Spatial Transcriptomic and Metabolomic Landscapes of Oral Submucous Fibrosis‐Derived Oral Squamous Cell Carcinoma and its Tumor Microenvironment

Abstract In South and Southeast Asia, the habit of chewing betel nuts is prevalent, which leads to oral submucous fibrosis (OSF). OSF is a well‐established precancerous lesion, and a portion of OSF cases eventually progress to oral squamous cell carcinoma (OSCC). However, the specific molecular mechanisms underlying the malignant transformation of OSCC from OSF are poorly understood. In this study, the leading‐edge techniques of Spatial Transcriptomics (ST) and Spatial Metabolomics (SM) are integrated to obtain spatial location information of cancer cells, fibroblasts, and immune cells, as well as the transcriptomic and metabolomic landscapes in OSF‐derived OSCC tissues. This work reveals for the first time that some OSF‐derived OSCC cells undergo partial epithelial–mesenchymal transition (pEMT) within the in situ carcinoma (ISC) region, eventually acquiring fibroblast‐like phenotypes and participating in collagen deposition. Complex interactions among epithelial cells, fibroblasts, and immune cells in the tumor microenvironment are demonstrated. Most importantly, significant metabolic reprogramming in OSF‐derived OSCC, including abnormal polyamine metabolism, potentially playing a pivotal role in promoting tumorigenesis and immune evasion is discovered. The ST and SM data in this study shed new light on deciphering the mechanisms of OSF‐derived OSCC. The work also offers invaluable clues for the prevention and treatment of OSCC.


Figure S1 | Histological Examination of OSF-derived OSCC Samples
(A) After H&E staining, representative fields (6.5 mm × 6.5 mm) of 4 OSF-derived OSCC were selected for ST and SM analysis in accordance with the guidance of senior pathologists.There are Tumor (red line surrounded), ISC (yellow line, only in P1 and P3), OSF (blue line), Stroma (brown line), Muscle (green line, P2-P4) and Vessel/Gland (green line, only in P1), Adjacent Epithelium (orange line, only in P2 and P4) regions in the 4 samples.Representative panoramic images (6.5 mm × 6.5 mm) and enlarged views of certain histopathological regions (from left to right: 40×, 100×, 200×, 400×) were presented (from left to right) with scale bars of 200 μm, 100 μm, 50 μm, and 20 μm, respectively.(B) Masson's Staining revealed the deposition of collagen fibers in OSF-derived OSCC. 4 cases of OSFderived OSCC showed significant blue staining of collagen fibers in the OSF region (blue line).However, no collagen fiber deposition was observed in the adjacent epithelial tissue of OSCC without OSF, which was used as a negative control.From left to right, representative panoramic images of the field of view (6.5 mm × 6.5 mm), and 40×, 100×, 200×, and 400× magnification images of the corresponding histopathological areas with scales of 200 μm, 100 μm, 50 μm, and 20 μm, respectively.

Figure S2 | ST Characteristics of OSF-derived OSCC Samples
(A) Schematic diagram showing the strategy of ST.In P1, frozen tissue section (6.5 mm × 6.5 mm) was divided into 4,316 spots (about 55 μm in diameter, 100 μm between 2 neighboring spots) for ST sequencing.After permeabilization, total RNA in each spot was captured by primers with specific spatial barcode attached on the 10x Visium Spatial Gene Expression Slide for reverse transcription.Eventually, cDNA was eluted from the slides for further sequencing.
(B) Violin plots displaying the average numbers of unique molecular identifiers (nUMI, top) and gene expression (nGene, bottom) in each spot of 4 samples.(C) 17,319 spots obtained from ST were performed unsupervised clustering analysis UMAP and the distribution of each spot in the UMAP plot was colored by patients.There was a balanced distribution among spots from 4 samples, indicating the absence of batch effect among 4 OSFderived OSCC patients.(D) Bar plots showing the proportion of 11 clusters (obtained from the UMAP analysis) in each patient.(E) 10 representative marker genes of each cluster were obtained through differential expressed gene (DEG) analysis.Heatmap showed the expression patterns of the top 10 marker genes in each cluster.

Figure S3 |Figure S4 |
Figure S3 | Cell Constitution Analysis of Each Spot in OSF-derived OSCC

Figure S5 |
Figure S5 | Spatial Landscape and Interaction of Infiltrated Immune Cells in OSF-derived OSCC

Figure S7 |
Figure S7 | Distribution of Differential Metabolites in OSF-derived OSCC in Global MetabolicFlux

Table S2 | Statistics of 10x Genomics Visium Spatial Transcriptomics
* Mean mito percent: Mean percentage of mitochondrial genes expressed in each spot.