Non-neoplastic Epithelial Cells Research Articles

Targeting canine mammary neoplastic epithelial cells with a reengineered anthrax toxin: first study.

Mammary tumors are the most frequent type of neoplasms in intact female dogs. New therapies that target neoplastic cells without affecting normal cells are highly sought. The Bacillus anthracis toxin has been reengineered to target tumor cells that express urokinase plasminogen activators and metalloproteinases. In previous studies carried out in our laboratory, the reengineered anthrax toxin had inhibitory effects on canine oral mucosal melanoma and canine osteosarcoma cells. In this study, five canine neoplastic epithelial cell lines (four adenocarcinomas and one adenoma) and one non-neoplastic canine mammary epithelial cell line were treated with different concentrations of reengineered anthrax toxin components. Cell viability was quantified using an MTT assay and half-maximal inhibitory concentration (IC50) values. Cell lines were considered sensitive when the IC50 was lower than 5000ng/ml. One canine mammary adenocarcinoma cell line and one mammary adenoma cell line showed significantly decreased viability after treatment, whereas the non-neoplastic cell line was resistant. We conclude that the reengineered anthrax toxin may be considered a targeted therapy for canine mammary neoplasms while preserving normal canine mammary epithelial cells.

Abstract 1467: Contribution of estrogen metabolites to never-smoking lung cancer

Abstract There has been a consistent rise in the incidence of non-small cell lung cancer (NSCLC) among individuals who have never smoked. Remarkably, over 50% of diagnosed female cases and 15-20% of male cases are never-smokers. Notably, NSCLC in never-smokers exhibits distinctive clinical and pathological features, notably a higher incidence of tumors with EGFR mutations, setting it apart from its counterpart in smokers. Despite the improved disease-free survival observed with EGFR-tyrosine kinase inhibitors, eventual relapse and limited responsiveness to immunotherapy are common, underscoring the urgency to comprehend the molecular mechanisms driving the escalating prevalence of NSCLC in never-smokers and develop targeted preventive and interceptive therapeutic strategies. The preponderance of females among NSCLC patients who never smoked has prompted an exploration into the potential contribution of estrogen to lung tumorigenesis. In contrast to prevailing research focused on estrogen receptor-mediated signaling, our investigations have yielded compelling evidence supporting the involvement of estrogen metabolism in lung carcinogenesis among never-smokers. This group is the first to report that estrogen is metabolized extensively in both mouse and human lungs, resulting in the generation of various derivatives, including the potential carcinogen 4-hydroxyestrogen (4-OHE). Notably, recent findings from our laboratory indicate that prolonged exposure of nonneoplastic human bronchial epithelial cells to 17β estradiol or 4-OHE, at concentrations approximating physiological levels, induces the accumulation of double-strand DNA breaks and cellular transformation. A comprehensive analysis was performed to further elucidate the mechanisms underlying the transformation process. Isolated transformed clones obtained after 9, 18, and 22 wks of exposure to 4-OHE were subjected to karyotyping, F.I.S.H., and RNA and exome sequencing. A longitudinal progression of aberrant chromosomal rearrangements and copy number alterations was observed, that increased with time of 4-OHE exposure. In addition, RNA-Seq analyses revealed the epithelial-mesenchymal transition pathway was highly enriched at the early timepoint (9 wks), while replicative stress-related signaling pathways were induced at all timepoints. To determine if the differentially expressed genes were also dysregulated in clinical lung cancer cases, TCGA lung adenocarcinoma (LUAD) data were examined. Interestingly, many of the genes upregulated after estrogen exposure were similarly overexpressed in the LUAD samples and often associated with a worse prognosis. In conclusion, our data support the role of estrogen metabolites in promoting lung cancer among never-smokers. (Supported by CA217161 and generous contributions from the Hellendall Family Foundation, Gregory G. Lawton and the Timothy and Aurora Hughes Cancer Research Fund.) Citation Format: Mitchell Cheung, Lisa A. Vanderveer, Daniel Krzizike, J. Nicholas Bodor, Joseph Treat, Joseph R. Testa, Margie L. Clapper. Contribution of estrogen metabolites to never-smoking lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1467.

Inhibitory Effects of Alpha-Connexin Carboxyl-Terminal Peptide on Canine Mammary Epithelial Cells: A Study on Benign and Malignant Phenotypes.

Mammary cancer is highly prevalent in non-castrated female dogs. Cell-to-cell communication is an important mechanism to maintain homeostasis, and connexins are proteins that assemble to form the communicating gap junctions. In many cancers, communication capacity is reduced; several approaches are being tested in order to increase the communication capacity in cancer cells and, therefore, alter their viability. This study analyzed the effects of the alpha-connexin carboxyl-terminal peptide (αCT1) on canine mammary non-neoplastic and neoplastic epithelial cells. Seven canine epithelial mammary cell lines were used. Among these, one was a normal canine epithelial mammary cell line (LOEC-NMG), two canine mammary adenomas (LOEC-MAd1 and LOEC-MAd2), and four canine mammary adenocarcinomas (LOEC-MCA1, LOEC-MCA2, LOEC-MCA3 and CF41). The αCT1 corresponds to a short Cx43 C-terminal sequence linked to an internalization sequence called the antennapedia. After 24 h of incubation, the medium containing different αCT1 peptide concentrations was added to the cells, and only the culture medium was used for control. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used to quantify cell viability before treatment and 48, 72, and 96 h after the treatment. Results showed that the normal mammary epithelial cell line (LOEC-NMG) was resistant to treatment with αCT1, which is consistent with a previous study on human mammary cell lines. One of the adenoma cell lines (LOEC-MAd2) was also resistant to treatment with αCT1, although the other (LOEC-MAd1) was susceptible to treatment, mostly at 72 h after treatment. Regarding the four canine adenocarcinoma cell lines, they differ regarding the susceptibility to the treatment with αCT1. Three cell lines, canine mixed adenocarcinoma (LOEC-MCA1), canine complex adenocarcinoma (LOEC-MCA2), and commercial canine mammary adenocarcinoma cell line CF41, were susceptible to treatment with αCT1, while one canine mammary adenocarcinoma cell line (LOEC-MCA3) was resistant to treatment. In most αCT1 treated cell lines, Cx43 was strongly detected in cell membranes by immunofluorescence. We propose that αCT1 restored the cell-to-cell communication capacity of neoplastic cells and induced inhibitory effects on cell viability.

27-hydroxycholesterol and DNA damage repair: implication in prostate cancer.

We previously reported that cholesterol homeostasis in prostate cancer (PC) is regulated by 27-hydroxycholesterol (27HC) and that CYP27A1, the enzyme that converts cholesterol to 27HC, is frequently lost in PCs. We observed that restoring the CYP27A1/27HC axis inhibited PC growth. In this study, we investigated the mechanism of 27HC-mediated anti-PC effects. We employed in vitro models and human transcriptomics data to investigate 27HC mechanism of action in PC. LNCaP (AR+) and DU145 (AR-) cells were treated with 27HC or vehicle. Transcriptome profiling was performed using the Affymetrix GeneChip™ microarray system. Differential expression was determined, and gene set enrichment analysis was done using the GSEA software with hallmark gene sets from MSigDB. Key changes were validated at mRNA and protein levels. Human PC transcriptomes from six datasets were analyzed to determine the correlation between CYP27A1 and DNA repair gene expression signatures. DNA damage was assessed via comet assays. Transcriptome analysis revealed 27HC treatment downregulated Hallmark pathways related to DNA damage repair, decreased expression of FEN1 and RAD51, and induced "BRCAness" by downregulating genes involved in homologous recombination regulation in LNCaP cells. Consistently, we found a correlation between higher CYP27A1 expression (i.e., higher intracellular 27HC) and decreased expression of DNA repair gene signatures in castration-sensitive PC (CSPC) in human PC datasets. However, such correlation was less clear in metastatic castration-resistant PC (mCRPC). 27HC increased expression of DNA damage repair markers in PC cells, notably in AR+ cells, but no consistent effects in AR- cells and decreased expression in non-neoplastic prostate epithelial cells. While testing the clinical implications of this, we noted that 27HC treatment increased DNA damage in LNCaP cells via comet assays. Effects were reversible by adding back cholesterol, but not androgens. Finally, in combination with olaparib, a PARP inhibitor, we showed additive DNA damage effects. These results suggest 27HC induces "BRCAness", a functional state thought to increase sensitivity to PARP inhibitors, and leads to increased DNA damage, especially in CSPC. Given the emerging appreciation that defective DNA damage repair can drive PC growth, future studies are needed to test whether 27HC creates a synthetic lethality to PARP inhibitors and DNA damaging agents in CSPC.

KDF1 Promoted Proliferation, Migration and Invasion of Lung Adenocarcinoma Cells through Activating STAT3 and AKT Pathway.

KDF1 has been reported to be correlated with carcinogenesis. However, its role and mechanism are far from clear. To explore the possible role and underlying mechanism of KDF1 in lung adenocarcinoma (LUAD), we investigated KDF1 expression in LUAD tissues and the influence of KDF1 in the phenotype of LUAD cells (A549 and PC-9) as well as the underlying mechanism. Compared to non-tumor lung epithelial cells, KDF1 was upregulated in the cancer cells of the majority of LUAD patients, and its expression was correlated with tumor size. Patients with enhanced KDF1 in cancer cells (compared with paired adjacent non-neoplastic lung epithelial cells) had shorter overall survival than patients with no increased KDF1 in cancer cells. Knockdown of KDF1 inhibited the migration, proliferation and invasion of LUAD cells in vitro. And overexpression of KDF1 increased the growth of the subcutaneous tumors in mice. In terms of molecular mechanisms, overexpression of KDF1 induced the expression of AKT, p-AKT and p-STAT3. In KDF1-overexpressing A549 cells, inhibition of the STAT3 pathway decreased the level of AKT and p-AKT, whereas inhibition of the AKT pathway had no effect on the activation of STAT3. Inhibition of STAT3 or AKT pathways reversed the promoting effects of KDF1 overexpression on the LUAD cell phenotype and STAT3 inhibition appeared to have a better effect. Finally, in the cancer cells of LUAD tumor samples, the KDF1 level was observed to correlate positively with the level of p-STAT3. All these findings suggest that KDF1, which activates STAT3 and the downstream AKT pathway in LUAD, acts as a tumor-promoting factor and may represent a therapeutic target.

Characterization of alkaline phosphatase cytochemistry in canine neoplastic and non-neoplastic pulmonary mass aspirates.

Distinguishing primary and secondary pulmonary neoplasms can be challenging via cytology, and a rapid, inexpensive diagnostic tool to differentiate these neoplasms is unavailable. Alkaline phosphatase cytochemistry (ALP-CC) has been used to identify primary pulmonary carcinomas in human patients, and we hypothesized it could be applied to canine lung aspirates. We aimed to characterize ALP-CC expression in fine-needle aspirate (FNA) samples of canine pulmonary neoplastic and non-neoplastic tumors. A retrospective case search was conducted to identify cases with contemporaneous cytology and histopathology reports from pulmonary lesions, including neoplastic and non-neoplastic etiologies. Slides prepared from pulmonary aspirates were stained for ALP-CC activity, and the percentage of ALP-CC-positive primary pulmonary epithelial tumors was determined. To characterize the ALP-CC expression in non-neoplastic cellular constituents of pulmonary FNA samples, mesothelial cells were also evaluated. Forty-eight canine cases met the inclusion criteria. ALP-CC-positive cells were seen in both neoplastic and non-neoplastic lesions. In non-neoplastic lesions, pulmonary epithelial cells were ALP-CC positive. Eighty-nine percent of primary pulmonary epithelial neoplasms were ALP-CC positive, and no ALP-CC positivity was noted in mesothelial cells. ALP-CC-positive neoplastic cells were seen in a metastatic amelanotic melanoma. Primary pulmonary epithelial neoplasms are frequently ALP-CC positive, but such positivity is not restricted to this tumor type. Non-neoplastic pulmonary epithelial cells can be ALP-CC positive, whereas mesothelial cells are negative.

A Novel Cell Membrane-Associated RNA Extraction Method and Its Application in the Discovery of Breast Cancer Markers.

Cell membrane-associated RNA (mem-RNA) has been demonstrated to be cell-specific and disease-related and are considered as potential biomarkers for disease diagnostics, drug delivery, and cell screening. However, there is still a lack of methods specifically designed to extract mem-RNA from cells, limiting the discovery and applications of mem-RNA. In this study, we propose the first all-in-one solution for high-purity mem-RNA isolation based on two types of magnetic nanoparticles, named MREMB (Membrane-associated RNA Extraction based on Magnetic Beads), which achieved ten times enrichment of cell membrane components and over 90% recovery rate of RNA extraction. To demonstrate MREMB's potential in clinical research, we extracted and sequenced mem-RNA of typical breast cancer MCF-7, MDA-MB-231, and SKBR-3 cell lines and non-neoplastic breast epithelial cell MCF-10A. Compared to total RNA, sequencing results revealed that membrane/secreted protein-encoding mRNAs and long noncoding RNAs (lncRNAs) were enriched in the mem-RNA, some of which were significantly overexpressed in the three cancer cell lines, including extracellular matrix-related genes COL5A1 and lncRNA TALAM1. The results indicated that MREMB could enrich membrane/secreted protein-coding RNA and amplify the expression differences of related RNAs between cancer and non-neoplastic cells, promising for cancer biomarker discovery.

Evaluation of Cytotoxic Activity of Epigenetic Drugs in Oesophageal Squamous Cell Carcinoma

Oesophageal squamous cell carcinoma (OESCC) is a poor prognosis cancer with current treatments demonstrating limited efficacy. Recently, the field of epigenetics has gained equal importance to that of genetics in cancer, including in oesophageal cancer. The increasing number of epigenetic drugs is driving the need to identify the most efficacious and potent of these drugs in various cancers. This study investigated both the metabolic activity and importantly, the long-term cytotoxic effects of different families of epigenetic drugs including DNA methyltransferase inhibitors (DNMTi: azacitidine, 5-aza-2ʹ-deoxycytidine, 5-fluoro-2ʹ-deoxycytidine, RG108, procaine hydrochloride, zebularine), histone deacetylase inhibitors (HDACi: valproic acid, panobinostat, vorinostat, belinostat, romidepsin, RGFP966 and santacruzamate A) and bromodomain and extra-terminal motif inhibitors (BETi: JQ1, OTX015, I-BET151A and I-BET762) in OESCC cells (KE-8 and KYSE-450) as well as non-neoplastic oesophageal epithelial cells (HET-1A). Effects on metabolic activity were measured using the MTT bio-reduction assay, while cytotoxic effects were assessed using the long-term clonogenic assay. Dose-dependent effects were observed with specific drugs from all three classes of epigenetic drugs. OESCC cells were most vulnerable to the FDA approved drugs (valproic acid, panobinostat, vorinostat, belinostat and romidepsin) in both the MTT bio-reduction and clonogenic assays. Interestingly, the non-neoplastic HET-1A epithelial cells demonstrated resistance to some DNMTi and HDACi drugs at concentrations that were cytotoxic to OESCC. Thus, our findings have identified a subset of epigenetic drugs that can impair the metabolic activity of OESCC and importantly, are cytotoxic. This may aid in the design of future therapeutic regimes for treatment of this drug resistant cancer.

Benzenesulfonyl thiazoloimines as unique multitargeting antibacterial agents towards Enterococcus faecalis

New efficient antimicrobial agents are urgently needed to combat invasive multidrug-resistant pathogens infections. Structurally unique benzenesulfonyl thiazoloimines (BSTIs) were exploited as novel potential antibacterial victors to confront terrific drug resistance. Some developed BSTIs exerted effectively antimicrobial efficacy against the tested strains. Notably, 2-pyridyl BSTI 14d exhibited good antibacterial activity against E. faecalis with MIC value of 1 μg/mL, which was superior to sulfathiazole and norfloxacin. The most active compound 14d not only showed rapid bactericidal properties and impeded E. faecalis biofilm formation to effectually relieve the development of drug resistance, but also performed low toxicity toward human red blood cells, human normal squamous epithelial cells and human non-neoplastic colon epithelial cells. Mechanistic investigation demonstrated that molecule 14d could exert efficient membrane destruction leading to the leakage of intracellular materials and metabolism inhibition, cause oxidative damage of E. faecalis through accumulation of excess reactive oxygen species and reduction of glutathione activity, and intercalate into DNA to hinder replication of DNA. Molecular docking indicated that the formation of 14d-dihydrofolate synthetase supramolecular complex could hinder the function of this enzyme. ADME analysis displayed that compound 14d possessed promising pharmacokinetic properties. These findings suggested that the newly developed benzenesulfonyl thiazoloimines with multitargeting antibacterial potential provided a new possibility for evading resistance.

Arglabin, an EGFR receptor tyrosine kinase inhibitor, suppresses proliferation and induces apoptosis in prostate cancer cells

Evidence for clinical efficacy of a semisynthetic derivative of arglabin in anticancer treatment prompted us to examine molecular mechanisms and cellular targets of arglabin. Arglabin, a sesquiterpene lactone isolated from Artemisia glabella was cytotoxic to different human cancer cell lines including those derived from advanced triple-negative breast, lung, androgen-dependent and androgen-independent prostate carcinomas. Noteworthy, arglabin was less toxic to non-neoplastic prostate epithelial cells indicating selectivity for cancer cells. At the molecular level, prior to any biochemical signs of cellular toxicity, arglabin reduced levels of cell-surface sulphanyl groups and inhibited phosphorylation of the redox-sensitive receptor tyrosine kinase EGFR, the only active RTK in PC-3 prostate cancer cells among 49 TRKs analyzed by the assay. Henceforth, arglabin inhibited the EGFR downstream signaling pathways mTORC1 and mTORC2. Accordingly, arglabin induced autophagosome formation and autophagic flux, inhibited phosphorylation of ribosomal protein S6 kinase beta-1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and impeded cell cycle progression and proliferation of PC-3 cells. In agreement with inhibition of the mTORC2 pathway, arglabin induced sustained actin polymerization, inhibited cell migration, and triggered apoptosis in vitro in 2D cell culture and colony formation assay and in vivo in prostate cancer xenografts grown on chick chorioallantoic membranes. Under physiological conditions, arglabin rapidly formed adducts with reduced glutathione (GSH). Moreover, thiol-based antioxidants GSH and β-mercaptoethanol abolished arglabin-induced cancer cell toxicity, whereas the non-thiol antioxidant trolox was ineffective pointing to a crucial role of interaction with cell-surface sulphanyl groups for arglabin cytotoxic activity against cancer cells.

Carbonic anhydrase XII as biomarker and therapeutic target in ovarian carcinomas.

Targeting the tumor-associated carbonic anhydrase XII (CA XII) is considered a promising strategy to improve cancer treatment. As such progress is highly demanded for ovarian carcinomas, the present study aimed to provide deeper information about their CA XII expression profile. A large collection of tissue specimens was stained immunohistochemically with a specific anti-CA XII antibody to evaluate the expression in neoplastic and non-neoplastic epithelial ovarian cells. In addition, flow cytometry was used to measure CA XII expression on tumor cells from malignant ascites fluid. Binding of the antibody revealed a significant CA XII expression in most ovarian carcinoma tissue samples and ascites-derived ovarian carcinoma cells. Moreover, CA XII was expressed at higher levels in ovarian carcinomas as compared to borderline ovarian tumors and non-neoplastic ovarian epithelia. Within the carcinoma tissues, high expression of CA XII was associated with higher tumor grading and a trend towards shorter overall survival. Our results indicate that CA XII plays a crucial role for the malignancy of ovarian carcinoma cells and emphasize the potential of CA XII as a diagnostic marker and therapeutic target in the management of ovarian carcinomas.

Cervical cytology: Radiation and other therapy effects

The different treatment options for carcinoma cervix include radiation, chemotherapy, and surgical treatments. Cytological analysis of smears is crucial for patient follow-up to determine response to therapy and to diagnose the persistence or recurrence of malignancy. Anatomical alterations and changes in cell morphology following radiation or chemotherapy make collecting and interpreting cervical cytology samples difficult. These issues can be mitigated by liquid-based cytology. Ionizing radiation is used in radiotherapy (RT) to kill cells. It is important that cytologists are aware of alterations in morphology of the cells. Radiation can cause cytoplasmic and nuclear changes. Cellular enlargement, vacuolation, granularity loss, and other changes linked with cell death are examples of cytoplasmic alterations. Nuclear enlargement and multinucleation are the most frequent nuclear alterations. These changes are determined by the amount of time that has passed since radiation. It should be emphasized that no one characteristic is pathognomonic. Post-irradiation dysplasia is a condition described as abnormal cellular changes in non-neoplastic epithelial cells after RT. Chemotherapy causes comparable alterations as radiation but impacts fewer cells. Busulfan and other chemotherapeutic treatments may produce morphological alterations, which cytologists must be aware of and able to identify. Immunosuppressive treatments, hormonal therapy, and tamoxifen are some of the other drugs that might cause changes in cervical morphology. Surgical methods used in the detection and treatment of cervical cancer may potentially cause alterations as a result of thermal damage and healing. For the treatment of cervical lesions, electrocautery and the loop electrosurgical excisional procedure are available. These procedures employ electric current ablation leading to ischemic changes in the cervical smear. Cytological analysis of smears following treatment with these modalities necessitates a comprehensive history, kind of therapy, and duration of treatment.

Abstract P5-12-13: Exploring the impact of the tumor microenvironment on nuclear morphometry: Lessons learned for sensitivity to cytotoxic treatment

Abstract Globally, breast cancer remains the leading cause of death in females. While systemic therapy for breast cancer may work effectively in the early phases, for more than 10% of primary and 50% of metastatic cases, the disease eventually progresses, resisting treatments. To overcome this issue, recognizing markers of resistance as early as possible is critical. Microenvironmental factors influence tumors, and increased stiffness of the extracellular matrix (ECM) is associated with cancer progression. Additionally, neighboring cells such as non-neoplastic epithelial cells(that may constitute an important portion of the tumor microenvironment -TME) are suspected to affect tumors, but details on their impact are lacking. Here, we asked whether the response to increased ECM stiffness differed depending on the degree of aggressiveness of triple negative breast cancers (TNBC) and the presence of the non-neoplastic epithelium. We investigated two morphometric parameters, nuclear circularity and area (used in cancer pathology), and tumors’ sensitivity to cisplatin to assess tumor aggressiveness- with TNBC cultured at normal (770 Pa) and increased (2000 and 3300 Pa) collagen I stiffness. Reduced nuclear circularity and increased heterogeneity for this parameter in the cancer cell population resulted from higher matrix stiffness and were associated with decreased sensitivity to the cytotoxic drug, cisplatin, notably in the more invasive cancer type. Interestingly, the impact of stiffness increase was reduced by the conditioned medium from the non-neoplastic cells. These findings warrant further investigations to identify whether changes in nuclear circularity that illustrate a global nuclear reorganization affect gene expression profiles linked to cell survival. Citation Format: Apekshya Chhetri. Exploring the impact of the tumor microenvironment on nuclear morphometry: Lessons learned for sensitivity to cytotoxic treatment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-13.

Polycystic Kidney in Adult Guinea Pig: A Case Report

Polycystic kidney disease (PKD) is a genetic or acquired disorder characterized by progressive distension of multiple tubular segments, manifested by fluid accumulation, and growth of non-neoplastic epithelial cells. A male, 2.5 years old, Dunkin Hartley guinea pig, which was kept as a retired breeder in a lab animal facility, TANUVAS, was found dead and subjected for necropsy. History of dullness, depression, anorexia and emaciation was reported. Grossly, both the kidneys were enlarged with multiple, variable sized, spherical transparent cysts with clear fluid. The gall bladder was slightly enlarged with thick bile and a few small, black coloured stones. The tissue samples were fixed in 10% neutral buffered formalin and processed through paraffin embedding technique. Histopathological examination of kidney revealed multiple, variable sized cysts mostly in the cortical areas and the cyst wall was lined by flattened epithelium. Partial to complete loss of glomerular tufts, glomerulosclerosis, tubular epithelial cells degeneration and necrosis were also observed. Some cysts were filled with eosinophilic fluid. Interstitial areas revealed the presence of mononuclear cells infiltration. The basement membrane which supported the cyst wall showed mild thickening by special staining like Masson’s trichrome and Periodic Acid Schiff. Liver showed multifocal, periportal fibrosis with mononuclear cells infiltration and bile duct hyperplasia. Lungs revealed pulmonary congestion, emphysema and bronchiolar epithelial cell degeneration with peribronchiolar mononuclear cells infiltration. Presence of multiple cysts with degenerative changes in adjacent tubules and glomeruli of both the kidneys might have lead to the death of the animal.

VIMENTIN EXPRESSION AND ITS CORRELATION WITH LYMPH NODE METASTASIS IN ORAL SQUAMOUS CELL CARCINOMA

Squamous cell carcinoma and Adenocarcinoma are common subtypes of carcinoma of oral cavity. More than six lakh cases of cancers of head and neck are being diagnosed globally every year and worldwide, it’s the 6th most common cancer. Head and Neck cancers mostly take origin from epithelial lining of oral cavity, oropharynx, larynx and hypopharynx and 90% of them morphologically is predominantly squamous cell carcinoma. OSCC has a poor prognosis, the reason being metastasis to lymph nodes and its recurrence. So, the identification of specific markers with metastasis to lymph nodes should be done for early and specific diagnosis. Normal and neoplastic mesenchymal cells contain vimentin as major protein constituent of intermediate filaments. Vimentin does not show expression usually in non-neoplastic epithelial cells, but shows expression in mesenchymal cells. Vimentin as a marker of epithelial to mesenchymal transitionis used in diagnosing lymph node metastasis in cases of OSCC.

A Cytological Study Enlightening the Unseen Effects of Concomitant Chemoradiotherapy in Contralateral Normal Buccal Mucosa of Oral Squamous Cell Carcinoma Patients.

Background/objectivesIn patients receiving concomitant chemoradiotherapy (CCRT) as a treatment for oral squamous cell carcinoma (OSCC), cytological changes were seen not only in neoplastic epithelial cells but the non-neoplastic epithelial cells are also affected, resulting in cytopathological atypical changes. The present study was designed to observe oral epithelial atypical cytopathologic changes induced in contralateral normal buccal mucosa in OSCC patients receiving CCRT.MethodsThe study included 150 patients with OSCC treated by CCRT whose details were collected from the Institute of Nuclear Medicine and Oncology Lahore (INMOL) Hospital Lahore. Cytological smears were obtained from the contralateral normal buccal mucosa of OSCC patients. The serial scrape smears were taken before, immediately after, on the 17th day (mid of treatment), and at the end of CCRT, whereas 20 patients were taken as normal healthy controls and were not exposed to CCRT. The smears were stained with hematoxylin and eosin and Papanicolaou stain. SPSS version 20 (Armonk, NY: IBM Corp.) was used for statistical analysis and p > 0.05 was considered to be significant.ResultsCCRT-induced oral epithelial atypical cytological changes were predominantly noted at end of therapy (19.7%) in the contralateral normal buccal mucosa. Nuclear atypia features were higher on the 17th day and end of treatment; whereas, epithelial atypia was mainly observed on the 17th day of CCRT (40%). A highly significant association was observed between epithelial atypia and radio-chemotherapy dose (p = 0.045), between CCRT-induced epithelial atypical cytological changes and days of treatment (p = 0.001), and between days of CCRT and nuclear atypia (0.000) accordingly. Atypia was not observed in any control group.ConclusionVarying degrees of oral epithelial atypical cytological changes may occur in otherwise normal contralateral mucosa of the patients receiving CCRT.

Cancer-associated fibroblasts induce an aggressive phenotypic shift in non-malignant breast epithelial cells via interleukin-8 and S100A8.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment have been associated with tumor progression in breast cancer. Although crosstalk between breast cancer cells and CAFs has been studied, the effect of CAFs on non-neoplastic breast epithelial cells is not fully understood to date. Here, we investigated the effect of CAFs on aggressive phenotypes in non-neoplastic MCF10A breast epithelial cells. CAFs induced epithelial-to-mesenchymal transition (EMT) and invasive phenotype in MCF10A cells. S100A8, a potential prognostic marker in several cancers, was markedly increased in MCF10A cells by CAFs. S100A8 was crucial for CAFs-induced invasive phenotype of MCF10A cells. Among cytokines increased by CAFs, interleukin (IL)-8 induced S100A8through transcription factors p65 NF-κB and C/EBPβ. In a xenograft mouse model with MCF10A cells and CAFs, tumor was not developed, suggesting that coinjection with CAFs may not be sufficient for in vivo tumorigenicity of MCF10A cells. Xenograft mouse tumor models with MDA-MB-231 breast carcinoma cells provided an in vivo evidence for the effect of CAFs on breast cancer progression as well as a crucial role of IL-8 in tumor growth and S100A8 expression in vivo. Using a tissue microarray of human breast cancer, we showed that S100A8 expression was correlated with poor outcomes. S100A8 expression was more frequently detected in cancer-adjacent normal human breast tissues than in normal breast tissues. Together, this study elucidated a novel mechanism for the acquisition of invasive phenotype of non-neoplastic breast cells induced by CAFs, suggesting that targeting IL-8 and S100A8 may be an effective strategy against breast cancer.

NQO1 as a Marker of Chemosensitivity and Prognosis for Colorectal Liver Metastasis.

This study aimed to evaluate how NAD(P)H: quinone oxidoreductase-1 (NQO1) affects survival after hepatectomy in patients with colorectal liver metastasis (CRLM). A retrospective analysis was conducted of 88 consecutive patients who underwent hepatectomy for CRLM. Of the 88 patients, preoperative chemotherapy was administered to 30 patients. Immunohistochemistry of the resected specimens was conducted using monoclonal anti-NQO1 antibody. NQO1-positive expression in tumor cells of CRLM was associated with worse overall survival (p=0.026) and was an independent adverse prognostic factor in multivariate analysis (hazard ratio=5.296, p=0.007). Among 30 patients who received preoperative chemotherapy, patients with loss of NQO1 expression in non-neoplastic epithelial cells of the bile ducts (NQO1 polymorphism: n=19) showed significantly better response to preoperative chemotherapy for CRLM (p=0.004). NQO1-positive expression in tumor cells of CRLM may be an adverse prognostic factor after hepatectomy for CRLM.

Multi-Omics Analysis to Characterize Cigarette Smoke Induced Molecular Alterations in Esophageal Cells.

Though smoking remains one of the established risk factors of esophageal squamous cell carcinoma, there is limited data on molecular alterations associated with cigarette smoke exposure in esophageal cells. To investigate molecular alterations associated with chronic exposure to cigarette smoke, non-neoplastic human esophageal epithelial cells were treated with cigarette smoke condensate (CSC) for up to 8 months. Chronic treatment with CSC increased cell proliferation and invasive ability of non-neoplastic esophageal cells. Whole exome sequence analysis of CSC treated cells revealed several mutations and copy number variations. This included loss of high mobility group nucleosomal binding domain 2 (HMGN2) and a missense variant in mediator complex subunit 1 (MED1). Both these genes play an important role in DNA repair. Global proteomic and phosphoproteomic profiling of CSC treated cells lead to the identification of 38 differentially expressed and 171 differentially phosphorylated proteins. Bioinformatics analysis of differentially expressed proteins and phosphoproteins revealed that most of these proteins are associated with DNA damage response pathway. Proteomics data revealed decreased expression of HMGN2 and hypophosphorylation of MED1. Exogenous expression of HMGN2 and MED1 lead to decreased proliferative and invasive ability of smoke exposed cells. Immunohistochemical labeling of HMGN2 in primary ESCC tumor tissue sections (from smokers) showed no detectable expression while strong to moderate staining of HMGN2 was observed in normal esophageal tissues. Our data suggests that cigarette smoke perturbs expression of proteins associated with DNA damage response pathways which might play a vital role in development of ESCC.

Long-term model of colitis-associated colorectal cancer suggests tumor spread mechanism and nature of cancer stem cells.

Although chemical-induced animal models of colorectal cancer (CRC) suggest a lot about the disease, more efforts are required to establish metastasis models. Azoxymethane (AOM) and dextran sodium sulfate (DSS)-treated (AOM/DSS) Crl:CD-1 mice were sacrificed after 10 or 20 weeks in our previous study, and most colon tumors exhibited intramucosal adenocarcinomas. Our observations were extended until 30 weeks to study a colitis-associated advanced CRC mouse model, and explore whether linker threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr) immunostaining-positive cells were involved in the progressive course of colitis-associated CRC as cancer stem cells. AOM/DSS mice were sacrificed at 10, 20 and 30 weeks after AOM administration. Following the histopathological analysis, immunohistochemical staining was performed for the following markers: CD34, podoplanin, β-catenin, E-cadherin, Ki67, Bmi1 and pSmad2/3L-Thr. Compared with AOM/DSS mice at 10 and 20 weeks, submucosal tumor infiltration and tumor invasion into vessels were markedly increased at 30 weeks. In the parts of colon tumors from AOM/DSS mice, particularly in mice at 30 weeks, the positive signal of E-cadherin was clearly reduced in the cell membranes. The percentage of Ki67-positive tumor cells in mucosal areas of AOM/DSS mice was higher than that in the sites of submucosal infiltration. In mucosal areas of colon tumors, pSmad2/3L-Thr-positive cells were scattered among tumor cells. At sites of submucosal infiltration and vessel invasion of these tumors, pSmad2/3L-Thr-positive cells were also observed among tumor cells. In colon tumors from AOM/DSS mice at 30 weeks, the percentage of pSmad2/3L-Thr-positive cells among the nuclear β-catenin-positive tumor cells was higher than that among the cytoplasmic β-catenin-positive tumor cells. For both non-neoplastic and neoplastic epithelial cells, pSmad2/3L-Thr-positive cells exhibited immunohistochemical co-localization with Bmi1. The present study developed an advanced CRC mouse model that exhibited tumor infiltration into the submucosa and invasion into vessels. The present study re-confirmed the theory that pSmad2/3L-Thr-positive cells may be cancer stem cells.