Keratin 5 basal cells are temporally regulated developmental and tissue repair progenitors in bladder urothelium.

Urothelium forms a distensible yet impermeable barrier, senses and transduces stimuli, and defends the urinary tract from mechanical, chemical, and bacterial injuries. Biochemical and genetic labeling studies support the existence of one or more progenitor populations with the capacity to rapidly regenerate the urothelium following injury, but slow turnover, a low mitotic index, and inconsistent methodologies obscure progenitor identity. The progenitor properties of basal keratin 5 urothelial cells (K5-UCs) have been previously investigated, but those studies focused on embryonic or adult bladder urothelium. Urothelium undergoes desquamation and apoptosis after birth, which requires postnatal proliferation and restoration. Therefore, we mapped the fate of bladder K5-UCs across postnatal development/maturation and following administration of cyclophosphamide to measure homeostatic and reparative progenitor capacities, respectively. In vivo studies demonstrate that basal K5-UCs are age-restricted progenitors in neonates and juveniles, but not in adult mice. Neonatal K5-UCs retain a superior progenitor capacity in vitro, forming larger and more differentiated urothelial organoids than adult K5-UCs. Accordingly, K5-UC transcriptomes are temporally distinct, with enrichment of transcripts associated with cell proliferation and differentiation in neonates. Induction of urothelial proliferation is sufficient to restore adult K5-UC progenitor capacity. Our findings advance the understanding of urothelial progenitors and support a linear model of urothelial formation and regeneration, which may have significant impact on therapeutic development or tissue engineering strategies.NEW & NOTEWORTHY Fate mapping reveals an important linear relationship, whereby bladder basal urothelial cells give rise to intermediate and superficial cells in an age-restricted manner and contribute to tissue repair. Neonatal basal cells reprise their role as superior progenitors in vitro and display distinct transcriptional signatures, which suggest progenitor function is at least partially cell intrinsic. However, the urothelium progenitor niche cannot be overlooked, since FGF7 rescues adult basal cell progenitor function.

Following the p63/Keratin5 basal cells in the sensory and non-sensory epithelia of the vomeronasal organ.

The vomeronasal organ (VNO) is a part of the accessory olfactory system, which detects pheromones and chemical factors that trigger a spectrum of sexual and social behaviors. The vomeronasal epithelium (VNE) shares several features with the epithelium of the main olfactory epithelium (MOE). However, it is a distinct neuroepithelium populated by chemosensory neurons that differ from the olfactory sensory neurons in cellular structure, receptor expression, and connectivity. The vomeronasal organ of rodents comprises a sensory epithelium (SE) and a thin non-sensory epithelium (NSE) that morphologically resembles the respiratory epithelium. Sox2-positive cells have been previously identified as the stem cell population that gives rise to neuronal progenitors in MOE and VNE. In addition, the MOE also comprises p63 positive horizontal basal cells, a second pool of quiescent stem cells that become active in response to injury. Immunolabeling against the transcription factor p63, Keratin-5 (Krt5), Krt14, NrCAM, and Krt5Cre tracing experiments highlighted the existence of horizontal basal cells distributed along the basal lamina of SE of the VNO. Single cell sequencing and genetic lineage tracing suggest that the vomeronasal horizontal basal cells arise from basal progenitors at the boundary between the SE and NSE proximal to the marginal zones. Moreover, our experiments revealed that the NSE of rodents is, like the respiratory epithelium, a stratified epithelium where the p63/Krt5+ basal progenitor cells self-replicate and give rise to the apical columnar cells facing the lumen of the VNO.

The value of oestrogen receptor, progesterone receptor and keratins 5 and 14 immunohistochemistry in the evaluation of epithelial proliferations at cauterised margins in breast-conserving surgery specimens.

In breast conservative surgery, it is sometimes difficult to decide whether the cauterised tissue at the inked margin represents normal / hyperplastic or neoplastic tissue. We retrospectively assessed the value of ER, PR, CK5 and CK14 IHC in clarifying the nature of cauterised tissues at the margins concerning 34 lesions of 23 patients. 27 cases belonged to lesions that could not be adequately classified on the basis of the HE stains. Two thirds of them could be classified as non-neoplastic or neoplastic and two thirds of the remaining could be favourised as neoplastic or non-neoplastic, with 3/27 cases remaining uncertain. All 4 IHC reactions were helpful in classifying the lesions in almost half of the cases. However, 3 or 4 immunostains were supportive of the classification in 19/27. The most useful stains were the keratins, generally demonstrating a matching pattern of cell labelling with CK5 and CK14. ER and PR were somewhat less useful in classifying uncertain lesions. Considering all the 27 questionable lesions, IHC with ER, PR, CK5 and CK14 clarified the lesions at the cauterised margins in 23 cases. Taken all these considerations into account, CK5, CK14, PR and ER IHC may help in distinguishing between cautery damaged neoplastic and non-neoplastic tissues. All four IHC may yield the best support for decision making, but CK5 and/or CK14 may be sufficient in their own. The essential approach is that the results must be interpreted with caution, in the context of the given patient's disease, to avoid misinterpretations.

Increase of KLK7, cytokeratin 5/6, and elafin expression in head and neck squamous cell carcinoma compared with lung squamous cell carcinoma.

Squamous cell carcinoma is the most common primary tumor in the head and neck epithelium and is the second most common primary tumor type in the lung. Although morphologically indistinguishable from each other with hematoxylin and eosin stain on histology, the tumors have different protein expression profiles. Using 24 formalin-fixed paraffin embedded squamous cell carcinomas of the lung and 24 squamous cell carcinomas in the head and neck, protein expression for cytokeratin 5/6, kallikrein 7, and elafin was evaluated by immunohistochemistry. All three proteins were found to evidence higher expression in head and neck squamous cell carcinoma as compared with that of squamous cell carcinoma of the lung. The differences in expression may help clinical differentiation between primary tumors of the lung from metastatic tumors to the lung from the oral/laryngeal cavities.

Single-cell resolution of human airway epithelial cells exposed to bronchiolitis obliterans-associated chemicals.

Bronchiolitis obliterans (BO) is a fibrotic lung disease characterized by progressive luminal narrowing and obliteration of the small airways. In the nontransplant population, inhalation exposure to certain chemicals is associated with BO; however, the mechanisms contributing to disease induction remain poorly understood. This study's objective was to use single-cell RNA sequencing for the identification of transcriptomic signatures common to primary human airway epithelial cells after chemical exposure to BO-associated chemicals-diacetyl or nitrogen mustard-to help explain BO induction. Primary airway epithelial cells were cultured at air-liquid interface and exposed to diacetyl, nitrogen mustard, or control vapors. Cultures were dissociated and sequenced for single-cell RNA. Differential gene expression and functional pathway analyses were compared across exposures. In total, 75,663 single cells were captured and sequenced from all exposure conditions. Unbiased clustering identified 11 discrete phenotypes, including 5 basal, 2 ciliated, and 2 secretory cell clusters. With chemical exposure, the proportion of cells assigned to keratin 5+ basal cells decreased, whereas the proportion of cells aligned to secretory cell clusters increased compared with control exposures. Functional pathway analysis identified interferon signaling and antigen processing/presentation as pathways commonly upregulated after diacetyl or nitrogen mustard exposure in a ciliated cell cluster. Conversely, the response of airway basal cells differed significantly with upregulation of the unfolded protein response in diacetyl-exposed basal cells, not seen in nitrogen mustard-exposed cultures. These new insights provide early identification of airway epithelial signatures common to BO-associated chemical exposures.NEW & NOTEWORTHY Bronchiolitis obliterans (BO) is a devastating fibrotic lung disease of the small airways, or bronchioles. This original manuscript uses single-cell RNA sequencing for identifying common signatures of chemically exposed airway epithelial cells in BO induction. Chemical exposure reduced the proportion of keratin 5+ basal cells while increasing the proportion of keratin 4+ suprabasal cells. Functional pathways contributory to these shifts differed significantly across exposures. These new results highlight similarities and differences in BO induction across exposures.

Transcriptomic and immunophenotypic characterization of two cases of adamantinoma-like Ewing sarcoma of the thyroid gland.

INTRODUCTION: Adamantinoma-like Ewing sarcoma (ALES) is a rare aggressive malignancy occasionally diagnosed in the thyroid gland. ALES shows basaloid cytomorphology, expresses keratins, p63, p40, frequently CD99, and harbours the t(11;22) EWSR1::FLI1 translocation. There is debate on whether ALES resembles more sarcoma or carcinoma. METHODS: We performed RNA sequencing from two ALES cases and compared findings with skeletal Ewing's sarcomas and nonneoplastic thyroid tissue. ALES was investigated by in situ hybridization (ISH) for high-risk human papillomavirus (HPV) DNA and immunohistochemistry for the following antigens: keratin 7, keratin 20, keratin 5, keratins (AE1/AE3 and CAM5.2), CD45, CD20, CD5, CD99, chromogranin, synaptophysin, calcitonin, thyroglobulin, PAX8, TTF1, S100, p40, p63, p16, NUT, desmin, ER, FLI1, INI1, and myogenin. RESULTS: An uncommon EWSR1::FLI transcript with retained EWSR1 exon 8 was detected in both ALES cases. Regulators of EWSR1::FLI1 splicing (HNRNPH1, SUPT6H, SF3B1) necessary for production of a functional fusion oncoprotein, as well as 53 genes (including TNNT1, NKX2.2) activated downstream to the EWSR1::FLI1 cascade, were overexpressed. Eighty-six genes were uniquely overexpressed in ALES, most of which were related to squamous differentiation. Immunohistochemically, ALES strongly expressed keratins 5, AE1/AE3 and CAM5.2, p63, p40, p16, and focally CD99. INI1 was retained. The remaining immunostains and HPV DNA ISH were negative. CONCLUSION: Comparative transcriptomic profiling reveals overlapping features of ALES with skeletal Ewing's sarcoma and an epithelial carcinoma, as evidenced by immunohistochemical expression of keratin 5, p63, p40, CD99, the transcriptome profile, and detection of EWSR1::FLI1 fusion transcript by RNA sequencing.

Keratin 5 in Lung Cancer Specimens: Comparison of Four Antibody Clones and KRT5 mRNA-ISH.

Recent improvements in the medical treatment of non-small cell lung carcinoma have made the histopathological distinction between adenocarcinomas (ACs) and squamous cell carcinomas (SCCs) increasingly important. One immunohistochemical marker of squamous differentiation is Keratin 5 (K5). Several K5 antibody clones are commercially available, and data from external quality assessment (NordiQC) have shown large variations in their performance. However, comparing antibody performance characteristics of optimized K5 immunohistochemical assays in lung cancer specimens is needed. Tissue microarrays comprising 31 SCCs, 59 ACs, 17 large cell carcinomas, 8 large cell neuroendocrine carcinomas, 5 carcinosarcomas, and 10 small cell carcinomas were included. Serial sections from the tissue microarrays were stained using optimized assays based on the K5 mouse monoclonal antibodies D5/16 B4 and XM26, and the K5 rabbit monoclonal antibodies SP27 and EP1601Y, respectively. The staining reactions were assessed using H-score (0-300). In addition, p40 immunohistochemistry and KRT5 mRNA-ISH analyses were conducted. Clone SP27 showed significantly higher analytical sensitivity than the other 3 clones. However, a distinct positive reaction was observed in 25% of the ACs using clone SP27 but not with the other clones. Clone D5/16 B4 displayed granular staining in 14 ACs, probably representing Mouse Ascites Golgi-reaction. A weak, scattered expression of KRT5 mRNA was seen in 71% of the ACs. In conclusion, the K5 antibody clones D5/16 B4, EP1601Y, and XM26 showed equal sensitivity in lung cancer specimens, but D5/16 B4 also showed nonspecific Mouse Ascites Golgi-reaction. Clone SP27 demonstrated superior analytical sensitivity but lower clinical specificity in the differential diagnosis of SCC versus AC.

TRPS1 expression in cytokeratin 5 expressing triple negative breast cancers, its value as a marker of breast origin.

The lack of oestrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 expression in breast cancer (BC) is the basis for the categorization of the tumour as triple negative breast carcinoma (TNBC). The majority of TNBCs are aggressive tumours with common metastases and decreased expression of markers that could help in identifying the metastatic lesion as of mammary origin. Breast markers, such as gross cystic disease fluid protein-15 (GCDPF-15), GATA binding protein 3 (GATA3), mammaglobin (MGB) and SOX10, are not uniquely specific to BC. Our aim was to evaluate trichorhinophalangeal syndrome type 1 (TRPS1) protein as a breast marker in a series of cytokeratin-5-expressing TNBC, mostly corresponding to basal-like TNBCs, previously characterized for the expression of other breast markers. One hundred seventeen TNBCs in tissue microarrays were immunostained for TRPS1. The cut-off for positivity was ≥ 10%. The reproducibility of this classification was also assessed. TRPS1 positivity was detected in 92/117 (79%) cases, and this exceeded the expression of previously tested markers like SOX10 82 (70%), GATA3 11 (9%), MGB 10 (9%) and GCDFP-15 7 (6%). Of the 25 TRPS1-negative cases, 11 were positive with SOX10, whereas 5 to 6 dual negatives displayed positivity for the other makers. The evaluation showed substantial agreement. Of the five markers compared, TRPS1 seems the most sensitive marker for the mammary origin of CK5-expressing TNBCs. Cases that are negative are most often labelled with SOX10, and the remainder may still demonstrate positivity for any of the 3 other markers. TRPS1 has a place in breast marker panels.

Arl13b controls basal cell stemness properties and Hedgehog signaling in the mouse epididymis.

Epithelial cells orchestrate a series of intercellular signaling events in response to tissue damage. While the epididymis is composed of a pseudostratified epithelium that controls the acquisition of male fertility, the maintenance of its integrity in the context of tissue damage or inflammation remains largely unknown. Basal cells of the epididymis contain a primary cilium, an organelle that controls cellular differentiation in response to Hedgehog signaling cues. Hypothesizing its contribution to epithelial homeostasis, we knocked out the ciliary component ARL13B in keratin 5-positive basal cells. In this model, the reduced size of basal cell primary cilia was associated with impaired Hedgehog signaling and the loss of KRT5, KRT14, and P63 basal cell markers. When subjected to tissue injury, the epididymal epithelium from knock-out mice displayed imbalanced rates of cell proliferation/apoptosis and failed to properly regenerate in vivo. This response was associated with changes in the transcriptomic landscape related to immune response, cell differentiation, cell adhesion, and triggered severe hypoplasia of the epithelium. Together our results indicate that the ciliary GTPase, ARL13B, participates in the transduction of the Hedgehog signaling pathway to maintain basal cell stemness needed for tissue regeneration. These findings provide new insights into the role of basal cell primary cilia as safeguards of pseudostratified epithelia.

Lycopene protects against Bisphenol A induced toxicity on the submandibular salivary glands via the upregulation of PPAR-γ and modulation of Wnt/ß-catenin signaling.

BACKGROUND AND AIM: The submandibular salivary glands (SMG) represent a suitable model for studying epithelial cell growth and differentiation. Bisphenol A (BPA) is a xenoestrogen, synthesized to produce polymers such as polycarbonates and epoxy resins. There are concerns about the occurrence of BPA in food, water as well as its appearance in human tissues and body fluids. Lycopene (LYC) is a carotenoid compound that exerts antioxidant and anti-inflammatory properties. This work was performed to study possible protective effect of LYC against BPA toxicity in SMG. MATERIAL AND METHODS: 40 albino rats were divided into 4 groups; Group I: served as controls. Group II: rats received LYC (4 mg/kg, p.o), Group III: rats received BPA (10 mg/kg, p.o) and Group IV: rats received LYC (4 mg/kg, p.o) and BPA (10 mg/kg, p.o). All drugs were administered for 45 days then under anesthesia, rats were sacrificed. The SMG specimens were taken for histological and biochemical studies. RESULTS: BPA resulted in a significant rise of malondialdehyde, tumor necrosis factor-α and interleukine-1ß. In contrast, the tissue levels of glutathione and PPAR-γ were significantly decreased. BPA activated Wnt/ß-catenin pathway evidenced by upregulating WNT3a, ß-catenin and c-myc expression. Moreover, SMG of BPA showed degenerative changes that affected the parenchymal and stromal elements of the glands. The immunohistochemical localization of cytokeratin 5,6 and 18 of BPA rats revealed weak immunostaining of the serous secretory cells, myoepithelial cells and ductal cells. Upon treatment with LYC, glutathione and PPAR-γ were restored. CONCLUSION: LYC acted as a protective agent against BPA-induced pathological changes in SMG.

Histopathologic and transcriptomic phenotypes of a conditional RANKL transgenic mouse thymus.

Although conventional knockout and transgenic mouse models have significantly advanced our understanding of Receptor Activator of NF-κB Ligand (RANKL) signaling in intra-thymic crosstalk that establishes self-tolerance and later stages of lymphopoiesis, the unique advantages of conditional mouse transgenesis have yet to be explored. A main advantage of conditional transgenesis is the ability to express a transgene in a spatiotemporal restricted manner, enabling the induction (or de-induction) of transgene expression during predetermined stages of embryogenesis or during defined postnatal developmental or physiological states, such as puberty, adulthood, and pregnancy. Here, we describe the K5: RANKL bigenic mouse, in which transgene derived RANKL expression is induced by doxycycline and targeted to cytokeratin 5 positive medullary thymic epithelial cells (mTECs). Short-term doxycycline induction reveals that RANKL transgene expression is significantly induced in the thymic medulla and only in response to doxycycline. Prolonged doxycycline induction in the K5: RANKL bigenic results in a significantly enlarged thymus in which mTECs are hyperproliferative. Flow cytometry showed that there is a marked enrichment of CD4+ and CD8+ single positive thymocytes with a concomitant depletion of CD4+ CD8+ double positives. Furthermore, there is an increase in the number of FOXP3+ T regulatory (Treg) cells and Ulex Europaeus Agglutinin 1+ (UEA1+) mTECs. Transcriptomics revealed that a remarkable array of signals-cytokines, chemokines, growth factors, transcription factors, and morphogens-are governed by RANKL and drive in part the K5: RANKL thymic phenotype. Extended doxycycline administration to 6-weeks results in a K5: RANKL thymus that begins to display distinct histopathological features, such as medullary epithelial hyperplasia, extensive immune cell infiltration, and central tissue necrosis. As there are intense efforts to develop clinical approaches to restore thymic medullary function in the adult to treat immunopathological conditions in which immune cell function is compromised following cancer therapy or toxin exposure, an improved molecular understanding of RANKL's involvement in thymic medulla enlargement will be required. We believe the versatility of the conditional K5: RANKL mouse represents a tractable model system to assist in addressing this requirement as well as many other questions related to RANKL's role in thymic normal physiology and disease processes.

Trophoblast cell-surface antigen 2 (TROP2) expression in triple-negative breast cancer.

BACKGROUND: Trophoblast cell-surface antigen 2 (TROP2) is related to tumor proliferation enhancement and poor prognosis. An antibody targeting TROP2 was developed to treat metastatic triple-negative breast cancer (TNBC) which has a limited treatment modality. To characterize the TROP2 expressing tumors in TNBC, we analyzed TROP2 expression in three cohorts; (1) primary tumor without neoadjuvant chemotherapy, (2) primary tumor with neoadjuvant chemotherapy, and (3) metastatic tumor. METHODS: A total of 807 TNBC cases were evaluated for TROP2 immunohistochemical expression. We evaluated the TROP2 H-score distribution in the three cohorts. Tumors were divided into two groups based on TROP2 expression (high vs. low). We analyzed the relationship between clinicopathologic features and markers, including epidermal growth factor receptor, cytokeratin 5/6, p53, and Ki-67, and prognostic significance at high vs. low TROP2 expression. RESULTS: There was no difference in TROP2 H-score distribution between the three cohorts. Moderate-to-strong membranous expression of TROP2 in at least 10% of tumor cells was present in 662 cases (82.0%) in Cohort 1, 59 cases (89.4%) in Cohort 2, and 23 cases (88.5%) in Cohort 3. There was no significant difference in clinicopathologic features between high vs. low TROP2 in all cohorts. TROP2 H-score was an independent poor prognostic factor for overall survival in Cohort 3. CONCLUSIONS: TNBC showed similar TROP2 expression regardless of neoadjuvant treatment or primary tumor/metastasis. Although the prognostic significance of TROP2 expression in metastatic TNBC has been revealed, further evaluation of the predictive value of TROP2 expression for targeted therapy is needed.

Expression of the pro-inflammatory P2Y14 receptor in the non-vasectomized and vasectomized human epididymis.

BACKGROUND: Vasectomy causes spermatozoa accumulation in the epididymis, which may cause epididymitis. Inflammation is triggered by alert molecules released following tissue stress or injury. These include uracil-diphosphate glucose (UDP)-glucose, which activates the pro-inflammatory P2Y14 receptor (P2Y14), and induces immune cell recruitment. However, little is known about P2Y14 in the epididymis and its potential activation following vasectomy. OBJECTIVES: (i) To localize P2Y14 in the human excurrent duct; and (ii) to examine the effect of vasectomy on P2Y14 protein and P2RY14 mRNA content, the production of selected cytokines and chemokines, and immune cell recruitment in the epididymis. MATERIAL AND METHODS: In situ hybridization, qRT-PCR, western blotting, immunohistochemistry, and immunofluorescence were performed in banked human epididymis samples. RESULTS: P2RY14 mRNA and P2Y14 protein were detected in epithelial cells in the efferent duct, epididymis and vas deferens in non-vasectomized men. Keratin 5 (KRT5)-positive basal cells were strongly labeled for P2Y14 in all epididymal segments. A progressive apical localization was detected in principal cells (negative for the proton pump V-ATPase) from the corpus to the cauda. A subset of V-ATPase-positive clear cells also showed strong P2Y14 labeling. Vasectomy induced an increase in P2RY14 mRNA in the corpus and cauda, and stronger apical labeling in principal cells in the corpus. CXCL10 mRNA increased in the cauda and CCL2 mRNA decreased in the corpus of vasectomized versus non-vasectomized men. No change in IL-8 and IL-1ß mRNA was detected. Numerous CD45+ leukocytes were detected in the interstitium of the corpus and cauda following vasectomy, while only a few were seen in non-vasectomized men. Several CD45+ leukocytes, some of which containing spermatozoa, were detected in the corpus lumen following vasectomy. DISCUSSION AND CONCLUSION: Our study indicates that vasectomy-induced spermatozoa congestion may lead to an inflamed-prone local environment characterized by potential activation of P2Y14 and recruitment of immune cells in the epididymis.

Oncocytic low-grade myoepithelial carcinoma ex pleomorphic adenoma - A rare case illustrating key learning points.

BACKGROUND: Oncocytic myoepithelial carcinoma ex pleomorphic adenoma neoplastic is a rare neoplastic event and may not display overt malignant radiological features. METHODS: Using routine histopathology and immunohistochemistry, we characterize a case of low-grade oncocytic carcinoma ex pleomorphic adenoma. RESULTS: The tumor arose in the left parotid gland in a 59 year old female. Computed tomography (CT) imaging demonstrated a well-defined, lobulated, enhancing lesion with relative central stellate hypoenhancement. Histologically, the tumor displayed a multi-nodular, non-destructive, invasive pattern, low mitotic activity (one mitotic figure per 10 high power fields) and a small remnant focus of pleomorphic adenoma. The neoplastic cells showed significant expression of cytokeratin 5/6, S-100 protein, smooth muscle actin and p63. CONCLUSION: Low-grade oncocytic carcinoma ex pleomorphic adenoma is a challenging histopathological diagnosis which can be established with use of immunohistochemistry, generous tumor sampling and recognition of the multi-nodular, non-destructive, pattern of invasion. In the absence of clear-cut tumor encroachment into external structures, its malignant nature may not be easily identified on pre-operative imaging.

Kinase Inhibition by PKC412 Prevents Epithelial Sheet Damage in Autosomal Dominant Epidermolysis Bullosa Simplex through Keratin and Cell Contact Stabilization.

Epidermolysis bullosa simplex (EBS) is a severe and potentially life-threatening disorder for which no adequate therapy exists. Most cases are caused by dominant sequence variations in keratin genes K5 or K14, leading to the formation of cytoplasmic keratin aggregates, profound keratinocyte fragility, and cytolysis. We hypothesized that pharmacological reduction of keratin aggregates, which compromise keratinocyte integrity, represents a viable strategy for the treatment of EBS. In this study, we show that the multikinase inhibitor PKC412, which is currently in clinical use for acute myeloid leukemia and advanced systemic mastocytosis, reduced keratin aggregation by 40% in patient-derived K14.R125C EBS-associated keratinocytes. Using a combination of epithelial shear stress assay and real-time impedance spectroscopy, we show that PKC412 restored intercellular adhesion. Molecularly, global phosphoproteomic analysis together with immunoblots using phosphoepitope-specific antibodies revealed that PKC412 treatment altered phosphorylated sites on keratins and desmoplakin. Thus, our data provide a proof of concept to repurpose existing drugs for the targeted treatment of EBS and showcase how one broad-range kinase inhibitor reduced keratin filament aggregation in patient-derived EBS keratinocytes and the fragility of EBS cell monolayers. Our study paves the way for a clinical trial using PKC412 for systemic or local application in patients with EBS.

Cytokeratins 5 and 17 Maintain an Aggressive Epithelial State in Basal-Like Breast Cancer.

Basal-like breast cancers (BLBC) are the most common triple-negative subtype (hormone receptor and HER2 negative) with poor short-term disease outcome and are commonly identified by expression of basal cytokeratins (CK) 5 and 17. The goal of this study was to investigate whether CK5 and CK17 play a role in adverse behavior of BLBC cells. BLBC cell lines contain heterogeneous populations of cells expressing CK5, CK17, and the mesenchymal filament protein vimentin. Stable shRNA knockdown of either CK5 or CK17 compared with non-targeting control in BLBC cells was sufficient to promote an epithelial-mesenchymal transition (EMT) gene signature with loss of E-cadherin and an increase in vimentin expression. Relative to control cells, CK5 and CK17 knockdown cells acquired a more spindle-like morphology with increased cell scattering and were more invasive in vitro. However, CK5 or CK17 knockdown compared with control cells generated decreased lymph node and lung metastases in vivo. Loss of CK5 or CK17 moderately reduced the IC50 dose of doxorubicin in vitro and led to increased doxorubicin efficacy in vivo. Single-cell RNA-sequencing of BLBC patient-derived xenografts identified heterogeneous populations of CK5/CK17, vimentin, and dual basal CK/vimentin-positive cells that fell on an EMT spectrum of epithelial, mesenchymal, and intermediate, respectively, whereas knockdown of CK5 transitioned cells toward a more mesenchymal score. IMPLICATIONS: This study supports that basal CKs 5 and 17 contribute to the adverse behavior of BLBC cells and could be an untapped source of therapeutic vulnerability for this aggressive disease.

Human-Induced Pluripotent Stem Cell‒Derived Keratinocytes, a Useful Model to Identify and Explore the Pathological Phenotype of Epidermolysis Bullosa Simplex.

Epidermolysis bullosa simplex (EBS), an autosomal dominant skin disorder, is characterized by skin fragility. Genetically, the majority of cases are related to missense sequence variations in two keratin genes K5 or K14, leading to cytolysis of basal keratinocytes (KCs) and intraepidermal blistering. Progress toward the identification of treatments has been hampered by an incomplete understanding of the mechanisms underlying this disease and availability of relevant and reliable in vitro models recapitulating the physiopathological mechanisms. Recent advances in stem cell field have fueled the prospect that these limitations could be overcome, thanks to the availability of disease-specific human induced pluripotent stem cells (hiPSCs). In this study, we generated hiPSC-derived KCs from patients carrying keratin gene K5-dominant sequence variations and compared them with nonaffected hiPSC-derived KCs as well as their primary counterparts. Our results showed that EBS hiPSC-derived KCs displayed proliferative defects, increased capacity to migrate, alteration of extracellular signal‒regulated kinase signaling pathway, and cytoplasmic keratin filament aggregates as observed in primary EBS KCs. Of interest, EBS hiPSC-derived KCs exhibited downregulation of hemidesmosomal proteins, revealing the different effects of keratin gene K5 sequence variations on keratin cytoskeletal organization. With a combination of culture miniaturization and treatment with the chaperone molecule 4-phenylbutyric acid, our results showed that hiPSC-derived KCs represent a suitable model for identifying novel therapies for EBS.

Multicenter study of diffuse pleural mesothelioma. Histopathological and immunohistochemical features.

The pathological diagnosis of diffuse pleural mesothelioma (DPM) contributes to treatment selection and clinical trials interpretation. To know its characteristics and evaluate the viability of comprehensive pathological diagnosis of DPM in Argentina we did a retrospective descriptive study of DPM cases reported from 2009 to 2018. We analyzed 398 cases corresponding to 238 (60%) men and 160 (40%) women, median age 66 years, from surgical biopsies (78%), small biopsies (16.5%) and surgical resections (5.5%). The 77% were epithelioid (E-DPM), 12% biphasic, 10% sarcomatoid, and 4 cases transitional variant. In E-DPM the main pattern was tubular in 36% and solid in 33%. There was a second pattern in 179 cases. Considering the main pattern and the second together, 48% presented tubular subtype and 48% solid subtype. Stroma, necrosis, and nuclear score showed significant differences between E-DPM and non-epithelioid mesotheliomas. Overall tumor grade was predominantly low in E-DPM, except for 42% of the solid main pattern. We recognized the transitional variant extensively in 4 cases and focally in 8. The immunohistochemical antibody panel used included pan-cytokeratin, calretinin, WT-1, cytokeratin 5, CEA and TTF-1. The expression of cytokeratin 5, calretinin and WT-1 was lower in the sarcomatoid type (43%, 87 and 37%) than in the epithelioid type (92%, 98% and 93%). This study highlights the tumor heterogeneity of DPM that shows the diagnostic difficulty, and the feasibility of evaluating histological aggressiveness in E-DPM, B-DPM and S-DPM in our country.

El diagnóstico patológico del mesotelioma pleural difuso (MPD) contribuye a la selección del tratamiento y a la interpretación de los ensayos clínicos. Para conocer sus características y evaluar la viabilidad del diagnóstico patológico de MPD en Argentina se realizó un estudio descriptivo retrospectivo de los casos de MPD informados de 2009 a 2018. Se analizaron 398 casos correspondientes a 238 (60%) hombres y 160 (40%) mujeres, mediana de edad de 66 años, a partir de biopsias quirúrgicas (78%), biopsias pequeñas (16.5%) y resecciones quirúrgicas (5.5%). El 77% fue epitelioide (E-MPD), 12% bifásicos, 10% sarcomatoides y 4 casos variante transicional. En E-MPD se encontró como patrón principal el tubular en 36% y el sólido en 33%. Hubo un segundo patrón en 179 casos. Considerando el principal y el segundo patrón en conjunto, el 48% presentó subtipo tubular y el 48% subtipo sólido. El estroma, la necrosis y el score nuclear mostraron diferencias significativas entre E-MPD y mesoteliomas no epitelioides. El grado general del tumor fue predominantemente bajo en E-MPD, a excepción del 42% del patrón principal sólido. Reconocimos la variante transicional en forma extensa en 4 casos y focalmente en 8. La expresión de citoqueratina 5, calretinina y WT-1 fue menor en el tipo sarcomatoide (43%, 87 y 37%) que en el tipo epitelioide (92%, 98% y 93%). Este estudio destaca la heterogeneidad tumoral de MPD que evidencia la dificu ltad en el diagnóstico y la viabilidad de evaluar la agresividad histológica en E-MPD, B-MPD y S-MPD en nuestro país.

Differential LysoTracker Uptake Defines Two Populations of Distal Epithelial Cells in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal degenerative lung disease of unknown etiology. Although in its final stages it implicates, in a reactive manner, all lung cell types, the initial damage involves the alveolar epithelial compartment, in particular the alveolar epithelial type 2 cells (AEC2s). AEC2s serve dual progenitor and surfactant secreting functions, both of which are deeply impacted in IPF. Thus, we hypothesize that the size of the surfactant processing compartment, as measured by LysoTracker incorporation, allows the identification of different epithelial states in the IPF lung. Flow cytometry analysis of epithelial LysoTracker incorporation delineates two populations (Lysohigh and Lysolow) of AEC2s that behave in a compensatory manner during bleomycin injury and in the donor/IPF lung. Employing flow cytometry and transcriptomic analysis of cells isolated from donor and IPF lungs, we demonstrate that the Lysohigh population expresses all classical AEC2 markers and is drastically diminished in IPF. The Lysolow population, which is increased in proportion in IPF, co-expressed AEC2 and basal cell markers, resembling the phenotype of the previously identified intermediate AEC2 population in the IPF lung. In that regard, we provide an in-depth flow-cytometry characterization of LysoTracker uptake, HTII-280, proSP-C, mature SP-B, NGFR, KRT5, and CD24 expression in human lung epithelial cells. Combining functional analysis with extracellular and intracellular marker expression and transcriptomic analysis, we advance the current understanding of epithelial cell behavior and fate in lung fibrosis.

Human alveolar type 2 epithelium transdifferentiates into metaplastic KRT5+ basal cells.

Loss of alveolar type 2 cells (AEC2s) and the ectopic appearance of basal cells in the alveoli characterize severe lung injuries such as idiopathic pulmonary fibrosis (IPF). Here we demonstrate that human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, transdifferentiate into basal cells in response to fibrotic signalling in the lung mesenchyme, in vitro and in vivo. Single-cell analysis of normal hAEC2s and mesenchymal cells in organoid co-cultures revealed the emergence of pathologic fibroblasts and basaloid cells previously described in IPF. Transforming growth factor-ß1 and anti-bone morphogenic protein signalling in the organoids promoted transdifferentiation. Trajectory and histologic analyses of both hAEC2-derived organoids and IPF epithelium indicated that hAEC2s transdifferentiate into basal cells through alveolar-basal intermediates that accumulate in proximity to pathologic CTHRC1hi/TGFB1hi fibroblasts. Our study indicates that hAEC2 loss and expansion of alveolar metaplastic basal cells in severe human lung injuries are causally connected through an hAEC2-basal cell lineage trajectory driven by aberrant mesenchyme.