Retrospective identification of rare cell populations underlying drug resistance connects molecular variability with cell fate
Posted on: 14 April 2020
Preprint posted on 19 March 2020
Pioneering a new technique called Rewind, Emert et al. connect differences in gene expression and MAPK signaling with cell fate outcomes to retrospectively identify the rare cell populations that arise during targeted cancer therapy.
Selected by NYUPeerReview
Categories: cancer biology, cell biology, genetics
Context
Resistance to cancer drugs remains a significant hurdle for effective treatment across many cancer types. This primarily stems from poor molecular understanding of how a subset of cancer cells survives treatment and facilitates cancer recurrence, while most cells are killed by drug treatment. While previous work shows that drug resistance is conferred by new mutations arising during treatment, existing heterogeneity among cells is believed to contribute as well, as rare populations can exist in a state “primed” for drug resistance. Thus, identification of molecular differences that distinguish these rare “primed” cancer cells can inform strategies for alternative therapeutic intervention or identify biomarkers that better predict prognosis following drug treatment. However, attempts to do so have largely been challenging from a technical standpoint, including a limited ability to (1) detect and isolate rare cells, and (2) monitor molecular processes over an extended time scale (such as in time lapse microscopy).
Impact
This preprint caught our attention because it described a clever system capable of overcoming the technical limitations highlighted above. The authors’ method, Rewind, not only allows for identification and tracking of rare cells of interest at different timepoints, but also enables their isolation for use in molecular assays. By introducing unique DNA barcodes into tens of thousands of cells, the authors can identify resistant cells surviving anti-cancer drug treatment through sequencing. They then can “rewind” and isolate clonally related cells in the pre-treatment population by FACS sorting for FISH probes targeting those same barcodes. To demonstrate how Rewind can be used, the authors asked if these resistant cells expressed different genes compared to the rest of the population, reflecting a “primed” state. They confirmed that “primed” cells express known markers associated with resistance, but they were also able to identify many new markers.
From a broad perspective, Rewind has the potential to address questions beyond initial events driving drug resistance and cancer model systems. Rewind could be used for instance to trace immune cell development or understand molecular details of senescence. The possibilities are endless! Rewind also comprises simple molecular techniques that are available to most research labs, making it a relatively accessible approach. We commend the authors for supporting reproducibility by providing highly detailed methods and protocols supplemented with links to pipelines and their raw data.
Key Findings
- The authors present Rewind, which is able to identify and isolate rare cells primed for chemotherapy resistance, present in only 0.05% of the cellular population, by tracking unique DNA barcodes through sequencing. These primed cells expressed previously identified resistance markers, like EGFR and AXL, thus validating their method. They additionally identified about 200 new resistance markers, such as ITGA3.
- Single molecule FISH analysis showed that about 87% of primed cells expressed many resistance markers simultaneously, indicating a coordinated primed signature rather than sporadic expression of resistance markers in the population.
- Using Rewind, the authors outlined transcriptional differences between populations of “highly resistant” primed cells, “less resistant” primed cells, and unprimed cells. This highlights the heterogeneity that exists within these rare primed populations, which may inform more effective interventions to prevent cancer recurrence.
- The authors explored the broad applicability of Rewind by asking several other questions relevant to vemurafenib resistance in a melanoma cell line. These include exploring induced resistance by DOT1L inhibition and how vemurafenib affects phosphorylation levels of kinases implicated in proliferation.
Questions for Authors
- The authors assayed the transcriptomes of the rare primed cells before vemurafenib treatment. What else can be done with these cells beyond RNA-Seq? Would it be possible to adapt this protocol for mass spectrometry to understand proteomic differences, or to use ATAC-Seq and ChIP-Seq to look into chromatin states and interactions?
- What is the sensitivity limit for tagging rare cells? How low can you go? How does it compare to other competing approaches?
- The authors define cells contributing the top 60 ranked barcodes after vemurafenib treatment as their resistant population, and further classify the barcodes ranked 1-30 as highly resistant, and barcodes 31-60 as less resistant. Was there specific rationale behind setting these thresholds, or were these chosen arbitrarily?
doi: https://doi.org/10.1242/prelights.18604
Read preprint
(No Ratings Yet)Have your say
Sign up to customise the site to your preferences and to receive alerts
Register hereAlso in the cancer biology category:
Integrin conformation-dependent neutrophil slowing obstructs the capillaries of the pre-metastatic lung in a model of breast cancer
Simon Cleary
Mitochondria-derived nuclear ATP surge protects against confinement-induced proliferation defects
Teodora Piskova
Spatial transcriptomics elucidates medulla niche supporting germinal center response in myasthenia gravis thymoma
Jessica Chevallier
Also in the cell biology category:
Motor Clustering Enhances Kinesin-driven Vesicle Transport
Sharvari Pitke
Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis
Ankita Walvekar
Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water
Safieh Shah, Benjamin Dominik Maier
Also in the genetics category:
Intracellular diffusion in the cytoplasm increases with cell size in fission yeast
Leeba Ann Chacko, Sameer Thukral
HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos
Anchel De Jaime Soguero
Significantly reduced, but balanced, rates of mitochondrial fission and fusion are sufficient to maintain the integrity of yeast mitochondrial DNA
Leeba Ann Chacko
preLists in the cancer biology category:
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
| List by | Helen Zenner |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
| List by | Osvaldo Contreras |
Single Cell Biology 2020
A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.
| List by | Alex Eve |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
Anticancer agents: Discovery and clinical use
Preprints that describe the discovery of anticancer agents and their clinical use. Includes both small molecules and macromolecules like biologics.
| List by | Zhang-He Goh |
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
| List by | Joseph Jose Thottacherry |
Also in the cell biology category:
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
preLights peer support – preprints of interest
This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.
| List by | preLights peer support |
The Society for Developmental Biology 82nd Annual Meeting
This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.
| List by | Joyce Yu, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
| List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
| List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
| List by | Ana Dorrego-Rivas |
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
| List by | Dey Lab, Samantha Seah |
1
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
| List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
| List by | Paul Gerald L. Sanchez and Stefano Vianello |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
| List by | Dey Lab |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
| List by | Sandra Malmgren Hill |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
| List by | Pablo Ranea Robles |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
| List by | Dey Lab |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
| List by | Sandra Franco Iborra |
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
| List by | Dey Lab, Amanda Haage |
Also in the genetics category:
BSDB/GenSoc Spring Meeting 2024
A list of preprints highlighted at the British Society for Developmental Biology and Genetics Society joint Spring meeting 2024 at Warwick, UK.
| List by | Joyce Yu, Katherine Brown |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University
This preList contains preprints discussed during the 'Semmelweis Symposium 2022' (7-9 November), organised around the 40th anniversary of international medical education at Semmelweis University covering a wide range of topics.
| List by | Nándor Lipták |
20th “Genetics Workshops in Hungary”, Szeged (25th, September)
In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf
| List by | Nándor Lipták |
2nd Conference of the Visegrád Group Society for Developmental Biology
Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)
| List by | Nándor Lipták |
EMBL Conference: From functional genomics to systems biology
Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020
| List by | Jesus Victorino |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
| List by | Sandra Malmgren Hill |
Zebrafish immunology
A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.
| List by | Shikha Nayar |
5 years
BENJAMIN EMERT
Wow, thank you for the thoughtful summary and discussion. Regarding your questions for the authors:
1. We’re hopeful that the approach can be combined with additional downstream assays (beyond RNA-seq, RNA-FISH and immunofluorescence) that are compatible with fixed cells (formaldehyde or methanol). With a first attempt at ATAC-seq (using the ATAC-SEE protocol) the tagmentation looked incomplete on the bioanalyzer and I haven’t gone back to try optimizing. I suspect that you could selectively amplify and sequence integrated barcodes from an ATAC-seq library so if the phenotype is not too rare, you could try connecting chromatin accessibility to fate using scATAC-seq (analogous to what was done in Biddy et al and Weinreb and Rodriguez-Fraticelli et al.).
We have not tried ChIP-seq with Rewind due to the cell number requirement. Also, another student in lab has had trouble doing ChIP-seq on cells post smFISH. CUT & RUN/CUT & TAG might be a good alternative for rare cell populations although from what I’ve read, you may need to start with a larger number of cells if they’re fixed.
We haven’t seriously considered combing proteomics with Rewind and I don’t know if our fixation and FISH protocols would need to be modified or are simply incompatible with mass spec.
2. In the DOT1L inhibitor experiments (figures 5 and 6) we isolated (by FACS) and identified (by imaging) primed cells that were at a frequency of <1:10,000 cells. That number is based on using probes targeting 30 barcodes out of 400,000 barcodes introduced at the start and is consistent with the frequency of FISH-positive cells we found by FACS and by imaging. I haven't tried isolating anything more rare than that. I believe this is slightly more sensitive than COLBERT or the method described in Feldman et al.
If you need to increase sensitivity/specificity further, you could try the following: A. Increase the length of the barcodes to fit more FISH probes. We have some designs to keep sequencing costs down. B. Use combinations of fluors to detect different barcodes (a la SeqFISH/MERFISH or Feldman et al 2019). Computationally shouldn't be too difficult since you should only need to register cells round to round, not spots. C. Enrich for the cells of interest ahead of time using some marker with high sensitivity but possibly low specificity D. Tolerate greater false positives in the Rewind sort then perform scRNA-seq to determine which cells actually carry the barcodes of interest.
3. That first threshold for defining which barcodes to probe was based on A. The frequency of colonies emerging from cells treated with the chosen concentration of drug. In general we wanted the expected frequency of resistance to be greater the the fraction of barcodes we probed. B. How many barcodes can we probe simultaneously while still feeling confident in our detection. Since the cells we were interested in are so rare initially, we optimized protocols and set thresholds to minimize false positives as much as possible. Other application may require limiting the false negatives.
In terms of splitting the barcodes into "highly resistant" and "less resistant", that decision was mostly arbitrary. In retrospect (or in future experiments), it might've made more sense to separate the rankings further or probe additional quantiles.
I hope I understood your questions and addressed them at least somewhat. Thank you again for taking the time to review our work 🙂