Combination therapy has emerged as a promising strategy for treating pancreatic ductal adenocarcinoma (PDAC). Our previous findings show that combination therapy can reprogram immunosuppressive mechanisms, particularly in the mesenchymal PDAC subtype. Specifically, the novel combination therapy (trametinib + nintedanib, T/N) induces immunomodulatory chemokine secretion, promotes cytotoxic T-cell infiltration, and sensitizes mesenchymal PDAC to PD-L1 inhibition (Falcomatà et al., 2022). To map therapy-induced changes in the tumor microenvironment (TME) landscape, we conducted a time-course experiment examining T/N combination with immune checkpoint blockade in vivo and in vitro. To assess dynamic transcriptional changes between the tumor cells and their TME in response to treatment, we employed lineage and RNA recovery (LARRY)-based barcoding.

To investigate the characteristics of resistant and sensitive clones, we barcoded mesenchymal PDAC clones using the LARRY barcode and characterized their genomic features with low-coverage whole-genome sequencing (lcWGS). A pool of 40 distinct clones was orthotopically implanted into mice. We divided the cohorts into two experimental arms: control and treated (T/N + anti-PD-L1). Mice were sacrificed at specific time points, and in vitro samples were collected simultaneously. Samples were analyzed using Chromium 3'-scRNA-seq, FFPE-based Flex gene expression, and Xenium. To enable barcode detection with Flex, we designed specific probes for each clone.

We successfully detected tumor barcodes using both 3'-scRNA-seq and Flex, thus allowing a clear distinction between tumor and non-tumor populations. Barcode analysis identified several treatment-resistant clones. Notably, two distinct populations emerged, including highly proliferative tumor cells with high Avil expression, predominantly composed of myc-amplified clones, and low-proliferative tumor cells with high Bgn expression, which showed more resistance to therapy. This analysis revealed gene signatures linked to resistance mechanisms against combinatorial therapy and immune checkpoint blockade.

Barcode-based tumor detection offers a powerful approach for tracking clonal dynamics, identifying resistant populations, and uncovering molecular mechanisms of therapy resistance in PDAC.