Trellis Single-Cell Screening Reveals Stromal Regulation of Patient-Derived Organoid Drug Responses

Abstract

Patient-derived organoids (PDOs) model personalized cancer therapy responses. How-ever, existing bulk PDO screening technologies cannot reveal drug response mechanisms or model how cells of the tumor microenvironment alter therapy performance. To address this, we developed a highly-multiplexed thiol-reactive organoid barcodingin situ(TOBis) mass cytometry platform to perform single-cell post translational modification (PTM) signaling analysis of colorectal cancer (CRC) PDOs and cancer-associated fibroblasts (CAFs) in response to clinical therapies. To compare patient- and microenvironment-specific treatment effects in thousands of single-cell PTM datasets, we developedTrellis— a highly-scalable, hierarchical tree-based treatment effect analysis method. Trellis analysis of>2,500 single-cell PTM PDO-CAF organoid cultures revealed that on-target cell-cycle blockage and DNA-damage drug effects are common, even in chemorefractory PDOs. However, drug-induced apoptosis is patient-specific. We found drug-induced apoptosis does not correlate with genotype or clinical staging, but does align with cell-intrinsic PTM signaling in PDOs. We observe that CAFs protect chemosensitive PDOs by shifting cancer cells into a slow-cycling cell-state in a patient-specific manner and show that CAF chemoprotection can be reversed by inhibiting YAP. These results reveal that PTM signaling flux is a major determinant of chemosensitivity and demonstrate CAFs regulate patient-specific drug responses by altering cancer cell-state.

SIGNAL-seq: Multimodal Single-cell Inter- and Intra-cellular Signalling Analysis

“…This sample preparation workflow is similar to standard intra-cellular flow and mass cytometry assays [10, 11]. To minimise non-specific background staining, permeabilised cells are blocked with BSA, dextran sulphate [4], and sheared salmon sperm DNA [5], and then stained with poly-A barcoded oligonucleotide-tagged antibodies targeting cytoplasmic and nuclear PTMs and intra- and extra-cellular proteins (previously validated using mass cytometry [10, 11, 12, 13]).…”

“…Deregulated PTM signalling can then alter cancer cell gene expression to drive phenotypic plasticity [20]. For example, in colorectal cancer (CRC) inter-cellular signalling from cancer associated fibroblasts (CAFs) can regulate cancer cell PTM signalling to polarise cancer cells from a chemosensitive proliferative colonic stem cell (proCSC) fate to a chemorefractory revival stem cell (revCSC) fate [12, 21].…”

“…single-cell PDO specific proCSC and revCSC signatures were computed by scoring each PDO epithelial cell for literature derived proCSC and revCSC CRC genes identified by Ramos Zapatero et al [12] with the score_genes function in Scanpy using default settings on Log-Normalised data. To derive refined single-cell proCSC and revCSC gene signatures, these two literature proCSC and revCSC gene scores were then correlated against the single-cell gene expression matrix for the control treated PDO epithelial cells to identify a more comprehensive list of genes significantly associated with either CRC stem cell phenotype (r >0.1, >3 SD above the mean for shuffled data).…”

A Single-cell Perturbation Landscape of Colonic Stem Cell Polarisation

“…Our results confirmed that TGF-β can induce revCSC-like cells in CRC organoids, but this process is rare (Figure S3C) and requires low PI3K signalling (Figure 5F). Moreover, we recently demonstrated that cancer associated fibroblasts (CAFs) can also induce a revCSC-like state in CRC patient-derived organoids (PDOs) that protects CRC cells from chemotherapies including fluorouracil, oxaliplatin, and irinotecan [38]. In this model, CAF-chemoprotection can also be overcome by inhibiting YAP signalling – further demonstrating the central role of YAP in revCSC identity.…”

Highlights from the 1st European cancer dependency map symposium and workshop

“…Chris Tape from the UCL Cancer Institute gave a talk on single‐cell analysis of drug response mechanisms inpatient‐derived organoids (PDOs) and cancer‐associated fibroblasts (CAFs), showing that CAFs alter PDO drug sensitivity by cell‐fate plasticity switching and mechanistic insights can drive rational drug re‐sensitisation [31]. Chris particularly emphasised that cell‐extrinsic cues can regulate cancer cell dependencies and that future DepMap efforts would benefit from incorporating elements of the tumour microenvironment.…”