Global analysis of contact-dependent human-to-mouse intercellular mRNA and lncRNA transfer in cell culture
Curation statements for this article:-
Curated by eLife
eLife assessment
The authors show that tunneling nanotubes or TNTs are used by cells to transfer full-length mRNAs. The data show that as much as 1% of the endogenous mRNA are passed between cells by this procedure. The transferred mRNA affect the transcriptome of the acceptor cells thus highlighting the significance of this nanotube mediated trafficking of mRNA between cells. We appreciate the difficulty of this exercise. The strength of the presented evidence could be questioned based on technical limitations.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Cell Biology (eLife)
Abstract
Full-length mRNAs transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the ‘transferome’) is unknown. Here, we analyzed the transferome in an in vitro human-mouse cell co-culture model using RNA-sequencing. We found that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells. Typically,<1% of endogenous mRNAs undergo transfer. Non-selective, expression-dependent RNA transfer was further validated using synthetic reporters. RNA transfer appears contact-dependent via TNTs, as exemplified for several mRNAs. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.
Article activity feed
-
-
Author Response
Reviewer #1 (Public Review):
Tunneling nanotubes, contrary to exosomes, directly connect remote cells and have been shown to allow the transfer of material between cells, including cellular organelles and RNAs. However, whether sorting mechanisms exist that allow to specifically transfer subspecies of RNAs, especially of mRNA, has not been shown, and the transcriptional consequences of RNA transfer have not been addressed yet.
Using cocultures (or mix or single cultures as controls) of human MCF7 breast cancer cell line, and immortalized mouse embryo fibroblasts (MEFs), followed by separation of human and mouse cells by cell sorting, the authors performed deep sequencing of the human mRNAs detected in mouse cells. An accurate analysis of the transferred material shows that all donor cell mRNAs transfer in a manner …
Author Response
Reviewer #1 (Public Review):
Tunneling nanotubes, contrary to exosomes, directly connect remote cells and have been shown to allow the transfer of material between cells, including cellular organelles and RNAs. However, whether sorting mechanisms exist that allow to specifically transfer subspecies of RNAs, especially of mRNA, has not been shown, and the transcriptional consequences of RNA transfer have not been addressed yet.
Using cocultures (or mix or single cultures as controls) of human MCF7 breast cancer cell line, and immortalized mouse embryo fibroblasts (MEFs), followed by separation of human and mouse cells by cell sorting, the authors performed deep sequencing of the human mRNAs detected in mouse cells. An accurate analysis of the transferred material shows that all donor cell mRNAs transfer in a manner that correlates with their expression level, with less than 1% of total mRNA being transferred in acceptor cells.
These results show that the process of RNA transfer is nonselective and that the consequences on the cells receiving the RNAs should depend on the phenotype of the sending cells.
Although we did not address this last point in the original paper, we concur with this statement since we presented evidence to this effect in our previous publication (Haimovich et al., 2017) and which we discussed in the in the original Discussion section (lines 498-508 in the original manuscript; lines 529-539 in the revised manuscript). We have now amended the Introduction (line 91 of the modified manuscript) to reflect this idea.
These results are complemented by the last part of the manuscript where the authors convincingly show that the coculture of the two cell lines results in significant transcriptomic changes in acceptor MEF cells that could become CAF-like cells.
Reviewer #2 (Public Review):
In this manuscript, the authors characterize the extent of RNA transfer between cells in culture, with an emphasis on trying to identify RNAs that are transferred through tunneling nanotubes (TNTs). They use an in vitro human-mouse cell co-culture model, consisting of mouse embryonic fibroblasts and human MCF7 breast cancer cells. They take advantage of the CD326 cell surface molecule, which is specifically expressed on MCF7 cells, to separate the two cell populations using magnetic beads conjugated to anti-CD326 antibodies, followed by deep sequencing to identify human RNAs present in mouse cells. They identify many 'transferred' RNAs. Further analysis of sequencing data together with experiments using synthetic reporters indicate that RNA transfer is non-selective, that the amount of transfer strongly correlates with the level of expression in donor cells, and does not appear to require specific RNA motifs. The authors also note that co-culture with MCF7 cells leads to significant changes in the MEF transcriptome.
The experiments are overall carefully designed, and the data are clearly and quite carefully presented to point out limitations in interpretation and to distinguish speculations from experimental conclusions.
We thank the reviewer for this comment.
It should however be kept in mind that it is unclear to what extent these limitations influence the conclusions reached. For example, the identification of transferred RNAs relies on the purity of the isolated cell populations ad, while the authors provide some supporting evidence for this, nevertheless potential caveats remain. For instance, the isolated MEF samples used for analysis appear to lack single MCF7 cells, but still contain components, labeled as 'double stained' and 'unstained' cells, which are uncharacterized. The authors present some arguments as to why these would not contribute to 'transferred' reads, but given the low level of detectable transferred RNAs, and the unclear origin of these components, whether they influence the results could be debatable.
It is unlikely that these populations contributed to the human mRNA signals in the MEFs, since the percentage of these populations was substantially higher in the “Mix” samples than in the “Co-culture” samples. We now added the following text (lines 174-181 in the revised manuscript) which clarifies this point: “In addition, we found small sub-populations of double-stained and unstained cells within the purified populations that we suspect are mostly MEFs (see Methods). These sub-populations were greater in the Mix-derived MEFs vs. the Co-culture-derived MEFs (i.e. 0.08% and 0.03% double-stained, and 2.8% and 2.67% unstained in Mix samples vs. 0% and 0.03% double-stained, and 1% and 1.9% unstained in the Co-culture samples). As a consequence, if these double-stained and unstained cells had contributed to the background of human reads in the MEFs, we would’ve expected to have many more human reads in the Mix-derived MEFs.” However, this was not the case, rather we observed a 6.6-fold increase in human RNA presence in the Co-culture-derived MEFs (versus that in the Mix-derived MEFs) after subtraction of the single culture background. In addition, we note that the level of detectable human RNAs in the MEFs is not low, rather it is the percentage of human RNA that undergoes transfer that is low.
Furthermore, the small number of replicates (2 replicates for the genome-wide studies and 1 replicate for most of the subsequent experiments) minimizes the confidence in the conclusions.
We apologize for not stating it clearly that the smFISH, RT-qPCR ,and quadrapod experiments were all performed in 2 replicates. This information has now been added to the figure legends.
In this context, it is also notable that the profile of transferred RNAs between the two replicates of co-cultured samples appears quite different by PCA analysis. It is thus conceivable that there might be specificity in the RNA 'transferome', influenced by unknown experimental variables, which is though masked when averaging those samples in subsequent analyses.
We have replied to Reviewer #1 on this issue. PCA analysis (Figure 2B) of the heat map data (Figure 2A) reveals the similarity between the different samples, whereby 78% of the variability in the data is revealed by PC1 and 6.7% by PC2. Given that PC2 measures only 6.7% of the variation in the data, it likely results from small differences in the individual co-culture samples (such differences are often observed within replicas of RNA-seq experiments) and not via major differences in the measured transferomes. This indicates that the co-culture samples were overall quite similar as can also be observed from the heat map shown in Figure 2A, as differentiated from the controls (e.g. Mix, Single culture). Thus, we do not believe that further replicas will greatly change the results showing the abundant presence of human RNAs in the mouse cells after subtraction of the Mix background. We included additional sentences in the text and figure legend to clarify this point (lines 208-212 in the revised manuscript).
While the manuscript emphasizes the role of TNTs in RNA transfer, the actual involvement of TNTs relies solely on the observation that potential TNTs form between co-cultured cells. Other means of transfer, such as through engulfment or phagocytosis of cell fragments, could still possibly contribute.
While it is possible that transfer might occur through other means, our earlier paper (Haimovich et al., 2017) showed that engulfed apoptotic bodies rarely contribute to mRNA transfer, even upon near-100% of donor cell death. Moreover, RNAs in apoptotic bodies found in acceptor cells can be clearly identified by smFISH, as the RNAs are tightly clumped together. Likewise, our quadrapod experiments (Figure 6-figure supplement 1) might have revealed RNA transfer if engulfment of cell fragments had occurred.
Furthermore, the dependence of mRNA transfer on direct cell-to-cell contact is demonstrated for 5 RNAs and extrapolated to transcriptome-wide RNA transfer, an assumption which might, or might not, be valid.
We concur that we extrapolate from the few validated examples and have now added the following text (line 604-611 in the revised manuscript): “We validated several examples of transferred mRNAs that transfer via a contact-dependent mechanism, likely TNTs (Figure 6 and Figure 6-figure supplements 1 and 3), and extrapolate from them to the entire transcriptome. Although it is possible that some or many mRNAs transfer by means other than TNTs, we think it unlikely, since the results on TNT-mediated cell-to-cell transfer in both this and our previous publication (Haimovich, 2017), as well as by others (Ortin-Martinez et al., 2021; Su and Igyarto, 2019), tested a variety of mRNAs from different families and which localize to various sub-cellular localizations. This indicates that the pathway we have uncovered is more general than the few examples presented here.” In addition, we now cite in the Discussion (lines 611-621 in the revised manuscript) a new pre-print recently posted to bioRxiv that shows similar results of mRNA transfer in a human-mouse cells co-culture model.
Finally, the results on gene expression changes induced by co-culture (Figures 7, 8) are of unclear relevance. As the authors point out, it is uncertain whether RNA transfer or other paracrine or adhesion-mediated signaling events, underlie these changes. It is therefore not easy to see how these results relate to the rest of the presented work. Furthermore, while the authors expand on the potential significance of changes observed in genes related to cancer-associated fibroblasts or to immunity-related genes, these remain speculative and untested.
We concur that the part of the paper regarding the consequences of co-culture (upon the endogenous transcriptome) does not clarify the specific contribution of the “transferome” to the phenomenon. Future co-culture studies measuring transcriptome-wide transfer using the quadrapod co-culture system versus cell-cell contact co-culture could be performed. Yet, to make the distinction between TNT-dependent and -independent effects when cells are in contact will require further mechanistic knowledge of TNT-mediated mRNA transfer, which is beyond the scope of this paper. Nevertheless, we believe that the data on the endogenous gene expression in co-culture is important and could be useful to the cancer research community outside the context of the transferome information.
Overall, the manuscript presents evidence indicating that RNA is transferred non-selectively in co-cultured cells, under specific conditions and between the cell types tested. The impact of the work is reduced by the lack of mechanistic understanding underlying this transfer and the uncertainty of whether this phenomenon has any subsequent physiological relevance.
Our global analysis of TNT-mediated transfer (the transferome) is only a second step towards understanding this important and only recently identified process (i.e. the first step). Obviously, we would be happy to gain more mechanistic insight and knowledge of physiological relevance. We are currently working on several projects to try and answer some of these questions, but as one can understand, these are technically challenging, and have not yet come to fruition.
-
eLife assessment
The authors show that tunneling nanotubes or TNTs are used by cells to transfer full-length mRNAs. The data show that as much as 1% of the endogenous mRNA are passed between cells by this procedure. The transferred mRNA affect the transcriptome of the acceptor cells thus highlighting the significance of this nanotube mediated trafficking of mRNA between cells. We appreciate the difficulty of this exercise. The strength of the presented evidence could be questioned based on technical limitations.
-
Reviewer #1 (Public Review):
Tunneling nanotubes, contrary to exosomes, directly connect remote cells and have been shown to allow the transfer of material between cells, including cellular organelles and RNAs. However, whether sorting mechanisms exist that allow to specifically transfer subspecies of RNAs, especially of mRNA, has not been shown, and the transcriptional consequences of RNA transfer have not been addressed yet.
Using cocultures (or mix or single cultures as controls) of human MCF7 breast cancer cell line, and immortalized mouse embryo fibroblasts (MEFs), followed by separation of human and mouse cells by cell sorting, the authors performed deep sequencing of the human mRNAs detected in mouse cells. An accurate analysis of the transferred material shows that all donor cell mRNAs transfer in a manner that correlates with …
Reviewer #1 (Public Review):
Tunneling nanotubes, contrary to exosomes, directly connect remote cells and have been shown to allow the transfer of material between cells, including cellular organelles and RNAs. However, whether sorting mechanisms exist that allow to specifically transfer subspecies of RNAs, especially of mRNA, has not been shown, and the transcriptional consequences of RNA transfer have not been addressed yet.
Using cocultures (or mix or single cultures as controls) of human MCF7 breast cancer cell line, and immortalized mouse embryo fibroblasts (MEFs), followed by separation of human and mouse cells by cell sorting, the authors performed deep sequencing of the human mRNAs detected in mouse cells. An accurate analysis of the transferred material shows that all donor cell mRNAs transfer in a manner that correlates with their expression level, with less than 1% of total mRNA being transferred in acceptor cells. These results show that the process of RNA transfer is nonselective and that the consequences on the cells receiving the RNAs should depend on the phenotype of the sending cells. These results are complemented by the last part of the manuscript where the authors convincingly show that the coculture of the two cell lines results in significant transcriptomic changes in acceptor MEF cells that could become CAF-like cells.
-
Reviewer #2 (Public Review):
In this manuscript, the authors characterize the extent of RNA transfer between cells in culture, with an emphasis on trying to identify RNAs that are transferred through tunneling nanotubes (TNTs). They use an in vitro human-mouse cell co-culture model, consisting of mouse embryonic fibroblasts and human MCF7 breast cancer cells. They take advantage of the CD326 cell surface molecule, which is specifically expressed on MCF7 cells, to separate the two cell populations using magnetic beads conjugated to anti-CD326 antibodies, followed by deep sequencing to identify human RNAs present in mouse cells. They identify many 'transferred' RNAs. Further analysis of sequencing data together with experiments using synthetic reporters indicate that RNA transfer is non-selective, that the amount of transfer strongly …
Reviewer #2 (Public Review):
In this manuscript, the authors characterize the extent of RNA transfer between cells in culture, with an emphasis on trying to identify RNAs that are transferred through tunneling nanotubes (TNTs). They use an in vitro human-mouse cell co-culture model, consisting of mouse embryonic fibroblasts and human MCF7 breast cancer cells. They take advantage of the CD326 cell surface molecule, which is specifically expressed on MCF7 cells, to separate the two cell populations using magnetic beads conjugated to anti-CD326 antibodies, followed by deep sequencing to identify human RNAs present in mouse cells. They identify many 'transferred' RNAs. Further analysis of sequencing data together with experiments using synthetic reporters indicate that RNA transfer is non-selective, that the amount of transfer strongly correlates with the level of expression in donor cells, and does not appear to require specific RNA motifs. The authors also note that co-culture with MCF7 cells leads to significant changes in the MEF transcriptome.
The experiments are overall carefully designed, and the data are clearly and quite carefully presented to point out limitations in interpretation and to distinguish speculations from experimental conclusions. It should however be kept in mind that it is unclear to what extent these limitations influence the conclusions reached. For example, the identification of transferred RNAs relies on the purity of the isolated cell populations and, while the authors provide some supporting evidence for this, nevertheless potential caveats remain. For instance, the isolated MEF samples used for analysis appear to lack single MCF7 cells, but still contain components, labeled as 'double stained' and 'unstained' cells, which are uncharacterized. The authors present some arguments as to why these would not contribute to 'transferred' reads, but given the low level of detectable transferred RNAs, and the unclear origin of these components, whether they influence the results could be debatable. Furthermore, the small number of replicates (2 replicates for the genome-wide studies and 1 replicate for most of the subsequent experiments) minimizes the confidence in the conclusions. In this context, it is also notable that the profile of transferred RNAs between the two replicates of co-cultured samples appears quite different by PCA analysis. It is thus conceivable that there might be specificity in the RNA 'transferome', influenced by unknown experimental variables, which is though masked when averaging those samples in subsequent analyses.
While the manuscript emphasizes the role of TNTs in RNA transfer, the actual involvement of TNTs relies solely on the observation that potential TNTs form between co-cultured cells. Other means of transfer, such as through engulfment or phagocytosis of cell fragments, could still possibly contribute. Furthermore, the dependence of mRNA transfer on direct cell-to-cell contact is demonstrated for 5 RNAs and extrapolated to transcriptome-wide RNA transfer, an assumption which might, or might not, be valid.
Finally, the results on gene expression changes induced by co-culture (Figures 7, 8) are of unclear relevance. As the authors point out, it is uncertain whether RNA transfer or other paracrine or adhesion-mediated signaling events, underlie these changes. It is therefore not easy to see how these results relate to the rest of the presented work. Furthermore, while the authors expand on the potential significance of changes observed in genes related to cancer-associated fibroblasts or to immunity-related genes, these remain speculative and untested.
Overall, the manuscript presents evidence indicating that RNA is transferred non-selectively in co-cultured cells, under specific conditions and between the cell types tested. The impact of the work is reduced by the lack of mechanistic understanding underlying this transfer and the uncertainty of whether this phenomenon has any subsequent physiological relevance.
-
-