Tumor Microenvironment

Tumor cells stimulate significant molecular, cellular and physical changes within their host tissues. The emerging tumor microenvironment (TME) is a complex and continuously evolving entity. The composition of the TME varies between tumor types, but hallmark features include immune cells, stromal cells, blood vessels, and extracellular matrix (ECM). It is believed that the “TME is not just a silent bystander, but rather an active promoter of cancer progression”. Early in tumor growth, a dynamic and reciprocal relationship develops between cancer cells and components of the TME to support cancer cell survival, local invasion and metastatic dissemination. To overcome a hypoxic and acidic microenvironment, the TME coordinate a program that promotes angiogenesis to restore oxygen/nutrient supply and remove metabolic waste. Tumors become infiltrated with diverse adaptive and innate immune cells that can perform both pro- and anti- tumorigenic effects (Figure 1). An expanding literature on the TME has identified new targets within it for therapeutic intervention.

Figure 1. Impact of immune cells within the TME.

The role of immune cells in the TME can be to either suppress tumor formation (anti-tumor microenvironment) or promote tumorigenesis (immune suppressive microenvironment). Depending on context and tumor type immune cells can both be pro or anti-tumorigenic.

Immune Cells

Immune cells are a critical component of the TME. Depending on the context, a dichotomy exists in the relationship between immune cells and TMEs: immune cells can either suppress tumor growth or promote it (Figure 1). Persistent inflammation due to chronic infection is a common mechanism underlying tumor formation in several types of cancer, including: colorectal, hepatocellular and cervical cancer. Broadly, immune cells fall into two categories: adaptive immune cells and innate immune cells. Adaptive immunity is activated by exposure to specific antigens and uses an immunological memory to “evaluate” the threat and enhance immune responses. T-cells, B-cells and NK cells belong to the adaptive immune response. Innate immunity is a non-specific defense mechanism that comes into play within hours of a foreign antigen entering the body. Cells that carry out an innate immune response include macrophages, neutrophils and dendritic cells.

T-cells

Each T-cell develops its own T cell receptor (TCR) that recognizes a specific antigen. Within the TME there are several distinct populations of T-cells that influence tumorigenesis. Cytotoxic T-cells (CD8+) detect abnormal tumor antigens expressed on cancer cells and target tumor cells for destruction. The presence of cytotoxic T-cells in the TME is often associated with positive prognosis in cancer patients. Aside from killing tumor cells, cytotoxic T-cells also suppress angiogenesis through the secretion of interferon gamma (IFN-γ). CD4⁺ T-cells differentiate into a variety of subtypes and thus coordinate a wide range of immune responses within the context of the TME. T helper 1 (Th-1) cells are proinflammatory CD4+ T-cells that support CD8+ cells through the secretion of interleukin-2 (IL-2) and IFN-γ. Increased levels of Th-1 cells within the TME are associated with positive outcomes in many types of cancer. Regulatory T cells (Tregs) are normally required to suppress inflammatory responses and control autoimmunity. In the context of the TME, Tregs are ubiquitous and promote tumor development and progression by dampening antitumor immune responses. For example, Tregs secrete IL-2 which modulates natural killer (NK) cell homeostasis and function. Additionally, Tregs cells directly support the survival of cancer cells through the secretion of growth factors, and indirectly through interaction with stromal cells such as fibroblasts and endothelial cells. The immune landscape within the TME falls into three main categories: immune infiltrated, immune excluded, and immune silent. In an immune infiltrated tumor, immune cells (e.g. cytotoxic T-cells) are homogenously distributed throughout the tumor indicating an active immune response. Alternatively, some tumors are classified as immune excluded were T-cells are at the periphery of tumors and have not infiltrated the TME. Finally, some tumors are categorized as “immune silent” and completely lack immune cell infiltrate indicating no immune response to the tumor.

B-cells

B-cells are specialized immune cells responsible for antibody production, antigen presentation and secretion of cytokines. Typically, B-cells concentrate at the margin of tumors and are commonly found in lymph nodes in close proximity to the TME. Compared to T-cells, the TME has relatively few infiltrating B-cells; however, recent studies have demonstrated that the presence and function of B-cells are important during tumorigenesis. Tumor infiltrating B-cells are important in the formation of “tertiary lymphoid structures”, which are ectopic lymphoid structures formed within the TME. Tertiary lymphoid structures allow close association between T and B cells and are a positive prognostic marker in breast cancer, melanoma and ovarian cancer. The anti-tumorigenic roles of B-cells, include antigen-presentation to T-cells, anti-tumor antibody production and secretion of cytokines that promote cytotoxic immune responses (e.g. IFN-γ). Alternatively, B-cells can have protumor effects, and their presence in the TME can be predictive of poor outcome in bladder cancer, prostate cancer, renal cell carcinoma. Regulatory B-cells promote tumor aggression through production of cytokines (e.g. IL-10 and transforming growth factor-beta (TGF-β)) that promote immune suppressive phenotypes in macrophages, neutrophils and cytotoxic T cells.

Natural Killer Cells

Natural killer cells naturally patrol the blood stream and seek out virally infected host cells or tumor cells. Functionally, NK cells can be broken down into two classes; they either directly participate in cell-mediated killing of tumor cells or they secrete inflammatory cytokines. NK cells are highly efficient at killing tumor cells within the circulation and can participate in blocking metastasis, but are less efficient at killing within the tumor microenvironment.

Macrophages

Macrophages are a critical component of the innate immune system that modulate immune responses through pathogen phagocytosis and antigen presentation. In addition, macrophages are critical in wound healing and tissue repair. Monocyte derived macrophages can be categorized into either inflammatory M1 macrophages that phagocytize and kill cells or immune-suppressive M2 macrophages that participate in wound healing. While both classes of macrophages can be found within a tumor, the TME promotes the M2 phenotype through hypoxia and the secretion of cytokines (e.g. IL-4) to support tumor growth and progression. Certain tumor types can be heavily infiltrated with macrophages and can make up to 50% of a tumors mass. Typically, high macrophage infiltration is associated with poor patient prognosis in many types of cancer, such as breast cancer, lung cancer and gastric cancer. Often macrophages are found to surround blood vessels in the TME where they secrete vascular endothelial growth factor (VEGF)-A and induce new blood vessel formation.

Neutrophils

Neutrophils make up to 70% of circulating leukocytes and provide the first line of defense against many pathogens. In the context of cancer, neutrophils can act to either suppress or promote tumor growth, depending on tumor type and stage of development. As a tumor begins to grow neutrophils are recruited to the TME and promote inflammation through release of cytokines and reactive oxygen species to promote tumor cell apoptosis. Later in tumor development neutrophils promote tumor growth through modification of the extracellular matrix, releasing VEGF and producing matrix metalloprotease (MMP) −9 to stimulate angiogenesis and ultimately tumor progression and local invasion.

Dendritic Cells

Dendritic cells (DCs) play a critical role in the immune system as antigen presenting cells; they recognize, capture and present antigens to T-cells (antigen presenting cells, APC) at secondary lymphoid organs (e.g. lymph nodes). Ultimately, DCs bridge a gap between adaptive and innate immunity to initiate pathogen-specific T-cell responses. The tumor microenvironment shapes the fate of DCs by providing environmental cues that either create an immune response to tumor cells or accept them. Cytokines secreted from the TME trigger DCs to tolerate the presence of tumor cells and block the induction of an immune response.

Stromal Cells

Cancer cells recruit supporting cells from nearby endogenous tissue stroma to promote critical steps in tumor formation. Stromal cell composition can vary significantly between tumors types and include: vascular endothelial cells, fibroblasts, adipocytes and stellate cells. Once recruited to the TME, stromal cells secrete many factors that influence angiogenesis, proliferation, invasion, and metastasis (Figure 2).

Figure 2. Defining the role of stromal cells in promoting cancer progression.

Cancer cells recruit stromal cells from neighboring tissue during tumorigenesis. In the TME, stromal cells and cancer are in a dynamic relationship promoting the cancer progression. The stromal cell composition varies between tumor types but includes endothelial cells, fibroblasts, adipocytes and stellate cells. The TME orchestrates angiogenesis, proliferation, invasion and metastasis through the secretion of growth factors and cytokines.

Endothelial Cells

Vascular endothelium is a thin monolayer of endothelial cells (ECs) and help to orchestrate the formation of blood vessels. Not only does vascular endothelium separate circulating blood from tissues, it also delivers water and nutrients, maintains metabolic homeostasis, carries immune cells and participates in the formation of new blood vessels. During the initial stages of tumor development cancer cells rely on passive diffusion for gas exchange and the transportation of nutrients. Once tumors reach 1–2mm³ in volume, insufficient oxygen and a build-up metabolic waste results in the TME becoming hypoxic and acidic. To overcome hypoxia and acidification, tumors must develop their own blood supply. A hypoxic TME leads to the activation hypoxia inducible factors (HIFs), transcription factors critical in coordinating cellular responses to low O₂. Vessel sprouting is a common mechanism used by tumors to co-opt existing blood vessels and induce the growth of new vessels. Specifically, HIFs initiate vessel sprouting by instructing ECs to secrete proangiogenic factors such as, platelet derived growth factor (PDGF), epidermal growth factor (EGF) and VEGF. In an autocrine and paracrine fashion, VEGF stimulates migration of ECs to form new blood vessel lumens. Next ECs secrete proteins to form new basement membranes. Blood vessels in the TME often fail to achieve the final stages of maturation, resulting in leaky vasculature.

Beyond angiogenesis, ECs are critical in promoting cancer cell migration, invasion and metastasis. ECs are highly plastic in nature and can change cell fate. During tumor progression, ECs undergo endothelial-mesenchymal transition to become cancer associated fibroblasts (CAFs). The transition from an EC to a CAF is organized by TGF-β and bone morphogenetic protein (BMP) and leads to loss of cell-to-cell connections, detachment and elongation, enhanced migration and loss of endothelial properties. CAF are critical in stimulating migration and invasion of tumor cells (see below). Metastasis is a multistep process that involves translocation of cancer cells from the primary TME to distant locations. Tumor cells must first escape the primary tumor site and enter the vasculature in a process known as intravasation. During intravasation, tumor cells adhere to ECs and this interaction changes the endothelial barrier and allows tumor cells to migrate between two ECs. Blood vessels formed in the TME are usually immature and lack proper cell-to-cell connections, enabling cancer cells to extravasate.

Cancer Associated Fibroblasts

Cancer associated fibroblasts (CAFs) are a major component of the tumor stroma and play a critical role in facilitating crosstalk between cancer cells and TME. While diverse in origin, CAFs often arise from tissue resident fibroblasts. CAFS are also formed from adipocytes, ECs, pericytes, stellate cells and bone marrow derive mesenchymal stem cells. Upon injury, fibroblasts that normally reside within tissues can become reversibly induced to form myofibroblasts, which actively participate in wound healing. Myofibroblasts are activated by TGF-β signaling and develop characteristics important in wound healing, such as proliferation, contractile properties, secretory phenotypes and extracellular matrix formation. Tumors have been aptly termed “wounds that never heal”. In the TME, cancer cells and other stroma cells secrete transforming growth factors to convert fibroblasts into CAFs, such as TGF-β, platelet derived growth factor (PGDF) and FGF2. A build-up of CAFs within the tumor microenvironment is often associated with poor prognosis in many cancer types. For example, in colorectal cancer the presence of CAFs is strongly associated with disease reoccurrence. Despite an association with poor prognosis, CAFs have been shown to both promote and restrain tumorigenesis. Alternatively, some cancer types, such as breast cancer and lung cancer, have improved prognosis and overall survival when they are highly desmoplastic.

Within the TME, CAFs produce the majority of extracellular components, including growth factors, cytokines and extracellular matrix. CAFs shape the TME in four main ways: tumor proliferation and metastasis, neoangiogenesis, ECM remodeling and immunosuppression. In tumors of epithelial origin, EMT or epithelial-mesenchymal transition is a critical step in metastasis were epithelial cells lose cell polarity and cell-to-cell adhesions and gain migratory and invasive phenotypes. One way CAFs control metastasis is through the secretion of TGF-β, a growth factor required for EMT and angiogenesis. To facilitate migration of cancer cells through the TME, CAFs secrete MMP-3 which degrades E-cadherin to promote cancer cell invasion. The ECM is also an important source of VEGF, which can be released by MMP-13 to promote angiogenesis. In general, CAFs promote an immunosuppressive phenotype through the production of immune modulatory chemokines and cytokines.

Adipocytes

Adipocytes are specialized cells within the body that regulate energy balance and are responsible for storing excess energy as fat. Adipocytes exert their effects on the TME through secretion of metabolites, enzymes, hormones, growth factors and cytokines. Within the context of the TME, adipocytes are in a dynamic and reciprocal relationship with tumor cells to support tumor progression. Breast tissue is largely composed of white adipose tissue (WAT); therefore, adipocytes are a critical player in the breast cancer TME. Breast cancer cells can stimulate adipocytes to undergo lipidlysis, which breaks down lipid stores making free fatty acids available uptake by the cancer cells. Breast cancer cells use free fatty acids for energy production, cell membrane formation, lipid bioactive molecules and exosomes. Leptin is an important hormone produced by adipocytes and promotes tumor progression directly by influencing breast cancer cell proliferation and indirectly by activation of macrophages. Adipocytes also play an important role in modifying ECM through secretion of metalloproteases, such as MMP-1, MMP-7, MMP-10, MMP-11 and MMP-14. More than 40% of cancer patients are overweight, making obesity is a major risk factor for many types of cancer, including: breast, pancreatic and ovarian. White adipose tissue is an endocrine organ and can promote breast cancer cells to metastasis to the liver and lungs through paracrine signaling.

Stellate Cells

Stellate cells are quiescent stromal cells of mesenchymal origin located within the liver and pancreas. Upon tissue injury, stellate cells become activated, enter into cell cycle and are induced to transform into myofibroblasts. A characteristic feature of stellate cells is the deposition of vitamin A in lipid droplets. In the liver, hepatic stellate cells (HSC) are normally located within perisinusoidal and portal areas and can constitute as much as 15% of liver mass. Hepatocellular carcinoma (HCC) is the predominant form of liver cancer and HSCs function to promote crosstalk within the TME. A key signaling molecule, TGF-β is produced by HCC and triggers HSCs to become activated. Once activated HSCs modify the ECM and produce proangiogenic factors such as VEGF-A and MMP-2. Lipid droplets are critical structures in HSCs used to produce new ECM and remodel it through the production of MMPs. Pancreatic ductal adenocarcinoma is the most common form of pancreatic cancer (95%), characterized by dense fibrotic tissue or desmoplasia. When pancreatic stellate cells (PSCs) are quiescent, they contribute to ECM modification through the production of ECM proteins (e.g. desmin, vimentin) and degradation enzymes. Vitamin A depletion results in PSC activation, leading to the secretion of cytokines and chemokines, enhanced migration and proliferation potential. Activated PSCs play a critical role in promoting the desmoplastic phenotype of PDAC tumors and their hypoxic microenvironments.

Non-cellular components of the TME

ECM

The extracellular matrix (ECM) has important functions within the TME; not only does it provide a physical scaffold for cells; it also is a key factor in promoting tumor cell dissemination. ECM is an important molecular component of the TME and composed of collagen, fibronectin, elastin, and laminin. Solid tumors contain large ECM deposits and constitute up to 60% of tumors mass. Large collagen deposits together with a high percentage of fibroblast infiltration result in desmoplasia, which is strongly linked to poor patient prognosis. Many cells within the TME secrete components of the ECM, although CAFs are the predominant source of ECM. MMPs are proteases that breakdown ECM proteins and are critical in remodeling ECM to promote tumor progression and metastasis. The ECM is a depot for cytokines and growth factors, which are released by proteases (e.g. MMPs). For example, the ECM can be a deposit for proangiogenic factors, like VEGF, FGF, PDGFB, TGFB.

Exosomes

Exosomes are microvesicles that range in size from 30nm to 200nm. Their contents reflect the original cell, including protein, RNA, DNA and lipids. Within the TME exosomes play a critical role in facilitating cross talk between cancer cells and stromal cells. Functionally, exosomes have been shown to promote inflammation, tumor progression, angiogenesis, and metastasis within the TME. Conditions of hypoxia appear to exacerbate exosome production by cancer cells and promote the transition of stromal cells into CAFs.

Therapeutically Targeting the TME

Over the last decade cancer treatment has undergone a revolution. Traditionally, drugs targeted tumors more broadly (e.g. chemotherapy) and now new therapeutic strategies target specific cells within the TME. Immune check-point blockade (ICB) was the first generation of antibody-based therapies to target immune cells in the TME. ICB works by blocking receptor/ligand interactions (e.g. CTLA4 and PD1) dulling T-cell activation and function. Patients who respond to ICB have significant clinical benefit, but the majority of patients do not respond to ICB. The identification of relevant biomarkers is required to recognize patients who are expected to benefit from immune check-point blockade therapy. Therapeutically targeting DC activation through the use of DC vaccination has been successfully used in the treatment of prostate cancer. The “Provenge” protocol involves harvesting monocytes from prostate cancer patients, differentiation into DC, activation with PAP antigen, and then re-introducing them back into patients. Provenge therapy can result in significant reduction in tumor burden of prostate cancer patients.

Growing tumors require the formation of new blood vessels to relieve oxygen deprivation and accumulating metabolic waste, therefore targeting angiogenesis was an attractive strategy. Antiangiogenic (AA) therapy has focused on targeting the VEGF/VEGFR signaling axis and has included: a neutralizing antibody to VEGF-A (Bevacizumab); decoy receptor for VEGF-A or B (Aflibercept); tyrosine kinase inhibitor (Sorafenib); and antibody that blocks VEGF binding its receptor (Ramucirumab). As a single agent most patients either do not respond to AA therapy or develop resistance. Successful integration of AA therapy into the clinic will likely require combination with other agents. For example, Bevacizumab in combination with PDL-1 has shown some success for the treatment of HCC and renal cancer.

While therapeutically targeting the TME is an attractive strategy for the treatment of cancer, existing FDA approved treatments have limited efficacy. As we continue to understand how the TME contributes to tumorigenesis, new therapeutic targets and strategies will be identified. Promising preclinical studies have shown potential for the use of CAR-NK cells, liver stellate cells and fibroblasts.