Establishment and characterization of a prostate cancer cell line from a prostatectomy specimen for the study of cellular interaction.

Though human prostate cancer (PCa) heterogeneity can best be studied using multiple cell types isolated from clinical specimens, the difficulty of establishing cell lines from clinical tumors has hampered this approach. In this proof-of-concept study, we established a human PCa cell line from a prostatectomy surgical specimen without the need for retroviral transduction. In a previous report, we characterized the stromal cells derived from PCa specimens. Here, we characterized the epithelial cells isolated from the same tumors. Compared to the ease of establishing prostate stromal cell lines, prostatic epithelial cell lines are challenging. From three matched pairs of normal and tumor tissues, we established one new PCa cell line, HPE-15. We confirmed the origin of HPE-15 cells by short tandem repeat microsatellite polymorphism analysis. HPE-15 cells are androgen-insensitive and express marginal androgen receptor, prostate-specific antigen and prostate-specific membrane antigen proteins. HPE-15 expresses luminal epithelial markers of E-cadherin and cytokeratin 18, basal cell markers of cytokeratin 5 and p63 and neuroendocrine marker of chromogranin A. Interestingly, HPE-15 Cells exhibited no tumorigenicity in different strains of immune-deficient mice but can become tumorigenic through interaction with aggressive cancer cell types. HPE-15 cells can thus serve as an experimental model for the study of PCa progression, metastasis and tumor cell dormancy.

Somatic alterations of TP53, ERBB2, PIK3CA and CCND1 are associated with chemosensitivity for breast cancers.

The correlation of genetic alterations with response to neoadjuvant chemotherapy (NAC) has not been fully revealed. In this study, we enrolled 247 breast cancer patients receiving anthracycline-taxane-based NAC treatment. A next generation sequencing (NGS) panel containing 36 hotspot breast cancer-related genes was used in this study. Two different standards for the extent of pathologic complete response (pCR), ypT0/isypN0 and ypT0/is, were used as indicators for NAC treatment. TP53 mutation (n = 149, 60.3%), PIK3CA mutation (n = 109, 44.1%) and MYC amplification (n = 95, 38.5%) were frequently detected in enrolled cases. TP53 mutation (P = 0.019 for ypT0/isypN0 and P = 0.003 for ypT0/is) and ERBB2 amplification (P < 0.001 for both ypT0/isypN0 and ypT0/is) were related to higher pCR rates. PIK3CA mutation (P = 0.040 for ypT0/isypN0) and CCND2 amplification (P = 0.042 for ypT0/is) showed reduced sensitivity to NAC. Patients with MAPK pathway alteration had low pCR rates (P = 0.043 for ypT0/is). Patients with TP53 mutation (-) PIK3CA mutation (-) ERBB2 amplification (+) CCND1 amplification (-), TP53 mutation (+) PIK3CA mutation (-) ERBB2 amplification (+) CCND1 amplification (-) or TP53 mutation (+) PIK3CA mutation (+) ERBB2 amplification (+) CCND1 amplification (-)had significantly higher pCR rates (P < 0.05 for ypT0/isypN0 and ypT0/is) than wild type genotype tumors. Some cancer genetic alterations as well as pathway alterations were associated with chemosensitivity to NAC treatment. Our study may shed light on the molecular characteristics of breast cancer for prediction of NAC expectations when breast cancer is first diagnosed by biopsy.

Mutational profiling of poorly differentiated and anaplastic thyroid carcinoma by the use of targeted next-generation sequencing.

AIMS: To characterise the mutational profiles of poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC) and to identify markers with potential diagnostic, prognostic and therapeutic significance. METHODS AND RESULTS: Targeted next-generation sequencing with a panel of 18 thyroid carcinoma-related genes was performed on tissue samples from 41 PDTC and 25 ATC patients. Genetic alterations and their correlations with clinicopathological factors, including survival outcomes, were also analysed. Our results showed that ATC had significantly higher mutation rates of BRAF, TP53, TERT and PIK3CA than PDTC (P = 0.005, P = 0.007, P = 0.005, and P = 0.033, respectively). Nine (69%) ATC cases with papillary thyroid carcinoma (PTC) components harboured BRAF mutations, all of which coexisted with a late mutation event (TP53, TERT, or PIK3CA). Nine cases with oncogenic fusion (six RET cases, one NTRK1 case, one ALK case, and one PPARG case) were identified in 41 PDTCs, whereas only one case with oncogenic fusion (NTRK1) was found among 25 ATCs. Moreover, all six cases of RET fusion were found in PDTC with PTC components, accounting for 33%. In PDTC/ATC patients, concurrent TERT and PIK3CA mutations were associated with poor overall survival after adjustment for TNM stage (P = 0.001). CONCLUSIONS: ATC with PTC components is typically characterised by a BRAF mutation with a late mutation event, whereas PDTC with PTC components is more closely correlated with RET fusion. TERT and concurrent PIK3CA mutations predict worse overall survival in PDTC/ATC patients.

Cytoplasmic SIRT1 inhibits cell migration and invasion by impeding epithelial-mesenchymal transition in ovarian carcinoma.

Sirtuin1 (SIRT1) is a mammalian NAD+-dependent type III deacetylase that plays paramount roles in diverse cellular processes. The nucleocytoplasmic shuttling of SIRT1 was discovered more than a decade ago, but the roles of subcellular SIRT1 localization in tumor progression remain unclear. Here, we report that cytoplasmic SIRT1 acts as a tumor suppressor in ovarian carcinoma. By creating ovarian carcinoma cell lines overexpressing wild-type SIRT1 and nuclear localization signals (NLSs) mutated SIRT1 together with both unbiased proteomic and acetylomic approaches and Transwell assays, we identified that mutations in the NLS sequences prevented SIRT1 from entering the nucleus, resulting in the predominant cytoplasmic localization of SIRT1; the cytoplasmic localization of SIRT1 suppressed the mesenchymal program, activated the epithelial program, and inhibited the migration and invasion of tumor cells, thus providing experimental evidence that SIRT1 functions as a tumor suppressor or oncogene may depend on its subcellular localization. Altogether, our findings may highlight a novel role of cytoplasmic SIRT1 in ovarian carcinoma, providing new possible insights for studies investigating the role of SIRT1 in tumor progression.

Isocitrate dehydrogenase1 mutation reduces the pericyte coverage of microvessels in astrocytic tumours.

INTRODUCTION: Tumour-associated angiogenesis is associated with the malignancy and poor prognosis of glioma. Isocitrate dehydrogenase (IDH) mutations are present in the majority of lower-grade (WHO grade II and III) and secondary glioblastomas, but their roles in tumour angiogenesis remain unclear. METHODS: Using magnetic resonance imaging (MRI), the cerebral blood flow (CBF) of IDH-mutated glioma was measured and compared with the IDH-wildtype glioma. The densities of microvessels in IDH-mutated and wildtype astrocytoma and glioblastoma were assessed by immunohistochemical (IHC) staining with CD34, and the pericytes were labelled with α-smooth muscle antigen (α-SMA), neural-glial antigen 2 (NG2) and PDGF receptor-ß (PDGFR-ß), respectively. Furthermore, glia-specific mutant IDH1 knock-in mice were generated to evaluate the roles of mutant IDH1 on brain vascular architectures. The transcriptions of the angiogenesis-related genes were assessed in TCGA datasets, including ANGPT1, PDGFB and VEGFA. The expressions of these genes were further determined by western blot in U87-MG cells expressing a mutant IDH1 or treated with 2-HG. RESULTS: The MRI results indicated that CBF was reduced in the IDH-mutated gliomas. The IHC staining showed that the pericyte coverages of microvessels were significantly decreased, but the microvessel densities (MVDs) were only slightly decreased in IDH-mutated glioma. The mutant IDH1 knock-in also impeded the pericyte coverage of brain microvessels in mice. Moreover, the TCGA database showed the mRNA levels of angiogenesis factors, including ANGPT1, PDGFB and VEGFA, were downregulated, and their promoters were also highly hyper-methylated in IDH-mutated gliomas. In addition, both mutant IDH1 and D-2-HG could downregulate the expression of these genes in U87-MG cells. CONCLUSIONS: Our results suggested that IDH mutations could reduce the pericyte coverage of microvessels in astrocytic tumours by inhibiting the expression of angiogenesis factors. As vascular pericytes play an essential role in maintaining functional blood vessels to support tumour growth, our findings imply a potential avenue of therapeutic strategy for IDH-mutated gliomas.

Perilipin 5 alleviates HCV NS5A-induced lipotoxic injuries in liver.

BACKGROUND: The homeostasis of lipid droplets (LDs) plays a crucial role in maintaining the physical metabolic processes in cells, and is regulated by many LD-associated proteins, including perilipin 5 (Plin5) in liver. As the putative sites of hepatitis C virus (HCV) virion assembly, LDs are vital to viral infection. In addition, the hepatic LD metabolism can be disturbed by non-structural HCV proteins, such as NS5A, but the details are still inexplicit. METHODS: HCV NS5A was overexpressed in the livers and hepatocytes of wild-type and Plin5-null mice. BODIPY 493/503 and oil red O staining were used to detect the lipid content in mouse livers and hepatocytes. The levels of lipids, lipid peroxidation and inflammation biomarkers were further determined. Immunofluorescence assay and co-immunoprecipitation assay were performed to investigate the relationship of Plin5 and NS5A. RESULTS: One week after adenovirus injection, livers expressing NS5A showed more inflammatory cell aggregation and more severe hepatic injuries in Plin5-null mice than in control mice, which was consistent with the increased serum levels of IL-2 and TNF-α (P < 0.05) observed in Plin5-null mice. Moreover, Plin5 deficiency in the liver and hepatocytes aggravated the elevation of MDA and 4-HNE levels induced by NS5A expression (P < 0.01). The triglyceride (TG) content was increased approximately 25% by NS5A expression in the wild-type liver and hepatocytes but was unchanged in the Plin5-null liver and hepatocytes. More importantly, Plin5 deficiency in the liver and hepatocytes exacerbated the elevation of non-esterified fatty acids (NEFAs) stimulated by NS5A expression (P < 0.05 and 0.01 respectively). Using triacsin C to block acyl-CoA biosynthesis, we found that Plin5 deficiency aggravated the NS5A-induced lipolysis of TG. In contrast, Plin5 overexpression in HepG2 cells ameliorated the NS5A-induced lipolysis and lipotoxic injuries. Immunofluorescent staining demonstrated that NS5A expression stimulated the targeting of Plin5 to the surface of the LDs in hepatocytes without altering the protein levels of Plin5. By co-IP, we found that the N-terminal domain (aa 32-128) of Plin5 was pivotal for its binding with NS5A. CONCLUSIONS: Our data highlight a protective role of Plin5 against hepatic lipotoxic injuries induced by HCV NS5A, which is helpful for understanding the steatosis and injuries in liver during HCV infection.

Fibrous hamartoma of infancy: radiologic features and literature review.

BACKGROUND: Fibrous hamartoma of infancy(FHI) is a rare benign lesion most frequently occurring within the first year of life. So far, just over 200 cases have been reported in the English literature, in which the radiologic findings of FHI have not been fully described. Herein, 2 adult cases of FHI receiving treatment in our hospital and the published cases searched on PubMed are reviewed, with the emphasis on the discussion of the spectrum of MR findings and their histologic correlation. CASE PRESENTATION: We present two adult cases who aged 47 years and 19 years with slow growing masses beginning from their childhood in the posterior craniocervical area. On CT and MR imaging, the tumours showed as the superficially located lesions with ill-defined margins that involved the subcutaneous layer and its underlying muscles. The size of the lesions were 21.3 × 16.7 × 16 cm in case 1 and 20.2 × 19.3 × 13.6 cm in case 2. The tumours demonstrated heterogeneous intensities/signals with the adipose tissue presenting as the disperse strands or small focus of fatty intensity/signal. Parallel or whirling appearance, and dilated vessels were delineated in the cases. Contrast enhancement was administered in case 1 and marked enhancement was found. CONCLUSIONS: The usually observed manifestation of FHI on CT and/or MR imaging is the strands of adipose/fibrous intensities traversing the lesions, with the characteristic parallel or whirling appearance in some cases. The tumours with ill-defined margins have the tendency to involve the underlying muscles. Some fibroblastic and adipocytic tumours should be ruled out in differential diagnosis.

Perilipin 5 improves hepatic lipotoxicity by inhibiting lipolysis.

UNLABELLED: Abnormal metabolism of nonesterified fatty acids (NEFAs) and their derivatives has been reported to be the main cause of intracellular lipotoxic injury. Normally, NEFAs are stored in lipid droplets (LDs) in the form of triglyceride (TG), which could reduce the lipotoxicity of cytosolic NEFAs. Previous studies have implicated that Perilipin 5 (Plin5), an LD-binding protein, regulates the storage and hydrolysis of TG in LD. However, its roles and underlying mechanisms in the liver remain unknown. Here we found that Plin5 expression was increased in steatotic livers. Using Plin5 knockout mice, we found that Plin5 deficiency resulted in reduced hepatic lipid content and smaller-sized LDs, which was due to the elevated lipolysis rate and fatty acid utilization. Plin5-deficient hepatocytes showed increased mitochondria proliferation, which could be explained by the increased expression and activity of PPARα stimulated by the increased NEFA levels. Meanwhile, Plin5-deficient livers also exhibited enhanced mitochondrial oxidative capacity. We also found that Plin5 deficiency induces lipotoxic injury in hepatocytes, attributed to lipid peroxidation. Mechanistically, we found that Plin5 blocks adipose triglyceride lipase (ATGL)-mediated lipolysis by competitively binding to comparative gene identification-58 (CGI-58) and disrupting the interaction between CGI-58 and ATGL. CONCLUSION: Plin5 is an important protective factor against hepatic lipotoxicity induced by NEFAs generated from lipolysis. This provides an important new insight into the regulation of hepatic lipid storage and relation between lipid storage and lipotoxicity.

Cideb facilitates the lipidation of chylomicrons in the small intestine.

Cell death-inducing DFF45-like effector b (Cideb), an endoplasmic reticulum (ER)- and lipid droplet (LD)-associated protein, has been shown to play a critical role in maintaining hepatic lipid homeostasis by promoting the lipidation and maturation of VLDL particles. Here, we observed that Cideb is expressed in the jejunum and ileum sections of the small intestine, and its expression was induced by high-fat diet. Intragastric gavage with lipids resulted in the retention of lipids in the intestine in Cideb-deficient mice. In addition, we observed that mice with Cideb deficiency exhibited reduced intestinal chylomicron-TG secretion and increased lipid accumulation in the enterocytes. The sizes of chylomicrons secreted from the small intestine of Cideb-deficient mice were also smaller than those from wild-type mice. Furthermore, the overexpression of Cideb increased TG secretion and reduced lipid accumulation in the enterocyte-like Caco-2 cells. In addition, we proved that Cideb was localized to the ER and LDs and could interact with ApoB48 in Caco-2 cells. Overall, these data revealed that Cideb plays an important role in controlling intestinal chylomicron lipidation.

Metformin suppresses lipid accumulation in skeletal muscle by promoting fatty acid oxidation.

BACKGROUND: Obesity is a major risk factor for metabolic syndrome, including insulin resistance (IR), type 2 diabetes mellitus (T2DM), and cardiovascular disease; ectopic fat deposition plays a key role in the development of these conditions. In insulin-resistant and/or T2DM patients, lipid accumulation is increased in skeletal muscle; the intramuscular accumulation of fatty acid metabolites is recognized to play a critical role in metabolic syndrome. Besides improving insulin sensitivity, the anti-diabetic drug metformin can reduce lipid accumulation in skeletal muscle; however, its mechanism of action remains unclear. METHODS: Ob/ob mice and C2C12 cells were used to explore the effects of metformin on the morphological and physiological changes of lipid droplets. To clarify the mechanism by which metformin regulates fatty acid metabolism, a cDNA microarray and quantitative real-time PCR were used to examine the effects of metformin on the transcriptome of C2C12 cells treated with 200 micromol/L oleic acid. RESULTS: Metformin could retard body weight gain, improve insulin sensitivity and reduce intramyocellular lipid accumulation in ob/ob mice. In C2C12 cells, metformin inhibited lipid accumulation, stimulated fatty acid oxidation, and decreased triglyceride synthesis. Twenty-seven differentially expressed genes, including 12 upregulated and 15 downregulated genes, were involved in fatty acid metabolism. Interestingly, several genes involved in acyl-CoA synthesis and fatty acid oxidation were also upregulated, such as Ppard, Acsbg1, Ascl3, and Mlycd. However, several genes related to lipolysis were downregulated, such as Ces1d and Cel. Moreover, several important genes related to lipid metabolism were also downregulated, such as Fabp4, Adipoq, and Apoc2. CONCLUSIONS: Metformin retards body weight gain, improves insulin sensitivity, and suppresses lipid accumulation in skeletal muscle by promoting fatty acid oxidation.

Autophagy involved in lipopolysaccharide-induced foam cell formation is mediated by adipose differentiation-related protein.

BACKGROUND: Autophagy is an essential process for breaking down macromolecules and aged/damaged cellular organelles to maintain cellular energy balance and cellular nutritional status. The idea that autophagy regulates lipid metabolism is an emerging concept with important implications for atherosclerosis. However, the potential role of autophagy and its relationship with lipid metabolism in foam cell formation remains unclear. In this study, we found that autophagy was involved in the lipopolysaccharide (LPS)-induced the formation of foam cells and was at least partially dependent on adipose differentiation-related protein (ADRP). METHOD: Foam cell formation was evaluated by Oil red O staining. Autophagic activity was determined by immunofluorescence and Western blotting. ADRP gene expression of ADRP was examined by real-time PCR (RT-PCR). The protein expression of ADRP and LC3 was measured using Western blotting analysis. Intracellular cholesterol and triglyceride levels in foam cells were quantitatively measured by enzymatic colorimetric assays. RESULTS: LPS promoted foam cell formation by inducing lipid accumulation in macrophages. The activation of autophagy with rapamycin (Rap) decreased intracellular cholesterol and triglyceride levels, whereas the inhibition of autophagy with 3-methyladenine (3MA) enhanced the accumulation of lipid droplets. Overexpression of ADRP alone increased the formation of foam cells and consequently autophagic activity. In contrast, the inhibitory effects of ADRP activity with siRNA suppressed the activation of autophagy. Taken together, we propose a novel role for ADRP in the regulation of macrophage autophagy during LPS stimulation. CONCLUSION: We defined a new molecular pathway in which LPS-induced foam cell formation is regulated through autophagy. These findings facilitate the understanding of the role of autophagy in the development of atherosclerosis.

Spontaneous Fusion with Transformed Mesenchymal Stromal Cells Results in Complete Heterogeneity in Prostate Cancer Cells.

Tumor cells gain advantages in growth and survival by acquiring genotypic and phenotypic heterogeneity. Interactions with bystander cells in the tumor microenvironment contribute to the progression of heterogeneity. We have shown that fusion between tumor and bystander cells is one form of interaction, and that tumor-bystander cell fusion has contrasting effects. By trapping fusion hybrids in the heterokaryon or synkaryon state, tumor-bystander cell fusion prevents the progression of heterogeneity. However, if trapping fails, fusion hybrids will resume replication to form derivative clones with diverse genomic makeups and behavioral phenotypes. To determine the characteristics of bystander cells that influence the fate of fusion hybrids, we co-cultured prostate mesenchymal stromal cell lines and their spontaneously transformed sublines with LNCaP as well as HPE-15 prostate cancer cells. Subclones derived from cancer-stromal fusion hybrids were examined for genotypic and phenotypic diversifications. Both stromal cell lines were capable of fusing with cancer cells, but only fusion hybrids with the transformed stromal subline generated large numbers of derivative subclones. Each subclone had distinct cell morphologies and growth behaviors and was detected with complete genomic hybridization. The health conditions of the bystander cell compartment play a crucial role in the progression of tumor cell heterogeneity.

Optical imaging of kidney cancer with novel near infrared heptamethine carbocyanine fluorescent dyes.

PURPOSE: We assessed the application of near infrared heptamethine carbocyanine dyes, including IR-783 and the synthetic analogue MHI-148, as optical imaging agents for the rapid detection of human kidney cancer. MATERIALS AND METHODS: The uptake, retention and subcellular localization of these organic dyes were investigated in cultured kidney cancer cells. Tumor specificity of dye uptake and retention was evaluated by whole body imaging of mice bearing human kidney cancer xenografts or freshly harvested clinical kidney cancer specimens. In addition, dye accumulation at the tissue and cellular levels was confirmed by ex vivo studies with results confirmed by fluorescence imaging of frozen tissue sections. Peripheral blood spiked with kidney cancer cells was stained to simulate the detection of circulating tumor cells. RESULTS: Preferential uptake and retention of carbocyanine near infrared dyes was observed in cultured human kidney cancer cells, human kidney cancer cell spiked whole blood, human kidney cancer xenografts and freshly harvested human kidney cancer tissues compared to normal kidney epithelial cells and normal host organs. CONCLUSIONS: We describe a new class of near infrared heptamethine carbocyanine dyes that show potential for detecting kidney cancer cells in circulating blood and kidney cancer cells in clinical specimens. Near infrared carbocyanine dyes can be further developed as dual modality agents for deep tissue imaging of localized and disseminated kidney cancer in patients.

MUC1 promotes cancer stemness and predicts poor prognosis in osteosarcoma.

Osteosarcoma (OS) is one of the most common primary bone malignancy. Combining chemotherapy and surgical treatment significantly improved clinical outcomes for osteosarcoma patients. Osteosarcoma stem cells (OSCs) are often more malignant than differentiated cancer cells and are a key determinant of responses to chemotherapy and radiation therapy, therefore, the removal of OSCs could be an effective therapeutic strategy. Myxoprotein 1 (MUC1) is aberrantly overexpressed in many human cancers and it promotes cancer stemness through activation of pluripotency networks. In this study, we observed elevated MUC1 in osteosarcoma and a depressed prognosis in patients with high MUC1 expression profiles. Our observations also revealed that MUC1 promoted OS stemness and tumor metastasis both in vivo and in vitro. These data led us to hypothesize that MUC1 may be a therapeutic target for patients with OS.

FAM107A as a tumor suppressor in esophageal squamous carcinoma inhibits growth and metastasis.

BACKGROUND: Sequence similarity Family 107 member A (FAM107A) has been recognized as a tumor suppressor of various malignancies, which suppresses tumor proliferation and metastasis. Its specific role in esophageal squamous cell carcinoma (ESCC) remains unclear. METHODS: Public datasets including Gene Expression Profiling Interactive Analysis (GEPIA) and Gene Expression Omnibus (GEO), quantitative real-time PCR (qRT-PCR), and Western blot were utilized for comparative analysis of FAM107A expression between ESCC and normal tissues. The link between FAM107A and clinicopathological features, as well as prognosis determined through χ2-test, log-rank analysis, and univariate and multivariate analyses, respectively. The impact of FAM107A on ESCC cell malignant behavior was confirmed through in vitro assays, including cell counting using the Cell Counting Kit-8 (CCK-8), clonal formation, wound healing, and transwell assays. Western blot analysis was employed to assess the effects of FAM107A on tumor epithelial-mesenchymal transition (EMT) and cell cycle-related proteins. Finally, xenograft tumors were developed to investigate the influence of FAM107A on ESCC growth in vivo. RESULTS: FAM107A exhibited low expression in ESCC tissues. Reduced FAM107A expression was associated with a poorer prognosis and unfavorable clinicopathological characteristics, such as degree of differentiation, T-stage, and N-stage. Overexpression of FAM107A suppressed ESCC cell proliferation, invasion, migration, the EMT process, and cell cycle progression. Finally, FAM107A overexpression inhibited tumor development in vivo. CONCLUSION: The decreased expression of FAM107A is indicative of a worse prognosis for ESCC patients. FAM107A exerts inhibitory impacts on malignant behavior and may hold promise as a therapeutic target for ESCC.

Breast Cancer MCF-7 Cells Acquire Heterogeneity during Successive Co-Culture with Hematopoietic and Bone Marrow-Derived Mesenchymal Stem/Stromal Cells.

During disease progression and bone metastasis, breast tumor cells interact with various types of bystander cells residing in the tumor microenvironment. Such interactions prompt tumor cell heterogeneity. We used successive co-culture as an experimental model to examine cancer-bystander cell interaction. RMCF7-2, a clone of the human breast cancer MCF-7 cells tagged with a red fluorescent protein, was tracked for morphologic, behavioral, and gene expression changes. Co-cultured with various types of hematopoietic cells, RMCF7-2 adopted stable changes to a rounded shape in suspension growth of red fluorescent cells, from which derivative clones displayed marked expressional changes of marker proteins, including reduced E-cadherin and estrogen receptor α, and loss of progesterone receptor. In a successive co-culture with bone marrow-derived mesenchymal stem/stromal cells, the red fluorescent clones in suspension growth changed once more, adopting an attachment growth, but in diversified shapes. Red fluorescent clones recovered from the second-round co-culture were heterogeneous in morphology, but retained the altered marker protein expression while displaying increased proliferation, migration, and xenograft tumor formation. Interaction with bystander cells caused permanent morphologic, growth behavioral, and gene expressional changes under successive co-culture, which is a powerful model for studying cancer cell heterogeneity during breast cancer progression and metastasis.

UCHL1 promotes cancer stemness in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a special kind of breast cancer with strong ability of invasion and metastasis. UCHL1 belongs to the ubiquitin carboxy-terminal hydrolase family and is found to be increased in a variety of malignancies, but its expression in TNBC is unknown. METHODS: First, we analyzed the expression of UCHL1 in 128 TNBC specimens and paired adjacent normal tissues from 17 TNBC patients undergoing curative resection by immunohistochemistry. Then, the relationship between UCHL1 and cancer stemness was investigated by cell flow cytometry, spheroid formation assays and western blot. Moreover, cell scratch assay and Transwell assays were performed to explore whether UCHL1 promotes the migration and invasion of TNBC cells. Finally, we constructed a xenografts model of TNBC cell lines to observe the effect of UCHL1 on tumorigenesis in vivo. RESULTS: UCHL1 was overexpressed in TNBC tissues and associated with poor prognosis. UCHL1 promoted stem cancer cells properties, including the percentage of CD44+/CD24- cells, sphere-forming ability and CSCs related markers. Furthermore, Scratch assay and Transwell assay proved that UCHL1 enhanced the migration and invasion of TNBC cells. The experimental results of xenografts model in nude mice showed that UCHL1 promoted tumorigenesis of TNBC in vivo. CONCLUSION: UCHL1 may play a role in the malignant progression of TNBC by maintaining the stemness and promoting cell invasion and is expected to become a potential therapeutic target for TNBC patients.

HTNV infection of CD8+ T cells is associated with disease progression in HFRS patients.

Hantaan viruses (HTNVs) are zoonotic pathogens transmitted mainly by rodents and capable of infecting humans. Increasing knowledge of the human response to HTNV infection can guide the development of new preventative vaccines and therapeutic strategies. Here, we show that HTNV can infect CD8+ T cells in vivo in patients diagnosed with hemorrhagic fever with renal syndrome (HFRS). Electron microscopy-mediated tracking of the life cycle and ultrastructure of HTNV-infected CD8+ T cells in vitro showed an association between notable increases in cytoplasmic multivesicular bodies and virus production. Notably, based on a clinical cohort of 280 patients, we found that circulating HTNV-infected CD8+ T cell numbers in blood were proportional to disease severity. These results demonstrate that viral infected CD8+ T cells may be used as an adjunct marker for monitoring HFRS disease progression and that modulating T cell functions may be explored for new treatment strategies.

Validating a targeted next-generation sequencing assay and profiling somatic variants in Chinese non-small cell lung cancer patients.

Non-small cell lung cancer (NSCLC) is featured with complex genomic alterations. Molecular profiling of large cohort of NSCLC patients is thus a prerequisite for precision medicine. We first validated the detection performance of a next-generation sequencing (NGS) cancer hotspot panel, OncoAim, on formalin-fixed paraffin-embedded (FFPE) samples. We then utilized OncoAim to delineate the genomic aberrations in Chinese NSCLC patients. Overall detection performance was powerful for mutations with allele frequency (MAF) ≥ 5% at >500 × coverage depth, with >99% sensitivity, high specificity (positive predictive value > 99%), 94% accuracy and 96% repeatability. Profiling 422 NSCLC FFPE samples revealed that patient characteristics, including gender, age, lymphatic spread, histologic grade and histologic subtype were significantly associated with the mutation incidence of EGFR and TP53. Moreover, RTK signaling pathway activation was enriched in adenocarcinoma, while PI(3)K pathway activation, oxidative stress pathway activation, and TP53 pathway inhibition were more prevalent in squamous cell carcinoma. Additionally, novel co-existence (e.g., variants in BRAF and PTEN) and mutual-exclusiveness (e.g., alterations in EGFR and NFE2L2) were found. Finally, we revealed distinct mutation spectrum in TP53, as well as a previously undervalued PTEN aberration. Our findings could aid in improving diagnosis, prognosis and personalized therapeutic decisions of Chinese NSCLC patients.

Cancer cell's neuroendocrine feature can be acquired through cell-cell fusion during cancer-neural stem cell interaction.

Advanced and therapy-resistant prostate tumors often display neural or neuroendocrine behavior. We assessed the consequences of prostate cancer cell interaction with neural cells, which are rich in the human prostate and resident of the prostate tumor. In 3-dimensional co-culture with neurospheres, red fluorescent human LNCaP cells formed agglomerates on the neurosphere surface. Upon induced neural differentiation, some red fluorescent cells showed morphology of fully differentiated neural cells, indicating fusion between the cancer and neural stem cells. These fusion hybrids survived for extended times in a quiescent state. A few eventually restarted cell division and propagated to form derivative hybrid progenies. Clones of the hybrid progenies were highly heterogeneous; most had lost prostatic and epithelial markers while some had acquired neural marker expression. These results indicate that cancer cells can fuse with bystander neural cells in the tumor microenvironment; and cancer cell fusion is a direct route to tumor cell heterogeneity.