# 3D bioengineered microfluidic platform for research on Ewing sarcoma bone metastasis

> **NIH NIH R21** · GEORGETOWN UNIVERSITY · 2024 · $401,115

## Abstract

Ewing sarcoma (ES) is a pediatric malignancy, which carries adverse prognosis in its metastatic form. Bone
metastases, although less common, associate with the worst clinical outcome. Yet, this form of the disease
remains understudied due to challenges in its modeling. Recently, we have shown that ES bone metastases are
triggered by severe hypoxia in primary tumors, which leads to the formation of polyploid cells, the progeny of
which, hereafter named HYP-4n cells, have a high propensity for bone metastasis. However, the mechanisms
underlying the ability of HYP-4n cells to colonize the bone remain unknown. This type of scientific question can
be addressed by functional genomics approaches, such as CRISPR screens. Yet again, such assays are challenging
in the setting of bone metastasis in vivo, due to the low number of osseous lesions developing in animal models
and their clonal nature. On the other hand, most of the existing in vitro models fail to faithfully recapitulate
heterocellular interactions occurring in the bone environment. Thus, we propose the 3D bicellular microfluidic
platform (3D-BMP), which mimics the conditions ES cell encounter during osseous dissemination, as a
research tool that will enable high-throughput genomic screens on bone metastasis. 3D-BMP consists of a
bioengineered microvessel covered with endothelial cells and embedded in the bone-mimicking matrix with
osteoblasts and osteoclasts. It allows for control of important parameters of the bone environment, i.e. matrix
stiffness, hydrostatic pressure and oxygen tension. As such, it enables testing for rapid structural and functional
changes occurring in the bone during the metastatic process and simultaneous analysis of multiple invading
colonies, both of which are not achievable in animal models. The goal of our project is to test the utility of 3D-
BMP in studies on the biology of ES bone metastasis, including large scale genomic assays. To this end, we will
use HYP-4n cells, with high propensity for bone metastasis, and their diploid counterparts derived from the same
ES cell lines cultured in normoxia (NOR-2n), which do not metastasize to bone, to optimize the experimental
conditions in the 3D-BMP to best reflect their differences in metastatic properties and test their interactions with
the bone environment (Aim 1). This model will be used to perform a focused CRISPRi drop-out assay targeting
genes the most highly upregulated in HYP-4n cells to select candidate molecules crucial for dissemination to the
bone tissues and survival in this environment (Aim 2). The selected targets will be first validated via 3D-BMP
and then tested in vivo, using an orthotopic xenograft model, to provide evidence for the biological relevance of
our platform. The proposed study will: 1) validate 3D-BMP as a model recapitulating key steps of osseous spread;
2) test the experimental pipeline involving CRISPR screen in 3D-BMP followed by in vivo validation as a research
tool for the identific...

## Key facts

- **NIH application ID:** 10976090
- **Project number:** 1R21CA294025-01
- **Recipient organization:** GEORGETOWN UNIVERSITY
- **Principal Investigator:** STELLA ALIMPERTI
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $401,115
- **Award type:** 1
- **Project period:** 2024-07-02 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10976090

## Citation

> US National Institutes of Health, RePORTER application 10976090, 3D bioengineered microfluidic platform for research on Ewing sarcoma bone metastasis (1R21CA294025-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10976090. Licensed CC0.

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