Imaging tools to enhance animal tumor models for cancer research and drug discovery

CHAPTER 4

Imaging tools to enhance animal tumor models for cancer research and drug discovery Hashem O. Alsaab*, Rami Alzhrani*,§, Atiah H. Almalki†, Yusuf S. Althobaiti‡, Samaresh Sau§, Arun K. Iyer§,¶ *

Department of Pharmaceutics & Pharmaceutical Technology, Taif University, Taif, Saudi Arabia Department of Medicinal Chemistry, Taif University, Taif, Saudi Arabia Department of Pharmacology & Toxicology, Taif University, Taif, Saudi Arabia § Use-inspired Biomaterials & Integrated Nano Delivery (U-BiND) Systems Laboratory Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, United States ¶ Molecular Imaging Program, Barbara Ann Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, MI, United States †

‡

Contents 1 Introduction 2 Imaging tools used in preclinical cancer models 2.1 Magnetic resonance imaging and hyperpolarized MRI 2.2 Computed tomography imaging 2.3 PET and SPECT imaging 2.4 Fluorescence imaging 2.5 Ultrasound imaging 3 Animal models and applications of imaging modalities 3.1 Ectopic tumor xenograft model 3.2 Orthotopic xenograft models 3.3 PDX model 4 Translational challenges, prospects, and conclusions References Further reading

76 78 78 80 80 83 84 84 87 88 89 91 96 106

Objective This chapter will focus on how newer imaging methodologies are supporting capture drug response in real time with imaging techniques. The chapter will have a subsection on models that are truly representative of the tumor microenvironment and highlight its applications, advantages, and translational challenges.

Animal Models in Cancer Drug Discovery https://doi.org/10.1016/B978-0-12-814704-7.00004-0

© 2019 Elsevier Inc. All rights reserved.

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1 Introduction In considering the several kinds of animal cancer models described in this chapter, it is imperative to understand that they are incomplete representations of the complex, diverse and multifaceted spectrum of genetic diseases that surround human cancer. Therefore, cancer is not a single disease, but by its very nature displays abundant intra- and inter-tumor heterogeneity both genotypically and phenotypically which is both dynamic and variable in nature, with most cancers utilizing multiple and excessive dysfunctions in survival and growth-regulatory pathways in the course of their adaptive evolution [1–3]. Over recent years, many studies employing global genomic sequencing analyses across cancer genomes and in specific types of cancers have indicated the molecular basis for this heterogeneity and the evolutionary diversity of human cancers. The results of these analyses have shown the substantial incidence, patterns, and variety of genetic alterations (somatic mutations, gene amplifications, deletions) and epigenetic alterations in human cancers and their nature in relation to tumor evolution [4–10]. Animal xenograft models of human cancer and the in vivo biological, pharmacodynamic/pharmacokinetic (PD/PK) and pharmacological data they can provide remain crucial components in: (i) knowing the pathophysiology of cancer involving new target identification; (ii) determining new therapeutic agents; (iii) investigating the use of novel therapeutic approaches in combination with established chemo- and radio-therapeutic regimens and approved targeted therapeutic agents; and (iv) in understanding mechanisms of intrinsic and acquired resistance to cytotoxic and targeted therapies. Despite variations in the types of animal models discussed in this chapter, tumor development is more rapid and homogeneous in murine models as compared to the heterogeneity of human cancers. While contributing significant practical advantages for drug discovery, development, translational biology, and biomarker evaluation of newly anticancer therapeutic agents [11–14], these animal models also have drawbacks like most animal models in other diseases. When utilized and interpreted properly to address specific experimental hypotheses, these models are helpful and valuable in oncology drug discovery. The huge need for applicable, cost-efficient, and pharmacologically relevant preclinical animal models of human cancer is clear because drug discovery and development is a high-cost and high-risk journey requiring average funding of $1 billion [15] and more than 10 years from a laboratory works to FDA final approval.

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The animal models of cancer exhibit a substantial experimental connection between fundamental discoveries occurring at the bench and the treatment of patients in the clinic. Whether researching the underlying genetic or epigenetic basis of cancer, the efficacy of new therapeutic options, or the specificity of new diagnostic imaging techniques, in vivo animal model tumor models allow hypotheses to be studied efficiently in terms of a complex biological system. Key characteristics of this complexity include a functional circulatory system, tumor-stromal interaction, and in some cases a fully functioning immune system [16]. Currently, many of these characteristics cannot be mimicked in a tissue culture dish, and so hypothesis validation with animal tumor models is still a crucial factor for clinical translation [17, 18]. Over recent decades, several noninvasive preclinical imaging modalities have been developed to increase the use of animal cancer models. The noninvasive factor of all technologies is critical, as it allows researchers to assess several aspects of tumor biology, anatomical or functional, dynamically in the same individual subject. In the context of basic sciences research, imaging enables scientists to assess aspects of tumor biology that are not clear and to efficiently evaluate the effects of experimental interventions. In the context of applied sciences research, imaging is valuable in standardizing the start of treatment without considering the extent of tumor burden. Imaging also evaluates tumor response to treatment over time. Therefore, the appropriate practice of noninvasive imaging both enhances the quality of experimental outcomes and reduces the number of subjects involved to result in statistically valid endpoints. Several preclinical imaging approaches are smaller scale versions of well-established clinical approaches, such as magnetic resonance imaging (MRI) or positron emission tomography (PET) [19, 20]. Other approaches are more experimental and at present are suitable only for imaging preclinical models. All imaging modalities have great advantages and disadvantages and choosing which one to utilize is mainly dependent on the biological hypothesis and questions needing to be answered. For instance, some modalities are superior to others for high-resolution anatomical imaging (e.g., MRI, computed tomography (CT), and ultrasound (US)), which can be helpful for evaluating tumor burden at deep tissue body sites. Other imaging modalities provide functional or metabolic information (e.g., PET and hyperpolarized MRI) and are thus able to confer unique information into the actual underlying mechanism of the molecular biology of a tumor in vivo, mainly irrespective of body location. While such limitations must be recognized in the context of the particular strengths, weaknesses, applications, and predictive power of

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various preclinical cancer models, the use of imaging modalities might support to solve the major problem contributing to the high attrition rates and long development process and times for cancer drugs [21].

2 Imaging tools used in preclinical cancer models 2.1 Magnetic resonance imaging and hyperpolarized MRI Preclinical MRI can yield images with excellent anatomic detail (as low as 30-μm resolution) [22] with unparalleled soft-tissue contrast. MRI is to some extent an insensitive imaging approach and commonly in vivo MRI signal is dependent on the spin relaxation of 1H from H2O, which is predominant in the body. In the existence of the magnetic field, the spins of 1H nuclei coordinate either parallel or antiparallel with the field. Signal results from the minimal excess of spins (a few parts per million) that coordinate with the field in the lower energy state and supply a net magnetization of the tested samples. Image contrast can also be produced in various ways, utilizing different radio wave frequencies, sequences, and magnetic gradients. That is why MRI is a flexible imaging approach, and it can measure diverse aspects of biology. Improved image contrast can be reached with the administration of a contrast agent such as gadolinium-based or iron oxide-based nanoparticles. These agents affect the relaxation rate of 1H protons in proximity and can be targeted to a specific tumor epitope, [23] accumulate in cells labeled with a reporter transgene, such as organic anion transporting protein, [24] or simply assembled in the extracellular space within the tumor microenvironment because of the EPR (enhanced permeability and retention) effect [25–29]. Other known examples of complicated MRI contrast mechanisms incorporate contrast-enhanced saturation transfer MRI, which has been utilized to noninvasively image protease activity in vivo, [30] the extracellular pH of tumors, [31] and tumor cell uptake of glucose, [32] and also diffusion-weighted MRI, which measures the diffusion rate of water through tissue (inversely proportional to cell viability) and is useful for measuring tumor response to therapy (Fig. 1) [33]. A new technique in the field of MRI, called hyperpolarized MRI, has changed the way that metabolism can be imaged in vivo. This strategy is 10,000 times more sensitive than conventional MRI, and it enables the quick detection of labeled molecules that are far less abundant in the body than H2O [34]. For tumor imaging, the technique depends on the giving of a nonendogenous molecular probe (commonly used 13C, a nucleus with magnetic spin) that has undergone a process called dynamic nuclear polarization [35].

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Fig. 1 (A) MR images representing a patient with recurrent glioblastoma responding to antiangiogenic therapy by reduction of abnormal tumor vessel calibers and a change in the direction of the vessel vortex curve estimated from a combined gradient-echo (GE) and spin-echo (SE) MR signal readout. (B) In vivo MRI monitoring of colon cancer xenografts with MFSN-Ctx based on the use of different injection methods. T 2-weighted MR images of tumors are shown at time intervals after intravenous (A) and intraperitoneal (B) injection. The white arrows indicate implanted tumor xenografts. The white dots from the left or right side of the image picture is a buffer solution reference. The inserted color images reflect changes of negative enhancement from red (black) to green (white) [4]. (A) Data from Emblem KE, Mouridsen K, Bjornerud A, Farrar CT, Jennings D, Borra RJH, Wen PY, Ivy P, Batchelor TT, Rosen BR, et al. Vessel architectural imaging identifies cancer patient responders to antiangiogenic therapy. Nat Med 2013;19:1178.

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This MRI-based technique is powerful as there is currently no other method to noninvasively image metabolism inside the tumor microenvironment in real time and clinical trials have explained that [36].

2.2 Computed tomography imaging Preclinical CT depends on collecting many individual X-ray images of a sample, taken at different angles, into a single three-dimensional (3D) and tomographic image [37]. CT is a high-resolution technique utilized for anatomic imaging, and as with planar X-ray imaging, image contrast is based on the relative variance in tissue electron density. That is, bone relative to softtissue and solid tumor masses in the lung respective to air space in the healthy lung. As a result, preclinical CT is comparatively poor at imaging tumors originating from soft tissue; though, radioisotope iodine or nanoparticlebased contrast agents can be utilized to enhance soft-tissue contrast in some tumor cases [38, 39]. CT imaging includes ionizing radiation; therefore, for specific applications, CT can be a high throughput imaging modality. Usually, CT is used to give anatomic context for PET and SPECT scans and to enhance the accuracy of radionuclide probe quantitation by precisely mapping tissue attenuation (Fig. 2) [40]. In addition, CT can be utilized to exactly map tumor vasculature in 3D and measure relative blood volume with tumor material ex vivo after doing terminal perfusion with a radiopaque agent that polymerizes quickly into a solid cast [41].

2.3 PET and SPECT imaging Both PET and SPECT imaging depends on the detection of an injected molecular probe labeled with a radioactive isotope. Currently, in the clinic, detectors encircle around the subject to detect either coincident annihilation photons emitted from a positron-electron annihilation event (PET) or single γ-ray emissions following passing via a collimator (SPECT). Both modalities allow the distribution of the probe to be mapped accurately in 3D space, showing the researcher where precisely in the body the administered radioisotope has accumulated. Both PET and SPECT are considered as the most sensitive preclinical imaging techniques that exist, as both can pass through tissue greatly unimpeded, and only trace amounts of the probe are needed to produce a robust signal (Fig. 3). A preclinical PET is at least an order of magnitude more sensitive than SPECT, as the collimation technology utilized by the latter results in more than 99% of emitted γ-rays going undetected. However, when SPECT is done with small aperture pinhole collimators,

Imaging tools to enhance animal tumor models for cancer research and drug discovery

CT

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Fig. 2 Evolution of xenograft growth at days 12 and 17 after inoculation of 0.5
106 C6 cells in nude mouse as seen on CT and on 18F-FDG PET/CT fusion images. Tumor ROIs are outlined in orange. White arrows indicate necrotic area at periphery of tumor, visible on 18 F-FDG PET image but not discriminable from viable tumor on CT image alone. Fiducial markers are indicated by blue arrows. Data from Deroose CM, De A, Loening AM, Chow PL, Ray P, Chatziioannou AF, Gambhir SS. Multimodality imaging of tumor xenografts and metastases in mice with combined small-animal PET, small-animal CT, and bioluminescence imaging. J Nucl Med 2007;48:295.

it yields images with higher spatial resolution than PET [42]. Combination of two modalities, such as PET/CT or SPECT/CT scanners, is now relatively common, and a CT image can be taken rapidly and coordinated with the PET or SPECT scan to result in a radionuclide image with anatomic context. CT can also enhance the quantitation of a radiolabelled probe by precisely mapping tissue attenuation of the radioactive signal [40].

3D PET/CT

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Fig. 3 Detection of pulmonary metastasis with 18F-FDG PET/CT. Transverse (A) and coronal (B) sections of coregistered 18F-FDG PET and CT images of SCID mouse 45 days after tail-vein injection of 0.7
106 A375M-Fluc melanoma cells. White arrows indicate hypermetabolic area seen on 18F-FDG PET image. Yellow arrows show water-density nodule in dorsal apex of right lung in CT image. PET/CT fusion image confirms registration of hypermetabolic image on anatomic reference (red arrow). Physiologic tracer uptake in heart (h), kidneys (k), and bladder (b) is marked. (C-D) MicroPET, fused microPET/CT, and axial images of 64Cu-DOTA-8-AOC-BBN(7–14)NH2 (C) and 64Cu-CB-TE2A-8-AOC-BBN(7–14)NH2 (D) in PC-3 tumor-bearing SCID mice at 20 h after administration. Lateral and axial projection images have been normalized to the highest pixel intensity for each respective image [3]. (A-B) Taken with permission from Deroose CM, De A, Loening AM, Chow PL, Ray P, Chatziioannou AF, Gambhir SS. Multimodality imaging of tumor xenografts and metastases in mice with combined small-animal PET, small-animal CT, and bioluminescence imaging. J Nucl Med 2007;48:295. (C-D) Taken with permission from Garrison JC, Rold TL, Sieckman GL, Figueroa SD, Volkert WA, Jurisson SS, Hoffman TJ. In vivo evaluation and small-animal PET/CT of a prostate cancer mouse model using 64Cu bombesin analogs: side-by-side comparison of the CB-TE2A and DOTA chelation systems. J Nucl Med 2007;48:1327.

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Currently, the direction of clinical effort is to combine PET/MRI scanners which are being under development [43]. PET/MRI scans will yield sensitive images of probe uptake with excellent soft-tissue contrast and less exposure to ionizing radiation relative to PET/CT [44]. Furthermore, PET/MRI scanners will mostly have less imaging throughput than PET/ CT and that could lead to minimizing the number of mice imaged per batch of the radiolabelled probe. Different types of radioisotopes with different characteristics can be used for PET or SPECT imaging of animal tumor models, for example, (1) 18F, a positron-emitting isotope of fluorine with a two-hour half-life (t½); and (2) 11C, which has a 20-min t½ and it is useful for minimizing a subject’s exposure to ionizing radiation [45].

2.4 Fluorescence imaging Preclinical in vivo fluorescence imaging is the most common and feasible experimental imaging modality, mainly because the signal can be detected easily with approximately inexpensive hardware setting [46, 47]. Also, it is safe because of nonradioactivity, and it is compatible with a range of both in vitro and ex vivo analysis techniques, allowing optimization and validation of the imaging approach. Preclinical fluorescence of tumor biology can be performed on both microscopic and macroscopic scales. Recent known examples are direct visualization of a tumor and stromal cells in response to chemotherapeutic treatment, [48] direct imaging of mammary cancer stem cell plasticity and the assessment of tumor subclones, [49] and direct visualization of interactions between tumor cells and tumor-associated immune cells [50]. In addition, whole-animal fluorescence typically uses conventional fluorescence excitation to produce a signal, whereby an excited label transgene releases the absorbed energy as red-shifted light and heat. In addition to fluorescent reporter transgenes, several conjugatable fluorescent moieties available for in vivo imaging have been discussed, and that include dyes, nanoparticles, and quantum dots [51–54]. Multiple fluorescent labels can be imaged in the same subject on condition that their wavelengths of emission are sufficiently distinct to be spectrally recognized by optical filters. As for whole-animal imaging, fluorescence in the red, far-red, and near-infrared part of the spectrum is superior to imaging with blue and green colors, as the excitation and emission wavelengths are more tissue penetrative and generate less tissue autofluorescence [54]. Significant efforts of research and industry are being conducted to bring this modality into the clinic, for example, to help surgeons identify cancerous lesions in patients and

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establish resectable boundaries [55]. Also, fluorescent endoscopy methods are also being developed using narrow and accessible fiberoptics to detect targeted probes bound to a tumor by laparoscopy [56] in the lumen of the colon [57] and the esophagus [58].

2.5 Ultrasound imaging US imaging modality is mainly useful for measuring tumor burden in soft tissue and for measuring aspects of tumor vascularity and perfusion [59]. Preclinical US scanners have a transducer to transmit high-frequency sound waves 10–70 MHz into tissue and detect their echoes, reflected from anatomical structures, to yield a near-instantaneous (hundreds of frames per second) and high-resolution 2D image. Multiple 2D images can be combined consequently to result in a 3D view and to precisely calculate tumor size and burden [60, 61]. US is helpful only for soft-tissue imaging as the sound waves that occur do not transmit through bone or air spaces. Besides that, the native contrast of US is also limited, and personal experience can be useful to fully interpret the data and the anatomic nature of scans. In addition, the US is useful for making functional measurements of tumor vasculature and blood flow. Tumor perfusion rates can be measured within a field of view by acoustically bursting the bubbles and then calculating the time spent to repopulate and regain contrast [62].

3 Animal models and applications of imaging modalities Animal models of cancer include: (i) ectopic xenografts of tumor-derived cell lines or tissue explants implanted into syngeneic or immunecompromised rodent hosts; (ii) orthotopic models in which tumor tissues or established tumor lines are implanted within the proper organ site or tissue; and (iii) patient-derived xenografts models (PDXs), one of the animal models based on propagating a fragment of a patient tumor to an immunocompromised mouse to mimic the histological and molecular characterization of the original tumor [63]. The strengths, weaknesses, applicability, and predictability of utilizing imaging modalities will be discussed in this part with these preclinical animal models [64]. Excellent and comprehensive reviews are available on the history and development of these specific types of preclinical tumor models in animals and their applications, advantages, and limitations in oncology drug discovery and development as shown in Table 1.

Table 1 Characteristics and applications of classes of animal models of cancer and their utility in cancer drug discovery Type of animal cancer model Features Utility Strengths and advantages Weaknesses and caveats

1. Ectopic xenograft models

2. Orthotopic tumor models

- Ectopic implanta- - Facile and rapid tion of cell lines - Pharmacological screening of NCEs or tissue fragments throughout dis- Nonphysiologic covery screens growth location - Tumor PK/PD relationships - Antitumor response efficacy - Implantation into - Assess survival endpoints organ of origin - Evaluate effects on - Reconstitutes primary tumor organ growth in proper microenvironment microenvironment - Local and meta- Assess influence on static spread local and metastatic tumor spread

- Reproducible - Cost and time effective tumor measurements - Applicable to many tumor cell types - Assess influences of immune surveillance and evasion (immunocompetent hosts) - High metastatic rates - Correct tumor microenvironment - Assessment of tumorstromal interactions - Assess antitumor efficacy of primary and metastases - Assess influence on immune surveillance and evasion (immunocompetent hosts)

Clinical predictability

- Limited histological - Limited to poor predictiveness for and phenotypic similarmost cancers ities to primary cancers with noted - Loss of tumor exceptions (e.g., heterogeneity oncogene driver - Low metastatic rates mutations) - Lack of native tumor microenvironment - More time and labor - Limitedmoderate intensive predictiveness - In vitro artificial selec- Useful to model tion of cell lines - Histological dissimilarclinical course of ities with human metastatic disease tumors - Loss of tumor heterogeneity - Imaging modalities needed for in situ assessment of tumor development Continued

Table 1 Characteristics and applications of classes of animal models of cancer and their utility in cancer drug discovery—cont’d Type of animal Clinical cancer model Features Utility Strengths and advantages Weaknesses and caveats predictability

3. Primary PDX models

- Highly predictive - Direct implanta- - Mid-later stage dis- - Preserves and stabilizes - Access to freshly and physiologi excised human tumors genetic, histological, covery screening tion/propagation cal; used for and phenotypic features of freshly excised - High front-end costs - Evaluate NCE “clinical trials in a of primary tumor human tumors and labor-intensive effects on original mouse” preparation human tumor - Maintains stromal and stem cell components - Limited engraftment ectopically or rates and long latency of primary tumor orthotopically for tumor development - Facilitates biomarker assessment - Potential to metastasize - Ease of tumor measurements

Modified from Ruggeri BA, Camp F, Miknyoczki S. Animal models of disease: pre-clinical animal models of cancer and their applications and utility in drug discovery. Biochem Pharmacol 2014;87(1): 150–161.

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3.1 Ectopic tumor xenograft model Ectopic model is generally defined as a xenograft mice model when a subcutaneous injection is given to animals using nonhuman or human cancer cells. The ectopic xenograft is the classical model of cancer which has been using utilized in oncology drug discovery study for evaluation and measurement. NCI had developed 60 cell lines which stemmed from eight organs which were used for developing ectopic xenograft model [65]. The ectopic model can be suitable and prognostic for evaluation of a new chemotherapeutic agent which is useful for translation selection of chemotherapeutic agents to clinical trial. It became an applicable and acceptable approach model in drug discovery, and biology of a cancer disease [66]. Ectopic models of cancer involved the injection of a tumor derived from cell lines or tissue culture subcutaneously into a syngeneic or immuno-deficient animal model. This model is valuable in evaluating the biological and pharmacological efficacy of the chemotherapeutic agents. Also, it is beneficial to study a particular molecular modulation receptor or signaling pathway for the assessment, evaluation, and screening of new chemical entities (NCEs) emerging from drug discovery paradigms. The ectopic model is widely used among researchers because it is easy and reproducible. The volume of the tumor (V) is estimated based on the tumor’s maximum length and the tumor’s minimum length.
V mm3 ¼ ðmaximumlengthÞ
ðminimumlengthÞ2 =1

3.1.1 Application of ectopic model In 2009, PET was used to assess Erlotinib on lung cancer [67]. PET was implemented with [11C]-erlotinib in ectopic mice xenografts of lung cancer cells. The highest uptake of [11C]-erlotinib uptake was in HCC827 as compared with A549, and NCI358 tumors were confirmed using PET. Also, PET and CT were used in ovarian cancer to assess the efficacy of Top216 [68]. In this study, mice were injected with cancer cells ectopically (in the flank). Top216 was shown to reduce cell proliferation in 2 h which was evaluated by PET/CT imaging technique. In another example, PET/CT imaging technique was also used to assess the effect of bombesin analogues on prostate cancer. Cancer cells were inoculated ectopically in the flank [20]. In addition, PET was also used to assess T84.66 on colon cancer. The colorectal cancer cells were implanted into the leg of nude mice. It was shown that 18F represents a new group of the antigen-specific probe for PET [19]. In another study, PET was used to evaluate the activity of Cetuximab which is a monoclonal antibody

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on epidermal growth factor receptor (EGFR) [69]. The model that was used in this study was a subcutaneous model. PET technique in this study revealed that tumor activity was increased with 64Cu-DOTAcetuximab in EGFR-positive tumors as compared to EGFR-negative tumors. MRI is also a powerful technique which has been used extensively in cancer drug discovery. It was used to monitor the development of colon cancer [70]. MRI was used to detect HER2/neu receptors in breast cancer [71]. It was found that Herceptin improved the intensity of the MRI signal even with low HER2/neu receptors in cells. Furthermore, fluorescence microscopy is considered as one of the powerful tools to study cancer biology and is used extensively in cancer drug discovery. Fluorescence imaging technique was used to assess the prostate and breast cancers cells [72]. Also, magneto-fluorescent nanoparticles (MFSN) were used to target EGFR positive colon cancer [70]. Another fluorescence technique was used to assess brain tumors.

3.2 Orthotopic xenograft models Orthotopic tumor implantation involves delivering carcinoma into a specific site that mimics the original site of a tumor in patients [73] either by injecting a suspension of tumor cell lines [74] or tumor fragments [75, 76]. This technique provides a microenvironment resembling the patient’s original one to have a realistic model of the invasive nature and metastasis of the studied tumor [75, 77, 78]. This model has been successfully established in several types of tumors affecting various tissues and organs such as breast [78], colon [75, 79, 80], pancreas [73, 81], ovaries [76, 77, 82], bladder [83–85], and prostate [86, 87]. Interestingly, these models have led to several drug discoveries. For instance, inhibition of metastasis and growth of tumors in the colon cancer model have been shown with the metalloproteinase inhibitors, batimastat [79] and CT1746 [88]. In pleural cancer utilizing this orthotopic technique, IFN-ɣ has been shown to limit metastasis and prolong survival [89]. Moreover, inhibition of metastasis of colon cancer has been found with TNP-470 utilizing this model [90, 91]. Recently, imaging systems and machines have been adopted to be suitable for use in rodents and small animals for preclinical uses. Multimodal imaging systems (e.g., SPECT/CT and PET/CT) can capture a 3D image but have low sensitivity and resolution, especially in deep tissue imaging and relatively slow acquisition time. An example of these systems is positron emission tomography-computed tomography (PET-CT) which has been used to evaluate an orthotopic model of endometrial carcinoma [92], glioma

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[93], prostate cancer [94, 95], lung cancer [96], glioblastoma multiforme [97], and pancreatic cancer [98, 99]. Fluorescence and bioluminescence imaging systems are useful in detecting various types of tumors in the orthotopic model, such as pancreatic cancer [100–103], ovarian cancer [104–106], and prostate cancer [107–109]. However, these imaging systems have a low resolution as well as light scattering which cannot be accurate in deep tissue imaging. Photoacoustic imaging is a new technique that can detect deep tissue with high resolution [110, 111]. This new imaging technique has been used to detect orthotopic sophisticated model, such as glioma [93, 112, 113].

3.3 PDX model In 1913, Murphy observed that a human tumor could grow in chick embryos, while not being feasible in adult chickens. After this discovery, researchers started to take the animal host into account [114]. It was discovered that the host immunity plays a significant role in transplantation success. It was a breakthrough to develop immune-deficient mice (Nude mouse) that lack both thymus (functional T cells) and natural killer (NK) cell function [115–117]. This discovery opens up avenues to apply multiple PDX tumor on animal models. PDXs is one of the animal models based on propagating a fragment of a patient tumor to an immunocompromised mouse to mimic the histological and molecular characterization of the original tumor [118–120]. Typically, the early stages of the resected patient’s tumor are used to establish PDX models [118, 120]. PDX model has become one of the most used models due to its similarity to human cancer biology more than cancer cell lines. To date, PDX model is considered the best tumor model due to its reflectance of human tumor heterogeneity, histology, gene expression, gene mutation and therapeutic response [121, 122]. Maintaining PDX intra-tumor heterogeneity, which depends on both cell autonomous (genetic and epigenetic heterogeneity) and noncell autonomous (such as stromal) heterogeneity is pivotal to mimic the patient responses to specific therapy or emergence of particular resistance to any of the newly developed therapies [123] (Table 1). The implantation site (subcutaneous, subrenal capsule and orthotopic) of the tumor plays a vital role in PDX engraftment success rate. Subcutaneous implantation is the preferred method of tumor implantation due its procedure simplicity and ease of tumor measurement [124, 125]. The microenvironment in the subcutaneous model does not reflect the real human microenvironment. The subrenal capsule is the ideal site of tumor

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implantation due to the hypervascularity of this region and provides more realistic tumor microenvironments. The major drawback of the subrenal capsule is the complexity of the surgery. Finally, the orthotopic PDX tumor implantation mimics the human tumor environments from which it is obtained. The complexity of this surgery is the major obstacle, for which a highly experienced surgeon is needed [126]. 3.3.1 Application of PDX in imaging for cancer Well-designed animal models open up avenues for discovering new tumor targets for imaging and therapy. Also, the animal models fill the translational gap between in vitro and clinical studies. The translation to the clinic passes through two steps. The first step can move in vitro to preclinical studies via inculcating two-dimensional cells into nude mice. The second step can move preclinical to clinical studies via using tumor fragments from a human tumor and then inculcated in an immunocompromised mouse to mimic the biological and histopathological characteristics of the original tumor; these models include genetically engineered mouse models and orthotopic PDX models [127–129]. Despite the recent huge advancement and diversity of animal models, no model will cover all human cancer aspects. Thus, in vivo imaging has substantially contributed to developing our understanding of the biology and characteristics of cancer [130, 131]. Also, live animal imaging contributes to reducing the required animals for a specific experiment where the same animal can act as its own control during the experiment. The right choice of the animal model and the imaging probe are complementary tools that can provide more accurate and detailed results. Recently, researchers started to conjugate an imaging probe to nanoparticles as a new tool for studying cancer biology [16, 132, 133]. Many animal techniques have been used to study tumor biology, such as PET, single photon emission computed tomography (SPECT), MRI, CT, US, and optical imaging [134–138]. Each imaging tool answers a specific question in tumor study, thus, it should be chosen carefully. Many imaging tools have been developed for humans, and then modified to adapt small animals as an imaging tool. One of these tools is microcomputed tomography (micro-CT) which works based on the differential absorption of the tissue to X-ray [139]. CT two provides high spatial resolution in which the tumor anatomy and phenotype can be studied. However, micro-CT lacks molecular specificity [140]. Yet micro-CT offers an excellent guide to evaluate the accumulation of the dye in the tumor site and the response of the tumor toward the applied therapies. The major

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drawback of micro-CT is its reliance on iodinated contrast agent and inability to image the same animal many times due to the damaging effect of radiation [141]. The micro-MRI machine functions by generating a magnetic field to provide a detailed three-dimensional image [142]. Moreover, micro-MRI provides high spatial resolution and can provide functional and anatomical tumor imaging. The strength of the magnetic field of micro-MRI enables high sensitivity in which the tumor development and vasculature can be tracked. An advantage of micro-MRI is that it does not have a radiation source; thus, the animal can be imaged multiple times [143–146]. PET and SPECT imaging tools are among the latest advanced technology that enable to utilize small animal in cancer imaging research efficiently. PET and SPECT imaging tools are based on emitting of gamma photons out of radiotracer molecule which its sensitivity in picomolar concentration that allows these two tools are tracking the interaction of radiotracer with the body molecules [147]. SPECT has different radiotracers that have multiple radioisotopes, and their half-life is long. These characteristics make SPECT accessible to many researchers [148]. SPECT is connected to a pinhole collimator that is attached to a rotating gamma camera. The pinhole collimator is responsible for providing a high spatial resolution image of the small animal [148]. Advanced technologies have participated in improving SPECT imaging resolution and efficiency in recent years. The principle of PET is like SPECT, but PET sensitivity is higher. Both PET and SPECT sensitivity are several times better than micro-MRI [148]. Also, PET and SPECT are sensitive, nontoxic, noninvasive, and easily quantified in the tumor area [145, 149]. The differences in both PET and SPECT can be found in Table 2 [150]. The high sensitivity of both PET and SPECT provide the capability of these two techniques to be used in monitoring tumor cell biology, progression, and metastasis. A drawback of both SPECT and PET is the inability to provide anatomical information that can be fulfilled by microMRI and micro-CT [151].

4 Translational challenges, prospects, and conclusions The ectopic model is the conventional xenograft of cancer which was utilized in tumor studies where cultured derived cancer cells embedded into normal or immune-deficient mice. Ectopic xenograft has many advantages over other tumor xenografts. Ectopic animal xenograft is reproducible, cheap, and appropriate to many cancer cell types. After the establishment

Table 2 Sources of tumor heterogeneity, their consequences in translational and basic cancer biology, and how they are currently represented in PDX models Implications for basic and Representation in current Source of heterogeneity translational research PDX models Future prospects

Cell-autonomous

Genomic clonal dynamics

Epigenetic/cellular clones

Minor KRAS subclones predict resistance to EGFR targeted therapies in colorectal cancer. Overall clonal diversity correlates with drug resistance in ovarian and oesophageal cancers [16, 17] Epigenetic “attractor states” increase the phenotypic heterogeneity within the tumor and hence widen the pool of cellular clones able to contribute to treatment resistance [20]

Genomic clones reconstructed in a panel of 15 breast cancer PDX models revealed ongoing clonal dynamics. Polyclonal engraftment was possible, but clonal selection was clearly evident [5] Colorectal and breast PDX models show five distinct cellular clonal phenotypes [6, 7]. “Type IV” quiescent clones were responsible for resistance to chemotherapy in colorectal cancer [7]

International collaborations, such as the EuroPDX consortium, should facilitate sharing of expertise and eventually lead to increased engraftment with less pronounced clonal selection [10] PDX models which better reflect the native tumor microenvironment should allow for more appropriate epigenetic clonal diversity

Table 2 Sources of tumor heterogeneity, their consequences in translational and basic cancer biology, and how they are currently represented in PDX models—cont’d Implications for basic and Representation in current Source of heterogeneity translational research PDX models Future prospects

Non cellautonomous

Stromal heterogeneity

Pro- and antitumor properties are attributed to different populations of cancer associated fibroblasts (CAF). Heterogeneous CAF or MSC populations could confer heterogeneity on the tumor bulk [26]

Immune infiltrate

The role of the immune infiltrate on tumor progression is highly complex. Checkpoint inhibitors and other immunotherapeutics are promising new treatment strategies in oncology

Human stromal components are replaced by murine equivalents on PDX passage. It is unclear how closely mouse fibroblasts mimic their human counterparts in supporting tumor growth. Human fibroblast cell lines are heterogeneous in their ability to promote treatment resistance [26, 27] Highly immunodeficient mice are used for PDX implantation. The NSG strain is characterized by a lack of mature lymphocytes, the absence of functional NK cells, defective macrophages, and defective dendritic cells [33]

Patient matched stromal components should be sourced whenever possible. Although human fibroblasts can be expanded in vitro, cell sorting may reduce engraftment efficiency of tumor cells and this should be considered

HuPDX immune models remain a substantial technical challenge. But the implications for study of tumor biology are profound

Continued

Table 2 Sources of tumor heterogeneity, their consequences in translational and basic cancer biology, and how they are currently represented in PDX models—cont’d Implications for basic and Representation in current Source of heterogeneity translational research PDX models Future prospects

Dysregulated ECM

Regulated ECM maintains tissue architecture and stem cell compartments. Loss of structure in cancer could contribute to oscillation between distinct transcriptional programs [21]

Matrigel is currently used to increase engraftment efficiency. Growth factors present in this murine basement membrane extract could support preferential engraftment of specific cell types. ECM is tissue specific, however in PDX models, ectopic implantation is commonly used [23, 25]

The ECM is tissue specific and orthotopic models should be considered where possible. Synthetic human alternatives to Matrigel should be investigated

Note: Strategies to improve the model, by better representation of both cell-autonomous (genomic and epigenomic clones etc.) and noncell-autonomous (stroma, immune infiltrate etc.) drivers of heterogeneity are proposed. Reproduced with permission from Cassidy J, Caldas C, Bruna A. Maintaining tumor heterogeneity in patient-derived tumor xenografts. Cancer Res 2015;75(15):2963–2968.

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of cancer cell lines for anticancer screening in NCI, xenograft models derived from these cell lines were developed. Ectopic model is usually used for drug discovery study, pharmacodynamics and pharmacokinetics of chemotherapeutic agents’ study, pharmacological, and biological research. Notably, it is valuable in early screening in measuring both the efficacy of the anticancer agent and tolerable in vivo because of their cost- and time-effectiveness, and wide-range of tumor cell selections. For example, alternative dosing study, kinetics of the drug research, dose-response, cytotoxicity and tolerability of chemotherapeutic agent research were suitable for this model [152]. However, these models do not encompass essential components of the pathological and physiological environment of the tumor which led to limited reliability of clinical outcomes [31]. Also, extended in vitro culture of tumors in such conditions that do not resample the microenvironment of the host promotes genetic changes, and gene transcriptional profiles in comparison with the original tumors in their primary microenvironment. Moreover, epigenetic heterogeneity of ectopic xenograft model which derived from artificial cell lines is not a generally illustrative model of an original tumor [66]. Therefore, 10% of chemotherapeutic agents that were tested in cancer clinical trials were eventually approved by the FDA [73–75]. The drug resistance in the clinical trial usually results from the differences between the ectopic model and clinical efficacy microenvironment conditions. These microenvironment conditions, which include chemokine, cytokines, and growth factors, particularly restricted the influence of ectopic model on differentiation, metastasis, and the proliferation of a tumor [1, 13, 41]. These differences in tumor models resulted in a discrepancy of outcomes as compared to the good results in the preclinical study of chemotherapeutic agents. Therefore, it has been suggested that preclinical xenograft should incorporate these microenvironment in such a way that these models would relate to clinical conditions to generate outcomes with an optimistic prediction rate [153]. Therefore, other models, such as PDX or genetically-modeled xenograft, have been introduced to improve the prediction rate as compared to a cell line derived xenograft [153]. There are several advantages of the orthotopic model. This orthotopic model can mimic the human metastasis, which is considered one of the main problems in tumor management, more efficiently for drug discovery and development [107, 154]. Moreover, the microenvironment resembling the original environment in the patients provides a more realistic and clinically relevant picture of tumor growth and characteristics as compared to s. c. models. For example, cisplatin showed efficacy in lung cancer using

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orthotopic but not ectopic model [155]. On the other hand, an ectopic model of colon cancer has shown dox to be effective while it is not effective in humans [156]. However, more detailed and well-controlled studies are needed to assess the current chemotherapies against known types of cancer utilizing and comparing these models. While the orthotopic model is attractive, it still has some drawbacks. One of the main limitations of this model is the technicality. One should accurately and precisely deliver the fragments or cell lines into the corresponding site in the mouse. This can be expensive, complex, and time-consuming as compared to the ectopic model. As compared to the ectopic model, the endpoints for therapy assessment are far more difficult to determine in an orthotopic model. Notably, complex imaging techniques are required to noninvasively detect and monitor orthotopic tumor models. Theoretically, PDX tumor biology triplicates the human tumor in which more accurate answers will be collected, and better-personalized therapy can be developed. Also, the PDX tumor biology is more consistence as it is obtained from the primary tumor compared to the conventional tumor model. Interestingly, the inter- and intra-tumor heterogeneity matches the human one in which PDX drug response and sensitivity replicate human response to the same drug [157]. One challenge of PDX tumor is that the animal model should be chosen carefully because not all PDX tumors grow in all animal models. Also, the more differentiated the PDX tumor, the better chance of growing it in the required animal model [158]. The host animal has an impact on the implanted PDX tumor. Researchers found that human tumor stromal components can be lost or replaced in the host animal. Thus, the PDX animal models are a mixture of both human and animal stroma. The stromal cross-reaction changes the nature of human tumors; therefore, these animal models might not accurately represent the clinical reality of human tumors [159]. Although PDX animal model has a bright future, many hurdles constrain its clinical applications; for example, the engraftment rate of success is low [159].

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