Bulk RNA Sequencing for Aggregated and Staggered Pattern Pancreatic Cancer Microenvironment Assembloid Model Constructed by SIAT (Spatial Imaging with Acoustic Tweezers) Platform

In this study, we designed SIAT (Spatial Imaging with Acoustic Tweezers) platform, which integrates dynamic holographic acoustic field manipulation with mass spectrometry imaging-based spatial metabolomics. By applying SIAT platform, we constructed pancreatic cancer microenvironment assembloid model which consisted with four cell types and eight organoids/spheroids in a contact-free construction manner. We used SIAT to assemble multicellular spheroids composed of pancreatic cancer cells, fibroblasts, and endothelial cells into two defined configurations: “Staggered Pattern” in which spheroids of the three cell types were interwoven in close proximity to mimic interdigitated sub-tumor microareas, and “Aggregated Pattern” in which spheroids of each cell type were clustered on one side, resembling segregated tumor–stroma compartments. Bulk RNA sequencing was performed to study the transcriptional difference between “Staggered Pattern” and “Aggregated Pattern”. RNA was isolated using MiniBEST Universal RNA Extraction Kit (Takara, Japan). RNA concentration was measured by NanoDrop OneC Spectrophotometers (ThermoFisher, USA). Libraries were prepared with the Fast RNA-seq Lib Prep Kit V2 (Abclonal, China) and Illumina NovaSeq X Plus. Sequencing data were processed and aligned to the human reference genome GRCh38 (hg38). Feature counts were generated and applied to further differential expressed gene analysis by edgeR package (4.0.16) in R (4.3.2).