Mariano Sebastian Viapiano - US grants

1033991
Palmer

The proposed research will develop a nanofiber-based sensor capable of quantifying oxygen distributions within and around populations of migrating glioblastoma populations. The PI has already shown that electrospun aligned fiber arrays can guide the migration of adherent human-derived tumor cells. By fabricating a "core-shell" nanofiber, oxygen-sensitive indicators can be added to the "core" of this fiber while the "shell" protects cells from exposure to the indicator. Confocal microscopy can interrogate oxygen content and enable experiments involving one or more neurospheres, how they interact with the surrounding [O2] and with each other.
A sensor system that recreates hypoxic, normoxic, and hyperoxic conditions within the human body while simultaneously encouraging migration known to be a component of tumor metastasis will be useful in the study of many human cancers. Furthermore, the sensing capability will lead to increased understanding of similar issues that arise in the creation and integration of new, normal human tissue during organ generation and replacement.

DESCRIPTION (provided by applicant): Malignant gliomas are the most common and deadly type of brain cancer, highly invasive and resistant to radiation and chemotherapy. Our goal is to understand and target the mechanisms that drive tumor invasion and promote survival in glioma cells. In recent work we identified and characterized a novel extracellular matrix (ECM) protein named fibulin-3, which is absent in normal brain but is abundant in gliomas and promotes tumor growth and invasion. From our preliminary results, we hypothesize that fibulin-3 acts as a diffusible factor in the ECM, promoting invasion and cell survival by mechanisms that may include activation of anti-apoptotic Notch signaling and the NF-kappaB pathway, increased expression of pro-invasive genes, and controlled degradation of the extracellular matrix. Accordingly, inhibition of fibulin-3 may reduce tumor invasion and make gliomas more sensitive to chemotherapeutics. To test these hypotheses, in Specific Aim 1 we propose to investigate the mechanisms by which fibulin-3 promotes tumor cell migration and survival. We will analyze the mechanisms of activation of the Notch pathway by fibulin-3, and the requirement of Notch signaling to mediate the effects of fibulin-3 on cell invasion and survival. In Specific Aim 2 we propose to analyze the mechanisms by which fibulin-3 may promote ECM degradation. We will analyze if fibulin-3 increases metalloprotease activity and degradation of the ECM by activating the pro-invasive NF-kappaB pathway and inhibiting the metalloprotease inhibitor TIMP3. Finally, in Specific Aim 3 we propose to evaluate the impact of suppressing fibulin-3 on tumor progression and response to chemotherapy. We will assay a novel system to induce the downregulation of fibulin-3 in the tumor and will analyze the effect this downregulation on tumor growth, invasion, animal survival, and sensitization of gliomas to a standard-of-care anti-neoplastic drug. Successful completion of these studies will establish the role of fibulin-3 in brain tumors and will provide new insights into the mechanisms that support brain tumor progression. These results may translate into novel strategies to disrupt tumor invasion and achieve more effective therapies. PUBLIC HEALTH RELEVANCE: Malignant brain tumors, known as gliomas, are one of the types of cancer with worst prognosis. This situation has not improved even after decades of research because these tumors resist conventional chemotherapy and escape novel therapies, thanks, in large part, to their ability to infiltrate in the brain. We propose to characterize and target a novel protein that is secreted by glioma cells but is absent in normal brain. This protein promotes brain tumor invasion and tumor survival, which are the two key processes that reduce the efficacy of current treatments. Therefore, our proposed research is highly relevant because it may lead to effective disruption of tumor progression and increased sensitivity of the tumor to conventional chemotherapeutics. We believe that these studies will have relevance for the development of more effective strategies and therapeutic reagents against malignant brain tumors.

? DESCRIPTION (provided by applicant): The goal of this project is to develop and validate novel biological reagents against glioblastomas (GBMs), which are one of the most common and the most aggressive kind of brain tumors. GBMs are highly invasive in brain tissue and their dispersion is exacerbated by cytotoxic and antiangiogenic therapies, resulting in a much worse clinical picture when the tumor recurs. Novel therapeutics addressing the biology of GBM should be able to disrupt invasion or vascularization and sensitize tumor cells, thus causing tumor disruption in multiple fronts when combined with conventional therapies. This project will focus on the protein fibulin-3 identified by our group, which is secreted by GBM cells and has unique localization and mechanisms in GBM but is absent from normal brain. Fibulin-3 is a novel activator of the Notch pathway and genetic targeting of this protein disrupts Notch signaling in the tumor and reduces GBM growth and resistance. We have produced a function-blocking antibody against fibulin-3 (mAb428.2) and in this project we will use it as a starting point to generate and validate a family of anti-fibulin-3 recombinant reagents. In Specific Aim 1, we propose to validate safety and efficacy of mAb428.2 against intracranial GBMs to advance it towards investigational new drug status. We hypothesize that anti-fibulin-3 will reduce tumor growth and vascularization with no significant peripheral toxicity or local off-target effects. We will study mAb428.2 specificity, cross-reactivity, biodistribution, safety and anti-tumor efficacy following FDA guidelines, aiming at completing pre-IND validation. In Specific Aim 2, we propose to develop and validate an anti-fibulin-3 single-chain immunotoxin as cytotoxic agent for GBM. We have already generated a single-chain variable fragment (scFv) derived from mAb428.2 and hypothesize that the smaller size of this fragment will improve access and toxicity against GBM. We will conjugate the scFv with Pseudomonas exotoxin and will validate this novel immunotoxin for specificity, safety and efficacy against intracranial GBM. Finally, using fibulin-3 scFv, in Specific Aim 3 we propose to generate and test a chimeric antigen receptor (CAR) against fibulin-3. We hypothesize that a fibulin-3-binding CAR expressed in T lymphocytes will be robustly activated in the tumor microenvironment and will result in strong anti-tumor response even in presence of tumor cell heterogeneity. This is a high-risk/high-reward concept for future immunotargeting of the tumor stroma, and the goal of this aim will be to establish a proof of feasibility and demonstrate that such CAR can be generated and specifically activated by fibulin-3. Successful completion of this project will result in a family f lead biological agents targeting the GBM microenvironment, with different levels of preclinical validation. These results will be used to advance the most promising candidate(s) for submission as investigational new drug(s) for GBM.

Glioblastomas (GBMs) are the most common and aggressive primary brain tumors in adults, remaining as one of the deadliest forms of cancer. These tumors are highly invasive, which makes their complete resection impossible and contributes to their recurrence and lethality. Changes in cell polarity are critical for tumor invasion in response to signals from the tumor microenvironment; however, these mechanisms have been largely overlooked in GBMs. Our goal in this project is to investigate a novel cell polarity target that may underlie the ability of GBM cells to invade in response to specific signals produced by neural cells. The proteins of the DLG family contribute to normal cell polarity by regulating the transport of signaling molecules between the apical and basolateral domains of normal cells. These proteins are downregulated in epithelial tumors and are thought to act as tumor suppressors. However, our preliminary work has revealed that the protein DLG5 has a striking upregulation in GBMs and contributes to tumor invasion and activation of the Sonic Hedgehog (SHH) pathway in GBM stem cells. Our central hypothesis is that DLG5 plays a unique tumor-promoting role in GBMs and regulates the ability of invasive GBM cells to respond to guidance signals (i.e., SHH ligand) originated from neural cells. To test this hypothesis, our first Aim is to determine the functions of DLG5 on GBM growth, dispersion, and tropism towards neural cells. Using an inducible-shRNA strategy we will study how the loss of DLG5 affects tumor growth, invasion, and interaction of GBM cells with oligodendrocyte precursors that are a major source of SHH ligand in adult brain. Our second aim is to determine if DLG5 specifically regulates pro- invasive SHH signaling in GBM stem cells. We will study the molecular association of DLG5 with components of the SHH pathway and will analyze how DLG5 knockdown affects this pathway in GBM cells in vitro and in vivo. To our knowledge, this is the first investigation of the DLG family of cell polarity proteins in GBMs, and the first study to propose that DLG5 may have a tumor-promoting function and is required for interactions between brain tumor cells and their unique neural microenvironment. Successful completion of the project will help understand the communication that occurs between glioma cells and neural cells during tumor dispersion, allowing us to formulate strategies to disrupt those interactions. Our studies will help disable tumor cells that remain in the brain after surgery, which could potentiate therapies to prevent recurrence and achieve long-term survival in patients with malignant gliomas.

PROJECT SUMMARY: Glioblastomas (GBMs) are aggressive brain tumors with poor response to conventional treatments. Novel therapies that stimulate immune responses are showing great promise to treat other solid tumors but have limited effect against GBM. This is due, in large part, to the presence of a sizable population of tumor-associated microglia/macrophages (TAMs) that are induced by the tumor cells to acquire an immunosuppressive, tumor- promoting phenotype. The suppression of innate and adaptive immunity caused by TAMs, coupled with the substantial intra-tumoral heterogeneity of GBM, facilitates the escape of malignant cells that cause unavoidable recurrence. Therefore, there is a dire need for novel targeted approaches that can create a persistent anti-tumor immune environment and at the same time overcome GBM heterogeneity. The extracellular matrix (ECM) is a unique target accessible outside the cells, widespread in the tumor, and less heterogeneous than the tumor cells that it surrounds. Accordingly, we propose a novel anti-tumor approach based on ECM targeting to reduce immunosuppression. Specifically, we will investigate the immunomodulatory functions and targeting value of the ECM protein fibulin-3, which is a major tumor-promoting factor secreted by GBM cells but absent from normal brain. Preliminary evidence suggests a positive correlation between fibulin-3 and immunosuppressive signals in GBM, as well as signs of immune activation following fibulin-3 disruption. Accordingly, we hypothesize that fibulin-3 promotes TAM immunosuppression, contributing to GBM immune escape and progression. To validate this hypothesis, our first Specific Aim is to investigate the effect of fibulin-3 downregulation on TAM polarization. We will first disrupt fibulin-3 signaling in GBM cells to analyze its regulatory effect on immunosuppressive cytokines and immune checkpoint ligands. Next, we will use a model for inducible downregulation of fibulin-3 in vitro and in vivo, to test if the loss of this protein enhances an inflammatory TAM phenotype, causing tumor-lytic effects. In our second Specific Aim, we will investigate whether fibulin-3 blockade enhances anti-tumor immunity. We have developed a function-blocking anti-fibulin-3 antibody that reduces the growth of GBM and, surprisingly, increases TAM infiltration and tumor necrosis. Using GBM / TAM co-cultures and intracranial xenograft models, we will investigate if anti-fibulin-3 can reduce immunosuppressive signals from GBM cells and rescue the undesirable tumor-promoting TAM phenotype into a desired anti-tumor phenotype, without negative systemic effects. Finally, using syngeneic GBMs models, we will test if anti-fibulin-3 can also increase the intratumoral infiltration of activated T cells for a sustained anti-tumor response. Successful completion of this project will validate fibulin-3 as a novel immunomodulatory factor in the microenvironment of GBM, which can be targeted to overcome the heterogeneity of GBM cells and potentiate anti-tumor immunity.