Novel alveolus-level heterogeneities of aerosol transport and its alteration by metastasis

Introduction: The distribution of inhaled aerosols is critical in drug delivery and transport of pollutants and pathogens in the lung (Ruge et al., Lancet Respir. Med., 2023). Clinical imaging techniques (e.g., CT, MRI) have low spatial resolution, and so have only examined aerosol transport in the lung at the lobe scale (e.g., apex vs. base regions). Histological methods offer a cellular resolution “snapshot” of the lung but cannot capture dynamic aerosol transport and deposition. Due to these limitations, it is generally assumed that aerosol deposition in alveoli is uniform.

To fill this technological gap, we recently developed a transparent “crystal” ribcage (Banerji & Grifno, et al., Nat. Methods, 2023) that enables whole organ to cellular scale visualization of the functional mouse lung during dynamic respiration and circulation. Using the crystal ribcage, we may now probe the real-time transport of aerosols in healthy and metastatic mouse lungs from the whole organ to single alveolus level. We found for the first time that certain alveoli regularly receive liquid aerosols directly through airspace deposition, which are then transported to neighboring alveoli through diffusion. We then report how this process is disrupted with the presence of metastatic tumors. This fundamental finding on lung microphysiology implies that normal lung biology and immunology may exhibit heterogeneity at the alveolus level due to differential exposure to pollutants and pathogens. Our ongoing research seeks to elucidate the origin of the mosaic pattern and its implications to better understand lung microphysiology and improve delivery of therapeutics to the lung.

Materials and

Methods: The lung-heart bloc was removed from reporter mTmG-C57/BL6 mice and positioned within a crystal ribcage imaging device as previously reported (Banerji & Grifno, et al., Nat. Methods, 2023). Dynamic lung respiration using negative pressure, as in spontaneous breathing, was applied using a custom ventilator-nebulizer system. To investigate aerosol spatiotemporal dynamics from the whole-lobe to alveolar scale in the crystal ribcage, fluorescent particles of variable mass, from soluble dyes (0.5, 10, 70 kDa) to nanoparticles (200 μm), whole bacterial pathogens (1 μm), and polystyrene beads (1-5 μm) were delivered to probe size-dependent particle deposition after inhalation. To probe how lung metastasis remodels the mosaic pattern, B16F10 mouse cancer cells were injected in the circulation via the tail vein. Stereomicroscope fluorescent microscopy or spinning disk confocal fluorescence microscopy of the lung surface was utilized during and after particle delivery at speeds up to 200 frames per second to examine the spatiotemporal distribution of aerosols within the distal alveoli in healthy and cancerous lungs.

Results, Conclusions, and Discussions: Utilizing our crystal ribcage platform (A), we probed the spatial distribution of aerosolized liquid droplets (5-10 μm) or dry polystyrene beads (1-5 μm) within alveoli (B-C). Previously, it was assumed that airflow and particle distribution was uniform. However, we found a highly spatially heterogeneous deposition, manifesting as a "mosaic" pattern, with semi-regular geometry (n = 20+ mice) across multiple doses of variable materials within the same lung (B). This heterogeneity persisted at the alveolar scale, where groups of 3-5 alveoli within the mosaic "tile" received abundant droplets, while neighboring alveoli in the "band" received negligible droplets (C). Post deposition, liquid droplets continued to diffuse through the tissue towards alveoli that did not receive droplets directly during inhalation. This diffusion was dependent on the molecular weight (MW) of the fluorophores dissolved in the droplets. We measured the diameter of each mosaic tile over time to compare the diffusion rate of aerosolized small vs. high MW dyes and found that small dyes diffused quickly (1-3 min) while heavy dyes diffused slowly (10+ min) (D-E). Lastly, in a mouse model of melanoma lung metastasis, we observed that the mosaic pattern “tile” geometry degraded with increasing tumor burden over 9 days (F), indicating that pulmonary transport of inhaled particles, therapeutics, and potentially airflow, is disrupted by tumor remodeling of the lung structure.

In summary, previous methods for studying pulmonary aerosol transport cannot probe alveolar-scale particle deposition. We used the crystal ribcage to capture the highly spatially heterogeneous deposition of aerosols into a “mosaic pattern”, and the temporal dynamics of subsequent diffusion post deposition. This patterning has never previously been characterized and implies that normal lung biology may be heterogeneous due to the differential distribution of inhaled pollutants, pathogens, therapeutics, and potentially gases in alveoli that may arise due to inertia-driven airflow heterogeneity. Lastly, we have found that the pattern breaks down during remodeling of lung structure in metastasis. The mosaic pattern and its breakdown implies that respiratory flow dynamics may have an unexpected influence on local lung biology and immunity and may be leveraged to optimize therapeutics for acute and chronic lung diseases

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