Improved Murine Model for the Intravital Microscopic Examination of Manifest Tumors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors utilized a mouse dorsal skinfold chamber in conjunction with intravital microscopy (IVM), a powerful tool for studying tumor angiogenesis and evaluating therapeutic interventions in advanced tumors. This is an interesting paper; however, several inconsistencies in the data should be addressed to strengthen the manuscript’s conclusions. The main points requiring clarification are:

Figure 3

1. How many mice were used in each group (PDS PDS® plate, Dualmesh® membrane, and Preclude®)? The variation is high in the Preclude® and Dualmesh® groups. Increasing the number of mice could clarify whether these materials affect leakage and help reduce variation caused by the procedure.
2. Please provide representative leakage images for each group.

Figure 7
3. Why does the tumor in the Dualmesh® group look so different compared to the other two groups?
4. Why is the striated muscle tightly close to the tumor in the Dualmesh® group, whereas in the other groups it appears separated?
5. Please label the figure to indicate sc: subcutis and m: striated muscle.

Additional points
6. To visualize vessels in the tumor, P53 and CD31 co-staining is recommended.
7. Does SCC-15 have a specific cell surface marker? The paper shows positive p53 staining in skin and adipose tissue. Does this indicate SCC-15 cells migrated to those areas, or is it due to local p53 expression?
8. In Figures 8–10, please indicate which areas were enlarged.
9. Providing videos of tumor vascular growth in the three groups would strengthen the study.

 

Comments on the Quality of English Language

None

Author Response

Review of reviewer 1     Cells-3865598.

The authors utilized a mouse dorsal skinfold chamber in conjunction with intravital microscopy (IVM), a powerful tool for studying tumor angiogenesis and evaluating therapeutic interventions in advanced tumors. This is an interesting paper; however, several inconsistencies in the data should be addressed to strengthen the manuscript’s conclusions. The main points requiring clarification are:

 

Figure 3

1. How many mice were used in each group (PDS PDS® plate, Dualmesh® membrane, and Preclude®)? The variation is high in the Preclude® and Dualmesh® groups. Increasing the number of mice could clarify whether these materials affect leakage and help reduce variation caused by the procedure.

Response: We thank the reviewer for this valuable comment. As indicated in line 105 of the manuscript, each experimental group consisted of four mice. We fully agree with the reviewer that the SEM values for individual measurements, particularly for macromolecular leakage, are relatively large. This is indeed a direct consequence of the limited number of animals included in the study.

The primary objective of the present work, however, was to establish a model for repetitive intravital microscopic analysis of established tumors. Accordingly, the focus was placed on the development and validation of the experimental approach rather than on generating data with minimal variability that would allow for comprehensive statistical evaluation of all described parameters.

Furthermore, animal welfare legislation in Germany requires that experimental protocols be precisely planned and fully described prior to approval. In particular, the number of animals as well as the type and conduct of interventions must be specified in advance. Any subsequent deviations from the approved protocol are not permissible. Consequently, retrospective increases in group size are not possible.

 

1. Please provide representative leakage images for each group.

Response: We thank the reviewer for this thoughtful comment. The analysis of macromolecular leakage was performed using the CapImage software, based on the determination of gray values inside and outside of the vessels. For this purpose, still images extracted from the recorded video files were analyzed.

We believe that a parallel presentation of the experimental findings in both diagrammatic form and as images would not lead to a clearer representation of the results; on the contrary, it would rather reduce the clarity of the message. An advantage of the software-based analysis is its higher dynamic range, which clearly exceeds that of the human eye.

In light of the variability of the obtained data, as already noted by the reviewer in comment 1, we therefore consider it more appropriate to provide a single, unambiguous representation of the results — in this case, in the form of a diagram.

 

Figure 7
3. Why does the tumor in the Dualmesh® group look so different compared to the other two groups?

Response: We thank the reviewer for this important observation. We assume that the optical variations observed in the HE staining of the tumors do not reflect physiological differences. Rather, they are likely attributable to minor variations within the processing workflow, starting from tissue collection at the end of the in vivo experiment through fixation, embedding, staining, and mounting of the final specimen.

 

4. Why is the striated muscle tightly close to the tumor in the Dualmesh® group, whereas in the other groups it appears separated?

Response: We thank the reviewer for this remark. This observation likewise does not reflect a physiological difference. Rather, the distance observed between striated muscle and the tumor in the PDS and Preclude groups should be regarded as a preparation artifact, which has no impact on the fundamental depiction of the tumor, its appearance, or its size.

 

5. Please label the figure to indicate sc: subcutis and m: striated muscle.

Response: We have prepared a revised version of Figure 7 and have incorporated the reviewer’s comments and suggestions as far as possible. In Figure 7, as well as in the subsequent histological figures, we refrained from labeling the subcutis, since this structure is not relevant for the conclusions of the study. Furthermore, in Figure 7 we have indicated the enlarged area, consistent with Figures 8–10, in order to ensure a uniform layout across all figures. The figure legends have been revised accordingly.

 

Additional points
6. To visualize vessels in the tumor, P53 and CD31 co-staining is recommended.

Response: We very much appreciate this valuable suggestion. Unfortunately, due to the limited time available for the revision of the manuscript, it is not possible to implement this change.

 

7. Does SCC-15 have a specific cell surface marker? The paper shows positive p53 staining in skin and adipose tissue. Does this indicate SCC-15 cells migrated to those areas, or is it due to local p53 expression?

Response: We thank the Reviewer for this thoughtful remark. To the best of our knowledge, no single, unequivocal surface marker exists that would allow for the clear identification of SCC-15; this can only be achieved through the combination of multiple markers, which is difficult to implement by means of immunohistochemistry. Since p53, as a transcription factor, is involved in a wide range of biological processes, its detection outside the actual tumor is not unexpected. This is also a well-documented finding in murine tissue, where p53 expression has, for example, been reported in hair follicles (Botchkarev et al., 2001, doi:10.1016/S0002-9440(10)64659-7). In our study, we used p53 to demonstrate the neoplastic nature of stained cells within the tumor area, a strategy that has also been applied in other studies (Kamat et al., 2022, doi:10.1007/s00784-022-04574-y). We consider the combination of anatomical context and positive p53 staining sufficient to clearly delineate the growing tumor. In this regard, we would also like to refer to our response to the comments of the second Reviewer on line 200 and Figure 8.

 

8. In Figures 8–10, please indicate which areas were enlarged.

Response: We thank the reviewer for this helpful comment. We have prepared revised versions of Figures 8, 9, and 10 in which the enlarged areas are clearly marked.

 

9. Providing videos of tumor vascular growth in the three groups would strengthen the study.

Response: We would like to thank the reviewer for this thoughtful comment. The observation of vascular growth by intravital microscopy does not represent a continuous process extending over long periods of time. Instead, the vascular system can only be examined at selected time points (in this study, immediately after preparation of the dorsal skinfold chamber and subsequently at three and six days post-preparation) over short observation intervals. The corresponding video recordings are then subjected to quantitative analysis, which allows, among other parameters, the determination of functional capillary density. Due to methodological constraints, however, it is unfortunately not feasible to present vascular growth as a continuous video, for example in the form of a time-lapse sequence.

Reviewer 2 Report

Comments and Suggestions for Authors

Review Cells-3865598.

 

In their manuscript ‘Improved murine model for the intravital microscopic examination of manifest tumors.’ the authors nicely present a promising technique for in-vivo assessment of squamous cell carcinoma.

Their study approach seems sound and the manuscript is well-written.

However, I do have some issues that I would like to see addressed.

 

Introduction:

 

Line 53: reference 16 and 19 do not seem to be dealing with the skinfold chamber.

 

Material and Methods:

 

Line 81: I suppose the PowerPlex21 was produced by Promega?

 

Lines 81-83: This sentence to me is more an explanation than a real description of materials and methods used in this study. Therefore it would be more logical to have this included in the discussion section where it would need further explanation why this difference is important to emphasize.

 

Lines 85 and 86: Spongostan is also used in veterinary medicine. The mere fact that this product is approved for use in humans doesn’t automatically mean that it can be used safely in other animals species.

 

Line 132: for clarity, perhaps specify that the pocket was created in the dorsal thoracic, lumbar or thoracolumbar region.

 

Line 137: ‘…inhalation anesthesia was discontinued…’ instead of ‘over’.

 

Line 137: I would suggest reporting that ‘neoplastic cells’ had been implanted. A tumor obviously is more than only the neoplastic cell population and according to the previous section, you implanted tumor cells and not a “whole tumor”.

 

Line 139: please clarify in which way ‘a manifest’ tumor was recognized? Perhaps ‘grossly visible’ is a more illustrative description.

 

Lines 145-146: what was the rationale behind shortening the length of the study? Since the authors are aiming at analyzing “advanced tumors”, a longer time frame seems more logical.

 

Line 149: since “hemodynamics” is also part of an (acute) inflammatory response. It might be clarifying to specify the ‘inflammatory parameters’ that were analyzed.

 

Line 149: please specify in which area these parameters were analyzed. Was that within the neoplasm, or in the regions of interest that the authors mention in the ‘Analysis of microcirculatory parameters’ section later in the manuscript (i.e. lines 172-173).

 

Lines 172-173: based on which criteria were these 4 regions of interest chosen? I had the impression that the intra-neoplastic vasculature was the main point of interest from a potential therapeutic point of view. Can the authors be sure that the vasculature and hemodynamics in the periphery of the tumor are predictive/representative of the intra-tumoral vessels?

Line 193: was the histological evaluation performed by a veterinary or human pathologist?

 

Line 200: the use of antibodies produced by mice on murine tissues might pose a challenge because of cross-reactivity with mouse cells and therefore precludes confirmation of human origin of the neoplastic cells which I think is shown in figure 8 (see comment there).

 

Results:

 

Line 222: ‘….after tumor cell implantation…’ instead of ‘cells’. Furthermore, the addition of ‘(see M&M)’ seems redundant.

 

Lines 237-238: I feel this sentence is not a result form the study but more of a reasoning. Therefore, it might be better situated in the discussion section.

 

Line 238: ‘Among the materials tested’ seems unnecessary, since obviously you cannot report results from material that was not used.

 

Line 239: the authors might consider adding ‘because it could be removed easily from the tumor’ as an explanation why this product was deemed ‘to be most suitable’.

 

Line 241: please specify ‘a few cases’.

 

Line 245: please specify the clinical presentation of these ‘irritative wound conditions’. Furthermore, from a linguistic perspective ‘…irritative wound conditions were found.’ might be a more order of words.

 

Line 258: since the authors measured the velocity of the blood cells, perhaps ‘However, the velocity of blood cells increased over time…..’ might be more appropriate (instead of ‘appeared to’).

 

Lines 298-300: than the PDS membrane?

 

Line 318: ‘underlying’ might be somewhat confusing, perhaps ‘surrounding’ or ‘associated’ might be more appropriate. Furthermore, from an anatomical/histological perspective ‘….cutis, subcutis and muscles…..’ might be a more appropriate order.

 

Figure 7: I have not detected the ‘m’ mentioned in the legends in any of the images.

The histological images used are clear and illustrative. Perhaps some tweaking using for instance Photoshop could get rid of the grey background.

Additionally (although also more cosmetic than of major relevance), positioning the epidermis at the top and the neoplasm below would seems more representative of the situation in-situ.

 

Figure 8: as already predicted in my comment on line 200, there seems to be immunoreactivity in murine cells too. Although somewhat difficult to assess at the magnifications used for images A, C and E, I think there is marked staining in for instance the bulbs of the hairs in the normal skin.

Since the histological appearance clearly shows a neoplasm of squamous epithelium, and there is basically no reason to doubt the origin, the cross reactivity is not a major problem but I think this p53 antibody cannot be used as confirmation of human origin of the neoplastic cells.

 

Line 336: the authors describe the presence of endothelial cells mainly in the peripheral (‘marginal’) area of the tumor. This seems not ideal since it seems to be especially the blood vessels within the neoplasm that the authors are interested in. I think this aspect might be discussed in the discussion section of the manuscript.

 

Discussion:

 

Line 366: perhaps ‘…..anatomical location.’ is more appropriate than ‘direction’?

 

Line 372: ‘… requirement was minimal adhesion……’.

 

Line 374: please explain why removal of the tumor without compromising the vasculature was ‘essential’. According to the manuscript the neoplasm was left in-situ in the subcutis during the experiment, so there was no removal phase there. Postmortal harvesting of the neoplasm and the surrounding tissue should also possible without the presence of a membrane. I agree that the membrane will have facilitated the removal from the deeper tissues, but I don’t think that qualifies as “essential”.

 

Line 380: the reported 3-4 mm size of the neoplasm seems to be a limitation with respect to the fact that the authors are mainly interested in advanced tumors that might have a different pathophysiology and morphology than the earlier stages of the same tumor. Perhaps the authors can elaborate on this aspect.

 

Line 391: a “full stop” is missing at the end of the sentence.

 

Lines 392-394: please specify ‘interaction between tumor cells and the surrounding host tissue’. I can see the advantages with respect to the actual hemodynamics in the current setting, but interaction in the sense of mediators, growth factors, matrix proteins (and proteinases) have not become apparent in the protocol used in this pilot study.

 

Lines 407-409: I don’t think that you can conclude that the (neo)vascularization was actually tumor-induced. The authors have induced a surgical wound, created a subcutaneous pocket (that need healing), and inserted foreign body material (that in itself could induce an inflammatory response that includes a vascular component). I think that, in order to be able to conclude that the (neo)vascularization is a purely neoplasm-driven process, a “negative control” in which the same procedures were followed with exception of the application of the neoplastic cells should have been included in order to definitively conclude that (most of) the newly formed blood vessels were induced by the tumor cells.

 

Lines 412-414: I think apoptosis should not lead to stimulation of vascularization.

 

Lines 420-422: See my earlier comment on lines 407-409. Indeed the application of foreign body material can lead to an inflammatory reaction which can include the formation of new blood vessels.

Even though these vessels can contribute to the vascularization of the neoplasm, the underlying molecular mechanism in an inflammatory environment might be different from a purely neoplasm-driven stimulation of angiogenesis and therefore might have different pathways that could potentially be targeted as a therapeutic approach.

So I think the inflammatory response could be an undesired component.

 

Lines 423-428: could the authors comment on the (potential) effect of the skin fold chamber device on the circulation within the tissue that is enclosed in the device.

It seems a relative large contraption that compressed the skin and part of the subcutaneous tissue that probably will have its effect on the circulation anyway. Therefore I think it might wise to be cautious with attributing al hemodynamic effects to presence of the tumor.

 

Line 437: The term ‘physiological’ seems awkward for neoplastic cells that are more or less by definition not-physiologic. Apart from that, I think this also applies to the not-physiological situation that is created by inserting foreign body material in the subcutis. Perhaps rephrasing this sentence might prevent confusion.

 

Lines 439-440: I think the statement that the used model is ‘based on implantation of pre-formed, manifest tumors’ is not correct. According to the materials and methods, neoplastic cell in a spongostan environment are, along with a membrane, implanted in the subcutis from which a tumor develops. This grossly visible neoplasm is then exposed in the skin fold chamber device.

This is different from the actual implantation of an already developed tumor in the subcutis.

Please rephrase to avoid confusion.

 

Line 442: what do the authors mean by ‘acute cell injection’? I think an injection often is an action of short duration.

 

Line 451: please explain the ‘atraumatic’ nature of the procedure used. It seems counterintuitive to use the term atraumatic when you apply cells in spongostan and a membrane after surgical incision of the skin and preparing a subcutaneous pocket.

 

Line 452: see earlier comment on line 420-422. I suppose induction of angiogenesis by the neoplasm itself would be preferable over the “inflammatory route”?

 

Line 455: what is the view of the authors on the fact that the establishment of a neoplasm in the mice takes 4-5 weeks in relation to ‘personalized in vivo testing’. Is this procedure not too time consuming?

Author Response

Review of reviewer 2         Cells-3865598.

 

In their manuscript ‘Improved murine model for the intravital microscopic examination of manifest tumors.’ the authors nicely present a promising technique for in-vivo assessment of squamous cell carcinoma.

Their study approach seems sound and the manuscript is well-written.

However, I do have some issues that I would like to see addressed.

 

 

Introduction:

 

Line 53: reference 16 and 19 do not seem to be dealing with the skinfold chamber.

Response: We thank the reviewer for this helpful remark. The comment applies accurately to reference 19, where repetitive observation of the periosteum by intravital microscopy without the use of the dorsal skinfold chamber is described. However, in reference 16 by Lunt et al. (2010), the dorsal skinfold chamber is indeed discussed, specifically in Chapter 4 of the publication. Since references 18 and 19 are not essential for illustrating the diversity of existing intravital microscopy models, we have removed these references accordingly.

Material and Methods:

 

Line 81: I suppose the PowerPlex21 was produced by Promega?

Response: Please refer to our answer to the next remark!

 

Lines 81-83: This sentence to me is more an explanation than a real description of materials and methods used in this study. Therefore it would be more logical to have this included in the discussion section where it would need further explanation why this difference is important to emphasize.

Response: We sincerely thank the reviewer for pointing this out. There are two OSCC cell lines with very similar names. To ensure clarity from the outset, we had included a note on this matter in the original text and also indicated that the identity of the cell line used in our study had been verified. However, as this clarification seems to introduce more confusion than it resolves, we have removed this passage from the revised manuscript in order to improve readability for the reader.

 

Lines 85 and 86: Spongostan is also used in veterinary medicine. The mere fact that this product is approved for use in humans doesn’t automatically mean that it can be used safely in other animals species.

Response: We thank the reviewer for this valuable comment. In response, we have revised and shortened the respective sentence as follows to improve clarity: “Spongostan™ is a medical device approved for human and animal use.”

 

Line 132: for clarity, perhaps specify that the pocket was created in the dorsal thoracic, lumbar or thoracolumbar region.

Response: We thank the reviewer for this helpful suggestion. To provide a more precise description of the experimental procedure, we have added the phrase “in the thoracolumbar region.”

 

Line 137: ‘…inhalation anesthesia was discontinued…’ instead of ‘over’.

Response: We thank the reviewer for this valuable comment. The respective sentence has been revised accordingly.

 

Line 137: I would suggest reporting that ‘neoplastic cells’ had been implanted. A tumor obviously is more than only the neoplastic cell population and according to the previous section, you implanted tumor cells and not a “whole tumor”.

Response: We thank the reviewer for this helpful remark. To improve our manuscript, we have deleted the clause “and the tumor had been implanted.”

 

Line 139: please clarify in which way ‘a manifest’ tumor was recognized? Perhaps ‘grossly visible’ is a more illustrative description.

Response: We thank the reviewer for this valuable comment. We have revised and expanded the respective sentence as follows: “a clearly visible and palpable manifest tumor.”

 

Lines 145-146: what was the rationale behind shortening the length of the study? Since the authors are aiming at analyzing “advanced tumors”, a longer time frame seems more logical.

Response: In contrast to previous studies combining tumor investigation with the dorsal skinfold chamber and intravital microscopy, our study is characterized by the fact that tumor growth was induced prior to the preparation of the dorsal skinfold chamber. This approach allows the tumor to develop entirely independently of the temporal limitations inherently associated with the restricted observation period of the dorsal skinfold chamber. As a result, the tumor can reach a size that, at least for the cell line used in this study, cannot be achieved when cells are directly injected into an already prepared dorsal skinfold chamber.

Response: We would like to emphasize that our study was designed as a pilot study (please see line 106 :” As this study was designed as a pilot study…”), primarily aiming to demonstrate the methodology and its feasibility. For this purpose, a shortened observation period in the dorsal skinfold chamber was sufficient, as the primary goal was to show that the tumor remained connected to the vascular system and continued to be perfused. In subsequent studies, in which the model may be applied, for example, to test pharmacological substances, an extended observation period will, of course, be highly relevant and valuable.

 

Line 149: since “hemodynamics” is also part of an (acute) inflammatory response. It might be clarifying to specify the ‘inflammatory parameters’ that were analyzed.

Response: We sincerely thank the reviewer for this helpful suggestion. To provide a more precise formulation, we have revised the text at this point as follows: “inflammatory parameters (leukocyte–endothelial cell interactions)”.

 

Line 149: please specify in which area these parameters were analyzed. Was that within the neoplasm, or in the regions of interest that the authors mention in the ‘Analysis of microcirculatory parameters’ section later in the manuscript (i.e. lines 172-173).

We agree with the reviewer that our initial wording was imprecise at this point. The analysis of the parameters was performed in the periphery of the growing tumor, as this is the region where the vascular changes are most pronounced and, in addition, the vessels can still be delineated with sufficient accuracy. Please also refer to our response to the following comment for further clarification.

 

Lines 172-173: based on which criteria were these 4 regions of interest chosen? I had the impression that the intra-neoplastic vasculature was the main point of interest from a potential therapeutic point of view. Can the authors be sure that the vasculature and hemodynamics in the periphery of the tumor are predictive/representative of the intra-tumoral vessels?

Response: We thank the reviewer for this valuable comment and agree that the tumor vasculature is an important therapeutic target. In our view, however, it is particularly the vessels in the tumor periphery that are most relevant for treatment. In advanced tumors, the central regions are often underperfused and necrotic areas are present, whereas the periphery remains supplied through the ingrowth of new vessels from the surrounding tissue, offering greater therapeutic opportunities.

Moreover, this focus also has practical reasons: tumor-induced angiogenesis creates a dense network of small capillaries, making it difficult to clearly distinguish individual vessels in the tumor core due to overlapping fluorescence signals. The manuscript has been revised accordingly.

 

Line 193: was the histological evaluation performed by a veterinary or human pathologist?

Response: The analysis of the histological data was performed by the authors FT, JW, and AK. These researchers are trained medical doctors/dentists and biologists.

 

Line 200: the use of antibodies produced by mice on murine tissues might pose a challenge because of cross-reactivity with mouse cells and therefore precludes confirmation of human origin of the neoplastic cells which I think is shown in figure 8 (see comment there).

Response: We thank the reviewer for this important comment and agree that it can be problematic to use antibodies raised in mice for the immunohistological investigation of murine tissues. However, the detection of p53 in our histological sections was not intended to demonstrate the human origin of the labeled cells, but rather to indicate the neoplastic character of the stained regions, as evidenced by the enhanced accumulation of this protein. The accumulation of p53 is frequently observed in oral squamous cell carcinoma and has also been used for diagnostic purposes (see, for example, Kamat et al., 2022, doi:10.1007/s00784-022-04574-y).

To demonstrate the human origin of the neoplastic cells in our study, we relied on the detection of cytokeratin (see Figure 9). The antibody used for this purpose was raised in rabbit, is human-specific, and therefore suitable for detecting the human cells. To avoid any ambiguity, we have clarified the wording in the revised version of the manuscript.

Results:

 

Line 222: ‘….after tumor cell implantation…’ instead of ‘cells’. Furthermore, the addition of ‘(see M&M)’ seems redundant.

Response: We thank the reviewer for this suggestion and changed the manuscript accordingly.

 

Lines 237-238: I feel this sentence is not a result form the study but more of a reasoning. Therefore, it might be better situated in the discussion section.

Response: We thank the reviewer for this comment. However, we believe that, for a better understanding of our chosen approach, it is appropriate at this point to deviate slightly from a purely result-oriented presentation and to provide a brief explanation of why different types of membranes were used. This aspect is discussed in more detail later in the Discussion section (lines 339–363). For improved readability, we have slightly rephrased the relevant sentence as follows: “PDS® plate proved to be most suitable, because it could be removed easily from the tumor, while the Dualmesh® membrane had a pronounced adherence to the tumor surface.”

 

Line 238: ‘Among the materials tested’ seems unnecessary, since obviously you cannot report results from material that was not used.

Response: We thank the reviewer for this suggestion and changed the manuscript accordingly.

 

Line 239: the authors might consider adding ‘because it could be removed easily from the tumor’ as an explanation why this product was deemed ‘to be most suitable’.

Response: We thank the reviewer for this suggestion and changed the manuscript accordingly.

 

Line 241: please specify ‘a few cases’.

Response: We thank the reviewer for this important remark and have added the missing information: “In two of the four animals examined in this group, small remnants of the membrane had to be left in place and could not be removed in order to avoid damaging the tumor tissue and the supplying vessels.” This information has been included in the revised manuscript.

 

Line 245: please specify the clinical presentation of these ‘irritative wound conditions’. Furthermore, from a linguistic perspective ‘…irritative wound conditions were found.’ might be a more order of words.

Response: We thank the reviewer for this important suggestion, which has helped to improve the manuscript. We have revised the text at this point as follows: “The dorsal skinfold chamber was well tolerated by all animals. In three of the 12 animals, mild irritations occurred in the area of the dorsal skin or wound margins, which presented as slight swelling or redness. However, these had no impact on the animals’ well-being or wound healing and resolved within two days.”

 

Line 258: since the authors measured the velocity of the blood cells, perhaps ‘However, the velocity of blood cells increased over time…..’ might be more appropriate (instead of ‘appeared to’).

Response: We thank the reviewer for this suggestion and changed the manuscript accordingly.

 

Lines 298-300: than the PDS membrane?

Response: We thank the reviewer for this important comment. We acknowledge that the wording at this point was unclear and have revised it accordingly in the updated version of the manuscript.

 

Line 318: ‘underlying’ might be somewhat confusing, perhaps ‘surrounding’ or ‘associated’ might be more appropriate. Furthermore, from an anatomical/histological perspective ‘….cutis, subcutis and muscles…..’ might be a more appropriate order.

Response: We thank the reviewer for this suggestion and changed the manuscript accordingly.

 

Figure 7: I have not detected the ‘m’ mentioned in the legends in any of the images.

Response: We thank the reviewer for this important suggestion. Based on additional comments and feedback, it was necessary to revise Figures 7–10. In this context, the corresponding figure legends were also adjusted and corrected. Any inconsistencies present in the previous version have been resolved in the revised manuscript.

 

The histological images used are clear and illustrative. Perhaps some tweaking using for instance Photoshop could get rid of the grey background.

Response: We thank the reviewer for the kind words regarding the quality of the figures and agree that it would be possible to improve the background of individual panels. However, our general policy when presenting data is to show unaltered original images. Modifying specific aspects of an image, such as the background using image-editing software, would require that all images be processed in the same way to maintain comparability. We consider such manipulation inappropriate with respect to the integrity of the original data. Moreover, we believe that all relevant details are clearly visible in the current version of the figures. Therefore, we have decided not to modify the images.

 

Additionally (although also more cosmetic than of major relevance), positioning the epidermis at the top and the neoplasm below would seems more representative of the situation in-situ.

Response: We appreciate this comment and agree with the reviewer that it would be possible to present the histological images in a different orientation. However, the orientation of the images does not affect the scientific message and, as the reviewer also notes, would be purely cosmetic. In our previous publications involving intravital microscopy data, we have presented histological images in the same orientation as in the current manuscript in the majority of cases. Therefore, we prefer to maintain this approach as a form of our “laboratory standard”.

 

Figure 8: as already predicted in my comment on line 200, there seems to be immunoreactivity in murine cells too. Although somewhat difficult to assess at the magnifications used for images A, C and E, I think there is marked staining in for instance the bulbs of the hairs in the normal skin.

Response: Please refer to our answer to the next point!

 

Since the histological appearance clearly shows a neoplasm of squamous epithelium, and there is basically no reason to doubt the origin, the cross reactivity is not a major problem but I think this p53 antibody cannot be used as confirmation of human origin of the neoplastic cells.

Response: We sincerely thank the reviewer for this valuable remark and fully agree that in Figure 8 p53 was also detected in areas of the normal skin, such as hair follicles. As already outlined above in response to the comment on line 200 of the manuscript, the detection of p53 in our study was intended to demonstrate the neoplastic nature of the stained cells rather than to provide evidence of their human origin. As the reviewer correctly points out, the positive staining for p53, in combination with the local arrangement and anatomical context, provides a clear picture that clearly delineates the growing tumor. We are grateful for this helpful observation and have revised the wording in the updated version of the manuscript to make our reasoning at this point more explicit.

 

Line 336: the authors describe the presence of endothelial cells mainly in the peripheral (‘marginal’) area of the tumor. This seems not ideal since it seems to be especially the blood vessels within the neoplasm that the authors are interested in. I think this aspect might be discussed in the discussion section of the manuscript.

Response: We thank the reviewer for this comment and would like to refer in this context to our response to the remark concerning lines 172–173.

 

Discussion:

 

Line 366: perhaps ‘…..anatomical location.’ is more appropriate than ‘direction’?

Response: We thank the reviewer for this suggestion and changed the manuscript accordingly.

 

Line 372: ‘… requirement was minimal adhesion……’.

Response: We thank the reviewer for this suggestion and changed the manuscript accordingly.

 

Line 374: please explain why removal of the tumor without compromising the vasculature was ‘essential’. According to the manuscript the neoplasm was left in-situ in the subcutis during the experiment, so there was no removal phase there. Postmortal harvesting of the neoplasm and the surrounding tissue should also possible without the presence of a membrane. I agree that the membrane will have facilitated the removal from the deeper tissues, but I don’t think that qualifies as “essential”.

Response: We thank the reviewer for this helpful remark. The use of different membranes is the key measure that enables the subsequent successful preparation of the dorsal skinfold chamber with a vascularized tumor inside. Our previous wording may have created a misleading impression. It is by no means essential—or intended—that the tumor itself be removed. Rather, our point was that without the use of membranes, the procedure we established could not be carried out.

At the same time, the use of different membranes represents a double-edged sword. While they are crucial for directing the vascular supply of the tumor, they also complicate the later preparation of the dorsal skinfold chamber. If the membranes were to remain on the established tumor during chamber preparation, subsequent intravital microscopic analysis would no longer be feasible. It was therefore essential that the membranes could be removed during preparation without causing any damage to the tumor or its supplying vasculature.

We have revised and expanded the corresponding passage in the updated version of the manuscript to improve the clarity of our discussion.

 

Line 380: the reported 3-4 mm size of the neoplasm seems to be a limitation with respect to the fact that the authors are mainly interested in advanced tumors that might have a different pathophysiology and morphology than the earlier stages of the same tumor. Perhaps the authors can elaborate on this aspect.

Response: We agree with the reviewer that the maximum tumor size feasible for preparation within the dorsal skinfold chamber represents only a relatively small tumor. This limitation, however, is inherent to the model and, in particular, to the dimensions of the dorsal skinfold chamber in the mouse. From a clinical perspective, it would certainly be desirable to conduct investigations in larger tumors. Nevertheless, our in vivo model for studying the vascular system of established tumors using intravital microscopy is, by its very nature, a model that requires certain constraints and simplifications to be applicable.

The intended experimental application of this model is the investigation of anti-angiogenic agents. To date, no model exists that allows the study of such agents at the level of the vascular system. Existing models typically permit only indirect analyses, for example by monitoring tumor regression. In contrast, our model goes a step further by enabling direct observation of the vascular system. While this represents only one specific aspect within the wide spectrum of possible therapeutic approaches, it is not feasible to develop a single in vivo model capable of evaluating all conceivable pharmacological treatment options equally comprehensively and precisely. Instead, multiple complementary models will always be required, each with inherent, model-specific compromises.

We have revised and expanded the corresponding passage in the updated version of the manuscript to improve the clarity of our discussion.

 

Line 391: a “full stop” is missing at the end of the sentence.

Response: We thank the reviewer for this suggestion and changed the manuscript accordingly.

 

Lines 392-394: please specify ‘interaction between tumor cells and the surrounding host tissue’. I can see the advantages with respect to the actual hemodynamics in the current setting, but interaction in the sense of mediators, growth factors, matrix proteins (and proteinases) have not become apparent in the protocol used in this pilot study.

Response: We agree with the reviewer that it is not possible to directly observe the effects of growth factors, mediators, or proteins by intravital microscopy; such effects can only be assessed indirectly through the changes they induce in the vascular system. We have revised this section of the Discussion in the updated version of the manuscript to accurately reflect this point.

 

Lines 407-409: I don’t think that you can conclude that the (neo)vascularization was actually tumor-induced. The authors have induced a surgical wound, created a subcutaneous pocket (that need healing), and inserted foreign body material (that in itself could induce an inflammatory response that includes a vascular component). I think that, in order to be able to conclude that the (neo)vascularization is a purely neoplasm-driven process, a “negative control” in which the same procedures were followed with exception of the application of the neoplastic cells should have been included in order to definitively conclude that (most of) the newly formed blood vessels were induced by the tumor cells.

Response: At this point, we must respectfully disagree with the reviewer. It is correct that some of the many steps required to ultimately obtain a manifest tumor within the dorsal skinfold chamber may themselves trigger angiogenesis. However, it has already been demonstrated that the preparation of the dorsal skinfold chamber alone does not induce changes in functional capillary density; in a chamber without an implant, test material or cells, no angiogenesis can be detected (see Rücker et al., 2006, doi:10.1016/j.biomaterials.2006.05.033). This is self-explanatory, as the vessels observed in an empty chamber are, of course, uninjured, and the wound margins generally lie outside the observation field.

We consider the influence of the different membranes on neovascularization to be minor and assume that the angiogenic effect is predominantly driven by the growing tumor itself, since it actively promotes neovascularization through the release of angiogenic factors.

 

Lines 412-414: I think apoptosis should not lead to stimulation of vascularization.

Response: At this point, we must respectfully disagree with the reviewer. There are indeed studies demonstrating a connection between apoptosis of tumor cells and the subsequent induction of angiogenesis. We have included the corresponding references in the revised version of the manuscript.

 

Lines 420-422: See my earlier comment on lines 407-409. Indeed the application of foreign body material can lead to an inflammatory reaction which can include the formation of new blood vessels.

Response: We thank the reviewer for this remark and would like to refer in this context to our response to the question concerning lines 407–409. We do not doubt that the implantation of different membranes may induce angiogenesis, as these are indeed foreign bodies being implanted. However, all materials used have been well established in clinical practice for a long time and do not elicit adverse reactions. Any potential foreign body reaction after implantation is expected to be minor and, compared to the strong angiogenesis induced by the growing tumor, not of relevance. Furthermore, within the overall context of the established model of a manifest tumor in the dorsal skinfold chamber, this question is of secondary importance, since the membranes had already been removed by the time intravital microscopic investigations were performed. Thus, the vascular changes observed at that point are exclusively influenced by the tumor itself.

 

Even though these vessels can contribute to the vascularization of the neoplasm, the underlying molecular mechanism in an inflammatory environment might be different from a purely neoplasm-driven stimulation of angiogenesis and therefore might have different pathways that could potentially be targeted as a therapeutic approach.

So I think the inflammatory response could be an undesired component.

Response: Please refer to our answer tot he question above.

 

Lines 423-428: could the authors comment on the (potential) effect of the skin fold chamber device on the circulation within the tissue that is enclosed in the device.

It seems a relative large contraption that compressed the skin and part of the subcutaneous tissue that probably will have its effect on the circulation anyway. Therefore I think it might wise to be cautious with attributing al hemodynamic effects to presence of the tumor.

Response: We thank the reviewer for this kind remark and would like to refer to our response to the question concerning lines 407–409. Our group has extensive experience with various intravital microscopy chamber models, some of which were developed by our team. We have published a number of studies using the dorsal skinfold chamber, several of which are also cited in the present manuscript. As shown in Figure 2, in our current studies we employ a chamber model with a markedly reduced design that is in no way comparable to the conventional large chamber frames.

As with many experimental models, the use of the dorsal skinfold chamber requires careful training and experience. All of our experiments are performed exclusively by experienced investigators who are well familiar with this technique. With correct preparation of the dorsal skinfold chamber, the microcirculation within the chamber remains unaffected and shows no alterations throughout the observation period.

 

Line 437: The term ‘physiological’ seems awkward for neoplastic cells that are more or less by definition not-physiologic. Apart from that, I think this also applies to the not-physiological situation that is created by inserting foreign body material in the subcutis. Perhaps rephrasing this sentence might prevent confusion.

Response: We thank the reviewer for this suggestion and changed the manuscript accordingly.

 

Lines 439-440: I think the statement that the used model is ‘based on implantation of pre-formed, manifest tumors’ is not correct. According to the materials and methods, neoplastic cell in a spongostan environment are, along with a membrane, implanted in the subcutis from which a tumor develops. This grossly visible neoplasm is then exposed in the skin fold chamber device.

This is different from the actual implantation of an already developed tumor in the subcutis.

Please rephrase to avoid confusion.

Response: We thank the reviewer for this suggestion and changed this sentence in the revised manuscript into “In contrast, our study establishes a model based on the implantation induction of pre-formed, manifest tumors before preparation of the skinfold chamber…”.

 

Line 442: what do the authors mean by ‘acute cell injection’? I think an injection often is an action of short duration.

Response: We thank the reviewer for this suggestion and changed this sentence in the revised manuscript into “Notably, models based on cell injection directly followed by an imaging procedure…“.

 

Line 451: please explain the ‘atraumatic’ nature of the procedure used. It seems counterintuitive to use the term atraumatic when you apply cells in spongostan and a membrane after surgical incision of the skin and preparing a subcutaneous pocket.

Response: We thank the reviewer for this suggestion and changed this clause in the revised manuscript into “enabling preparation of the skinfold chamber without disturbing the tumor vasculature“.

 

Line 452: see earlier comment on line 420-422. I suppose induction of angiogenesis by the neoplasm itself would be preferable over the “inflammatory route”?

Response: We thank the reviewer for this helpful remark. To improve our manuscript, we have deleted the clause “and promoting angiogenesis through a local inflammatory response.”

Line 455: what is the view of the authors on the fact that the establishment of a neoplasm in the mice takes 4-5 weeks in relation to ‘personalized in vivo testing’. Is this procedure not too time consuming?

Response: We fully agree with the reviewer that, in the context of tumor diseases, a time span of 4–5 weeks can indeed be considered long, and that many therapeutic approaches require an immediate response. However, this also depends on the specific tumor entity, and it is therefore conceivable that such a period may be available to identify a meaningful therapeutic option that is minimally burdensome for the patient while offering a high chance of cure. This will certainly not apply to advanced tumors, but rather to early-detected neoplasms, which can be well modeled within our experimental system.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

None