Hon S. Leong

Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles

Viral nanoparticles are a novel class of biomolecular agents that take advantage of the natural circulatory and targeting properties of viruses to allow the development of therapeutics, vaccines and imaging tools. We have developed a multivalent nanoparticle platform based on the cowpea mosaic virus (CPMV) that facilitates particle labeling at high density with fluorescent dyes and other functional groups. Compared with other technologies, CPMV-based viral nanoparticles are particularly suited for long-term intravital vascular imaging because of their biocompatibility and retention in the endothelium with minimal side effects. The stable, long-term labeling of the endothelium allows the identification of vasculature undergoing active remodeling in real time. In this study, we describe the synthesis, purification and fluorescent labeling of CPMV nanoparticles, along with their use for imaging of vascular structure and for intravital vascular mapping in developmental and tumor angiogenesis models. Dye-labeled viral nanoparticles can be synthesized and purified in a single day, and imaging studies can be conducted over hours, days or weeks, depending on the application.

Re-Examining the Size/Charge Paradigm: Differing In Vivo Characteristics of Size and Charge-Matched Mesoporous Silica Nanoparticles

The combination of nanoparticle (NP) size, charge, and surface chemistry (e.g., extent of modification with polyethylene glycol (PEG)) is accepted as a key determinant of NP/cellular interactions. However, the influence of spatial arrangement and accessibility of the charged molecules on the NP surface vis-à-vis the average surface charge (zeta (ζ) potential) is incompletely understood. Here we demonstrate that two types of mesoporous silica nanoparticles (MSNP) that are matched in terms of primary and hydrodynamic particle size, shape, pore structure, colloidal stability, and ζ potential, but differ in surface chemistry, viz. the spatial arrangement and relative exposure of surface amines, have profoundly different interactions with cells and tissues when evaluated in vitro and in vivo. While both particles are ∼50 nm in diameter, PEGylated, and positively charged (ζ = +40 mV), PEG-PEI (MSNPs modified with exposed polyamines), but not PEG-NMe3(+) (MSNP modified with distributed, obstructed amines) rapidly bind serum proteins, diverse cells types in vitro, and endothelial and white blood cells in vivo (ex ovo chick embryo model). This finding helps elucidate the relative role of surface exposure of charged molecules vs ζ potential in otherwise physicochemically matched MSNP and highlights protein corona neutrality as an important design consideration when synthesizing cationic NPs for biological applications.

KISS1R Induces Invasiveness of Estrogen Receptor- Negative Human Mammary Epithelial and Breast Cancer Cells

Kisspeptins (KPs), peptide products of the KISS1 metastasis-suppressor gene, are endogenous ligands for a G protein-coupled receptor (KISS1R). KISS1 acts as a metastasis suppressor in numerous human cancers. However, recent studies have demonstrated that an increase in KISS1 and KISS1R expression in patient breast tumors correlates with higher tumor grade and metastatic potential. We have shown that KP-10 stimulates invasion of estrogen receptor α (ERα)-negative MDA-MB-231 breast cancer cells via transactivation of the epidermal growth factor receptor (EGFR). Here, we report that either KP-10 treatment of ERα-negative nonmalignant mammary epithelial MCF10A cells or expression of KISS1R in MCF10A cells induced a mesenchymal phenotype and stimulated invasiveness. Similarly, exogenous expression of KISS1R in ERα-negative SKBR3 breast cancer cells was sufficient to trigger invasion and induced extravasation in vivo. In contrast, KP-10 failed to transactivate EGFR or stimulate invasiveness in the ERα-positive MCF7 and T47D breast cancer cells. This suggested that ERα negatively regulates KISS1R-dependent breast cancer cell migration, invasion, and EGFR transactivation. In support of this, we found that these KP-10-induced effects were ablated upon exogenous expression of ERα in the MDA-MB-231 cells, by down-regulating KISS1R expression. Lastly, we have identified IQGAP1, an actin cytoskeletal binding protein as a novel binding partner of KISS1R, and have shown that KISS1R regulates EGFR transactivation in breast cancer cells in an IQGAP1-dependent manner. Overall, our data strongly suggest that the ERα status of mammary cells dictates whether KISS1R may be a novel clinical target for treating breast cancer metastasis.

BRCA2 inhibition enhances cisplatin-mediated alterations in tumor cell proliferation, metabolism, and metastasis

Tumor cells have unstable genomes relative to non‐tumor cells. Decreased DNA integrity resulting from tumor cell instability is important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti‐cancer agents is therefore an attractive therapeutic strategy. BRCA2 is involved in homologous recombination repair. BRCA2 defects increase cancer risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2‐targeting second‐generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast cancer cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck cancer cells, but induced minimal cisplatin sensitivity in non‐tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency in vivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is a potential therapeutic anti‐cancer agent.

SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases

Sorting nexin 9 is a multifunctional scaffold protein that coordinates endocytic trafficking, activation of RhoGTPases, and actin nucleation via N-WASP to modulate cancer cell invasion and metastasis.

Probiotic Lactobacillus rhamnosus reduces organophosphate pesticide absorption and toxicity to Drosophila melanogaster

ABSTRACT Organophosphate pesticides used in agriculture can pose health risks to humans and wildlife. We hypothesized that dietary supplementation with Lactobacillus, a genus of commensal bacteria, would reduce absorption and toxicity of consumed organophosphate pesticides (parathion and chlorpyrifos [CP]). Several Lactobacillus species were screened for toleration of 100 ppm of CP or parathion in MRS broth based on 24-h growth curves. Certain Lactobacillus strains were unable to reach stationary-phase culture maxima and displayed an abnormal culture morphology in response to pesticide. Further characterization of commonly used, pesticide-tolerant and pesticide-susceptible, probiotic Lactobacillus rhamnosus strain GG (LGG) and L. rhamnosus strain GR-1 (LGR-1), respectively, revealed that both strains could significantly sequester organophosphate pesticides from solution after 24-h coincubations. This effect was independent of metabolic activity, as L. rhamnosus GG did not hydrolyze CP and no difference in organophosphate sequestration was observed between live and heat-killed strains. Furthermore, LGR-1 and LGG reduced the absorption of 100 μM parathion or CP in a Caco-2 Transwell model of the small intestine epithelium. To determine the effect of sequestration on acute toxicity, newly eclosed Drosophila melanogaster flies were exposed to food containing 10 μM CP with or without supplementation with live LGG. Supplementation with LGG simultaneously, but not with administration of CP 3 days prior (prophylactically), mitigated CP-induced mortality. In summary, the results suggest that L. rhamnosus may be useful for reducing toxic organophosphate pesticide exposure via passive binding. These findings could be transferable to clinical and livestock applications due to affordability and practical ability to supplement products with food-grade bacteria. IMPORTANCE The consequences of environmental pesticide pollution due to widespread usage in agriculture and soil leaching are becoming a major societal concern. Although the long-term effects of low-dose pesticide exposure for humans and wildlife remain largely unknown, logic suggests that these chemicals are not aligned with ecosystem health. This observation is most strongly supported by the agricultural losses associated with honeybee population declines, known as colony collapse disorder, in which pesticide usage is a likely trigger. Lactobacilli are bacteria used as beneficial microorganisms in fermented foods and have shown potentials to sequester and degrade environmental toxins. This study demonstrated that commonly used probiotic strains of lactobacilli could sequester, but not metabolize, organophosphate pesticides (parathion and chlorpyrifos). This Lactobacillus-mediated sequestration was associated with decreased intestinal absorption and insect toxicity in appropriate models. These findings hold promise for supplementing human, livestock, or apiary foods with probiotic microorganisms to reduce organophosphate pesticide exposure.

Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

BackgroundMembrane Type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease implicated in metastatic progression ostensibly due to its ability to degrade extracellular matrix (ECM) components and allow migration of cells through the basement membrane. Despite in vitro studies demonstrating this principle, this knowledge has not translated into the use of MMP inhibitors (MMPi) as effective cancer therapeutics, or been corroborated by evidence of in vivo ECM degradation mediated by MT1-MMP, suggesting that our understanding of the role of MT1-MMP in cancer progression is incomplete.MethodsMCF-7 and MDA-MB 231 breast cancer cell lines were created that stably overexpress different levels of MT1-MMP. Using 2D culture, we analyzed proMMP-2 activation (gelatin zymography), ECM degradation (fluorescent gelatin), ERK signaling (immunoblot), cell migration (transwell/scratch closure/time-lapse imaging), and viability (colorimetric substrate) to assess how different MT1-MMP levels affect these cellular parameters. We also utilized Matrigel 3D cell culture and avian embryos to examine how different levels of MT1-MMP expression affect morphological changes in 3D culture, and tumourigenecity and extravasation efficiency in vivo.ResultsIn 2D culture, breast cancer cells expressing high levels of MT1-MMP were capable of widespread ECM degradation and TIMP-2-mediated proMMP-2 activation, but were not the most migratory. Instead, cells expressing low levels of MT1-MMP were the most migratory, and demonstrated increased viability and ERK activation. In 3D culture, MCF-7 breast cancer cells expressing low levels of MT1-MMP demonstrated an invasive protrusive phenotype, whereas cells expressing high levels of MT1-MMP demonstrated loss of colony structure and cell fragment release. Similarly, in vivo analysis demonstrated increased tumourigenecity and metastatic capability for cells expressing low levels of MT1-MMP, whereas cells expressing high levels were devoid of these qualities despite the production of functional MT1-MMP protein.ConclusionsThis study demonstrates that excessive ECM degradation mediated by high levels of MT1-MMP is not associated with cell migration and tumourigenesis, while low levels of MT1-MMP promote invasion and vascularization in vivo.

Neonicotinoid-induced pathogen susceptibility is mitigated by Lactobacillus plantarum immune stimulation in a Drosophila melanogaster model

Pesticides are used extensively in food production to maximize crop yields. However, neonicotinoid insecticides exert unintentional toxicity to honey bees (Apis mellifera) that may partially be associated with massive population declines referred to as colony collapse disorder. We hypothesized that imidacloprid (common neonicotinoid; IMI) exposure would make Drosophila melanogaster (an insect model for the honey bee) more susceptible to bacterial pathogens, heat stress, and intestinal dysbiosis. Our results suggested that the immune deficiency (Imd) pathway is necessary for D. melanogaster survival in response to IMI toxicity. IMI exposure induced alterations in the host-microbiota as noted by increased indigenous Acetobacter and Lactobacillus spp. Furthermore, sub-lethal exposure to IMI resulted in decreased D. melanogaster survival when simultaneously exposed to bacterial infection and heat stress (37 °C). This coincided with exacerbated increases in TotA and Dpt (Imd downstream pro-survival and antimicrobial genes, respectively) expression compared to controls. Supplementation of IMI-exposed D. melanogaster with Lactobacillus plantarum ATCC 14917 mitigated survival deficits following Serratia marcescens (bacterial pathogen) septic infection. These findings support the insidious toxicity of neonicotinoid pesticides and potential for probiotic lactobacilli to reduce IMI-induced susceptibility to infection.

Prostate extracellular vesicles in patient plasma as a liquid biopsy platform for prostate cancer using nanoscale flow cytometry

Background Extracellular vesicles released by prostate cancer present in seminal fluid, urine, and blood may represent a non-invasive means to identify and prioritize patients with intermediate risk and high risk of prostate cancer. We hypothesize that enumeration of circulating prostate microparticles (PMPs), a type of extracellular vesicle (EV), can identify patients with Gleason Score≥4+4 prostate cancer (PCa) in a manner independent of PSA. Patients and Methods Plasmas from healthy volunteers, benign prostatic hyperplasia patients, and PCa patients with various Gleason score patterns were analyzed for PMPs. We used nanoscale flow cytometry to enumerate PMPs which were defined as submicron events (100-1000nm) immunoreactive to anti-PSMA mAb when compared to isotype control labeled samples. Levels of PMPs (counts/μL of plasma) were also compared to CellSearch CTC Subclasses in various PCa metastatic disease subtypes (treatment naïve, castration resistant prostate cancer) and in serially collected plasma sets from patients undergoing radical prostatectomy. Results PMP levels in plasma as enumerated by nanoscale flow cytometry are effective in distinguishing PCa patients with Gleason Score≥8 disease, a high-risk prognostic factor, from patients with Gleason Score≤7 PCa, which carries an intermediate risk of PCa recurrence. PMP levels were independent of PSA and significantly decreased after surgical resection of the prostate, demonstrating its prognostic potential for clinical follow-up. CTC subclasses did not decrease after prostatectomy and were not effective in distinguishing localized PCa patients from metastatic PCa patients. Conclusions PMP enumeration was able to identify patients with Gleason Score ≥8 PCa but not patients with Gleason Score 4+3 PCa, but offers greater confidence than CTC counts in identifying patients with metastatic prostate cancer. CTC Subclass analysis was also not effective for post-prostatectomy follow up and for distinguishing metastatic PCa and localized PCa patients. Nanoscale flow cytometry of PMPs presents an emerging biomarker platform for various stages of prostate cancer.

Fibroblast Growth Factor Receptor-Dependent and -Independent Paracrine Signaling by Sunitinib-Resistant Renal Cell Carcinoma

ABSTRACT Antiangiogenic therapies, such as sunitinib, have revolutionized renal cell carcinoma (RCC) treatment. However, a precarious understanding of how resistance emerges and a lack of tractable experimental systems hinder progress. We evaluated the potential of primary RCC cultures (derived from tumors and tumor grafts) to signal to endothelial cells (EC) and fibroblasts in vitro and to stimulate angiogenesis ex vivo in chorioallantoic membrane (CAM) assays. From 65 patients, 27 primary cultures, including several from patients with sunitinib-resistant RCC, were established. RCC cells supported EC survival in coculture assays and induced angiogenesis in CAM assays. RCC-induced EC survival was sensitive to sunitinib in half of the tumors and was refractory in tumors from resistant patients. Sunitinib sensitivity correlated with vascular endothelial growth factor (VEGF) production. RCC induced paracrine extracellular signal-regulated kinase (ERK) activation in EC which was inhibited by sunitinib in sensitive but not in resistant tumors. As determined by fibroblast growth factor receptor substrate 2 (FRS2) phosphorylation in fibroblasts, RCC broadly induced low-level fibroblast growth factor receptor (FGFR) signaling. Whereas ERK activation in EC was uniformly inhibited by combined VEGF/platelet-derived growth factor (PDGF)/FGF receptor inhibitors, paracrine ERK activation in fibroblasts was blocked in only a fraction of tumors. Our data show that RCC activates EC through VEGF-dependent and -independent pathways, that sunitinib sensitivity correlates with VEGF-mediated ERK activation, and that combined inhibition of VEGF/PDGF/FGF receptors is sufficient to inhibit mitogenic signaling in EC but not in fibroblasts.