Arming Mesenchymal Stromal/Stem Cells Against Cancer: Has the Time Come?

Since mesenchymal stromal/stem cells (MSCs) were discovered, researchers have been drawn to study their peculiar biological features, including their immune privileged status and their capacity to selectively migrate into inflammatory areas, including tumors. These properties make MSCs promising cellular vehicles for the delivery of therapeutic molecules in the clinical setting. In recent decades, the engineering of MSCs into biological vehicles carrying anticancer compounds has been achieved in different ways, including the loadingof MSCs with chemotherapeutics or drug functionalized nanoparticles (NPs), genetic modifications to force the production of anticancer proteins, and the use of oncolytic viruses. Recently, it has been demonstrated that wild-type and engineered MSCs can release extracellular vesicles (EVs) that contain therapeutic agents. Despite the enthusiasm for MSCs as cyto-pharmaceutical agents, many challenges, including controlling the fate of MSCs after administration, must still be considered. Preclinical results demonstrated that MSCs accumulate in lung, liver, and spleen, which could prevent their engraftment into tumor sites. For this reason, physical, physiological, and biological methods have been implemented to increase MSC concentration in the target tumors. Currently, there are more than 900 registered clinical trials using MSCs. Only a small fraction of these are investigating MSC-based therapies for cancer, but the number of these clinical trials is expected to increase as technology and our understanding of MSCs improve. This review will summarize MSC-based antitumor therapies to generate an increasing awareness of their potential and limits to accelerate their clinical translation

TRAIL receptors are expressed in both malignant and stromal cells in pancreatic ductal adenocarcinoma

: This study assesses the expression of all TNF-related apoptosis-inducing ligand (TRAIL) receptors in pancreatic ductal adenocarcinoma (PDAC) tumor tissue. We aimed to include TRAIL receptor expression as an inclusion parameter in a future clinical study using a TRAIL-based therapy approach for PDAC patients. Considering the emerging influence of PDAC desmoplastic stroma on the efficacy of anti-PDAC therapies, this analysis was extended to tumor stromal cells. Additionally, we performed PDAC stroma characterization. Our retrospective cohort study (N=50) included patients with histologically confirmed PDAC who underwent surgery. The expression of TRAIL receptors (DR4, DR5, DcR1, DcR2, and OPG) in tumor and stromal cells was evaluated by immunohistochemistry (IHC). The amount of tumor stroma was assessed by anti-vimentin IHC and Mallory's trichrome staining. The prognostic impact was determined by the univariate Cox proportional hazards regression model. An extensive expression of functional receptors DR4 and DR5 and a variable expression of decoy receptors were detected in PDAC tumor and stromal cells. Functional receptors were detected also in metastatic tumor and stromal cells. A poor prognosis was associated with low or absent expression of decoy receptors in tumor cells of primary PDAC. After assessing that almost 80% of tumor mass was composed of stroma, we correlated a cellular-dense stroma in primary PDAC with reduced relapse-free survival. We demonstrated that TRAIL functional receptors are widely expressed in PDAC, representing a promising target for TRAIL-based therapies. Further, we demonstrated that a low expression of DcR1 and the absence of OPG in tumor cells, as well as a cellular-dense tumor stroma, could negatively impact the prognosis of PDAC patients

Impact of soluble tumor necrosis factor-related apoptosis-inducing ligand released by engineered adipose mesenchymal stromal cells on white blood cells

Background aims: The proapoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is physiologically expressed by immune cells and performs regulatory functions in infections, autoimmune diseases and cancer, where it acts as a tumor suppressor. Adipose-derived mesenchymal stromal cells (AD-MSCs) also may play immunomodulatory roles in both primary and acquired immune responses. We have previously demonstrated the efficacy of an anticancer gene therapy based on AD-MSC engineered to secrete a soluble TRAIL variant (sTRAIL) against pancreatic cancer. However, the impact of AD-MSC sTRAIL on leukocyte subsets has been not yet considered also to predict a possible immunotoxicity profile in the clinical translation of this cell-based anticancer strategy. Methods: Monocytes, polymorphonuclear cells and T lymphocytes were freshly isolated from the peripheral blood of healthy donors. Immunophenotype and functional (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors were tested by flow cytometry. The viability of white blood cells treated with sTRAIL released by gene-modified AD-MSC or co-cultured with AD-MSC sTRAIL was then evaluated by both metabolic assays and flow cytometry. In addition, cytokine profile in co-cultures was analyzed by multiplex enzyme-linked immunosorbent assay. Results: Monocytes and polymorphonuclear cells showed high positivity for DR5 and DcR2, respectively, whereas T cells revealed negligible expression of all TRAIL receptors. Irrespective of TRAIL receptors' presence on the cell membrane, white blood cells were refractory to the proapoptotic effect displayed by sTRAIL secreted by gene-modified AD-MSC, and direct cell-to-cell contact with AD-MSC sTRAIL had negligible impact on T-cell and monocyte viability. Cytokine crosstalk involving interleukin 10, tumor necrosis factor alpha, and interferon gamma secreted by T lymphocytes and vascular endothelial growth factor A and interleukin 6 released by AD-MSC was highlighted in T-cell and AD-MSC sTRAIL co-cultures. Conclusions: In summary, this study demonstrates the immunological safety and thus the clinical feasibility of an anticancer approach based on AD-MSC expressing the proapoptotic molecule sTRAIL.Background aims: The proapoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is physiologically expressed by immune cells and performs regulatory functions in infections, autoimmune diseases and cancer, where it acts as a tumor suppressor. Adipose-derived mesenchymal stromal cells (AD-MSCs) also may play immunomodulatory roles in both primary and acquired immune responses. We have previously demonstrated the efficacy of an anticancer gene therapy based on AD-MSC engineered to secrete a soluble TRAIL variant (sTRAIL) against pancreatic cancer. However, the impact of AD-MSC sTRAIL on leukocyte subsets has been not yet considered also to predict a possible immunotoxicity profile in the clinical translation of this cell-based anticancer strategy. Methods: Monocytes, polymorphonuclear cells and T lymphocytes were freshly isolated from the peripheral blood of healthy donors. Immunophenotype and functional (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors were tested by flow cytometry. The viability of white blood cells treated with sTRAIL released by gene-modified AD-MSC or co-cultured with AD-MSC sTRAIL was then evaluated by both metabolic assays and flow cytometry. In addition, cytokine profile in co-cultures was analyzed by multiplex enzyme-linked immunosorbent assay.Results: Monocytes and polymorphonuclear cells showed high positivity for DR5 and DcR2, respectively, whereas T cells revealed negligible expression of all TRAIL receptors. Irrespective of TRAIL receptors' pres-ence on the cell membrane, white blood cells were refractory to the proapoptotic effect displayed by sTRAIL secreted by gene-modified AD-MSC, and direct cell-to-cell contact with AD-MSC sTRAIL had negligible impact on T-cell and monocyte viability. Cytokine crosstalk involving interleukin 10, tumor necrosis factor alpha, and interferon gamma secreted by T lymphocytes and vascular endothelial growth factor A and inter-leukin 6 released by AD-MSC was highlighted in T-cell and AD-MSC sTRAIL co-cultures. Conclusions: In summary, this study demonstrates the immunological safety and thus the clinical feasibility of an anticancer approach based on AD-MSC expressing the proapoptotic molecule sTRAIL.(c) 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/

MRI of placenta accreta: diagnostic accuracy and impact of interventional radiology on foetal-maternal delivery outcomes in high-risk women

To assess accuracy and reproducibility of MRI diagnosis of invasive placentation (IP) in high-risk patients and to evaluate reliability of MRI features. Secondary aim was to evaluate impact of interventional radiology (IR) on delivery outcomes in patients with IP at MRI

Giosuè Carducci prosatore

Questo volume su Giosuè Carducci prosatore raccoglie i contributi presentati al XVII Convegno internazionale di Letteratura italiana "Gennaro Barbarisi", tenutosi a Palazzo Feltrinelli (Gargnano del Garda) dal 29 settembre al 1° ottobre 2016. Si è trattato di una proficua occasione di incontro, di studio e di approfondimento su un tema forse poco frequentato, soprattutto in tempi recenti, ma ricco di sollecitazioni per una più articolata e storicamente fondata definizione della personalità di un autore così significativo nel panorama della cultura italiana fra Otto e primo Novecento; non soltanto sul versante della poesia (un primato sancito dal premio Nobel nel 1906) ma anche, e forse ancora di più, su quello della prosa saggistica, degli scritti di polemica, delle curatele editoriali, delle ricerche erudite, fino alle prove di alta oratoria e all'epistolografia

Correction to: Solving patients with rare diseases through programmatic reanalysis of genome-phenome data

In the original publication of the article, consortium author lists were missing in the articl

Solving patients with rare diseases through programmatic reanalysis of genome-phenome data.

Funder: EC | EC Seventh Framework Programm | FP7 Health (FP7-HEALTH - Specific Programme "Cooperation": Health); doi: https://doi.org/10.13039/100011272; Grant(s): 305444, 305444Funder: Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness); doi: https://doi.org/10.13039/501100003329Funder: Generalitat de Catalunya (Government of Catalonia); doi: https://doi.org/10.13039/501100002809Funder: EC | European Regional Development Fund (Europski Fond za Regionalni Razvoj); doi: https://doi.org/10.13039/501100008530Funder: Instituto Nacional de Bioinformática ELIXIR Implementation Studies Centro de Excelencia Severo OchoaFunder: EC | EC Seventh Framework Programm | FP7 Health (FP7-HEALTH - Specific Programme "Cooperation": Health)Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP's Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics

Efficacia e Sicurezza di Modelli Pre-clinici Combinatori di Chemioterapia e Terapia Genica per Bersagliare il Tumore del Pancreas ed il suo Microambiente

Introduzione. Il tumore del pancreas (PC), ed in particolare l’adenocarcinoma duttale, mostra ancora una scarsa risposta alle terapie disponibili. Un elemento peculiare è l’abbondante stroma fibrotico, composto da fibroblasti associati al tumore (TAF), cellule immunitarie, citochine e fattori di crescita immersi nella matrice extracellulare (ECM), con un ruolo chiave nella progressione e farmacoresistenza del PC (Korc et al., 2007). Sforzi significativi si sono così focalizzati sullo sviluppo di terapie capaci di bersagliare sia cellule tumorali che TAF (Sun et al., 2018). Da anni, stiamo sviluppando una terapia genica antitumorale a base di cellule stromali/staminali mesenchimali da tessuto adiposo (AD-MSC) secernenti la molecola pro-apoptotica solubile TRAIL (sTRAIL) per bersagliare cellule tumorali di PC (Spano et al., 2019; Rossignoli et al., 2019). Qui, abbiamo investigato in vitro ed in vivo l’impatto sinergico di un approccio combinatorio tra il chemioterapico Gemcitabina (Gem) e le AD-MSC sTRAIL sia su cellule tumorali che su TAF di PC. Inoltre, per verificare la sicurezza di tale terapia, abbiamo valutato l’impatto citotossico delle AD-MSC sTRAIL su tessuti sani ed in particolare sui leucociti. Materiali e metodi. Saggi in vitro su linee di PC: la sensibilità di linee cellulari di PC (BxPC-3, MIA PaCa-2 e PA-TU-8988T) a sTRAIL, da solo o in combinazione con Gem, è stata valutata con CellTiter Glo in 2D. Un microambiente di PC in vivo-like è stato riprodotto caricando linee di PC in un bioreattore 3D e la mortalità cellulare è stata quantificata dopo trattamento con Gem & AD-MSC sTRAIL. Modelli animali in vivo: modelli murini ortotopici di PC sono stati sviluppati mediante impianto di linee di PC nel pancreas di topi NOD/SCID. L’effetto sinergico dell’approccio combinatorio sulla crescita tumorale è stato verificato mediante ecografia. Sezioni di tumore in paraffina sono state valutate con H&E e IHC per citocheratina 7 e 8/18. Citotossicità su TAF di PC: TAF primari sono stati isolati e testati per la sensibilità a sTRAIL, da solo o in combinazione con Gem, tramite CellTiter Glo. RNA-Seq sul microambiente di PC: i TAF sono stati co-coltivati con una linea di PC, sortati mediante FACS e analizzati con RNA-Seq. Immunotossicità: vitalità e proliferazione di cellule immunitarie (monociti, neutrofili e linfociti T) da sangue periferico di donatori sani trattate con sTRAIL o co-coltivate con AD-MSC sTRAIL sono state valutate con saggi metabolici e FACS. Risultati. Le linee cellulari di PC mostrano differenti livelli di sensibilità a Gem o sTRAIL da soli e la combinazione Gem & sTRAIL ha rivelato un effetto sinergico sulle cellule di PC in 2D ed in 3D. La sinergia tra Gem e AD-MSC sTRAIL è stata confermata nei modelli murini ortotopici di PC con una significativa degenerazione del tessuto tumorale rispetto al controllo e alla sola Gem. I TAF primari di PC sono resistenti all’effetto citotossico di sTRAIL da solo, mentre il pre-trattamento con Gem ne ripristina la sensibilità. Studiando l’interazione stroma-tumore, l’RNA-Seq ha rivelato un aumento significativo dell’espressione di geni correlati all’ECM, come MMP2, POSTN, COL1A1 e COL12A1, nel tumore dopo co-coltura con i TAF. Infine, studi sulla sicurezza immunologica hanno confermato che i leucociti sono refrattari all’effetto di sTRAIL e il contatto diretto con le AD-MSC sTRAIL ha un impatto trascurabile sulla vitalità di monociti e cellule T. Allo stesso modo, la proliferazione delle cellule T non è influenzata sia da sTRAIL che dalle AD-MSC sTRAIL. Conclusioni. Questi dati hanno dimostrato la sicurezza ed il potenziale terapeutico di combinare terapia genica e chemioterapia per bersagliare sia la componente tumorale che stromale dell’adenocarcinoma pancreatico duttale.Introduction. Pancreatic cancer (PC), and in particular the ductal adenocarcinoma, has still a poor response to available therapies. A hallmark of this malignancy is the pronounced fibrotic stroma, composed by tumor-associated fibroblasts (TAF), immune cells, cytokines and growth factors stored in the extracellular matrix (ECM), with a key role in PC progression and drug resistance (Korc et al., 2007). For these reasons, efforts have been focused on the development of novel therapies able to target both malignant cells and TAF (Sun et al., 2018). Since years, we have developed an anticancer gene therapy based on human adipose mesenchymal stromal/stem cells (AD-MSC) secreting the pro-apoptotic soluble (s)-TRAIL to mostly target PC malignant cells (Spano et al., 2019; Rossignoli et al., 2019). Here, by in vitro and in vivo models, we investigated the potential synergistic impact of a novel combinatory approach between chemotherapeutic Gemcitabine (Gem) and AD-MSC sTRAIL on both PC tumor and TAF. Moreover, to initially verify the safety of this strategy, we assessed the impact of AD-MSC sTRAIL on normal tissues and in particular against white blood cells. Material and methods. In vitro assay on PC cell lines: sensitivity of PC cell lines (BxPC-3, MIA PaCa-2 and PA-TU-8988T) to sTRAIL, alone or in combination with Gem, was evaluated by CellTiter Glo assay in 2D. PC in vivo-like microenvironment was reproduced loading PC cell lines in 3D bioreactor and cell death was quantified after Gem & AD-MSC sTRAIL combinatory treatment. In vivo animal model: PC orthotopic xenotransplant murine models were developed by implantation of PC cell lines in the pancreas of NOD/SCID mice. Synergistic effect of the combinatory approach on tumor growth was verified by Ultrasound imaging. Paraffin embedded tumor sections were evaluated by H&E staining and Cytokeratin 7 and 8/18 IHC. Cytotoxicity study on PC TAF: primary TAF isolated by mechanical and enzymatic digestion were tested for sensitivity to sTRAIL, alone or in combination with Gem, by CellTiter Glo. RNA-Seq on PC microenvironment: TAF were co-cultured with a PC cell line, subsequently sorted by FACS and analysed by RNA-Seq. Immunotoxicity study: viability and proliferative rate of immune cells (monocytes, neutrophils and T lymphocytes) isolated from peripheral blood of healthy donors and treated with sTRAIL or co-cultured with AD-MSC sTRAIL were evaluated by metabolic assays and FACS. Results. PC cell lines showed different sensitivity levels to Gem or sTRAIL alone. Gem & sTRAIL combinatory approach revealed a synergistic cytotoxic effect on PC cells both in 2D and 3D systems. Synergy between Gem and AD-MSC sTRAIL was confirmed in the orthotopic PC murine models, showing a significant tumor tissue degeneration compared to control and Gem alone. Primary PC TAF were refractory to the sTRAIL cytotoxic effect as single agent, however pre-treatment with Gem was able to restore sTRAIL sensitivity. Investigating tumor-stroma crosstalk, gene expression profiling revealed a significant increase in expression of genes related to ECM, such as MMP2, POSTN, COL1A1 and COL12A1, in PC tumor cells after co-culture with TAF. Finally, immunological safety study confirmed that white blood cells were refractory to the pro-apoptotic effect displayed by sTRAIL and the direct cell-to-cell contact with AD-MSC sTRAIL had negligible impact on monocyte and T cell viability. Similarly, proliferation capability of T cells was not significantly affected by sTRAIL alone, as well as by co-culture with AD-MSC sTRAIL. Conclusions. These data demonstrated the safety and therapeutic potential of combining a gene therapy approach with chemotherapy to target both the tumor and stromal compartment in pancreatic ductal adenocarcinoma