Future R&D
Personalized therapy
Personalized medicine and precision cancer therapy aim to customize appropriate treatment decisions based on individual cases. Recent advances in genotypic methods, like DNA profiling and next generations sequencing (NGS), are increasingly available and have identified thousands of mutations that are important to cancer generation and progression. Most efforts in personalized medicine have focused on tailoring the treatment to the specific patient based on his genomic data. However, the correlation between cancer cell genetics and clinical results is complex. Due to modulations of tumor microenvironment and the existence of non-DNA genetic variations, including epigenetic modifications and lineage-specific changes, the understanding on how mutations affect drug response is limited. Not all mutations can be matched with known treatment options and the genotypic methods do not always identify the best cancer therapeutics. Moreover, molecular data alone has not proven sufficient to predict both the efficacy and the toxicity of an anti-cancer drug.
Due to the above limitations, it is clear that genomics data has to be supplemented with additional phenotypic/functional information in order to optimally guide the treatment for each patient. Experimental preclinical drug testing, in which patient cancer cells are exposed to multiple treatments ex-vivo, as therapeutic guide for individual patients, is needed.
Such preclinical testing for drug response before patient treatment is a great challenge, because of the lack of representative patient tumor models that can recapitulate the key features of the original tumors.
Commonly used human cancer models include cancer cell lines and primary patient-derived tumor xenografts (PDTXs). The cell lines do not always recapitulate the genetic heterogeneity of the original tumors, since they may have undergone genetic changes. PDTXs are generated by transplanting patient derived material into immune-deficient mice. As in-vivo models, PDTXs mimic the biological characteristics of the human tumor better than in-vitro cellular models. However, success rate of transplantation is relatively low, culturing time is long (3 months), PDTXs may undergo mouse-specific tumor evolution, and the approach is expensive and resource consuming.
Recently, progress in 3D culture technologies has led to the development of novel and more physiological cancer models. Once embedded into a 3D matrix in media that contains growth factors and small molecule inhibitors, tumor-derived patient cells can be grown with high efficiency into self-organizing structures termed Organoids.
Patient derived organoids (PDOs) are a 3D culture of cancerous cells derived from tumor tissues, similar to the original specimen in terms of both genome and function. It has been shown that PDOs can emulate the pathological characteristics of primary tissue at the organ level and better simulate the tumor in vivo.
PDOs can be cultured from cells derived from biopsies or resection samples, the culture time is relatively short, and the media composition and extracellular cellular matrix (ECM) components can be selected according to the source tissue. Organoids were established from various types of primary tumor cells including colon, esophagus, pancreas, stomach, liver, endometrium, ovary and breast, as well as from metastatic colon, prostate and breast cancer biopsy samples. Moreover, PDOs recapitulate the in vivo environment, reproducing patient responses to drugs in the clinic.
Although organoids have been established efficiently from patient-derived tumor tissue and were found as a physiologically relevant ex-vivo model, their integration into patient-focused approaches is very slow, mainly due to technical difficulties, and the absence of advanced commercial technologies for easy, efficient and rapid generation, culturing and testing of PDOs.
In recent years, a new technology; the Hydrogel Microstructure Array (HMA) was developed at the Jerome Schottenstein Center. Our researchers routinely design and fabricate HMAs of numerous of nanoliter wells and various shapes and sizes. Individual cells can be positioned in these wells where spheroids or organoids can be self-generated and monitored in a high throughput mode. By integration of our HMA platform with organoids technology, we intend to match optimal personalized treatment to each patient.
In collaboration with interested hospitals, the Schottenstein Center intends to utilize the HMA technology and the TOC platform as tools for personalized tumor therapy. The HMA technology will be used to generate and analyze PDOs derived from primary patient tissue in 3D cell culture, aiming to make patient-specific response predictions prior to initiation of treatment in the clinic.
The goal of this study is to develop ex-vivo preclinical tumor models based on PDOs in HMA imaging platform, to accurately and efficiently mimic tumor micro-environment for testing personalized medicine.
Stem cell research
Directed differentiation of stem cells towards a specific cell fate is a key issue for developmental biology and regenerative medicine. Naturally, as stem cells may differentiate into different lineages producing a diverse population of cells, it is essential to study the progeny at the resolution of the individual cell. In the case of neural differentiation, the identity and functional maturity of each cell can only be determined using functional parameters such as neurotransmitter release and specific gene expression. The Cell Retaining methodologies will be used to explore the effect of a large set of differentiation factors in controllable amounts and temporal sequences in a highly parallelized and automated fashion. The monitoring of cell differentiation will be performed by combined optical and electrochemical assays, as well as by transcriptome analysis. Specifically, we will study the differentiation of neurospheres into dopaminergic neurons as well as the differentiation of human limbal adult stem cells into neuronal lineages and pancreatic insulin-secreting beta-cells. The latter are of particular interest, since the production of autologous beta cells from adult stem cells could lead to a new cellular therapy of diabetes. The neurospheres, which are used routinely by one of our European academic collaborators, are composed of around 200 undifferentiated cells, obtained from rat brain at days 14-15 of embryogenesis. Their development can be directed toward becoming dopaminergic neuronal cells by a treatment with a complex mixture of various factors at an adequate time. The differentiation of neurospheres into dopaminergic (DA) neurons will be tested by a) direct microscopic observation of the morpho-type of the neurospheres; b) quantitative fluorescence analysis of a fluorescent promoter for tyrosine hydroxylase (TH, a dopamine neuron marker) and by catecholamine sensing electrodes; and c) transcriptome analysis of the neurospheres undergoing differentiation.
Traumatic and ischemic brain diseases
In order to enable the identification of new targets for therapeutic vascular protection in brain injury, brain endothelial cell dysfunction, that mediates brain injury aggravation, will be monitored. A recognized marker of the endothelium dysfunction is nitric oxide (NO) production, which is decreased, while superoxide anion production is increased.
A key challenge in studying endothelial cells from mammalian tissues is the cellular functional heterogeneity which compromises the ability to interpret data obtained from a large bulk of cells. While the molecular tools for the analysis of individual cells have been established in our group, functional assays that can deliver information on individual cell physiology are still missing. The use of the Cell Retaining methodology will allow the analysis of functional parameters in a large population of endothelial cells at the resolution of an individual cell. It will also facilitate the collection of individual cells for molecular assays after being tested for physiological parameters over time. As such, this approach is likely to deliver novel insights into endothelial cell pathophysiology. By using the present platform, we will study the hypoxic response on primary brain capillary and artery endothelial cells. Through NO and superoxide anion electrochemical and optical detection, this technology will allow, for the first time, to identify the individual cells that display the highest stress response. Such cells will be then collected on an individual basis using optical tweezers, and submitted to single-cell transcriptome analysis.
Vascular pathologies
The loss of endothelial barrier function and the subsequent increase in vascular permeability to low density lipoprotein (LDL) may be central events in the etiology of common and debilitating vascular pathologies such as migraine.
The production of free radicals by endothelial cells lining the vasculature within the central nervous system (CNS) has been associated with impaired endothelial barrier function. A direct link between endothelial free radical production and the release of inflammatory mediators by glial cells within the CNS has also been proposed.
The Cell Retainers will be used as a novel platform to facilitate the specific, sensitive and real-time analysis of free radical production by human middle cerebral artery endothelial cells (MCAECs). Free radical production will be stimulated by inflammatory mediators released from glial cells, co-cultured with MCAECs. The CR will enable the identification of endothelial cells that show high levels of free radical production, and could thus impair the endothelial barrier function. These cells will be selected, sub-cultured into monolayers and used in subsequent in vitro protein permeability studies.
The CR methodology will be also used in conjunction with specific endothelial membrane receptor antagonists to identify inflammatory mediators produced by glial cells that may have detrimental effects on the endothelial barrier function.