European Nuclear Medicine Guide
Knowledge of fundamental biological processes is essential for effective clinical practice in nuclear medicine and to inspire future developments in our discipline. This chapter provides a very concise description and explanation of selected biological processes that are often explored by nuclear medicine. The authors are aware that many biological themes and concepts are not addressed in the chapter due to space constraints. Cell proliferation and apoptosis are included, since they regulate physiological growth and homeostasis of all organs and their alterations promote the pathogenesis of many diseases. Angiogenesis, hypoxia, and glucose metabolism are related to the delivery of nutrients, oxygen, and energy production in normal and cancer cells. The interaction of cell surface receptors with their natural ligands triggers a number of cellular responses to external stimuli in both normal and pathological tissues. Finally, metastatic dissemination and immune evasion of cancer cells may provide targets for innovative diagnostic and therapeutic approaches in nuclear medicine.
Replication of normal cells occurs through a series of temporally ordered events that constitute the cell cycle. In the presence of growth-promoting signals, cells leave the quiescent phase (G0) and enter the first phase of the cell cycle (G1) during which they prepare for DNA replication. In the following S phase, DNA replication occurs, and the correct duplication and assembly of DNA is ensured in the subsequent G2 phase. Finally, cells enter the fourth phase of mitosis (M) that leads to cell division and formation of two identical daughter cells. Cell cycle progression is regulated by positive and negative feedback loops involving cyclin-dependent kinases (CDKs), cyclins, CDK inhibitors, and CDK substrates [1] Cyclins are key regulatory proteins that are expressed and degraded at specific times during each cell cycle. They bind to and activate CDKs that in turn trigger phosphorylation of distinct sets of substrates and allow cell cycle progression. This process is negatively regulated by CDK inhibitors that bind to CDK-cyclin complexes and inhibit their protein kinase activity. The cell cycle is also being controlled by external signals that can have mitogenic or anti-mitogenic effects usually acting during G1 phase. For instance, signalling by receptor tyrosine kinases (RTKs) induces expression of cyclin D and activation of G1-CDK promoting G1/S transition, whereas Tumour Growth factor β (TGF-β) receptor transduces signals that may induce up-regulation of CDK inhibitors blocking cell cycle progression.
When cell cycle regulation is altered, normal cells may become malignant. Activation of oncogenes or loss of function of tumour suppressor genes results in dysregulation of the cell cycle and continuous progression of cancer cells throughout the cell cycle. Hence, unlimited and uncontrolled proliferation is one of the acquired capabilities of cancer cells.
Apoptosis or programmed cell death is a highly regulated multistep process leading to selective cell death and elimination. Two main pathways initiate apoptosis: one is mediated by death receptors on the cell surface, and the other is mediated by mitochondria [2]. Both pathways lead to activation of specific enzymes, termed caspases, which cleave cellular substrates and cause the characteristic biochemical and morphological changes of apoptosis including DNA fragmentation, phosphatidylserine membrane exposure, and formation of apoptotic bodies.
The death receptor pathway, also known as the extrinsic apoptotic pathway, mediates apoptosis in several different cell types including activated lymphocytes. The binding of death receptors, such as CD95 (APO-1/Fas) and TNF-related apoptosis-inducing ligand (TRAIL) receptor, with their own cognate ligands induces the recruitment of adaptor proteins resulting in the activation of caspase 8 that in turn cleaves, and thereby activates downstream effector caspases such as caspase 3.
In the mitochondrial pathway, also known as the intrinsic pathway, apoptosis is initiated by the release of apoptogenic factors such as cytochrome c and apoptosis inducing factor (AIF) from the mitochondrial intermembrane space. These, in turn, activate caspase 9 through the formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex. Then, the proteolytic cascade propagates to effector caspases. The release of cytochrome c and other apoptogenic factors into the cytoplasm is under the control of Bcl-2 family members. This family includes both pro-apoptotic proteins, such as Bax and Bak, and anti-apoptotic proteins, such as Bcl-2 and Bcl-xL. Pro-apoptotic proteins increase the permeability of the mitochondrial membrane and thereby allow the release of cytochrome c, whereas anti-apoptotic proteins prevent its release.
Angiogenesis is multistep processes leading to the formation of new blood vessels from pre-existing capillaries. This physiological process can be performed through two different mechanisms, endothelial sprouting or intussusceptive microvascular growth (IMG), enables the growth and development of tissues and can occur in response to ischemic injuries, wound healing, or inflammation. Angiogenesis is also required for tumour growth and metastatic dissemination [3].
In the first phases of angiogenesis, endothelial cells are activated by angiogenic factors produced by stromal or tumour cells so that they can proliferate, degrade the extracellular matrix, migrate, and eventually assemble to form a new blood vessel [4]. This final step is accompanied by maturation and stabilization of new vessels occurring through basement membrane formation and recruitment of pericytes. However, in tumours, newly formed capillaries are tortuous, irregularly fenestrated, and not always functional [5]. The whole process is regulated by a balance between several activators and inhibitors. The main driver of angiogenesis is vascular endothelial growth factor (VEGF), that upon binding to its cognate receptor (VEGFR subtype 2) can induce endothelial cell proliferation and migration. The expression of VEGF is primarily stimulated by hypoxia through activation of the transcription factor Hypoxia Inducible Factor 1α (HIF-1α). Other important biomarkers of angiogenesis are αvβ3 integrin and matrix metalloproteinases. Integrin αvβ3 is early and highly up-regulated in activated endothelial cells in response to pro-angiogenic growth factors, binds with high affinity to components of extracellular matrix such as vitronectin and fibronectin, and promotes endothelial cell migration. Matrix metalloproteinases are enzymes that can degrade all components of the extracellular matrix and thus enhance migration of endothelial cells.
Normal tissues depend on oxygen supply for efficient adenosine triphosphate (ATP) generation. Oxygen status in tissues is determined by the balance between the rate of oxygen consumption and the rate of oxygen supply from the blood. Hypoxia occurs when oxygen delivery to tissues and cells is insufficient to cover their demand. This condition may present with different grades of severity depending on the relative decrease of oxygen concentration and duration. Hypoxia usually induces a cascade of adaptive cellular responses through mainly two oxygen-responsive signalling pathways that involve the HIF family of transcription factors and the unfolded protein response (UPR) [6]. Activation of HIF-1α and HIF-2α results in transcription of a pool of genes involved in angiogenesis, metabolic adaptation, tolerance to acidosis, and survival. On the other hand, severe hypoxia, as do other stress stimuli, increases the levels of unfolded proteins in the endoplasmic reticulum (ER) and activates the UPR pathways resulting in inhibition of protein synthesis, enhanced protein degradation in the ER, and induction of apoptosis or autophagy.
During exponential growth, tumours may become hypoxic due to oxygen diffusion limitations in the absence of an efficient vascular network. The diffusion range of oxygen in tissues is up to 200 μm. Therefore, tumour regions that are far from functional capillaries may become hypoxic.
In the presence of oxygen, normal mammalian cells convert glucose to pyruvate through glycolysis, and then pyruvate is completely degraded to carbon dioxide in the mitochondria through oxidative phosphorylation. Under anaerobic conditions, pyruvate produced by glycolysis in normal cells is redirected away from mitochondrial oxidation and is reduced to lactate. Cancer cells preferentially convert glucose to lactic acid through the glycolytic pathway even in the presence of oxygen, an alteration of energy metabolism known as aerobic glycolysis or the Warburg effect. This glycolytic phenotype is not only the result of metabolic adaptation to proliferative requirements and microenvironmental conditions, but it is also induced by genetic alterations of cancer cells. In fact, activation of oncogenes or loss of function of suppressor genes, in addition to drive tumorigenesis, causes a reprogramming of glucose metabolism [7].
Activation of the PI3K/AKT pathway by aberrant signalling from receptor tyrosine kinases, loss of function of PTEN, or activating mutations in the PI3K complex itself, is one of the mechanisms underlying the glycolytic phenotype. When active, AKT is able to up-regulate the expression of glucose transporters on the plasma membrane and phosphorylate key glycolytic enzymes.
At the transcriptional level, HIF-1 virtually activates all genes involved in the glycolytic cascade from glucose transporters to pyruvate kinase and lactate dehydrogenase A. A number of factors may modulate the transcriptional activity of HIF-1. Under hypoxic conditions, HIF-1 is stabilized in its active spatial conformation and amplifies the transcription of genes involved in glycolysis. Under normoxic conditions, HIF-1 can be activated by a variety of oncogenic signalling pathways and by mutations in von-Hippel Lindau (VHL), succinate dehydrogenase (SDH), and fumarate (FH) tumour suppressor genes. Moreover, high levels of the oncogenic transcription factor Myc induce up-regulation of several glucose transporters and glycolytic enzymes. Unlike HIF-1, Myc regulates genes involved in glutaminolysis, an additional metabolic pathway of energy production.
Cell surface receptors are transmembrane proteins that are able to receive external signals and transduce them inside the cell. Each type of receptor binds select endogenous ligands as well exogenous molecules. The binding has most of the time a nanomolar range affinity, is saturable and usually reversible.
One important class of receptors are G-protein coupled receptors that use guanine nucleotide-binding proteins (G-proteins) to trigger an intracellular signalling cascade[8]. When a ligand binds to a G-protein coupled receptor, it induces a conformational change in the receptor that in turn activates a G-protein by promoting exchange of GDP with GTP. When active, G-protein dissociates from the receptor and splits into its subunits that continue a downstream signalling cascade using various second messenger systems. An example of a G-protein coupled receptor is the somatostatin receptor, that upon binding with its native ligand and depending on the receptor subtype and effector system, induces a variety of cellular responses including growth arrest, apoptosis, inhibition of hormone and growth factors secretion, and blockade of angiogenesis[9]. One feature of these receptors their high susceptibility to desensitization and internalization. Receptor tyrosine kinases are another important class of cell surface receptor. These transmembrane proteins comprise an extracellular ligand binding domain, a hydrophobic transmembrane domain, and an intracellular domain with protein-tyrosine kinase activity. Most growth factors including EGF, VEGF, and NGF bind to receptor tyrosine kinases[10]. Ligand binding induces dimerization and autophosphorylation of the receptor at multiple tyrosine residues, and the active receptor is able to recruit additional proteins that in turn propagate a signal.
Ligand-gated ion channels are an additional type of cell surface receptor that mediate fast synaptic transmission. Binding of a neurotransmitter to these receptor causes a conformational change of the receptor that opens an ion channel, allows a rapid flow of selected ions across the plasma membrane, and results in subsequent changes in trans-membrane potentials that can elicit an excitatory or inhibitory response.
Cancer cells have the ability to invade the surrounding tissues and migrate to distant organs. This process usually requires multiple steps, namely detachment of cancer cells from the primary tumour mass, migration of cancer cells to the surrounding tissues, intravasation, dissemination into the bloodstream, extravasation, and tumour growth at distant sites[11]. Several receptors and signalling pathways, including those modulating cell survival, adhesion, and migration, are involved in metastatic dissemination. Furthermore, physical and functional interactions of cancer cells with components of microenvironment modulate several steps in metastasis formation.
Among the receptors involved in tumour invasion, the urokinase-type plasminogen activator receptor (uPAR) is one of the most extensively studied. This receptor binds with high affinity to urokinase-plasminogen activator (uPA) and its proenzyme (pro-uPA). By degrading directly or indirectly all components of the extracellular matrix, uPA promotes cancer cell migration and invasion. Several integrins are involved in the metastatic cascade, and they integrate the extracellular matrix with the intracellular cytoskeleton and mediate adhesion, invasion, and metastatic colonization. A prominent role is played by αvβ3, αvβ5, and α5β1 integrins that recognize the amino acid sequence Arg- Gly-Asp (RGD) in the protein structure of their endogenous ligands, most of which are components of extracellular matrix. The chemokine receptor CXCR4, upon binding with stromal cell-derived factor-1α, its native ligand, promotes homing of cancer cells at distant sites by modulating chemotaxis, gene transcription, cell survival, and proliferation.
Cancer cells can evade immune surveillance by inhibiting the native capacity of immune cells to recognize and destroy abnormal and foreign cells. In this manner, cancer cells cannot be killed by immune cells and can proliferate and disseminate throughout the body. One mechanism adopted by cancer cells to overcome immune response is to express plasma membranes proteins that, by interacting with their co-receptors on immune cells, limit their functional activity[12]. Therapeutic disruption of these inhibitory interactions by specific antibodies restores the ability of immune cells to recognize and destroy tumour cells. Currently, three immune checkpoints have been exploited as suitable targets for blockade therapy in cancer patients: the programmed death 1 (PD-1) receptor, its endogenous ligand programmed death ligand 1 (PD-L1), and the cytotoxic T-lymphocyte associated antigen 4 (CTLA-4).
PD-1 is a member of the B7 family of co-receptors and is expressed on the surface of T-lymphocytes, B-lymphocytes, and natural killer cells. PD-1 binds to PD-L1 and acts as a negative regulator of T-cell activity by limiting their tumour cell killing function. PD-L1 is present on the surface of tumour cells and antigen presenting cells and has been identified as the main driver of PD-1 mediated immune resistance of cancer cells.
CTLA-4 is a transmembrane co-receptor expressed on the surface of activated T-lymphocytes, where its major function is to regulate the amplitude of T-cell response to antigen. CTLA-4 and its homologue CD28 share the same ligands, CD80 and CD86. It has been proposed that CTLA-4 reduces the activation of T-cells by interacting with CD80 and CD86 ligands thus preventing their binding to the co-stimulatory receptor CD28. The inhibition of CTLA-4 checkpoint releases the brake of effector T-cells and amplifies immune response to tumour cells.
The brief description of selected biological processes provided in this chapter may be considered as a quick reference guide for a rapid introduction of nuclear medicine physicians to this matter. The authors are aware that the chapter is not an exhaustive compilation of all biological processes that can be probed with nuclear medicine techniques, and we foresee a continuous expansion and update of this content.