Tumor Cell Morphology Research Articles

Abstract C39: High-content screening to capture tumor dynamics in response to microenvironmental perturbations

Abstract Tumor populations are in constant flux as a result of dynamic pressures of their surrounding microenvironment. Measuring the phenotypic response of multiple cell types exposed to physiological relevant tumor conditions has been a challenge due to the heterogenous nature of the tumor microenvironment. Further, traditional cell biology assays for assessing cell viability do not allow for additional downstream analyses and use surrogate measures for cell number, which may fail to provide a complete picture of cellular events. Here, we present a method to utilize high-content screening to generate quantitative, robust data sets characterizing cellular phenotypic responses (i.e. birth, death, morphology) of tumor cells to co-occurring perturbations of drug, oxygen, glucose, ECM stiffness, and stromal cell co-culture. Using an Operetta high-content imaging platform, we are able to perform multiple analyses on the same experimental setup to quantitatively assess the evolutionary dynamics of heterogenous cell populations under multiple environmental perturbations. We are able to differentiate between the effect of microenvironmental conditions on a decrease in cell birth versus increase in cell death, which has significant implications for predicting tumor growth kinetics and drug treatment response. We applied this platform to analyze isogenic lung tumor cells that are either sensitive or resistant to erlotinib, a commonly used tyrosine receptor kinase inhibitor in the treatment of lung cancer. We identified specific microenvironmental contexts where the resistant populations have a fitness advantage over the sensitive population, irrespective of the resistance mechanism. Furthermore, our experimental workflow allows us to capture and quantify cell morphology, an additional measure for overall cell health. We observed significant shifts in specific cell morphological parameters in response to alterations in tumor microenviromental conditions such as low oxygen and increased drug concentration. This novel application of a high-content screening platform allows for the quantification of multiple cellular phenotypes in response to over a hundred different microenvironmental contexts, thus providing deeper insight into the effect of tumor microenvironmental heterogeneity on evolving tumor cell populations. Citation Format: Colleen M. Garvey, Oscar Chen, Jasmine Foo, Shannon M. Mumenthaler. High-content screening to capture tumor dynamics in response to microenvironmental perturbations. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C39.

Abstract 2877: Effects of metabolic stressors on cellular differentiation

Abstract Poor differentiation (the divergence of tumor cell morphology from the cellular morphology of the normal tissue of origin) is associated with unfavorable responses to chemotherapy and poor clinical outcomes in solid tumors. Recurrent somatic mutations in the genes encoding for the metabolic enzymes isocitrate dehydrogenase (IDH) 1 and 2 have recently been identified in patients with acute myeloid leukemias, chrondrosarcomas, cholangiocarcinomas and glioblastomas. Point mutations in IDH lead to production of 2-hydroxyglutarate (2HG). 2HG acts as a potent inhibitor of enzymes involved in the removal of chromatin marks associated with transcriptional repression (DNA and Histone H3K9 methylation). Excess 2HG leads to impaired differentiation but very little is known about the underlying mechanisms. Here we use a well established model of cellular differentiation, the differentiation of 10T1/2 fibroblasts into myofibers by the lineage-specifying transcription factor MyoD to study the mechanisms by which IDH mutations impair differentiation. We show that 5-azacytidine induced differentiation of 10T1/2 cells is impaired when mutant IDH2 is expressed and that IDH2 mutations impair the ability of MyoD expression to reprogram 10T1/2 cells into myofibers. We propose that the metabolic landscape of undifferentiated cells determines the ability of the lineage determining factor MyoD to trigger differentiation and that the undifferentiated state of cancer cells is dependent on their unique metabolic profile. Further work aims to a) define the effects of mutant IDH1/2 on the ability of MyoD to program muscle differentiation, b) determine the nature of the tumorigenic effects of IDH1/2 mutations in 10T1/2 cells and c) identify molecular pathways that can overcome differentiation blocks upon activation. Insight from these experiments will outline the mechanism(s) by which metabolic alterations, in the form of 2HG, impair differentiation and drive tumorigenesis. They will also serve as a platform for the identification of metabolic alterations that trigger differentiation. These mechanisms could prove to be novel and readily targetable processes in an array of poorly differentiated and aggressive cancer subtypes where other pharmacological approaches have failed. Citation Format: Juan Manuel Schvartzman, Craig B. Thompson. Effects of metabolic stressors on cellular differentiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2877.

Effect of fentanyl on expression of VEGF-A and MMP-9 in subcutaneous tumor of human gastric cancer in nude mice

Objective To evaluate the effect of fentanyl on the expression of vascular endothelial growth factor A (VEGF-A) and matrix metallopeptidase-9 (MMP-9) in the subcutaneous tumor of human gastric cancer in nude mice. Methods Thirty SPF male BALB/C nude mice, aged 4-5 weeks, weighing 15-20 g, in which the model of subcutaneous tumor of human gastric cancer cell line MGC-803 was established, were randomly divided into 6 groups (n=5 each) using a random number table: control group (C group), normal saline group (NS group) and fentanyl 0.05, 0.10, 0.20 and 0.40 mg/kg groups (F1-4 groups). The mice in group C received no treatment.Fentanyl 0.05, 0.10, 0.20 and 0.40 mg/kg were intraperitoneally injected once a day for 14 consecutive days in F1-4 groups, respectively, while the equal volume of normal saline 1.5 ml/kg was given instead of fentanyl in group NS.The nude mice were sacrificed on 1 day after the end of administration, and the tumor tissues were obtained for examination of the ultrastructure of subcutaneous tumor (with a transmission electron microscope) and for detection of the expression of VEGF-A and MMP-9 (by immunohistochemistry and Western blot) and expression of VEGF-A and MMP-9 mRNA (by real-time reverse transcriptase polymerase chain reaction). Results No abnormality in the morphology of the subcutaneous tumor cells was observed in C and NS groups.The swollen nucleus, chromatin margination, nuclear fragmentation and apoptotic bodies were found in the subcutaneous tumor cells in F1-4 groups.Compared with group C, the expression of VEGF-A and MMP-9 protein and mRNA was significantly down-regulated in F1-4 groups (P 0.05). There was no significant difference in the expression of VEGF-A and MMP-9 protein and mRNA among F1-4 groups (P>0.05). Conclusion The mechanism by which fentanyl inhibits the growth and metastasis of subcutaneous tumor cells of human gastric cancer is related to down-regulation of VEGF-A and MMP-9 expression in nude mice. Key words: Fentanyl; Stomach neoplasms; Vascular endothelial growth factor A; Matrix metalloproteinase 9

Gastrin-releasing peptide receptor (GRPr) promotes EMT, growth, and invasion in canine prostate cancer.

The gastrin-releasing peptide receptor (GRPr) is upregulated in early and late-stage human prostate cancer (PCa) and other solid tumors of the mammary gland, lung, head and neck, colon, uterus, ovary, and kidney. However, little is known about its role in prostate cancer. This study examined the effects of a heterologous GRPr agonist, bombesin (BBN), on growth, motility, morphology, gene expression, and tumor phenotype of an osteoblastic canine prostate cancer cell line (Ace-1) in vitro and in vivo. The Ace-1 cells were stably transfected with the human GRPr and tumor cells were grown in vitro and as subcutaneous and intratibial tumors in nude mice. The effect of BBN was measured on cell proliferation, cell migration, tumor growth (using bioluminescence), tumor cell morphology, bone tumor phenotype, and epithelial-mesenchymal transition (EMT) and metastasis gene expression (quantitative RT-PCR). GRPr mRNA expression was measured in primary canine prostate cancers and normal prostate glands. Bombesin (BBN) increased tumor cell proliferation and migration in vitro and tumor growth and invasion in vivo. BBN upregulated epithelial-to-mesenchymal transition (EMT) markers (TWIST, SNAIL, and SLUG mRNA) and downregulated epithelial markers (E-cadherin and β-catenin mRNA), and modified tumor cell morphology to a spindle cell phenotype. Blockade of GRPr upregulated E-cadherin and downregulated VIMENTIN and SNAIL mRNA. BBN altered the in vivo tumor phenotype in bone from an osteoblastic to osteolytic phenotype. Primary canine prostate cancers had increased GRPr mRNA expression compared to normal prostates. These data demonstrated that the GRPr is important in prostate cancer growth and progression and targeting GRPr may be a promising strategy for treatment of prostate cancer. Prostate 76:796-809, 2016. © 2016 Wiley Periodicals, Inc.

Mammary-type Myofibroblastoma: Clinicopathologic Characterization in a Series of 143 Cases.

Mammary-type myofibroblastoma (MTMF) is a benign mesenchymal neoplasm initially described to occur in the breast. MTMF is typically CD34 and desmin positive and genetically has rearrangement or deletion of 13q14, resulting in loss of Rb expression by immunohistochemistry (IHC). Although the wider anatomic distribution of MTMF is increasingly recognized, no large series with clinicopathologic information has been reported to date. Archival cases were retrieved, and the diagnosis of MTMF was confirmed. Hematoxylin and eosin-stained slides and IHC slides were reviewed when available (CD34, Desmin, Rb, SMA, S100, EMA, MDM2, CDK4). The patient age, sex, tumor anatomic location and size, preceding symptoms, and margin status were recorded when possible. Clinical follow-up data were requested for tumor recurrence, metastasis, and patient status at last follow-up. A total of 143 cases of MTMF comprised this study, affecting 94 (66%) male and 49 (34%) female individuals. Mean tumor size was 6.6 cm (range, 1 to 22 cm). Anatomic locations included: inguinal/groin region (65; 45%), breast (15; 10%), chest wall/axilla (7; 5%), trunk (17; 12%), lower (18; 13%) and upper (2; 1%) extremities, or intra-abdominal/retroperitoneal (14; 10%). MTMFs were characterized by spindle cells with relatively short, stubby nuclei and a variable adipocytic component. Hyalinization and myxoid stroma were common. Less common morphologic features included nuclear atypia, epithelioid tumor cell morphology, and neurilemmoma-type nuclear palisading. CD34 and desmin were positive in 89% and 91%, respectively, and were both negative in 3%. Rb expression was lost in 92% (57/62). No cases with follow-up data available had tumor recurrence, although 1 case was reportedly a recurrence itself. In summary, MTMF appears more common at extramammary sites than in the breast and can cause diagnostic difficulty when atypia or epithelioid morphology is present or when located in an unusual anatomic location. MTMF is frequently positive for CD34 and desmin by IHC; however, rare cases are negative for both. There is no evidence of any significant recurrence risk for MTMF, even in the presence of positive resection margins. The degree of morphologic overlap between spindle cell lipoma, cellular angiofibroma, and MTMF, in combination with shared genetics and slightly overlapping anatomic distribution, raises the question of whether or not these tumors are truly distinct entities or instead represent points along a single spectrum of genetically related tumors.

Liquid-based versus conventional cytology in solid pediatric neoplasm: Comparison of their diagnostic and morphological spectra.

Background:Conventional cytology (CC) is a well-established and effective methodology for the evaluation of pediatric tumors. Liquid-based cytology (LBC) is a better technique of smear preparation and is at present the standard protocol in cervical cytology samples.Aims:In the present study, we analyzed both smear preparatory techniques in fine-needle aspiration (FNA) samples from solid pediatric neoplasms in terms of adequacy and efficacy with objectives to study the changes in background and morphology of tumor cells in LBC smears.Materials and Methods:This was a prospective observational study conducted in a tertiary care teaching hospital, which included 52 pediatric patients with clinical diagnosis of malignancy and an assessable lump. Both conventional aspiration cytology and LBC smears were prepared as per standard protocols (SurePath BD™) from FNA samples of cases and examined.Results and Conclusion:On comparing the diagnostic efficacy of cytology smears prepared by both techniques, LBC alone was diagnostic in 80.8% of the cases and conventional smear (CS) alone was diagnostic in 71.2% of the cases (agreement was 83.7%, P = 0.3). Cellular morphology was better preserved in LBC and interpretation was easier. There was a lower inadequacy rate in LBC and none of the samples was inadequate due to poor morphology in LBC smears (P = 0.0003). LBC showed an overall 40% improvement in inadequate cases by CS. LBC has been recommended as a complement to CC in nongynecological samples. Improved morphology and lower inadequacy rate make LBC a finer technique compared to CS in pediatric tumors as well.

Genetic characteristics of non-Hodgkin lymphoma in ethnic Uighur people, and their clinical significance.

The incidence of non-Hodgkin lymphoma (NHL) in China is increasing and is attracting attention as a topic of research. The percentage of NHL cases in ethnic Uighur people is also gradually increasing. We therefore recruited Uighur people with NHL to investigate the correlation between genetic alternations and clinical/pathological features in an attempt to determine their clinical significance. A total of 60 NHL patients were recruited from our hospital for a microscopic examination of their tumor cell morphology. Further analysis of chromosome karyotypes revealed the relationship between genetic alternations and clinical/pathological features. Microscopic examination revealed increased numbers of tumor cells with altered morphology. The recruited patients all exhibited abnormal karyotypes. Chromosomal breakages were detected at 14q32, 18q21, 6q21-25, +3, +, +18, and short tandem repeat 17 (str17) in 18.3, 25, 25, 18.3, 15, and 21.7% of patients, respectively. Karyotype change was not related to age, gender, performance status score, or pathological type (P > 0.05), but was correlated with clinical stage, average lactate dehydrogenase (LDH) level, extra-lymphatic metastasis, median survival time, and efficacy of radio- or chemotherapy (P < 0.05). Independent risk factors for genetic change in Uighur NHL patients included clinical stage, average LDH level, extra-lymphatic metastasis, median survival time, and efficacy of radio- or chemotherapy (P < 0.05). Uighur NHL patients exhibited genetic changes including t(14:18), 6q21-25, +3, +7, +18, and str17. Clinical stage, average LDH level, extra-lymphatic metastasis, median survival time, and efficacy of radio- or chemotherapy were all independent risk factors for NHL.

Immunoregulation and genotype in colorectal cancer

Meeting abstracts Classical methods for determining malignant disease prognosis are based upon characterizing the morphology and location of tumor cells. While this analysis provides important information about a patient's disease it fails to capture the biological complexity of the tumor

Abstract 1605: Dielectrophoretic microwell array system for detection circulating tumor cells in patients with solid tumors and single cell analysis of detected circulating tumor cells

Abstract Background Detection and analysis of circulating tumor cells (CTCs) is expected to be a non-invasive real-time monitoring of response to therapy and tumor cell status, however it remains technically challenging. Besides, biological feature of CTCs is still unclear. We have developed a capture system for detection and molecular characterization of single CTCs based on a high-density dielectrophoretic microwell array technology. Material and methods CTC enrichment was performed by density gradient centrifugation with RosetteSep®. After CTC enrichment, samples were loaded into the cell entrapment chamber, followed by application of 20 Vp-p AC voltage (1 MHz for 3 minutes), so as to entrap cells in the microwells using the dielectrophoretic force. Then samples were fixed and labeled with DAPI, FITC-anti-cytokeratin (CK), PE-anti-CD45 antibodies, and detected by image-based analysis using a fluorescence microscope. The results of the CTC enumeration were compared with those by CellSearch® system. In this study, we investigated the DAPI+, CK-, CD45- cells with tumor cell morphology in addition to cells with classical definition of CTCs (DAPI+, CK+, CD45-). Those cells were picked up by micropipette, and isolated single cells were subjected to whole genome amplification followed by sequencing in 50 cancer-related genes on the Ion Torrent PGM platform. Fifteen patients with metastatic breast cancer and 6 patients with metastatic non-small lung cancer were examined in this study. Results The number of CK-positive(+) and CK-negative(-) cells (DAPI+, CD45-) detected by our system ranged from 1 - 38 CTCs (median: 11 / 3 mL), and 100% (21 / 21) of the samples were above the threshold level (≧ 1 / 3 mL). On the other hand, with CellSearch® system, 57.9% of the samples had a ≧ 2 / 7.5 mL threshold level (median: 2 / 3 mL). In 2 blood samples from the healthy donors, both systems detected 0 CTC. Single cell sequencing was performed on 9 of the 21 samples. All analyzed samples contained cells with at least one mutation in 50 cancer-related mutations, indicating that those isolated single cells were possible CTCs including CK-negative(-) cells. Discussion We established a platform enable us to capture and characterize CTCs at the single-cell level. The results of the present study show that single cells isolated by our system are useful for further genomic analysis. In addition, the results of mutation analysis of isolated single CTCs suggested that CTCs have mutational heterogeneity in cancer-related genes. Further investigation is going to be conducted to evaluate the capability of capture and characterize CTCs in other types of cancer. Citation Format: Takeshi Sawada, Atsushi Morimoto, Tatsu Shimoyama, Toshinari Yamashita, Toshifumi Mogami, Kazuki Iijima, Mayu Yunokawa, Shintaro Kanda, Yasuyuki Akiyama, Koji Katayama, Masaru Watanabe, Yasuhiro Koh, Kenji Tamura, Tomohide Tamura, Toru Futami, Fumiaki Koizumi. Dielectrophoretic microwell array system for detection circulating tumor cells in patients with solid tumors and single cell analysis of detected circulating tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1605. doi:10.1158/1538-7445.AM2015-1605

Total Synthesis of Myrmekioside A, a Mono‐O‐alkyl‐diglycosylglycerol from Marine Sponge Myrmekioderma sp.

AbstractMyrmekioside A, which was isolated from the marine sponge Myrmekioderma sp. as a member of the family of natural mono‐O‐alkyl‐diglycosylglycerols, and which has a strong reversion effect on the tumor cell morphology of ras‐transformed cells at 5 μg/mL, was synthesized for the first time in 17 steps and 14 % overall yield. The β‐glycosidic linkages in myrmekioside A were successfully constructed by the neighbouring‐group‐participation approach using trichloroacetimidates and thioglycosides as glycosyl donors. The 2R absolute configuration at C‐2 of the glycerol backbone was derived from (S)‐1,2‐isopropylideneglycerol (8). This synthetic approach may be applicable to the preparation of other myrmekioside analogues featuring different sugars and alkyl chains for further structure–activity relationship studies.

Quantitative analysis of drug-induced complement-mediated cytotoxic effect on single tumor cells using atomic force microscopy and fluorescence microscopy.

In the antibody-based targeted therapies of B-cell lymphomas, complement-mediated cytotoxicity (CMC) is an important mechanism. CMC is activated after the binding of drugs (monoclonal antibodies) to tumor cells. The activation of CMC ultimately leads to the lysis of tumor cells. However, it remains poorly understood how CMC alters the morphology and mechanics of single tumor cells at the nanoscale. In recent years, nanoscopic observations of cellular behaviors with the use of atomic force microscopy (AFM) have contributed much to the field of cell biology. In this work, by combining AFM with fluorescence microscopy, the detailed changes in cellular ultra-microstructures and mechanical properties during the process of CMC were quantitatively investigated on single tumor cells. AFM imaging distinctly showed that the CMC effect could lead to the formation of nano holes on the tumor cells. Quantitative analysis of AFM images indicated that cell surface became lower and rougher after the CMC process. The cellular mechanics measurements showed that during the process of CMC cells firstly softened and finally stiffened, which was validated by dynamically monitoring the mechanical changes of single living cells during CMC. The experimental results provide novel insights into the antibody-dependent CMC.

Abstract 2841: Characterization of ALK fusions in circulating tumor cells (CTCs) of NSCLC

Abstract Background: Many NSCLC patients have insufficient tumor sample or have high co-morbidities preventing access to tissue biopsies for FISH or IHC to determine ALK fusion and subsequent eligibility to Crizotinib therapy. We developed a gene/protein assay for the ALK fusion and examined CTCs and CTC subpopulation incidence and molecular characterization in newly diagnosed NSCLC patients. Methods: 10 samples from newly diagnosed NSCLC patients prior to therapy were recruited and blood specimens were collected and shipped to Epic Sciences. 2/10 had known ALK rearrangement through tissue analysis. All nucleated cells were plated onto glass slides and subjected to IF staining and CTC identification by fluorescent scanners and algorithmic analysis. CTCs, defined as classic (CK+ CD45- w/intact DAPI nuclei and morphologically distinct), apoptotic (CK+, CD45-, non-intact nuclei) and CK- (CK-, CD45-, intact and distinct) were identified. Samples were characterized with ALK IF to assess expression. Patients with known ALK rearrangements in tissue also had their CTCs assessed with ALK FISH. Results: Assays for the ALK protein and ALK gene rearrangement were developed and specificity confirmed utilizing H2228 (ALK positive) &amp; A549 (ALK negative) cells spiked into donor blood and run through the Epic Assay. Expression of ALK fusions was visualized in CTCs of 1/2 patients with known ALK rearrangements and 0/8 patients with no ALK rearrangements. ALK fusion protein expression was seen in classic, apoptotic, small and CK- CTCs. Conclusion: ALK fusion protein and ALK rearrangement assessment with FISH utilizing the Epic CTC platform demonstrates sensitivity and specificity to identification of patients eligible to Crizotinib. The identification of ALK in CTC subpopulations identifies unique tumor cell morphology and suggests evidence of epithelial plasticity. Further clinical studies are ensuing to determine the sensitivity and specificity of ALK in CTCs and the relevance of CTC subpopulations related to Crizotinib therapy and NSCLC progression. Citation Format: David Lu, Rachel Krupa, Natalee Bales, Jessica Louw, Dena Marrinucci, Ryan Dittamore. Characterization of ALK fusions in circulating tumor cells (CTCs) of NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2841. doi:10.1158/1538-7445.AM2014-2841

Combination therapy with low molecular weight heparin and Adriamycin results in decreased breast cancer cell metastasis in C3H mice.

The aim of this study was to investigate the effect of low molecular weight heparin (LMWH) against breast cancer cell invasion and metastasis in C3H mice and the underlying mechanism. The C3H mouse breast cancer model was established, and the mice were then randomly divided into four groups: normal saline group, LMWH group, Adriamycin positive control group and the combination group (LMWH combined with Adriamycin). Twelve days after inoculation, drug treatment was initiated. During the one-month period of drug administration, tumor growth curves were recorded. At the end of the treatment period, the mice were sacrificed and the solid tumor tissue and lung were removed. Hematoxylin and eosin (H&E) staining was used to observe the overall changes in tumor cell morphology and lung metastasis following the treatment. A terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay was used for detection of apoptosis in tumor cells, and immunohistochemical (IHC) analysis was used to determine the expression of vascular endothelial growth factor (VEGF). The tumor growth curves demonstrated that the overall growth of the combination group was less compared with that of the other three groups, indicating that LMWH inhibited the growth of the tumors. H&E staining showed that the area of tumor cell necrosis in the combination group was significantly greater compared with that in the other groups, and less metastasis was observed in the lung. The results from the TUNEL staining demonstrated that there was an increase in the number of blue-black apoptotic cells, and the expression of VEGF was significantly reduced in the combination group compared with the other three groups. Therefore, this indicates that LMWH, combined with Adriamycin significantly reduced the growth of breast cancer cells in C3H mice. The results suggest that the mechanism may be associated with breast cancer cell apoptosis and inhibition of VEGF expression.

The MicroRNA-23b/27b/24 Cluster Promotes Breast Cancer Lung Metastasis by Targeting Metastasis-suppressive Gene Prosaposin

MicroRNAs (miRNAs) have been shown to function as key regulators of tumor progression and metastasis. Recent studies have indicated that the miRNAs comprising the miR-23b/27b/24 cluster might influence tumor metastasis, although the precise nature of this regulation remains unclear. Here, expression of the miR-23b/27b/24 cluster is found to correlate with metastatic potential in mouse and human breast cancer cell lines and is elevated in metastatic lung lesions in human breast cancer patients. Ectopic expression of the miRNAs in the weakly metastatic mouse 4TO7 mammary tumor cell line had no effect on proliferation or morphology of tumor cells in vitro but was found to increase lung metastasis in a mouse model of breast cancer metastasis. Furthermore, gene expression profiling analysis of miRNA overexpressing 4TO7 cells revealed the direct targeting of prosaposin (PSAP), which encodes a secreted protein found to be inversely correlated with metastatic progression in human breast cancer patients. Importantly, ectopic expression of PSAP was able to suppress the metastatic phenotype in highly metastatic 4T1 and MDA-MB-231 SCP28 cells, as well as in cells ectopically expressing miR-23b/27b/24. These findings support a metastasis-promoting function of the miR-23b/27b/24 cluster of miRNAs, which functions in part through the direct inhibition of PSAP.

Recent developments in prognostic and predictive testing in uveal melanoma

To provide an update on the rapidly evolving methods for assessing prognosis and predicting response to targeted molecular therapy in uveal melanoma. The techniques for assessing prognosis in uveal melanoma have evolved from simple physical features, such as tumor size, location, and cell morphology, to the slightly more sophisticated counting of chromosomal gains and losses. More recently, gene expression profiling has provided a highly accurate and biologically informative gold standard for molecular prognostication. The latest step in the evolution of molecular testing has been the recent discovery of major driver mutations that allow predictive testing of response to targeted molecular therapies. Mutations in GNAQ and GNA11 are early events that promote cell proliferation, and these mutations are sensitive to MAPK kinase, PKC, and AKT inhibitors. Mutations in BAP1, SF3B1, and EIF1AX are later events that are largely mutually exclusive. Mutations in BAP1 are strongly associated with metastasis, whereas those in SF3B1 and EIF1AX are associated with good prognosis. Uveal melanomas with BAP1 mutations demonstrate sensitivity to epigenetic modulators, such as histone deacetylase inhibitors. Clinical trials are now available to evaluate the efficacy of these targeted molecular agents in patients with uveal melanoma. Molecular prognostic testing and enrollment of high-risk patients into clinical trials of targeted molecular therapy are rapidly becoming the standard of care in the management of uveal melanoma.

Nodular histiocytic/mesothelial hyperplasia: a clinicopathologic analysis of 7 cases

To analyze the clinicopathologic and immunohistochemical features of nodular histiocytic/mesothelial hyperplasia (NHMH) and to improve the knowledge of this disease. Seven cases of NHMH were collected and the clinicopathologic and immunohistochemical data were analyzed with review of the literature. Seven male patients aged from 1.5 to 5.0 years (mean 2.8). The main clinical symptom was an inguinal mass.Grossly, main pathological changes were the mural nodule or free nodule in lumen, with diameter of 0.1-0.5 cm.Histologically, the tumor cell morphology was relatively single, cohesive polygonal or oval cells which were arranged in solid sheets or nests, usually with ovoid or deeply grooved nuclei and a moderate amount of pale pink cytoplasm in the nodular collection area. The nuclei had delicate chromatin and no obvious atypia, and mitosis was incidentally found. A few scattered lymphocytes were found in the stroma. The cyst wall was lined by a single layer of mesothelial cells.Immunohistochemically, the most cells in nodular lesion were strongly positive for the histiocytic marker CD68, vimentin and α1-antichymotrypsin, while lining mesothelial cells on the wall were positive for calretinin, MC, WT1, CK5/6, CKpan and EMA. NHMH is a rare and benign tumor-like lesion, and easy to be misdiagnozed, which should be distinguished from neuroendocrine tumors, Langerhans cell histiocytosis, seminoma, mesothelioma and so on. The correct diagnosis of this lesion depends on the clinical characteristics, morphology and immunohistochemistry.

Differentiation of Cancer Cell Origin and Molecular Subtype by Plasma Membrane N-Glycan Profiling

In clinical settings, biopsies are routinely used to determine cancer type and grade based on tumor cell morphology, as determined via histochemical or immunohistochemical staining. Unfortunately, in a significant number of cases, traditional biopsy results are either inconclusive or do not provide full subtype differentiation, possibly leading to inefficient or ineffective treatment. Glycomic profiling of the cell membrane offers an alternate route toward cancer diagnosis. In this study, isomer-sensitive nano-LC/MS was used to directly obtain detailed profiles of the different N-glycan structures present on cancer cell membranes. Membrane N-glycans were extracted from cells representing various subtypes of breast, lung, cervical, ovarian, and lymphatic cancer. Chip-based porous graphitized carbon nano-LC/MS was used to separate, identify, and quantify the native N-glycans. Structure-sensitive N-glycan profiling identified hundreds of glycan peaks per cell line, including multiple isomers for most compositions. Hierarchical clusterings based on Pearson correlation coefficients were used to quickly compare and separate each cell line according to originating organ and disease subtype. Based simply on the relative abundances of broad glycan classes (e.g., high mannose, complex/hybrid fucosylated, complex/hybrid sialylated, etc.), most cell lines were readily differentiated. More closely related cell lines were differentiated based on several-fold differences in the abundances of individual glycans. Based on characteristic N-glycan profiles, primary cancer origins and molecular subtypes could be distinguished. These results demonstrate that stark differences in cancer cell membrane glycosylation can be exploited to create an MS-based biopsy, with potential applications toward cancer diagnosis and direction of treatment.

Altered features and increased chemosensitivity of human breast cancer cells mediated by adipose tissue-derived mesenchymal stromal cells

BackgroundMesenchymal stromal cells (MSCs) represent heterogeneous cell population suitable for cell therapies in regenerative medicine. MSCs can also substantially affect tumor biology due to their ability to be recruited to the tumor stroma and interact with malignant cells via direct contacts and paracrine signaling. The aim of our study was to characterize molecular changes dictated by adipose tissue-derived mesenchymal stromal cells (AT-MSCs) and the effects on drug responses in human breast cancer cells SKBR3.MethodsThe tumor cells were either directly cocultured with AT-MSCs or exposed to MSCs-conditioned medium (MSC-CM). Changes in cell biology were evaluated by kinetic live cell imaging, fluorescent microscopy, scratch wound assay, expression analysis, cytokine secretion profiling, ATP-based viability and apoptosis assays. The efficiency of cytotoxic treatment in the presence of AT-MSCs or MSCs-CM was analyzed.ResultsThe AT-MSCs altered tumor cell morphology, induced epithelial-to-mesenchymal transition, increased mammosphere formation, cell confluence and migration of SKBR3. These features were attributed to molecular changes induced by MSCs-secreted cytokines and chemokines in breast cancer cells. AT-MSCs significantly inhibited the proliferation of SKBR3 cells in direct cocultures which was shown to be dependent on the SDF-1α/CXCR4 signaling axis. MSC-CM-exposed SKBR3 or SKBR3 in direct coculture with AT-MSCs exhibited increased chemosensitivity and induction of apoptosis in response to doxorubicin and 5-fluorouracil.ConclusionsOur work further highlights the multi-level nature of tumor-stromal cell interplay and demonstrates the capability of AT-MSCs and MSC-secreted factors to alter the anti-tumor drug responses.

A novel rapid-onset high-penetrance plasmacytoma mouse model driven by deregulation of cMYC cooperating with KRAS12V in BALB/c mice

Our goal is to develop a rapid and scalable system for functionally evaluating deregulated genes in multiple myeloma (MM). Here, we forcibly expressed human cMYC and KRAS12V in mouse T2 B cells (IgM+B220+CD38+IgD+) using retroviral transduction and transplanted these cells into lethally irradiated recipient mice. Recipients developed plasmacytomas with short onset (70 days) and high penetrance, whereas neither cMYC nor KRAS12V alone induced disease in recipient mice. Tumor cell morphology and cell surface biomarkers (CD138+B220−IgM−GFP+) indicate a plasma cell neoplasm. Gene set enrichment analysis further confirms that the tumor cells have a plasma cell gene expression signature. Plasmacytoma cells infiltrated multiple loci in the bone marrow, spleen and liver; secreted immunoglobulins; and caused glomerular damage. Our findings therefore demonstrate that deregulated expression of cMYC with KRAS12V in T2 B cells rapidly generates a plasma cell disease in mice, suggesting utility of this model both to elucidate molecular pathogenesis and to validate novel targeted therapies.

Epigenetic reprogramming reverses the malignant epigenotype of the MMP/TIMP axis genes in tumor cells

Cancer progression is characterized by extensive tumor invasion into the surrounding extracellular matrix (ECM) and migration to metastatic sites. The increased proteolytic degradation of the ECM during tumor invasion is directly dependent on the activity of matrix metalloproteinases (MMPs), counter-balanced by tissue inhibitors of matrix metalloproteinases (TIMPs). In this study, we found that unbalanced expression of MMP/TIMP axis genes in tumors was correlated with aberrant epigenotypes in the various gene promoters. The malignant epigenotypes could be therapeutically corrected by a simple defined factor-mediated reprogramming approach. Correction of the abnormal epigenotypes by nuclear remodeling leads to a rebalance in the gene expression profile, an alteration in tumor cell morphology, attenuation of tumor cell migration and invasion in vitro, and reduced tumorigenicity in nude mice. We further identified the downregulation of the MKK-p38 MAPK signal pathway as an important underlying mechanism for reduced tumorigenicity in this epigenetic reprogramming model. These data demonstrate that the malignant phenotypes seen in cancer can be corrected by a nuclear remodeling mechanism, thus highlighting a novel non-chemotherapeutic, non-radiotherapeutic approach for the treatment of cancer.