Ethel J. Ngen

Radiotherapy-induced brain injury (RIBI) is a chronic side effect that affects brain tumor survivors treated with radiotherapy. Neuroinflammation is a key contributor to RIBI. Thus, imaging methods capable of noninvasively monitoring neuroinflammation are needed. Although positron emission tomography (PET)-based radiotracers exist for imaging neuroinflammation, PET involves ionizing radiation which could be detrimental to pediatric patients already facing the risk of RIBI. Here, we evaluated the feasibility of developing contrast-enhanced T₁W MRI as a predictive biomarker of neuroinflammation in RIBI. Four groups of eight-week-old female BALB/c mice were stereotactically irradiated at 80 Gy and monitored longitudinally for neuroinflammation using ¹¹C-DPA-713 PET; ¹¹C-CPPC PET; gadoteridol-based contrast-enhanced T₁-weighted MRI; and TSPO, CD68, IBA1 immunohistochemistry. Our results showed that contrast-enhanced T₁W MRI was as effective as ¹¹C-DPA-713 PET; ¹¹C-CPPC PET and immunohistochemistry (P < 0.05, n = 3) in predicting neuroinflammation, by detecting subtle changes in the blood-brain barrier permeability that affected neuroinflammation changes.

Objective: Radiotherapy-induced brain injury (RIBI) is a chronic side effect that affects up to 90% of brain tumor survivors treated with radiotherapy. Here, we used multiparametric magnetic resonance imaging (MRI) to identify noninvasive and clinically translatable biomarkers of RIBI. Method: 8-week-old female, immune competent BALB/c mice were stereotactically irradiated with a single dose of 80 Gy, at a dose rate of 1.7 Gy/minute. The irradiated mice were then monitored longitudinally with MRI, behavioral tests of learning and memory, and immunohistochemistry, in comparison to nonirradiated mice. Results: Three types of MRI biomarkers of RIBI were identified. A contrast-enhanced T ₁ -weighted MRI biomarker was identified as being best suited to detect the onset of injury, by detecting changes in the blood-brain barrier (BBB) permeability. Maximum BBB permeability (18.95 ± 1.75) was detected with contrast-enhanced T ₁ -weighted MRI at 1-month postirradiation in irradiated mice ( P < 0.0001, n = 3). Interestingly, maximum neuroinflammation (24.14 ± 6.72) was also detected using IBA1 and CD68 immunohistochemistry at 1-month postirradiation in irradiated mice ( P = 0.0041, n = 3). This simultaneous maximum BBB permeability and neuroinflammation detection also coincided with the detection of the onset of transient cognitive impairment, detected using the fear-conditioning behavioral test at 1-month postirradiation in irradiated mice compared to nonirradiated mice ( P = 0.0017, n = 10). A T ₂ -weighted MRI hyperintensity biomarker was also identified, and determined to be best suited to detect intermediate injury. Maximum T ₂ -weighted MRI hyperintensity (3.97 ± 2.07) was detected at 2-month postirradiation in the irradiated mice compared to nonirradiated mice ( P = 0.0368, n = 3). This T ₂ -weighted MRI hyperintensity also correlated with maximum astrogliosis (9.92 ± 4.21), which was also detected at 2-month postirradiation using GFAP immunohistochemistry in the irradiated mice compared to nonirradiated mice ( P = 0.0215, n = 3). Finally, T ₂ -weighted and T ₂ *-weighted MRI hypointensity biomarkers were identified as being best suited to detect late injury, from 4-month postirradiation. These biomarkers correlated with increased iron deposition from late vascular damage, which was validated with Perls' Prussian blue histology ( P < 0.05, n = 3). These hypointense MRI biomarkers of late injury also preceded significant weight loss, severe cognitive impairment, and decreased survival in the irradiated mice compared to the nonirradiated mice. Conclusions: Here, we identified 3 types of translational MRI biomarkers of RIBI that could enable the noninvasive longitudinal evaluation of potential RIBI prophylactic and therapeutic agents. These translational MRI biomarkers could also play a pivotal role in the management of RIBI in brain tumor survivors.

Purpose To develop a prostate-specific membrane antigen (PSMA)-targeted radiotherapeutic for metastatic castration-resistant prostate cancer (mCRPC) with optimized efficacy and minimized toxicity employing the β-particle radiation of ¹⁷⁷Lu. Methods We synthesized 14 new PSMA-targeted, ¹⁷⁷Lu-labeled radioligands (¹⁷⁷Lu-L1–¹⁷⁷Lu-L14) using different chelating agents and linkers. We evaluated them in vitro using human prostate cancer PSMA(+) PC3 PIP and PSMA(−) PC3 flu cells and in corresponding flank tumor models. Efficacy and toxicity after 8 weeks were evaluated at a single administration of 111 MBq for ¹⁷⁷Lu-L1, ¹⁷⁷Lu-L3, ¹⁷⁷Lu-L5 and ¹⁷⁷Lu-PSMA-617. Efficacy of ¹⁷⁷Lu-L1 was further investigated using different doses, and long-term toxicity was determined in healthy immunocompetent mice. Results Radioligands were produced in high radiochemical yield and purity. Cell uptake and internalization indicated specific uptake only in PSMA(+) PC3 cells. ¹⁷⁷Lu-L1, ¹⁷⁷Lu-L3 and ¹⁷⁷Lu-L5 demonstrated comparable uptake to ¹⁷⁷Lu-PSMA-617 and ¹⁷⁷Lu-PSMA-I&T in PSMA-expressing tumors up to 72 h post-injection. ¹⁷⁷Lu-L1, ¹⁷⁷Lu-L3 and ¹⁷⁷Lu-L5 also demonstrated efficient tumor regression at 8 weeks. ¹⁷⁷Lu-L1 enabled the highest survival rate. Necropsy studies of the treated group at 8 weeks revealed subacute damage to lacrimal glands and testes. No radiation nephropathy was observed 1 year post-treatment in healthy mice receiving 111 MBq of ¹⁷⁷Lu-L1, most likely related to the fast renal clearance of this agent. Conclusions ¹⁷⁷Lu-L1 is a viable clinical candidate for radionuclide therapy of PSMA-expressing malignancies because of its high tumor-targeting ability and low off-target radiotoxic effects.

Magnetic nanoparticle (MNP)-induced hyperthermia is currently being evaluated for localized prostate cancer. We evaluated the feasibility of tumor-selective delivery of prostate-specific membrane antigen (PSMA)-targeted MNPs in a murine model with high-resolution magnetic resonance imaging (MRI) after intravenous administration of MNPs at a concentration necessary for hyperthermia. A PSMA-targeted MNP was synthesized and evaluated using T2-weighted MRI after intravenous administration of 50 mg/kg of the MNP. Significant contrast enhancement (P < 0.0002, n = 5) was observed in PSMA(+) tumors compared to PSMA(-) tumors 24 h and 48 h after contrast agent administration. Mice were also imaged with near-infrared fluorescence imaging, to validate the MRI results. Two-photon microscopy revealed higher vascular density at the tumor periphery in keeping with peripheral accumulation of PSMA-targeted MNPs. These results suggest that the delivery of PSMA-targeted MNPs to PSMA(+) tumors is both actively targeted and passively mediated.

Cell-based therapies are currently being developed for applications in both regenerative medicine and in oncology. Preclinical, translational, and clinical research on cell-based therapies will benefit tremendously from novel imaging approaches that enable the effective monitoring of the delivery, survival, migration, biodistribution, and integration of transplanted cells. Magnetic resonance imaging (MRI) offers several advantages over other imaging modalities for elucidating the fate of transplanted cells both preclinically and clinically. These advantages include the ability to image transplanted cells longitudinally at high spatial resolution without exposure to ionizing radiation, and the possibility to co-register anatomical structures with molecular processes and functional changes. However, since cellular MRI is still in its infancy, it currently faces a number of challenges, which provide avenues for future research and development. In this review, we describe the basic principle of cell-tracking with MRI; explain the different approaches currently used to monitor cell-based therapies; describe currently available MRI contrast generation mechanisms and strategies for monitoring transplanted cells; discuss some of the challenges in tracking transplanted cells; and suggest future research directions.

Brain tumors are among the most lethal types of tumors. Therapeutic response variability and failure in patients have been attributed to several factors, including inadequate drug delivery to tumors due to the blood–brain barrier (BBB). Consequently, drug delivery strategies are being developed for the local and targeted delivery of drugs to brain tumors. These drug delivery strategies could benefit from new approaches to monitor the delivery of drugs to tumors. Here, we evaluated the feasibility of imaging 4-[bis(2-chloroethyl)amino]-l-phenylalanine (melphalan), a clinically used DNA alkylating agent, using chemical exchange saturation transfer magnetic resonance imaging (CEST MRI), for theranostic applications. We evaluated the physicochemical parameters that affect melphalan’s CEST contrast and demonstrated the feasibility of imaging the unmodified drug by saturating its exchangeable amine protons. Melphalan generated a CEST signal despite its reactivity in an aqueous milieu. The maximum CEST signal was observed at pH 6.2. This CEST contrast trend was then used to monitor therapeutic responses to melphalan in vitro. Upon cell death, the decrease in cellular pH from ∼7.4 to ∼6.4 caused an amplification of the melphalan CEST signal. This is contrary to what has been reported for other CEST contrast agents used for imaging cell death, where a decrease in the cellular pH following cell death results in a decrease in the CEST signal. Ultimately, this method could be used to noninvasively monitor melphalan delivery to brain tumors and also to validate therapeutic responses to melphalan clinically.

This paper reports the damaging effects of magnetic iron-oxide nanoparticles (MNP) on magnetically labeled cancer cells when subjected to oscillating gradients in a strong external magnetic field. Human breast cancer MDA-MB-231 cells were labeled with MNP, placed in the high magnetic field, and subjected to oscillating gradients generated by an imaging gradient system of a 9.4T preclinical MRI system. Changes in cell morphology and a decrease in cell viability were detected in cells treated with oscillating gradients. The cytotoxicity was determined qualitatively and quantitatively by microscopic imaging and cell viability assays. An approximately 26.6% reduction in cell viability was detected in magnetically labeled cells subjected to the combined effect of a static magnetic field and oscillating gradients. No reduction in cell viability was observed in unlabeled cells subjected to gradients, or in MNP-labeled cells in the static magnetic field. As no increase in local temperature was observed, the cell damage was not a result of hyperthermia. Currently, we consider the coherent motion of internalized and aggregated nanoparticles that produce mechanical moments as a potential mechanism of cell destruction. The formation and dynamics of the intracellular aggregates of nanoparticles were visualized by optical and transmission electron microscopy (TEM). The images revealed a rapid formation of elongated MNP aggregates in the cells, which were aligned with the external magnetic field. This strategy provides a new way to eradicate a specific population of MNP-labeled cells, potentially with magnetic resonance imaging guidance using standard MRI equipment, with minimal side effects for the host.

Stem cell therapies are being developed for radiotherapy-induced brain injuries (RIBI). Magnetic resonance imaging (MRI) offers advantages for imaging transplanted stem cells. However, most MRI cell-tracking techniques employ superparamagnetic iron oxide particles (SPIOs), which are difficult to distinguish from hemorrhage. In current preclinical RIBI models, hemorrhage occurs concurrently with other injury markers. This makes the evaluation of the recruitment of transplanted SPIO-labeled stem cells to injury sites difficult. Here, we developed a RIBI model, with early injury markers reflective of hippocampal dysfunction, which can be detected noninvasively with MRI and behavioral tests. Lesions were generated by sub-hemispheric irradiation of mouse hippocampi with single X-ray beams of 80 Gy. Lesion formation was monitored with anatomical and contrast-enhanced MRI and changes in memory and learning were assessed with fear-conditioning tests. Early injury markers were detected 2 weeks after irradiation. These included an increase in the permeability of the blood–brain barrier, demonstrated by a 92 ± 20 % contrast enhancement of the irradiated versus the non-irradiated brain hemispheres, within 15 min of the administration of an MRI contrast agent. A change in short-term memory was also detected, as demonstrated by a 40.88 ± 5.03 % decrease in the freezing time measured during the short-term memory context test at this time point, compared to that before irradiation. SPIO-labeled stem cells transplanted contralateral to the lesion migrated toward the lesion at this time point. No hemorrhage was detected up to 10 weeks after irradiation. This model can be used to evaluate SPIO-based stem cell-tracking agents, short-term.

Stem cell therapies are currently being investigated for the repair of brain injuries. Although exogenous stem cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) prior to transplantation provides a means to noninvasively monitor stem cell transplantation by magnetic resonance imaging (MRI), monitoring cell death is still a challenge. Here, we investigate the feasibility of using an MRI dual-contrast technique to detect cell delivery, cell migration and cell death after stem cell transplantation. Human mesenchymal stem cells were dual labelled with SPIONs and gadolinium-based chelates (GdDTPA). The viability, proliferation rate and differentiation potential of the labelled cells were then evaluated. The feasibility of this MRI technique to distinguish between live and dead cells was next evaluated using MRI phantoms and in vivo using both immune-competent and immune-deficient mice, following the induction of brain injury in the mice. All results were validated with bioluminescence imaging. In live cells, a negative (T₂/T₂*) MRI contrast predominates and is used to track cell delivery and cell migration. Upon cell death, a diffused positive (T₁) MRI contrast is generated in the vicinity of the dead cells and serves as an imaging marker for cell death. Ultimately, this technique could be used to manage stem cell therapies.