Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer

  1. Andrea Sacchetti
  2. Miriam Teeuwssen
  3. Mathijs Verhagen
  4. Rosalie Joosten
  5. Tong Xu
  6. Roberto Stabile
  7. Berdine van der Steen
  8. Martin M Watson
  9. Alem Gusinac
  10. Won Kyu Kim
  11. Inge Ubink
  12. Harmen JG Van de Werken
  13. Arianna Fumagalli
  14. Madelon Paauwe
  15. Jacco Van Rheenen
  16. Owen J Sansom
  17. Onno Kranenburg
  18. Riccardo Fodde

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Abstract

Phenotypic plasticity represents the most relevant hallmark of the carcinoma cell as it bestows it with the capacity of transiently altering its morphological and functional features while en route to the metastatic site. However, the study of phenotypic plasticity is hindered by the rarity of these events within primary lesions and by the lack of experimental models. Here, we identified a subpopulation of phenotypic plastic colon cancer cells: EpCAM lo cells are motile, invasive, chemo-resistant, and highly metastatic. EpCAM lo bulk and single-cell RNAseq analysis indicated (1) enhanced Wnt/β-catenin signaling, (2) a broad spectrum of degrees of epithelial to mesenchymal transition (EMT) activation including hybrid E/M states (partial EMT) with highly plastic features, and (3) high correlation with the CMS4 subtype, accounting for colon cancer cases with poor prognosis and a pronounced stromal component. Of note, a signature of genes specifically expressed in EpCAM lo cancer cells is highly predictive of overall survival in tumors other than CMS4, thus highlighting the relevance of quasi-mesenchymal tumor cells across the spectrum of colon cancers. Enhanced Wnt and the downstream EMT activation represent key events in eliciting phenotypic plasticity along the invasive front of primary colon carcinomas. Distinct sets of epithelial and mesenchymal genes define transcriptional trajectories through which state transitions arise. pEMT cells, often earmarked by the extracellular matrix glycoprotein SPARC together with nuclear ZEB1 and β-catenin along the invasive front of primary colon carcinomas, are predicted to represent the origin of these (de)differentiation routes through biologically distinct cellular states and to underlie the phenotypic plasticity of colon cancer cells.

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  1. Version published to 10.7554/elife.61461 on eLife
  2. Version published to 10.1101/2020.07.24.219238v2 on bioRxiv
  3. eLife

    ###Reviewer #3:

    This work by Sacchetti et al. describes how phenotypic plasticity contributes to local invasion and metastasis formation in colon cancer cells. Based on human classical colon carcinoma cell lines and cell sorting they identified a subpopulation of colon cancer cells that are CD44hi/EpCAMlo cells which have enhanced phenotypic plasticity that underlies enhanced invasion and metastatic behavior. In these EpCAMlo cells elevated ZEB1 expression has been identified. Increased WNT signaling results in elevated expression of the EMT associated transcription factor ZEB1. The EpCAMlo expression status is linked with the CMS4 subgroup of human malignant colon cancer. Overall this is an interesting and well written paper for which I offer a few supportive questions/remarks.

    Major comments:

    1. Page 6: The miR-200 family of miRNAs is targeting the mRNA of transcription factors ZEB1 and ZEB2 in epithelial cells but this is not transcriptional regulation

    2. A clear association of EpCAMlo cells and elevated ZEB1 expression is identified. Conditional knockdown of ZEB1 results in a strongly decreased number of EpCAMlo cells. For now, it is not clear if ZEB1 KD results in the death of these EpCAMlo cells or that the mesenchymal gene signature is controlled by ZEB1. The functional contribution of ZEB1 as an EMT inducer should be experimentally proven as for now the role of ZEB1 is not clear.

    3. The importance of the role of EMT is not well established so far in the manuscript in relation to resistance to chemotherapeutic drugs and metastasis. Conditional KD of ZEB1 in metastasis and therapy resistance assays should be added otherwise the title and the claims made in the abstract should be tuned down.

    4. The use of AKP organoids brings further relevance to this research manuscript. Are these EpCAMlo cells also present in the AKP organoids and what is the endogenous expression status of ZEB1 in the AKP organoids?

    5. Why have the authors maintained the conditional expression of ZEB1 induced in the AKP-Z organoid transplantation experiments? This is driving the epithelial cells in a locked mesenchymal state - which is not compatible with the earlier observed plasticity with the EpCAMlo cells in SW480 and HCT116 cells. Also mesenchymal to epithelial transition is generally believed to be essential for metastasis formation. The experimental outcome of these experiments is not relevant and the authors should consider temporal ZEB1 expression control in transplanted AKP-Z organoids.

    6. The data depicted in Fig 10A & B are confusing and deserve a better explanation. How is it possible that EpCAMlo and EpCAMhi sorted cells show overlapping single cell expression profiles upon t-sne plotting in particular for the SW480 cells. This is very contradictory as the authors claim earlier in the manuscript that EpCAMlo cells have a more mesenchymal gene expression profile which is then confirmed with the 'EMT signature' analysis. Is there a difference between EpCAM protein expression and EpCAM mRNA expression?

    7. The Heatmap from the EMT signature shown in figure 10B is representing which cell line?

    Overall the authors link the gene expression signature of EpCAMlo with the colon cancer consensus molecular subtype CMS4 which has the worst relapse free and overall survival (Dienstmann R et al. 2017; 17, Nat Rev Cancer 79-92). There are multiple lines of evidence that the mesenchymal signature in CMS4 colon cancers is due to profound infiltration of stromal cells (CAFs, immune cells), extracellular matrix remodeling, TGF-beta pathway activation and not the consequence of EMT in cancers cells (e.g. Calon et al. 2015; DOI: 10.1038/ng.3225). It is of course possible that a few epithelial cells in this inflammatory context are undergoing a partial EMT but there is little evidence and this likely will happen in a minority of cells. Together, the authors should revise their manuscript regarding (partial) EMT and the CMS4 and put their findings in a more critical context.

  4. eLife

    ###Reviewer #2:

    The manuscript by Fodde et al investigates the presence of a population of colorectal cancer cells within commonly used human cell lines that have a propensity to form metastasis to the liver and lung. These cells are marked as being CD44HiEpCamlo and have increased expression of the EMT marker Zeb1. They show that this population of EpCam-low cells is able to drive metastatic colonisation and that this is likely due to levels of Zeb1. These cells have a signature similar to the CMS4 group of colorectal cancers, which are highly invasive.

    The manuscript is generally well written and presented in a stepwise and straightforward manner so is relatively easy to follow.

    There is a lot of data presented in this paper with 10 primary figures and a number of supplementary figures. I would encourage the authors to look at which data needs presenting and ask whether some of the earlier figures in particular could be combined and the paper streamlined...its by the time you get to the really interesting data in the organoid transplantation and scRNA seq there has been a lot to get through already.

    There are some questions I have about the experimental data and presentation:

    1. Whilst the authors investigate the expression of EpCam and CD44 in cell lines, is there any evidence of this EpCam-low population in primary human tumours? or primary tumours in the mouse? I appreciate that finding these cells in human could be rate limiting, but what about in tumours that are generated in mice and are metastatic - specifically I am thinking about the recent work in colon showing that Notch signalling drives colonic to liver metastasis (Jackstadt et al 2019) - do the Notch active cells in this model have lower EpCam levels?

    2. For the FACS plots could the authors include their complete gating and FMO control gating strategy in the supplementary. It would be helpful to be able to confirm that the shifts the authors are describing are real.

    3. In figure 2, can the authors quantify the protein expression of Ecad and Zeb1? In one of the panels of the CD44 high EpCam low (SW480 cells) there seems to be cells with quite high levels of EpCam - having a quantified measure of these proteins in the two populations would be important here.

    4. It was very interesting that the different populations gave rise to different metastatic rates following injection through the spleen. Do the authors have information on whether this is because the different populations move out of the spleen and into the liver at different rates (so initiation/seeding) is different or is this a consequence of proliferation i.e. both cell populations colonise the liver, but only the EpCam-low population sticks around and colonises the tissue? Further to this, can the authors delete Zeb1 in the EpCam-low cells (as they have done in vitro) and show that colonisation is Zeb1 dependent - this latter point would not be considered essential given the following overexpression experiments.

    5. Much of the metastatic quantification is done through IVIS imagine (from what I can see) - have the authors pathologically quantified the number and size of tumours following ZEB1 overexpression in AKP derived metastasis with histology?

    6. The authors concede that the continuous activation of Zeb1 following transplantation of AKP organoids (pg9 of the PDF) could be the reason that metastatic colonisation is not as impressive as hoped - have the authors considered pulling Dox to initiate metastatic colonisation of the liver and then withdrawing Dox to favour proliferation following metastatic seeding? It would be interesting to know whether the timing of Zeb1 expression is important for this phenotype.

    7. As Wnt signalling is important in the establishment of the EpCam-low population, have the authors inhibited this pathway (either at the ligand level or through inhibiting b-cat transcription) to confirm that the population is Wnt responsive?

    8. Finally, linked to point 7. In the scRNA sequencing, in the populations that have increased EMT and EMT-gene expression, does this correlate to a Wnt/B-catenin signature on a single cell level?

  5. eLife

    ###Reviewer #1:

    Sacchetti and co-workers have employed established human colorectal cancer cell lines to identify a subpopulation of colorectal cancer (CRC) cells (CD44 high/EpCAM low) which represent cells with high tumorigenicity and malignancy in vitro and in vivo. These cells can also be found in patient-derived tumor organoids and in patient samples. Using bulk and single cell RNA sequencing and subsequent functional validation they go on to demonstrate that enhanced canonical Wnt signaling mediates the expression of the EMT transcription factor ZEB1 and with it an EMT-like process. Consistent with this observation, this cell population exhibits higher drug resistance as compared to the parental cells or to CD44 high/EpCAM high cells. They finally employ a number of cutting-edge computational analysis to classify several subgroups within the EMT cell subpopulation which seem to represent various stages of the EMT continuum, and thus may exhibit various degrees of cell plasticity. The particular gene expression signatures of the identified subpopulations also correlate with poor clinical outcome and with the CMS4 subclass of poor prognosis CRC.

    Overall, the manuscript is presented in a straightforward and concise manner, the experimental approaches are thoughtfully designed and appropriately controlled. However, some of the results, in particular of the first part, are not specifically novel. The correlation between CRC invasion and nuclear -catenin and ZEB1 has been reported before, as actually appropriately cited by the authors. Moreover, the migratory and invasive and pro-metastatic and drug-resistant phenotype of ZEB1-expressing, EMT-like cancer cells have been shown before and are as expected. Finally, as detailed below, the mechanisms regulating the homeostasis of the EpCAM-low and EpCAM-high cells in cell culture and in organoids in vitro and in cancers in vivo remain elusive. While the novel insights into the potential trajectories of the genesis of the various subpopulations and the respective gene signatures is exciting, the functional validation of these signatures for the definition of cell plasticity and the actual establishment and functional validation of an identifiable gene signature for cell plasticity has not been directly addressed. Along these lines, the report goes with the mainstream literature in using the term "cell plasticity" with a rather vague description. Is it defined by EMT in general or only by a specific hybrid stage of EMT, by therapy resistance, by differentiation potential, by the reversibility of processes, by stemness, etc.? How can it be functionally tested? The manuscript, as it stands, is not adding tangible data and information on how to identify cell plasticity and what it means in terms of identifying and assessing novel therapeutic targets.

    Specific comments:

    Introduction: the literature on the role of Prrx1 in EMT/MET and the need of MET for metastatic outgrowth should be mentioned already in the Introduction. The discovery and functional characterization of the various EMT stages should also be mentioned already in the Introduction, not only in the Discussion. Finally, the term cell plasticity should be defined in the Introduction, at least how it is used in the following chapters.

    Figure 1/Suppl.1: "similarly variable"? There is a variability of 0 - 99.6% for the levels of the CD44 -igh/EpCAM-low subpopulation in the different CRC cell lines. Notably, there is no correlation of the levels of this subpopulation with the CMS classification of CRC origin, as is claimed later with CMS4.

    Why do the EpCAM-low cells get lost during long-term culture and turn into EpCAM-high, E-cadherin-high cells? How then is the homeostasis between the EpCAM-high and low populations maintained in the parental cells which have been cultured for decades? Also, almost all single cell cones of EpCAM-low cells turn into EpCAM-high over time. Why are some maintaining the EpCAM -ow status? Is there a difference in gene expression or epigenetic imprints? Has the fetal calf serum been stripped of TGF or does it still contain TGF which could induce an EMT?

    Figure 5E, text: the reversibility of EMT by a MET is here used as equal to cell plasticity. Is this a correct definition of cell plasticity (see also above)? The EpCAM-low status seems rather unstable and not metastable in vitro and in vivo, this may not represent the homeostasis of EMT induction and its reversion and thus not true cell plasticity.

    Figure 6: The induction of an EMT by ZEB1 is not new or unexpected as is the increase of metastasis, even though the latter is not statistically significant here. The "excuse" that the incidence of metastasis could be higher, when ZEB1 expression would have been stopped by removing Dox, could have been actually tested. This would be a more meaningful experiment.

    Figure 7: RNA sequencing identifies Wnt signaling to be enhanced in EpCAM-low cells. GSK inhibition induces the expression of ZEB1 (as known before), yet this works only in HCT116 and not in SW480 cells, which actually show an induction of Wnt signaling. The results seem to indicate that there is not just a mere enhancement of Wnt signaling and that other changes/pathways are required as well. What about other cell lines?

    Is the prognostic and predictive value for the gene signature only true for CMS4 CRCs or for all subtypes? Does the EpCAM -ow signature and the signatures of the various EMT stages correlate with CMS subtypes, therapy resistance and clinical outcome? This is not really clear from the data presented.

    The scRNA sequencing seems to reflect the EMT full and hybrid stages. The computational analysis is impressive and exciting, the potential trajectories offer a working model which could be experimentally tested by functional validation of the subgroups to finally pinpoint the cell populations with the highest cell plasticity. And most importantly, what defines cell plasticity at the molecular and cellular level? Is it Wnt signaling or something in addition? Here, the reader is left without a clear picture (see also comment on Discussion, below).

    Text: Seurat33 = Stuart33.

    Discussion: What is the mechanistic basis for the "further enhancement" of Wnt signaling? Is it the dose of Wnt signaling or is it the combination with other signaling pathways which cooperate with Wnt transcriptional control, such as Hippo or TGF signaling? There could be a hint from the RNA sequencing data to distinguish these possibilities. Do the target gene lists change with the enhancement of Wnt signaling?

  6. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    This manuscript is in revision at eLife.

    ###Summary:

    While all reviewers see merit in aspects of the work, and indeed the consensus that there were elements of novelty and interest in this manuscript, they felt that novel advances were limited as presented. Briefly, the manuscript falls into two parts; it is too long with too much data presented and we recommend focus on potentially the most exciting/novel part, ie. the RNAseq / sc and computational analyses, and extending this to provide further functional validation. Some of the earlier figures reflect quite well understood biology (EMT, Zeb1, Wnt etc in EMT), and would require much more work to tighten up the conclusions; therefore, it was felt that even if these were improved, the data would likely confirm a lot of what we know already. It is true that the role of EMT is controversial - but what is presented in the first part of the manuscript does not add much definitive new data to inform that debate, and indeed the authors' submission letter refers to their 'confirmatory' nature.

  8. Version published to 10.1101/2020.07.24.219238v1 on bioRxiv