Cell vertices in epithelia comprise specialized tricellular junctions (TCJs) that seal the paracellular space between three adjoining cells [1,2]. Although TCJs play fundamental roles in tissue homeostasis, pathogen defense, and in sensing tension and cell shape [3-5], how they are assembled, maintained and remodeled is poorly understood. In Drosophila the transmembrane proteins Anakonda (Aka [6]) and Gliotactin (Gli [7]) are TCJ components essential for epithelial barrier formation. Additionally, the conserved four-transmembranedomain protein M6, the only myelin proteolipid protein (PLP) family member in Drosophila, localizes to TCJs [8,9]. PLPs associate with cholesterol-rich membrane domains and induce filopodia formation [10,11] and membrane curvature [12], and Drosophila M6 acts as a tumor suppressor [8], but its role in TCJ formation remained unknown. Here we show that M6 is essential for the assembly of tricellular, but not bicellular occluding junctions, and for barrier function in embryonic epithelia. M6 and Aka localize to TCJs in a mutually dependent manner and are jointly required for TCJ localization of Gli, whereas Aka and M6 localize to TCJs independently of Gli. Aka acts instructively and is sufficient to direct M6 to cell vertices in the absence of septate junctions, while M6 is required permissively to maintain Aka at TCJs. Furthermore, M6 and Aka are mutually dependent for their accumulation in a lowmobility pool at TCJs. These findings suggest a hierarchical model for TCJ assembly, where Aka and M6 promote TCJ formation through synergistic interactions that demarcate a distinct plasma membrane microdomain at cell vertices. Results and Discussion M6 co-localizes with Aka and Gli at tricellular junctions in embryonic epitheliaWe analyzed the distribution of M6 in embryos using a GFP protein trap insertion into the M6 locus, yielding GFP-tagged variants of five of the six annotated M6 isoforms (M6 CA06602 , referred to as GFP::M6; Fig. 1A [13]), or using an antiserum raised against two peptides present in five M6 protein isoforms (Fig. S1A). Anti-M6 immunostaining detected M6 protein in epithelia, including epidermis, salivary glands, tracheae, hindgut, midgut, Malpighian tubules, as well as in the nervous system of stage 14 embryos (Fig. S1D-I). In the epidermis of stage 15 embryos, M6 was detectable at lateral cell membranes (Fig. S1B,C). Compared to anti-M6 staining, GFP::M6 CA06602 displayed stronger accumulation at TCJs (Fig. S1B), suggesting that anti-M6 preferentially detects M6 isoforms or trafficking intermediates that are not GFP-positive in GFP::M6 CA06602 embryos. GFP::M6 accumulated at vertices at the level of septate junctions (SJs) marked by ; Fig. 1B), and co-localized with the TCJ components Aka and Gli (Fig. 1C). In living embryos GFP::M6 was enriched 7.56 +/-0.66-fold (n=10) at vertices relative to bicellular contacts,

Pulmonary sarcomatoid carcinoma (PSC) has highly aggressive biological behaviour and poor clinical outcomes, raising expectations for new therapeutic strategies. We characterized 179 PSC by immunohistochemistry, next-generation sequencing and in silico analysis using a deep learning algorithm with respect to clinical, immunological and molecular features. PSC was more common in men, older ages and smokers. Surgery was an independent factor (p < 0.01) of overall survival (OS). PD-L1 expression was detected in 82.1% of all patients. PSC patients displaying altered epitopes due to processing mutations showed another PD-L1-independent immune escape mechanism, which also significantly influenced OS (p < 0.02). The effect was also maintained when only advanced tumour stages were considered (p < 0.01). These patients also showed improved survival with a significant correlation for immunotherapy (p < 0.05) when few or no processing mutations were detected, although this should be interpreted with caution due to the small number of patients studied. Genomic alterations for which there are already approved drugs were present in 35.4% of patients. Met exon 14 skipping was found more frequently (13.7%) and EGFR mutations less frequently (1.7%) than in other NSCLC. In summary, in addition to the divergent genomic landscape of PSC, the specific immunological features of this prognostically poor subtype should be considered in therapy stratification.

Precision oncology and immunotherapy have revolutionized the treatment of advanced non-small-cell lung cancer (NSCLC). Emerging studies show that targeted therapies are also beneficial for patients with driver alterations such as epidermal growth factor receptor (EGFR) mutations in early-stage NSCLC (stages I–IIIA). Furthermore, patients with elevated programmed death-ligand 1 (PD-L1) expression appear to respond favorably to adjuvant immunotherapy. To determine the frequency of genomic alterations and PD-L1 status in early-stage NSCLC, we retrospectively analyzed data from 2066 unselected, single-center patients with NSCLC diagnosed using next-generation sequencing and immunohistochemistry. Nine-hundred and sixty-two patients (46.9%) presented with early-stage NSCLC. Of these, 37.0% had genomic alterations for which targeted therapies have already been approved for advanced NSCLC. The frequencies of driver mutations in the early stages were equivalent to those in advanced stages, i.e., the rates of EGFR mutations in adenocarcinomas were 12.7% (72/567) and 12.0% (78/650) in early and advanced NSCLC, respectively (p = 0778). In addition, 46.3% of early-stage NSCLC cases were PD-L1-positive, with a tumor proportion score (TPS) of ≥1%. With comparable frequencies of driver mutations in early and advanced NSCLC and PD-L1 overexpression in nearly half of patients with early-stage NSCLC, a broad spectrum of biomarkers for adjuvant and neoadjuvant therapies is available, and several are currently being investigated in clinical trials.

Wittek et al.2 SummaryCell vertices in epithelia comprise specialized tricellular junctions (TCJs) that seal the paracellular space between three adjoining cells [1,2]. Although TCJs play fundamental roles in tissue homeostasis, pathogen defense, and in sensing tension and cell shape [3][4][5], how they are assembled, maintained and remodeled is poorly understood. In Drosophila the transmembrane proteins Anakonda (Aka [6]) and Gliotactin (Gli [7]) are TCJ components essential for epithelial barrier formation. Additionally, the conserved four-transmembranedomain protein M6, the only myelin proteolipid protein (PLP) family member in Drosophila, localizes to TCJs [8,9]. PLPs associate with cholesterol-rich membrane domains and induce filopodia formation [10,11] and membrane curvature [12], and Drosophila M6 acts as a tumor suppressor [8], but its role in TCJ formation remained unknown. Here we show that M6 is essential for the assembly of tricellular, but not bicellular occluding junctions, and for barrier function in embryonic epithelia. M6 and Aka localize to TCJs in a mutually dependent manner and are jointly required for TCJ localization of Gli, whereas Aka and M6 localize to TCJs independently of Gli. Aka acts instructively and is sufficient to direct M6 to cell vertices in the absence of septate junctions, while M6 is required permissively to maintain Aka at TCJs. Furthermore, M6 and Aka are mutually dependent for their accumulation in a lowmobility pool at TCJs. These findings suggest a hierarchical model for TCJ assembly, where Aka and M6 promote TCJ formation through synergistic interactions that demarcate a distinct plasma membrane microdomain at cell vertices. Results and Discussion M6 co-localizes with Aka and Gli at tricellular junctions in embryonic epitheliaWe analyzed the distribution of M6 in embryos using a GFP protein trap insertion into the M6

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