NanoFlares for the detection, isolation, and culture of live tumor cells from human blood.

Metastasis portends a poor prognosis for cancer patients. Primary tumor cells disseminate through the bloodstream before the appearance of detectable metastatic lesions. The analysis of cancer cells in blood­so-called circulating tumor cells (CTCs)­may provide unprecedented opportunities for metastatic risk assessment and investigation. NanoFlares are nanoconstructs that enable live-cell detection of intracellular mRNA. NanoFlares, when coupled with flow cytometry, can be used to fluorescently detect genetic markers of CTCs in the context of whole blood. They allow one to detect as few as 100 live cancer cells per mL of blood and subsequently culture those cells. This technique can also be used to detect CTCs in a murine model of metastatic breast cancer. As such, NanoFlares provide, to our knowledge, the first genetic-based approach for detecting, isolating, and characterizing live cancer cells from blood and may provide new opportunities for cancer diagnosis, prognosis, and personalized therapy.

Synthetic high-density lipoprotein-like nanoparticles as cancer therapy.

High-density lipoproteins (HDL) are diverse natural nanoparticles that carry cholesterol and are best known for the role that they play in cardiovascular disease. However, due to their unique targeting capabilities, diverse molecular cargo, and natural functions beyond cholesterol transport, it is becoming increasingly appreciated that HDLs are critical to cancer development and progression. Accordingly, this chapter highlights ongoing research focused on the connections between HDL and cancer in order to design new drugs and targeted drug delivery vehicles. Research is focused on synthesizing biomimetic HDL-like nanoparticles (NP) that can be loaded with diverse therapeutic cargo (e.g., chemotherapies, nucleic acids, proteins) and specifically targeted to cancer cells. Beyond drug delivery, new data is emerging that HDL-like NPs may be therapeutically active in certain tumor types, for example, B cell lymphoma. Overall, HDL-like NPs are becoming increasingly appreciated as targeted, biocompatible, and efficient therapies for cancer, and may soon become indispensable agents in the cancer therapeutic armamentarium.

Peptide amphiphile nanofiber delivery of sonic hedgehog protein to reduce smooth muscle apoptosis in the penis after cavernous nerve resection.

INTRODUCTION: Erectile dysfunction (ED) is a serious medical condition that affects 16-82% of prostate cancer patients treated by radical prostatectomy and current treatments are ineffective in 50-60% of prostatectomy patients. The reduced efficacy of treatments makes novel therapeutic approaches to treat ED essential. The secreted protein Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle and apoptosis that is decreased in cavernous nerve (CN) injury and diabetic ED models. Past studies using Affi-Gel beads have shown SHH protein to be effective in suppressing apoptosis caused by CN injury. AIM: We hypothesize that SHH protein delivered via novel peptide amphiphile (PA) nanofibers will be effective in suppressing CN injury-induced apoptosis. METHODS: Adult Sprague Dawley rats (n=50) were used to optimize PA injection in vivo. PA with SHH protein (n=16) or bovine serum albumin (BSA) (control, n=14) was injected into adult rats that underwent bilateral CN cut. Rats were sacrificed at 2, 4, and 7 days. Alexa Fluor-labeled SHH protein was used to determine the target of SHH signaling (n=3). MAIN OUTCOME MEASURES: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and semiquantitative immunohistochemical analysis for SHH protein and cluster differentiation protein three (CD3) were performed. RESULTS: SHH-PA caused a 25% and 16% reduction in apoptosis at 4 and 7 days after CN injury and a 9.3% and 19% increase in SHH protein at 4 and 7 days after CN injury. CD3 protein was not observed in SHH-PA-treated penis. In vitro, 73% of SHH protein diffused from PA within 6 days. Labeled SHH was observed in smooth muscle. CONCLUSIONS: PA technology is effective in delivering SHH protein to the penis and SHH is effective in suppressing CN injury-induced apoptosis. These results suggest substantial translational potential of this methodology and show that only a short duration of SHH treatment is required to impact the apoptotic index.

Analysis of testosterone effects on sonic hedgehog signaling in juvenile, adolescent and adult sprague dawley rat penis.

INTRODUCTION: Smooth muscle apoptosis is a major contributing factor to erectile dysfunction (ED) development in prostatectomy and diabetic patients and animal models. A critical regulator of penile smooth muscle and apoptosis is Sonic hedgehog (SHH). The SHH protein is decreased in ED models and SHH treatment of cavernous nerve (CN) injured rats prevents smooth muscle apoptosis. A close association between androgen deficiency and ED has been suggested in the literature, but few studies have examined the molecular effects on penile smooth muscle and on known signaling mechanisms that regulate morphology. Aim. Examine testosterone and SHH interaction in eugonadal adult, adolescent and juvenile rats by performing castration studies and treatment with supraphysiological testosterone. METHODS: The eugonadal adult Sprague Dawley rats were either treated with testosterone for 7 or 14 days (N = 14) or were castrated for 4 or 7 days (N = 12). The juvenile rats were treated with testosterone for 8 days (N = 7). The adolescent rats were castrated and sacrificed at P88 (N = 8). The control rats had empty vehicle (N = 22) or sham surgery (N = 20). MAIN OUTCOME MEASURES: The active form of SHH protein and mRNA were quantified by semi-quantitative immunohistochemical analysis and real-time reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Testosterone treatment did not alter SHH signaling in juvenile rats. Shh mRNA increased 3.2-fold and SHH protein increased 1.2-fold in rats castrated during puberty. In adult rats, castration decreased Shh mRNA 3.2-fold but did not alter SHH protein. Testosterone supplement in adult rats increased Shh mRNA 2.3-fold and decreased SHH protein 1.3-fold. CONCLUSIONS: SHH signaling is independent of testosterone in normal juvenile rats and is sensitive to testosterone during adolescence, while testosterone supplement in the adult adversely impacts SHH signaling in a very similar manner to that observed with CN injury.

The role of hedgehog-interacting protein in maintaining cavernous nerve integrity and adult penile morphology.

INTRODUCTION: Sonic hedgehog (SHH) is an essential regulator of smooth muscle apoptosis in the penis that has significant clinical potential as a therapy to suppress post-prostatectomy apoptosis, an underlying cause of erectile dysfunction (ED). Thus an understanding of how SHH signaling is regulated in the adult penis is essential to move the field of ED research forward and to develop new treatment strategies. We propose that hedgehog-interacting protein (HIP), which has been shown to bind SHH protein and to play a role in SHH regulation during embryogenesis of other organs, is a critical regulator of SHH signaling, penile morphology, and apoptosis induction. AIMS: We have examined HIP signaling in the penis and cavernous nerve (CN) during postnatal differentiation of the penis, in CN-injured, and a diabetic model of ED. METHODS: HIP localization/abundance and RNA abundance were examined by immunohistochemical (IHC) analysis and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in Sprague-Dawley rats between the ages of 7 and 92 days old, in CN-injured Sprague-Dawley rats and in BioBreeding/Worcester diabetic rats. HIP signaling was perturbed in the pelvic ganglia and in the penis and TUNEL assay was performed in the penis. CN tie, lidocaine, and anti-kinesin experiments were performed to examine HIP signaling in the CN and penis. RESULTS: In this study we are the first to demonstrate that HIP undergoes anterograde transport to the penis via the CN, that HIP perturbation in the pelvic ganglia or the penis induces apoptosis, and that HIP plays a role in maintaining CN integrity, penile morphology, and SHH abundance. CONCLUSIONS: These studies are significant because they show HIP involvement in cross-talk (signaling) between the pelvic ganglia and penis, which is integral for maintenance of penile morphology and they suggest a mechanism of how nerves may regulate target organ morphology and function.

Pre-metastatic cancer exosomes induce immune surveillance by patrolling monocytes at the metastatic niche.

Metastatic cancers produce exosomes that condition pre-metastatic niches in remote microenvironments to favor metastasis. In contrast, here we show that exosomes from poorly metastatic melanoma cells can potently inhibit metastasis to the lung. These "non-metastatic" exosomes stimulate an innate immune response through the expansion of Ly6Clow patrolling monocytes (PMo) in the bone marrow, which then cause cancer cell clearance at the pre-metastatic niche, via the recruitment of NK cells and TRAIL-dependent killing of melanoma cells by macrophages. These events require the induction of the Nr4a1 transcription factor and are dependent on pigment epithelium-derived factor (PEDF) on the outer surface of exosomes. Importantly, exosomes isolated from patients with non-metastatic primary melanomas have a similar ability to suppress lung metastasis. This study thus demonstrates that pre-metastatic tumors produce exosomes, which elicit a broad range of PMo-reliant innate immune responses via trigger(s) of immune surveillance, causing cancer cell clearance at the pre-metastatic niche.

Synthetic high-density lipoproteins as targeted monotherapy for chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) remains incurable despite the introduction of new drugs. Therapies targeting receptors and pathways active specifically in malignant B cells might provide better treatment options. For instance, in B cell lymphoma, our group has previously shown that scavenger receptor type B-1 (SR-B1), the high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL), is a therapeutic target. As evidence suggests that targeting cholesterol metabolism in CLL cells may have therapeutic benefit, we examined SR-B1 expression in primary CLL cells from patients. Unlike normal B cells that do not express SR-B1, CLL cells express the receptor. As a result, we evaluated cholesterol-poor synthetic HDL nanoparticles (HDL NP), known for targeting SR-B1, as a therapy for CLL. HDL NPs potently and selectively induce apoptotic cell death in primary CLL cells. HDL NPs had no effect on normal peripheral blood mononuclear cells from healthy individuals or patients with CLL. These data implicate SR-B1 as a target in CLL and HDL NPs as targeted monotherapy for CLL.

Effects of an invasive cattail species (Typha x glauca) on sediment nitrogen and microbial community composition in a freshwater wetland.

Sediments from Cheboygan Marsh, a coastal freshwater wetland on Lake Huron that has been invaded by an emergent exotic plant, Typhaxglauca, were examined to assess the effects of invasion on wetland nutrient levels and sediment microbial communities. Comparison of invaded and uninvaded zones of the marsh indicated that the invaded zone showed significantly lower plant diversity, as well as significantly higher aboveground plant biomass and soil organic matter. The sediments in the invaded zone also showed dramatically higher concentrations of soluble nutrients, including greater than 10-fold higher soluble ammonium, nitrate, and phosphate, which suggests that Typhaxglauca invasion may be impacting the wetland's ability to remove nutrients. Terminal restriction fragment length polymorphism analyses revealed significant differences in the composition of total bacterial communities (based on 16S-rRNA genes) and denitrifier communities (based on nirS genes) between invaded and uninvaded zones. This shift in denitrifiers in the sediments may be ecologically significant due to the critical role that denitrifying bacteria play in removal of nitrogen by wetlands.

Pathways for Modulating Exosome Lipids Identified By High-Density Lipoprotein-Like Nanoparticle Binding to Scavenger Receptor Type B-1.

Exosomes are produced by cells to mediate intercellular communication, and have been shown to perpetuate diseases, including cancer. New tools are needed to understand exosome biology, detect exosomes from specific cell types in complex biological media, and to modify exosomes. Our data demonstrate a cellular pathway whereby membrane-bound scavenger receptor type B-1 (SR-B1) in parent cells becomes incorporated into exosomes. We tailored synthetic HDL-like nanoparticles (HDL NP), high-affinity ligands for SR-B1, to carry a fluorescently labeled phospholipid. Data show SR-B1-dependent transfer of the fluorescent phospholipid from HDL NPs to exosomes. Modified exosomes are stable in serum and can be directly detected using flow cytometry. As proof-of-concept, human serum exosomes were found to express SR-B1, and HDL NPs can be used to label and isolate them. Ultimately, we discovered a natural cellular pathway and nanoparticle-receptor pair that enables exosome modulation, detection, and isolation.

Sonic hedgehog protein is decreased and penile morphology is altered in prostatectomy and diabetic patients.

Erectile dysfunction (ED) is a debilitating medical condition and current treatments are ineffective in patients with cavernous nerve (CN) injury, due to penile remodeling and apoptosis. A critical regulator of penile smooth muscle and apoptosis is the secreted protein sonic hedgehog (SHH). SHH protein is decreased in rat prostatectomy and diabetic ED models, SHH inhibition in the penis induces apoptosis and ED, and SHH treatment at the time of CN injury suppresses smooth muscle apoptosis and promotes regeneration of erectile function. Thus SHH treatment has significant translational potential as an ED therapy if similar mechanisms underlie ED development in patients. In this study we quantify SHH protein and morphological changes in corpora cavernosal tissue of control, prostatectomy and diabetic patients and hypothesize that decreased SHH protein is an underlying cause of ED development in prostatectomy and diabetic patients. Our results show significantly decreased SHH protein in prostatectomy and diabetic penis. Morphological remodelling of the penis, including significantly increased apoptotic index and decreased smooth muscle/collagen ratio, accompanies declining SHH. SHH signaling is active in human penis and is altered in a parallel manner to previous observations in the rat. These results suggest that SHH has significant potential to be developed as an ED therapy in prostatectomy and diabetic patients. The increased apoptotic index long after initial injury is suggestive of ongoing remodeling that may be clinically manipulatable.

Regeneration of the cavernous nerve by Sonic hedgehog using aligned peptide amphiphile nanofibers.

SHH plays a significant role in peripheral nerve regeneration and has clinical potential to be used as a regenerative therapy for the CN in prostatectomy patients and in other patients with neuropathy of peripheral nerves. Efforts to regenerate the cavernous nerve (CN), which provides innervation to the penis, have been minimally successful, with little translation into improved clinical outcomes. We propose that, Sonic hedgehog (SHH), is critical to maintain CN integrity, and that SHH delivered to the CN by novel peptide amphiphile (PA) nanofibers, will promote CN regeneration, restore physiological function, and prevent penile morphology changes that result in erectile dysfunction (ED). We performed localization studies, inhibition of SHH signaling in the CN, and treatment of crushed CNs with SHH protein via linear PA gels, which are an innovative extended release method of delivery. Morphological, functional and molecular analysis revealed that SHH protein is essential to maintain CN architecture, and that SHH treatment promoted CN regeneration, suppressed penile apoptosis and caused a 58% improvement in erectile function in less than half the time reported in the literature. These studies show that SHH has substantial clinical application to regenerate the CN in prostatectomy and diabetic patients, that this methodology has broad application to regenerate any peripheral nerve that SHH is necessary for maintenance of its structure, and that this nanotechnology method of protein delivery may have wide spread application as an in vivo delivery tool in many organs.

Elevated atmospheric CO2 alters soil microbial communities associated with trembling aspen (Populus tremuloides) roots.

Global atmospheric CO(2) levels are expected to double within the next 50 years. To assess the effects of increased atmospheric CO(2) on soil ecosystems, cloned trembling aspen (Populus tremuloides) seedlings were grown individually in 1 m(3) open bottom root boxes under either elevated (720 ppm, ELEV) or ambient CO(2) (360 ppm, AMB). After 5 years, soil cores (40 cm depth) were collected from the root boxes and divided into 0-20 cm and 20-40 cm fractions. ELEV treatment resulted in significant decreases in both soil nitrate and total soil nitrogen in both the 0-20 cm and 20-40 cm soil fractions, with a 47% decrease in soil nitrate and a 50% decrease in total soil nitrogen occurring in the 0-20 cm fraction. ELEV treatment did not result in a significant change in the amount of soil microbial biomass. However, analysis of indicator phospholipid fatty acids (PLFA) indicated that ELEV treatment did result in significant increases in PLFA indicators for fungi and Gram-negative bacteria in the 0-20 cm fraction. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to analyze the composition of the soil bacterial communities (using primers targeting the 16SrRNA gene) and the soil fungal communities (using primers targeting the intergenic transcribed spacer region). T-RFLP analysis revealed shifts in both bacterial and fungal community structure, as well as increases in both bacterial and fungal species richness with ELEV treatment. These results indicated that increased atmospheric CO(2) had significant effects on both soil nutrient availability and the community composition of soil microbes associated with aspen roots.