The breast tumor microenvironment
At the Garvan Institute, current work is focusing more on defining the landscape of the breast tumor microenvironment at single-cell resolution, according to Dr. Swarbrick, a senior research fellow and head of the Tumour Progression Laboratory there.
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“Breast cancers ... are complex cellular ecosystems, and it’s really the sum of the interactions between the cell types that play major roles in determining the etiology of disease and its response to therapy,” he said. “So I think that going forward toward a new age of diagnostics and therapeutics, there’s wonderful potential in capitalizing on the tumor microenvironment for new developments, but this has to be built on a really deep understanding of the tumor microenvironment, and – I might say – a new taxonomy of the breast cellular environment.”
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Therefore, in an effort to address “this limitation in our knowledge base,” his lab is also working toward development of a breast cell atlas.
A fresh tissue collection program was established to collect early breast cancer tissues at the time of surgery, metastatic biopsies, and metastatic lesions from autopsies. The tissues are quickly dissociated into their cellular components and they undergo massively parallel capture and sequencing using the 10x genomics platform, he said.
Thousands of cells per case are analyzed using single-cell RNA sequencing (RNA-seq), as well as “RAGE-seq” and “CITE-seq,” which are performed in parallel to the RNA sequencing to address some of the limitations of the RNA sequencing alone and to “try to gain a multi-omic insight into the cell biology,” he explained.
RAGE-seq, which Dr. Swarbrick and his team developed, “is essentially a method to do targeted long-read sequencing in parallel to the short-read sequencing that we use for RNA-seq,” and CITE-seq is “a really fantastic method developed at the New York Genome Center that essentially allows us to gather proteomic data in parallel to the RNA data,” he said.
Based on findings from the analyses of about 125,000 cells from 25 patients, a map was created that showed the cell clusters identified by both canonical markers and gene expression signatures.
“We find the cell types we would expect to be present in a breast cancer,” he said.
The map shows clusters of myeloid, epithelial-1 and -2, cancer-associated fibroblast (CAF)-1 and -2, endothelial, T Reg, B, and CD8 and CD4 T cells.
Next, each cell type is quantified in each patient, and a graphic representation of the findings shows large variability in the proportions of each cell type in each patient.
“Ultimately, our goal is to be able to relate the frequencies of cell types and molecular features to each other, but also to clinical-pathological features from these patients,” he noted.
A closer look at the findings on an individual case level demonstrates the potential for development of better therapies.
For example, a case involving a high-grade triple-negative invasive ductal carcinoma exhibited each of the cell types found overall.
“One of the things that strikes us early on is we see a number of malignant epithelial populations,” he said, noting that proliferation is one of the drivers of the heterogeneity, but that heterogeneity was also seen for “other clinically relevant features such as basal cytokeratins,” which were heterogeneously expressed in different cell-type clusters.
“This was kind of paralleled in the immunohistochemistry results that we obtained from this patient,” he said. “We could also apply other clinically used tests that we’ve developed on bulk (such as PAM50 intrinsic subtyping) and ask whether they can be applied at the single-cell resolution.
“We think that these are going to be great tools to try to now get in and understand the significance of this heterogeneity and try to identify the lethal cells within this patient, and potentially therapeutic strategies to eradicate those cells,” he added.