One of the fundamental attributes of cancer cells is their capability to proliferate even with no associated signaling. This is what Hanahan and Weinberg called sustaining proliferative signaling and can be achieved by increasing the production of growth factors, stimulating normal cells to provide cancer ones with growth factors, activating protein in the downstream signaling pathway, and increasing the number of receptors on the cell or either modifying them to ease cancer cell signaling. In cancer cells, the process of intracellular communication may be taken over by specific mutations in otherwise normal genes, resulting in an abnormal gene called oncogene. Oncogenes generate abnormal genes products such as oncoproteins leading to a malignant cell behavior. These mutations may be produced in kinases, including EGFR mutations and c-Kit mutations, among others, that can disturb the signal transmitting function [1]. PI3K-Akt signaling, MAP-kinase pathway, mTOR pathway, cellular senescence, or oncogenes and tumor suppressor genes might contribute in this step [3]. This is key in targeted drug therapy as there is an interaction of membrane receptor-based tyrosine kinase and PI3K-Akt and RAS/ERK pathways and many drugs being used to inhibit signaling in different ways. Molecular-based imaging modalities have been used to image these molecular pathways. For example, some studies have demonstrated the extraordinary effect against gastrointestinal stromal tumors (GIST) of a tyrosine kinase inhibitor imatinib drug (Gleevec). GIST has been related to a transmembrane receptor, the oncogenic protein c-Kit. ¹⁸F-fluorodeoxyglucose or ¹⁸F-FDG, an analogue of glucose labeled with a positron emitter F-18, has been used in molecular imaging for assessing the therapeutic response of this drug in GIST patients. ¹⁸F-FDG has shown the effect of blocking the specific tyrosine kinase activity associated with c-Kit [1]. Depending on the drug being used and its molecular target, a certain type of radiopharmaceutical can be applied to assess treatment response. For example, other tyrosine kinase inhibitor drugs, sunitinib and sorafenib, have been used in a type of kidney cancer. These drugs are targeting a specific angiogenic growth factor called vascular endothelial growth factor or VEGF. Other tracers such as ¹⁸F-desatibib, ⁶⁸Ga-Fab’₂ herceptin, and ¹⁸F-fluorodihydrotestosterone or ¹⁸F-FDHT have been used to image different targets: mutant ABL in resistant chronic myeloid leukemia (CML), Her2 in breast cancer, and androgen receptor (AR) in prostate cancer, respectively.

Cell proliferation in normal cells is a controlled process where many signals (pro- and anti-growth) coordinate the cycle phases. For example, the G₁ phase is the most vulnerable cell cycle vital due to the fact that it is at this point where extrinsic mitogenic signals may facilitate more mutations and therefore enable the development of cancer cells [4]. The rapid growth of malignant tissue can be measured by computed tomography imaging (CT) to evaluate changes in volume. Molecular imaging with specific tracers linked to proliferative processes such as the accelerated synthesis of DNA is more suited for this. Many tracers have been tested to image this mechanism. 2-¹⁸F-fluorothymidine or ¹⁸F-FLT is probably the most interesting and extensively used radiopharmaceutical in this setting. ¹⁸F-FLT is a pyrimidine-based tracer taken by the cell via passive diffusion and facilitated transport by Na+-dependent carriers and then phosphorylated by thymidine kinase 1 (TK1) and finally trapped in the cell. In quiescent cells, TK1 activity is virtually absent, but in proliferating cells its activity is increased, particularly in the S phase of the cell cycle. This radiolabeled tracer has shown very promising results in monitoring response to treatment. In one study performed by Pio et al., 14 breast cancer patients were evaluated 2 weeks after the first cycle of treatment. Levels of the cancer antigen 27.29 (CA27.29), which is a soluble form of a mucin-like glycoprotein abundantly expressed on most carcinoma cells, showed very good correlation with ¹⁸F-FLT uptake, as shown in Fig. 1.2 [5]. Another study evaluated ¹⁸F-FLT in early response evaluation of high-grade non-Hodgkin lymphoma in 22 patients that were treated with combined immunochemotherapy (R-CHOP) or chemotherapy (CHOP) alone showing that a rapid reduction in FLT uptake was shown 7 days after initiation of therapy. However, there was no significant change in FLT uptake following the administration of rituximab alone [6].

The way antiproliferative signals usually work in normal cells is by inducing the G₀ phase or a postmitotic state [4]. However, most cancer cells evade these signals so they can proliferate. This characteristic is known as evading growth suppressors [3].

The two most representative tumor suppressors are p53 and the retinoblastoma protein (Rb) which regulate the cell cycle. While the protein p53 operates as a central control of apoptosis when there is a DNA damage, the Rb protein decides whether a cell should go or not through its cell cycle; in other words, it inhibits the pathway through the restriction checkpoint in G₁ [3, 7]. A protein such as p53 has been thought to participate in cancer cells metabolism as a modulator, especially by facilitating oxidative phosphorylation and reducing glycolysis. Therefore, when p53 is inactive due to a mutation, a process of glycolysis is then favored. This process can be studied with ¹⁸F-FDG as this is a marker of glucose metabolism as some authors have shown a correlation between p53 and ^18F-FDG [8–10]. When any of the tumor suppressors or processes is dysregulated, then the cycle progresses and consequently the cell proliferation is ongoing.

Another important cancer hallmark is activating invasion and metastasis, responsible for the spreading of neoplastic cells from the primary site into surrounding tissues and distant organs. It is believed, though not clear, that this process involves modifications in the cells so they can be attached to other cells and to the extracellular matrix (ECM) [3]. This may include different stages: local tissue invasion, intravasation, transition via blood and lymphatics, and colonization of foreign tissue [3]. Matrix metalloproteinases (MMPs) consist of a family if zinc-dependent endopeptidases for degrading ECM constituents, playing an important role in tumor invasion and metastases. The overproduction and uncontrolled function of MMPs have been correlated to many different tumors (brain, colon, melanoma, breast, etc.) [11]. Cross talk between cancer cells and cells of neoplastic stroma introduces the idea that metastatic process requires input from the surrounding tissue, so they do not originate from a cell-autonomous model [3, 12]. Imaging MMP expression with noninvasive imaging techniques such as PET, single-photon emission tomography (SPECT), and optical imaging may offer important information to predict metastatic potential of a tumor. Some synthetic sulfonamide-based MMP inhibitors including ⁶⁴Cu-DOTA-CTT, ⁶⁴Cu-DOTA-STT, and ¹²³I-CGS 27023A, among others, have been used as radiotracers to image overexpression of MMPs in tumors [11, 13].

The formation of new blood vessels, also called angiogenesis, is an essential process of tumor growth and metastatic dissemination [14]. Generally, angiogenesis is strongly controlled by a balance between stimulatory and inhibitory signaling molecules. However, when this balance is disrupted, a rapid proliferation of new vessels can happen, for instance, when a primary tumor releases angiogenic growth factors. The tumor-associated neovasculature is a multistep mechanism including signaling input from multiple angiogenic growth factors like VEGF, known as “angiogenic switch,” and also the integrin signaling pathway [3]. Integrins are a group of cell adhesion molecules that are present on both neoplastic cells and newly formed vessels. Among the different integrin molecules already known, α_vβ₃ is the most studied in angiogenesis. Angiogenesis is therefore essential to the survival and growth of neoplastic tumors. Inducing angiogenesis eases tumor expansion mainly by delivering of oxygen and nutrients and production of growth factors for the cancer cell [14]. Angiogenesis has attracted some attention in therapy as a target for chemotherapeutic drugs. In fact, the first anti-angiogenic drug proven in humans was bevacizumab, a humanized monoclonal antibody which targets VEGF [15].

These processes can be imaged with radiolabeled tracers such as ⁶⁴Cu(DOTA)-VEGF₁₂₁ (targeting VEGF) or ¹⁸F-galato-RGD (targeting integrin α_vβ₃) for PET imaging [16, 17]; ¹²⁵I-VEGF and ¹¹¹In-perfluorocarbon for SPECT imaging [18]; NP-RGD for SPECT imaging; RDG-Gd³⁺ for magnetic resonance imaging (MRI); RGD MBs and anti-VEGFR-2-Ab-microbubbles for ultrasound (US) or RGD-QD705; and QD-VEGF(DEE) for optical imaging (OI) [19].

The perfusion of tumors and their surrounding tissues is considered an essential physiological characteristic of tumor microenvironment as it is meant to be very important for planning and monitoring treatment response. Many imaging modalities including CT, MRI, and radionuclide techniques have shown their utility in understanding perfusion not only in oncology but also in heart pathologies (myocardium viability) and neurodegenerative disorders (dementia). Several tracers have been used to study blood flow, hypoxia, and neovascularization in primary tumors to investigate tumor perfusion. For example, ¹⁵O-H₂O has turned out to be one of the most promising tracers to explain tumor perfusion in breast, kidney, colon, or lung cancer [20–22]; ⁶²Cu^IIPTSM or other more commonly used PET perfusion imaging agents such as ⁸²Rb-RbCl, ¹³N-NH₃, and ⁶⁴Ga-citrate have shown potential in brain, myocardium, and kidney, although with limitations [1]. Different CT techniques and novel MRI sequences are being used to learn more and quantify perfusion in neoplastic tissues and will be detailed in Sect. 1.2.

The programmed cell death (apoptosis) is a key process in cancer development and progression. In normal cells when an event such as an irreparable DNA damage is produced, they tend to switch on apoptosis. The ability of cancer cells to avoid apoptosis and continue with their proliferation is one of the fundamental features of cancer and is a major target of cancer therapy development [2, 3]. A signaling dysregulation in cancer cells promotes the overexpression of antiapoptotic proteins and mutes the production of proapoptotic proteins [23]. Moreover, by altering the necrosis and normal cellular autophagy, cancer cells may resist cell death [3]. A radiolabeled molecule to image apoptosis called ¹⁸F-ML10 has shown promising results in brain metastasis from non-small cell lung cancer patients treated with radiation therapy with promising results [24].

Related to apoptosis, angiogenesis, tumor invasion, and metastasis, hypoxia is another very interesting feature that has impact on tumor environment. Hypoxia is a pathological condition defined as a reduction of tissue oxygenation and may lead to an insufficient supply of oxygen and nutrients to cells. In oncology, its severity will be dependent on the phenotype of cancer. For instance, cervical cancers are known to be highly hypoxic [1]. It has been exhibited in many studies that hypoxic neoplasias have worse prognosis for disease-free survival after treatment with chemotherapy as cytotoxicity aimed to be produced with certain anticancer drugs decreases in hypoxic tissues [25, 26]. Furthermore, hypoxic cells show significantly more resistance to radiotherapy treatment so affecting the radiation sensitivity [27–30]. Several hypoxia-selective PET tracers have been used with different results, but it is worth highlighting ¹⁸F-fluoromisonidazole (¹⁸F-MISO) and ⁶⁴Cu^II-ATSM which are being tested in many studies with interesting outcomes [31, 32]. ¹⁸F-MISO has affinity for hypoxic cells with functional nitroreductase enzymes; therefore, it accumulates in activated cells but not in necrotic cells [33]. Many studies have shown excellent correlation between ¹⁸F-MISO uptake and the oxygenation status of non-small cell lung neoplasms, head and neck cancer, gliomas, and cervical cancer [34].

Another interesting radiopharmaceutical is ¹²⁴I-cG250, an iodine-based radiolabeled tracer, which has shown the potential in evaluating hypoxia in certain tumors. cG250 is an antibody reacting against carbonic anhydrase-IX, which is overexpressed in hypoxic conditions, especially in clear cell renal carcinomas, the most common and aggressive renal tumor [35, 36]. Divgi et al. studied 26 patients with renal masses who were scheduled to undergo surgical resection. They concluded that PET with 124I-cG250 could identify accurately clear cell renal carcinoma; therefore, a negative PET scan would be highly predictive of a less aggressive phenotype helping surgical planning (Fig. 1.3) [37].

Dynamic contrast-enhanced and diffusion-weighted MRI sequences are also contributing in assessing hypoxia in tumor cells and the response of chemotherapy in these neoplasias [38–41].

In addition to all those oncologic features, two new hallmarks have been recently introduced in oncology: deregulating cellular energetics and avoiding immune destruction, together with some enabling characteristics such as the genome instability and mutation generating random mutations and the tumor-promoting inflammation that favors tumor progression through various ways (Fig. 1.4) [3].

Cancer cells must make adjustments in their energy production in order to maintain the uncontrolled proliferation. Many mechanisms including using alternative metabolic pathways, adapting their glucose metabolism, and upregulating glucose transporters (GLUT) translate into an increase glucose uptake and utilization shown in many neoplasms, easily corroborated by molecular imaging, particularly by ¹⁸F-FDG-PET [3, 42]. ¹⁸F-FDG, as an analogue of glucose, is transported into cells by glucose transporter proteins and then phosphorylated by hexokinase to form FDG-6-phospate. However, it cannot be degraded via the glycolysis pathway nor dephosphorylated by glucose-6-phosphatase like glucose is. Therefore, FDG-6-phosphate gets trapped within the cell, and in this sense the more FDG there is in the cells, the greater the uptake in the tumor.

Many studies have suggested that a continuously active immune system recognizes and eliminates the majority of cancer cells before settling to form a tumor mass [3, 43, 44]. So the immune system is considered a fundamental process preventing tumor formation.

However, this is an unresolved issue because the role of the immune system is not yet clear. Hanahan and Weinberg and other authors introduced the concept of an elimination phase where the immune system recognizes and eliminates cancer cells. Then, the immune system is able to control the cancer cell growth but is not able to eliminate other cancer cells being in an equilibrium phase. Finally, tumor cells or clones not detected or destroyed by the immune system keep on growing [3, 45].

Each of the alterations that are produced in cancer cells of these hallmarks and tumor microenvironment characteristics are associated with fundamental key molecules that might be targeted with certain probes or tracers and imaged.

By definition, a biomarker is a biological molecule used as an imprint of a biological condition. It is a feature used to measure and evaluate, in an objective way, biological or pathogenic processes. Also it can be used as a treatment response marker, to detect or isolate a particular cell type or even a genetic level.

Biomarkers can be classified accordingly to the information gathered [46]:

Therefore, biomarkers can be of tremendous value, being an essential piece of predictive and preventive medicine mainly in cardiology, neurology, and oncology, not only by adding understanding to these pathological processes but also by offering personalized therapy targeting cancer hallmarks [3].

Depending on what we want to image and/or treat, we have to acknowledge the target. Based on the hallmarks of cancer previously commented, many biomarkers have been studied including VEGF or integrin vectors for evaluating angiogenesis, radiolabeled glucose for the abnormal cellular energetics, thymidine analogues for proliferative signaling, receptor- [47] or enzyme-based [48] imaging, antibodies, selective anti-inflammatory drugs, telomerase inhibitors for the replicative immortality of cancer cells, and apoptotic markers, among endless others.

All of the many signaling targets have been made possible thanks to a numerous researchers by implementing translational studies from laboratory, preclinical, clinical, or population cohorts into clinical applications after an extensive testing in experimental models [49].

Imaging biomarkers, or molecular imaging, have many advantages including being considered a noninvasive method; it can be quantified [50, 51], is reliable, and offers reproducible data allowing multiparametric imaging. It represents a group of methods with specific probes, contrast agents, or MR pulse sequences [49] that image particular targets or pathways. All this infinitive information can be very useful to clinicians in the daily practice.

Besides cancer-related biomarkers, one of the fields where molecular imaging has an interesting impact is in cardiac studies. Vulnerable atherosclerotic plaque can be studied with coronary angiography by looking at the coronary lumen [52] requiring catheterization or at the coronary arterial wall with intravascular ultrasound (IVUS) [53, 54], though in these cases, it is considered to be an invasive imaging method. Other intravascular imaging techniques such as coronary computed tomography and cardiac magnetic resonance have been used for coronary and carotid lumen and wall [55] imaging. Optical coherence tomography (OI) [56] and near-infrared spectroscopy used for tissue spectral contrast [57] are being investigated.

In neurology, MRI and CT have been used to study brain infarction size and extent, lesion size, brain activity in multiple sclerosis and structural atrophy, and β-amyloid plaques with PET [58–60].

Section 1.2 of this chapter will briefly go through some of the molecular imaging techniques and its biomarkers and the unthinkable possibilities that imaging may offer in cancer biology.

Molecular imaging is a relatively “new” area that integrates molecular biology and state-of-the-art medical imaging, arisen in the last 15–20 years as a nexus between molecular biology and in vivo imaging. As already stated, it is defined as a noninvasive technique able to visualize the natural microenvironment in real time, measure pharmacokinetics and pharmacodynamics of pharmaceuticals, and measure physiological or pathological inside living subjects at a cellular, molecular, or genetic level, among others [61–64].

Compared with conventional imaging techniques, molecular imaging has many advantages including 3D images of living organism thus enabling research into physiological or pathological situations such as signal transduction pathways, cell metabolism, and gene expression.

MI is a multidisciplinary branch of knowledge including molecular biology, genetics, biochemistry, physiology, physics, mathematics, pharmacology, immunology, and medicine [64]. Five imaging modalities are available for molecular imaging: CT, MRI, optical imaging, US, and radionuclide imaging which includes PET and SPECT.

Challenging problems remain to be solved such as the developing of new probes, translational research to the clinical arena, and multidisciplinary approach.

Each of the different imaging techniques has their different strengths and weaknesses that will be explained in the following section.