications/losses, to chromosomal rearrangements such as structural variations. Especially gross chromosomal aberrations involving whole chromosomes or are likely to occur during mitosis, a crucial phase in the cell cycle at which the DNA needs to be correctly distributed over both daughter cells.
The fundamental principles of cell division have been studied in different model systems, invaluable in untangling the genetic components, signaling pathways and timing of the components that orchestrate correct cell division. However, to study cell division in a model system that is most representative for human tumors, including 3 dimensional growth and heterogeneity, compatible with high resolution live-cell imaging, we studied patient derived tumor organoids (PDOs). This model allows to study chromosomal instability (CIN), focusing on chromosomal missegregations and mitotic errors during tumor cell proliferation.
Tumors of esophageal origin, such as esophageal adenoma carcinoma’s (EAC) are of specific interest to study the impact of CIN, since EACs seem not driven by sequential acquisition of a specific driver mutations. Rather, EACs progress through multiple combinations of chromosomal alterations, including copy-number changes and large structural variants.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

We initiated a collaboration with EAC expert, Rebecca Fitzgerald to obtain esophageal organoids for in-depth studies on CIN. We set up multiple organoids lines, with samples representing different aspects on the genomically instability spectrum. I.e. large alterations in chromosome number vs a near normal genetic content, but rampant with structural variants.

1) Multiple genetic fluorescent reporters were introduced to visualize and quantify chromosomal behavior, as well as fluorescent membrane markers to monitor correct formation of daughter cells. This led to the observation that EAC organoids show a range of mitotic errors including anaphase bridges, micronuclei formation or cytokinetic failures.

In a collaborative effort with insights from other cancers 2) an in-depth analysis of CIN phenotypes via live-cell imaging and DNA sequencing was combined to conclude that cellular heterogeneity in terms of chromosomal copy number variations due to ongoing CIN can be used to predict cancer survival across tissues of different origin and disease stage. Our results were incorporated in a larger collaborative effort and published accordingly.

Furthermore we followed up on the exciting discovery that 3) EAC tumors can harbor a specific form of gene amplification; extrachromosomal circular DNA (ecDNA). Improved sequencing methodology/analysis demonstrate that some tumor types are driven by ecDNAs. Their circular nature avoids DNA damage responses and displays non-mendelian inheritance, potentially leading to a rapid increase in copy number.
DNA analysis by our collaborators identified two EAC lines suspected to carry ecDNA with the oncogene KRAS. As bulk sequencing provides an average of copy-numbers, I implemented two novel methodologies to determine cell-to-cell variation. First, to quantify the number of ecDNA copies 4) I developed 2 DNA FISH probes against the KRAS gene. Second, to measure ERK activity, 5) I introduced a FRET biosensor. There is significant interest in the wider Life Science community on ecDNA biology and the functional consequence of KRAS ecDNAs will become part of a scientific publication that is in preparation.

These results have been presented at external meeting; the Oncode community, Oncode Microfluidics Workshop and regularly at departmental meetings within the UMCU. I initiated regular meetings with the pathology department of the UMCU to explore implementation ecDNA KRAS FISH probes in a diagnostic setting.

Overview and dissemination:
1&2 - reported in (departmental) meetings and a publication
3 - reported at meetings and follow up with pathology dept. and with a bioinformatics group investigating clinical relevance and improved detection.
4 - exploited knowledge of developing in-house gene specific DNA probes, and creation of KRAS probes. Pipeline has been presented at meetings.
5 - Ongoing investigation (conjuncted to 4) to single cell dissect gene amplifications to single cell signaling. Presented both internally and externally.

My data and results were presented at the University of Cambridge (UK), also via regular virtual meeting with the Fitzgerald lab, and two members of her lab have visited our lab, where I provided training in live-cell microscopy and patient organoid model culturing.
I engaged in teaching multiple students (bachelor and master) during this project in lab work and a literature thesis. Additionally, I have discussed my work and career path during the PhD career event of the Graduate School of Life Sciences.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

This project initially focused on DNA damage upon CIN in EAC organoids, but refocused on the unique situation to study the occurrence of KRAS ecDNAs in esophageal cancers, as well as their impact on oncogenic signaling networks to drive tumor progression. The work in this project has greatly advanced the state of the art by the following results.
1. Quantification of various mitotic error phenotypes in EAC via live-imaging of genetic fluorescent reporters in organoids
2. DNA sequencing of EAC organoid lines and analyzing their chromosomal heterogeneity score in bulk, as a result of CIN levels, extrapolated to patient outcome
3. Discovering EAC organoids carrying ecDNA copies with KRAS
4. Establishing methodology to measure ERK signaling in real time in single cells of EAC organoids
5. Developed DNA FISH probes for KRAS ecDNA detection and quantification per single tumor cell.
In this project I have created access to organoid lines from a novel tissue of origin for our laboratory. I employed live-cell microscopy to FRET biosensors with DNA FISH linking DNA alterations and cellular signaling. This project has sparked discussions on ecDNA emergence and propagation, exposure of ecDNA to the cytoplasm with DNA damage responses, and whether EACs that carry KRAS ecDNAs should be effectively treated with tailored treatments. Hence, my explorations with our pathology department whether DNA FISH probes against KRAS ecDNAs can be applied for diagnostic purposes.
This work has led to a preliminary collaboration with the de Ridder group, experts in detecting circular DNA fragments via liquid biopsies. This project influenced their research on a fundamental level and applied perspective for diagnostic purposes, in collaboration with their spin off company, Cyclomics.
Taken together, I am excited to continue my study on the biology of ecDNAs with an interdisciplinary combination of (single) cell DNA sequencing, live-cell microscopy and clinical perspective to increase our understanding of ecDNAs in cancer biology and potentially improve ecDNA detection for patient stratification towards optimal therapeutic interventions.

Esophageal organoids show different genomic instability phenotypes