Zdravka Medarova

Background: In our earlier work, we identified microRNA-10b (miR10b) as a master regulator of the viability of metastatic tumor cells. This knowledge allowed us to design a miR10b-targeted therapeutic consisting of an anti-miR10b antagomir conjugated to ultrasmall iron oxide nanoparticles (MN), termed MN-anti-miR10b. In mouse models of breast cancer, we demonstrated that MN-anti-miR10b caused durable regressions of established metastases with no evidence of systemic toxicity. As a first step towards translating MN-anti-miR10b for the treatment of metastatic breast cancer, we needed to determine if MN-anti-miR10b, which is so effective in mice, will also accumulate in human metastases. Results: In this study, we devised a method to efficiently radiolabel MN-anti-miR10b with Cu-64 (⁶⁴Cu) and evaluated the pharmacokinetics and biodistribution of the radiolabeled product at two different doses: a therapeutic dose, referred to as macrodose, corresponding to ⁶⁴Cu-MN-anti-miR10b co-injected with non-labeled MN-anti-miR10b, and a tracer-level dose of ⁶⁴Cu-MN-anti-miR10b, referred to as microdose. In addition, we evaluated the uptake of ⁶⁴Cu-MN-anti-miR10b by metastatic lesions using both in vivo and ex vivo positron emission tomography–magnetic resonance imaging (PET–MRI). A comparable distribution of the therapeutic was observed after administration of a microdose or macrodose. Uptake of the therapeutic by metastatic lymph nodes, lungs, and bone was also demonstrated by PET–MRI with a significantly higher PET signal than in the same organs devoid of metastatic lesions. Conclusion: Our results demonstrate that PET–MRI following a microdose injection of the agent will accurately reflect the innate biodistribution of the therapeutic. The tools developed in the present study lay the groundwork for the clinical testing of MN-anti-miR10b and other similar therapeutics in patients with cancer.

RNA interference represents one of the most appealing therapeutic modalities for cancer because of its potency, versatility, and modularity. Because the mechanism is catalytic and affects the expression of disease-causing antigens at the post-transcriptional level, only small amounts of therapeutic need to be delivered to the target in order to exert a robust therapeutic effect. RNA interference is also advantageous over other treatment modalities, such as monoclonal antibodies or small molecules, because it has a much broader array of druggable targets. Finally, the complementarity of the genetic code gives us the opportunity to design RNAi therapeutics using computational, rational approaches. Previously, we developed and tested an RNAi-targeted therapeutic, termed MN-anti-miR10b, which was designed to inhibit the critical driver of metastasis and metastatic colonization, miRNA-10b. We showed in animal models of metastatic breast cancer that MN-anti-miR10b accumulated into tumors and metastases in the lymph nodes, lungs, and bone, following simple intravenous injection. We also found that treatment incorporating MN-anti-miR10b was effective at inhibiting the emergence of metastases and could regress already established metastases in the lymph nodes, lungs, and bone. In the present study, we extend the application of MN-anti-miR10b to a model of breast cancer metastatic to the brain. We demonstrate delivery to the metastatic lesions and obtain evidence of a therapeutic effect manifested as inhibition of metastatic progression. This investigation represents an additional step towards translating similar RNAi-targeted therapeutics for the systemic treatment of metastatic disease.

Chemotherapy, a major cancer treatment approach, suffers seriously from multidrug resistance (MDR), generally caused by innate DNA repair proteins that reverse the DNA modification by anti-cancer therapeutics or trans-membrane efflux proteins that pump anti-cancer therapeutics out of the cytosol. This project focused on finding microRNAs that can regulate MDR proteins by managing corresponding mRNA levels through post-transcriptional regulation based on nucleotide sequence matching. Screening was done with bioinformatics databases for unpublished/unexplored microRNAs with high nucleotide sequence correspondence to two representative MDR proteins, MGMT (a DNA repair protein) and ABCB1 (an efflux protein), revealing microRNA-4539 and microRNA-4261 respectively. To investigate the enhancement of chemotherapeutics in cancer cells, high MGMT expressing glioblastoma (T98G) and a high ABCB1 expressing triple-negative breast cancer cell line (MDA-MB-231-luc) were treated with varying concentrations of chemotherapeutics and corresponding miRNAs. Newly identified MDR-related miRNAs (MDRmiRs) enhanced the response to anti-cancer therapeutics and resulted in effective cell death. In this study, we demonstrated that therapeutic miRNAs could be identified based on the nucleotide sequence matching of miRNAs to targeted mRNA and the same approach could be employed for the screening of therapeutic candidates to regulate specific target proteins in diverse diseases.

Prior research has shown that critical differences between non-metastatic and metastatic tumor cells are at the level of microRNA. Consequently, harnessing these molecules for the treatment of metastatic cancer could have significant clinical impact. In the present study, we set out to identify metastasis-specific microRNAs which drive metastatic colonization of distant organs. Using a murine model of metastatic breast cancer, we employed a directed approach in which we screened for microRNAs that are differentially expressed between the primary tumors and metastatic lesions but concordantly expressed in all of the metastatic lesions irrespective of the tissue that is colonized. Of the identified targets, we focused on miR-710, which was consistently and significantly downregulated in the metastatic lesions relative to the primary tumors. The level of downregulation was independent of the distant organ that is involved, suggesting that miR-710 plays a fundamental role in metastatic colonization. Computational target prediction suggested a pleiotropic role for miR-710 in apoptosis, migration and invasion, and stemness. Using a previously validated oligonucleotide delivery system, we introduced miR-710 mimics into 4T1 metastatic breast adenocarcinoma cells and assessed the resultant phenotypic effects. We demonstrated significant inhibition of cell viability, migration, and invasion. We also showed that the treatment profoundly enhanced cell senescence, reduced stemness, and influenced markers of epithelial to mesenchymal transition, as evidenced by enhanced E-cadherin and reduced vimentin expression. This knowledge represents a first step towards harnessing a similar approach to discover novel microRNA targets with therapeutic potential in metastasis.

The recent past has seen impressive progress in the treatment of various malignancies using immunotherapy. One of the most promising approaches involves immune checkpoint inhibitors. However, the clinical results with these agents have demonstrated variability in the response. Pancreatic cancer, in particular, has proven resistant to initial immunotherapy approaches. Here, we describe an alternative strategy that relies on combining gemcitabine and a novel programmed death-ligand 1 (PD-L1) inhibitor, termed MN-siPDL1. MN-siPDL1 incorporates small interfering RNA against PD-L1 (siPDL1) conjugated to a magnetic nanocarrier (MN). We show that noninvasive magnetic resonance imaging (MRI) could be used to monitor therapeutic response. Combination therapy consisting of gemcitabine and MN-siPDL1 in a syngeneic murine pancreatic cancer model resulted in a significant reduction in tumor growth and an increase in survival. Following optimization, a 90% reduction in tumor volume was achieved 2 weeks after the beginning of treatment. Whereas 100% of the control animals had succumbed to their tumors by week 6 after the beginning of treatment, there was no mortality in the experimental group by week 5, and 67% of the experimental animals survived for 12 weeks. This method could provide therapeutic benefit against an intractable disease for which there are no effective treatments and which is characterized by a mere 1% 5-year survival.

Traditional cancer therapy has relied on a strictly cytotoxic approach that views non-metastatic and metastatic tumor cells as identical in terms of molecular biology and sensitivity to therapeutic intervention. Mounting evidence suggests that in fact non-metastatic and metastatic tumor cells differ in key characteristics that could explain the capacity of the metastatic cells to not only escape the primary organ but also to survive while in the circulation and to colonize a distant organ. Here, we lay out a framework for a new multi-pronged therapeutic approach. This approach involves modifying the local microenvironment of the primary tumor to inhibit the formation and release of metastatic cells; normalizing the microenvironment of the metastatic organ to limit the capacity of metastatic tumor cells to invade and colonize the organ; remediating the immune response to tumor neoantigens; and targeting metastatic tumor cells on a systemic level by restoring critical and unique aspects of the cell’s phenotype, such as anchorage dependence. Given the limited progress against metastatic cancer using traditional therapeutic strategies, the outlined paradigm could provide a more rational alternative to patients with metastatic cancer.

Traditionally, cancer therapy has relied on surgery, radiation therapy, and chemotherapy. In recent years, these interventions have become increasingly replaced or complemented by more targeted approaches that are informed by a deeper understanding of the underlying biology. Still, the implementation of fully rational patient-specific drug design appears to be years away. Here, we present a vision of rational drug design for cancer that is defined by two major components: modularity and image guidance. We suggest that modularity can be achieved by combining a nanocarrier and an oligonucleotide component into the therapeutic. Image guidance can be incorporated into the nanocarrier component by labeling with a specific imaging reporter, such as a radionuclide or contrast agent for magnetic resonance imaging. While limited by the need for additional technological advancement in the areas of cancer biology, nanotechnology, and imaging, this vision for the future of cancer therapy can be used as a guide to future research endeavors.

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.

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.