Genomic dissection of tumor heterogeneity and progression

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The first aim of this project is to use single cell sequencing to understand the complexities of cell types and cell to cell heterogeneity that is present within pediatric solid tumors. In this work, we are focusing on generating comprehensive gene expression profiling of the cells present within tumors that occur in patients with NF1. To date we have collected and analyzed surgical specimens from Plexiform Neurofibromas (PN), Atypical Neurofibromas (AN) and Malignant Peripheral Nerve Sheath Tumors (MPNST). Over the past year, we have generated high gene coverage sequencing on 350,000 single cells from 12 patients with NF1 nerve tumors including histologically validated PN, ANF and MPNST. Our largest dataset generated to date includes single-cell sequencing of PN and ANF. From these experiments, we capture the landscape of cellular heterogeneity within these tumors. Within PNs we have identified at least 21 unique cell populations including a variety of stromal and immune cell types. This rich dataset details the transcriptional profile of each of these populations and highlights both known and novel cell types. Following the same procedure, we have generated scRNAseq data from 300,000 cells from three MPNST tumors and 10 PDX MPNST models. Important findings from this project are multiple clusters of transcriptionally distinct cells that compose the tumor cluster as well as a large population of immune suppressive macrophages. Currently, we are using this dataset to describe the multitude of cell to cell interactions within these tumors to dissect potential tumor specific vulnerabilities. Given the difficulty in obtaining multiple sequential biopsy specimens from patients with solid tumors, in the past year we have undertaken a project to assay cell free DNA to assess disease status in NF1 patients. In this work we have developed an assay that marries low pass whole genome sequencing with NF1 specific targeted capture deep sequencing of selected genes. Currently we are evaluating our assays value using samples collected on the actively enrolling clinical trial SARC031 (NCT03433183) "MEK Inhibitor Selumetinib (AZD6244) in Combination with the mTOR Inhibitor Sirolimus for Patients With MPNST". Initial studies correlating the changes observed in the circulating tumor DNA with the changes observed on re-staging imaging are underway. Secondary efforts will pair the circulating tumor DNA with an on-treatment tumor biopsy to observe correlation and description of the global genomic changes in the circulating tumor DNA to discover mechanisms of tumor evolution. The second aim of this work is to develop novel barcoding strategies married with single cell sequencing that can be used in preclinical model systems to model tumor cell resistance and survival. Single cell sequencing has the ability to dissect the gene expression profile of thousands of cells in parallel but is limited in its ability to track populations of cells under a selective pressure. Within the current year we completed pilot experiments that highlighted the need to add a feature to our experimental system which enabled tracking a particular cell over time and as it replicated. To accomplish this, we have incorporated a unique sequence "DNA barcode" into each cell within a pool of cells (greater than 10 million unique barcodes in 10 separate pools). Importantly, our vector generates a polyadenylated expressed gene that is compatible with capture using single cell RNA sequencing methods. Experiments have been completed which demonstrated that we are able to detect the expressed DNA barcodes within the scRNAseq data over time and with cell population expansion. Current experiments are employing the single cell barcoding strategy to understand the heterogenous tumor cell responses to topoisomerase inhibitors and small molecules targeting epigenetic modifiers. We anticipate that this will enable discernment of the expression profiles of individual surviving cells and definition of new therapeutic targets.