Neutrophils and Inflammation in Colorectal Cancer with CRI Lloyd J. Old STAR Ivan Zanoni

The environment within our gut is extremely complex. Many different cells, molecules, and bacteria interact, and these relationships govern the immune activity (or lack thereof) within our intestines. Normally, these feedback loops keep cancer-causing inflammation in check, but if these finely tuned mechanisms are disrupted, colorectal cancer can develop. Once colorectal tumors do form, the altered environment in the gut can suppress our immune system and enable cancer cells to grow and the disease advance.

Neutrophils are important immunological actors in the gut environment, but unlike T cells and other immune cells, their precise roles have yet to be fully clarified. To that end, Ivan Zanoni, PhD, a Cancer Research Institute Lloyd J. Old STAR at Boston Children’s Hospital and Harvard Medical School, is working to characterize several factors that influence the activity of neutrophils and determine whether they will act in a way that helps or hinders tumors.

Ultimately, Zanoni—a principal investigator in the Departments of Immunology and Gastroenterology at Boston Children’s Hospital as well as an associate professor and member of the Immunology Program at Harvard Medical School—aims to define how immune and non-immune cells in our gut integrate the various signals from the body as well as gut bacteria in the context of cancer, and how these pathways can be harnessed to help people with colorectal cancer.

Recently, we spoke with him to learn more about this promising avenue of cancer immunotherapy research.

TRANSCRIPT

Arthur Brodsky, PhD

Hello, I'm Dr. Arthur Brodsky, assistant director of scientific content at the Cancer Research Institute, and today I'm grateful to be joined by Dr. Ivan Zanoni, a CRI Lloyd J. Old STAR at Boston Children's Hospital and Harvard Medical School. Welcome, Dr. Zanoni!

Ivan Zanoni, PhD

Thank you, thank you for having me.

Arthur Brodsky, PhD

Your CRI STAR work focuses on a group of immune cells known as neutrophils. What are neutrophils, and what do we know about the roles that they play in immune responses, especially in the context of cancer?

Ivan Zanoni, PhD

Neutrophils are the most abundant cell type in our blood, and they are basically involved in every immune response, and during every inflammatory process they play very important roles. They respond very rapidly to an invasion of the pathogen or when a tissue insult. They do this to protect us against pathogen invasion. For this reason, they can produce many different molecules, mediators that potently contain a possible pathogen. But at the same time also may affect and alter our own tissues. Now, tumor development, for several years, is seen as a type of inflammatory disease in which the immune system plays a very, very fundamental part. The immune system works as a two-edged sword, and so do neutrophils. These cells can play anti-tumor roles, but they can also become pro-tumor. Tumors really produce a lot of molecules that we don't fully understand yet, that are able to change this activity of the neutrophils from anti-tumor to pro-tumor. That's why their biology is so important to study. Ideally, if we can revert their function back to anti-tumor, we may use a very potent ally against tumors.

Arthur Brodsky, PhD

That's a really important point you mentioned, that we think of the immune system as purely protective. But because of how powerful the immune system is, it also has to have a way to turn itself off, which in cancer can sometimes go awry. As you mentioned, there's so many factors, and we don't quite know what tips the balance. So, you're exploring a lot of these different factors that affect neutrophils' activity, and recently found that molecules known as type 3 interferons can impact neutrophils in a way that promotes the development and growth of tumors, specifically colorectal cancer tumors. Fortunately, though, you also found that blocking the activity of these type 3 interferons could disrupt this effect. Why is this information important, and how might you build on it moving forward?

Ivan Zanoni, PhD

Yes, exactly. The growth of tumors is very complicated. There are many factors that are involved. The tumors themselves can produce different molecules or can alter their microenvironment. Not only the tumor cells, but also what is around these cells. We are particularly focused on colorectal cancer, as you mentioned, and that's a very unique environment, because it's in our gut. And in our gut there are many microorganisms that live there called the microbiota, but they can also induce inflammation if they enter into contact with immune cells. It's a very unique environment, and the colorectal tumors learned how to 'grow up' in this in this context. One of the molecules that is most specific of these tissues, like mucosal barriers, like the gut, the lungs – let's talk about the gut – are these interferons, these type 3 interferons. And these interferons have a very potent anti-viral role in the gut. We were working on that, we were working on colitis, we were working on colorectal cancer, and we asked how these interferons, that are called type 3 interferons because there are three different families, may affect specifically the response of neutrophils during colorectal cancer. As you said, we found that if we get rid of the capacity of these interferons to change the activity of neutrophils we protect against colorectal cancer, meaning that now we can potentially target – to come to your question of how we want to exploit it – we can potentially target this pathway in neutrophils, exactly to do what we were saying before: to revert neutrophils from pro-tumor cells to anti-tumor cells. We are studying this both in mouse models in vivo and in vitro, and we identified the specific type of neutrophils that play these pro-tumor roles and whose activities changed when they do not see any more type 3 interferons.

Arthur Brodsky, PhD

That's great to hear, I look forward to seeing your progress and how that approach goes. In addition to type 3 interferons, you're also looking at how certain fat molecules, which are called oxidized lipids, how they impact the metabolism of neutrophils and subsequently the overall level of inflammation within our gut. What do you hope that this avenue of investigation might yield?

Ivan Zanoni, PhD

Yes, in recent years we went back to the metabolism of the immune cells. The immune response is very complicated. There are so many different aspects of it, as it is for the tumor roles. Many groups, including my group, really started to look how, during an inflammatory response, the metabolism of different cellular immune cells may change. And these changes in the metabolism really affect the capacity of the cells to respond and to exert specific functions. Going back to this unique environment in the gut, we know that during colorectal cancer development, very often there is an inflammatory process that is going on. So, we know that among the molecules that are present there, there are these lipids that are oxidized because of the inflammatory process. Studying other cell types before we found that these lipids can change the activity of several types of immune cells. And now we're interested in understanding if oxidized lipids may also affect the biology of neutrophils in the context of colorectal cancer. We have preliminary studies that demonstrated this is the case. How these phospholipids, these oxidized lipids, may affect the broad or anti-tumor functions of neutrophils, we are actively looking into this right now.

Arthur Brodsky, PhD

Very interesting. We've been talking about two specific areas, the interferons and the lipids that you're looking at. But the gut, and really all tissues, are complicated. But especially the gut. You have microbes, you have our food, you have all these nutrients coming in, you have our immune cells, you have the epithelial cells, it's a very complicated situation, as you alluded to. So, as you're trying to explore this situation and determine the mechanisms and what are the causes and effects, what are some of the biggest challenges with respect to answering these questions? Are there any technological hurdles that you're addressing at the same time in order to get to these answers?

Ivan Zanoni, PhD

Yes, the system is very complicated. For a long period of time, we sought to study immune cells, like in the void. So we give only one stimulus, and then we look at how they responded. But now it's clear that the situation is way more complicated, and these cells receive multiple stimuli. This is what we want to do, to understand how these cells like the neutrophils respond in this very complex environment in which they will receive multiple information from the tumor, from the inflammation, from the from the bugs that live in our gut, from these oxidized lipids. Of course, we need a lot of technology to do these types of studies. And there are many new technologies that really helped us to do this. There is single cell RNA sequencing and many others, and we are intensively collaborating with other groups to further develop other technologies. We are working with people at Northeastern to develop single cell proteomics, thinking really to move it toward the study of these immune cells in the context of the tumor. There are technical challenges also because neutrophils are very unique cells. They die very fast in vitro. It's hard to study them and start to modify their genetics, things that we can do with other cells, for other cell types. So, there are many technological hurdles, but the technology is advancing very fast and we are trying to use everything out there and helping develop more to try to understand the biology of these cells.

Arthur Brodsky, PhD

Is it reasonable to assume also that as you develop these new technologies that allow you to investigate the cells in new ways, that maybe they could it could have other applications beyond just cancer biology?

Ivan Zanoni, PhD

Yeah, absolutely. That's the beauty of the technology, that sometimes you develop something for a completely different reason, and then it is proposed for other reasons. But this is part of what is happening constantly, everywhere in the world.

Arthur Brodsky, PhD

That's great to hear. As a CRI STAR, you'll be receiving $1.25 million over the next five years to pursue your high-risk, high-reward research. Can you explain why this STAR funding is so important for you, and specifically what will this support enable you to pursue that you wouldn't have been able to otherwise?

Ivan Zanoni, PhD

It's a very unique type of funding. Our research is extremely expensive. We get money from foundations, from the NIH to perform our studies. But usually, we write a project with some aim, some objectives that we want to reach. And we have to stick to those objectives very tightly because we have been given money to do that type of research. But research is a very complicated field. Sometimes you have an idea, you follow an idea, you do some experiments, and you find something that was unexpected but very interesting at the same time. And so the CRI STAR award really gives us the opportunity to use the money that we've been given to do these type of discoveries and study. We have a very high-risk project. We hypothesize some possible results, but we will have to test, to do the actual experiment, look at the data, and then with this grant, we have the possibility to really follow what will be most important and most interesting in our data. It's a very unique funding source that is given to the researcher more than the project. Of course, we will work in the context of colorectal cancers, but we have this flexibility that is very important. We have already some new preliminary results that were completely unexpected and that we are starting to dissect right now, thanks to this STAR award from the CRI.

Arthur Brodsky, PhD

That's a really important point to appreciate that. Yes, we have the evidence and we have hypotheses and you go in a direction that you think will work, but you never really know until you do it. And then, like you said, you get there and you might come up with some new discovery that leads a new direction, it's great that you'll be able to follow the data and the science where it leads. Speaking of, what do you hope to accomplish over the next five years as a CRI STAR? And just zooming out a little bit, how do you hope that your work will impact the field overall?

Ivan Zanoni, PhD

Yeah, exactly. It is linked to what I said before. There's the freedom to really look at the data and being able to follow new possible direction related to this project that maybe was not foreseen before. It is really important. What I would really like to do with this grant is to study how these cells work as hubs that receive multiple information, multiple stimuli, and change their responses according to the stimuli. Understanding these molecular mechanisms, which we really think we will be able to do, is key to highlighting new possible therapeutic targets. This is the real challenge we have, but also what we believe we have all the tools and preliminary data to do: understand exactly at the molecular level what is happening and define new possible targets that can be targeted in the next five years or so. For the field,  it's more complicated. But this idea of the integration of the multiple stimuli, I think that it may apply to many different cell types. And we have this preliminary data showing that if we give our type 3 interferons, but then we also give these oxidized lipids, things change. Starting to dissect these types of questions I think will be very important. We are looking at this in neutrophils and colorectal cancer, but this may apply to other tissues, other types of tumors, other cell types.

Arthur Brodsky, PhD

Absolutely. Yeah, I mean immunotherapy has made a lot of great strides in some cancer types, but other cancers, especially like colorectal cancer, there hasn't been as much progress for the patients, so hopefully approaches like yours – we'll need new approaches and new targets and new strategies that hopefully work like yours will uncover and we can help more patients.

Ivan Zanoni, PhD

That's the hope. That's why we are working so hard to get our data.

Arthur Brodsky, PhD

I can't wait to see what you find. I just want to thank you again very much for joining me today, Dr. Zanoni, and I wish you the best of luck in your research.

Ivan Zanoni, PhD

Thank you thank you, it was really a pleasure.