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Abstract Human pluripotent stem cell (hPSC)-derived cardiomyocytes provide a promising regenerative cell therapy for cardiovascular patients and an important model system to accelerate drug discovery. However, cost-effective and time-efficient platforms must be developed to evaluate the quality of hPSC-derived cardiomyocytes during biomanufacturing. Here, we develop a non-invasive label-free live cell imaging platform to predict the efficiency of hPSC differentiation into cardiomyocytes. Autofluorescence imaging of metabolic co-enzymes is performed under varying differentiation conditions (cell density, concentration of Wnt signaling activator) across five hPSC lines. Live cell autofluorescence imaging and multivariate classification models provide high accuracy to separate low (< 50%) and high (≥ 50%) differentiation efficiency groups (quantified by cTnT expression on day 12) within 1 day after initiating differentiation (area under the receiver operating characteristic curve, 0.91). This non-invasive and label-free method could be used to avoid batch-to-batch and line-to-line variability in cell manufacturing from hPSCs. 

Abstract Breast cancer metastasis occurs via blood and lymphatic vessels. Breast cancer cells ‘educate’ lymphatic endothelial cells (LECs) to support tumor vascularization and growth. However, despite known metabolic alterations in breast cancer, it remains unclear how lymphatic endothelial cell metabolism is altered in the tumor microenvironment and its effect in lymphangiogenic signaling in LECs. We analyzed metabolites inside LECs in co-culture with MCF-7, MDA-MB-231, and SK-BR-3 breast cancer cell lines using $$^1\hbox {H}$$ 1 H nuclear magnetic resonance (NMR) metabolomics, Seahorse, and the spatial distribution of metabolic co-enzymes using optical redox ratio imaging to describe breast cancer-LEC metabolic crosstalk. LECs co-cultured with breast cancer cells exhibited cell-line dependent altered metabolic profiles, including significant changes in lactate concentration in breast cancer co-culture. Cell metabolic phenotype analysis using Seahorse showed LECs in co-culture exhibited reduced mitochondrial respiration, increased reliance on glycolysis and reduced metabolic flexibility. Optical redox ratio measurements revealed reduced NAD(P)H levels in LECs potentially due to increased NAD(P)H utilization to maintain redox homeostasis. $$^{13}\hbox {C}$$ 13 C -labeled glucose experiments did not reveal lactate shuttling into LECs from breast cancer cells, yet showed other $$^{13}\hbox {C}$$ 13 C signals in LECs suggesting internalized metabolites and metabolic exchange between the two cell types. We also determined that breast cancer co-culture stimulated lymphangiogenic signaling in LECs, yet activation was not stimulated by lactate alone. Increased lymphangiogenic signaling suggests paracrine signaling between LECs and breast cancer cells which could have a pro-metastatic role. 

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ’s effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.