T32 Trainees and Project Summaries

Year 1 Trainees – 7/1/23 – 6/30/24

Year 2 Trainees – 7/1/24 – 6/30/25

T32 Trainees 2023- 2024

Vamsi Mangena, PhD (Imaging / Molecular Pathology)

PGY 1
Appointment Date: August 1, 2023
Project Title: Glioblastoma-cortical organoids model state-specific genetic transfer in the neuroglial tumor microenvironment
Primary Mentor: Mario Suva, M.D., Ph.D. (MGH)
Secondary Mentor: Daniel Cahill, MD, PhD (MGH)
Institution: Massachusetts General Hospital, Molecular Pathology, 149 13^th Street, Room 7350, Boston, MA, 02129

• Vamsi Mangena Statement of Research Interest

  Gliomas are incurable cancers of the brain and spinal cord. Recent advances in glioma biology have highlighted the importance of heterogeneity and intercellular communication within these tumors. Appropriate models for studying glioma behavior and progression that adequately recapitulate these key features are necessary for next-generation therapeutic development. For this reason, patient-derived tumor xenografts are widely used in vivo model systems for studying gliomas. However, the lack of complementary in vitro glioma models that better capture the molecular and phenotypic spectrum of the human disease remains a critical bottleneck in the field.

  We have developed a novel class of in vitro glioma models ("glioma-cortical organoids") by allografting patient derived glioma cells into mature human cortical organoids (derived from normal human stem cells). We have developed robust protocols to generate pediatric and adult glioma-cortical organoid models, including those involving otherwise intractable IDH-mutant gliomas. From a phenotypic perspective, we observe that glioma cells infiltrate into human brain organoids and form structures that emulate glioma biology (such as the formation of tumor microtubes and glioma-neural synapses). From a molecular perspective, we observe that malignant cells in glioma-brain organoid models recreate a spectrum of cell states that is more faithful to patient tumors than traditional, genetically matched cell line models.

  These models have become an enabling technology generating new biological insights into glioma biology. Most strikingly, we have observed transfer of reporter molecules (e.g., fluorescent proteins) from glioma cells to a subset of non-malignant cells in glioma-cortical organoid models. Based on this observation, we further discovered that there is widespread transfer of endogenous material from malignant to non-malignant cells, a previously underappreciated phenomenon.

  Further avenues of study that I am interested in exploring during this fellowship include 1) how this intercellular crosstalk influences malignant cell states, 2) consideration of likely mechanisms for this crosstalk, 3) possible therapeutic or genetic approaches for inhibiting this crosstalk, 4) the biological impact of intercellular crosstalk on glioma progression, and 5) the potential for observing this mechanism of transfer across non-glioma cancer types.

  In sum, we have developed glioma-cortical organoid models that form a functionally integrated tumor microenvironment and provide significantly improved in vitro disease modeling and therapeutic testing capabilities for human gliomas. I am seeking to apply these models towards a greater understanding of cell-type specific intercellular crosstalk in the glioma microenvironment, specifically in the context of emerging trends in cancer neuroscience.

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Joseph Amick, PhD (Imaging / Molecular Pathology)

PGY 4
Appointment Date: August 1, 2023
Project Title: Molecular Regulators of Blood-Brain Barrier Permeability
Primary Mentor: Chenghua Gu, PhD (HMS)
Secondary Mentor: Jean J. Zhao, PhD
Institution: Harvard Medical School, Department of Neurobiology, Armenise Bldg 315 220 Longwood Ave Boston, MA 02115

• Joseph Amick Statement of Research Interest

  Vertebrate endothelial cells, which form the inner lining of blood vessels, exhibit tissue-specific features to support the diverse functions of the body’s organs. Of the body’s many organs, the brain is especially metabolically demanding and neural function depends on the proper chemical environment. Therefore, endothelial cells of the brain possess unique features to meet the particular challenges posed by the brain. These features began to be uncovered in the 1960s by electron microscopy; they include specialized tight junctions, which restrict the movement of molecules between cells, and very low rates of vesicular transport between the blood and brain (transcytosis). Brain endothelial cells also express low levels of leukocyte adhesion molecules, which limits immune cell entry into the central nervous system, and high expression of transporters that influx and efflux substrates between the brain and blood to meet the brain’s energetic demands. These features define the Blood-Brain Barrier (BBB), the stringent divider between brain tissue and bloodstream. The BBB is critical for maintaining the proper environment for central nervous system health and function; conversely, the BBB limits the delivery of therapeutics to the central nervous system. Greater understanding of the mechanisms underlying BBB function, therefore, has broad potential implications for human health. While the BBB’s unique features have been characterized, much remains to be learned about their molecular basis and regulation.

  To better understand the molecular basis of brain endothelial cells’ ability to form the BBB, the Gu lab, and other groups, have performed comparative transcriptomic profiling of brain endothelial cells versus peripheral endothelial cells (such as lung endothelial cells) using single-cell RNAseq. We reasoned that because BBB properties are unique to brain endothelial cells compared to peripheral endothelial cells, if we identify molecular signatures of CNS endothelial cells, we will begin to understand how the BBB functions. This work identified a group of nearly 200 genes highly expressed in brain endothelial cells, but have low or undetectable expression in peripheral endothelial cells. We view these genes as candidate regulators of BBB function.

  These transcriptomic studies have given us information about genes expressed specifically in brain endothelial cells. However, there is a gap between this recently-discovered list of transcripts and the ultrastructural observations by electron microscopy of low rates of transcytosis and specialized tight junctions. My research interest is in bridging this gap by investigating at the cellular level how these genes function in brain endothelial cells to give rise to these classically-observed features.

  To investigate candidate BBB regulators, we developed a viral delivery platform to acutely ablate candidate genes in adult mice. In this approach, a viral construct encoding guide RNA targeting a gene of interest is packaged into a brain endothelial-specific Adeno-Associated Virus (AAV). The virus is injected into Cas9 knock-in mice. This combination results in potential acute ablation of the target gene in CNS ECs of adult mice, with just three weeks from virus injection to BBB leakage analysis. With this approach, I have identified genes whose knockdown results in BBB leakage. My next aim is to understand how these genes regulate BBB permeability. To accomplish this, I am using both well-established tools like electron microscopy, and developing new approaches to label cellular structures, membranes and endocytic pathways in brain endothelial cells. I anticipate these efforts will provide new insights into the cell biology of brain endothelial cells and reveal mechanisms that restrict BBB permeability.

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Gustavo Alencastro Veiga Cruzeiro, PhD (Tumor Microenvironment)

PGY 5
Appointment Date: August 1, 2023
Project Title: Determining how brain tumor cells utilize neuronal activity to invade and resist targeted therapy
Primary Mentor: Mariela G. Filbin, MD, PhD (DFCI)
Secondary Mentor: Rosalind Segal, MD, PhD
Institution: Dana Farber Cancer Institute, Pediatric Oncology, Filbin Lab, 360 Longwood Avenue, Longwood Center, LC6101, Boston, MA 02215

• Gustavo Cruzeiro Statement of Research Interest

  Brain tumors are the most common solid tumors in childhood. A deadly type of pediatric brain tumor called "pediatric-type diffuse high-grade glioma" shares molecular similarities with neurodevelopmental disorders. While the neuroscience field has made significant contributions to understanding neurodevelopmental disorders and developing new therapies, childhood brain tumors have received less attention. However, when examined through single-cell transcriptomics, childhood brain tumors exhibit molecular signatures similar to normal brain cells. This suggests that these tumors may partially resemble neurons, astrocytes, oligodendrocytes, and their progenitors. Additionally, brain cancer cells can exploit signals from the normal brain microenvironment to promote proliferation, invasion, and resistance to therapy.

  Applying cancer neuroscience tools and concepts can help us understand the behavior of childhood brain tumors and potentially lead to new treatment options. However, many research proposals aimed at developing novel therapies face challenges in progressing to clinical trials. By leveraging the T32 Cancer Neuroscience training grant, I will acquire expertise in assessing how brain tumor cells utilize neuronal activity to invade the brain or resist targeted therapy. I will also seek guidance from the program faculties involved in relevant trials for pediatric brain tumors to develop innovative therapy strategies and improve the translation of my findings.

  As a basic scientist with a strong background in genomics, transcriptomics, and pediatric brain tumor biology, I will seize the opportunity offered by the T32 Cancer Neuroscience training program. My goal is to deepen my understanding of aggressive pediatric brain tumors and develop novel treatment strategies to improve patient outcomes. The program will provide access to highly experienced staff and core facilities at the Dana-Farber Cancer Institute/Harvard Cancer Center, as well as invaluable mentorship from principal investigators.

  Since joining the Filbin lab at Dana-Farber Cancer Institute, I have already gained extensive knowledge and skills in studying cancer neuroscience using preclinical models of pediatric high-grade gliomas. I have expertise in culturing neurons, mouse brain slices, and patient-derived cell lines. I routinely perform live confocal/widefield microscopy to assess calcium transients and execute preclinical studies, including stereotactic injections. To further enhance my knowledge and expertise, I will actively engage in a variety of opportunities. These include enrolling in specialized courses, attending seminars, participating in retreats, networking, and bridging with the Harvard neuroscience community my cancer biology background. By regularly meeting with my advisory panel, I will ensure continuous guidance and support. My focus will extend beyond theoretical learning. I am eager to gain hands-on experience with advanced methodologies such as two-photon microscopy and cranial windows to develop proficiency in intravital imaging techniques, which will enable me to conduct in-depth assessments of invasion patterns and neuronal activity in brain tumor cells. Ultimately, my aspiration is to establish myself as an independent scientist, making significant contributions in the field.

  Dr. Filbin has established a supportive environment for my project and has engaged collaborators such as Dr. Michelle Monje and Dr. Rosalind Segal. These collaborators will assist me in addressing critical aspects of my project, providing conceptual brainstorming on cancer neuroscience, neurobiology, and glioma biology.

  Long-term research interest goal: My long-term research interest and career goal are to lead my own laboratory in the field of pediatric brain tumors. Leveraging my educational background and prior research experience in pediatric brain tumors with the new concepts and skills I will develop with the accomplishment of this award, I ultimately aim to establish my independent research group focused on three specific areas from basic to translational research focused on pediatric brain tumors, including but not limited to high-grade gliomas: (1) Using single-cell transcriptomics to dissect the cellular architecture, (2) Using cancer neuroscience tools to understand the diffuse behavior and cellular connectivity, (3) Using the basis of #1 and #2 to develop innovative therapeutic strategies to treat patients.

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Jessica Kenison-White, PhD (Neuro Immunology)

PGY 2
Appointment Date: September 1, 2023
Appointment End: August 31, 2024
Project Title:  Neuro-immune cell-cell communication in the context of glioblastoma
Primary Mentor: Francisco J. Quintana, PhD (BWH)
Secondary Mentor: Vijay Kuchroo, DVM, PhD (BWH)
Institution: Brigham and Women’s Hospital, Ann Romney Center for Neurologic Diseases, Department of Neurology, Francisco Quintana Laboratory, 60 Fenwood Road, BTM Room 10032, Boston, MA 02115

• Jessica Kenison-White Statement of Research Interest

  Glioblastoma (GBM) is the most common form of brain and CNS cancer, accounting for nearly 50% of all cases5. Despite the development of immunotherapies which have revolutionized the treatment of many forms of cancer, GBM survival prognosis is very poor, and response rates to all therapies, including surgical resection, radiotherapy, chemotherapy, and immunotherapy is low, with a five-year survival rate of less than 7%5-9. Thus, novel therapies for the treatment of GBM are urgently needed.

  Inflammation plays a major role in the development and progression of GBM. Indeed, the GBM tumor microenvironment (TME) is composed of many subsets of CNS-infiltrating adaptive immune cells, including cytotoxic and regulatory T cells, B cells, macrophages, monocytes and dendritic cells10-12. These immune cells interact directly with tumor cells, as well as with CNS-resident cells including astrocytes and microglia to promote anti- or pro-tumor responses13-15. However, our understanding of mechanisms of cell-cell communication between tumor cells and immune cells, and their roles in GBM pathogenesis is still limited.

  Our lab has recently developed a forward genetic screening platform called SPEAC-seq16 which can be used to identify cell-cell interaction mechanisms between two cells of interest, based on their co-encapsulation in droplets where one cell is perturbed by a genome-wide CRISPR/Cas9 library, and a partner cell expresses a reporter of a cellular state such as EGFP. By co-culturing GBM tumor cells with immune cells like dendritic cells and macrophages, SPEAC-seq will allow us to identify cell interaction mechanisms by which myeloid cells, or other immune cells, interact with tumor cells, as our lab recently did to identify amphiregulin as an important regulator of astrocyte responses16. Moreover, our lab has shown that GBM tumor cells directly suppress immune cells in the TME, at least in part, through the production of anti-inflammatory glioma-derived kynurenine which activates aryl hydrocarbon receptor (AhR) signaling and suppresses local immune cells including macrophages, dendritic cells and T cells17. Further, I have shown that glioma cells highly express activated AhR18 and that AhR expression in other tumor types drives expression of immune checkpoint molecules like PD-L1 and is critical for tumor growth in vivo1. Thus, in this project, I propose to apply SPEAC-seq to the study of GBM, to define mechanisms of disease pathogenesis and identify candidate therapeutic targets, including gathering important mechanistic information about existing therapeutic candidates such as the AhR. My Specific Aims are:

  Aim 1: Interrogate mechanisms of cell-cell communication between GBM tumor cells and immune cells in the TME. To study mechanisms of cell-cell communication in the GBM microenvironment and identify mechanisms driving local immunosuppression I propose to: 1) analyze by SPEAC-seq the effect of gene perturbations on interactions between GBM tumor cells, tumor-associated macrophages and dendritic cells; 2) validate the translational relevance of candidate mechanisms by performing immunofluorescence staining of target proteins in human clinical samples and using available epidemiologic data; and 3) functionally validate candidate mechanisms modulating tumor-myeloid cell interactions using CRISPR-driven cell-specific in vivo gene perturbation studies19-22 in GBM implanted mice.

  Aim 2: Evaluate candidate therapeutics for the treatment of GBM. Based on targets identified in Aim 1, I propose to: 1) evaluate the therapeutic efficacy of modulating novel targets in tumor cells and local TME dendritic cells and macrophages using nanoliposome-mediated delivery of small molecules2 or nanoliposome-mediated delivery of mRNAs targeting those pathways. As an example, given our previous data implicating the role of AhR in tumor progression, we may target the AhR using nanoliposome-delivered mRNA encoding for the AhR repressor (AhRR).

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Taylor Uccello, PhD 

PGY 1
Appointment Date: September 1, 2023
Project Title: Enhancing Radiotherapy for Medulloblastoma using Gamma Aminobutyrate Inhibition
Primary Mentor: Rakesh K. Jain, PhD (MGH)
Secondary Mentor: Humsa Venkatesh, PhD (BWH)
Institution: Massachusetts General Hospital, Radiation Oncology, E.L Steele Laboratories, 100 Blossom Street, Cox 7, Boston MA 02114

• Taylor Uccello Statement of Research Interest

  I have been fortunate to work with multiple mentors during my scientific training who have shaped my longterm career ambitions. My PhD advisor, Dr. Scott Gerber, became successful by combining two separate disciplines once thought to have divergent goals, but because of his work, are now intertwined. Dr. Gerber’s research examining the interconnection between radiation therapy (RT) and the immune response changed the paradigm of how radiotherapy is viewed. More recently, I have had the pleasure of joining Dr. Rakesh Jain’s research group at MGH. Similarly, Dr. Jain leveraged his background in chemical engineering and mathematical modeling to provide unprecedented insights into the tumor microenvironment (TME). Dr. Jain’s seminal findings on how the abnormal tumor vasculature fuels tumor progression and confers resistance to various cancer treatments, including radiation, chemo- and immuno-therapy, and how normalizing tumor vessels can overcome this resistance has had a major impact on basic and translational cancer research. His bench-to-bedside and back approach has led to several therapeutic advancements.

  Inspired and influenced by my mentors and their interdisciplinary research strategies, I began actively establishing my own research niche exploring the interplay between the nervous and immune systems to study oncological diseases – a nascent field with promising therapeutic applications. Notably, exploring the contribution of neurons to tumor growth led to the emergence of well characterized neurotransmitters as major modulators of extracranial cancers. Indeed, nerves have been shown to promote cancer cell proliferation and induce immunosuppression by directly acting on immune cells. Yet, the role of this crosstalk and its bidirectionality in central nervous system tumors remains largely unexplored. Excitingly, discoveries in this sphere could rapidly translate to the clinic due to the availability of numerous FDA-approved antagonists for various neurotransmitters: i.e., beta blockers against noradrenergic signaling and bicuculline against GABAergic signaling.

  During my doctorate work, I identified intratumoral nerves as a vastly understudied area of the TME that enforces immunosuppression in rectal cancer and fuels tumor progression. More specifically, I determined that adrenergic neurotransmitters such as norepinephrine reduce efficacy of RT in murine models and demonstrated that blocking beta-adrenergic receptors using propranolol enhances RT response. Since joining MGH as a postdoctoral fellow, I have shifted from noradrenergic neurohormones to focus my research on GABAergic signaling in the TME of pediatric medulloblastoma (MB) - a devastating malignancy with limited treatment options. My preliminary studies show that (a) overexpression of GABA receptors in human MB samples correlates with worsened patient outcome, and (b) GABA receptors are expressed on immune cells in the TME of mice bearing MB tumors. Furthermore, I have shown that GABA stimulation in vitro induces immunosuppression by reducing CD8+ T cell proliferation, activation and cytokine release while increasing inhibitory checkpoint molecule (PD-1) expression, as well as polarizing monocytes towards an immunosuppressive M2 phenotype (IL-10+, IL-35+, PDL-1+). Ultimately, GABA receptor A blockade in vivo resulted in increased overall survival and decreased symptoms of disease in an orthotopic model of Group 3 MB, suggesting a potential clinical application of modulating the GABAergic pathway in these tumors.

  I now plan to examine the mechanism of GABA-induced immunosuppression and determine if GABA blockade synergizes with standard of care (radiotherapy and chemotherapy) for MB – using various molecular, genetic and pharmacological approaches available in Dr. Jain’s Lab as well as his neuroscience collaborators’. My specific hypothesis is that targeting GABA via the GABA A or B receptors (or a combination of both) will alleviate immunosuppression in MB while synergizing with radiotherapy and protecting the brain against normal tissue toxicity.  To address this hypothesis, I will first characterize intratumoral GABA and its immunosuppressive mechanisms in genetically engineered mouse models of MB and secondly investigate therapeutic synergy between GABAergic antagonism and RT in orthotopic models of MB. My rapidly translatable project will establish neurotransmitter therapy as a promising avenue for targeting brain cancers and will thus inform clinical trials to enhance MB therapy and improve quality of life.

  My long-term research ambitions are to leverage my findings from both my doctoral and post-doctoral research to establish a translatable research program focused on modulating immunosuppression in solid tumors by abrogating local neurotransmitter/immune and neurotransmitter/tumor interactions. This complex interplay between nerves and immune cells has a promising clinical potential as an alternative way of alleviating immunosuppression to activate a robust anti-tumor immune response. My experience in adrenergic and GABAergic signaling in intra- and extra- cranial solid malignancies has prepared me to explore alternative neurotransmitters in the context of immunosuppression in various tumor types.

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Tamar Berger, MD, PhD (Cancer Therapeutics)

PGY 8
Appointment Date: November 1, 2023
Appointment End: October 31, 2024
Project Title: Identifying Novel Tumor Suppressor Genes in Glioblastoma
Primary Mentor: William Kaelin, MD (DFCI)
Secondary Mentor: Rameen Beroukhim, MD, PhD (DFCI)
Institution: Dana Farber Cancer Institute, Medical Oncology, Kaelin Laboratory, 450 Brookline Avenue, Mayer 452, Boston, MA 02215

• Tamar Berger Statement of Research Interest

  Glioblastoma (GBM) is the most common and aggressive primary brain cancer in adults. Despite numerous clinical trials, the standard-of-care treatment has not significantly changed since 2005, and average overall survival remains stagnant at 14.6 months. One reason for the lack of treatment efficacy is extensive intratumoral heterogeneity, whereby known targetable mutations are present in only a subset of GBM cells (tumor subclones).

  Targeting early mutations (“truncal mutations”) that are shared by all cancer subclones is more likely to be efficacious. In GBM, the most prevalent truncal events include chromosome 7 gains (often multiple whole-arm copies), monoallelic loss of chromosome 9p, or monoallelic loss of chromosome 10. At least 80% of GBM tumors have one or more of these events. Unfortunately, the fact that these truncal events involve entire chromosome arms has made it difficult to unambiguously identify the relevant gene (or more likely, genes) on each arm that contribute to tumorigenesis. The overarching goal of my research is to identify the genes that are the pathogenic targets of the truncal chromosomal changes in GBM and to identify therapeutic vulnerabilities arising from such chromosomal changes.

  My preliminary work identified two novel tumor suppressor gene candidates in GBM, both of which function to regulate glutamate metabolism. Specifically, I used The Cancer Genome Atlas (TCGA) to identify genes on chromosomes 9p and 10 that have reduced expression in GBM patients with copy-loss relative to copy-neutral tumors. I found that expression of the glutamate importer SLC1A1 (on chromosome 9p) is reduced in GBM patients with chromosome 9p loss and that expression of the glutamate dehydrogenase enzyme GLUD1 (on chromosome 10) is reduced in GBM patients with chromosome 10 loss. I then performed a whole-genome CRISPR-activation screen in GBM patient-derived glioma stem cells (“neurospheres”) with loss of both chromosomes 9p and 10 to identify genes that promote a fitness disadvantage following transcriptional activation. I found a statistically significant depletion of sgRNAs targeting the promoter regions of SLC1A1 and GLUD1, suggesting that transcriptional activation of these genes promotes a fitness disadvantage in GBM and implicating them as candidate haploinsufficient tumor suppressor genes on chromosomes 9p and 10. Based on my preliminary data, I hypothesize that the truncal loss of SLC1A1 on chromosome 9p and GLUD1 on chromosome 10 promotes tumorigenesis by reprogramming glioma glutamate metabolism. Additionally, since the loss of chromosomes 9p and 10 are large-scale genomic events, I further predict that deletion of two or more genes on each chromosome work in combination to promote tumorigenesis. I propose to test these hypotheses in the following aims:

  Aim 1: Test the hypothesis that SLC1A1 and GLUD1 function as haploinsufficient tumor suppressor genes in GBM. My preliminary work used whole-genome CRISPR-activation technology in GBM neurospheres with chromosome 9p and 10 loss to identify that SLC1A1 (chromosome 9p) and GLUD1 (chromosome 10) result in a fitness disadvantage when induced, suggesting that these are putative haploinsufficient tumor suppressor genes in GBM. I hypothesize that copy-loss of SLC1A1 and GLUD1 promotes a fitness advantage in GBM by reprogramming glioma glutamate metabolism. 1A) I will use CRISPR-activation technology in GBM neurospheres with chromosome 9p loss to test if activation of SLC1A1 alone or in combination with the known chromosome 9p TSGs CDKN2A/2B, decreases tumorigenesis in vitro and in vivo. 1B) I will use CRISPRactivation technology in GBM neurospheres with chromosome 10 loss to test if activation of GLUD1 either alone or in combination with the known chromosome 10 tumor suppressor PTEN decreases tumorigenesis in vitro and in vivo.1C) I will use CRISPR-interference technology in non-cancerous neural stem cells to test if transcriptional suppression of SLC1A1 and GLUD1 promote tumorigenesis in vitro and in vivo.

  Aim 2: Test the hypothesis that two or more genes on chromosomes 9p and 10 promote tumorigenesis through combinatorial effects. I will use combinatorial CRISPR-knockout technology in non-cancerous immortalized neural stem cells to test if chromosome 9p and 10 loss promotes tumorigenesis through deletion of two or more tumor suppressor genes. In a complimentary set of experiments, I will use combinatorial CRISPR activation technology in GBM neurospheres with chromosomes 9p and 10 loss to determine if transcriptional activation of one or more genes per chromosome results in a fitness disadvantage. The experiments I propose will advance our understanding of truncal alterations in GBM and hopefully identify new drug targets.

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T32 Trainees 2024 - 2025

Brian Andersen, MD, PhD

PGY 9
Appointment Date: September 1, 2024
Project Title: The role of FPR1 and calcium signaling in glioblastoma cell necroptosis
Primary Mentor: Francisco J. Quintana, PhD (BWH)
Secondary Mentor:    
Institution: Brigham and Women’s Hospital, Ann Romney Center for Neurologic Diseases, Department of Neurology, Francisco Quintana Laboratory, 60 Fenwood Road, BTM Room 10032, Boston, MA 02115

• Brian M. Andersen Project Summary

  GBM is the most lethal primary brain malignancy³. Immunosuppression in the GBM TME is a major barrier to immune-targeted therapies, but our understanding of mechanisms of immune regulation in the GBM TME is limited. I recently used RABID-seq², a viral barcode interaction tracing approach to analyze TME cell-cell communication in GBM clinical samples and preclinical models at single-cell resolution. I further investigated interactions between malignant GBM cells and non-malignant astrocytes using single-cell and bulk RNA-seq analyses, human organotypic GBM cultures, in vivo cell-specific CRISPR/Cas9-driven genetic perturbations and human and mouse experimental systems. This work has culminated in the identification of an annexin A1- formyl peptide receptor 1 (ANXA1-FPR1) bi-directional astrocyte-GBM communication pathway that limits tumor-specific immune responses. FPR1 inhibits immunogenic necroptosis (a type of programmed immunogenic cell death) in tumor cells, while ANXA1 suppresses NF-kB and inflammasome activation in astrocytes. ANXA1 expression in astrocytes and FPR1 expression in cancer cells are associated with shorter survival and earlier recurrence in GBM patients (Andersen et al., under review). The inactivation of astrocyte- glioma ANXA1-FPR1 signaling enhanced T-cell and tumor-associated macrophage responses, increasing infiltration by tumor-specific CD8+ T cells while limiting T-cell exhaustion. Notably, inhibition of FPR1, a Gi- linked g-protein coupled receptor, is known to increase calcium influx into other cell types4, and increasing calcium levels lowers the threshold for necroptosis in other tumor cell types5. I hypothesize that promoting GBM cell necroptosis via FPR1 inhibition and calcium influx offer a novel opportunity to reverse immunosuppression in the GBM TME. To test this hypothesis, I propose to complete the following aims for my Cancer Neuroscience T32 project:

  SPECIFIC AIM 1: Investigate the role of glioma calcium flux in the induction of necroptosis. Necroptosis is a form of immunogenic programmed cell death that provokes T-cell responses against other tumor types, and gene expression studies suggest that necroptosis in GBM is associated with extended survival. Moreover, I recently discovered that knockdown of Fpr1 in mouse glioma cells and blockade of FPR1 in human GBM cells leads to increased necroptosis in vivo and in vitro. The mechanisms by which FPR1 antagonism leads to necroptosis remain unknown, but data from other labs suggest that increased intracellular calcium levels lower the threshold for necroptosis. Therefore, I propose to:

  • Perform live calcium imaging of human GBM cells from the Dana-Farber center for Patient-Derived Models in the presence or absence of FPR1 blocking antibodies;

  • Establish the threshold for necroptosis in human GBM cells by inducing calcium influx, in the presence and absence of FPR1 manipulation; and

  • Quantify glioma cell necroptosis and T- and dendritic cell responses in mice implanted with syngeneic glioma expressing chemogenetic receptors for the induction of calcium signaling.

SPECIFIC AIM 2: Test the preclinical efficacy of T-0080, a potent CNS-penetrant FPR1 inhibitor with activity against human and mouse FPR1.

My preliminary data indicate that upon FPR1 blockade, necroptosis of GBM cells and astrocyte inflammasome responses are boosted. In addition, mice bearing GL261 syngeneic glioma cells with CRISPR/Cas9-mediated inactivation of Fpr1 have extended survival. A collaborating group recently developed T-0080, a potent, CNS- penetrant FPR1 inhibitor that blocks the binding site with ANXA1. T-0080 inhibits FPR1 signaling in mouse and human cells, suggesting it could be developed as a small molecular inhibitor to boost T-cell responses in GBM. I propose to treat mice bearing the genetically engineered mouse GBM model cell line SB28 with T-0080 to:

• • Quantify glioma cell necroptosis and T- and dendritic cell responses; and

  • Measure tumor burden and survival time, in the presence or absence of an anti-PD1 blocking antibody, which is ineffective as a monotherapy against SB28 and GBM.

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Alexandra Vaccaro Santiago, PhD

PGY 8
Appointment Date: February 1, 2025
Project Title: Searching for Neuronal Signals Linking Sleep Loss to Gut Tumor Dynamics
Primary Mentor: Dragana Rogulja, PhD.  (HMS)
Secondary Mentor: 
Institution: Harvard Medical School, Department of Neurobiology, 210 Longwood Ave., Armenise 345, Boston, MA, 02115

• Alexandra Vaccaro Santiago Statement of Research Interest

  I am a postdoctoral fellow in the Rogulja lab in the Neurobiology Department at Harvard Medical School. I’m interested in understanding the physiological function(s) of sleep, and more specifically how sleep disruption influences the course of pathological conditions such as cancer.

  Over the course of my graduate and postdoctoral work, I have acquired a versatile skillset in genetics, behavior, biochemistry and molecular biology using two animal models, fruit flies and mice. My work in the Rogulja lab has focused on better understanding how poor sleep decreases longevity and impairs quality of life. I showed that sleep restriction leads to gut oxidation, which can cause premature death (Vaccaro et al. 2020). Such gut damage can trigger the proliferation of intestinal stem cell, as a form of tissue repair and regeneration mechanism. However, excessive proliferation of these cells can contribute to tumor formation. I recently found that lack of sleep promotes a substantial increase in intestinal stem cell proliferation, which suggests a role for sleep in preventing tumor formation. Given that sleep problems are so prevalent in the general population and clinically connected to many types of cancers, these findings argue for the importance of my work to understand how poor sleep contributes to tumor formation.

  The enteric nervous system is referred to as “the second brain" due to its extensive network of neurons in the gastrointestinal (GI) tract which release multiple neurotransmitters and neurotrophic factors essential for normal gut function. For example, acetylcholine and serotonin released by the enteric neurons have been implicated in regulating proliferation, apoptosis, and differentiation of cells in the intestine. I hypothesize that dysregulation of these signaling pathways contributes to cancer development in the GI tract. I will search for neuronal signals that inform the gut when sleep is insufficient and determine how these signals influence tumor formation, growth, and metastasis in the gut. To identify these signals, I will look through the neurotransmitter systems, neural circuits connecting the brain and the gut, and neuroendocrine pathways, and will observe whether and how altering them impacts cancer development and progression in the GI tract.

  My familiarity with cancer biology and complementary fields is still limited, as both my doctoral and postdoctoral training focused on sleep/circadian rhythms in the context of aging, longevity and neurodegeneration. This T32 training program's structured curriculum led by top researchers in the field will provide me with the tools to master the principles of cancer neuroscience. Moreover, participation in the program will allow me to interact and build strong collaborative relationships with other trainees in different areas of cancer neuroscience.

  My long-term goal is to lead an independent research project within the Rogulja lab, which will make important contributions towards understanding the complex interactions between the nervous system and cancer, as well as provide new avenues for the development of innovative cancer therapies. This environment will allow me to pursue my scientific interests and follow wherever my scientific curiosity leads me, while contributing to the education of the next generation of scientists. Receiving this T32 grant will enable me to start a training in cancer biology, a field completely new to me, and will facilitate my transition to becoming more independent.

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