Biobanked Glioblastoma Patient-Derived Organoids as a Precision Medicine Model to Study Inhibition of Invasion.

Glioblastoma (GBM) is highly resistant to treatment and invasion into the surrounding brain is a cancer hallmark that leads to recurrence despite surgical resection. With the emergence of precision medicine, patient-derived 3D systems are considered potentially robust GBM preclinical models. In this study, we screened a library of 22 anti-invasive compounds (i.e., NF-kB, GSK-3-B, COX-2, and tubulin inhibitors) using glioblastoma U-251 MG cell spheroids. We evaluated toxicity and invasion inhibition using a 3D Matrigel invasion assay. We next selected three compounds that inhibited invasion and screened them in patient-derived glioblastoma organoids (GBOs). We developed a platform using available macros for FIJI/ImageJ to quantify invasion from the outer margin of organoids. Our data demonstrated that a high-throughput invasion screening can be done using both an established cell line and patient-derived 3D model systems. Tubulin inhibitor compounds had the best efficacy with U-251 MG cells, however, in ex vivo patient organoids the results were highly variable. Our results indicate that the efficacy of compounds is highly related to patient intra and inter-tumor heterogeneity. These results indicate that such models can be used to evaluate personal oncology therapeutic strategies.

Plasmonic gap-enhanced Raman tag nanorods for imaging 3D pancreatic spheroids using surface-enhanced Raman spectroscopy and darkfield microscopy.

Plasmonic gap-enhanced Raman tags (GERTs) are new emerging nanoprobes that, based on their unique surface-enhanced Raman spectroscopy (SERS) signal, can play a major role in complex imaging and detection of biological systems. GERTs are generated from a metal core nanostructure and layered with one or more metal nanosized layers, encasing a Raman active molecule. The advantages of GERTs are enhanced surface plasmon and electromagnetic resonance, as well as inherent protection of the Raman active molecule from environmental deterioration that could reduce their spectroscopic signatures over time. In this study, we used in vitro three-dimensional (3D) spheroid cultures to demonstrate these advantages. 3D spheroids mimic the in vivo tumor microenvironment better than 2D culture, with abundant extracellular matrix and hypoxia inducing variability of pH and enzymatic reactions. Here, we report the use of GERTs in large pancreatic 3D spheroids (>500 µm in apparent diameter) for complex penetration visualization. Our combined imaging technique of enhanced darkfield microscopy and SERS was able to identify the presence and distribution of the GERTs within the 3D spheroid structure. The distribution of GERTs 2 hours after the nanorods' incubation indicated accumulation, generally in the outermost layer of the spheroids but also, more randomly, in non-uniform patterns in deep layers of the 3D spheroids. These observations bring into question the mechanism of uptake and flow of the nanoparticles in function of their incubation time while demonstrating the promising potential of our approach. Additionally, the SERS signal was still detectable after 24 hours of incubation of GERTs with the 3D culture, indicating the stability of the Raman signal.

Raman spectroscopy using plasmonic and carbon-based nanoparticles for cancer detection, diagnosis, and treatment guidance.Part 1: Diagnosis.

Optical techniques, including Raman, photothermal and photoacoustic microscopy and spectroscopy, have been intensively explored for the sensitive and accurate detection of various diseases. Rapid advances in lasers, photodetectors, and nanotechnology have led to the development of Raman spectroscopy, particularly surface-enhanced Raman scattering (SERS), as a promising imaging modality that can help diagnose many diseases. This review focuses on the major recent advances in Raman spectroscopy and SERS-enhancing contrast nanoagents, as well as their potential to transition from a proof-of-concept approach to a cancer detection tool in vitro and in vivo.

Raman spectroscopy using plasmonic and carbon-based nanoparticles for cancer detection, diagnosis, and treatment guidance. Part 2: Treatment.

Raman spectroscopy and surface-enhanced raman scattering (SERS) have the potential to improve the detection and monitoring of various diseases, particularly cancer, with or without the support of multifunctional active nanosystems. This review is focused on the recent advances that have made Raman a major tool for treatment guidance for surgical tumor resection or for analytical monitoring of various therapies, such as photodynamic therapy, photothermal therapy, and drug delivery. The potential of Raman spectroscopy and nanosytems to further improve cancer treatments is also discussed.

The role of surface chemistry in the cytotoxicity profile of graphene.

Graphene and its derivative, because of their unique physical, electrical and chemical properties, are an important class of nanomaterials being proposed as foundational materials in nanomedicine as well as for a variety of industrial applications. A major limitation for graphene, when used in biomedical applications, is its poor solubility due to its rather hydrophobic nature. Therefore, chemical functionalities are commonly introduced to alter both its surface chemistry and biochemical activity. Here, we show that surface chemistry plays a major role in the toxicological profile of the graphene structures. To demonstrate this, we chemically increased the oxidation level of the pristine graphene and compared the corresponding toxicological effects along with those for the graphene oxide. X-ray photoelectron spectroscopy revealed that pristine graphene had the lowest amount of surface oxygen, while graphene oxide had the highest at 2.5% and 31%, respectively. Low and high oxygen functionalized graphene samples were found to have 6.6% and 24% surface oxygen, respectively. Our results showed a dose-dependent trend in the cytotoxicity profile, where pristine graphene was the most cytotoxic, with decreasing toxicity observed with increasing oxygen content. Increased surface oxygen also played a role in nanomaterial dispersion in water or cell culture medium over longer periods. It is likely that higher dispersity might result in graphene entering into cells as individual flakes ~1 nm thick rather than as more cytotoxic aggregates. In conclusion, changes in graphene's surface chemistry resulted in altered solubility and toxicity, suggesting that a generalized toxicity profile would be rather misleading. Copyright © 2016 John Wiley & Sons, Ltd.

Genomic and Transcriptomic Profiling of Brain Metastases.

Brain metastases (BM) are the most common brain tumors in adults occurring in up to 40% of all cancer patients. Multi-omics approaches allow for understanding molecular mechanisms and identification of markers with prognostic significance. In this study, we profile 130 BM using genomics and transcriptomics and correlate molecular characteristics to clinical parameters. The most common tumor origins for BM were lung (40%) followed by melanoma (21%) and breast (15%). Melanoma and lung BMs contained more deleterious mutations than other subtypes (p < 0.001). Mutational signatures suggested that the bulk of the mutations were gained before metastasis. A novel copy number event centered around the MCL1 gene was found in 75% of all samples, suggesting a broader role in promoting metastasis. Unsupervised hierarchical cluster analysis of transcriptional signatures available in 65 samples based on the hallmarks of cancer revealed four distinct clusters. Melanoma samples formed a distinctive cluster in comparison to other BM subtypes. Characteristics of molecular profiles did not correlate with survival. However, patients with self-identified black race or those who did not receive radiation correlated with poor survival. These data identify potential new drivers of brain metastatic progression. Our data also suggest further investigation of sociodemographic and clinical features is needed in BM cohorts.

Gold nanorods enhance different immune cells and allow for efficient targeting of CD4+ Foxp3+ Tregulatory cells.

Gold nanoparticles (AuNPs) hold great promise in nanomedicine, yet their successful clinical translation has not been realized. Some challenges include effective AuNP targeting and delivery to improve modulation of immune cells of interest while limiting potential adverse effects. In order to overcome these challenges, we must fully understand how AuNPs impact different immune cell subsets, particularly within the dendritic cell and T cell compartments. Herein, we show that polyethylene glycol coated (PEG) gold nanorods (AuNRs) and PEG AuNRs covered with a thin layer of silver (AuNR/Ag) may enhance the immune response towards immune suppression or activation. We also studied the ability to enhance CD4+ Foxp3+ Tregs in vitro using AuNRs functionalized with interleukin 2 (IL2), a cytokine that is important in Treg development and homeostasis. Our results indicate that AuNRs enhance different immune cells and that NP composition matters in immune targeting. This knowledge will help us understand how to better design AuNRs to target and enhance the immune system.

Brain Tumor Biobank Development for Precision Medicine: Role of the Neurosurgeon.

Neuro-oncology biobanks are critical for the implementation of a precision medicine program. In this perspective, we review our first year experience of a brain tumor biobank with integrated next generation sequencing. From our experience, we describe the critical role of the neurosurgeon in diagnosis, research, and precision medicine efforts. In the first year of implementation of the biobank, 117 patients (Female: 62; Male: 55) had 125 brain tumor surgeries. 75% of patients had tumors biobanked, and 16% were of minority race/ethnicity. Tumors biobanked were as follows: diffuse gliomas (45%), brain metastases (29%), meningioma (21%), and other (5%). Among biobanked patients, 100% also had next generation sequencing. Eleven patients qualified for targeted therapy based on identification of actionable gene mutations. One patient with a hereditary cancer predisposition syndrome was also identified. An iterative quality improvement process was implemented to streamline the workflow between the operating room, pathology, and the research laboratory. Dedicated tumor bank personnel in the department of neurosurgery greatly improved standard operating procedure. Intraoperative selection and processing of tumor tissue by the neurosurgeon was integral to increasing success with cell culture assays. Currently, our institutional protocol integrates standard histopathological diagnosis, next generation sequencing, and functional assays on surgical specimens to develop precision medicine protocols for our patients. This perspective reviews the critical role of neurosurgeons in brain tumor biobank implementation and success as well as future directions for enhancing precision medicine efforts.

3D cultures for modeling nanomaterial-based photothermal therapy.

Photothermal therapy (PTT) is one of the most promising techniques for cancer tumor ablation. Nanoparticles are increasingly being investigated for use with PTT and can serve as theranostic agents. Based on the ability of near-infrared nano-photo-absorbers to generate heat under laser irradiation, PTT could prove advantageous in certain situations over more classical cancer therapies. To analyze the efficacy of nanoparticle-based PTT, preclinical in vitro studies typically use 2D cultures, but this method cannot completely mimic the complex tumor organization, bioactivity, and physiology that all control the complex penetration depth, biodistribution, and tissue diffusion parameters of nanomaterials in vivo. To fill this knowledge gap, 3D culture systems have been explored for PTT analysis. These models provide more realistic microenvironments that allow spatiotemporal oxygen gradients and cancer cell adaptations to be considered. This review highlights the work that has been done to advance 3D models for cancer microenvironment modeling, specifically in the context of advanced, functionalized nanoparticle-directed PTT.

Tracking Gold Nanorods' Interaction with Large 3D Pancreatic-Stromal Tumor Spheroids by Multimodal Imaging: Fluorescence, Photoacoustic, and Photothermal Microscopies.

Pancreatic cancer is one of the most complex types of cancers to detect, diagnose, and treat. However, the field of nanomedicine has strong potential to address such challenges. When evaluating the diffusion and penetration of theranostic nanoparticles, the extracellular matrix (ECM) is of crucial importance because it acts as a barrier to the tumor microenvironment. In the present study, the penetration of functionalized, fluorescent gold nanorods into large (>500 µm) multicellular 3D tissue spheroids was studied using a multimodal imaging approach. The spheroids were generated by co-culturing pancreatic cancer cells and pancreatic stellate cells in multiple ratios to mimic variable tumor-stromal compositions and to investigate nanoparticle penetration. Fluorescence live imaging, photothermal, and photoacoustic analysis were utilized to examine nanoparticle behavior in the spheroids. Uniquely, the nanorods are intrinsically photoacoustic and photothermal, enabling multi-imaging detection even when fluorescence tracking is not possible or ideal.

Quantification of cellular associated graphene and induced surface receptor responses.

The use of graphene for biomedical and other applications involving humans is growing and shows practical promise. However, quantifying the graphitic nanomaterials that interact with cells and assessing any corresponding cellular response is extremely challenging. Here, we report an effective approach to quantify graphene interacting with single cells that utilizes combined multimodal-Raman and photoacoustic spectroscopy. This approach correlates the spectroscopic signature of graphene with the measurement of its mass using a quartz crystal microbalance resonator. Using this technique, we demonstrate single cell noninvasive quantification and multidimensional mapping of graphene with a detection limit of as low as 200 femtograms. Our investigation also revealed previously unseen graphene-induced changes in surface receptor expression in dendritic cells of the immune system. This tool integrates high-sensitivity real-time detection and monitoring of nanoscale materials inside single cells with the measurement of induced simultaneous biological cell responses, providing a powerful method to study the impact of nanomaterials on living systems and as a result, the toxicology of nanoscale materials.

Modifying Dendritic Cell Activation with Plasmonic Nano Vectors.

Dendritic cells (DCs) can acquire, process, and present antigens to T-cells to induce an immune response. For this reason, targeting cancer antigens to DCs in order to cause an immune response against cancer is an emerging area of nanomedicine that has the potential to redefine the way certain cancers are treated. The use of plasmonically active silver-coated gold nanorods (henceforth referred to as plasmonic nano vectors (PNVs)) as potential carriers for DC tumor vaccines has not been presented before. Effective carriers must be able to be phagocytized by DCs, present low toxicity, and induce the maturation of DCs-an early indication of an immune response. When we treated DCs with the PNVs, we found that the cell viability of DCs was unaffected, up to 200 µg/ml. Additionally, the PNVs associated with the DCs as they were phagocytized and they were found to reside within intracellular compartments such as endosomes. More importantly, the PNVs were able to induce expression of surface markers indicative of DC activation and maturation, i.e. CD40, CD86, and MHC class II. These results provide the first evidence that PNVs are promising carriers for DC-based vaccines and warrant further investigating for clinical use.