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. Author manuscript; available in PMC: 2023 Oct 1.
Published in final edited form as: Invest Radiol. 2022 Jun 22;57(10):655–663. doi: 10.1097/RLI.0000000000000893

Next Generation Cancer Magnetic Resonance Imaging with Tumor-Targeted Alkylphosphocholine Metal Analogs

Ray R Zhang 1,2, Cynthia Choi 3, Christina L Brunnquell 4,6, Reinier Hernandez 1,4, Anatoly N Pinchuk 1, Joseph G Grudzinski 1, Paul A Clark 2, Alan B McMillan 1, Anjon Audhya 5, Justin Jeffrey 5, John S Kuo 2,5,7, Jamey P Weichert 1,5
PMCID: PMC9469686  NIHMSID: NIHMS1802964  PMID: 36069439

Abstract

Objectives:

In an effort to exploit the elevated need for phospholipids displayed by cancer cells relative to normal cells, we have developed tumor-targeted alkylphosphocholines (APC) as broad-spectrum cancer imaging and therapy agents. Radioactive APC analogs have exhibited selective uptake and prolonged tumor retention in over 50 cancer types in preclinical models as well as over 15 cancer types in over a dozen clinical trials. In order to push the structural limits of this platform, we recently added a chelating moiety capable of binding gadolinium and many other metals for cancer-targeted magnetic resonance imaging (MRI), PET imaging, and targeted radionuclide therapy. The aim of this work was to synthesize, characterize, and validate the tumor selectivity of a new broad-spectrum, tumor-targeted, macrocyclic MRI chelate, Gd-NM600, in xenograft and orthotopic tumor models. A secondary aim was to identify and track the in vivo chemical speciation and spatial localization of this new chelate Gd-NM600 in order to assess its Gd deposition properties.

Materials and Methods:

T1relaxivities of Gd-NM600 were characterized in water and plasma at 1.5T and 3.0T. Tumor uptake and subcellular localization studies were performed using transmission electron microscopy. We imaged 8 different preclinical models of human cancer over time and compared the T1-weighted imaging results to that of a commercial macrocyclic Gd chelate, Gd-DOTA. Finally, MALDI-MSI was used to characterize and map the tissue distribution of the chemical species of Gd-NM600.

Results:

Gd-NM600 exhibits high T1 relaxivity (approximately 16.4 s-1/mM at 1.5T), excellent tumor uptake (3.95 %ID/g at 48 hours), prolonged tumor retention (7 days) and MRI conspicuity. Moreover, minimal tumor uptake saturability of Gd-NM600 was observed. Broad-spectrum tumor-specific uptake was demonstrated in eight different human cancer models. Cancer cell uptake of Gd-NM600 via endosomal internalization and processing was revealed with transmission electron microscopy. Importantly, tissue mass spectrometry imaging successfully interrogated the spatial localization and chemical speciation of Gd compounds and also identified breakdown products of Gd species.

Conclusion:

We have introduced a new macrocyclic cancer-targeted Gd chelate that achieves broad-spectrum tumor uptake and prolonged retention. Furthermore, we have demonstrated in vivo stability of Gd-NM600 by ultrahigh resolution MS tissue imaging. A tumor-targeted contrast agent coupled with the enhanced imaging resolution of MRI relative to PET may transform oncologic imaging.

Introduction

Most clinically approved contrast agents for MRI are paramagnetic gadolinium chelates, which only transiently enhance tumors during their renal excretion phase, and are typically limited by relatively low T1 relaxivities (1–4). The low sensitivity of MRI contrast agents (detectable at μM-nM concentrations) presents a barrier to targeted MRI of molecular processes. Accordingly, whereas receptor-targeting strategies for PET and SPECT imaging have enjoyed marked success in tumor imaging, receptor-based MRI agents have failed to achieve sufficient contrast signal due to inadequate levels of even the most highly-expressed receptors (3, 5). Cancer uptake of these non-targeted contrast agents is passive and transient, via blood-brain barrier disruption and/or the enhanced permeability and retention (EPR) effect (2, 4, 6). Therefore, uptake more accurately reflects aberrations in vascularity, permeability, and tissue structure rather than molecular differences between cancer and normal tissue, thus contributing to diagnostic inaccuracies (5, 7, 8). Moreover, the recent controversy surrounding off-target gadolinium deposition that has led to Food and Drug Administration warnings on all commercial Gd chelates, as well as the suspension of some linear chelates by the European Medicine Agency (9). Although more evidence suggests that the linear chelates do deposit more in brain structures and other organs compared to macrocyclic chelates, some of the linear chelate agents that have been suspended in Europe have the highest relaxivities (MultiHance; Bracco diagnostic Inc, Milan, Italy) and thus superior diagnostic accuracy of cancer compared to its macrocyclic Gd chelates (9).

We report here, a novel tumor-targeted, macrocyclic Gd chelate that demonstrates superior relaxivity and selective uptake and prolonged retention by numerous cancer cells and models in vitro and in vivo compared to commercial macrocyclic Gd chelates. This novel agent circumvents many limitations of conventional MRI contrast agents through an observed high-capacity tumor cell uptake mechanism (10–20). Gd-NM600, which demonstrates significantly higher and prolonged tumor enhancement relative to commercial macrocyclic MRI agents, also possesses significantly higher relaxivity and stability than commercial linear Gd chelates.

Many of these favorable characteristics are attributable to the small-molecule alkylphosphocholine platform (21). Tumor-targeted APC analogs developed in our lab have demonstrated extensive broad-scope tumor uptake and retention in numerous nuclear medicine imaging clinical trials, targeted radiotherapy clinical trials, and in preclinical fluorescence guided surgical studies (12, 13, 15–20, 22–25). Due to the success of the precursor analogs, we synthesized and explored the biodistribution properties of Gd-NM600, a DOTA chelate of the tumor targeting APC molecule conjugated with gadolinium, in order to assess its potential for targeted MRI of cancer. Indeed, substitution of the iodine atom in the prior generation APC analogs with a DOTA chelate did not perturb the tumor avidity of this molecular platform and, in fact, afforded several benefits including faster tumor uptake kinetics and faster blood clearance properties.

The favorable characteristics of Gd-NM600 including broad-spectrum tumor targeting, high relaxivity, faster kinetics, and macrocyclic chelate stability poise this agent for clinical translation. This agent may lead to improvements in diagnostic accuracy in several body regions including the in head and neck, spinal cord, breast, liver, and musculoskeletal imaging where MRI outperforms other imaging modalities in terms of accuracy, and soft-tissue resolution for purposes of local cancer staging, as well as whole-body staging of non-FDG avid malignancies (26–29).

Lastly, because the chemical speciation of Gd chelates remains an important area of clinical relevance, we also developed a mass spectrometry methodology that would allow us to interrogate the chemical speciation with high sensitivity and specificity and spatial localization of our agent. We also demonstrate that this method can be used to characterize the chemical speciation of commercial linear and macrocyclic Gd chelates, which would enhance our understanding of the behavior of Gd chelates and the mechanism of gadolinium deposition. This methodology will enhance our understanding of the behavior of Gd chelates used extensively in MRI, and may finally shed light on the mechanisms of gadolinium deposition.

Materials and Methods

Animals

All animal studies were performed in compliance with protocols approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee. Studies were performed on mice bearing orthotopic glioblastoma or glioblastoma stem cell (U87, n=7 and GSC 12.1, n=6, respectively), αβ-overexpressing triple negative breast cancer (n=4), flank prostate cancer (PC3, n=3), flank non-small cell lung cancer (A549, n=9), flank colorectal cancer (HT29, n=3), flank pancreatic cancer (MiaPaca, n=3), and flank glioblastoma (U87, n=12). For flank xenograft models, 1–2×106 cells were injected into the right flank of nude athymic mice, and tumors were grown to a diameter of 5–15mm. For orthotopic xenograft glioblastoma models, 1–2×106 U87 or glioblastoma stem cells (GSC 12.1) were stereotactically injected in the right striatum of immunodeficient NOD/SCID mice (30–32). For breast cancer models, 106 αβ-overexpressing triple negative breast cancer cells were prepared as previously described and injected into the mammary ducts (33). APC analogs were formulated for injection as previously reported (10).

Relaxivity Measurements

Relaxivity of Gd-NM600 was measured at 1.5T and 3.0T (GE Signa HDxt and Signa PET/MR, GE Healthcare, Waukesha, WI) at 37°C. Gd-NM600 samples were prepared at concentrations of 0.125–1 mM in water, human plasma, and 0.125 mM human serum albumin. For relaxivity measurements, conical tubes containing 0.125–1 mM Gd-NM600 were warmed to 37°C in an MRI-compatible circulating water bath. For T1 measurement, an inversion recovery pulse sequence with inversion time TI=50–750ms, repetition time TR=4000ms/5000ms (1.5T/3.0T), and echo time TE=8–9ms was utilized. For T2 measurement, a spin-echo sequence with TR=5000ms and TE=50–600ms was utilized. T1 and T2 times were estimated using nonlinear least squares fitting of the signal magnitude vs. inversion time and echo time, respectively. Longitudinal (r1) and transverse (r2) relaxivities of Gd-NM600 were estimated from the slope of the linear relationship between relaxation rate and agent concentration.

Tumor Imaging

Gd-NM600 was administered intravenously (0.12g/kg=0.11mmol/kg, up to 0.18g/kg=0.165mmol/kg) in eight rodent models (n=5) of human cancer: mouse models include an orthotopic glioblastoma, a triple negative breast cancer, and flank xenografts of prostate cancer, non-small cell lung cancer, colorectal cancer, pancreatic cancer, and glioblastoma, and a rat flank glioblastoma xenograft model. In vivo MR imaging of mice was performed on a 4.7T preclinical scanner (Agilent Technologies, Santa Clara, CA) pre-injection and at multiple time points for up to seven days following i.v. contrast administration. T1-weighted tumor imaging was performed with a 2D fast spin echo pulse sequence, and tumor R1 maps were estimated using 3D SPGR acquisitions with variable flip angles and B1 field correction(34). A T1-weighted 3D SPGR scan was used to image the abdomen and visualize biodistribution of Gd-NM600 in the heart, liver, and kidneys. To compare uptake of Gd-NM600 to a clinical agent, Gd-DOTA, a flank xenograft glioma model (U87) was used (n=3). To assess tumor-specific uptake, the tumor to muscle T1-weighted signal ratio was computed across multiple time points in two models. To demonstrate the broad avidity of Gd-NM600 for multiple cancers, Several additional tumor models were investigated to further demonstrate the broad-spectrum tumor uptake of Gd-NM600. These models include colorectal flank xenograft models (HT29), a flank xenograft prostate cancer model (PC3), an orthotopic αβ-overexpressing triple negative breast cancer model(35), pancreatic cancer (MiaPaca), and two orthotopic brain cancer models (histologically confirmed U87 and a cancer stem cell model, GSC 12.1).

Tumor Saturation Imaging Study

To investigate saturation of uptake and contrast loading in tumor cells, an in vivo study involving serial daily injections and imaging and Gd-NM600 was performed. T1-weighted tumor imaging on 6 nude athymic female mice bearing A549 flank xenografts was performed prior to administration of Gd-NM600. Mice were administered Gd-NM600 via tail vein (0.12g/kg), and underwent T1-weighted imaging 24hr after administration. Immediately following imaging, mice received another equal dose of Gd-NM600.

MALDI-Mass Spectrometry Imaging Sample Preparation

Matrix-assisted Laser Desorption and Ionization is a soft ionization technique that harnesses a deposited matrix which absorbs laser energy and ionizes neighboring molecules in such a fashion that preserves the chemical form of compounds of interest (39, 40). Coupled with a mass analyzer, MALDI can serve as a powerful tool to not only identify compounds in tissues with high mass accuracy, but also spatially map their distribution (39). Notably, this method can identify Gd deposits with high specificity due to the unique isotopic fingerprint of Gd.

Mice bearing A549-flank xenografts were injected with either Gd-NM600 (n=3), or the formulation alone (n=3). Six animals bearing A549 flank xenografts were injected with 0.12g/kg of Gd-NM600 (n=3) or vehicle alone (n=3), and tissues were harvested at 48 hours post-injection. Animals were perfused with 4mL of IV saline immediately after sacrifice in order to clear the compound out of the blood pool. Flash frozen organs (tumor, liver, kidney, and lung) were embedded in 100 mg/mL gelatin (Becton, Dickinson, and Company) and stored at −80°C. Organs were sectioned at 12 μm thickness on a cryostat (Microtom HM 525, Thermo Scientific) at −20°C. Sections were thaw mounted onto indium tin oxide coated glass slides (Delta Technologies). 2,5-dihydroxybenzoic acid (DHB, Acros Organics) matrix (40 mg/mL in 50:50 methanol:H2O) was applied using the TM Sprayer (HTX Technologies, LLC, Carrboro, NC, USA) automatic sprayer system. The TM sprayer method used 12 passes with 30 s dry time (rotation and offset), 3 mm spacing, 1250 velocity, 80°C temperature, and 0.1 mL/min flow rate. Matrix covered samples were stored in a dry box at −20°C until analysis.

MALDI-Mass Spectrometry Imaging Instrumentation

MALDI-Mass spectrometry imaging (MALDI-MSI) was performed in positive ion mode on a MALDI LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with a N2 laser. LTQ Tune software (Thermo Scientific) was used to select the imaging region and step size, and Xcaliber (Thermo Scientific, Waltham) was used to select the instrument parameters. Imaging was performed on three control mice and three mice dosed with the Gd-NM600 compound at 75 μm raster step size, from 130–2000 mass to charge ratio (m/z) at 60K resolution. Two microscans were averaged at each pixel. ImageQuest software (Thermo Scientific) was used to view raw data and export the raw data to an imzML format. MSiReader software (1) was used to generate images unique to mice dosed with the compound (experimental mice). Briefly, m/z elements that were present in at least 10% of the interrogated zone (experimental mice) and in less than 5% of the reference zone (control mice) or in over 5% of the reference zone with a ratio greater than 2 were selected. Images for these m/z were then identified and manually inspected.

For MALDI mass spectra of Gd-DOTA, and Gd-BOPTA (MultiHance), a mixture of these compounds formulated at 1 μmol in distilled water and 2,5-dihydroxybenzoic acid (DHB, Acros Organics) matrix (40 mg/mL) was used to obtain the MALDI mass spectra.

Statistical Analysis

Statistical analyses were performed with Microsoft Excel or GraphPad Prism 5.0. Paired t-tests were used to determine differences in Gd-NM600 uptake based on imaging and biodistribution data. All p<0.05 were considered statistically significant.

Results

Gd-NM600 exhibits high relaxivity

The longitudinal and transverse relaxivities of Gd-NM600 in water, human plasma, and 0.125 mM human serum albumin were measured (r1 and r2, respectively). At 1.5T, the measured longitudinal relaxivity was between 14.9–16.5 s−1/mM; at 3.0T, it ranged from 8.7–10.0 s−1/mM (Fig 1). The transverse relaxivity ranged from 32.8–41.2 s−1/mM at 1.5T and 23.7–33.2 s−1/mM at 1.5T and 3.0T, respectively (Fig 1). Transverse relaxivity was always lowest in water compared to other preparations, while longitudinal relaxivity was more consistent across all preparations. The significantly higher longitudinal relaxivity of this agent compares favorably to current clinical agents with r1 values ranging from 3.5–7.0 s−1/mM at 1.5T and 2.5–6.5 s−1/mM at 3.0T (36).

Fig. 1. Longitudinal relaxivity (r1) and transverse relaxivity (r2) of Gd-NM600.

Fig. 1.

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(a, c) Relaxivity was determined by linear regression of relaxation rate (R1 or R2) versus sample concentration at each field strength and for each solvent. (b) r1 values at 1.5T and 3.0T in water, albumin, and plasma. Gd-NM600 was found to have greater r1 values at 1.5T than at 3.0T, and when prepared in water compared to plasma and albumin. The longitudinal relaxivity in water (16.4 s-1/mM) compares favorably against Gd-BOPTA (MultiHance), with the highest relaxivity (6.3–7.9 s-1/mM) of the commercial agents. (d) r2 values at 1.5T and 3.0T in water, albumin, and plasma. Gd-NM600 was found to have greater r2 values when prepared in plasma and albumin compared to water.

Transmission electron microscopy confirms selective uptake in cancer cells

After as little as 15 minutes of incubation and at all time points thereafter, Gd-NM600 uptake is observed in U87 cells (Figure 2a). At early time points, the agent accumulates in early endosomes. As these compartments mature and acidify, they become more electron dense, and ultimately fuse with lysosomes. These electron-dense clusters corresponding to accumulation in endosomes and lysosomes are observed at a reduced extent in skin fibroblast control cells. Notably, no dense clusters corresponding to gadolinium are observed in the nucleus (Figure 2c), consistent with previous studies that show no nuclear uptake of APCs and APC-like molecules (12, 37, 38). Surface transmission electron microscopy confirmed high metal contrast inside the U87 cancer cells (Figure 2d).

Fig. 2. Uptake of Gd-NM600 in U87 glioblastoma cells and SK fibroblasts in vitro.

Fig. 2.

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(a) Transmission electron microscopy images of a U87 cell treated with 1μM Gd-NM600. Accumulation in early and mature endosomes (red/blue arrows) leads to electron dense spheres corresponding to intracellular Gd-NM600. Scale bar = 1μm. (b) Untreated control U87 cell shows no electron dense regions. Scale bar = 1μm. (c) Uptake of Gd-DOTA-APC in U87 after 24 hour incubation compared to minimal uptake in SK fibroblasts at the same time point. No nuclear uptake is observed (N denotes nucleus). Scale bar = 2μm. (d) Scanning transmission electron microscopy of U87 cells treated with 1μM Gd-NM600confirms the presence of the heavy metal Gd within cells. Scale bar = 200nm.

T1-weighted imaging and biodistribution demonstrate tumor-specific uptake and retention in multiple cancer models

In vivo uptake of Gd-NM600 was characterized with pilot studies using two flank xenograft models of human cancer, A549 (non-small cell lung cancer) and U87 (glioma), injected with 0.165 mmol/kg. T1-weighted MR images and T1 maps were acquired in all subjects prior to intravenous contrast administration and imaging was repeated at multiple time points up to seven days (Fig 3). Flank tumors of both models showed tumor-specific enhancement over the 7-day period. Tumor enhancement, was maintained for up to 4 and 7 days in A549 and U87, respectively (p<0.05). Specifically, the tumor to control (muscle) signal ratio at 24 hours post Gd-NM600 administration was 1.7 and 1.9 times that in pre-injection imaging for A549 and U87 tumors, respectively. T1 maps acquired at pre-injection and 48 hour time points confirmed that a significant increase in the whole-tumor median R1 relaxation rate was observed at 48 hours post-contrast in both tumor models (increased to approximately 2.2 times pre-injection R1 in both tumor models, p<0.05). In comparison, gadoterate meglumine (Dotarem®), Gd chelated by DOTA, was investigated in mouse flank tumor xenografts. Three nude mice with U87 flank xenografts were scanned with a T1-weighted sequence prior to administration of Gd-DOTA, immediately following contrast administration, and at multiple time points up to 24 hours. After Gd-DOTA administration, the T1-weighted tumor to muscle signal ratio rapidly increased over the course of five minutes, reduced over the course of one hour, and returned to baseline signal levels by 24 hours (Fig 3d–e). Gd-DOTA administration increased ratio of tumor to muscle T1-weighted signal from 1.15 pre-injection to 1.67 five minutes post-contrast. With Gd-NM600 administration, tumor to muscle signal ratio increased from 1.24 pre-injection to a maximum of 2.12 at 24 hours post-contrast. These results indicate increased uptake and prolonged retention of Gd-NM600 in cancer cells that is not observed with the extracellular macrocyclic agent Gd-DOTA.

Fig. 3. Tumor uptake of Gd-NM600 in U87 and A549 and comparative uptake dynamics of Gd-DOTA.

Fig. 3.

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(a) Representative T1-weighted images of two tumor models pre-injection and at multiple time points up to 7 days following delivery of Gd-NM600. Tumor location indicated by white arrow. (b) Tumor to muscle ratio of T1-weighted signal increased over the course of 24–48 hours and remained significantly enhanced compared to pre-injection for up to 4 and 7 days for A549 and U87 xenografts, respectively. Error bars reflect standard deviation among three subjects (*p<0.05 compared to pre-injection, A549; #p<0.05 compared to pre-injection, U87). (c) Quantification of R1 relaxation rate (R1=1/T1) revealed a significant increase of greater than 2x in whole-tumor median R1 rate in both tumor models 48 hours after contrast administration. (d) Representative T1-weighted images of Gd-DOTA uptake in a single subject with flank U87 xenograft model. (E) Time course of T1-weighted signal enhancement after delivery of Gd-NM600 and Gd-DOTA in U87 (N=3 for each model) shows that Gd-NM600 enhancement is greater and more prolonged, indicating that uptake reflects specific targeting and incorporation of the contrast agent in cancer cells. (†p<0.05 compared to pre-injection, Gd-DOTA; #p<0.05 compared to pre-injection, Gd-NM600)

Prolonged T1-weighted signal enhancement was observed with in vivo T1-weighted imaging in mice with colorectal (HT29), prostate cancer (PC3), pancreatic flank xenografts, and orthotopic αβ-overexpressing triple negative breast cancer (35), between 24 and 96 hours following Gd-NM600 administration (Fig 4). The uptake observed in a wide variety of tested cancer types suggests a broad-spectrum uptake mechanism.

Fig. 4. Tumor-specific uptake of Gd-NM600 in multiple human cancer models.

Fig. 4.

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(a) Representative axial T1-weighted images of three flank xenograft models including HT29 (colorectal), MiaPaca (pancreatic), and PC3 (prostate) at various time points pre-injection and up to three days post-contrast. White arrows indicate the location of the cancer. (b) T1-weighted images of two orthotopic tumor models, a triple-negative breast cancer (TNB) implanted in the mammary fat pad and a U87 glioblastoma in the brain. Uptake was observed at 24 and 48 hours, and U87 tumors were confirmed with H&E staining. (c) Perfused tissues from nude athymic mice bearing U87 flank xenografts (N=3) were collected 48 hours following Gd-NM600 administration. Gd uptake was measured with high-resolution (magnetic-sector) inductively-coupled plasma mass spectrometry (ICP-MS) following acid digestion (52–54). Other than the organs of clearance, the tumor had the highest uptake of any organ.

Biodistribution in U87 flank tumor-bearing mice was assessed both ex vivo and in vivo. Gd content in the tumor (3.95% injected dose per gram, (%ID/g)) was higher than in all other tissues except the organs of clearance, the liver and kidney (Supplemental figure S1). Ex vivo biodistribution of bulk administration of Gd-NM600 was quite similar to that of trace levels of 64Cu-DOTA-APC, including in tumors, despite three orders of magnitude difference in amount of agent delivered (10−6 mol vs. 10−9 mol, Supplemental Figure S2a). In vivo biodistribution of Gd-NM600 and 64Cu-DOTA-APC in a flank-bearing U87 rat model demonstrated excellent localization of both PET signal and T1-weighted enhancement on simultaneous PET/MR imaging with the co-administration of these two agents at different mass doses (Supplemental Figure S2b–d). Ex vivo biodistribution was compared to in vivo observations of Gd-NM600 in the blood pool, liver, and kidneys. T1-weighted imaging of the abdomen demonstrated Gd-NM600 clearance from the liver, kidneys, and blood over the 7-day period (Supplemental Figure S2). In contrast, 24 hours following Gd-DOTA administration, little observable agent remained in circulation (Supplemental Figure S1b). High uptake in the liver and kidneys was observed, with maximum liver signal 24 hours and maximum kidney uptake one hour following contrast administration.

Chemical Speciation of Gadolinium Chelates by MALDI-MSI of Gd-DOTA APC

MALDI-MSI of the tissues demonstrated the intact chemical species of Gd-NM600 in all three tissues in the treated animals, but not in the control animals (Figure 5a–c). Overlay of the histology and the distribution of the Gd-NM600 revealed homogenous signal in the kidneys and liver, and heterogeneous signal in the tumor (Figure 5c). The unique isotopic distribution of the Gd species can be identified in the mass spectra (Figure 5d). Notably, a rim of high signal around the tumor periphery was observed in the tumor tissues (Figure 5a), suggesting the highest abundance of the compound at the edge of the tumor margin, most likely representing actively dividing tumor cells.

Figure 5. MALDI-MSI images of Gd-NM600 in mice bearing A549 flank xenografts.

Figure 5.

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MALDI-MSI and histological overlays of Gd-NM600 in whole A549 tumor (a), whole kidney (b), and liver tissue (c) in an animal injected with Gd-NM600(left), and an animal injected with vehicle (right). The white bar indicates 2 mm for (a) and (b) and 1 mm for (c). The color bar maximum was set at 50% of the maximum normalized intensity of all three biological replicates (only biological replicate 2 is shown here). (d) An averaged mass spectra from the tumor region is shown, with the Gd peak corresponding to the mass peaks of the compound Gd-NM600 highlighted. The isotopic distribution of the Gd provides a specific Gd mass signature.

To assess if MALDI-MSI is useful to understand the deposition of commercial chelates, we also employed this method for commercial chelates. Gd-DOTA, and Gd-BOPTA (MultiHance) at the same concentration were incorporated into a mixture with the same matrix and analyzed using MALDI-MSI (Supplemental Figure S3a–b). Identification of these compounds was achieved with high mass accuracy. Again, Gd’s unique isotopic distribution is seen in the mass spectra. The results suggest that this methodology could be employed to understand the chemical species of Gd that is being deposited in normal tissues. This methodology can be used to identify Gd species with high mass accuracy and specificity, and also assess chemical integrity of the chelated species of gadolinium. Further work is underway to assess the chemical speciation of our Gd-APC chelates at longer time points using MALDI-MSI.

Gd-NM600 tumor uptake mechanism demonstrates low saturability

We hypothesized that the uptake mechanism of Gd-NM600 in tumor cells would exhibit minimal saturability. This would potentially allow more efficient tumor uptake in hopes of overcoming MRI’s low sensitivity to molecular-targeted contrast agents. To test this hypothesis, six animals bearing A549 flank xenografts were administered three times the standard mass dose (3×0.12g/kg) of Gd-NM600 over the course of three days (one administration per day). Twenty-four hours after each contrast injection, T1-weighted imaging was performed. All six animals in this study tolerated the increased total dose of 0.36g/kg (0.34mmol/kg) Gd-NM600 over 72 hours. Tumor enhancement increased over the three days of post-contrast imaging and did not reach saturation even after three consecutive days of contrast delivery (Figure 6). The average tumor to muscle ratio increased from 1.89× to 2.43× the baseline value over three days. In comparison, this ratio slowly decreased from 1.90× to 1.41× the baseline value in the three animals administered a single mass dose of Gd-NM600. In this study, complete saturation of Gd-NM600 uptake was not observed over three consecutive days of contrast delivery.

Fig. 6. Continuous uptake of multiple Gd-DOTA-APC doses.

Fig. 6.

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(a) In a representative subject bearing an A549 flank xenograft, non-saturating tumor enhancement after consecutive Gd-NM600 administration (i.v. after pre-injection imaging and after imaging at 24 and 48 hours) was observed over the course of three days on T1-weighted axial imaging. (b) Tumor to muscle T1-weighted signal ratio continually increased in subjects delivered consecutive daily doses of Gd-NM600 (N=6), while contrast enhancement slowly decreased after 24 hours in subjects administered a single dose (N=3). White arrow highlights location of the tumor.

Discussion

To date, tumor-targeted MRI contrast agents have demonstrably failed due to low relaxivities, and insufficient ability to obtain sufficient concentrations of Gd in tumors. This challenge is further complicated by deposition of Gd in normal human tissues, which is more evident with the linear Gd chelates owing to decreased stability compared to macrocylic chelates. This report introduces a new small molecule, macrocyclic Gd contrast agent that exhibits high relaxivity, broad-spectrum tumor-targeting, and prolonged tumor retention properties. Gd-NM600 can image multiple cancer models with higher contrast than commercial Gd chelates. Gd-NM600 demonstrates a high longitudinal relaxivity in water of 16.4s−1/mM at 1.5T, which is approximately 4.2-fold higher relaxivity compared to Gd-DOTA (41, 42), which has a longitudinal relaxivity of 3.9s−1/mM at 1.5T (41, 42). The significantly higher relaxivities of this agent coupled with its cancer selectivity and prolonged retention may improve the diagnostic accuracy for local staging of cancers as well as metastases over current commercial Gd contrast agents, as crossover studies have shown improved cancer visualization, definition, and contrast enhancement are seen when MRI agents with higher relaxivities are used instead of lower relaxivity agents (43–47). Gd-NM600 may also permit whole-body staging of cancers in superior soft tissue resolution compared to FDG, especially in cancers that do not display FDG avidity as the uptake and retention of these alkylphosphocholine analogs is driven by an altogether different mechanism.

Furthermore, Gd-NM600 appears to exhibit high capacity uptake and minimal saturability by cancer cells at clinically relevant dose levels. Unlike many other targeted agents which bind to cell receptors, thus limiting their sensitivity, our APC analogs enter cells via outer membrane lipid rafts overexpressed in cancer cells and are subsequently incorporated into endosomes as seen by TEM imaging. This high-capacity mechanism allows for high concentrations of Gd to accumulate within cancer cells, which translates into elevated and persistent tumor contrast enhancement. This distinctive mechanism of endosomal targeting through lipid rafts circumvents traditional receptor-based approaches, which have failed due to inadequate concentrations of even the highest-expressed receptors on cell surfaces to achieve T1-weighted contrast enhancement (3, 5). Importantly, the high capacity uptake and prolonged tumor retention (exceeding a week) allows for some additional clinical applications of this agent, including motion management for MRI-guided radiation therapy, and neutron-capture therapy.

Long-term retention of Gd MRI agents remains a contentious issue given the observed deposition of Gd in the globus pallidus and dentate nucleus after multiple long term exposures (48, 49). Macrocyclic Gd-NM600 was designed to offer better thermodynamic and kinetic stability relative to linear chelates (41). As the debate continues on the clinical relevance of Gd deposition in normal brain tissues, we have demonstrated the utility of (MALDI-MSI) to enhance our understanding of the chemical speciation and spatial distribution of our own and other Gd MRI agents. However, it is important to weigh the risks and benefits with any clinical procedure. As these cancer targeted agents are designed to diagnose and stage cancer, we offer that the benefits of more appropriate treatment and overall survival afforded by more accurate imaging outweigh any potential risks associated with potential gadolinium deposition, which have not been clearly, if at all, demonstrated. Moreover, enhanced tumor selectively and multifold increased relaxivities of Gd-NM600 will significantly decrease the doses needed for accurate diagnostic staging while minimizing the off-target gadolinium burden and deposition. Furthermore, histological analysis of Gd deposits in the dentate nucleus and globus pallidus have failed to demonstrate pathological correlates such as reactive, inflammatory, or fibrotic changes (50, 51).

Even so, we have developed a highly sensitive and specific method of mapping Gd species using mass spectrometry in order to better understand the issue of Gd deposition. MALDI-MSI of Gd-NM600 in tissues of highest uptake demonstrates that the Gd in these tissues remains unchanged from the original parent structure, suggesting that the agent appears to be stable up to the first week after administration. Further work is underway to characterize the actual metabolites as well as any Gd species that are deposited at longer time points of more than a week in an effort to understand how Gd-chelates interact with normal tissue.

Conclusion

In this study, we report a new cancer-targeted MRI contrast agent that demonstrates high relaxivity, and tumor-specific uptake and retention in six preclinical flank xenograft cancer models and three orthotopic cancer models. This novel tumor-targeted macrocyclic agent, NM600, outperformed commercial Gd contrast agents in our studies by providing higher relaxivities, and superior and prolonged tumor conspicuity. Its advantages include a 4.2-fold higher longitudinal relaxivity compared to Gd-DOTA and higher cancer specificity due to the APC targeting moiety, while maintaining the stability of a macrocyclic DOTA chelate. The higher relaxivities and tumor specificity may permit accurate detection of smaller soft-tissue tumors and metastases at reduced doses compared with current clinical Gd chelates. We have observed a distinct, high-capacity, cancer-targeting mechanism of these APC chelates that is not receptor-mediated and common to a broad array of cancers. Additionally, we demonstrate a specific and sensitive method to identify and spatially map the presence and the chemical form of Gd containing compounds, including parent Gd-NM600, in tissue samples through MALDI-MSI analysis. Overall, Gd-NM600 exhibits broad-spectrum tumor targeting and prolonged retention capabilities, high relativities, and macrocylic chelate stability that may improve diagnostic accuracy and staging of cancers of the soft tissue over current oncologic imaging modalities. Finally, a tumor selective contrast agent with prolonged tumor retention may play an important role in MR-guided external beam radiotherapy and motion management associated with this newer technology.

Supplementary Material

Supplemental Digital Content Documents

Acknowledgements:

R.R.Z. is partially supported by the University of Wisconsin MD/PhD program via T32 GM008692. We are grateful to the University of Wisconsin Department Of Radiology Research and Development Pilot Grant program, Dept. of Neurological Surgery, The Carbone Cancer Center Small Animal Imaging and Radiotherapy Facility and the University of Wisconsin Carbone Cancer Center Support Grant (P30 CA014520) for supporting this work. PAC and JSK were partly supported by the Roger Loff Memorial Fund for GBM Research. We also thank Dr. Caroline Keller, Dr. Lingjun Li for their mass spectrometry work and use of their core facility, Dr. Martin Schafer at the Wisconsin State Lab of Hygiene for use of Magnector sector instrumentation and analysis, and Dr. Vincent Cryns for his labs triple negative breast cancer model. We also thank Dr. Thomas Grist (UW), and Dr. Scott Reeder (UW) for early discussions and feedback as well as manuscript preview by Dr. Michael Tweedle (OSU).

Conflicts of interest and sources of funding:

RRZ was partially supported by the University of Wisconsin MD/PhD program via an NIH T32 GM008692 grant as well as Department of Radiology and Neurological Surgery pilot funding. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. JPW is a cofounder and ANP, JJG and RH have served as consultants of Archeus Technologies which holds the licensing rights to Gd-NM600. Some of the information contained in the manuscript was presented at the bi-annual Contrast Media Research meeting in 2015 and 2017.

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