An integrated multiomics analysis of rectal cancer patients identified POU2F3 as a putative druggable target and entinostat as a cytotoxic enhancer of 5-fluorouracil.

Rectal cancer (RC) accounts for one-third of colorectal cancers (CRC), and 40% of these are locally advanced rectal cancers (LARC). The use of neoadjuvant chemoradiotherapy (nCRT) significantly reduces the rate of local recurrence compared to adjuvant therapy or surgery alone. However, after nCRT, up to 40%-60% of patients show a poor pathological response, while only about 20% achieve a pathological complete response. In this scenario, the identification of novel predictors of tumor response to nCRT is urgently needed to reduce LARC mortality and to spare poorly responding patients from unnecessary treatments. Therefore, by combining gene and microRNA expression datasets with proteomic data from LARC patients, we developed an integrated network centered on seven hub-genes putatively involved in the response to nCRT. In an independent validation cohort of LARC patients, we confirmed that differential expression of NFKB1, TRAF6 and STAT3 is correlated with the response to nCRT. In addition, the functional enrichment analysis also revealed that these genes are strongly related to hallmarks of cancer and inflammation, whose dysfunction may causatively affect LARC patient's response to nCRT. Furthermore, by constructing the transcription factor-module network, we hypothesized a protective role of POU2F3 gene, which could be used as a new drug target in LARC patients. Finally, we identified and tested in vitro entinostat, a histone deacetylase inhibitor, as a chemical compound that could be combined with a classical therapeutic regimen in order to design more efficient therapeutic strategies in LARC management.

Preclinical three-dimensional colorectal cancer model: The next generation of in vitro drug efficacy evaluation.

Colorectal cancer (CRC), the third most common cancer diagnosed in both men and women in the United States, shows a highly ineffective therapeutic management. In these years neither substantial improvements nor new therapeutic approaches have been provided to patients. Performing the early lead discovery phases of new cancer drugs in cellular models, resembling as far as possible the real in vivo tumor environment, may be more effective in predicting their future success in the later clinical phases. In this review, we critically describe the most representative bioengineered models for anticancer drug screening in CRC from the conventional two-dimensional models to the new-generation three-dimensional scaffold-based ones. The scaffold aims to replace the extracellular matrix, thus influencing the biomechanical, biochemical, and biological properties of cells and tissues. In this scenario, we believe that reconstitution of tumor condition is mandatory for an alternative in vitro methods to study cancer development and therapeutic strategies.

Decellularized colorectal cancer matrix as bioactive microenvironment for in vitro 3D cancer research.

Three-dimensional (3D) cancer models are overlooking the scientific landscape with the primary goal of bridging the gaps between two-dimensional (2D) cell lines, animal models and clinical research. Here, we describe an innovative tissue engineering approach applied to colorectal cancer (CRC) starting from decellularized human biopsies in order to generate an organotypic 3D-bioactive model. This in vitro 3D system recapitulates the ultrastructural environment of native tissue as demonstrated by histology, immunohistochemistry, immunofluorescence and scanning electron microscopy analyses. Mass spectrometry of proteome and secretome confirmed a different stromal composition between decellularized healthy mucosa and CRC in terms of structural and secreted proteins. Importantly, we proved that our 3D acellular matrices retained their biological properties: using CAM assay, we observed a decreased angiogenic potential in decellularized CRC compared with healthy tissue, caused by direct effect of DEFA3. We demonstrated that following a 5 days of recellularization with HT-29 cell line, the 3D tumor matrices induced an over-expression of IL-8, a DEFA3-mediated pathway and a mandatory chemokine in cancer growth and proliferation. Given the biological activity maintained by the scaffolds after decellularization, we believe this approach is a powerful tool for future pre-clinical research and screenings.

Recent Advancements in Hydrogel Biomedical Research in Italy.

Hydrogels have emerged as versatile biomaterials with remarkable applications in biomedicine and tissue engineering. Here, we present an overview of recent and ongoing research in Italy, focusing on extracellular matrix-derived, natural, and synthetic hydrogels specifically applied to biomedicine and tissue engineering. The analyzed studies highlight the versatile nature and wide range of applicability of hydrogel-based studies. Attention is also given to the integration of hydrogels within bioreactor systems, specialized devices used in biological studies to culture cells under controlled conditions, enhancing their potential for regenerative medicine, drug discovery, and drug delivery. Despite the abundance of literature on this subject, a comprehensive overview of Italian contributions to the field of hydrogels-based biomedical research is still missing and is thus our focus for this review. Consolidating a diverse range of studies, the Italian scientific community presents a complete landscape for hydrogel use, shaping the future directions of biomaterials research. This review aspires to serve as a guide and map for Italian researchers interested in the development and use of hydrogels in biomedicine.

Patient Derived Organoids (PDOs), Extracellular Matrix (ECM), Tumor Microenvironment (TME) and Drug Screening: State of the Art and Clinical Implications of Ovarian Cancer Organoids in the Era of Precision Medicine.

Ovarian cancer (OC) has the highest mortality rate of all gynecological malignancies due to the high prevalence of advanced stages of diagnosis and the high rate of recurrence. Furthermore, the heterogeneity of OC tumors contributes to the rapid development of resistance to conventional chemotherapy. In recent years, in order to overcome these problems, targeted therapies have been introduced in various types of tumors, including gynecological cancer. However, the lack of predictive biomarkers showing different clinical benefits limits the effectiveness of these therapies. This requires the development of preclinical models that can replicate the histological and molecular characteristics of OC subtypes. In this scenario, organoids become an important preclinical model for personalized medicine. In fact, patient-derived organoids (PDO) recapture tumor heterogeneity with the possibility of performing drug screening. However, to best reproduce the patient's characteristics, it is necessary to develop a specific extracellular matrix (ECM) and introduce a tumor microenvironment (TME), which both represent an actual object of study to improve drug screening, particularly when used in targeted therapy and immunotherapy to guide therapeutic decisions. In this review, we summarize the current state of the art for the screening of PDOs, ECM, TME, and drugs in the setting of OC, as well as discussing the clinical implications and future perspectives for the research of OC organoids.

Establishment of a human 3D pancreatic adenocarcinoma model based on a patient-derived extracellular matrix scaffold.

Pancreatic cancer is likely to become one of the leading causes of cancer-related death in many countries within the next decade. Surgery is the potentially curative treatment for pancreatic ductal adenocarcinoma (PDAC), although only 10%-20% of patients have a resectable disease after diagnosis. Despite recent advances in curative surgery the current prognosis ranges from 6% to 10% globally. One of the main issues at the pre-clinical level is the lacking of model which simultaneously reflects the tumour microenvironment (TME) at both structural and cellular levels. Here we describe an innovative tissue engineering approach applied to PDAC starting from decellularized human biopsies in order to generate an organotypic 3D in vitro model. This in vitro 3D system recapitulates the ultrastructural environment of native tissue as demonstrated by histology, immunohistochemistry, immunofluorescence, mechanical analysis, and scanning electron microscopy. Mass spectrometry confirmed a different extracellular matrix (ECM) composition between decellularized healthy pancreas and PDAC by identifying a total of 110 non-redundant differently expressed proteins. Immunofluorescence analyses after 7 days of scaffold recellularization with PANC-1 and AsPC-1 pancreatic cell lines, were performed to assess the biocompatibility of 3D matrices to sustain engraftment, localization and infiltration. Finally, both PANC-1 and AsPC-1 cells cultured in 3D matrices showed a reduced response to treatment with FOLFIRINOX if compared to conventional bi-dimensional culture. Our 3D culture system with patient-derived tissue-specific decellularized ECM better recapitulates the pancreatic cancer microenvironment compared to conventional 2D culture conditions and represents a relevant approach for the study of pancreatic cancer response to chemotherapy agents.

The Study of the Extracellular Matrix in Chronic Inflammation: A Way to Prevent Cancer Initiation?

Bidirectional communication between cells and their microenvironment has a key function in normal tissue homeostasis, and in disease initiation, progression and a patient's prognosis, at the very least. The extracellular matrix (ECM), as an element of all tissues and cellular microenvironment, is a frequently overlooked component implicated in the pathogenesis and progression of several diseases. In the inflammatory microenvironment (IME), different alterations resulting from remodeling processes can affect ECM, progressively inducing cancer initiation and the passage toward a tumor microenvironment (TME). Indeed, it has been demonstrated that altered ECM components interact with a variety of surface receptors triggering intracellular signaling that affect cellular pathways in turn. This review aims to support the notion that the ECM and its alterations actively participate in the promotion of chronic inflammation and cancer initiation. In conclusion, some data obtained in cancer research with the employment of decellularized ECM (dECM) models are described. The reported results encourage the application of dECM models to investigate the short circuits contributing to the creation of distinct IME, thus representing a potential tool to avoid the progression toward a malignant lesion.

An investigation on [5 fluorouracil and epigallocatechin-3-gallate] complex activity on HT-29 cell death and its stability in gastrointestinal fluid.

Recently an enhancement of the sensitivity of colorectal cancer (CRC) cells by 5-fluorouracil (5FU) due to the concurrent treatment with epigallocatechin-3-gallate (EGCG) has been found. In the present paper, to investigate on this aspect, adenocarcinoma cells HT29 were treated with 5FU, EGCG and an equimolar mixture of 5FU and EGCG ([5FU+EGCG]) and cell viability was determined. While 5FU exhibits a clear activity, EGCG alone does not express any activity. However by treating the cells with [5FU+EGCG] a strong effect of EGCG is evidenced: the sensitivity of HT29 cells to 5FU was increased by 12-fold. A simulation of the behavior of [5FU+EGCG] in different compartments of the gastrointestinal digestion model was also performed. 5FU and EGCG solubilized into a mixture of digestive fluids analyzed by mass spectrometry did not lead to signals of 5FU, EGCG and the related complex, while by diluting the solution they become detectable. On the contrary, when 5FU and EGCG are submitted to the step-by-step digestion model procedure, the analysis did not show the presence of 5FU, EGCG and [5FU+EGCG]. This behaviour could be ascribed to the instability of these compounds due to the too severe digestion conditions and/or to the complexity of the matrix which could lead in ESI conditions to the suppression of the signals of the analytes of interest.

Tumor Cells and the Extracellular Matrix Dictate the Pro-Tumoral Profile of Macrophages in CRC.

Tumor-associated macrophages (TAMs) are major components of the tumor microenvironment. In colorectal cancer (CRC), a strong infiltration of TAMs is accompanied by a decrease in effector T cells and an increase in the metastatic potential of CRC. We investigated the functional profile of TAMs infiltrating CRC tissue by immunohistochemistry, flow cytometry, ELISA, and qRT-PCR and their involvement in impairing the activation of effector T cells. In CRC biopsies, we evidenced a high percentage of macrophages with low expression of the antigen-presenting complex MHC-II and high expression of CD206. Monocytes co-cultured with tumor cells or a decellularized tumor matrix differentiated toward a pro-tumoral macrophage phenotype characterized by decreased expression of MHC-II and CD86 and increased expression of CD206 and an abundant release of pro-tumoral cytokines and chemokines. We demonstrated that the hampered expression of MHC-II in macrophages is due to the downregulation of the MHC-II transactivator CIITA and that this effect relies on increased expression of miRNAs targeting CIITA. As a result, macrophages become unable to present antigens to CD4 T lymphocytes. Our data suggest that the tumor microenvironment contributes to defining a pro-tumoral profile of macrophages infiltrating CRC tissue with impaired capacity to activate T cell effector functions.

Intrinsic and Extrinsic Modulators of the Epithelial to Mesenchymal Transition: Driving the Fate of Tumor Microenvironment.

The epithelial to mesenchymal transition (EMT) is an evolutionarily conserved process. In cancer, EMT can activate biochemical changes in tumor cells that enable the destruction of the cellular polarity, leading to the acquisition of invasive capabilities. EMT regulation can be triggered by intrinsic and extrinsic signaling, allowing the tumor to adapt to the microenvironment demand in the different stages of tumor progression. In concomitance, tumor cells undergoing EMT actively interact with the surrounding tumor microenvironment (TME) constituted by cell components and extracellular matrix as well as cell secretome elements. As a result, the TME is in turn modulated by the EMT process toward an aggressive behavior. The current review presents the intrinsic and extrinsic modulators of EMT and their relationship with the TME, focusing on the non-cell-derived components, such as secreted metabolites, extracellular matrix, as well as extracellular vesicles. Moreover, we explore how these modulators can be suitable targets for anticancer therapy and personalized medicine.

Recellularized Colorectal Cancer Patient-derived Scaffolds as in vitro Pre-clinical 3D Model for Drug Screening.

Colorectal cancer (CRC) shows highly ineffective therapeutic management. An urgent unmet need is the random assignment to adjuvant chemotherapy of high-risk stage II and stage III CRC patients without any predictive factor of efficacy. In the field of drug discovery, a critical step is the preclinical evaluation of drug cytotoxicity, efficacy, and efficiency. We proposed a patient-derived 3D preclinical model for drug evaluation that could mimic in vitro the patient's disease. Surgically resected CRC tissue and adjacent healthy colon mucosa were decellularized by a detergent-enzymatic treatment. Scaffolds were recellularized with HT29 and HCT116 cells. Qualitative and quantitative characterization of matched recellularized samples were evaluated through histology, immunofluorescences, scanning electron microscopy, and DNA amount quantification. A chemosensitivity test was performed using an increasing concentration of 5-fluorouracil (5FU). In vivo studies were carried out using zebrafish (Danio rerio) animal model. Permeability test and drug absorption were also determined. The decellularization protocol allowed the preservation of the original structure and ultrastructure. Five days after recellularization with HT29 and HCT116 cell lines, the 3D CRC model exhibited reduced sensitivity to 5FU treatments compared with conventional 2D cultures. Calculated the half maximal inhibitory concentration (IC50) for HT29 treated with 5FU resulted in 11.5 µM in 3D and 1.3 µM in 2D, and for HCT116, 9.87 µM in 3D and 1.7 µM in 2D. In xenograft experiments, HT29 extravasation was detected after 4 days post-injection, and we obtained a 5FU IC50 fully comparable to that observed in the 3D CRC model. Using confocal microscopy, we demonstrated that the drug diffused through the repopulated 3D CRC scaffolds and co-localized with the cell nuclei. The bioengineered CRC 3D model could be a reliable preclinical patient-specific platform to bridge the gap between in vitro and in vivo drug testing assays and provide effective cancer treatment.

Patient-Derived Scaffolds of Colorectal Cancer Metastases as an Organotypic 3D Model of the Liver Metastatic Microenvironment.

The liver is the most common site for colorectal cancer (CRC) metastasis and there is an urgent need for new tissue culture models to study colorectal cancer liver metastasis (CRLM) as current models do not mimic the biological, biochemical, and structural characteristics of the metastatic microenvironment. Decellularization provides a novel approach for the study of the cancer extracellular matrix (ECM) as decellularized scaffolds retain tissue-specific features and biological properties. In the present study, we created a 3D model of CRC and matched CRLM using patient-derived decellularized ECM scaffolds seeded with the HT-29 CRC cell line. Here, we show an increased HT-29 cell proliferation and migration capability when cultured in cancer-derived scaffolds compared to same-patient healthy colon and liver tissues. HT-29 cells cultured in CRLM scaffolds also displayed an indication of epithelial-mesenchymal transition (EMT), with a loss of E-cadherin and increased Vimentin expression. EMT was confirmed by gene expression profiling, with the most represented biological processes in CRLM-seeded scaffolds involving demethylation, deacetylation, a cellular response to stress metabolic processes, and a response to the oxygen level and starvation. HT-29 cells cultured in cancer-specific 3D microenvironments showed a reduced response to treatment with 5-fluorouracil and 5-fluorouracil combined with Irinotecan when used at a standard IC50 (as determined in the 2D culture). Our 3D culture system with patient-derived tissue-specific decellularized ECM better recapitulates the metastatic microenvironment compared to conventional 2D culture conditions and represents a relevant approach for the study of CRLM progression and assessing the response to chemotherapy agents.

Crude extract of Origanum vulgare L. induced cell death and suppressed MAPK and PI3/Akt signaling pathways in SW13 and H295R cell lines.

Oregano (Origanum vulgare L.) is a common aromatic plant used in Mediterranean and Asian Regions for treating respiratory diseases, painful menstruation, rheumatoid arthritis, etc. Recently its role as an anticancer plant has been suggested, although oregano has been never evaluated into adrenocortical tumour cell models. This study analysed for the first time the anticancer effects of a crude extract of wild mountain oregano (Origanum vulgare L.) in SW13 and H295R cell lines. The crude extract was characterised by GC/MS and the toxic effects of oregano were first analysed by brine shrimp lethality assay. Our findings demonstrated that oregano decreased cell viability, survival, modified cell cycle and induced cell death (through necrotic process) and that the effects can be attributed to a blockade of MAPK and PI3 K/Akt pathways. These results suggest that oregano extract exerts anticancer activities in adrenocortical tumour cell lines, providing evidence for further research in higher models.

EF24 (a Curcumin Analog) and ZSTK474 Emphasize the Effect of Cabozantinib in Medullary Thyroid Cancer.

XL184 is a small-molecule kinase inhibitor recently included in first-line systemic therapy for patients with advanced, progressive medullary thyroid cancer (MTC). EF24 is a curcumin analog with a high bioavailability, and ZSTK474 is an inhibitor of the phosphatidylinositol 3-kinase signaling pathway. We investigated the effect of these compounds, alone and in combination, in two rearranged during transfection (RET)-mutated TT and MZ-CRC-1 MTC cell lines and in six mostly RET wild-type human MTC primary cultures. Low IC50 values demonstrated the efficacy of the drugs, whereas the combination index revealed an important synergistic effect of combinations of XL184 + ZSTK474 and XL184 + EF24. Cell-cycle changes and the induction of apoptosis or necrosis were modulated by single compounds or combinations thereof. Both XL184 and EF24, alone or combined, were effective in reducing calcitonin secretion. Western blot and in-cell Western analysis showed that the compounds prompted a decrease in general reactivity to phosphorylated antibodies. Our data confirm XL184 alone as the reference drug for RET-mutated MTC, but we also demonstrated that EF24 alone is effective in inhibiting MTC cell viability. We tested the combinations XL184 + ZSTK474 and XL184 + EF24 too, finding that they act synergistically, irrespective of RET mutation status.

miR-194 as predictive biomarker of responsiveness to neoadjuvant chemoradiotherapy in patients with locally advanced rectal adenocarcinoma.

AIMS: Curative surgery remains the primary form of treatment for locally advanced rectal cancer (LARC). Recent data support the use of preoperative chemoradiotherapy (pCRT) to improve the prognosis of LARC with a significant reduction of local relapse and an increase of overall survival. Unfortunately, only 20% of the patients with LARC present complete pathological response after pCRT, whereas in 20%-40%, the response is poor or absent. METHODS: We investigated the expression level of miR-194 in n=38 patients with LARC using our public microRNA (miRNA) expression dataset. miR-194 expression was further validated by real-time quantitative PCR (qRT-PCR) and in situ hybridisation (ISH). Protein-protein interaction network and pathway enrichment analysis were performed on miR-194 targets. RESULTS AND DISCUSSION: Using biopsy samples collected at diagnosis, mir-194 was significantly upregulated in patients responding to treatment (p value=0.016). The data was confirmed with qRT-PCR (p value=0.0587) and ISH (p value=0.026). Protein-protein interaction network and pathway enrichment analysis reveal a possible mechanism of susceptibility to pCRT involving Wnt pathway via its downstream mediator TRAF6. Finally, we interrogated the Comparative Toxicogenomics Database database in order to identify those chemical compounds able to mimic the biological effects of miR-194 as new possible therapeutic option in LARC treatment. The present study combining miRNA expression profiling with integrative computational biology identified miR-194 as predictive biomarker of response to pCRT. Using known and predicted drug mechanism of action, we then identified possible chemical compounds for further in vitro validation.

Combination training in aging individuals modifies functional connectivity and cognition, and is potentially affected by dopamine-related genes.

BACKGROUND: Aging is a major co-risk factor in many neurodegenerative diseases. Cognitive enrichment positively affects the structural plasticity of the aging brain. In this study, we evaluated effects of a set of structured multimodal activities (Combination Training; CT) on cognitive performances, functional connectivity, and cortical thickness of a group of healthy elderly individuals. CT lasted six months. METHODOLOGY: Neuropsychological and occupational performances were evaluated before and at the end of the training period. fMRI was used to assess effects of training on resting state network (RSN) functional connectivity using Independent Component Analysis (ICA). Effects on cortical thickness were also studied. Finally, we evaluated whether specific dopamine-related genes can affect the response to training. PRINCIPAL FINDINGS: Results of the study indicate that CT improves cognitive/occupational performances and reorganizes functional connectivity. Intriguingly, individuals responding to CT showed specific dopamine-related genotypes. Indeed, analysis of dopamine-related genes revealed that carriers of DRD3 ser9gly and COMT Val158Met polymorphisms had the greatest benefits from exposure to CT. CONCLUSIONS AND SIGNIFICANCE: Overall, our findings support the idea that exposure to a set of structured multimodal activities can be an effective strategy to counteract aging-related cognitive decline and also indicate that significant capability of functional and structural changes are maintained in the elderly.