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Supplementary Figure S1. PPP2R2A mRNA levels in the excised tumors from 5637 bearing mouse xenografts. Supplementary Figure S2. PPP2R2A mRNA and protein levels upon transient silencing.Supplementary Figure S3. PPP2R2A protein levels upon transient overexpression. Supplementary Table S1. Bioinformatics prediction of the top networks regulated by PPP2R2A. Supplementary Table S2. Bioinformatics prediction of the top canonical pathways regulated by PPP2R2A. Supplementary Table S3. Xcelligence Cell Index and P values of siPPP2R2RA-treated versus control BC cells. Supplementary Table S4. Xcelligence Cell Index and P values of PPP2R2A-overexpressing versus control BC cells, all treated with miR-21 mimic.

Bladder cancer represents a disease associated with significant morbidity and mortality. MiR-21 has been found to have oncogenic activity in multiple cancers, including bladder cancer, whereas inhibition of its expression suppresses tumor growth. Here, we examine the molecular network regulated by miR-21 in bladder cancer and evaluate the effects of i.v. and i.p. administration of a novel miR-21 chemical inhibitor in vivo. LNA miR-21 reduced the oncogenic potential of bladder cancer cells, whereas the MKAD-21 chemically modified antisense oligo against miR-21 dose-dependently blocked xenograft growth. I.v. administration of LNA miR-21 was more effective in suppressing tumor growth than was i.p. administration. Integration of computational and transcriptomic analyses in a panel of 28 bladder cancer lines revealed a 15-gene signature that correlates with miR-21 levels. Protein Phosphatase 2 Regulatory Subunit Balpha (PPP2R2A) was one of these 15 genes and was experimentally validated as a novel miR-21 direct target gene. Gene network and molecular analyses showed that PPP2R2A is a potent negative regulator of the ERK pathway activation and bladder cancer cell proliferation. Importantly, we show that PPP2R2A acts as a mediator of miR-21–induced oncogenic effects in bladder cancer. Integrative analysis of human bladder cancer tumors and a large panel of human bladder cancer cell lines revealed a novel 15-gene signature that correlates with miR-21 levels. Importantly, we provide evidence that PPP2R2A represents a new miR-21 direct target and regulator of the ERK pathway and bladder cancer cell growth. Furthermore, i.v. administration of the MKAD-21 inhibitor effectively suppressed tumor growth through regulation of the PPP2R2A–ERK network in mice. Mol Cancer Ther; 17(7); 1430–40. ©2018 AACR.

Bladder cancer represents a disease associated with significant morbidity and mortality. MiR-21 has been found to have oncogenic activity in multiple cancers, including bladder cancer, whereas inhibition of its expression suppresses tumor growth. Here, we examine the molecular network regulated by miR-21 in bladder cancer and evaluate the effects of i.v. and i.p. administration of a novel miR-21 chemical inhibitor in vivo. LNA miR-21 reduced the oncogenic potential of bladder cancer cells, whereas the MKAD-21 chemically modified antisense oligo against miR-21 dose-dependently blocked xenograft growth. I.v. administration of LNA miR-21 was more effective in suppressing tumor growth than was i.p. administration. Integration of computational and transcriptomic analyses in a panel of 28 bladder cancer lines revealed a 15-gene signature that correlates with miR-21 levels. Protein Phosphatase 2 Regulatory Subunit Balpha (PPP2R2A) was one of these 15 genes and was experimentally validated as a novel miR-21 direct target gene. Gene network and molecular analyses showed that PPP2R2A is a potent negative regulator of the ERK pathway activation and bladder cancer cell proliferation. Importantly, we show that PPP2R2A acts as a mediator of miR-21–induced oncogenic effects in bladder cancer. Integrative analysis of human bladder cancer tumors and a large panel of human bladder cancer cell lines revealed a novel 15-gene signature that correlates with miR-21 levels. Importantly, we provide evidence that PPP2R2A represents a new miR-21 direct target and regulator of the ERK pathway and bladder cancer cell growth. Furthermore, i.v. administration of the MKAD-21 inhibitor effectively suppressed tumor growth through regulation of the PPP2R2A–ERK network in mice. Mol Cancer Ther; 17(7); 1430–40. ©2018 AACR.

Introduction: Circulating tumor cells (CTCs) are considered as a “liquid biopsy” that allows the assessment of tumor changes over time. Tumor cells may escape from the immune system through the activation of PD-1/PD-L1 axis. Targeting these molecules with monoclonal antibodies has shown encouraging results at many types of cancers, including NSCLC. In the current study we investigated the expression of PD-1/PD-L1 molecules on the CTCs isolated from NSCLC patients treated with Nivolumab. Methods: CTCs were isolated based on their size using the ISET platform from 27 patients before treatment, after 1 cycle and 3 cycles. CTCs were detected with Giemsa staining and immunofluorescence (IF) experiments, using either pancytokeratin (A45-B/B3) (CK7)/PD-1/CD45 or (A45-B/B3)(CK7)/PD-L1/CD45 combination of antibodies and analysis with the ARIOL system. Spiking experiments using the NSCLC cell lines: H460, H1299, HCC827 and SKMES in normal blood were used to evaluate the detection method. Results: Giemsa evaluation in Nivolumab-treated patients at baseline (25 evaluable samples), after the 1st (9 evaluable samples) and the 3rd (8 evaluable patients) cycle of treatment showed that CTCs could be detected in 48% (12/25), 33.3% (3/9) and 50% (4/8) of patients, respectively. IF could also reveal the presence of CK-positive cells in 44.4% (12/27), 22% (2/9) and 75% (6/8) patients, respectively. PD-1 (+) CTCs were detected in 33.3% (4/12) of patients at baseline, in, 0% after the 1 and 16.7% (1/6) of patients after the 3rd cycle. The same percentages were identified for PD-L1 expression in the same cohort of patients. The expression of PD-1 at baseline was associated with poorer OS (p=0.022) and PFS (p=0.011), while the expression of PD-L1 was associated with shorter PFS (p=0.011). Multivariate analysis revealed that the presence of CK-positive cells is an independent prognostic factor for OS (p=0.028) Conclusion: Nivolumab reduced the number of PD-1- and PD-L1-expressing CTCs in advanced NSCLC patients. Furthermore the expression of both markers at baseline is associated with the clinical outcome. Citation Format: Galatea Kallergi, Anastasios Koutsopoulos, Eleni Lagoudaki, Despoina Agouraki, Eleni-Kiriaki Vetsika, Anastasia Voumvouraki, Stuart S. Martin, Filippos Koinis, Christos Stournaras, Vassilis Georgoulias, Athanasios Kotsakis. Detection of PD-L1 and PD-1 positive circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) patients treated with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1587.

Introduction: Circulating tumour cells (CTCs) are responsible for the metastatic dissemination of the tumor. They have been shown to express Programmed Death-Ligand1 (PD-L1) to escape from the immune system surveillance through its ligation with the PD-1receptor on the surface of effector immune cells. We investigated the expression of PD-1/PD-L1 on CTCs isolated from NSCLC patients treated with chemotherapy. Methods: CTCs were isolated based on their size using the ISET platform from 30 stage IV chemo-naïve NSCLC patients (before and after chemotherapy). CTCs were detected after staining with Giemsa and immunofluorescence (IF). Double and triple staining experiments with different combination of antibodies: [Cytokeratins(CK)/PD-1/CD45 and CK/PD-L1/CD45] were performed and the samples were analyzed with the ARIOL system. Results Giemsa staining showed that twenty-three (77%) out of 30 and six (54.5%) out of 11 patients had detectable CTCs at baseline and after the 3rd cycle of front-line chemotherapy. IF staining revealed seventeen out of 30 (56.7%) patients positive for CTCs at baseline level and 8 out of 11 (72.7%) samples after the 3rd cycle of treatment. PD-1 and PD-L1 expression was observed in 53% (9/17) and in 47% of the CTC-positive patients at baseline; in addition, 13% (1/8) and 63% (5/8) patients had PD-1 and PD-L1, respectively after the 3rd cycle. Among the total number of detected CTCs, 67% were PD-1(+) at baseline and 25% after the 3rd cycle (p=0.069). In addition, 26% and 80% were PD-L1(+) at baseline and after the 3rd cycle, respectively. Patients with more than 3 PD-1 positive CTCs showed shorter PFS (p=0.022). Primary tissue from ten of the examined patients was also available. More than 5% of PD-L1 (+) tumor infiltrating lymphocytes (TILs) were observed in 20% (2/10) of the patients. More than 5% PD-L1 positive cells in the primary tumor were observed in 20%. However the two group of the patients were different. In addition both patients with PD-L1 (+) TILs harvested CTCs with PD-1 expression and one of them had also PD-L1 positive CTCs. Conclusion: PD-1- and PD-L1-positive CTCs could be detected before and during 1st line treatment in metastatic NSCLC. This expression was related to patients’ prognosis, implying that these molecules can be served as targets for metastasis restoration. Furthermore the expression of PD-1 on CTCs suggests a bilateral cross-talk between tumor and immune cells. Citation Format: Galaktea Kallergi, Eleni Kyriaki Vetsika, Despoina Aggouraki, Eleni Lagoudaki, Anastasios Koutsopoulos, Filippos Koinis, Panagiotis Katsarlinos, Maria Trypaki, Christos Stournaras, Vassilis Georgoulias, Athanasios Kotsakis. Evaluation of PD-L1/PD-1 on circulating tumor cells (CTCs) and on primary tumor in advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1726. doi:10.1158/1538-7445.AM2017-1726

Background: Programmed cell death-1 (PD-1), an inhibitory immune check-point, plays a pivotal role in tumor immune escape. The interaction of PD-1 with its ligand (PD-L1) results in T cells exhaustion, and the blockade of this interaction can partially restore T cell function. Recently, antibodies targeting PD-1 and PD-L1 have been approved for treatment of advanced Non Small Cell Lung Cancer (NSCLC). In this pilot study, we aimed to investigate the effect of anti-PD1 treatment or chemotherapy on the frequencies of circulating PD-1+ T cells and PD-L1+ immunosuppressive cells in NSCLC patients. Patients & Methods: Peripheral blood samples were collected from 35 advanced NSCLC patients before initiation of treatment and after 3 cycles. Twelve treatment-naïve patients received front-line chemotherapy, whereas 23 patients received anti-PD1 treatment in the second-line setting. Flow cytometry was used to quantify PD-1- and PD-L1-expressing immune cells. Changes in the frequencies of these cells were compared between the two settings and correlated with the clinical outcome. Results: Chemotherapy had no effect on the percentages of PD-1+CD4+ and PD-1+CD8+ T cells after 3 cycles, whereas there was a significant decrease in PD-1+CD4+ and PD-1+CD8+ T cells in patients who received 3 administrations of anti-PD1 antibody (p=0.007 and p=0.05, respectively). Moreover, the levels of PD-1-CD4+ (p=0.009) and PD-1-CD8+ (p=0.009) were augmented in response to anti-PD-1 therapy. The frequencies of both peripheral CD4+ Tregs (CD3+CD4+CD25highCD127-/lowCD152+FoxP3+) and granulocytic MDSCs (G-MDSC; CD14-CD15+CD33+CD11b+HLA-DR-Lin-) expressing PD-L1 were decreased following anti-PD1 therapy (p=0.01 and p=0.02, respectively). In contrast, chemotherapy affected only the PD-L1+CD4+ Tregs, but not the PD-L1+G-MDSC, by increasing their levels after 3 cycles (p=0.04). Anti-PD-1 treatment induced a superior reduction of the PD-1+CD4+, PD-1+CD8+ T cells, PD-L1+CD4+ Tregs and PD-L1+G-MDSCs percentages compared to the effect of first line chemotherapy (p=0.04, p=0.05, p=0.002 and p=0.01, respectively). Furthermore, a significant decrease in PD-1+CD8+ T cells, PD-L1+CD4+ Tregs and PD-L1+G-MDSCs after 3 doses of anti-PD-1 was observed in patients who experienced stable disease compared to baseline (p=0.006, p=0.05 and p=0.03, respectively). At the time of response evaluation to chemotherapy, the percentage of the PD-L1+CD4+ Tregs after 3 cycles was significantly inferior compared to baseline, in disease progressors (p=0.04). Conclusion: These data indicate that although chemotherapy affected the levels of PD-L1+CD4+ Tregs, anti-PD1 therapy seems to exert an effect on both PD1+ T cells and PD-L1+ immunosuppressive cells. Additional studies are needed in a larger cohort in order to document its impact on their clinical relevance in NSCLC patients. This study is ongoing and updated data will be presented at the meeting. Citation Format: Eleni-Kyriaki Vetsika, Galatea Kallergi, Despoina Aggouraki, Zaharoula Lyristi, Aristeidis Koukos, Despoina Kourougkiaouri, Filippos Koinis, Vassilis Georgoulias, Athanasios Kotsakis. Effect of anti-PD-1 therapy on immune cells in the peripheral blood of non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 619. doi:10.1158/1538-7445.AM2017-619

Background: The determination of Ki67 in the primary tumor has prognostic value in early breast cancer (BC). We evaluated Ki67 expression in circulating tumor cells (CTCs) from patients with early BC undergoing adjuvant chemotherapy and correlated Ki67 positivity with patient outcome. Methods: Ki67 expression in CTCs was evaluated by immunofluorescent analysis in paired blood samples of patients with early BC (n=166) obtained before and after adjuvant chemotherapy. Ki67 expression was also evaluated in CTC-positive patients at 6 - 24 months after the end of chemotherapy (n=31). Cytospins of peripheral blood mononuclear cells were double stained with A45-B/B3 cytokeratin and Ki67 antibodies. The proliferation index (PI) of CTCs was defined as the ratio of Ki67-positive CTCs/total CTCs. Results: CTCs were detected in 53 (32%) patients before and/or after chemotherapy. Ki67-positive [Ki67(+)] CTCs were identified in 79% of CTC-positive patients, 25% presenting exclusively Ki67(+) CTCs and 21%, exclusively Ki67(-) CTCs. The mean value of Ki67(+) CTCs/patient remained unchanged pre- and post-chemotherapy [(mean±SE): pre- vs post-chemotherapy 2.5±0.7 vs 4.2±2, respectively; p= 0.900]. Similarly, the PI among the total CTCs detected pre- and post-chemotherapy was 59% and 60%, respectively. Ten (19%) of 53 CTC-positive and 9 (8%) of 113 CTC-negative patients relapsed (p = 0.039). In addition, all CTC-positive patients who relapsed harbored Ki67(+) CTCs before and/or after chemotherapy. Interestingly, 70% of them experienced early disease recurrence, ranging from 6-29 months after the initiation of adjuvant chemotherapy. Furthermore, 38.5% of patients with exclusively Ki67(+) CTCs relapsed compared to none among patients with exclusively Ki67(-) CTCs (p = 0.041). Of the 31 CTC-positive patients evaluated during follow-up, 39% remained CTC-positive. However, only 33.3% of them harbored Ki67(+) CTCs, 8.3% had exclusively Ki67(+) CTCs and 66.7% exclusively Ki67(-) CTCs. The mean value of Ki67(+) CTCs/patient was significantly reduced on the follow-up samples [(mean±SE): follow-up vs pre-chemotherapy, 1.35±1.3 vs 2.5±0.7, respectively; p=0.014 and follow-up vs post-chemotherapy, 1.35±1.3 vs 4.2±2, respectively; p= 0.026]. Conclusions: Ki67 expression on CTCs is predictive of early relapse in patients with early BC. Ki67 expression is not decreased by adjuvant chemotherapy, whereas it is reduced early during follow-up, possibly due to adjuvant hormone therapy and/or anti-HER2 therapy. The above results suggest that additional therapy is needed for patients with early BC and Ki67(+) CTCs to prevent early disease recurrence. Citation Format: Agelaki S, Spiliotaki M, Politaki E, Spanaki A, Kassiou L, Koinis F, Georgoulias V, Mavroudis D. Ki67-positive CTCs are associated with early disease relapse in patients with early breast cancer undergoing adjuvant chemotherapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-06.

Background: The assessment of Ki67 in the primary tumor represents a prognostic marker with potential predictive implications in early BC. We evaluated Ki67 expression in CTCs from patients with early BC and assessed the effect of adjuvant chemotherapy as well as Ki67 expression in the primary tumor. Methods: Ki67 was evaluated in CTCs of 97 patients with early BC pre- and post- adjuvant chemotherapy by the use of immunofluorescence analysis. Cytospins of peripheral blood mononuclear cells were double stained with A45-B/B3 cytokeratin mouse antibody and a Ki67 rabbit antibody. Ki67 staining of the primary tumor was also performed for 13 CTC-positive patients. A proliferation index (PI) in CTCs was considered as the ratio of Ki67-positive CTCs/total CTCs. A PI of up to 14% was defined as 'low' whereas a PI above 14% was defined as 'high'. Results: CTCs were detected in 26 (26.8%) patients before and/or after chemotherapy. Seven (27%) of 26 CTC-positive and 8 (11%) of 74 CTC-negative patients relapsed (p = 0.047). Ki67-positive CTCs were identified in 20 (76.9%) of 26 patients, whereas in 1 (3.9%) and 5 (19.2%) patients, exclusively Ki67-positive and Ki67-negative CTCs, respectively, were detected. Seven (33%) of 21 Ki67-positive patients relapsed in contrast to none among the exclusively Ki67-negative patients (p = 0.13). A total of 154 and 161 CTCs were detected pre- and post-chemotherapy, respectively; the PI in CTCs was 56% and 55%, respectively. In 8 patients with detectable CTCs at both time points, the PI was 65% and 49% pre- and post-chemotherapy, respectively. In 5 (62.5%) out of 8 patients, the PI remained high, in 2 (25%) increased and in 1 patient no Ki67–positive CTCs were detected post-chemotherapy. Seventeen patients were CTC-positive at baseline [HER2 positive, n=5; triple negative, n=1; hormone receptor positive, n=11]. A concordance in Ki67 staining between the primary tumor and CTCs was recorded in 10 (77%) out of 13 patients. Moreover, in 2 (67%) of 3 patients with exclusively Ki67-negative CTCs, low Ki67 expression was also observed in the primary tumor. Interestingly, 2 out of 5 patients with HER2 positive primary relapsed and both had high PI in their CTCs, whereas 2 out the 3 HER2 positive patients that did not relapse had low CTC proliferation index. Similarly, the triple negative patient had low PI in her CTCs and has not relapsed after 4 years of follow up. Conclusions: Adjuvant chemotherapy fails to decrease the proliferation index in CTCs. Ki67 expression in CTCs seems to reflect Ki67 expression in the primary tumor and could be predictive of patient outcome. Citation Format: Agelaki S, Spiliotaki M, Spanaki A, Kassiou L, Tzardi M, Koinis F, Kafousi M, Georgoulias V, Mavroudis D. The proliferation index of circulating tumor cells (CTCs) is not influenced by the administration of adjuvant chemotherapy in early breast cancer (BC) and seems to reflect Ki67 expression of the primary tumor. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-16.

Background: The immune system can either prevent and control the tumor growth or promote tumor escape. Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature cells with immune suppressive properties and their overexpression seems to confer a worse prognosis in cancer patients. Objective: The aim of the study was to investigate the presence of new phenotypically immature subpopulations of MDSCs which could be associated with advanced/metastatic NSCLC patients' clinical outcome. Materials & Methods: Circulating subtypes of MDSCs (monocytic and granulocytic), as well as the CD4+ , CD8+ , dendritic (DC), monocytes and B cells were assessed in peripheral blood of of chemotherapy-naive patients with advanced/metastatic NSCLC (n=134) and healthy controls (n=28) were analyzed using flow cytometry. The phenotypic characterization of MDSCs subpopulations was determined in strictly immature myeloid cells. A correlation between the levels of MDSCs and other tested immune cells as well as their association with overall (OS) and progression- free survival (PFS) was evaluated; high expression of MDSCs was defined as the percentage of the cells above the 90% percentile of the controls. Results: Two monocytic [M-MDSC: CD14+CD15-CD11b+CD33+HLA-DR-Lin- and CD14+CD15+CD11b+CD33+HLA-DR-Lin-] and a granulocytic [G-MDSC: CD14-CD15+CD11b+CD33+HLA-DR-Lin-] subpopulation of MDSCs were significantly increased (p Conclusion: The data provide evidence that the increased frequency of two new M-MDSC subpopulations in patients with advanced/metastatic NSCLC is associated with an unfavorable clinical outcome. Citation Format: Eleni Kyriaki Vetsika, Filippos Koinis, Marianthi Gioulmpasani, Despoina Aggouraki, Anna Koutoulaki, Eirini Skalidaki, Dimitris Mavroudis, Vassilis Georgoulias, Athanasios Kotsakis. High frequency of a circulating monocytic subpopulation of myeloid-derived suppressor cells predicts worst clinical outcome in untreated non-small lung cancer patients. [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 3664. doi:10.1158/1538-7445.AM2014-3664