Loss of cIAP1 in Endothelial Cells Limits Metastatic Extravasation through Tumor-Derived Lymphotoxin Alpha

Round 1

Reviewer 1 Report

I have carefully read the manuscript titled "Loss of cIAP1 in endothelial cells limits metastatic extravasation through tumor-derived lymphotoxin alpha" and on the basis of my knowledge in the field, I absolutely confirm the high level of the research. Furthermore, methods are remarkably adequate with the aim of the study and with the data here presented. The results are well supported by a careful reading of the literature in the field along with a well-exploited statistical analysis. Before publication, I strongly recommend a careful reading of the text for the presence of many typos. 

Author Response

Response to Reviewer 1 comments.

We thank the reviewer for their compliment about the design of our study. We will carefully proof-read the text as suggested.

Reviewer 2 Report

The subject of the article is pertinent and interesting, once the authors show that the loss of cIAP1 alters endothelial cells from responding to signals from the tumor cells to change shape and allow for tumor cells to pass through the endothelial barrier.

General comments:

1. The introduction are very good. However, the objective of the work is not clear, neither in the introduction nor in the abstract. Please clarify the main objectives of the work.
2. The discussion must have de clinic relevance of the works, pleased reformulated.
3. In my opinion the Conclusion needs to be reformulated, especially the first sentences, which are strange.

Author Response

Comments to Reviewer 2:

The subject of the article is pertinent and interesting, once the authors show that the loss of cIAP1 alters endothelial cells from responding to signals from the tumor cells to change shape and allow for tumor cells to pass through the endothelial barrier.

General comments:

1. The introduction are very good. However, the objective of the work is not clear, neither in the introduction nor in the abstract. Please clarify the main objectives of the work.

Response 1:

We thank the reviewer for their constructive criticism to further clarify the purpose and findings in our study. 

An objective has been added to the abstract and introduction. In the abstract, we have added:

In this study, we determined whether Smac mimetics play a role in metastasis, specifically in circulation, tumor extravasation and growth in a metastatic site. Reports suggest targeting the loss of IAPs through use of Smac mimetics, alter the ability of the tumor cell to metastasize but have not defined a role for the immune compartment or stroma in affecting the ability of tumor cells to metastasize. To define whether the immune compartment or stroma can affect the ability of tumor cells to migrate, we utilized syngeneic tumor models in a late stage model of metastasis.

In the introduction, we have added:

In this study, we determine the role of Smac mimetics in the tumor microenvironment. Use of Smac mimetics is mimicked by the loss of cIAP1 or cIAP2 throughout the entire mouse without altering the expression level of IAPs in the tumor cell. The loss of cIAP1 but not cIAP2 in the endothelium obstructs tumor cell extravasation into the lung. Surprisingly, loss of cIAP1 does not enhance endothelium cell death during tumor extravasation and the results can be recapitulated using Smac mimetics (Birinapant). Our data show that lymphotoxin alpha and not TNF secreted from the tumor cell requires cIAP1 expression in the endothelium to induce tumor cell extravasation. These results suggest that application of Smac mimetics in the clinics might not only contribute to tumor killing by activating the immune system but also by preventing tumor metastasis of individual cancer types.

1. The discussion must have de clinic relevance of the works, pleased reformulated.

Response 2:

We have included the clinical relevance of the study in the discussion. This is highlighted in the following text below:

Besides birinapant (NCT00993239, NCT01681368), there are several other Smac mimetic compounds that have reached clinical trials. Among them are LCL161 (NCT01098838), GDC-0152 (NCT00977067, NCT01226277, NCT01908413) and Debio1143 (NCT01078649) and the results of these clinical trials has been recently summarized (Morrish et al. 2020). These clinical trials show that in a range of different tumor types, Smac mimetic compounds are unlikely to be effective as a single agent in reducing the primary tumor load. Clinical trials with Smac mimetics in combination with current chemotherapeutics show more promise in efficacy although cytokine release syndrome may be a limiting factor, potentially due to loss of IAPs driving cytokine production in innate immune cells(Wong et al. 2014). Debio1143 in combination with high-dose  cisplatin  chemoradiotherapy showed increased survival at 18 months in advanced squamous cell carcinoma of the head and neck (Sun et al. 2020). Expression profiling of triple negative breast cancer patients treated with birinapant and paclitaxel show high expression of TNF, RIPK1 and STX37 mRNA are indicative of response (Bardia et al. 2018). Whether these identified markers will translate to tumors inhibited in metastasis by Smac mimetics is unknown. No clinical trial has been designed to specifically examined the potential of Smac mimetics to reduce metastasis as an endpoint, however, without identifying which tumor types or potential markers of sensitivity, constructing such a trial will be difficult. Yet, in searching for therapies to  prolong survival, reducing metastasis may be one essential aspect worth investigating. 

1. In my opinion the Conclusion needs to be reformulated, especially the first sentences, which are strange.

Response 3:

Indeed, we apologize for the poorly written conclusion section and have re-written it as follows:

Our study shows the loss of cIAP1 but not cIAP2 alters the ability of endothelial cells to respond to permeability factors. This results in a decreased number of tumor cells extravasating through the endothelial barrier in a tumor-derived lymphotoxin alpha manner. Taken together, these findings identify that Smac mimetics can alter the stroma compartment of the tumor microenvironment,  reducing metastasis to the lung.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors state that functional inhibition of cIAP1, but not cIAP2, suppresses metastatic extravasation. They also confirm that gene expression of LTA, a ligand for TNFR, affects survival. The authors provide useful information, but the following points need to be revised.

1. It has been previously reported that Smac mimetic is involved in cancer cell invasion and metastasis (PMID: 31521127, PMID: 15911110). The novelty of the present results should be more clearly demonstrated.

2. The reasons why inhibition of cIAP1, but not cIAP2, is involved in metastatic extravasation should be more concretely demonstrated.

3. It should be noted in the discussion whether inhibition of metastatic extravasation by loss of cIAP1 is caused on other cancer types.

4. The TCGA analysis process should be described in more detail in the Method section. In particular, you should indicate the criterion. How did you separate high from low?

5. Add an explanation for "Comp A" in Figure 1A to the legend.

6. In Figure 1, the concentrations of the drugs should be indicated in the legend.

7. In Figure 2, it is shown that deletion of cIAP1 reduces the number and size of tumors in tissues. Which data show the invasion inhibition effect? In Figure 4 and Figure 5, The effect of the barrier is observed, but the number of tumor cells that invaded the tissue is unknown. Even if the number of invasion is the same, if growth is inhibited in the tissue, the result would be the same as shown in this article.

8. Other tissue images used in Figure 2C should be included as supplementary data.

9. In Figure 3, only the lung was observed, but is the same true for other tissues?

10. The molecular mechanism of how cIAP1 inhibits invasion needs to be discussed. More experiments on molecular mechanisms should be added.

11. line 256, "we we" => "we"

12. How about TCGA data for cIAP1?

Author Response

Please see attached file.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

The authors clearly answer to my revisions.

Author Response

Thank you for your critical input.