Applying activity-based nanotechnologies towards lung cancer interception

Introduction:: Chronic inflammation is strongly associated with cancer development, especially in the lungs which face continuous environmental exposure. The pro-inflammatory cytokine IL-1b is a key mediator of the inflammatory response in lung cancer, and its systemic inhibition has been shown to reduce the incidence of and mortality from lung cancer in patients three to seven years later [1]. This observation suggests there is a window of opportunity for cancer interception in certain patient populations via enforcing perturbations in the IL-1b pathway. As IL-1b is produced in an inactive pro-form and requires proteolytic processing for activation, the immune-related IL-1b-cleaving enzymes serve as critical upstream determinants of inflammation [2]. Herein, we utilized activity-based nanotechnology platforms to monitor changes in IL-1b-related protease activity in response to antibody treatments and during the pathogenic progression of murine lung adenocarcinoma (LUAD).

Materials and Methods:: LUAD tumors were developed in mice from the Kras/Trp53-mutant model with lentiviral incorporation to express SIINFEKL antigen (KPS). Mice were injected intraperitoneally with control IgG, IL-1b antibody, PD-1 antibody, or both IL-1b and PD-1 antibody at 10 mg/kg/dose three times a week, starting five weeks after tumor induction. To measure the activity of target proteases at the site of disease, after each of the first two treatments, a panel of 20-plex activity-based nanosensors (ABNs) was administered intratracheally. ABNs are designed such that kidney-clearable mass-encoded reporter fragments are linked to the nanosensor scaffold via defined peptide sequences and released when cleaved by local target protease cleavage [3]. Urine was subsequently collected for mass spectrometry of the panel of reporters. Lungs were inflated with 50% OCT for collection and cryosectioning at timepoints of 6, 8, and 10 weeks. Sections were stained with activity zymography probes (AZPs) and antibodies for cell and protease markers. AZPs consist of a negatively charged reporter masked by a peptide substrate sequence that prevents it from binding to tissue until cleaved by localized target proteases [4]. Quantification and visualization were performed with QuPath and GraphPad Prism.

Results, Conclusions, and Discussions:: We observed that IL-1b, as well as proteases that recognize and cleave IL-1b (e.g. proteinase-3 and cathepsin B), were highly expressed in KPS tumors as early as 6 weeks (Fig. 1a). ABNs that were cleaved in vivo by lung proteases released mass-encoded reporters, many of which differed in detection frequency following IL-1b and PD-1 blockade in the tumor-bearing mice (Fig. 1b). Our ABN panel revealed particularly robust changes in response to two proteases-of-interest (POI); a reporter for granzyme B was elevated while another POI exhibited reduced reporter release (p_adj < 0.05), suggesting that the IL-1b processing pathway is important to cancer development and is disrupted upon treatment (Fig. 1c). The AZP forms of the hits uses electrostatic interaction to bind to tissue upon cleavage and further supports the high protease activity in situ in tumors, highlighting a promising target for cancer interception and improved immunotherapy efficacy (Fig. 1d,e). Proteases play important roles in regulating tumor-associated IL-1b inflammation. We designed substrates that could monitor lung cancer progression and treatment response, as well as probe the tumor-immune microenvironment. By understanding the interplay of cells in protease and cytokine production, we can develop novel approaches to mitigate inflammation and slow down or prevent progression of lung cancer.

Acknowledgements (Optional): : Research was supported by Janssen Pharmaceuticals/Johnson & Johnson and National Science Foundation Graduate Research Fellowship Program (NSF GRFP). We thank the Swanson Biotechnology Histology Core for their help processing tissue samples.

References (Optional): : [1] Ridker, P. M., MacFadyen, J. G., Thuren, T., Everett, B. M., Libby, P., Glynn, R. J., ... & Gersh, B. (2017). Effect of interleukin-1β inhibition with canakinumab on incident lung cancer in patients with atherosclerosis: exploratory results from a randomised, double-blind, placebo-controlled trial. The Lancet, 390(10105), 1833-1842.

[2] van Kempen, L. C. L., de Visser, K. E. & Coussens, L. M. Inflammation, proteases and cancer. Eur. J. Cancer 42, 728–734 (2006).

[3] Kirkpatrick, J. D., Warren, A. D., Soleimany, A. P., Westcott, P. M., Voog, J. C., Martin-Alonso, C., ... & Bhatia, S. N. (2020). Urinary detection of lung cancer in mice via noninvasive pulmonary protease profiling. Science translational medicine, 12(537), eaaw0262.

[4] Amini, A. P., Kirkpatrick, J. D., Wang, C. S., Jaeger, A. M., Su, S., Naranjo, S., ... & Bhatia, S. N. (2022). Multiscale profiling of protease activity in cancer. Nature Communications, 13(1), 5745.

[5] Soleimany, A. P., Kirkpatrick, J. D., Su, S., Dudani, J. S., Zhong, Q., Bekdemir, A., & Bhatia, S. N. (2021). Activatable zymography probes enable in situ localization of protease dysregulation in cancer. Cancer research, 81(1), 213-224.