Investigating Tumor Microenvironment Drivers of Drug-Resistant Non-Small Cell Lung Cancer

Introduction: Non-small cell lung cancer (NSCLC) is a global health challenge. Despite significant advances, patients succumb to NSCLC most often because the disease becomes multi-drug resistant (MDR). One of the mechanisms by which this disease attains MDR is due to factors from the tumor microenvironment (TME) that promote cell survival even under pressure from otherwise powerful chemotherapy and targeted drug therapies. To combat this drug resistance in NSCLC, the Pritchard Lab has developed the dual-switch gene drive therapy which involves using cancer cells to kill other cancer cells in their vicinity by engineering the cells to produce an enzyme that can convert a pro-drug into a chemotherapeutic. Ensuring that this therapy is successful once it gets to clinical trials requires testing where it can potentially fail. The goal of this project is to test this therapy in 3D cell culture systems as they closely mimic cell interactions found in real tissues. We use a 3D cell-cell interactions culture model and different 3D cell-extracellular matrix (ECM) biomaterials to study how drug resistance to this therapy may occur.

Materials and

Methods: To mimic 3D cell-cell interaction culture models, a mixture of PC9 osimertinib-sensitive, PC9 osimertinib-resistant and PC9 gene drive cells obtained from Pritchard lab is used and the cells are cultured in 3D spheroids at different ratios. We use a U-bottom plate to form the spheroids. After 3 days of culture, the formed spheroids are encapsulated in 3D matrigel, or 3D collagen gel then we start the dual-switch gene drive treatment to assess its efficacy in those environments. A spinning disk confocal microscope is used to determine live and dead cells within the spheroids and the images are quantified to determine the response of the cells to the therapy (figure 1).

Results, Conclusions, and Discussions: Preliminary results have revealed a striking phenomenon: drug-resistant cells are more difficult to eradicate in 3D matrigel (figure 3) compared to 3D collagen gel (figure 2). The results have also shown that even though drug-resistant cells tend to proliferate at the start of the gene drive treatment, they eventually start succumbing to the treatment (figure 2). These findings suggest that 3D cell-cell contacts between the drug-resistant cells may help them survive the gene drive treatment. Additionally, when using different biomaterials for drug screening, the response from cells may differ based on the microenvironmental cues that the cells receive from those biomaterials. For instance, matrigel contains more extracellular matrix proteins and growth factors unlike collagen gel which contains only collagen I. Our ongoing work is optimizing the timing of the gene drive treatment drugs, the ratio of the three PC9 cell populations within spheroids to effectively eradicate the drug-resistant cells within a 3D environment using the gene drive cells, pairing 3D environments and a mouse model. The 3D environments will include synthetic hydrogels engineered to mimic the diseased lung tissue's protein composition. These hydrogels will incorporate two key features: integrin-binding peptides and Matrix Metalloproteinase (MMP)- degradable peptides. This research has the potential to help develop more effective therapies for NSCLC patients and ultimately bridge the gap between preclinical and clinical results.

Acknowledgements (Optional):