Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. Here, we describe an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment, or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation, while being both time-saving and cost-effective.
Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. Here, we describe an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment, or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation, while being both time-saving and cost-effective.
The prevalence of “Long COVID”, including among vaccinated patients, is just one of the conundrums that indicate how much remains unknown about the lung’s response to viral infection, particularly to SARS-CoV-2 for which the lung is the point of entry. Therefore, we used an in vitro human lung system to enable a prospective, unbiased, sequential single cell level analysis of pulmonary cell responses following infection by multiple strains of SARS-CoV-2. By starting with human induced pluripotent stem cells (hiPSCs) and emulating lung organogenesis, three-dimensional lung organoids were generated and infected in which several unexpected but pertinent insights emerged. First, SARS-CoV-2 tropism is much broader than previously believed: most lung cell types can be infected, if not through a canonical receptor-mediated route (e.g., via ACE2) then via a non-canonical “backdoor” endocytosis/micropinocytosis route. Such entry can be abrogated by FDA-approved endocytosis blockers, suggesting novel adjunctive therapies. Regardless of route-of-entry, the virus triggers a heretofore unrecognized lung epithelial cell-intrinsic autonomous innate immune response involving interferons and cytokine/chemokine production in the absence of hematopoietic cells or their derivatives. The virus can spread rapidly throughout human lung organoid cell cultures resulting in mitochondrial apoptosis mediated by the pro-survival protein Bcl-xL. This host cytopathic response to the virus may help explain persistent inflammatory signatures in a dysfunctional pulmonary environment of long COVID. The host response to the virus is, in part, dependent on the presence of pulmonary Surfactant Protein-B (SP-B), which plays an unanticipated role in signal transduction, viral resistance, dampens systemic inflammatory cytokine production, and minimizes the induction of apoptosis.
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles surfactant deficient RDS. Using a novel multi-cell type, human induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that inflammatory cytokine/chemokine production and interferon (IFN) responses are dynamically regulated autonomously within the lung following SARS-CoV-2 infection, an intrinsic defense mechanism mediated by surfactant proteins (SP). Single cell RNA sequencing revealed broad infectability of most lung cell types through canonical (ACE2) and non-canonical (endocytotic) viral entry routes. SARS-CoV-2 triggers rapid apoptosis, impairing viral dissemination. In the absence of surfactant protein B (SP-B), resistance to infection was impaired and cytokine/chemokine production and IFN responses were modulated. Exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion restored resistance to SARS-CoV-2 and improved viability.
In pancreatic ductal adenocarcinoma (PDAC), glutamine is a critical nutrient that drives a wide array of metabolic and biosynthetic processes that support tumor growth. Despite this established dependency, the targeting of specific enzymes involved in glutamine metabolism is yet to yield any clinical benefit. Here, we have examined the therapeutic potential of 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist that broadly inhibits glutamine metabolism. We found that DON treatment significantly blocks PDAC tumor growth and attenuates metastasis. Interestingly, we link the effectiveness of DON in PDAC to asparagine (Asn) metabolism. By inhibiting asparagine synthetase (ASNS), DON significantly reduces intracellular Asn production and Asn supplementation rescues the anti-proliferative effects of DON. We discern that PDAC cells upregulate expression of ASNS as a metabolic adaptation and that modulating ASNS levels can impact DON efficacy. Strikingly, in patient-derived organoids, DON responsiveness is inversely correlated with ASNS expression, a feature that is not observed for other metabolic enzymes targeted by DON. We find that treatment with L-asparaginase (ASNase), an enzyme that catabolizes free Asn, synergizes with DON to impact the viability of PDAC cells. Finally, we identify that a combination therapy of DON and ASNase has a significant impact on metastasis. These results shed light on the mechanisms that drive the effects of glutamine mimicry and point to the utility of co-targeting adaptive responses to control PDAC progression.
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