uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

Abstract High intratumoral levels of urokinase‐type plasminogen activator (uPA)‐plasminogen activator inhibitor‐1 (PAI‐1) heteromers predict impaired survival and treatment response in early breast cancer. The pathogenetic role of this protein complex remains obscure. Here, we demonstrate that heteromerization of uPA and PAI‐1 multiplies the potential of the single proteins to attract pro‐tumorigenic neutrophils. To this end, tumor‐released uPA‐PAI‐1 utilizes very low‐density lipoprotein receptor and mitogen‐activated protein kinases to initiate a pro‐inflammatory program in perivascular macrophages. This enforces neutrophil trafficking to cancerous lesions and skews these immune cells toward a pro‐tumorigenic phenotype, thus supporting tumor growth and metastasis. Blockade of uPA‐PAI‐1 heteromerization by a novel small‐molecule inhibitor interfered with these events and effectively prevented tumor progression. Our findings identify a therapeutically targetable, hitherto unknown interplay between hemostasis and innate immunity that drives breast cancer progression. As a personalized immunotherapeutic strategy, blockade of uPA‐PAI‐1 heteromerization might be particularly beneficial for patients with highly aggressive uPA‐PAI‐1high tumors.


th Aug 2020 1st Editorial Decision
24th Aug 2020 Dear Prof. Reichel, Thank you for the submission of your manuscript to EMBO Molecular Medicine, and please accept my apologies for the delay in getting back to you. We have now received feedback from two of the three reviewers who agreed to evaluate your manuscript. Given that referee #1 has unfortunately not returned his/her report so far despite several chasers, and that both referees 2 and 3 are overall positive, we prefer to make a decision now in order to avoid further delay in the process. Should referee #1 provide a report, we will send it to you, with the understanding that we would not ask you for extensive experiments in addition to the ones required in the enclosed reports.
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In the event of acceptance, this file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication.
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EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection. Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status. I look forward to receiving your revised manuscript.  The technical quality of the present manuscript is high and the employed model systems are adequate. The novelty of the described findings are medium as uPA-PAI1 has already been associated with neutrophil trafficking by the authors elsewhere and effects of WX-340 on breast cancer progression have already been described by others. An application of WX-340 in therapy needs lengthy approval thus a direct medical impact is not given.
Referee #2 (Remarks for Author): The authors of the manuscript "uPA-PAI-1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils" show that tumors release urokinase-type plasminogen activator (uPA)-plasminogen activator inhibitor-1 (PAI-1) heteromers and initiate a pro-inflammatory program in perivascular macrophages leading to the attraction of pro-tumorigenic neutrophils. Inhibition of uPA-PAI-1 heteromerization by WX-340 decreased neutrophil trafficking and had prophylactic and therapeutic effects in vivo.
The manuscript is well written and technically sound.
Certain effects such as the effect of uPA-PAI1 on neutrophil trafficking especially compared to single proteins are rather weak and the depicted assays ( Figure 1c) lack positive controls. The authors might want to critically discuss this.
It would be interesting to compare the effect of uPA-PAI1 to single proteins alone in further assay such as the cremaster expression of ICAM and VCAM after injection.
The prophylactic effect of the inhibitor could be tested in a model of induced breast cancer to underline its efficacy and applicability. Some typos such as microvacular (page6) and their (which should be "the" on page 7)...

Referee #3 (Comments on Novelty/Model System for Author):
The findings are interesting but it very technical without clear e explanation for general readership.It need expanding of the text especially the results for this to becomes more clear to a reader who does not have immunological expertise or imaging of the particular technology used by the authors.
Referee #3 (Remarks for Author): In this manuscript, Uhl et al. present evidence for the role of urokinase-type plasminogen activator (uPA)-plasminogen activator inhibitor-1 (PAI-1) heteromers in breast cancer. The authors demonstrate the potential for clinical relevance by associating uPA and PAI-1 protein levels with both extravascular and intravascular neutrophils, from breast cancer cohort patients, that were diagnosed with G1 breast cancer. uPA-PAI-1 heteromers are part of the fibrinolytic system, impacting cell adhesion and migration, angiogenesis, signal transduction and apoptosis. uPA-PAI-1 can recruit both macrophages and neutrophils and tumor derived uPA-PAI-1 protein expression, is known to impact breast cancer and influence metastasis. The authors utilized a mouse peritoneal assay(s), along with multi-channel flow cytometry, to track neutrophil motility in response to uPA-PAI-1 heteromers treatment. In response to uPA-PAI-1 exposure neutrophils, increased transmigration activity, in the intertumoral space thus promoting early tumor growth and metastasis. In vivo, uPA-PAI-1 exposure increases the number of both macrophage and neutrophils into the peritoneal cavity, neither B and or T lymphocytes were similarly recruited after treatment. The activity of uPA-PAI-1 does not depend on activating endothelial cells expression of either ICAM and or VCAM, for transmigration activity. Rather uPA-PAI-1 heteromers activity increase ICAM and VCAM expression in a neutrophil specific manner through the supposed activity of TNF. The authors present data that increase TNF production arises from the macrophages in response to uPA-PA-1 exposure. This would suggest that uPA-PAI activation is upstream of TNF as it increases ICAM and VCAM expression in neutrophils but not in the endothelial cells. Moreover, uPA-PAI-1 activity in vivo, that influenced the NE neutrophils subtype rather than MMP9 and or VEGF subtype. The NE neutrophils stimulate 4T1 proliferation in vitro and in vivo uPA-PAI-1 inhibitor (WX340) decreased neutrophil recruitment delaying 4T1 cells tumor growth and decreasing metastasis. General comments: In general, the manuscript has very intriguing results and mostly well control. Description(s) of the experimental methodology was often lacking in details within the text causing one to pause and ask was the specific assay been conducted in vivo or in vitro? Description of muscle cremaster models using the Ly-6G+ CX3CR-1-cells is poorly described in this manuscript it is referenced as published, should be introduce for general readership. Some results where conflated in one experimental system to conclude similar findings in a second experimental system and suggesting the impact on cellular activity was the same without providing raw data for independent assessment of these conclusions. Tumors biology lack tracking of tumor growth curves either daily or weekly. Histomorphogenic analysis of tumors and metastasis lesion was absent to ensure the arguments that this was due to less neutrophil recruitment. Validation of the IHC analysis to make conclusion in regards blocking growth and or preventing metastasis was not convincing. In all the manuscript has made a very important observation(s) and conclusions that merit considerations for publication. This would require major editing and response to several major and minor points so that impact of the work is broadly accepted.
Major Specific points: 1. Fig. 1B it is not clear why the author depleted the neutrophils with Ly-6B yet identify the neutrophils as Cd11bhi/GR-1hi/CD115 neg, the raw FACS data needs to be shown. Ly-6B is a general granulocyte marker also found in eosinophils and basophils. Ly-6B is also found in activate macrophages and uPA-PAI-1 activated macrophage recruitment to the peritoneal cavity. So it is misleading to called it a "neutrophil depletion" when it also depletes macrophages as shown by the figure 1A. 2. The neutralizing assay is not detailed in M&M and which antibody was used to do the depletion. 3. Fig 1C As this model is heavily dependent on many of its I conclusion on microcopy imaging it is quite unclear why the cells are label Green (GR1 positive) when utilizing the Ly-6G+ GFP-using the Cx3CR-1GFP+ mice. This highlight the difficult the author creates by not describing their model for this particular manuscript with little details and relying on previous published work to call the cells neutrophils. This manuscript should stand alone without having to go read another manuscript. 4. A single image is not representative of the quantified data shown for adherence and transmigration assays for either the macrophages and or the neutrophils the data is not impressive showing two cells. 5. Fig 2a. Cremasteric assay no reference provide in text for this manuscript. Immunofluorescent green for UPA and PAI are they labels with green tags, is unclear. Upon TNF treatment recruitment of neutrophils are pink and green the protein moved it is just not clear at all or are the uPA-PAI-1 heterodimerization occurring in the neutrophils or another cell subtype, this is not at all clear. 6. Fig 2C an in in vitro assay with RAW 264.7 exposed to uPA-PAI-1 heteromers is not the same thing as macrophages derived from an in vivo source. Especially if the arguments are that macrophages are been recruit to assist the neutrophils too attach and trans migrate. As they provide the chemokines and our TNF production as the source to elicit such action in vivo, that is trans migration. 7. Fig2D results using an endothelial in vitro assay vs what was shown in Fig2B in vivo are not consistent, in one assay the uPA-PAI-1 heteromers increase ICAM/VCAM but in the other I assay it does not? If the author were attempting to imply that the in vivo assay requires macrophage recruitment for uPA-PA-1 to the endothelial cell for both ICAM/VAM to be up regulated only in vivo but not in vitro this was not clear at all in the text or the explanations of the results. Therefore, culture of the bEND.3 with the RAW cells would potentially address this issue when exposed to either TNF and or uPA-PAI-1 heteromers. 8. Fig1E require images for independent assessment that cell is specifically interacting with one cell surface receptor but not the other. 9. Fig EV4 needs to show IHC results of the neutrophil population in G1, G2 and G3 breast cancer samples. For independent assessment of authors conclusions. 10. Figure 3A a single IHC for uPA-PAI-1 is not valid for this analysis and will require showing of multiple samples with wider range of the tumor showing. 11. The author do not address the expression for uPA-PAI-1 has been shown that can also come from human tumors and not just the infiltrating neutrophils. 12. Figure 3b the cohort analyzed for the METABRIC data set need to be clarify as to which subtype of breast cancer were analyzed. Where there any differences between Luminal A and or B, Her 2 and or basal/TNBC subtypes. No p-value and or HR are shown diminishing the significance of the study. Simple correlation are not indicate of clinical relevance. 13. Figure 3d it is not clear why the inhibitor WX-340 was not added to the neutrophils plus 4t1 cells or when NE inhibitor was added to show combinatorial effects. 14. 15. Fig4C the staining for uPA and or PAI-1 are unimpressive and do not appear to be specific but rather a wide expression pattern is shown to most every cell. 16. The author does not rule out the effects on lack of macrophage recruitment to the tumor biology his needs to be addressed. 17. Fig4D tumor growth kinetics are lacking along with histomorphogenic analysis of tumors. 18. Figure 4F effects on metastasis by 4T1 cells is very robust to multiple organs yet these results cannot be independently asses by the data shown. 19. IHC and HE comparison are requires and timing of the experiment metastasis is also lacking.   Answer: According to the reviewer's suggestion, we now show that the effect of uPA-PAI-1 heteromers on the endothelial expression of ICAM-1/CD54 and VCAM-1/CD106 is more pronounced as compared to the single proteins. These novel data are included and discussed in the revised version of our manuscript (Appendix Fig. S1b; p. 6, 2 n d paragraph; p. 13, 2 nd paragraph).

2.) The neutralizing assay is not detailed in M&M and which antibody was used to do the depletion.
Answer: The experimental approach to deplete neutrophils is now described in more detail in the Materials and Methods section of the revised version of our manuscript (p. 26, 1 st paragraph). Answer: According to the reviewer's suggestion, technical details regarding our multi-channel in vivo microscopy experiments in CX 3 CR-1 +/GFP ('monocyte-reporter') mice are now described in more detail in the revised version of our manuscript. Briefly, CX 3 CR-1 +/GFP mice exhibit GFP low classical monocytes and GFP high non-classical monocytes. In the cremaster muscle of male CX 3 CR-1 +/GFP+ mice, neutrophils were identified as Ly-6G + CX 3 CR-1cells, classical monocytes as Ly-6G -CX 3 CR-1 low cells, and non-classical monocytes as Ly-6G -

4.) A single image is not representative of the quantified data shown for adherence and transmigration assays for either the macrophages and or the neutrophils the data is not impressive showing two cells.
Answer: According to the reviewer's suggestion, we now also show representative in vivo microscopy images from the unstimulated control group in the revised version of our manuscript (Fig. 1c).

5.) Fig 2a. Cremasteric assay no reference provide in text for this manuscript.
Immunofluorescent green for UPA and PAI are they labels with green tags, is unclear. Upon Here, uPA and PAI-1 were detected in the perivenular space of inflamed tissue as well as -to a lesser extent -on the microvascular endothelium of postcapillary venules.
However, we cannot clearly state to what extent intracellular and extracellular uPA-PAI-1 heteromerization occurs in inflamed tissue. This is now clearer in the revised version of our manuscript and a reference to these findings is provided in the manuscript text (p. 13, 2 nd paragraph; Figure legend 2). Fig 2C an in in vitro assay with RAW 264.7 exposed to uPA-PAI-1 heteromers is not the same thing as macrophages derived from an in vivo source. Especially if the arguments are that macrophages are been recruit to assist the neutrophils too attach and transmigrate. As they provide the chemokines and our TNF production as the source to elicit such action in vivo, that is transmigration.

6.)
Answer: According to the reviewer's suggestion, we now also show that recombinant murine uPA-PAI-1 heteromers induce the release of TNF from peritoneal macrophages isolated from WT mice (Appendix Fig. S2a; p. 7, 1 st paragraph).

Fig1E require images for independent assessment that cell is specifically interacting with one cell surface receptor but not the other.
Answer: In addition to the quantitative flow cytometry data, confocal microscopy images of primary mouse neutrophils exposed to recombinant murine uPA-PAI-1 heteromers or PBS binding to ICAM-1/CD54-Fc or VCAM-1/CD106-Fc are now provided in the revised version of our manuscript (Appendix Fig. S2c). In these novel images, enhanced and clustered binding of neutrophils to ICAM-1/CD54, but not to VCAM-1/CD106, is visualized (p. 7, 2 nd paragraph).

Fig EV4 needs to show IHC results of the neutrophil population in G1, G2 and G3 breast cancer samples. For independent assessment of authors conclusions.
Answer: According to the reviewer's suggestion, representative images of neutrophil infiltration in G1, G2, and G3 breast cancer samples is now shown in the revised version of our manuscript (Fig. EV4). 10. Figure 3A a single IHC for uPA-PAI-1 is not valid for this analysis and will require showing of multiple samples with wider range of the tumor showing.
Answer: According to the reviewer's suggestion, more representative images of neutrophil infiltration in breast cancer samples is now shown in the revised version of our manuscript ( Fig. EV4; see above).

The author do not address the expression for uPA-PAI-1 has been shown that can also come from human tumors and not just the infiltrating neutrophils.
Answer: We appreciate the notion of this reviewer that not only neutrophils release uPA and PAI-1, but also breast cancer cells. This point is now clearer in the revised version of our manuscript (p. 11, 2 nd paragraph; Appendix Fig. S3a). Answer: We thank the reviewer for this essentially important comment and apologize that this was not mentioned in the original version of our manuscript. P-values and HR are now shown in Fig. 3b and Fig. EV5b. Further, cross-tabulation and Chi-squared analysis revealed no significant association between high RNA expression of PLAU or SERPINE1 in the tumor and the molecular breast cancer subtype as defined by the 3-gene-classifier.

Figure
Accordingly, we conclude that the significantly impaired overall survival of stage 0-1 breast cancer patients with PLAU or SERPINE1 high expressing tumors is not confounded by enrichment of a distinct molecular subtype. These results have been added to the revised version of our manuscript in form of a mosaic plot in Fig. EV5c.

Figure 3d it is not clear why the inhibitor WX-340 was not added to the neutrophils plus 4t1 cells or when NE inhibitor was added to show combinatorial effects.
Answer: We appreciate the reviewer's suggestion to analyze the effect of compound WX-340 on the proliferation of 4T1 breast cancer cells in more detail. In a novel set of experiments, application of WX-340 did not significantly alter the proliferation 4T1 tumor cells exposed to the supernatant of uPA-PAI-1-primed neutrophils. Similarly, additional application of WX-340 did not significantly change the proliferation of 4T1 tumor cells upon inhibition of NE. These data are now included in the revised version of our manuscript (Appendix Fig. S2d).

Fig4a not clear what cells are been used either the text or the figure legends. Similar for Fig4b not clear if this is in the context of the tumor in vivo model or not.
Answer: In Fig. 4a, the effect of WX-340 on the binding of recombinant mouse uPA protein to PAI-1 protein was quantitatively assessed in vitro using ELISA analyses. In   Here, we show that antibody-mediated depletion of neutrophils severely compomised tumor growth in 4T1 breast cancer, whereas antibody-mediated depletion of classical monocytes exhibited a lower, but significant tumor-suppressive effect than depleting neutrophils. These novel data are now included in the revised version of our manuscript (Appendix Fig. S3c; Appendix Tab. S2).

Fig4D tumor growth kinetics are lacking along with histomorphogenic analysis of tumors.
Answer: According to the reviewer's suggestion, tumor growth kinetics are now shown in the revised version of our manuscript (Appendix Fig. S3b).
18. Figure 4F effects on metastasis by 4T1 cells is very robust to multiple organs yet these results cannot be independently asses by the data shown. To enable the more independent assessment of the effect of WX-340 on tumor metastasis in lungs and brain, we provide representative images of immunohistochemically stained tissue sections in the revised version of our manuscript (Appendix Fig. S4b). 19. IHC and HE comparison are requires and timing of the experiment metastasis is also lacking.
Answer: As mentioned above, we now also provide representative images of immunohistochemically stained tissue sections as well as a schematic illustration of the experimental protocol of our tumor metastasis experiments (Appendix Fig. S4a, b). Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the two referees who re-reviewed your manuscript. As you will see, they are now supportive of publication, and I am therefore pleased to inform you that we will be able to accept your manuscript, once the following minor points will be addressed: 1) Referees' comments: Please address the remaining point from referee #1 regarding the reference to add and discuss.
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-Please upload the figures separately as independent files (without legends). Please make sure the figures are big enough to be fully visible/readable. -The manuscript text refers to figure EV9, please clarify. 4) Checklist: please provide information in sections E/11-12. Please also provide information in section F/18. Regarding section F/19, please clarify to which dataset you are referring to. 5) Thank you for providing 'The paper explained'. I slightly edited the text to shorten it, please let me know if you agree with the following: PROBLEM: Breast cancer is the most common oncological disorder in women worldwide. High intratumoral levels of heteromers of the serine protease urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor-1 (PAI-1) predict impaired survival and treatment response already in early stages of breast cancer. Although these single proteins are well known to control tissue perfusion by regulating clot formation as key components of the fibrinolytic system, the pathogenetic role of this protein complex in breast cancer remains obscure. RESULTS: Utilizing patient data and different syngeneic mouse models of breast cancer, we demonstrate that heteromerization of uPA and PAI-1 multiplies the potential of the single proteins to attract pro-tumorigenic neutrophils. To this end, tumor-released uPA-PAI-1 utilizes the very low density lipoprotein receptor and intracellular mitogen-activated protein kinases to initiate a proinflammatory program in perivascular macrophages in the proximity of malignant tumors. This enforces neutrophil trafficking to cancerous lesions and skews these immune cells towards a protumorigenic phenotype, thus supporting tumor growth and metastasis. Blockade of uPA-PAI-1 heteromerization by a novel small-molecule inhibitor interfered with these events and effectively prevented tumor progression. IMPACT: Our findings identify a therapeutically targetable, hitherto unknown interplay between hemostasis and innate immunity that drives breast cancer progression. As a personalized immunotherapeutic strategy, blockade of uPA-PAI-1 heteromerization might be particularly beneficial for patients with highly aggressive uPA-PAI-1high tumors. 6) Thank you for providing a 'For more information' section. Please note that the English webpage was not accessible, however the German one was (https://www.femtelle.de/). 7) Thank you for providing a nice synopsis picture. Please upload it separately as a png or jpeg file 550px wide x 400px high. 8) As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here. Please note that the Authors checklist will be published at the end of the RPF. The aut hors have address all concerns from this reviewer. Th aut hors great ly improve bot h the qualit y of result s and readership.

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