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Uncovering novel cancer therapeutics using the Exoneural Medicines Platform: The role of innervation in cancerShan Lou, Alexandria Fink, Jay Wang, Jesse Turner, Garmen Yuen, Monica Thanawala, Alexandra Lantermann, Jenny Shu, Hongyue Dai, Pearl Huang, Jonathan [email protected] Cygnal Therapeutics 325 Vassar St, Suite 2B, Cambridge, MA 02139
AbstractPeripheral nerves were first described as a component of tumors in the late 19th century. In the mid-20th century early preclinical studies indicated that tumor cells could recruit innervation from spinal cord ganglia and that stress promotes tumorigenesis. Within the last 10 years improvements in technologies allowing neuronal tracking and regulation of neuronal function have uncovered a role for both autonomic and sensory innervation in multiple tumor types. Initial studies have implicated this biology in prostate, pancreatic, gastric and breast cancer, (among others) and point to a role for nerves in initiation, maintenance and metastasis of tumors. The stimulation of tumor cell growth by neural growth factors, the recruitment of neurites by tumor cells, the invasion and migration of tumor cells along nerves (perineural invasion), and the role of innervation in driving tumor angiogenesis are just a few examples of how peripheral nerves interact with the tumor microenvironment. Indeed, an argument could be made that innervation of tumors should be considered a hallmark of cancer.
We describe here a biological platform that can define and decode the role of neural signaling in cancer. This platform, which we call Exoneural Medicines Platform ™, has 6 technical components: 1) co-culture models of primary neurons with both tumor and immune cells, 2) advanced imaging modalities to define the neural component of tumors, 3) AAV and transgenic tools that allow regulation of neurons proximal to tumors in vivo and in vitro, 4) neural-focused functional genomics, 5) neural-focused bioinformatics, and 6) a neuropharmacopeia compound library for probing relevant biological pathways and identification of advanced starting points for drug discovery. We show here how the platform is being employed to characterize this exciting new space in cancer biology and identify new drivers of disease.
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In house Tissue clearing and imaging pipeline
Before During Final
Neurites Blood Vessels
NeuritesBlood Vessels
Pancreatic PDX model Human patient biopsy Melanoma cell line xenograft
Neurites Blood Vessels
Tumor samples were sectioned into 2 mm slices, followed by fixation, polymerization, lipid removal and staining to show structures of blood vessels and nerve fibers
Blood vessels Neurites Blood vessels Neurites
SCLC SCLC + neurons
Flow chart of neuron/cancer cell coculture development
Real-Time Cancer cell growth detection
Lenti-CMV-nGFP
Neuron
nGFP Tuj1 NeuN
Figure showing faster growth of SCLC in coculture of neurons compared with monoculture in otherwise the same growth condition
Heat map showing effects of neurons on cancer cell growth
nGFP Tuj1 NeuN
Pancreatic PDX cell line
DRG neuron
Coculture of rat cortical neurons with SCLC showed direct contact of neurites with cancer cells
Transmission EM showing direct contact between cancer cells and neurons (arrow: protein density at intersection of the 2 cell types)
Coculture assay
Neurons Cancer cells
DAPI GFP Tuj1 NeuN
+ SCLC + PCaNeurons Alone
Compartmentalized chamber
Compartmentalized chambers separate neuron soma and cancer cells while allowing neurite growth cone exposure to cancer cell media as well as cancer cell perineural invasion after contact with neurites.
Neurite growth into the cancer cell compartment can be quantified. Comparison across several different cancer cell lines indicate different levels of neurite growth effects
Nerve labelingTumor implantationTissue analysis
10-14 daysNon-tumor
targeting ganglia
Non-tumor targeting ganglia
Tumor targeting ganglia
AAV intratumoral injection
AAV virusParticles
Spinal cord
DRG/SCG
Neuromanipulation
We can retrogradely label the tumor-innervating neurons by injecting AAV virus into the tumor. Only the ganglia which extend axons into the tumor are labeled with reporter fluorescence.
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DAPIGFPNeuNTuj1h
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-DTR
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Vehicle DTA
Multiple AAV tools can be used to manipulate the tumor-innervating neurons by either ablating the neurons or activating them. Graphs showing proof of concept experiments in vitro.
Explant culture
Dorsal root ganglia (DRG) and Superior cervical ganglia (SCG) are dissected and cultured ex vivo, extending long neurites radially.
DAPI GFP Tuj1 NeuN
When cocultured the ganglia explants, cancer cells can migrate into the center of the ganglia directionally. Cancer cell motility and frequency inside the ganglia can be used to quantitate migration
→ Focused CRISPR library with sgRNAs targeting neuronal genes, transporters and ion channels: ~11,000 sgRNAs targeting ~1800 genes, 6 sgRNAs per gene
Day 1Plate cells
Day 2Transduce cells with CRISPR library
Day 3Startselection
Day 7Collect transduced cells
Baseline
In vitro screen
In vivo screen
Next Generation Sequencing
Data analysis
Extract genomic DNA
B. CRISPR screen identified the autism and schizophrenia-associated CYFIP protein for pancreatic tumor growth
A. Schematic of in vitro and in vivo CRISPR screening
CRISPR screening using highly innervated models
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NGS identifies lower read counts for individual sgRNAs targeting CYFIP1 in in vitro and in vivo replicates than in baseline replicates.
The autism and schizophrenia-associated CYFIP1 protein is required for pancreatic tumor growth and presents a potential therapeutic target. Poster LB-C05
MAGECK-VISPR algorithm was used to analyze the performance of the sgRNAs targeting each gene in the library. In vitro and in vivo performance plots for each sgRNA targeting CYFIP1 is show for BxPC3, PAXF1997 and PAXF1657 cell line models. Disease-specific survival analysis of TCGA Pancreatic adenocarcinoma (PAAD) patient data with low (red), moderate (green), or high (blue) expression of CYFIP1
Growth kinetics of subcutaneously implanted PAXF1657 cells expressing doxycycline inducible non-targeting control (NTC) or CYFIP1 sgRNAs.
Tumor innervation revealed by CLARITY
