Materials e Methods (Part 4) - Outline of Research Objectives [Corrected 2].docx

**MATERIALS & METHODS** **[4 Outline of Research Objectives:]** The primary purpose of the PhD thesis is to provide pre-clinical data on the potential use of ATRA in the personalized treatment of Gastric Cancer. In addition, the project aims to define the molecular mechanisms and gene-networks und...

**MATERIALS & METHODS** **[4 Outline of Research Objectives:]** The primary purpose of the PhD thesis is to provide pre-clinical data on the potential use of ATRA in the personalized treatment of Gastric Cancer. In addition, the project aims to define the molecular mechanisms and gene-networks underlying the expected anti-tumor activity exerted by ATRA in specific subgroups of Gastric Cancer using non-oriented gene-expression approaches based on RNA-sequencing. A further goal is to develop a novel diagnostic tool to be used for the selection of Gastric Cancer patients who may benefit from ATRA-based therapies. A final and long-term goal of the project is to develop rationale therapeutic combinations between ATRA and compounds targeting specific components of the gene-networks identified in the previous points. **[4.1 Specific Aim 1:]** ATRA-sensitivity of Gastric Cancer cell lines and the definition of the associated genomic profiles show how Gastric Cancer is a relatively heterogeneous disease that can be classified into different groups^1^. Gastric Cancer heterogeneity is partially recapitulated by immortalized cell lines. The first goal of Aim 1 is to carry out RNA-Sequencing analyses to evaluate the transcriptomic profiles of our panel of Gastric Cancer cell lines and the effects on gene-transcription activated by ATRA. A second partial goal is to apply a classification based on the molecular profile of our panel of GC cell lines, in order to identify different sub-groups and individually assess their ATRA sensitivity. *4.1.1 Experimental Design -- Aim 1* Using the mRNA extracted from 15 Gastric Cancer cell lines belonging to our laboratory the goal is to perform RNA-Sequencing studies through the use of NextSeq-500 Illumina. The samples are treated with vehicle and ATRA, in order to evaluate the transcriptional profile resulting from differential analysis following treatment with ATRA. With the aim of carrying out the clustering and identifying the subgroups according to the molecular profile, the methods described in the review by Wang et al.^2^ are taken into consideration. Among these, the most suitable is the one available in Tan et al.^3^, as it uses different unsupervised and unbiased clustering techniques. **[4.2 Specific Aim 2:]** Using bioinformatics methodologies and our panel of Gastric Cancer cell lines, the goal is to develop a new gene-expression model capable of predicting ATRA sensitivity in stomach tumors. The long-term goal is to identify a minimal gene-expression signature to be used as a diagnostic tool for the selection of Gastric Cancer patients who may benefit from ATRA-based treatments. *4.2.1 Experimental Design -- Aim 2* Considering our panel of gastric cell lines (27 Gastric Cancer cell lines), which is larger than the one used for the transcriptomic analyses (15 Gastric Cancer cell lines), each cell line is evaluated for its response to the anti-proliferative action of ATRA, using a score reflecting the in vitro sensitivity to ATRA (AUC/ATRA-score). The new gene-expression model is developed using the methods explained in the next chapter, which are based on bioinformatics procedures and computational techniques. To pursue this goal, the cell lines are profiled for their sensitivity to the anti-proliferative action of ATRA, using the transcriptomic expression data of the corresponding 27 Gastric Cancer cell lines retrieved from CCLE (Cancer Cell Line Encyclopaedia). The new model predicting ATRA-sensitivity in a tumor-independent fashion is validated and optimized in our panel of Gastric Cancer cell lines using the associated basal gene-expression profiles and bio-computational approaches. The generation of a predictive model is necessary to guarantee specificity and optimal results in Gastric Cancer. **[4.3 Specific Aim 3:]** In vitro, short-term tissue cultures of immortalized Gastric Cancer cell lines are useful tools to study the anti-tumor activity of ATRA, as they often recapitulate the major biological characteristics of the tumors they derive from. However, the in vitro data obtained in cell lines must be confirmed in other models that more closely reflect real life. We developed/implemented a model based on short-term tissue-slice cultures^4^, which permits the evaluation of the activity of pharmacological agents ex-vivo on primary-tumor specimens. This model is used to test the response of primary breast tumors to ATRA. The plan is to apply short-term tissue cultures to establish ATRA anti-tumor activity on samples derived from Gastric Cancer patients in terms of cell growth inhibition and apoptosis. With this model, it is possible to establish a correlation between the in vitro responses to ATRA anti-proliferative and apoptotic effects determined in tissue-slice cultures. For this purpose, RNA-sequencing experiments in tissue slices exposed to vehicle and ATRA are performed. Hence, this aim includes validation of the genomic profiles associated with ATRA sensitivity and determination of the genomic effects exerted by the retinoid in Gastric Cancer tissue slices. This will be done by applying the novel gene-expression model developed in Aim 2, which validates the gene-networks modulated by ATRA. The data deriving from both Aim 2 and Aim 3 are likely to result in the identification of novel targets of pharmacological intervention in view of the development of ATRA-based therapeutic combinations. *4.3.1 Experimental Design -- Aim 3* The plan is to use freshly isolated surgical samples from several Gastric Cancer patients which were obtained over the course of the project. Surgical samples must be processed within 24 hours from the collection. With a Krumdick tissue-slicer, the dissection of the core samples in tissue slices is executed (200 µm of thickness). Slices are incubated for 48-72 hours in an optimized culture medium containing the vehicle (DMSO) or ATRA (0.1-1.0 micromolar). At the end of the treatment, slices are fixed in formalin, embedded in paraffin and evaluated for: a. **Growth inhibition**. To define this parameter, the quantitative expression of Ki67 (percentage of immunohistochemistry positive cells) is determined by a pathologist. The goal is to look for significant reductions in Ki67 levels after treatment with ATRA. This analysis is blinded as for treatment. In addition, measurement of a number of RNAs coding for proliferation associated genes using PCR technologies is performed. b. **Apoptosis**. The potential pro-apoptotic action of ATRA is evaluated as described under point a) for Ki67, using an antibody targeting activated caspase-3, a biomarker associated with the early phases of apoptosis. After this step, slices are used for RNA extraction. The goal is to perform oriented RNA-sequencing experiments in these tissue slices exposed to vehicle and ATRA. A major goal is to confirm the predictive power of the novel gene-expression model (Aim 2) in primary Gastric Cancer samples. In particular, the priority is to establish correlations between the computational model and the in vitro sensitivity of each tumor to the anti-proliferative and/or apoptotic actions of ATRA. As for the second goal of Aim 3, the focus is to determine ATRA-dependent perturbations on the gene expression profiles of Gastric Cancer and to identify genes differentially regulated by the retinoid, using standard computational analyses of the RNA-sequencing data. **[4.4 Expected Outcomes, Risks and Innovation:]** *4.4.1 Expected Outcomes* The study will provide insights into the therapeutic potential of ATRA in specific groups of Gastric Cancers. In addition, a diagnostic tool to be used in the clinics for the selection of patients who may benefit from ATRA-based therapeutic strategies, will be developed and validated. Finally, we will identify therapeutic targets for the design of innovative ATRA-based drug combinations. *4.4.2 Risk Analysis, possible problems and solutions* The proposed study\'s aims are feasible, and no technical problems are foreseen regarding the high-throughput genomic studies required. However, no systematic data in the literature on the anti-tumor action of ATRA in Gastric Cancer are available. The data obtained on the cell lines may support the idea that ATRA favours rather than blocks the proliferation and survival of Gastric Cancer cells. If this is the case, the observation may redirect the research project on inverse-agonists of the retinoid receptors. *4.4.3 Significance and Innovation* ATRA is a non-conventional anticancer agent differing from classic chemotherapeutics. Its anti-tumor activity has been established in pre-clinical models of different tumor types, although there are very few studies evaluating ATRA therapeutic potential in Gastric Cancer. This project explores the sensitivity of Gastric Cancer to ATRA with a systematic approach. The study is expected to provide insights into the possible clinical development of a novel type of anti-cancer agent that could act in synergy with other therapies. The emerging theme of personalized medicine calls for the development of accurate diagnostic tools capable of predicting the sensitivity of individual patients to a given therapeutic agent. It is foreseen that completion of the project will provide the rationale for the design of phase I/II trials based on ATRA or derived retinoids in gastric cancer. **[REFERENCES:]** 1\. Nguyen, P. H. *et al.* All-trans retinoic acid targets gastric cancer stem cells and inhibits patient-derived gastric carcinoma tumor growth. *Oncogene* **35**, 5619--5628 (2016).2. Wang, Q., Liu, G. & Hu, C. Molecular Classification of Gastric Adenocarcinoma. *Gastroenterol. Res.* **12**, 275--282 (2019).3. Tan, I. B. *et al.* Intrinsic Subtypes of Gastric Cancer, Based on Gene Expression Pattern, Predict Survival and Respond Differently to Chemotherapy. *Gastroenterology* **141**, 476-485.e11 (2011).4. Centritto, F. *et al.* Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression. *EMBO Mol. Med.* **7**, 950--972 (2015).