Pediatric Sarcomas: The Next Generation of Molecular Studies

Abstract

Pediatric sarcomas constitute one of the largest groups of childhood cancers, following hematopoietic, neural, and renal lesions. Partly because of their diversity, they continue to offer challenges in diagnosis and treatment. In spite of the diagnostic, nosologic, and therapeutic gains made with genetic technology, newer means for investigation are needed. This article reviews emerging technology being used to study human neoplasia and how these methods might be applicable to pediatric sarcomas. Methods reviewed include single cell RNA sequencing (scRNAseq), spatial multi-omics, high-throughput functional genomics, and clustered regularly interspersed short palindromic sequence-Cas9 (CRISPR-Cas9) technology. In spite of these advances, the field continues to be challenged by a dearth of properly annotated materials, particularly from recurrences and metastases and pre- and post-treatment samples.

Keywords: CRISPR-Cas9; high-throughput functional genomics; molecular genetics; pediatric sarcoma; scRNA seq; spatial multi-omics; specimen annotation; technology.

Figure 1

Overview of single-cell RNA sequencing. Tumor tissue is disaggregated into constituent single cells which can be isolated through various methods. Isolated single cells are lysed, and cellular RNA is converted into cDNA. cDNA from individual cells is ligated to cell-specific oligonucleotide barcodes after which all cDNA is pooled and sequenced collectively via next-generation sequencing. The transcriptomes of each cell are then segregated computationally using the barcode sequences, enabling the analysis of single-cell gene expression profiles.

Figure 2

Overview of spatial genomics, transcriptomics, and proteomics methodologies. Tissue sections are typically visualized either through a “tiling”-based approach or through the visualization of “barcoded” probes.

Figure 3

Schematic of high-throughput genetic screening using clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations and single-cell RNA sequencing. Tumor cell lines that constitutively express the Cas9 protein are infected with lentiviruses that each contain (1) a single guide RNA (sgRNA) specific for a single gene, and (2) a unique oligonucleotide barcode specific for the sgRNA. In the context of a CRISPR-i or CRISPR-a screen, the Cas9 protein expressed by the cell line has been modified to inhibit or activate transcription, respectively. In this context, the sgRNAs target the regulatory regions of genes. In the context of a gene knockout screen, however, the Cas9 protein expressed by the cell line cleaves the genomic region targeted by the sgRNA, rendering the targeted gene inactive. Following the CRISPR-based perturbation, cells can be further manipulated (e.g., treated with a drug) after which they undergo single-cell RNA sequencing. Using the sgRNA-specific and cell-specific barcodes, the expression profile associated with each perturbation can then be delineated.