Poster BB6 - Three-dimensional matrix stiffness drives Piezo1 activity in oral squamous cell carcinoma spheroids and derived small extracellular vesicles

Introduction: Biophysical cues such as extracellular matrix (ECM) stiffness are key stimuli affecting tumor progression in vivo. While the influence of ECM stiffness on regulating cell behavior has been extensively studied, it remains unclear how cells in a 3D tumor microenvironment (TME) perceive matrix mechanical stiffness stimuli and translate them into intracellular signals driving cancer. Additionally, how stiffness influences intercellular communications in the microenvironment for tumor progression also remains unknown. Mechanosensitive Piezo1 ion channels, upregulated in many malignancies, mostly of epithelial origin, are major transducers of physical stimuli into biochemical responses. Small extracellular vesicles (sEVs) secreted by cells, mediate intercellular communication facilitating interplay and rewiring between different components of the TME, and play a role in priming the premetastatic niche in cancer. Here, we engineered a 3D spheroid culture environment with varying biomechanical properties to study the effect of static matrix stiffness stimuli on Piezo1 activation in oral squamous cell carcinoma (OSCC) tumors. Additionally, the influence of matrix stiffness on the secretion and biomolecular cargo of sEVs including the employment of Piezo1 and CD44 signatures from parental spheroids was also studied.

Materials and

Methods: Matrix stiffness was defined as soft and stiff for 3 and 12 mg/mL Matrigel, respectively, and characterized by atomic force microscopy (AFM) and shear rheology. Cal27 spheroids were generated on soft and stiff matrices using the liquid overlay technique in sEV-depleted media. Day 14 spheroids were fixed and stained with DAPI, Piezo1-FITC, and CD44-APC and imaged using a Nikon A1R confocal microscope with multichannel z-stacks performed across the height of the spheroid. For flow cytometric analysis, day 14 spheroids were dissociated into single cells and stained with Piezo1 and AF594-secondary or CD44-APC. Flow data were acquired using Cytek Aurora and analyzed using FlowJo. Total RNA was extracted using mirVana miRNA Isolation kit from spheroid samples for next-generation RNA sequencing and analyzed using bioinformatics. sEVs were extracted from day 14 spheroid culture media using a label-free lab on a chip dielectrophoretic device. Nanoparticle tracking analysis (NTA) was performed using Nanosight NS300. Extracted sEVs samples were lysed and analyzed for protein markers of interest using Western blot. Single sEVs immobilized on EV profiler chips using protein and non-protein-dependent capture methods were stained for CD63-Cy38, Piezo1-FITC, and CD44-APC. Direct stochastic optical reconstruction microscopy (dSTORM) was performed with a Nanoimager S Mark II and analyzed using CODI. Imaging flow cytometry (iFCM) was performed for sEVs stained with Piezo1-FITC, CD44-APC, and CD63-FITC using ImageStreamX Mark II and analyzed using IDEAS.

Results, Conclusions, and Discussions: Mechanical characterization of hydrogels indicated a 3-fold increase in stiffness at the intermolecular level and 8-fold increase at the bulk level using AFM and rheology, respectively. Morphological analysis using brightfield and confocal microscopy indicated a significant increase in spheroid size with stiffness. Cellular flow cytometry demonstrated an increase in Piezo1 and CD44 expressions with increasing stiffness. RNA-sequencing indicated upregulation of a selection of genes involved in mechanoreception, ECM organization, ion channel transport, and tumorigenesis in stiff ECM spheroids. NTA indicated a 2-fold increase in particle concentration between soft (EVsoft) and stiff (EVstiff) cultures. Western blot indicated the presence of parental spheroidal markers Piezo1 and CD44 along with EV-specific markers CD63, TSG101, and HSP70 in both EVsoft and EVstiff groups. dSTORM and iFCM were used for further validation of Piezo1 and CD44 marker presence in spheroid-derived sEVs.
Overall, our results demonstrate that varying ECM stiffness in a spheroid culture platform plays a role in enhancing spheroid growth and malignant phenotypes by employing Piezo1 and TRPV4 mechanoreceptors. This suggests that physical and biochemical cues exhibit dynamic interplay in OSCC. The upregulation of multiple genes involved with ion channel transport also provides preliminary evidence of the presence of mechano-electric crosstalk wherein biomechanical cues from the ECM influence the bioelectric state of cancer cells.
The role and utility of mechanical cues including ECM stiffness in cancer diagnosis, prognosis, and therapy has led to the relatively recent coining of terms such as “mechanopathology” and “mechanomedicine”. The utility of a 3D culture system over traditional 2D systems in the study furthers our understanding of how OSCC progression is influenced by the stiffening of tumor tissues. Our results also provide promise for future model studies to investigate the influence of the knockdown of specific mechanoreception genes on tumor growth in vivo. Furthermore, the effect of 3D ECM stiffness on the production and composition of sEVs and the employment of biomechanical Piezo1 and CD44 signatures from parental spheroids under distinct stiffness stimuli provides the premise for exploring sEVs-Piezo1 as a potential therapeutic target or prognostic marker against OSCC in liquid biopsy.

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