Decipher novel cytotoxic T cell resistant mechanisms in lung cancer

Background
To this day, non-small cell lung cancer (NSCLC) is still the leading cause of cancer related deaths worldwide. In the past few years, immunotherapies that aim to stimulate the cancer killing potential of cytotoxic T lymphocytes (CTLs), have transformed the field of cancer therapies. Unfortunately, response rates remain <25% in unselected NSCLC patients, indicating the need to uncover and tackle CTL-killing resistant mechanisms (CRMs) installed by the tumor. These CRMs can be tumor cell extrinsic, installed within the non-malignant tumor microenvironment or tumor cell intrinsic. Typical examples of the latter are PD-L1 upregulation, loss of β2-microglobulin (β2M) and insensitivity to IFNɣ. Here we aimed to unravel novel tumor cell intrinsic CRMs by generating β2M+ CTL-killing resistant tumor cells, still responsive to IFNɣ.

Methods
To that end, we used the KrasG12Dp53-/- KP line to generate an eGFP and ovalbumin+ KP-eO line. In vitro we subjected this line for 12hrs to OT-I CTL mediated killing (T:E of 1:10). Next, leftover KP-eO cells were expanded and subjected to OT-I CTLs again, for 6 rounds in total, until the completely CTL-resistant KP-eO-R6 was generated. In vivo, 5x10e5 KP-eO cells were injected intravenously. Four weeks later, eGFP+ cells were isolated from the lungs, subjected to an OT-I killing assay in vitro after which the killing resistant KP-eO-Rx line was generated. After FACS sorting of β2M and IFNɣ responsive KP-eO-R6 and KP-eO-Rx cells, we used RNAseq to decipher transcriptional differences between them and the KP-eO CTL-killing sensitive (wild type) line.
Results
Upon GSEA and subsequent KEGG pathway analysis we could define two significantly upregulated genes; GSTA2 in KP-eO-R6 and GSTM3 in KP-eO-Rx and -R6.

Conclussion
Both genes have previously been linked to evasion of apoptosis but not resistance to immunotherapy. Currently we are evaluating if we can erase the resistance of KP-eO-R6 and/or KP-eO-Rx to OT-I mediated killing by knocking out GSTA2 and GSTM3 (alone or both). Moreover, we are deciphering if the KP-eO wild type, KP-eO R6 and KP-eO Rx lines differently grow and mold the tumor microenvironment and ability to stimulate T cells in the periphery upon re-engraftment in mice. Our results can reveal valid biomarkers for patient selection prior to immunotherapy as well as novel bullseyes to target on the battlefield against cancer.