Search

Abstract Vaccination against an airborne pathogen is very effective if it induces also the development of mucosal antibodies that can protect against infection. The mRNA‐based vaccine‐encoding SARS‐CoV‐2 full‐length spike protein (BNT162b2, Pfizer/BioNTech) protects also against infection despite being administered systemically. Here, we show that upon vaccination, cognate IgG molecules are also found in the saliva and are more abundant in SARS‐CoV‐2 previously exposed subjects, paralleling the development of plasma IgG. The antibodies titer declines at 3 months from vaccination. We identified a concentration of specific IgG in the plasma above which the relevant IgG can be detected in the saliva. Regarding IgA antibodies, we found only protease‐susceptible IgA1 antibodies in plasma while they were present at very low levels in the saliva over the course of vaccination of SARS‐CoV‐2‐naïve subjects. Thus, in response to BNT162b2 vaccine, plasma IgG can permeate into mucosal sites and participate in viral protection. It is not clear why IgA1 are detected in low amount, they may be proteolytically cleaved.

Background and Aims: The SARS-CoV-2 pandemic has overwhelmed the treatment capacity of the health care systems during the highest viral diffusion rate. Patients reaching the emergency department had to be either hospitalized (inpatients) or discharged (outpatients). Still, the decision was taken based on the individual assessment of the actual clinical condition, without specific biomarkers to predict future improvement or deterioration, and discharged patients often returned to the hospital for aggravation of their condition. Here, we have developed a new combined approach of omics to identify factors that could distinguish coronavirus disease 19 (COVID-19) inpatients from outpatients. Methods: Saliva and blood samples were collected over the course of two observational cohort studies. By using machine learning approaches, we compared salivary metabolome of 50 COVID-19 patients with that of 270 healthy individuals having previously been exposed or not to SARS-CoV-2. We then correlated the salivary metabolites that allowed separating COVID-19 inpatients from outpatients with serum biomarkers and salivary microbiota taxa differentially represented in the two groups of patients. Results: We identified nine salivary metabolites that allowed assessing the need of hospitalization. When combined with serum biomarkers, just two salivary metabolites (myo-inositol and 2-pyrrolidineacetic acid) and one serum protein, chitinase 3-like-1 (CHI3L1), were sufficient to separate inpatients from outpatients completely and correlated with modulated microbiota taxa. In particular, we found Corynebacterium 1 to be overrepresented in inpatients, whereas Actinomycetaceae F0332, Candidatus Saccharimonas, and Haemophilus were all underrepresented in the hospitalized population. Conclusion: This is a proof of concept that a combined omic analysis can be used to stratify patients independently from COVID-19.

Despite advances in their diagnosis and treatment, pediatric cancers remain among the leading causes of death in childhood. The development of immunotherapies and other forms of targeted therapies has significantly changed the prognosis of some previously incurable cancers in the adult population. However, so far, the results in pediatric cohorts are disappointing, which is mainly due to differences in tumor biology, including extreme heterogeneity and a generally low tumor mutational burden. A central role in the limited efficacy of immunotherapeutic approaches is played by the peculiar characteristics of the tumor microenvironment (TME) in pediatric cancer, with the scarcity of tumor infiltration by T cells and the abundance of stromal cells endowed with lymphocyte suppressor and tumor-growth-promoting activity. Thus, progress in the treatment of pediatric solid tumors will likely be influenced by the ability to modify the TME while delivering novel, more effective therapeutic agents. In this review, we will describe the TME composition in pediatric solid tumors and illustrate recent advances in treatment for the modulation of immune cells belonging to the TME.

Breast implants are extensively employed for both reconstructive and esthetic purposes. However, the safety of breast implants with textured surfaces has been questioned, owing to a potential correlation with anaplastic large-cell lymphoma and the recurrence of breast cancer. This study investigates the immune response elicited by different prosthetic surfaces, focusing on the comparison between macrotextured and microtextured breast implants. Through the analysis of intraoperatively harvested periprosthetic fluids and cell culture experiments on surface replicas, we demonstrate that macrotextured surfaces elicit a more pronounced chronic-like activation of leucocytes and an increased release of inflammatory cytokines, in contrast to microtextured surfaces. In addition, in vitro fluorescent imaging of leucocytes revealed an accumulation of lymphocytes within the cavities of the macrotextured surfaces, indicating that the physical entrapment of these cells may contribute to their activation. These findings suggest that the topography of implant surfaces plays a significant role in promoting a chronic-like inflammatory environment, which could be a contributing factor in the development of lymphomas associated with a wide range of implantable devices., (© 2024 Vinci et al.)

Background: Malignant Pleural Mesothelioma (MPM) is an aggressive cancer of the mesothelial lining associated with exposure to airborne non-degradable asbestos fibers. Its poor response to currently available treatments prompted us to explore the biological mechanisms involved in its progression. MPM is characterized by chronic non-resolving inflammation; in this study we investigated which inflammatory mediators are mostly expressed in biological tumor samples from MPM patients, with a focus on inflammatory cytokines, chemokines and matrix components., Methods: Expression and quantification of Osteopontin (OPN) was detected in tumor and plasma samples of MPM patients by mRNA, immunohistochemistry and ELISA. The functional role of OPN was investigated in mouse MPM cell lines in vivo using an orthotopic syngeneic mouse model., Results: In patients with MPM, the protein OPN was significantly more expressed in tumors than in normal pleural tissues and predominantly produced by mesothelioma cells; plasma levels were elevated in patients and associated with poor prognosis. However, modulation of OPN levels was not significantly different in a series of 18 MPM patients receiving immunotherapy with durvalumab alone or with pembrolizumab in combination with chemotherapy, some of whom achieved a partial clinical response. Two established murine mesothelioma cell lines: AB1 and AB22 of sarcomatoid and epithelioid histology, respectively, spontaneously produced high levels of OPN. Silencing of the OPN gene ( Spp1 ) dramatically inhibited tumor growth in vivo in an orthotopic model, indicating that OPN has an important promoting role in the proliferation of MPM cells. Treatment of mice with anti-CD44 mAb, blocking a major OPN receptor, significantly reduced tumor growth in vivo ., Conclusion: These results demonstrate that OPN is an endogenous growth factor for mesothelial cells and inhibition of its signaling may be helpful to restrain tumor progression in vivo . These findings have translational potential to improve the therapeutic response of human MPM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor CP declared a past co-authorship with the author ME., (Copyright © 2023 Digifico, Erreni, Mannarino, Marchini, Ummarino, Anfray, Bertola, Recordati, Pistillo, Roncalli, Bossi, Zucali, D’Incalci, Belgiovine and Allavena.)

The incidence of periprosthetic joint infections (PJIs) is ~2% of total procedures and it is expected to rise due to an ageing population. Despite the large burden PJI has on both the individual and society, the immune response to the most commonly isolated pathogens, i.e., Staphylococcus aureus and Staphylococcus epidermidis , remains incompletely understood. In this work, we integrate the analysis of synovial fluids from patients undergoing hip and knee replacement surgery with in-vitro experimental data obtained using a newly developed platform, mimicking the environment of periprosthetic implants. We found that the presence of an implant, even in patients undergoing aseptic revisions, is sufficient to induce an immune response, which is significantly different between septic and aseptic revisions. This difference is confirmed by the presence of pro- and anti-inflammatory cytokines in synovial fluids. Moreover, we discovered that the immune response is also dependent on the type of bacteria and the topography of the implant surface. While S. epidermidis seems to be able to hide better from the attack of the immune system when cultured on rough surfaces (indicative of uncemented prostheses), S. aureus reacts differently depending on the contact surface it is exposed to. The experiments we performed in-vitro also showed a higher biofilm formation on rough surfaces compared to flat ones for both species, suggesting that the topography of the implant could influence both biofilm formation and the consequent immune response.

Background:  Pediatric renal cancer survivors have higher rate of chronic renal disease and hypertension. These patients have similar survival rates when treated according to either Children's Oncology Group (COG) or International Society of Pediatric Oncology (SIOP) protocols. We aimed to compare the late outcome of these two approaches., Methods:  We performed a meta-analysis of all studies from 2000 to 2021; database search using keywords: long-term outcomes OR late effects, nephrectomy, pediatric renal cancer. For each protocol, data were collected, and the "pooled" outcomes were compared. Continuous and dichotomous variables were obtained with a 95% odds ratio., Results:  Sixteen studies with a total of 715 pediatric renal cancer survivors were analyzed. The mean follow-up time was 17.4 (standard deviation 5.6) years. Reduced renal function and hypertension were the most encountered long-term complications. The mean estimated glomerular filtration rate was similar in both protocols (101.62 vs. 101.70 mL/min/1.73 m 2 ), while the prevalence of hypertension was 23% in COG and 10% in SIOP. The prevalence of secondary malignancy was 1.1% in COG and 6.7% in SIOP (1.1% vs. 6.7%, p ≤ 0.001). Chronic kidney disease was similar in both groups., Conclusion:  A high prevalence of hypertension was observed among pediatric renal cancer survivors, as well as an increased risk of a secondary tumor. These results emphasize the importance of long-term follow-up into adulthood, to promptly diagnose any long-term side effects of the treatment. Thanks to the increased overall survival, future protocols will pay attention to the reduction of long-term sequelae., Competing Interests: None declared., (Thieme. All rights reserved.)

KRAS mutations characterize pancreatic cell transformation from the earliest stages of carcinogenesis, and are present in >95% of pancreatic ductal adenocarcinoma (PDAC) cases. In search of novel biomarkers for the early diagnosis of PDAC, we identified the proteins secreted by the normal human pancreatic cell line (HPDE) recently transformed by inducing the overexpression of the KRASG12V oncogene. We report a proteomic signature of KRAS-induced secreted proteins, which was confirmed in surgical tumor samples from resected PDAC patients. The putative diagnostic performance of three candidates, Laminin-C2 (LAMC2), Tenascin-C (TNC) and Pentraxin-3 (PTX3), was investigated by ELISA quantification in two cohorts of PDAC patients (n = 200) eligible for surgery. Circulating levels of LAMC2, TNC and PTX3 were significantly higher in PDAC patients compared to the healthy individuals (p < 0.0001). The Receiver Operating Characteristics (ROC) curve showed good sensitivity (1) and specificity (0.63 and 0.85) for LAMC2 and PTX3, respectively, but not for TNC, and patients with high levels of LAMC2 had significantly shorter overall survival (p = 0.0007). High levels of LAMC2 and PTX3 were detected at early stages (I−IIB) and in CA19-9-low PDAC patients. In conclusion, pancreatic tumors release LAMC2 and PTX3, which can be quantified in the systemic circulation, and may be useful in selecting patients for further diagnostic imaging.

The spread of biofilms on medical implants represents one of the principal triggers of persistent and chronic infections in clinical settings, and it has been the subject of many studies in the past few years, with most of them focused on prosthetic joint infections. We review here recent works on biofilm formation and microbial colonization on a large variety of indwelling devices, ranging from heart valves and pacemakers to urological and breast implants and from biliary stents and endoscopic tubes to contact lenses and neurosurgical implants. We focus on bacterial abundance and distribution across different devices and body sites and on the role of environmental features, such as the presence of fluid flow and properties of the implant surface, as well as on the interplay between bacterial colonization and the response of the human immune system.

Immune cells in the tumor micro-environment (TME) establish a complex relationship with cancer cells and may strongly influence disease progression and response to therapy. It is well established that myeloid cells infiltrating tumor tissues favor cancer progression. Tumor-Associated Macrophages (TAMs) are abundantly present at the TME and actively promote cancer cell proliferation and distant spreading, as well as contribute to an immune-suppressive milieu. Active research of the last decade has provided novel therapeutic approaches aimed at depleting TAMs and/or at reprogramming their functional activities. We reported some years ago that the registered anti-tumor agent trabectedin and its analogue lurbinectedin have numerous mechanisms of action that also involve direct effects on immune cells, opening up new interesting points of view. Trabectedin and lurbinectedin share the unique feature of being able to simultaneously kill cancer cells and to affect several features of the TME, most notably by inducing the rapid and selective apoptosis of monocytes and macrophages, and by inhibiting the transcription of several inflammatory mediators. Furthermore, depletion of TAMs alleviates the immunosuppressive milieu and rescues T cell functional activities, thus enhancing the anti-tumor response to immunotherapy with checkpoint inhibitors. In view of the growing interest in tumor-infiltrating immune cells, the availability of antineoplastic compounds showing immunomodulatory effects on innate and adaptive immunity deserves particular attention in the oncology field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Allavena, Belgiovine, Digifico, Frapolli and D’Incalci.)

A considerable proportion of cancer patients are resistant or only partially responsive to immune checkpoint blockade immunotherapy. Tumor-Associated Macrophages (TAMs) infiltrating the tumor stroma suppress the adaptive immune responses and, hence, promote tumor immune evasion. Depletion of TAMs or modulation of their protumoral functions is actively pursued, with the purpose of relieving this state of immunesuppression. We previously reported that trabectedin, a registered antitumor compound, selectively reduces monocytes and TAMs in treated tumors. However, its putative effects on the adaptive immunity are still unclear. In this study, we investigated whether treatment of tumor-bearing mice with trabectedin modulates the presence and functional activity of T-lymphocytes. In treated tumors, there was a significant upregulation of T cell-associated genes, including CD3, CD8, perforin, granzyme B, and IFN-responsive genes (MX1, CXCL10, and PD-1), indicating that T lymphocytes were activated after treatment. Notably, the mRNA levels of the Pdcd1 gene, coding for PD-1, were strongly increased. Using a fibrosarcoma model poorly responsive to PD-1-immunotherapy, treatment with trabectedin prior to anti-PD-1 resulted in improved antitumor efficacy. In conclusion, pretreatment with trabectedin enhances the therapeutic response to checkpoint inhibitor-based immunotherapy. These findings provide a good rational for the combination of trabectedin with immunotherapy regimens., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

We aimed to explore whether variants of SARS-CoV-2 (Chinese-derived strain (D614, lineage A), Italian strain PV10734 (D614G, lineage B.1.1) and Alpha strain (lineage B.1.1.7)) were able to infect monocytes (MN) and monocyte-derived macrophages (MDM) and whether these infected cells may, in turn, be vectors of infection. For this purpose, we designed an in vitro study following the evolution of MN and MDM infection at different time points in order to confirm whether these cells were permissive for SARS-CoV-2 replication. Finally, we investigated whether, regardless of viral replication, the persistent virus can be transferred to non-infected cells permissive for viral replication. Thus, we co-cultured the infected MN/MDM with permissive VERO E6 cells verifying the viral transmission. This is a further in vitro demonstration of the important role of MN and MDM in the dissemination of SARS-CoV-2 and evolution of the COVID-19 disease.

Myeloid cells infiltrating tumors are gaining ever growing attention in the last years because their pro-tumor and immunosuppressive functions are relevant for disease progression and therapeutic responses. The functional ambiguity of tumor-associated macrophages (TAMs), mostly promoting tumor evolution, is a challenging hurdle. This is even more evident in the case of cancer stem cells (CSCs); as active participants in the specialized environment of the cancer stem cell niche, TAMs initiate a reciprocal conversation with CSCs. TAMs contribute to protect CSCs from the hostile environment (exogenous insults, toxic compounds, attacks from the immune cells), and produce several biologically active mediators that modulate crucial developmental pathways that sustain cancer cell stemness. In this review, we have focused our attention on the interaction between TAMs and CSCs; we describe how TAMs impact on CSC biology and, in turn, how CSCs exploit the tissue trophic activity of macrophages to survive and progress. Since CSCs are responsible for therapy resistance and tumor recurrence, they are important therapeutic targets. In view of the recent success in oncology obtained by stimulating the immune system, we discuss some macrophage-targeted therapeutic strategies that may also affect the CSCs and interrupt their malevolent alliance., (© 2021. The Author(s).)

Background: Surgical site infection represents the most severe complication in prosthetic breast reconstruction. Risk profiling represents a useful tool for both clinicians and patients., Materials and Methods: In our hospital, 534 breast reconstructions with tissue expander implants, in 500 patients, were performed. Several clinical variables were collected. In our study, we evaluated the different inflammatory markers present in the periprosthetic fluid and we compared them with the ones present in plasma., Results: The surgical site infection rate resulted to be 10.5%, and reconstruction failed in 4.5% of the cases. The hazard ratio for complications was 2.3 in women over 60 (CI: 1.3-4.07; p = 0.004), 2.57 in patients with expander volume ≥ 500 cc (CI: 1.51-4.38; p < 0.001), 2.14 in patients submitted to previous radiotherapy (CI: 1.05-4.36; p < 0.037), and 1.05 in prolonged drain use (CI: 1.03-1.07; p < 0.001). 25-OH, PCT, and total protein were less concentrated, and ferritin and LDH were more concentrated in the periprosthetic fluid than in plasma ( p < 0.001). CRP ( p = 0.190) and β -2 microglobulin ( p = 0.344) did not change in the two fluids analyzed. PCT initial value is higher in patients who underwent radiotherapy, and it could be related to the higher rate of their postoperative complications. Patients with a tissue expander with a volume ≥ 500 cc show an increasing trend for CRP in time ( p = 0.009)., Conclusions: Several risk factors (prolonged time of drains, age older than 60 years, and radiotherapy) have been confirmed by our study. The study of markers in the periprosthetic fluid with respect to their study in plasma could point toward earlier infection detection and support early management., Competing Interests: The authors declare that they have no conflicts of interest to disclose., (Copyright © 2021 Andrea Lisa et al.)

Nowadays, it is well accepted that inflammation is a critical player in cancer, being, in most cases, the main character of the process. Different types of tumor arise from sites of infection or chronic inflammation. This non-resolving inflammation is responsible for tumor development at different levels: it promotes tumor initiation, as well as tumor progression, stimulating both tumor growth and metastasis. Environmental factors, lifestyle and infections are the three main triggers of chronic immune activation that promote or increase the risk of many different cancers. In this review, we focus our attention on tumor onset; in particular, we summarize the knowledge about the cause and the mechanisms behind the inflammation-driven cancer development.

In the last decade, it has been well-established that tumor-infiltrating myeloid cells fuel not only the process of carcinogenesis through cancer-related inflammation mechanisms, but also tumor progression, invasion, and metastasis. In particular, tumor-associated macrophages (TAMs) are the most abundant leucocyte subset in many cancers and play a major role in the creation of a protective niche for tumor cells. Their ability to generate an immune-suppressive environment is crucial to escape the immune system and to allow the tumor to proliferate and metastasize to distant sites. Conventional therapies, including chemotherapy and radiotherapy, are often not able to limit cancer growth due to the presence of pro-tumoral TAMs; these are also responsible for the failure of novel immunotherapies based on immune-checkpoint inhibition. Several novel therapeutic strategies have been implemented to deplete TAMs; however, more recent approaches aim to use TAMs themselves as weapons to fight cancer. Exploiting their functional plasticity, the reprogramming of TAMs aims to convert immunosuppressive and pro-tumoral macrophages into immunostimulatory and anti-tumor cytotoxic effector cells. This shift eventually leads to the reconstitution of a reactive immune landscape able to destroy the tumor. In this review, we summarize the current knowledge on strategies able to reprogram TAMs with single as well as combination therapies.

Malignant Pleural Mesothelioma (MPM) is an aggressive tumor of the pleural lining that is usually identified at advanced stages and resistant to current therapies. Appropriate pre-clinical mouse tumor models are of pivotal importance to study its biology. Usually, tumor cells have been injected intraperitoneally or subcutaneously. Using three available murine mesothelioma cell lines with different histotypes (sarcomatoid, biphasic, epithelioid), we have set up a simplified model of in vivo growth orthotopically by inoculating tumor cells directly in the thorax with a minimally invasive procedure. Mesothelioma tumors grew along the pleura and spread on the superficial areas of the lungs, but no masses were found outside the thoracic cavity. As observed in human MPM, tumors were highly infiltrated by macrophages and T cells. The luciferase-expressing cells can be visualized in vivo by bioluminescent optical imaging to precisely quantify tumor growth over time. Notably, the bioluminescence signal detected in vivo correctly matched the tumor burden quantified with classical histology. In contrast, the subcutaneous or intraperitoneal growth of these mesothelioma cells was considered either non-representative of the human disease or unreliable to precisely quantify tumor load. Our non-invasive in vivo model of mesothelioma is simple and reproducible, and it reliably recapitulates the human disease.

Background: Thyroid carcinoma includes several variants characterized by different biological and clinical features: from indolent microcarcinoma to undifferentiated and aggressive anaplastic carcinoma. Inflammation plays a critical role in thyroid tumors. Conditions predisposing to cancer, as well as oncogene activity, contribute to the construction of an inflammatory microenvironment that facilitates thyroid tumor progression. Moreover, oncogene-induced senescence, a mechanism tightly connected with inflammation, and able to restrain or promote cancer progression, is involved in thyroid cancer. The interactions between thyroid tumor cells and the microenvironment are not completely clarified., Methods: We characterize in vitro the interplay between macrophages and senescent thyrocytes and tumor-derived cell lines, modeling early and late thyroid tumor stages, respectively. Purified peripheral blood-derived human monocytes were exposed to thyroid cell-derived conditioned medium (CM) and assessed for phenotype by flow cytometry. The factors secreted by thyroid cells and macrophages were identified by gene expression analysis and ELISA. The protumoral effect of macrophages was assessed by wound healing assay on K1 thyroid tumor cells. The expression of PTGS2 and M2 markers in thyroid tumors was investigated in publicly available datasets., Results: Human monocytes exposed to CM from senescent thyrocytes and thyroid tumor cell lines undergo M2-like polarization, showing high CD206 and low MHC II markers, and upregulation of CCL17 secretion. The obtained M2-like macrophages displayed tumor-promoting activity. Among genes overexpressed in polarizing cells, we identified the prostaglandin-endoperoxide synthase enzyme (PTGS2/COX-2), which is involved in the production of prostaglandin E2 (PGE2). By using COX-2 inhibitors we demonstrated that the M2-like polarization ability of thyroid cells is related to the production of PGE2. Co-expression of PTGS2 and M2 markers is observed a significant fraction of human thyroid tumors., Conclusions: Our results demonstrate that both senescent thyrocytes and thyroid tumor cell lines trigger M2-like macrophage polarization that is related to PGE2 secretion. This suggests that the interaction with the microenvironment occurs at both early and late thyroid tumor stages, and favors tumor progression. The co-expression of PTGS2 gene and M2 markers in human thyroid carcinoma highlights the possibility to counteract tumor growth through COX-2 inhibition.

Cancer stem cells (CSCs) have been involved in the maintenance, progression and relapse of several tumors, but their origin is still elusive. Here, in vitro transformed human fibroblasts (cen3tel cells) and the tumorsphere assay were used to search for and possibly characterize CSCs in transformed somatic cells. Cen3tel cells formed spheres showing self-renewal capacity and Sox2 overexpression, suggesting that they contained a subset of cells with CSC-like features. Sphere cells displayed deregulation of a c-MYC/miR-34a circuitry, likely associated with cell protection from apoptosis. Gene expression profiles of sphere cells revealed an extensive transcriptional reprogramming. Genes up-regulated in tumorspheres identified processes related to tumorigenesis and stemness, as cholesterol biosynthesis, apoptosis suppression, interferon and cytokine mediated signalling pathways. Sphere cells engrafted into NSG mice more rapidly than adherent cells, but both cell populations were tumorigenic. These results indicate that, during transformation, human somatic cells can acquire CSC properties, confirming the high plasticity of tumor cells. However, CSC-like cells are not the only tumorigenic population in transformed cells, indicating that the CSC phenotype and tumorigenicity can be uncoupled.

Background: Lurbinectedin is a novel anticancer agent currently undergoing late-stage (Phase II /III) clinical evaluation in platinum-resistant ovarian, BRCA1/2-mutated breast and small-cell lung cancer. Lurbinectedin is structurally related to trabectedin and it inhibits active transcription and the DNA repair machinery in tumour cells., Methods: In this study we investigated whether lurbinectedin has the ability to modulate the inflammatory microenvironment and the viability of myeloid cells in tumour-bearing mice., Results: Administration of lurbinectedin significantly and selectively decreased the number of circulating monocytes and, in tumour tissues, that of macrophages and vessels. Similar findings were observed when a lurbinectedin-resistant tumour variant was used, indicating a direct effect of lurbinectedin on the tumour microenviroment. In vitro, lurbinectedin induced caspase-8-dependent apoptosis of human purified monocytes, whereas at low doses it significantly inhibited the production of inflammatory/growth factors (CCL2, CXCL8 and VEGF) and dramatically impaired monocyte adhesion and migration ability. These findings were supported by the strong inhibition of genes of the Rho-GTPase family in lurbinectedin-treated monocytes., Conclusions: The results illustrate that lurbinectedin affects at multiple levels the inflammatory microenvironment by acting on the viability and functional activity of mononuclear phagocytes. These peculiar effects, combined with its intrinsic activity against cancer cells, make lurbinectedin a compound of particular interest in oncology.

Despite the accepted dogma that TRAIL kills only tumor cells and spares normal ones, we show in this study that mononuclear phagocytes are susceptible to recombinant TRAIL via caspase-dependent apoptosis. Human resting monocytes and in vitro-differentiated macrophages expressed substantial levels of the functional TRAIL receptors (TRAIL-R1 and TRAIL-R2), while neutrophils and lymphocytes mostly expressed the non-signaling decoy receptor (TRAIL-R3). Accordingly, exclusively monocytes and macrophages activated caspase-8 and underwent apoptosis upon recombinant TRAIL treatment. TRAIL-Rs were up-regulated by anti-inflammatory agents (IL-10, glucocorticoids) and by natural compounds (Apigenin, Quercetin, Palmitate) and their treatment resulted in increased TRAIL-induced apoptosis. In mice, the only signaling TRAIL-R (DR5) was preferentially expressed by blood monocytes rather than neutrophils or lymphocytes. In both mice and humans, Tumor-Associated Macrophages (TAM) expressed functional TRAIL-R, while resident macrophages in normal tissues did not. As a proof of principle, we treated mice bearing a murine TRAIL-resistant fibrosarcoma with recombinant TRAIL. We observed significant decrease of circulating monocytes and infiltrating TAM, as well as reduced tumor growth and lower metastasis formation. Overall, these findings demonstrate that human and murine monocytes/macrophages are, among leukocytes, uniquely susceptible to TRAIL-mediated killing. This differential susceptibility to TRAIL could be exploited to selectively target macrophages in tumors., Competing Interests: The authors declare no competing financial interests.

Myeloid cells infiltrating the tumor microenvironment, especially tumor-associated macrophages (TAMs), are essential providers of cancer-related inflammation, a condition known to accelerate tumor progression and limit the response to anti-tumor therapies. As a matter of fact, TAMs may have a dual role while interfering with cancer treatments, as they can either promote or impair their functionality. Here we review the connection between macrophages and anticancer therapies; moreover, we provide an overview of the different strategies to target or re-program TAMs for therapeutic purposes.

Cancer cells use two major types of movement: Mesenchymal, which is typical of cells of mesenchymal origin and depends on matrix metalloproteinase (MMP) activity, and amoeboid, which is characteristic of cells with a rounded shape and relies on the activity of Rho-associated kinase (ROCK). The present authors previously demonstrated that, during neoplastic transformation, telomerase-immortalized human fibroblasts (cen3tel cells) acquired a ROCK-dependent/MMP independent mechanism of invasion, mediated by the downregulation of the ROCK cellular inhibitor Round (Rnd)3/RhoE. In the present study, cen3tel transformation was also demonstrated to be paralleled by downregulation of Snail, a major determinant of the mesenchymal movement. To test whether Snail levels could determine the type of movement adopted by mesenchymal tumor cells, Snail was ectopically expressed in tumorigenic cells. It was observed that ectopic Snail did not increase the levels of typical mesenchymal markers, but induced cells to adopt an MMP-dependent mechanism of invasion. In cells expressing ectopic Snail, invasion became sensitive to the MMP inhibitor Ro 28-2653 and insensitive to the ROCK inhibitor Y27632, suggesting that, once induced by Snail, the mesenchymal movement prevails over the amoeboid one. Snail-expressing cells had a more aggressive behavior in vivo , and exhibited increased tumor growth rate and metastatic ability. These results confirm the high plasticity of cancer cells, which can adopt different types of movement in response to changes in the expression of specific genes. Furthermore, the present findings indicate that Rnd3 and Snail are possible regulators of the type of invasion mechanism adopted by mesenchymal tumor cells.

Targeted therapies were rationally designed to inhibit molecular pathways in tumor cells critically involved in growth and survival; however, many drugs used in targeted therapies may affect the immune system. In addition, selected conventional chemotherapeutic agents have also been reported to be endowed with direct or indirect effects on immunity, for instance via immunogenic death of tumors. Thus, cancer therapies may have off-target effects, some of which are directed to the immune system. Here, we will review some of these effects in specific therapeutic approaches. We will examine the modulation of the immune contexture in human sarcoma and melanoma induced by anti-angiogenic therapies and by BRAF inhibitors, respectively. We will then discuss how the anti-tumor agent trabectedin is selectively cytotoxic to cells of the monocytic-macrophage lineage and how these immune-related effects can be part of the response to treatment.

Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process. Just after telomerase catalytic subunit (hTERT) expression, cen3tel telomeres shortened despite the presence of telomerase activity. At a later stage and concomitantly with transformation, cells started elongating telomeres, which reached a mean length greater than 100kb in about 900 population doublings. Super-telomeres were stable and compatible with cell growth and tumorigenesis. Telomere extension was associated with increasing levels of telomerase activity that were linked to the deregulation of endogenous telomerase RNA (hTERC) and exogenous telomerase reverse transcriptase (hTERT) expression. Notably, the increase in hTERC levels paralleled the increase in telomerase activity, suggesting that this subunit plays a role in regulating enzyme activity. Telomeres ranging in length between 10 and more than 100kb were maintained in an extendible state although TRF1 and TRF2 binding increased with telomere length. Super-telomeres neither influenced subtelomeric region global methylation nor the expression of the subtelomeric gene FRG1, attesting the lack of a clear-cut relationship between telomere length, subtelomeric DNA methylation and expression in human cells. The cellular levels of the telomeric proteins hTERT, TRF1, TRF2 and Hsp90 rose with transformation and were independent of telomere length, pointing to a role of these proteins in tumorigenesis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Tumor-associated macrophages (TAMs) and other myeloid cells that infiltrate neoplastic lesions promote tumor progression and are associated with poor patient prognosis. We have recently demonstrated that trabectedin, a licensed and commercially available anticancer agent, is selectively cytotoxic for TAMs and their circulating precursors (monocytes). The macrophage-depleting effect of trabectedin is a key component of its antitumor activity.

There is widespread interest in macrophages as a therapeutic target in cancer. Here, we demonstrate that trabectedin, a recently approved chemotherapeutic agent, induces rapid apoptosis exclusively in mononuclear phagocytes. In four mouse tumor models, trabectedin caused selective depletion of monocytes/macrophages in blood, spleens, and tumors, with an associated reduction of angiogenesis. By using trabectedin-resistant tumor cells and myeloid cell transfer or depletion experiments, we demonstrate that cytotoxicity on mononuclear phagocytes is a key component of its antitumor activity. Monocyte depletion, including tumor-associated macrophages, was observed in treated tumor patients. Trabectedin activates caspase-8-dependent apoptosis; selectivity for monocytes versus neutrophils and lymphocytes is due to differential expression of signaling and decoy TRAIL receptors. This unexpected property may be exploited in different therapeutic strategies., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribosylation) play essential roles in several biological processes, among which neoplastic transformation and telomere maintenance. In this paper, we review the poly(ADP-ribosylation) process together with the highly appealing use of PARP inhibitors for the treatment of cancer. In addition, we report our results concerning poly(ADP-ribosylation) in a cellular model system for neoplastic transformation developed in our laboratory. Here we show that PARP-1 and PARP-2 expression increases during neoplastic transformation, together with the basal levels of poly(ADP-ribosylation). Furthermore, we demonstrate a greater effect of the PARP inhibitor 3-aminobenzamide (3AB) on cellular viability in neoplastically transformed cells compared to normal fibroblasts and we show that prolonged 3AB administration to tumorigenic cells causes a decrease in telomere length. Taken together, our data support an active involvement of poly(ADP-ribosylation) in neoplastic transformation and telomere length maintenance and confirm the relevant role of poly(ADP-ribosylation) inhibition for the treatment of cancer.

We have developed a cellular system constituted of human telomerase immortalized fibroblasts that gradually underwent neoplastic transformation during propagation in culture. We exploited this cellular system to investigate gene and miRNA transcriptional programs in cells at different stages of propagation, representing five different phases along the road to transformation, from non-transformed cells up to tumorigenic and metastatic ones. Here we show that gene and miRNA expression profiles were both able to divide cells according to their transformation phase. We identified more than 1,700 genes whose expression was highly modulated in cells at at least one propagation stage and we found that the number of modulated genes progressively increased at successive stages of transformation. These genes identified processes significantly deregulated in tumorigenic cells, such as cell differentiation, cell movement and extracellular matrix remodeling, cell cycle and apoptosis, together with upregulation of several cancer testis antigens. Alterations in cell cycle, apoptosis, and cancer testis antigen expression were particular hallmarks of metastatic cells. A parallel deregulation of a panel of 43 miRNAs strictly connected to the p53 and c-Myc pathways and with oncogenic/oncosuppressive functions was also found. Our results indicate that cen3tel cells can be a useful model for human fibroblast neoplastic transformation, which appears characterized by complex and peculiar alterations involving both genetic and epigenetic reprogramming, whose elucidation could provide useful insights into regulatory networks underlying cancerogenesis.

Studying neoplastic transformation of telomerase immortalized human fibroblasts (cen3tel), we found that the transition from normal to tumorigenic cells was associated with the loss of growth contact inhibition, the acquisition of an epithelial-like morphology and a change in actin organization, from stress fibers to cortical bundles. We show here that these variations were paralleled by an increase in N-cadherin expression and relocalization of different adhesion molecules, such as N-cadherin, α-catenin, p-120 and β-catenin. These proteins presented a clear membrane localization in tumorigenic cells compared to a more diffuse, cytoplasmic distribution in primary fibroblasts and non-tumorigenic immortalized cells, suggesting that tumorigenic cells could form strong cell-cell contacts and cell contacts did not induce growth inhibition. The epithelial-like appearance of tumorigenic cells did not reflect a mesenchymal-epithelial transition; in fact, cen3tel cells expressed vimentin and did not express cytokeratins at all transformation stages. Moreover, they did not express epithelial proteins such as occluding and claudin-1. In contrast, ZO-1 showed higher levels and a more defined membrane localization in tumorigenic cells compared to non-tumorigenic cells; this confirms its role in adherens junction formation in mesenchymal cells and is in agreement with the strong cell-cell contact formation by neoplastically transformed cells. Finally, we found α-catenin and ZO-1 nuclear localization in non-transformed cells, suggestive of possible additional roles of these proteins besides cell junction formation.

Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

Background: Mesenchymal and amoeboid movements are two important mechanisms adopted by cancer cells to invade the surrounding environment. Mesenchymal movement depends on extracellular matrix protease activity, amoeboid movement on the RhoA-dependent kinase ROCK. Cancer cells can switch from one mechanism to the other in response to different stimuli, limiting the efficacy of antimetastatic therapies., Methodology and Principal Findings: We investigated the acquisition and molecular regulation of the invasion capacity of neoplastically transformed human fibroblasts, which were able to induce sarcomas and metastases when injected into immunocompromised mice. We found that neoplastic transformation was associated with a change in cell morphology (from fibroblastic to polygonal), a reorganization of the actin cytoskeleton, a decrease in the expression of several matrix metalloproteases and increases in cell motility and invasiveness. In a three-dimensional environment, sarcomagenic cells showed a spherical morphology with cortical actin rings, suggesting a switch from mesenchymal to amoeboid movement. Accordingly, cell invasion decreased after treatment with the ROCK inhibitor Y27632, but not with the matrix protease inhibitor Ro 28-2653. The increased invasiveness of tumorigenic cells was associated with reduced expression of Rnd3 (also known as RhoE), a cellular inhibitor of ROCK. Indeed, ectopic Rnd3 expression reduced their invasive ability in vitro and their metastatic potential in vivo., Conclusions: These results indicate that, during neoplastic transformation, cells of mesenchymal origin can switch from a mesenchymal mode of movement to an amoeboid one. In addition, they point to Rnd3 as a possible regulator of mesenchymal tumor cell invasion and to ROCK as a potential therapeutic target for sarcomas.

The adenine misincorporated by replicative DNA polymerases (pols) opposite 7,8-dihydro-8-oxoguanine (8-oxo-G) is removed by a specific glycosylase, leaving the lesion on the DNA. Subsequent incorporation of C opposite 8-oxo-G on the resulting 1-nt gapped DNA is essential for the removal of the 8-oxo-G to prevent G-C to T-A transversion mutations. By using model DNA templates, purified DNA pols beta and lambda and knockout cell extracts, we show here that the auxiliary proteins replication protein A and proliferating cell nuclear antigen act as molecular switches to activate the DNA pol lambda- dependent highly efficient and faithful repair of A:8-oxo-G mismatches in human cells and to repress DNA pol beta activity. By using an immortalized human fibroblast cell line that has the potential to induce cancer in mice, we show that the development of a tumoral phenotype in these cells correlated with a differential expression of DNA pols lambda and beta.