Epithelial cells
Both canonical and new gene markers were used to identify luminal and basal epithelial cells, Ms. Seth noted.
Among the known markers were KRT18 for luminal epithelial cells and KRT5, KRT6B, KRT14, and KRT15 for basal epithelial cells. Among the new markers were SLC39A6, EFHD1 and HES1 for the luminal epithelial cells, and CITED4, CCK28, MMP7, and MDRG2 for the basal epithelial cells.
“We went on and validated these markers on the tissue section using methods like spatial transcriptomics,” she said, explaining that this “really helps capture the RNA expression spatially,” and can resolve the localization of cell types markers in anatomical structures.
For these cells, the expression of the newly identified gene markers was mostly confined to ducts and lobules.
In addition, an analysis of cell states within the luminal epithelial cells showed four different cell states, each of which have “different kinds of genes that they express, and also different pathways that they express, suggesting that these might be transcriptomally different,” Ms. Seth said.
Of note, these cells and cells states are not biased to a specific condition or patient, suggesting that they are coming from all of the patients, she added.
Two of the four cell states – the secretory and hormone responsive states – have previously been reported, but Ms. Seth and her colleagues identified two additional cell states that may have different biological functions and are present in the different anatomical regions of the breast.
Fibroblasts
Fibroblasts, the cells of the connective tissue, were the most abundant cell type. Like the epithelial cells, both canonical collagen markers (COL6A3, MMP2, FBN1, FBLN2, FBN, and COL1A1) and newly identified gene markers (TNXB, AEBP1, CFH, CTSK, TPPP3, MEG3, HTRA1, LHFP, and OGN) were used to identify them.
Endothelial cells
Breast tissue is highly vascular, so endothelial cells, which line the walls of veins, arteries, and lymphatic vessels, are plentiful.
“Again, for both these cell types, we identified them using the canonical marker CD31, and we identified some new gene markers,” she said, noting that the new markers include CCL21, CLDN5, MMRN1, LYVE1, and PROX1 for lymphatic endothelial cells, and RNASE1 and IFI27 for vascular endothelial cells.
Two different groups – or states – of vascular endothelial cells were identified, with each expressing “very different genes as well as very different pathways, again suggesting that they might have different biological functions, which we are still investigating,” she said.
Additional findings and future directions
In addition to stromal cells, some immune cells were also seen. These included T cells that came mostly from two patients, as well as macrophages and monocytes, which comprised the most abundant immune cell population.
Of note, all of these cells are also found in the tumor microenvironment, but they are in a transformed state. For example cancer-associated fibroblasts, tumor endothelial cells, tumor-associated macrophages, and tumor-associated adipocytes have been seen in that environment, she said.
“So what we are trying to do with this project is ... learn how these cells are, and how these cells behave in the normal ecosystem,” she explained, noting that the hope is to provide a valuable reference for the research community with new insights about how normal cell types are transformed in the tumor microenvironment.
In an effort to overcome the adipocyte-associated limitation of the technology, adipocytes are “now being isolated by single nucleus RNA sequencing.”
“This [sequencing] technology has helped us identify multiple cell states within a cell type; and most of these cell states may have different biological functions, which probably can be investigated by spatial transcriptomic methods,” she said.
Spatial transcriptomics also continue to be used for validation of the new gene markers identified in the course of this research, she noted.