CD71-Mediated Effects of Soluble Vasorin on Tumor Progression, Angiogenesis and Immunosuppression

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents an important and novel finding that soluble Vasorin (sVASN) binds to CD71 and is internalized via CD71-mediated endocytosis in cancer cells (CAN-19), endothelial cells, and T cells. The authors further demonstrate that sVASN promotes phosphorylation of STAT3, and facilitates cancer cell proliferation through activation of the YAP1-TAZ and mTOR-AKT pathways. Additionally, sVASN is shown to enhance angiogenesis via the VEGF signaling pathway. The study offers significant mechanistic insights with strong implications for clinical translation.

Several concerns and suggestions should be addressed to improve the clarity and rigor of the presentation:

1. Figure 1A, 1D, 1E, 1F, S1E, and S1G: Please clearly label each band to indicate the specific antibody used (e.g., anti-His, anti-Flag). This will help readers accurately interpret the data.
2. Figure 3B: For clarity, it is recommended to write “CD71 blockade” or “CD71 blocking peptide” instead of the abbreviation “CD71 blk,” which may be unclear to readers unfamiliar with the shorthand.
3. Figure 11A: The labeling is currently unclear. Please specify which protein is being stained in the figure to ensure accurate interpretation.

4. Additionally, prior studies have reported that VASN can bind to TGFβ and potentially suppress its signaling, which is known to play a critical role in cancer progression. It would be helpful if the authors could briefly address this point in the Discussion—either to clarify whether this pathway is involved in their current model, or to distinguish their findings from this known function of VASN.

Author Response

1. Summary
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files. 

2. Point-by-point response to Comments and Suggestions for Authors
Comments 1: Figure 1A, 1D, 1E, 1F, S1E, and S1G: Please clearly label each band to indicate the specific antibody used (e.g., anti-His, anti-Flag). This will help readers accurately interpret the data.
Response 1: Thank you for pointing this out. We have labeled the specific antibodies next to each band in accordance with the reviewer's comments, including the co-IP results in Figure 8E. In this way, it is indeed clearer and more straightforward, making it easier to interpret the data.
Comments 2: Figure 3B: For clarity, it is recommended to write “CD71 blockade” or “CD71 blocking peptide” instead of the abbreviation “CD71 blk,” which may be unclear to readers unfamiliar with the shorthand.
Response 2: Agree. We have modified all "CD71 blk" to "CD71 blockade" in accordance with the reviewer's suggestion, including in Figure 3C, Figure 8, Figure 9, Figure 10, Figure 11, and Figure 13, Figure S7A-B, Figure S8B-C, which are highlighted in red letters in the Figure legends.
Comments 3: Figure 11A: The labeling is currently unclear. Please specify which protein is being stained in the figure to ensure accurate interpretation.
Response 3: Thanks for the suggestion. Indeed, labeling the specific stained proteins is very helpful for comprehension. However, we haven't been able to find the relevant information from the instruction manual provided by the manufacturer or its website (Abcam, ab204726, Angiogenesis Assay Kit, In Vitro). We further reviewed multiple articles published with the use of this kit. Regrettably, none of them contained the relevant content we were seeking. According to the instruction manual, the fluorescent dye is labeled as “Staining Dye Concentrate”, which we speculate that it might be the tube staining dye Calcein or its modified components. This dye has already been used in several other tube formation assay kits, such as the Endothelial Cell Tube Formation Assay Kit from Corning (Cat. No. 354216), and the Endothelial Tube Formation Assay Kit (In Vitro Angiogenesis) from Cell Biolabs, Inc. (Catalog Number BA-200). Although we cannot make sure whether this dye is Calcein or not, we have also labeled the “tube staining dye” on the Figure to ensure accurate interpretation.
Comments 4: Additionally, prior studies have reported that VASN can bind to TGFβ and potentially suppress its signaling, which is known to play a critical role in cancer progression. It would be helpful if the authors could briefly address this point in the Discussion—either to clarify whether this pathway is involved in their current model, or to distinguish their findings from this known function of VASN.
Response 4: Thanks for the suggestion and it is very valuable. We have added this part of the content in the Discussion section and highlighted in red letters in line 421-431. The content is as follows: “TGF-β paradox”, described as the unique characteristic of functionally switching roles from tumor suppressor to metastasis promoter, has attracted much attention in the field of cancer research. The TGF-β paradox may be better elucidated by the involvement of sVASN, which has the ability to bind to TGF-β and disrupt its signaling cascade. In normal or near-physiological cellular contexts, TGF-β exerts a pivotal tumor-suppressive function by inhibiting cell proliferation and suppressing tumorigenesis through well-defined molecular mechanisms. However, during tumor progression, an excessive amount of sVASN antagonizes the TGF-β signaling pathway and is internalized into the cells through CD71 to regulate the malignant progression of tumors. As a consequence, the tumor-suppressive effects of TGF-β are abrogated and astonishingly transforms into a driver of tumor invasion and metastasis." To emphasize the role of CD71 in this process, we designed this study to mitigate the possible impact of the TGF-β signaling pathway on the tumor phenotype. Specifically, the cell lines selected for this research predominantly exhibit low expression levels of TGF-β receptors and high expression of CD71, including Ishikawa, U251, U118, HaCaT, hCMEC/D3, among others (seeing the figure below). Our findings significantly expand the understanding of the mechanisms underlying VASN-mediated tumor promotion, uncovering a novel pathway that operates independently of the TGF-β signaling cascade.

                    

3. Additional clarifications
The panel arrangements of Figure 1 and Figure 8 have been adjusted, which are highlighted in red letters in the figure legends. The textual annotations in several Figures have been slightly modified (changes from CD71 blk to CD71 blockade), with the Figure panels highlighted. We've added Figure S4 to the supplementary materials, matching Figure 4 in the main text, and highlighted in red letters for easy identification. With the addition of Figure S4 to the supplementary materials, the figure numbering of the supplementary materials has been updated, and all revised figures are clearly highlighted for quick reference. To match the modified figures and additional content, we’ve made minor edits to the main text. The changed parts are clearly highlighted for easy review.  

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Please see the attachment. 

Comments for author File: Comments.pdf

Author Response

1. Summary
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files. 

2. Point-by-point response to Comments and Suggestions for Authors
Comments 1: Why was overexpression chosen as the method to study VASN-binding proteins, and why were the specific cell lines used not introduced? What was the rationale for selecting CD71 in particular?
Response 1: Thank you for the questions. 

    i. The study primarily aims to elucidate the process and underlying mechanisms by which sVASN, cleavage by ADAM17, is re-internalized into cells, encompassing both adjacent and distant cells. To this end, our efforts were focused on the capture of sVASN-binding proteins, with a particular emphasis on those located on the cell membrane. The rationale for employing overexpression methods to investigate VASN-binding proteins is as follows: 1) We need artificial tag proteins to distinguish between endogenous and exogenous VASN, so the overexpression method is the best choice. 2) CD71 is generally highly expressed in all types of cell lines, probably due to its important function as a transferrin receptor. In contrast, VASN is predominantly highly expressed in specific cancerous or endothelial cells. Therefore, overexpression of VASN allows for a more comprehensive and unambiguous determination of its binding interactions. 3) As both VASN and CD71 are membrane proteins, we have also performed the binding assays of VASN or CD71 in the form of cell surface, and found that cell surface VASN can bind to CD71 as well, albeit with relatively low efficiency.

    ii. We are sorry for not describing this part clearly in the original manuscript and we have added this description in this version. We chose HEK293T cells as the cell line for co-IP assays, as it is a classic and commonly used choice for such analysis. HEK293T cells are highly amenable to transfection and can efficiently uptake exogenous DNA, thereby enabling the effective introduction of target genes into the cells. Moreover, their rapid growth rate and strong proliferative ability allow for the acquisition of a large number of cells in a short time, which significantly enhances the repeatability of the experiments. Additionally, the relatively simple profile of endogenous proteins expressed by HEK293T cells greatly reduces interference with the experimental results. This feature enables a clearer detection of the interaction between the target protein and its interacting partners, ultimately improving the accuracy and reliability of the experimental outcomes. 

    iii. CD71 was identified as a binding protein of sVASN through IP-MS analysis of the supernatant of hepatocellular carcinoma cells, as described in line 76-78. Among the numerous identified proteins, we focused on CD71 because of its crucial role as a cell membrane protein in the processes of endocytosis and protein transportation. As a well-known transferrin receptor, CD71 is involved in various cellular uptake mechanisms, making it a highly plausible candidate for mediating the internalization of sVASN. Our subsequent studies have also demonstrated that sVASN is endocytosed into different cells through CD71 to exert its functions. Our findings not only elucidate the molecular mechanism by which sVASN re-internalized into cells but also provide potential targets for future therapeutic interventions.
Comments 2: Why not perform endogenous IP? Is CD71 directly interacting with VASN?
Response 2: Thanks for the suggestions.

    i. In this revised version, we have comprehensively supplemented detailed information about the IP-MS methods, as illustrated in Figure S1A. Actually, we indeed performed endogenous IP by using a VASN-specific monoclonal antibody to capture the binding proteins of sVASN from HepG2 hepatocellular carcinoma cell lysates previously. Following endogenous IP, the silver-stained protein bands were subjected to mass spectrometry analysis (MS), which led to the identification of CD71. Having identified CD71 through the integration of endogenous IP combined with mass spectrometry, we carried out the exogenous co-IP assays to further confirm the direct binding of sVASN to CD71.

    ii. In this study, we have analyzed the binding sites, binding domains, and kinetic simulations of sVASN to CD71 by means of co-IP, LSCM, SPR, ELISA assays, and molecular docking experiments. The results of all these experiments collectively demonstrated the binding of sVASN to CD71. Thus, we have compelling evidence to conclude that CD71 and VASN directly interact.
Comments 3: "Why were different cell lines used in Figure 3A and 3B?
Response 3: Thank you for the questions. Our objective was to ascertain that sVASN undergoes endocytosis into diverse cell types via CD71 present on the cell surface. To this end, we selected multiple cell types for comprehensive verification. The experiments depicted in Figure 3A and Figure 3B were validated across multiple cell lines. 
Comments 4: In Figure 3A, the knockdown efficiency of CD71 in Ishikawa cells was too low, and there was no change in VASN when comparing the control non-targeting (ctrl NC) and the non-targeting control (VASN NC).
Response 4: Thank you for the questions. 

    i. Despite the knockdown efficiency of CD71 in Ishikawa cells was lower than that in U251 cells, the statistics results of multiple biological replicate experiments clearly demonstrated the effective downregulation of CD71 (Figure 3A, column 5 VS 6, p=0.0067; column 7 VS 8, p=0.0009). Additionally, upon treatment with si-CD71, the endocytosis of exogenous sVASN was notably reduced in Ishikawa cells (Figure 3A, column 1 VS 2, p=0.1611; column 3 VS 4, p=0.0004). We speculated that the reason for the poor knockdown efficiency of CD71 is likely attributable to its critical role in iron uptake. Upon CD71 knockdown, cells may activate compensatory mechanisms, thereby limiting the effectiveness of the silencing, which need to be confirmed in the future. 

    ii. The results of the control non-targeting (ctrl NC) and non-targeting control (VASN NC) also demonstrated that exogenous sVASN addition led to a significant increase in intracellular VASN levels (Figure 3A, column 1 VS 3, p=0.0361). 
Comments 5: Fig3： In Figure 1, a mutant of VASN that binds to CD71 was identified, so why not compare the mutant with the wild-type (WT) VASN?"
Response 5: Thanks for the suggestion. The reason we did not compare the VASN mutant identified in Figure 1, with the wild-type VASN is that the mutant forms lack biological function (seeing the figure below). 

In this study, we were more concerned about the biological functions of sVASN after being endocytosed into cells. Hence, we selected the wild-type sVASN as the subject of our research rather than the mutant. Although not the central focus of this article, we have also investigated the endocytosis of the VASN mutant form in U251 cells and Ishikawa cells, as illustrated in the following figures, which were not shown in the main manuscripts.

 
 
Comments 6: Fig3D, "Was western blot (WB) validation performed for the CD71 knockout (KO) cells?"
Response 6: Thanks for the suggestion and it is very valuable. Western blot (WB) verification was conducted for the U251 KO cell line depicted in Figure 3D, and we have added this part of the content in the Supplementary Materials Figure S3C and highlighted in red letters. The results indicated an effective knockout of CD71. 
Comments 7: Fig4A: "Regarding the ER localization - was appropriate validation performed? LAMP1 should typically exhibit punctate staining patterns in cellular localization assays, while the Golgi apparatus should localize adjacent to the nucleus. Were specific organelle markers used as probes to corroborate these findings?"
Response 7: Thanks for the suggestions. The organelle staining assays were executed based on the following two references [1,2]. Antibodies selection and staining procedures were carried out in accordance with the organelle markers described in the study by Burr et al. Our results revealed that LAMP1 and GM130 exhibited distinct staining patterns across various cell lines. Specifically, in CAL-27 and HepG2 cells, LAMP1 was distinctly observed as punctate pattern in the cytoplasm, while GM130 was linearly distributed near the nucleus, which was consistent with what the reviewer mentioned (seeing the figures below and Supplementary Materials Figure S4). Although HCT116 cells, being smaller in size compared to CAL-27 and HepG2 cells, presented less pronounced staining characteristics, they provided a more informative visualization of the subcellular localization of sVASN following endocytosis. Therefore, confocal microscopy images of HCT116 cells were selected for presentation in the manuscript to best illustrate the sublocalization of sVASN within the cellular context.

[1]. Burr, M.L.; Sparbier, C.E.; Chan, Y.C.; Williamson, J.C.; Woods, K.; Beavis, P.A.; Lam, E.Y.N.; Henderson, M.A.; Bell, C.C.; Stolzenburg, S.; et al. CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. Nature 2017, 549, 101-105, doi:10.1038/nature23643.

[2]. Grant, B.D.; Donaldson, J.G. Pathways and mechanisms of endocytic recycling. Nat Rev Mol Cell Biol 2009, 10, 597-608, doi:10.1038/nrm2755.

CAL-27 cells

HepG2 cells

Comments 8: Fig4A: "Can it simply demonstrate the degradation pathway of VASN?"
Response 8: Thank you for the question. The results presented in Figure 4A are primarily intended to elucidate the transport pathway of exogenous sVASN internalized into cells. According to the confocal microscopy findings, sVASN predominantly localizes within the endoplasmic reticulum and the Golgi apparatus. Although this single piece of evidence supports this process, numerous studies in the literature have shown that the primary route for exogenous proteins to enter cells is through degradation, a mechanism that helps to avoid immune responses. Based on this, we reached the following conclusion: from the perspective of the transport route of exogenous protein, 1) sVASN is likely to undergo subsequent modifications in the endoplasmic reticulum and the Golgi; 2) exogenous sVASN enters into the lysosomes for degradation. Relevant studies are ongoing, which represent separate stories.
Comments 9: Fig5E: "Why not use endogenous co-IP or GST-pull-down to demonstrate the interaction between STAT3 and VASN?"
Response 9: Thank you for pointing this out. The reason we did not employ endogenous co-IP to verify the binding between STAT3 and VASN was also to differentiate between endogenous VASN and exogenous sVASN. Our main objective was to investigate how sVASN re-internalizes into cells via CD71 and subsequently translocates to the nucleus by binding to STAT3. Structurally, these functions are attributed to soluble form of VASN. Hence, we made a clear distinction between the endogenous and exogenous forms. Additionally, we refrained from conducting further GST pull-down experiments because the binding of STAT3 and VASN had already been confirmed in previous studies (ref. 33). Consequently, in this study, we solely utilized the exogenous co-IP experimental method for verification. The relevant references are provided as follows.
Comments 10: Fig6A: "Conversely, can VASN promote the nuclear translocation of STAT3? Is there any relationship between CD71 and the nuclear translocation of STAT3?"
Response 10: Thank you for the questions. 

    i. Our results indicate that sVASN can facilitate the nuclear translocation of STAT3, and the nuclear translocation of sVASN was dependent on the phosphorylated STAT3 at tyrosine 705 site. STAT3 and sVASN exhibit a synergistic effect during the nuclear translocation. STAT3 aids sVASN in translocating into the nucleus, and sVASN increases the nuclear translocation of STAT3. This reciprocal interaction triggers the activation of a series of genes, ultimately leading to alterations in the cell phenotype.

    ii. Currently, we lack sufficient experimental data to establish a connection between CD71 and the nuclear translocation of STAT3. Based on the existing findings, CD71 serves as a prerequisite for sVASN to enter the nucleus. Specifically, sVASN, whether in the form of exogenous sVASN or VASN cleaved by ADAM17, firstly need to be endocytosed into cells via CD71 before it binds to STAT3 and subsequently translocate into the nucleus. Blocking CD71 impedes the entry of sVASN into cells, thereby affecting its nuclear translocation as well. Thus, there exists a sequential relationship between them. Nevertheless, whether CD71 influences the nuclear translocation of STAT3 through other pathways remains to be further explored. This undoubtedly represents a highly intriguing scientific question.
Comments 11: In the manuscript the authors used many cell lines belonging to different diseases, so I don't know what the author's research purpose is?
Response 11: Thank you for the questions. Our study provides abundant evidence that sVASN undergoes endocytosis into cells via CD71 on the cell surface, and subsequently translocates into the nucleus through its binding to STAT3, ultimately leading to alterations in the cell phenotype. Significantly, this biological process is independent of cell type, relying solely on the presence of CD71 on the cell surface, thereby representing a universal phenomenon. The employment of multiple cell lines was not only aimed at confirming the universality of the mechanism but also stemmed from a comprehensive consideration of the tumor microenvironment. In the complex tumor microenvironment, the interactions among tumor cells, endothelial cells, and immune cells play pivotal roles in driving tumor progression. Recognizing this, we conducted these investigations across tumor cells, immune cells, and endothelial cells to explore the functions of sVASN mediated by CD71 on the cell surface. 

Collectively, the central objective of this paper is to elucidate that the entry of sVASN into cells through CD71 on the cell surface, which promotes tumor malignant progression, is a widespread occurrence across multiple cell types. By targeting and intervening in this process, it may be feasible to modulate tumor progression, thereby paving the way for the development of a novel dual-targeted treatment strategy centered around the sVASN-CD71 binding interaction.
3. Additional clarifications
The panel arrangements of Figure 1 and Figure 8 have been adjusted, which are highlighted in the figure legends. The textual annotations in several Figures have been slightly modified (changes from CD71 blk to CD71 blockade), with the Figure panels highlighted in red letters. We have added a section related to TGF-β in the "Discussion" part, included two cited references. We've added Figure S4 to the supplementary materials, matching Figure 4 in the main text, and highlighted in red letters for easy identification. With the addition of Figure S4 to the supplementary materials, the figure numbering of the supplementary materials has been updated, and all revised figures are clearly highlighted in red letters for quick reference. To match the modified figures and additional content, we’ve made minor edits to the main text. The changed parts are clearly highlighted in red letters for easy review. 

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

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