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Aghi Lab Newsletter
Volume 5 - January 2021
“Cancer is a noun but in the body it acts like a verb...”
Dr. Manish Aghi, MD, Ph.D., directs a research lab and operates on brain tumor patients at UCSF. His surgical practice and lab emphasize (1) glioblastoma (GBM), an aggressive brain tumor resistant to current treatments; (2) metastases of cancers that spread to the brain from other organs; (3) pituitary adenomas, benign tumors that dramatically impact a patient’s quality of life upon reaching critical size; and (4) medulloblastoma, a devastating pediatric brain tumor. The lab's primary focus is the microenvironment of these tumors, recognizing that many cancer treatments fail because they do not recognize cancer for what it is, a dynamic organ with a complex interplay between tumor cells and their microenvironment. Integrating Dr. Aghi's lab and clinical practice maximizes the impact of studying human tissues in the lab and implementing concepts from the lab into clinical trials to help patients.

The COVID-19 pandemic made 2020 a challenging year.  After scaling down our operations in the spring, we resumed research with social distancing and transitioning our in person meetings to review data to an online format during which our 12-person research team of postdoctoral fellows, students, and volunteers gathered to discuss the research highlighted below.
Screen shot from one of the weekly Aghi lab meetings over zoom to review experimental data
Current Research
Using CRISPR libraries and 3D bioengineered models of glioblastoma to define novel druggable mediators of invasion
The poor prognosis of glioblastoma is largely due to glioblastoma cell invasion, which enables escape from surgical resection and drives inevitable recurrence, typically 2 cm from the location at diagnosis.  This invasion ultimately proves fatal as tumor cells invade the brain’s essential real estate and sever its intricate synapses, with reoperation exerting equally devastating effects on quality of life. In a sense, GBM is a grenade that has exploded, piercing the brain with its shrapnel of tumor cells. Through a five year NIH R01 grant awarded to the Aghi lab, we are collaborating with the Kumar lab in the UC Berkeley bioengineering department to use 3D engineered culture models of invasion and CRISPR libraries targeting the druggable human genome to identify novel druggable mediators of invasion in GBM. This project has already yielded data examining the ways that GBM gene expression changes between the core and invasive edges of the tumor, providing many intriguing insights as we continue to figure out the complexities of this aggressive type of tumor.
Shown is a 3D bioengineered model of parenchymal invasion in which tumor cells are loaded into a cell reservoir and are then watched under a microscope for their migration out of the reservoir and into a hydrogel that models the brain parenchyma. By isolating the most invasive cells, we can determine factors mediating invasion, findings that can be validated using the CRISPR library targeting the druggable human genome.
Role of c-Met/β1 integrin complex in the metastatic cascade
Metastasis causes 90% of human cancer deaths with a median survival of fewer than six months following diagnosis. The five metastasis steps include local invasion, intravasation, extravasation, colonization at a distal site, and proliferation. A paper from previous Aghi lab MD/PhD student Arman Jahangiri in Proceedings of the National Academy of Sciences defined the role of a novel C-met/β1 intern protein complex in driving resistance to anti-angiogenic therapy and cancer metastases. In a follow up paper currently being revised, UCSF neurosurgery resident Darryl Lau and previous Aghi lab managers Harsh Wadhwa and Sweta Sudhir established that such a complex leads to increased intravasation of cancer cells into the bloodstream. Current lab manager Alexander Chang is now working to explore the mechanism of the said phenomenon by testing to identify the downstream actors affected by this complex and if this effect is common between breast cancer lineages with different receptors. Interestingly, we are finding that this complex can be blocked by OS2996, a β1 integrin blocking antibody developed by Oncosynergy, a company that former Aghi lab postdoc Shawn Carbonell developed after exciting pilot data developed in the Aghi lab. OS2996 is currently the subject of a phase I clinical trial in glioblastoma and this work suggests it also has potential as an anti-metastatic agent.

Activation of a c-Met1/β1 integrin complex leads to increased activation of stem cell and mesenchymal genes, leading to increased intravasation and binding affinity for certain tissue types. 
Identifying cancer-associated fibroblasts in glioblastoma and characterizing their effects on tumor stem cells
While pro-tumoral cancer-associated fibroblasts (CAFs) have been identified in some cancers, CAFs had been presumed absent in glioblastoma given the lack of brain fibroblasts.  Saket Jain, a postdoc in the Aghi lab, follows up on research conducted by prior Aghi Lab HHMI fellow Jonathan Rick and Rushikesh Joshi to define the protumoral effect exerted by these unique cells. Single-cell RNA-seq suggested that these cells and pericytes shared mesenchymal stem cell lineage. Our research has shown that CAFs are recruited to the tumor microenvironment via platelet-derived growth factor (PDGF) and TGF-β and enrich the GBM  stem cell (GSC) population. Glioblastoma CAFs also induce hypertrophied vessels and M2 macrophage polarization, the latter through unique CAF production of the EDA fibronectin variant, which binds macrophage toll-like receptor 4 (TLR4) in a targetable manner. We also found that glioblastoma CAFs were enriched in the subventricular zone, which houses neural stem cells that produce GSCs. Depleting CAFs in GSC-derived xenografts slowed in vivo growth. These findings are among the first to identify glioblastoma CAFs and reveal their involvement with GSCs, making them an intriguing future therapeutic target. 
GBM cells secrete chemoattractants which draw perivascular progenitor cells towards the tumor.  The GBM cells then transform these progenitor cells into CAFs.
Using retroviral gene therapy to reverse the immunodepleted glioblastoma microenvironment
Despite its encouraging initial results, replicating retroviral gene therapy for glioblastoma failed to demonstrate efficacy in a randomized clinical trial. Initial results suggested that viral therapies promote an anti-tumor immune response. However, the GBM microenvironment is incredibly immunosuppressed, contributing to the lack of efficacy seen in clinical trials investigating single-agent systemic immunotherapies. The Aghi lab is addressing these limitations by using local retroviral delivery of immunomodulator gene(s) directly to the GBM microenvironment. This approach reduces systemic immunotherapies' side effects and allows for combination treatments. The Aghi lab is investigating the local delivery of single and combination immunotherapies directly to the GBM tumor microenvironment in this vein. UCSF medical student Alex Haddad, postdoctoral fellow and medical student Jordan Spatz, Ph.D., and  Brown medical student Elaina Wang are working on different locally delivered therapies to generate a robust anti-tumor immune response. Alex published a paper from the Aghi lab in Scientific Reports in 2020 that suggested the possibility that these approaches could be personalized to individual tumors.
Cytolytic score is a way of assessing intratumoral immune cell infiltrate. For glioblastoma, cytolytic score impacts prognosis in a manner opposite that seen with other cancers, underscoring the unique way in which immmune cells interact with glioblastoma.  The Aghi lab will explore using metrics like this to identify personalized strategies for glioblastoma.
Defining pro-tumoral effects of tumor-associated neutrophils in glioblastoma
Tumor-associated neutrophils (TANs) can assume distinct pro-tumoral and anti-tumoral phenotypes in a wide array of solid tumors. In patients with glioblastoma (GBM), the elevated neutrophil-to-lymphocyte ratio in peripheral blood correlates with worse outcomes. Additionally, increased neutrophil infiltration into the tumor microenvironment correlates with higher grade glioma. In light of these clinical findings, we have conducted in vitro, and transcriptomic analyses on TANs sorted from neurosurgical patients at UCSF. Our data demonstrate that TANs support the glioblastoma stem-like cell (GSC) niche through various paracrine pathways. Aghi Lab Junior Specialist Angad Beniwal is currently using pre-clinical mouse models to investigate further the mechanisms associated with this apparent pro-tumoral activity, hoping further to elucidate the role of neutrophils in the tumor microenvironment to understand better the complex challenge that is aggressive brain cancer. 
Tumor associated neutrophils (green) in the perivascular niche of glioblastoma
Single cell sequencing reveals driver genetic changes in the formation of pituitary adenomas
Pituitary adenomas are among the most common primary brain tumors and comprise 15% of all brain neoplasms. In the Aghi lab, postdoctoral fellow Saket Jain is using single-cell RNA sequencing to investigate cellular heterogeneity in 15 pituitary adenomas. Copy number variation analysis highlighted the loss of chromosomes 2 or 15 appearing across all clones (“early” changes) and other changes such as loss of chromosome 19 appearing in some clones (“late” changes). Saket has also identified 3-4 clusters of tumor cells per case: (1) a secretory hormone/neuropeptide secretion cluster; (2) a proliferative cluster; (3) a metabolic cluster; and (4) a pro-angiogenic cluster. Non-tumor cell clusters identified included (1) antigen-presenting and processing cells; (2) fibroblast cells; and (3) neuronal cells. This work, combined with single-cell ATAC-seq to data-mine the epigenetic changes in these tumors at the single cell level, will help us define pituitary adenomas' molecular fingerprint and provide insights that could be utilized in the clinic to manage these tumors better. 
Copy number variation analysis showing chromosomal alterations in patient pituitary tumors
Defining Metabolic Alterations in Invasive Glioblastoma Cells
The poor prognosis of GBM caries is largely due to cellular invasion, which enables escape from resection and drives inevitable recurrence. Numerous factors have been proposed as the primary driving forces behind GBM’s ability to invade adjacent tissues, including shifts in its cellular metabolism. To satisfy the need for constant growth and proliferation, invasive GBM cells undergo significant metabolic reprogramming. Joseph Garcia, a UCSF medical student funded by an NIH diversity supplement in the Aghi lab, is using  a series of system-wide unbiased screens to determine the specific metabolic alterations that occur in GBM’s most invasive cells. Following the screens, our team will probe for specific mechanisms using targeted assays. By understanding the metabolic factors that drive tumor invasion, we hope to identify novel therapeutic targets for GBM. 
System-wide screens will be used to assess regional differences in glioblastoma metabolism
Restoring MHC-I Expression and T-Cell Chemotaxis in Medulloblastoma
Medulloblastoma is an aggressive brain tumor that predominantly impacts children. Current treatments can be toxic and neurologically devastating. As such, immunotherapies are promising alternatives. These tumors are notoriously immunosuppressed, with a dearth of anti-tumor processes occurring in the tumor microenvironment. MYCN-amplified Group 3 medulloblastoma is particularly aggressive, with a devastating prognosis. Using a murine model of MYCN-amplified Group 3 medulloblastoma, we seek to uncover and reverse the MYCN-induced chemokine abnormalities in medulloblastoma to drive immune cells towards the tumor site. Of particular interest is restoring tumor expression of CXCL10, though several other cytokines are also under investigation. We are also working on upregulating MHC-I expression on tumor cells to enhance T-cell mediated tumor-killing. EZH2 inhibition is a clinically promising option that we are focusing on and pathways involving MYCN, interferon-gamma, TNF-alpha, and NF-kB. 

Ultimately, we hope to enhance T-cell migration, invasion, and tumor-killing, while upregulating MHC-I expression on tumor cells to initiate a robust immune response to these devastating tumors. This project was initiated by Aghi lab postdoc and UCSF neurosurgery resident Taemin Oh and is now being continued by UCSF medical student and yearlong research fellow Ryan Phelps.

Immunomodulation of medulloblastoma
Using Genetic Perturbation to Study a Novel Glioblastoma Therapeutic Target 

Research by Aghi lab postdoc and  UCSF neurosurgery resident Michael Safaee with assistance from Sabraj Gill, an Aghi Lab research associate, revealed that CD97 is highly expressed in glioblastoma and may have roles in tumor invasion, angiogenesis, immune cell suppression, and immune evasion. Michael and Sabraj are targeting the gene with CRISPR interference to study CD97's role in tumor invasiveness, angiogenesis, tumor cell metabolism, and regulation of immune cell populations in glioblastoma. Proximity-dependent biotinylation labeling is being used to characterize the extracellular binding partners of CD97 in vivo. The lab is also studying an Fc fusion protein targeting CD97 as a potential therapeutic in collaboration with industry.

Defining Immunologic Changes Driving Breast Cancer Metastases to the Brain
Breast cancer is the most common malignancy in women in the United States, and brain metastases in these patients are associated with poor prognosis. Immunoediting is a critical component of metastatic tumor cell elimination, and tumor clones that develop immune-escape mechanisms are associated with progression and metastatic dissemination. Ramin Morshed, an Aghi lab postdoc and UCSF Neurosurgery resident, is currently working with syngeneic mouse models of metastatic breast cancer to identify genetic changes in brain metastatic cancer cells that lead to immune system evasion. By identifying perturbations in gene expression, antigen presentation, and immune cell infiltrate into breast cancer brain metastases, immune escape mechanisms and potential novel targets for immunotherapy may be identified.
Clinical Studies 2020
Aside from the basic science work described above, physician-scientist members of our team published several clinical studies in 2020, two of which are highlighted below:

1. UCSF medical student Matheus Perreira published a study showing the efficacy and safety of transsphenoidal surgery for elderly patients with pituitary adenomas.  This study was published in Neurosurgical Focus.
2. UCSF medical student Alex Haddad published a study finding that null cell pituitary adenomas were more aggressive than gonadotroph pituitary adenomas in 1166 cases from UCSF. This study was published in Neurosurgical Focus.
3. Former Aghi lab Howard Hughes research fellow Ankush Chandra published a study of glioblastoma in the elderly and found that outcomes were independent of tumor location in a large cohort of patients from the publicly available SEER database. This study was published in World Neurosurgery.
For More Information
For more information about research in the Aghi Lab or how to support our efforts, visit our website at To schedule a tour, please contact Anders Yang in the UCSF Development Office at 415-502-8309. If you do not wish to receive further fundraising communications from UCSF, please contact: Record Manager, UCSF Box 0248, San Francisco, CA 94143-0248 or email or call 1-888-804-4722.
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