Current Instrumentation and Technologies in Modern Radiobiology Research—Opportunities and Challenges

Author’s Accepted Manuscript Current instrumentation and technologies in modern radiobiology research – opportunities and challenges Eric Ford, Jim Deye www.elsevier.com/locate/enganabound

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S1053-4296(16)30017-0 http://dx.doi.org/10.1016/j.semradonc.2016.06.002 YSRAO50555

To appear in: Seminars in Radiation Oncology Cite this article as: Eric Ford and Jim Deye, Current instrumentation and technologies in modern radiobiology research – opportunities and challenges, Seminars in Radiation Oncology, http://dx.doi.org/10.1016/j.semradonc.2016.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Current instrumentation and technologies in modern radiobiology research – opportunities and challenges Eric Ford1 and Jim Deye2 1 Department of Radiation Oncology, University of Washington Medical Center, Seattle, WA and 2 Radiation Research Program, National Cancer Institute, Bethesda, MD

Correspondence to: Eric Ford, PhD FAAPM Associate Professor Department of Radiation Oncology University of Washington Medical Center Seattle, WA [email protected]

Abstract There is a growing awareness of the gaps in the technical methods employed in radiation biology experiments. These quality gaps can have a substantial impact on the reliability and reproducibility of results as outlined in several recent meta-studies. This is especially true in the context of the newer laboratory irradiation technologies. These technologies allow for delivery of highly localized dose distributions and increased spatial accuracy but also present increased challenges of their own. In this article we highlight some of the features of the new technologies and the experiments they support; this includes: image-guided localized radiation systems, micro-irradiator systems using carbon nanotubes and physical radiation modifiers such as gold nanoparticles. We discuss the key technical issues related to the consistency and quality of modern radiation biology experiments including: dosimetry protocols that are essential to all experiments, quality assurance approaches, methods to validate physical radiation targeting including immunohistochemical assays and other biovalidation approaches. We highlight the future needs in terms of education and training and the creation of tools for cross-institutional benchmarking quality in preclinical studies. The demands for increased experimental rigor are challenging but can be met with an awareness and a systematic approach which ensures quality.

1. Introduction: Challenges in modern radiobiology research Though the technology that is available for radiobiology offers increasingly sophisticated avenues of research (see section 2), it also presents new challenges. A recent review of the literature by Stone et al1 found significant concerns with preclinical data concerning the efficacy of 10 drug-radiation combinations presented in 125 publications prior to 2015. While the preponderance of concerns were related to the biological aspects of these studies, it was also noted that necessary radiation parameters were either “not reported (or were unclear-)” to an extent that compromised the reproducibility of the experiment. This was true for both the in vitro and in vivo studies. While the spatial and temporal precision of new technologies exacerbates the importance of any uncertainties in the radiation delivery and calibration, an NCI co-sponsored workshop2 pointed out that there is an increasing separation of the radiation physics and biology disciplines. Even for traditional radiobiology that does not push the boundaries of experimental techniques, this disconnect has often resulted in the use of equipment that is not properly utilized or calibrated and publications of radiobiology results often lacking important dosimetry details. Additionally, beyond the obvious need for proper use and calibration of irradiators, there is a growing awareness of more subtle effects including radiation interactions (physical, atomic and nuclear) with the materials around the sample(s) as well as mechanical and environmental stresses on the cells being exposed in-vitro and in-vivo. Examples of such complexities that can modify molecular responses to irradiation are strong magnetic or other non-ionizing fields as employed in MR or ultrasound guided radiotherapy3, environmental stresses on cells being tested in high throughput irradiators4 or physiological effects in animals that can alter radiation response. In order to assess such possible perturbations, Fowler et al.4 and others5 have pursued the concept of “bio-validation” that entails the use of known biological endpoints and dose response relationships in order to ascertain the completeness of the dosimetry characterization of the system. Clearly the increased sophistication of modern radiobiology demands an increased focus on the quality and reliability of experimental techniques. There are many dimensions to this, and this article attempts to present an overview of the key issues. In navigating this discussion Table 1 may be helpful. It presents a brief annotated summary, with references, of six key aspects in the utilization of modern technologies in radiobiology.

2. New Technology and New Biology The gap in techniques and standards discussed above can only be fully appreciated in the context of the new technologies for biology research that have emerged in the past ten years. New technologies are being applied on both the physical and biological fronts. Pre-clinical radiobiology is increasingly using tools such as genetically engineered mouse models (GEMMS), Crispr-mediated genetic constructs, patient-derived xenografts (PDXs) and orthotopic models in 2

order to bridge the gaps across systems (cells to humans) and scale (nano to macro), while new technologies for irradiation have adapted radiation research to this new biological frontier. As described in this issue of Seminars in Radiation Oncology, 21 st Century preclinical research addresses combined modalities involving radiotherapy and therefore it must try to understand drug – radiation interactions on the cellular and sub-cellular levels as they evolve over time, which leads to the need for time-resolved, spatially-precise delivery of the radiation component. Particularly important in this context are the techniques that have been developed to deliver precision radiation in the laboratory setting. These come in many flavors. Perhaps the simplest approach is to modify existing laboratory devices, using for example lead shields and the like, an approach that many studies have employed. A somewhat more sophisticated approach is to use clinical systems to provide localized irradiation in animal models. This has been reported with various systems such as CyberKnife6,7, GammaKnife8-11 or Tomotherapy12,13 and linear accelerators [e.g.14-16]. Most of these approaches, however, are limited when used with small biological specimens and are typically restricted to relatively large fields (e.g. whole brain irradiation) limiting their use in small animal models. An additional factor is the time required to deliver the dose which can also effect the radiobiological endpoints 17,18. In 2005 a growing interest in dedicated radiation systems specifically designed for preclinical research prompted the NIH to fund the development of a number of such systems. As reviewed recently19 the goal was to provide more precise irradiation techniques that mimic clinical practice. Numerous designs were proposed and pursued20-27 with reports on customized solutions continuing to appear28. However, the technology that is now commercialized and most widely available consists of an x-ray tube system coupled with an on-board CT and radiography system for image-guidance22,27. The tube operates at high kVp and filtration providing a suitable depth dose. The beam is restricted with a passive collimation system and this device is adjustable on some units. The on-board CT29 provides for image-guidance with the rodent under anesthesia or otherwise immobilized. Table 2 lists this and other recently developed technologies along with references and indications of their radiobiology relevance and level of complexity. Figure 1 shows several examples of advanced irradiator technology. Most specialized irradiation technologies offer three main advantages: 1) Delivery of highly localized dose distributions. The treatment beams can be made extremely small (down to 0.5 mm) or even smaller 26 . In some systems multiple beams and/or arcs can be used to make the dose even more conformal. When low energies are used, the edge of the beam can be extremely sharp. It is also possible to calculate and visualize the dose distribution on the CT scan30,31. 2) Increased spatial accuracy with on-board imaging and targeting On-board image guidance provides the ability to locate the dose in a living subject with high precision and accuracy. An accuracy of < 0.5 mm has been measured using histological sections32. On-board imaging coupled with precise radiation delivery 3

significantly expands the possibilities for research and clinical translation. For a review of technology circa 2010 see Verhaegen et al.33. Transgenic models often develop multifocal disease making imaging-based assessment of tumor burden very useful. 3) Molecular and functional imaging for localization On-board imaging including both CT and, more recently, bioluminescent tomography imaging34-36 facilitates targeting of individual lesions and comparisons within a single animal (e.g. bilateral flank xenograft models). Newer mouse models 37 of a single tumor formation also benefit greatly from on-board imaging. 38,39

The precision irradiator technologies described above have enabled important advances in various areas of radiation biology as exemplified in the articles in this issue along with many of their included references. Further examples can be drawn from two sites in particular: lung and brain. Though there is a rich history of radiobiology studies of lung tumors40, the availability of image-guidance on irradiation devices opens new experimental possibilities. This includes the study of radiation response in transgenic models15,37 and the exploration of high-dose-perfraction irradiation schemes18,23,41. Precision image-guidance is crucial for these studies since classic murine lung tumor models are characterized by development of multifocal disease. Precision irradiator technology also has enabled the study of normal lung tissue at a refined level including radiation response studies42,43, adult stem-cell-mediated effects44, and dosevolume-dependent response such as the release of cytokines45. GBM models are another example of studies which require these advanced technologies. As discussed by Felix et al28, while some tumors may have equivalent results whether treated in heterotopic or orthotopic models, the radiobiology of glioblastoma strongly depends on the host tissue microenvironment which leads to conflicting data between the two engraftment methods. Another area where precision irradiators have had an impact is combination of radiation and immune therapies46. This approach seeks to enhance in-field tumor control as well as promote distant responses—the so-called abscopal effect. Immune augmentation strategies include growth factors, cytokines, and immune checkpoint inhibitors. Preclinical models have been important in studying these processes47-50 and the ability to deliver localized radiation to animal model systems will likely be crucial in future investigations. The immune-response phenotype appears to have a relatively sensitive radiation dose-response threshold47,51. This underscores the need for control and accuracy of the radiation dosimetry in experiments. Finally, precision irradiator technology is playing a role in investigations of radiation enhancement therapies using biomolecular tools such as gold nanoparticles 52,53. Gold nanoparticles can act as radiation sensitizers due to their localized dose enhancement effect. Animal models are crucial in further understanding these processes. Experiments have shown an increased survival of mice with an orthotopic glioma tumor suggestive of a disruption of the blood brain barrier (BBB) post-20 Gy irradiation54. Recent experiments suggest that this effect may be caused by a dose enhancement to the vascular endothelial cells 55. Efforts are ongoing 4

and the ability to perform in vivo experiments with low-energy x-ray beams will be crucial to further understanding. 3. Improving consistency and quality The brief survey of recent studies above highlights the exciting results that are emerging as a result of high precision irradiation techniques. However, in order to form the basis of human studies, pre-clinical radiobiology demands high statistical confidence and reproducible results from multi-institutional studies. Hence concerns about consistency and quality assurance of the irradiation become paramount56. Even when the delivered dose is only a surrogate for the resulting bio-effect, it is still essential that the physical dose is well characterized and traceable to national physical standards1,2 since it is the one common element in the translation between the different irradiator systems. In some instances non-uniform dose effects are dealt with through standard operating procedures (SOPs)57 that require adherence to algorithms or are driven by software that compensates (at least partially) for these effects. Checklists and published SOPs have also been suggested for ensuring and communicating such characterizations2,58. However such an approach may present a “black box” to the user and thus result in significant errors in the biological ends points if not rigorously followed 59,60. Regarding delivered radiation dose, Zoetelief et al.58 presents a protocol that is essential to all radiobiology dosimetry, while Verhaegan et al.33 present an excellent review of the complexities surrounding the characterization and validation of small animal radiotherapy research platforms. These include beam delivery requirements (targeting, beam energy, dose distribution, biological effectiveness) as well as imaging requirements (spatial resolution, imaging dose, magnification, detector timing and dose response, artifacts). As noted in a recent Nature commentary61 “The lack of rigor that currently exists around generation and analysis of preclinical data is reminiscent of the situation in clinical research about 50 years ago.”. In radiation research trials these clinical standards were raised in part through the formation of the Radiological Physics Center (now the IROC http://rpc.mdanderson.org/RPC/home.htm) that employs clinically relevant quality control specifications and anthropomorphic phantoms so as to proscribe the radiation methodologies used by all participants in human clinical trials. An educational structure is also needed to support the development of the next generation of scientists in the specifics of radiobiology research and techniques62. It appears that the time has arrived for the creation of equivalent benchmarks for preclinical studies. This might include biologically relevant phantoms (in-vivo and in-vitro) in order to standardize and compare results between radiobiology researchers16,60,63. For high throughput research increased automation should make it easier to document, control and reproduce results across centers. This includes both systems to ensure the dosimetry of beams64,65 as well as targeting accuracy66. One concrete initiative that may address this need is a proposed multiinstitutional consortium to be funded by the NCI (See NOT-CA-16-005 http://grants.nih.gov/grants/guide/notice-files/NOT-CA-16-005.html). An important element of 5

this announcement is the assurance of NIST-traceable radiation dosimetry and consistent delivery methods across the consortium awardees. The demands for increased experimental rigor come at a time when modern radiobiology is confronted with greater demands on many fronts and while the resources to address these needs are dwindling67. However, without the proper diligence toward and resources for radiation standardization and consistency, the necessary preclinical investigations discussed in this special issue will not lead to any more definitive clinical trial designs than numerous past efforts. The future is hopeful but only if it is built on a strong foundation.

Figure 1: Examples of advanced technology and applications for laboratory irradiation. Panel A&B: A micro-beam irradiation platform based on carbon nanotube technology (A) and irradiation of the mouse brain (B) showing H2Ax staining (red). See reference 38 and 73. Panel C-E: An X-ray tube based irradiation system with on-board CT imaging (C), localized irradiation of the mouse brain (D) and resulting ablation of neural stem cells in the hypothalamic proliferative zone (E) with targeting confirmed via H2Ax staining (green). See reference 39.

6

Table 1: Key topics in precision radiator technology and related references.

Topic

Summary

Reference

Protocols for basic dosimetry

Protocol for x-ray dosimetry specific to laboratory systems (EURADOS collective)

58

Basic protocol for dose calibration of lowenergy x-ray beams Establishment of dosimetry centers

68

59,69

image quality and image-guidance capabilities of a cone-beam CT based small-animal image-guided irradiation unit

29

tumor visualization and accurate target localization for small field, high dose irradiation

23

Validation of radiation targeting

This may be accomplished with histopathology, immunohistochemical assays for DNA damage (e.g. H2Ax), and other endpoints.

6,32,70,71

QA of new precision irradiator technologies

High throughput device for QA for precision irradiators

65

Comprehensive quality assurance phantom for the small animal radiation research platform

63,72

Monthly Quality Management Program Assessing the Consistency of Robotic Image –guided Small Animal Radiation Systems

64

Integration of imaging

System for measuring and ensuring the accuracy of isocenter targeting Bio-validation

behavior with absorbed dose escalation for the production of intracellular reactive oxygen species, physical DNA double strand breaks, and modulation of the cellular double strand break pathway Biovalidation has value when considering new or more complex radiation technologies. For example the reference here on histological biovalidation of synchrotron beams (ref 5) and also carbon nanotube-based microbeams (ref 71). The

66

4

5,73

7

latter includes a longitudinal study of tumor and normal tissue response with apoptosis and proliferation assays. consideration of bio-effects due to imaging dose. More study is needed, but may be important effects above 10 cGy (see discussion in reference).

Modality comparison for small animal radiotherapy: A simulation study

Educational and training need

33

74

current state of basic (pre-clinical) research in radiation oncology from the perspective of relevance to the modern clinical practice of radiation oncology, as well as the education of our trainees and attending physicians in the biological sciences

75

state of radiobiology as well as future research opportunities in radiation oncology from both a physician and radiobiologist perspective

76

core physics curriculum for radiation oncology residents.

77

Specific issues in small animal dosimetry and irradiator calibration.

69

Training courses funded by governmental agencies. An example is the NIH-funded “Integrated Course in Biology and Physics of Radiation Oncology” (R25)

62

8

Table 2. Summary of Technologies for Radiation Biology Experiments Classification X ray

Linear Accelerator

Isotopic

Radiation modifiers

#

Technology

references

Primary use(s)

Complexity

standard kV

58,68

large fields, few samples

S

kV with imaging

22,27

H

carbon nano-tube

26,73

synchrotron

78,79

Image-guided focal in vivo x-ray irradiation Cell culture experiments, microbeams, in vivo microbeams Microbeams

Cyberknife

6,7

H

Tomotherapy

12,13

Clinical linac

14-16

Cs 137 : single specimen Cs 137: multispecimen Ir 192

80

Small field in vivo irradiation, esp. CNS Small field in vivo irradiation, esp. CNS In vivo and in vitro irradiation. Larger animals. Sometimes with image-guidance. Whole body or cell cultures Whole-body irradiation

H

Co 60 (Gamma Knife) Gold nanoparticles

8-11

BNCT

83

Small field radiation. Modified clinical HDR system Small field in vivo irradiation, esp. CNS Radiosensitization esp. at lower energies Increased bioeffectiveness

57

21,81

52,53,82

#

V

V

H M

S M

M V V

S = standard, M = moderate, H = high, V = very high

9

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