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Evaluation of Tumor Microenvironment in an Animal Model using a Nanoparticle Contrast Agent in Computed Tomography Imaging

Posted Dec 31, 2010 Updated Apr 7, 2023
By Ketan B. Ghaghada PhD , Cristian T. Badea PhD , Lohitash Karumbaiah PhD , Nicole Fettig BS , Ravi V. Bellamkonda PhD , G.A. Johnson PhD , Ananth Annapragada PhD
12 min read

Rationale and Objectives

Non-invasive longitudinal imaging of tumor vasculature could provide new insights into the development of solid tumors, facilitating efficient delivery of therapeutics. In this study, we report three-dimensional imaging and characterization of tumor vascular architecture using a nanoparticle contrast agent and high-resolution computed tomography (CT) imaging.

Materials and Methods

Five Balb/c mice implanted with 4T1/Luc syngeneic breast tumors cells were used for the study. The nanoparticle contrast agent was systemically administered and longitudinal CT imaging was performed pre-contrast and at serial time points post-contrast, for up to 7 days for studying the characteristics of tumor-associated blood vessels. Gene expression of tumor angiogenic biomarkers was measured using quantitative real-time polymerase chain reaction.

Results

Early-phase imaging demonstrated the presence of co-opted and newly developed tumor vessels. The co-opted vessels demonstrated wall-permeability and “leakiness” characteristics evident by an increase in extravascular nanoparticle-based signal enhancement visible well beyond the margins of tumor. Diameters of tumor-associated vessels were larger than the contralateral normal vessels. Delayed-phase imaging also demonstrated significant accumulation of nanoparticle contrast agent both within and in areas surrounding the tumor. A heterogeneous pattern of signal enhancement was observed both within and among individual tumors. Gene-expression profiling demonstrated significant variability in several angiogenic biomarkers both within and among individual tumors.

Conclusions

The nanoparticle contrast agent and high-resolution CT imaging facilitated visualization of co-opted and newly developed tumors vessels as well as imaging of nanoparticle accumulation within tumors. The use of this agent could provide novel insights into tumor vascular biology and could have implications on the monitoring of tumor status.

Solid tumors undergo significant changes in their architecture during growth and development. Angiogenesis, the growth of new blood vessels, is one of the hallmarks of growing tumors . The tumor blood vessels show distinctly different patterns compared to blood vessels found in normal tissues . The presence of large pores in the endothelial lining of the vessels and the absence of continuous pericyte and smooth muscle cell coverage are two of the commonly observed traits of “leaky” tumor blood vessels . These abnormalities in tumor vessels often lead to significant changes in the transport processes within the tumor interstitial space, affecting the delivery of nutrients and therapeutics . Rapidly growing tumors also represent a large source of growth factors such as vascular endothelial growth factor (VEGF) and its receptors (eg, VEGFR-1 and VEGFR-2), which are then systemically distributed . The increased levels of growth factors and their receptors are known to have implications for blood vessel permeability, rapid proliferation, and the growth and metastatic potential of tumors. Furthermore, although tumors of different origin are expected (and known) to demonstrate marked variability in tumor-architecture and biomarker levels, inter- and intratumor heterogeneity has been observed even within the same tumor type . This heterogeneity could have significant implications in personalized tumor treatment.

Non-invasive monitoring of tumor architecture coupled with gene-expression profiling could therefore enhance our understanding of growing tumors and its interactions with the surrounding host tissue. Dynamic and longitudinal imaging could also provide insights into the functional characteristics of these tumor blood vessels. One methodology that has been used in preclinical models of solid tumors is noninvasive micro–computed tomography (CT) imaging . The three-dimensional nature of the technique combined with high spatial resolution provides excellent assessment of tumors. The linear relationship between image signal enhancement and contrast agent concentration enables accurate quantitative assessment of tumor vasculature and its micro-environment. However the pre-clinical utility of CT imaging for investigating tumor vasculature has been limited because of two major limitations of conventional contrast agents: their rapid systemic clearance from the blood pool and low vessel conspicuity from high propensity to rapidly extravasate into the tumor extravascular space. In this work, we evaluated the tumor micro-environment using a nanoparticle contrast agent and CT imaging. Specifically, we investigated whether a long circulating blood-pool nanoparticle CT contrast agent can enable 1) visualization of tumor vasculature and 2) demonstration of tumor vessel “leakiness” by imaging nanoparticle extravasation into the tumor interstitial space. Early phase imaging was performed to investigate the feasibility of tumor vascular imaging. Longitudinal delayed-phase imaging was performed to study tumor vessel permeability as well as the transport and accumulation of nanoparticles in tumors. These studies were performed during the National Cancer Institute–sponsored Cancer Imaging Summer Camp, held at Washington University, St. Louis, in June 2009.

Materials and methods

Fabrication of Nanoparticle Contrast Agent

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In Vivo Studies

Animal tumor model

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Micro-CT setup

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Imaging study

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Table 1

Imaging Time Points and Experimental Parameters Used in the Study

Animal Number Animal weight (g) Iodine Dose (mg I/g) Imaging Time Points Pre 0 hours 1 day 2 days 3 days 4 days 5 days 6 days 7 days T-1 16 2.9 X X X X X X T-2 19 3.2 X X X X T-3 20 3.2 X X X X X T-4 18 2.9 X X X X T-5 19 3.0 X X X X

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Quantitative Real-time Polymerase Chain Reaction

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Immuno-histology

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Image Analysis

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Data Analysis

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Statistical Analysis

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Results

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Figure 1, In vivo pharmacokinetic profile and biodistribution of nanoparticle contrast agent. The nanoparticle contrast agent demonstrated long blood residence times. The signal returned to baseline at about 120 hours (5 days) after administration. The agent is primarily cleared by the liver and spleen as indicated by steady signal increase in these organs (a) . Negligible signal enhancement is observed in the kidney, bladder, and muscle indicating in vivo stability of the nanoparticle contrast agent (b) .

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Figure 2, Early-phase imaging of co-opted and neo-angiogenic tumor vessels. Top row : coronal three-dimensional volume-rendered images demonstrating co-opted vessels ( white arrows ) and newly developed tumor vessels (green) in early phase imaging (immediately after nanoparticle contrast agent administration). Both the co-opted and newly developed vessels traced their origin back to major venous structures such as the jugular vein (origin of vessel labeled with white arrow ) or the inferior vena cava (origin of vessel labeled with green arrow ). Corresponding blood vessels on the contralateral side ( blue arrow ) were either absent or not clearly demonstrated. Bottom row : coronal maximum intensity projection images showing an expanded view of the tumor and surrounding regions demonstrating the tumor vasculature.

Figure 3, (a) Analysis of tumor vessel attenuation in precontrast and postcontrast images. The nanoparticle contrast agent enabled visualization of tumor vasculature as demonstrated by statistical differences in precontrast and postcontrast measurements ( P < .05). (b) Longitudinal analysis of tumor signal enhancement. The nanoparticle contrast agent enabled visualization of tumor vessel leakiness as demonstrated by significant signal enhancement in tumor at delayed time points ( P < .05).

Figure 4, Dynamic characteristics of co-opted tumor vessels. Coronal volume-rendered images demonstrating the architecture of co-opted tumor vessels ( a, b ) ( yellow arrow ). The contralateral vessel demonstrated limited development ( blue arrow ). The path of the contralateral vessel could not be traced below the thoracic region on two-dimensional images. Dynamic monitoring of nanoparticle contrast agent extravasation from the co-opted vessel ( c–g ) demonstrated significant extravasation of nanoparticle contrast agent from the co-opted vessel, well beyond the margins of tumor, thus implicating the highly permeable and “leaky” nature of the co-opted vessel. The tumor also demonstrate heterogeneous pattern of signal enhancement suggesting highly permeable intratumoral vessels.

Figure 5, Development of tumor venous system. Coronal maximum intensity projection image comparing the structures of tumor-associated vessels (TV) versus contralateral normal vessels (NV). The tumor venous structures were highly dilated and tortuous compared to their counterparts on the contra-lateral side. Micro–computed tomography imaging was performed immediately after the injection of the nanoparticle contrast agent.

Table 2

Comparison of Vessel Diameters Between Tumor-associated Vessels and Contralateral Normal Vessels

Vessel Origin - Inferior Vena Cava Vessel Origin - Jugular Vein Tumor Identification Tumor vessel diameter (μm) Contralateral vessel diameter (μm) Co-opted vessel diameter (μm) Contra-lateral vessel diameter (μm) T-1 617 ± 66 429 ± 67 448 ± 49 338 ± 9 T-2 696 ± 28 472 ± 3 442 ± 30 381 ± 17 T-3 654 ± 61 473 ± 117 445 ± 32 231 ± 37 T-4 805 ± 42 467 ± 33 194 ± 35 Not visible T-5 642 ± 82 445 ± 27 336 ± 72 231 ± 41

Two types of vessel were analyzed based on their origin. For tumors vessels that originated from inferior vena cava, size analysis was performed for vessel segments surrounding the tumor tissue. Identical contralateral normal vessels were used for comparison. For co-opted vessel originating from the jugular vein, size analysis was performed for vessel segments in the thoraco-abdominal region. Identical contralateral normal vessels were used for comparison. In most animals, the contralateral vessels originating from the jugular vein was not clearly demonstrated below the abdominal region. Values are reported as mean and standard deviation with three to five measurements per vessel.

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Figure 6, Dynamics of tumor signal enhancement. Coronal three-dimensional volume-rendered images demonstrating the extravasation and accumulation of nanoparticle contrast agent within the tumor ( yellow arrow ). Immediately after administering the nanoparticle contrast agent, the overall body and tumor vasculature is nicely demonstrated. Tumor accumulation of nanoparticle contrast agent was observed as early as 24 hours. No image-detectable nanoparticle contrast agent signal was observed from the blood-pool at post-120 hours (day 5) as confirmed by the absence of any vessels or heart signal on post-120 hour image. However, the tumor is clearly enhanced. The only other organs enhanced are the liver and spleen, which are the organs for nanoparticle contrast agent clearance.

Figure 7, Signal enhancement pattern in tumors. Coronal thick-slab (2 mm) maximum intensity projection images demonstrating signal enhancement patterns at different locations in two tumors. Imaging was performed on day 5 (1 a–h ) and day 7 (2 a–h ) after nanoparticle contrast agent administration. Heterogeneous signal enhancement pattern was observed between the tumors as well as within each tumor. Peripheral enhancement indicating nanoparticle contrast agent extravasation from highly permeable vessels was observed. A second inner circular ring of enhancement was observed within the tumor surrounding nonenhanced central core (1 c, d). A region of high nanoparticle contrast agent accumulation, corresponding to large signal enhancement was also observed near the surface ( blue arrow ).

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Figure 8, Comparison of computed tomography (CT) image enhancement with immunohistology. (a) CT image demonstrating negligible enhancement in the tumor central core and significant enhancement surrounding it. (b) Representative hematoxylin and eosin (H&E) staining demonstrating the presence of a central necrotic center and viable region surrounding it; (c) CD-31 staining demonstrates the presence of extensive vessel growth mainly on the periphery of tumor (20× magnification). (d, f) Mac-2 staining demonstrates infiltration of macrophages in the vessel lumen. Negligible macrophages were seen in the tumor interstitial. (e) Representative H&E staining for (d) and (f) .

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Figure 9, Gene-expression profiling of tumors using quantitative real-time polymerase chain reaction (qRT-PCR). Each tumor was divided into four quadrants based on anatomical markers that were added immediately after the final session of computed tomography imaging. The gene expression levels of various angiogenic biomarkers were then determined for each quadrant using qRT-PCR. Expression levels were normalized to muscle. Error bars represent standard errors.

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Discussion

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Acknowledgments

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Academic Radiology, Volume 18, Issue 1
Journals General Radiology
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