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Thesis
Peer Reviewed

Breast Pericytes, Fibroblasts, and Adipocytes Distinguished at Single Cell Resolution

Nee, Kevin
Advisor(s): Kessenbrock, Kai

UC Irvine Electronic Theses and Dissertations (2022)

Despite nearly 5000 years of study and treatment, breast cancer remains the most common form of cancer in women. Breast cancer is the leading cause of malignant mortality in women, and its treatments are a significant cause of morbidity. Women with mutations in the gene Breast Cancer 1 (BRCA1) have a 70% chance of developing breast cancer in their lifetimes. While the molecular subtyping of breast cancer has formed the basis of targeted therapies, the majority of breast cancers that develop in women with a BRCA1 mutation will be ‘triple-negative’, with no targeted therapies, poor prognosis, and decreased overall survival. Changes in the BRCA1 breast epithelium have been observed before tumors are detected, however, BRCA1 mutations are carried in all cells of mutation carriers, not just the epithelium. The mesenchymal stromal cells pericytes, fibroblasts, and adipocytes have been observed to be an important, albeit enigmatic contributor to breast tumorigenesis and metastasis. These less well studied connective tissue stromal cells and the preneoplastic changes that occur in them before tumorigenesis are the focus of this dissertation. The overarching hypothesis of this dissertation is that the breast microenvironment is distorted in women with BRCA1 mutations, and that stromal cells contribute to cancer initiation. A persistent challenge in elucidating the roles of pericytes, fibroblasts, and adipocytes in the breast cancer microenvironment has been the lack of distinguishing molecular phenotypic markers of these cell types. To overcome this challenge, our lab has performed single cell RNA sequencing (scRNAseq) of more than 200,000 stromal and epithelial cells from a 22-patient cohort of high-risk BRCA1 mutation carrier and non-mutation carrier patient surgical breast samples. I show that scRNAseq has the resolution to distinguish fibroblasts and pericytes from one another, and develop a novel flow cytometry strategy to isolate these cells for the first time from the human breast and find that they have distinctive differentiation capacities. Using our scRNAseq data of preneoplastic breasts I identify pro-proliferative changes in stromal cell communications with epithelial cells in BRCA+/mut tissues. We test these signals from pericytes and fibroblasts and find that they have the potential to alter the cell proliferation and cell fate of breast epithelium. Finally, to determine the transcriptomic profile of adipocytes, which are not amenable to standard methods of droplet based single-cell sequencing, I develop a method to isolate the nuclei from adipocytes and submit them to scRNAseq. Using this method, I distinguish vast differences between the adipocytes of human breast tissues and those of murine mammary glands. As a body of work, this dissertation emphasizes the importance of mesenchymal stromal cells in the breast microenvironment and highlights the individually important roles they play in breast cancer tumorigenesis.

Cover page: Breast Pericytes, Fibroblasts, and Adipocytes Distinguished at Single Cell Resolution

Article
Peer Reviewed

Experimental Considerations for Single-Cell RNA Sequencing Approaches

UC Irvine Previously Published Works (2018)

Single-cell transcriptomic technologies have emerged as powerful tools to explore cellular heterogeneity at the resolution of individual cells. Previous scientific knowledge in cell biology is largely limited to data generated by bulk profiling methods, which only provide averaged read-outs that generally mask cellular heterogeneity. This averaged approach is particularly problematic when the biological effect of interest is limited to only a subpopulation of cells such as stem/progenitor cells within a given tissue, or immune cell subsets infiltrating a tumor. Great advances in single-cell RNA sequencing (scRNAseq) enabled scientists to overcome this limitation and allow for in depth interrogation of previously unexplored rare cell types. Due to the high sensitivity of scRNAseq, adequate attention must be put into experimental setup and execution. Careful handling and processing of cells for scRNAseq is critical to preserve the native expression profile that will ensure meaningful analysis and conclusions. Here, we delineate the individual steps of a typical single-cell analysis workflow from tissue procurement, cell preparation, to platform selection and data analysis, and we discuss critical challenges in each of these steps, which will serve as a helpful guide to navigate the complex field of single-cell sequencing.

Cover page: Experimental Considerations for Single-Cell RNA Sequencing Approaches

Creative Commons 'BY' version 4.0 license

Article
Peer Reviewed

The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline

UCLA Previously Published Works (2015)

Removal of cytosine methylation from the genome is critical for reprogramming and transdifferentiation and plays a central role in our understanding of the fundamental principles of embryo lineage development. One of the major models for studying cytosine demethylation is the mammalian germ line during the primordial germ cell (PGC) stage of embryo development. It is now understood that oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is required to remove cytosine methylation in a locus-specific manner in PGCs; however, the mechanisms downstream of 5hmC are controversial and hypothesized to involve either active demethylation or replication-coupled loss. In the current study, we used the aorta-gonad-mesonephros (AGM) organ culture model to show that this model recapitulates germ line reprogramming, including 5hmC reorganization and loss of cytosine methylation from Snrpn and H19 imprinting control centers (ICCs). To directly address the hypothesis that cell proliferation is required for cytosine demethylation, we blocked PI3-kinase-dependent PGC proliferation and show that this leads to a G1 and G2/M cell cycle arrest in PGCs, together with retained levels of cytosine methylation at the Snrpn ICC, but not at the H19 ICC. Taken together, the AGM organ culture model is an important tool to evaluate mechanisms of locus-specific demethylation and the role of PI3-kinase-dependent PGC proliferation in the locus-specific removal of cytosine methylation from the genome.

Cover page: The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline

Article
Peer Reviewed

The Sm protein methyltransferase PRMT5 is not required for primordial germ cell specification in mice

UCLA Previously Published Works (2015)

PRMT5 is a type II protein arginine methyltransferase with roles in stem cell biology, reprograming, cancer and neurogenesis. During embryogenesis in the mouse, it was hypothesized that PRMT5 functions with the master germline determinant BLIMP1 to promote primordial germ cell (PGC) specification. Using a Blimp1-Cre germline conditional knockout, we discovered that Prmt5 has no major role in murine germline specification, or the first global epigenetic reprograming event involving depletion of cytosine methylation from DNA and histone H3 lysine 9 dimethylation from chromatin. Instead, we discovered that PRMT5 functions at the conclusion of PGC reprograming I to promote proliferation, survival and expression of the gonadal germline program as marked by MVH. We show that PRMT5 regulates gene expression by promoting methylation of the Sm spliceosomal proteins and significantly altering the spliced repertoire of RNAs in mammalian embryonic cells and primordial cells.

Cover page: The Sm protein methyltransferase PRMT5 is not required for primordial germ cell specification in mice

Article
Peer Reviewed

Targeting tumor–stromal interactions in triple-negative breast cancer using a human vascularized micro-tumor model

UC Irvine Previously Published Works (2024)

Triple-negative breast cancer (TNBC) is highly aggressive with limited available treatments. Stromal cells in the tumor microenvironment (TME) are crucial in TNBC progression; however, understanding the molecular basis of stromal cell activation and tumor-stromal crosstalk in TNBC is limited. To investigate therapeutic targets in the TNBC stromal niche, we used an advanced human in vitro microphysiological system called the vascularized micro-tumor (VMT). Using single-cell RNA sequencing, we revealed that normal breast tissue stromal cells activate neoplastic signaling pathways in the TNBC TME. By comparing interactions in VMTs with clinical data, we identified therapeutic targets at the tumor-stromal interface with potential clinical significance. Combining treatments targeting Tie2 signaling with paclitaxel resulted in vessel normalization and increased efficacy of paclitaxel in the TNBC VMT. Dual inhibition of HER3 and Akt also showed efficacy against TNBC. These data demonstrate the potential of inducing a favorable TME as a targeted therapeutic approach in TNBC.

Cover page: Targeting tumor–stromal interactions in triple-negative breast cancer using a human vascularized micro-tumor model

Article
Peer Reviewed

Defining the emergence of myeloid-derived suppressor cells in breast cancer using single-cell transcriptomics

UC Irvine Previously Published Works (2020)

Myeloid-derived suppressor cells (MDSCs) are innate immune cells that acquire the capacity to suppress adaptive immune responses during cancer. It remains elusive how MDSCs differ from their normal myeloid counterparts, which limits our ability to specifically detect and therapeutically target MDSCs during cancer. Here, we sought to determine the molecular features of breast cancer-associated MDSCs using the widely studied mouse model based on the mouse mammary tumor virus (MMTV) promoter-driven expression of the polyomavirus middle T oncoprotein (MMTV-PyMT). To identify MDSCs in an unbiased manner, we used single-cell RNA sequencing to compare MDSC-containing splenic myeloid cells from breast tumor-bearing mice with wild-type controls. Our computational analysis of 14,646 single-cell transcriptomes revealed that MDSCs emerge through an aberrant neutrophil maturation trajectory in the spleen that confers them an immunosuppressive cell state. We establish the MDSC-specific gene signature and identify CD84 as a surface marker for improved detection and enrichment of MDSCs in breast cancers.

Cover page: Defining the emergence of myeloid-derived suppressor cells in breast cancer using single-cell transcriptomics

Article
Peer Reviewed

In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse.

UCLA Previously Published Works (2015)

Methylation of cytosines (5(me)C) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5(me)C levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo.

Cover page: In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse.

Article
Peer Reviewed

PGC Reversion to Pluripotency Involves Erasure of DNA Methylation from Imprinting Control Centers followed by Locus-Specific Re-methylation

UCLA Previously Published Works (2015)

Primordial germ cells (PGCs) are fate restricted to differentiate into gametes in vivo. However, when removed from their embryonic niche, PGCs undergo reversion to pluripotent embryonic germ cells (EGCs) in vitro. One of the major differences between EGCs and embryonic stem cells (ESCs) is variable methylation at imprinting control centers (ICCs), a phenomenon that is poorly understood. Here we show that reverting PGCs to EGCs involved stable ICC methylation erasure at Snrpn, Igf2r, and Kcnqot1. In contrast, the H19/Igf2 ICC undergoes erasure followed by de novo re-methylation. PGCs differentiated in vitro from ESCs completed Snrpn ICC erasure. However, the hypomethylated state is highly unstable. We also discovered that when the H19/Igf2 ICC was abnormally hypermethylated in ESCs, this is not erased in PGCs differentiated from ESCs. Therefore, launching PGC differentiation from ESC lines with appropriately methylated ICCs is critical to the generation of germline cells that recapitulate endogenous ICC erasure.

Cover page: PGC Reversion to Pluripotency Involves Erasure of DNA Methylation from Imprinting Control Centers followed by Locus-Specific Re-methylation

Article
Peer Reviewed

MORC1 represses transposable elements in the mouse male germline.

UCLA Previously Published Works (2014)

The Microrchidia (Morc) family of GHKL ATPases are present in a wide variety of prokaryotic and eukaryotic organisms but are of largely unknown function. Genetic screens in Arabidopsis thaliana have identified Morc genes as important repressors of transposons and other DNA-methylated and silent genes. MORC1-deficient mice were previously found to display male-specific germ cell loss and infertility. Here we show that MORC1 is responsible for transposon repression in the male germline in a pattern that is similar to that observed for germ cells deficient for the DNA methyltransferase homologue DNMT3L. Morc1 mutants show highly localized defects in the establishment of DNA methylation at specific classes of transposons, and this is associated with failed transposon silencing at these sites. Our results identify MORC1 as an important new regulator of the epigenetic landscape of male germ cells during the period of global de novo methylation.

Cover page: MORC1 represses transposable elements in the mouse male germline.

Article
Peer Reviewed

Preneoplastic stromal cells promote BRCA1-mediated breast tumorigenesis

UC Irvine Previously Published Works (2023)

Women with germline BRCA1 mutations (BRCA1^+/mut) have increased risk for hereditary breast cancer. Cancer initiation in BRCA1^+/mut is associated with premalignant changes in breast epithelium; however, the role of the epithelium-associated stromal niche during BRCA1-driven tumor initiation remains unclear. Here we show that the premalignant stromal niche promotes epithelial proliferation and mutant BRCA1-driven tumorigenesis in trans. Using single-cell RNA sequencing analysis of human preneoplastic BRCA1^+/mut and noncarrier breast tissues, we show distinct changes in epithelial homeostasis including increased proliferation and expansion of basal-luminal intermediate progenitor cells. Additionally, BRCA1^+/mut stromal cells show increased expression of pro-proliferative paracrine signals. In particular, we identify pre-cancer-associated fibroblasts (pre-CAFs) that produce protumorigenic factors including matrix metalloproteinase 3 (MMP3), which promotes BRCA1-driven tumorigenesis in vivo. Together, our findings demonstrate that precancerous stroma in BRCA1^+/mut may elevate breast cancer risk through the promotion of epithelial proliferation and an accumulation of luminal progenitor cells with altered differentiation.

Cover page: Preneoplastic stromal cells promote BRCA1-mediated breast tumorigenesis