Patient‐Derived Tumor Organoids Combined with Function‐Associated ScRNA‐Seq for Dissecting the Local Immune Response of Lung Cancer

Abstract In vitro models coupled with multimodal approaches are needed to dissect the dynamic response of local tumor immune microenvironment (TIME) to immunotherapy. Here the patient‐derived primary lung cancer organoids (pLCOs) are generated by isolating tumor cell clusters, including the infiltrated immune cells. A function‐associated single‐cell RNA sequencing (FascRNA‐seq) platform allowing both phenotypic evaluation and scRNA‐seq at single‐organoid level is developed to dissect the TIME of individual pLCOs. The analysis of 171 individual pLCOs derived from seven patients reveals that pLCOs retain the TIME heterogeneity in the parenchyma of parental tumor tissues, providing models with identical genetic background but various TIME. Linking the scRNA‐seq data of individual pLCOs with their responses to anti‐PD‐1 (αPD‐1) immune checkpoint blockade (ICB) allows to confirm the central role of CD8+ T cells in anti‐tumor immunity, to identify potential tumor‐reactive T cells with a set of 10 genes, and to unravel the factors regulating T cell activity, including CD99 gene. In summary, the study constructs a joint phenotypic and transcriptomic FascRNA‐seq platform to dissect the dynamic response of local TIME under ICB treatment, providing a promising approach to evaluate novel immunotherapies and to understand the underlying molecular mechanisms.

assembled MoSMAR-chip, reaction chip, transfer coverslip and the connectors for placing transfer coverslip.D and E) Images of the "spot-cover" procedure with the transfer coverslip pre-loading (D) and docking to the reaction chip (E) for reagent delivery.PCC analysis between the average expression levels of function-related genes for CD8 + T cells in pLCOs and the killing index.10 genes with good correlation were chosen as a geneset to calculate T cell activation index (Tai).F) Comparison of average Tai in P5 and P7 pLCOs under two treatment conditions (n = 69).G) GO enrichment analysis of the DEGs between the Tai high (>0.5)and other CD8 + T cells.

Figure S2 .
Figure S2.Supplementary illustration and optimization of the automated single cell/organoid distribution instrument.A) The major composition of YOLOv4-based neural network classifier adopted in our system.B) The operation logic map of automated single cell distribution instrument.C-E) Optimization of the automated single-cell distribution instrument

Figure S3 .
Figure S3.Extended data of Figure 2. A) Scheme of the FascRNA-seq procedure for single organoid and images showing single cells in the microwells at the indicated steps.Scale bars: 50 μm.B) Violin plots of gene expression features of the four unsupervised clusters in Figure 2J.C) Pseudo-trajectory and gene expression analysis along the inferred trajectory.

Figure S4 .
Figure S4.Extended data of Figure 3. A) tSNE visualization of single-cell landscape derived from 171 pLCOs, color labeled by unsupervised seurat clusters (left) or sample IDs (right).B) Heatmap of the CNV patterns of all epithelial cells.Red means amplification and blue indicates

Figure S6 .
Figure S6.Characterization of parenchyma infiltrating CD8 + T cells (PITs) derived from pLCOs.A) tSNE visualization of 191 CD8 + T cells from all pLCOs.Data point of each cell was labeled by treatment conditions (top), patient (bottom) and the categories of killing index (Ki) (middle, High: Ki ≥ 0.5; Medium: 0.25 < Ki < 0.5; Low: Ki ≤ 0.25).B) Featureplots of 191 T cells derived from pLCOs color labeled with expression levels of T cell function-related

Figure S8 .
Figure S8.Cell-cell interactions received by CD8 + T cells in individual pLCOs.A and B) Heatmaps showing the cell-cell interactions in individual pLCOs received by CD8 + T cells in P5 (A) and P7 (B).The heatmaps are color labeled by the possibility of interactions (i.e., communication score) calculated by CellChat.The bars on the top represent the killing index of individual organoids.C) Pie charts showing the abundance of the three types of interactions.D) Comparison of the communication scores in individual organoids under the two treatment conditions from P5 (n = 38) and P7 (n = 31).E) Sankey plots of intercellular communications between various cell types and T cells in P5SO21 and P7SO21 organoids.The color label represents the positive or negative related ligand-receptor interactions in tumor immunity.F) T

Figure S9 .
Figure S9.Specific features of TIME for individual patients.A) Sankey plot showing cellular interaction between Mphs and ECs or T cells.Cells from all the P4 and P5 organoids were pooled together for the analysis.B,C and D) Sankey plots of intercellular communication landscapes in P4 (B), P5 (C), and P6 (D).Cells from the same tumor sample were pooled