Immunomodulatory activity of argentatins A and B isolated from guayule.

Argentatins are secondary metabolites synthesized by guayule (Parthenium argentatum A. Gray) with numerous potential medical applications. In addition to inhibiting insect growth, they are endowed with several pharmacological properties including antimicrobial and antitumorigenic activity. However, their potential as immunomodulators remains unexplored. The aim of the present study was to investigate whether argentatins can modulate the function of the immune system. Human mesenchymal stem cells were treated with argentatins and the production of several anti- and proinflammatory cytokines was evaluated. The effect of argentatins on the polarization of CD4+ T-lymphocytes and macrophages was also assessed. Results demonstrated that argentatins can modulate the production of proinflammatory cytokines and the polarization of cellular phenotypes, including Th2 lymphocytes and M1 macrophages. These findings suggest that argentatins are promising therapeutic agents in autoimmune or allergic diseases, and open new perspectives for the investigation of argentatins in immune response and in the development of more targeted and effective immunomodulatory therapies.

Novel Blood Biomarkers for Response Prediction and Monitoring of Stereotactic Ablative Radiotherapy and Immunotherapy in Metastatic Oligoprogressive Lung Cancer.

Up to 80% of patients under immune checkpoint inhibitors (ICI) face resistance. In this context, stereotactic ablative radiotherapy (SABR) can induce an immune or abscopal response. However, its molecular determinants remain unknown. We present early results of a translational study assessing biomarkers of response to combined ICI and SABR (I-SABR) in liquid biopsy from oligoprogressive patients in a prospective observational multicenter study. Cohort A includes metastatic patients in oligoprogression to ICI maintaining the same ICI due to clinical benefit and who receive concomitant SABR. B is a comparative group of oligometastatic patients receiving only SABR. Blood samples are extracted at baseline (T1), after the first (T2) and last (T3) fraction, two months post-SABR (T4) and at further progression (TP). Response is evaluated by iRECIST and defined by the objective response rate (ORR)-complete and partial responses. We assess peripheral blood mononuclear cells (PBMCs), circulating cell-free DNA (cfDNA) and small RNA from extracellular vesicles. Twenty-seven patients could be analyzed (cohort A: n = 19; B: n = 8). Most were males with non-small cell lung cancer and one progressing lesion. With a median follow-up of 6 months, the last ORR was 63% (26% complete and 37% partial response). A decrease in cfDNA from T2 to T3 correlated with a good response. At T2, CD8+PD1+ and CD8+PDL1+ cells were increased in non-responders and responders, respectively. At T2, 27 microRNAs were differentially expressed. These are potential biomarkers of response to I-SABR in oligoprogressive disease.

The effect of physical exercise on anticancer immunity.

Regular physical activity is associated with lower cancer incidence and mortality, as well as with a lower rate of tumour recurrence. The epidemiological evidence is supported by preclinical studies in animal models showing that regular exercise delays the progression of cancer, including highly aggressive malignancies. Although the mechanisms underlying the antitumorigenic effects of exercise remain to be defined, an improvement in cancer immunosurveillance is likely important, with different immune cell subtypes stimulated by exercise to infiltrate tumours. There is also evidence that immune cells from blood collected after an exercise bout could be used as adoptive cell therapy for cancer. In this Perspective, we address the importance of muscular activity for maintaining a healthy immune system and discuss the effects of a single bout of exercise (that is, 'acute' exercise) and those of 'regular' exercise (that is, repeated bouts) on anticancer immunity, including tumour infiltrates. We also address the postulated mechanisms and the clinical implications of this emerging area of research.

Fluorometric Quantification of Total Cell-Free DNA as a Prognostic Biomarker in Non-Small-Cell Lung Cancer Patients Treated with Immune Checkpoint Blockade.

The present study aimed to investigate the potential of basal cell-free fluorometric DNA (cfDNA) quantification as a prognostic biomarker in advanced non-small cell lung cancer (NSCLC) patients treated with an Immune Checkpoint Blockade (ICB). A discovery and validation cohort of 61 and 31 advanced lung cancer patients treated with ICB were included in this study. Quantification of cfDNA concentration was performed before the start of the treatment and patients were followed up for a median of 34 (30-40) months. The prognostic predicted value of cfDNA was evaluated based on ROC, and Cox regression was conducted via univariate and multivariate analyses to estimate the hazard ratio. We observed that a cfDNA cut-off of 0.55 ng/µL before the ICB determines the overall survival of patients with a log rank p-value of 3.3 × 10-4. That represents median survivals of 3.8 vs. 17.5 months. Similar results were obtained in the validation cohort being the log rank p-value 3.8 × 10-2 with median survivals of 5.9 vs. 24.3. The univariate and multivariate analysis revealed that the cut-off of 0.55 ng/µL before ICB treatment was an independent predictive factor and was significantly associated with a better survival outcome. High cfDNA concentrations identify patients with advanced NSCLC who do not benefit from the ICB. The determination of cfDNA is a simple test that could select a group of patients in whom new therapeutic strategies are needed.

Association of Circular RNA and Long Non-Coding RNA Dysregulation with the Clinical Response to Immune Checkpoint Blockade in Cutaneous Metastatic Melanoma.

Cutaneous melanoma (CM) is the most lethal form of skin cancer if it becomes metastatic, where treatment options and survival chances decrease dramatically. Immunotherapy treatments based on the immunologic checkpoint inhibitors programmed death cell protein 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) constituted a main breakthrough in the treatment of metastatic CM, particularly for the achievement of long-term benefits. Even though it is a very promising therapy, resistance to primary immune checkpoint blockade (ICB) arises in about 70% of CM patients treated with a CTLA-4 inhibitor, and 40-65% of CM patients administered with a PD-1-targeting treatment. Some long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) are implicated in triggering pro- and anti-tumorigenic responses to various cancer treatments. The relationship between lncRNAs, circRNAs and ICB immunotherapy has not been explored in cutaneous metastatic melanoma (CMM). The aim of this pilot study is to evaluate the potential role of circRNA and lncRNA expression variability as pre-treatment predictor of the clinical response to immunotherapy in CMM patients. RNA-seq from 12 formalin-fixed paraffin-embedded (FFPE) samples from the metastatic biopsies of CMM patients treated with nivolumab was used to identify response-associated transcripts. Our findings indicate that specific lncRNAs and circRNAs, probably acting as competitive endogenous RNAs (ceRNAs), are involved in the regulatory networks of the immune response against metastatic melanoma that these patients have under treatment with nivolumab. Moreover, we established a risk score that yields predictions of the overall survival (OS) and progression-free survival (PFS) of CMM patients with high accuracy. This proof-of-principle work provides a possible insight into the function of ceRNAs, contributing to efforts to decipher the complex molecular mechanisms of ICB cancer treatment response.

Application of low-intensity pulsed therapeutic ultrasound on mesenchymal precursors does not affect their cell properties.

Ultrasound is considered a safe and non-invasive tool in regenerative medicine and has been used in the clinic for more than twenty years for applications in bone healing after the approval of the Exogen device, also known as low-intensity pulsed ultrasound (LIPUS). Beyond its effects on bone health, LIPUS has also been investigated for wound healing of soft tissues, with positive results for various cell processes including cell proliferation, migration and angiogenesis. As LIPUS has the potential to treat chronic skin wounds, we sought to evaluate the effects produced by a conventional therapeutic ultrasound device at low intensities (also considered LIPUS) on the migration capacity of mouse and human skin mesenchymal precursors (s-MPs). Cells were stimulated for 3 days (20 minutes per day) using a traditional ultrasound device with the following parameters: 100 mW/cm2 with 20% duty cycle and frequency of 3 MHz. At the parameters used, ultrasound failed to affect s-MP proliferation, with no evident changes in morphology or cell groupings, and no changes at the cytoskeletal level. Further, the migration and invasion ability of s-MPs were unaffected by the ultrasound protocol, and no major changes were detected in the gene/protein expression of ROCK1, integrin ß1, laminin ß1, type I collagen and transforming growth factor ß1. Finally, RNA-seq analysis revealed that only 10 genes were differentially expressed after ultrasound stimulation. Among them, 5 encode for small nuclear RNAs and 2 encode for proteins belonging to the nuclear pore complex. Considering the results overall, while the viability of s-MPs was not affected by ultrasound stimulation and no changes were detected in proliferation/migration, RNA-seq analysis would suggest that s-MPs do respond to ultrasound. The use of 100 mW/cm2 intensity or conventional therapeutic ultrasound devices might not be optimal for the stimulation the properties of cell populations. Future studies should investigate the potential application of ultrasound using variations of the tested parameters.

Ultrasound Therapy: Experiences and Perspectives for Regenerative Medicine.

Ultrasound has emerged as a novel tool for clinical applications, particularly in the context of regenerative medicine. Due to its unique physico-mechanical properties, low-intensity ultrasound (LIUS) has been approved for accelerated fracture healing and for the treatment of established non-union, but its utility has extended beyond tissue engineering to other fields, including cell regeneration. Cells and tissues respond to acoustic ultrasound by switching on genetic repair circuits, triggering a cascade of molecular signals that promote cell proliferation, adhesion, migration, differentiation, and extracellular matrix production. LIUS also induces angiogenesis and tissue regeneration and has anti-inflammatory and anti-degenerative effects. Accordingly, the potential application of ultrasound for tissue repair/regeneration has been tested in several studies as a stand-alone treatment and, more recently, as an adjunct to cell-based therapies. For example, ultrasound has been proposed to improve stem cell homing to target tissues due to its ability to create a transitional and local gradient of cytokines and chemokines. In this review, we provide an overview of the many applications of ultrasound in clinical medicine, with a focus on its value as an adjunct to cell-based interventions. Finally, we discuss the various preclinical and clinical studies that have investigated the potential of ultrasound for regenerative medicine.

Benefits of exercise and immunotherapy in a murine model of human non-small-cell lung carcinoma.

BACKGROUND: Lung cancer has the highest incidence and mortality rate in the world. One of the most promising new cancer therapies in recent years is immunotherapy, which is based on the blockade of immune checkpoints such as programmed cell death protein 1 (PD-1). Exercise training is beneficial to maintain and improve the quality of life of cancer patients, and it might also modulate the anti-tumoral efficiency of some chemotherapeutic agents. However, the potential of exercise combined with immunotherapy as a cancer therapy remains to be elucidated. Here, we examined the effects of exercise on tumor growth and its possible adjuvant effects when combined with anti-PD-1 immunotherapy (nivolumab) in a patient derived xenograft (PDX) model of non-small-cell lung carcinoma (NSCLC). METHODS: We generated a PDX model using NOD-SCID gamma mice with subcutaneous grafts from tumor tissue of a patient with NSCLC. Animals were randomly assigned to one of four groups: non-exercise + isotype control (n=5), exercise + isotype control (n=5), non-exercise + nivolumab (n=6) or exercise + nivolumab (n=6). The animals undertook an 8- week moderate-intensity training regimen (treadmill aerobic exercise and strength training). Immunotherapy (nivolumab) or an isotype control was administered 2 days/week, for 6 weeks. Several tumor growth and microenvironment parameters were measured after the intervention. RESULTS: Improvements in aerobic capacity and muscle strength (p=0.027 and p=0.005) were noted in exercised animals. Exercise alone reduced the tumor growth rate with respect to non-exercised mice (p=0.050). The double intervention (exercise + nivolumab) increased tumor necrosis and reduced apoptosis with respect to controls (p=0.026; p=0.030). All interventions achieved a reduction in proliferation compared with the control group (p=0.015, p=0.011, and p=0.011). Exercise alone increased myeloid tumor infiltrates (mostly neutrophils) with respect to the nivolumab only group (p=0.018). Finally, Vegf-a expression was higher in the nivolumab groups (in combination or not with exercise) than in exercise + isotype control group (p=0.045 and p=0.047, respectively). No other significant effects were found. CONCLUSIONS: Our results would suggest that aerobic and strength training should be studied as an adjuvant to cancer immunotherapy treatment.

Functional Assays of Stem Cell Properties Derived from Different Niches.

It has been described that adult tissues contain mesenchymal stem cell populations. The specific areas where stem cells reside are known as niches. Crosstalk between cells and their niche is essential to maintain the correct functionality of stem cell. MSCs present a set of abilities such as migration, invasion, and angiogenic potentials, which make them ideal candidates for cell-based therapies. In order to test the regenerative capacity of these cells, we have described a methodology for the collection and for the evaluation of these mesenchymal precursors from different niches.

Unhealthy Stem Cells: When Health Conditions Upset Stem Cell Properties.

The stem cell field has grown very rapidly during the last decade, offering the promise of innovative therapies to treat disease. Different stem cell populations have been isolated from various human adult tissues, mainly from bone marrow and adipose tissue, but many other body tissues harbor a stem cell population. Adult tissue stem cells are invariably found in discrete microenvironments termed niches, where they play key roles in tissue homeostasis by enabling lifelong optimization of organ form and function. Some diseases are known to strike at the stem cell population, through alterations in their specific microenvironments, making them non-viable. Furthermore, it has been shown that a transformed stem cell population could prompt the development of certain cancers. This review focuses on the potential negative aspects of a range of diseases on the activity of stem cells and how their potential use in cell therapies may be affected.

Importance and regulation of adult stem cell migration.

Cell migration is an essential process throughout the life of vertebrates, beginning during embryonic development and continuing throughout adulthood. Stem cells have an inherent ability to migrate, that is as important as their capacity for self-renewal and differentiation, enabling them to maintain tissue homoeostasis and mediate repair and regeneration. Adult stem cells reside in specific tissue niches, where they remain in a quiescent state until called upon and activated by tissue environmental signals. Cell migration is a highly regulated process that involves the integration of intrinsic signals from the niche and extrinsic factors. Studies using three-dimensional in vitro models have revealed the astonishing plasticity of cells in terms of the migration modes employed in response to changes in the microenvironment. These same properties can, however, be subverted during the development of some pathologies such as cancer. In this review, we describe the response of adult stem cells to migratory stimuli and the mechanisms by which they sense and transduce intracellular signals involved in migratory processes. Understanding the molecular events underlying migration may help develop therapeutic strategies for regenerative medicine and to treat diseases with a cell migration component.

Adipose stem cells from obese patients show specific differences in the metabolic regulators vitamin D and Gas5.

Adipose tissue is a significant source of mesenchymal stem cells for regenerative therapies; however, caution should be taken as their environmental niche can affect their functional properties. We have previously demonstrated the negative impact of obesity on the function of adipose-derived stem cells (ASCs). Here we have evaluated other possible properties and targets that are altered by obesity such as the recently described long non-coding molecule Gas5, which is involved in glucocorticoid resistance. Using ASCs isolated from obese (oASCs) and control subjects (cASCs), we have analyzed additional metabolic and inflammatory conditions that could be related with their impaired therapeutic potential and consequently their possible usefulness in the clinic.

Membrane Blebbing Is Required for Mesenchymal Precursor Migration.

Mesenchymal precursors (MPs) present some advantageous features, such as differentiation and migration, which make them promising candidates for cell therapy. A better understanding of MP migration characteristics would aid the development of cell delivery protocols. Traditionally, cell migration is thought to occur only through the formation of lamellipodia. More recently, contractility-driven bleb formation has emerged as an alternative mechanism of motility. Here we report that MPs derived from different tissues present spontaneously dynamic cytoplasmic projections in sub-confluent culture, which appear as a combination of lamellipodia with blebs in the leading edge. Upon initial seeding, however, only bleb structures could be observed. Immunofluorescence revealed the presence of pERM, RhoA and F-actin during the blebbing process. Results from migration assays in the presence of blebbistatin, a myosin II inhibitor, showed that bleb formation correlated with migratory capacity, suggesting a functional role for blebs in migration. Bleb formation might be a useful mechanism to improve cell migration in cellular therapy protocols.

Transcriptional profiling of interleukin-2-primed human adipose derived mesenchymal stem cells revealed dramatic changes in stem cells response imposed by replicative senescence.

Inflammation is a double-edged sword with both detrimental and beneficial consequences. Understanding of the mechanisms of crosstalk between the inflammatory milieu and human adult mesenchymal stem cells is an important basis for clinical efforts. Here, we investigate changes in the transcriptional response of human adipose-derived stem cells to physiologically relevant levels of IL-2 (IL-2 priming) upon replicative senescence. Our data suggest that replicative senescence might dramatically impede human mesenchymal stem cell (MSC) function via global transcriptional deregulation in response to IL-2. We uncovered a novel senescence-associated transcriptional signature in human adipose-derived MSCs hADSCs after exposure to pro-inflammatory environment: significant enhancement of the expression of the genes encoding potent growth factors and cytokines with anti-inflammatory and migration-promoting properties, as well as genes encoding angiogenic and anti-apoptotic promoting factors, all of which could participate in the establishment of a unique microenvironment. We observed transcriptional up-regulation of critical components of the nitric oxide synthase pathway (iNOS) in hADSCs upon replicative senescence suggesting, that senescent stem cells can acquire metastasis-promoting properties via stem cell-mediated immunosuppression. Our study highlights the importance of age as a factor when designing cell-based or pharmacological therapies for older patients and predicts measurable biomarkers characteristic of an environment that is conducive to cancer cells invasiveness and metastasis.

Low-Intensity Pulsed Ultrasound Improves the Functional Properties of Cardiac Mesoangioblasts.

Cell-based therapy is a promising approach for many diseases, including ischemic heart disease. Cardiac mesoangioblasts are committed vessel-associated progenitors that can restore to a significant, although partial, extent, heart structure and function in a murine model of myocardial infarction. Low-intensity pulsed ultrasound (LIPUS) is a non-invasive form of mechanical energy that can be delivered into biological tissues as acoustic pressure waves, and is widely used for clinical applications including bone fracture healing. We hypothesized that the positive effects of LIPUS on bone and soft tissue, such as increased cell differentiation and cytoskeleton reorganization, could be applied to increase the therapeutic potential of mesoangioblasts for heart repair. In this work, we show that LIPUS stimulation of cardiac mesoangioblasts isolated from mouse and human heart results in significant cellular modifications that provide beneficial effects to the cells, including increased malleability and improved motility. Additionally, LIPUS stimulation increased the number of binucleated cells and induced cardiac differentiation to an extent comparable with 5'-azacytidine treatment. Mechanistically, LIPUS stimulation activated the BMP-Smad signalling pathway and increased the expression of myosin light chain-2 together with upregulation of ß1 integrin and RhoA, highlighting a potentially important role for cytoskeleton reorganization. Taken together, these results provide functional evidence that LIPUS might be a useful tool to explore in the field of heart cell therapy.

Targeting endothelial junctional adhesion molecule-A/ EPAC/ Rap-1 axis as a novel strategy to increase stem cell engraftment in dystrophic muscles.

Muscular dystrophies are severe genetic diseases for which no efficacious therapies exist. Experimental clinical treatments include intra-arterial administration of vessel-associated stem cells, called mesoangioblasts (MABs). However, one of the limitations of this approach is the relatively low number of cells that engraft the diseased tissue, due, at least in part, to the sub-optimal efficiency of extravasation, whose mechanisms for MAB are unknown. Leukocytes emigrate into the inflamed tissues by crossing endothelial cell-to-cell junctions and junctional proteins direct and control leukocyte diapedesis. Here, we identify the endothelial junctional protein JAM-A as a key regulator of MAB extravasation. We show that JAM-A gene inactivation and JAM-A blocking antibodies strongly enhance MAB engraftment in dystrophic muscle. In the absence of JAM-A, the exchange factors EPAC-1 and 2 are down-regulated, which prevents the activation of the small GTPase Rap-1. As a consequence, junction tightening is reduced, allowing MAB diapedesis. Notably, pharmacological inhibition of Rap-1 increases MAB engraftment in dystrophic muscle, which results into a significant improvement of muscle function offering a novel strategy for stem cell-based therapies.

Association of polymorphisms in TRAIL1 and TRAILR1 genes with susceptibility to lymphomas.

The TRAILR1/TRAIL system is implicated in the induction of the extrinsic apoptotic pathway and constitutes an emerging target in cancer therapeutics. The objective of this study is to assess lymphoma risk associated with certain polymorphisms in TRAILR1 and TRAIL1 genes. DNA was extracted from 381 subjects (190 lymphoma cases and 191 matched controls) and genotyped for polymorphisms rs20576, rs2230229 and rs20575 in TRAILR1 and rs12488654 in TRAIL gene. In contrast to TRAILR1 polymorphisms, the genotype distribution of rs12488654 in TRAIL gene was different between cases and controls, A allele carriers (CA/AA) being much more common in the cases with different lymphoma types (follicular, 45 %; diffuse large B cell, 45.2 % and Hodgkin lymphomas, 40 %) than in controls (15.7 %) (odds ratio (OR), 3.5; CI, 2.1­5.9; p<0.001; OR, 3.5; CI, 1.6­7.9; p=0.001; OR, 2.9; CI, 1.1­7.5; p=0.027, respectively). This effect was consistently independent of the association with the TRAILR1 polymorphisms studied, as demonstrated by linkage disequilibrium and haplotype analyses. This study is the first one to report an association between a TRAIL polymorphism and lymphoma risk and suggests a possible role of TRAIL in B cell lymphomagenesis.

The potential of stem cells in the treatment of cardiovascular diseases.

Although the adult mammalian heart was once believed to be a post-mitotic organ without any capacity for regeneration, recent findings have challenged this dogma. A modified view assigns to the mammalian heart a measurable capacity for regeneration throughout life. The ultimate goals of the cardiac regeneration field have been pursued by multiple strategies, including understanding the developmental biology of cardiomyocytes and cardiac stem and progenitor cells, applying chemical genetics, and engineering biomaterials and delivery methods that facilitate cell transplantation. Successful stimulation of endogenous regenerative capacity in injured adult mammalian hearts can benefit from studies of natural cardiac regeneration.