Tumor Micro-environment

The PLGA Program is investigating how the tumor micro-environment affects the growth and development of pediatric low-grade gliomas by asking questions such as why do tumors that express the KIAA1549-BRAF fusion genes only occur in the cerebellum?  Tumors are mixtures of different cell types, including diverse types  of cancer and non-cancer cells. This has implications for cancer treatment as distinct cancer cell types may respond differently to individual therapies. Moreover, immune cells present within a tumor can be harnessed to attack the cancer cells, an approach used in many other cancers such as melanoma.  The Program’s researchers are taking two approaches to exploring the cellular diversity of tumors: developing culture systems for growing primary tumors in the laboratory and performing single cell RNA sequencing on individual tumor cells.

Cultures that mimic the 3D environment of tumor cells are often superior to 2D monolayer cultures in propagating tumors and in maintaining the phenotype observed in vivo.  The laboratory of Rosalind Segal, MD, PhD has developed a 3D in vitro culture method for successfully growing PLGA patient tumor cells on mouse brain slices for up to two weeks.  In collaboration with Nathanael Gray, PhD, and Michael Eck, MD, PhD, they are using the slice culture assay to test preclinical compounds on their ability to impede tumor growth.  While advancing the ability to study PLGA cells, the system is limited by the slice lifespan and the inability to extract the tumor cells after the culture period. Therefore Dr. Segal’s lab is developing a 3D hydrogel matrix culture system to facilitate the long term propagation and study of these cells. Working with the Bio-Engineering Program at MIT, they have developed a hydrogel matrix that is tuned for supporting the propagation of pediatric brain tumor cells.  Primary tumor cells are supported in the hydrogel, and then cultured together with murine astrocytes prepared from infratentorial brain regions.  This will allow them to grow the cells in the laboratory for longer times, enabling more effective testing of candidate therapies directly on authentic tumor cells.

Defining cell types in pediatric low-grade astrocytomas is important for the development of effective treatments of both primary and recurrent tumors.   Single cell RNA sequencing (scRNAseq) is a powerful approach to characterizing individual cells in tumors. Pratiti Bandopadhayay, MD, MBBS, Rameen Beroukhim, MD, PhD, and Keith Ligon, MD, PhD developed technologies to 1) disaggregate PLGAs and sort cells according to expression of a glial progenitor marker, and 2) determine BRAF fusion status at the single cell level, among cells subject to single cell RNA sequencing. These technologies have allowed them to robustly distinguish between cancer cells and non-cancer cells, and to determine transcriptional profiles of each cell type at the single-cell level.

They found that the cancer cells formed two clusters, one containing radial glia/oligodendroglial precursor-like cells, and the other resembling more mature astrocytes.  These clusters contrast with higher-grade tumors whose constituent cells are more similar immature neural progenitors, and include both astrocytic and oligodendroglial components. Consistent with the understanding of low-grade astrocytomas, the majority of the cancer cells were not in the cell cycle.

In addition to the BRAF-mutant cells, they identified multiple, non-cancer cells with the tumors. They found that non-cancer cells formed three major clusters of immune cells: microglia, T-cells and macrophages.   Intriguingly, they found that the expression of immune checkpoint receptors and ligands varied among the tumor samples, a result that may be useful for developing immune therapies for pediatric tumors.

Selected Publications

Plant AS, Koyama S, Sinai C, Solomon IH, Griffin GK, Ligon KL, Bandopadhayay P, Betensky R, Emerson R, Dranoff G, Kieran MW, Ritz J
Immunophenotyping of pediatric brain tumors: correlating immune infiltrate with histology, mutational load, and survival and assessing clonal T cell response.
J Neurooncology. April 2018/137(2):269-278..