An Antitumor Immune Response Is Evoked by Partial-Volume Single-Dose Radiation in 2 Murine Models.

PURPOSE: This study examined tumor growth delay resulting from partial irradiation in preclinical mouse models. METHODS AND MATERIALS: We investigated 67NR murine orthotopic breast tumors in both immunocompetent and nude mice. Treatment was delivered to 50% or 100% of the tumor using a 2 × 2 cm collimator on a microirradiator. Radiation response was modulated by treatment with anti-CD8 and anti-intercellular adhesion molecule (anti-ICAM) antibodies. Similar experiments were performed using the less immunogenic Lewis lung carcinoma mouse model. Tumor growth delay and γ-H2AX phosphorylation were measured, and immune response was assessed by immunofluorescence and flow cytometry at 1 and 7 days after radiation therapy. Tumor expression of cellular adhesion molecules was also measured at different times after radiation therapy. RESULTS: Partial irradiation led to tumor responses similar to those of fully exposed tumors in immunocompetent mice, but not in nude mice. After a single dose of 10 Gy, infiltration of CD8+ T cells was observed along with increased expression of ICAM. The response to 10 Gy in hemi-irradiated tumors was abrogated by treatment with either anti-CD8 or anti-ICAM antibodies. Similar responses were obtained in the less immunogenic Lewis lung carcinoma mouse model delivering 15 Gy to half the tumor volume. Treatment with FTY720, a compound that inhibits T-cell egress from lymph nodes, did not affect tumor response at the time of CD8+ T cells infiltration in the nonirradiated area of the tumor. This result indicated that the most likely source of these cells is the irradiated portion of the hemi-irradiated tumors. In addition, a significant abscopal effect was observed after partial irradiation with a single dose of 10 Gy in the 67NR model. CONCLUSIONS: In these models, radiation controls tumor growth both directly through cell killing and indirectly through immune activation. This outcome raises the possibility that this effect could be induced in the clinic.

Phosphorylation state of Ser165 in α-tubulin is a toggle switch that controls proliferating human breast tumors.

Engineered overexpression of protein kinase Cα (PKCα) is known to phosphorylate Ser165 in α-tubulin resulting in stimulated microtubule dynamics and cell motility, and activation of an epithelial-mesenchymal transition (EMT) in non-transformed human breast cells. Here it is shown that endogenous phosphorylation of native α-tubulin in two metastatic breast cell lines, MDA-MB-231-LM2-4175 and MDA-MB-468 is detected at PKC phosphorylation sites. α-Tubulin mutants that simulated phosphorylated (S165D) or non-phosphorylated (S165 N) states were stably expressed in MDA-MB-231-LM2-4175 cells. The S165D-α-tubulin mutant engendered expression of the EMT biomarker N-cadherin, whereas S165 N-α-tubulin suppressed N-cadherin and induced E-cadherin expression, revealing a 'cadherin switch'. S165 N-α-tubulin engendered more rapid passage through the cell cycle, induced shorter spindle fibers and exhibited more rapid proliferation. In nude mice injected with MDA-MB-231-LM2-4175 cells, cells expressing S165 N-α-tubulin (but not the S165D mutant) produced hyper-proliferative lung tumors with increased tumor incidence and higher Ki67 expression. These results implicate the phosphorylation state of Ser165 in α-tubulin as a PKC-regulated molecular switch that causes breast cells to exhibit either EMT characteristics or hyper-proliferation. Evaluation of genomic databases of human tumors strengthens the clinical significance of these findings.

Pazopanib radio-sensitization of human sarcoma tumors.

Recent data in our laboratory indicate that engagement of host-derived microenvironmental elements impact tumor response to single high dose radiation therapy (SDRT). In these studies we showed that microvascular endothelial damage plays a critical role in tumor response as regulator of direct lethal damage of SDRT. Using a genetic model of Acid Sphingomyelinase (ASMase)-deficient mice we showed that activation of this enzyme by SDRT-induced damage in the endothelium is mandatory for tumor cure. ASMase activation triggers ceramide-mediated apoptosis, and therein microvascular dysfunction, which increased the vulnerability of tumor cells to lethal damage by radiation. Angiogenic factors repressed this activity while a monoclonal antibody targeting VEGF, de-repressed ASMase activity and radiosensitized tumor endothelium when delivered immediately prior to SDRT. In this study, we tested the effect of SDRT in combination with the short-acting anti-angiogenic agent, Pazopanib (anti-VEGFR-1/2/3, PDGF-α/ß and c-kit), in two xenograft models of human sarcoma. Pre-treatment with a single dose of Pazopanib increased SDRT-induced ASMase activity and endothelial dysfunction in vitro and in vivo, enhancing SDRT tumor cure, and exhibiting critical dependence on timing relative to SDRT exposure, suggesting a mechanism of action identical to that demonstrated for anti-VEGF/VEGFR2 antibodies. These results demonstrate the ability of Pazopanib to shift the response towards tumor cure and could therefore have a significant impact on clinical trial development in combination with SDRT for sarcoma cancer patients.

PEGylated dendritic polyglycerol conjugate targeting NCAM-expressing neuroblastoma: Limitations and challenges.

Neural cell adhesion molecule (NCAM) is found to be a stem-cell marker in several tumor types and its overexpression is known to correlate with increased metastatic capacity. To combine extravasation- and ligand-dependent targeting to NCAM overexpressing-cells in the tumor microenvironment, we developed a PEGylated NCAM-targeted dendritic polyglycerol (PG) conjugate. Here, we describe the synthesis, physico-chemical characterization and biological evaluation of a PG conjugate bearing the mitotic inhibitor paclitaxel (PTX) and an NCAM-targeting peptide (NTP). PG-NTP-PTX-PEG was evaluated for its ability to inhibit neuroblastoma progression in vitro and in vivo as compared to non-targeted derivatives and free drug. NCAM-targeted conjugate inhibited the migration of proliferating endothelial cells, suggesting it would be able to inhibit tumor angiogenesis. The targeting conjugate provided an improved binding and uptake on IMR-32 cells compared to non-targeted control. However, these results did not translate to our in vivo model on orthotopic neuroblastoma bearing mice.

Wilms Tumor NCAM-Expressing Cancer Stem Cells as Potential Therapeutic Target for Polymeric Nanomedicine.

Cancer stem cells (CSC) form a specific population within the tumor that has been shown to have self-renewal and differentiation properties, increased ability to migrate and form metastases, and increased resistance to chemotherapy. Consequently, even a small number of cells remaining after therapy can repopulate the tumor and cause recurrence of the disease. CSCs in Wilms tumor, a pediatric renal cancer, were previously shown to be characterized by neural cell adhesion molecule (NCAM) expression. Therefore, NCAM provides a specific biomarker through which the CSC population in this tumor can be targeted. We have recently developed an NCAM-targeted nanosized conjugate of paclitaxel bound to a biodegradable polyglutamic acid polymer. In this work, we examined the ability of the conjugate to inhibit Wilms tumor by targeting the NCAM-expressing CSCs. Results show that the conjugate selectively depleted the CSC population of the tumors and effectively inhibited tumor growth without causing toxicity. We propose that the NCAM-targeted conjugate could be an effective therapeutic for Wilms tumor. Mol Cancer Ther; 16(11); 2462-72. ©2017 AACR.

Targeting NCAM-expressing neuroblastoma with polymeric precision nanomedicine.

Neural cell adhesion molecule (NCAM) expression is known to be associated with an aggressive biological behavior, increased metastatic capacity and expression of stem-cell markers in several tumor types. NCAM was also found to be expressed on tumor endothelial cells while forming new capillary-like tubes, but not on normal endothelial cells. An NCAM-targeted polymer-drug conjugate can be used both to target tumors expressing high levels of NCAM as well as the angiogenic vessels and cancer stem cells populations characterized by NCAM expression within tumors. Here, we describe the design, synthesis, physico-chemical characterization and the biological evaluation of an NCAM-targeted conjugate of polyglutamic acid with paclitaxel that was developed and evaluated on neuroblastoma, a high NCAM-expressing tumor. This conjugate inhibited tumor growth to a higher extent compared to the control conjugates and was less toxic than free paclitaxel. The dose of the conjugate could be increased at least twice than the maximum tolerated dose of paclitaxel to achieve better activity without aggravating toxicity. This work presents evidence that NCAM targeting can highly increase the efficacy of nanomedicines in the appropriate tumor models.

In vivo comparative study of distinct polymeric architectures bearing a combination of paclitaxel and doxorubicin at a synergistic ratio.

Nowadays, combination therapy became a standard in oncology. In this study, we compare the activity of two polymeric carriers bearing a combination of the anticancer drugs paclitaxel (PTX) and doxorubicin (DOX), which differ mainly in their architecture and supramolecular assembly. Drugs were covalently bound to a linear polymer, polyglutamic acid (PGA) or to a dendritic scaffold, polyglycerol (PG) decorated with poly(ethylene glycol) (PEG), forming PGA-PTX-DOX and PG-PTX-bz-DOX-PEG, respectively. We explored the relationship between the polymeric architectures and their performance with the aim to augment the pharmacological benefits of releasing both drugs simultaneously at the tumor site at a synergistic ratio. We recently designed and characterized a PGA-PTX-DOX conjugate. Here, we describe the synthesis and characterization of PG dendritic scaffold bearing the combination of PTX and DOX. The performance of both conjugates was evaluated in a murine model of mammary adenocarcinoma in immunocompetent mice, to investigate whether the activity of the treatments is affected by the immune system. Drug conjugation to a nano-sized polymer enabled preferred tumor accumulation by extravasation-dependent targeting, making use of the enhanced permeability and retention (EPR) effect. Both PGA-PTX-DOX and PG-PTX-bz-DOX-PEG nano-sized conjugates exhibited superior anti-tumor efficacy and safety compared to the combination of the free drugs, at equivalent concentrations. However, while PGA-PTX-DOX was more efficient than a mixture of each drug conjugated to a separate PGA chain, as was previously shown, PG-PTX-bz-DOX-PEG had similar activity to the mixture of the PG-PTX-bz-PEG and PG-DOX-PEG conjugates. Our results show that both conjugates are potential candidates as precision combination nanomedicines for the treatment of breast cancer.

Anticancer polymeric nanomedicine bearing synergistic drug combination is superior to a mixture of individually-conjugated drugs.

Paclitaxel and doxorubicin are potent anticancer drugs used in the clinic as mono-therapies or in combination with other modalities to treat various neoplasms. However, both drugs suffer from side effects and poor pharmacokinetics. These two drugs have dissimilar physico-chemical properties, pharmacokinetics and distinct mechanisms of action, toxicity and drug resistance. In order to target both drugs selectively to the tumor site, we conjugated them at a synergistic ratio to a biocompatible and biodegradable polyglutamic acid (PGA) backbone. Drugs conjugation to a nano-sized polymer enabled preferred tumor accumulation by passive targeting, making use of the enhanced permeability and retention (EPR) effect. The rational design presented here resulted in co-delivery of combination of the drugs and their simultaneous release at the tumor site. PGA-paclitaxel-doxorubicin nano-sized conjugate exhibited superior anti-tumor efficacy and safety compared to the combination of the free drugs or a mixture of the drugs conjugated to separate polymer chains, at equivalent concentrations. This novel polymer-based multi-drug nano-sized conjugate allowed for true combination therapy since it delivered both drugs to the same target site at the ratio required for synergism. Using mice bearing orthotopic mammary adenocarcinoma, we demonstrate here the advantage of a combined polymer therapeutic bearing two synergistic drugs on the same polymer backbone, compared to each drug bound separately to the backbone.

Overcoming obstacles in microRNA delivery towards improved cancer therapy.

MicroRNAs (miRNAs) are small noncoding RNAs found to govern nearly every biological process. They frequently acquire a gain or a loss of function in cancer, hence playing a causative role in the development and progression of cancer. There are major obstacles on the way for the successful delivery of miRNA, which include low cellular uptake of the RNA and endosomal escape, immunogenicity, degradation in the bloodstream, and rapid renal clearance. The delivered miRNA needs to be successfully routed to the target organ, enter the cell and reach its intracellular target in an active form. Consequently, in order to exploit the promise of RNA interference, there is an urgent need for efficient methods to deliver miRNAs. These can be divided into three main categories: complexation, encapsulation, and conjugation. In this review, we will discuss the special considerations for miRNA delivery for cancer therapy, focusing on nonviral delivery systems: lipid, polymeric, and inorganic nanocarriers.

Administration, distribution, metabolism and elimination of polymer therapeutics.

Polymer conjugation is an efficient approach to improve the delivery of drugs and biological agents, both by protecting the body from the drug (by improving biodistribution and reducing toxicity) and by protecting the drug from the body (by preventing degradation and enhancing cellular uptake). This review discusses the journey that polymer therapeutics make through the body, following the ADME (absorption, distribution, metabolism, excretion) concept. The biological factors and delivery system parameters that influence each stage of the process will be described, with examples illustrating the different solutions to the challenges of drug delivery systems in vivo.

Physicochemical parameters affecting liposomal bisphosphonates bioactivity for restenosis therapy: internalization, cell inhibition, activation of cytokines and complement, and mechanism of cell death.

Partial inactivation and transient depletion of monocytes/macrophages by liposomal bisphosphonates (LIP-BPs) is widely experimented in various inflammatory disorders including restenosis. Previous studies on activation of cytokines by LIP-BPs are limited to certain cell lines. Moreover, the correlation between in vitro and in vivo studies and complement (C) activation has not been reported. We report here a comprehensive study on the bioactivity of LIP-BPs on various cells' internalization and proliferation, mechanism of cell death, cytokines (in vitro and in vivo) and C activation (in the rat, rabbit and pig). The role of the following parameters has been determined i) drug type (clodronate/alendronate); ii) vesicles size (60-800nm); iii) charge (neutral/negative/ positive); and iv) cell culture type (various cell lines and primary cultures). It was found that monocyte/macrophage inhibition and cytokine activation depend on the cell type, with a limited correlation to the bioactivity obtained in the rat and rabbit models of restenosis. Negatively charged liposomes (85+/-20nm) effectively depleted rabbit's monocytes (67% depletion), with a minor activation of cytokines and no C activation. It is concluded that cell culture studies are insufficient for assessing cytokine activation, and that by controlling LIP-BP properties (size, charge and drug type) optimal bioactivity could be achieved.

Additive-free albumin nanoparticles of alendronate for attenuating inflammation through monocyte inhibition.

AIMS: Particulated dosage forms of bisphosphonates, such as polymeric nanoparticles and liposomes, deplete circulating monocytes and attenuate inflammation. The aim of this work was to develop a novel formulation of albumin nanoparticles with no crosslinkers that encapsulate the bisphosphonate, alendronate and, further, to examine its bioactivity in vitro and in vivo. RESULTS: The novel formulation was prepared by desolvation of human serum albumin in acidic pH induced by alendronate, which enables an electrostatic interaction between albumin and the acidic drug. The mean particle size of the negatively charged nanoparticle was 250-300 nm and drug-entrapment efficiency was 49%. The formulation can be filter sterilized and lyophilized for increased stability. Alendronate nanoparticles exhibited significant inhibitory effects on RAW264 macrophage growth and a significant attenuation of stenosis in rats. CONCLUSION: It is concluded that bioactive nanoparticles of human albumin can be formulated without crosslinkers and potentially toxic additives.