CONFERENCE ABSTRACTS
CIRCULATING TUMOUR CELLS IN BREAST AND OVARIAN CANCER: SIZE-BASED ISOLATION AND EX VIVO EXPANSION
Introduction/Background
Circulating tumour cells (CTCs) play a crucial role in cancer dissemination and cellular extravasation leading to metastasis. There are only a limited number of CTCs per clinically/ethically allowed cancer patient`s blood draw and expanding this population of cells in vitro is crucial in order to provide a reliable number of cells to analyse CTC biology. CTCs can grow in a hypoxic environment and the activation of hypoxia-inducible factor (HIF-1a) results in increased cell survival and cellular proliferation, leading to cancer progression. Our aim was to optimise cell culture conditions using cobalt chloride (CoCl2) as a chemical inducer of hypoxia that would allow us to examine growth of cells in real time. Primary ovarian cancer cells would be used for the hypoxia optimisation and conditions adapted ovarian/breast CTC cultures in vitro.
Methodology
Primary ovarian cancer cells were cultured in modified media supplemented with various concentrations of CoCl2 for HIF1 a induction (50, 100, 150 and 200 uM). Cell viability and the expression of HIF-1a, PHH3, EpCAM and HER2 were examined in these cells using either ELISA, Immunoblotting or Immunofluorescence techniques. CTCs were isolated from breast and ovarian cancer patients using the ScreenCell® Cyto R device and cultured in specially modified media optimised for CTC culture supplemented with 20% FCS, growth factors and additives including: FGF-2, FGF-10, Nicotinamide, Y-27632, Primocin and CoCl2. EpCAM and HER2 were examined in cultured and expanded CTCs using Immunofluorescence techniques.
Results
HIF-1a expression was induced and cell proliferation and viability were maintained in the primary ovarian cancer cells at a concentration of 100 mM of CoCl2. Subsequently this concentration was used for the culturing of isolated CTCs. Using this condition, CTCs were successfully cultured and expanded for more than nine weeks. Based on the morphological and phenotypical characterisation, two phenotypes of CTCs were isolated from a breast cancer patient; epitheliallike expressed EpCAM and quasi-mesenchymal express HER2
Mohamed 2020
Aims
To evaluate systemic inflammation in TBI by exploration of the inflammasome pathway, a component of the innate immune system that regulates and induces inflammation. We examine the pathway at baseline in TBI compared with healthy control children, and in vitro in response to both LPS stimulation and melatonin therapy. Melatonin has protective effects against NLRP3 inflammasome activation and has therapeutic implications.
Methods
Whole blood was sampled from children with TBI (n=10) within 24 hours of injury and compared to healthy age-matched controls (n=8) at baseline, following stimulation with bacterial endotoxin (LPS) (10ng/ml) and melatonin treatment (10–3M). Granulocytes were delineated as CD66b+ and FSC, SSC-A. Measurements of mean channel fluorescence (MCF) of CD11b and TLR4 expression on FACS Canto II were recorded and analysed with FloJo software v10. Gene Expression of NLRP3 via rtPCR was recorded in 10 patients and 10 controls at baseline and following LPS and melatonin treatment.
Results
Granulocyte CD11b expression was lower in children with TBI compared to controls (p=0.04) Both upregulated CD11b with LPS stimulation. Melatonin significantly decreased this LPS upregulation. There was no significant difference in baseline TLR4 expression between TBI and controls, but LPS upregulation of TLR4 was decreased by melatonin in the TBI cohort. Inflammasome was upregulated via NLRP3 expression in children with TBI compared to controls (p= 0.02). Melatonin significantly decreased LPS-induced upregulation of NLRP3 only in controls.
Conclusion
Inflammation is altered in TBI compared to controls with altered responsiveness to melatonin treatment following LPS stimulation. The inflammasome is downregulated in children immediately following TBI. Selective inhibition of systemic inflammation targeting the inflammasome may have a
future immunomodulatory role as a target in treating TBI.
Ryan 2019
Aims
To evaluate components of the innate immune system, the inflammsome, in mild Traumatic Brain Injury (TBI) and the correlation with symptom burden 2 weeks from injury. We examine activation of the pathway at presentation, and also at 2 weeks from injury compared with healthy control children.
Methods
Whole blood was sampled from children with mild TBI within 24 hours of injury and at two weeks from injury and compared to healthy paediatric controls at baseline. RNA was isolated and cDNA was synthesized. Gene Expression of NLRP3 and IL-1β via rtPCR was recorded in 22 patients and 5 controls at baseline and 15 patients at 2 weeks. The Post Concussive Symptom Inventory was administered at 2 weeks. A change from pre-injury baseline was recorded.
Results
Inflammasome was upregulated via NLRP3 expression in children with TBI compared to controls across groups however this did not correlate with symptoms at 2 weeks. Higher IL-1β transcription levels at presentation were positively correlated by Pearson correlation (p = 0.029) with higher symptom scores at 2 weeks.
Conclusion
Inflammation is altered in TBI compared to controls The NLRP3 component of inflammasome while elevated does not correlate with symptom burden. IL-1β gene transcription does. IL-1β holds promise in predicting symptom burden following mTBI. Selective inhibition of systemic inflammation targeting the inflammasome may have a future immunomodulatory role as a target in treating mTBI.
Ryan 2019
Perez
2019
Background
Ovarian cancer is a major cause of cancer death in women worldwide with less than 40% of women surviving beyond 5 years post diagnosis. This is due mainly to late diagnosis and the development of recurrent chemoresistant disease. High grade serous is the most common and most lethal subtype of ovarian cancer. While chemotherapy response rates are initially good in ovarian cancer, patients often develop recurrent, chemoresistant disease. There is a need for novel biomarkers for improved diagnostics and patient stratification in ovarian cancer. Neuromedin U (NmU) is a neuropeptide found to have distinct roles in various cancers depending on context and cancer type. In breast cancer NmU is associated with more aggressive disease, HER2-targeted therapy resistance and poorer prognosis. NmU protein expression is transiently increased in the ovary during ovulation and increased NmU mRNA expression has been observed previously in ovarian cancer. The aim of this study was to assess the role of NmU in high grade serous ovarian cancer.
Design
Cases were identified from the Discovary bioresource in the Department of Obstetrics and Gynaecology in Trinity College Dublin. All patients gave informed consent and ethical approval was received. Two tissue microarrays were constructed; five 0.6mm cores were selected from representative tumour regions from (n=102) high grade serous stage 3-4 FFPE tissue blocks. Immunohistochemistry was performed using a NmU antibody (1:50 NmU; HPA025926, Atlas Antibodies, Stockholm, Sweden). NmU protein expression was then evaluated and scored as a product of intensity (scored 0-3) and percentage of cells stained (0-10%=1, 10-40%=2, 40-70%=3, 70-100%=4) Statistical analysis was performed using SPSS Software (IBM). Kaplan Meier survival curves were used with calculated log-ranks for univariate analysis. Data was censored for cases where an event had not occurred by the time of analysis. The criterion for statistical significance applied was p <0.05.
Results
NmU was over-expressed in ovarian high grade serous carcinoma tissue compared to benign controls. Increased NmU expression correlated with a significant progression free survival advantage (p=0.004) and increased disease-free interval (p=0.022) in the evaluable patient cohort (n=88).
Conclusions
These findings suggest that NmU may have a role in the pathogenesis of high grade serous carcinoma and may represent a novel prognostic and potentially diagnostic biomarker for this disease worthy of further investigation.
OC-0381: LESSONS FROM ISOGENIC MODELS OF RADIORESISTANT PROSTATE CANCER CELLS
Purpose or Objective
Evidence of the multiple effects of radiation on the molecular biology of prostate cancer cells is accumulating, but new frontier molecular biology is still required to achieve the personalisation of prostate cancer radiotherapy. We hypothesised that the induction of a radioresistant phenotype in response to fractionated radiation and exposure to an hypoxic environment share a common molecular response.
Material and Methods
A panel of 22Rv1 prostate cancer cells of differing radiosensitivities was developed through exposure to 60Gy in 2Gy dose fractions and exposure to 0.5% oxygen levels for 24 hours. The impact of these exposure on clonogenic survival to radiation was determined using clonogenic assays. The molecular response was examined through miRNA and high content Digiwest protein profiling technologies. Differentially expressed miRNAs and proteins were independently validated using RT-PCR, Western Blots and relationship with radioresistance was examined using clonogenic assays.
Results
Exposure to fractionated radiation significantly increased the clonogenic survival capacity of 22Rv1 cells, where compared to unexposed, wild type (WT) cells. The clonogenic survival of these cells was similar to that of hypoxic WT-22Rv1 cells. Three candidate miRNAs were associated with a radioresistant phenotype across the models: miR200a, miR210 and miR4284. Protein profiling identified 64 significantly differentially expressed proteins in RR22Rv1, when compared to WT-22RV1 cells, including the androgen receptor, p53, YB-1, members of the Notch (Notch-1, Notch-3), apoptosis (bcl-xL) and DNA repair (PARP, ATR) signalling pathways. Interdependence between Notch-3, YB-1 and miR-4284 expression levels were identified though treatment with Notch, YB-1 and p53 inhibitors. These agents were associated with a modification in the clonogenic survival capacity of these cells following radiation exposure.
Conclusion
This study identifies a novel, therapeutically actionable, molecular network associated with radioresistance in prostate cancer.
Inder
2018
PROFILING OF RADIORESISTANT PROSTATE CANCER CELLS IDENTIFIES DEREGULATION OF KEY PROTEIN
Introduction
Radiotherapy (RT) has a prominent role in the management of prostate cancer patients. Novel molecular radiobiology is required to enhance the decision-making process for radiotherapy. This study aimed to identify the impact of fractionated radiation exposure on the protein profile of prostate cancer cells.
Methods
The protein profiles of a previously generated isogenic model of radioresistant 22Rv1 prostate cancer cells through exposure to 30 X 2-Gy dose fractions was performed using the Digiwest High Con-tent Protein Profiling technology. Differentially expressed proteins between the radioresistant (RR-22Rv1) and wild-type (WT-22Rv1) cells were independently validated using Western Blots. Relationship with radioresistance was examined using clonogenic assays.
Results
The expression levels of 64 proteins were significantly different in RR-22Rv1 when compared to WT-22RV1 cells, including the androgen receptor, p53, YB-1, members of the Notch (Notch-1, Notch-3), apoptosis (bcl-xL) and DNA repair (PARP, ATR) signalling pathways. Treatment with Notch, YB-1 and PARP inhibitor modified the clonogenic survival capacity of these cells following radiation exposure.
Conclusion
This study identifies candidate proteins with potential for the development of a protein-based prognostic assay for radiotherapy prostate cancer patients.
Inder
2017
TLR4 AND MAD2: DRIVERS OF CHEMORESISTANCE IN OVARIAN CANCER
Background
MAD2 is a key protein involved in the spindle assembly checkpoint and knockdown of this protein has been shown to facilitate paclitaxel resistance through the induction of senescence. Additionally low MAD2 immunohistochemical (IHC) staining has been associated with reduced progression free survival (PFS). Conversely the TLR4-MyD88 pathway is known to mediate paclitaxel resistance through the upregulation of pro-tumourigenic cytokines and pro-survival genes and blockage of this pathway has been shown to restore chemosenstivity to paclitaxel. Furthermore high TLR4 IHC staining is associated with reduced PFS.
Design
A2780 and SKOV-3 cells were transfected with siRNA targeting MAD2. MAD2 and TLR4 expression was then analysed by RT-PCR. SKOV-3 cells were subsequently transfected with siRNA targeting MAD2 and then treated with 1µM paclitaxel. Cell viability was then assessed using the CCK-8 assay. β-galactosidase activity was also measured in SKOV-3 cells following transfection using the Senescence β-Galactosidase Staining kit.
Results
In this study a novel link between TLR4 and MAD2 was observed. Specifically, when MAD2 was knocked down in both A2780 and SKOV-3 cells, a 3 fold upregulation of TLR4 mRNA expression was observed. MAD2 knockdown in SKOV-3 cells was also shown to render SKOV-3 cells highly resistant to paclitaxel. These cells displayed enlarged cell and nuclear size and an increase in β-galactosidase activity. Additionally, TLR4 and MAD2 expression was analysed by IHC in a tissue microarray cohort of high grade serous, stage 2-4 ovarian cancer patients (n=36). Patients with a TLR4 high and MAD2 low phenotype had significantly reduced DFI (p=0.002) and PFS (p=0.002), compared to those possessing any other phenotype, a reduction in patient overall survival was also observed but this was just below significance (p=0.081).
Conclusion
The results demonstrate the development of a lethal phenotype which is highly resistant to paclitaxel chemotherapy and a synergy between the MAD2 senescence
Bates
2016
Background
MyD88 and MAD2 are two potential prognostic biomarkers that have been investigated in ovarian cancer. High MyD88 and Low MAD2 IHC staining is associated with reduced PFS, both markers are also linked to paclitaxel chemoresistance.
Objectives
The main objective of this study was to assess the in vitro relationship between MAD2 and MyD88, through alteration of MAD2, MyD88 or its receptor TLR4 in two ovarian cancer cell lines using siRNA targeting MAD2, TLR4 or MyD88 and a MyD88 overexpression plasmid vector. Following overexpression/siRNA knockdown procedures, MyD88, TLR4 and MAD2 expression was assessed through qPCR and Western Blot analysis. Mir-433, Mir-21 and Mir-146a gene expression was also assessed by qPCR. Furthermore the effect of TLR4/MyD88 knockdown on chemoresponse was assessed in SKOV-3 cells using the CCK-8 assay.
Results/Discussion
It was found that knockdown or overexpression of MyD88 in SKOV-3 or A2780 cells respectively or knockdown of TLR4 in SKOV-3 cells had no effect on MAD2 expression or the expression of Mir-21, Mir-433 and Mir-146a. Interestingly however knockdown of MAD2 in both cell lines induced a 3 fold increase in TLR4 expression, furthermore knockdown of TLR4 in SKOV-3 cells was shown to restore chemosensitivity to paclitaxel.
Conclusion
The results demonstrate a potential in vitro link between TLR4 and MAD2 and support a role for TLR4 in paclitaxel chemoresistance.
Bates
2015
Background
MyD88 and MAD2 are two potential prognostic biomarkers that have been investigated in ovarian cancer. High MyD88 and Low MAD2 IHC staining is associated with reduced PFS, both markers are also linked to paclitaxel chemoresistance.
Objectives
The main objective of this study was to assess the in vitro relationship between MAD2 and MyD88, through alteration of MAD2, MyD88 or its receptor TLR4 in two ovarian cancer cell lines using siRNA targeting MAD2, TLR4 or MyD88 and a MyD88 overexpression plasmid vector. Following overexpression/siRNA knockdown procedures, MyD88, TLR4 and MAD2 expression was assessed through qPCR and Western Blot analysis. Mir-433, Mir-21 and Mir-146a gene expression was also assessed by qPCR. Furthermore, the effect of TLR4/MyD88 knockdown on chemoresponse was assessed in SKOV-3 cells using the CCK-8 assay.
Results/Discussion
It was found that knockdown or overexpression of MyD88 in SKOV-3 or A2780 cells respectively or knockdown of TLR4 in SKOV-3 cells had no effect on MAD2 expression or the expression of Mir-21, Mir-433 and Mir-146a. Interestingly however knockdown of MAD2 in both cell lines induced a 3-fold increase in TLR4 expression, furthermore, knockdown of TLR4 in SKOV-3 cells was shown to restore chemosensitivity to paclitaxel.
Conclusion
The results demonstrate a potential in vitro link between TLR4 and MAD2 and support a role for TLR4 in paclitaxel chemoresistance.