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Ashish Kamat: Hello, and welcome to UroToday's Bladder Cancer Center of Excellence. I'm Ashish Kamat, Professor of Urologic Oncology and Cancer Research, and the Director of the bladder program at MD Anderson Cancer Center in Houston. And it is a great pleasure to welcome a dear friend, a colleague, and more importantly for today's purpose, a true expert in the field of bladder cancer. In fact, we call her the queen of bladder cancer. Dr. Molly Ingersoll, who's joining us live currently from Paris, has been doing a lot of work in bladder cancer and very appropriately is now the Research Director at both the Institut Pasteur and the Institute Cochin. And Molly is going to talk to us today about mucosal immunity in the bladder with specific reference to sex differences and how that affects biology. So Molly, with that, the stage is yours.

Molly Ingersoll: Great. Thank you. That was a very nice introduction, Ashish, and absolutely I think of you as more than a colleague, certainly a really good friend, so I am really happy to have this opportunity. So as Ashish said, I'm between two institutes right now, trying to make a bridge between a sort of fundamental research and more clinical-directed research. And essentially in our lab, what's important to understand is that we are really interested in understanding the immune response to tumor growth and the immune response that takes place in the context of immunotherapy. And one question that we are really curious about more recently in the last few years is really how does sex influence this.

And so when I say we are interested in the immune response, we have this picture here of a tumor, and there are all these different aspects that contribute to it. But because we're really immunologists by training, what we focus on mostly is the cellular response that happens, the cells that are inside the tumor, the cells that are recruited to the tumor, the cytokines and other signals that they make to indicate that there is inflammation or there's no inflammation happening during tumor growth in immunotherapy.

And really, if we want to get a little bit more specific, one of our main areas of research is trying to understand how memory develops after immunotherapy. So the idea is, how does our immune response know to kill these specific tumor cells? And we're really quite interested, again, in understanding how are those differences between the sexes. And so one question we asked ourselves very early on is, can we identify T-cells that are able to recognize tumor cells? And I mentioned that a lot of our work is really fundamental, and for this, we use preclinical models, so animal models of bladder cancer. And what's important is our lab also asks questions about inflammation in other contexts in the bladder, such as urinary tract infection, and it's actually a finding that we had from urinary tract infection that got us interested in understanding the immune response in cancer.

And essentially what that is, is when we infected female animals, what we saw is there was a strong innate immune response. So really in the first 24 hours, we have innate immune cells like neutrophils or monocytes that come into the bladder, and you can see these numbers right here, and we know that this response diminishes to some extent after two days. But what's interesting is we infected male mice at the same time as these female mice, so female in red and male mice in blue, and what you can see is that the very first response in the first 24 hours is different between female mice and male mice. And I just want to point out these are the total numbers of these cells that have come into the bladder, and this is on a log scale so these are pretty large differences.

And so this got us interested in understanding whether or not these same differences exist in the context of tumors in the bladder. And one of the first experiments that we did really was to understand whether or not there was any sort of influence of BCG on the response to tumor cells. And this follows from a study that came out of a lab at Institut Pasteur that looked at the response, the memory response, essentially, to tumors in individuals that were vaccinated or not with BCG prior. So essentially they already had immunity to BCG, and then they received BCG for treatment of their non-muscle invasive bladder cancer.

And so we were curious to understand if this improved memory response was because of a difference in immune cell infiltration. And to do this, we use an animal model where we give mice a carcinogen in the water. So this is a commonly used model. This has been used for, my gosh, maybe close to 60 years now. And essentially what we do is the animals drink that and they develop tumors in their bladder. And one of the very first observations in this particular experimental setup is those male mice actually developed larger tumors compared to female mice, and we are showing that here by showing bladder weight. So what we do is these animals will receive vaccinations with BCG or not, we wait about 12 weeks for them to develop their tumors, and then we treat them with intravesical BCG.

And this point right here I'll come to later, but essentially what we can do is we can transfer in T-cells that we know are specific for an antigen, for a protein that's on the surface of the tumor. So the first thing that we see when we compare naive animals to animals that have consumed this carcinogen, it's called BVN, we see that the bladder weight increases. And it increases in female mice, but it increases more in male mice, so this shows us that they have larger tumors overall. But then when we looked essentially at the animals that had received a vaccination or not, BCG right at the beginning, so whether they had immunity or not, we saw that this really had no impact on the infiltration of all immune cells. So this is sort of a pan-marker. This is our way of looking globally at the immune response.

And you can see in the columns, it's no vaccination, vaccination in female mice and male mice.  We saw there were no differences in the resident immune cells, and macrophages are a key component of the immune cell population that is constantly in the bladder. There's no difference in this infiltration of these innate cells like we saw in the infection scenario. So that was essentially the same at the level of neutrophils. And then when we were considering globally the T-cell response, which we know we need for memory, we did not see any differences there. So it's just really quite surprising to us, and it made it a little bit more difficult to understand those previous observations that having immunity to BCG actually is better for an individual.

So in just trying to understand exactly what's going on, one of the things we focus on is the upregulation of checkpoint molecules or checkpoint inhibitor molecules, which we know now are good targets for immunotherapy for bladder cancer and other cancers. And so essentially what we did is we can measure how many of these molecules are on the surface of the cells, and what we can do is we can look at the difference between naive animals that have never seen this carcinogen and animals that have been exposed to 12 weeks and have developed tumors in their bladder.

And what we see globally is an increase in the marker PD-L1 on monocytes, which are again an innate immune cell that comes into the bladder, macrophages, which are resident in that tissue already, and the two types of dendritic cells that are commonly associated with initiating an immune response specific to the tumor. So we see that molecule increases compared to a naive scenario, so that's essentially in response to tumor growth and potentially treatment. But again, there are no differences between male and female animals, and so we can rule out PD-L1 as a reason for differences in the response between the sexes. And then when we look at PD1, which is typically expressed on T-cells, what we see again is this augmentation, this increase in the molecule on the surface of these cells, but again, no differences between male and female animals.

So then we went on essentially really to understand globally whether there was an immune response difference at the level of the cells. And so we looked again at these same populations that I already described for infection and essentially the cells that we just looked at for their level of checkpoint inhibitor molecules. And what we see again, while we have a big increase, for instance, these tumors are full of neutrophils, they are full of eosinophils, which is really sort of a curious question for anyone who's interested in a lesser well-studied immune cell, they are full of these monocytes that come from circulation, and they accumulate macrophages and DCs. But again, we saw no real differences between the sexes to explain our observations in humans.

And then what we did is we took a step back and we looked at those tumor-specific T-cells that we had put into the animal. And essentially what we saw is, globally, T-cells were the same, CD4, CD8 T-cells, those numbers were about the same, and we counted how many were present in the tumor. And it's only here, at the level of these T-cells that are specific for our tumors, where we see that there are more of these T-cells in male mice compared to female mice. So essentially what we were able to say is perhaps the reason that, generally, very generally speaking, male patients do better or respond better to therapy compared to female patients with non-muscle invasive bladder cancer, or perhaps in this scenario of BCG immunity. We think that maybe one of the reasons for this is that we have a better infiltration of T-cells that know the tumor that can come in and can kill those cells. And so this is a really exciting line of research for us. This is something that we are following up on and working with some clinical partners to see if we can verify this in humans.

And so essentially what our work tells us is that there is a really diverse population of immune cells that come into the tumor while it grows and in the context of therapy. We know that BCG-specific immunity can impact infiltration differently, depending on the model. So I only showed you one model, but we have a second one and we do see some differences there. And so for anyone who is thinking about preclinical research or thinking about how to apply what they've read in the literature to medicine, it's really important to understand that we can have really different results from different models.

And finally, we think that we can conclude that tumor-specific T-cells infiltrate male tumors better than females in mice, and that really leads us to try to understand more globally what mediates these differences in T-cell infiltration, and really, as I said before, does this occur in humans? How are PD-1 and other checkpoints, how's that regulated in men versus women? Is it the same or not, and ultimately what's going to be the best immunotherapeutic approach to treat men versus to treat women? Will it be the same? Are there small changes we can make in scheduling drugs, for instance? And I think that those are important questions to bring both to the laboratory and to the clinic.

I think it's also equally important to point out the individuals who've done a lot of this work. This is Matthieu Rousseau who led this project, and this is a number of master's students who came in. So this is Anna, Conan, Hannah, [Delie 00:11:53], and [Idris 00:11:55] who came in to help Matthieu with these studies. And so with that, I'd really be interested to hear any questions that you have for me, Ashish.

Ashish Kamat: That was great, Molly, as usual. You managed to take a complex topic and distill it down to a level that even I can understand. So thank you for are doing that. I do have one question. I mean, it's more to kind of set it straight, because I know you obviously thought about this and we've talked about this before. But when you are looking at these models and you are looking at the patient population, essentially, could you share with our audience your thoughts on how this relates to, I guess, the animal model in which the patient, for example, has been exposed to different hormones of women, but is now in a postmenopausal setting? Because most of our bladder cancer patients that are female are in that postmenopausal setting. Could you share that with our audience a little bit?

Molly Ingersoll: Absolutely. I think that's a fantastic question. Just generally in terms of biology, when we are talking about responses to infectious and non-infectious diseases in patient populations, so what's important is a majority of our studies are actually in young animals, and so they should have sort of peak estrogen and testosterone levels, respectively, in female and male mice, or, well, both, in fact. And we know from work in the area of UTI, but also to some degree in bladder cancer, that levels of both estrogens and androgens can impact tumor growth, the immune response, and I would hypothesize also a response to drugs. So I think while our model has helped us address some first questions, I think a better model would be to somehow bring aging into the environment or potentially to imagine modifying hormone levels with antiandrogens for example, or by supplements. And that's something that we've done already in the context of infection and are aiming to do in these preclinical bladder cancer models.

Ashish Kamat: Right, so, again, since you brought up infections, and I know you've done a lot of work in that realm, there's an emerging body of evidence that the use of antibiotics changing the microbiome, both in men and women, especially in women, does affect the response to systemic I-O therapy, and that's more for metastatic disease and melanoma and lung, and I suspect it will hold true in the bladder as well. Could you share some of your thoughts on that? I know it's not related to this topic, but having you here with you doing a lot of work on the microbiome and infections, I'd hate to not ask you that question.

Molly Ingersoll: Yeah. Those studies that are coming out are incredibly exciting, understanding the impact of short-term antibiotics, sort of maybe in the months before receiving therapy or understanding the long-term impact of antibiotics on the microbiota and whether or not that predisposes us to a more or less permissive tumor-developing environment. I think that's incredibly interesting. I think if we want to understand that better, we really need to incorporate metabolic studies into what we are doing. So there is really some nice research that shows that antibiotics can be processed by the microbiome and changed into other things, for example, changed into more or less efficacious drugs, and it depends on the kind of who's there in your microbiome. And then certainly, chemotherapeutic agents can be changed by the microbiome as well as, exactly as you mentioned, these responses to I-O.

The challenge I see in front of us is to incorporate all of these different parameters into one model, and I think that's not going to be possible right now. I think what we need to do is collectively sort of split up some of these different parameters that might be important and work together to see if we can come up with a working model. I don't think one lab is going to be able to accomplish everything. But I think what has come now is we cannot neglect the microbiome and its contribution to disease development, progression, and response to therapy. And one thing, if I have the chance to push this, I would love to see more studies that involve looking directly at the bladder or the urinary microbiome, as well as the influence of the gut microbiome, because I think that is, particularly in the context of bladder cancer, that's going to become very, very important because a lot of therapies are local, so of course, they're interacting with the bladder microbiome.

Ashish Kamat: Yeah, no, and in some ways, you could even say that BCG is a microbiome-altering agent-

Molly Ingersoll: Absolutely.

Ashish Kamat: ... just by nature of what it is, right? And I'm absolutely with you. I think we need to push for more research. It's not that research hasn't occurred in this field. Essentially over the years, essentially, as you know, when you look at BCG vaccination in kids, there has been a huge amount of work done showing a correlation between the positive PPD and the BCG scar and improved mortality, more so for girls than for boys, but the tools haven't been there to kind of dissect out what happens. And then of course, when we have minds like yours and the tools available coming together, we have researched such as this that comes out. So it's great that you could join us today and share your perspective on this. In closing, I'll hand the stage back to you. Any closing thoughts on this topic that you want to share with our audience, the stage is yours again.

Molly Ingersoll: Oh, wow. Oh my goodness. This is my chance to advocate for all the things that I think are crucial. I'll share one thing that I think is important that I've mentioned a few times now in presenting this work. I sometimes wonder if our focus on immunology has made us miss other things, miss the contributions of the tumor itself, or the stroma, or I come back to metabolism again. And I think that if we can examine our work that way and we can say, you know what, I might be missing things because I don't have the right expertise, that's my cue that we need to be working together. We need to be forming larger consortiums that can really come at these big biological questions from a lot of different aspects. And so I'm all for collective, collaborative research, and I think anyone who's interested in that, this would be in a fantastic field because I have found urologists, physician-scientists to be incredibly collaborative and incredibly eager to work together to address these questions.

Ashish Kamat: Dr. Ingersoll, thank you so much for taking the time. It's always great to have you in any of these discussions, and congratulations once again, a smooth transition, and I know you're going to bite off more than you can chew-

Molly Ingersoll: Yeah.

Ashish Kamat: ... but that's what makes it fun, right?

Molly Ingersoll: That's what makes it fun, yes. That and a glass of wine, no?

Ashish Kamat: Take care.

Molly Ingersoll: Great, thank you very much.