Tumor Cell Nuclei Research Articles

EZH2 regulates pancreatic cancer cells through E2F1, GLI1, CDK3, and Mcm4

Pancreatic cancer (PC) is one of the most common malignant tumors in digestive tract. To explore the role of epigenetic factor EZH2 in the malignant proliferation of PC, so as to provide effective medical help in PC. Sixty paraffin sections of PC were collected and the expression of EZH2 in PC tissues was detected by immunohistochemical assay. Three normal pancreas tissue samples were used as controls. The regulation of EZH2 gene on proliferation and migration of normal pancreatic cell and PC cell were determined by MTS, colony forming, Ki-67 antibody, scratch and Transwell assays. Through differential gene annotation and differential gene signaling pathway analysis, differentially expressed genes related to cell proliferation were selected and verified by RT-qPCR. EZH2 is mainly expressed in the nuclei of pancreatic tumor cells, but not in normal pancreatic cells. The results of cell function experiments showed that EZH2 overexpression could enhance the proliferation and migration ability of PC cell BXPC-3. Cell proliferation ability increased by 38% compared to the control group. EZH2 knockdown resulted in reduced proliferation and migration ability of cells. Compared with control, proliferation ability of cells reduced by 16%-40%. The results of bioinformatics analysis of transcriptome data and RT-qPCR demonstrated that EZH2 could regulate the expression of E2F1, GLI1, CDK3 and Mcm4 in normal and PC cells. The results revealed that EZH2 might regulate the proliferation of normal pancreatic cell and PC cell through E2F1, GLI1, CDK3 and Mcm4.

Annexins A2 and A5 are potential early biomarkers of hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is a highly lethal liver cancer with late diagnosis; therefore, the identification of new early biomarkers could help reduce mortality. We determine the tissue and plasma status of five annexins during hepatocarcinogenesis by diethylnitrosamine-induced cirrhosis-HCC. We found that Anxa5 was the earliest upregulated gene at week 12 after HCC initiation, while Anxa1 and Anxa2 were upregulated in advanced HCC stages (weeks 18 and 22). Furthermore, the protein level of Annexin A1, A2, A5 and A10 was increased from the early stages. Immunofluorescence and subcellular fractionation revealed Annexin A1, A2, and A5 in the cytoplasm and nuclei of tumor cells. Notably, increased plasma levels of Annexin A5 significantly (r2 = 0.8203) correlated with Annexin A5 levels in liver tissue from week 12 and gradually increased until week 22. Using the TCGA database, we found that the expression of ANXA2 (HR = 1.7, p = 0.0046) and ANXA5 (HR = 1.8, p = 0.00077) was associated with poor survival in HCC patients. In conclusion, we have identified Annexin A1 and A5 as potentially useful early biomarkers for poor prognosis in HCC patients.

First Analysis of Expression of Napsin-A in Ovine Pulmonary Adenomatosis of Sheep and Goat at Duhok Governorate

Ovine Pulmonary Adenomatosis is a chronic respiratory disease of sheep or contagious lung cancer of sheep, and less commonly of goats. A total of 4061 lung samples were selected randomly from slaughtered sheep and goats. All samples of lungs with typical lesions were fixed in 10% neutral buffered formalin and examination is performed by macrocsopical and microscopical observation. To perform IHC technique, we used TMA with a polymer system by application of specific markers Napsin A. Results showed 35 lungs suspected from natural cases are diagnosed as OPA. Histologically results showed that the presence of multiple proliferations foci of neoplastic cells in the epithelial cell of alveoli and bronchi, these proliferations appears either papillary or as impression of solid growths appearance or acinar shape which is classified as classical and atypical form. The current study found that the expression of Nnapsin A was observed in 85.71% of the OPA as a granular cytoplasmic staining but there was staining in the nucleus of tumor cells. In conclusion, these findings indicate for the first time that Napsin A is extremely specific for adenocarcinoma of lung in primary origin and use of this important biomarker is guaranteed to help disconnect primary pulmonary adenocarcinoma for diagnosis from other types of lung cancer.

In situ isolation of nuclei or nuclear proteins from adherent cells: a simple, effective method with less cytoplasmic contamination

BackgroundIsolation of nuclei or nuclear proteins is a prerequisite for western blot, nuclear proteome profiling, and other evaluations of nuclear proteins. Here, we developed a simple method for in situ isolation of nuclei or nuclear proteins by in situ removing the extranuclear part of adherent cells via a classical nonionic detergent triton X-100.ResultsFirst, the feasibility of our method was confirmed by confocal microscopy, atomic force microscopy, scanning electron microscopy, dynamic light scattering, immunofluorescence imaging, and time-lapse dynamic observation. Next, the optimal concentration range (approximately 0.1–1% for ~ 10 min) of triton X-100 and the optimal treatment time (< 30 min) of 0.1–1% Triton X-100 for our method were determined via western blotting of eight extra-/intra-nuclear proteins. Subsequently, the effectiveness, sensitivity, and cytoplasmic contamination of our method were tested by investigating the levels of phosphorylated p65 (a NF-κB subunit) in the nuclei of endothelial or tumor cells treated with/without lipopolysaccharide (LPS) via western blotting and by comparing with a commercial nuclear protein extraction kit (a classical detergent-based method). The data show that compared with the commercial kit our method obtained a higher yield of total nuclear proteins, a higher pP65 level in both control and LPS groups, and much lower content of GAPDH (as a reference for cytoplasmic contamination) in nuclei.ConclusionsThe in situ isolation of nuclei or nuclear proteins from adherent cells in this study is a simple, effective method with less cytoplasmic contamination. This method/strategy has the potential of improving the quality of downstream evaluations including western blotting and proteomic profiling.

Cooperative NF-κB and Notch1 signaling promotes macrophage-mediated MenaINV expression in breast cancer

Metastasis is a multistep process that leads to the formation of clinically detectable tumor foci at distant organs and frequently to patient demise. Only a subpopulation of breast cancer cells within the primary tumor can disseminate systemically and cause metastasis. To disseminate, cancer cells must express MenaINV, an isoform of the actin regulatory protein Mena, encoded by the ENAH gene, that endows tumor cells with transendothelial migration activity, allowing them to enter and exit the blood circulation. We have previously demonstrated that MenaINV mRNA and protein expression is induced in cancer cells by macrophage contact. In this study, we discovered the precise mechanism by which macrophages induce MenaINV expression in tumor cells. We examined the promoter of the human and mouse ENAH gene and discovered a conserved NF-κB transcription factor binding site. Using live imaging of an NF-κB activity reporter and staining of fixed tissues from mouse and human breast cancer, we further determined that for maximal induction of MenaINV in cancer cells, NF-κB needs to cooperate with the Notch1 signaling pathway. Mechanistically, Notch1 signaling does not directly increase MenaINV expression, but it enhances and sustains NF-κB signaling through retention of p65, an NF-κB transcription factor, in the nucleus of tumor cells, leading to increased MenaINV expression. In mice, these signals are augmented following chemotherapy treatment and abrogated upon macrophage depletion. Targeting Notch1 signaling in vivo decreased NF-κB signaling activation and MenaINV expression in the primary tumor and decreased metastasis. Altogether, these data uncover mechanistic targets for blocking MenaINV induction that should be explored clinically to decrease cancer cell dissemination and improve survival of patients with metastatic disease.

Synthesis of Magnetic Nanocarrier Conjugated by Folate Based on Tragacanth and In Vitro Investigation of their Efficiency on Breast Cancer Cells

AbstractIn this research, a novel nanocarrier is synthesized based on tragacanth as a natural polysaccharide. For this purpose, carboxymethyl tragacanth (CMT) is first synthesized from tragacanth and then it is functionalized with isocyanate (IS) groups. In another parallel synthesis, magnetite nanoparticles (MNPs) are prepared by the co‐precipitation method and coated with SiO2 to form MNPs coated SiO2 (MNPs@SiO2). Then MNPs@SiO2 is reacted with CMT‐IS to form Fe3O4@SiO2/CMT. In the second step, the MNPs@SiO2/CMT surface is aminated by reaction with 3‐amino propyl triethoxy silane (APTES) and then it is reacted with folic acid (FA) as a targeting agent to form MNPs@SiO2/CMT/FA nanocarrier. The synthesized nanocarrier is characterized by FT‐IR, SEM, EDX, TEM, XRD, VSM, TGA, and zeta potential analysis and then it is loaded with doxorubicin (DOX) as a typical anti‐cancer drug. The in vitro drug release studies are carried out from the DOX‐loaded nanocarrier. The pH sensitivity of the DOX‐loaded nanocarrier is examined attwo different pHs. The effect of DOX‐loaded nanocarrier is investigated on MCF7 human breast cancer cells and the rate of cancer cell death is determined by MTT assay and drug IC50. The changes in the morphology of the tumor cell nuclei are observed using fluorescence microscopy.

Abstract 3598: Cooperative NF-KappaB and Notch1 signaling promotes a macrophage-mediated metastatic phenotype in breast cancer

Abstract Metastasis is a multistep process that leads to the formation of clinically detectable tumor foci at distant organs and, frequently, to patient demise. Only a subpopulation of breast cancer cells within the primary tumor can disseminate systemically and cause metastasis. To disseminate, cancer cells must express MenaINV, an isoform of the actin-regulatory protein, Mena, encoded by the ENAH gene that endows tumor cells with transendothelial migration activity allowing them to enter and exit the blood circulation. We have previously demonstrated that MenaINV mRNA and protein expression is induced in cancer cells by macrophage contact. Here, we report on the discovery of the precise mechanism by which macrophages induce MenaINV expression in tumor cells. We examined the promoter of the human and mouse ENAH gene and discovered a conserved NF-κB transcription factor binding site. Using live imaging of an NF-κB activity reporter and staining of fixed tissues from mouse and human breast cancer we further determined that, for maximal induction of MenaINV in cancer cells, NF-κB needs to cooperate with the Notch1 signaling pathway. Mechanistically, Notch1 signaling does not directly increase MenaINV expression, but instead enhances and sustains NF-κB signaling through retention of p65, an NF-κB transcription factor, in the nucleus of tumor cells, leading to increased MenaINV expression. In mice, these signals are augmented following chemotherapy treatment and abrogated upon macrophage depletion. Targeting Notch1 signaling in vivo decreased NF-κB signaling and MenaINV expression in the primary tumor and decreased metastasis. Altogether, these data uncover mechanistic targets for blocking MenaINV induction that should be explored clinically to decrease cancer cell dissemination and improve survival of patients with metastatic disease. Citation Format: Camille L. Duran, George S. Karagiannis, Xiaoming Chen, Ved P. Sharma, David Entenberg, John S. Condeelis, Maja H. Oktay. Cooperative NF-KappaB and Notch1 signaling promotes a macrophage-mediated metastatic phenotype in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3598.

Borrelia Infected Neoplastic Glial Cells in Five Patients With Glioblastoma Multiformae

Glioblastoma multiformae (GBM) is a primary malignancy of brain astrocytes. Transcranial stereotactic biopsy specimens from five deceased and anonymous patients with GBM were retrieved from archival storage two years after the death of the patients. All patients were residents of the Long Island, New York metropolitan region where borrelia burgdorferi and borrelia miyamotoi infections are hyperendemic. Fluorescence in situ hybridization (FISH) using two DNA probes specific for genes of burgdorferi borrelia and an additional FISH method study with two DNA probes specific for miyamotoi borrelia each and individually produced high signal borrelia DNA hybridizations inside malignant cells in brain biopsies from all five decedents. Bright signal bacterial DNA hybridizations inside live borrelia spirochetes were found at all levels in serial sections of tumor biopsy fragments in low magnification imaging and outright invasion of the nuclei of individual tumor cells in high resolution imaging. Herein is the first report of a cluster of GBM patients with universal borrelia microbe invasion of neoplastic glial cells.

Reproductive Toxicity Evaluation of Graphene-Based Tumor Cell Nucleus-Targeting Fluorescent Nanoprobes on Reproduction and Offspring Health.

A new graphene-based fluorescent nanoprobe for tumor cell nucleus (GTTNs) was synthesized in our laboratory that penetrates the cell membrane and particularly targets cancer cell nucleus and displays tremendous potential for clinical applications. Although acute and subacute toxicity studies have been conducted on GTTNs, a primary result could be drawn that GTTNs appear to have almost no acute and subacute toxicity. However, as an important part of safety evaluation, the influences on reproductive and offspring developmental toxicity are still absent. In this study, male mice were injected intravenously with GTTNs, and the survival status, histopathology of the testes and epididymides, proliferation and apoptosis of testicular tissue, and sperm motility of mice were measured. To evaluate the short- and long-term fertility in male mice, different male mice resided with untreated female mice on days 1 and 30 after the end of the last treatment, and the offspring health parameters were assessed by measuring pup numbers, body weight, and organ indexes of the pups. The results indicated that GTTNs-exposed male mice retained good fertility, healthy structure of testes and epididymides, and production of healthy sperm. Meanwhile, there were no significant differences between the offspring and the control group. In consideration of GTTNs with broad prospects for biomedical applications, our results contribute a basis for further understanding of its biosafety.

Evaluating p53 nuclear expression and prostate cancer progression in a predominantly African American cohort.

217 Background: Increased utilization of next-generation sequencing has revealed evidence of early TP53 alterations in localized prostate cancer (PCa), suggesting a growing clinical potential for p53 immunohistochemistry (IHC). Prior research by Harmon and Gesztes et al. (Radiology, 2021 &amp; Scientific Reports, 2022) support the clinical utility of detecting p53 overexpression in core biopsies and radical prostatectomies (RP) to predict underlying TP53 alterations, biochemical recurrence (BCR), and metastasis. However, to date, no established consensus exists evaluating p53 “overexpression” in PCa. This study aimed to compare two fundamental approaches to assess p53 expression across various specimens with regard to PCa progression. A secondary study aim explored whether ethnic differences in p53 expression exist. Methods: This retrospective cohort study comprised 84 randomly selected PCa patients (46 metastatic; 38 non-metastatic) enrolled between 1996 – 2021 at the DC VA Medical Center. Representative sections of core biopsies (60%), RP (17%), prostate chips (7%), and metastatic deposits (16%) were examined, where (%) equals a share of the total cohort. IHC was performed on selected sections with a p53 monoclonal antibody (Biocare Medical; p53 Tumor Suppressor Protein; clone DO-7). p53 nuclear expression was manually scored according to the highest intensity (0 absent, 1+ weak, 2+ moderate, or 3+ marked) and percentage (0%, &lt;1%, 1-5%, and &gt;5%) of tumor cell nuclei observed in one index block per patient. All slides were reviewed by two pathologists who were blinded to clinical data. Fisher’s exact test was used to test the association between p53 positivity and categorical variables. Results: Of 84 men included, 63 (75%) self-identified as African American (AA). The median follow-up time was 6.2 years. Thirty-four patients (40%) exhibited p53 nuclear expression, of which 18 patients (21%) showed marked (3+) nuclear intensity. Notably, 11 of 18 (61%) 3+ patients displayed &lt;5% p53 expression. All patients with &gt;5% p53 positivity showed marked (3+) p53 staining. The presence of marked p53 staining, regardless of percentage, was found to be significantly associated with a higher Gleason score ( p=0.0002), higher PSA at the time of biopsy ( p&lt;0.0001), BCR ( p=0.0007) and metastasis ( p&lt;0.0001). Importantly, marked p53 staining was identified only in patients who developed metastatic disease. No significant differences were observed in marked p53 expression between AA and Caucasian men. Conclusions: In prostate cancer, marked (3+) p53 nuclear intensity (regardless of the percentage) is associated with disease progression with no observable ethnic predilection. Therefore, careful assessment of intensity is essential to avoid under-detection (when percent criteria are applied) and maximize the number of patients who may benefit from enhanced clinical interventions.

Polyhedral Oligomeric Silsesquioxane-Based Nanoparticles for Efficient Chemotherapy of Glioblastoma.

Glioblastoma (GBM) is the most common lethal brain tumor with dismal treatment outcomes and poor response to chemotherapy. As the regulatory center of cytogenetics and metabolism, most tumor chemotherapeutic molecules exert therapeutic effects in the nucleus. Nanodrugs showing the nuclear aggregation effect are expected to eliminate and fundamentally suppress tumor cells. In this study, a nanodrug delivery system based on polyhedral oligomeric silsesquioxane (POSS) is introduced to deliver drugs into the nuclei of GBM cells, effectively enhancing the therapeutic efficacy of chemotherapy. The nanoparticles are modified with folic acid and iRGD peptides molecules to improve their tumor cell targeting and uptake via receptor-mediated endocytosis. Nuclear aggregation allows for the direct delivery of chemotherapeutic drug temozolomide (TMZ) to the tumor cell nuclei, resulting in more significant DNA damage and inhibition of tumor cell proliferation. Herein, TMZ-loaded POSS nanoparticles can significantly improve the survival of GBM-bearing mice. Therefore, the modified POSS nanoparticles may serve as a promising drug-loaded delivery platform to improve chemotherapy outcomes in GBM patients.

Primary Non Hodgkin Lymphoma on the left breast of a 53 year old woman: A rare case report

Primary breast lymphoma (PBL) is a rare but distinct subtype of extranodal lymphoma, comprising 0.5% of breast malignancies, approximately 1% of all non-Hodgkin's lymphomas (NHL) and &lt;3% of extranodal lymphomas. Because the cases are rare, the diagnosis is difficult to establish by routine histopathological examination alone. This paper reports a case of a 53-year-old woman, complaining of a lump in the left breast. The lump is said to have been noticed since approximately 2 years ago on the nipple. The patient said that initially the lump was the size of a grape and then realized that it had enlarged since 3 weeks ago with a more chewy texture. Ultrasound of the left mammary, heterogeneous solid lesion with indistinct borders with dilatation of the drawerous duct with the appearance of intraductal calcification filling the superolateral and superomedial quadrants of the left mammary suggestively malignant (BIRADS 5). The microscopic appearance of the FNAB suggested a malignant round cell tumor. Based on the results of the biopsy and immunohistochemical examination, the results were positive CD 45, CD 20 positive, CD 3 negative, and Ki67 positive in 90% of tumor cell nuclei. It was concluded that it was a non-Hodgkin's lymphoma, with a large diffuse pattern. The patient was reported did not survive.

Effect of Morus alba extract sanggenon C on growth and proliferation of glioblastoma cells

Glioblastoma is the most common primary cranial malignancy, and chemotherapy remains an important tool for its treatment. Sanggenon C(San C), a class of natural flavonoids extracted from Morus plants, is a potential antitumor herbal monomer. In this study, the effect of San C on the growth and proliferation of glioblastoma cells was examined by methyl thiazolyl tetrazolium(MTT) assay and 5-bromodeoxyuridinc(BrdU) labeling assay. The effect of San C on the tumor cell cycle was examined by flow cytometry, and the effect of San C on clone formation and self-renewal ability of tumor cells was examined by soft agar assay. Western blot and bioinformatics analysis were used to investigate the mechanism of the antitumor activity of San C. In the presence of San C, the MTT assay showed that San C significantly inhibited the growth and proliferation of tumor cells in a dose and time-dependent manner. BrdU labeling assay showed that San C significantly attenuated the DNA replication activity in the nucleus of tumor cells. Flow cytometry confirmed that San C blocked the cell cycle of tumor cells in G_0/G_1 phase. The soft agar clone formation assay revealed that San C significantly attenuated the clone formation and self-renewal ability of tumor cells. The gene set enrichment analysis(GSEA) implied that San C inhibited the tumor cell division cycle by affecting the myelocytomatosis viral oncogene(MYC) signaling pathway. Western blot assay revealed that San C inhibited the expression of cyclin through the regulation of the MYC signaling pathway by lysine demethylase 4B(KDM4B), which ultimately inhibited the growth and proliferation of glioblastoma cells and self-renewal. In conclusion, San C exhibits the potential antitumor activity by targeting the KDM4B-MYC axis to inhibit glioblastoma cell growth, proliferation, and self-renewal.

Cumulative antitumor effect of bismuth lipophilic nanoparticles and cetylpyridinium chloride in inhibiting the growth of lung cancer.

To determine the combined antitumor effect of bismuth lipophilic nanoparticles (BisBAL NP) and cetylpyridinium chloride (CPC) on human lung tumor cells. The human lung tumor cells A549 were exposed to 1-100 µM BisBAL NP or CPC, either separately or in a 1:1 combination. Cell viability was measured with the PrestoBlue assay, the LIVE/DEAD assay, and fluorescence microscopy. The integrity and morphology of cellular microtubules were analyzed by immunofluorescence. A 24-h exposure to 1 µM solutions reduced A549 growth with 21.5% for BisBAL NP, 70.5% for CPC, and 92.4% for the combination (p < 0.0001), while a 50 µM BisBAL NP/CPC mixture inhibited cell growth with 99% (p < 0.0001). BisBAL NP-curcumin conjugates were internalized within 30 min of exposure and could be traced within the nucleus of tumor cells within 2 h. BisBAL NP, but not CPC, interfered with microtubule organization, thus interrupting cell replication, similar to the action mechanism of docetaxel. The growth inhibition of A549 human tumor cells by BisBAL NP and CPC was cumulative as of 1 µM. The BisBAL NP/CPC combination may constitute an innovative and cost-effective alternative for treating human lung cancer.

What is the contribution of tumor capsule thickness and tumor nucleus diameter to differential diagnosis in encapsulated variant thyroid papillary carcinomas?

Objective: Capsule thickness and papillary carcinoma nuclear features are common diagnostic criteria for invasive follicular variant papillary thyroid carcinoma (IEFVPTC), non-invasive follicular thyroid neoplasm with papillary nuclear features (NIFT-P), and encapsulated papillary thyroid carcinoma (E-PTC). Our goal in this study is to evaluate these two histomorphological criteria using computerized image analysis systems and to determine whether they can serve as auxiliary criteria for distinguishing these diagnostic groups. Materials and methods: 10 patients were selected from IEFVPTC, NIFT-P, and E-PTC diagnostic groups. Slides containing the largest tumor diameter were scanned and digitized. Using computerized image analysis systems, tumor capsule thickness was measured from 4 areas of 4 equal quadrants, and the average tumor capsule thickness (TCT) was determined. Furthermore, from these 4 equal quadrants, the nucleus diameter of 10 clearly distinguishable cells was measured to determine the average tumor cell nucleus diameter (ND). Results: TCT and ND histomorphological criteria were statistically significant among the diagnostic groups. For IFVPTC diagnosis, both TCT and ND were found to be significant diagnostic tools. We demonstrated that ND can easily be used as a diagnostic tool for NIFT-P, whereas more caution is required for TCT. Conclusion: Differentiating papillary nuclear feature-containing follicular variant thyroid papillary carcinomas is challenging. In this study with a limited number of patients, we demonstrated that the TCT and ND criteria can, to some extent, contribute to differential diagnosis between diagnostic groups. Future studies with larger patient groups and more advanced computerized image analysis programs will yield more promising results.

Morphological characteristics of testicular interstitial cell tumors in dogs

The study presents an assessment of variability of histological structure, measurement, and comparison of size of the neoplasm obtained by ultrasonographic examination and pathological examination, as well as the morphometric dimensions of nuclei and cytoplasm of testicular interstitial cell tumors in dogs. The study involved 35 dogs with neoplasms of 46 testes, where 11 animals had interstitial cell tumors in both testes. Insignificant differences of the size of these neoplasms were revealed (p 0.05) using ultrasonography and pathoanatomical measurement methods. Hence, it allows using both methods to assess the size of interstitial cell tumors. In the study, interstitial cell tumor was detected in both testes at once in 50 % of cases in dogs, however, this might be due to specific characteristics of the sample, and further research is required. In the course of scientific work, a morphological study showed the presence of variability in histological structure of interstitial cell tumors, which can lead to incorrect interpretation of the morphological picture and misdiagnosis, e. g. adipocyte-like morphology of interstitial cell tumors have morphological similarity to a benign neoplasm from adipose tissue - lipoma. In addition, there was an extremely pronounced difference in size of cytoplasm (from 23.6 to 148.4 m; average 66.21 22.42 m) and nuclei (from 9 to 57.6 m; average 23.19 7.10 m) in tumor cells. It proves the presence of pronounced anisocytosis and anisokariosis, which should indicate malignancy of the neoplasm, however, testicular interstitial cell tumors extremely rarely metastasize in practice and according to numerous studies.

Localization of Fusobacterium nucleatum in oral squamous cell carcinoma and its possible directly interacting protein molecules: A case series.

While 15 to 20% of cancers are associated with microbial infection, the relationship between oral microorganisms and oral squamous cell carcinoma (OSCC) remains unclear. The location of bacteria in a tumor is closely related to its carcinogenic mechanism. The aim of this study was to analyse bacterial diversity in clinical OSCC tissue samples and tumor distant normal tissues, locate target bacteria, and search for proteins that may interact with target bacteria. The 16S rDNA method was used to analyse bacterial diversity in clinical OSCC tissue samples and tumor distant normal tissues. Correlations between Fusobacterium abundance and clinicopathological characteristics were analysed using the χ2 test. The position of target bacteria was analysed by fluorescence in situ hybridization (FISH), and the expression of CK, CD31, CD45, CD68, cyclin D1, β-catenin, E-cadherin, NF-κB, and HIF-1α was analysed by immunohistochemistry (IHC) in OSCC tumor tissues and tumor distant normal tissues. The 16S rDNA results showed that the detected amount of Fusobacterium in OSCC tumor tissues was significantly larger than that in tumor distant normal tissues. High expression of Fusobacterium was significantly correlated with the lifestyle-related oral risk habits, including smoking (p=0.036) and alcohol consumption (p=0.022), but did not correlate with patient sex, age, tumor laterality, tumor size, grade or TNM stage. Fusobacterium nucleatum was enriched in tumor stroma, where CD31+ blood vessels and inflammatory cells (including CD45+ leukocytes and CD68+ macrophages) were densely distributed. Cyclin D1 was mainly expressed in the nucleus of tumor cells. β-catenin was expressed in the tumor cell membrane and was positively expressed in tumor interstitial vascular endothelial cells. E-cadherin was mainly expressed in tumor cell membranes. NF-κB was positively expressed in the cytoplasm of tumor cells, tumor interstitial cells and myo-fibrocytes. HIF-1α was mainly expressed in the cytoplasm of tumor interstitial cells. HIF-1α was highly expressed where Fusobacterium nucleatum was densely distributed. According to our study, the detected amount of Fusobacterium in OSCC tumor tissues was significantly larger than that in tumor distant normal tissues, and Fusobacterium nucleatum might aggravate inflammation and hypoxia by interacting with NF-κB and HIF-1α in OSCC.

A multifunctional non-viral vector for the delivery of MTH1-targeted CRISPR/Cas9 system for non-small cell lung cancer therapy

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system adapted from bacteria is a programmable nuclease-based genome editing tool. The long-lasting effect of gene silencing or correction is beneficial in cancer treatment. Considering the need to broaden the practical application of this technology, highly efficient non-viral vectors are urgently required. We prepared a multifunctional non-viral vector that could actively target tumor cells and deliver CRISPR/Cas9 plasmids into nuclei of cancer cells. Protamine sulfate (PS) which contains nuclear localization sequence was utilized to condense plasmid DNA and facilitate nuclei-targeted delivery. Liposome-coated protein/DNA complex avoided the degradation of nuclease in blood circulation. The obtained PS@Lip/pCas9 was further modified with distearoyl phosphoethanolamine-polyethylene glycol-hyaluronic acid (HA) to endow the vector ability to actively target tumor cell. Results suggested that PS@HA-Lip could deliver CRISPR/Cas9 plasmids into nuclei of tumor cells and induce genome editing effect. With the disruption of MTH1 (mutT homolog1) gene, the growth of non-small cell lung cancer was inhibited. Moreover, cell apoptosis in tumor tissue was promoted, and liver metastasis of non-small cell lung cancer (NSCLC) was reduced. Our study has provided a therapeutic strategy targeting MTH1 gene for NSCLC therapy. Statement of significanceCRISPR/Cas9 as a powerful tool for genome editing has drawn much attention. The long-lasting effect possesses unique advantage in cancer treatment. Non-viral vectors have high loading capacity, high safety and low immunogenicity, playing an important role in CRISPR/Cas9 delivery. In our study, a multifunctional non-viral vector for the efficient delivery of CRISPR/Cas9 plasmid was constructed. With the active targeting ligand and nuclei-targeting component, the cargo was efficiently delivered into cell nuclei and exerted genome editing effect. By using this vector, we successfully inhibited the growth and induced the apoptosis of non-small cell lung cancer by disrupting MTH1 expression with good safety. Our work provided an efficient non-vial vector for CRISPR/Cas9 delivery and explored the possibility for cancer treatment.

Deep Learning–Based Nuclear Morphometry Reveals an Independent Prognostic Factor in Mantle Cell Lymphoma

Blastoid/pleomorphic morphology is associated with short survival in mantle cell lymphoma (MCL), but its prognostic value is overridden by Ki-67 in multivariate analysis. Herein, a nuclear segmentation model was developed using deep learning, and nuclei of tumor cells in 103 MCL cases were automatically delineated. Eight nuclear morphometric attributes were extracted from each nucleus. The mean, variance, skewness, and kurtosis of each attribute were calculated for each case, resulting in 32 morphometric parameters. Compared with those in classic MCL, 17 morphometric parameters were significantly different in blastoid/pleomorphic MCL. Using univariate analysis, 16 morphometric parameters (including 14 significantly different between classic and blastoid/pleomorphic MCL) emerged as significant prognostic factors. Using multivariate analysis, Biologic MCL International Prognostic Index (bMIPI) risk group (P=0.025), low skewness of nuclear irregularity (P=0.020), and high mean of nuclear irregularity (P=0.047) emerged as independent adverse prognostic factors. Additionally, a morphometric score calculated from the skewness and mean of nuclear irregularity (P=0.0038) was an independent prognostic factor in addition to bMIPI risk group (P=0.025), and a summed morphometric bMIPI score was useful for risk stratification of patients with MCL (P=0.000001). These results demonstrate, for the first time, that a nuclear morphometric score is an independent prognostic factor in MCL. It is more robust than blastoid/pleomorphic morphology and can be objectively measured.

Hippo-YAP signaling activation and cross-talk with PI3K in oral cancer: A retrospective cohort study.

This study aimed to investigate the clinical and prognostic relevance of the Hippo-YAP transactivators YAP1 and TAZ in oral squamous cell carcinoma, and their possible relationship with PI3K/mTOR pathway activation. Immunohistochemical analysis of YAP1, TAZ, PIK3CA (p110α), p-AKT (Ser473), and p-S6 (Ser235) was performed in paraffin-embedded tissue specimens from 165 OSCC patients. Correlations between protein expression and clinical data were further assessed. YAP1 expression was detected in both cytoplasm and nucleus of tumor cells, whereas TAZ expression was only found in the nucleus. Nuclear YAP1 was significantly associated with tumor size (p= 0.03), neck lymph node metastasis (p= 0.02), TNM stage (p= 0.02), and poor differentiation (p= 0.04). Nuclear TAZ was associated with tobacco (p= 0.03) and alcohol consumption (p= 0.04), and poor tumor differentiation (p= 0.04). There was a positive significant correlation between nuclear and cytoplasmic YAP1, nuclear TAZ, p110α expression, and mTORC1 activation p-S6 (S235). Combined expression of nuclear and cytoplasmic YAP1 was prognostic in both univariate and multivariate analyses. Active nuclear YAP1 was significantly and independently associated with poor disease-specific (p= 0.005, HR=2.520; 95%CI=1.319-4.816) and overall survival (p= 0.015, HR=2.126; 95%CI=1.155-3.916). Nuclear YAP1 is an independent predictor of poor survival in oral squamous cell carcinoma.