Polyclonality and Metabolic Heterogeneity in a Colorectal Tumor Model

The monoclonal origin of cancer is widely accepted, although numerous studies suggest that some are of polyclonal origin. Loss-of checkpoints in transformed cells gives rise to carcinomas comprising a wide diversity of cell types that fulfill distinct, even complementary, metabolic functions, contrasting with a hypothetical monoclonal origin. Here, using a Drosophila intestinal tumor model, we show that, despite an identical genetic background, these tumors 1), comprise a conserved set of different metabolic-specialized clusters; 2), are always polyclonal and derive from several clones characterized by distinct metabolic specificity; 3) depend on motility of the founder clones for their growth; 4) share metabolic needs similar to those of human cancers. In summary, our study indicates that, in this model, tumor formation always requires assembly between founder clones potentially providing distinct cellular functions, as visualized by their metabolic heterogeneity. Thus, this polyclonal assembly would constitute a critical step of tumor progression. To investigate metabolism in tumors, single and group of 6 tumors were dissected and sequenced. Tumorous flies were heat-shocked at 37°C for 2-hrs 2-3 days after adult eclosion. At least 21-dpi flies were selected based on GFP fluorescence through the abdomens. Tumors were dissected on ice in Ringer buffer, rinsed twice ice-cold Ringer baths and the extracellular matrix was digested by gentle pipetting for 3-5 minutes with 20µL 5X Trypsin (PAN BIOTECH P10-022100) twice diluted. Efficient digestion was promptly checked under fluorescent dissecting microscope. Consequently, for each sample, 1X Ringer buffer was added up to 1.5mL and each tube was filtered through 30µm filters (Sysmex CellTrics). Tubes were centrifugated for 4 minutes at 1000G, at 4°C; 1mL of supernatant was discarded and the same volume of fresh 1X Ringer buffer was added. A second centrifugation (4 minutes, 1000G, 4°C) was done and after carefully removing all supernatant, pellets were gently resuspended in 30µL 1X Ringer buffer for sequencing. The whole sample preparation procedure did not exceed 20 minutes. Cell suspension were loaded into the Chromium Controller using a Chromium Next GEM Single Cell 3’ v3.1 kit (10x Genomics) to generate a droplet emulsion. cDNA were purified and libraries were prepared, according to the manufacturer recommendations. Single cell libraries were sequenced on a NextSeq 2000 Intrument (Illumina) using a P2-100 cycles kit. FastQ files were analyzed using the Cell Ranger software (10X Genomics, version 6.0.1), including alignment, filtering and quantitation of reads on the Drosophila genome r6.42 and generation of feature-barcode matrices. We used the Seurat R pipeline 10 for sample quality control and analysis. The R script used to evaluate sample quality, generate clusters, umaps, heatmaps and GO analysis is available at: https://github.com/Pdelam/Delamotte-et-al.-2024.git