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The failure of cancer therapies in clinical settings is often attributed to the lack of a relevant tumor model and pathological heterogeneity across tumor types in the clinic. The objective of this study was to develop a robust in vivo tumor model that better represents clinical tumors for the evaluation of anti-cancer therapies. We successfully developed a simple mouse tumor model based on 3D cell culture by injecting a single spheroid and compared it to a tumor model routinely used by injecting cell suspension from 2D monolayer cell culture. We further characterized both tumors with cellular markers for the presence of myofibroblasts, pericytes, endothelial cells and extracellular matrix to understand the role of the tumor microenvironment. We further investigated the effect of chemotherapy (doxorubicin), nanomedicine (Doxil®), biological therapy (Avastin®) and their combination. Our results showed that the substantial blood vasculature in the 3D spheroid model enhances the delivery of Doxil® by 2.5-fold as compared to the 2D model. Taken together, our data suggest that the 3D tumors created by simple subcutaneous spheroid injection represents a robust and more vascular murine tumor model which is a clinically relevant platform to test anti-cancer therapy in solid tumors.

Ovarian cancer is an aggressive tumor owing to its ability to metastasize from stage II onward. Herein, lipid nanoparticles (LNPs) that encapsulate combination of small interfering RNAs (siRNAs), polo-like kinase-1 (PLK1), and eukaryotic translation-initiation factor 3c (eIF3c), to target different cellular pathways essential for ovarian cancer progression are generated. The LNPs are further modified with hyaluronan (tNPs) to target cluster of differentiation 44 (CD44) expressing cells. Interestingly, hyaluronan-coated LNPs (tNPs) prolong functional activity and reduce growth kinetics of spheroids in in vitro assay as compared to uncoated LNPs (uNPs) due to ≈1500-fold higher expression of CD44. Treatment of 2D and 3D cultured ovarian cancer cells with LNPs encapsulating both siRNAs result in 85% cell death and robust target gene silencing. In advanced orthotopic ovarian cancer model, intraperitoneal administration of LNPs demonstrates CD44 specific tumor targeting of tNPs compared to uNPs and robust gene silencing in tissues involved in ovarian cancer pathophysiology. At very low siRNA dose, enhanced overall survival of 60% for tNPs treated mice is observed compared to 10% and 20% for single siRNA-, eIF3c-tNP, and PLK1-tNP treatment groups, respectively. Overall, LNPs represent promising platform in the treatment of advanced ovarian cancer by improving median- and overall-survival.

Hematological malignancies are a group of diseases characterized by clonal proliferation of blood-forming cells. Malignant blood cells are classified as myeloid or lymphoid cells depending on their stem cell origin. Lymphoid malignancies are characterized by lymphocyte accumulation in the blood stream, in the bone marrow, or in lymphatic nodes and organs. Several of these diseases are associated with chromosomal translocations, which cause gene fusion and amplification of expression, while others are characterized with aberrant expression of oncogenes. Overall, these genes play a major role in development and maintenance of malignant clones. The discovery of antisense oligonucleotides and RNA interference (RNAi) mechanisms offer new tools to specifically manipulate gene expression. Systemic delivery of inhibitory oligonucleotides molecules for manipulation of gene expression in lymphocytes holds a great potential for facilitating the development of an oligonucleotides -based therapy platform for lymphoid blood cancer. However, lymphocytes are among the most difficult targets for oligonucleotides delivery, as they are resistant to conventional transfection reagents and are dispersed throughout the body, making it difficult to successfully localize or deliver oligonucleotides payloads via systemic administration. In this review, we will survey the latest progress in the field of oligonucleotides based nanomedicine in the heterogeneous group of hematological malignancies with special emphasis on RNA based strategies. We will describe the most advanced non-viral nanocarriers for RNA delivery to malignant blood cells. We will also discuss targeted strategies for cell specific delivery of RNA molecules using nanoparticles and the therapeutic benefit of manipulating gene function in hematological malignancies. Finally, we will focus on the ex vivo, in vivo, and clinical trial strategies, that are currently under development in hematological malignancies - strategies that might increase the arsenal of drugs available to hematologists in the upcoming years.

The use of nanoparticles as anticancer drug carriers has been studied for over 50 years. These nanoparticles that can carry drugs are now termed "nanomedicines". Since the approval of the first FDA "nanodrug", DOXIL in 1995, tremendous efforts have been made to develop hundreds of nanomedicines based on different materials. The development of drug nanocarriers (NCs) for cancer therapy is especially challenging and requires multidisciplinary approach. Not only is the translation from a lab scale production of the NCs to clinical scale a challenge, but tumor biology and its unique physiology also possess challenges that need to be overcome with cleverer approaches. Yet, with all the efforts made to develop new strategies to deliver drugs (including small molecules and biologics) for cancer therapy, the number of new NCs that are reaching clinical trials is extremely low. Here we discuss the reasons most of the NCs loaded with anticancer drugs are not likely to reach the clinic and emphasize the importance of understanding tumor physiology and heterogeneity, the use of predictive animal models, and the importance of sharing data as key denominators for potential successful translation of NCs from a bench scale into clinical modality for cancer care.

Cancer cells are rapidly evolving due to their unstable genome, which contributes to the development of new cancer clones with different gene expression profile (GEP). Manipulating the expression of the genes vital for the progression of the disease is essential to overcome its heterogeneity. However, targeting overexpressed genes, retrieved from GEP analysis, would be efficient for a specific kind of a malignancy. Alternatively, manipulating the expression of genes that are part of a fundamental mechanism in the cell would be effective against a wide range of malignancies. To test this hypothesis we characterized, using RNAi approaches, the therapeutic potential of the housekeeping eIF3c gene in five different cancer cell lines NCI-ADR/RES (NAR), HeLa, MCF7, HCT116 and B16F10. eIF3c is one of the core subunit of the eukaryote translation initiation factor (eIF) 3 complex, which has a crucial role in the translation initiation process. In this study, we demonstrated that eIF3c is vital to translation initiation in vivo, as its downregulation decreases the global protein synthesis and causes a polysome run-off. In addition, reducing the expression of eIF3c mediates G0/G1 or G2/M arrest in a tissue dependent manner, which leads to a reduction in cell proliferation and eventually to cell death. Moreover, we demonstrated the efficiency of the hyaluronan (HA)-coated lipid-based nanoparticles (LNPs) platform to deliver eIF3c-siRNAs to mouse melanoma cells. Taking together, our results emphasize the importance of seeking ubiquitously expressed housekeeping genes such as eIF3c rather than tumor associated overexpressed genes as therapeutic targets for the heterogeneous malignancies.

RNA interference (RNAi), a natural cellular mechanism for RNA-guided regulation of gene expression could in fact become new therapeutic modality if an appropriate efficient delivery strategy that is also reproducible and safe will be developed. Numerous efforts have been made for the past eight years to address this challenge with only mild success. The majority of these strategies are based on cationic formulations that condense the RNAi payload and deliver it into the cell cytoplasm. However, most of these formulations also evoke adverse effects such as mitochondrial damage, interfering with blood coagulation cascade, induce interferon response, promote cytokine induction and activate the complement. Herein, we present a strategy that is devised from neutral phospholipids and cholesterol that self-assembled into lipid-based nanoparticles (LNPs). These LNPs were then coated with the glycosaminoglycan, hyaluronan (HA). HA-LNPs bound and internalized specifically into cancer cells compared with control, non-coated particles. Next, loaded with siRNAs against the multidrug resistance extrusion pump, p-glycoprotein (P-gp), HA-LNPs efficiently and specifically reduced mRNA and P-gp protein levels compared with control particles and with HA-LNPs loaded with control, non-targeted siRNAs. In addition, no cellular toxicity or cytokine induction was observed when these particles were cultured with human Peripheral Blood Mononuclear Cells (PBMCs). The HA-LNPs may offer an alternative approach to cationic lipid-based formulations for RNAi delivery into cancer cells in an efficient and safe manner.

Cervical cancer is a type of slow-growing cancer associated with high mortality rates. Early detection can enable lifesaving early intervention. Current cervical premalignant lesion detection methods suffer from both high miss rates and excessive referrals for unnecessary biopsies. Herein, coherent light backscatter and modifications in reflected white-light spectra were measured to specifically discriminate between cervical tumors and normal squamous epithelial tissues resected from a mouse xenograft model. The combined measurements resulted in 92% sensitivity and 93% specificity in discrimination between the two tissues. These methods can be used to develop a noninvasive portable optical probe for sensitive and objective detection of precancer and cancer epithelial lesions in the cervix and other accessible epithelial tissues.

Purpose: The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), are involved in a wide range of biological activities, including cell proliferation, motility, invasion, and angiogenesis. The HGF/SF-Met signaling pathway is frequently activated in a variety of cancers, and uncontrolled Met activation correlates with highly invasive tumors and poor prognosis. In this study, we investigated the inhibitory effect of a novel soluble splice variant of Met on the HGF/SF-Met pathway. Experimental Design: Using our alternative splicing modeling platform LEADS, we have identified a novel splice variant of the Met receptor, which encodes a truncated soluble form of the receptor. This variant was produced as a recombinant Fc-fused protein named Cgen-241A and was tested in various cell-based assays representing different outcomes of the HGF/SF-Met pathway. Results: Cgen-241A significantly inhibited HGF/SF-induced Met phosphorylation as well as cell proliferation and survival. In addition, Cgen-241A showed a profound inhibitory effect on cell scattering, invasion, and urokinase up-regulation. The inhibitory effects of Cgen-241A were shown in multiple human and nonhuman cell types, representing different modes of Met activation. Furthermore, Cgen-241A showed direct binding to HGF/SF. Conclusions: Taken together, our results indicate that Cgen-241A is a potent antagonist of the HGF/SF-Met pathway, underlining its potential as a therapeutic agent for the treatment of a wide variety of human malignancies that are dependent on this pathway.

Metastatic lung cancer is one of the most common cancers leading to mortality worldwide. Current treatment includes chemo- and pathway-dependent therapy aiming at blocking the spread and proliferation of these metastatic lesions. Nanomedicine is an emerging multidisciplinary field that offers unprecedented access to living cells and promises the state of the art in cancer detection and treatment. Development of nanomedicines as drug carriers (nanocarriers) that target cancer for therapy draws upon principles in the fields of chemistry, medicine, physics, biology, and engineering. Given the zealous activity in the field as demonstrated by more than 30 nanocarriers already approved for clinical use and given the promise of recent clinical results in various studies, nanocarrier-based strategies are anticipated to soon have a profound impact on cancer medicine and human health. Herein, we will detail the latest innovations in therapeutic nanomedicine with examples from lipid-based nanoparticles and polymer-based approaches, which are engineered to deliver anticancer drugs to metastatic lung cells. Emphasis will be placed on the latest and most attractive delivery platforms, which are developed specifically to target lung metastatic tumors. These novel nanomedicines may open new avenues for therapeutic intervention carrying new class of drugs such as RNAi and mRNA and the ability to edit the genome using the CRISPER/Cas9 system. Ultimately, these strategies might become a new therapeutic modality for advanced-stage lung cancer.

RNAi therapeutics is a powerful tool for treating diseases by sequence-specific targeting of genes using siRNA. Since its discovery, the need for a safe and efficient delivery system for siRNA has increased. Here, we have developed and characterized a delivery platform for siRNA based on the natural polysaccharide starch in an attempt to address unresolved delivery challenges of RNAi. Modified potato starch (Q-starch) was successfully obtained by substitution with quaternary reagent, providing Q-starch with cationic properties. The results indicate that Q-starch was able to bind siRNA by self-assembly formation of complexes. For efficient and potent gene silencing we monitored the physical characteristics of the formed nanoparticles at increasing N/P molar ratios. The minimum ratio for complete entrapment of siRNA was 2. The resulting complexes, which were characterized by a small diameter (~ 30 nm) and positive surface charge, were able to protect siRNA from enzymatic degradation. Q-starch/siRNA complexes efficiently induced P-glycoprotein (P-gp) gene silencing in the human ovarian adenocarcinoma cell line, NCI-ADR/Res (NAR), over expressing the targeted gene and presenting low toxicity. Additionally, Q-starch-based complexes showed high cellular uptake during a 24-hour study, which also suggested that intracellular siRNA delivery barriers governed the kinetics of siRNA transfection. In this study, we have devised a promising siRNA delivery vector based on a starch derivative for efficient and safe RNAi application.

Lipid-based nanoparticles (LNPs) hold great promise as delivery vectors in the treatment of cancer, inflammation, and infections and are already used in clinical practice. Numerous strategies based on LNPs are being developed to carry drugs into specific target sites. The common denominator for all of these LNPs-based platforms is to improve the payloads' pharmacokinetics, biodistribution, stability and therapeutic benefit, and to reduce to minimal adverse effects. In addition, the delivery system must be biocompatible and non-toxic and avoid undesirable interactions with the immune system. In order to achieve optimal benefits from these delivery strategies, interactions with the immune system must be thoroughly investigated. This report will center on the interactions of LNPs with different subsets of leukocytes and will detail representative examples of suppression or activation of the immune system by these carriers. By understanding the interactions of LNPs with the innate and the adaptive arms of the immune system it might be possible to attain improved therapeutic benefits and to avoid immune toxicity.

Increasing exposure of biological systems to large amounts of polycyclic aromatic hydrocarbons is of great public concern. Organisms have an array of biological defense mechanisms, and it is believed that mucosal gel (which covers the respiratory system, the gastrointestinal tract, etc.) provides an effective chemical shield against a range of toxic materials. However, in this work, we demonstrate, for the first time, that, upon complexation of polyaromatic hydrocarbons with mucins, enhanced bioavailability and, therefore, toxicity are obtained. This work was aimed to demonstrate how complexation of various highly hydrophobic polycyclic aromatic hydrocarbons with representative mucin glycoprotein could lead to the formation of previously undescribed materials, which exhibit increased toxicity versus pristine polycyclic aromatic hydrocarbons. In the present work, we show that a representative mucin glycoprotein, bovine submaxillary mucin, has impressive and unprecedented capabilities of binding and solubilizing water-insoluble materials in physiological solution. The complexes formed between the mucin and a series of polycyclic aromatic hydrocarbons were comprehensively characterized, and their toxicity was evaluated by both in vivo and in vitro assays. In addition, the bioavailability and membrane-penetration capabilities were tested using an internalization assay. Our results provide, for the first time, evidence of an unknown route by which hydrophobic materials may achieve higher bioavailability, penetrating some of the biological defense systems, in the form of water-soluble complexes with mucosal proteins.