Following a cancer diagnosis, patients are inevitably subjected to severe stress as they endure weeks of therapy and worry about their prognosis. But how does chronic stress influence cancer progression and metastasis? Dr. Xue-Yan He, Assistant Professor of Cell Biology and Physiology at Washington University, is one of the researchers whose lab is looking into deciphering the influence of stress on tumour progression at a molecular level.
In this episode, Dr. Xue-Yan He discusses her recent research on the impact of stress on the tumour microenvironment, with a focus on the altered balance between neutrophils and lymphocytes and its role in promoting tumour spread. Tune in for an in-depth discussion on the interplay between stress and cancer biology, and its implications for understanding tumour progression.
This study, published in Cancer Cell (Volume 42, Pages 474–486), was selected for the EACR’s Highlights in Cancer Research, showcasing its significance to the field.
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Our host is Alexandra Boitor, EACR Scientific Officer.
Episode transcript
Alexandra: I would like to start this conversation by clarifying a few key concepts from your paper. First of all, can you please define what chronic stress is?
Xue-Yan: Thank you for inviting me for this conversation. Very happy to share with you what we have done in this paper. So, to define chronic stress, we actually have some readouts in the mouse experiment that we can measure the stress hormone level from the blood samples of the mice. For example, we can measure the level of glucocorticoid, which is a typical stress hormone released when people or mice are under stress from the adrenal gland. So, we saw an incredibly increased level of glucocorticoid, which means the mice were undergoing chronic stress.
Alexandra: Before getting into the details of the results of your study, can you briefly describe the stress inducing assays that you’ve used and explain how they relate to the human post diagnosis stress that patients experience.
Xue-Yan: Yes, so in this paper, we mainly use a model called physical restrained stress. In this model, we restrain the mice for two hours per day. So, that one is a very widely used model for people in the neuron field. This is a well established model to measure depression or to mimic depression of patients. We also have another model called chronic unpredictable stress model. In this model, we have 12 different kinds of stressors, which we chose randomly two stressors per day to kind of stress or surprise the mice. So, that one is to mimic different stress we are experiencing in our daily life, including like chronic pain or a wet housing, mimicking the different housing conditions people are experiencing, or overnight illumination, mimic a different, rhythm, like the circadian, people may have different jet lag or different time experience, to mimic all these kind of different stressors. We have done some experiment we call the behaviour test, where a camera can record the behaviour of the mice. This abnormal behaviour from the mice shows that the mice are undergoing like depression or anxiety related phenotype, meaning that this model can best mimic what the cancer patients have gone through.
Alexandra: I understand, it sounds really interesting. And what were the main consequences of this chronic stress that you’ve managed to mimic in the lab? What are the consequences that you’ve noticed in your model?
Xue-Yan: I would say there are two big parts of consequencing. The first one is when we only stress the mice, the consequence we got is of course the stress hormone going up and the mice are showing abnormal depression like phenotype. Another big consequence is when we combine the stress model with tumour model. So, we saw basically the consequence of how stress can promote tumour progression, including primary tumour growth and metastasis. So, this is the big consequence we got from the paper. It shows how chronic stress promotes breast cancer metastasis to the lung.
Alexandra: If I remember correctly from your study, the tumours increased two to four times in size within the same time frame as compared to the control, wasn’t it?
Xue-Yan: It was the increase of metastasis by two to four fold. So, we mainly focus on the metastasis. Of course we saw the promotion of primary tumour growth, but were more interested in how the stress can promote metastasis because that is a major factor in cancer patients. We want to study how the metastasis or the metastatic organ can be unaffected by the stress. We saw the increase of lung metastasis foresight, including both the numbers and area of the metastatic lesion. That is two to four fold.
Alexandra: One thing I don’t remember reading in your paper, and I was actually quite curious, is whether you managed to look at cell invasion, whether you’ve noticed an increase in cell invasion or just an increase in metastases. Do you know how this increase in metastases relates to cell invasion? Is it that more cells are prone to invading from the primary site or is it that the secondary sites become more prone to developing metastases, have maybe a composition of their environment that’s more prone to allowing tumours to develop?
Xue-Yan: This is a very good question. So, we also were interested to understand which step has the stress affected the tumour metastasis. So, the model that we used is called tumour resection model, or tumour removal model. So in this model, we first inject the primary tumour. And in this primary phase, we didn’t stress any of the mice. And then when the primary tumour got to a certain size, we performed the surgery on these mice to remove all the primary tumours. So, we did this to mimic what cancer patients have gone through. They probably were not aware of the primary tumour until they were diagnosed and get the surgery.
And then after the surgery, we started to stress the mice. So, that means everything happens before the surgery, like the primary tumour grows, cancer cell invasion, or get into the bloodstream, are the same in these mice. So, we started to stress the mice after the surgery, and we found the chronic stress can modify the secondary organ. Or like the metastatic organ, which is the lung in our breast cancer model. So, it’s possible that the stress hormone can affect the cancer cell invasion, which has been shown by the other group in the previous paper. But in our model, we saw mostly the stress hormone can generate a prometastatic niche in the lung. That is the reason why the cancer cells are more favourable to go to the lung and form metastasis there.
Alexandra: You keep referring to the stress hormone and previous studies, such as one published from the Bentires’ Group from University of Basel in 2019, have linked corticosterone to breast cancer metastases by activating the tyrosine kinase ROR1 in cancer cells. Can the prometastatic effects observed in your study be attributed to the observed increase in cortisol levels? And are those effects induced by acting directly on the cancer cells?
Xue-Yan: We actually have read that paper a lot when I was doing my study, but the mechanism we found is different from that paper. So, we actually found, at least in our model system, the effect from glucocorticoid was not directly on the cancer cells. So, I’m not saying that they were wrong, it’s just a different model may work differently. So, what we did is to generate a cell line, using CRISPR Cas9, to deplete or knock out the glucocorticoid receptor on the cancer cells. So, when we use these cancer cells without any receptor for the glucocorticoid, these cancer cells won’t react to the stress hormone. And we found that when we inject these cancer cells into the stressed mice, they still have more metastasis, meaning that even the cancer cells don’t respond to the hormone, but the other cells, the non tumour cells, may also respond to the hormone. So, that’s why we think that in our model system, it was not the cancer cells, but the cells in the tumour microenvironment, who can sense the stress hormone, like glucocorticoid, and trigger the metastasis. And we found that the glucocorticoid acting on the neutrophils, that that’s in the later part.
Alexandra: I think my next question kind of leads to that. You’ve mentioned already that the lung was one of the body parts that were affected by the stress you’ve induced in your mouse models. And you said there were changes to the microenvironment. So, if you could get into a bit more detail regarding the changes that you’ve noticed in the tumour microenvironment following chronic stress exposure?
Xue-Yan: We actually found a lot of changes in the lung microenvironment, which we can call it tumour microenvironment, or the pre-metastatic microenvironment. For example, we saw the increased infiltration of neutrophils. We saw fewer T cells, especially the effector T cells or the cytotoxic CD8+ T cells in the lung tissue. We also saw an increase of extracellular matrix protein like fibronectin deposition in the lung tissue. So, a lot of these changes, if we combine them together, have made the lung tissue as a favourable or pre- metastatic, pro- metastatic, environment for the cancer cell to stay and to grow. I also want to address here that apart from giving the mice the stress model to mimic the stress in the patient, we also tried to inject the stress hormone, like dexamethasone, which is a synthetic glucocorticoid. So, when we give the mice the glucocorticoid, it can also increase the metastasis, or it can also change the microenvironment of the lung tissue. So, it means that the stress hormone is doing something in our bodies.
Alexandra: Have you tried to reverse it? Are there any inhibitors of the cortisol available that you could give your mouse models and see whether that would decrease the amount of metastasis even though the animals were stressed?
Xue-Yan: Instead of make them happier, we were trying to find the target to really cure them. I can talk about the mechanism that we found and then that’s how we found the way of stress intervention, not really stress intervention, but try to cure them. So, we first found that the stress hormone can trigger the neutrofils or activate the neutrophils to form the NETs, which is the neutrophil extracellular trap. And then we found if we target these NETs by injecting the DNase I, we can eliminate all the NETs in their circulating system or also in the lung tissue.
These NETs are triggered or induced by the stress hormone. If we target the NETs, we can rescue the stress induced metastasis. So even though the mice were undergoing stress, the stress induced metastasis can be largely reversed or inhibited by targeting the NETs. The second way that we were trying to cure these mice is to block the glucocorticoid receptor, specifically in the neutrophils. So, what we did is to use a genetic engineered mouse model to deplete the glucocorticoid receptor, specifically on the neutrophils. It is because the neutrophil will use this receptor to sense the hormone and then formylase, or get activated. So, when we knocked out this stress hormone receptor in the neutrophils, we can also block or prevent the stress induced metastasis. So, this is the way that we are trying to do some kind of therapy instead of to make them happier. But clinically I think people have tried the beta blocker, which is like a blocker of stress hormone. So, when people combine the beta blocker with chemotherapy in breast tumour cancer patients, they have some positive effects as well. So, I think in clinic, that one could work as well.
Alexandra: If I may take a step back, can you please explain into a bit more detail what NETs are and why they are important in this context for those listeners that may not be so familiar with this field of research.
Xue-Yan: Yeah, of course. So, the NETs is the short term of neutrophil extracellular trap. NETs have been found since 2004, but it was first found as a way of neutrophil to eliminate the pathogens. So neutrophils, when they are activated by this like LPS or some products from the bacteria, they can use this pathway, like PAD4 independent pathway, to spread out its chromatin and to form this DNA scaffold in the extracellular space. And this DNA scaffold is not only in the DNA, but also being coated with many enzymes and the citrullinated histones, which the neutrophils can take advantage and to digest all these pathogens. So, NETs have been firstly found in 2004, but it was found NETs are doing something in cancer in 2013. That people showing that the NET structures have been shown the tumour samples. And then our lab, the Mikala (Egeblad)’s lab, was one of the first labs showing NETs are functional in the tumour. We found that the cancer cells can also activate the neutrophils to form a NET. And the NETs can reversely promote the invasion of the cancer cells and promote metastasis.
So, the role of NETs in tumour progression has been shown by more and more people. And it’s like the field is explosive now. So, the NETs are functional, not only in the metastatic side, but can also protect the circulating cancer cells not to be killed by the T cells or NK cells. The NETs can also help the primary cancer cells undergoing invasion. They can generate a prometastatic niche by inhibiting T cells, by regenerating the microenvironment, by remodeling ECM proteins. So, there are a lot of mechanisms described by people of how NETs are promoting cancer. So, it’s very complicated. I’m just talking several examples that I can remember now. What we found very unique is that we found the first time that the stress hormone can also induce the neutrophil to form NETs. And these stress induced NETs are actually using different pathways. They are not PAD4 dependent. They are using the glucocorticoid receptor pathways. And these stress induced NETs can also modify the pro- metastatic niche in the lungs. They can inhibit T cell function. They can remodel the ECM proteins as well. So, that’s how they have these pro-implementary or pro-tumourigenesis functions.
Alexandra: And did you look if NET levels in cancer patients resemble those observed in mice?
Xue-Yan: Actually, the NET levels in cancer patients are closely associated with the tumour progression. So, what we wanted to do is to link the NET level with the stress level, but that is very challenging because the stress hormone, like the cortisol in patients, or the glucocorticoid in mice, are actually under the rhythm, meaning that if you take the blood samples from the patient in the morning, versus the blood samples in the evening, the glucocorticoid levels are different. So, we were unable to get this information, like to know when the blood samples were taken. We cannot really make this connection. That was one of the shortage of the paper, but we actually tried a lot to do some like clinical relevance of our study. What we did is to get a tumour sample from the stressed mice, and we performed the RNA seq on this tumour sample.
So, we got a list of the genes and we call this gene list a stress signature. And when we applied these stress signature genes into the TCGA database of the breast cancer patient samples, we found that when the breast cancer patients are having a similar trend of the stress related genes, meaning they were probably more stressed than the other ones, they actually showed very poor overall survival, meaning that this stress gene signature probably can predict the survival of the cancer patients. So, this is something that we tried to make this study more clinically relevant.
Alexandra: You’ve noticed there’s a difference in the diurnal/nocturnal rate of NETs that you can find, like morning versus evening. And I know that you’re looking at the tumour microenvironment, but I was wondering whether this could be related in some way to Nicola Aceto’s research, that shows there are differences in the level of cell invasion, and ability of cells to metastasise, based on the diurnal/nocturnal rate of the body as well.
Xue-Yan: Like how the change of circadian rhythm can affect the tumour behaviour?
Alexandra: Thank you for so nicely rephrasing my question. Thank you.
Xue-Yan: We actually think it’s very related. The reason why is because the stress is very related with the circadian rhythm. It is because the glucocorticoid receptor itself, when it gets activated, can directly bind to a clock gene called PER1. So, it means that the stress hormone can directly control the expression of the clock complex, meaning they can control the circadian as well. What we found in this paper, and in the data that we didn’t show, is that the circadian of neutrophils, as well as other white blood cells, has been shifted under stress. You see the curve of the rhythm, right? Then we saw a shift of this rhythm. So, the neutrophils, according to previous studies, they have different abilities to form NETs in the morning versus in the night, because the neutrophils in the mice, they are released during the night as the fresh or young neutrophils, and then during the daytime they will get aged. These aged neutrophils are more prone to undergo NETosis. So what we found, this is shifting of this circadian may indicate that there is an abnormal regulation of the neutrophils undergoing aging phenotype. So, these abnormal aging neutrophils, triggered by the stress, may make them more prone to form these NETs.
And I agree with the previous paper showing that the cancer cells do have different behaviour according to different timeframes. That’s very possible, but we didn’t do this in our paper, but we show this time shifting in the neutrophils matters a lot in mediating cancer metastasis.
Alexandra: So, it really sounds like the circadian rhythm might influence several aspects of cancer progression.
Xue-Yan: Yes.
Alexandra: Thank you for so nicely talking us through your research and your results. I’ve got one last question if I may, and if it’s something that you could give away, what do you have planned next for this study?
Xue-Yan: I would say this project has opened up a lot of opportunities for questions that we haven’t answered. For example, we show that the stress has affected the immune system like the neutrophils. But the stress can also affect our nervous system. So, when people are undergoing stress, the whole sympathetic nerve system can be affected by the stress. So, what are this nervous system working in promoting cancer progression is largely unknown. And this is a very new field that people are jumping into. This is a very interesting future direction. One of the interesting future directions that both Mikala and I are interested in.
And the next part of this project, we would like to find some interventions that can really take advantage of the signaling pathway in the neutrophils and develop more intervention ways to block stress induced NETs, not only by blocking the glucocorticoid receptor, but what about the downstream targets of glucocorticoid? Is there any pharmaceutical intervention that we can use to better treat stressed cancer patients, is also one of the directions that we’re going now.
Alexandra: Sounds like a very exciting and busy future for your research and it sounds like you might want to, maybe not completely transition, but try out the field of neuro oncology as well, in addition to immuno-oncology.
Xue-Yan: I think that would be a very interesting like cross-talk with different fields.
Alexandra: It very much sounds like it. Well, thank you very much for your time and for explaining us your research. It was great hearing about your study and a pleasure talking to you.
Xue-Yan: Thank you so much for the invitation. I hope I have addressed all your questions, and if anything, just feel free to email me.
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