Vasculopathy and Increased Vascular Congestion in Fatal COVID-19 and Acute Respiratory Distress Syndrome.

Rationale: The leading cause of death in coronavirus disease 2019 (COVID-19) is severe pneumonia, with many patients developing acute respiratory distress syndrome (ARDS) and diffuse alveolar damage (DAD). Whether DAD in fatal COVID-19 is distinct from other causes of DAD remains unknown. Objective: To compare lung parenchymal and vascular alterations between patients with fatal COVID-19 pneumonia and other DAD-causing etiologies using a multidimensional approach. Methods: This autopsy cohort consisted of consecutive patients with COVID-19 pneumonia (n = 20) and with respiratory failure and histologic DAD (n = 21; non-COVID-19 viral and nonviral etiologies). Premortem chest computed tomography (CT) scans were evaluated for vascular changes. Postmortem lung tissues were compared using histopathological and computational analyses. Machine-learning-derived morphometric analysis of the microvasculature was performed, with a random forest classifier quantifying vascular congestion (CVasc) in different microscopic compartments. Respiratory mechanics and gas-exchange parameters were evaluated longitudinally in patients with ARDS. Measurements and Main Results: In premortem CT, patients with COVID-19 showed more dilated vasculature when all lung segments were evaluated (P = 0.001) compared with controls with DAD. Histopathology revealed vasculopathic changes, including hemangiomatosis-like changes (P = 0.043), thromboemboli (P = 0.0038), pulmonary infarcts (P = 0.047), and perivascular inflammation (P < 0.001). Generalized estimating equations revealed significant regional differences in the lung microarchitecture among all DAD-causing entities. COVID-19 showed a larger overall CVasc range (P = 0.002). Alveolar-septal congestion was associated with a significantly shorter time to death from symptom onset (P = 0.03), length of hospital stay (P = 0.02), and increased ventilatory ratio [an estimate for pulmonary dead space fraction (Vd); p = 0.043] in all cases of ARDS. Conclusions: Severe COVID-19 pneumonia is characterized by significant vasculopathy and aberrant alveolar-septal congestion. Our findings also highlight the role that vascular alterations may play in Vd and clinical outcomes in ARDS in general.

Therapeutically reprogrammed nutrient signalling enhances nanoparticulate albumin bound drug uptake and efficacy in KRAS-mutant cancer.

Nanoparticulate albumin bound paclitaxel (nab-paclitaxel, nab-PTX) is among the most widely prescribed nanomedicines in clinical use, yet it remains unclear how nanoformulation affects nab-PTX behaviour in the tumour microenvironment. Here, we quantified the biodistribution of the albumin carrier and its chemotherapeutic payload in optically cleared tumours of genetically engineered mouse models, and compared the behaviour of nab-PTX with other clinically relevant nanoparticles. We found that nab-PTX uptake is profoundly and distinctly affected by cancer-cell autonomous RAS signalling, and RAS/RAF/MEK/ERK inhibition blocked its selective delivery and efficacy. In contrast, a targeted screen revealed that IGF1R kinase inhibitors enhance uptake and efficacy of nab-PTX by mimicking glucose deprivation and promoting macropinocytosis via AMPK, a nutrient sensor in cells. This study thus shows how nanoparticulate albumin bound drug efficacy can be therapeutically improved by reprogramming nutrient signalling and enhancing macropinocytosis in cancer cells.

Improving nanotherapy delivery and action through image-guided systems pharmacology.

Despite recent advances in the translation of therapeutic nanoparticles (TNPs) into the clinic, the field continues to face challenges in predictably and selectively delivering nanomaterials for the treatment of solid cancers. The concept of enhanced permeability and retention (EPR) has been coined as a convenient but simplistic descriptor of high TNP accumulation in some tumors. However, in practice EPR represents a number of physiological variables rather than a single one (including dysfunctional vasculature, compromised lymphatics and recruited host cells, among other aspects of the tumor microenvironment) - each of which can be highly heterogenous within a given tumor, patient and across patients. Therefore, a clear need exists to dissect the specific biophysical factors underlying the EPR effect, to formulate better TNP designs, and to identify patients with high-EPR tumors who are likely to respond to TNP. The overall pharmacology of TNP is governed by an interconnected set of spatially defined and dynamic processes that benefit from a systems-level quantitative approach, and insights into the physiology have profited from the marriage between in vivo imaging and quantitative systems pharmacology (QSP) methodologies. In this article, we review recent developments pertinent to image-guided systems pharmacology of nanomedicines in oncology. We first discuss recent developments of quantitative imaging technologies that enable analysis of nanomaterial pharmacology at multiple spatiotemporal scales, and then examine reports that have adopted these imaging technologies to guide QSP approaches. In particular, we focus on studies that have integrated multi-scale imaging with computational modeling to derive insights about the EPR effect, as well as studies that have used modeling to guide the manipulation of the EPR effect and other aspects of the tumor microenvironment for improving TNP action. We anticipate that the synergistic combination of imaging with systems-level computational methods for effective clinical translation of TNPs will only grow in relevance as technologies increase in resolution, multiplexing capability, and in the ability to examine heterogeneous behaviors at the single-cell level.

Development of flow cytometry assays for measuring cell-membrane enzyme activity on individual cells.

Background: Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable. Methods: We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry. One assay measured live-cell natural processing of FPS and binding of its fluorescent product onto individual-cell membrane-bound enzymes. The other assay measured processing of specifically-bound and glutaraldehyde-crosslinked FPS, and consequent generation of its coupled fluorescent product onto individual-cell membrane-bound enzymes. Results: Confocal-microscopy imaging indicated that proteolytic processing of FPS selectively occurred on and labeled cell membrane of individual cells. The new assays measured specific increases of cell-associated FPS fluorescent product in substrate-concentration-, temperature- and time-dependent manners. A large proportion of processed FPS fluorescent products remained cell-associated after cell washing, indicating their binding to cell-membrane expressing enzymes. The assays measured higher levels of cell-associated FPS fluorescent product on wild-type than ADAM10-knockout mouse fibroblasts and on human monocytes than lymphocytes, which correlated with ADAM10 presence and expression levels on cell membrane, respectively. Furthermore, the enzyme activity assays could be combined with fluorescent anti-ADAM10 antibody staining to co-label and more directly associate enzyme activity and ADAM10 protein levels on cell membrane of individual cells. Conclusions: We report on two novel assays for measuring cell-membrane anchored enzyme activity on individual cells, and their potential use to directly study specific biology of cell-surface-expressing proteases.

Single-Cell Intravital Microscopy of Trastuzumab Quantifies Heterogeneous in vivo Kinetics.

Cell-to-cell heterogeneity can substantially impact drug response, especially for monoclonal antibody (mAb) therapies that may exhibit variability in both delivery (pharmacokinetics) and action (pharmacodynamics) within solid tumors. However, it has traditionally been difficult to examine the kinetics of mAb delivery at a single-cell level and in a manner that enables controlled dissection of target-dependent and -independent behaviors. To address this issue, here we developed an in vivo confocal (intravital) microscopy approach to study single-cell mAb pharmacology in a mosaic xenograft comprising a mixture of cancer cells with variable expression of the receptor HER2. As a proof-of-principle, we applied this model to trastuzumab therapy, a HER2-targeted mAb widely used for treating breast and gastric cancer patients. Trastuzumab accumulated to a higher degree in HER2-over expressing tumor cells compared to HER2-low tumor cells (~5:1 ratio at 24 h after administration) but importantly, the majority actually accumulated in tumor-associated phagocytes. For example, 24 h after IV administration over 50% of tumoral trastuzumab was found in phagocytes whereas at 48 h it was >80%. Altogether, these results reveal the dynamics of how phagocytes influence mAb behavior in vivo, and demonstrate an application of intravital microscopy for quantitative single-cell measurement of mAb distribution and retention in tumors with heterogeneous target expression. © 2019 International Society for Advancement of Cytometry.