Abstract
Purpose: Radiation-induced cardiotoxicity is a significant concern in thoracic oncology patients. However, the basis for this disease pathology is not well characterized. We developed a novel mouse model of radiation-induced cardiotoxicity to investigate pathophysiologic mechanisms and identify clinically targetable biomarkers of cardiac injury.
Experimental Design: Single radiation doses of 20, 40, or 60 Gy were delivered to the cardiac apex of female C57BL/6 mice ages 9–11 weeks, with or without adjacent lung tissue, using conformal radiotherapy. Cardiac tissue was harvested up to 24 weeks post-radiotherapy for histologic analysis. Echocardiography and Technetium-99m sestamibi single photon emission computed tomography (SPECT) at 8 and 16 weeks post-radiotherapy were implemented to evaluate myocardial function and perfusion. Mouse cardiac tissue and mouse and human plasma were harvested for biochemical studies.
Results: Histopathologically, radiotherapy resulted in perivascular fibrosis 8 and 24 (P < 0.05) weeks post-radiotherapy. Apical perfusion deficits on SPECT and systolic and diastolic dysfunction on echocardiography 8 and 16 weeks post-radiotherapy were also observed (P < 0.05). Irradiated cardiac tissue and plasma showed significant increases in placental growth factor (PlGF), IL6, and TNFα compared with nonradiated matched controls, with greater increases in cardiac cytokine levels when radiotherapy involved lung. Human plasma showed increased PlGF (P = 0.021) and TNFα (P = 0.036) levels after thoracic radiotherapy. PlGF levels demonstrated a strong correlation (r = 0.89, P = 0.0001) with mean heart dose.
Conclusions: We developed and characterized a pathophysiologically relevant mouse model of radiation-induced cardiotoxicity involving in situ irradiation of the cardiac apex. The model can be used to integrate radiomic and biochemical markers of cardiotoxicity to inform early therapeutic intervention and human translational studies.
Translational Relevance
With the rising life expectancy of oncology patients, the long-term clinical consequences of radiation-mediated cardiotoxicity are becoming increasingly prevalent. Furthermore, the lack of effective diagnostic and therapeutic strategies has revealed a deficiency in the current data on the relevant pathophysiology. We developed a novel mouse model of partial heart irradiation, which revealed radiation-induced cardiac injury to be histologically characterized by perivascular fibrosis and vessel wall thickening. To define histopathologic and functional correlates, we implemented echocardiography and Technetium-99m sestamibi single photon emission computed tomography and observed systolic and diastolic dysfunction and myocardial perfusion deficits after radiation. In addition, biochemical studies identified placental growth factor and TNFα as circulating markers of cardiac injury, and informed translational human studies in thoracic radiation patients, which corroborated these findings. Our model can uniquely facilitate the translation of preclinical findings into clinical practice, guiding targeted diagnostic and therapeutic interventions to decrease cardiovascular morbidity and mortality associated with treatment.
Introduction
Radiotherapy has proven to be highly effective in controlling or eradicating solid tumors, with over half of all patients with cancer expected to receive radiotherapy at some point in their disease course (1). In the case of thoracic radiotherapy, a significant number of patients are at an increased risk of developing some form of cardiac disease due to incidental dose delivery to the heart (2–4). As cardiovascular disease is now the most common nonmalignant cause of death among cancer survivors (5), there is a strong incentive to elucidate the causative pathophysiologic mechanisms of radiation-induced heart disease (RIHD).
RIHD is postulated to result from a series of events including direct DNA damage, increased oxidative stress, vascular endothelial cell injury, inflammation, and fibrosis (6–9). However, our current understanding of such processes has been limited by a lack of physiologically relevant translational small animal models. Mezzaroma and colleagues described the use of a mouse model for the assessment of cardiomyopathy post-radiotherapy; however, the model requires irradiation of the entire thorax (10). A more physiologically relevant mouse model was reported by Sievert and colleagues involving irradiation of the entire heart and a portion of lung tissue (11). However, whole heart irradiation is uncommon in clinical practice and also limits the ability to histologically and radiologically characterize the spatial patterning of radiotherapy-induced damage. We hypothesized that a more clinically relevant mouse model of radiotherapy-induced cardiotoxicity would be useful for investigating relevant pathophysiology and could be generated by the irradiation of only a portion of the heart. The mouse model described in this study allows for targeted, in situ partial heart (PH) irradiation alone or with simultaneous partial lung (PHL) irradiation. In addition, conformal radiotherapy was utilized to deliver high doses of radiotherapy with minimal skin toxicity and to better mimic radiotherapy administered in the clinic.
Despite extensive investigation, the role of sensitive imaging measures and biomarkers in signaling radiotherapy-induced cardiotoxicity has yet to be established. Early identification of radiomic and biochemical markers in thoracic radiotherapy patients with increased risk of cardiac complications would be useful in reducing treatment-associated morbidity and mortality through the implementation of more aggressive and targeted therapeutic interventions for RIHD. With respect to imaging, most clinical and preclinical data have relied on echocardiography to measure left ventricular ejection fraction (LVEF; ref. 12); however, this approach fails to provide insight into the basis for mechanistic changes in cardiac function. With increasing evidence implicating vascular damage as an important mechanism of radiotherapy-induced cardiac injury (13, 14), one radiomic approach is to implement Technetium-99m (Tc-99m) sestamibi single photon emission computed tomography (SPECT) to evaluate for myocardial perfusion defects. In addition, from a biochemical perspective, several studies have demonstrated value in using circulating myocardial injury markers (troponin, pro-brain natriuretic peptide), inflammatory markers (high-sensitivity C-reactive protein, growth differentiation factor, myeloperoxidase, cytokines), and vascular pathology markers [placental growth factor (PlGF)] to identify disease development following chemotherapy or radiotherapy in patients with breast cancer, lung cancer, and lymphoma (15–18). Therefore, we used our model of radiotherapy-induced cardiotoxicity to evaluate radiologic and biochemical markers of cardiac injury, and leveraged our findings to guide subsequent human plasma biomarker identification studies. Our model is a valuable tool for understanding causative underlying pathophysiology and establishing predictive toxicity markers that signal the onset and progression of RIHD, before it becomes irreversible.
Here we report results from three overarching objectives of this study: (i) to develop a novel mouse model of radiotherapy-induced cardiotoxicity involving PH irradiation, (ii) to characterize the pathophysiologic mechanisms of cardiotoxicity in the model, and (iii) to use the model to identify biomarkers of cardiac injury.
Materials and Methods
Mice
Female 9- to 11-week-old C57BL/6 mice (The Jackson Laboratory) were maintained in our institution's animal facilities. All experimental procedures were conducted in accordance with the guidelines and protocols approved by the Institutional Animal Care and Use Committee. Mice were subjected to image-guided radiotherapy using the Small Animal Radiation Research Platform (SARRP, Xstrahl Life Sciences) and irradiated with a single dose of 20, 40, or 60 Gy. Mice were euthanized at various time points specified below to collect plasma and heart and lung tissues for further analysis. Three or more mice were used in each group for any given study.
Cone-beam CT imaging and radiotherapy
For these studies, all mice received a single 200 μL tail vein injection of a blood pool contrast agent (FenestraVC, MediLumine Inc.) to visualize the heart on cone-beam CT (CBCT) imaging prior to irradiation. Each mouse was imaged and irradiated while immobilized with 2.5% isoflurane anesthesia with medical air as the carrier gas (VetEquip). Following 5 minutes in an induction chamber, the fully anesthetized mouse was placed on a customized platform in the ventral recumbent position. The head of the mouse was placed in a face mask that allows the gas to be scavenged (Xerotec Inc.). The platform was placed off the stage of the SARRP. Using the SARRP Control Interface, a CBCT image was initiated with the X-ray tube operating at 65 kV, 0.5 mA with aluminum filtration approximately 5–15 minutes after FenestraVC administration. The images were reconstructed with Xstrahl' MuriSlice Software. Dosimetry was performed by measuring EBT2 gafchromic film exposure, at depth using solid-water phantom material, with a Microtek Artixcam M1 camera. The dose-exposure calibration was done using a cesium irradiator with calibration dose rate.
To carry out PH irradiation, an isocenter was selected on the contrast-enhanced CBCT at the posterolateral cardiac wall [left ventricular (LV) wall] approximately 2–3 mm superior to the cardiac tip for targeted radiotherapy using the software. Once the isocenter was determined, the robotic stage moved the animal to the proper location. Single doses of 20, 40, or 60 Gy of photon radiotherapy were precisely delivered to the cardiac apex with or without including adjacent lung tissue, using a 3 × 3 mm2 collimated beam operating at 220 kV, 13 mA with copper filtration. Each dose was calculated and prescribed using tissue segmentation and heterogeneity corrections for soft tissue and air densities. The dose rate was 1.65 Gy/minute.
γ-H2AX and Masson trichrome staining
Details of sample processing and staining are given in Supplementary Materials and Methods. Quantification of cardiac fibrosis on trichrome staining at 8 and 24 weeks post-radiotherapy was performed using the Aperio ImageScope 12.4.3—Pathology Slide Viewing Software. Five sections separated by 100 μmol/L were used for fibrosis quantification of each mouse heart. For each section, whole heart fibrosis was calculated by drawing an area of interest around the entire stained cardiac tissue. In addition, the thickness of cardiac vessels in the apical half of the heart (irradiated region) were calculated by measuring the distance from the lumen of the vessel to the edge of the perivascular fibrotic or connective tissue. Depending on vessel size and uniformity, 4–20 measurements were taken per vessel and then averaged.
Biochemical marker and cytokine quantification
Mouse cardiac tissue homogenates were analyzed for troponin T, troponin I, PlGF, IL2, IL6, and TNFα levels. Human and mouse plasma samples were also analyzed for these marker levels. Details are given in Supplementary Materials and Methods.
Mouse plasma collection
Whole blood was collected into heparinized collecting tubes (Thermo Fisher Scientific, 02-668-10) also containing EDTA, from cardiac puncture in terminal experiments or the tail vein of the mice in nonterminal experiments at 1, 2, 4, and 8 weeks post-radiotherapy. Plasma was obtained by centrifugation for 10 minutes at 1,500 rpm using a refrigerated centrifuge. Plasma samples were aliquoted and stored at −80°C.
Human plasma collection and sample selection
A random selection of human plasma samples from patients who were treated with thoracic radiotherapy and who were enrolled in a prospective longitudinal cohort study (IRB#808014) conducted at our institution from June 2015 until February 2018 were obtained. Details of the study population and methods have been reported previously (16). Briefly, blood samples were collected pre- and post-radiotherapy (median, 20 days) and stored at −80°C. A total of 14 patients with lung cancer and 32 patients with breast cancer had plasma samples available both pre- and post-radiotherapy and were used in this study. Only 4 patients with mediastinal lymphoma had pre- and post-radiotherapy plasma samples available, with most samples yielding undetectable biomarker levels on analysis. These samples were therefore excluded from the study.
Quantitative echocardiography
Ultrasound examination of the LV was performed at 4, 8, and/or 16 weeks post-radiotherapy using a Fujifilm VisualSonics Ultrasound System (VisualSonics Inc) and using an MS400 (18-38 MHZ) transducer. Mice were lightly anesthetized with an intraperitoneal injection of 0.005 mL/g of 2% Avertin (2,2,2-Tribromoethanol, Sigma-Aldrich). Hair was removed from the anterior chest using chemical hair remover (Nair), and the animals were placed on a warming pad in a left lateral decubitus position to maintain normothermia (37°C), monitored by a rectal thermometer. Ultrasound gel was applied to the chest. Care was taken to maintain adequate contact while avoiding excessive pressure on the chest. Two-dimensional long-axis, short-axis M-Mode images were obtained to evaluate LV systolic function. Transmitral inflow pattern and tissue Doppler were obtained in modified four-chamber apical view to evaluate LV diastolic function. After completion of the imaging studies, mice were allowed to recover from anesthesia and returned to their cages. M-Mode Images were analyzed for LV structure and function-related parameters and Pulse wave and tissue Doppler images were analyzed for diastolic function–related parameters using Vevo Lab software (Visual Sonics Inc). Analyses were performed blinded to treatment group.
Tc-99m sestamibi SPECT
SPECT imaging was performed at 8 weeks post-radiotherapy under continuous isoflurane gas anesthesia by the nose cone. While under anesthesia, mice were injected via tail vein with 4–7 milliCurie (volume <10% body weight of animal) of a radioactive tracer, Tc-99m sestamibi (Nuclear Diagnostic Products), and scanned on the small animal SPECT system (MILabs USPECT) approximately 5–10 minutes after injection. Mice were placed in the approved radioactive decay room until they were no longer radioactive (10 half-lives of the isotope).
Images were reconstructed with a reconstruction program (U-SPECT-Rec2.51g) provided by MiLabs. The reconstruction parameters were: 0.4 mm voxel size, four subsets, six iterations, 0.25 FWHM Gaussian postfilter for all images.
LV wall perfusion was qualitatively and quantitatively assessed using MIM Software Inc. Quantitative assessment was carried out by subdividing LV walls into apical and basal components for the anterior, inferior, lateral, and septal walls. The software was used to contour the specific LV wall segments and quantify regional tracer uptake. Individual LV wall tracer concentration values obtained were normalized to activity of tracer injected or blood pool tracer concentration in separate analyses.
Statistical analysis
Statistical analyses were performed with GraphPad Prism version 8 (GraphPad Software) and Microsoft Excel. Data are presented as means ± SEM. Results were considered statistically significant when P < 0.05 by the appropriate test (unpaired Student t test). A one-sample t test was used to assess for significant fold changes in marker levels pre- and post-radiotherapy. Linear regression was used to evaluate for significant correlations between fold change in marker levels pre -and post-radiotherapy and mean heart radiotherapy dose. Fisher exact test was utilized for comparison of fold changes in marker levels pre- and post-radiotherapy in patients with lung cancer versus patients with breast cancer. The threshold was set at >30% change over pre-radiotherapy levels (16).
Results
Mouse model for radiotherapy-induced localized heart damage
The first objective of this study was to develop a reproducible mouse model of targeted PH irradiation. The treatment schemes implemented are depicted in Fig. 1. Radiotherapy planning was done on contrast-enhanced CBCT images 5–10 minutes after FenestraVC tail vein injection (Supplementary Fig. S1). PH irradiation was first performed at 20 Gy (n = 12), a typical dose for small animal thoracic radiotherapy studies, using a single 3 × 3 mm2 beam angled at 15o to the horizontal (Supplementary Fig. S2A). To evaluate direct DNA damage post-radiotherapy and radiotherapy targeting, sections of heart and lung tissue were stained for γ-H2AX (n = 4). The phosphorylation of histone H2AX at serine 139 (γ-H2AX) is a well-known, early effect of radiotherapy exposure, and the presence of γ-H2AX is considered a sensitive and quantitative marker of DNA double-strand breaks. Results from the immunofluorescence staining of heart tissue for γ-H2AX at 1 hour post-radiotherapy confirmed DNA damage is restricted to the 3 × 3 mm2 area in the lower third of the heart (cardiac apex; Supplementary Fig. S2B), leaving the surrounding tissues intact. As the radiotherapy target volume was significantly smaller than that in previously reported studies, we hypothesized that higher doses would not only be well tolerated, but might also be necessary to elicit significant, detectable cardiac injury within a practical timeframe. Indeed, no qualitative abnormalities on the histopathologic analysis described below were identified after 20 Gy PH radiotherapy at 8 and 24 weeks of follow-up (n = 4 each). Therefore, subsequent studies were conducted at doses ≥ 40 Gy.
Schemes of cardiac irradiation. All mice received the contrast agent FenestraVC through tail vein injection, followed by CBCT and radiation delivery. The radiation beam was targeted at the apex of the heart with a 3 × 3 mm2 collimator at a dose of 20, 40, or 60 Gy, with conformal radiotherapy (RT) implemented at the latter two doses. Radiation was alternatively targeted at the apex of the heart and a portion of the left lung at a dose of 40 Gy using two opposing AP/PA 20 Gy beams.
To deliver doses of 40 Gy (n = 20) and 60 Gy (n = 20) while minimizing skin toxicity and ulceration, we used a novel radiotherapy plan based on our treatment scheme for 20 Gy PH radiotherapy, involving conformal radiotherapy delivered over a 75o arc, again using a 3 × 3 mm2 beam (Fig. 2A and B). This treatment scheme reduced the maximum dose delivered to the skin almost 2-fold (Supplementary Fig. S2B). Finally, to evaluate the potential indirect effects of lung irradiation on cardiac injury, PHL irradiation to 40 Gy (n = 20) involving approximately 40% of the left lung volume was carried out using two opposing anterior-posterior posterior-anterior (AP/PA) 3 × 3 mm2 beams, each delivering a 20 Gy dose to the isocenter selected at the LV apical wall (Fig. 2C and D). The increase in radiotherapy dose delivery to the lung with this treatment scheme can be seen in Supplementary Fig. S2B. Corresponding staining at 1 hour after conformal radiotherapy to 40 and 60 Gy revealed localized DNA damage extending up to 1 mm beyond the 3 × 3 mm2 border in the lower third of the heart (Supplementary Fig. S2B), in the area of dose fall-off. Results from lung tissue staining after conformal radiotherapy confirmed the absence of lung tissue DNA damage (Fig. 2D and E). Conversely, clear and localized DNA damage confined to a 3 × 3 mm2 area in the left lung was detected in the radiotherapy scheme involving AP/PA beams (Fig. 2F).
Dose plan in MuriPlan and corresponding immunohistochemical γ-H2AX staining of heart and lung tissue. A, Axial, sagittal, and frontal views of radiotherapy planning and delivery to the cardiac apex at a dose of 60 Gy with a 3 × 3 mm2 collimator and conformal radiotherapy over a 75o arc. B, Positive staining in the cardiac apex and negative staining in the lungs confirms selective PH radiotherapy targeting. C, Axial, sagittal, and frontal views of radiotherapy planning and delivery to the cardiac apex at a dose of 40 Gy using two 20 Gy 3 × 3 mm2 vertical opposing beams. D, Positive staining in both the cardiac apex and in the mid-left lung confirms PHL radiotherapy targeting. The red dotted circles outline the cardiac silhouette. The red dotted boxes indicate the 3 × 3 mm2 collimator. Scale bar, 1 mm.
Mean heart dose for 40 Gy PH-, 40 Gy PHL-, and 60 Gy PH-treated mice were 5.7 Gy, 4.8 Gy, and 8.7 Gy, respectively. Corresponding values for mean partial heart dose (dose radiotherapy beam targeting) were 39, 38, and 59 Gy. No deaths were observed in any of the treatment schemes and all the mice continued gaining weight post-radiotherapy (Supplementary Fig. S3).
Development of cardiac fibrosis and vascular damage
Radiotherapy-induced damage can develop chronically, and in most normal tissues is characterized by collagen deposition and excessive formation of fibrous tissue, at which point damage is considered irreversible. Therefore, we evaluated the levels of fibrosis in cardiac tissue at 8 and 24 weeks post-radiotherapy using Masson trichrome staining. Figure 3A shows representative trichrome staining at 8 weeks post-radiotherapy, and Fig. 3B shows results from fibrosis quantification at 8 and 24 weeks post-radiotherapy. Although increased at 8 weeks post-radiotherapy, fibrosis levels only reached statistical significance at 24 weeks post-radiotherapy: 0 Gy (% fibrosis = 1.0), 40 Gy PH (1.4%, P = 0.079), 40 Gy PHL (2.0%, P = 0.022), and 60 Gy PH (1.5%, P = 0.017). Surprisingly, and in contrast to studies reporting the development of diffuse myocardial fibrosis in irradiated areas of tissue, we found that fibrosis was primarily located in perivascular regions (Fig. 3C), with only mild interstitial fibrosis concentrated in the lower half of the heart (roughly the area of irradiation). To further characterize differences in the cardiac vasculature between irradiated and nonirradiated mice, we quantified cardiac vessel wall thickness in the area of irradiation at 8 and 24 weeks post-radiotherapy. Compared with control (11.4 μm ± 1.4), average vessel wall thickness was most increased at 24 weeks after 40 Gy PH (13.4 μm ± 1.2, P = 0.347), 40 Gy PHL (18.4 μm ± 0.7, P = 0.005), and 60 Gy PH RT (28.9 μm ± 1.7, P < 0.001; Fig. 3D).
Masson trichrome staining of cardiac tissue at 8 and 24 weeks post-radiotherapy. A, Representative trichrome stains from 0 and 60 Gy cohorts at 8 weeks post-radiotherapy. Cardiac tissue is characterized by perivascular fibrosis superimposed on scattered interstitial fibrosis. Scale bar, 100 μm. B, Quantification of fibrosis per measured area (n = 4/group). P = 0.022 and 0.017 for 40 Gy PHL and 60 Gy PH cohorts, respectively. C, Representative vessels from each radiotherapy cohort showing perivascular fibrosis, vessel wall thickening, and lumen narrowing in irradiated cohorts 24 weeks post-radiotherapy. D, Quantification of vessel wall thickness in the lower half of the heart (irradiated region; n = 4/group). At 8 weeks, *, P = 0.022 and **, P = 0.002. At 24 weeks, *, P = 0.013; **, P = 0.001 (60 Gy PH) and 0.005 (control); and ***, P < 0.001 (control and 40 Gy PH). Scale bar, 20 μm.
Dose- and time-dependent mechanistic cardiac dysfunction
Current recommendations for managing potential cardiac dysfunction in patients receiving cardiotoxic therapies heavily rely on LVEF monitoring with serial echocardiograms. As such, we implemented echocardiography at 4 weeks after 60 Gy radiotherapy (Supplementary Fig. S4), and also at 8 and 16 weeks in all cohorts (Fig. 4A and B) to evaluate functional changes in the heart. Qualitatively, radiotherapy resulted in dose-dependent, progressive cardiac dysfunction, as indicated by hypokinesis at the cardiac apex (Supplementary Video S1). There were no significant differences in heart rate across groups.
Quantitative echocardiography. A, Comparison of cardiac systolic and diastolic functional parameters between irradiated and nonirradiated groups 8 weeks post-radiotherapy (post-RT; n = 8, 5, 8, 8 per group). B, Corresponding comparison at 16 weeks post-radiotherapy (n = 8, 5, 4, 4 per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (details in Supplementary Table S1). E/e′, ratio of mitral peak velocity of early filling (E) to early diastolic mitral annular velocity (e′).
LVEF and E/e′ [ratio of mitral peak velocity of early filling (E) to early diastolic mitral annular velocity (e′)] were used as measures of systolic and diastolic dysfunction, respectively (Supplementary Table S1). Compared with nonirradiated mice (LVEF = 68.0% ± 1.2, 67.3% ± 1.2, and 69.7% ± 3.2 at 4, 8, and 16 weeks post-radiotherapy, respectively), LVEF was decreased at 8 (62.2% ± 0.6, P = 0.012) and 16 (62.8% ± 4.0, P = 0.006) weeks after 40 Gy PH radiotherapy, at 8 (63.8% ± 1.5, P = 0.090) and 16 (63.8% ± 4.0, P = 0.011) weeks after 40 Gy PHL radiotherapy, and at 4 (61.9% ± 2.0, P = 0.068), 8 (53.5% ± 2.3, P < 0.001), and 16 (55.2% ± 3.6, P < 0.001) weeks after 60 Gy PH radiotherapy. Compared with control (E/e′ = 28.2 ± 2.2 and 27.5 ± 4.5 at 8 and 16 weeks post-radiotherapy, respectively), E/e′ was significantly increased, indicative of diastolic dysfunction, at 16 weeks after 40 Gy PHL radiotherapy (38.8 ± 4.9, P = 0.002) and at 8 (36.4 ± 0.9, P = 0.002) and 16 (40.1 ± 2.3, P < 0.001) weeks after 60 Gy PH radiotherapy.
To evaluate for progressive, time-dependent LVEF and E/e′ changes post-radiotherapy, we analyzed changes within each treatment group. For LVEF, we only observed significant, progressive LVEF decreases in the 60 Gy PH radiotherapy cohort from 4 to 8 weeks post-radiotherapy (P = 0.016). E/e′, however, significantly increased from 8 to 16 weeks after 40 Gy PH radiotherapy (P = 0.026) and 40 Gy PHL radiotherapy (P = 0.011), and 4 to 8 (P = 0.030) and 4 to 16 (P = 0.022) weeks after 60 Gy radiotherapy. The remaining parameters measured with echocardiography are shown in Supplementary Fig. S5.
Dose- and site-dependent decrease in myocardial perfusion
It is well established that SPECT imaging with Tc-99m sestamibi is a useful imaging modality to identify LV wall perfusion deficits (19). Therefore, we carried out cardiac SPECT imaging at 8 weeks post-radiotherapy to provide insight into the physiologic basis for the observed echocardiographic abnormalities. Compared with control, dose-dependent LV apical perfusion deficits were observable post-radiotherapy (Fig. 5A). Upon segmentation and contouring of the LV basal and apical walls (Supplementary Fig. S6), quantification of tracer uptake normalized to tracer activity injected revealed a modest fold-change reduction in myocardial perfusion in the LV base, with greater fold-change reduction in the LV apex after 40 Gy PH (0.839 ± 0.1, P = 0.342), 40 Gy PHL (0.602 ± 0.04, P = 0.006), and 60 Gy PH (0.494 ± 0.03, P = 0.002) radiotherapy compared with control (1.0 ± 0.1; Fig. 5B; Supplementary Table S2A). Similar results were observed when LV wall tracer uptake was normalized to blood pool tracer concentration (Supplementary Table S2B).
Tc-99m sestamibi SPECT. A, From top to bottom row, representative frontal view of control mouse and 40 Gy PH, 40 Gy PHL, and 60 Gy PH radiotherapy mice 8 weeks post-radiotherapy (post-RT). Red arrows highlight differences in apical LV perfusion. B, Basal and apical LV tracer uptake at 8 weeks post-radiotherapy (n = 4/group). *, P < 0.05; **, P < 0.01, (details in Supplementary Table S2).
Cardiomyocyte and vascular stress and systemic inflammatory response
Analysis of biomarkers of heart damage in situ and in circulating blood was another objective of this study. Thus, we evaluated irradiated segments of cardiac tissue from mice and plasma from both humans and mice for known and novel markers of cardiac damage, vascular injury, and inflammation at various timepoints post-radiotherapy (Fig. 6; Supplementary Table S3; Supplementary Fig. S7). In irradiated cardiac tissue, troponin T and PlGF significantly decreased and increased, respectively, at various timepoints between 1 and 8 weeks after 40 Gy PHL and after 60 Gy PH radiotherapy, with PlGF also significantly increased after 40 Gy PH radiotherapy. The inflammatory markers evaluated, IL2, IL6, and TNFα, showed the most significant increases in cardiac tissue compared with control following concomitant PHL radiotherapy (Fig. 6A). These findings prompted a corresponding analysis to be conducted in the plasma of patients who received breast (mean heart dose 1.5 Gy) and lung (mean heart dose 8.6 Gy) radiotherapy, which showed significant increases in PlGF (P = 0.021) and TNFα (P = 0.036) post–lung radiotherapy (Fig. 6B). In addition, the fold change in PlGF was found to be significantly correlated with mean heart dose in the lung cancer subgroup (r = 0.89, P = 0.0001; Fig. 6B). Corresponding analysis in the breast cancer subgroup did not reveal significant correlations (data not shown here), and when compared with the lung cancer subgroup, showed significantly decreased occurrence of PlGF (P = 0.0118) and TNFα (0.0008) increases post-radiotherapy (Fig. 6C). Data from plasma analysis in mice and additional plasma biomarker analysis in humans are shown in Supplementary Fig. S7.
Biochemical analysis of irradiated mouse heart tissue segments and human plasma. A, Effects of focal cardiac radiotherapy on markers of cardiovascular injury and inflammation at 1, 2, 4, and 8 weeks after 40 Gy PH, 40 Gy PHL, and 60 Gy PH radiotherapy (n = 3 or 4 per group unless otherwise noted). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (details in Supplementary Table S3). Cardiac tissue levels of troponin I were above detection range of assay. B, Effects of thoracic radiotherapy in patients with lung cancer on PlGF and TNFα and correlation of fold change in these markers with mean heart dose. C, Comparison of PlGF and TNFα changes in patients with lung versus breast cancer. Data are from 14 patients with lung cancer and 32 patients with breast cancer. Missing data are due to undetectable marker levels in several plasma samples.
Discussion
In this study, we have developed and implemented a novel mouse model of image-guided radiotherapy-induced cardiac damage to study the molecular and cellular pathophysiology of RIHD, and to identify sensitive biomarkers of disease development. Using a contrast agent to improve visualization of cardiac substructures on CBCT imaging prior to radiotherapy, we were able to carry out PH irradiation similar to that encountered in clinical practice. In addition, we were able to use the model to demonstrate dose- and site-dependent radiotherapy-induced damage on radiologic, biochemical, and histologic analyses.
To our knowledge, this is the first mouse model of PH irradiation which implements cardiac conformal radiotherapy, building upon other existing models and offering several unique advantages. First, the model only requires a single tail vein injection of the FenestraVC contrast agent, which aids in radiotherapy beam targeting through improving visualization of cardiac borders. While necessary for most current SARRP instruments, contrast agent injection may prove to be unnecessary in future studies as preliminary implementation of a more advanced SARRP instrument has already shown adequate unenhanced CT resolution, thus increasing the potential feasibility and cost-effectiveness of this model. Second, the use of cardiac conformal radiotherapy in a mouse is itself a novel technological innovation, because it enables high-dose delivery without significant skin toxicity. The PH radiotherapy design not only mimics the cardiac radiotherapy exposure of thoracic radiation oncology patients, but it may also enable the investigation of targeted radioablation for refractory cardiac arrhythmias, a nononcologic use of radiotherapy that is rapidly being incorporated into clinical practice. Finally, although not tested in this study, our model is also amenable to fractionated delivery of radiotherapy, which may further increase its clinical relevance.
Prior mouse models of RIHD have been valuable in determining dose-dependent effects of cardiac dysfunction and have provided insights into the pathophysiologic parameters involved in disease progression (20). However, they have been limited by the fact that they involved either whole thorax or whole heart and partial lung radiotherapy. In addition to such treatment plans not being used clinically, there is also an increased risk of death of the irradiated mice at early timepoints, before long-term toxicities such as fibrosis, which can take months to develop depending on the dose and irradiated area, have manifested. Lee and colleagues carried out PH irradiation in Tie2Cre; p53(FL/−) and Tie2Cre; p53(FL/+) mice; however, due to significant dose constraints (12 Gy), no cardiac damage was detectable on imaging or histology of wild-type mice over 8 weeks of follow-up (21). It should be noted that in our experiments, no mice died because of radiotherapy toxicity over 24 weeks of follow-up despite the very high radiotherapy doses eliciting detectable cardiac damage. This finding underscores a volume and dose effect on mortality after thoracic radiotherapy. It also affords the opportunity to study long-term effects of radiotherapy-induced damage, which is especially useful in studies of cardiovascular damage, which often develops more chronically than damage in other organs.
An important feature of preclinical models of radiotherapy-induced cardiotoxicity is the ability to histologically evaluate cardiac tissue (8, 22), an analysis which can rarely be done in humans. A novel, previously unreported histologic finding from our study is the appearance of significant fibrosis primarily in perivascular regions and largely absent from other areas. Trichrome staining revealed clear vessel wall thickening and morphologic changes, with only scattered areas of interstitial fibrosis and no evidence of replacement fibrosis, suggesting a potential degree of cardiomyocyte radioresistance relative to cardiac vascular endothelium. If validated in human patients, this finding may be useful in guiding prioritization selection of dosimetric constraints in thoracic radiotherapy planning, that is, mean heart dose versus mean left anterior descending arterial dose (23). In addition, we were able to elucidate the topography of the radiotherapy-induced damage, which at higher doses and with radiation exposure of lung tissue extended beyond the small area of radiotherapy, consistent with both direct and systemic components to the radiotherapy-induced damage. Indeed, while localized cardiac damage, as evidenced by increased vessel wall thickness in the irradiated portion of the heart, was greatest after 60 Gy PH, total cardiac fibrosis was actually greatest after lower dose PHL radiotherapy. Finally, it is worth noting that significantly higher doses of radiotherapy were necessary to elicit histologic abnormalities when compared with previous studies of whole heart or whole thorax radiotherapy, which report histopathologic changes at doses ranging from 16 to 24 Gy (10, 11, 24). Given the smaller volume of irradiated tissue, and the relative sparing of large vessels and valves (ascending aorta and valve) reported in this model, this provides evidence in support of both volume of radiotherapy and vessel/valve radiation exposure as significant driving factors in the development of cardiac injury, and further characterization of the dose–response curve and MTD/lowest significant dose could be useful. As this study exclusively involves radiotherapy of the cardiac apex (chosen primarily to minimize dose to critical cardiac vessels and structures during initial development of the model, and to achieve maximal lung radiotherapy sparing in the PH treatment scheme), future studies involving radiotherapy of the cardiac base and ascending aorta would be valuable in elucidating potential differences in resulting cardiotoxicity.
At present, there is great interest in implementing radiologic studies to analyze both acute and chronic effects of radiation-induced cardiotoxicity and identify sensitive biomarkers of disease development. Through the use of quantitative echocardiography, we were able to identify systolic and diastolic dysfunction. However, studies have reported mixed results in both humans (12, 16) and animal models (10, 25) with echocardiography post-radiotherapy, whose value is often limited to characterizing cardiac function (12, 16). SPECT imaging represents an attractive diagnostic approach, as it has demonstrated significant utility in assessing myocardial perfusion (19, 26). The gross perfusion deficits in the area of radiotherapy, and the global decrease in perfusion observed in our study, provide evidence for a significant vascular component to radiotherapy-induced cardiac injury, and was corroborated histologically by vessel wall thickening and fibrosis. Further evaluation of SPECT for the diagnosis of cardiac perfusion abnormalities and risk stratification of thoracic radiotherapy patients would be a useful undertaking for the detection of subclinical cardiotoxicity.
In this study, we also analyzed levels of several markers of myocardial injury, vascular damage, and inflammation. While troponin has routinely been used to diagnose cardiac injury of varying etiologies, studies have failed to demonstrate consistent and significant associations between increased troponin levels and radiotherapy-induced cardiac damage (23, 27, 28). One explanation for this is that, as our study demonstrates, cardiac injury may be primarily mediated by vascular endothelial cell injury and associated inflammation, as opposed to direct myocardial damage. PlGF, a VEGF homolog, has been shown to play crucial roles in atherogenic lesion formation and cardiac vessel wall thickening, and studies have reported its potential role in the progression of cardiovascular disease in the general population (29, 30). More recently, blood PlGF levels have also been evaluated prospectively in thoracic radiotherapy patients, and after radiotherapy PlGF was found to be increased in patients with lymphoma and lung cancer, and associated with mean heart radiation dose, V5, and V30 (percent volume of heart receiving 5 Gy and 30 Gy, respectively). In addition, acute increases in inflammatory cytokines after radiotherapy are well characterized in multiple tissues (18, 31, 32). As such, we conducted a multiparametric analysis of PlGF, IL2, IL6, IL10, and TNFα, which showed significant increases in the cardiac tissue and/or plasma of irradiated mice compared with that of nonirradiated mice. These findings were corroborated in preliminary human plasma studies, with PlGF and TNFα showing significant increases post–lung radiotherapy and PlGF fold changes significantly correlating with mean heart dose, These results not only support the clinical relevance of our model but also highlight the potential for use of these markers to identify cardiac injury in clinical care. Furthermore, as we observed greater marker level changes (particularly PlGF level changes) in patients with lung cancer (higher mean heart doses) compared with patients with breast cancer, future studies could investigate the specificity of PlGF increases for cardiac radiotherapy by evaluating plasma samples pre- and post–noncardiac radiotherapy (liver, intestine, prostate, etc.).
A particularly noteworthy observation was the greater increase in inflammatory markers following PHL irradiation (40 Gy radiotherapy) compared with higher-dose PH irradiation alone (60 Gy conformal radiotherapy). This suggests that lung irradiation may indirectly cause cardiac damage by stimulating a more robust inflammatory response. To date, numerous studies in humans have demonstrated a significant relationship between pulmonary toxicity (e.g., radiation pneumonitis) and the release of inflammatory cytokines following lung radiotherapy (33–35). However, similar data on the potential indirect effects of lung radiotherapy on the heart are lacking. Future investigation in cytokine (e.g., IL6) knockout mice may be useful in elucidating a causative role of inflammatory mediators. In addition, future histopathologic analysis of lung tissue after PH and PHL radiotherapy could more robustly correlate cardiac dysfunction and the degree of pulmonary injury. Nevertheless, when taken together with our radiologic and histologic findings, our results provide evidence for a strong vascular component to radiation-mediated cardiotoxicity, and a potential role of lung irradiation in eliciting an inflammatory cascade causing cardiac damage.
Our study has a number of limitations, one being the fact that radiation was delivered to a beating heart and breathing mouse, creating some degree of variability in radiotherapy targeting and delivery. In addition, to elicit detectable cardiac damage within a practical timeframe, we delivered radiotherapy at doses of 40–60 Gy, which are significantly greater than heart radiotherapy doses patients typically receive; nevertheless, now that we have established and characterized a novel model, lower-dose radiotherapy experiments with longer periods of follow-up (1–2 years) could potentially offer more clinically relevant insights. Another limitation is the use of single fractionation radiotherapy. However, future studies could implement multiple fractionation radiotherapy, or even newer radiotherapy modalities to characterize differences in pathophysiology. Furthermore, the model is amenable to the study of radiosensitizers and radioprotectors (such as antifibrotic agents, including anti-TGFβ antibody and anti-inflammatory agents including anti-IL6 antibody), which could eventually be used to directly influence clinical care. Finally, given the limited number of patients included in the human plasma biomarker analysis, further validation in larger cohorts is needed prior to implementation in the clinic.
In conclusion, we have developed a novel mouse model of PH irradiation with or without lung irradiation as a new preclinical tool to study clinically relevant radiotherapy-induced cardiotoxicity. In addition, we used our model to gain insight into relevant pathophysiologic mechanisms, and identified significant changes in myocardial function and perfusion and vascular and inflammatory biomarkers after radiotherapy. Thus, the model can be used for identification of clinically targetable biologic mediators and ultimately contribute to better strategies for surveillance and treatment of disease.
Authors' Disclosures
S.J. Feigenberg reports personal fees from Varian Medical Systems outside the submitted work. B. Ky reports grants and other from Roche, and other from Cytokinetics and American College of Cardiology outside the submitted work. No disclosures were reported by the other authors.
Authors' Contributions
A.D. Dreyfuss: Conceptualization, resources, data curation, formal analysis, funding acquisition, validation, investigation, methodology, writing–original draft, writing–review and editing. D. Goia: Data curation, formal analysis, methodology, writing–review and editing. K. Shoniyozov: Conceptualization, resources, data curation, writing–review and editing. S.V. Shewale: Resources, Data curation, formal analysis, writing–review and editing. A. Velalopoulou: Data curation, methodology, writing–review and editing. S. Mazzoni: Resources, data curation, methodology. H. Avgousti: Data curation, formal analysis, writing–review and editing. S.D. Metzler: Conceptualization, resources, formal analysis, supervision, methodology, writing–review and editing. P.E. Bravo: Conceptualization, resources, data curation, software, formal analysis, supervision, methodology, writing–review and editing. S.J. Feigenberg: Conceptualization, resources, data curation, formal analysis, supervision, validation, methodology, writing–review and editing. B. Ky: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, methodology, writing–review and editing. I.I. Verginadis: Conceptualization, resources, data curation, software, formal analysis, supervision, validation, investigation, methodology, project administration, writing–review and editing. C. Koumenis: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, methodology, project administration, writing–review and editing.
Acknowledgments
The authors acknowledge the Small Animal Imaging Facility and the Veterinary School of Medicine at the University of Pennsylvania (Pennsylvania, PA) for their invaluable contributions to this study. The authors also thank the University of Pennsylvania Diabetes Research Center for the use of the Biomarkers Core (P30-DK19525). A.D. Dreyfuss was supported by a fellowship from the National Center for Advancing Translational Sciences of the NIH (TL1TR001880). B. Ky was supported by R01 HL 118018 and B. Ky and S.J. Feigenberg by R01 HL148272. This work was partially supported by a grant to C. Koumenis from the Cardio-Oncology Translational Center of Excellence of the Abramson Comprehensive Cancer Center and by developmental funds from the Department of Radiation Oncology at the University of Pennsylvania (Pennsylvania, PA).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Footnotes
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
Clin Cancer Res 2021;27:2266–76
- Received September 30, 2020.
- Revision received December 3, 2020.
- Accepted February 1, 2021.
- Published first February 4, 2021.
- ©2021 American Association for Cancer Research.
References
Volume 27, Issue 8
