Il ruolo dell’infiammazione nello sviluppo di adenomi in pazienti con Poliposi Adenomatosa Familiare associata al gene APC.

APC-associated Adenomatous Polyposis (AAP) is an autosomal dominant hereditary colorectal cancer (CRC) syndrome associated with germline mutations in the APC tumor suppressor gene. AAP is characterized by the development in the colon of hundreds to thousands of adenomatous polyps starting from the teen years. Currently, there are no approved chemo-preventive agents for AAP individuals. Several clinical studies using Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), have shown a notable reduction in the size and number of polyps. Indeed, inflammation is directly related to adenoma formation and CRC development. Recently, a low inflammatory dietary pilot study was conducted at the Fondazione IRCCS Istituto Nazionale dei Tumori of Milan (INT) on a group of 34 patients with AAP, with the aim to reduce the high local inflammation on the rectal mucosa. Patients Derived Organoids (PDOs) from AAP adenomas have been developed to study the effects produced by inflammatory microenvironment on epithelial cells. AAP PDOs are an innovative in vitro model that reproduces most of the characteristics of the adenomas. Material and Methods 34 patients, diagnosed with AAP, under surveillance at INT were enrolled in the pilot dietary intervention study. Participants accepted to follow a six months low inflammatory diet, providing fecal and blood samples at three different times: baseline (T0), three months of diet (T1), end of dietary intervention (T2). Local and systemic inflammation markers such as fecal and serum Calprotectin, Insulin and Insulin Growth Factor-1, were evaluated. Formalin Fixed and Paraffin Embedded (FFPE) biopsies were collected from 16 patients at T0 and T2 for immunohistochemistry (IHC) evaluation of COX-2 and 15-PGDH. Four 3D-Patients Derived Organoids (PDOs) cultures from colorectal adenomas of AAP patients were generated, immunophenotypically and molecularly characterized. Gene expression profiles of FAP PDOs maintained under lymphoid and myeloid pro-inflammatory conditions were carried out with the Nanostring PanCancer Immune Profile panel. Results Changes in the expression of stool and serum Calprotectin were observed on 24 patients at different samplings (p-values: 0.012 and 0.002 respectively). All the tissue samples showed high COX-2 immunoreactivity and no significant differences in COX-2 expression were observed between T0 and T2 in normal colonic crypts, adenomatous crypts, stromal infiltrating immune cells and adenoma infiltrating immune cells. Coherently, 15-PGDH expression was almost null in most of healthy crypts at T0. After the dietary intervention, a significant increase in 15-PGDH IHC expression was observed in the normal colonic crypts of the matched T0-T2 biopsies (Wilcoxon Signed Rank: 0.02). Cell Cycle analyses of FAP PDOs cultured under pro-inflammatory conditions (grown in presence of a cocktail of interleukins - IL, including IL-17a, IL-22 and IL-6) were similar for FAP1, FAP2 and FAP3 PDOs, while FAP4 presented a higher percentage of cells in G2/M phase. Differential gene expression (DGE) analysis of FAP PDOs cultured under ILs vs Standard conditions using an Immune Profile panel highlighted that 28 genes were significantly increased (log2FC > 2 and FDR cut off 0.05). DMBT1 was the most differentially expressed gene and showed more than 3-fold increase in FAP3, 9-fold in FAP2, and 7.5-fold in FAP1 PDOs, no changes were observed in FAP4 PDOs. Results After 3 months of diet AAP subjects reduced their local and systemic inflammation, as also confirmed by the increase of 15-PGDH observed on rectal AAP biopsies collected from patients at the end of the dietary intervention. The reconstruction of a lymphoid pro-inflammatory condition for AAP PDO growth highlighted that IL-17a, IL-22 and IL-6 cocktail could have a protective role for epithelial cells composing the AAP colonic mucosa at the very early steps of CRC progression through the activation of the tumor suppressor gene DMBT1.

Il progetto di dottorato che ho portato avanti in questi anni è costituito da due parti. Nella prima parte sono state eseguite analisi immunoistochimiche su biopsie ottenute dal colon di 34 soggetti con poliposi adenomatosa familiare associata al gene APC (AAP). I pazienti arruolati in uno studio pilota, rivolto a ridurre i livelli di infiammazione della mucosa colica hanno eseguito due colonscopie prima dell’intervento dietetico e alla fine dello stesso. I risultati hanno mostrato una riduzione dei marcatori di infiammazione locale alla fine dell’intervento dietetico antiinfiammatorio. La seconda parte del progetto è stata incentrata sullo sviluppo, la caratterizzazione e lo studio di quattro modelli cellulari 3D (Organoidi) derivati da pazienti con AAP (FAP1-4). Gli Organoidi sono stati generati mettendo a punto le condizioni ottimali per la loro crescita, sono stati validati dal punto di vista immunofenotipico e molecolare. Al fine di studiare gli effetti dell’infiammazione sugli Organoidi FAP che rappresentano la componente epiteliale della mucosa colica, due condizioni pro-infiammatorie sono state ricreate in vitro. La prima condizione basata l’uso di Interleuchine di derivazione linfoide, in particolare linfociti Th17 e Th22. La seconda basata sulla co-coltura di Organoidi FAP e una linea mieloide (THP1) in grado di secernere citochine tipiche dei macrofagi. Le analisi svolte sul ciclo cellulare e sull’espressione genica degli Organoidi FAP1-4 ha mostrato che i due ambienti pro-infiammatori condizionavano la crescita degli Organoidi, così come l’espressione di geni correlati alla proliferazione e alla salvaguardia delle cellule in un ambiente infiammatorio.