In this study, we investigated the expression pattern of angiogenesis related genes, at single cell level across different cell types in the tumor microenvironment. This analysis revealed that there are distinct subsets of these angiogenesis related genes that are overexpressed in different cell types, that give rise to various biological processes related to angiogenesis (Fig. 3A, B, and C).
Through inspection of the interaction network of the overexpressed genes (Fig. 3A, B, and C), and domain knowledge, we choose top 10 key genes to discuss here in detail. Also, here we discuss in more detail, the cell types that these genes are overexpressed in.
Important Cell Types In Angiogenesis
It is well-established that the endothelial cell is of utmost importance in the development of solid tumor and therapeutic response. Increased angiogenic activities have been proven to be an important cancer-associated hallmark feature. Formation of new blood vessels is essential for extravasation and massive infiltration of immune cells into the tumor milieu. Directed migration of leukocytes across the vascular endothelium and their retention within the tumor mass is mediated through the constitutive expression of adhesion molecules on the surface of endothelial cells and the local production of chemokines inside the tumor. The interaction between immune cells and cancer endothelium is thus a critical step in orchestrating immunosurveillance of tumors (13).
During vasculogenesis, formation of new vessels is achieved by differentiation of endothelial progenitor cells (EPCs) into mature vascular endothelial cells that is derived by signals from the local environment. Many of these pro-angiogenic factors that are initially released into the extracellular fluids show significant paracrine reaction in endothelial cells. These endothelial cells initiate migration in a concentration gradient of pro-angiogenic factors and remain attached to the blood vessels to generate new vessels. In the tumor tissues, endothelial cells are primarily blood vessels (14).
Immune cells are another essential part in regulation of the tumor vascular development. More generally, the immune system is identified to protect host against invading pathogens and eliminate the tumor cells. T cells are an important subset of immune cells with critical roles in anti-tumor immune activities. The two most common types of T cells infiltrating the tumor microenvironment are helper CD4 + T cells, and cytotoxic CD8 + T cells (15). They generate a prominent anti-tumor response by secretion of some mediators, particularly cytokines such as TNFα (tumor necrosis factor-alpha), interleukin (IL)-2, IL-6, IL-1, and IFN-γ (interferon-gamma), and by direct interaction with and killing of tumor cells (16, 17). Immunity within the tumor is dependent on both populations of effector and regulatory immune cells. Studies have highlighted a regulatory role for some B cell and T cell subsets specialized for immune suppression. A variety of regulatory B (Breg) and regulatory T (Treg) cells exists and expands during the tumor promotion. These immune cells produce an orchestra of immunosuppressive cytokines (IL-10, IL-35, IL-37 and transforming growth factor-beta [TGF-β]) and thereby confer a suitable niche to promote cancer progression. Myeloid-derived suppressor cells (MDSCs) as a heterogeneous population of cells represents a different kind of regulatory cells that derive from multipotent progenitor cells and expand during a variety of pathologic conditions including cancer. MDSCs are implicated in the escape of tumor cells from antitumor immunity through several underlying mechanisms that suppress the immune response of CD8 + T cells and enhance tumor growth. For instance, studies indicate that MDSCs suppress immune cell activities through nitric oxide synthase production and secreting high levels of reactive oxygen species (ROS). Additionally, these immunosuppressive cells are required to ensure protective immunity, preventing the overactivation of inflammatory pathways, and to maintain homeostasis of immune system (18–20).
The innate immune system is another player particularly important in response to cellular stresses and fight against tumors. Innate immunity is composed of macrophages, dendritic cells (DC), and natural killer cells (NK). NK cells are large effector lymphocytes found in spleen tissue, bone marrow, and peripheral blood that participate in the early control of neoplastic cells. In response to cytokine signals, NK cells are recruited to inflammatory sites as well. As a component of the innate immune system, NK cells have several non-specific protective mechanisms against cancer cells that mainly involve the secretion of high amounts of IFNγ, granulocyte-macrophage colony-stimulating factor (GM-CSF), TNF-α, and other inflammatory mediators (21, 22). DCs are immunostimulatory antigen presenting cells able of stimulating proinflammatory Th1 cells, and efficient cytotoxic T cell responses. Macrophages are required to maintain tissue repair and homeostasis by engulfing and digesting cellular debris. They also employ multiple strategies most notably phagocytosis to resist microbial invasion (23).
Tumor development and growth relies not only on the alterations in cancer cells but also on some microenvironment factors that play significant roles. Breast cancer consists of several cell types with different functions (Ref: Tumor initiating cancer stem cells from human breast cancer cell lines). Myoepithelial cells are a type of cells in mammary gland duct that were thought to be primarily responsible to contract and eject the milk produced by epithelial cells from the lumen. However, they have now proven to play a role in some other physiological functions like promotion of proliferation of neighboring cells. They also secrete various molecules such as growth factors and cytokines. Myoepithelial cells appear to have a dual function in tumor progression. These cells show a strong role in the construction of basement membrane and when interact with immunoreactive cells trigger the release of extracellular matrix remodeling enzymes and cause degradation of the basement membrane. Thus, it can be suggested that the damaged myoepithelial cells are associated with tumor invasion which may be mediated by the effects of these proteolytic enzymes. Importantly, while epithelial cells are susceptible to undergo oncogenic transformation, myoepithelial cells are usually less likely. This suggests that myoepithelial cells can also act as an autocrine tumor suppressor. Moreover, myoepithelial cells are known to be a reservoir of diverse proteinase inhibitors and angiogenic inhibitors while still having a low amount of proteinases and angiogenic factors (24, 25). Gene expression profiling experiments have proved that myoepithelial tumors seem to be associated with a favorable outcome and are classified as low-grade neoplasms. Indeed, these cells have been shown to exert pleiotropic tumor-suppressive effects in both clinical and preclinical studies (26).
Currently, therapies targeting the interaction of VEGF with its receptor and blocking the release of VEGF from tumor epithelial cells are promising candidates to suppress progestin-dependent breast tumors. Furthermore, attempts are being made to develop strategies targeting the selective progesterone receptors that result in the inhibition of the neo-angiogenic process in breast cancer cells (27).
Disruption of epithelial gene expression pattern has important implications in many areas of cancer biology such as invasion, and migration. During malignant progression, epithelial cells undergo morphological and functional changes known as epithelial-mesenchymal transition (EMT) that is the loss of epithelial features and acquisition of mesenchymal cell phenotype with increased migratory capacity (28). The EMT process includes degradation of intercellular adhesion proteins associated with disassembly of cell-cell contacts and the cleavage of basement membrane and remodeling of extracellular matrix leading to individual cell mobility (29, 30). Therefore, reprogramming epithelial cells to motile mesenchymal cells can cause unwanted outcomes like tumor outgrowth and metastasis (31).
Cancer-associated fibroblast (CAFs), a major part of tumor stroma, is another regulator of tumor neo-angiogenesis. As such, the heterogeneous population of tumor stromal fibroblasts, which include activated fibroblasts and the myofibroblasts with characteristics of fibroblasts and smooth muscle cells, differentiate from normal tissue fibroblasts and has important roles in various tumor entities by producing increased amounts of soluble factors (e.g., cytokines and growth factors) and via direct cell-to-cell contacts. These cells are also crucial for extracellular matrix (ECM) deposition and its modification and function as key determinants in the malignant transformation of epithelial cells since CAF inhibition is proved to be a promising tool in epithelial cancer types. It has been shown that CAF density correlates with poor prognosis and distant metastasis in cancer patients whereby they provide potential oncogenic signals including invasion, angiogenesis, and therapeutic resistance through paracrine and autocrine effects. Studies demonstrate that inhibition of CAF function provides a survival benefit for patients with pancreatic cancer compared with chemotherapy alone and that the effects were more pronounced in preventing tumor progression than tumor invasion (32). As such, further understanding of interactions between CAFs and cancer cells and CAFs-driven neo-angiogenesis may reveal a novel approach for cancer treatment.
Additional insights into cellular composition of tumor infiltrates have revealed a distinct lineage of cells called perivascular-like (PVL) cells with unique biology. PVL cell is found to resemble pericytes or smooth muscle cells and clustered into two subsets, immature PVL and differentiated PVL cells. Each cell subset has a specific gene expression signature which may explain the diverse morphologies and the functional properties of the known PVL cells consistent with extracellular matrix regulation.
Interaction between tumor stromal subtypes and immune cells is thought to play fundamental roles in the tumor microenvironment. This biophysical crosstalk can influence several aspects of cancer pathogenesis. It has been indicated that the relationship of stromal and immune cells serves as a regulatory mechanism to control immune responses within solid tumors. In a recent research work, dataset of human triple-negative breast cancer (TNBC) stroma composition obtained by single-cell analysis confirmed signaling between the stromal cells and immune cells that mediates immune suppression and impacts on response to therapy. Inflammatory-like CAFs and differentiated PVL contacts with immune cells has been shown to result in cytotoxic T cell dysfunction and exclusion, respectively. Such findings highlight the potential of developing stromal-targeted anti-cancer therapies which deserves further investigation (33).
Important genes in angiogenesis
1. ACKR1: ACKR1 (Atypical chemokine receptor) the chemokine system's regulator. This system is essential in angiogenesis, cell proliferation, and migration.
ACKR1 controls chemokine accessibility by cleaning, transporting, or stocking chemokines. This gene has an inhibitory effect on tumor development. It is a repository for chemokines, decreasing their concentration in the bloodstream and repressing tumor angiogenesis and metastasis by cleaning angiogenetic chemokines. The tetraspanin CD82/KAI F interacts with this gene (34).
2. AQP1: AQP1 encodes a water channel protein that is a small integral membrane protein. Cell migration requires the construction of ion channels and transporters critical components. AQ1 is Involved in cell migration by creating osmotic water flow across the plasma membrane following actin's polymerization.
Also, Overexpression of AQP-1 causes fibroblast growth, which is sufficient to increase invasion through the extracellular matrix. (35).
3. EGR1: EGR1 is a transcriptional regulator that, by initiating the expression of E-cadherin transcriptional inhibitors, contributes mainly to tumor invasion and metastasis (SNAIL and SLUG).
EGR1 contributes to tumor angiogenesis by binding to the HIF1 promoter in hypoxic conditions and causes HIF1 expression. Also, EGR1 by binding to the VEGFA promoter and increasing angiogenesis, directly initiates VEGFA expression in lung cancer cells (36).
4. ID1-ID3: ID family of helix-loop-helix proteins has no DNA binding activity, which negatively regulates the transcription factors in actively proliferating cells. They handle various cellular processes such as cellular growth, senescence, differentiation, angiogenesis. MMP9 seems to be all three Id genes (Id1, Id2, and Id3).
Id gene expression influences endothelial cells through downregulated expression of the angiogenesis inhibitor thrombospondin-1 (TSP-1 is an extracellular matrix glycoprotein that inhibits tumor growth and metastases) and VEGF receptor-mediated functions (VEGFR1,2) on myeloid precursor cells and circulating endothelial precursor cells (CEPs) (37).
5. IGFBP: This gene is a member of the Insulin-like growth factor-binding protein (IGFBP). It is bound to both types of IGFs( I and II) and, by blood plasma distribution, modify their interaction with cell surface receptors and, as a result, suppresses or induces the growth-promoting effects of IGF.
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IGFBP1: Insulin-like growth factor binding protein-1 (IGFBP-1) belongs to the IGF system, which plays in average growth and development and proliferation, invasion, migration, and adhesion in tumor cells. The biological action of IGFBP-1 in cancer is related to its phosphorylation state and IGF-dependent and independent mechanisms (38).
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IGFBP3: IGFBP-3 can bind IGF-1 and IGF-2 and block their access to the IGF-1 receptor (IGF1R). It is also active in the cellular environment. IGFBP-3 production is stimulated by transforming growth factor β (TGF-β). A role for IGFBP-3 in mediating TGF-β inhibitory activity is suggested. This process works in some breast cancer cells (39).
6. PECAM1: PECAM1 (Platelet and Endothelial Cell Adhesion Molecule 1) encoded a member of the immunoglobulin superfamily. In the endothelium, PECAM-1 plays a role as an adhesion molecule and contributes to intracellular signaling pathways in several cell adhesive mechanisms and endothelial nitric oxide synthase (eNOS). There is an essential relationship between PECAM-1 and endoglin, an important molecule during angiogenesis, expression. PECAM-1 isoforms may be involved in modulating endothelial cell adhesion mechanisms, expression, and activity of eNOS, endoglin, and angiogenesis (40).
7. PGRN: Progranulin (Pgrn) is a growth factor that induces the survival and proliferation of cells by activating mitogen-activated protein kinase (MAPK) and phosphatidyl 3-kinase (PI3K) pathways as well as can increase the expression of cyclins D and B in the cell cycle (41).
8. RGCC (RGC32): This gene activates the cell cycle. P53 induces it in response to DNA damage or by sublytic levels of C5b-9 (complement system) proteins that cause Response Gene to Complement (RGC)-32 by activating the Akt and CDC2 kinases. Tumor cells express RGC-32, which has two roles in cancer; it acts as a tumor stimulant or a tumor suppressor (42).
9. TMSB4X: Thymosin β4 (Tβ4), as a multifunctional peptide, plays a critical role in various processes such as proliferation, angiogenesis, anti-apoptosis, and inflammation. Tβ4 induces angiogenesis, enhance endothelial progenitor cell, cell viability, and promotes cell migration, as well as the formation of capillary-like structures in cells. Tβ4 upregulates VEGF expression (43).
10. TYMP: TYMP encodes the enzyme thymidine phosphorylase. Thymidine phosphorylase is a growth factor that can up-regulation growth on endothelial cells and promotes angiogenesis. Some of TYMP activity mediates through cytokines like interleukin 10 (IL-10), essential fibroblast growth factor, and tumor necrosis factor α (TNFα).
It enhances angiogenesis by stimulating endothelial cell migration through the thymidine metabolite deoxy-D-ribose, stimulating endothelial cell migration (44).