International Medical Conference Endometriosis 2025:
Endometriosis 2025: Your Mother Should Know, Your Doctor Should Know Better!
Iron Meets its Match: Endometriosis Cells' Pro-Fibrotic Counterattack - Polona Safaric Tepes, PhD
Our next speaker is Dr. Palona Cha ish is a postdoctoral research fellow at Northwell Health Feinstein Institute for Medical Research. She earned her doctorate in biomedicine from the University of Luana Slovenia where she focused on cancer biology. Dr. Sha TE has contributed to research on nuclear receptor coactivators and rare fusion driven neoplasms and has been actively involved in endometriosis studies.
Thank you so much for introduction and for pronouncing my last name very, very well. You're like a Slovenian. So ladies and gentlemen, today I'm going to talk about a battle and it's a battle between a micronutrient that all of you know very well and the meat lovers here likes to eat, which is iron and endometriosis cells. You see endometriosis cells developed a special counter attack strategy to fight against iron toxic effects. And I'll show you how. But first I would like to start with a personal story. As Dr. Kin mentioned today that scientists and MDs should collaborate together. We should go to the OR and see how surgery looks like and how the tissue looks like and vice versa. They should come to our labs to see what we're doing. I was lucky enough to be invited by Dr. Seskin and Dr. Chu to be part of one of a quite long surgery a year ago that was quite complicated.
And boy, I can tell you the things that I saw, it was really striking. I mean, you realize that endometriosis is not a joke. There is a lot of, I would call it almost a bloody mess because there is a lot of blood happening there and it's really like you have chocolate cysts and everything that is there. And that day got me thinking, what is the role of all this blood there and what is the role of iron? So I started really to investigate that. And if you think about the Samsung theory on its own, it's also a bloody mess because the tissues, the cells, the erythrocytes are coming in, the peritoneal cavity, the lies they release hemoglobin and of course release free iron and that's the toxic part of it. But the question is of course, how is this iron really toxic? So you see iron usually comes in two forms, either is ferric, which is FA three plus and it's insoluble.
So this is not really useful for the cells to absorb it. And it comes also in FA two plus, which is Ferris iron. And this is soluble and it's very useful for the cells. So when you have a high iron environment in peral cavity, the cells uptake this FA three plus and immediately transform it into FA two plus, which they use it for normal reactions in the cell like enzymatic reactions, respiration, and of course also DNA replication and proliferation. And just to tell you how important iron is, so the life on earth started with iron and one of the first trace elements that cells incorporated was iron. And it's used now in one third of the enzymatic reactions and enzymes to work. So it's really important, but on the other hand it also acts like a dynamite, like explosive and can literally blow up the whole cell.
So cells need to really keep this iron on a tight control because one wrong move it can blow up the cell. And how does it do it? I'm going to try to simplify a quite complicated fentanyl reaction. So FA two plus in the cell very easily reacts with hydrogen peroxide, which is very abundant in the cell. And in fact a fun fact is that the reaction that is converting FA three plus into FA two plus produces hydrogen peroxide. So it's very easily to engage in that reaction and up a little bit backward and reaction. So FA two plus gets converted in FA three plus and it's producing these highly reactive hydroxy radicals. And these are the bad guys. They interate with DNA and it's causing mutations and also they're very carcinogenic. And in the worst cancer scenario, the cell die from fortosis, which is iron made at cell death.
So we spoke about fortosis right now, but what this really is so fortosis is a newly discovered type of cell death that was only discovered about a decade ago by Dixon right here in New York. And it's actually triggered again by iron. So the excess of iron, it sparks the deadly domino effect that is per acidizing fatty acids and it's causing the cell membrane to erupt. And as this is not bad enough, it gets worse because the cellar content then spills in the extracellular space and all this excess of iron and all this cell debris is activating other cells to also undergo fortosis. So it's really like a domino effect and that's why they really need to keep fortosis under tight regulation. So how they do it, there are many ways. One of the very important ways is antioxidants and we are going to quickly touch on a couple of antioxidants.
They're very important because of the data that I'm going to show. So the first, here we go, the first antioxidant is SL C3 A two, which is a transporter that brings in the building blocks for glutathione, which are then used by GPX four, that's another enzyme and it's a master antioxidant enzyme inside the cells. Then another actor is FS P one and the name already says osis suppressor protein one. So it's like a guard, it's like a firefighter inside the membrane that is protecting the membrane against lipid peroxidation peroxidation utilizing COASE Q 10. But the question here is how do all these antioxidants like this one and many other ones get called into action? Well, there is a master regulator, actually there are a bunch of them and we're only going to talk about two of them. And these are the transcription factors. So you see the transcription factors when there is a high stress in the cell, they rush into the nucleus, bind to the specific DNA elements and transcribe thousands and thousands of antioxidant genes.
And NRF two is one of them. But NCOA seven is the second. And NCO seven actually has a special talent, which this crowd will kind of appreciate because besides binding to the antioxidant responsive genes, it also binds to estrogen responsive genes. So whenever estrogen receptor is activated, NCOA seven goes into the nucleus and binds to the estrogen receptor genes and transcribe many, many of them elements. So it has to dual role. Now let's quickly zoom out what we know about endometriosis and iron. So our colleagues from, or I'll say alumni from found conference like Dr. Donne, which I met last year, I'm not sure if he's going to come here this year, but he published already in 2002 an amazing figure here showing how endometriosis cells in endometrial lesion are accumulating high concentration of iron. And there's already known that there's high iron, high ferritin and high hemoglobin levels in the peritoneal follicular fluid and endo lesions.
And in addition, another alumni who's I think going to get a prize this year, Dr. VE Chelini, he published already in nineties that women with endometriosis have heavier menstrual flow, they have longer flow duration and shorter length of cycle. So the main question still persists and it has been published recently in many papers. How does endometriosis tolerate the high levels of iron? So we try to address this question and we use multiple methods. First we start with the human samples. And as Linda mentioned very well today, we try to use as much as possible human In our studies. We recruited through rose study patients that are diagnosed with endometriosis, they're surgically diagnosed and patients that are healthy controls, they're not surgically confirmed. They are healthy controls, but they have no history of endometriosis or symptoms. And I call them here we isolate their menstrual effluence and or collected it.
And I called them in my talk DXs, the cells that come from here, DX and ct. So we isolate menstrual tissue and did transcriptomics a single cell RN aic and we also grew stromal cells and then use three different techniques to analyze and try to understand this question. So let's go to the first one, those response curves. So we treated the stromal cells from 10 endometriosis patients, so DS and 10 healthy controls with increasing concentrations of hydrogen peroxide. And what was striking that a sublet concentration of 0.5 millimolar of hydrogen peroxide killed most of the controls, but 80% of stromal cells survive this high oxidative stress and which kind of suggests that endometriosis cells have this remarkable ability to resist oxidative stress. Next, of course, we wanted to understand if you treat these cells with high concentration of iron, what happened to them And results were quite remarkably similar.
So seven DXs showed a very high resistance to stress induced ous, stress induced by iron while controls at a very high concentration 1.22 million that's really high, they died and diagnosed survives. So in conclusion, it looks like that that cells the X cells, they not only survive in high oxidative environment, but they also have some kind of mechanism to protect themselves from stress induced by iron. So of course the first question here was why they're doing that. So to address this question, we decided to investigate two key mechanisms. One is that endometriosis cells can produce more antioxidant enzymes and the second is that they have higher expression or activity of these master regulators. So transcription factors. And we decided to first check for in our already published single cell RNAC data from diagnosed and controlled patients. And we saw that the first antioxidant was very highly expressed in DX and not in control.
Next GPX four also had a very high expression in DX and not in controls. And lastly, the FSP one co-factor binding protein. Quin CQ 10 was also very highly abundant. And this is just couple of antioxidants, there are many more. So we are just going to go into that. But this crowd know very well that mRNA sick data do not always correspond to the protein level and we always have these issues. So therefore we went to test their protein expression level and results were even more remarkable because what we see here on the top level are controls and on the bottom are diagnosed. And you can see that all the proteins that we just saw there are very highly, highly expressed in diagnosed, but not in controls. And in that point, I couldn't just shake the feeling that if endometriosis cells have this high, they're antioxidant rock stars and we know that oxidants and antioxidants can pass from one cell to another in different cargoes and different using exosomes and things like that.
Is it possible that endometriosis cells are secreting some kind of survival kits to other cells to kind of protect them from oxidative stress? So that's why we utilized mass spectometry to analyze the secum of the cells and we treated controls and diagnosed with iron for eight days. And after that we analyzed their secretion with my spectrometry and we found that DX so diagnosed boosted with iron, secreted many proteins that were associated with intact exosomes, which means that there is high chance that these cells are secreting exosomes and they're passing information to neighboring cells. And the top secreted 70 top secreted proteins showed to be antioxidant molecules and they tend to be proteins that are involved in synthesis of a very specific amino acids seleno aluminum metabolism. And guess which is the biggest enzyme that is using seleno amino acids GPX four. So which kind of confirmed what we saw before and we also confirmed this single cell with the RNAC data on the same sample that this was true.
So that was quite encouraging. But of course the question was instead of getting answers, which just got more questions and of course what would happen if we would take away this antioxidant power and reduce amount of GX four in the endometrial stromal cells, would that synthesize them to oxidative stress? So we treated the cells with a very specific inhibitor of GPX four RSL three oh, it doesn't work here. And we found that the anter is the cells still survives in the endometriosis but not in controls with increasing concentration of GPX inhibitor, which means that this is most likely that because GPX four is highly overexpressed. But of course the next question is who is responsible for such a high expression of GP X four? And that's when we decided to start looking in the master regulators.
The next one, of course, the last question was in the last piece of the puzzle, are there any master regulators like transcription factors that are over overexpressed in diagnosed and not in controls that are responsible for this high expression of antioxidant proteins? And we found that with the single cell RNA CIG data that indeed NRF two and NCO seven are highly expressed on the mRNA level level. But actually when we check the protein expression, the results were even more striking. And we found that these master regulators must be a winner of this antioxidant contest because they're really high expressed in endos but not in controls. And I believe that you agree with me that an CO seven is definitely a bigger winner, and this is even more important because if these transcription factor is binding to antioxidant responsive elements, what are the chances that is also binding to estrogen responsive elements at the same time?
I would say that to close out my talk for the keen observers in the crowd, probably you were all asking, what about these two bands here in the controls? Because they're kind of looking similar as todo down there. So for those one, I have an answer for you and there is an explanation and the explanation is in the way how we obtain these samples. So after a while we decided, you know what? Let's just not try to grow stromal cells from menstrual effluent. Let's also try to grow them from blood clots because they're full of this iron-rich gelatinous environment there. So we decided to grow them. And guess what was the results of it? 10 are all controls, all diagnosed, grew stromal cells from the blood clots, but only two out of 10 controls grew stromal cells from the blood clots while all the cells grew it from the menstrual effluent.
And with that, let's go now back to our western blot. So the CT six, which you see here in the purple and CT six blood clot is this control that grew out of blood clot. And you can see next suite is diagnosed, right? So you can see that it expresses the antioxidants, so almost same degree on the protein level than the diagnosed. And even more so control number two is the menstrual affluent of the second control that grew blood clot. And it shows very similar results right here. So in conclusion, diagnosed endometrial TH cells have high baseline expression of antioxidant enzymes, which might protect them from fortosis. And the question here is also is resistance to oxidative stress, a risk factor for development of endometriosis? And with that, I would like to also thank of course all my team. I would like to thank Dr.
Gregerson and Dr. Matt and of course Dr. Kin to give opportunity to give a talk here. And also of course all our team members and in the lab rose team and Northrop pathology department. And also I would like to really thank for that our lab is supporting everyone from postdocs to also PIs. Everyone can come here and give a talk. So I really appreciate that. And you'll see here in the background, this is the end. This is a tissue. This is actually a biopsy of endometrial endometrial biopsy from Dr. Kin that he sent it to us. And we stain this one with fsp one protein in yellow, and you can see high expression in both stromal and gland. So epithelial department. So it's really interesting, but we didn't have time to go into this data. So thank you very much. And that will be all from here.
