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Rosa T. Branca, Zackary I. Cleveland, Boma Fubara, Challa S. S. R. Kumar, Robert R. Maronpot, Carola Leuschner, Warren S. Warren, and Bastiaan Driehuys
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Early and specific detection of metastatic cancer cells in the lung (the most common organ targeted by metastases) could significantly improve cancer treatment outcomes. However, the most widespread lung imaging methods use ionizing radiation and have low sensitivity and/or low specificity for cancer cells. Here we address this problem with an imaging method to detect submillimeter-sized metastases with molecular specificity. Cancer cells are targeted by iron oxide nanoparticles functionalized with cancer-binding ligands, then imaged by high-resolution hyperpolarized 3He MRI. We demonstrate in vivo detection of pulmonary micrometastates in mice injected with breast adenocarcinoma cells. The method not only holds promise for cancer imaging but more generally suggests a fundamentally unique approach to molecular imaging in the lungs.

Keywords

  • hyperpolarized gas MRI
  • superparamagnetic iron oxide nanoparticles
  • luteinizing hormone-releasing hormone

In 2009, ≈1,400,000 people in the United States were diagnosed with cancer, and as many as 562,000 died of the disease (1). Despite impressive increases in the number of cancer drugs and treatments, cancer survival rates have remained low for the past 20 years. Survival in cancer patients depends strongly on cancer containment and is thus inversely correlated with the incidence of metastases (2). Although all of the causes are not fully known, cancer metastases are particularly opportunistic in the lungs and are found in 20–54% of all patients who die of the disease (3). Because the presence of lung metastases will alter cancer management, their early and specific identification could provide a timely and powerful tool for improving patient outcomes.

Detection of lung metastases by current preclinical or clinical imaging techniques has substantial limitations. X-ray computed tomography (CT) permits clinical imaging of pulmonary nodules as small as 1 to 2 mm but lacks the specificity to distinguish benign lesions from cancerous tumors (4, 5). Positron emission tomography with fluorodeoxyglucose (FDG-PET) can differentiate between benign and cancerous lesions, but its low spatial resolution limits its use and reduces its specificity for malignancy in lesions smaller than 5 mm (6, 7). Moreover, both modalities use ionizing radiation, which represents a serious concern in repeated scanning, particularly in young adult patients and children (8). An equally compelling need exists for new preclinical molecular imaging of xenograft murine models. Although they replicate human disease imperfectly, these models provide an expeditious means to explore the biologic determinants of metastases and evaluate novel therapies, while readily permitting histologic correlation. Such preclinical studies would benefit equally from noninvasive longitudinal imaging with better resolution than current methods (≈1.2 mm detection limit for PET and 0.85 mm for micro-CT (9).

Here, we introduce a fundamentally unique, minimally invasive, and specific approach to cancer detection in the lung by combining two MRI technologies—hyperpolarized (HP) 3He and functionalized superparamagnetic iron oxide nanoparticles (SPIONs). SPIONs are a particularly promising class of MR contrast agents, because they generate strong local magnetic susceptibility gradients that rapidly dephase nearby transverse magnetization and thereby produce localized dark spots in an MR image (10–12). Their effect is so strong that a small number of SPION particles can dephase a large number of surrounding spins, allowing even single cells to be detected (13, 14). Different versions of these iron oxide particles (10–100 nm in size) are commercially available and are used to image tumors in the liver and to detect metastasic invasion of lymph nodes (15). Moreover, this contrast agent can be easily functionalized with biologically active ligands to endow them with a high degree of molecular targeting specificity (16, 17). Size and coating are, in this case, key factors to escape macrophage recognition and to improve targeting efficacy (16). Although functionalized SPIONs are not yet approved for clinical use, a great variety of particles have been shown to accumulate selectively in cancer cells (17).

MRI is a well-established diagnostic tool for studying most organs, but this imaging modality is particularly challenged by the lung. The lung’s tissues constitute only 20–25% of the total lung volume, and in the gas exchange regions this density is reduced to 10% (18). The lung’s low density thus generates weak intrinsic MRI signals, but more importantly, its many air–tissue interfaces give rise to substantial field gradients (a few mT/m at 2 T), such that the small available tissue signal decays rapidly with a T2* on the order of 1 ms for humans at 1.5 T (19) and <0.8 ms for small animals at 2.0 T (20). This rapid decay makes it virtually impossible to observe any additional 1H spin dephasing caused by SPIONs in the lung. Fortunately, the recent innovations of HP 3He and 129Xe MRI now permit high-resolution imaging of the lung both clinically (21–25) and preclinically (26–29). The low density of these gases is overcome by increasing their nuclear polarization by 4 to 5 orders of magnitude through laser-based optical pumping techniques (30). Unlike immobilized tissue protons, these gases are much less affected by the lung’s susceptibility gradients because their high diffusivity averages away most of the field gradients experienced at the alveolar walls. This motional narrowing gives the HP gases a transverse relaxation time in the native lungs that is much longer than that of protons

[T2,Xe* ≈50.4 ms (31), T2, He* ≈26.8 ms in humans at 1.5 T (32)], providing a much longer temporal window for SPIONs to induce dephasing.

SPIONs generate a magnetic field gradient that induces a broad phase spread in the transverse magnetization of the HP gases. Although motional narrowing may still average out this dephasing in a single alveolus, the averaging cannot extend across multiple alveoli. This causes a transition to the static dephasing regime and to a macroscopic signal loss similar to that produced by an aggregation of magnetically labeled cells in tissue (33), with a signal decay more likely proportional to the square of the echo time (TE). The long T2* of the HP gases in the native lung permit images to be acquired at longer TE than for 1H MRI, thereby permitting dephasing of a larger surrounding volume of the magnetization. Combined with the high spatial resolution and signal-to-noise ratio (SNR) with which the lung can be imaged using HP gases, they provide an ideal medium with which to detect SPION contrast.

The use of untargeted iron oxide particles, injected intravenously, has already been shown to alter the 3He MRI scan contrast in relation to pulmonary perfusion (34). In this work, we expand on this mechanism by using SPIONs functionalized with luteinizing hormone–releasing hormone (LHRH) to selectively target breast cancers that have metastasized to the lung. LHRH-SPIONs have previously been demonstrated to have low affinity for healthy lung tissue but to accumulate by receptor-mediated endocytosis in lung metastases (72 pg of Fe per cell) that overexpress the LHRH receptor (35). These particles, which have an uncoated size of 12 nm and a hydrodynamic size smaller than 50 nm, are designed for prolonged circulation (more than 12 h of plasma life time) and low opsonization that permits the LHRH-SPIONs to escape macrophage recognition (35). After injection, LHRH-SPIONs accumulate in metastatic cancer cells, forming clusters smaller than 0.5 μm (35) that can then be detected using HP 3He MRI.

In this study, we examined the lungs of nude mice inoculated orthotopically, in the mammary fat pads, with human breast adenocarcinoma (MDA-MB-231, n = 4), or s.c. with either human breast carcinoma cells (MDA-MB-435s, n = 4) or prostate cancer cells (PC3, n = 4). Subcutaneous inoculations of these cancer cell lines in nude mice usually produce a low rate of pulmonary metastases, whereas orthotopic implantations result in a higher rate (36). The metastases, when present, are targeted by LHRH-SPIONs with equal affinity as the primary tumor (35, 37). To detect these metastatic nodules using our protocol, the animals were imaged using HP 3He 24–48 h after an i.p. injection of LHRH-SPIONs (Fig. 1). Intraperitoneal rather than i.v. injections were used to avoid the possibility of SPION cluster aggregation in the pulmonary capillary beds, which could generate false-positive image findings. Immediately after the MRI experiments, the mice were killed by an overdose of pentobarbital, and their lungs were processed for histopathology to identify tumor sites and to provide a gold standard against which 3He MRI could be compared.

Fig. 1. Diagram illustrating the detection of breast and prostate cancer metastases in a mouse lungs. (A) Forty-eight hours before the imaging session, mice receive an i.p. injection of LHRH-SPIONs. (B) Cancer cells overexpressing LHRH receptors on their cell membrane take up LHRH-SPIONs through receptor-mediated endocytosis, causing SPIONs to accumulate in the cell, where they form submicron-sized clusters. (C) The iron uptake is then detected using HP 3He MRI. HP 3He transverse magnetization is rapidly dephased by SPIONs that have clustered in the metastatic lesions. As a result, metastatic nodules are readily visualized in MR images as regions of attenuated signal intensity.

Fig. 1.
Diagram illustrating the detection of breast and prostate cancer metastases in a mouse lungs. (A) Forty-eight hours before the imaging session, mice receive an i.p. injection of LHRH-SPIONs. (B) Cancer cells overexpressing LHRH receptors on their cell membrane take up LHRH-SPIONs through receptor-mediated endocytosis, causing SPIONs to accumulate in the cell, where they form submicron-sized clusters. (C) The iron uptake is then detected using HP 3He MRI. HP 3He transverse magnetization is rapidly dephased by SPIONs that have clustered in the metastatic lesions. As a result, metastatic nodules are readily visualized in MR images as regions of attenuated signal intensity.

Results

A convenient method for demonstrating SPION-induced dephasing of 3He MRI in the lung is to exploit the nonspecific uptake of SPIONs in the right cranial mediastinal lymph node, which sits directly under the right cranial lobe of the lung. This uptake is caused by the drainage of the nanoparticles from the i.p. cavity to the lymphatic system (38). When the contrast agent is administrated i.p., some of the SPIONs accumulate into the right cranial mediastinal lymph node and produce a strong susceptibility effect that dephases the 3He spins in the nearby lung parenchyma, causing a local signal loss. An example is shown in Fig. 2: the top row (Fig. 2A) shows a control animal that received no SPIONs, whereas the middle row (Fig. 2B) shows an animal with SPIONs in the lymph node and the associated 3He signal attenuation. The presence of iron in the lymph node was confirmed by histologic examination, as shown in Fig. 2 D and E. Iron in this lymph node was detected by3He MRI in all tumor-bearing animals that did not develop lung metastases. Histologic examination of the healthy lung tissues in all animals revealed no iron and indicates that these particles escaped recognition by the lung macrophages, consistent with prior studies (35).

Fig. 2. Effect of SPION on HP 3He images. (A) HP images (TE = 1 ms) from a control mouse that did not receive LHRH-SPION injection. (B) Images from a human prostate mouse model (TE = 1 ms) after injection of LHRH-SPIONs. Although the lung parenchyma is free of metastases, the iron uptake in the right cranial mediastinal lymph node caused by the i.p. injection produces a clear signal defect in the helium images (yellow circles). (C) 3D volume rendering of the HP helium image dataset. (D) H&E examination of the excised lung tissue (magnification, 1×). (E) Magnification (40×) of the same area, showing the lymph node and the iron uptake.

Fig. 2.
Effect of SPION on HP 3He images. (A) HP images (TE = 1 ms) from a control mouse that did not receive LHRH-SPION injection. (B) Images from a human prostate mouse model (TE = 1 ms) after injection of LHRH-SPIONs. Although the lung parenchyma is free of metastases, the iron uptake in the right cranial mediastinal lymph node caused by the i.p. injection produces a clear signal defect in the helium images (yellow circles). (C) 3D volume rendering of the HP helium image dataset. (D) H&E examination of the excised lung tissue (magnification, 1×). (E) Magnification (40×) of the same area, showing the lymph node and the iron uptake.

The nonspecific SPION uptake in the lymph node was also used to demonstrate the enhancement of SPION contrast with increasing TE. Fig. 3 shows that, although some 3He signal attenuation is already observable at short TE (1 ms), it is significantly enhanced at longer TE (4 ms). This pattern was observed in all experiments, indicating that the sensitivity of this technique (i.e., the contrast-to-noise ratio) is enhanced at longer TE. Moreover, the signal void seen on 3He MRI is larger than the actual cluster of iron particles that gives rise to it. In this example, the SPIONs were found to be loosely clustered over a 300-μm area but gave rise to a signal void with a 1,200-μm diameter on 3He imaging.

Fig. 3. Detection sensitivity. (A) HP 3He lung images from a prostate tumor model mouse at TE = 1 ms. (B) Same image slice at TE = 4 ms. The signal loss in the right cranial lobe, due to the nonspecific iron uptake in the right cranial mediastinal lymph node, is clearly enhanced at longer TE, where it reaches a size of 1.2 mm. (C) Magnification of the signal loss area (TE = 4 ms). (D and E) Magnification of the same area (20× and 100×) in the histologic slide, which reveals a lymph node of 300 μm.

Fig. 3.
Detection sensitivity. (A) HP 3He lung images from a prostate tumor model mouse at TE = 1 ms. (B) Same image slice at TE = 4 ms. The signal loss in the right cranial lobe, due to the nonspecific iron uptake in the right cranial mediastinal lymph node, is clearly enhanced at longer TE, where it reaches a size of 1.2 mm. (C) Magnification of the signal loss area (TE = 4 ms). (D and E) Magnification of the same area (20× and 100×) in the histologic slide, which reveals a lymph node of 300 μm.

Fig. 4 shows an example of 3D radial 3He images acquired at TE = 4 ms from a control animal and from a mouse bearing a human breast adenocarcinoma xenograft, after the injection of the LHRH-SPION. Whereas the control animal showed high signal intensity throughout the lungs, the tumor-bearing animal showed a pronounced signal loss that affected the entire right lung. Comparison of 3He MRI against H&E histology confirmed that 3He signal voids occurred in the region of the lung parenchyma that contained disseminated micrometastases (Fig. 5A). Additionally, Prussian blue staining revealed that these micrometastases had been heavily targeted by LHRH-SPIONs (Fig. 5C). Further immunohistochemical staining confirmed that SPIONs did not accumulate in healthy lung tissue (Fig. 5D), which are free of LHRH receptors (Fig. 5F), but were confined to cancer cells with high LHRH receptor expression (Fig. 5 E–H).