CAF-like cells remodel the Ewing sarcoma tumor microenvironment

From the Lawlor lab, Seattle Children’s Hospital and the Cancer Basic Biology Program of the Cancer Consortium

Humans don’t exist in a vacuum: our environment plays a key role in our well-being. As it turns out, it is also important to the survival and proliferation of tumor cells.

“The role of the tumor microenvironment, including adjacent non-tumor cells and the extracellular matrix, has been shown to be critical for promoting metastasis and disease progression in many cancer types,” explained Dr. Elizabeth Lawlor, professor of pediatrics and the associate director of the Ben Towne Center for Childhood Cancer Research at Seattle Children’s Research Institute. Research in the Lawlor lab focuses on Ewing sarcoma, an aggressive tumor that occurs in the bone or soft tissues around bones and typically manifests in children and young adults.

“Cancer-associated fibroblasts (CAFs), normal stromal cells that have been reprogrammed by tumors, have a particularly key role in remodeling the tumor microenvironment by depositing extracellular matrix and inhibiting immune cells. But until recently, the tumor microenvironment has not been studied in much detail in Ewing sarcoma,” said Dr. Lawlor. In a new study published in Clinical Cancer Research, researchers from the Lawlor lab and their collaborators identified “CAF-like” cells that deposit pro-tumorigenic extracellular matrix proteins to remodel the Ewing sarcoma tumor microenvironment. Drs. Emma Wrenn and April Apfelbaum, a current postdoc and a former graduate student in the Lawlor lab, respectively, co-led the study.

“Recent work in the field has found that Ewing sarcoma tumor cells exist along a spectrum of activity for the driving oncogene, EWS::FLI1. High EWS::FLI1 activity can promote oncogenesis and proliferation, but interestingly, new findings show cells with lower EWS::FLI1 activity can be more metastatic. EWS::FLI1 normally represses mesenchymal identity genes that can increase metastasis, so lower EWS::FLI1 levels can release the brake on these genes,” explained Dr. Wrenn.

The researchers used transcriptomic profiling of Ewing sarcoma cell lines and patient tumors to identify NT5E (CD73), whose transcription is inhibited by EWS::FLI1, as a cell-surface marker for EWS::FLI1-low tumor cells. NT5E encodes a poorly characterized phosphatase that plays a role in immunoregulation. Phenotypic and immunofluorescent profiling of CD73+ cells revealed that subpopulations of CD73+ cells are enriched along tumor borders and invasive fronts. To interrogate the relationship between phenotype and gene expression in individual cells, the researchers collaborated with Dr. Scott Furlan at Fred Hutch and performed single-cell proteogenomic profiling of Ewing sarcoma cells. This analysis indicated that a subpopulation of CD73+ cells, much like CAFs in carcinomas, deposit extracellular matrix proteins including tenascin-C, collagens, and proteoglycans.

Left: Fluorescent staining of nuclei and TNC (ECM) protein) on an Ewing sarcoma patient biopsy. Right: a model showing the hybrid state of Ewing sarcoma tumor cells reported in this study
Researchers identified “CAF-like” cells that deposit pro-tumorigenic extracellular matrix proteins to remodel the Ewing sarcoma tumor microenvironment. The activating and repressive properties of EWS::FLI1 can be dissociated in individual tumor cells, resulting in cells with hybrid transcriptional states. These hybrid states that activate mesenchymal, pro-metastatic genes but also keep EWS::FLI1’s pro-growth oncogenic signature may represent especially aggressive tumor cells. Image provided by Dr. Emma Wrenn

EWS::FLI1 represses the matrisomal gene program, so it was unsurprising that the EWS::FLI1-low “CAF-like” cells turned on these mesenchymal identity genes. Unexpectedly, however, these “CAF-like” CD73+ cells showed no depletion in EWS::FLI1 transcript or protein. This suggests that the activating and repressive properties of EWS::FLI1 can be dissociated in individual tumor cells with hybrid transcriptional states. “These ‘hybrid states’ may represent especially aggressive tumor cells that activate mesenchymal, pro-metastatic genes but also keep EWS::FLI1’s pro-growth oncogenic signature,” said Dr. Wrenn.

The large degree of heterogeneity among both patient samples and cell lines also came as a surprise to the researchers. “Ewing sarcomas are somewhat homogeneous genetically – they are all driven by the same type of transcription factor fusions, and rarely have additional driver mutations. But across the nine cell lines and many patient tumor samples studied here, we observed huge transcriptional differences and a spectrum of distinct cell states,” explained Dr. Lawlor. “This heterogeneity has been underappreciated, and probably contributes to difficulties treating Ewing sarcoma in the clinic. Work is underway in the lab to better understand how this heterogeneity is generated, so we can try to find treatments that target multiple transcriptional states to more effectively destroy the entire tumor.”

The results from this study have generated additional questions for the researchers to pursue. “We want to better understand the specific functions of these ‘CAF-like’ cells,” said Dr. Lawlor. The team is interested in characterizing the types of matrix the “CAF-like” cells deposit, and whether they promote drug resistance, growth, or metastasis. They also want to know if there are subpopulations within the “CAF-like” cells, consistent with those observed in bona fide CAFs, and whether “CAF-like” cells affect the behavior of local immune cells or immune-cell therapies like CAR-Ts.

“Secondly, we want to understand the upstream signals that create this heterogeneity in the first place,” said Dr. Lawlor. “Why do Ewing sarcoma cells switch into, and out of, this extracellular matrix-secreting state? Is that switch necessary for successful metastasis and colonization? If we understood that, we may be able to design treatments that trap cells in one state or the other, preventing this tumor microenvironment remodeling and making tumor cells more sensitive to therapy.”

Dr. Lawlor highlighted the key roles of Drs. Scott Furlan and Erin Rudzinski in generating the CITE-seq datasets and analyzing histologic sections from archived clinical samples from patients who were treated at Seattle Children’s Hospital, respectively. “This project was greatly accelerated by collaborations throughout the UW, Fred Hutch, and Seattle Children’s communities,” said Dr. Lawlor. “By integrating the single cell studies from our lab models with patient tumor material, we were able to validate our observations and make much stronger conclusions about the presence and role of these cell states in Ewing sarcoma.”

This work was supported by the National Institutes of Health, National Cancer Institute, AACR-QuadW Foundation Sarcoma Research Fellowship in Memory of Willie Tichenor, Sam Day Foundation, 1M4Anna Foundation, Lafontaine-U-Can-Cer-Vive Foundation, and the Flow Cytometry Shared Resource of the Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium.

The Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium members Drs. Elizabeth Lawlor and Scott Furlan contributed to this work.

Wrenn ED, Apfelbaum AA, Rudzinski ER, Deng X, Jiang W, Sud S, Van Noord RA, Newman EA, Garcia NM, Miyaki A, Hoglund VJ, Bhise SS, Kanaan SB, Waltner OG, Furlan SN, Lawlor ER. 2023. Cancer-associated fibroblast-like tumor cells remodel the Ewing sarcoma tumor microenvironment. Clin Cancer Res. CCR-23-1111. Epub ahead of print.