Mimicking angiogenic microenvironment of alveolar soft-part sarcoma in a microfluidic coculture vasculature chip

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Mimicking angiogenic microenvironment of alveolar soft-part sarcoma in a microfluidic coculture vasculature chip

Significance

The in vitro model of alveolar soft-part sarcoma (ASPS) comprehensively mimicked the complex angiogenesis ASPS environment with abundant pericytes-rich and perfusable vascular networks. Compared to the conventional avascular tumor spheroid models, our ASPS-on-a-chip, comprising a mouse ASPS spheroid and pericyte-endothelial cell coculture, replicates the in vivo tumor vasculature that is rich in pericytes, enabling the study of cellular interactions before metastasis. Besides, the ASPS-on-a-chip provided functional and morphological similarity as the in vivo mouse model, which helps to investigate the role of Rab27a and Sytl2 in intracellular signaling during tumor vessel formation. These findings are crucial for developing targeted therapies that disrupt the cellular communication within the tumor microenvironment, potentially offering broad avenues for ASPS treatment.

Abstract

Alveolar soft-part sarcoma (ASPS) is a slow-growing soft tissue sarcoma with high mortality rates that affects adolescents and young adults. ASPS resists conventional chemotherapy; thus, decades of research have elucidated pathogenic mechanisms driving the disease, particularly its angiogenic capacities. Integrated blood vessels that are rich in pericytes (PCs) and metastatic potential are distinctive of ASPS. To mimic ASPS angiogenic microenvironment, a microfluidic coculture vasculature chip has been developed as a three-dimensional (3D) spheroid composed of mouse ASPS, a layer of PCs, and endothelial cells (ECs). This ASPS-on-a-chip provided functional and morphological similarity as the in vivo mouse model to elucidate the cellular crosstalk within the tumor vasculature before metastasis. We successfully reproduce ASPS spheroid and leaky vessels representing the unique tumor vasculature to assess effective drug delivery into the core of a solid tumor. Furthermore, this ASPS angiogenesis model enabled us to investigate the role of proteins in the intracellular trafficking of bioactive signals from ASPS to PCs and ECs during angiogenesis, including Rab27a and Sytl2. The results can help to develop drugs targeting the crosstalk between ASPS and the adjacent cells in the tumoral microenvironment.

Tumor-on-a-chip (ToC) is a promising approach to develop tumor models (1, 2) that simplify the self-organization of cells in static cell cultures (3) and the pathophysiological tumor microenvironment (4). ToCs have been explored over the last decade because these chips recapitulate human physiology more accurately than animal models (5–7). The integration of cell culture and microfluidic technology enables the design of a specific tumoral microenvironment suitable for elucidating cell responses to the environment (8), the evaluation of drug efficacy in drug screening (9–11), and the test of therapeutic applications (12) prior to human clinical trials.
Furthermore, the combination of a three-dimensional (3D) tissue culture (a tumor spheroid) with a microfluidic coculture vasculature chip can recapitulate specific cellular pathways, human tissue microenvironments, and it can potentially assemble as a 3D solid tumor (3, 8, 13) and capillary blood vessels (8, 13). Even if transgenic mice are the most common models to recapitulate tumors of immunocompromised patients with specific gene mutations (14–18), animal models involve ethical problems (19–21) that do not exist with in vitro preclinical platforms. Most importantly, elucidating molecular mechanisms such as cell–cell and cell–microenvironment interactions is not possible using animal models.
Herein, we developed a microfluidic coculture vasculature chip and focused on replicating a rare solid tumor, namely alveolar soft-part sarcoma (ASPS) (22–25), and its ability to recruit blood vessels through angiogenesis. Integrated blood vessels rich in pericytes and metastatic capacity are essential characteristics of ASPS suitable to be analyzed with a ToC approach. In addition, ASPS resists conventional chemotherapy (26), and thus, decades of research have elucidated pathogenic mechanisms driving the disease, particularly its angiogenic behavior. By using the proposed ToC preclinical model, tissue-level physiological interactions and manifestations of the disease are more similar to in vivo conditions of ASPS than in animal models, improving our understanding of ASPS pathophysiology. Such results could help in ASPS drug development, and the coculture vasculature chip could be a ready-to-use platform for preclinical drug screening of ASPS treatments.
Hence, the ASPS angiogenic microenvironment was mimicked using a mouse ASPS spheroid in a microfluidic coculture vasculature chip. Pericytes (PCs) in the layer of endothelial cells (ECs) positively influenced the formation of a mouse ASPS spheroid, recruiting tumor vasculature through the platelet-derived growth factor β (Pdgfb) signaling pathway (27) since PCs contribute to angiogenic initiation and vessel stability in both tumor and non-tumor cells. In addition, the present study confirmed that Pdgfb and glycoprotein non-metastatic melanoma protein B (Gpnmb) (28) pathways increased ASPS angiogenesis with the participation of Rab27a and Sytl2 that facilitate intracellular transport of cytoplasmic vesicles (25, 29, 30). Rab27a and Sytl2 are direct transcriptional targets of ASPSCR1-TFE3, which is overexpressed in ASPS (25). The ASPS-on-a-chip provides the vascularized and perfusable ASPS spheroid within a microfluidic chip which is different from a widely-used avascular tumor models. The distinctive blood vessel characteristics that are rich in PCs are well constructed by coculturing PCs, and ECs in a microfluidic chip where the perfusable vascular networks are connected to the core of the spheroid. The microfluidic coculture vasculature chip faithfully resembles the vascularized ASPS solid tumor with abundant PCs-rich tumor networks, being a robust drug screening platform for ASPS and contributing to developing drugs that target the cellular crosstalk during the ASPS angiogenesis process.

https://www.pnas.org/doi/10.1073/pnas.2312472121

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Ie : GPNMB



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