Alana Ross

Near-infrared fluorescence molecular imaging of amyloid beta species and monitoring therapy in animal models of Alzheimer’s disease

Significance Drug development for Alzheimer’s disease (AD) has been largely unsuccessful to date. Although numerous agents are reportedly effective in vitro, only an inadequate number of them have been tested in vivo, partially because of the lack of reliable and cost-efficient imaging methods to monitor their in vivo therapeutic effectiveness. Several amyloid beta (Aβ)-specific PET tracers have been used for clinical studies. However, their application for monitoring drug treatment in small animals is limited. Near-infrared fluorescence (NIRF) molecular imaging is a cheap, easy-to-use, and widely available technology for small animal studies. In this report we demonstrate, for the first time to our knowledge, that NIRF imaging can be used to monitor the loading changes of Aβs in an AD mouse model. Near-infrared fluorescence (NIRF) molecular imaging has been widely applied to monitoring therapy of cancer and other diseases in preclinical studies; however, this technology has not been applied successfully to monitoring therapy for Alzheimer’s disease (AD). Although several NIRF probes for detecting amyloid beta (Aβ) species of AD have been reported, none of these probes has been used to monitor changes of Aβs during therapy. In this article, we demonstrated that CRANAD-3, a curcumin analog, is capable of detecting both soluble and insoluble Aβ species. In vivo imaging showed that the NIRF signal of CRANAD-3 from 4-mo-old transgenic AD (APP/PS1) mice was 2.29-fold higher than that from age-matched wild-type mice, indicating that CRANAD-3 is capable of detecting early molecular pathology. To verify the feasibility of CRANAD-3 for monitoring therapy, we first used the fast Aβ-lowering drug LY2811376, a well-characterized beta-amyloid cleaving enzyme-1 inhibitor, to treat APP/PS1 mice. Imaging data suggested that CRANAD-3 could monitor the decrease in Aβs after drug treatment. To validate the imaging capacity of CRANAD-3 further, we used it to monitor the therapeutic effect of CRANAD-17, a curcumin analog for inhibition of Aβ cross-linking. The imaging data indicated that the fluorescence signal in the CRANAD-17–treated group was significantly lower than that in the control group, and the result correlated with ELISA analysis of brain extraction and Aβ plaque counting. It was the first time, to our knowledge, that NIRF was used to monitor AD therapy, and we believe that our imaging technology has the potential to have a high impact on AD drug development.

GLP-1R–Targeting Magnetic Nanoparticles for Pancreatic Islet Imaging

Noninvasive assessment of pancreatic β-cell mass would tremendously aid in managing type 1 diabetes (T1D). Toward this goal, we synthesized an exendin-4 conjugated magnetic iron oxide–based nanoparticle probe targeting glucagon-like peptide 1 receptor (GLP-1R), which is highly expressed on the surface of pancreatic β-cells. In vitro studies in βTC-6, the β-cell line, showed specific accumulation of the targeted probe (termed MN-Ex10-Cy5.5) compared with nontargeted (termed MN-Cy5.5). In vivo magnetic resonance imaging showed a significant transverse relaxation time (T2) shortening in the pancreata of mice injected with the MN-Ex10-Cy5.5 probe compared with control animals injected with the nontargeted probe at 7.5 and 24 h after injection. Furthermore, ΔT2 of the pancreata of prediabetic NOD mice was significantly higher than that of diabetic NOD mice after the injection of MN-Ex10-Cy5.5, indicating the decrease of probe accumulation in these animals due to β-cell loss. Of note, ΔT2 of prediabetic and diabetic NOD mice injected with MN-Cy5.5 was not significantly changed, reflecting the nonspecific mode of accumulation of nontargeted probe. We believe our results point to the potential for using this agent for monitoring the disease development and response of T1D to therapy.

A Theranostic Small Interfering RNA Nanoprobe Protects Pancreatic Islet Grafts From Adoptively Transferred Immune Rejection

Islet transplantation has recently emerged as an acceptable clinical modality for restoring normoglycemia in patients with type 1 diabetes mellitus (T1DM). The long-term survival and function of islet grafts is compromised by immune rejection–related factors. Downregulation of factors that mediate immune rejection using RNA interference holds promise for improving islet graft resistance to damaging factors after transplantation. Here, we used a dual-purpose therapy/imaging small interfering (si)RNA magnetic nanoparticle (MN) probe that targets β2 microglobulin (B2M), a key component of the major histocompatibility class I complex (MHC I). In addition to serving as a siRNA carrier, this MN-siB2M probe enables monitoring of graft persistence noninvasively using magnetic resonance imaging (MRI). Human islets labeled with these MNs before transplantation into B2M (null) NOD/scid mice showed significantly improved preservation of graft volume starting at 2 weeks, as determined by longitudinal MRI in an adoptive transfer model (P < 0.05). Furthermore, animals transplanted with MN-siB2M–labeled islets demonstrated a significant delay of up to 23.8 ± 4.8 days in diabetes onset after the adoptive transfer of T cells relative to 6.5 ± 4.5 days in controls. This study demonstrated that our approach could protect pancreatic islet grafts from immune rejection and could potentially be applied to allotransplantation and prevention of the autoimmune recurrence of T1DM in islet transplantation or endogenous islets.

Combining miR-10b–Targeted Nanotherapy with Low-Dose Doxorubicin Elicits Durable Regressions of Metastatic Breast Cancer

The therapeutic promise of microRNA (miRNA) in cancer has yet to be realized. In this study, we identified and therapeutically exploited a new role for miR-10b at the metastatic site, which links its overexpression to tumor cell viability and proliferation. In the protocol developed, we combined a miR-10b-inhibitory nanodrug with low-dose anthracycline to achieve complete durable regressions of metastatic disease in a murine model of metastatic breast cancer. Mechanistic investigations suggested a potent antiproliferative, proapoptotic effect of the nanodrug in the metastatic cells, potentiated by a cell-cycle arrest produced by administration of the low-dose anthracycline. miR-10b was overexpressed specifically in cells with high metastatic potential, suggesting a role for this miRNA as a metastasis-specific therapeutic target. Taken together, our results implied the existence of pathways that regulate the viability and proliferation of tumor cells only after they have acquired the ability to grow at distant metastatic sites. As illustrated by miR-10b targeting, such metastasis-dependent apoptotic pathways would offer attractive targets for further therapeutic exploration.

Sequence-dependent combination therapy with doxorubicin and a survivin-specific small interfering RNA nanodrug demonstrates efficacy in models of adenocarcinoma

The clinical management of cancer reflects a balance between treatment efficacy and toxicity. While typically, combination therapy improves response rate and time to progression compared with sequential monotherapy, it causes increased toxicity. Consequently, in cases of advanced cancer, emerging guidelines recommend sequential monotherapy, as a means to enhance quality of life. An alternative approach that could overcome nonspecific toxicity while retaining therapeutic efficacy, involves the combination of chemotherapy with targeted therapy. In the current study, we tested the hypothesis that combination therapy targeting survivin (BIRC5) and low‐dose doxorubicin (Dox) will show enhanced therapeutic potential in the treatment of cancer, as compared to monotherapy with Dox. We demonstrate in both in vitro and in vivo models of breast cancer that combination therapy with a low dose of Dox and an anti‐survivin siRNA nanodrug (MN‐siBIRC5) is superior to mono‐therapy with either low‐ or high‐dose Dox alone. Importantly, therapeutic efficacy showed prominent sequence dependence. Induction of apoptosis was observed only when the cells were treated with Dox followed by MN‐siBIRC5, whereas the reverse sequence abrogated the benefit of the drug combination. In vivo, confirmation of successful sequence dependent combination therapy was demonstrated in a murine xenograft model of breast cancer. Finally, to determine if the observed effect is not limited to breast cancer, we extended our studies to a murine xenograft model of pancreatic adenocarcinoma and found similar outcomes as shown for breast cancer.

Targeted imaging of breast tumor progression and therapeutic response in a human uMUC-1 expressing transgenic mouse model.

The ability to monitor breast cancer initiation and progression on the molecular level would provide an effective tool for early diagnosis and therapy. In the present study, we focused on the underglycosylated MUC‐1 tumor antigen (uMUC‐1), which is directly linked to tumor progression from pre‐malignancy to advanced malignancy in breast cancer and has been identified as the independent predictor of local recurrence and tumor response to chemotherapy. We investigated whether changes in uMUC‐1 expression during tumor development and therapeutic intervention could be monitored non‐invasively using molecular imaging approach with the uMUC‐1‐specific contrast agent (MN‐EPPT) detectable by magnetic resonance and fluorescence optical imaging. This was done in mice that express human uMUC‐1 tumor antigen (MMT mice) and develop spontaneous mammary carcinoma in a stage‐wise fashion. After the injection of MN‐EPPT there was a significant reduction in average T2 relaxation times of the mammary fat pad between pre‐malignancy and cancer. In addition, T2 relaxation times were already altered at pre‐malignant state in these mice compared to non‐tumor bearing mice. This indicated that targeting uMUC‐1 could be useful for detecting pre‐malignant transformation in the mammary fat pad. We also probed changes in uMUC‐1 expression with MN‐EPPT during therapy with doxorubicin (Dox). We observed that tumor delta‐T2s were significantly reduced by treatment with Dox indicating lower accumulation of MN‐EPPT. This correlated with a lower level of MUC‐1 expression in the Dox‐treated tumors, as confirmed by immunoblotting. Our study could provide a very sensitive molecular imaging approach for monitoring tumor progression and therapeutic response.

Immune rejection after pancreatic islet cell transplantation: in vivo dual contrast-enhanced MR imaging in a mouse model.

PURPOSE To detect adoptively transferred immune attack in a mouse model of islet cell transplantation by using a long-circulating paramagnetic T1 contrast agent, a protected graft copolymer (PGC) that is covalently linked to gadolinium-diethylenetriaminepentaacetic acid with fluorescein isothiocyanate (Gd-DTPA-F), which accumulates in the sites of inflammation that are characterized by vascular disruption. MATERIALS AND METHODS All animal experiments were performed in compliance with institutional guidelines and approved by the subcommittee on research animal care. Six nonobese diabetic severe combined immunodeficiency mice received transplanted human islet cells under the kidney capsule and adoptively transferred 5 × 10(6) splenocytes from 6-week-old nonobese diabetic mice. These mice also served as control subjects for comparison of pre- and postadoptive transfer MR imaging results. Mice that received phosphate-buffered saline solution only were included as nonadoptive-transfer control subjects (n = 2). In vivo magnetic resonance (MR) imaging was performed before and 17 hours after intravenous injections of PGC-Gd-DTPA-F, followed by histologic examination. Statistical differences were analyzed by means of a paired Student t test and repeated two-way analysis of variance. RESULTS MR imaging results showed significantly greater accumulation of PGC-Gd-DTPA-F in the graft area after immune attack initiated by adoptive transfer of splenocytes compared with that of the same area before the transfer (T1, 137.2 msec ± 39.3 and 239.5 msec ± 17.6, respectively; P < .001). These results were confirmed at histologic examination, which showed considerable leakage of the contrast agent into the islet cell interstitium. CONCLUSION PGC-Gd-DTPA-F-enhanced MR imaging allows for the in vivo assessment of vascular damage of the graft T cell challenge.

Therapy targeted to the metastatic niche is effective in a model of stage IV breast cancer

Treatment of stage IV metastatic breast cancer patients is limited to palliative options and represents an unmet clinical need. Here, we demonstrate that pharmacological inhibition of miRNA-10b - a master regulator of metastatic cell viability – leads to elimination of distant metastases in a mouse model of metastatic breast cancer. This was achieved using the miRNA-10b inhibitory nanodrug, MN-anti-miR10b, which consists of magnetic nanoparticles, conjugated to LNA-based miR-10b antagomirs. Intravenous injection of MN-anti-miR10b into mice bearing lung, bone, and brain metastases from breast cancer resulted in selective accumulation of the nanodrug in metastatic tumor cells. Weekly treatments of mice with MN-anti-miR-10b and low-dose doxorubicin resulted in complete regression of pre-existing distant metastases in 65% of the animals and a significant reduction in cancer mortality. These observations were supported by dramatic reduction in proliferation and increase in apoptosis in metastatic sites. On a molecular level, we observed a significant increase in the expression of HOXD10, which is a known target of miRNA-10b. These results represent first steps into the uncharted territory of therapy targeted to the metastatic niche.

Magnetic particle imaging of islet transplantation in the liver and under the kidney capsule in mouse models

Background Islet transplantation (Tx) represents the most promising therapy to restore normoglycemia in type 1 diabetes (T1D) patients to date. As significant islet loss has been observed after the procedure, there is an urgent need for developing strategies for monitoring transplanted islet grafts. In this report we describe for the first time the application of magnetic particle imaging (MPI) for monitoring transplanted islets in the liver and under the kidney capsule in experimental animals. Methods Pancreatic islets isolated from Papio hamadryas were labeled with superparamagnetic iron oxides (SPIOs) and used for either islet phantoms or Tx in the liver or under the kidney capsule of NOD scid mice. MPI was used to image and quantify islet phantoms and islet transplanted experimental animals post-mortem at 1 and 14 days after Tx. Magnetic resonance imaging (MRI) was used to confirm the presence of labeled islets in the liver and under the kidney capsule 1 day after Tx. Results MPI of labeled islet phantoms confirmed linear correlation between the number of islets and the MPI signal (R2=0.988). Post-mortem MPI performed on day 1 after Tx showed high signal contrast in the liver and under the kidney capsule. Quantitation of the signal supports islet loss over time, which is normally observed 2 weeks after Tx. No MPI signal was observed in control animals. In vivo MRI confirmed the presence of labeled islets/islet clusters in liver parenchyma and under the kidney capsule one day after Tx. Conclusions Here we demonstrate that MPI can be used for quantitative detection of labeled pancreatic islets in the liver and under the kidney capsule of experimental animals. We believe that MPI, a modality with no depth attenuation and zero background tissue signal could be a suitable method for imaging transplanted islet grafts.

Predictive imaging of chemotherapeutic response in a transgenic mouse model of pancreatic cancer

The underglycosylated mucin 1 tumor antigen (uMUC1) is a biomarker that forecasts the progression of adenocarcinomas. In this study, we evaluated the utility of a dual‐modality molecular imaging approach based on targeting uMUC1 for monitoring chemotherapeutic response in a transgenic murine model of pancreatic cancer (KCM triple transgenic mice). An uMUC1‐specific contrast agent (MN‐EPPT) was synthesized for use with magnetic resonance imaging (MRI) and fluorescence optical imaging. It consisted of dextran‐coated iron oxide nanoparticles conjugated to the near infrared fluorescent dye Cy5.5 and to a uMUC1‐specific peptide (EPPT). KCM triple transgenic mice were given gemcitabine as chemotherapy while control animals received saline injections following the same schedule. Changes in uMUC1 levels following chemotherapy were monitored using T2‐weighted MRI and optical imaging before and 24 hr after injection of the MN‐EPPT. uMUC1 expression in tumors from both groups was evaluated by histology and qRT‐PCR. We observed that the average delta‐T2 in the gemcitabine‐treated group was significantly reduced compared to the control group indicating lower accumulation of MN‐EPPT, and correspondingly, a lower level of uMUC1 expression. In vivo optical imaging confirmed the MRI findings. Fluorescence microscopy of pancreatic tumor sections showed a lower level of uMUC1 expression in the gemcitabine‐treated group compared to the control, which was confirmed by qRT‐PCR. Our data proved that changes in uMUC1 expression after gemcitabine chemotherapy could be evaluated using MN‐EPPT‐enhanced in vivo MR and optical imaging. These results suggest that the uMUC1‐targeted imaging approach could provide a useful tool for the predictive assessment of therapeutic response.