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Aghi Lab Newsletter
Volume 6 - January 2022
“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. The lab's primary focus is the microenvironment of brain 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. 

In 2021, the Aghi Lab continued to adapt to the challenges from the COVID-19 pandemic. After a year of virtual discussions and social distancing protocols, we resumed research and  in-person meetings at full capacity, with the work of our research team of postdoctoral fellows, students, and volunteers  highlighted below.
The Aghi Lab shares a meal celebrating lab members' birthdays.

Recent Publications and Conferences

The Aghi Lab presented research at the 2021 Society for Neuro-Oncology Conference in Boston, MA. 
Current Research
Using CRISPR libraries and 3D bioengineered models of glioblastoma to define novel druggable mediators of invasion
The poor prognosis of glioblastoma (GBM) is largely due to GBM 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 GBM cells invade the brain’s essential real estate and sever its intricate synapses. 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 to identify novel druggable mediators of invasion in GBM. This project has already provided many translational insights.
Shown are 3D invasion assays for GBM cells treated with 4 different drugs targeting draggable genome candidates that emerged from the CRISPR library screen we performed.  These candidates are now being evaluated to treat GBM tumors in mice.
Role of c-Met/β1 integrin complex in the metastatic cascade
Metastasis causes 90% of cancer deaths with a median survival below six months following diagnosis. 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 integrin protein complex in driving resistance to anti-angiogenic therapy and cancer metastases. In a follow up 2021 paper published in JCI Insight, UCSF neurosurgery resident Darryl Lau and previous Aghi lab managers Harsh Wadhwa, Sweta Sudhir, and Alexander Chang and current Aghi lab manager Ananya Pappu established that this complex increases intravasation of cancer cells into the bloodstream. Ananya is now working to identify downstream actors affected by this complex. Utilizing a biotinylation labeling system, we identified the AHNAK scaffolding protein as interacting with the c-Met/β1 integrin complex and driving its signaling.
Greater levels of c-Met1/β1 integrin complex were detected in brain metastases from breast cancer.  Greater levels of c-Met1/β1 integrin complex (shown with red tissue staining) were also detected in brain metastases from the more aggressive triple negative breast cancers (TNBCs), which lack hormone-receptor (estrogen-receptor and/or progesterone-receptor positive) and HER2, than in luminal A breast cancer, which is hormone-receptor positive and HER2 negative.
In vivo CRISPR Screen for Glioblastoma Immunotherapy Resistance and Sensitizing Genes
Immunotherapy, which stimulates an immune response against the tumor, has been revolutionary in the treatment of some malignancies, but, unfortunately, clinical trials exploring immunotherapies for GBM have been disappointing to date. This failure has been attributed to GBM’s low mutational burden and systemic and local immune dysfunction. To improve these therapies, a more comprehensive understanding of molecular determinants of GBM's responsiveness to immunotherapy is needed. The emergence of CRISPR-Cas technology has enabled high-throughout, genome-wide genetic screens, such as in vivo ‘‘cell fitness screens” under different treatment conditions to identify ‘‘context-specific’’ genes governing treatment resistance. Under funding from the Neurosurgery Research Education Foundation (NREF), UCSF Neurosurgery resident Jacob Young is utilizing an in vivo genome-wide CRISPR screen in the setting of syngeneic tumors treated with immunotherapy to reveal immune-sensitizing candidate genes. 
Role of CD39 in Glioblastoma Immunosuppression
While successful in other cancers, immunotherapy has been ineffective for GBM. In addition to its immunosuppressive environment, GBM T cells become rapidly exhausted, greatly reducing their cytotoxic capabilities. The Aghi Lab is working to elucidate the role of one these markers, CD39, with the use of specially bred CD39 knockout mice. CD39 has yet to be rigorously tested in GBM. Western Michigan University medical student Allison Zheng is investigating its role in the GBM microenvironment via studies in cultured cells and mice. These studies hope to clarify how CD39 affects T-cell function, and its potential role in T cell exhaustion.
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 GBM 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 identify CAFs in GBM and define their protumoral effects. Using single-cell RNA-seq, we identified CAFs in GBM. We found that CAFs are recruited to the GBM microenvironment via platelet-derived growth factor (PDGF) and TGF-β and enrich the GBM stem cell (GSC) population. GBM CAFs also induce hypertrophied vessels and M2 macrophage polarization, the latter through unique CAF production of the EDA fibronectin variant. We also found that GBM CAFs were enriched in the subventricular zone, which houses neural stem cells that produce GSCs. Depleting CAFs in GSC-derived mouse tumors slowed growth. 
On the left, a t-SNE plot of cells from 12 patient GBMs reveals a cluster of cells expressing cancer associated fibroblast (CAF) genes. On the right we demonstrate that these cells lack the chromosomal alterations seen in tumor cells and are identified in each of 12 patients.
Using retroviral gene therapy to reverse the immunodepleted glioblastoma microenvironment
The Aghi lab is investigating retroviral delivery of immunomodulatory genes directly to the GBM microenvironment. We are particularly interested in immune cytokines and costimulatory ligands. One such molecule is RLI, an Il-15 superagonist, which has shown promise in the Aghi lab in curing or prolonging survival in several mouse models of GBM, with treated tumors exhibiting an increased number of immune cells. George Washington University medical student Eric Chalif and UCSF residents Alexander Haddad and Ramin Morshed are testing this agent in combination with several other promising molecules, including cytosine deaminase, an enzyme that was previously approved for phase III clinical testing and was delivered within a replicating retroviral vector. 
A full array of intratumoral immune cells rises two weeks after intratumoral treatment of mouse glioblastoma with RRV-RLI treatment before dropping by the time the tumors reach endpoint.
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 GBM patients, 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 GBM stem-like cell (GSC) niche through various paracrine pathways. Meeki Lad, a Rutgers New Jersey Medical School student in the Aghi Lab, is continuing former Junior Specialist Angad Beniwal's work by utilizing mouse models to investigate further the mechanisms associated with this pro-tumoral activity, hoping to elucidate the role of neutrophils in the GBM microenvironment. 
Neutrophil depletion of mice with intracranial glioblastomas leads to a reduction in T-cell related gene expression and a reduced antitumoral T-cell response
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). Pathway analysis revealed elevated Wnt signaling in adenomas and MALAT1, a draggable long non-coding RNA, expressed in most adenoma cells. This work will help us define pituitary adenomas' molecular fingerprint and provide insights that could be utilized in the clinic to manage these tumors better.
t-SNE plot showing expression of MALAT1, a draggable long non-coding RNA, in the majority of pituitary adenoma cells from 12 non-functional pituitary adenomas
Defining Metabolic Alterations in Invasive Glioblastoma Cells
The poor prognosis GBM caries is largely due to its invasive capacity, 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 energy demands required to grow and invade, cancer cells undergo significant metabolic reprogramming. Funded by a diversity supplement to an Aghi Lab R01, UCSF medical student Joseph Garcia has utilized system wide high-throughput screens to identify the transsulfuration pathway, which protects cells against oxidative stress, as being up regulated in invasive GBM cells. The Aghi Lab is currently investigating how this pathway facilitates invasion using targeted assays and inhibiting transsulfuration pathway enzymes to mitigate tumor cell invasion
Shown is the metabolic profiling of samples from patient tumor core vs. edge compared to core vs. edge samples from tumor cells invading 3D devices.  Profiling revealed that samples from the edge profiled similarly and distinctly than samples from the core, and 3D devices modeled the same metabolic changes occurring with invasion in patient GBMs. 
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 immunosuppressed, with a dearth of anti-tumor processes occurring in the tumor microenvironment. MYCN-amplified Group 3 medulloblastoma carries a particularly devastating prognosis. Using a mouse model, we seek to reverse MYCN-induced chemokine abnormalities in medulloblastoma to drive immune cells towards the tumor. Of particular interest is restoring tumor expression of CXCL9 & CXCL10 to recruit CD4+ and CD8+ T-cells. We have also upregulated MHC-I expression on tumor cells to 'uncloak' the tumor and enhance T-cell mediated tumor-killing through simultaneous MYCN & EZH2 inhibition.  This project was initiated by Aghi lab postdoc and UCSF neurosurgery resident Taemin Oh and is now being concluded by UCSF medical student and yearlong research fellow Ryan Phelps.
Inhibition of MCYN and EZH2 promotes T-cell killing of medulloblastoma by up regulating MHC-I expression on the surface of tumor cells
Defining the roles of CD97 in glioblastoma biology

Research by Aghi lab postdoc and  UCSF neurosurgery resident Michael Safaee with assistance from medical student Elaina Wang, Aghi Lab Junior Specialist Sabraj Gill, and medical student Allison Zheng revealed that CD97 is highly expressed in glioblastoma and may have roles in tumor invasion, angiogenesis, immune cell suppression, and immune evasion. In a paper accepted for publication in Scientific Reports, the team targeted CD97 with CRISPR interference to study CD97's role in tumor invasiveness, angiogenesis, tumor cell metabolism, and regulation of immune cell populations in glioblastoma. They also studied an Fc fusion protein targeting CD97 as a potential therapeutic in collaboration with an industry partner.

SIngle-cell sequencing of patient GBMs was used to compare CD97-expressing GBM cells to non-CD97-expressing GBM cells. Ontologic analysis of the top 25 genes with higher expression in CD97+ versus CD97- single cells reveal associations with second-messenger signaling and extracellular matrix organization.
Clinical Studies 2021
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. Medical student Meeki Lad published a study showing that graduating neurosurgery residents accumulate over six times as many spinal cases in their training as compared to orthopaedic residents.  This study was published in Journal of Neurosurgery: Spine.
2. Medical student Eric Chalif published a study detailing how giant pituitary adenomas greater than 4cm in diameter have better outcomes at high-volume facilities compared to low-volume facilities. This study was published in Journal of Neurosurgery.
3. Aghi Lab alumni and current neurosurgery resident Rushikesh Joshi published a study in Journal of Neurosurgery identifying risk factors for postoperative diabetes insipidus after over 1000 pituitary surgeries at UCSF.
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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