Probing functioning lung at the cellular resolution in health and disease

Summary: The lung is the site of many pathophysiologies due to air-borne pathogens, pollutants, and primary and metastatic cancer, and its diverse microenvironment is continuously exposed to chemical, mechanical, biological, and immunological stresses. The lack of effective therapeutics for many major pulmonary diseases, exemplified by the ongoing COVID-19 pandemic, demonstrates the urgent need to better understand the cellular dynamics of disease pathogenesis, and to identify new therapeutic targets. The majority of our understanding of pulmonary diseases relies on fixed/frozen specimens from patients and in vivo models, which only provide a snapshot of the lung's pathophysiology, and hence incapable of capturing the dynamic and early stage events in disease progression and response to therapy. The current in vitro and ex vivo models also lack the cellular diversity and complex biophysical and immunological environment in the lung. This lack of technologies to probe the lung cellular dynamics with high spatial and temporal resolutions is a major obstacle underlying our limited understanding of the following key dynamic events in health and disease: (i) real-time dynamics of respiration (e.g., gas transport) and circulation (e.g., vascular integrity), (ii) trafficking of immune and cancer cells, and their sequestration, extravasation, and differentiation, (iii) dynamics of cellular communication via biochemical (e.g., cytokine) and biophysical (e.g., shear stresses) factors and, (iv) transmission of air-borne pathogen, and the host response dynamics. To study these dynamic events, we propose to develop a transformative platform to mechanistically probe lung (patho)physiology in real-time and at the cellular resolution. This platform, termed LungEx, includes the long-term ex vivo maintenance of mouse and human lungs in near-physiological conditions that is equipped with a novel transparent ribcage, termed "crystal" ribcage, enabling real-time volumetric optical microscopy. Utilizing this platform, we will, for the first time, visualize the dynamics of lung pathophysiology in real-time, at the cellular resolution, and over nearly the entire surface of the lung while the respiratory/circulatory functions are fully preserved. Additionally, LungEx allows precise control of the physical and biochemical parameters of respiration/circulation as well as biochemical and optogenetic manipulation through the crystal ribcage, which will enable establishing causal links between physical, biological, and immunological determinants of lung diseases. Leveraging on our preliminary data and diverse collaborators, we will demonstrate the capabilities of the Mouse LungEx in probing the immune response to lung metastasis from the earliest stages of cancer cell seeding to established tumors. We will also demonstrate the unprecedented capabilities of Human LungEx to probe the spatial heterogeneities of the host response to viral infections such as SARS-CoV-2, and the very early time course of the immune response. In addition to these two key lung diseases, the Mouse and Human LungEx will transform virtually all areas of pulmonary research such as disease pathogenesis, drug development and delivery, aging, and transplantation.