Objective 1. To determine whether vitamin D inhibits another important pro-tumor action of CAFs such as increasing the resistance of tumor cells to drugs commonly used in the treatment of CHD.

Objective 2. To study whether the effects of vitamin D on normal fibroblasts (NFs) and CAFs described by the VitDColon group (Ferrer-Mayorga et al. 2017; Ferrer-Mayorga et al. 2019) are modulated by the gut microbiota.

Objective 3. To analyze the alterations produced by the NPC2 protein in CAFs.

Objective 1. To study the metastatic properties in organoids already available and in those generated during the project.

Objective 2. Study of global expression alterations (RNA-seq, proteomics, metabolomics) in metastatic organoids.

Objective 3. Molecular classification of organoids and prognostic power of the SEC6 signature.

Objective 4. To study the sensitivity of organoids to chemotherapy.

Objective 1. To evaluate the concordance of the determination of low-frequency RAS mutations in plasma, using the OncoBEAM technique, versus the reference test in FFPE tissue samples.

Objective 2. Use plasma cfDNA to capture alterations occurring in genes of signaling pathways implicated in CC, specifically EGFR, ERBB2, PIK3CA and MAP2K1, to unveil putative mechanisms of resistance to standard targeted therapies, such as anti-EGFR, anti-BRAF and anti-HER2 agents.

Objective 3. To study the role of protein phosphatases (PP2A) versus kinases that modulate these signaling pathways.

Objective 4. Identify potential miRNAs regulating the expression of PP2A complex subunits and their endogenous inhibitors.

Objective 5. To analyze a panel of biomolecules involved in inflammation such as IL-6, IL-8, IL-20, IL-1β, IL-22, PD-L1, CA19-9, MMPs (MMP-2, MMP-9, etc.), CEA, CA19-9, occludin, pMAPKs, STAT1 and STAT3, using a magnetic bead-based multiplex immunoassay (Luminex).

Objective 6. Validate the colonic organoids obtained by the VitDColon group and identify possible new therapeutic targets associated with them following the strategies developed by the CIB-CSIC group and described in WP 2.

Objective 1. To establish a prospective cohort of 200 patients with stage II and III CHD by performing continuous sample and data collection.

Objective 2. The biological relevance of our study will be determined by analyzing the possible associations between the data provided by the microbiota study, the levels of sncRNAs, the imaging tests analyzed and the clinicopathological characteristics of the tumor and the patients.

Objective 3. Exosomes will be isolated from patients’ peripheral blood samples using the ExoQuick™ kit (System Bioscience).

Objective 4. Serial imaging tests will be analyzed by radiomic study of the liver at the time of diagnosis and during the follow-up period and/or until the appearance of liver metastases.

Objective 5. An analysis of all the results obtained (images, sncRNAs levels in exosomes and clinicopathological, epidemiological and microbiota characteristics, including also 25-hydroxyvitamin D3 levels in blood and VDR expression in patients’ tumors) will be carried out to develop a bioinformatics algorithm with predictive value for the development of liver metastases.

Objective 1. To evaluate the changes induced in a panel of DC cells by elevated diabetic fatty acid mixture (MAD) and co-culture with human adipose tissue explants.

Objective 2. The effects of adipose tissue explants or MAD on the migratory and invasive capacity of tumor cells will be evaluated using wound closure assays and transwells.

Objective 3. To assess whether vitamin D interferes with lipolysis induced in adipose tissue by tumor cells (Seahorse), fat capture by tumor cells (microscopy), migratory and invasive capacity of tumor cells (functional assays), and changes induced in the immunogenicity of tumor cells (exposed antigen analytical cytometry). changes induced in the immunogenicity of tumor cells (analytical cytometry of exposed antigens).