RESUMO
In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin ß1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.
Assuntos
Ácidos Graxos , Neoplasias de Mama Triplo Negativas , Microambiente Tumoral , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Humanos , Feminino , Ácidos Graxos/metabolismo , Camundongos , Linhagem Celular Tumoral , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Metástase NeoplásicaRESUMO
Recent studies have found discordant mechanosensitive outcomes when comparing 2D and 3D, highlighting the need for tools to study mechanotransduction in 3D across a wide spectrum of stiffness. A gelatin methacryloyl (GelMA) hydrogel with a continuous stiffness gradient ranging from 5 to 38 kPa was developed to recapitulate physiological stiffness conditions. Adipose-derived stem cells (ASCs) were encapsulated in this hydrogel, and their morphological characteristics and expression of both mechanosensitive proteins (Lamin A, YAP, and MRTFa) and differentiation markers (PPARγ and RUNX2) were analyzed. Low-stiffness regions (â¼8 kPa) permitted increased cellular and nuclear volume and enhanced mechanosensitive protein localization in the nucleus. This trend was reversed in high stiffness regions (â¼30 kPa), where decreased cellular and nuclear volumes and reduced mechanosensitive protein nuclear localization were observed. Interestingly, cells in soft regions exhibited enhanced osteogenic RUNX2 expression, while those in stiff regions upregulated the adipogenic regulator PPARγ, suggesting that volume, not substrate stiffness, is sufficient to drive 3D stem cell differentiation. Inhibition of myosin II (Blebbistatin) and ROCK (Y-27632), both key drivers of actomyosin contractility, resulted in reduced cell volume, especially in low-stiffness regions, causing a decorrelation between volume expansion and mechanosensitive protein localization. Constitutively active and inactive forms of the canonical downstream mechanotransduction effector TAZ were stably transfected into ASCs. Activated TAZ resulted in higher cellular volume despite increasing stiffness and a consistent, stiffness-independent translocation of YAP and MRTFa into the nucleus. Thus, volume adaptation as a function of 3D matrix stiffness can control stem cell mechanotransduction and differentiation.
Assuntos
Adipogenia/genética , Diferenciação Celular/efeitos dos fármacos , Mecanotransdução Celular/genética , Osteogênese/genética , Citoesqueleto de Actina/genética , Actomiosina/genética , Aciltransferases , Adipogenia/efeitos dos fármacos , Amidas/farmacologia , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Encapsulamento de Células/métodos , Núcleo Celular/química , Tamanho Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Gelatina/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Lamina Tipo A/genética , Células-Tronco Mesenquimais/citologia , Miosina Tipo II/genética , PPAR gama/genética , Piridinas/farmacologia , Transativadores/genética , Fatores de Transcrição/genética , Quinases Associadas a rho/genéticaRESUMO
3D culture of stem cells can improve therapeutic effects. However, there is limited research on how to deliver cultured stem cell spheroids to the desired target. Here, we developed lotus seedpod-inspired hydrogel (LoSH) containing microwells for culture and delivery of stem cell spheroids. Human adipose-derived stem cells (hADSCs) inside the square microwells (200 or 400⯵m in width with various depths) spontaneously formed spheroids with high viability (94.08⯱â¯1.56%), and fibronectins conjugated to the hydrogel successfully gripped the spheroids, similar to the funiculus gripping seeds in the lotus seedpod. The spheroids slightly bound to the LoSH surface at 37⯰C were detached by the expansion of LoSH at lower temperature of 4⯰C. After spheroid formation, LoSH was placed on the target substrate upside-down, expanded at 4⯰C for 10â¯min, and removed from the target. As a result, the spheroids within the microwell were successfully transferred to the target substrate with high transfer efficiency (93.78⯱â¯2.30%). A delivery of spheroids from LoSH to full-thickness murine skin wound with chimney model showed significant enhancement of the number of SMA-positive vessels at day 21 compared to the group received the same number of spheroids by injection. Together, our findings demonstrate LoSH as a one-step platform that can culture and deliver spheroids to a large target area, which will be useful for various biomedical applications.