N-cadherin dynamically regulates pediatric glioma cell migration in complex environments

  1. Dayoung Kim
  2. James M Olson
  3. Jonathan A Cooper

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Abstract

Pediatric high-grade gliomas are highly invasive and essentially incurable. Glioma cells migrate between neurons and glia, along axon tracts, and through extracellular matrix surrounding blood vessels and underlying the pia. Mechanisms that allow adaptation to such complex environments are poorly understood. N-cadherin is highly expressed in pediatric gliomas and associated with shorter survival. We found that inter-cellular homotypic N-cadherin interactions differentially regulate glioma migration according to the microenvironment, stimulating migration on cultured neurons or astrocytes but inhibiting invasion into reconstituted or astrocyte-deposited extracellular matrix. N-cadherin localizes to filamentous connections between migrating leader cells but to epithelial-like junctions between followers. Leader cells have more surface and recycling N-cadherin, increased YAP1/TAZ signaling, and increased proliferation relative to followers. YAP1/TAZ signaling is dynamically regulated as leaders and followers change position, leading to altered N-cadherin levels and organization. Together, the results suggest that pediatric glioma cells adapt to different microenvironments by regulating N-cadherin dynamics and cell-cell contacts.

Summary

Pediatric gliomas invade the brain by migrating between nerve cells or exploiting extracellular matrix along blood vessels. This research reveals cross-talk between YAP1/TAZ signaling and N-cadherin that regulates leader-follower cell phenotypes and migration efficiency in neural and extracellular matrix environments.

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  1. Version published to 10.1101/2023.04.04.535599v2 on bioRxiv
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    Reply to the reviewers

    1. General Statements We appreciate the insightful reviewer comments. Both reviewers alluded to the logical lack of connection between two themes in the original paper. Specifically, we showed that N-cad differentially regulates migration in different environments, and that leader and follower cells differ phenotypically, but did not connect the two themes. In this revised version, we've performed additional experiments and undertaken a comprehensive reorganization of both the manuscript and figures. The major changes are outlined below:
    • Figure 4 A-C has been moved to Figure 6 F-H.
    • Figure 5 has been moved to Figure S3 F-H.
    • Figure 6 F has been moved to Figure 7 A.
    • Figure 6 G-H have been moved to Figure 7 D-E.
    • Figure 6 I-J have been moved to Figure S5 A-B.
    • Figure 7 C-F have been moved to Figure S5 C-F.
    • Added transcriptome profiling of control and N-cad-depleted cells and of leader and follower cells (Figures 6 E, S1 H and S4 C-D, Tables S2 and S3). We have incorporated additional figures (Figure 4 and 5 in the revised manuscript) to support the idea that the amount of N-cad at the cell surface is regulated by endocytic recycling, thereby stimulating glioma migration in the different local environments. Furthermore, our new findings showed that YAP1/TAZ regulates the surface level of N-cad during glioma migration (Figure 8). We trust that these additions contribute to the clarity and robust justification of our paper.

    Similar to other types of tumors, our findings revealed that pediatric high-grade gliomas migrate collectively, possibly contributing to a more aggressive invasion than single cells. In this study, we found that N-cad mediates this collective glioma migration. Interestingly, within these migrating groups, leader and follower cells dynamically interchange positions during migration, accompanied by changes their phenotypic characteristics. This suggests that differences in phenotypes, including N-cad recycling, proliferation and YAP activation, may be predominantly regulated by cell-extrinsic factors rather than being predetermined by genetic or epigenetic factors. Moreover, our new RNA-sequencing results indicate minimal difference between leader and follower cells, except for the upregulation of YAP response and wound healing migration genes in leader cells. Although genomic alterations still possibly encode the leader-follower exchange, our findings strongly suggest that the activation of YAP1 and glioma migration are regulated by the cellular context, specifically where cells are located within the group.

    Contrary to our initial findings suggesting a positive feedback loop between N-cad endocytosis and nuclear YAP1, our revised data indicates that nuclear YAP appears to be independent of N-cad. We observed that homotypic interactions with N-cad present in the surrounding environment, such as neurons (Figure 6 C-D) or N-cad extracellular domain-coated surface (Figure 7 B-C), did not affect nuclear YAP1. However, YAP1/TAZ depletion decreased N-cad expression and altered its localization at the surface (Figure 8). This leads us to propose a revised model where nuclear YAP1 stimulates surface N-cad, thereby facilitating the distinct modes of migration on ECM and neurons (Figure 8 I).

    1. Point-by-point description of the revisions

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, Kim and colleagues describe the role of N-Cadherin during pediatric glioma migration. They compare cell lines that have similar transcripts but different levels of N-Cadherin protein and find that N-Cadherin levels influence the route of migration - whether it be on ECM or other tissues. They also describe molecular feedback between N-Cadherin and YAP in leader vs follow cells of their systems. The data are clear, well presented, and convincing; and the conclusions described by the manuscript are mostly justified. My major criticism of the manuscript is that the line of questioning undertaken does not appear well justified. At many points, I was left asking "but why are they doing this?" and I could not understand the rationale for some of the experiments that were performed (even if they were performed well). The manuscript opens by validly describing how gliomas are highly invasive, poorly understood and that N-Cadherin was highly expressed in comparison to other adhesion proteins. This opened the path for the questions and experiments performed that contributed to Figures 1-3, which I thought were interesting. From there on, I found the logic of the story unclear and poorly justified. For example, I do not know why leader and follower cells were justified - when it had nothing to do with N-Cadherin which was the focus of the work prior. And then, having rightly concluded in Figure 4 that the data suggested that leader and follower cells dynamically exchange positions rather than being pre-determined, they went onto further figures focusing on differences between leader and follower cells, which left my quite confused. I am likewise confused by the model proposed in that, they authors describe that the difference between leader and follower cells contributes to a nuclear YAP/N-Cad endocytosis feedback loop that feeds into the speed of migration. Yet, the authors describe earlier that leader and follower cells frequently exchange positions, with no evidence that they are pre-determined. How do the authors square these seemingly conflicting points? And further, what is the relevance of this to understanding the differing modes of migration (on ECM or other tissues)? On this issue, I suggest authors re-consider whether the order of figures or logic of the story is appropriate (perhaps consider moving some figures to supplement?), and to clearly justify in the text the elements that are being addressed. Overall, I think the messaging, logic and justification could be use significant improvement; the experiments however are well performed, and the figures are very clear and nicely presented, and I don't have any qualms about them.

    We appreciate your insightful comments, recognizing the need for logical and justifiable improvements in certain sections of our previous manuscript. Please see Section 1, General Statements, for an explanation of changes made.

    Minor Comments

    1. Not required, but the authors may wish to consider putting t=0 pictures of the experiments in the supplement as supportive evidence for the circles of the initial seeding location they show in Fig 1.

    We provide the t=0 images in Figure S1 N and O.

    1. I assume the title of the second results section should say "migration speed" rather than "speed migration"

    The new title of the second results section is “N-cad stimulates and inhibits migration through intercellular homotypic interaction”.

    1. Fig. 4D - Are both example cell pictures leaders? If so, I'm not sure why two have been provided; I'm guessing the bottom set are supposed to be follower cells. If so, please label as appropriate. (And if not, a representative set of pictures from a follower cell should be provided).

    We have enhanced the clarity of the labels. We provide representative high magnification images of leader and follower cells. The updated figure can be found in Figure 5 A.

    1. Figure 5 Legend - the title of this figure is too definitive, and exaggerates further than the main text does, which was correct in saying that the experiments only suggest that N-Cadherin endocytosis might regulate the localisation of b-catenin and p120-catenin. Probably I would go further and say that there is no experimental evidence provided that even suggests that in the first place, and that this is a hypothesis that remains to be tested. The authors should inhibit endocytosis specifically (rather than just depleting N-Cad) and see the effect, to justify their conclusion.

    We appreciated your points and concerns. Following your earlier suggestion, we have moved the figure to the supplementary section (Figure S3 F-H). Moreover, we have addressed the reciprocal regulation of N-cad and catenins by knocking down p120-, β- or α-catenin. Our new findings showed that p120-, β- or α-catenin depletion decrease the amount of N-cad at the cell surface, not steady-state protein level, resulting in decreased migration on astrocytes but increased migration on ECM (see Figure 4). These findings support the idea that catenins play a role in glioma migration according to the environment by altering surface N-cad level. With that, we updated the figure title to “Catenins regulate N-cad surface levels to stimulate or inhibit migration.”

    Reviewer #1 (Significance (Required)):

    The manuscript provides a characterised of invasive glioma migration that was previously lacking. It also provides interesting observations related to the role of N-Cadherin for different modes of migration (on ECM or on tissues) that will be of interest for further exploration. It makes a good advance in terms of addressing a highly invasive cell type that has poor prognosis. I anticipate that now this initial characterisation has been performed, authors and others will be interested in gaining a deeper understanding as to how these two modes of migration are controlled, how there might be interplay between them and how such findings contribute to its highly invasive nature. I have expertise in collective cell migration and directed cell migration.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary In the submitted manuscript, Kim et al. describe various aspects of N-cadherin function in the collective migration of PBT-05 cells, a pediatric high-grade glioma line, on laminin, 3D-matrigel, neurons or astrocytes. N-cadherin promotes the collective migration on neurons or astrocytes, whereas it suppresses the migration on laminin or 3D-matrigel. The authors also show that N-cadherin is actively internalized and recycled in the leader, but not follower, cells of the collective, which induce the nuclear accumulation of YAP/TAZ proteins. YAP/TAZ proteins are shown to regulate the collective migration.

    Thank you for the comments. Please see Section 1, General Statements, for a summary of changes made. Please also note that our new experiments failed to show that N-cad levels or traffic regulate YAP/TAZ nuclear accumulation. Rather, YAP/TAZ are regulated by cell density independent of N-cad, and YAP/TAZ regulate N-cad protein levels and traffic independent of changes in N-cad RNA levels

    Major comments

    1. In Fig. 1G, N-cadherin knockdown seems to affect the distribution of astrocytes. The authors should stain a marker for astrocytes, instead of actin, and the red alone images should be provided.

    Astrocytes were cultured for 4 days to generate 3D scaffolds before adding the glioma spheroid, essentially as described (Gritsenko et al., Histochem Cell Biol, 2017). Co-cultures were stained for human-specific vimentin (glioma) or actin (glioma and astrocytes) (see Figure 1 G and separate channels in new Figure S1 P). There do not appear to be major changes in astrocyte organization outside the migration front, but we lack a way to stain for astrocytes specifically and cannot visualize astrocytes under the glioma cells. It remains possible that astrocytes may be affected differently by contact with control and N-cad-deficient glioma cells. However, we added a new experiment, assaying migration on decellularized astrocyte ECM. While N-cad stimulated migration on astrocytes it inhibited migration on astrocyte ECM (Figures 1 I and J and S1 Q). Thus N-cad stimulates glioma migration on astrocyte cells and not their ECM.

    1. The colocalization between N-cadherin and Rab11 may not be high in Figs. 4F and S2B. It is unclear whether the majority of the internalized N-cadherin is recycled to the plasma membrane. In Fig. S2B, the internalized N-cadherin may be located mainly at the early endosomes before transported to the recycling endosomes (Is it 20 min after the N-cadherin antibody internalization?). First, the authors should analyze the colocalization between the N-cadherin and Rab11 at 30-40 min after the internalization. If the colocalization with Rab11 would be still low at that time point, some of the internalized N-cadherin might be degraded in the lysosomes. To test this possibility, the authors should analyze the colocalization between N-cadherin and LAMP1 under the treatment with a lysosome inhibitor.

    At steady state, N-cad co-localized better with Rab5 than with Rab11 or LAMP1 (Figure 5 C-D). In kinetics experiments, N-cad antibodies were internalized for 40 min. They colocalized better with Rab5 or EEA1 than with Rab11 or LAMP1. When we allowed recycling for an additional 20 min, the surface level of N-cad antibodies partially recovered in leader cells more than follower cells (see Figures 5 G and S3 D). We tested whether treatment with lysosomal inhibitors would increase co-localization of N-cad with Rab11 in recycling endosomes. Surprisingly, however, Chloroquine or Bafilomycin A1 decreased the amount of internalized N-cad antibody in leader and follower cells, and long-term treatment did not increase total N-cad levels. Therefore, the fate of internalized N-cad in follower cells remains unclear.

    1. When N-cadherin is depleted, dissociated single cells are increased, but these cells are not well characterized. A high magnification image of the dissociated single cells is required. In addition, the migration speed of the dissociated single cells should be measured.

    We didn’t quantify single cell migration because only a minority of cells separate from the collective even when N-cad is depleted. Therefore, we quantified migration directionality and speed for cells at or near the front of collective migration (Figure 2 D-I). We have updated the image of single cells, providing representative high-magnification images in Figure S1 N and O.

    1. In Fig. S2D, treatment with Pitstop-2 alone or Dyngo-4a alone is required. Dynamin is also involved in clathrin-independent endocytosis and N-cadherin is reported to be internalized via caveolin-1-mediated endocytosis as well as clathrin-mediated during neuronal migration. It would be better to clarify which type of endocytosis occurs in the leader cells.

    We have removed results showing inhibition of cell migration and N-cad endocytosis by Pitstop-2 and Dyngo-4a from the paper. Treatment with either chemical alone had much less effect on internalization or migration than adding both together (see figure below). This is hard to explain. Pitstop-2 should inhibit clathrin-coated pit formation and Dyngo-4a should inhibit clathrin and caveolin-mediated endocytosis. Caveolin-1 and 2 transcripts were not detected in our cells (Table S2). There may be some other form of clathin-independent endocytosis. Interpretation is also challenging since these inhibitors will inhibit endocytosis of many receptors, not just N-cad. Accordingly, we have removed these results in the revised manuscript.

    1. In Fig. 2, N-cadherin depletion disturbs the migration directionality. Is this a result from disruption of cell polarity? To test this, the position of centrosome or Golgi or lamellipodia in the leader cells should be analyzed. (OPTIONAL)

    We elected not to perform this analysis.

    1. I cannot understand the significance of Fig. 5F and 5G. If the authors would speculate that alpha- and beta-Catenins may transduce the intracellular signaling from the internalized N-cadherin, the authors should perform the knockdown experiments of the Catenins and analyze whether it may affect the nuclear accumulation of YAP/TAZ. (OPTIONAL)

    We agree. In the initial manuscript, we showed that N-cad depletion altered the localization of p120-, β-, and α-catenin (previously shown in Figure 5 F-G). For better clarity and logic, these figures have been moved to Figure S2 H in the revised manuscript. Additionally, to test whether catenins regulate N-cad and YAP1, we depleted p120-, β-, or α-catenin using shRNA. We found that downregulation of p120-, β-, or α-catenin decreased N-cad surface levels, consequently slowing migration on astrocytes and stimulating migration on laminin (Figure 4). In other words, depleting catenins altered migration in parallel with the changes in N-cad surface level. Catenin depletion also increased single-cell dissociation, reduced the crowding of leader and follower cells, and increased nuclear YAP1 (see figure below). These findings suggest that the main role of p120-, β-, or α-catenin is to regulate surface N-cad. Since this result does not support a role for catenins transducing an N-cad signal to YAP1, we have not included it in the paper.

    Minor comments

    1. The quantitative data is required in Fig. 5E.

    Quantitative data from three independent experiment are now presented in Figure S2 G.

    1. Vinculin is associated with the cadherin-catenin complex and it may not be a good loading control (Fig. 3C and 3L).

    The Western blot data has been updated and is now presented in new Figure 3 B and 3 F, with β-tubulin as a loading control.

    **Referees cross-commenting**

    I totally agree with the other Reviewers' comments and evaluation. As the reviewer-1 pointed out, I also think the experiments are well performed, but it would lack logic at least in part (see my comment-6). In addition, as the reviewer-3 pointed out, the linking mechanism of N-cadherin homophilic interaction with YAP/TAZ signaling is important to improve this manuscript

    We hope the revisions have improved the logical flow. We have also added new results showing that YAP/TAZ regulate N-cad protein levels and localization but not N-cad RNA. N-cad is not needed for cell density-dependent regulation of YAP1 localization. The model is shown in Figure 8 I.

    Reviewer #2 (Significance (Required)):

    Strength N-cadherin has multiple function in cancer and neuronal migration, and both positive and negative effects of N-cadherin on cancer cell migration have been reported. In this regard, different behaviors of N-cadherin in the leader and follower cells of the collective are interesting and may explain the controversial previous results.

    Limitation This study reveals various aspects of N-cadherin function in the collective migration of the glioma cell line, but it is unclear whether these findings are applied to pediatric high-grade gliomas in vivo.

    Thus, this study is a potentially important and informative to cell biologists and researchers in cancer biology, although this reviewer also found several weak points that should be improved.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, the authors explore the role of N-cadherin in the migratory/infiltrative behavior of human pediatric brain tumor cells, in light of their surrounding microenvironment. Their in-depth phenotype analysis allows to document the behavior of migrating cells and revisit the concept of leading/follower migratory cells (somehow more commonly applied to endothelial cells). They suspected that the YAP/TAZ pathway might modulate N-cadherin endocytosis and vice versa, using imagery-based cell tracking.

    Major comments

    1. To control for co-culture models, migration should be evaluated on decellularized matrices from astrocyte and neuron cultures.

    We thank for your suggestion. We tested glioma migration on astrocyte-derived decellularized matrices. The mouse astrocytes we used are known to produce various extracellular matrices with a composition similar to Matrigel, except for laminin α5. (Gritsenko et al., J Cell Sci, 2018). N-cad shRNA cells migrated faster on decellularized ECM than control (Figure 1 I-J and S1 Q). This result agrees with N-cad depletion increasing migration on ECM but is opposite to migration on astrocytes.

    1. N-cadherin was stably knocked down with shRNA, which raises the question of adaptative/compensatory mechanisms. First, one could ask what happen in knockout conditions. Similarly, transient siRNA-mediated silencing might help to strengthen the findings. Second, is there any impact of Ncad knock down on alternate adhesive receptors (either cell-cell or cell-ECM). This should be verified with bulk RNAseq.

    Transient knockdown with N-cad siRNA also increased migration on laminin-coated surface (Figure S1 L-M). Unfortunately, N-cad depletion with siRNA was short-lived, precluding its use for long-term assays, like coculture with neurons or astrocytes. To test whether there is any impact of N-cad knockdown on alternative adhesion receptors, we performed RNA-Seq (Figure S1 H, Table S2). We found N-cad depletion did not alter expression of other cell-cell and cell-ECM adhesive receptors except CDH3 (2.8-fold increase compared with 7-fold decrease in CDH2). Integrin expression was unchanged.

    1. It would be interesting to evaluate the impact of N-cadherin/N-cadherin homotypic interactions on YAP/TAZ signaling, using for instance N-cad-coated surface.

    We observed that the homotypic interaction of N-cad with surrounding neurons and astrocytes did not hinder the accumulation of nuclear YAP1 in leader cells (Figure 6 C-D). To further support the idea that N-cad does not directly regulate YAP1 signaling, we have now measured YAP1 localization in cells migrating over N-cad ECD. The new data confirms that N-cad does not directly regulate YAP1 localization (Figure 7 B-C).

    1. along this line, the impact of mechanical cues (stiffness, 2D vs 3D) is not explored.

    We appreciate your suggestion. It is possible that different mechanical and cytoskeletal cues between leader and follower cells affect YAP1 signaling. In this study, we would like to focus more on the role of N-cad-mediated cell adhesions in YAP signaling.

    Minor comments

    1. Levels of N-cadherin expression in normal Astro and Neurons to compare with pediatric brain cancer cells (S1C)

    A new western blot analysis to show N-cad levels in DMG, PHGG and mouse cerebellar neurons and astrocytes has been added to Figure S1 F.

    1. Low versus high density culture conditions should be controlled and its further impact on the YAP/Ncad endocytosis route should be supported experimentally, or to be omitted from their proposed model.

    We previously used different size of micropattern discs to control low or high cell density. Smaller cell clusters, with more edge cells and hence fewer cell-cell interactions, had higher nuclear YAP1 (Figure 7 D-E). We have repeated this experiment, including N-cad ECD antibodies to measure N-cad endocytosis. Smaller cell clusters had higher N-cad antibody internalization (Figure 7 F). Together with our evidence that leader cells have higher YAP1 and more N-cad internalization than followers, and that YAP/TAZ knockdown inhibits N-cad internalization, these results high YAP/TAZ in leader cells regulates N-cad internalization.

    Reviewer #3 (Significance (Required)):

    This paper presents robust image analysis of human pediatric brain tumor migration in the context of the different microenvironment that they might encounter (matrices, neurons, astrocytes). This study brings new concepts on the way N-cadherin might contribute to tumor cell migratory behavior based on the nature of the interactions in which N-cadherin is involved. As a limitation, it remains unclear the mechanism by which N-cadherin endocytosis is driven.

    We now discuss the limitations of the study as follows:

    “The mechanisms by which YAP1 regulates N-cad levels and trafficking remain to be explored. YAP1 is widely expressed in human brain tumors and strongly associated poor survival. Leader cells expressed higher levels of YAP1-response and wound-healing gene transcripts, but transcript levels of N-cad and proteins known to regulate cadherin traffic, such as p120-catenin, Rab5/11 and Rac1, were similar. Therefore, N-cad is likely regulated at the level of protein synthesis or turnover. More endosomal N-cad recycled to the surface of leader than follower cells, implying that follower cells might divert more N-cad for lysosomal degradation, but our attempts to interfere with N-cad endocytosis or degradation specifically were unsuccessful. Further understanding of the mechanism and function of N-cad recycling for glioma cell migration will require cargo-specific ways to selectively regulate endocytosis and recycling”.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, the authors explore the role of N-cadherin in the migratory/infiltrative behavior of human pediatric brain tumor cells, in light of their surrounding microenvironment. Their in-depth phenotype analysis allows to document the behavior of migrating cells and revisit the concept of leading/follower migratory cells (somehow more commonly applied to endothelial cells). They suspected that the YAP/TAZ pathway might modulate N-cadherin endocytosis and vice versa, using imagery-based cell tracking.

    Major comments:

    1. To control for co-culture models, migration should be evaluated on decellularized matrices from astrocyte and neuron cultures.
    2. N-cadherin was stably knocked down with shRNA, which raises the question of adaptative/compensatory mechanisms. First, one could ask what happen in knockout conditions. Similarly, transient siRNA-mediated silencing might help to strengthen the findings. Second, is there any impact of Ncad knock down on alternate adhesive receptors (either cell-cell or cell-ECM). This should be verified with bulk RNAseq.
    3. It would be interesting to evaluate the impact of N-cadherin/N-cadherin homotypic interactions on YAP/TAZ signaling, using for instance N-cad-coated surface.
    4. along this line, the impact of mechanical cues (stiffness, 2D vs 3D) is not explored.

    Minor comments:

    1. Levels of N-cadherin expression in normal Astro and Neurons to compare with pediatric brain cancer cells (S1C)
    2. Low versus high density culture conditions should be controlled and its further impact on the YAP/Ncad endocytosis route should be supported experimentally, or to be omitted from their proposed model.

    Significance

    This paper presents robust image analysis of human pediatric brain tumor migration in the context of the different microenvironment that they might encounter (matrices, neurons, astrocytes).

    This study brings new concepts on the way N-cadherin might contribute to tumor cell migratory behavior based on the nature of the interactions in which N-cadherin is involved.

    As a limitation, it remains unclear the mechanism by which N-cadherin endocytosis is driven.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    In the submitted manuscript, Kim et al. describe various aspects of N-cadherin function in the collective migration of PBT-05 cells, a pediatric high-grade glioma line, on laminin, 3D-matrigel, neurons or astrocytes. N-cadherin promotes the collective migration on neurons or astrocytes, whereas it suppresses the migration on laminin or 3D-matrigel. The authors also show that N-cadherin is actively internalized and recycled in the leader, but not follower, cells of the collective, which induce the nuclear accumulation of YAP/TAZ proteins. YAP/TAZ proteins are shown to regulate the collective migration.

    Major comments

    1. In Fig. 1G, N-cadherin knockdown seems to affect the distribution of astrocytes. The authors should stain a marker for astrocytes, instead of actin, and the red alone images should be provided.
    2. The colocalization between N-cadherin and Rab11 may not be high in Figs. 4F and S2B. It is unclear whether the majority of the internalized N-cadherin is recycled to the plasma membrane. In Fig. S2B, the internalized N-cadherin may be located mainly at the early endosomes before transported to the recycling endosomes (Is it 20 min after the N-cadherin antibody internalization?). First, the authors should analyze the colocalization between the N-cadherin and Rab11 at 30-40 min after the internalization. If the colocalization with Rab11 would be still low at that time point, some of the internalized N-cadherin might be degraded in the lysosomes. To test this possibility, the authors should analyze the colocalization between N-cadherin and LAMP1 under the treatment with a lysosome inhibitor.
    3. When N-cadherin is depleted, dissociated single cells are increased, but these cells are not well characterized. A high magnification image of the dissociated single cells is required. In addition, the migration speed of the dissociated single cells should be measured.
    4. In Fig. S2D, treatment with Pitstop-2 alone or Dyngo-4a alone is required. Dynamin is also involved in clathrin-independent endocytosis and N-cadherin is reported to be internalized via caveolin-1-mediated endocytosis as well as clathrin-mediated during neuronal migration. It would be better to clarify which type of endocytosis occurs in the leader cells.
    5. In Fig. 2, N-cadherin depletion disturbs the migration directionality. Is this a result from disruption of cell polarity? To test this, the position of centrosome or Golgi or lamellipodia in the leader cells should be analyzed. (OPTIONAL)
    6. I cannot understand the significance of Fig. 5F and 5G. If the authors would speculate that alpha- and beta-Catenins may transduce the intracellular signaling from the internalized N-cadherin, the authors should perform the knockdown experiments of the Catenins and analyze whether it may affect the nuclear accumulation of YAP/TAZ. (OPTIONAL)

    Minor comments

    1. The quantitative data is required in Fig. 5E.
    2. Vinculin is associated with the cadherin-catenin complex and it may not be a good loading control (Fig. 3C and 3L).

    Referees cross-commenting

    I totally agree with the other Reviewers' comments and evaluation. As the reviewer-1 pointed out, I also think the experiments are well performed, but it would lack logic at least in part (see my comment-6). In addition, as the reviewer-3 pointed out, the linking mechanism of N-cadherin homophilic interaction with YAP/TAZ signaling is important to improve this manuscript

    Significance

    Strength

    N-cadherin has multiple function in cancer and neuronal migration, and both positive and negative effects of N-cadherin on cancer cell migration have been reported. In this regard, different behaviors of N-cadherin in the leader and follower cells of the collective are interesting and may explain the controversial previous results.

    Limitation

    This study reveals various aspects of N-cadherin function in the collective migration of the glioma cell line, but it is unclear whether these findings are applied to pediatric high-grade gliomas in vivo.

    Thus, this study is a potentially important and informative to cell biologists and researchers in cancer biology, although this reviewer also found several weak points that should be improved.

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    Referee #1

    Evidence, reproducibility and clarity

    Kim et al review

    In this manuscript, Kim and colleagues describe the role of N-Cadherin during pediatric glioma migration. They compare cell lines that have similar transcripts but different levels of N-Cadherin protein and find that N-Cadherin levels influence the route of migration - whether it be on ECM or other tissues. They also describe molecular feedback between N-Cadherin and YAP in leader vs follow cells of their systems. The data are clear, well presented, and convincing; and the conclusions described by the manuscript are mostly justified. My major criticism of the manuscript is that the line of questioning undertaken does not appear well justified. At many points, I was left asking "but why are they doing this?" and I could not understand the rationale for some of the experiments that were performed (even if they were performed well). The manuscript opens by validly describing how gliomas are highly invasive, poorly understood and that N-Cadherin was highly expressed in comparison to other adhesion proteins. This opened the path for the questions and experiments performed that contributed to Figures 1-3, which I thought were interesting. From there on, I found the logic of the story unclear and poorly justified. For example, I do not know why leader and follower cells were justified - when it had nothing to do with N-Cadherin which was the focus of the work prior. And then, having rightly concluded in Figure 4 that the data suggested that leader and follower cells dynamically exchange positions rather than being pre-determined, they went onto further figures focusing on differences between leader and follower cells, which left my quite confused.

    I am likewise confused by the model proposed in that, they authors describe that the difference between leader and follower cells contributes to a nuclear YAP/N-Cad endocytosis feedback loop that feeds into the speed of migration. Yet, the authors describe earlier that leader and follower cells frequently exchange positions, with no evidence that they are pre-determined. How do the authors square these seemingly conflicting points? And further, what is the relevance of this to understanding the differing modes of migration (on ECM or other tissues)? On this issue, I suggest authors re-consider whether the order of figures or logic of the story is appropriate (perhaps consider moving some figures to supplement?), and to clearly justify in the text the elements that are being addressed. Overall, I think the messaging, logic and justification could be use significant improvement; the experiments however are well performed, and the figures are very clear and nicely presented, and I don't have any qualms about them.

    Minor Comments

    • Not required, but the authors may wish to consider putting t=0 pictures of the experiments in the supplement as supportive evidence for the circles of the initial seeding location they show in Fig 1.
    • I assume the title of the second results section should say "migration speed" rather than "speed migration"
    • Fig. 4D - Are both example cell pictures leaders? If so, I'm not sure why two have been provided; I'm guessing the bottom set are supposed to be follower cells. If so, please label as appropriate. (And if not, a representative set of pictures from a follower cell should be provided).
    • Figure 5 Legend - the title of this figure is too definitive, and exaggerates further than the main text does, which was correct in saying that the experiments only suggest that N-Cadherin endocytosis might regulate the localisation of b-catenin and p120-catenin. Probably I would go further and say that there is no experimental evidence provided that even suggests that in the first place, and that this is a hypothesis that remains to be tested. The authors should inhibit endocytosis specifically (rather than just depleting N-Cad) and see the effect, to justify their conclusion.

    Significance

    The manuscript provides a characterised of invasive glioma migration that was previously lacking. It also provides interesting observations related to the role of N-Cadherin for different modes of migration (on ECM or on tissues) that will be of interest for further exploration. It makes a good advance in terms of addressing a highly invasive cell type that has poor prognosis. I anticipate that now this initial characterisation has been performed, authors and others will be interested in gaining a deeper understanding as to how these two modes of migration are controlled, how there might be interplay between them and how such findings contribute to its highly invasive nature.

    I have expertise in collective cell migration and directed cell migration.

  6. Version published to 10.1101/2023.04.04.535599v1 on bioRxiv