Preclinical Animal Model and Non-invasive Imaging in Apoptosis

Abstract

This chapter deals with preclinical studies involving animal model systems and non-invasive methodologies toward understanding the role of proteases in apoptotic pathways and their potential as therapeutic targets. Several critical diseases are associated with imbalance in the apoptotic machinery including neurodegeneration and cancer, and hence an effective strategy to target these molecules might be a tractable solution for combating these ailments. Proapoptotic proteases and their binding partners therefore have always been of special interest for designing and evaluating the efficacy of several drug-like molecules (both activators and inhibitors). This chapter is focused on discussing the development of few such molecules with specific examples. It also vividly describes the different in vivo model systems that are essential for rigorous screening of these molecules at different stages of drug development. Current role and future prospects of preclinical translational imaging platforms (PET, SPECT, CT and MRI) and their utility in clinical trials are also outlined in this chapter.

1 Introduction

Basic research has played a significant role in delineating the pathophysiological processes underlying various diseases as well as in determining potential leads for translational applications. The understanding of intricate pathways provides clue towards identifying the key players or potential molecular targets for therapeutic intervention. However, it takes a series of rigorous steps for a molecule of interest to emerge as a drug. This long process initiates with identification of a macromolecule, followed by designing small molecules for targeting the macromolecule with an aim at modulating its activity with desired characteristics. A multidisciplinary approach including in silico, biochemical, pharmacological, ex vivo, in vivo imaging and animal model studies are required for target identification i.e. finding potential ‘leads’ from innumerable ‘hits’. These ‘leads’ are further taken forward through several steps of preclinical trials prior to successful clinical development (Fig. 6.1).

Fig. 6.1

Schematic representation of steps towards drug discovery with an average time line

The strategies to cure and control disease progression due to impaired apoptosis led to targeting of proteases involved in this pathway. Less effective conventional methods that include activating proapoptotic signals by administration of cytotoxic and chemotherapeutic agents to induce cell damage are making way for more targeted therapy. Specific targeting of diseased cells can be achieved in multiple ways such as through activating or inhibiting proapoptotic proteins or designing antagonists of antiapoptotic molecules. Several such molecules (antisense oligonucleotides, peptidomimetics and synthetic compounds) are being currently explored as drug targets. The previous chapter vividly describes different laboratory protocols with relevant examples to understand and characterize the role of proapoptotic proteases in cell death. Here, we continue this effort with a step further toward discovering their potential in treatment of different diseases such as cancer and neurodegenerative disorders using in vivo animal model system and non-invasive preclinical imaging.

Although in vitro and ex vivo studies, for example, experiments with tumor cell lines, are essential in lead identification and optimization, they are not sufficient to mimic the complex disease environment in human systems [1, 2]. Moreover, this system fails to answer questions related to drug distribution, drug uptake, and pharmacokinetics and so on, which are essential steps for clinical development. To fill in this huge gap between fundamental studies and clinical applications, preclinical in vivo model systems have played a pivotal role over the last few decades. Different animal model systems have been used for understanding the complexities of a disease, its progression and effects of drugs in a three-dimensional scaffold. Both nude and SCID (severe combined immunodeficiency) mouse xenograft model systems are routinely used to study initiation and progression of different diseases. Advancement in the field of genetic engineering has also led to the use of transgenic mouse models in elucidating pathophysiological conditions [3–5]. A major shift in the focus of drug discovery in the modern era has identified various limitations that are associated with murine models, which consequently led way to exploring alternative models such as zebrafish and canines with spontaneous cancer [6–8]. Thus, a requirement for establishing a perfect model system becomes imperative where molecular characterization of the disease along with effect of the candidate drugs and drug-like molecules can be tested effectively. However, in this chapter, the most widely used mouse model systems have been described in context of apoptotic molecules and their associated diseases.

Non-invasive imaging in small animal model system is extremely useful toward elucidation of molecular events in disease progression, recurrent disease management and monitoring of response to drugs [9]. With increasing interest in harnessing apoptotic molecules for understanding and treatment of various diseases, an upsurge in advancement of imaging techniques has been observed in the past decade to monitor cell death. Various fluorescent and radiolabeled agents for PET/SPECT imaging have been specifically designed to study activation of proteases (mainly caspases) for direct visualization and quantitative evaluation of apoptosis in vivo. Development of several studies in animal model systems combined with tremendous advancement in the field of small animal imaging therefore showcase very high potential to translate basic research into clinical medicine.

2 Animal Model

In vitro and ex vivo studies have provided a wealth of information in biomedical research with critical leads in drug designing and development [10, 11] in the last few decades. However, they are limited by their inability to simulate the cellular milieu during a physiological process. This led to extensive research toward development of an appropriate animal model system so as to understand the intricacies of a complex cellular mechanism or pathophysiology of a disease.

Studies on animals led to significant advancement in translational research. Understanding of a human disease at the molecular level has been made possible either by studying spontaneous diseases in animal counterparts or by inducing different factors in normal animals for development of a disease of interest [12]. A varied array of species ranging from bacteria, protozoa, insects, zebra fish, guinea pigs, rabbits, felines, dogs and non-human primates [6, 7, 13–17] have been used in studying molecular mechanisms of different human ailments. Mouse has so far been the most popular and widely used model system for in vivo studies of human diseases [12].

2.1 Mouse as a Laboratory Model System

Quite intriguingly, mouse was used as a tool to study human anatomy way back in the second century AD by Galen, a Greek physician [18]. After a long recess, mouse regained its prominence as a popular animal model in experiments ranging from properties of air by Robert Hooke in the mid seventeenth century, to studies on inheritance in early 1900s by William Castle [19]. Owing to several advantages such as small size, easy and inexpensive maintenance, abundant breeding, optimal gestation period and most importantly ability to be genetically manipulated, mouse has become the most popular (∼70 %) laboratory animal with nearly 450 inbred varieties for studying human physiology, cellular pathways and effects of drugs on diseases [20, 21]. Here we will look into various applications of mouse model system in understanding the role of proapoptotic proteases in modulating neurodegenerative diseases and cancer.

Although the laboratory mouse bears several similarities with domestic mouse, artificial breeding has led to marked differences in certain characteristics that are required for study of specific diseases or macromolecules in cellular pathways. Currently, there are different types of genetic modifications available in mouse models. Therefore, strategically devising proper sets of experiments is the key toward understanding the regulatory steps in a cellular process or pathophysiology of a disease. The different types of laboratory mice that have been bred, maintained and used for biomedical research are discussed as follows:

2.1.1 Nude Mice

The pioneer in breeding nude mice was Miroslav Holub (1923–1998), an eminent Czech immunologist [22]. The basis for generating nude mice is to study tumor biology and obtain xenografts. They are made immunodeficient by genetic removal of FOXN1 gene [23] leading to absence of thymus. This results in hairless phenotype from which they derive their name. These mice do not produce T lymphocytes, therefore do not exhibit major types of immune responses that include

• (a) Generation of antibody due to lack of CD4⁺ helper T cells

• (b) Cell-mediated immune responses due to absence of one or both CD4⁺ and/or CD8⁺ T cells

• (c) Rejection of grafts that require both CD4⁺ and CD8⁺ T cells

• (d) Elimination of virus-infected or cells exhibiting malignancy due to lack of CD8⁺ cytotoxic T cells

Overall, the nude mice do not exhibit immune response toward foreign tissues or tumor grafts and have been a popular tool to be used in imaging and transplantation.

Nude mice served a popular experimental model for study of genesis, progression and treatment of cancer in the past few decades. However, precise experimental control is required for obtaining reproducible data. Several parameters need to be taken into careful consideration including tumor type, origin, kind of mouse host and its age for propagation of different human malignancies. Studies in nude mice have provided interesting findings regarding metastatic processes as well. Attempts have also been made to study spontaneous tumors in these animals but their frequency of occurrence compared to the thymus-bearing counterparts remains questionable. Nevertheless, for over 25 years, the nude mice system has provided a wealth of information on tumor biology and progression of cancer and other diseases [24]. The nude mice, although widely used, sometimes produce leaky phenotypes with presence of a few residual T lymphocytes. As a consequence, they are losing their prominence in translational research and making way for SCID mice.

2.1.2 Severe Combined Immunodeficient (SCID) Mice

Scid mice are homozygous for an autosomal recessive mutation (scid) on chromosome 16 and exhibit an apparent detention in the initial developmental stages of B and T cells. The pro-B cells that are formed in the bone marrow, do not mature into pre-B and B cells. Similarly, although early T-lineage cells (Thy-l + IL-2R +) are found in thymus, absence of functional CD3⁺ T cells are not observed. These conditions arise due to loss of function of PRKDC gene [25] which leads to impairment in resolving DNA strand breaks in developing T and B lymphocytes by the DNA repair enzyme (DNA-dependent protein kinase catalytic subunit) that the gene encodes [26]. All other nonlymphoid blood cells exhibit normal differentiation. SCID mice are being routinely used for transplantation and xenografting tumors as well as normal and malignant tissues. In addition, they are also used to test vaccines [27, 28].

The major application of SCID mice lies in engraftment of xenogeneic tumors. Although the quality of xenografts are similar to that of nude mice, in some conditions such as retinoblastoma, acute lymphoblastic leukemia, lung metastasis etc [29, 30], SCID show improved engraftment. Thus for several human tumors SCID mouse has established itself as a model with high efficacy to study initiation, progression metastasis and effect of drugs. However, as observed in case of nude mice, SCID unfortunately is also not completely devoid of leaky phenotypes as 15–25 % of young adult mice and almost all old mice develop a few clones of B and T cells.

A variant of SCID called NSG, or NOD scid gamma (NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl/SzJ), where the genetic background is derived from inbred NOD (non obese diabetic) mouse strain, is among the most immunodeficient inbred mice varieties described till date [31]. First developed in the Jackson laboratory [32], these mice do not contain mature T, B and natural killer (NK) cells. NSGs have impaired cytokine signaling pathways as well as defective innate immune system [33]. These multiple immunodeficiencies allow them to engraft a variety of primary human cells as well as to mimic complex cellular networks and diseases.

2.1.3 Transgenic Mice

The idea of introducing a foreign gene material into mouse germ line revolutionized the field of genetic engineering and translational research. This discovery has given a new dimension to the study of specific genes as well as human diseases in a living system. Apoptosis research as well as preclinical studies targeting diseases such as cancer and neurodegeneration have benefited tremendously with the advent of this technology [34, 35]. This advancement came hand-in-hand with emergence of technologies such as gene cloning, chromosomal mapping and DNA sequencing [36].

Transgenic mice can be generated mainly via three possible ways:

• (a) Delivery of foreign DNA by retroviral infection of mouse embryos at various developmental stages. However, due to technical complications, this technique has not become very popular [36].

• (b) The second method is very commonly used where foreign genetic material is injected into the pronuclei of fertilized one-cell mouse embryos leading to random integration of multiple copies of the transgene. This is followed by transfer of the embryos in oviducts of foster mothers that eventually produce the transgene [37, 38].

• (c) Finally, another way of procuring transgenic mice is through introduction of ‘loss or gain of function’ mutations of varied sizes at loci of interest in mouse embryonic stem cell (ES) [37, 38].

Detailed protocol for developing transgenic mice is provided at the end of this section.

2.1.4 Knockout Mice

These ‘designer mice’ have become an integral part of preclinical and clinical research where a particular gene of interest is made non-functional by knocking it out. This simple method aids in understanding the importance of a gene in an intricate cellular process and complexities of a disease. This is achieved using the homologous recombination technology [39] where mutations are introduced in pluripotent mouse stem cell lines (ES) that subsequently get transmitted to the progeny [40–42]. Knockout technology has been tremendously used in the last few decades for understanding apoptotic pathway and the diseases it is associated with [43]. Oncomice is such a variant of transgenic mice that has been genetically designed to develop cancer by expressing prominent oncogenes, way back in the 1980s. Oncomice has been phenomenal in providing in-depth understanding of different stages of cancer as well as to devise strategies for therapeutic intervention [44].

3 Proteases in Preclinical and Clinical Research

Proteases are tightly regulated during tissue development to maintain homeostasis and proper cellular functions. However, genetic aberrations, exo- as well as endogenous factors, abnormal (both less and excessive) proteolytic activity that occurs in the cellular milieu, often lead to several diseases including cancer, inflammation, neurodegeneration as well as microbial infections [45]. Modulating activity of specific proteases so as to control or eliminate a disease condition has been one of the major foci of pharmaceutical research [46]. History of drug design involving proteases goes way back to the mid twentieth century when two drugs against thrombosis (heparin and warfarin-a vitamin K analog) became available that indirectly regulate thrombin activity. Two major breakthroughs in targeting proteases is development of ACE (peptidyl dipeptidase A) inhibitors against cardiovascular diseases [47] and HIV protease inhibitors [10]. Although, designing a protease modulator (mostly inhibitors) seems straightforward, it requires an in-depth understanding of the structure, dynamics and activation mechanism of the protease. Inhibitors can either target the protease directly, allosterically or indirectly through inactivation of downstream protease molecules [48–52]. Moreover, absence of any direct correlation between expression levels and altered activity of an enzyme adds to the complexity. Recently, tremendous efforts are being made to circumvent these barriers through an interdisciplinary holistic approach including in silico drug design, structural probes, proteomics and other high throughput approaches that act as an excellent support system for in vivo studies. This technical advancement has led to a huge upsurge in the overall clinical and pharmaceutical research involving proteases with more than 50 proteases being characterized as potential targets. A majority of these targets were identified in knockout studies in mouse model systems [47, 53–59] reiterating the indispensability of this modality in preclinical and clinical research.

3.1 Different Strategies to Modulate Protease Activities

Targeting a protease though challenging can be achieved through multiple ways. Based on the requirement for a particular disease, an inhibitor or an activator may be designed. Moreover, allosteric regulators also play important roles in modulating a protease activity. Inhibitors in turn, can be reversible or irreversible. Studies have shown that reversible non-covalent inhibitors have better effect in the intracellular milieu compared to their irreversible counterparts as the former provide more specificity and lesser side effects. For designing reversible, non covalent inhibitors, transition state analogs are used as templates [60, 61]. On the other hand, allosteric modulators are considered better than the orthosteric ones due to greater sub-site specificity and lower-dose requirement. In a unique approach called ‘tethering’, a cysteine residue in the vicinity of the active site of caspases-3 and -7 was reversibly attached via a disulfide bond to a thiol group of the potential small molecule inhibitor(s) [62, 63]. This led to allosteric inhibition of the executioner caspases and freezing them in a proenzyme conformation. Similarly, enhancing the activity of a protease is possible indirectly through inhibiting its inhibitor. One such brilliant example is SMAC mimetics or small molecule inhibitors of IAP proteins, which in turn release inhibition on caspases thus facilitating apoptosis [34, 48]. These molecules have shown cancer specific apoptosis both in mammalian cell culture as well as in xenograft tumor model system in mice and are currently in clinical trials.

3.2 Mouse Models Involving Proteases of the Apoptotic Pathway

The proteases in the apoptotic pathway have been extensively explored in biomedical research. A few successful stories emanating from these efforts are substrates of caspases and granzyme B [64, 65]. In addition, SMAC mimetics and inhibitors of cathepsin K are under clinical trials. While SMAC analogs are devised to combat cancer, few inhibitors of cathepsin K are currently being tested against osteoporosis, osteoarthritis and bone metastasis [66]. Understanding the intricacies of a cellular pathway as well as effects of drugs or drug-like molecules on a pathological condition requires a living model system. As mentioned earlier, knockout studies have been an extremely important tool for understanding the role of proteases in a particular disease and devising strategies for therapeutic intervention. A slightly modified model system where an animal disease model is combined with a rodent knockout model has led to specifically understand the role of the target protease, for example, cathepsins in pancreatic cancer [67]. Knockouts have also been used as a model system to understand role of proteases in neurodegeneration [35]. Apart from these, tumor xenograft models in rodents, mainly mice have given interesting insights into initiation, progression and metastasis of cancer.

Here we would discuss general strategies involving in vivo tumor xenograft and knockout model systems for targeting proapoptotic proteases or their interacting partners in diseases such as cancer or neurodegeneration using two specific examples.

• Case I: Designing mimetics of a proapoptotic molecule to relieve inhibitory effect of its antiapoptotic binding partner on caspases:

An enormous amount of research endeavors to understand the protein-protein interactions involving apoptotic proteases resulted in a few molecules to enter clinical trials. One such molecule is a mimetic of proapoptotic SMAC/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low pI) that relieves inhibition of inhibitor of apoptosis proteins (IAPs) on upstream caspases [68, 69]. It has been observed that IAPs have been associated with different cancers such as breast, prostrate, lung, renal and bladder carcinomas [70–73]. Moreover, this antiapoptotic family of proteins including XIAP and cIAPs facilitate metastatic progression in several cancers [74, 75]. At the cellular level, antiapoptotic property of IAPs is manifested through their ability to interact with upstream caspases which make them potential therapeutic targets against cancer. SMAC/DIABLO is a mitochondrial protein that gets released into the cytosol upon apoptotic trigger and relieves the inhibition of IAPs on caspases by interacting with IAPs through their N-terminal tetrapeptide (AVPI) motif. While the BIR3 (Baculovirus Inhibitor of apoptosis protein Repeat domain 3) of IAPs are known to bind caspase-9, BIR2 domain inhibits caspase-3 and -7 [76]. Therefore, agonists of SMACs (peptidomimetics and small molecule analogs) can be tested as therapeutic targets against several types of cancers where over-expression of IAPs has been observed. It has been proven that SMAC analogs induce apoptosis in tumor cells and reduce tumor growth in mice [77–82].

Overall Strategy

The mimetics of ‘AVPI’ have been developed using structure guided design either by, ‘shape-based screening’ of a virtual drug library or by designing small molecule analogs of ‘AVPI’ by molecular docking using SMAC-XIAP complex structure as a template (PDB IDs: 1G73 and 1TW6) [83, 84]. A few best molecules were selected based upon binding score and were further tested for their drug-like properties. A 100 total ‘leads’ were shortlisted which were combined to get a final 71 ‘hits’ to be tested in ex vivo model systems. In the other work, structure-guided chemical synthesis of a series of small molecule analogs was performed directly. In both the cases, a battery of ex vivo studies were performed such as cytotoxicity, caspase activation, cell phase distribution and IAP inhibition assays to screen these molecules for in vivo testing of their efficacy. Xenografts were generated in nude mice for studying the effect of the synthesized SMAC analog on prostrate and breast tumor xenograft models. The mice were subjected to treatment intraperitoneally with different doses of the ‘hit’ compound. The animals were sacrificed post experiment, and the tumors were removed, a series of experiments that followed provided vital information on the ability of the compounds to reduce the weight and volume of the tumors. Elaborate preclinical pharmacokinetic studies and ADME (absorption, distribution, metabolism, and excretion) analyses were performed in various animal models to identify the best molecule to take forward toward clinical trials.

• Case II: Knock out of a caspase gene: Implications in neurodegenerative diseases [ 35 ]

Caspases are the most prominent proteases involved in programmed cell death and therefore their deregulation leads to several diseases that include neurodegeneration. Caspase 6, like any other protease of the family exists as a proenzyme which gets processed into active dimeric protease by upstream caspases -7, -8 and -10 post apoptotic signal. In addition, self-activation in caspase 6 has also been observed [85]. Although predominantly an executioner protease, caspase 6 has also been found as an initiator of the caspase cascade. This statement is supported by the observation that caspase 6 is present in brains of Huntington and Alzheimer patients long before apparent cell death and in turn has the ability to activate caspase-2, -3 and -8 [86–89]. In addition, inhibition of β-amyloid precursor protein at caspase 6 recognition site (residue 664), rescues Alzheimer-like phenotype [90–97]. Caspase 6 activation is also associated with axonal degeneration in an Alzheimer’s Disease (AD) mouse model [98]. Similarly, cleavage of a mutant Huntington (mhtt) at a particular residue (AA 586) leads to pathogenesis of Huntington’s disease (HD). It has also been reported in the literature that prevention of this cleavage in mice prevents neurotoxicity and aberrant neuronal behaviour [99–103]. These observations strongly implicate caspase 6 in progression of neurological diseases and therefore designing caspase 6 inhibitors could be a successful strategy to combat those ailments. Since understanding the role of caspase 6 in normal brain development is the first step in this process, this study performs behavioural and neuropathological characterization of mouse brain lacking caspase 6 (caspase 6-/-) [35].

The first step in this process was to precisely design, develop and validate a caspase 6-/- mouse model system (refer to step-by-step protocol below). These knockout mice were then used for several experiments pertaining to caspase 6 mediated axonal degeneration and excitotoxicity, determination of Mendelian ratio and size of the cerebellar cortex, so as to understand the effect of caspase 6 on normal functions of the brain, determination of body weight as well as motor coordination. All these studies in caspase (6-/-) knockouts confirm the role of caspase 6 activation primarily in AD and/or HD. This suggests designing caspase 6 inhibitors would be a promising therapeutic strategy against Alzheimer’s and other neurodegenerative diseases.

4 General Protocol for Generation of Tumor Xenograft and Knockout Mice

4.1 Tumor Xenograft in Nude Mice: A Step-by-Step Protocol

A flowchart providing a pictorial representation of xenograft generation is shown in Fig. 6.2a.

Fig. 6.2

Flowcharts illustrating generation of tumor xenograft and knockout mice. (a) Generation of knock-out mice. The steps (designated as a, b, c etc.) in the figure are provided in detail in the text. (b) Generation of xenograft bearing mice. Step 1 in the figure represents the following (from left to right): cryo-preserved tumor samples, subcutaneous transplantation of the tumor in donor mouse with a trocar and follow the growth of the tumor to an optimum size. Step 2 (from left to right): Select the tumor bearing mouse and remove the tumor with a scalpel blade, transfer tumor mass in a medium with suitable antibiotics, cut the tumor into several small pieces, place a small piece under the skin of recipient mouse using forceps and scissors, growth of tumor in recipient mouse (tumor is encircled in red)

• Step 1. Preparation of donor tumor-bearing mouse:

  • (a) Thaw the cryo-preserved tumor sample and transplant it subcutaneously using sterile trocar with proper aseptic care

  • (b) Observe the tumor growth daily

  • (c) As the tumor diameter reaches ∼1.5 cm, the tumor can be used for transplantation in other mice. This tumor bearing mouse is known as donor mouse

• Step 2. Tumor transplantation:

  Precautions: The person carrying out tumor transplantation must wear sterile protective gears i.e. long surgical lab-coat, cap, mask, shoe covers, and surgical latex gloves. This procedure should be conducted in laminar hood. The ultraviolet lamp in laminar hood should be turned on 15–30 min prior to initiation of the procedure.

  • (a) Select the donor tumor-bearing mouse

  • (b) Sacrifice the donor tumor-bearing mouse by cervical dislocation and dip it in a jar containing absolute alcohol for approximately 3 min

  • (c) Remove the mouse from jar and wipe the excess alcohol using a paper towel

  • (d) Pull the skin over the tumor and detach it from the tumor with the help of scalpel blade, if required

  • (e) Remove the tumor mass completely and put it into suitable medium with broad spectrum antibiotics (streptomycin and penicillin) in a petri-plate

  • (f) Transfer the donor mouse into the discard-bag and dispose it following proper procedure of biological waste disposal

  • (g) Cut the tumor into small pieces of approximately 2 cubic mm with the help of sterile forceps and scalpel blade. Make as many pieces as number of mice needed for the study along with an additional piece for histological examination of the tumor

  • (h) Anaesthetize the recipient mice using isoflurane (2-chloro-2-(difluoromethoxy)-1, 1, 1-trifluoro-ethane) gaseous anesthesia

  • (i) Pick up one mouse and place it on a sterile paper towel in a position so that its dorsal side faces the sky

  • (j) Dip a small piece of cotton gauze in absolute alcohol (ethanol) and wipe the skin on the posterior side of the back

  • (k) Lift this skin with blunt bent sterile forceps and make a small incision (approximately 3 mm) with 1.5 cm blunt-end scissors. Insert the scissors under the skin through this incision towards the right hind limb area. Gently open the scissors to widen the skin pocket and then quickly bring it back to closed position

  • (l) Remove the scissors from the skin pocket still holding the skin at the incision using forceps with left hand. With the right hand, pick up one piece of tumor from the petri-plate using blunt bent forceps and insert it deep into the skin pocket that has already been made. After releasing the tumor into the pocket, remove the forceps and roll it over the pocket skin in a sweeping movement so as to push the tumor piece to the interior of the pocket

  • (m) Release the skin at the incision and put 1 drop of 100× antibiotic cocktail on the incision followed by one or two drops of tissue adhesive

  • (n) Transfer the mouse to the recovery cage. Maximum recovery-time required with isoflurane is 2–3 min

  • (o) Repeat the procedure from step 11–14 for transplant in the next mouse. This procedure takes 3–5 min of time

  Carry out tumor transplants in animals required for one study in single sitting, using the tumor from the same donor mouse.

4.2 Protocol for Generating Knockout Mouse

An overview of the experimental procedure is shown in Fig. 6.2b

• Step 1. Acquiring DNA of Interest:

  • (a) Screen genomic library that is generated preferably from 129Sv-derived embryonic stem (ES) cell lines. The advantage of 129Sv over ES cell lines derived from other strains is that they are more reliable at forming germ cell line colonies [104]. The 129Sv cells that dramatically changed the field of transgenic mouse technology was derived from a cross breed of 129 substrain and another inbred strain [105]. Alternatively, use the conventional method of PCR amplification of the gene of interest from genomic databases

  • (b) Insert the PCR amplified region of genomic DNA that encompasses the DNA of interest into a bacterial plasmid with compatible restriction sites. This DNA of interest should also have a selectable drug resistance marker

  • (c) Linearize the DNA using suitable restriction enzymes

• Step 2. Development of ES cells:

  • (a) Insert the DNA of interest into embryonic stem cells via electroporation.

  • (b) This piece of DNA replaces a part of the normal gene in the mouse through homologous recombination.

  • (c) Grow the cells in suitable medium with proper antibiotics. Cells that uptake the plasmid with the antibiotic resistant insert will grow and the rest will die. Hand-pick ∼500–600 colonies and expand them in plates

  • (d) Perform DNA extraction (using standard molecular biology protocols) from individual colonies Perform Southern Blot experiment or PCR to identify the colonies with integrated DNA at the proper loci

These clones will be further used to generate the knockout mouse.

• Step 3. Generation of Knockout mouse:

  • (a) Grow the ES cells harboring the gene of interest in a suitable culture medium

  • (b) Inject ES cells into mouse blastocysts (having brown and black phenotypes due to different strains of donor and recipient mouse respectively) so that it becomes an integral part of the embryonic tissue. Transfer these blastocysts into the uterus of pseudo-pregnant recipient female mouse

  • (c) Pups that are born with integrated ES cells are chimeras with black and brown stripes owing to the respective coat colors of the donor and recipient. This will help in selecting the chimeras easily

  • (d) Breed female chimeras with black male for approximately 8 weeks

  • (e) Select the brown offspring’s (suggests modified ES cells have contributed to the germ line)

  • (f) Perform genetic tests to identify mice with knockout gene. Breed desired colonies and initiate phenotypic analysis

5 Current Scenario and Future Perspectives

The ever expanding biomedical literature and successes in preclinical and clinical trials underscore the importance of animal models in basic as well as translational research. Techniques to utilize this unique research tool has been extensively modified and fine-tuned to cater to existing as well as novel research ideas. However, although mouse models have been successful in aiding the basic biomedical research, it requires more fidelity and reproducibility for using it in preclinical settings. For example, a rigorous screening procedure should precede preclinical studies with any mouse model. The mouse model should undergo a quality control check to ensure its ability to mimic genetics of a human system or pathophysiology of a disease. Secondly, the histopathology of the model should precisely match that of human tumors and it needs to be evaluated by a scientist with expertise both in human and animal pathology. Another interesting way of establishing credibility of mouse models is through back-validation i.e. studying the effect of drugs in mice whose effects have already been established in humans. This global and transparent approach would definitely lead toward development of better mouse models that would efficiently simulate human physiology as well as diseased conditions.

Although transgenic rodent models have proved their mettle in biomedical and cancer research, another less explored yet excellent alternative is use of spontaneous tumor models that provide perfect environment to test novel therapeutics. Some of these promising immune competent and syngeneic models are genetically engineered mice (GEM) and companion (pet) animals that naturally develop cancers [7]. An appropriate use of both xenograft and spontaneous models, taking into consideration their pros and cons, might help understand complex physiological processes as well as combat different diseases with higher precision and efficacy.

6 Noninvasive Apoptosis Imaging

6.1 Introduction

In vivo noninvasive imaging being a potential tool for visualizing and understanding pathophysiological processes, plays an important role in development of novel diagnostic and therapeutic molecules. Apoptosis is an evolutionary conserved and tightly regulated biological phenomenon that is crucial for maintenance of cellular homeostasis [106]. It is a fundamental process observed in normal as well as in cells exposed to cytotoxic agents used in anticancer therapy regime. Apoptosis induced by anticancer drug is often seen as an indicator of therapy response. Hence in vivo apoptosis imaging is an active area of research since last decade, and efforts are being directed towards making use of it as a prognostic marker [107]. Since, apoptotic switch is highly regulated by a subset of proteases that are activated by exogenous and endogenous factors, these proteins have become an integral part of apoptosis imaging.

6.2 Imaging Techniques

The field of diagnostic imaging includes a variety of non-invasive modalities that are pivotal for visualization and assessment of molecular events in several diseases such as neoplastic and neurodegenerative processes. Imaging modalities including x-ray computed tomography (CT), positron emission tomography (PET), single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI) have routine application in imaging laboratory animals as well as in clinics [108–111]. However modalities such as optical (bioluminescence and fluorescence) and cerenkov imaging are still at preclinical stage and gradually leaping towards clinical applications [112, 113]. Most of these tools are complementary to each other and help visualization and precise measurement of structural changes, biochemical, physiological and molecular processes. Currently dual or hybrid imaging (HDI) is preferred over single imaging modality as it provides an advantage to visualize molecular/biochemical/functional events along with structural information. However, multi-modality requires precise image co-registration algorithm besides complementarity of the technologies. The two most commonly used imaging combinations or ‘fusion imaging’ systems are PET-CT and SPECT-CT where molecular/functional/biochemical data can be co-registered on anatomical platform and read together to enhance our understanding of the underline molecular processes in both preclinical and clinical settings [114].

The above mentioned imaging techniques provide a large amount of information without using any invasive tool. Moreover, these procedures do not alter the disease process or cause unacceptable discomfort to the animal. Although these procedures are painless, sedation is often desirable to reduce associated anxiety and stress so as to enable acquisition of good diagnostic data with minimal repeats. The use of anesthetic agents also controls stress or pain associated with handling of tumor bearing animals or other disease models. However, the most challenging aspect of these preclinical systems is to achieve sub millimeter spatial resolution, which is plausible with the use of high-end detector materials, efficient signal transmission electronics, appropriate data acquisition and processing algorithms.

In vivo preclinical imaging has high impact in drug development as it can precisely monitor disease progression and therapeutic response longitudinally in the same subject. In vivo experiments are typical longitudinal studies where each individual animal serves as its own control thus enhancing the reproducibility and accuracy of the data. PET Tracers (e.g. Fluorine-18 based), SPECT tracers (e.g. ^99mTechnetium and ¹²⁵Iodine), contrast agents, bioluminescent markers/reporters (luciferins, proluciferins etc.) are the most often used reagents in these imaging studies [115–118].

The selection of an imaging technique mainly depends upon the disease to be monitored and its pathophysiological impact. Translation from preclinical evaluation to clinical studies has been highly facilitated by these present generation imaging modalities with the imaging protocols being easily applicable in clinical setup. They are routinely used in characterizing newer and more realistic models of human diseases such as invasive disease in the tissue of origin as well as transgenic mouse models. Another important reason for present interest in preclinical imaging is making the process of drug discovery and development more dependable. The process of drug discovery is both time consuming and resource intensive mostly with uncertain outcomes. Therefore to reduce the failure rate of drug-like molecules in the later stage of clinical trials, incorporation of advanced translational imaging technologies in preclinical studies is essential. These cutting-edge translational imaging platforms help predict and understand the importance and limitations of the animal model system in a particular clinical evaluation [119]. The different imaging modalities are described below in detail.

6.2.1 Positron Emission Tomography

Small animal PET imaging has become an indispensable preclinical tool in the last decade due to advanced technological development in instrumentation and detector technology. It has tremendous potential to translate basic understanding derived from animal imaging to clinical medicine. PET is being used increasingly to advance the understanding of cellular and molecular processes that are altered in cancer initiation and progression [109]. Compared with other molecular imaging technologies, PET enables higher sensitive and quantitative measurements of biological and biochemical processes in vivo through specific labeling of organic compounds (or close analogs) with positron emitters, such as ¹⁸F [120, 121]. Human PET scanner has revolutionized biomedical research since its introduction in mid-1970s. Simultaneously, there was a strong interest in developing preclinical scanners due to inability of the human system to image a mouse, which is ∼2000 times lighter. Over a period of time, scanners for imaging animals have been developed from analog to today’s completely digital systems with advanced solid state detector materials [122].

Instrumentation

Advanced microPET scanner has one-to-one coupled Phoswich Avalanche Photodiode (APD) detector. This detector is optically coupled with a pair of scintillation crystals that are made up of materials with nanosecond decay time. This configuration provides high spatial resolution and advanced coupling of scintillator to APD enhances energy resolution. This combination of scintillation detectors efficiently attenuates high energy (511 keV) gamma ray photons. However, the short half-life of PET radioisotope is a challenging factor in terms of synthesis of molecules and further execution of the animal imaging studies. Hence availability of a medical cyclotron onsite is highly desirable. Among all PET radionuclides, ¹⁸F has sufficiently long half-life, hence there is a huge interest in developing ¹⁸F-based molecules [121]. There are also several long-lived radioisotopes such as ⁶⁴Cu and ¹²⁴I having half-life of 12.7 h and 4.2 days respectively [123]. These isotopes can be shipped from production site to various preclinical labs. Usefulness of these radionuclides is in metabolic/accumulation/clearance studies where tracer accumulation over a longer period of time is of interest. Alternatively, there are also generator systems consisting of a long-lived parent radionuclide (⁶²Zn/⁶²Cu and ⁶⁸Ge/⁶⁸Ga generator) that are supplied directly to the preclinical labs as per the research requirements. The parent radioisotope continuously decays into a short-lived daughter radionuclide (⁶²Cu, T_1/2 = 9.7 min; ⁶⁸Ga, T_1/2 = 68 min) [124]. The use of long-lived radiotracers or availability of generator systems facilitates the preclinical imaging without having an on-site cyclotron. Typical injected tracer dosages for mice are in the range of 50- to 350-μCi. The limiting factor in selection of dose in human studies is a radiation dose, but in case of animals it is count rate capability of the scanner, specific activity and volume of injected dose [125].

6.2.2 Single Photon Computed Tomography (SPECT)

There has been a huge advancement and application of micro-SPECT systems in preclinical research in the last decade. Most important advantage of SPECT over PET is easy availability of ^99mTc (Technetium), which is the most common SPECT radioisotope. The half-life logistics and labeling versatility of ^99mTc adds to the utility of SPECT technology in research setup. Besides, multiple radioisotope imaging can be done simultaneously using SPECT, which enables performing studies with 2 to 3 different radioisotopes having distinguishable gamma emissions [110].

Instrumentation

SPECT imaging device is basically a gamma ray detector, which can localize the distribution pattern of systemically administered radiotracer noninvasively. The SPECT instrumentation has significantly improved in the last decade in terms of performance characteristics and its diagnostic/research analytical qualities. Initial gamma cameras had smaller field of view, low sensitivity and resolution as compared to present generation systems. Advanced digital μSPECT scanners have solid-state CZT (Cadmium Zink Telluride) detector due to its superior energy resolution. The detector is mounted on a rotating gantry, which is common for SPECT and CT scanner. These preclinical scanners possess detachable SPECT and CT assembly, which significantly improve the scanner utility. Availability of common animal imaging beds allows its use with multiple scanners of different modalities. The collimator present in this system assists gamma cameras in locating the site of emission and also plays significant role in improving image resolution. Similar to clinical scanners, preclinical scanners are also well equipped with high resolution parallel hole, single and multiple pinhole low energy collimators, which are useful for imaging animals using different research protocols. The achievable resolution using this scanner is ∼0.5 mm after data reconstruction. The extraordinary energy resolution of this preclinical system allows multi isotope studies simultaneously.

6.2.3 microCT

microCT owes its popularity as an indispensable imaging tool in preclinical studies to high-quality spatial and temporal resolution. This significant technological advancement has made capture of detailed anatomical images possible so as to precisely monitor the progress of a disease condition in small animals [108]. Earlier its application was limited to high-contrast structures such as bones. Currently, technological advancements and use of better contrast agents made in vivo application of microCT possible such as studying soft tissue structures as well as vasculature. This anatomical imaging modality in preclinical research is required for distinguishing structural abnormalities and evaluating the location and extent of disease. Precise location of any lesion can be achieved by co-registration with PET and SPECT image data. The other applications of microCT include mapping of tumor vascularity, visualization of bone metastasis and evaluating novel contrast agents. microCT scanner provides images with ∼50 μm resolution and allows faster whole body image acquisition. High-resolution (HIRES) scanning acquisition mode is used for specimen imaging with a resolution of 15–30 μm.

Although, the modality provides images with excellent resolution, its major setback is the high radiation dose that is prescribed for the animals under investigation. This might lead to disruption of biological networks, immune system and functioning of major cellular pathways thus interfering with the results of the study such as tumor size. Proper control studies need to be designed to circumvent this problem and maximal utilization of this technique.

Instrumentation

X-ray is the major source for Computed Tomography (CT) imaging. Unlike PET and SPECT, where the subject under probe emits radiation (source), microCT requires external X-ray for imaging. Here, the animal under test is placed in the centre of the scanner and X-rays from a focused radiation source is rotated around the animal under investigation [126]. The two parameters viz. current and voltage that are measured in milliamperes (mA) and kilovolts (kV) respectively determine the strength and number of x-rays produced by the X-ray tube. Increasing the current on the machine increases the number of x-rays produced and hence enhances image contrast. The strength of the X-ray is varied at different rates depending on the density of tissue it is focusing on. Tissue density that influences the absorption of x-rays results in contrast differences in the image. Since the x-ray source is rotated around the test animal, an array of two-dimensional images are acquired, which are later combined to produce 3D images using suitable computer software.

The different major imaging modalities are shown in Fig. 6.3.

Fig. 6.3

Cartoon depicting different translational preclinical imaging modalities (Courtesy: TriFoil Imaging, CA, USA)

6.3 Other Imaging Modalities

6.3.1 Micro Magnetic Resonance Imaging (microMRI)

Based on the principles of nuclear magnetic resonance (NMR) spectroscopy [127, 128], MRI is used in preclinical setting for generating images of different soft tissues. The major difference between microMRI and MRI is the strength of the magnet that is used to generate the magnetic field, which is higher in the former. With its excellent spatial and contrast resolutions as well as better safety features (radiation-free), the application of microMRI is somewhat limited due to its astronomical cost, longer scanning time (often hours) and inability to study real time processes and fluids. Currently, it is used for imaging of brain and small tumors in combination with other imaging modalities such as microPET/SPECT.

6.3.2 Cerenkov Luminescence Imaging (CLI)

Almost known for a century, CLI has recently been adopted as an imaging modality for biomedical research. This developing optical imaging tool uses several common medical isotopes for its functionality [112]. Charged particles passing through a dielectric medium at a speed greater than light emit Cherenkov radiation. This modality combines the principles of luminescence with applications of PET radioisotopes and several therapeutic radionuclides for molecular imaging. High signal to noise ratio, ability to perform imaging on multiple subjects parallely makes it a promising tool in small animal imaging. However, several technical challenges need to be overcome such as low signal intensity, before it can be widely used in preclinical and clinical settings.

6.3.3 Optical Imaging

As its name suggests, optical imaging is based on two major principles: fluorescence and bioluminescence [118, 129]. While fluorescence imaging requires a fluorophore such as GFP and YFP (green and yellow fluorescent protein), bioluminescence utilizes enzymatic reactions based on chemiluminescence. However, due to autofluorescence of tissues below 700 nm, near infra-red probes are being used to minimize this interference in fluorescence based assays. Although both these tools are fast, simple and extremely sensitive, their application in preclinical research is limited due to extremely poor penetration ability and hence they find their primary application in studies of biological molecules.

A list of specific features and applications of different imaging modalities is provided in Table 6.1.

Table 6.1 List of features and applications of different imaging modalities

7 Protocol for Animal Handling and Imaging

Studies with mouse will be described here with a general protocol applicable for various imaging modalities. The schematic flow of small animal imaging is illustrated in Fig. 6.4.

Fig. 6.4

Stepwise self-explanatory illustration of small animal imaging procedure

7.1 Immobilization of the Animal

For imaging studies, it is important for the animal to be immobile during the entire process and therefore anesthesia is the first and foremost step. However, the body temperature of the animals needs to be maintained using heated pads, beds and heating lamps. Extreme care need to be taken for recovery of animals post imaging procedure. Scientists conducting studies that require animals injected with radioactive contrast materials should ensure special handling of the animals after the experiment.

Anesthesia

The mice under investigation will be exposed to an inhalational anesthetic, isoflurane which will be given at an approximate dosage of 2–5 % in an induction chamber. During the entire procedure, this anesthetic state will be maintained in the imaging instrument by supplying 1–2 % isoflurane through a vaporizer-connected nose cone. For studies requiring imaging for a short period of time (30 min or less), injectable anesthetics are used such as Ketamine/Xylazine which are usually administered intraperitoneally at the rate of 100–200 mg or 5–16 mg per kg body weight respectively [130].

Following anesthesia, certain physiological parameters such as electrocardiogram (ECG) and respiratory rate need to be measured using ECG and respiratory sensor pads for PET, CT, SPECT and MRI imaging following proper protocols [111, 131].

7.2 mPET, mCT, mSPECT Imaging

Prior to image acquisition, animals are given injections of contrast agents which might either be iodine-based tracers (CT) or radio tracers (mPET, mSPECT). The role of appropriate tracers is crucial for obtaining high resolution images of target organ/system. Based on the requirement for some radiotracers, overnight fasting is required for optimal tracer distribution. Minimal amount of tracers (in micro- or nanograms) will be administered to the animal so that it does not evoke any pharmacological effect. The tracer materials are either injected directly or introduced with the aid of a catheter in the tail vein of the conscious or anesthetized mouse. In case there is no anesthesia, restraining is required for the procedure. Sometimes, to increase dilation of the tail vein, it is immersed for a minute in warm water or wiped with absolute alcohol prior to administration of the tracer material. As a precautionary measure, the syringe containing the radiotracer is properly shielded to minimize exposure of the administrator. The entire procedure of the tracer uptake and completion of the experiment takes approximately 30 min to 2 h.

Prior to imaging by any of the above-mentioned modalities, the mouse is placed on an absorbent paper towel that is plastic-coated at the bottom to contain any excreted material, Although the approximate time for imaging varies between few minutes to a couple of hours, some studies might require repetition of the same experiment with the same animal several times over a period of several weeks.

For fusion imaging using PET/SPECT and CT, sometimes fiducial markers with low radioactivity are placed on the skin (after hair removal at that region) of the animal so as to mark the anatomic site of interest while acquisition of both PET/SPECT and CT images. This is important in overlaying PET/SPECT and CT images for analysis of the data with higher precision.

Some protocols also demand blood sampling at various time points to study kinetics of novel tracers in the animal system.

7.3 Imaging with Other Modalities (MRI and Optical)

Although the overall procedure is very much similar for all the modalities, there are certain specific requirements for MRI and optical imaging. Since MRI is associated with high magnetic field, care should be taken to use compatible tools and accessories during the entire process. Special training is required for personnel involved in these imaging studies.

In case of optical imaging (both fluorescence and bioluminescence), hair is removed from where signal is expected for optimum light transmission and image acquisition. The use of nude mice is highly recommended in this case to reduce the signal interference. For both bioluminescence as well as fluorescence imaging, anesthetized animals are placed in a light-tight box and imaged with a CCD camera for the time period required for the experiment (typically between few seconds to an hour). Suitable imaging materials are used based on the requirement of the study. For example, in bioluminescence, the animal is injected with a reporter gene with luciferin whereas, a fluorescent-tagged red/near-infrared emitting optical contrast agent or mouse antibodies are used for fluorescence imaging. Present systems are now well equipped with anatomic imaging modality for improved understanding of target sites.

7.4 Post-imaging Care and Record-Keeping

After the experiment, proper care should be taken to house the animals in the vivarium. This is important for regular monitoring and follow-up experiments. Prior to returning the animals, their cages should be treated with disinfectants to eliminate any scope for infection. The cages housing animals with radioactive tracers should contain a proper radioactive label that will provide the details such as the date, name of the isotope, dose, and the approximate date when there will permissible radioactive burden. Regular monitoring of the radioactivity level will be performed by a survey meter till their safe release in general housing areas.

8 In vivo Apoptosis Imaging with Specific Apoptosis Targets

Non-invasive imaging of apoptosis is of great interest both in biomedical research as well as in clinical settings where real-time monitoring of several markers of apoptosis and effects of drugs or radiation is possible.

In programmed cell death, both the receptor mediated extrinsic as well as the mitochondrial pathways converge downstream with respect to activation of caspase-3. Different apoptotic markers can therefore be labeled to follow the pathways both in normal cells as well as in tumors. The common tracers and probes used in different apoptotic imaging studies are shown in Fig. 6.5. Moreover, effect of chemotherapy and other drugs can also be monitored in small animal model systems. Different steps in this pathway can be studied using individual or fusion imaging techniques that include microPET, microSPECT, CT, microMRI, and optical imaging [111].

Fig. 6.5

Cartoon representing various probes and tracers for apoptosis imaging. The tracers are shown as orange symbols of different shapes

8.1 Phosphatidylserine Imaging

The most popular method of visualizing apoptosis in small animals is through imaging of radiolabeled annexin V [132]. Annexin V is a protein belonging to the annexin family that has high affinity for phosphatidylserine or PS (an anionic phospholipid). Although, it is found on the inner side of plasma membrane in viable cells, apoptotic induction leads to activation of γ-scramblase which flips PS to the outer side of plasma membrane allowing it to interact with annexin V. In addition, drop in membrane potential enhances annexin V binding to PS in a dose-dependent manner [133]. Therefore, an abundance of PS on the cell surface of apoptotic cells post caspase-3 activation, makes annexin V an excellent probe for in vivo detection of programmed cell death. ^99mTc-labelled Annexin V derivatives have been routinely used in monitoring apoptosis and necrosis in cell death related disorders [134, 135]. ^99mTc with its optimal radionuclidic properties, easy availability and low cost is one of the most popular labels for non invasive imaging such as SPECT [136].

^99mTc-annexin V was first used in a mouse lymphoma model that was treated with cyclophosphamide. Post chemotherapy, these animals showed a significant increase in annexin V uptake (300 %) after 20 h compared to control mice [137]. In another more recent study it was used for monitoring apoptosis in a hereditary breast cancer mouse model after docetaxel treatment [138]. Immunohistochemical analysis of the sensitive tumors provided important clues on apoptotic changes that occurred due to the treatment. Moreover, in the early 2000s, ^99mTc-annexin V has been used in clinical trials involving cancer patients with leukemia, small cell lung cancer, lymphoma and so on [139]. Although extensively used, several setbacks such as uptake variability in different subjects, slow pharmacokinetics and low signal to noise ratio limit its use in clinical studies. Furthermore, annexin V is incapable of distinguishing apoptosis from necrosis since phosphatidylserine is also exposed in the latter.

Due to its several drawbacks, annexin V has given way to many other radiolabeled molecules. For example, ^99mTc-labeled C2A domain of synaptotagmin I has been used to study apoptosis in small lung cell carcinoma [140]. More specific binding to target cells and better contrast has made it a more promising candidate than annexin V in microPET and SPECT imaging. Further research has led to development of several peptides and small molecule based probes to target the anionic phosphatidylserine. These small molecules and peptides have the advantages of better clearance from circulation, enhanced target specificity and tumor homing. One such study describes monitoring of xenograft tumor model in nude mice by optical imaging after administration of a fluorescent-based 9-mer peptide with promising results [141]. In addition, ApopSense molecule that harbours a fluorine atom is an excellent probe to study the apoptotic process [142, 143] due to its preferential accumulation in the cytoplasm of apoptotic cells. The fluorine atom makes it most suitable for radiolabeling with its radioisotope (¹⁸F) to be monitored by PET imaging.

8.1.1 Morphology of Plasma Membrane

Apart from phosphatidylserine imaging, changes in the morphology of plasma membrane due to membrane acidification, loss of membrane potential and activation of γ-secretase during early apoptotic process have been monitored using small molecule ‘Aposense’ probes (in several cancer models [142, 144, 145]). One such novel probe, ¹⁸F-ML-10 that is selectively taken up by radio or chemotherapy treated apoptotic cells in a tumor, suggests loss of mitochondrial membrane potential, caspase activation and degradation of DNA [143]. One severe drawback however is lack of understanding in the mechanism of uptake of these compounds.

Studying the fate of caspases (primarily the executioner caspase-3) is another possibility in understanding the dynamism of apoptotic process. ¹⁸F radiolabeled sulfonamide derivatives of Isatin (¹H-indole-2,3-dione) that have high metabolic stability and considerable lipophilicity are the best molecules to trace executioner caspases by microPET [146–148] as they bind to caspases-3/-7 with nanomolar affinity. However, due to the stringent requirement of higher lipophilicity or a cell penetration moiety, these molecules are limited only to preclinical studies. Lack of selectivity of several caspase inhibitors prevents their effective use in the clinical and preclinical studies. Recently, this problem has been circumvented with the development of irreversible active site inhibitor probes of caspases that show negligible or no activity against other cysteine proteases such as cathepsins. These molecules were utilized in optical imaging of caspases with a near infrared fluorescent probe and peptide transduction domain [149]. Design of probes such as TcapQ(647) comprising activatable caspase recognition (DEVD tetrapeptide) sequences have taken this technique one step ahead where the probe is attached with a fluorophore- quencher pair that gets activated only post recognition by caspases. This molecule successfully probed the fate of caspases in different xenograft murine models [150, 151]. Other techniques that have been explored include fluorescent nanoparticles alone or in combination with activatable caspases [152, 153]. However, further research to reduce non-specific recognition of proteases will lead to their wider application in biomedical research as well as in clinics. Imaging of caspase activation with tagged reporter genes that have also been extensively experimented with a few successful endeavors, led their way to the market. One such example is Caspase-Glo 3/7 that has been developed by Promega [154, 155], where a luciferase acts as a reporter.

9 Future Perspectives

Non-invasive imaging has revolutionized biomedical and translational research in the current decade. With introduction of fusion imaging modalities and significant technological advancements, it has transcended itself from being a mere visualization tool to an indispensable preclinical and clinical aide in designing, quantifying and testing preventive interventions. With its ability to build three dimensional images of different forms of tissues, organs, bones and vasculature, it has taken up a more responsible task of understanding the intricacies of cellular networks and pathophysiological processes. Along with other biological pathways, apoptosis imaging has become a popular tool for studying progress and treatment of associated diseases in preclinical settings. However, better comprehension of various molecular features of cell death is required for development of better imaging agents with enhanced specificity and optimal pharmacokinetic properties. With all-round advancement including technology and probe efficacy, apoptosis imaging can be one of the most advanced tools to study the molecules involved in this pathway as well as its association with several disease conditions. Effective amalgamation of apoptosis imaging with other important biological processes will provide a global picture of pathophysiological conditions which will certainly improve clinical decision making in apoptosis-related diseases and interventions.