A Model of Lung Tumor Angiogenesis in a Biomimetic Poly(ethylene glycol)-based Hydrogel System

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior.

In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration.

Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines.

Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.