Peter Tolias

Patient-specific 3D microfluidic tissue model for multiple myeloma.

In vitro culturing of primary multiple myeloma cells (MMC) has been a major challenge as this plasma cell malignancy depends on the bone marrow environment for its survival. Using a microfluidic platform to emulate the dynamic physiology of the bone marrow microenvironment, we report here a new approach for culturing difficult to preserve primary human MMC. The system uses a three-dimensional ossified tissue to mimic the tumor niche and recapitulate interactions between bone marrow cells and osteoblasts (OSB). To this end, the human fetal OSB cell line hFOB 1.19 was cultured in an eight-chamber microfluidic culture device to facilitate the seeding of mononuclear cells from bone marrow aspirates from three multiple myeloma patients. Optical microscopy, used for real-time monitoring of mononuclear cell interactions with the ossified tissue, confirmed that these are drawn toward the OSB layer. After 3 weeks, cocultures were characterized by flow cytometry to evaluate the amount of expansion of primary MMC (with CD138(+) and CD38(+)CD56(+) phenotypes) in this system. For each of the three patients analyzed, bone marrow mononuclear cells underwent, on an average, 2 to 5 expansions; CD38(+)CD56(+) cells underwent 1 to 3 expansions and CD138(+) cells underwent 2.5 to 4.6 expansions. This approach is expected to provide a new avenue that can facilitate: (1) testing of personalized therapeutics for multiple myeloma patients; (2) evaluation of new drugs without the need for costly animal models; and (3) studying the biology of multiple myeloma, and in particular, the mechanisms responsible for drug resistance and relapse.

Ex Vivo Maintenance of Primary Human Multiple Myeloma Cells through the Optimization of the Osteoblastic Niche

We previously reported a new approach for culturing difficult-to-preserve primary patient-derived multiple myeloma cells (MMC) using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment and a culture medium supplemented with patient plasma. In the current study, we used this biomimetic model to show, for the first time, that the long-term survival of OSB is the most critical factor in maintaining the ex vivo viability and proliferative capacity of MMC. We found that the adhesion and retention of MMC to the tissue scaffold was meditated by osteoblastic N-cadherin, as one of potential mechanisms that regulate MMC-OSB interactions. However, in the presence of MMC and patient plasma, the viability and osteogenic activity of OSB became gradually compromised, and consequently MMC could not remain viable over 3 weeks. We demonstrated that the long-term survival of both OSB and MMC could be enhanced by: (1) optimizing perfusion flow rate and patient-derived plasma composition in the culture medium and (2) replenishing OSB during culture as a practical means of prolonging MMC’s viability beyond several weeks. These findings were obtained using a high-throughput well plate-based perfusion device from the perspective of optimizing the ex vivo preservation of patient-derived MM biospecimens for downstream use in biological studies and chemosensitivity analyses.

Use of surface-enhanced Raman scattering as a prognostic indicator of acute kidney transplant rejection

We report an early, noninvasive and rapid prognostic method of predicting potential acute kidney dysfunction using surface-enhanced Raman scattering (SERS). Our analysis was performed on urine samples collected prospectively from 58 kidney transplant patients using a He-Ne laser (632.8 nm) as the excitation source. All abnormal kidney function episodes (three acute rejections and two acute kidney failures that were eventually diagnosed independently by clinical biopsy) consistently exhibited unique SERS spectral features in just one day following the transplant surgery. These results suggested that SERS analysis provides an early and more specific indication to kidney function than the clinically used biomarker, serum creatinine (sCr).

Single-test parallel assessment of multiple genetic disorders

We advocate a new paradigm for genetic diagnosis based on using customized array panels, each of which groups multiple genes and mutations associated with clinical profiles that are common to particular syndromic diseases. This parallel approach, based on a single-test multigene multiplexing strategy, compared with traditional sequential testing by gene-by-gene genetic analysis, drastically reduces the time and cost of diagnosis while maintaining accuracy and reliability. Faster diagnosis enables early decision-making to facilitate better patient management and outcomes at reduced costs to the healthcare system.

Antidepressant-induced suicidality: how translational epidemiology incorporating pharmacogenetics into controlled trials can improve clinical care

Randomized controlled trials (RCTs) have been a staple of the drug development process for several decades. Here, we review the origins of RCTs and their adoption within drug development, highlighting shortcomings that tend to be ignored and possible solutions offered from personalized medicine. While RCTs play an important role in development of therapeutics, we underscore how if used indiscriminately, their adverse effects may outweigh the benefits. As an example, we focus on the development of antidepressants and how a severe adverse drug response - suicidal ideation - can be overlooked. We conclude with a discussion of how pharmacogenetics may address some of the deficiencies of RCTs, bringing the focus of drug response back to the individual patient rather than the population, using as an example the discovery of genetic markers associated with antidepressant-induced suicidal ideation.

The need to assess drugs selected from cancer genomic data prior to patient treatment

Using cancer biology to guide drug selection Research over the past decade has demonstrated that different types of tumors can have common genetic alterations, making them treatable with the same drug. An example is the drug Herceptin, approved by the US FDA in 1998 to target breast cancers that overexpress the HER2 gene as a biomarker. HER2 overexpression has also been found in gastric and ovarian cancer, prompting the FDA to approve the drug for treatment of gastric cancer in 2010. This was the first example of how biomarker-based drug selection simplifies therapeutic intervention by identifying patients that will respond to a drug. Biomarkers can also lead to more effective treatment by identifying circumstances when more than one drug should be used, particularly when the cancer is derived from more than a single clone. Scientists at the University of California, San Francisco (CA, USA) have found that the selection of two drugs targeting two different molecules is more effective in decreasing tumor size and blocking metastasis in mice [1]. The two-target approach was demonstrated with neuro endocrine pan creatic tumors and the authors suggest it may have broad application for treating a wide variety of cancers. An important observation was recently made regarding the structural organization of a tumor with respect to the unique number of cancer cells (originating from different clones) that reside within it. Apparently, taking a biopsy from just one part of a tumor may not give a full picture of its genetic landscape which illuminates why attempts at using single biopsies to identify biomarkers to which personalized cancer treatments can be targeted have met with limited success. Cancer researchers at the UK’s London Research Institute and their collaborators carried out a genome-wide analysis of the genetic variation between different regions of the same kidney cancer tumor. Alarmingly, they found that approximately two-thirds of the uncovered genetic alterations were not shared across other biopsies from the same tumor [2]. Their results demonstrate that cancer cells of a kidney tumor are not homogeneous and can be derived from multiple clones within the same tumor. This suggests the need for better characterization of tumor biopsies prior to selection of the best combination therapy.

Chromosomal microarrays: influential players in the diagnosis of developmental disorders

precise locations spanning the genome. These slides are laser scanned and analyzed with software that quantifies copy number differences between a test and a reference sample. Initially, bacterial artificial chromosomes were the probes of choice for this technique, known as array-based comparative genomic hybridization (array-comparative genomic hybridization). Theses arrays contain DNA isolated from bacterial artificial chromosomes that range in size from 150–200 kb, which are spotted directly onto the array [7].

MERRF and MELAS: current gene therapy trends and approaches

The mitochondrion is a unique organelle that predominantly functions to produce useful cellular energy in the form of adenosine triphosphate (ATP). Unlike other non-nuclear eukaryotic organelles (with the exception of chloroplasts), mitochondria have two lipid membranes that enclose their own mitochondrial DNA (mtDNA) and ribosomes for protein production. Similar to nuclear DNA, mtDNA is equally susceptible to mutations that may be classified as either pathogenic or nonpathogenic. Myoclonic Epilepsy with Ragged Red Fibers (MERRF) and Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes (MELAS) are mitochondrial diseases originating from pathogenic point mutations located within mtDNA. Currently, there is no cure and patient care primarily focuses on treating each disease’s associated symptoms. When considering the multiple barriers existing between the extracellular surface of the plasma membrane and the location of the mtDNA within the mitochondrial matrix, developing a pharmacological therapeutic that can both overcome these barriers and correct an mtDNA causing mitochondrial disease remains difficult at best. Interestingly, the field of gene therapy may provide an opportunity for effective therapeutic intervention by introducing a genetic payload (to a particular cellular gene) to induce the correction. This review primarily focuses on understanding the principles of mitochondrial biology leading to the mtDNA diseases, MERRF and MELAS, while providing a landscape perspective of gene therapy research devoted to curing these diseases.

Abstract 344: Downregulation of osteoblastic N-cadherin decreases primary multiple myeloma cell - osteoblast interactions

Introduction: Recent reports have shown that the adhesion molecule N-cadherin, a mesenchymal cadherin associated with epithelial-to-mesenchymal transition, is expressed in a large percentage of castration resistant prostate cancer tumors, allowing the interaction between N-cadherin expressing osteoblasts (OSB) and prostate cancer cells via homophilic binding, and can promote osteoclastogenesis and osteolytic disease. There is also evidence that in multiple myeloma (MM), direct adhesive interactions between MM cells and the bone marrow stroma are responsible for the development of drug resistance and relapse. This phenomenon termed “cell adhesion-mediated drug resistance” (CAMDR) is thought to be one of the major mechanisms by which multiple myeloma cells (MMC) escape the cytotoxic effects of therapeutic agents. We recently demonstrated the technical feasibility of culturing difficult to preserve primary human MMC by reconstructing a physiological relevant 3D microfluidic bone-like tissue microenvironment. Using this platform, we found that OSB played a key role in the ex vivo maintenance and expansion of MMC. In this study, we evaluated the role of osteoblastic N-cadherin in regulating MMC-OSB interactions as a first step to elucidate the role of this molecule in the development of CAMDR in MM. Materials and Methods: Knockdown of N-cadherin protein expression in the human OSB cell line hFOB 1.19 was achieved using shRNA lentiviral technology. Patient-derived bone marrow mononuclear cells (BMMC) containing malignant MMC were cocultured in the microfluidic device with either N-cadherin+OSB or with a mixture composed of N-cadherin-OSB and N-cadherin+OSB. Monitoring and quantification of MMC-OSB interactions was performed using time lapsed images and fluorescence microscopy. Results and Conclusions: Real-time images showed that CD138+ MMC were preferentially adhered to N-cadherin+ OSB. The retention of CD138+ MMC, based on the scaffold constitution, was determined by flow cytometric analyses as the CD138+ MMC/OSB ratio. Notably, after a 2 day coculture period, only 32.78% ± 4.15% CD138+ MMC was retained in the tissue scaffold containing N-cadherin-OSB compared to 70.21% ± 4.64% retention in the mock transfected OSB group. For the first time, we demonstrated here that osteoblastic N-cadherin is responsible, at least in part, for the development of strong interactions between MMC with stromal elements from the tumor microenvironment. Our data suggest a new therapeutic intervention area in the modulation of N-cadherin expression by OSB as a way to render MMC more susceptible to drug treatments and potentially decrease CAMDR. Citation Format: Wenting Zhang, Yexin Gu, Qiaoling Sun, David S. Siegel, Peter Tolias, Zheng Yang, Woo Lee, Jenny Zilberberg. Downregulation of osteoblastic N-cadherin decreases primary multiple myeloma cell - osteoblast interactions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 344. doi:10.1158/1538-7445.AM2015-344

Fiber-Optic Probe Enabled by Surface-Enhanced Raman Scattering for Early Diagnosis of Potential Acute Rejection of Kidney Transplant

We have explored the use of a fiber-optic probe with surface-enhanced Raman scattering (SERS) sensing modality for early, noninvasive and, rapid diagnosis of potential renal acute rejection (AR) and other renal graft dysfunction of kidney transplant patients. Multimode silica optical fiber immobilized with colloidal Ag nanoparticles at the distal end was used for SERS measurements of as-collected urine samples at 632.8 nm excitation wavelength. All patients with abnormal renal graft function (3 AR episodes and 2 graft failure episodes) who were clinically diagnosed independently show common unique SERS spectral features in the urines collected just one day after transplant. SERS-based fiber-optic probe has excellent potential to be a bedside tool for early diagnosis of kidney transplant patients for timely medical intervention of patients at high risk of transplant dysfunction.