Integrating single-cell and bulk RNA sequencing reveals CK19 + cancer stem cells and their specific SPP1 + tumor-associated macrophage niche in HBV-related hepatocellular carcinoma.

PURPOSE: Cytokeratin 19-positive cancer stem cells (CK19 + CSCs) and their tumor-associated macrophages (TAMs) have not been fully explored yet in the hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: Single-cell RNA sequencing was performed on the viable cells obtained from 11 treatment-naïve HBV-associated HCC patients, including 8 CK19 + patients, to elucidate their transcriptomic landscape, CK19 + CSC heterogeneity, and immune microenvironment. Two in-house primary HCC cohorts (96 cases-related HBV and 89 cases with recurrence), TCGA external cohort, and in vitro and in vivo experiments were used to validate the results. RESULTS: A total of 64,581 single cells derived from the human HCC and adjacent normal tissues were sequenced, and 11 cell types were identified. The result showed that CK19 + CSCs were phenotypically and transcriptionally heterogeneous, co-expressed multiple hepatics CSC markers, and were positively correlated with worse prognosis. Moreover, the SPP1 + TAMs (TAM_SPP1) with strong M2-like features and worse prognosis were specifically enriched in the CK19 + HCC and promoted tumor invasion and metastasis by activating angiogenesis. Importantly, matrix metalloproteinase 9 (MMP9) derived from TAM_SPP1, as the hub gene of CK19 + HCC, was activated by the VEGFA signal. CONCLUSIONS: This study revealed the heterogeneity and stemness characteristics of CK19 + CSCs and specific immunosuppressive TAM_SPP1 in CK19 + HCC. The VEGFA signal can activate TAM_SPP1-derived MMP9 to promote the invasion and metastasis of CK19 + HCC tumors. This might provide novel insights into the clinical treatment of HCC patients.

Comparison of Two Human Skin Cell Isolation Protocols and Their Influence on Keratinocyte and Fibroblast Culture.

For the development of advanced therapies, the use of primary cells instead of cell lines is preferred. The manufacture of human tissue-engineered skin substitutes requires efficient isolation and culture protocols allowing a massive expansion of the cells in culture from an initial specimen of a minimal size. This study compared two skin cell isolation protocols, routinely applied in two clinical laboratories. Epithelial (keratinocytes) and dermal (fibroblasts) cells were isolated and cultured from three human skin biopsies (N = 3). The two-step digestion protocol (LOEX-Protocol) firstly used thermolysin to enzymatically disrupt the dermal-epidermal junction while, for the one-step digestion protocol (UPCIT-Protocol), mechanical detachment with scissors was applied. Then, the epidermal and dermal layers were digested, respectively, to achieve cell isolation. The cell size, viability, yield and growth were analyzed over five passages (P). The colony-forming efficiency (CFE) and Keratin 19 (K19) expression of epithelial cells were also assessed after P0 and P1. Regarding the dermal cells, no significant differences were observed in the tested parameters of isolation and culture. However, for the epithelial cells, viability was higher (93% vs. 85%) and the number of cells extracted per cm2 of skin was 3.4 times higher using the LOEX-Protocol compared to the UPCIT-Protocol. No significant difference was observed for any parameter once the keratinocytes were cultured from P1 to P4. The CFE and K19 expression decreased from P0 to P1 in both protocols, probably due to the culture process. This study shows that both protocols enable the efficient isolation of skin dermal and epithelial cells and subsequent culture to produce grafts destined for the treatment of patients.

Keratin 19 binds and regulates cytoplasmic HNRNPK mRNA targets in triple-negative breast cancer.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (HNRNPK) regulates pre-mRNA processing and long non-coding RNA localization in the nucleus. It was previously shown that shuttling of HNRNPK to the cytoplasm promotes cell proliferation and cancer metastasis. However, the mechanism of HNRNPK cytoplasmic localization, its cytoplasmic RNA ligands, and impact on post-transcriptional gene regulation remain uncharacterized. RESULTS: Here we show that the intermediate filament protein Keratin 19 (K19) directly interacts with HNRNPK and sequesters it in the cytoplasm. Correspondingly, in K19 knockout breast cancer cells, HNRNPK does not localize in the cytoplasm, resulting in reduced cell proliferation. We comprehensively mapped HNRNPK binding sites on mRNAs and showed that, in the cytoplasm, K19-mediated HNRNPK-retention increases the abundance of target mRNAs bound to the 3' untranslated region (3'UTR) at the expected cytidine-rich (C-rich) sequence elements. Furthermore, these mRNAs protected by HNRNPK in the cytoplasm are typically involved in cancer progression and include the p53 signaling pathway that is dysregulated upon HNRNPK knockdown (HNRNPK KD) or K19 knockout (KRT19 KO). CONCLUSIONS: This study identifies how a cytoskeletal protein can directly regulate gene expression by controlling the subcellular localization of RNA-binding proteins to support pathways involved in cancer progression.

Scoring System and Diagnosis of Papillary Thyroid Carcinoma Using Human Bone Marrow Endothelium Marker-1, Cytokeratin 19, and Galectin-3.

Background: The increasing incidence of papillary thyroid carcinoma (PTC) and the inadequacy of routine histologic examination in its diagnosis necessitate the application of ancillary studies like immunohistochemistry. This research aimed to investigate the scoring system and diagnosis of PTC with cytokeratin 19 (CK19), human bone marrow endothelium marker-1 (HBME-1), and galectin-3. Methods: This experimental laboratory study was performed at Babol University of Medical Sciences, Mazandaran, Iran from April 2017 to March 2019. Neoplastic and nonneoplastic tissue samples of 100 cases with a diagnosis of PTC were selected by convenience sampling. CK19, HBME-1, and galectin-3 immunohistochemistry markers were used on tissue samples. Analysis was performed using the t test and the chi-square test, as well as the receiver operator characteristic (ROC) curve (significance level P < 0.05). Results: The CK19 staining was observed in all 100 (100%) non-neoplastic tissues, but HBME-1 and galectin-3 were positive in 36 (36%) and 14 (14%) of non-neoplastic tissues, respectively. The intensity scores of all the markers and their total had significantly different means in PTC and non-neoplastic tissues (P < 0.001). A significant difference was observed between the total score of each marker and the total score of their combination (P < 0.001). The combination of all 3 markers with an 11.5 0 cut-off for the total score showed the most sensitive (0.99) and specific (1.00) results. Conclusion: Interpreting CK19, HBME-1, and galectin-3 with the aid of the proposed scoring system was fruitful. HBME-1 and galectin-3 can be used individually or in combination for the diagnosis of PTC.

Keratin19 promotes pancreatic cancer progression and poor prognosis via activating the Hedgehog pathway.

Pancreatic cancer is a serious threat to human health, with strong invasiveness, rapid progression and poor prognosis. Tumors expressing keratin 19 (K19) have stronger invasiveness and a worse prognosis. However, the role and mechanism of K19 in pancreatic cancer have remained largely elusive. In the present study, K19 expression was detected in pancreatic cancer tissues, its effect on proliferation, apoptosis and metastasis of pancreatic cancer at the cellular, in vivo preclinical and clinical levels was evaluated and its effect on the Hedgehog pathway was analyzed. K19 was significantly overexpressed in pancreatic cancer, promoted pancreatic cancer proliferation and metastasis, inhibited tumor cell apoptosis and was associated with poor prognosis. Mechanistically, these effects were mediated through the activation of the Hedgehog pathway. In conclusion, K19 may be a novel target molecule for pancreatic cancer treatment.

Increased Hes1 expression to regulate differentiation of liver epithelial progenitor cells into cholangiocytes / 西安交通大学学报(医学版)

【Objective】 To use hairy enhancer of split 1 (Hes1) to regulate the differentiation of liver epithelial progenitor cells (LEPCs) into cholangiocytes. 【Methods】 The vectors, pTet-on and pTRE2hyg-Hes1, were transfected into LEPCs. The expression of Hes1 was induced by doxycycline (DOX) with different concentrations (0, 0.1, 1, 5, 10, 50, 100 and 500 μg/mL). The expressions of Hes1, molecular markers of hepatocyte and cholangiocyte, glutathione synthetase (Gss), keratin 19 (Krt19) and hepatic nuclear factor 1β (HNF1β) in LEPCs were verified by Western blotting, RT-PCR, Real-time PCR, immunocytochemistry and immunofluorescence. 【Results】 The expression of Hes1 in LEPCs transfected by pTet-on/pTRE2hyg-Hes1 was increased by 11.21 fold when induced by DOX at 10 ug/mL, which drove the LEPCs to differentiate into biliary epithelial cells. With increasing expression of Hes1, cholangiocyte markers, Krt19 and HNF1β, were significantly upregulated, while the hepatocyte marker, Gss, was obviously downregulated. 【Conclusion】 DOX at 10 μg/mL may induce a suitably up-regulated expression of Hes1 in LEPCs double-transfected by pTet-on and pTRE2hyg-Hes1, and the suitable high-expression rather than over-expression of Hes1 can regulate LEPCs to differentiate into cholangiocytes.

Study on the correlation of cyfra21-1 and modified ultrasound in connective tissue disease associated with interstitial lung disease / 中华风湿病学杂志

Objective:To investigate the correlation between cytokeratin 19 fragment(CYFRA21-1), modified ultrasound B-line and connective tissue disease associated with interstitial lung disease (CTD-ILD).Methods:The data of 112 patients with CTD hospitalized in the Department of Rheumatology and Immunology of the Second Hospital of Fujian Medical University from September 2019 to December 2021 were retrospectively collected. Sixty patients in the CTD-ILD group and 52 patients in the connective tissue disease without interstitial lung disease (CTD-noILD) group were included. The t-test and χ2 test were used to compare the demographic characteristics and tumor-associated antigens of the two groups of patients. Modified ultrasound score and HRCT Warrick score were evaluated by Pearson correlation analysis. In addition, the relationship between CYFRA21-1, modified ultrasound score and Warrick score were evaluated, and the diagnostic efficacy of CYFRA21-1 and modified ultrasound of CTD-ILD was evaluated and analyzed by binary logistic regression analysis. Results:Patients in the CTD-ILD group had higher CYFRA21-1 concentrations than the CTD-no-ILD group[5.74(4.25, 9.79) ng/ml vs. 2.79(2.21, 3.23) ng/ml, Z=45.94, P<0.001], patients in the CTD-ILD group had higher modified ultrasound scores than the CTD-no-ILD group [44.5(36.5, 60.0) vs. 5.0 (3.2, 6.8), P<0.001]. Modified ultrasound score was positively correlated with Warrick score ( r=0.93, P<0.001) and CYRFA21-1 was positively correlated with modified ultrasound score ( r=0.39, P=0.042). The sensitivity of CYFRA21-1 in determining CTD-ILD was 81.7% and the specificity was 92.3% [ AUC (95% CI)=0.88(0.81, 0.95), P<0.001], the sensitivity of modified ultrasound B-line to determine CTD-ILD was 96.4% and the specificity was 92.9% [ AUC (95% CI)=0.99 (0.97, 1.00), P<0.001]. History of smoking[ OR(95% CI)=9.26(1.11, 77.12), P=0.040] and elevated CYFRA21-1 concentration[ OR(95% CI)=19.40(4.89, 76.95), P<0.001] were risk factors for CTD-ILD. Conclusion:CYFRA21-1 is expected to be a serum marker indicating concomitant ILD in patients with CTD. Modified ultrasound B-line to determine concomitant ILD in CTD patients has good diagnostic utility and can reflect the severity of pulmonary fibrosis in CTD-ILD patients.

The value of enhanced CT combined with serum CYFER21-1 and NSE in the diagnosis of lymph node metastasis in patients with NSCLC / 中国医师杂志

Objective:To investigate the value of preoperative enhanced CT combined with serum cytokeratin fragment 19 (CYFER21-1) and neuron-specific enolase (NSE) in the diagnosis of lymph node metastasis in patients with non-small cell lung cancer (NSCLC).Methods:160 patients with NSCLC admitted to Linyi Cancer Hospital from October 2018 to October 2021 were retrospectively selected. All patients received surgical treatment in our hospital, and 84 patients with lymph node metastasis (metastatic group) and 76 patients without lymph node metastasis (non-metastatic group) were confirmed after surgery. The features of enhanced CT images and serum CYFER21-1 and NSE levels were compared between the two groups before operation, and the value of each index in the diagnosis of lymph node metastasis in patients with NSCLC alone and in combination was analyzed by receiver operating characteristic (ROC) curve.Results:The proportions of patients with lesion diameter ≥3.0 cm, pleural depression, lymph node enlargement shown by CT, lymph node short diameter ≥10 mm, lymph node boundary ambiguity and lymph node enhancement in metastatic group were significantly higher than those in non-metastatic group, with statistical significance (all P<0.05). Serum CYFER21-1 and NSE levels in metastatic group were significantly higher than those in non-metastatic group, with statistical significance (all P<0.05). The area under curve (AUC) of CYFER21-1 and NSE levels in the diagnosis of lymph node metastasis in NSCLC patients were 0.652 and 0.845, respectively, and the diagnostic cut-off values were 4.81 ng/ml and 24.14 ng/ml, respectively. The sensitivity and specificity of CYFER21-1+ NSE+ enhanced CT in the diagnosis of lymph node metastasis in NSCLC patients were 91.67% and 94.74%. Conclusions:Preoperative enhanced CT is of certain clinical value in the diagnosis of lymph node metastasis in NSCLC patients. Combined with serum CYFER21-1 and NSE levels, enhanced CT can further improve the sensitivity and specificity of diagnosis.

Carcinomas assemble a filamentous CXCL12-keratin-19 coating that suppresses T cell-mediated immune attack.

Cancer immunotherapy frequently fails because most carcinomas have few T cells, suggesting that cancers can suppress T cell infiltration. Here, we show that cancer cells of human pancreatic ductal adenocarcinoma (PDA), colorectal cancer, and breast cancer are coated with transglutaminase-2 (TGM2)-dependent covalent CXCL12-keratin-19 (KRT19) heterodimers that are organized as filamentous networks. Since a dimeric form of CXCL12 suppresses the motility of human T cells, we determined whether this polymeric CXCL12-KRT19 coating mediated T cell exclusion. Mouse tumors containing control PDA cells exhibited the CXCL12-KRT19 coating, excluded T cells, and did not respond to treatment with anti-PD-1 antibody. Tumors containing PDA cells not expressing either KRT19 or TGM2 lacked the CXCL12-KRT19 coating, were infiltrated with activated CD8+ T cells, and growth was suppressed with anti-PD-1 antibody treatment. Thus, carcinomas assemble a CXCL12-KRT19 coating to evade cancer immune attack.

Lung and lymph node metastases from hepatocellular carcinoma: Comparison of pathological aspects.

BACKGROUND & AIMS: Extrahepatic metastasis from hepatocellular carcinoma (HCC) is a catastrophic event, yet organ-specific pathological characteristics of metastatic HCC remain unclear. We aimed to characterize the pathological aspects of HCC metastases to various organs. METHODS: We collected intrahepatic HCC (cohort 1, n = 322) and extrahepatic metastatic HCC (cohort 2, n = 130) samples. Clinicopathological evaluation and immunostaining for K19, CD34, αSMA, fibroblast-associated protein (FAP), CAIX, VEGF, PD-L1, CD3, CD8, Foxp3, CD163 and epithelial-mesenchymal transition (EMT)-related markers were performed. RESULTS: Independent factors for extrahepatic metastasis included BCLC stage B-C, microvascular invasion (MVI), vessels encapsulating tumour clusters (VETC)-HCC, K19 and FAP expression, and CD163+ macrophage infiltration (cohort 1, P < .05 for all). Lung metastases (n = 63) had the highest proportion of VETC-HCC and macrotrabecular-massive (MTM)-HCC. Lymph node metastases (n = 19) showed significantly high rates of EMT-high features, K19 expression, fibrous tumour stroma with αSMA and FAP expression, high immune cell infiltration, PD-L1 expression (combined positive score), CD3+, CD8+, Foxp3+ T cell and CD163+ macrophage infiltration (adjusted P < .05 for all). In both cohorts, EMT-high HCCs showed higher rates of K19 expression, fibrous tumour stroma, high immune cell infiltration, PD-L1 expression and CD3+ T cell infiltration, whereas EMT-low HCCs were more frequent among VETC-HCCs (P < .05 for all). Overall phenotypic features were not significantly different between paired primary-metastatic HCCs (n = 32). CONCLUSIONS: Metastatic HCCs to various organs showed different pathological features. VETC and MTM subtypes were related to lung metastasis, whereas K19 expression, EMT-high features with fibrous tumour stroma and high immune cell infiltration were related to lymph node metastasis.

Value of different tumor markers for the diagnosis and prognosis prediction of colon cancer in patients / 中国基层医药

Objective:To comprehensively analyze the clinical values of serum tumor markers of colon cancer, including carbohydrate antigen 724 (CA724), cytokeratin 19 fragment antigen (CYFRA21-1), carbohydrate antigen199 (CA199) and carcinoembryonic antigen (CEA) in the diagnosis and prognosis prediction of colon cancer in patients.Methods:The clinical data of 160 patients with colon cancer who received treatment in Zhuji Central Hospital from January 2018 to December 2020 (observation group) and the clinical data of 156 patients with benign colon polyps who concurrently received physical examination (control group) were retrospectively analyzed. All patients underwent CA724, CYFRA21-1, CA199 and CEA tumor marker screening. The levels of tumor markers, the positive rate of a single tumor marker, and the positive rate of a combination of four markers were compared between the control and observation groups. The levels of tumor markers were compared among different pathological stages. The levels of serum tumor markers were compared among patients with different prognoses based on 1-year follow-up data.Results:CA199-positve rate, CEA-positive rate, CYFRA21-1-positve rate, CA724-positive rate, and the positive rate of a combination of four tumor markers were 85.63% (137/160), 86.88% (139/160), 71.88% (115/160), 85.00% (136/160), and 95.63%(153/160), respectively, which were significantly higher than those in the control group ( χ2 = 8.64, 10.28, 8.33, 9.93, 7.27, all P < 0.001). Serum CA199, CEA, CYFRA21-1 and CA724 levels in patients with stage III-IV colon cancer were (58.96 ± 13.59) U/mL, (38.69 ± 11.84) μg/L, (14.78 ± 3.68) μg/L, (23.68 ± 5.38) U/mL, respectively, which were significantly higher than those in patients with stage I-II colon cancer [(48.35 ± 9.03) U/mL, (23.96 ± 12.25) μg/L, (9.57 ± 2.53) μg/L, (13.02 ± 4.32) U/mL, t = 10.29, 12.02, 8.47, 10.54, all P < 0.001). One-year follow-up results showed that serum levels of CA199, CEA, CYFRA21-1, CA724 in patients with recurrence and metastasis of colon cancer were (38.68 ± 3.04) U/mL, (17.12 ± 4.96) μg/L, (8.94 ± 2.32) μg/L, (11.22 ± 1.94) U/mL, which were significantly higher than those in patients without recurrence of colon cancer [(30.02 ± 2.95) U/mL, (3.75 ± 1.06) μg/L, (3.06 ± 1.15) μg/L, (6.28 ± 1.53) U/mL, t = 8.73, 11.02, 7.72, 7.57, all P < 0.001]. Conclusion:Serum levels of CEA, CA199, CA724 and CYFRA21-1 can be used as important indicators for diagnosis and prognosis prediction of colon cancer.

Histological Heterogeneity of Primary Liver Cancers: Clinical Relevance, Diagnostic Pitfalls and the Pathologist's Role.

Primary liver cancers (PLCs) mainly comprise hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (iCCA), and cHCC-CCA. Combined HCC-CCA and small duct type iCCA show similar clinical presentations, and their histological features are more complex than seen in HCC. Therefore, while their treatment strategy differs, it is difficult to properly diagnose these tumors. Currently, HCC is the only tumor that can be treated by liver transplantation. In addition, small duct type iCCA harbors IDH1/2 mutations and FGFR2 fusions, which can be used for targeted therapy. Thus, improving diagnostic accuracy is crucial. A further point to note is that PLCs often present as multiple liver tumors, and they can be a combination of different types of PLCs or HCCs. In the case of HCCs, two different scenarios are possible, namely intrahepatic metastasis, or multicentric occurrence. Therefore, it is essential to characterize the type of multiple liver tumors. This review aims to clarify the pathological features of HCC, iCCA and cHCC-CCA, including their diagnostic pitfalls and clinical relevance. It is designed to be of use to clinicians who are dealing with PLCs, to provide a better understanding of the pathology of these tumors, and to enable a more accurate diagnosis and optimal treatment choice.

Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells.

A cytoskeletal protein keratin 19 (K19) is highly expressed in breast cancer but its effects on breast cancer cell mechanics are unclear. In MCF7 cells where K19 expression is ablated,we found that K19 is required to maintain rounded epithelial-like shape and tight cell-cell adhesion. A loss of K19 also lowered cell surface E-cadherin levels. Inhibiting internalization restored cell-cell adhesion of KRT19  knockout cells, suggesting that E-cadherin internalization contributed to defective adhesion. Ultimately, while K19 inhibited cell migration and invasion, it was required for cells to form colonies in suspension. Our results suggest that K19 stabilizes E-cadherin complexes at the cell membrane to maintain cell-cell adhesion which inhibits cell invasiveness but provides growth and survival advantages for circulating tumor cells.

Changes and clinical significance of serum matrix metalloproteinase-9, squamous cell carcinoma antigen, cytokeratin-19 fragment, carcinoembryonic antigen and neuron-specific enolase levels in peripheral lung cancer / 中国基层医药

Objective:To investigate the changes and clinical significance of serum matrix metalloproteinase-9 (MMP-9), squamous cell carcinoma antigen (SCC), cytokeratin 19 fragment (CYFRA21-1), carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE) in peripheral lung cancer.Methods:Sixty-eight patients with peripheral lung cancer who received treatment in Luqiao Hospital of Taizhou Enze Medical Center (Group) between January 2017 and January 2020 were included in the observation group. Sixty-five patients with benign lung diseases who concurrently received treatment in the same hospital were included in the observation group 1, and another 65 healthy participants who concurrently received physical examination were included in the control group. Serum levels of MMP-9, CYFRA21-1, SCC, NSE and CEA were compared among the three groups. The sensitivity and specificity of using these indicators alone and in combination in the diagnosis of peripheral lung cancer were compared.Results:Serum levels of MMP-9, CYFRA21-1, SCC, NSE and CEA in the observation group (14.98 ± 2.10) ng/mL, (17.13 ± 2.71) ng/mL, (1.98 ± 0.41) μg/mL, (24.13 ± 2.10) ng/mL and (17.10 ± 2.10) ng/mL, respectively, which were significantly higher than those in the observation group 1 [(9.12 ± 1.41) ng/mL, (10.12 ± 1.58) ng/mL, (1.37 ± 0.31) μg/mL, (16.31 ± 1.78) ng/mL, (12.13 ± 1.79) ng/mL] and control group [(5.10 1 ± 0.68) ng/mL, (6.02 ± 0.94) ng/mL, (0.71 ± 0.11) μg/mL, (11.10 ± 1.02) ng/mL, (8.13 ± 1.02) ng/mL] ( F1 = 932.781, F2 = 737.100, F3 = 368.591, F4 = 989.851, F5 = 462.291, all P < 0.05). Serum levels of MMP-9, CYFRA21-1, SCC, NSE and CEA in patients with stage I-II peripheral lung cancer were (11.12 ± 2.10) ng/mL, (9.12 ± 1.85) ng/mL, (1.52 ± 0.21) μg/mL, (18.12 ± 3.02) ng/mL, (7.52 ± 1.02) ng/mL, respectively, which were significantly lower than those in patients with stage III-IV peripheral lung cancer [(15. 89 ± 2.18) ng/mL, (21.56 ± 2.11) ng/mL, (2.04 ± 0.31) μg/mL, (28.15 ± 2.62) ng/mL, (15.12 ± 1.55) ng/mL, t1 = 9.013, t2 = 25.146, t3 = 7.714, t4 = 14.586, t5 = 22.705, all P < 0.05]. The sensitivity (83.33%) and specificity (86.67%) of combined detection of all indicators were significantly higher than those of single detection of MMP-9 (50.00%, 59.68%), CEA (50.00%, 61.29%), CYFRA21-1 (66.67%, 58.06%), SCC (50.00%, 54.84%) or NSE (66.67%, 58.06%) (all P < 0.05). Conclusion:Serum levels of MMP-9, CYFRA21-1, SCC, NSE and CEA in patients with peripheral lung cancer are significantly increased, which has an important value in the diagnosis of peripheral lung cancer. The combined detection of the above indicators can increase the diagnostic accuracy of peripheral lung cancer in the clinic.

Morphology, Motility, and Cytoskeletal Architecture of Breast Cancer Cells Depend on Keratin 19 and Substrate.

Cancer cells gain motility through events that accompany modulation of cell shape and include altered expression of keratins. However, the role of keratins in change of cancer cell architecture is not well understood. Therefore, we ablated the expression of keratin 19 (K19) in breast cancer cells of the MDA-MB-231 cell line and found that cells lacking K19 become more elongated in culture, with morphological reversion toward the parental phenotype upon transduction of KRT19. Also, the number of actin stress fibers and focal adhesions were significantly reduced in KRT19 knockout (KO) cells. The altered morphology of KRT19 KO cells was then characterized quantitatively using digital holographic microscopy (DHM), which not only confirmed the phenotypic change of KRT19 KO cells but also identified that the K19-dependent morphological change is dependent on the substrate type. A new quantitative method of single cell analysis from DHM, via average phase difference maps, facilitated evaluation of K19-substrate interactive effects on cell morphology. When plated on collagen substrate, KRT19 KO cells were less elongated and resembled parental cells. Assessing single cell motility further showed that while KRT19 KO cells moved faster than parental cells on a rigid surface, this increase in motility became abrogated when cells were plated on collagen. Overall, our study suggests that K19 inhibits cell motility by regulating cell shape in a substrate-dependent manner. Thus, this study provides a potential basis for the altered expression of keratins associated with change in cell shape and motility of cancer cells. © 2020 International Society for Advancement of Cytometry.

Activation of extracellular signal-regulated kinase is associated with hepatocellular carcinoma with aggressive phenotypes.

AIM: Sorafenib inhibits multiple kinase signaling pathways, including the rat sarcoma virus (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and is a promising therapy for hepatocellular carcinoma (HCC). However, the role of ERK activation in HCC remains unclear. This study was designed to investigate the potential link between ERK activation and aggressive HCC phenotypes. METHODS: We evaluated nuclear ERK expression by immunohistochemistry in 154 resected HCC nodules from 136 patients. We then investigated the associations of ERK expression with the clinicopathological characteristics of HCC, c-MET expression, and the molecular subclass biomarkers Ki-67, keratin 19 (KRT19, CK19, or K19), and sal-like protein 4. Multivariate Cox regression analysis was carried out to determine independent prognostic factors for overall survival and recurrence-free survival. The effects of ERK activation by hepatocyte growth factor (HGF) on eight HCC cell lines were further examined. RESULTS: High-level nuclear expression of ERK was observed in 20 (13%) of 154 nodules and was significantly associated with higher serum alpha-fetoprotein levels (P = 0.034), poorer differentiation (P = 0.003), a higher Ki-67 index (P < 0.001), high-level expression of c-MET (P = 0.008), KRT19 (P = 0.002), or sal-like protein 4 (P < 0.001), and shorter overall survival (multivariate hazard ratio 3.448; P = 0.028) and recurrence-free survival (multivariate hazard ratio 2.755; P = 0.004). HCC cells treated with hepatocyte growth factor showed enhanced cell proliferation together with ERK activation and upregulated KRT19 expression, both of which were inhibited by sorafenib. CONCLUSIONS: High-level ERK activation is associated with a KRT19-positive highly proliferative subtype of HCC with a dismal prognosis. These findings support the key role of the hepatocyte growth factor/c-MET/ERK axis in HCC progression.

Clinico-Radio-Pathological and Molecular Features of Hepatocellular Carcinomas with Keratin 19 Expression.

Hepatocellular carcinoma (HCC) is a heterogeneous neoplasm, both from the molecular and histomorphological aspects. One example of heterogeneity is the expression of keratin 19 (K19) in a subset (4-28%) of HCCs. The presence of K19 expression in HCCs has important clinical implications, as K19-positive HCCs have been associated with aggressive tumor biology and poor prognosis. Histomorphologically, K19-positive HCCs demonstrate a more infiltrative appearance, poor histological differentiation, more frequent vascular invasion, and more intratumoral fibrous stroma than K19-negative conventional HCCs. From the molecular aspect, K19-positive HCCs have been matched with various gene signatures that have been associated with stemness and poor prognosis, including the G1-3 groups, S2 class, cluster A, proliferation signature, and vascular invasion signature. K19-positive HCCs also show upregulated signatures related to transforming growth factor-ß pathway and epithelial-to-mesenchymal transition. The main regulators of K19 expression include hepatocyte growth factor-MET paracrine signaling by cancer-associated fibroblast, epidermal growth factor-epidermal growth factor receptor signaling, laminin, and DNA methylation. Clinically, higher serum alpha-fetoprotein levels, frequent association with chronic hepatitis B, more invasive growth, and lymph node metastasis have been shown to be characteristics of K19-positive HCCs. Radiologic features including atypical enhancement patterns, absence of tumor capsules, and irregular tumor margins can be a clue for K19-positive HCCs. From a therapeutic standpoint, K19-positive HCCs have been associated with poor outcomes after curative resection or liver transplantation, and resistance to systemic chemotherapy and locoregional treatment, including transarterial chemoembolization and radiofrequency ablation. In this review, we summarize the currently available knowledge on the clinico-radio-pathological and molecular features of K19-expressing HCCs, including a detailed discussion on the regulation mechanism of these tumors.

Silver Sulfadiazine's Effect on Keratin-19 Expression as Stem Cell Marker in Burn Wound Healing.

BACKGROUND: Burn wounds are one of the causes of cutaneous injury that involve both epidermal and dermal layers of skin. Silver sulfadiazine (SSD) has been widely used to treat burn wounds, however recent studies have found the treatment to have some drawbacks, such as cellular toxicity effects. Cutaneous wound regeneration is known to start from the basal layer of the epidermal epithelial cells, which are enriched with highly proliferative cells. Keratin-19 (K19) is one of the epidermal stem cell biomarkers found in the skin. This study aims to explore the expression of K19 in burn wound tissue and to investigate the effect of SSD on its expression. METHODS: We created a burn wound model in Sprague Dawley rats and randomly divided them into control and SSD groups. Wound closure was evaluated (visitrak) overtime series followed by histological evaluation of K19 expression in the wound tissue (immunohistochemistry staining). RESULTS: Our model successfully represents full-thickness damage caused by a burn wound. The SSD group showed a faster reduction of wound surface area (wound closure) compared to the control group with the peak at day 18 post wounding (p < 0.05). K19 expression was found in both groups and was distributed on epidermal layers, hair follicles and dermis of granulation tissue showing similar patterns. CONCLUSION: Topical application of SSD on burn wounds showed superiority in wound closure and is likely to have no harmful effect on epidermal stem cells. However, further study is required to investigate the effect of silver species on cell viability and toxicity effects during long term treatment.

Heterogeneity of Epigenetic and Epithelial Mesenchymal Transition Marks in Hepatocellular Carcinoma with Keratin 19 Proficiency.

OBJECTIVE: Keratin 19 (K19) expression is a potential predictor of poor prognosis in patients with hepatocellular carcinoma (HCC). To clarify the feature of K19-proficient HCC, we traced epigenetic footprints in cultured cells and clinical materials. PATIENTS AND METHODS: In vitro, KRT19 promoter methylation was analyzed and 5-aza-2'-deoxycytidine with trichostatin A (TSA) treatment was performed. Among 564 surgically resected HCCs, the clinicopathological relevance of K19-proficent HCCs was performed in comparison with hepatocytic (HepPar-1 and arginase-1), epithelial-mesenchymal transition (E-cadherin and vimentin), biliary differentiation-associated (K7 and NOTCH-1) markers, and epigenetic markers (KRT19 promoter/long interspersed nucleotide element-1 [LINE-1] methylation status). RESULTS: KRT19 promoter methylation was clearly associated with K19 deficiency and 5-aza-2'-deoxycytidine with TSA treatment-stimulated K19 re-expression, implicating DNA methylation as a potential epigenetic process for K19 expression. After excluding HCCs with recurrence, TNM stage as IIIB or greater, preoperative therapy, transplantation, and combined hepatocellular cholangiocarcinoma, we assessed 125 of 564 HCC cases. In this cohort, K19 expression was found in 29 HCCs (23.2%) and corresponded with poor survival following surgery (p = 0.025) and extrahepatic recurrence-free survival (p = 0.017). Compared with K19-deficient HCCs, lower KRT19 promoter methylation level was observed in K19-proficient HCCs (p < 0.0001). Conversely, HCC with genome-wide LINE-1 hypermethylation was frequently observed in K19-proficient HCCs (p = 0.0079). Additionally, K19 proficiency was associated with K7 proficiency (p = 0.043), and reduced E-cadherin and HepPar-1 expression (p = 0.043 and p < 0.0001, respectively). CONCLUSIONS: K19-proficient HCC exhibited poor prognosis owing to extrahepatic recurrence, with molecular signatures differing from those in conventional cancer stem cells, providing novel insights of the heterogeneity underlying tumor development.

BRAFV600E-induced KRT19 expression in thyroid cancer promotes lymph node metastasis via EMT.

Keratin 19 (KRT19) is a type I cytokeratin that serves an important role in multiple types of cancer; however, little is known regarding its role in thyroid cancer. Therefore, the aim of the current study was to investigate the role of KRT19 in thyroid cancer. Using The Cancer Genome Atlas database, the expression and clinical significance of KRT19 in thyroid cancer tissues were investigated. The effect of KRT19 in thyroid cancer cell lines was also investigated in vitro. The results demonstrated that KRT19 expression was higher in thyroid cancer when compared with normal thyroid tissues, and was associated with lymph node metastasis, tumor stage and tumor-node-metastasis stage. Knockdown of KRT19 inhibited the proliferation and migration of thyroid cancer cell lines. In addition, increased KRT19 expression was observed only in tumors harboring the B-Raf Proto-Oncogene, Serine/Threonine Kinase (BRAF)V600E mutation, and BRAFV600E overexpression induced KRT19 expression. Furthermore, KRT19 exerted its phenotype via promoting epithelial-mesenchymal transition (EMT). In conclusion, the results of the current study suggest that BRAFV600E-induced KRT19 expression may promote thyroid cancer metastasis via EMT.