Exploring the Immunomodulatory Potential of Pancreatic Cancer-Derived Extracellular Vesicles through Proteomic and Functional Analyses.

Pancreatic cancer (PC) has a poor prognosis and displays resistance to immunotherapy. A better understanding of tumor-derived extracellular vesicle (EV) effects on immune responses might contribute to improved immunotherapy. EVs derived from Capan-2 and BxPC-3 PC cells isolated by ultracentrifugation were characterized by atomic force microscopy, Western blot (WB), nanoparticle tracking analysis, and label-free proteomics. Fresh PBMCs from healthy donors were treated with PC- or control-derived heterologous EVs, followed by flow cytometry analysis of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated or untreated PBMCs was performed, and the IFN-γ concentration was measured by ELISA. Notably, most of the proteins identified in Capan-2 and BxPC-3 EVs by the proteomic analysis were connected in a single functional network (p = 1 × 10-16) and were involved in the "Immune System" (FDR: 1.10 × 10-24 and 3.69 × 10-19, respectively). Interestingly, the treatment of healthy donor-derived PBMCs with Capan-2 EVs but not with BxPC-3 EVs or heterologous control EVs induced early activation of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated PBMCs was consistent with their activation by Capan-2 EVs, indicating IFN-γ among the major upstream regulators, as confirmed by ELISA. The proteomic and functional analyses indicate that PC-EVs have pleiotropic effects, and some may activate early immune responses, which might be relevant for the development of highly needed immunotherapeutic strategies in this immune-cold tumor.

Prognostic value of gender and primary tumor location in metastatic colon cancer.

Sex might influence prognosis in patients affected by colorectal cancer. We retrospectively studied a cohort of patients affected by metastatic colon cancer (mCC) stratified by sex and primary tumor location. RAS mutational status was also included in the analysis. Overall, 616 patients met the eligibility criteria, 261 women and 355 men. Neither gender, nor RAS mutational status influenced overall survival (OS) in the entire population. As expected, patients with right-sided colon cancer (RCC) had a significant shorter OS compared to those with left-sided colon cancer (LCC) (21.3 vs 33.1 months, p= 0.002). When the analysis was performed stratifying for gender, RCC retained worse prognosis among men (OS 20.5 vs 33.9 months, p= 0.008), but not among women (p= 0.132). Similarly, the presence of RAS mutations had no prognostic effect in women, but was significantly associate with shorter survival in men (OS 29.5 vs 33.7 months, p= 0.046). In addition, when comparing clinical outcome of women or men according to sidedness and RAS mutational status, RCC was associated with dismal prognosis only in men with RAS mutated tumor (OS 17.2 vs 32.3 months, p= 0.008). Our study highlights the importance of gender in the outcome of patients with mCC.

Anti-Inflammatory, Antioxidant, and WAT/BAT-Conversion Stimulation Induced by Novel PPAR Ligands: Results from Ex Vivo and In Vitro Studies.

Activation of peroxisome proliferator-activated receptors (PPARs) not only regulates multiple metabolic pathways, but mediates various biological effects related to inflammation and oxidative stress. We investigated the effects of four new PPAR ligands containing a fibrate scaffold-the PPAR agonists (1a (αEC50 1.0 µM) and 1b (γEC50 0.012 µM)) and antagonists (2a (αIC50 6.5 µM) and 2b (αIC50 0.98 µM, with a weak antagonist activity on γ isoform))-on proinflammatory and oxidative stress biomarkers. The PPAR ligands 1a-b and 2a-b (0.1-10 µM) were tested on isolated liver specimens treated with lipopolysaccharide (LPS), and the levels of lactate dehydrogenase (LDH), prostaglandin (PG) E2, and 8-iso-PGF2α were measured. The effects of these compounds on the gene expression of the adipose tissue markers of browning, PPARα, and PPARγ, in white adipocytes, were evaluated as well. We found a significant reduction in LPS-induced LDH, PGE2, and 8-iso-PGF2α levels after 1a treatment. On the other hand, 1b decreased LPS-induced LDH activity. Compared to the control, 1a stimulated uncoupling protein 1 (UCP1), PR-(PRD1-BF1-RIZ1 homologous) domain containing 16 (PRDM16), deiodinase type II (DIO2), and PPARα and PPARγ gene expression, in 3T3-L1 cells. Similarly, 1b increased UCP1, DIO2, and PPARγ gene expression. 2a-b caused a reduction in the gene expression of UCP1, PRDM16, and DIO2 when tested at 10 µM. In addition, 2a-b significantly decreased PPARα gene expression. A significant reduction in PPARγ gene expression was also found after 2b treatment. The novel PPARα agonist 1a might be a promising lead compound and represents a valuable pharmacological tool for further assessment. The PPARγ agonist 1b could play a minor role in the regulation of inflammatory pathways.

CAR-T-Derived Extracellular Vesicles: A Promising Development of CAR-T Anti-Tumor Therapy.

Extracellular vesicles (EVs) are a heterogenous population of plasma membrane-surrounded particles that are released in the extracellular milieu by almost all types of living cells. EVs are key players in intercellular crosstalk, both locally and systemically, given that they deliver their cargoes (consisting of proteins, lipids, mRNAs, miRNAs, and DNA fragments) to target cells, crossing biological barriers. Those mechanisms further trigger a wide range of biological responses. Interestingly, EV phenotypes and cargoes and, therefore, their functions, stem from their specific parental cells. For these reasons, EVs have been proposed as promising candidates for EV-based, cell-free therapies. One of the new frontiers of cell-based immunotherapy for the fight against refractory neoplastic diseases is represented by genetically engineered chimeric antigen receptor T (CAR-T) lymphocytes, which in recent years have demonstrated their effectiveness by reaching commercialization and clinical application for some neoplastic diseases. CAR-T-derived EVs represent a recent promising development of CAR-T immunotherapy approaches. This crosscutting innovative strategy is designed to exploit the advantages of genetically engineered cell-based immunotherapy together with those of cell-free EVs, which in principle might be safer and more efficient in crossing biological and tumor-associated barriers. In this review, we underlined the potential of CAR-T-derived EVs as therapeutic agents in tumors.

Resveratrol Derivative Exhibits Marked Antiproliferative Actions, Affecting Stemness in Pancreatic Cancer Cells.

Pancreatic cancer (PC) is one of the deadliest malignancies, with an increasing incidence and limited response to current therapeutic options. Therefore, more effective and low-toxic agents are needed to improve PC patients' outcomes. Resveratrol (RSV) is a natural polyphenol with multiple biological properties, including anticancer effects. In this study, we explored the antiproliferative activities of newly synthetized RSV analogues in a panel of PC cell lines and evaluated the physicochemical properties of the most active compound. This derivative exhibited marked antiproliferative effects in PC cells through mechanisms involving DNA damage, apoptosis induction, and interference in cell cycle progression, as assessed using flow cytometry and immunoblot analysis of cell cycle proteins, PARP cleavage, and H2AX phosphorylation. Notably, the compound induced a consistent reduction in the PC cell subpopulation with a CD133+EpCAM+ stem-like phenotype, paralleled by dramatic effects on cell clonogenicity. Moreover, the RSV derivative had negligible toxicity against normal HFF-1 cells and, thus, good selectivity index values toward PC cell lines. Remarkably, its higher lipophilicity and stability in human plasma, as compared to RSV, might ensure a better permeation along the gastrointestinal tract. Our results provide insights into the mechanisms of action contributing to the antiproliferative activity of a synthetic RSV analogue, supporting its potential value in the search for effective and safe agents in PC treatment.

Anti-Inflammatory and Antioxidant Effects Induced by Allium sativum L. Extracts on an Ex Vivo Experimental Model of Ulcerative Colitis.

Inflammatory bowel diseases (IBDs) are chronic and multifactorial inflammatory conditions of the colonic mucosa (ulcerative colitis), characterized by increased and unbalanced immune response to external stimuli. Garlic and its bioactive constituents were reported to exert various biological effects, including anti-inflammatory, antioxidant and immunomodulatory activities. We aimed to evaluate the protective effects of a hydroalcoholic (GHE) and a water (GWE) extract from a Sicilian variety of garlic, known as Nubia red garlic, on an ex vivo experimental model of ulcerative colitis, involving isolated LPS-treated mouse colon specimens. Both extracts were able to counteract LPS-induced cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, nuclear factor-kB (NF-kB), and interleukin (IL)-6 gene expression in mouse colon. Moreover, the same extracts inhibited prostaglandin (PG)E2, 8-iso-PGF2α, and increased the 5-hydroxyindoleacetic acid/serotonin ratio following treatment with LPS. In particular, GHE showed a better anti-inflammatory profile. The anti-inflammatory and antioxidant effects induced by both extracts could be related, at least partially, to their polyphenolic composition, with particular regards to catechin. Concluding, our results showed that GHE and GWE exhibited protective effects in colon, thus suggesting their potential use in the prevention and management of ulcerative colitis.

Optimizing the Choice for Adjuvant Chemotherapy in Gastric Cancer.

Advances in the management of gastric cancer have improved patient survival in the last decade. Nonetheless, the number of patients relapsing and dying after a diagnosis of localized gastric cancer is still too high, even in early stages (10% in stage I). Adjuvant systemic chemotherapy has been proven to significantly improve outcomes. In the present article we have critically reviewed the clinical trials that guide the current clinical practice in the adjuvant treatment of patients affected by resectable gastric cancer, focusing on the different approaches worldwide, i.e., adjuvant chemotherapy, adjuvant chemoradiotherapy, and perioperative chemotherapy. We also delineate the clinical-pathological characteristics that are commonly taken into account to identify patients at a higher risk of recurrence and requiring adjuvant chemotherapy, and also describe novel biomarkers and therapeutic agents that might allow personalization of the treatment.

High Blood Concentration of Leukocyte-Derived Extracellular Vesicles Is Predictive of Favorable Clinical Outcomes in Patients with Pancreatic Cancer: Results from a Multicenter Prospective Study.

Pancreatic cancer (PC) is one of the leading causes of cancer-related death worldwide. Identification of novel tumor biomarkers is highly advocated in PC to optimize personalized treatment algorithms. Blood-circulating extracellular vesicles hold promise for liquid biopsy application in cancer. We used an optimized flow cytometry protocol to study leukocyte-derived EVs (CD45+) and PD-L1+ EVs in blood from 56 pancreatic cancer patients and 48 healthy controls (HCs). Our results show that PC patients presented higher blood levels of total EVs (p = 0.0003), leukocyte-derived EVs (LEVs) (p = 0.001) and PD-L1+ EVs (p = 0.01), as compared with HCs. Interestingly, a blood concentration of LEVs at baseline was independently associated with improved overall survival in patients with borderline resectable or primary unresectable PC (HR = 0.17; 95% CI 0.04-0.79; p = 0.02). Additionally, increased blood-based LEVs were independently correlated with prolonged progression-free survival (HR = 0.10; 95% CI 0.01-0.82; p = 0.03) and significantly associated with higher disease control rate (p = 0.02) in patients with advanced PC receiving standard chemotherapy. Notably, a strong correlation between a decrease in blood LEVs concentration during chemotherapy and disease control was observed (p = 0.005). These intriguing findings point to the potential of LEVs as novel blood-based EV biomarkers for improved personalized medicine in patients affected by PC.

A grape (Vitis vinifera L.) pomace water extract modulates inflammatory and immune response in SW-480 cells and isolated mouse colon.

Grape (Vitis vinifera L.) pomace is a residue derived from the winemaking process, which contains bioactive compounds displaying noteworthy health-promoting properties. The aim of the present study was to investigate the phenolic composition and protective effects of a water extract of grape pomace (WEGP) in colorectal cancer cell line SW480 and in isolated mouse colon exposed to Escherichia coli lipopolysaccharide (LPS). The extract decreased SW-480 cell viability, as well as vascular endothelial factor A (VEGFA), hypoxia-induced factor 1α (HIF1α), and transient receptor potential M8 (TRPM8) LPS-induced gene expression. Moreover, the extract inhibited mRNA levels of nuclear factor kB (NFkB), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)α, interleukin (IL)-6, IL-1ß, IL-10, inducible nitric oxide synthase (iNOS), and interferon (IFN)γ, in isolated colon. Conversely, WEGP increased the gene expression of antioxidant catalase (CAT) and superoxide dismutase (SOD), in the same model. The modulatory effects exerted by WEGP could be related, at least in part, to the phenolic composition, with particular regards to the catechin level. Docking calculations also predicted the interactions of catechin toward TRPM8 receptor, deeply involved in colon cancer; thus further suggesting the grape pomace as a valuable source of bioactive extracts and phytochemicals with protective effects in the colon.

Anesthetics and Long Term Cancer Outcomes: May Epigenetics Be the Key for Pancreatic Cancer?

Knowledge shows a divergence of results between preclinical and clinical studies regarding anesthesia and postoperative progression of cancer. While laboratory and animal data from then 2000s onwards raised much enthusiasm in this field of research leading to several clinical investigations worldwide, data from randomized trials seem to have killed off hope for many scientists. However several aspects of the actual knowledge should be reevaluated and there is space for new strategies of investigation. In this paper, we perform a critical review of actual knowledge and propose new research strategies with a special focus on anesthetic management and repurposed anesthetic adjuvants for pancreatic cancer.

Benzothiazole Derivatives Endowed with Antiproliferative Activity in Paraganglioma and Pancreatic Cancer Cells: Structure-Activity Relationship Studies and Target Prediction Analysis.

The antiproliferative effects played by benzothiazoles in different cancers have aroused the interest for these molecules as promising antitumor agents. In this work, a library of phenylacetamide derivatives containing the benzothiazole nucleus was synthesized and compounds were tested for their antiproliferative activity in paraganglioma and pancreatic cancer cell lines. The novel synthesized compounds induced a marked viability reduction at low micromolar concentrations both in paraganglioma and pancreatic cancer cells. Derivative 4l showed a greater antiproliferative effect and higher selectivity index against cancer cells, as compared to other compounds. Notably, combinations of derivative 4l with gemcitabine at low concentrations induced enhanced and synergistic effects on pancreatic cancer cell viability, thus supporting the relevance of compound 4l in the perspective of clinical translation. A target prediction analysis was also carried out on 4l by using multiple computational tools, identifying cannabinoid receptors and sentrin-specific proteases as putative targets contributing to the observed antiproliferative activity.

Blood Circulating CD133+ Extracellular Vesicles Predict Clinical Outcomes in Patients with Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is one of the most incident and lethal malignancies worldwide. Recent treatment advances prolonged survival in patients with metastatic colorectal cancer (mCRC). However, there are still few biomarkers to guide clinical management and treatment selection in mCRC. In this study, we applied an optimized flow cytometry protocol for EV identification, enumeration, and subtyping in blood samples of 54 patients with mCRC and 48 age and sex-matched healthy controls (HCs). The overall survival (OS) and overall response rate (ORR) were evaluated in mCRC patients enrolled and treated with a first line fluoropyrimidine-based regimen. Our findings show that patients with mCRC presented considerably higher blood concentrations of total EVs, as well as CD133+ and EPCAM+ EVs compared to HCs. Overall survival analysis revealed that increased blood concentrations of total EVs and CD133+ EVs before treatment were significantly associated with shorter OS in mCRC patients (p = 0.001; and p = 0.0001, respectively). In addition, we observed a correlation between high blood levels of CD133+ EVs at baseline and reduced ORR to first-line systemic therapy (p = 0.045). These findings may open exciting perspectives into the application of novel blood-based EV biomarkers for improved risk stratification and optimized treatment strategies in mCRC.

Flow Cytometry Detection of Anthracycline-Treated Breast Cancer Cells: An Optimized Protocol.

The use of anthracycline derivatives was approved for the treatment of a broad spectrum of human tumors (i.e., breast cancer). The need to test these drugs on cancer models has pushed the basic research to apply many types of in vitro assays, and, among them, the study of anthracycline-induced apoptosis was mainly based on the application of flow cytometry protocols. However, the chemical structure of anthracycline derivatives gives them a strong autofluorescence effect that must be considered when flow cytometry is used. Unfortunately, the guidelines on the analysis of anthracycline effects through flow cytometry are lacking. Therefore, in this study, we optimized the flow cytometry detection of doxorubicin and epirubicin-treated breast cancer cells. Their autofluorescence was assessed both by using conventional and imaging flow cytometry; we found that all the channels excited by the 488 nm laser were affected. Anthracycline-induced apoptosis was then measured via flow cytometry using the optimized setting. Consequently, we established a set of recommendations that enable the development of optimized flow cytometry settings when the in vitro assays of anthracycline effects are analyzed, with the final aim to reveal a new perspective on the use of those in vitro tests for the further implementation of precision medicine strategies in cancer.

Protective Effects Induced by a Hydroalcoholic Allium sativum Extract in Isolated Mouse Heart.

The aim of the present study was to investigate the possible protective effects of a garlic hydroalcoholic extract on the burden of oxidative stress and inflammation occurring on mouse heart specimens exposed to E. coli lipopolysaccharide (LPS), which is a well-established inflammatory stimulus. Headspace solid-phase microextraction combined with the gas chromatography-mass spectrometry (HS-SPME/GC-MS) technique was applied to determine the volatile fraction of the garlic powder, and the HS-SPME conditions were optimized for each of the most representative classes of compounds. CIEL*a*b* colorimetric analyses were performed on the powder sample at the time of delivery, after four and after eight months of storage at room temperature in the dark, to evaluate the color changing. Freshly prepared hydroalcoholic extract was also evaluated in its color character. Furthermore, the hydroalcoholic extract was analyzed through GC-MS. The extract was found to be able to significantly inhibit LPS-induced prostaglandin (PG) E2 and 8-iso-PGF2α levels, as well as mRNA levels of cyclooxygenase (COX)-2, interleukin (IL)-6, and nuclear factor-kB (NF-kB), in heart specimens. Concluding, our findings showed that the garlic hydroalcoholic extract exhibited cardioprotective effects on multiple inflammatory and oxidative stress pathways.

Drug Repurposing, an Attractive Strategy in Pancreatic Cancer Treatment: Preclinical and Clinical Updates.

Pancreatic cancer (PC) is one of the deadliest malignancies worldwide, since patients rarely display symptoms until an advanced and unresectable stage of the disease. Current chemotherapy options are unsatisfactory and there is an urgent need for more effective and less toxic drugs to improve the dismal PC therapy. Repurposing of non-oncology drugs in PC treatment represents a very promising therapeutic option and different compounds are currently being considered as candidates for repurposing in the treatment of this tumor. In this review, we provide an update on some of the most promising FDA-approved, non-oncology, repurposed drug candidates that show prominent clinical and preclinical data in pancreatic cancer. We also focus on proposed mechanisms of action and known molecular targets that they modulate in PC. Furthermore, we provide an explorative bioinformatic analysis, which suggests that some of the PC repurposed drug candidates have additional, unexplored, oncology-relevant targets. Finally, we discuss recent developments regarding the immunomodulatory role displayed by some of these drugs, which may expand their potential application in synergy with approved anticancer immunomodulatory agents that are mostly ineffective as single agents in PC.

Effects of growth hormone-releasing hormone receptor antagonist MIA-602 in mice with emotional disorders: a potential treatment for PTSD.

Anxiety and depression have been suggested to increase the risk for post-traumatic stress disorders (PTSD). A link between all these mental illnesses, inflammation and oxidative stress is also well established. Recent behavior studies by our group clearly demonstrate a powerful anxiolytic and antidepressant-like effects of a novel growth hormone releasing hormone (GHRH) antagonist of MIAMI class, MIA-690, probably related to modulatory effects on the inflammatory and oxidative status. In the present work we investigated the potential beneficial effects of MIA-602, another recently developed GHRH antagonist, in mood disorders, as anxiety and depression, and the possible brain pathways involved in its protective activity, in adult mice. MIA-602 exhibited antinflammatory and antioxidant effects in ex vivo and in vivo experimental models, inducing anxiolytic and antidepressant-like behavior in mice subcutaneously treated for 4 weeks. The beneficial effect of MIA-602 on inflammatory and oxidative status and synaptogenesis resulting in anxiolytic and antidepressant-like effects could be related by increases of nuclear factor erythroid 2-related factor 2 (Nrf2) and of brain-derived neurotrophic factor (BDNF) signaling pathways in the hippocampus and prefrontal cortex. These results strongly suggest that GHRH analogs should be tried clinically for the treatment of mood disorders including PTSD.

Exosomes as Pleiotropic Players in Pancreatic Cancer.

Pancreatic cancer (PC) incidence is rising and due to late diagnosis, combined with unsatisfactory response to current therapeutic approaches, this tumor has an extremely high mortality rate. A better understanding of the mechanisms underlying pancreatic carcinogenesis is of paramount importance for rational diagnostic and therapeutic approaches. Multiple lines of evidence have showed that exosomes are actively involved in intercellular communication by transferring their cargos of bioactive molecules to recipient cells within the tumor microenvironment and systemically. Intriguingly, exosomes may exert both protumor and antitumor effects, supporting or hampering processes that play a role in the pathogenesis and progression of PC, including shifts in tumor metabolism, proliferation, invasion, metastasis, and chemoresistance. They also have a dual role in PC immunomodulation, exerting immunosuppressive or immune enhancement effects through several mechanisms. PC-derived exosomes also induce systemic metabolic alterations, leading to the onset of diabetes and weight loss. Moreover, exosomes have been described as promising diagnostic and prognostic biomarkers for PC. Their potential application in PC therapy as drug carriers and therapeutic targets is under investigation. In this review, we provide an overview of the multiple roles played by exosomes in PC biology through their specific cargo biomolecules and of their potential exploitation in early diagnosis and treatment of PC.

Tgf-ß1 transcriptionally promotes 90K expression: possible implications for cancer progression.

The 90K protein, also known as Mac-2 BP or LGALS3BP, can activate the immune response in part by increasing major histocompatibility (MHC) class I levels. In studies on a non-immune cell model, the rat FRTL-5 cell line, we observed that transforming growth factor (TGF)-ß1, like γ-interferon (IFN), increased 90K levels, despite its immunosuppressive functions and the ability to decrease MHC class I. To explain this paradoxical result, we investigated the mechanisms involved in the TGF-ß1 regulation of 90K expression with the aim to demonstrate that TGF-ß1 utilizes different molecular pathways to regulate the two genes. We found that TGF-ß1 was able to increase the binding of Upstream Stimulatory Factors, USF1 and USF2, to an E-box element, CANNTG, at -1926 to -1921 bp, upstream of the interferon response element (IRE) in the 90K promoter. Thyrotropin (TSH) suppressed constitutive and γ-IFN-induced 90K expression by decreasing USF binding to the E-box. TGF-ß1 was able to overcome TSH suppression at the transcriptional level by increasing USF binding to the E-box. We suggest that the ability of TGF-ß1 to increase 90K did not result in an increase in MHC class I because of a separate suppressive action of TGF-ß1 directly on the MHC class I gene. We propose that the increased levels of 90K may play a role, rather than in immune response, in the context of the TGF-ß1-induced changing of the cellular microenvironment that predisposes to cell motility and cancer progression. Consistently, analyzing the publicly available cancer patient data sets cBioPortal, we found that 90K expression directly correlated with TGF-ß1 and USFs and that high levels of 90K were significantly associated with increased mortality in patients affected by different types of cancer.

Screening of Benzimidazole-Based Anthelmintics and Their Enantiomers as Repurposed Drug Candidates in Cancer Therapy.

Repurposing of approved non-antitumor drugs represents a promising and affordable strategy that may help to increase the repertoire of effective anticancer drugs. Benzimidazole-based anthelmintics are antiparasitic drugs commonly employed both in human and veterinary medicine. Benzimidazole compounds are being considered for drug repurposing due to antitumor activities displayed by some members of the family. In this study, we explored the effects of a large series of benzimidazole-based anthelmintics (and some enantiomerically pure forms of those containing a stereogenic center) on the viability of different tumor cell lines derived from paraganglioma, pancreatic and colorectal cancer. Flubendazole, parbendazole, oxibendazole, mebendazole, albendazole and fenbendazole showed the most consistent antiproliferative effects, displaying IC50 values in the low micromolar range, or even in the nanomolar range. In silico evaluation of their physicochemical, pharmacokinetics and medicinal chemistry properties also provided useful information related to the chemical structures and potential of these compounds. Furthermore, in view of the potential repurposing of these drugs in cancer therapy and considering that pharmaceutically active compounds may have different mechanisms of action, we performed an in silico target prediction to assess the polypharmacology of these benzimidazoles, which highlighted previously unknown cancer-relevant molecular targets.