Zohreh Amoozgar

Recent advances in stealth coating of nanoparticle drug delivery systems

Modifying surfaces of nanoparticles (NPs) with polyethylene glycol (PEG), the so-called PEGylation, is the most commonly used method for reducing premature clearance of NPs from the circulation. However, several reports point out that PEGylation may negatively influence the performance of NPs as a drug carrier. Alternative surface modification strategies, including substitute polymers, conditional removal of PEG, and biomimetic surface modification, may provide solutions for the limitations of PEG.

Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival

Significance Improving survival of patients with glioblastoma (GBM) using antiangiogenic therapy remains a challenge. In this study we show that dual blockade of angiopoietin-2 and vascular endothelial growth factor delays tumor growth and enhances survival benefits through reprogramming of tumor-associated macrophages toward an antitumor phenotype as well as by pruning immature tumor vessels. The antitumor immunomodulatory potential of this dual blockade supports clinical testing of this approach for GBM with other immunotherapeutic approaches such as checkpoint blockers. Inhibition of the vascular endothelial growth factor (VEGF) pathway has failed to improve overall survival of patients with glioblastoma (GBM). We previously showed that angiopoietin-2 (Ang-2) overexpression compromised the benefit from anti-VEGF therapy in a preclinical GBM model. Here we investigated whether dual Ang-2/VEGF inhibition could overcome resistance to anti-VEGF treatment. We treated mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBM xenografts with antibodies inhibiting VEGF (B20), or Ang-2/VEGF (CrossMab, A2V). We examined the effects of treatment on the tumor vasculature, immune cell populations, tumor growth, and survival in both the Gl261 and MGG8 tumor models. We found that in the Gl261 model, which displays a highly abnormal tumor vasculature, A2V decreased vessel density, delayed tumor growth, and prolonged survival compared with B20. In the MGG8 model, which displays a low degree of vessel abnormality, A2V induced no significant changes in the tumor vasculature but still prolonged survival. In both the Gl261 and MGG8 models A2V reprogrammed protumor M2 macrophages toward the antitumor M1 phenotype. Our findings indicate that A2V may prolong survival in mice with GBM by reprogramming the tumor immune microenvironment and delaying tumor growth.

Low molecular-weight chitosan as a pH-sensitive stealth coating for tumor-specific drug delivery

When a nanoparticle is developed for systemic application, its surface is typically protected by poly(ethylene glycol) (PEG) to help prolonged circulation and evasion of immune clearance. On the other hand, PEG can interfere with interactions between nanocarriers and target cells and negatively influence the therapeutic outcomes. To overcome this challenge, we propose low molecular-weight chitosan (LMWC) as an alternative surface coating, which can protect the nanomedicine in neutral pH but allow cellular interactions in the weakly acidic pH of tumors. LMWCs with a molecular weight of 2-4 kDa, 4-6.5 kDa, and 11-22 kDa were produced by hydrogen peroxide digestion and covalently conjugated with poly(lactic-co-glycolic acid) (PLGA). Nanoparticles created with PLGA-LMWC conjugates showed pH-sensitive cell interactions, which enabled specific drug delivery to cells in a weakly acidic environment. The hydrophilic LMWC layer reduced opsonization and phagocytic uptake. These properties qualify LMWCs as a promising biomaterial for pH-sensitive stealth coating.

Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages

Significance Inhibition of the VEGF/VEGF receptor (VEGFR) pathway has failed to increase overall survival in phase III trials in patients with glioblastoma (GBM). Previously we identified the angiopoietin-2 (Ang-2)/TEK receptor tyrosine kinase (Tie-2) pathway as a potential driver of resistance to VEGF inhibition in GBM. Here we show that dual inhibition of VEGFRs and Ang-2 inhibits tumor growth and prolongs vessel normalization compared with VEGFR inhibition alone, resulting in improved survival in murine GBM models. Furthermore, by blocking macrophage recruitment, we demonstrate that macrophages contribute to the beneficial effects of dual therapy. Glioblastomas (GBMs) rapidly become refractory to anti-VEGF therapies. We previously demonstrated that ectopic overexpression of angiopoietin-2 (Ang-2) compromises the benefits of anti-VEGF receptor (VEGFR) treatment in murine GBM models and that circulating Ang-2 levels in GBM patients rebound after an initial decrease following cediranib (a pan-VEGFR tyrosine kinase inhibitor) administration. Here we tested whether dual inhibition of VEGFR/Ang-2 could improve survival in two orthotopic models of GBM, Gl261 and U87. Dual therapy using cediranib and MEDI3617 (an anti–Ang-2–neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth and increasing U87 necrosis, effectively reducing viable tumor burden. Consistent with their vascular-modulating function, the dual therapies enhanced morphological normalization of vessels. Dual therapy also led to changes in tumor-associated macrophages (TAMs). Inhibition of TAM recruitment using an anti–colony-stimulating factor-1 antibody compromised the survival benefit of dual therapy. Thus, dual inhibition of VEGFR/Ang-2 prolongs survival in preclinical GBM models by reducing tumor burden, improving normalization, and altering TAMs. This approach may represent a potential therapeutic strategy to overcome the limitations of anti-VEGFR monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells.

Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps

Treatment with DNase I–coated nanoparticles prevents metastasis by targeting neutrophil extracellular traps induced by cancer cells in a mouse model. Metastasis caught in a NET Neutrophil extracellular traps, or NETs, are DNA structures that are produced by neutrophils in response to infection and can promote the spread of cancer in the presence of infection. Park et al. discovered that even in the absence of infection, metastatic breast cancer cells can stimulate neutrophils to form NETs, which further support the spread of metastasis. The authors also demonstrated an approach to breaking this vicious cycle using nanoparticles coated with DNase I, an enzyme that breaks down DNA NETs. This treatment was effective in reducing lung metastases in mice, demonstrating the potential of NETs as a therapeutic target. Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These are pathogen-trapping structures generated by expulsion of the neutrophil’s DNA with associated proteolytic enzymes. NETs produced by infection can promote cancer metastasis. We show that metastatic breast cancer cells can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with deoxyribonuclease I (DNase I) blocked these processes. Treatment with NET-digesting, DNase I–coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-promoting tumor-host interaction and a potential therapeutic target.

Decitabine Enhances Lymphocyte Migration and Function and Synergizes with CTLA-4 Blockade in a Murine Ovarian Cancer Model

A mechanistic basis for the synergy between decitabine and anti–CTLA-4 was found. This provides a rationale for initiating trials of combination therapy in ovarian cancer, in which many patients do not benefit from immune checkpoint blockade alone. The lack of second-line treatment for relapsed ovarian cancer necessitates the development of improved combination therapies. Targeted therapy and immunotherapy each confer clinical benefit, albeit limited as monotherapies. Ovarian cancer is not particularly responsive to immune checkpoint blockade, so combination with a complementary therapy may be beneficial. Recent studies have revealed that a DNA methyl transferase inhibitor, azacytidine, alters expression of immunoregulatory genes in ovarian cancer. In this study, the antitumor effects of a related DNA methyl transferase inhibitor, decitabine (DAC), were demonstrated in a syngeneic murine ovarian cancer model. Low-dose DAC treatment increases the expression of chemokines that recruit NK cells and CD8+ T cells, promotes their production of IFNγ and TNFα, and extends the survival of mice bearing subcutaneous or orthotopic tumors. While neither DAC nor immune checkpoint blockade confers durable responses as a monotherapy in this model, the efficacy of anti–CTLA-4 was potentiated by combination with DAC. This combination promotes differentiation of naïve T cells into effector T cells and prolongs cytotoxic lymphocyte responses as well as mouse survival. These results suggest that this combination therapy may be worthy of further consideration for improved treatment of drug-resistant ovarian cancer. Cancer Immunol Res; 3(9); 1030–41. ©2015 AACR.

Rapidly Photo-Cross-Linkable Chitosan Hydrogel for Peripheral Neurosurgeries

Restoring continuity to severed peripheral nerves is crucial to regeneration and enables functional recovery. However, the two most common agents for coaptation, sutures and fibrin glues, have drawbacks such as inflammation, pathogenesis, and dehiscence. Chitosan-based adhesives are a promising alternative, reported to have good cytocompatibility and favorable immunogenicity. A photo-cross-linkable hydrogel based on chitosan is proposed as a new adhesive for peripheral nerve anastomosis. Two Az-chitosans were synthesized by conjugating 4-azidobenzoic acid with low (LMW, 15 kDa) and high (HMW, 50-190 kDa) molecular weight chitosans. These solutions formed a hydrogel in less than 1 min under UV light. The LMW Az-chitosan was more tightly cross-linked than the HMW variant, undergoing significantly less swelling and possessing a higher rheological storage modulus, and both Az-chitosan gels were stiffer than commercial fibrin glue. Severed nerves repaired by Az-chitosan adhesives tolerated longitudinal forces comparable or superior to fibrin glue. Adhesive exposure to intact nerves and neural cell culture showed both Az-chitosans to be nontoxic in the acute (minutes) and chronic (days) time frames. These results demonstrate that Az-chitosan hydrogels are cytocompatible and mechanically suitable for use as bioadhesives in peripheral neurosurgeries.

The PARP1 inhibitor BMN 673 exhibits immunoregulatory effects in a Brca1-/- murine model of ovarian cancer

Familial breast and ovarian cancer are often caused by inherited mutations of BRCA1. While current prognoses for such patients are rather poor, inhibition of poly-ADP ribose polymerase 1 (PARP1) induces synthetic lethality in cells that are defective in homologous recombination. BMN 673 is a potent PARP1 inhibitor that is being clinically evaluated for treatment of BRCA-mutant cancers. Using the Brca1-deficient murine epithelial ovarian cancer cell line BR5FVB1-Akt, we investigated whether the antitumor effects of BMN 673 extend beyond its known pro-apoptotic function. Administration of modest amounts of BMN 673 greatly improved the survival of mice bearing subcutaneous or intraperitoneal tumors. We thus hypothesized that BMN 673 may influence the composition and function of immune cells in the tumor microenvironment. Indeed, BMN 673 significantly increases the number of peritoneal CD8(+) T cells and NK cells as well as their production of IFN-γ and TNF-α. These data suggest that the cell stress caused by BMN 673 induces not only cancer cell-intrinsic apoptosis but also cancer cell-extrinsic antitumor immune effects in a syngeneic murine model of ovarian cancer. BMN 673 may therefore serve as a promising adjuvant therapy to immunotherapy to achieve durable responses among patients whose tumors harbor defects in homologous recombination.

Photocrosslinkable chitosan modified with angiopoietin-1 peptide, QHREDGS, promotes survival of neonatal rat heart cells.

Myocardial infarction (MI) results in the death of cardiomyocytes (CM), which causes scar formation and pathological remodeling of the heart. The delivery of healthy myocytes or bone marrow cells reduces pathological remodeling after MI, however, current cell injection methods have low cell survival rates and high cell loss. The main objective of this work was to develop a novel hydrogel that can promote survival of CMs. Photocrosslinkable azidobenzoic acid modified chitosan (Az-chitosan) was conjugated with the angiopoietin-1-derived peptide, QHREDGS. This novel peptide is thought to mediate attachment and survival responses of CM to angiopoietin-1 via integrin binding. Thin layers of Az-chitosan, Az-chitosan-QHREDGS, and Az-chitosan-DGQESHR (scrambled peptide control) were spin coated on glass slides and photocrosslinked with application of UV light (365 nm). Neonatal rat heart cells cultured up to 5 days, demonstrated significantly higher attachment and viability on Az-chitosan-QHREDGS compared to cells on other hydrogel controls. Surfaces were also stained for the CM-specific marker troponin I, demonstrating significantly higher percentage of CMs on Az-chitosan-QHREDGS compared to Az-chitosan. The cells cultivated on Az-chitosan-QHREDGS demonstrated significantly lower levels of caspase 3/7 activation after taxol treatment in comparison to cells cultivated on the control hydrogels, glass substrate, or Az-chitosan linked to RGD, an established integrin binding peptide that did not protect against apoptosis. Thus, Az-chitosan-QHREDGS supports attachment and survival of neonatal rat heart cells.

Targeting myeloid cells using nanoparticles to improve cancer immunotherapy.

While nanoparticles have traditionally been used to deliver cytotoxic drugs directly to tumors to induce cancer cell death, emerging data suggest that nanoparticles are likely to generate a larger impact on oncology through the delivery of agents that can stimulate antitumor immunity. Tumor-targeted nanocarriers have generally been used to localize chemotherapeutics to tumors and thus decrease off-target toxicity while enhancing efficacy. Challengingly, tumor heterogeneity and evolution render tumor-intrinsic approaches likely to succumb to relapse. The immune system offers exquisite specificity, cytocidal potency, and long-term activity that leverage an adaptive memory response. For this reason, the ability to manipulate immune cell specificity and function would be desirable, and nanoparticles represent an exciting means by which to perform such manipulation. Dendritic cells and tumor-associated macrophages are cells of the myeloid lineage that function as natural phagocytes, so they naturally take up nanoparticles. Dendritic cells direct the specificity and potency of cellular immune responses that can be targeted for cancer vaccines. Herein, we discuss the specific criteria needed for efficient vaccine design, including but not limited to the route of administration, size, morphology, surface charge, targeting ligands, and nanoparticle composition. In contrast, tumor-associated macrophages are critical mediators of immunosuppression whose trans-migratory abilities can be exploited to localize therapeutics to the tumor core and which can be directly targeted for elimination or for repolarization to a tumor suppressive phenotype. It is likely that a combination of targeting dendritic cells to stimulate antitumor immunity and tumor-associated macrophages to reduce immune suppression will impart significant benefits and result in durable antitumor responses.