Identification of cells exhibiting elevated ERBB network activity in KRas/ Myc-driven lung tumours

Our lab has identified a completely unexpected requirement for ERBB signalling in KRAS-driven Lung Adenocarcinoma (LuAd). We have shown that this requirement is present from the very outset of lung tumour initiation, and, crucially, we have found a pronounced increase in ERBB signalling during progression from low grade adenocarcinoma to locally invasive disease. We hypothesize that this increased activity, which manifests as strongly elevated expression of multiple EGF/ERBB-family ligands, such as Amphiregulin (AREG), and paracrine activation of ERBB2 on infiltrating macrophages, may be exploited for early detection of KRAS- driven malignancy. Alongside a more general increase in AREG expression as tumours progress to higher grade disease, we find individual cells that express extremely high levels of AREG mRNA. We refer to these cells as AREG super-expressers and their lineage identity is unknown. Cell morphology suggests they maybe epithelial, however, unlike the bulk of tumours, these cells fail to express the lung epithelial marker SPC and staining for cytokeratins is inconclusive. Their detection at and adjacent to the tumour:normal interface suggests that they maybe disseminating tumour cells undergoing EMT. Alternatively, they may belong to another lung-resident or infiltrating population, induced to express AREG through direct contact with tumour cells. Immediately adjacent to progressing tumours we find large numbers of F4/80-positive macrophages that stain strongly for tyrosine phosphorylated ERBB2 (p-Tyr-ERBB2), suggesting paracrine activation in response to local production of ERBB ligands. The aim of this part of the work is to identify the cell lineage of the AREG superexpressers and to investigate the effect of ERBB-inhibition using the multi-ERBB inhibitor Neratinib. Overall design: scRNA-seq was performed on whole-tumour cell suspensions from lungs of SPC-Cre+ KrasG12D+/-;MYC+/+ (KM) genetically engineered mice 8-9 weeks after transgene induction. Three days prior to culling, mice received daily (3 doses total) intraperitoneal injections of neratinib in peanut oil (80mg/kg), peanut oil, or were left untreated (n=3 per group).