<p>Pancreatic cancer has a tough early diagnosis and awful prognosis among all human cancers. Tumor heterogeneity can hinder the exploration of tumor progression because it can induce various discrepancies in tumor growth rate, invasion and metastasis ability, and drug susceptibility. Here, we proposed 3D-hetero-type multicellular spheroids (3D-HMS) and investigated the spatial distribution of lipids during the spatiotemporal progression of pancreatic cancer. The results showed that the malignant degree of 3D-HMS increased during the tumor progression, including an increase in the positively expressed micro-region of CK, the absence of FN positive expression, and significant spatial heterogeneity of cell states, which were consistent with the growth state and internal structure of solid tumor tissues in pancreatic cancer. Additionally, spatial distributions of lipids in 3D-HMS exhibited spatiotemporal heterogeneity. MSI-based 3D-HMS construction and lipid analysis will provide a new insight into the occurrence of metastasis, spatiotemporal progression, as well as early cancer detection.</p>