GU Cancers 2021 | scRNA-seq of UTUC reveals immune TME heterogeneity
Peter Reisz, MD, Memorial Sloan-Kettering Cancer Center, New York, NY, outlines the use of single-cell RNA sequencing (scRNA-seq) of upper tract urothelial cancer (UTUC) to reveal significant heterogeneity of the tumor and immune microenvironment at ASCO GU 2021. Prior investigations have used bulk profiling to identify molecular subtypes, however, this does not allow for analysis of the significant heterogeneity present in urothelial tumors. The study found high luminal gene expression across samples, as previously demonstrated with bulk profiling, and that the immune tumor microenvironment (TME) was heterogenous, with increased macrophage infiltration in high-grade disease. This interview took place during the 2021 Genitourinary Cancers Symposium.
Transcript (edited for clarity)
I’ve been working primarily with Dr Jonathan Coleman. He’s one of the faculty at Memorial Sloan Kettering and his research has focused primarily on the molecular characterization of upper tract urothelial carcinoma, and he’s published extensively in the past on his findings using bulk sequencing, bulk DNA and RNA sequencing of these upper tract tumors, and so our next step was to use this newer technology, this single-cell RNA sequencing to get a higher resolution sequencing picture of this disease and to explore some of the interesting components, including the tumor immune microenvironment, which as we know across cancer types plays a larger role than we previously thought in how cancer’s progress and respond to therapy and things like that...
I’ve been working primarily with Dr Jonathan Coleman. He’s one of the faculty at Memorial Sloan Kettering and his research has focused primarily on the molecular characterization of upper tract urothelial carcinoma, and he’s published extensively in the past on his findings using bulk sequencing, bulk DNA and RNA sequencing of these upper tract tumors, and so our next step was to use this newer technology, this single-cell RNA sequencing to get a higher resolution sequencing picture of this disease and to explore some of the interesting components, including the tumor immune microenvironment, which as we know across cancer types plays a larger role than we previously thought in how cancer’s progress and respond to therapy and things like that. So, this is an exciting technology, and we were finally at a place where we could apply it.
So we were able to obtain nine endoscopically obtained samples from the operating room, and so this is fresh tissue obtained directly from the operating room and then brought directly to the lab and sequenced using the 10X genomics RNA sequencing, single-cell sequencing platform. And so once we had that data, then we approached it with our computational biologist, David Kuo, and so the first thing we wanted to look at where the molecular subtypes present in these samples. Prior work in urothelial carcinoma of the bladder has shown that there are five or six distinct molecular subtypes that dictates how these tumors respond to therapy and things like that and their unique characteristics such as a luminal papillary subtype versus a basal subtype or a neuroendocrine subtype. And so, we wanted to see if our tumors, how they fell in that. So, prior bulk sequencing work has shown us that upper tract tumors are generally luminal papillary and of that subtype.
So, our single-cell analysis also corroborated that and showed that all of the tumors are predominantly expressing luminal papillary markers. However, it showed that they were also expressing other subtype markers as well, suggesting that these tumors are highly heterogeneous. And that’s important to know because from the bulk sequencing findings, everything is just averaged out where with the single-cell sequencing, you can really tease apart the heterogeneity, which is important. But overall, from that analysis, we found that these tumors are predominantly luminal papillary, which is what we would expect. So, our next step, we took those same samples and analyzed them for the proportion of immune cells present throughout the samples and we compare those by low-grade and high-grade histology. And the notable findings were that one, there’s a high level of heterogeneity across all samples in terms of the populations of T-cells or macrophages present, it’s highly variable.
But the one statistically significant finding that we did have was that high-grade tumors appeared to have a higher infiltration of macrophages, and so that’s kind of our big hypothesis generating finding from this data. We know that tumor-associated macrophages can create an immunosuppressive environment that can promote tumor growth and so our next steps will be to explore the higher resolution what types of macrophages are present, how they’re interacting with the T-cell populations in the dendritic cell populations and that requires kind of obviously a detailed computational analysis, which is ongoing and now we have some more samples sequenced. So, we’re excited to pivot into the next step of this project, looking at the complexity.
The overall takeaways from this project are one, that this process of sequencing upper tract disease from these tiny endoscopic biopsies is number one, it’s feasible. And we’ve been able to be successful at a high rate, even despite some unique limitations to single-cell sequencing and then second of all, this confirms that these urothelial tumors are very heterogeneous and that we need to take that into account as we consider these tumors in terms of treatment and such. And then the third thing is really exploring the immune microenvironment, what kind of immune cells are present within the tumor and around the tumor, and how is that affecting progression, recurrence, things that matter to patients, treatment response, for example. So, we’re excited to continue our work, exploring that and hope to include that in our publication down the road.
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