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Unlike other epithelial cancer types, circulating tumor cells (CTCs) are less frequently detected in the peripheral blood of non-small cell lung cancer (NSCLC) patients using epithelial marker-based detection approaches despite the aggressive nature of NSCLC. Here, we demonstrate hexokinase-2 (HK2) as a metabolic function-associated marker for the detection of CTCs. In 59 NSCLC patients bearing cytokeratin-positive (CKpos) primary tumors, HK2 enables resolving cytokeratin-negative (HK2high/CKneg) CTCs as a prevalent population in about half of the peripheral blood samples with positive CTC counts. However, HK2high/CKneg tumor cells are a minority population in pleural effusions and cerebrospinal fluids. Single-cell analysis shows that HK2high/CKneg CTCs exhibit smaller sizes but consistent copy number variation profiles compared with CKpos counterparts. Single-cell transcriptome profiling reveals that CK expression levels of CTCs are independent of their epithelial-to-mesenchymal transition (EMT) status, challenging the long-standing association between CK expression and EMT. HK2high/CKneg CTCs display metastasis and EGFR inhibitor resistance-related molecular signatures and are selectively enriched in patients with EGFRL858R driver oncogene mutation as opposed to EGFR19Del , which is more frequently found in patients with prevalent CKpos CTCs in the blood. Consistently, treatment-naïve patients with a larger number or proportion of HK2high/CKneg CTCs in the blood exhibit poor therapy response and shorter progression-free survival. Collectively, our approach resolves a more complete spectrum of CTCs in NSCLC that can potentially be exploited to identify patient prognosis before therapy.

Accurate prediction of chemo- or targeted therapy responses for patients with similar driver oncogenes through a simple and least-invasive assay represents an unmet need in the clinical diagnosis of non-small cell lung cancer. Using a single-cell on-chip metabolic cytometry and fluorescent metabolic probes, we show metabolic phenotyping on the rare disseminated tumor cells in pleural effusions across a panel of 32 lung adenocarcinoma patients. Our results reveal extensive metabolic heterogeneity of tumor cells that differentially engage in glycolysis and mitochondrial oxidation. The cell number ratio of the two metabolic phenotypes is found to be predictive for patient therapy response, physiological performance, and survival. Transcriptome analysis reveals that the glycolytic phenotype is associated with mesenchymal-like cell state with elevated expression of the resistant-leading receptor tyrosine kinase AXL and immune checkpoint ligands. Drug targeting AXL induces a significant cell killing in the glycolytic cells without affecting the cells with active mitochondrial oxidation.

Protein hydrolysates from the goat placenta provide multiple benefits, such as immune system enhancement, antioxidant activities, and reductions in uric acid levels. Despite these benefits, their industrial applications have been underexplored. This study aimed to prepare extract protein hydrolysates (GPERPs) from residual goat placenta extract (GPER) and assess their functional properties, focusing on how different drying methods influence these properties. The essential amino acid contents were 30.94% for the GPER and 34.11% for the GPERPs. Moreover, all the essential amino acids were present, and the amino acid score (AAS) for each exceeded 1.0 in the GPERPs. The foaming properties of the spray-dried GPERPs (95.56 ± 5.89%) were significantly greater than those of the freeze-dried GPERPs (49.13 ± 4.17%) at pH values of 4.0~10.0. The emulsion stability (ES) of the spray-dried GPERPs (453.44 ± 8.13 min) was notably greater than that of the freeze-dried GPERPs (245.58 ± 7.12 min). Furthermore, the water retention capacity (WRC) of the freeze-dried GPERPs (201.49 ± 6.12%) was significantly greater than that of the spray-dried GPERPs (103.35 ± 7.13%), except at pH 10.0 (101.44 ± 8.13%). Similarly, at pH values of 6.0, 8.0, and 10.0, the oil retention capacity (ORC) of the freeze-dried GPERPs (715.58 ± 12.15%) was significantly greater than that of the spray-dried GPERPs (560.56 ± 11.15%), although the opposite trend was noted under acidic conditions. In terms of the antioxidant activity, the ability of the goat placenta extract residual protein hydrolysates (GPERPs) to scavenge DPPH radicals and superoxide anion radicals increased with the increasing peptide powder concentration, and the maximum scavenging rates of the DPPH radicals (39.5 ± 0.56%) and superoxide anions (81.2 ± 0.54%) in the freeze-dried peptide powder were greater than those in the spray-dried peptide powder. These findings contribute to the understanding of the physicochemical and antioxidant properties of GPERPs under various drying methods and provide fundamental data for the development of functional foods based on GPERPs. [ABSTRACT FROM AUTHOR]

Circulating tumor cells are formed by cancer cells in the primary or metastatic foci spreading to circulatory system, which is of great value in the early diagnosis of tumor, metastasis and recurrence monitoring and evaluation of efficacy, especially the important target of metastasis intervention. In vitro culture of CTCs can provide a model for the screening of metastatic drugs and improve the accuracy of CTCs phenotypic analysis. However, CTCs are difficult to be cultured in vitro and become a bottleneck in the field of research. In the future, the primary task of this field is to identify the molecular characteristics of the CTCs subgroups (metastatic-CTC) that actually lead to metastasis, and then to build the intervention of drugs in vitro screening platform, thereby enhancing the efficacy of metastatic intervention. In this paper, the extraction of CTCs, culture methods, as well as in vitro research progress were reviewed in order to promote the progress of the field.

In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions (1-8). A homeostatic makeup of these functional phenotypes dictates the overarching effect of [...], Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (~1 x [10.sup.4] cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen-specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen-specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.