3D Bioprinted In Vitro Model: A New Research Tool for Investigating Bidirectional Communication in Chondrosarcoma - Chondrosarcoma (CHS), the second most common bone sarcoma, resists chemotherapy, immunotherapy, and radiotherapy and tends to metastasize, posing clinical challenges. In the CHS tumor microenvironment, mesenchymal stem cells (MSCs) and tumor cells act as localized signaling hubs, engaging in continuous bidirectional communication through spatially organized gradients of cytokines and chemokines. These interactions likely induce MSC phenotypic changes that promote tumor growth and metastasis, though the exact mechanisms remain unclear. Current in vitro models fail to replicate these complex interactions, as conditioned medium transfer disrupts continuous signaling, and conventional 3D hydrogel co-culture systems allow paracrine factors to homogenize, erasing essential spatial gradients. To address this gap, we developed EXPECT (EXtrusion Patterned Embedded ConstruCTs), a temperature-sensitive hydrogel system that enables long-term, spatially organized cell-cell communication in 3D. EXPECT uses extrusion bioprinting to embed cell-laden channels and leverages mild temperature actuation to control cell migration. Preliminary studies show EXPECT supports migration along defined axes for up to 36 days, preserving paracrine gradients and enabling the study of sustained MSC-CHS interactions. We hypothesize that temperature actuation in EXPECT preserves spatial gradients, enhancing bidirectional communication between MSCs and CHS cells. This will promote CHS growth, MSC chemotaxis, and phenotypic shifts in MSCs toward a CHS-like profile. Aim 1 investigates cellular level bidirectional crosstalk between MSCs and CHS spheroids within EXPECT. We will co-culture spheroids under temperature actuation for 30 days, hypothesizing that sustained crosstalk drives MSC migration, CHS metabolism, matrix remodeling, and gene expression changes. Aim 2 explores molecular pathways affected by MSC-CHS communication using single-cell RNA sequencing. We expect temperature actuation to activate migratory pathways like PI3K/PIP3 in MSCs, leading to gene expression changes tied to cytoskeletal remodeling, paracrine signaling, and ECM modification, promoting a CHS-like MSC phenotype. EXPECT fills a critical gap in CHS research by enabling studies of sustained MSC-CHS interactions that mimic in vivo conditions. This platform may reveal previously unrecognized crosstalk and MSC phenotypic changes that promote tumor progression, providing new therapeutic targets and improving drug testing models.
