Alfredo Quinones-Hinojosa - US grants

DESCRIPTION (provided by applicant): Preliminary work from our lab suggests that in humans the subventricular zone (SVZ) astrocytes function as neural stem cells and that the cytoarchitecture of the SVZ in humans is different from that of other mammals. We will perform experiments to determine the cytoarchitecture of the human SVZ and fates of its new cells by doing a serial reconstruction of the adult human SVZ with electron microscopy and by finding the exact location of adult human SVZ neural stem cells with immunocytochemistry techniques. This information is essential to raise hypothesis regarding the function of proliferative cells in the SVZ. We will learn if cells with the characteristics of transient amplifying cells, of young neurons or young oligodendrocytes, exist in the SVZ. Our second aim is to describe the SVZ-Olfactory Bulb (OB) system in adult human brain. We will study other possible pathways for adult human neuronal migration and we will characterize the adult human OB and tract. We will focus in the identification of young neuronal precursors using specific markers to explore possible migratory routes in the adult human brain and we will also investigate whether there is an intrinsic population of stem cells within the OB and how they may possibly migrate to supply other regions of the brain.

[unreadable] DESCRIPTION (provided by applicant): The subventricular zone (SVZ) has been shown to be the largest germinal region in the adult rodent brain. Recent studies have also demonstrated not only that a similar neurogenic region exists in the SVZ of the adult human brain, but also that the cytoarchitecture is different in the human SVZ: a hypocellular gap and a ribbon of astrocytes is present under the ependyma. It remains unknown whether neural stem cells in the human brain are precisely localized in this ribbon of astrocytes lining the adult human SVZ and whether these adult neural stem cells have the ability to migrate. What exactly regulates the migratory capacity of stem cells or the transformation of brain tumors is not known, but epidermal growth factor (EOF) has been implicated in this process. Within this context we will pursue the following Specific Aims: Aim 1: To precisely localize the adult human neural stem cells with respect to the ependyma and to compare the adult human SVZ to the fetal human SVZ and to the SVZ of patients with gliomas. The hypothesis is that stem cells are concentrated in the band of astrocytes parallel to the ependyma and that this ribbon of astrocytes is 1) more prominent when patients have gliomas that are adjacent and/or part of the subventricular zone; 2) there is a larger population of CD 133+cells within this ribbon of astrocytes in patients with brain tumors 3) this ribbon of astrocytes does not exist in the fetal human SVZ. Aim 2: To establish the role of EGF in the migratory ability of adult, fetal, and human brain cancer stem cells using both in vitro migration assays and an in vivo rodent model. The hypothesis is that human adult stem cells have the ability to migrate and that their manipulation with EGF can change them to a more aggressive migratory behavior that resembles that of the fetal human brain and/or high grade tumors. This project has direct relevance to public health. If adult neural stem cells have the ability to migrate we will further elucidate the human brain's capacity for self-repair and may be able to figure out ways to make these cells migrate to replace those lost following extensive neuronal damage or disease. In addition, neural stem cells and cancer stem cells may share similar mechanisms of migration and invasion and understanding this mechanism may lead to better treatments of brain tumors. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM), the most common and devastating intracranial malignant tumor accounts for 20% of all primary brain tumors and has a median survival rate of only 14 months. Cancer cells often disseminate far from primary tumors and individual glioma cells migrate from the gross tumor into the surrounding parenchyma, making complete surgical resection nearly impossible. This migratory capacity of malignant gliomas represents the greatest challenge to any potential therapy in spite of advances in surgery, chemotherapy and radiotherapy and growth of the remaining invasive cells leads to a recurrence incidence of 99%. What exactly regulates the migratory capacity of brain tumor cells is not fully understood and need to be studied. The main goal of this proposal is to understand the link between known pro- migratory signals such as epidermal growth factor (EGF) and Slit proteins with cell volume regulation. EGF and Slit proteins may play an important role in the modulation of invasive and migratory ability of GBM derived stem cells through Akt pathway that in turn regulates the activation of ion cotransport NKCC1. We propose to study invasive patterns and cell volume changes resulting in the extension of a leading process of a migrating cell, using various cell migration assays and measuring intracellular anion concentration. The results obtained from this work will help us understand the downstream signaling pathways involved in the activation of cascade mechanism responsible for brain tumor cell migration. Further, such knowledge will undoubtedly result in better therapeutic alternatives to current sub-optimal treatments for this devastating disease. PUBLIC HEALTH RELEVANCE: Glioblastoma multiforme (GBM) is the most common and devastating primary malignant tumor. Our project aims to study the migration of GBM-derived Brain Tumor Stem Cells (BTSCs). BTSCs are thought to be responsible for maintaining the bulk of the tumor and to induce recurrence after surgical resection, nevertheless the molecular mechanisms that regulate their migration are not known. In this study, we propose to understand the role of pro-migratory signals in brain tumor invasion in order to increase the available targets to prevent brain tumor dispersal.

? DESCRIPTION (provided by applicant): Glioma is the most common adult primary brain cancer, with inevitable recurrence and finite survival times. Safe maximal resection for glioma patients remains the standard of care based on best evidence medicine (coupled with adjuvant therapies such as radiation and chemotherapy). Multiple studies have shown clear relief of symptoms, improvement of life quality, survival advantage, and delayed recurrence for patients undergoing safe maximal extent of resection (EOR). This is highly beneficial to high-grade glioma (glioblastoma - GBM) patients, and is even more critical for low-grade glioma patients who enjoy years of improved survival. However, it has been extremely challenging to visually distinguish cancer from non-cancer brain tissue intra- operatively even by very experienced surgeons with the best clinically available technologies. On one hand, remaining cancer quickens recurrence, increases resistance to adjuvant therapies, and worsens survival; on the other hand, resection of normal functional brain (e.g. speech and motor areas) can lead to poor functional status and worse survival outcomes. Currently the lack of effective intra-operative guidance technologies prevents neurosurgeons from achieving maximal safe EOR despite of its clear survival advantage. The objective of this proposal is to develop and evaluate the ability of a high-speed, high-resolution, non- invasive and label-free optical coherence tomography (OCT) imaging technology, along with a novel tissue optical property quantification algorithm, to distinguish cancer from non-cancer in real time with high sensitivity/specificity. Our preliminary data (recently published in Science Translational Medicine) suggests exciting potential of OCT for identifying brain cancer vs. non-cancer. To fully investigate the capability and potential of OCT for label-free, quantitative and real-time assessment of brain cancer in an intra-operative setting, we propose the following aims: In Aim 1, we will develop a high- speed OCT imaging platform to identify human brain cancer infiltration with minimized motion and blood artifacts. We will also develop a novel processing algorithm for rapid and robust optical property retrieval from volumetric OCT imaging data, and a method of constructing a color-coded optical property map to provide a direct visual cue for distinguishing cancer versus non-cancer at high resolution. In Aim 2, we will perform the first systematic evaluation of OCT using ex vivo brain tissues from 30 GBM and 30 low-grade brain cancer patients, and a novel in vivo murine brain cancer model (implanted with patient-derived GBM cell lines). Using histopathological analyses as the gold standard, we will establish the first quantitative OCT diagnostic thresholds and determine the OCT sensitivity/specificity in brain cancer identification. In Aim 3, we will address the feasibiliy of brining real-time intra-operative capabilities of OCT into the operating room (OR) by conducting a pilot in vivo OCT imaging study with an additional 30 GBM and 30 low-grade brain cancer patients. This pilot study should pose minimal risk to the patient as the imaging light intensity is low, all imaging data will be collected in a sterile, non-contact manner, and the pilo study will not influence any clinical decisions nor the extent of resection. In summary, our proposed study will be the first to provide quantitative and real-time brain cancer tissue identification using a color-coded optical property map, the first to provide systematic evaluation of the translational OCT technology (involving 120 patients), and the first to provide non-invasive, label-free, non- contact and real-time differentiation of brain cancer versus non-cancer in the OR. These advances will open doors for future large-scale clinical trials to guide brain surgery and increase extent of resection, not only for glioma but also for patients with other types of cancers (such as cancers metastasized to the brain, oral, cervical and GI cancers), thereby improving patient survival.

? DESCRIPTION (provided by applicant): Glioblastoma (GBM) is the most common primary brain tumor in adults, and accounts for 20% of all primary brain tumors. GBM has a median survival rate of only 14.6 months despite current best treatment practices including surgery and chemoradiation. A significant reason for this morbidity and mortality is the ability of GBM to invade normal brain parenchyma, making localized treatment ineffective. There is increasing evidence of a small subset of cells, brain tumor initiating cells (BTICs) that are responsible for the disease's treatment resistance. In order for treatment to be effective, these invading cells need to be targeted. One promising approach involves the use of mesenchymal stem cells (MSCs), which have been found to migrate preferentially to and home in on cancer cells. Moreover, MSCs can be engineered to synthesize and release anti-tumor proteins, like bone morphogenic protein 4 (BMP4), which affects BTICs. MSCs can be obtained from bone marrow (BM- MSC) and adipose tissue (AMSCs). BM-MSCs are difficult to obtain, have limited ex vivo proliferation capacity, and decrease in effectiveness with donor age. Unlike BM-MSCs, AMSCs are more abundant in supply, easier to obtain from fat tissue, express higher levels of surface markers implicated in cell migration, and have been shown to resist oncogenic transformation. AMSCs may therefore be a better option. The viral gene delivery method, though commonly used to modify AMSCs, is associated with insertional mutagenesis and immunogenicity, and, therefore, has potentially limited translational ability for use in human patients. Biodegradable, polymeric nanoparticles enable effective non-viral gene delivery to multiple cell types, including human AMSCs (hAMSCs), while avoiding the problems typical of viruses. In this grant, we propose a novel technology to combine Freshly-extracted Adipose Tissue (F.A.T.) and nanoparticles to non-virally engineer the primary hAMSCs contained within F.A.T without prior culture to secrete anti-cancer proteins while maintaining the cells' ability to migrate toward tumo cells. Our overall hypothesis is that nanoparticle-modified hAMSCs obtained from F.A.T. retain their tumor suppressive characteristics in a clinically relevant in vivo human GBM model. To test this hypothesis, we will pursue the following specific aims: (1) To effectively deliver exogenous genes of interest to Freshly-extracted Adipose Tissue (F.A.T.) from patients via lyophilized biodegradable nanoparticles. (2) To determine if nanoparticle-modified BMP4-secreting hAMSCs retain an anti-glioma effect in vitro. (3) To determine the safety and efficacy of nanoparticle-modified BMP4-secreting hAMSC treatment in combination with targeted radiation therapy on human GBM in an in vivo murine model. Aim 1 involves investigation and optimization of a unique technology of combining nanoparticles with F.A.T. from our patients. For aims 2 and 3, using nanoparticles already tested in commercial hAMSCs, we will now investigate the modification of primary hAMSCs that have been isolated and cultured prior to adding the nanoparticles. The techniques to be used in vitro and in vivo in this proposal have been developed and further characterized by our teams. In vitro studies will be conducted using new advancements in the fields of microfluidics and nanobiotechnology. In vivo studies will employ a mammalian xenograft model that engrafts human GSC-derived GBM, which bests recapitulates human GBM. Further, in the in vivo studies, animal subjects will be treated with radiation using Small Animal Radiation Research Platform (SARRP), thus recreating traditional conformal beam radiotherapy for humans on the scale of a mouse. The results of this study will determine whether nanoparticle-modified hAMSCs can provide a treatment that is safe and effective for not only patients with GBM, but many types of primary and metastatic brain cancers. For future clinical application, the nanoparticles could be administered either to hAMSCs obtained from patient fat after culturing for a few days or then given IV as a treatment or to F.A.T. with the resulting engineered hAMSCs re- administered during surgery. This may lead to clinical trials, with a revolutionary new way of treating patients with brain cancer and facilitating personalized medicine.

PROJECT SUMMARY Gliomas are aggressive brain cancers. Treatment is surgical resection of the tumor supplemented by radio and chemotherapy. Patient prognosis is best when gross total tumor resection is achieved. However, gross total resection is a challenge for gliomas that diffuse extensively and microscopically into the adjacent normal brain parenchyma. Neurosurgeons augment their ability to visualize tumorous tissue though imaging technology like MRI, 5ALA (contrast agent), and/or OCT (optical coherence tomography technique to visualize cancer) that enhances contrast of physical structures associated with the diseased tissue. Because details and spatial information are limited, especially when approaching the periphery of the tumor, there is significant need for the development of alternative approaches that can complement imaging modalities by providing microscopic observation of pathological features of tissue and guide resection maneuvers; there is a need to determine intraoperative pathological molecular characteristics to determine how thorough the resection should be and/or future local therapies to be considered at the time of surgery. The traditional approach to assess tissue pathology is the optical observation of cellular morphology through microscopy but it has limited applicability during surgery because it requires time-consuming laboratory testing and it gives the surgeon no intraoperative molecular information. We propose the use of desorption electrospray ionization mass spectrometry (DESI-MS) as an intraoperative pathology tool to provide diagnostic and prognostic information that can improve the surgical decision-making process. DESI-MS detects the underlying molecular changes occurring in cancerous cells and tissue and can do so within minutes working on minimally processed tissue smears. DESI sprays tissue with a mist of charged droplets to generate and then mass-measure ions. We target 2-hydroxyglutarate to assess the presence of IDH mutations in the tissue and membrane phospholipids and N-acetylaspartate to estimate tumor infiltration (i.e. tumor cell percentage), at locations of particular interest to the surgeon. Neither measurement is provided in current surgical or diagnostic protocols, but these compounds are markers associated with improved surgical outcomes and best patient prognosis. This proposal seeks to test intraoperative DESI-MS during surgical glioma resection cases executed at Mayo Clinic under the supervision of PIs Quiñones-Hinojosa and Chaichana to (i) further the development of the DESI-MS technology, the feasibility of which was demonstrated in a preliminary intraoperative study conducted at Indiana University School of Medicine under PI Cooks, (ii) collect an extensive set of data that will allow us to strongly link intraoperative molecular findings with preoperative imaging and postoperative diagnostics for validation, and (iii) set the foundation of follow-up clinical studies measuring prognostic utility.