When studying TRAIL expression in the tumour stroma, Dr. Massimiliano Mazzone and Dr. Carla Riera-Domingo made a surprising discovery with great therapeutic potential: this immunoregulator acts as a gatekeeper of the vasculature at the premetastatic niche.

Listen to this episode to find out what implications this has for the formation of the premetastatic niche and the therapeutic value this novel finding holds.

This study has been published in Science Advances 9 (12), eadd5028. DOI: 10.1126/sciadv.add5028 and was included in the EACR’s Highlights in Cancer Research.

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Our guests in this episode:
  • Massimiliano Mazzone, Professor at VIB-KU Leuven in Belgium
  • Carla Riera-Domingo, former member of the Mazzone Lab and currently a researcher at IMEC, Belgium

And our host: Alexandra Boitor, EACR Scientific Officer.

Episode transcript

Alexandra: In this episode, we will be taking a slightly different approach to our regular conversations by introducing a new type of episode, the Research Focus. In these episodes, we will be discussing some of the most interesting and impactful recent papers in cancer research, with no other than the authors themselves.

Despite significant advances in cancer research and therapy over the past couple of decades, metastasis remains the leading cause of mortality for patients diagnosed with cancer. This is, at least in part, because we still have many unknowns when it comes to understanding this process. Dr. Carla Riera Domingo and her supervisor, Dr.
Massimiliano Mazzone, are some of the researchers looking into deciphering the process of metastasis.

Their paper, Breast Tumors Interfere with Endothelial TRAIL, a premetastatic niche to promote cancer cell seeding, published in Science Advances last year and highlighted in the November edition of the EACR’s Highlights in Cancer Research, focuses on the factors that promote the formation of a premetastatic niche, consequently facilitating the later stages of metastasis, extravasation, and colonisation of distal organs. To be a bit more precise, Carla and Max are studying the endothelial cells in the lung.

Hi, Carla and Max. Your recent paper is looking at how endothelial cells from the lung are involved in the formation of the premetastatic niche in this organ. You so nicely explain in your paper why TRAIL is important in the process of colonising the lungs, and I’d very much like to discuss that in more detail in a moment.

But first, I would like to ask you what made you direct your attention to studying the tumour necrosis factor related apoptosis inducing ligand, or TRAIL for short.

Max: Well, I can say that for us, it was our mission was to study TRAIL in the first in the tumour stroma. Actually we were quite surprised when we started to characterise the expression of TRAIL in different immunocells and in the endothelial cell compartment.
And we found that the endothelium had 25 times more TRAIL than the immune cells. So, for us, it was quite striking and shocking that this protein that has been emphasised for its role in the immune system as an immunoregulator was actually 25 times higher in endothelial cells, rather than in the cells where so far most people have been focusing.

And then I said to Carla, okay, it’s probably the time to understand what the TRAIL is doing in the endothelial cells, in the blood vessels, because at the same time, we saw that together with TRAIL, with the ligand, also the receptor, DR5, was highly expressed by the same cell, by the endothelium. And this, for us, was the first hint to start our study.

Alexandra: The role of vasculature lining in cancer progression from angiogenesis and other functions they play as elements of the tumour microenvironment has been discussed previously in the literature from, for instance, by Fares et al. in their comprehensive review, Molecular Principles of Metastasis: a Hallmark of Cancer Revisited, published in 2020, or in 2021 the review paper Therapeutic Targeting of the Tumour Microenvironment from Professor Johanna Joyce’s lab. Which is the element of novelty that your paper brings?

Max: We think that the big novelty of this study is that, with TRAIL, we identify a gatekeeper that is keeping very tight and very solid the vasculature at the premetastatic niche. Now, what we know more after our study is that the tumour is capable of hijacking this barrier distantly downregulating the level of TRAIL on endothelial cells, and this is, as you said, favoring the consequent process that is leading to metastasis, including cancer cell extravasation from the blood into the premetastatic niche.

Alexandra: Interesting. Which would you say was the key experiment in your study?

Carla: As Max mentioned, we observed that endothelial cells actually express higher levels of TRAIL than the other immune cells we analysed, but one of the key experiments was when we were trying to analyse TRAIL protein expression by flow cytometry, and in those experiments, we first tried to detect TRAIL at the cell surface, which is where it’s normally located, and we barely detected it there. Then we thought, okay, let’s check the intracellular pools, and to our surprise, it was there where we hit the jackpot, and we discovered that TRAIL actually is retained intracellularly in endothelial cells, which is quite unique.

And then, of course, it was also very key to generate the conditional TRAIL knockout mice that allowed us to deplete TRAIL only in endothelial cells and further validate the function of TRAIL in this subtype. So these two, I would say, were the key experiments in our study.

Alexandra: Thank you. My understanding is that the focus of this paper and the main hypothesis, I would say, is that changes in TRAIL expression are one of the initiating events in the formation of the premetastatic niche. Am I right?

Carla: Yes, that’s what we postulate.

Alexandra: And then you investigate the molecular mechanisms that are at play in preparing the secondary site for being invaded and colonised. But before getting into the mechanistic details, which I’m very eager to hear more about, how is TRAIL inhibited by cancer cells?

Carla: What we discovered is that cancer cell derived factors, and in particularly, we identified the proangiogenic molecules VEGF-A and PIGF, they naturally suppress TRAIL expression in endothelial cells in the premetastatic niche. And we believe that this is also mechanistically taking place through the pro-proliferative cascade, MEK/MAPK kinase signaling.

Another observation we made is that actually TRAIL is mostly expressed by quiescent endothelial cells. However, when endothelial cells start to proliferate, which would be for instance, under the effect of these proangiogenic factors, TRAIL expression is almost completely abrogated. This also explains why endothelial cells are the premetastatic niche, which is usually a healthy adult endothelium where endothelial cells are mostly quiescent is very high, whereas in the primary tumour where the levels of these proangiogenic molecules are very high and the endothelium is typically proliferating, the level of TRAIL is very low.

So, indeed, it’s this switch from high levels of TRAIL to low levels of TRAIL which impacts endothelial cell characteristics, awakens them from quiescence, and destabilises this fortress that Max was mentioning in order to allow cancer cell seeding.

Alexandra: Moving on to my second question, how does the inhibition of TRAIL signaling promote metastasis? My understanding is that there are two main mechanisms that act simultaneously. On one hand, TRAIL silencing compromises vasculature integrity in the lung, and then on the other hand, decreased expression of TRAIL induces significant changes to the transcriptional program in lung endothelial cells. Do you want to elaborate a bit more on this?

Carla: Yes, so indeed, in quiescent endothelial cells, which express high levels of both the ligand TRAIL and the receptor, we found that those two interact intracellularly. And this uniquely blocks the action of DR5. However, when TRAIL is depleted, DR5 signaling is unleashed, and this promotes, on one hand the activation of the caspase cascade, which induces apoptosis, and on the other hand, it also induces the activation of p38 and NF-kB and this translates into an increase in the expression of adhesion molecules. So in this context, the endothelial cells become a subset of them die, creating holes in a way, and another subset is more sticky. So when a cancer cell arrives, it’s more prone to attach to it and then cross the barrier.

We actually later on also proved that when we treat our mice with antibodies blocking the adhesion molecules, we are able to rescue the increasing metastasis observed in the absence of TRAIL. So it’s actually via this increase of adhesion molecule expression that cancer cells attach more to the endothelium and then also thanks to the higher apoptotic rate, they can also cross the endothelium.

Alexandra: And then what determines if the absence or presence of TRAIL would lead to apoptosis due to caspase activation and/or to an inflammatory phenotype?

Max: Well, this for me was a stunning observation. As Carla said, so far what was known in the TRAIL field is that if you give TRAIL from the outside, cancer cells might die. The TRAIL from the extracellular environment can be a recombinant TRAIL that we generate in the lab, therefore can represent a putative drug, or can be the TRAIL that is produced by an immunocell. Like for example, a natural killer cell and it’s released and will actually attach to the TRAIL receptor on the cancer cell and perhaps induce apoptosis.

Now, what we have seen as we are insisting from the start of this interview is that in endothelial cells, the ligand TRAIL, and the receptors are present in the same context, in the same cell. So this leads to a very interesting mechanism whereby the presence of TRAIL is actually, as Carla said before, is blocking intracellularly the receptor. So in this case is the presence of the ligand intracellularly that is preventing a pro-apoptotic or a pro-inflammatory signaling. Now, when the tumour, the cancer cell, the breast tumour is releasing VEGF, vascular endothelial growth factor, that has been mentioned before by Carla, then the TRAIL on the distant blood vessels is down-regulated.

And now DR5, the TRAIL receptor, is free of acting and transducing the pro-apoptotic, pro-inflammatory signal. So in other words, in an autocrine manner, when both receptor and ligand are expressed by the same cell, we disclose for the first time that this leads to the block of the pro-apoptotic, pro-inflammatory program of TRAIL receptors. Whereas when TRAIL is seen in a paracrine stimulation from the outside, then the target cells will actually elicit upon stimulation, the pro-apoptotic and pro-inflammatory program.

Alexandra: Thank you for explaining that. Which is the evidence in humans that TRAIL regulation is involved in patient metastasis?

Carla: We analysed single cell RNA-seq databases publicly available, and we observed that, corroborating our results, endothelial cells express high levels of TRAIL. We also observed in some patients where endothelial cells clustered according to their distance to the lesion. We also saw that in endothelial cells that were proximal to the lesion the levels of TRAIL were very low, whereas in the rest of the endothelial cells that were more distant to the lesion, the levels of TRAIL were higher, which mirrors exactly what we observed in our study. So, this supports the evidence that TRAIL regulation is also involved in patient metastasis.

And on top of that, we also corroborated it at the protein level in a subset of patients where we extracted the metastasis versus the peripheral lung parenchyma. And moreover, very interestingly in those subsets where TRAIL is highly expressed in the distant parenchyma, the expression levels of these adhesion molecules I was mentioning before, is very low.
And in contrast, in the subset of cells where TRAIL is lower and are proximal to the metastatic lesion, the level of adhesion molecules is very high, which completely mimics our phenotype. So we believe that this is at least a proof of concept that TRAIL regulation may be involved in patient metastasis.

Alexandra: Antagonists of the TRAIL receptors have previously been tried in clinical trials, as TRAIL has been previously shown to be used by natural killer cells in the fight against cancer but didn’t show the desired anti-tumoural effect. In your opinion, in light of these new roles that TRAIL seems to play in cancer progression, should attention be directed again towards TRAIL treatments?

Max: Well, absolutely. Our research is a lesson, right? It is a lesson that in those cases where TRAIL agonists have been given to induce apoptosis, there is a problem. And the problem is the genetic flexibility of the cancer cell that can erase mechanisms to resist TRAIL. In contrast, we know that the stroma is more homogeneous among patients.
And now based on our study, we can suggest, for example, that for those patients that do not respond to exogenous TRAIL to TRAIL agonist, we should actually intervene in a different way.

And the different ways are actually proposed by the paper itself and are actually substantially two. In the paper, we show that by giving lipid nanoparticle, LNP, that are loaded with a transcript that codify for TRAIL and goes only in the endothelium, we actually reestablish the block by TRAIL in the endothelial cells that I was mentioning before. So we reestablish the TRAIL that the tumour is trying to repress. And by reestablishing this TRAIL, as I said before, we reestablish autocrine interaction between the TRAIL and the DR5.

So beside this strategy that is consisting of the delivery of LNP with TRAIL, we could actually conceive a strategy where instead of acting on the modulation of the ligand, we could actually think in terms of TRAIL receptor inhibition, and prevent the overall signaling of TRAIL and therefore prevent what you have mentioned before, prevent the pro-apoptotic and the pro-inflammatory cascade that the endothelial cells of the premetastatic niche are destroying this gatekeeping mechanism and is favouring the formation of metastasis.

Alexandra: Thank you very much. This is indeed a very interesting conversation and I’m happy to learn so much more about TRAIL and the role that it plays in the formation of the premetastatic niche. Unfortunately, our time for today is coming closely towards an end, but I wouldn’t want to leave this conversation without asking you a final question that’s been on my mind ever since I’ve read your paper.

How much do you think your results could be generalised? Would this apply to the development of lung metastases from other types of cancer, or would TRAIL be relevant in priming the premetastatic niche from other organs than the lung?

Carla: This is a very interesting question. So nowadays it’s known that vascular beds from different organs also differ in their expression of several molecules and TRAIL is possibly one of them. So we believe, and we have proven, that these results can be generalised at least in those organs where endothelial cells express high levels of TRAIL, and in our paper, we’ve demonstrated that this is also the case in liver metastasis of breast cancer.

So definitely we believe that these results could be generalised at least to a certain extent when in vascular beds that express TRAIL and where TRAIL has this function. When it comes to different kinds of cancer, of course it always depends on the response and the features of each cancer type, but we also believe that it could be generalised to different types of cancer.

Alexandra: Thank you very much, Max and Carla, for taking the time to answer some of my questions today and for allowing us a glimpse into your research, which is so interesting.

We really hope that you enjoyed listening to this fantastic research story and that you learned something new from us today. If you have any follow up questions regarding this conversation, please don’t hesitate to contact Max and Carla.

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