Oliver Mrowczynski

Interleukin-13 conjugated quantum dots for identification of glioma initiating cells and their extracellular vesicles

Cadmium selenide (CdSe) based quantum dots modified with polyethylene glycol and chemically linked to interleukin-13 (IL13) were prepared with the aim of identifying the high affinity receptor (IL13Rα2) which is expressed in glioma stem cells and exosomes secreted by these cancer stem cells. IL13 conjugated quantum dots (IL13QD) were thoroughly characterized for their physicochemical properties including particle size and surface morphology. Furthermore, the specific binding of the IL13QD to glioma cells and to glioma stem cells (GSC) was verified using a competitive binding study. The exosomes were isolated from the GSC conditioned medium and the expression of IL13Rα2 in the GSC and exosomes was verified. The binding property of IL13QD to the tumor associated exosomes was initially confirmed by transmission electron microscopy. The force of attraction between the quantum dots and U251 glioma cells and the exosomes was investigated by atomic force microscopy, which indicated a higher force of binding interaction between the IL13QD and IL13Rα2 expressing glioma cells and exosomes secreted by glioma stem cells. Flow cytometry of the IL13QD and exosomes from the culture media and cerebrospinal fluid (CSF) of patients with glioma tumors indicated a distinctly populated complex pattern different from that of non-targeted quantum dots and bovine serum albumin (BSA) conjugated quantum dots confirming specific binding potential of the IL13QD to the tumor associated exosomes. The results of this study demonstrate that IL13QD can serve as an ex vivo marker for glioma stem cells and exosomes that can inform diagnosis and prognosis of patients harboring malignant disease. STATEMENT OF SIGNIFICANCE Functionalized quantum dots are flexible semiconductor nanomaterials which have an immense application in biomedical research. In particular, when they are functionalized with biomolecules like proteins or antibodies, they have the specialized ability to detect the expression of receptors and antigens in cells and tissues. In this study we designed a cytokine (interleukin-13) functionalized quantum dot to detect a cancer associated receptor expressed in cancer stem cells and the extracellular vesicles (exosomes) secreted by the cancer cells themselves. The binding pattern of these cytokine modified quantum dots to the cancer stem cells and exosomes alters the physical properties of the complex in the fixed and suspended form. This altered binding pattern can be monitored by a variety of techniques, including transmission electron microscopy, atomic force microscopy and flow cytometry, and subsequent characterization of this quantum dot binding profile provides useful data that can be utilized as a fingerprint to detect cancer disease progression. This type of functionalized quantum dot fingerprint is especially useful for invasive cancers including brain and other metastatic cancers and may allow for earlier detection of disease progression or recurrence, thus saving the lives of patients suffering from this devastating disease.

HFE genotype affects exosome phenotype in cancer

Neuroblastoma is the third most common childhood cancer, and timely diagnosis and sensitive therapeutic monitoring remain major challenges. Tumor progression and recurrence is common with little understanding of mechanisms. A major recent focus in cancer biology is the impact of exosomes on metastatic behavior and the tumor microenvironment. Exosomes have been demonstrated to contribute to the oncogenic effect on the surrounding tumor environment and also mediate resistance to therapy. The effect of genotype on exosomal phenotype has not yet been explored. We interrogated exosomes from human neuroblastoma cells that express wild-type or mutant forms of the HFE gene. HFE, one of the most common autosomal recessive polymorphisms in the Caucasian population, originally associated with hemochromatosis, has also been associated with increased tumor burden, therapeutic resistance boost, and negative impact on patient survival. Herein, we demonstrate that changes in genotype cause major differences in the molecular and functional properties of exosomes; specifically, HFE mutant derived exosomes have increased expression of proteins relating to invasion, angiogenesis, and cancer therapeutic resistance. HFE mutant derived exosomes were also shown to transfer this cargo to recipient cells and cause an increased oncogenic functionality in those recipient cells.

Exosomes and their implications in central nervous system tumor biology

Exosomes are 20-100 nm cellular derived vesicles that upon discovery, were thought to be a form of cellular recycling of intracellular contents. More recently, these vesicles are under investigation for their purported significant roles in intercellular communication in both healthy and diseased states. Herein, we focus on the secretion of exosomes associated with glioblastoma, as most exosome studies on brain tumors have been performed in this tumor type. However, we included exosomes secreted from other forms of brain tumors for comparison as available. Exosomes contain intracellular content that can be transferred to other cells in the tumor or to cells of the immune system and endothelial cells. These recipient cells may subsequently take on oncogenic properties, including therapeutic resistance, cancer progression, and angiogenesis. Genetic components (DNA, RNA and miRNA) of the cell of origin may be included in the secreted exosomes. The presence of genetic material in the exosomes could serve as a biomarker for mutations in tumors, potentially leading to novel treatment strategies. In the last decade, exosomes have been identified as having a major impact on multiple aspects of medicine and tumor biology, and appear to be primed for a critical position in cancer diagnosis, prognosis, and treatment.

Development of tannin-inspired antimicrobial bioadhesives

Tissue adhesives play an important role in surgery to close wounds, seal tissues, and stop bleeding, but existing adhesives are costly, cytotoxic, or bond weakly to tissue. Inspired by the water-resistant adhesion of plant-derived tannins, we herein report a new family of bioadhesives derived from a facile, one-step Michael addition of tannic acid and gelatin under oxidizing conditions and crosslinked by silver nitrate. The oxidized polyphenol groups of tannic acid enable wet tissue adhesion through catecholamine-like chemistry, while both tannic acid and silver nanoparticles reduced from silver nitrate provide antimicrobial sources inherent within the polymeric network. These tannin-inspired gelatin bioadhesives are low-cost and readily scalable and eliminate the concerns of potential neurological effect brought by mussel-inspired strategy due to the inclusion of dopamine; variations in gelatin source (fish, bovine, or porcine) and tannic acid feeding ratios resulted in tunable gelation times (36 s-8 min), controllable degradation (up to 100% degradation within a month), considerable wet tissue adhesion strengths (up to 3.7 times to that of fibrin glue), excellent cytocompatibility, as well as antibacterial and antifungal properties. The innate properties of tannic acid as a natural phenolic crosslinker, molecular glue, and antimicrobial agent warrant a unique and significant approach to bioadhesive design. STATEMENT OF SIGNIFICANCE This manuscript describes the development of a new family of tannin-inspired antimicrobial bioadhesives derived from a facile, one-step Michael addition of tannic acid and gelatin under oxidizing conditions and crosslinked by silver nitrate. Our strategy is new and can be easily extended to other polymer systems, low-cost and readily scalable, and eliminate the concerns of potential neurological effect brought by mussel-inspired strategy due to the inclusion of dopamine. The tannin-inspired gelatin bioadhesives hold great promise for a number of applications in wound closure, tissue sealant, hemostasis, antimicrobial and cell/drug delivery, and would be interested to the readers from biomaterials, tissue engineering, and drug delivery area.

Tumor targeted delivery of doxorubicin in malignant peripheral nerve sheath tumors

Peripheral nerve sheath tumors are benign tumors that have the potential to transform into malignant peripheral nerve sheath tumors (MPNSTs). Interleukin-13 receptor alpha 2 (IL13Rα2) is a cancer associated receptor expressed in glioblastoma and other invasive cancers. We analyzed IL13Rα2 expression in several MPNST cell lines including the STS26T cell line, as well as in several peripheral nerve sheath tumors to utilize the IL13Rα2 receptor as a target for therapy. In our studies, we demonstrated the selective expression of IL13Rα2 in several peripheral nerve sheath tumors by immunohistochemistry (IHC) and immunoblots. We established a sciatic nerve MPNST mouse model in NIH III nude mice using a luciferase transfected STS26T MPNST cell line. Similarly, analysis of the mouse sciatic nerves after tumor induction revealed significant expression of IL13Rα2 by IHC when compared to a normal sciatic nerve. IL13 conjugated liposomal doxorubicin was formulated and shown to bind and internalized in the MPNST cell culture model demonstrating cytotoxic effect. Our subsequent in vivo investigation in the STS26T MPNST sciatic nerve tumor model indicated that IL13 conjugated liposomal doxorubicin (IL13LIPDXR) was more effective in inhibiting tumor progression compared to unconjugated liposomal doxorubicin (LIPDXR). This further supports that IL13 receptor targeted nanoliposomes is a potential approach for treating MPNSTs.

MLN8237 treatment in an orthoxenograft murine model for malignant peripheral nerve sheath tumors

OBJECTIVEMalignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas arising from peripheral nerves. MPNSTs have increased expression of the oncogene aurora kinase A, leading to enhanced cellular proliferation. This makes them extremely aggressive with high potential for metastasis and a devastating prognosis; 5-year survival estimates range from a dismal 15% to 60%. MPNSTs are currently treated with resection (sometimes requiring limb amputation) in combination with chemoradiation, both of which demonstrate limited effectiveness. The authors present the results of immunohistochemical, in vitro, and in vivo analyses of MLN8237 for the treatment of MPNSTs in an orthoxenograft murine model.METHODSImmunohistochemistry was performed on tumor sections to confirm the increased expression of aurora kinase A. Cytotoxicity analysis was then performed on an MPNST cell line (STS26T) to assess the efficacy of MLN8237 in vitro. A murine orthoxenograft MPNST model transfected to express luciferase was then developed to assess the efficacy of aurora kinase A inhibition in the treatment of MPNSTs in vivo. Mice with confirmed tumor on in vivo imaging were divided into 3 groups: 1) controls, 2) mice treated with MLN8237, and 3) mice treated with doxorubicin/ifosfamide. Treatment was carried out for 32 days, with imaging performed at weekly intervals until postinjection day 42. Average bioluminescence among groups was compared at weekly intervals using 1-way ANOVA. A survival analysis was performed using Kaplan-Meier curves.RESULTSImmunohistochemical analysis showed robust expression of aurora kinase A in tumor cells. Cytotoxicity analysis revealed STS26T susceptibility to MLN8237 in vitro. The group receiving treatment with MLN8237 showed a statistically significant difference in tumor size compared with the control group starting at postinjection day 21 and persisting until the end of the study. The MLN8237 group also showed decreased tumor size compared with the doxorubicin/ifosfamide group at the conclusion of the study (p = 0.036). Survival analysis revealed a significantly increased median survival in the MLN8237 group (83 days) compared with both the control (64 days) and doxorubicin/ifosfamide (67 days) groups. A hazard ratio comparing the 2 treatment groups showed a decreased hazard rate in the MLN8237 group compared with the doxorubicin/ifosfamide group (HR 2.945; p = 0.0134).CONCLUSIONSThe results of this study demonstrate that MLN8237 is superior to combination treatment with doxorubicin/ifosfamide in a preclinical orthoxenograft murine model. These data have major implications for the future of MPNST research by providing a robust murine model as well as providing evidence that MLN8237 may be an effective treatment for MPNSTs.

The predictive potential of hyponatremia for glioblastoma patient survival

Glioblastoma is a devastating malignancy with a dismal survival rate. Currently, there are limited prognostic markers of glioblastoma including IDH1, ATRX, MGMT, PTEN, EGFRvIII, and others. Although these biomarkers for tumor prognosis are available, a surgical biopsy must be performed for these analyses, which has morbidity involved. A non-invasive and readily available biomarker is sought after which provides clinicians prognostic information. Sodium is an electrolyte that is easily and quickly obtained through analysis of a patient’s serum. Hyponatremia has been shown to have a predictive and negative prognostic indication in multiple cancer types, but the role of glioblastoma patients’ serum sodium at the time of diagnosis in predicting glioblastoma patient survival has not been determined. We assessed whether hyponatremia at the time of glioblastoma diagnosis correlates to patient survival and show that in our cohort of 200 glioblastoma patients, sodium, at any level, did not significantly correlate to glioblastoma survival, unlike what is seen in multiple other cancer types. We further demonstrate that inducing hyponatremia in an orthotopic murine model of glioblastoma has no effects on tumor progression and survival.

Percutaneous Radiofrequency Ablation for the Treatment of Peripheral Nerve Sheath Tumors: A Case Report and Review of the Literature

Peripheral nerve sheath tumors (PNSTs) may arise sporadically or in the presence of genetic disorders, including neurofibromatosis (NF) types 1 and 2, schwannomatosis, and in patients with large genetic deletions involving the CDKN2A gene. Surgical resection is the treatment of choice for symptomatic PNSTs and offers patients a potential cure; however, pre-existing conditions or tumor location may limit a patient’s surgical options. Radiofrequency ablation (RFA) may provide an alternative therapeutic strategy for the treatment of selected PNSTs that are not amenable to surgical resection. Here, we present a case report of a 49-year-old patient with multiple neurofibromas who underwent RFA treatment of two symptomatic retroperitoneal neurofibromas and review previously reported cases of percutaneous treatment of PNSTs.

Craniopharyngiomas: A systematic review and evaluation of the current intratumoral treatment landscape

Cushing once described craniopharyngiomas as the most forbidding tumor; and, despite surgical advances decades later, craniopharyngioma resection is still extremely complex due to its location and infiltration into local structures, making gross total resection challenging. Adjuvant treatments include radiation and chemotherapy, but intratumoral therapy may emerge as an adjuvant treatment for craniopharyngiomas. Here, we present a review of the literature on this treatment modality; and, summarize the available reported cases to underline usefulness and effectiveness of this treatment method. Our review of the literature included all articles from MEDLINE/PubMed and Ovid from 1974 to 2017. All articles were assessed for relevancy before inclusion into this review. Although the role for intratumoral therapy is unclear, multiple studies have reported efficacy in the treatment of craniopharyngiomas, and current results are promising. Out of the intratumoral agents utilized, intratumoral alpha interferon seems to provide the best response and least side effects for the treatment of craniopharyngiomas. The use of intratumoral therapy has led to delay in treatment with definitive surgery or radiation, both of which are associated with significant morbidities, detrimental in developmental years of childhood. Out of the intratumoral agents utilized, intratumoral alpha interferon seems to provide the best response and least side effects for the treatment of craniopharyngiomas. These findings need to be explored further with randomized controlled trials, outlining a standard dosing regimen. Furthermore, trials in craniopharyngioma patients with these combination therapies must be performed to determine the optimal therapeutic regimen for the successful treatment of these patients.

Intra-cerebrospinal fluid antibiotics to treat central nervous system infections: A review and update

Central nervous system infections can be complications of neurosurgical procedures or can occur spontaneously, and occasionally lead to devastating neurological complications, increased rate of mortality, and lengthier stays in the hospital, subsequently increasing costs. The use of intrathecal antibiotics to bypass the blood brain barrier and provide effective concentrations to the central nervous system has been described as an adjunct treatment option. However, the regimens of antibiotics utilized intrathecally have not been standardized. Our review of the literature included all articles from MEDLINE/PubMed and Ovid from inception to 2017 and after removing duplicates and checking for relevancy, the final number of articles yielded was 200. This review summarizes the use of antibiotics intrathecally to treat CNS infections, the dosages, therapeutic efficacies, and highlights significant side effects. The current rates of mortality in patients suffering from CNS infections is high, thus intrathecal antibiotic therapy should be considered as a potential therapeutic strategy in this patient population. Multiple antibiotics have demonstrated safety and efficacy when used intrathecally, and further studies, including clinical trials, need to be performed to elucidate their full therapeutic potential and outline proper dosing regimens.