Abstract Cancer-associated fibroblasts (CAFs) play a pivotal role in cancer progression by enhancing extracellular matrix remodeling, cancer cell invasion, and mediating inflammatory environment. Therefore, CAFs have become targets for cancer therapies. However, a major hurdle to developing such therapies are the challenges of studying CAFs in culture - specifically it remains unclear which CAF functions and phenotypes are sustained during in vitro culture and the signaling pathways driving distinct CAF phenotypes. We investigated whether CAF phenotypes and functions are sustained or changed during 2D CAF culture under in vitro systems, our analysis included early passage, late passage, and immortalized cultures, as well as CAFs in coculture with cancer cell lines. Transcriptomic analysis revealed that inflammatory-related gene expression programs were suppressed in late passage and immortalized CAFs compared with early passage equivalents. Direct co-culture with lung cancer cells can revert the inflammatory gene expressions in immortalized CAFs such that they partially resemble early passage CAFs. In contrast, indirect coculture with lung cancer cells - in which CAFs could not form cell-cell contacts with cancer cells - had a less significant effect on immortalized CAF inflammatory gene expression but could induce TGFβ regulated genes. Interestingly, inhibition of the TGFβ signaling pathway potentiated the inflammatory gene expression network induced by direct co-culture, suggesting that the signaling mediated by direct cellular interaction is antagonistic to the TGFβ signaling pathway. These data further our understanding of cancer cell – CAF crosstalk and will help to develop in vitro assays that more closely maintain the state of CAFs observed in tissue. Citation Format: Yutaka Naito, Robert Hynds, David Novo, Probir Chakravarty, Gavin Kelly, Charles Swanton, Kazufumi Honda, Erik Sahai, TRACERx Consortium. Tracking the transcriptome of lung cancer-associated fibroblasts within the TRACERx lung study from patient to culture models reveals phenotypic plasticity and instructive cues from cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 674.