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Prostate cancer is the second most common cancer in men worldwide. Gleason grading is an important predictor of prostate cancer outcomes and is influential in determining patient treatment options. Clinical decisions based on a Gleason score of 7 are difficult as the prognosis for individuals diagnosed with Gleason 4+3 cancer is much worse than for those diagnosed with Gleason 3+4 cancer. Laser capture microdissection (LCM) is a highly precise method to isolate specific cell populations or discrete microregions from tissues. This report undertook a detailed molecular characterization of the tumor microenvironment in prostate cancer to define the proteome in the epithelial and stromal regions from tumor foci of Gleason grades 3 and 4. Tissue regions of interest were isolated from several Gleason 3+3 and Gleason 4+4 tumors using telepathology to leverage specialized pathology expertise to support LCM. Over 2,000 proteins were identified following liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of all regions of interest. Statistical analysis revealed significant differences in protein expression (>100 proteins) between Gleason 3 and Gleason 4 regions—in both stromal and epithelial compartments. A subset of these proteins has had prior strong association with prostate cancer, thereby providing evidence for the authenticity of the approach. Finally, validation of these proteins by immunohistochemistry has been obtained using an independent cohort of prostate cancer tumor specimens. Implications: This unbiased strategy provides a strong foundation for the development of biomarker protein panels with significant diagnostic and prognostic potential. Mol Cancer Res; 15(3); 281–93. ©2017 AACR.

Background: Circulating tumour cells (CTCs) are silent precursors of metastatic disease that utilise various mechanisms to survive in circulation and metastasise to distal sites. Classical CTC detection relies on EpCam affinity-based technologies; however, CTCs are highly heterogeneous and often undergo EMT. Recent research highlights the ability of platelets and neutrophils to ‘cloak' CTCs and crosstalk to aid in their proliferation and survival in circulation. Aims: Assess the role of platelets and neutrophils in the characterisation of CTCs. Methods: Cell lines: Breast cancer (MCF-7) and ovarian cancer (SKOV-3, OVCAR3, OAW42) cells were exposed to healthy donor isolated platelets and neutrophils for 24h. EMT and immune evasion gene assays, flow cytometry analysis and cell proliferation assays were performed following co-culture. Cell line-blood spike-in experiments were performed for gating strategy optimisation.Patient samples: Blood specimens were prospectively collected from breast and ovarian cancer patients (central venous and peripheral blood). CTCs and immune cells were then isolated using a ClearCell FX microfluidic device. FX isolated cells were immunophenotyped by either immunofluorescence (IF) or flow cytometry. Single cells were index sorted using BD FACS Melody for subsequent scRNAseq. Results: Platelet cloaking altered EpCam, PLEK2, CCL2 and TWIST1 mRNA expression in MCF-7 cells. Platelet and neutrophil co-culture altered EpCam, PAI-1 and PD-L1 expression in ovarian cancer cell lines. Healthy neutrophils co-cultured with ovarian cancer cells increased cell viability, while induction of NETosis increased cancer cell proliferation. CTCs isolated from peripheral blood in breast and ovarian patients were EpCam/panCK+/CD45- by IF. Immunophenotyped CTCs were identified as EpCam+/E-Cadherin+/CD45-, N-Cadherin+/CD45- and as PD-L1+/EpCam+/E-Cadherin+/CD45- cells by flow cytometry. We also identified EpCam+/CD45+/CD66b+ cells in ovarian central blood. Isolated classical and non-classical CTCs were sorted based on their different immunophenotypes for downstream scRNAseq using BD Precise Whole Transcriptome Assays. Conclusion: Platelets and neutrophils alter the expression of markers used in the identification of CTCs. Neutrophils were found to increase the proliferation of cancer cells as well as increase PD-L1 expression. We identified a heterogeneous population of CTCs across different patients and found that CTCs from central venous blood have increased numbers of non-classical CTCs (CD66b+/EpCam+ cells). Molecular scRNAseq signatures of CTC subsets form the basis for identifying the most clinically relevant CTCs in circulation. Our approach may improve diagnostic accuracy, allow for immunophenotyping of cloaked CTCs, and provide prognostic information. Citation Format: Mark P. Ward, Bashir M. Mohamed, Laura Kane, Mark Bates, Janina Berghoff, Cathy L. Spillane, Tanya Kelly, John Kennedy, Feras A. Saadeh, Karsten Hokamp, Noreen Gleeson, Orla Sheils, Cara Martin, Michael Gallagher, Sean Hannify, Eric P. Dixon, Sharon A. O'Toole, John J. O'Leary. Influence of platelets and neutrophils on circulating tumour cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 764.

Background: Globally, prostate cancer (PrCa) is the fourth most common cancer type. Obesity and inflammation have been shown to play significant roles in PrCa disease progression. Obesity and a high body mass index (BMI) are associated with increased PrCa-specific mortality in patients with advanced disease. Furthermore, proinflammatory cytokines can aid metastatic potential and promote angiogenesis. The ExPeCT (Exercise, Prostate Cancer and Circulating Tumor Cells) trial seeks to examine the effectiveness of a structured exercise program in modulating inflammatory mediators and obesity in patients with metastatic PrCa. Methods: ExPeCT (CTRIALIE 15-21 (ClincalTrials.gov identifier NCT02453139)) is a multicenter, randomized trial for patients with metastatic PrCa (n=67). Participants were randomized to either control or exercise arms. Participants in the exercise arm completed six months of prescribed aerobic exercise, which was monitored using percentage heart-rate reserve. Serum samples were collected for all participants at baseline (T0), three months (T3), and six months (T6), and assayed for 16 interlinked adipokines and cytokines using the Meso Scale Discovery platform. An interim statistical analysis was performed (n=26) comparing median change in serum analyte levels between control (n=13) and exercise (n=13) arms using non-parametric Wilcoxon rank-sum tests. Results: Among 26 patients included in our interim analysis, mean age at baseline was 71 years, median BMI was 29.1 kg/m2, and median waist circumference (WC) was 107 cm, with no significant differences between arms (all p>0.3). Between T0 and T6, WC decreased by a median of 3.8 cm in the exercise group and 2.6 cm in the control group (p=0.412), with a similar trend for BMI. Interim serum cytokine analysis showed a 3-fold increase in IL-10 levels in the exercise arm at T3 when compared to the control arm (p=0.036). No significant change in IL-10 levels was recorded at T6 between arms (p=0.776). Similarly, CXCL8 (IL-8) levels were increased by 1.8-fold at T3 in the exercise arm in comparison to the control arm (p=0.017), with no significant change reported at T6 (p=0.191). While changes were evident in serum TNFα, IL-6, VEGF, IL-17a, MMP9 and CCL5 (RANTES) levels, these did not reach significance. Differences in levels of adipokines leptin and resistin were also reported. A 1.5-fold increase in resistin expression was observed in the exercise arm at T6; however, it was not significant (p=0.293). A 2-fold decrease in leptin in the control arm relative to the exercise arm at T6 was also detected (p=0.676). Analysis of adiponectin, MMP2, and CCL2 is ongoing. Conclusion: Our interim analysis of ExPeCT trial participants demonstrated a significant increase in serum CXCL8 and IL-10 levels after three months of a supervised exercise intervention. These preliminary data suggest that a structured exercise program has the potential to modify inflammatory status in patients with metastatic PrCa. Citation Format: Lauren Brady, Grainne Sheill, Anne-Marie Baird, Emma H. Allott, Tatjana Vlajnic, John Greene, Orla Casey, Brian Hayes, Emer Guinan, Juliette Hussey, Fidelma Cahill, Mieke Van Hemelrijck, Nicola Peat, Sarah Rudman, Moya Cunningham, Liam Grogan, Thomas Lynch, Rustom P. Manecksha, John McCaffrey, Orla Sheils, Dearbhaile M. O’Donnell, John O’Leary, Ray McDermott, Stephen P. Finn. Examining the link between obesity, inflammation, and exercise in patients with metastatic prostate cancer—An interim analysis from the ExPeCT trial [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A057.

Annually, ovarian cancer (OC) affects 240,000 women worldwide and is the most lethal gynaecological malignancy. Such mortality is predominantly associated with the development of an intrinsic and acquired resistance to chemotherapy, the lack of targeted therapies and the lack of biomarkers predicting therapeutic response. Our clinical data demonstrates that increased miR-433 expression in primary high grade serous OC (HGSOCs) is significantly associated with poor PFS (n=46, p=0.024). Interestingly, the IHC analysis of two miR-433 targets: MAD2 [Furlong et al., 2012 PMID:22069160] and HDAC6 shows that low IHC levels of both proteins is also significantly associated with worse outcome (p=0.002 and 0.002 respectively; n=43). Additionally, the analysis of miR 433 in the publicly available TCGA dataset corroborates that high miR-433 is significantly correlated with worse OS for patients presenting with OC (n=558 and p=0.027). In vitro, in a panel of OC cell lines, higher miR-433 and lower MAD2 and HDAC6 levels were associated with resistance to paclitaxel. To further investigate the role of miR-433 in the cellular response to chemotherapy, we generated an OC cell line stably expressing miR-433, or miR-control. MTT viability assays and Western Blot analyses established that miR-433 cells were more resistant to paclitaxel treatment (50nM) compared to miR-controls. Importantly, we have shown for the first time that miR 433 induced senescence, exemplified by a flattened morphology and down-regulation of phosphorylated Retinoblastoma (p-Rb), a molecular marker of senescence. Surprisingly, miR 433 induced senescence was independent from two well recognised senescent drivers: namely p53/p21 and p16. To explore this further we performed an in silico analysis of seven microRNA platforms which indicated that miR 433 potentially targets Cyclin-dependent kinase CDK6, which promotes sustained phosphorylation of Rb and thus cell cycle progression. In vitro, the overexpression of pre-miR-433 resulted in diminished CDK6 expression demonstrating a novel interaction between miR-433 and CDK6. In conclusion, this study demonstrates that high miR-433 expression predicts poor outcome in OC patients by putatively rendering OC cells resistant to paclitaxel treatment through the induction of cellular senescence identifying this microRNA as a potential marker of chemoresponse. Founding: Health Research Board :HRA- POR/2011/113 Citation Format: Karolina Weiner-Gorzel, Malgorzata Milewska, Danniel Sharpe, Caitlin Beggan, Nazia Faheem, Aloysius McGoldrick, Valerie Toh, Patricia Fitzpatrick, Sharon O'Toole, John O'Leary, Stephen F. Madden, Madeline Murphy, Elaine Kay, Malcolm Kell, Amanda McCann, Fiona Furlong. MiR-433 induces cellular senescence rendering ovarian cancer cells less likely to undergo chemotherapy-induced apoptosis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A34.

Introduction: Germline mutations in DNA repair genes BRCA1/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality, the inhibition of two major DNA repair pathways. Therefore, there is a need to characterize ovarian cell lines for their BRCA1/2 status for in vitro studies of novel drugs. Methods: The BRCA1/2 genes were sequenced in 43 ovarian cell lines, mRNA expression of BRCA1/2 and gene methylation of BRCA1 were also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 10 cell lines. Results: Sequencing of BRCA1/2 revealed 18 and 33 different alterations respectively, mostly non-pathogenic polymorphisms. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G>A and is the only deleterious BRCA1/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations which restore the function of the protein. BRCA1 was methylated in three cell lines (A1847, OVCAR8, EF021) and there was a corresponding decrease in gene expression in these cell lines. The deleterious mutant SNU-251 was relatively resistant to PARP inhibition, compared to wild-type cells. However, the methylated cell lines A1847 and OVCAR8 were more sensitive to PARP inhibition than wild-type cells. Conclusions: The incidence of BRCA1/2 deleterious mutations 1/43 cell lines (2.3%) is much lower than the population incidence. The reversion mutations suggest that there is a selective pressure against BRCA1/2 mutations in cell culture. PARP inhibitors may be useful in patients with BRCA1 methylation and reduced gene expression in their ovarian tumour. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 78. doi:1538-7445.AM2012-78

Ovarian cancer is the leading cause of gynaecological cancer death worldwide. This high mortality is caused in large part by development of chemoresistant recurrent disease. In primary tumours differentiation status of the tumour is considered a key prognostic factor however in recurrent disease this is no longer the case, suggesting that in recurrent tumours the differentiation status of the tumour has no bearing on the response to chemotherapy. The origins of recurrent disease may be explained by the cancer stem cell (CSC) theory. CSCs are a minority population of cancer cells with stem like properties including enhanced proliferation. The adaptor molecule myeloid differentiation-primary response gene (88) (MyD88), is a key constituent of the toll like receptor 4 (TLR4) pathway. In normal circumstances TLR pathways mediate the body's response to pathogens; however MyD88 has been recently suggested as a possible marker of cancer stemness in ovarian cancer. This work investigates the involvement of MyD88 in the CSC response to chemotherapy, differentiation stimulus and hypoxia treatment. Cell line experiments were carried out in two CSC lines: NTera2 and 2102Ep. NTera2 cells readily differentiate in response to retinoic acid (RA) treatment while 2102Ep cells are nullipotent and resist differentiation via RA. NTera2 cells are more chemosensitive than 2102Ep cells. Cells were incubated in the presence of both retinoic acid and a platinum based chemotherapy drug, both in isolation and in various combinations. RNA was isolated from treated cells, cDNA was synthesised and interrogated for TLR4/MyD88 expression via Q-PCR. To assess whether MyD88 was necessary and/or sufficient for the normal cell response, MyD88 was knocked down in cells via siRNA transfection, and overexpressed via insertion of an overexpression plasmid. The effect of these alterations on the cell lines response to chemotherapy, differentiation stimulus and hypoxia was then measured. The downstream effect of ablation of the MyD88 signal was also assessed using an Affymetrix array, and the chemokine and cytokine expression profile of both cell lines in all treatments was assessed using Quantibody arrays from RayBiotech. MyD88 expression was shown to be affected by chemotherapy, differentiation stimulus and hypoxic treatments. Furthermore when cells were pre-treated with differentiation stimulus or chemotherapy, their normal responses were altered. So far, our work has shown that ablation of the MyD88 signal in nullipotent 2102Ep cells removes their ability to resist differentiation via RA. To close this mechanism we have also shown that overexpression of MyD88 in pluripotent NTera2 cells allows them to resist differentiation via RA. Functional analysis of the role of MyD88 in chemotherapy and hypoxia resistance is currently being assessed. This and the mechanisms involved will be detailed, along with the chemokine/cytokine profile of treated cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3325. doi:1538-7445.AM2012-3325

Background: Tumorgenicity studies show that Cancer Stem Cells (CSCs) but not their differentiated counterparts efficiently generate tumors. Therapeutically targeting CSCs could remove the malignant potential from tumours, while circumventing chemoresistance, relapse and metastasis. CSCs are difficult to study as they usually represent a small fraction of tumors. We will present a systematic approach we developed to investigate CSCs within any malignancy. We will present the data obtained thus far in the application of this system to the study of Ovarian CSCs. Methods: We developed a four phase model for investigating CSCs: Screening - Validation - Characterisation - Identification of novel therapeutic targets. Six cell lines modelling various stages of ovarian cancer (A2780, A2780cis, IGROV-1, IGROV(CDDP), SK-OV-3 and 59M) and one cell line (HIO-80) modelling non malignant ovarian surface epithelium were screened for the presence of CSCs and somatic stem cells respectively. Three flow cytometry based screens were implemented; ALDEFLUOR, Hoechst Side Population and Cell Surface Protein Screens. Cells of interest were isolated via Fluorescence Activated Cell Sorting. Mouse Tumorgenicity and Asymmetric Division Assays have been developed to validate putative CSCs (pCSCs) as true CSCs. Hypoxic growth, chemotherapeutic and matrigel invasion assays have all been developed to investigate the role of Ovarian CSCs in these commonly observed phenotypes of Ovarian cancer. Results: Six pCSC populations have been identified. No overlap has been observed between the three screening approaches. ALDEFLUOR identified pCSCs within A2780 and A2780cis. Hoechst Side Population identified pCSCs within IGROV-1 and IGROV(CDDP). CD44/CD117 identified pCSCs within SK-OV-3 and 59M. No somatic stem cells were identified within HIO-80. To date pCSC and non-pCSCs have been sorted from two cell lines. In both models the isolated pCSCs regenerated the non-pCSC phenotype. Both the isolated pCSCs and non-pCScs from each model are resistance to cisplatin and paclitaxel. In both cases the parent lines were chemoresistant, so this finding is not unexpected. All pCSCs are currently being validated via Mouse Tumourgenicity and Single Cell Asymmetric Division Assays. All validated CSCs will be described. Conclusion: A systematic approach has been established for the isolation and study of CSCs. Each ‘type’ of ovarian CSC appears to be mutually exclusive. This may reflect different stages/histologies of ovarian disease, or perhaps the selective conditions of tissue culture. pCSCs have been identified within chemosensitive cell lines. Investigation of the differences between chemoresistant and chemosensitive CSCs could elucidate the mechanisms behind chemoresistant relapse. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3382. doi:1538-7445.AM2012-3382

Introduction: The MEK/ERK pathway is mutated in approximately 7% of human cancers. Extracellular ligands signal via RAS to recruit RAF kinase and activate MEK and its downstream effectors. This pathway is involved in a range of biological pathways including cell proliferation, differentiation and senescence. The BRAF isoform is the most potent activator of MEK and is frequently mutated in cancer. A single thymine to adenine base pair change at position 1799 accounts for greater than 95% of somatic mutation in BRAF. BRAFV600E can gain 500-fold increased activation, stimulating the constitutive activation of the MEK/ERK pathway in the absence of extracellular signalling. The strategic position of MEK within the pathway makes it a promising target for drug development. Although MEK is downstream of both RAF and RAS, RAF but not RAS mutation has been demonstrated to predict sensitivity to MEK inhibition. Materials and Methods: Human thyroid cell lines were SNP typed for the T1799A mutation using a custom TaqMan® SNP genotyping assay. The optimal IC50 and length of treatment for the MEK inhibitor PD0325901 for each cell line were determined via a series of MTT assays. Each cell line was subjected to PD0325901 treatment and surviving cells were harvested for mRNA and protein extraction. A comparative study for relative expression of MAP2K1 (MEK1), MAP2K2 (MEK2), MAPK3 (ERK1), MAPK1 (ERK2) and the downstream target ELK1 was performed using TaqMan assays with GAPDH as the endogenous control. Results: Human thyroid cell line 8505c carries the T1799A mutation and N-Thy-Ori cell lines carry wild type BRAF. BRAFV600E cells had visually evident patches of inhibition following PD0325901 treatment which were not evident in media-only treated controls or in the BRAFWT cell line. In BRAFV600E, all 5 genes were down-regulated in PD0325901 treated cells compared to their media-only treated counterparts. This pattern was not seen with the BRAFWT cells. BRAFV600E cells exhibited this pattern following both a single treatment and two successive treatments with PD0325901. Discussion: This pattern of gene expression is consistent with the hypothesis that resistance to small molecule MEK inhibition occurs via upregulation of an alternative pathway or activation of a redundant signalling pathway rather than through reactivation off the BRAF/MEK/ERK pathway. It also reinforces the theory that MEK inhibition induces differential effects in target cells dependent upon BRAF status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B135.