Abstract Background: Functional and molecular changes often precede gross anatomical changes in cancer, so early assessment of a tumor’s functional and molecular response to therapy can help reduce a patient’s exposure to the side effects of ineffective chemotherapeutics or other treatment strategies. Clear-cell renal cell carcinoma (ccRCC) is an aggressive and hyper-vascular form of renal cancer that is often treated with anti-angiogenic and Notch Inhibition therapies, which target the vasculature feeding the disease. The purpose of this work is to show that ultrasound microvascular imaging can provide indications of response to antiangiogenic and Notch Inhibition therapies prior to measurable changes in tumor size. Methods: Mice bearing 786-O ccRCC xenograft tumors were treated with SU (Sunitnib malate, Selleckchem, TX), an antiangiogenic drug, and a combination of SU and the Notch inhibitor GSI (Gamma secretase inhibitor, PF-03084014, Pfizer, New York, NY) therapies (n=8). A 3D ultrasound system (SonoVol Inc., Research Triangle Park, NC), in addition to microbubble ultrasound contrast agents, was used to obtain a measurement of microvascular density over time and assess the response of the tumors to the therapies. CD31 immunohistochemistry was performed to serve as a gold standard for comparison against imaging results. Statistical tests included: Spearman correlation to compare imaging and histology; Kruskal-Wallis analysis with Tukey multiple comparison post-test to determine if the vessel density or tumor volume were significantly different between the treatment groups; and receiver operating characteristic (ROC) curve analysis to determine sensitivity/specificity for separating treated/untreated groups. Results: Data indicated that ultrasound-derived microvascular density can detect response to antiangiogenic and Notch inhibition therapies a week prior to changes in tumor volume. Furthermore, the imaging measurements of vasculature are strongly correlated with physiological characteristics of the tumors as measured by histology (p=0.75). Moreover, data demonstrated that ultrasound measurements of vascular density can determine response to therapy and classify between-treatment groups 1 week after the start of treatment with a high sensitivity and specificity of 94% and 86%, respectively. Conclusion: This work shows vascular density measurements that are strongly correlated with histology can be obtained using ultrasound, and that imaging-derived vessel density metrics may be a better tool for assessing the response of ccRCC to antiangiogenic and Notch inhibition therapies than anatomical size measurements. Note: This abstract was not presented at the meeting. Citation Format: Juan D. Rojas, Virginie Papadopoulou, Tomasz Czernuszewicz, Rajalekha Rajamahendiran, Anna Chytil, Yun-Chen Chiang, Diana Chong, Victoria L. Bautch, Wendy K. Rathmell, Stephen Aylward, Ryan Gessner, Paul Dayton. Early treatment response detected in a murine clear cell renal cell carcinoma model in response to combination therapy with antiangiogenic and notch inhibition therapy using a non-invasive imaging tool [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1958.
Functional and molecular changes often precede gross anatomical changes, so early assessment of a tumor's functional and molecular response to therapy can help reduce a patient's exposure to the side effects of ineffective chemotherapeutics or other treatment strategies.Our intent was to test the hypothesis that an ultrasound microvascular imaging approach might provide indications of response to therapy prior to assessment of tumor size.Mice bearing clear-cell renal cell carcinoma xenograft tumors were treated with antiangiogenic and Notch inhibition therapies. An ultrasound measurement of microvascular density was used to serially track the tumor response to therapy.Data indicated that ultrasound-derived microvascular density can indicate response to therapy a week prior to changes in tumor volume and is strongly correlated with physiological characteristics of the tumors as measured by histology ([Formula: see text]). Furthermore, data demonstrated that ultrasound measurements of vascular density can determine response to therapy and classify between-treatment groups with high sensitivity and specificity.Results suggests that future applications utilizing ultrasound imaging to monitor tumor response to therapy may be able to provide earlier insight into tumor behavior from metrics of microvascular density rather than anatomical tumor size measurements.
This study presents a novel dual-modality imaging system for assessing cancer progression in rodents. The system incorporates bioluminescence imaging (BLI), used to assess tumor growth, and contrast-enhanced ultrasound (CEUS), used to assess anatomical information and map microvasculature. The combination of the two modalities has previously been shown to reduce inter-user variability of BLI quantification, and in this work, we demonstrate that a dual BLI/US system can provide a more holistic assessment of disease.NSG (NOD/scid/gamma) female mice were implanted with luc-tagged lymphoma cells (BCBL-1, RRID: CVCL_0165, 1x105 cells, intraperitoneal (IP) injection, N = 8 mice) and imaged using the US and BLI hybrid modality system (SonoVol, Inc.), and a BLI-alone system (Perkin Elmer, Inc.) for comparison to a widely available commercial BLI system. BLI sensitivity was evaluated using a weakly luminescent tritium phantom to find the shortest exposure required to detect signal. In vivo studies consisted of an IP injection of D-luciferin (250 µL at 15 mg/mL) and serial captures of images with exposure times of 60 s every 3 min. Acoustic Angiography (AA), a high-resolution CEUS technique, was used to acquire 3D volumes in the abdomen surrounding the tumor site to assess angiogenesis-induced vascular remodeling associated with tumor growth.In vitro BLI sensitivity experiments showed that the dual-modality system required an exposure of 3 sec to detect signal (p < 0.05) and the BLI-alone system required an exposure of 1 sec (p< 0.05). For in vivo studies, the change in luminescence occurring between week 2 and 3 post-cell implantation was calculated (a surrogate measurement for tumor growth), and the difference in signal was 17.15 ± 10.1 photons/sec and 16.04 ± 7.6 for the dual-modality and BLI-alone systems, respectively. Images of the vascular remodeling arising during the first two weeks of tumor growth were captured with AA and demonstrated an increase in perfusion in the vicinity of BLI signal by a factor of 1.4 ± 0.38, with vascular remodeling being evident even at the periphery of BLI signal.This work demonstrates that non-invasive measurements of in vivo microvascular remodeling can be precisely mapped to changes in tumor growth with a hybrid modality system. The system has comparable sensitivity to a BLI-alone system and provides similar assessments of longitudinal tumor growth. Adding quantitative metrics for vascular remodeling to the widely used luminescent imaging could provide a more comprehensive assessment for tumor functional status than either modality could individually. This should prove valuable when using antiangiogenic therapies because changes in vasculature will precede cell death, and the ability to monitor both the cells and their blood supply might help to elucidate underlying biological processes.Citation Format: Juan D. Rojas, Rajalekha Rajamahendiran, Tomasz J. Czernuszewicz, Brian Velasco, Jonathan Perdomo, Max Harlacher, Graeme O'Connell, James Butler, Blossom Damania, Paul A. Dayton, Ryan C. Gessner. Tracking angiogenesis induced microvascular changes in a lymphoma model via a new high throughput non-invasive dual modality imaging platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1935.
Background: Preclinical ultrasound (US) and contrast-enhanced ultrasound (CEUS) imaging have long been used in oncology to noninvasively measure tumor volume and vascularity. While the value of preclinical US has been repeatedly demonstrated, these modalities are not without several key limitations that make them unattractive to cancer researchers, including: high user-variability, low throughput, and limited imaging field-of-view (FOV). Herein, we present a novel robotic preclinical US/CEUS system that addresses these limitations and demonstrates its use in evaluating tumors in 3D in a rodent model.Methods: The imaging system was designed to allow seamless whole-body 3D imaging, which requires rodents to be imaged without physical contact between the US transducer and the animal. To achieve this, a custom dual-element transducer was mounted on a robotic carriage, submerged in a hydrocarbon fluid, and the reservoir sealed with an acoustically transmissive top platform. Eight NOD/scid/gamma (NSG) female mice were injected subcutaneously in the flank with 8×109 786-O human clear-cell renal cell carcinoma (ccRCC) cells. Weekly imaging commenced after tumors reached a size of 150 mm3 and continued until tumors reached a maximum size of 1 cm3 (∼4-5 weeks). An additional six nude athymic female mice were injected subcutaneously in the flank with 7 × 105 SVR angiosarcoma cells to perform an inter-operator variability study. Imaging consisted of 3D B-mode (conventional ultrasound) of the whole abdomen (< 1 min), as well as contrast-enhanced acoustic angiography (< 10 min) to measure blood vessel density (BVD). Tumors were manually segmented in 3D (< 2 min) and inter-operator and inter-reader reliability was assessed with Krippendorff's alpha.Results: Wide-field US images reconstructed from 3D volumetric data showed superior FOV over conventional US. Several anatomical landmarks could be identified within each image surrounding the tumor, including the liver, small intestines, bladder, and inguinal lymph nodes. Tumor boundaries were clearly delineated in both B-mode and BVD images, with BVD images showing heterogeneous microvessel density at later timepoints suggesting tumor necrosis. Excellent agreement was measured for both inter-reader and inter-operator experiments, with alpha coefficients of 0.914 (95% CI: 0.824-0.948) and 0.959 (0.911-0.981), respectively.Conclusion: We have demonstrated a novel preclinical US imaging system that can accurately and consistently evaluate tumors in rodent models. The system leverages cost-effective robotic technology, and a new scanning paradigm that allows for easy and reproducible data acquisition to enable wide-field, 3D, multi-parametric ultrasound imaging.Note: This abstract was not presented at the meeting.Citation Format: Tomasz Czernuszewicz, Virginie Papadopoulou, Juan D. Rojas, Rajalekha Rajamahendiran, Jonathan Perdomo, James Butler, Max Harlacher, Graeme O'Connell, Dzenan Zukic, Paul A. Dayton, Stephen Aylward, Ryan C. Gessner. A preclinical ultrasound platform for widefield 3D imaging of rodent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1955.
Abstract This study presents a novel dual-modality imaging system for assessing cancer progression in rodents. The system incorporates bioluminescence imaging (BLI), used to assess tumor growth, and contrast-enhanced ultrasound (CEUS), used to assess anatomical information and map microvasculature. The combination of the two modalities has previously been shown to reduce inter-user variability of BLI quantification, and in this work, we demonstrate that a dual BLI/US system can provide a more holistic assessment of disease. NSG (NOD/scid/gamma) female mice were implanted with luc-tagged lymphoma cells (BCBL-1, RRID: CVCL_0165, 1x105 cells, intraperitoneal (IP) injection, N = 8 mice) and imaged using the US and BLI hybrid modality system (SonoVol, Inc.), and a BLI-alone system (Perkin Elmer, Inc.) for comparison to a widely available commercial BLI system. BLI sensitivity was evaluated using a weakly luminescent tritium phantom to find the shortest exposure required to detect signal. In vivo studies consisted of an IP injection of D-luciferin (250 µL at 15 mg/mL) and serial captures of images with exposure times of 60 s every 3 min. Acoustic Angiography (AA), a high-resolution CEUS technique, was used to acquire 3D volumes in the abdomen surrounding the tumor site to assess angiogenesis-induced vascular remodeling associated with tumor growth. In vitro BLI sensitivity experiments showed that the dual-modality system required an exposure of 3 sec to detect signal (p < 0.05) and the BLI-alone system required an exposure of 1 sec (p< 0.05). For in vivo studies, the change in luminescence occurring between week 2 and 3 post-cell implantation was calculated (a surrogate measurement for tumor growth), and the difference in signal was 17.15 ± 10.1 photons/sec and 16.04 ± 7.6 for the dual-modality and BLI-alone systems, respectively. Images of the vascular remodeling arising during the first two weeks of tumor growth were captured with AA and demonstrated an increase in perfusion in the vicinity of BLI signal by a factor of 1.4 ± 0.38, with vascular remodeling being evident even at the periphery of BLI signal. This work demonstrates that non-invasive measurements of in vivo microvascular remodeling can be precisely mapped to changes in tumor growth with a hybrid modality system. The system has comparable sensitivity to a BLI-alone system and provides similar assessments of longitudinal tumor growth. Adding quantitative metrics for vascular remodeling to the widely used luminescent imaging could provide a more comprehensive assessment for tumor functional status than either modality could individually. This should prove valuable when using antiangiogenic therapies because changes in vasculature will precede cell death, and the ability to monitor both the cells and their blood supply might help to elucidate underlying biological processes. Citation Format: Juan D. Rojas, Rajalekha Rajamahendiran, Tomasz J. Czernuszewicz, Brian Velasco, Jonathan Perdomo, Max Harlacher, Graeme O'Connell, James Butler, Blossom Damania, Paul A. Dayton, Ryan C. Gessner. Tracking angiogenesis induced microvascular changes in a lymphoma model via a new high throughput non-invasive dual modality imaging platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1935.
Background: Functional and molecular changes often precede gross anatomical changes in cancer, so early assessment of a tumor's functional and molecular response to therapy can help reduce a patient's exposure to the side effects of ineffective chemotherapeutics or other treatment strategies. Clear-cell renal cell carcinoma (ccRCC) is an aggressive and hyper-vascular form of renal cancer that is often treated with anti-angiogenic and Notch Inhibition therapies, which target the vasculature feeding the disease. The purpose of this work is to show that ultrasound microvascular imaging can provide indications of response to antiangiogenic and Notch Inhibition therapies prior to measurable changes in tumor size.Methods: Mice bearing 786-O ccRCC xenograft tumors were treated with SU (Sunitnib malate, Selleckchem, TX), an antiangiogenic drug, and a combination of SU and the Notch inhibitor GSI (Gamma secretase inhibitor, PF-03084014, Pfizer, New York, NY) therapies (n=8). A 3D ultrasound system (SonoVol Inc., Research Triangle Park, NC), in addition to microbubble ultrasound contrast agents, was used to obtain a measurement of microvascular density over time and assess the response of the tumors to the therapies. CD31 immunohistochemistry was performed to serve as a gold standard for comparison against imaging results. Statistical tests included: Spearman correlation to compare imaging and histology; Kruskal-Wallis analysis with Tukey multiple comparison post-test to determine if the vessel density or tumor volume were significantly different between the treatment groups; and receiver operating characteristic (ROC) curve analysis to determine sensitivity/specificity for separating treated/untreated groups.Results: Data indicated that ultrasound-derived microvascular density can detect response to antiangiogenic and Notch inhibition therapies a week prior to changes in tumor volume. Furthermore, the imaging measurements of vasculature are strongly correlated with physiological characteristics of the tumors as measured by histology (p=0.75). Moreover, data demonstrated that ultrasound measurements of vascular density can determine response to therapy and classify between-treatment groups 1 week after the start of treatment with a high sensitivity and specificity of 94% and 86%, respectively.Conclusion: This work shows vascular density measurements that are strongly correlated with histology can be obtained using ultrasound, and that imaging-derived vessel density metrics may be a better tool for assessing the response of ccRCC to antiangiogenic and Notch inhibition therapies than anatomical size measurements.Note: This abstract was not presented at the meeting.Citation Format: Juan D. Rojas, Virginie Papadopoulou, Tomasz Czernuszewicz, Rajalekha Rajamahendiran, Anna Chytil, Yun-Chen Chiang, Diana Chong, Victoria L. Bautch, Wendy K. Rathmell, Stephen Aylward, Ryan Gessner, Paul Dayton. Early treatment response detected in a murine clear cell renal cell carcinoma model in response to combination therapy with antiangiogenic and notch inhibition therapy using a non-invasive imaging tool [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1958.
Abstract Background: Preclinical ultrasound (US) and contrast-enhanced ultrasound (CEUS) imaging have long been used in oncology to noninvasively measure tumor volume and vascularity. While the value of preclinical US has been repeatedly demonstrated, these modalities are not without several key limitations that make them unattractive to cancer researchers, including: high user-variability, low throughput, and limited imaging field-of-view (FOV). Herein, we present a novel robotic preclinical US/CEUS system that addresses these limitations and demonstrates its use in evaluating tumors in 3D in a rodent model. Methods: The imaging system was designed to allow seamless whole-body 3D imaging, which requires rodents to be imaged without physical contact between the US transducer and the animal. To achieve this, a custom dual-element transducer was mounted on a robotic carriage, submerged in a hydrocarbon fluid, and the reservoir sealed with an acoustically transmissive top platform. Eight NOD/scid/gamma (NSG) female mice were injected subcutaneously in the flank with 8×109 786-O human clear-cell renal cell carcinoma (ccRCC) cells. Weekly imaging commenced after tumors reached a size of 150 mm3 and continued until tumors reached a maximum size of 1 cm3 (∼4-5 weeks). An additional six nude athymic female mice were injected subcutaneously in the flank with 7 × 105 SVR angiosarcoma cells to perform an inter-operator variability study. Imaging consisted of 3D B-mode (conventional ultrasound) of the whole abdomen (< 1 min), as well as contrast-enhanced acoustic angiography (< 10 min) to measure blood vessel density (BVD). Tumors were manually segmented in 3D (< 2 min) and inter-operator and inter-reader reliability was assessed with Krippendorff’s alpha. Results: Wide-field US images reconstructed from 3D volumetric data showed superior FOV over conventional US. Several anatomical landmarks could be identified within each image surrounding the tumor, including the liver, small intestines, bladder, and inguinal lymph nodes. Tumor boundaries were clearly delineated in both B-mode and BVD images, with BVD images showing heterogeneous microvessel density at later timepoints suggesting tumor necrosis. Excellent agreement was measured for both inter-reader and inter-operator experiments, with alpha coefficients of 0.914 (95% CI: 0.824-0.948) and 0.959 (0.911-0.981), respectively. Conclusion: We have demonstrated a novel preclinical US imaging system that can accurately and consistently evaluate tumors in rodent models. The system leverages cost-effective robotic technology, and a new scanning paradigm that allows for easy and reproducible data acquisition to enable wide-field, 3D, multi-parametric ultrasound imaging. Note: This abstract was not presented at the meeting. Citation Format: Tomasz Czernuszewicz, Virginie Papadopoulou, Juan D. Rojas, Rajalekha Rajamahendiran, Jonathan Perdomo, James Butler, Max Harlacher, Graeme O'Connell, Dzenan Zukic, Paul A. Dayton, Stephen Aylward, Ryan C. Gessner. A preclinical ultrasound platform for widefield 3D imaging of rodent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1955.
Noninvasive in vivo imaging technologies enable researchers and clinicians to detect the presence of disease and longitudinally study its progression. By revealing anatomical, functional, or molecular changes, imaging tools can provide a near real-time assessment of important biological events. At the preclinical research level, imaging plays an important role by allowing disease mechanisms and potential therapies to be evaluated noninvasively. Because functional and molecular changes often precede gross anatomical changes, there has been a significant amount of research exploring the ability of different imaging modalities to track these aspects of various diseases. Herein, we present a novel robotic preclinical contrast-enhanced ultrasound system and demonstrate its use in evaluating tumors in a rodent model. By leveraging recent advances in ultrasound, this system favorably compares with other modalities, as it can perform anatomical, functional, and molecular imaging and is cost-effective, portable, and high throughput, without using ionizing radiation. Furthermore, this system circumvents many of the limitations of conventional preclinical ultrasound systems, including a limited field-of-view, low throughput, and large user variability.
