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Elena Durendez-Saez
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Lunch & Poster Display session (ID 58)
- Event: ELCC 2019
- Type: Poster Display session
- Track:
- Presentations: 3
- Moderators:
- Coordinates: 4/11/2019, 12:30 - 13:00, Hall 1
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25P - Lung tumorspheres as a drug screening platform against cancer stem cells (ID 551)
12:30 - 13:00 | Author(s): Elena Durendez-Saez
- Abstract
Background
Treatment resistance and metastasis are linked to cancer stem cells (CSCs). This population represents a promising target, but remains unexplored in lung cancer. The main objective of this study was to characterize lung CSCs and discover new therapeutic strategies.
a9ded1e5ce5d75814730bb4caaf49419 Methods
The study was performed on NSCLC cells from 8 resected patients and 12 cell lines. Suspension cultures (tumorspheres) were established for CSCs enrichment and differentiated tumor cells were cultured as monolayers (2D). The CSCs properties of tumorspheres were assessed in vitro and in vivo. The expression of 60 CSC-related genes was analyzed by RTqPCR and the expression of 12 proteins was evaluated by immunoblot (IB) and immunofluorescence (IF). High-throughput screening was performed using Prestwick and Myria libraries. Selected drugs were administered intraperitoneally to NOD/SCID mice with tumors induced by NSCLC patient and H1650 tumorspheres.
20c51b5f4e9aeb5334c90ff072e6f928 Results
Lung tumorspheres showed unlimited exponential growth (>30 passages), great tumor initiation potential, differentiation capacity, and high resistance to chemotherapy agents, but not to salinomycin. Tumorspheres had significantly higher expression of CSC-related genes (ALDH1A1, KLF4, NANOG, CD44, CD90, CDKN1A, JUNB, MDM2), invasion promoters (MMP9, SNAI1, ITGA6), and Notch (NOTCH1, NOTCH3, DLL4, JAG1) and Wnt (CTNNB1, GSK3B) components than their paired adherent-cultured cells. IB confirmed the overexpression of proteins encoded by CD44, NANOG, CDKN1A, SNAI1, ITGA6, and NOTCH3, and IF showed different localization patterns on lung adenocarcinoma tumorspheres compared with the 2D cultures. Three novel drugs [Disulfiram (DSF), Compound 1 (COMP1) and Compound 2 (COMP2)] with greater cytotoxic potential against lung tumorspheres than monolayer cells were identified. These results were validated in vivo, demonstrating the capacity of these drugs to reduce tumor growth in mice.
fd69c5cf902969e6fb71d043085ddee6 Conclusions
Tumorspheres are a useful culture platform for CSCs characterization in a simple and cost-effective way. We found three drugs which are able to diminish the formation and viability of tumorspheres, constituting promising therapies against lung CSCs. Supported by CB16/12/00350, PI12-02838, and PI15-00753 from ISCIII.
b651e8a99c4375feb982b7c2cad376e9 Legal entity responsible for the study
Fundación de Investigación Hospital General Universitario de Valencia.
213f68309caaa4ccc14d5f99789640ad Funding
Centro de Investigación Biomédica en Red de Oncología (CIBERONC) and Instituto de Salud Carlos III (ISCIII).
682889d0a1d3b50267a69346a750433d Disclosure
All authors have declared no conflicts of interest.
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28P - Applicability of ctDNA at diagnosis and during the monitoring of EGFR-mutated patients (ID 557)
12:30 - 13:00 | Author(s): Elena Durendez-Saez
- Abstract
Background
Identification of activating mutations in the EGFR enabled to change the therapeutic approach for NSCLC patients as therapeutic targets. Liquid biopsy is a new option for analysis of biomarkers with valuable prognostic and predictive information, allowing the detection of EGFR mutations with a reproducible and minimally invasive approach. The aim of this study was to correlate EGFR mutational status in ctDNA at diagnosis and follow-up of NSCLC patients as a complementary/alternative to tissue-based molecular profiling.
a9ded1e5ce5d75814730bb4caaf49419 Methods
Study included 20 patients with EGFR-mutated advanced NSCLC. Blood samples were collected at diagnosis, during follow-up and at progression. CtDNA was obtained from plasma and EGFR mutations were assessed by BEAMing dPCR (Sysmex®). Concordance between tissue and plasma EGFR mutation status was calculated as the number of positive plasma samples out of the total number of tissue samples. Cases at which T790M were first detected in blood were compared to date of progression as determined by radiological imaging in standard practice.
20c51b5f4e9aeb5334c90ff072e6f928 Results
A total of 262 plasma samples from 20 patients were analyzed, 75% percent of patients were females; 60% had never smoked; with a median of 18 months of follow- up (range: 5.13-61.77). The Positive Percent Agreement (PPA) at baseline for EGFR del19 and L858R mutation status between plasma and tissue was 75%; in 12 cases the clearance of the primary mutation happens during the first four to eight weeks after initiation of EGFR TKI. Furthermore, eight patients with radiological progression presented the resistance mutation T790M (66%), remarking that the majority of these tumors presented again the sensitizing mutation. In 6 of these patients plasma T790M-positivity was detected an average of 16 weeks (range: 4-24) prior to radiological progression. These results suggest that periodic monitoring of EGFR status in ctDNA could provide information regarding diagnoses, response or resistance to TKI-treatment.
fd69c5cf902969e6fb71d043085ddee6 Conclusions
Analyses of EGFR mutations in cfDNA have important clinical applicability and can be a useful alternate biomarker of diagnoses and early detection of TKIs resistance mechanisms.
b651e8a99c4375feb982b7c2cad376e9 Legal entity responsible for the study
Fundación de Investigación del Hospital General Universitario de Valencia.
213f68309caaa4ccc14d5f99789640ad Funding
Work supported in part by AstraZeneca (ISSIRES 0110); CIBERONC (CB16/12/00350); López-Trigo Grant.
682889d0a1d3b50267a69346a750433d Disclosure
All authors have declared no conflicts of interest.
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32P - Exosomes in NSCLC: Analysis of its cargo as a source of biomarkers (ID 544)
12:30 - 13:00 | Presenting Author(s): Elena Durendez-Saez
- Abstract
Background
Exosomes are membranous vesicles around 40-130 nm secreted by cells, carrying key information to distant tissues. Exosomes have been detected in different types of clinical samples and may play a key role in NSCLC. They intervene in several processes such as angiogenesis, metastatic niche formation, immunosuppression; being relevant in stem cell differentiation. The main objective of this study was to analyze exosomal cargo from NSCLC cell lines and primary cultures under two conditions: suspension cultures with cancer stem cells features (3D tumorspheres) and adherent cultures (2D).
a9ded1e5ce5d75814730bb4caaf49419 Methods
Cell cultures were established from NSCLC resected patients and cell lines. Exosomes isolation was performed by ultracentrifugation. Characterization was carried out by NTA, electron microscopy, immunoblot and flow cytometry. Exosomal DNA was extracted to determine the mutational status of EGFR and RAS genes by BEAMing dPCR (Sysmex®). Transcriptomic analysis has been carried out from exosomal RNA with Clariom D Human microarrays (Affymetrix®), (p ≤ 0.01).
20c51b5f4e9aeb5334c90ff072e6f928 Results
Regarding exosomes characterization, NTA and electron microscopy showed an exosome size from 108-125 nm. Exosomes surface markers were detected by immunoblot and flow cytometry. Mutational analysis of EGFR and RAS genes shown the same pattern displayed by the origin cells. Transcriptomic analysis showed an expression of mRNAs, miRNAs and precursors significantly different between 3D and 2D exosomes. Afterwards, their targets were identified and a pathway enrichment analysis was performed to know in which biological processes (cancer-related) are involved. Significant differential expressions were also found between ADC vs SCC-derived exosomes. Interestingly, 7 exosomal miRNAs (miR-200c, 29a, 339, 224, 31, 21, 33a) had already been identified as overexpressed in NSCLC tissue by our group. Moreover, miR-339 and miR-21 were related to prognosis (p < 0.05) in ADC.
fd69c5cf902969e6fb71d043085ddee6 Conclusions
Differences in exosomal cargo have been observed between: i) 3D vs 2D cultures and ii) ADC vs SCC. In addition, the same mutational pattern was detected in exosomes as compared with parental cultures. Therefore, exosomes can be a useful source for biomarkers analysis in NSCLC.
b651e8a99c4375feb982b7c2cad376e9 Legal entity responsible for the study
Fundación de Investigación del Hospital General Universitario de Valencia (FIHGUV).
213f68309caaa4ccc14d5f99789640ad Funding
Supported by grant GV/2018/026 & Asociación Española contra el Cáncer (AECC Valencia).
682889d0a1d3b50267a69346a750433d Disclosure
All authors have declared no conflicts of interest.
cffcb1a185b2d7d5c44e9dc785b6bb25
