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. This overview encompasses the following subjects: (tumour) cell biology and metabolism, with a particular focus on disparities in cell cycle regulation and proliferation, programmed cell death by apoptosis, and energy production. Moreover, the paragraph will address specific tumour cell-relevant mechanisms, including angiogenesis, the impact of hypoxia, invasion and metastasis. Finally, molecular biological aspects, such as surface receptors and immune checkpoint proteins, provide targets for (innovative) diagnostic and therapeutic approaches in nuclear medicine. Physiological and radiobiological aspects are addressed in more detail in the subsequent chapters.
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 transition from the quiescent phase (G0) to the first phase of the cell cycle (G1) during which they prepare for DNA replication. The subsequent synthesis (S) phase is characterised by the process of DNA replication. This is followed by the G2 phase, where the correct duplication and assembly of DNA is ensured. The final mitosis (M) is characterised by cell division, resulting in the formation of two genetically identical daughter cells.
Cell cycle progression is subject to the regulation of positive and negative feedback loops involving cyclin-dependent kinases (CDKs), cyclins, CDK inhibitors, and CDK substrates. Cyclins, which are key regulatory proteins, are expressed and degraded at specific times during each cell cycle phase. They bind to and activate CDKs, which in turn trigger phosphorylation of distinct sets of substrates and allow cell cycle progression. This process is negatively regulated by CDK inhibitors, which bind to CDK-cyclin complexes and inhibit their protein kinase activity.
The cell cycle is also being controlled by external signals that can exert mitogenic or anti-mitogenic effects, usually acting during G1 phase. For instance, the induction of cyclin D and subsequent activation of G1-CDK by signalling via receptor tyrosine kinases (RTKs) promotes G1/S transition. Conversely, the upregulation of CDK inhibitors transduced by transforming growth factor β (TGF-β) signalling has been shown to block cell cycle progression.
The transformation of normal cells into malignant cells can be initiated by alterations in cell cycle regulation. Such alterations may be caused by the disruption of cell cycle checkpoints, changes in CDK activity, or modifications in transcriptional control. Tumour initiation can thereby occur through activation of oncogenes or loss of function of tumour suppressor genes by mutation or epigenetic changes. The resulting cell cycle dysregulation is characterised by the uncontrolled proliferation of cancer cells. Consequently, sustaining proliferative signalling emerges as a hallmark of cancer cells [1, 2].
Apoptosis or programmed cell death is a highly regulated multistep process leading to selective cell death and elimination. Two main pathways initiate apoptosis: the extrinsic pathway, mediated by death receptors on the cell surface, and the intrinsic pathway, mediated by mitochondria. Both 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 extrinsic apoptotic pathway is activated by ligand binding to death receptors of the tumour necrosis factor (TNF) family, such as CD95 (APO-1/Fas) and TNF-related apoptosis-inducing ligand (TRAIL) receptor. The recruitment of adaptor proteins is then induced, leading to the activation of caspase 8, which in turn cleaves and activates downstream effector caspases such as caspase 3. In immune cells and hepatocytes, apoptosis is often triggered via this extrinsic signalling pathway.
The intrinsic pathway is initiated by intracellular stress such as DNA damage, hypoxia, or nutrient deprivation. The release of apoptogenic factors, such as cytochrome c and apoptosis inducing factor (AIF) from the mitochondrial intermembrane space into the cytoplasma, occurs and activates caspase 9 through the formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex. The proteolytic cascade then propagates to effector caspases. The release of cytochrome c and other apoptogenic factors into the cytoplasm is controlled by members of the Bcl-2 family. 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, allowing the release of cytochrome c, whereas anti-apoptotic proteins prevent its release. [3]
An imbalance in apoptosis has been linked to a variety of diseases. The promotion of tumour growth and the reduction of therapeutic responses are consequences of the inhibition of apoptosis. However, in addition to its well-documented anti-cancer properties, apoptosis can also have pro-cancerous functions, operating through both extrinsic and intrinsic pathways in the cell [4].
Angiogenesis is a multi-step process leading to the formation of new blood vessels from pre-existing capillaries. This physiological process can be achieved by several mechanisms: (1) endothelial sprouting from pre-existing vessels, (2) intussusceptive microvascular growth (IMG), (3) vasculogenic mimicry of tumour cells, and (4) vessel co-option of tumour cells. Angiogenesis enables tissue growth and development of tissues, and can occur in response to hypoxia, tissue injury, organ development, inflammation, and tumour growth and metastasis.
In the initial stages of angiogenesis, endothelial cells are activated by angiogenic factors produced by stromal or tumour cells, allowing them to proliferate, degrade the extracellular matrix, migrate, and eventually assemble to form a new blood vessel. This final step is accompanied by maturation and stabilisation of the new vessel through basement membrane formation and recruitment of pericytes. However, in tumours, newly formed capillaries are tortuous, irregularly fenestrated, and not always functional. The whole process is regulated by a balance between several activators and inhibitors. The main driver of angiogenesis is vascular endothelial growth factor (VEGF), which can induce endothelial cell proliferation and migration by binding to its cognate receptor (VEGFR subtype 2). VEGF expression is primarily stimulated by hypoxia through activation of the transcription factor hypoxia inducible factor 1α (HIF-1α). [5, 6]
Other important biomarkers of angiogenesis include integrin αvβ3 and matrix metalloproteinases. The integrin αvβ3 is early and highly upregulated in activated endothelial cells in response to pro-angiogenic growth factors, binds with high affinity to extracellular matrix components, such as vitronectin and fibronectin, and promotes endothelial cell migration. [7] Matrix metalloproteinases are enzymes that can degrade all components of the extracellular matrix and thereby promoting endothelial cell migration. [8]
Tumours often undergo uncontrolled and rapid angiogenesis due to the overexpression of pro-angiogenic factors. [9]
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 typically induces a cascade of adaptive cellular responses through mainly two oxygen-responsive signalling pathways involving (1) the hypoxia inducible factor (HIF) family of transcription factors and (2) the unfolded protein response (UPR). 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. [10-12]
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 not only drives tumorigenesis, but also leads to a reprogramming of glucose metabolism. For example, activation of the phosphatidylinositol 3-kinases/protein kinase B (PI3K/AKT) pathway by aberrant signalling from receptor tyrosine kinases, loss of function mutations of phosphatase and tensin homolog (PTEN), or activating mutations in the PI3K complex itself, is one of the mechanisms underlying the glycolytic phenotype. When active, AKT is able to upregulate the expression of plasma membrane glucose transporters on the and phosphorylate key glycolytic enzymes.
At the transcriptional level, hypoxia inducible factor 1 (HIF-1) virtually activates all genes involved in the glycolytic cascade, from glucose transporters to pyruvate kinase and lactate dehydrogenase A. Under hypoxic conditions, HIF-1 is stabilised 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 upregulation of several glucose transporters and glycolytic enzymes. Unlike HIF-1, Myc regulates genes involved in glutaminolysis, an additional metabolic pathway of energy production. [13,14]
Cell surface receptors are transmembrane proteins capable of receiving external signals and transducing them within the cell. Each type of receptor binds selected endogenous ligands as well as exogenous molecules. The binding has mostly nanomolar affinity, is saturable and usually reversible. There are three major classes of surface receptors: (1) G protein-coupled, (2) enzyme-coupled, and (3) ligand-gated ion channels.
G protein-coupled receptors (GPCR) use guanine nucleotide-binding proteins (G proteins) to trigger an intracellular signalling cascade. When a ligand binds to a GPCR, it induces a conformational change in the receptor, which in turn activates a G protein by promoting the exchange of GDP for GTP. When active, the G protein dissociates from the receptor and splits into its subunits, which continue a downstream signalling cascade using various second messenger systems. [15] An example of a GPCR is the somatostatin receptor, which 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 factor secretion, and blockade of angiogenesis [16]. These receptors are characterised by their high susceptibility to desensitization and internalization. [17]
Within the enzyme-linked receptors, the receptor tyrosine kinases (RTKs) are a significant subclass. These transmembrane proteins consist of 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. Ligand-binding induces dimerisation and autophosphorylation of the receptor at multiple tyrosine residues, and the active receptor is capable of recruiting additional proteins that in turn transduce a signal. [18].
Ligand-gated ion channels composed of multiple subunits that form a pore allowing ion flow across the plasma membrane. Binding of a neurotransmitter to this receptor causes a conformational change in the receptor that opens an ion channel, allowing a rapid flow of selected ions across the plasma membrane and resulting in subsequent changes in trans-membrane potentials that can elicit an excitatory or inhibitory response. [19]
In tumours, cell surface receptors play a critical role in several hallmarks of cancer, for example (1) overexpression or aberrant activation of growth factor receptors induces sustained proliferative signalling; (2) altered signalling bypasses growth suppressors; (3) integrins and adhesion molecules activate invasion and metastasis; (4) enhanced VEGFR expression promotes vascularisation; or (5) immune checkpoint proteins evade immunogenic destruction (see next paragraph).
Some 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 into the surrounding tissues, intravasation, dissemination into the bloodstream, extravasation, and tumour growth at distant sites. Several receptors and signalling pathways are involved in metastatic dissemination, including those that modulate cell survival, adhesion, and migration. Furthermore, physical and functional interactions of cancer cells with components of the microenvironment modulate several steps in metastasis formation. [20]
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. [21]
In addition, several integrins are involved in the metastatic cascade, integrating the extracellular matrix with the intracellular cytoskeleton and mediating adhesion, invasion, and metastatic colonisation. A prominent role is played by the integrins αvβ3, αvβ5, and α5β1, which recognise 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 (CXCL12), its native ligand, promotes homing of cancer cells at distant sites by modulating chemotaxis, gene transcription, cell survival, and proliferation. [24]
Cancer cells can evade immune surveillance by inhibiting the native ability of immune cells to recognise and destroy abnormal and foreign cells. In this manner, several 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 immune checkpoint proteins on their surface, which limit the functional activity of immune cells by interacting with their co-receptors. Therapeutic disruption of these inhibitory interactions by specific antibodies restores the ability of immune cells to recognise and destroy tumour cells. To date, mainly three immune checkpoints have been studied 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). [25]
PD-1 is a member of the B7 family of co-receptors and is expressed on the surface of lymphocytes. 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 (e.g. metastatic melanoma, non-small cell lung carcinoma, bladder cancer) and antigen-presenting cells and has been identified as the main driver of PD-1-mediated immune resistance of cancer cells. [26,27]
CTLA-4 is a transmembrane co-receptor expressed on the surface of activated T lymphocytes, where its main function is to regulate the amplitude of the 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, thereby preventing their binding to the co-stimulatory receptor CD28. Inhibition of CTLA-4 checkpoint releases the brake of effector T cells and enhances the immune response to tumour cells. [28]
The brief description of selected biological processes provided in this chapter can 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 studied using nuclear medicine techniques, and we anticipate that this content will be continually expanded and updated.
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