Epithelio-mesenchymal Transition Research Articles

Abstract CT217: First-line durvalumab and tremelimumab with chemotherapy in RAS-mutated metastatic colorectal cancer: a phase 1b-2 trial

Abstract Metastatic colorectal cancer (mCRC) remains a difficult-to-treat disease. While patients with microsatellite instable metastatic colorectal cancer (mCRC) benefit from immune checkpoint blockade, chemotherapy with targeted therapies remains the only therapeutic option for microsatellite stable (MSS) tumors. The single arm, phase IB/II MEDITREME trial evaluated the safety and efficacy of durvalumab plus tremelimumab combined with mFOLFOX6 chemotherapy in first line, in 57 patients with RAS-mutant unresectable mCRC. Safety was the primary objective of phase IB; no safety issue was observed. The phase II primary objective of efficacy in terms of 3-month PFS in MSS patients was met, with 3-month PFS of 90.7% [95% CI: 79.2-96%]. For secondary objectives, response rate was 64.5%, median PFS was 8.2 months [95% CI: 5.9-8.6] and overall survival was not reached in MSS patients. We observed higher tumor mutational burden and lower genomic instability in responders. Integrated transcriptomic analysis underlined that high immune signature, and low epithelio-mesenchymal transition were associated with better outcome. Immunomonitoring showed induction of neoantigen and NY-ESO1 and TERT blood tumor specific T cell response, associated with better PFS. The combination of durvalumab-tremelimumab with mFOLFOX6 was tolerable with promising clinical activity in MSS mCRC. Clinicaltrials.gov identifier: NCT03202758. Citation Format: Marion Thibaudin, Jean-David Fumet, Emeric Limagne, Léa Hampe, Susy Daumoine, Valentin Derangère, Caroline Laheurte, Olivier Adotevi, Caroline Truntzer, François Ghiringhelli. First-line durvalumab and tremelimumab with chemotherapy in RAS-mutated metastatic colorectal cancer: a phase 1b-2 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT217.

Towards understanding chronic kidney disease

Chronic kidney disease (CKD) is a global health problem affecting almost 15% of the population worldwide. After renal injury, there is a nephron loss and remaining nephrons ensure the glomerular filtration rate (GFR) with compensatory hyperplasia and hypertrophy: This is called the nephron reduction. After nephron reduction, renal function will gradually decline and lead to chronic end-stage renal failure. Whatever the initial cause of the renal injury, recent data suggest there are common molecular mechanisms at the origin of CKD progression. Moreover, the renal lesions are very reproducible with glomerulosclerosis, tubular atrophy and partial epithelio-mesenchymal transition, interstitial fibrosis and vascular abnormalities. The physiopathology of CKD progression is unclear but some hypotheses have been described: i) the nephron "overwork", supported by recent works showing that the nephron reduction leads to hyperfiltration by the remaining nephrons and the stability of the GFR; ii) the "podocyte adaptation" theory, reflected by the importance of the podocytopathy in CKD progression and the crucial role of residual proteinuria in renal lesion development; iii) the activation of EGFR signaling pathways in surgical nephron reduction model and its involvement in CKD progression. Finally, CKD progression remains poorly understood and further studies will be necessary to discover new CKD molecular pathways and to develop new therapeutic insight in CKD management.

Survivin-positive circulating tumor cells as a marker for metastasis of hepatocellular carcinoma

BACKGROUNDCirculating tumor cells (CTCs) and survivin are indicators for tumor stage and metastasis, as well as epitheliomesenchymal transition, in various cancers, including hepatocellular cancer (HCC). AIMTo explore the potential of survivin-positive CTCs, specifically, as a marker for tumor progression in HCC patients.METHODSWe examined the survivin expression pattern in CTCs obtained from 179 HCC patients, and investigated the in vitro effects of survivin silencing and overexpression on the proliferation and invasion of HCC cells. CTC count and survivin expression in patient samples were examined using RNA in situ hybridization.RESULTSAll 179 patients were positive for CTC markers, and 94.41% of the CTCs were positive for survivin. The CTC and survivin-positive CTC counts were significantly higher in the HCC patients than in the normal controls, and were significantly associated with tumor stage and degree of differentiation. Further, survivin overexpression was found to induce HepG2 cell proliferation, reduce apoptosis, and improve invasive ability.CONCLUSIONSurvivin shows upregulated expression (indicative of anti-apoptotic effects) in HCC. Thus, survivin-positive CTCs are promising as a predictor of HCC prognosis and metastasis, and their accurate measurement may be useful for the management of this cancer.

MWCNTs induced epithelio-mesenchymal transition of BEAS-2B cells

MWCNTs induced epithelio-mesenchymal transition of BEAS-2B cells

IMP3 Expression in Ovarian Serous Tumours: Correlation with Epithelial-Mesenchymal Transition-Related Markers (E-Cadherin and Vimentin) and Clinicopathological Factors

Introduction: Ovarian epithelial cancer is an aggressive malignancy of which Ovarian Serous Carcinoma (OSC) represents a common type. The insulin-like growth factor mRNA-binding protein 3 (IMP3) is one of mRNA-binding protein family that is overexpressed in a variety of human cancers, including ovarian carcinomas. Being associated with adverse prognostic outcome in these cancers, which makes this protein a reliable biomarker to distinguish some cancers from their benign mimetic lesions. Aim: To analyse IMP3 expression in serous ovarian tumours and correlates the expression in malignant tumours with Epithelio-Mesenchymal Transition (EMT) related markers (Epithelial(E)-cadherin and vimentin) and clinicopathological factors to assess the clinical application of IMP3 in the diagnosis, prognosis and possible application in targeted therapy of such tumours. Materials and Methods: This was a cross-sectional research that was done at the Departments of Pathology, Obstetric and Surgery, Zagazig and Banha Universities Hospitals, Egypt. Fifty-nine formalin-fixed paraffin-embedded specimens (including 43 cases OSC, 6 cases border line serous tumour and 10 cases benign serous cystadenoma) were collected from January 2014 to December 2018. Each case was stained immunohistochemically with IMP3, E-cadherin and Vimentin. Markers expressions were statistically analysed using SPSS software (version 19.0; SPSS, Chicago, IL). The p-values ≤0.05 was regarded statistically significant. Results: IMP3 was detected in 76.7% (33/43) of the studied OSC. IMP3 was in a significant association with advanced FIGO (International Federation of Gynaecology and Obstetrics) stage and with lymph node metastases (p=0.02 and 0.019, respectively). E-cadherin was predominantly lost in 53.5% (23/43) of the studied OSCs and was significantly associated with advanced FIGO stage (p=0.004). Vimentin was detected in 79.1% (34/43) of the studied OSCs and was in association with high grades and advanced stage (p=0.018 and 0.007, respectively). An inverse significant correlation was detected between IMP3 and E-cadherin expressions (Spearman correlation (r)=-0.480, p-value=0.001), while positive significant correlation was detected between IMP3 and Vimentin expressions (Spearman correlation (r)=0.393, p-value=0.009). Conclusion: IMP3 is an oncogene unregulated in OSC. It is a prognostic marker associated with tumour aggressiveness. Moreover, IMP3 could promote tumour invasion and metastasis via EMT in OSCs.

PO-238 Clinicopathological significance of EMT markers in thymic epithelial tumours

IntroductionThymic epithelial tumours (TETs) are the relatively rare tumours originated from thymus. TETs are histologically categorised according to the WHO classification based on the morphology of epithelial tumour cells and proportion of lymphocytic involvement. Epithelio-mesenchymal transition (EMT) has reported to play pivotal roles in tumour progression including invasion/metastasis, drug resistance, and cancer stemness. However, the precise clinicopathological associations with EMT characteristics in TETs remain to be elusive. Herein, we have examined EMT markers in TETs integrated with their clinicopathological information.Material and methodsA total of 109 patients with TETs were surgically resected between 2002 and 2017 in our institution. Tissue samples were collected from the paraffine-embedded tumour blocks of these patients. The 2 mm cores in diameter of the most representative areas of the tumours were selected by the pathologist and assembled to make tissue microarrays. Immunohistochemical (IHC) stainings for E-cadherin (E) and vimentin (V) were performed. P63 and pan-cytokeratin were additionally stained to detect epithelial tumour cells. The expressions of these molecules were scored and quantified to categorise the activation level of EMT into three groups based on the combination of E and V levels: Full; E-V+, Partial; E+V+ or E-V-. Null; E+V-. For statistical analyses, chi-square tests and Cox hazard models were applied. P-value less than 0.05 was considered significant.Results and discussionsThe median follow-up time was 54.3 months (range, 0.3–178.7). Predominant WHO histological subtypes were as follows: A;8, AB;18, B1;30, B2;30, B3;6. C;15, others;2. Pathologic stage distributions by TNM classfication were as follows: 1;80, 2;2, 3;13, 4a;7, 4b;4. The positive rates of E-cadherin or vimentin expression in epithelial tumour cells were 54.2% and 57.9%, respectively. EMT activation was significantly exhibited in indolent predominant WHO subtype, and in the early stage of pT factor and the Masaoka classification (p=0.0005, 0.0493, and 0.0366, respectively). Multivariate analyses of overall survival time including pT, pN, pM, age, and EMT levels showed that pT factor was significantly prognostic (p=0.0218), but not for EMT.ConclusionEMT activation mechanisms in TETs were inversely correlated with their histological subtype and the primary tumour progression. Further studies are necessary to elucidate tumour progression mechanisms in TETs.

P3.02-046 EGFR-Grb2-GEP100 Complex Promoted Its Invasive and Metastatic Potential via Arf6 Pathway in Lung Adenocarcinoma

Invasive and metastatic activities are the most challenging hallmark of cancer. We previously showed that GEP100-Arf6 pathway signals from stimulated ErbB family receptors was pivotal for epithelio-mesenchymal transition (EMT) and induced cancer invasive activity leading to nodal and distant metastasis. Here we have examined the enhancing effect of Grb2 on the binding of GEP100 with EGFR leading to Arf6 activation and EMT, and the significance of EGFR-Grb2-GEP100 binding complex on the invasive and metastatic potentials in lung cancer cells as well as in clinical settings.

Is Epithelio-Mesenchymal transition actively involved in tissue folding?

Is Epithelio-Mesenchymal transition actively involved in tissue folding?

Clinicopathologic significance of epithelio-mesenchymal transition in human lung adenocarcinomas: An integrative analysis, inclusive of genetic alterations, on 256 surgically resected cases

IntroductionPrior studies have shown that the activation of epithelio-mesenchymal transition (EMT) in tumor cells induces their invasive and metastatic properties. In our study, we aimed to elucidate the clinicopathologic significance of EMT in lung adenocarcinomas. Patients and methodsClinical samples were obtained from 256 cases of lung adenocarcinomas that were consecutively resected from 2001 to 2007 at Kyoto University Hospital. Tissue microarrays of these samples were immunohistochemically stained for E-cadherin and vimentin, and classified into three groups, named “Full”, “Partial”, “Null”, based on the combined expression of these two markers, which is indicative of tumor EMT activation level.DNA was extracted from surgical specimens, and the mutations in the hot-spot exons of EGFR, ALK, K-ras, and p53 were detected by single-strand conformation polymorphism or direct sequencing. ResultsGroup “Full” exhibited the highest positivity in terms of local lymph-vascular involvements and lymph node metastases, followed by groups “Partial” and “Null,” respectively. Significant differences in overall and disease-free survivals (OS, DFS) were seen among these 3 groups. Poorly differentiated adenocarcinomas and heavy smoking status had strong associations with the EMT level. Tumors harboring mutant EGFRs showed less EMT activation. In statistical analyses, EMT was a significant factor both in logistic regression models for the prediction of lymph node metastases, as well as, in Cox hazard models for OS and DFS for exclusion of node metastasis. ConclusionsThe activation of EMT in human lung adenocarcinomas plays pivotal roles in their malignant progression. EMT is an appropriate target for the improvement of prognosis. MicroabstractThe significance of epithelio-mesenchymal transition in lung adenocarcinoma remains controversial in clinical settings. We retrospectively studied the combination of epithelial and mesenchymal markers on 256 patients. The EMT activation levels showed a trend for pathologic local invasiveness, node metastases, and poor survival. These findings suggest that EMT is the challenging target for the malignant progression, and is worth further investigation.

P3.01-048 Cigarette Smoking is Associated with Epithelio-Mesenchymal Transition in Human Adenocarcinoma: Topic: Functional Biology in Lung Cancer

Cigarette smoking (CS) is well known to cause lung cancer. In addition to the mechanisms of tumorigenesis of lung cancer with CS, a lot of evidences are currently accumulating that CS induces epithelio-mesenchymal transition (EMT) in tumor cells. The correlation of CS with the malignant properties of lung cancer remained elusive in clinical settings. Here we examined the clinical significance of CS with regard to EMT and malignant progression in human lung adenocarcinoma. Clinical samples were obtained from the 239 cases of resected lung adenocarcinoma which were consecutively operated from January 2001 to December 2007 in our institution. Pathological stage distribution of the cases by TNM classification (WHO, 7th edition) was below: 1A: 118, 1B: 71, 2A: 22, 2B: 4, 3A: 23, 3B: 1. Smoking history was taken from all the patients, then their smoking status were classified into 3 groups according to the Brinkman Index (Non;0, n=109: Light;1∼400, n=29: Heavy; 401∼, n=101). The samples were immunostained against E-cadherin and Vimentin using tissue microarrays of resected specimens to assess the activation level of EMT. Then, we classified into 3 groups: the group ‘N’, E-cadherin(E+) and Vimentin(V-), “null” EMT activation; the group ‘F’, E-/V+, ‘full’ EMT; the group ‘P’, E-/V- or E+/V+, ‘partial’ EMT. The numbers of the group F/ P/ N were 38/ 93/ 108, respectively. Furthermore, DNA samples were extracted from frozen surgical samples and the mutations for the hot-spot exons of EGFR, K-ras, and p53 were detected by SSCP or direct sequencing methods. The differences of survival duration, pathological invasive factors, DNA mutations and EMT activation level were statistically analyzed among smoking groups. Significant difference was found in 5-year survival rate among 3 smoking groups: Non, 89.1%; Light, 89.5%; Heavy, 61.7%. Smoking status was significantly associated with EMT activation, DNA mutation status, local invasive factors, and lymph-node metastasis. In the tumors harboring either wild-type K-ras or wild-type p53, heavy smoking was associated with EMT activation (p<0.0001, p=0.0002, respectively), whereas no correlation with regard to EGFR status. Smoking amounts had a significant association with EMT activation level and malignant progression of human adenocarcinoma. Heavy smoking was related with EMT activation of the tumors either with wild-type K-ras or p53.

An early-screening biomarker of endometrial carcinoma: NGAL is associated with epithelio-mesenchymal transition.

neutrophilgelatinase-associated lipocalin is currently one of the most interesting and enigmatic proteins involved in the development of malignancies. In this study, we found that the expression of neutrophilgelatinase-associated lipocalin was up-regulated in endometrial cancer tissues and cell lines, significantly increased in early-grade ones, suggesting it may serve as a biomarker for early-stage screening for endometrial carcinoma. Moreover, neutrophilgelatinase-associated lipocalin was up-regulated in Ishikawa cells under going epithelio-mesenchymal transition induced by epidermal growth factor (5 ng/ml). Up-regulation of neutrophilgelatinase-associated lipocalin may correlate with the down-regulation of E-cadherin expression, up-regulation of Vimentin expression, enhanced cell migration, invasion and proliferation, which are the typical hallmarks of epithelio-mesenchymal transition processes. neutrophilgelatinase-associated lipocalin may play a dual role during tumorigenetic and developmental processes of endometrial carcinoma. These results suggested neutrophilgelatinase-associated lipocalin to be a potential molecular target in the early diagnosis and treatment of endometrial carcinoma. Further studies are warranted to clarify the molecular mechanisms behind the expression and function of neutrophilgelatinase-associated lipocalin and epithelio-mesenchymal transition.

Abstract 1587: GEP100-Arf6 pathway enhanced by Grb2 expression plays important roles for node-metastasis of lung cancer

Abstract BACKGROUND Invasive and metastatic activities are the most challenging hallmark of cancer in clinical settings. Our previous reports have shown that GEP100 activates Arf6 by its binding to activated ErbB family receptors, such as EGFR. They also have revealed the phosphorylation at specific tyrosines in the C-terminal EGFR is necessary for the binding to the plekstrin homology (PH) domain of GEP100, which tyrosines are well known to be Grb2-binding sites as well. Additionally, this GEP100-Arf6 signal pathway is pivotal for epithelio-mesenchymal transition (EMT) leading to invasion and metastasis in various types of tumors. Here we have examined the augmentation effect of Grb2 on the binding of GEP100 with EGFR and the Arf6 activation. Furthermore, the significance of co-expression of Grb2 and GEP100 in clinical settings was analyzed MATERIALS AND METHODS GST-tagged proteins including PH domain of GEP100 or SH2/SH3 domain of Grb2 inserted into pGEX vector were expressed in bacteria with glutathione-beads purification. They were used for the mutual binding assays. A549 cells which HA-Grb2 or HA alone vector was transfected, were starved for 18hours and stimulated with EGF. And, their lysates were applied for the immunoprecipitation assay against GEP100. Then, Arf6 activities of these cells were examined using the pulldown assay with GST-tagged GGA protein. Furthermore the in vitro invasive activities of those cells were measured by Matrigel invasion assays. The expression levels of Grb2 and GEP100 of the tumor cells by immunohistochemical staining methods were examined in resected human lung adenocarcinoma specimens. The expression data of the two molecules integrated with their clinicopathological factors and EMT status information previously published were analyzed with regard to their invasive and metastatic activities. RESULTS Our results revealed that endogenous Grb2 was immunoprecipitaed with GEP100. These two molecules were physically associated through the PH domain of GEP100 and both the SH2 and N-terminal SH3 domain of Grb2, not its C-terminal SH3 domain. Exogenously aberrant expression of Grb2 in A549 lung cancer cells enhanced the association between activated EGFR and GEP100, consequently, Arf6 activation, and in vitro invasive activity, according to the expression level of Grb2. Among 239 lung adenocarcinoma specimens on tissue microarrays, 131 (55%) and 65 (27%) cases of patients were positive for Grb2 and GEP100, respectively. Tumors with double-positive for Grb2 and GEP100 (45 cases) showed significantly more aggressive EMT status (p = 0.0116) and higher node-metastatic potential (p = 0.0082, node-positive/negative; 12/33 to 7/77) than the double-negative one (84 cases). CONCLUSION Grb2 augments the binding of GEP100 to EGFR leading to Arf6 activation, and promotes lung cancer invasion and metastasis via GEP100-Arf6 pathway. Citation Format: Toshi Menju, Shigeto Nishikawa, Koji Takahashi, Shinya Neri, Takao Nakanishi, Hiroyuki Cho, Kei Shikuma, Terumasa Sowa, Makoto Sonobe, Stephan M. Feller, Hisataka Sabe, Hiroshi Date. GEP100-Arf6 pathway enhanced by Grb2 expression plays important roles for node-metastasis of lung cancer. [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 1587.

Biopathological Significance of TLR9 Expression in Cancer Cells and Tumor Microenvironment Across Invasive Breast Carcinomas Subtypes.

Toll-like receptors (TLRs) are pattern recognition receptors mainly expressed by cells of the immune system but also by epithelial tumor cells. Little is known about expression patterns of TLR genes in breast tumors, and their clinical significance is unclear. The aim of our study was to investigate expression of TLRs pathway components in pre-invasive breast lesions and invasive breast carcinomas (IBCs). We used RT-PCR assays to quantify mRNA levels of the 10 TLR genes and genes involved in TLR pathways in 350 breast tumors from patients with known clinical/pathological status and long-term outcome. Sets of 158 breast samples were also analyzed by immunochemistry including; 40 early noninvasive breast lesions, 38 IBCs and 80 triple negative carcinomas subtype (TNCs). We identified TLR9 as the major TLR gene family member upregulated in breast tumors and more particularly in TNCs. Immunohistochemical studies demonstrated that TLR9 protein was expressed in tumor epithelial and stromal cells of the TLR9 mRNA-overexpressing tumors. TLR9 overexpression appears very early during breast carcinogenesis. High TLR9 levels were associated with favorable outcome in the TNC sub-group. TLR9 overexpression was associated with alterations of down-stream components of the TLR9 signaling pathway, epithelio-mesenchymal transition (EMT) induction and EGFR pathway deregulation. TNCs with TLR9 overexpression were significantly correlated with development of a fibrous and inflammatory microenvironment with variable status of nuclear phosphoSTAT3. Our results suggest that TLR9 could play a role in TNC carcinogenesis and could be useful as predictive biomarker and therapeutic target.

Abstract P4-01-12: Circulating tumor cells (CTC) and endothelial cells (CEC) changes in HER2 negative metastatic breast cancer (MBC) patients treated with first line weekly paclitaxel and bevacizumab: Preliminary results of a prospective cohort from the French Breast Cancer

Abstract Background: increased levels of circulating tumor cells (CTC) are associated with worse progression-free survival (PFS) and overall survival (OS) in patients (pts) with MBC (Bidard FC et al, Lancet Oncol 2014). The failure of chemotherapy to reduce CTCs to levels below five CTCs per 7.5 mL whole blood at first follow-up after initiating a new systemic therapy for MBC predicts shorter time to progression and OS. It has been hypothesized that bevacizumab could modify CTC prognostic value due to extravasation or epithelio-mesenchymal transition induction. CEC variations to predict benefit of anti-angiogenic treatment is still controversial. Patients &amp; methods: the French cohort COMET is a prospective study including first line HER2 negative pts receiving weekly paclitaxel and bevacizumab according to EMEA approved combination. The aim of this cohort is to evaluate clinical, biological and radiological parameters associated with pts outcome (CTC, CEC, VEGFA levels, ctDNA, pharmacogenomic polymorphisms, metabolomic parameters, visceral fat assessed by initial CTscan, serum estradiol level, and quality of life). We present here the first planned analysis on 206 patients evaluated for CTC and CEC using the FDA cleared CellSearch method. Results: inclusions started in 09/2012. At time of analysis, 219 patients were included, 211 were evaluable for CTC at baseline (failure rate 4%) and 207 for CEC at baseline (failure rate 5%). Due to short follow-up, 173 pts and 166 pts were evaluable for both CTC and CEC at baseline and first day of second cycle of CT (D1C2) respectively. At baseline, 100/211 (47%) pts had ≥ 5 CTC (median 4 (range 0-30,000) and 30% had no detectable CTC (0 CTC). After one cycle of chemotherapy (D28) 38 pts (22%) had still ≥ 5 CTC: 37 pts with initial high level and only one patient with low CTC at baseline had increased CTC above 5. Median number CEC was 21 (0)-2231) at baseline and 22 (1-881) at D1C2. CEC increased in 16% and decreased in 15% of the cases. CTC &amp; CEC changes after one cycleCTC BaselineCTC at D1C2CEC BaselineCEC at D1C2≥ 5≥ 537 (21%)&amp;gt;20&amp;gt;2065 (39%)≥ 5&amp;lt;545 (26%)&amp;gt;20≤ 2025 (15%)&amp;lt;5≥ 51 (&amp;lt;1%)≤ 20&amp;gt;2027 (16%)&amp;lt;5&amp;lt;590 (52%)≤ 20≤ 2049 (30%)Total173166 CTC number at baseline line and CTC D1C2 were correlated (p&amp;lt;0.01) (Spearman test). There was no correlation between CEC at baseline or at D1C2 with CTC or CTC changes. Final analysis will be completed when 206 couples for both CTC and CEC at baseline and D1C2 will be available. Accrual is still ongoing. Conclusion: this 22% rate of failure to reduce CTC &amp;lt; 5 after one cycle of first line CT in a homogeneously bevacizumab-treated cohort of MBC patients did not differ from previous series. As second lines of chemotherapy do not improve the poor prognosis of this group of patients according to the SWOG 500 study results (Smerage et al, JCO 2014), trials of novel therapeutic agents should be considered at the time of progression. Citation Format: Jean-Yves Pierga, Isabelle Vaucher, Maya Gutierrez, Olivier Tredan, Séverine Guiu, Gilles Romieu, Anthony Goncalves, Marc Debled, Christelle Levy, Jean-Marc Ferrero, Christelle Jouannaud, Elisabeth Luporsi, Marie-Ange Mouret-Reynier, Florence Dalenc, Bernard Asselain, Jerome Lemonnier, Francois-Clement Bidard. Circulating tumor cells (CTC) and endothelial cells (CEC) changes in HER2 negative metastatic breast cancer (MBC) patients treated with first line weekly paclitaxel and bevacizumab: Preliminary results of a prospective cohort from the French Breast Cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-12.

Rho kinase activity controls directional cell movements during primitive streak formation in the rabbit embryo.

During animal gastrulation, the specification of the embryonic axes is accompanied by epithelio-mesenchymal transition (EMT), the first major change in cell shape after fertilization. EMT takes place in disparate topographical arrangements, such as the circular blastopore of amphibians, the straight primitive streak of birds and mammals or in intermediate gastrulation forms of other amniotes such as reptiles. Planar cell movements are prime candidates to arrange specific modes of gastrulation but there is no consensus view on their role in different vertebrate classes. Here, we test the impact of interfering with Rho kinase-mediated cell movements on gastrulation topography in blastocysts of the rabbit, which has a flat embryonic disc typical for most mammals. Time-lapse video microscopy, electron microscopy, gene expression and morphometric analyses of the effect of inhibiting ROCK activity showed – besides normal specification of the organizer region – a dose-dependent disruption of primitive streak formation; this disruption resulted in circular, arc-shaped or intermediate forms, reminiscent of those found in amphibians, fishes and reptiles. Our results reveal a crucial role of ROCK-controlled directional cell movements during rabbit primitive streak formation and highlight the possibility that temporal and spatial modulation of cell movements were instrumental for the evolution of gastrulation forms.

Parameters affecting efficiency of in ovo electroporation of the avian neural tube and crest.

Many variations in avian in ovo transfection of the neural tube/crest have been reported, but never compared quantitatively. Genome integrating pT2K-CAGGS-GFP and pCAGGS-T2TP transposase plasmids were co-electroporated into quail E2 embryo trunk neural tube and the proportion of GFP-expressing neural cells was counted 1 and 7 days later. Electroporation efficiency increased with plasmid concentration and pulse number but plateaued at, respectively, above 1.25 µg/µL and 3 pulses. Bilateral electroporation transfected more cells than unilateral but less than that anticipated by doubling the unilateral treatment. Holding the concentration of GFP plasmid constant and varying the transposase plasmid concentration revealed an optimum ratio of, in this case, 4:1 (1.2 µg/µL:0.3 µg/µL). Leaving transfected embryos to E9 confirmed that expression was maintained in vivo with the transposase system, but declined with non-integrated plasmid. Transfection of neural crest cells was low if electroporated less than 6-8 hr before emigration. We propose this indicates loss of epithelial integrity well prior to exit. We suggest this event be termed epithelio-mesenchymal transition sensu stricto, whereas the term delamination be reserved for the later emigration from the neural epithelium. Co-electroporation in ovo must take into account plasmid(s) concentration and ratio, pulse number, pulse directionality, and timing.

Epithelio-mesenchymal transitional attributes in oral sub-mucous fibrosis

Evaluating molecular attributes in association with its epithelial and sub-epithelial changes of oral sub-mucous fibrosis is meaningful in exploring the plausibility of an epithelio-mesenchymal transition (EMT) and malignant potentiality of this pathosis. In this study histopathological and histochemical attributes for basement membrane and connective tissue in biopsies of oral sub-mucous fibrosis (n=55) and normal oral mucosa (n=16) were assessed and expressions of p63, E-cadherin, β-catenin, N-cadherin and TWIST were analyzed immunohistochemically. The p63 and its isoforms (TA and ∆N), PARD3, E-cadherin and β-catenin were also assessed transcriptomically by q-PCR and EMT players like TWIST1, ZEB1, MMP9 and micro-RNA 205 were searched in gene expression microarrays. Oral epithelium demonstrating impairment in progressive maturation in oral sub-mucous fibrosis concomitantly experienced an increase in basement membrane thickness and collagen deposition along with alteration in target molecular expressions. In comparison to non-dysplastic conditions dysplastic stages exhibited significant increase in p63 and p63∆N expressions whereas, E-cadherin and β-catenin exhibited loss from the membrane with concurrent increase in cytoplasm. Further the N-cadherin and TWIST were gained remarkably along with the appearance of nuclear accumulation features of β-catenin. The microarray search had noticed the up-regulation of TWIST1, ZEB1 and MMP9 along with down regulation of micro-RNA 205. The simultaneous increase in basement membrane thickness and sub-epithelial collagen deposition were the plausible indicators for increased matrix stiffness with expected impact on oral epithelial functional homoeostasis. This was corroborated with the increase in expressions of epithelial master regulator p63 and its oncogenic isoform (∆N) along with membranous loss of E-cadherin (EMT hallmark) and its associate β-catein and gain of mesenchymal markers like N-cadherin and TWIST. These also became indicative for the induction of epithelial to mesenchymal transitional mechanism in oral sub-mucous fibrosis when connoted here with the relevant modulation in expressions of EMT regulators.

Cell Composition of the Primary Culture of Fetal Liver

Fetal liver is known as a source of multipotent mesenchymal stromal cells. These cells are routinely isolated by adhesion to plastic, but thus prepared culture is contaminated by other cells. For instance, primary cell culture of from rat fetal liver, apart from fibroblasts with phenotypic characteristics of mesenchymal stromal cells, contained skeletal muscle precursors, myofibroblasts, and epitheliocytes expressing cytokeratin-19 (the latter was also detected in some fibroblast-like cells probably undergoing epithelio-mesenchymal transition). During passaging, fibroblasts become practically the only cell type in the culture.

Expression of snail, twist, and Zeb1 in malignant mesothelioma

We examined 56 malignant mesotheliomas, 117 lung adenocarcinomas, and 34 metastatic lung adenocarcinomas with antibodies to transcription factors involved in epitheliomesenchymal transition. The tumors were stained immunohistochemically with antibodies zeb1, twist, and snail. Malignant mesotheliomas exhibited a stronger expression of zeb1 and twist than lung adenocarcinomas (p < 0.001 for both). Metastatic adenocarcinomas displayed a more frequent expression of zeb1 and twist (p < 0.001 for both) than lung adenocarcinomas. Patients with snail positive mesotheliomas experienced a better survival (p = 0.046), whereas in lung adenocarcinomas, this trait predicted worse survival (p = 0.024). Biphasic mesotheliomas had a more frequent expression of snail or zeb1 than epithelioid and sarcomatoid mesotheliomas as a group (p = 0.034 and p = 0.005, respectively). E-cadherin was more commonly present in epithelioid mesotheliomas (p = 0.002). Cases with snail positivity showed a significantly lower apoptotic index (p = 0.039). Malignant mesotheliomas display strong twist and zeb1 expression, which may be an indication of transdifferentiation between the epithelioid and sarcomatoid cell types with biphasic mesotheliomas representing tumors in active transition. Metastatic adenocarcinomas show a higher expression of zeb1 and twist than primary lung tumors, confirming our previous findings and highlighting their significance in the metastatic process. Snail expression is associated with poor prognosis in lung adenocarcinomas, but an opposite effect was seen in mesothelioma, a fact which may be related to the different cellular origin of epithelial and mesothelial cells.

Snail promotes an invasive phenotype in lung carcinoma

BackgroundSnail is a transcriptional factor which is known to influence the epitheliomesenchymal transition (EMT) by regulating adhesion proteins such as E-cadherin and claudins as well as matrix metalloproteases (MMP).MethodsTo evaluate the functional importance of snail, a transciptional factor involved in EMT in lung tumors, we investigated its expression in a large set of lung carcinomas by immunohistochemistry. Expression of snail and effects of snail knockdown was studied in cell lines.ResultsNuclear snail expression was seen in 21% of cases this being strongest in small cell lung carcinomas (SCLC). There was significantly greater snail expression in SCLC compared to squamous cell or adenocarcinoma. Positive snail expression was associated with poor survival in the whole material and separately in squamous cell and adenocarcinomas. In Cox regression analysis, snail expression showed an independent prognostic value in all of these groups. In several cell lines knockdown of snail reduced invasion in both matrigel assay and in the myoma tissue model for invasion. The influence of snail knockdown on claudin expression was cell type specific. Snail knockdown in these cell lines modified the expression of MMP2 and MMP9 but did not influence the activation of these MMPs to any significant degree.ConclusionsThe results show that snail plays an important role in the invasive characteristics of lung carcinoma influencing the survival of the patients. Snail knockdown might thus be one option for targeted molecular therapy in lung cancer. Snail knockdown influenced the expression of claudins individually in a cell-line dependent manner but did not influence MMP expressions or activations to any significant degree.