Preclinical Modelling of PDA: Is Organoid the New Black?

Pancreatic ductal adenocarcinoma (PDA) is a malignancy of the exocrine pancreas with the worst prognosis among all solid tumours, and soon to become the second leading cause of cancer-related deaths. A more comprehensive understanding of the molecular mechanisms underlying this disease is crucial to the development of diagnostic tools as well as to the identification of more effective therapies. High-frequency mutations in PDA occur in "undruggable" genes, and molecular subtyping based on bulk transcriptome analysis does not yet nominate valid therapeutic intervention strategies. Genome-wide sequencing studies have also demonstrated a considerable intra- and inter-patient's genetic heterogeneity, which further complicate this dire scenario. More than in other malignancies, functionalization of the PDA genome and preclinical modelling at the individual patient level appear necessary to substantially improve survival rates for pancreatic cancer patients. Traditional human PDA models, including monolayer cell cultures and patient-derived xenografts, have certainly led to valuable biological insights in the past years. However, those model systems suffer from several limitations that have contributed to the lack of concordance between preclinical and clinical studies for PDA. Pancreatic ductal organoids have recently emerged as a reliable culture system to establish models from both normal and neoplastic pancreatic tissues. Pancreatic organoid cultures can be efficiently generated from small tissue biopsies, which opens up the possibility of longitudinal studies in individual patients. A proof-of-concept study has demonstrated that patient-derived PDA organoids are able to predict responses to conventional chemotherapy. The use of this three-dimensional culture system has already improved our understanding of PDA biology and promises to implement precision oncology by enabling the alignment of preclinical and clinical platforms to guide therapeutic intervention in PDA

Influence of structure and solubility of chain transfer agents on the RAFT control of dispersion polymerisation in scCO2

Reversible addition-fragmentation chain transfer (RAFT) dispersion polymerisation of methyl methacrylate (MMA) is performed in supercritical carbon dioxide (scCO2) with 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) present as chain transfer agent (CTA) and surprisingly shows good control over PMMA molecular weight. Kinetic studies of the polymerisation in scCO2also confirm these data. By contrast, only poor control of MMA polymerisation is obtained in toluene solution, as would be expected for this CTA which is better suited for acrylates. In this regard, we select a range of CTAs and use them to determine the parameters that must be considered for good control in dispersion polymerisation in scCO2. A thorough investigation of the nucleation stage during the dispersion polymerisation reveals an unexpected “in situtwo-stage” mechanism that strongly determines how the CTA works. Finally, using a novel computational solvation model, we identify a correlation between polymerisation control and degree of solubility of the CTAs. All of this ultimately gives rise to a simple, elegant and counterintuitive guideline to select the best CTA for RAFT dispersion polymerisation in scCO

Covid-19 and the role of smoking: the protocol of the multicentric prospective study COSMO-IT (COvid19 and SMOking in ITaly).

The emergency caused by Covid-19 pandemic raised interest in studying lifestyles and comorbidities as important determinants of poor Covid-19 prognosis. Data on tobacco smoking, alcohol consumption and obesity are still limited, while no data are available on the role of e-cigarettes and heated tobacco products (HTP). To clarify the role of tobacco smoking and other lifestyle habits on COVID-19 severity and progression, we designed a longitudinal observational study titled COvid19 and SMOking in ITaly (COSMO-IT). About 30 Italian hospitals in North, Centre and South of Italy joined the study. Its main aims are: 1) to quantify the role of tobacco smoking and smoking cessation on the severity and progression of COVID-19 in hospitalized patients; 2) to compare smoking prevalence and severity of the disease in relation to smoking in hospitalized COVID-19 patients versus patients treated at home; 3) to quantify the association between other lifestyle factors, such as e-cigarette and HTP use, alcohol and obesity and the risk of unfavourable COVID-19 outcomes. Socio-demographic, lifestyle and medical history information will be gathered for around 3000 hospitalized and 700-1000 home-isolated, laboratory-confirmed, COVID-19 patients. Given the current absence of a vaccine against SARS-COV-2 and the lack of a specific treatment for -COVID-19, prevention strategies are of extreme importance. This project, designed to highly contribute to the international scientific debate on the role of avoidable lifestyle habits on COVID-19 severity, will provide valuable epidemiological data in order to support important recommendations to prevent COVID-19 incidence, progression and mortality

Loss of FGFR4 promotes the malignant phenotype of PDAC

Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients

Generation of Pancreatic Organoid-Derived Isografts

This protocol is a procedure for generating orthotopic isografts using mouse pancreatic cancer organoids. These isografts can be used to track the evolution of pancreatic ductal adenocarcinoma (PDA) from a preinvasive lesion to a metastatic disease and therefore represent a suitable model for identification of determinants of PDA progression. For complete details on the use and execution of this protocol, please refer to Boj et al. (2015) and Filippini et al. (2019)

Combinatorial Effect of Magnetic Field and Radiotherapy in PDAC Organoids: A Pilot Study

Pancreatic ductal adenocarcinoma (PDAC) is highly refractory to systemic treatment, including radiotherapy (RT) either as alone or in combination with chemotherapy. Magnetic resonance (MR)-guided RT is a novel treatment technique which conjugates the high MR imaging contrast resolution to the possibility of re-adapting treatment plan to daily anatomical variations. Magnetic field (MF) might exert a biological effect that could be exploited to enhance radiation effect. The aim of the present study was to lay the preclinical basis of the MF effect by exploring how it modifies the response to radiation in organoid cultures established from PDAC. The short-term effect of radiation, alone or in combination with MF, was evaluated in patient-derived organoids (PDOs) and monolayer cell cultures. Cell viability, apoptotic cell death, and organoid size following exposure to the treatment were evaluated. PDOs demonstrated limited sensitivity at clinically relevant doses of radiation. The combination of radiation and MF demonstrated superior efficacy than monotherapy in almost all the PDOs tested. PDOs treated with combination of radiation and MF were significantly smaller in size and some showed increased cell death as compared to the monotherapy with radiation. Long-time exposure to 1.5T MF can increase the therapeutic efficacy of radiation in PDAC organoids

Influence of structure and solubility of chain transfer agents on the RAFT control of dispersion polymerisation in scCO2

Reversible addition–fragmentation chain transfer (RAFT) dispersion polymerisation of methyl methacrylate (MMA) is performed in supercritical carbon dioxide (scCO(2)) with 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) present as chain transfer agent (CTA) and surprisingly shows good control over PMMA molecular weight. Kinetic studies of the polymerisation in scCO(2) also confirm these data. By contrast, only poor control of MMA polymerisation is obtained in toluene solution, as would be expected for this CTA which is better suited for acrylates. In this regard, we select a range of CTAs and use them to determine the parameters that must be considered for good control in dispersion polymerisation in scCO(2). A thorough investigation of the nucleation stage during the dispersion polymerisation reveals an unexpected “in situ two-stage” mechanism that strongly determines how the CTA works. Finally, using a novel computational solvation model, we identify a correlation between polymerisation control and degree of solubility of the CTAs. All of this ultimately gives rise to a simple, elegant and counterintuitive guideline to select the best CTA for RAFT dispersion polymerisation in scCO(2)

Therapeutic potential of combined BRAF/MEK blockade in BRAF-wild type preclinical tumor models

Background: Mounting evidence suggests that RAF-mediated MEK activation plays a crucial role in paradox MAPK (re)activation, leading to resistance and therapeutic failure with agents hitting a single step along the MAPK cascade. Methods: We examined the molecular and functional effects of single and combined BRAF (dabrafenib), pan-RAF (RAF265), MEK (trametinib) and EGFR/HER2 (lapatinib) inhibition, using Western Blot and conservative isobologram analysis to assess functional synergism, and explored genetic determinants of synergistic interactions. Immunoprecipitation based assays were used to detect the interaction between BRAF and CRAF. The Mann-Whitney U test was used for comparing quantitative variables. Results: Here we demonstrated that a combination of MEK and BRAF inhibitors overcomes paradoxical MAPK activation (induced by BRAF inhibitors) in BRAF-wt/RAS-mut NSCLC and PDAC in vitro. This results in growth inhibitory synergism, both in vitro and in vivo, in the majority (65%) of the cellular models analyzed, encompassing cell lines and patient-derived cancer stem cells and organoids. However, RAS mutational status is not the sole determinant of functional synergism between RAF and MEK inhibitors, as demonstrated in KRAS isogenic tumor cell line models. Moreover, in EGFR-driven contexts, paradoxical MAPK (re)activation in response to selective BRAF inhibition was dependent on EGFR family signaling and could be offset by simultaneous EGFR/HER-2 blockade. Conclusions: Overall, our data indicate that RAF inhibition-induced paradoxical MAPK activation could be exploited for therapeutic purposes by simultaneously targeting both RAF and MEK (and potentially EGFR family members) in appropriate molecular contexts. KRAS mutation per se does not effectively predict therapeutic synergism and other biomarkers need to be developed to identify patients potentially deriving benefit from combined BRAF/MEK targeting