Functional visualization of NK cell-mediated killing of metastatic single tumor cells

  1. Hiroshi Ichise
  2. Shoko Tsukamoto
  3. Tsuyoshi Hirashima
  4. Yoshinobu Konishi
  5. Choji Oki
  6. Shinya Tsukiji
  7. Satoshi Iwano
  8. Atsushi Miyawaki
  9. Kenta Sumiyama
  10. Kenta Terai
  11. Michiyuki Matsuda

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is unknown. Here, we have combined ultra-sensitive bioluminescence imaging with intravital two-photon microscopy involving genetically encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hr of arrival, but not thereafter. Intravital dynamic imaging revealed that 50% of NK-tumor cell encounters lead to tumor cell death in the first 4 hr after tumor cell arrival, but after 24 hr of arrival, nearly 100% of the interactions result in the survival of the tumor cell. During this 24-hr period, the probability of ERK activation in NK cells upon encountering the tumor cells was decreased from 68% to 8%, which correlated with the loss of the activating ligand CD155/PVR/Necl5 from the tumor cell surface. Thus, by quantitatively visualizing, the NK-tumor cell interaction at the early stage of metastasis, we have revealed the crucial parameters of NK cell immune surveillance in the lung.

Article activity feed

  1. Version published to 10.7554/elife.76269 on eLife
  6. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

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

    The authors aimed to understand the control and the elimination of disseminated tumor cells by NK cells within the lung, their main question being how pulmonary NK cells are able to prevent tumor cells from colonization in the lung.

    To dissect this question, Hiroshi Ichise and colleagues took advantage of the ultra-sensitive bioluminescence whole body imaging system combined with intravital two-photon microscopy technology involving genetically-encoded biosensors tumor or NK cells to explore the behavior and functional competences of NK cells in an experimental lung metastasis model.

    First, the authors have monitored the fate of intravenously injected B16-Akaluc cells from 5 min to 10 days and observe that tumor cells decrease rapidly within the first 12-24 hours. In parallel, they performed asialoGM1+ and NK1.1+ cells depletion by injection of depleting anti-aGM1 and anti-NK1.1 antibodies in order to see the involvement of these populations on the elimination of the disseminated tumor cells. They conclude that a rapid decrease of the tumor cells is mediated by NK cells. Consisting with this first data, the authors observe also the same early NK cells mediated impact on two other syngenic mouse tumor cell lines : the BRAFV600E melanoma and the colon adenocarcinoma MC-38.

    In a second part, the authors dissected NK cell dynamic behaviors in the pulmonary capillaries by taking advantage of the NKp46iCRExrosa26dtTomato mice where NKp46+ cells are fluorescents and performed 2P intravital imaging to follow the in situ the NKp46+ cells behavior. They could nicely observe that NK cells arrive from the capillaries and patrol on the lung epithelial cells in a stall-crawl-jump manner. Moreover, they also show that the attachment to the pulmonary capillaries is mediated by LFA-1. In the presence of B16F10 tumor model, they observe that NK cells stay longer in the capillaries and increase their duration time of crawling indicating that NK cells stay in contact longer with tumor cells.

    The authors then explored the NK-mediated tumor killing in the lung by measuring tumor cell apoptosis using B16F10-SCAT3 cells (which leads to visualize caspase 3 activation) and Ca2+ influx in tumor cells expressing two Ca2+ sensors, GCaMP6s and R-GECO. They could observe casp3 activation but also Ca+ influx on tumor cells within few minutes after encountering NK cells. They also observe that evasion of NK cell surveillance is mediated by Nectin-5 and Nectin-2 expressed on tumor cells.

    Then, they focus on NK cell activation by looking at ERK activation. To do so, they have isolated NK cells from Tg mice expressing a FRET-based ERK biosensor and performed in vitro killing assay against B16-R-GECO tumor cells but also in vivo experiments. For the in vivo experiments, they have developed reporter mice whose NK cells express the FRET biosensor for ERK. They observe that ERK-dependent NK cell activation contributes to the elimination of disseminated tumor cells within the first few hours but not after 24hours. Indeed, theu observe that B16F10-Akaluc tumor cells are equally eliminated when injected 24h after a first injection of B16F10 or PBS in mice. The authors concluded that tumor cell acquire the capacity to evade NK cell surveillance after 24h rather than a hypothesis toward NK cells loose tumoricidal activity over time.

    Finally, the authors have explored their last result on the potential tumor cell evasion of the NK cell surveillance. They show that this NK cell evasion is mediated by the shedding of cell surface Necl-5. They next show that clivage of extracellular domain of Necl-5 was mediated by thrombin in vitro and that anti-coagulation factors such as Warfarin, Edoxaban or Dabigatran Etexilate promote tumor elimination as observed by the bioluminescence experiments. This loss prevents the NK cell signaling needed for effective killing of tumor targets.

    However, most of the results remain correlations and have not been formally demonstrated or miss controls.

    B16F10 is a well known and characterized NK cell target in a in vivo model so the first part is not really knew except the in situ behavior of NK cells within the lung capillaries. The new mecanism of thrombin-mediated shedding of Necl-5 causing evasion from NK Cell surveillance is really concentrated on the last figure (Fig N{degree sign}6) and some supplemental experiments are mandatory and needed to really confirm this affirmation.

    Response: We deeply appreciate the reviewer’s effort to evaluate our work. The reviewer criticizes that the mechanism is well known except “the in situ behavior of NK cells within the lung capillaries.” Indeed, this is what we wish to emphasize in our work. Nobody has ever seen how NK cells kill metastatic tumor cells in the lung. There is a big GAP between in vitro tissue culture experiments and in vivo macroscopic counting of metastatic nodules. Most researchers do not even know when and where in the lung NK cells kill metastatic tumor cells. Live imaging is a powerful approach to address such questions.

    Reviewer #1 (Significance (Required)):

    There are several points to address to improve the significance of these data.

    **Major points**

    1) A global point : 3 mice/group is to small to analyse and interprete data because of the heterogeneity of the mice. Mean +/- SEM have to represented instead of SD.

    Response: For the sake of animal welfare, researchers are asked to use minimal number of mice. Moreover, only one mouse can be observed in each imaging session, which takes several hours. In most experiments we performed two independent experiments with three mice each. We believe, the number is appropriate for this type of experiment. In the case of small number of samples, we think SD is better than SEM.

    2) The authors used the well known polyclonal anti-asialoGM1 Ab to deplete NK cells. AsialoGM1 is also expressed by ILC1, T, NKT and gd+T cells but also basophils (Trambley J et al., Asialo GM1(+) CD8(+) T cells play a critical role in costimulation blockade-resistant allograft rejection. JCI, 1999). The authors checked the involvement only for the basophils. They have to check the depletion of each of these populations specifically in the lung to assume that the depletion impact only the NK cells or they must change their conclusion on the entire manuscrit and say that not only NK cells is responsible and involved in the control of the disseminated tumor cells but maybe also ILC1, NKT and or gd+T cells.

    Response: We obtained similar observations by using BALB/c nu/nu mice, which lack T cells. Therefore, we can exclude the contribution of T cells at least in the acute phase (*3) Lines 133 to 136 : The authors say that they « did not observe any significant difference in the relative increase of the bioluminescence signal between the control and αAGM1-treated mice, implying that NK cells eliminate disseminated melanoma cells primarily in the acute phase (Response: After 24 hrs, the slope of increment of bioluminescence intensity (BLI) did not change significantly betweenαAGM1-treated mice and control mice. In both mice, the doubling times of melanoma cells are approximately one day.

    4) Fig S3A-B : The authors say that basophils express aGM1 so they performed basophils involvement on the elimination of B16F10 tumor cells with depleting aCD200R3 mab. They also checked the involvement of neutrophils and monocytes. They observed that basophils, neutrophils and monocytes are not involved on the B16F10 elimination. But what is the hypohesis to assess the role of neutrophils and monocytes ? Moreover, they did not explore Basophil roles in the other models including MC-38, BRAFV600E and 4T1 tumor cells.

    Response: We depleted neutrophils and monocytes because antibody-mediated removal of leukocytes could have non-specifically increased the survival of tumor cells. As for expanding the number of experiments with different cell lines, we are afraid but it is too much burden, considering the period required for the experiments and animal welfare.

    5a) Fig 1D : Missing control : the author must add the WT Balbc + a-AGM1 as control.

    Response: We have this data, which will be included in the revised paper.

    5b) Lines 154 to 156 : the authors say that « T cell immunity does not contribute to tumor cell reduction » because tumor cells are eliminated in the nu/nu mice as efficiently as in the WT Balbc mice. This is not correct because they are looking in a window that correspond to innate immunity activation (up to 24h) so they cannot talk about T cell immunity, the adpative response will come more later around 8 days after.

    Response: Yes, we are focusing on the early phase of the rejection of metastatic tumor cells. We will rephrase the sentences.

    6) Line 159 : (refer to point #2) To affirm that NK cells is critical and involved in the elimination of the disseminated tumor, authors have to perform experiment in a model of NK cell deficiency. The most relevant nowaday is the NKp46ICRExrosa26DTA mice that are deficients in NK cells but also ILC1 cells. Indeed, the authors have used the NKp46iCre mice model for other questions.

    Response: As the reviewer stated, the contribution of NK cells in the rejection of metastatic tumors is very well known. We do not think we need to repeat the experiments by using other genetically modified mouse lines, which will take at least one year. We wish to emphasize again that the new findings of our paper are in the in vivo imaging.

    7a) Fig 2F : IC missing

    Response: According to the reviewer's suggestion, we will perform control experiments with an isotype control.

    7b) Lines 181-182 : Authors conclude that the effect of anti-LFA-1 on NK cells adhesion to the pulmonary endothelial cells is mediated primarily by LFA-1. It is not totally true because it is partially mediated as observed in the fig 2F. So authors should change their conclusion and precise that the involvement is partially mediated by LFA-1.

    Response: We will rephrase the result section in the revised paper.

    8) Fig S5B-C-D and S7: The authors talk about tumor cell death. But they are analyzing Ca2+ influx in vitro so it is a little bit different from the cell death. I'm wondering how the cell death is mesured espacially in the fig S5D and S7?

    Response: Under microscopes, apoptosis can be easily recognized by the appearance of blebs. We will include videos in the revised paper.

    9) Fig 4H and lines 232-233 : the authors conclude that « damage to tumor cells is dependent on the engagement of DNAM-1 on NK cells ». There is any experiment performed to affirm this point so the authors cannot maintain this conclusion. First, the authors only analyzed Ca2+ influx at a specific time point. So this result only show that Nectin-5 and/or Nectin-2 expressed by B16F10 is involved in the Ca2+ influx following NK cell contact but there is any data on DNAM-1 contribution. So, the role on the NK cells and specifically DNAM-1+ NK cells have not been adressed here. To answer to that question, the author have to perform in vivo model of engrafted WT vs Necl-5/2 ko B16F10 in a WT vs DNAM1 deficient NK cells mouse model to ascertain the contribution of Necl-5/2-DNAM-1 on NK cells. Moreover, survival curve and bioluminescent experiments would be very appreciated.

    Response: We have shown the data with Necl-5/Nectin-2-deficientB16F10 cells in Fig. S7. I understand the importance of the experiment with the DNAM-1-deficient mice. But the introduction of another knockout mouse line cannot be performed easily. Instead, we will tone down the conclusion on the requirement of signaling from Necl-5/Nectin-2 to DNAM-1.

    10) Lines 253-254 : the authors talk about tumor apoptosis but they are looking at Ca2+ influx. So, they should change their conclusion or show killing experiment.

    Response: In Figure S7, we have shown that the sustained Ca2+ influx is a useful surrogate marker for apoptosis. We will include this information explicitly in the revised paper.

    11) Fig 6 : the authors conclude that the trombin dependent shedding of Necl-5 causes evasion of NK cells surveillance. Moreover, all experiments are correlations and do not implicate in the same experiment Necl-5, DNAM-1+ NK cells and trombin or anti-coagulation factors. So, as in the comment #9, to adress this point, the authors should inject WT vs Necl-5 deficient B16F10-Akaluc into WT vs NK cell depleted mice and monitor the bioluminescence of the tumor cells within 24h following injection of anti coagulation factors as in the fig 6H. Moreover, the monitoring of the survival curve and the number of the lung metastasis would be also very important and informative to really answer to this point.

    Response: We will try the requested experiments during revision.

    **Minor points**

    1) Fig 2E: The authors assess the involvement of LFA-1 and MAC-1 on the NK cells attachement to the the pulmonary endothelial cells. But there is other adhesion molecules that are known to be expressed by NK cells as for example CR4 (CD11c/CD18). So, the attachement of NK cells could be also due to this molecule.

    Response: We agree. The text will be modified to suggest the involvement of other adhesion molecules.

    2) Lines 190 to 197 : Authors should put this methodology part in the « material and method » in order to be more clear on the message they want to deliver.

    Response: We will modify the text according to the suggestion.

    3) line 228 : There is any hypothesis or explanation regarding the use of Necl5/Necl2 deficient B16F10. Why authors decided to go and explore this pathway ? Authors could add some transition sentence and explanation to help readers.

    Response: We will refer to previous papers suggesting the role of DNAM-1 and its ligands, Necl-5 and nectin-2.

    4) The author could performed the same experiment as in Fig S7D and assessed ERK activation of DNAM+ vs - NK cells against WT vs Necl-5/Necl-2KO R-GEKO B16F10 cells.

    Response: We will try the suggested experiments.

    5) Line 283 : Thanks to reformulate the sentence. Check the firgures associated with the text.

    Response: We will correct this error. The figures will be Fig. 5E and 5F.

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

    The authors use in vivo imaging techniques to investigate the killing of lung metastasis by NK cells. They demonstrate that the cleavage of CD155 may result in resistance of killing by NK cells and suggest that this could be an immune evasion mechanism of metastatic tumor cells.

    Overall, the subject is highly relevant, and the in vivo imaging is an interesting and highly relevant technique. However, the message, that tumor cells escape the killing by NK cells by cleavage of CD155 is interesting, but not yet fully supported by the data.

    **Major comments:**

      • Figure 6: To support their main claim the authors would need to transfect the tumor cells with a CD155 mutant, which cannot be cleaved by Thrombin and show that these tumor cells can no longer escape NK cell-mediated killing. This experiment is straight forward and feasible. Another important experiment along this line would be the use the CD155/CD112 deficient tumor cells (Which the authors use in figure 4) in the experiments shown in figure 1. One would expect that tumor control by NK cells within the first 24h is absent when using these tumor cells.* Response: We previously made five CD155 mutants, which could be resistant to thrombin-mediated cleavage, and re-expressed in CD155/CD112 deficient tumor cells. However, none of the mutants was not killed by NK cells both in vivo and in vitro. It appears that the potential thrombin-cleavage site(s) reside in the recognition site by DNAM-1. We will include this observation in the discussion.
    • Figure 5: The demonstration that ERK is activated in this in vivo setting is novel. However, ERK activation is not DNAM-1 specific and the ERK inhibitor is significantly less effective that the depletion of NK cells. Therefore, the relevance of these data to the main message of the manuscript is unclear and the figure could be omitted.*

    Response: We agree that the modest effect of MEKi implies that ERK activation is dispensable for NK activation. However, ERK activation is a useful marker of NK cell activation. The data shown here vividly show the timing of NK cell activation and following tumor cell killing. Because the in vivo dynamics of NK cell activation and tumor cell killing is the most important message of this work, we wish to show this data.

    • In general, the issue of NK cell exhaustion should be addressed in more detail. The experiments do not address serial killing activity of NK cells and more data is needed to show that it is not an exhaustion of NK cells but the cleavage of CD155 from the tumor cells that prevents further killing.*

    Response: We believe, Fig. 5G clearly shows that NK cells are not exhausted 24 hours after tumor cell injection.

    **Minor comments:**

    • Figure 1C: The relevance of this experiment needs to be better explained.*

    Response: We will rephrase the result section in the revised paper.

    • Figure 3A: What does SHG stand for?*

    Response: It is shown in line 625, M&M section. We will show the statement that SHG stands for second harmonic generation channel in the figure legend.

    • Figure 3: Please add a statistical analysis for these experiments.*

    Response: We will include P values in the revised paper.

    • Figure 4: The use of the caspase-3 and the calcium sensors may detect different cytotoxic mechanisms used by the NK cells. While caspase-3 can be activated by death receptor and perforin/granzyme B mediated killing, the calcium sensor may report mostly on perforin mediated membrane damage. These killing mechanisms have different kinetics and are differentially used during serial killing by NK cells. This should be addressed (at least in the discussion).*

    Response: We thank this invaluable comment. We will include this discussion.

    Reviewer #2 (Significance (Required)):

    Investigating the in vivo cytotoxicity of NK cells against tumor cells by using live imaging technologies is highly relevant for the understanding of the dynamic relationship between tumor and killer cells. Therefore, the subject of this manuscript and the technologies used are very relevant, as in vivo killing activities do not always translate to the in vivo setting.

    Response: We thank the reviewer for the favorable comment.

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

    **Summary**

    Ichise et al., present a solid work describing the modality and time frame of action of NK control over seeding metastatic cells within the lung vasculature. Th authors use a variety of technique able to dissect how NK patrol lung vasculature, that they interact with cancer cells as they interact with the endothelial cells and they activate a ERK dependent activation leading to calcium influx in cancer cells leading to their death. The data support the notion that this NK control occur over an early time frame, 4h after cancer cells arrival and is mediated by Necl expression on cancer cells. After this time point cancer cells show a thrombin dependent loss of Necl expression on their surface and therefore become resistant to NK control.

    **Comments:**

    The data presented are supporting the conclusions. This work utilizes a variety of elegant strategy combining reporter strategy with in vivo imaging to assess the phenomenon of interaction, ERK activation, Calcium Inflax and Apoptosis activation directly in the lung.

    In term of experiments, I found the work thorough and complete.

    The data a presented well overall and the statistics seems adequate.

    I only have few suggestions:

    Supplementary Figure S3, show the use of antiLy6G to deplete neutrophils in the lungs of C57BL/6 mice injected with melanoma B16F10 cells. It was recently shown that this antibody is not efficient in depleting neutrophils in this background, but only lead neutrophils to internalise the Ly6G so they cannot be detected by FACS. As shown in Boivin et al 2020 http://doi.org/10.1038/s41467-020-16596-9) neutrophils depletion in C57BL/6 mice can be achieved by using antiGr1 antibody. Therefore, if the authors aim to show this additional control, which I also agree is really good to have, I suggest performing the experiment accordingly to the best-known practice.

    Response: We will perform the suggested experiment.

    Figure 1E: in the text the experiment is described as 4T1 Akaluc cells were inoculated into the foot pad of BALB/c mice with either control antibody or αAGM1, but the legend states that mice subcutaneously injected with B16 Akaluc cells into footpad.

    As B16 melanoma cells are not in BALB/c background, I assume the legend needs to be corrected as the cells should be 4T1, however I wonder if injecting 4T1 breast cancer cells in the footpad could have let to the substantial growth required for lung metastasis without impairing the animal mobility. Could it be that cells where actually injected in the fat pad of the mice and this is just a misspelling in the text?

    In this case, the different in the tissue residence NK cells could also potentially explain why 4T1 are not cleared in the fat pad like the B6 cells are in the footpad.

    The authors should comment on the difference in the in clearance of the cells at the injection site in Figure 1C VS Figure 1E.

    Response: We apology the erratum in the legend.

    Figure 1C was performed to examine whether NK cells in the lung could be exhausted or inert 14 days after the inoculation of B16F10 cells. In this experiment, Akaluc-expressing B16F10 cells were inoculated to monitor the bioluminescence for 24 hrs.

    In figure 1E, we used Akaluc-expressing 4T1 breast cancer cells because 4T1 cells inoculated into footpad can be spontaneously metastasized to the lung (Kamioka et al., 2017). We observed the bioluminescence of 4T1 cells in the lung for up to 20 days.

    Ref: Kamioka, Y., Takakura, K., Sumiyama, K., and Matsuda, M. (2017). Intravital FRET imaging reveals osteopontin-mediated polymorphonuclear leukocyte activation by tumor cell emboli. Cancer Sci 108, 226-235.

    Reviewer #3 (Significance (Required)):

    The present work is highly relevant to the field of cancer metastasis. While it is known that NK are responsible for the first line of defence against metastatic seeding, most of the studies focuses on how they are suppressed or influenced by other immune cells. The present study provides a very accurate description of their mechanism of action, how they depend in the interaction with the endothelial cells and highlight the novel aspect of thrombin in inducing cancer cells NK resistance. What cause thrombin activation is the next relevant question, by in my opinion this study is complete and important.

    My field of expertise is cancer metastasis and their interaction with the immune system and I personally enjoy very much reading this work.

    Response: We thank the reviewer for favorable comments and appreciate the effort to evaluate our work.

  7. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Summary

    Ichise et al., present a solid work describing the modality and time frame of action of NK control over seeding metastatic cells within the lung vasculature. Th authors use a variety of technique able to dissect how NK patrol lung vasculature, that they interact with cancer cells as they interact with the endothelial cells and they activate a ERK dependent activation leading to calcium influx in cancer cells leading to their death. The data support the notion that this NK control occur over an early time frame, 4h after cancer cells arrival and is mediated by Necl expression on cancer cells. After this time point cancer cells show a thrombin dependent loss of Necl expression on their surface and therefore become resistant to NK control.

    Comments:

    The data presented are supporting the conclusions. This work utilizes a variety of elegant strategy combining reporter strategy with in vivo imaging to assess the phenomenon of interaction, ERK activation, Calcium Inflax and Apoptosis activation directly in the lung. In term of experiments, I found the work thorough and complete. The data a presented well overall and the statistics seems adequate. I only have few suggestions:

    Supplementary Figure S3, show the use of antiLy6G to deplete neutrophils in the lungs of C57BL/6 mice injected with melanoma B16F10 cells. It was recently shown that this antibody is not efficient in depleting neutrophils in this background, but only lead neutrophils to internalise the Ly6G so they cannot be detected by FACS. As shown in Boivin et al 2020 http://doi.org/10.1038/s41467-020-16596-9) neutrophils depletion in C57BL/6 mice can be achieved by using antiGr1 antibody. Therefore, if the authors aim to show this additional control, which I also agree is really good to have, I suggest performing the experiment accordingly to the best-known practice.

    Figure 1E: in the text the experiment is described as 4T1 Akaluc cells were inoculated into the foot pad of BALB/c mice with either control antibody or αAGM1, but the legend states that mice subcutaneously injected with B16 Akaluc cells into footpad. As B16 melanoma cells are not in BALB/c background, I assume the legend needs to be corrected as the cells should be 4T1, however I wonder if injecting 4T1 breast cancer cells in the footpad could have let to the substantial growth required for lung metastasis without impairing the animal mobility. Could it be that cells where actually injected in the fat pad of the mice and this is just a misspelling in the text? In this case, the different in the tissue residence NK cells could also potentially explain why 4T1 are not cleared in the fat pad like the B6 cells are in the footpad.

    The authors should comment on the difference in the in clearance of the cells at the injection site in Figure 1C VS Figure 1E.

    Significance

    The present work is highly relevant to the field of cancer metastasis. While it is known that NK are responsible for the first line of defence against metastatic seeding, most of the studies focuses on how they are suppressed or influenced by other immune cells. The present study provides a very accurate description of their mechanism of action, how they depend in the interaction with the endothelial cells and highlight the novel aspect of thrombin in inducing cancer cells NK resistance. What cause thrombin activation is the next relevant question, by in my opinion this study is complete and important.

    My field of expertise is cancer metastasis and their interaction with the immune system and I personally enjoy very much reading this work.

  8. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The authors use in vivo imaging techniques to investigate the killing of lung metastasis by NK cells. They demonstrate that the cleavage of CD155 may result in resistance of killing by NK cells and suggest that this could be an immune evasion mechanism of metastatic tumor cells. Overall, the subject is highly relevant, and the in vivo imaging is an interesting and highly relevant technique. However, the message, that tumor cells escape the killing by NK cells by cleavage of CD155 is interesting, but not yet fully supported by the data.

    Major comments:

    1. Figure 6: To support their main claim the authors would need to transfect the tumor cells with a CD155 mutant, which cannot be cleaved by Thrombin and show that these tumor cells can no longer escape NK cell-mediated killing. This experiment is straight forward and feasible. Another important experiment along this line would be the use the CD155/CD112 deficient tumor cells (Which the authors use in figure 4) in the experiments shown in figure 1. One would expect that tumor control by NK cells within the first 24h is absent when using these tumor cells.
    2. Figure 5: The demonstration that ERK is activated in this in vivo setting is novel. However, ERK activation is not DNAM-1 specific and the ERK inhibitor is significantly less effective that the depletion of NK cells. Therefore, the relevance of these data to the main message of the manuscript is unclear and the figure could be omitted.
    3. In general, the issue of NK cell exhaustion should be addressed in more detail. The experiments do not address serial killing activity of NK cells and more data is needed to show that it is not an exhaustion of NK cells but the cleavage of CD155 from the tumor cells that prevents further killing.

    Minor comments:

    1. Figure 1C: The relevance of this experiment needs to be better explained.
    2. Figure 3A: What does SHG stand for?
    3. Figure 3: Please add a statistical analysis for these experiments.
    4. Figure 4: The use of the caspase-3 and the calcium sensors may detect different cytotoxic mechanisms used by the NK cells. While caspase-3 can be activated by death receptor and perforin/granzyme B mediated killing, the calcium sensor may report mostly on perforin mediated membrane damage. These killing mechanisms have different kinetics and are differentially used during serial killing by NK cells. This should be addressed (at least in the discussion).

    Significance

    Investigating the in vivo cytotoxicity of NK cells against tumor cells by using live imaging technologies is highly relevant for the understanding of the dynamic relationship between tumor and killer cells. Therefore, the subject of this manuscript and the technologies used are very relevant, as in vivo killing activities do not always translate to the in vivo setting.

  9. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The authors aimed to understand the control and the elimination of disseminated tumor cells by NK cells within the lung, their main question being how pulmonary NK cells are able to prevent tumor cells from colonization in the lung.

    To dissect this question, Hiroshi Ichise and colleagues took advantage of the ultra-sensitive bioluminescence whole body imaging system combined with intravital two-photon microscopy technology involving genetically-encoded biosensors tumor or NK cells to explore the behavior and functional competences of NK cells in an experimental lung metastasis model. First, the authors have monitored the fate of intravenously injected B16-Akaluc cells from 5 min to 10 days and observe that tumor cells decrease rapidly within the first 12-24 hours. In parallel, they performed asialoGM1+ and NK1.1+ cells depletion by injection of depleting anti-aGM1 and anti-NK1.1 antibodies in order to see the involvement of these populations on the elimination of the disseminated tumor cells. They conclude that a rapid decrease of the tumor cells is mediated by NK cells. Consisting with this first data, the authors observe also the same early NK cells mediated impact on two other syngenic mouse tumor cell lines : the BRAFV600E melanoma and the colon adenocarcinoma MC-38.

    In a second part, the authors dissected NK cell dynamic behaviors in the pulmonary capillaries by taking advantage of the NKp46iCRExrosa26dtTomato mice where NKp46+ cells are fluorescents and performed 2P intravital imaging to follow the in situ the NKp46+ cells behavior. They could nicely observe that NK cells arrive from the capillaries and patrol on the lung epithelial cells in a stall-crawl-jump manner. Moreover, they also show that the attachment to the pulmonary capillaries is mediated by LFA-1. In the presence of B16F10 tumor model, they observe that NK cells stay longer in the capillaries and increase their duration time of crawling indicating that NK cells stay in contact longer with tumor cells.

    The authors then explored the NK-mediated tumor killing in the lung by measuring tumor cell apoptosis using B16F10-SCAT3 cells (which leads to visualize caspase 3 activation) and Ca2+ influx in tumor cells expressing two Ca2+ sensors, GCaMP6s and R-GECO. They could observe casp3 activation but also Ca+ influx on tumor cells within few minutes after encountering NK cells. They also observe that evasion of NK cell surveillance is mediated by Nectin-5 and Nectin-2 expressed on tumor cells.

    Then, they focus on NK cell activation by looking at ERK activation. To do so, they have isolated NK cells from Tg mice expressing a FRET-based ERK biosensor and performed in vitro killing assay against B16-R-GECO tumor cells but also in vivo experiments. For the in vivo experiments, they have developed reporter mice whose NK cells express the FRET biosensor for ERK. They observe that ERK-dependent NK cell activation contributes to the elimination of disseminated tumor cells within the first few hours but not after 24hours. Indeed, theu observe that B16F10-Akaluc tumor cells are equally eliminated when injected 24h after a first injection of B16F10 or PBS in mice. The authors concluded that tumor cell acquire the capacity to evade NK cell surveillance after 24h rather than a hypothesis toward NK cells loose tumoricidal activity over time. Finally, the authors have explored their last result on the potential tumor cell evasion of the NK cell surveillance. They show that this NK cell evasion is mediated by the shedding of cell surface Necl-5. They next show that clivage of extracellular domain of Necl-5 was mediated by thrombin in vitro and that anti-coagulation factors such as Warfarin, Edoxaban or Dabigatran Etexilate promote tumor elimination as observed by the bioluminescence experiments. This loss prevents the NK cell signaling needed for effective killing of tumor targets. However, most of the results remain correlations and have not been formally demonstrated or miss controls. B16F10 is a well known and characterized NK cell target in a in vivo model so the first part is not really knew except the in situ behavior of NK cells within the lung capillaries. The new mecanism of thrombin-mediated shedding of Necl-5 causing evasion from NK Cell surveillance is really concentrated on the last figure (Fig N{degree sign}6) and some supplemental experiments are mandatory and needed to really confirm this affirmation.

    Significance

    There are several points to address to improve the significance of these data.

    Major points

    1. A global point : 3 mice/group is to small to analyse and interprete data because of the heterogeneity of the mice. Mean +/- SEM have to represented instead of SD.

    2. The authors used the well known polyclonal anti-asialoGM1 Ab to deplete NK cells. AsialoGM1 is also expressed by ILC1, T, NKT and gd+T cells but also basophils (Trambley J et al., Asialo GM1(+) CD8(+) T cells play a critical role in costimulation blockade-resistant allograft rejection. JCI, 1999). The authors checked the involvement only for the basophils. They have to check the depletion of each of these populations specifically in the lung to assume that the depletion impact only the NK cells or they must change their conclusion on the entire manuscrit and say that not only NK cells is responsible and involved in the control of the disseminated tumor cells but maybe also ILC1, NKT and or gd+T cells.

    3. Lines 133 to 136 : The authors say that they « did not observe any significant difference in the relative increase of the bioluminescence signal between the control and αAGM1-treated mice, implying that NK cells eliminate disseminated melanoma cells primarily in the acute phase (< 24hrs) of lung metastasis » Please comment because the depletion of asGM1+ cells impact also the growth of the tumor until 8 days (fig 1B-E-G)

    4. Fig S3A-B : The authors say that basophils express aGM1 so they performed basophils involvement on the elimination of B16F10 tumor cells with depleting aCD200R3 mab. They also checked the involvement of neutrophils and monocytes. They observed that basophils, neutrophils and monocytes are not involved on the B16F10 elimination. But what is the hypohesis to assess the role of neutrophils and monocytes ? Moreover, they did not explore Basophil roles in the other models including MC-38, BRAFV600E and 4T1 tumor cells.

    5a) Fig 1D : Missing control : the author must add the WT Balbc + a-AGM1 as control.

    5b) Lines 154 to 156 : the authors say that « T cell immunity does not contribute to tumor cell reduction » because tumor cells are eliminated in the nu/nu mice as efficiently as in the WT Balbc mice. This is not correct because they are looking in a window that correspond to innate immunity activation (up to 24h) so they cannot talk about T cell immunity, the adpative response will come more later around 8 days after.

    1. Line 159 : (refer to point #2) To affirm that NK cells is critical and involved in the elimination of the disseminated tumor, authors have to perform experiment in a model of NK cell deficiency. The most relevant nowaday is the NKp46ICRExrosa26DTA mice that are deficients in NK cells but also ILC1 cells. Indeed, the authors have used the NKp46iCre mice model for other questions.

    7a) Fig 2F : IC missing

    7b) Lines 181-182 : Authors conclude that the effect of anti-LFA-1 on NK cells adhesion to the pulmonary endothelial cells is mediated primarily by LFA-1. It is not totally true because it is partially mediated as observed in the fig 2F. So authors should change their conclusion and precise that the involvement is partially mediated by LFA-1.

    1. Fig S5B-C-D and S7: The authors talk about tumor cell death. But they are analyzing Ca2+ influx in vitro so it is a little bit different from the cell death. I'm wondering how the cell death is mesured espacially in the fig S5D and S7?

    2. Fig 4H and lines 232-233 : the authors conclude that « damage to tumor cells is dependent on the engagement of DNAM-1 on NK cells ». There is any experiment performed to affirm this point so the authors cannot maintain this conclusion. First, the authors only analyzed Ca2+ influx at a specific time point. So this result only show that Nectin-5 and/or Nectin-2 expressed by B16F10 is involved in the Ca2+ influx following NK cell contact but there is any data on DNAM-1 contribution. So, the role on the NK cells and specifically DNAM-1+ NK cells have not been adressed here. To answer to that question, the author have to perform in vivo model of engrafted WT vs Necl-5/2 ko B16F10 in a WT vs DNAM1 deficient NK cells mouse model to ascertain the contribution of Necl-5/2-DNAM-1 on NK cells. Moreover, survival curve and bioluminescent experiments would be very appreciated.

    3. Lines 253-254 : the authors talk about tumor apoptosis but they are looking at Ca2+ influx. So, they should change their conclusion or show killing experiment.

    4. Fig 6 : the authors conclude that the trombin dependent shedding of Necl-5 causes evasion of NK cells surveillance. Moreover, all experiments are correlations and do not implicate in the same experiment Necl-5, DNAM-1+ NK cells and trombin or anti-coagulation factors. So, as in the comment #9, to adress this point, the authors should inject WT vs Necl-5 deficient B16F10-Akaluc into WT vs NK cell depleted mice and monitor the bioluminescence of the tumor cells within 24h following injection of anti coagulation factors as in the fig 6H. Moreover, the monitoring of the survival curve and the number of the lung metastasis would be also very important and informative to really answer to this point.

    Minor points

    1. Fig 2E: The authors assess the involvement of LFA-1 and MAC-1 on the NK cells attachement to the the pulmonary endothelial cells. But there is other adhesion molecules that are known to be expressed by NK cells as for example CR4 (CD11c/CD18). So, the attachement of NK cells could be also due to this molecule.

    2. Lines 190 to 197 : Authors should put this methodology part in the « material and method » in order to be more clear on the message they want to deliver.

    3. line 228 : There is any hypothesis or explanation regarding the use of Necl5/Necl2 deficient B16F10. Why authors decided to go and explore this pathway ? Authors could add some transition sentence and explanation to help readers.

    4. The author could performed the same experiment as in Fig S7D and assessed ERK activation of DNAM+ vs - NK cells against WT vs Necl-5/Necl-2KO R-GEKO B16F10 cells.

    5. Line 283 : Thanks to reformulate the sentence. Check the firgures associated with the text.

  10. Version published to 10.1101/2021.01.15.426784 on bioRxiv