Funding Status: Minimally Funded

This grant has been minimally funded and can proceed with research.

Lorem Sit Amet Dolor
Researcher: Lorem Sit Amet

123 abc lane, Townsville, ZZ 00000, USA
Funding Progress: $§ / $§§§§§

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INNOVATIVE MODELS OF THE BRAIN MICROENVIRONMENT TO IDENTIFY NEW TREATMENTS FOR MEDULLOBLASTOMA
Translational
Brain Tumors (General), Medulloblastoma, Childhood (Brain Cancer)
Lay Summary

Brain tumors are the leading cause of disease related mortality in children. Survival rates for children diagnosed with medulloblastoma (MB), the most common malignant pediatric brain tumor, have stagnated for decades. Despite aggressive treatment approaches, a significant proportion of patients relapse, which is almost always fatal. Very little is currently known of the biology and the mechanisms underpinning medulloblastoma relapse. We urgently need a deeper understanding of how tumors relapse and become resistant to therapy to achieve better outcomes for children. 

Our preliminary work suggests that tumor-driven changes in the brain microenvironment contribute to MB relapse, in particular the surrounding supportive scaffold and brain vessels. Precisely how tumor cells interact with the dynamically changing tumor microenvironment and blood vessel network and the impact of this on MB response to therapy remains unknown. We are proposing to apply our innovative pre-clinical models to examine the dynamic interactions between MB cells and their environment. This work generates a dramatically improved understanding of how specific environmental cues influence the therapeutic response of MB. Understanding the mechanisms underpinning relapse positions us to identify new drug targets and propose combination therapies that will enhance survival by reducing the likelihood of relapse.

Executive Summary

Background:

Brain tumors are the leading cause of disease related mortality in children1, with Medulloblastoma (MB) representing the most common malignant pediatric brain tumor1. Multimodal therapy incorporating maximal surgical resection with craniospinal irradiation and chemotherapy give rise to five-year overall survival rates of 70-75% for average-risk disease and 60-70% for high-risk disease1. Nonetheless, survivors frequently relapse conferring an almost uniformly fatal prognosis2,3. Extensive genomic characterisation has defined four biologically and clinically distinct sub-groups of MB: Wingless (WNT), Sonic hedgehog (SHH), Group 3 (Gp3) and Group 4 (Gp4) and up to 12 subtypes4-9. While these studies have contributed to significant advance of our understanding of primary MB, very little is still known regarding the biology underpinning relapsed MB. Comparative studies of diagnostic versus relapsed MB have shown sub-group and subtype affiliation were largely maintained, with divergent clonal selection leading to the emergence of specific molecular alterations10-13. How these genetic alterations impact the biology of MB cells and drive tumor relapse is not clear. Developing a deeper biological understanding of the mechanisms underlying treatment response, resistance and relapse is urgently needed for the identification of novel targeted treatment strategies.

Pre-clinical mouse models currently play a central role in the discovery, development, design and delivery of novel drug treatments, with the inherent assumption that pre-clinical efficacy of novel targeted therapies will translate into clinically relevant outcomes. However, this can only be achieved if pre-clinical mouse models closely recapitulate the genetics and biology of the human disease14,15. Various mouse models recapitulating the MB molecular sub-groups have been developed. Commonly used mouse models include predisposed germline, conditional inducible and transposon-based genetically engineered mouse models (GEMMs). The field also employs orthotopic transplantation models using both mouse and human cell lines, in addition to Patient Derived Orthotopic Xenografts (PDOX)16. PDOX MB models are widely used as the gold standard for pre-clinical testing of novel therapeutics, maintaining the characteristics of the primary human tumors from which they were derived in terms of histology, immunohistochemistry, gene expression, DNA methylation, copy number and mutational profiles17

Many of these pre-clinical PDOX in vivo studies have led to clinical trials for children diagnosed with MB18-22. While these studies have facilitated clinical translation of a number of novel agents, there are still a number of disadvantages to their use. Firstly, PDOX models in immunodeficient mice do not adequately capture the intact human immune component. Developments in humanized mice that support the human immune system may eventually overcome this, however it is not yet known if the species-to-species differences in interactions between tumor cells and other aspects of the tumor microenvironment (TME) will impact tumor growth23. Additionally, tumor development in vivo is slow, expensive and not readily scalable, with most studies utilising end-point readouts such as survival, precluding the assessment of the dynamic biological interplay between tumor and host throughout therapy. This represents one of the most significant hurdles to progressing our knowledge of why brain tumor relapses occur - the lack of pre-clinical models that adequately facilitate this level of analysis. There is an urgent need for physiologically relevant cell culture models to bridge this gap.

Cell culture models are an excellent pre-clinical model for brain cancer, with tumor-derived cell lines widely used for high-throughput screening of novel therapies. Of the established MB cell lines, the most widely published examples are D283MED, D341MED, D425MED, UW228-2 and DAOY, all of which can be grown either as a 2D monolayer on hard, flat tissue culture surface or as multicell aggregates in media suspension24,25. However, less than half of all MB cell lines have been sub-grouped25 making it difficult to ascertain the translational value of data obtained from these models. 3D tumor spheres derived from sub-grouped PDOX models of Gp3 MB have been recently established, and more accurately recapitulate certain physiological characteristics of the brain tumor microenvironment (TME), such as nutrient and oxygen gradients and the cell-cell interactions26-31. Whilst, sub-grouped 3D tumor spheroids represent an important advance for high-throughput screening, they still lack several essential components of the brain specific TME.

The brain TME consists of a dynamic and complex mixture of cancer cells surrounded by extracellular matrix (ECM), diverse non-cancerous cells including endothelial cells (ECs), pericytes, fibroblasts and immune cells and a number of brain-resident cell types including astrocytes, neurons and microglia32,33. This complex tumor ecosystem provides a dynamic array of key signals shown to drive proliferation, invasion and therapy resistance. These can be broadly classified into ECM composition, ECM mechanics, topographical cues, interstitial fluid and stromal-cell interactions 34. Crosstalk between SHH MB cells, tumor-associated astrocytes and their surrounding ECM was recently shown to be required for establishing the appropriate TME to support survival and proliferation35-37. These findings suggest that targeting of astrocytes may represent an additional strategy for therapeutic intervention in SHH driven MB. Very little is known about the other aspects of the TME of MB, including the tumor ECM bio-chemical and bio-physical characteristics. ECM remodelling drives mechanical changes to the TME, including increases in stiffness, interstitial and tumor pressure, which in turn promote tumor growth and alterations in tumor vasculature32,33,38. This vasculature represents another unique aspect of the brain TME, known as the BBB, a highly specialised selective barrier. Maintained by a multicellular structure called the neurovascular unit (NVU)39, the BBB is disrupted during tumor progression and is referred to as the blood brain-tumor barrier (BBTB). The BBTB represents a major obstacle for drug delivery to the CNS and contributes to suboptimal drug accumulation within brain tumor tissue40-42. Given the TME is central to mediating tumor progression, defining therapeutic response, and tumor relapse 38, it is imperative to deconstruct the complexity and dynamic nature of the MB TME. This will allow for the identification and testing of therapies targeting to the MB TME and ultimately offer a broader more comprehensive approach to managing MB.

PI Genovesi has developed a novel systems pharmacogenomics approach to define novel therapeutic approaches which would be effective across SHH, Gp3 and Gp4 MB sub-groups (Genovesi et al., Genome Medicine, accepted May 2021). This led to work with collaborators demonstrating that Palbociclib (CDK4/6i), approved for the treatment of breast cancer, is a highly effective treatment for SHH and Gp3 MB21. However, we have shown that, like with other cancers where the drug is used, tumors relapse upon withdrawal of Palbociclib (CDK4/6i). We have observed significant alterations in ECM signaling and processes underlying tumor vascularisation in our SHH and Gp3 PDOX MB models undergoing treatment with Palbociclib (CDK4/6i) and in those tumors that relapsed. These data suggest that TME-dependent remodelling is dictating the response to Palbociclib (CDK4/6i) and driving tumor relapse. 

Goal:

The goal of this integrative research program is to identify and characterize dynamic changes in brain ECM and tumor vasculature throughout the MB growth and whilst undergoing therapy with Palbociclib (CDK4/6i). To do this, we will address the following Aims:

1. Identify extracellular matrix parameters that drive pro-survival mechanosensitive signaling in MB

2. To determine the functionality of the BBTB using a bio-engineered vasculature and novel zebrafish models of MB

This project builds on the momentum and expertise of a group of interdisciplinary researchers with exceptional and complementary track records in cancer cell biology and advanced imaging technologies (Co-I Stehbens), vascular biology and zebrafish disease models (Co-I Lagendijk), mechano-biology (Co-I Stehbens, Lagendijk), brain tumor biology and translational research (PI Genovesi) and computational cancer biology (Co-I Davis). Our work will identify novel players that drive adaptation of the ECM and tumor vasculature in the TME and that promote tumor survival and resistance to therapy. Characterising these dynamic changes and integrating this information with data obtained using our existing pre-clinical PDOX models will provide unprecedented insight into the role of the TME in driving MB biology and accelerate the discovery of novel therapies targeted to the MB ecosystem.

 

We hypothesize that medulloblastoma tumor survival, therapy resistance and relapse is promoted by and intricately linked to co-evolved mechanical changes in the tumor microenvironment including alterations in the extracellular matrix and a compromised blood vessel network.

 

Design and methods:

Our team aims to develop innovative pre-clinical brain-mimetic models which integrate the biophysical and vasculature components of the brain TME. We will harness our multi-disciplinary expertise to combine these models with state-of-the-art microscopy to understand how dynamic alterations in the brain TME drive the growth, therapeutic response and relapse of MB. Incorporating high resolution live-cell imaging to identify cell-cell and cell-matrix interactions, will allow detailed analysis of distinctive phenotypes associated with TME-dependent patterns, and subsequent correlation to the tumor’s specific transcriptomic profile. We will leverage advances in bioengineering to generate a tuneable platform to systematically examine the interactions of 3D brain-ECM containing microenvironment with PDOX cells ex vivo. We will also leverage advances in microfluidics to systematically examine vascular morphology and BBTB functionality of tumor co-developed vasculature in a brain-matrix mimetic TME ex vivo and in vivo using zebrafish PDOX models. The novel integration of these brain mimetic TME model systems with zebrafish in vivo MB PDOX models sets the stage for mechanistic studies deciphering the TME’s role in MB progression. Once established, this integrative brain-mimetic pipeline brain mimetic model can be repurposed for other pediatric brain tumor types. Together, the development of these models offers a valid, highly adaptable alternative as an experimental pre-clinical MB model, with the potential to overcome limitations related to mouse models (discussed in research design, Aim 2.2). The incorporation of specific parameters (ECM composition, mechanics, tumor cell-vasculature interactions) into these engineered models to recreate the MB TME allows for more precise manipulation and rapid hypothesis testing.

Clinical Significance:

There is currently no cure for relapsed MB. Patient advocates and our consumer partners have emphasized the health and psychological burden of tumor relapse; the risk of the tumor returning represents their greatest fear for their child following therapy. Several clinical trials are currently underway investigating CDK4/6i in children diagnosed with various brain tumors. We have continued to explore the response of PDOX tumors treated with Palbociclib, and our results indicate relapse remains an issue. Our research into the mechanisms underlying therapeutic resistance and relapse are extremely timely and significant. We will define the relationships of MB cells and their associated TME with tumor relapse, and describe important druggable mechanisms of resistance - likely to be relevant across cancer types more broadly. This project will create a breakthrough in our understanding of tumor relapse and is a crucial step to improving the clinical utility of this drug for multiple cancer types.

We will build an essential foundation for guiding international clinical trials and developing new combinations of therapies for MB. Phase 1 clinical studies are currently underway evaluating three agents hypothesized to increase clinical efficacy of CDK4/6i when combined as a doublet. Our work discovering targetable mechano-transduction pathways to selectively ablate cells responsible for driving relapse will address an important need. This will provide biological rationale for the selection of therapies to inform the clinic of therapeutic combinations with CDK4/6i. Not only will these insights inform the use of Palbociclib and other CDK4/6i in MB; they will also impact the use of CDK4/6i in other cancers of the brain where therapy failure may be caused by similar mechanisms.

Our work will advance our understanding of the functionality of the BBTB to allow for improved delivery of therapeutics. The BBTB is more permeable than the BBB, however its heterogeneous permeability throughout the tumor contributes to suboptimal drug accumulation. It is imperative to investigate successful drug distribution across all regions of a tumor including those with intact vasculature. Our work will facilitate a greater understanding of tumor vasculature formation and function with respect to drug delivery and the mechanisms impacting integrity of these vessels. This will help elucidate whether suboptimal drug delivery plays a role in tumor relapse following Palbociclib therapy. This data is not only crucial for ongoing clinical trials with this class of agents, but for the realistic translation of any agent into the clinic and ultimately improving patient prognosis.

 

Description of Research Proposal

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Budget

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer gravida non felis non euismod. Fusce finibus aliquet consequat. Nam ac metus bibendum, iaculis purus sed, suscipit ligula. Proin et nisi libero. Mauris non urna urna. Nullam augue eros, fringilla sed mauris vitae, porta tincidunt risus. Aliquam sed tincidunt sem. Quisque lacinia quam tortor, imperdiet efficitur odio iaculis in. Sed ultricies condimentum volutpat. Vivamus dignissim faucibus porta.

Curabitur ut ipsum non odio malesuada vulputate. Morbi maximus, est eu lobortis molestie, tortor sapien hendrerit nisi, in cursus odio diam ut odio. Fusce pulvinar volutpat velit. Aliquam erat volutpat. Integer rhoncus mollis suscipit. Praesent non ipsum mollis, finibus nunc a, scelerisque nibh. In feugiat iaculis velit, eu semper lacus dignissim nec. Praesent vitae nisi leo. Cras venenatis dictum magna ut semper. Sed eget eros nibh. Sed vitae quam sed dolor faucibus elementum. Curabitur interdum porttitor finibus. Nullam tincidunt odio lectus, sit amet rhoncus libero dapibus sed. Sed mollis egestas enim, vel porta tortor volutpat eget.

Morbi orci urna, ornare non pretium eget, pulvinar eget magna. Ut consectetur efficitur varius. Fusce ac aliquet mauris, at mattis ligula. Quisque est libero, interdum id orci et, ornare luctus diam. Proin commodo lectus id accumsan blandit. Nulla eu turpis interdum, luctus ante ac, imperdiet tellus. In semper enim eu tristique aliquam.

Collaborations and Conflicts of Interest

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Curabitur ut ipsum non odio malesuada vulputate. Morbi maximus, est eu lobortis molestie, tortor sapien hendrerit nisi, in cursus odio diam ut odio. Fusce pulvinar volutpat velit. Aliquam erat volutpat. Integer rhoncus mollis suscipit. Praesent non ipsum mollis, finibus nunc a, scelerisque nibh. In feugiat iaculis velit, eu semper lacus dignissim nec. Praesent vitae nisi leo. Cras venenatis dictum magna ut semper. Sed eget eros nibh. Sed vitae quam sed dolor faucibus elementum. Curabitur interdum porttitor finibus. Nullam tincidunt odio lectus, sit amet rhoncus libero dapibus sed. Sed mollis egestas enim, vel porta tortor volutpat eget.