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Background Cytokine-Induced Killer (CIK) cells are ex vivo expanded T cells with NK cell phenotype. They express both CD3 and CD56 antigens, and exert a potent antitumor activity against a variety of tumors. Several clinical trials demonstrated the safety and the feasibility of CIK cell therapy, with very low side effects and minimal graft-versus-host toxicity. In this study, we developed a GMP-compliant protocol for robust large-scale expansion of CIK cells using G-Rex® gas-permeable static culture flasks. Materials and Methods CIK cells were obtained by stimulating healthy donor PBMCs with GMP-grade IFN-γ, IL-2 and CD3 mAbs, and were cultured in G-Rex6® or G-Rex®6M well plates. CIK cells in G-Rex6® were split only once at day 7 to reduce cell density, whereas the number of CIK cells culterd in G-Rex®6M was not adjusted. In both culture conditions, fresh IL-2 was provided every 3–4 days. We compared these two culture protocols with the culture in standard flasks. Phenotype was analyzed by flow cytometry and cytotoxicity was assessed against several tumor cell lines by calcein-release assay. Results CIK cells cultured in G-Rex6® well plates showed an outstanding cell expansion compared to G-Rex®6M well plates or standard culture flasks, with a 400-fold expansion and a mean of 109 total cells obtained per single well in 14 days, starting from just 2.5 × 106 cells per well. Moreover, the cultures in G-Rex6® were characterized by an higher percentage of CD3+CD56+ cells, as compared to G-Rex®6M or standard culture flasks. Cells cultured in all devices had a comparable expression of NKG2D, NKp30, NKp44, 2B4 receptors. Importantly, CIK cells expanded in G-Rex®6 were as cytotoxic as cells expanded in standard culture flasks. Conversely, CIK cells cultured in G-Rex®6M showed a remarkable reduction of cytotoxicity against tumor cell targets, thus suggesting that cell density during expansion could affect CIK cell activity. Conclusions We propose a GMP-compliant protocol for robust large-scale production of CIK cells. G-Rex® system allows to obtain large amounts of CIK cells highly enriched in the CD3+CD56+ subset and endowed with high cytotoxic activity; this can be accomplished with just a single cell culture split at day 7, which dramatically reduces the culture manipulation as compared to the standard culture flasks. Notably, this strategy can be further and easily scalable to produce CIK cells for clinical immunotherapy applications. Disclosure Information A. Ventura: None. P. Palmerini: None. A. Dalla Pieta: None. R. Sommaggio: None. G. Astori: None. K. Chieregato: None. M. Tisi: None. C. Visco: None. O. Perbellini: None. M. Ruggeri: None. E. Cappuzzello: None. A. Rosato: None.

Background Cytokine-Induced Killer (CIK) cells are a population of effector cells that represents a promising tool for adoptive cell therapy. They are easily expandable ex-vivo, safe, and exert cytotoxicity against a broad range of tumor histotypes.1 We recently reported that they have a relevant expression of FcγRIIIa (CD16a), which can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their cytotoxicity in an antigen-specific manner, to improve their antitumor activity.2 Indeed, the engagement of CD16a on CIK cells leads to a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian cancer both in vitro and in vivo. Based on this observation, we investigated whether CIK cells can be specifically retargeted against B-cell malignancies by combination with anti-CD20 mAbs, namely Rituximab® (RTX) and Obinutuzumab® (OBI). Materials and Methods CIK cells were obtained from peripheral blood mononuclear cells of healthy donors, and stimulated in vitro with IFN-γ, CD3 mAb and IL-2 for 14 days; fresh IL-2 was provided every 3–4 days. CIK cell phenotype was analyzed by multicolor flow cytometry; cytotoxic activity was assessed by calcein AM-release assay against B-cell lines, primary samples and patient-derived xenografts (PDX) obtained from B-cell lymphoma patients after written informed consent. Results The combination with both RTX and OBI significantly increased specific CIK cells lysis against several CD20-expressing lymphoma B cell lines, primary tumors from B-cell lymphoma patients and an established PDX, compared to the combination with a control mAb (cetuximab, CTX). NK-depletion demonstrated that the mAb-mediated cytotoxicity is accountable to the CIK cells fraction within the bulk population since no difference in the lytic activity was detectd in the absence of NK cells. In addition, these results are further supported by in vivo preliminary experiments where the treatment with CIK cells in combination with OBI extensively reduced the growth of PDX and increased mice survival, compared to CIK cells or OBI administered alone. Conclusions Here we proved that CIK cells can be retargeted with clinical-grade mAbs against CD20-expressing lymphomas. These data indicate that the combination of CIK cells with mAbs can represent a novel approach for the treatment of haematological malignancies. References Franceschetti M, Pievani A, Borleri G, Vago L, Fleischhauer K, Golay J, et al. Cytokine-induced killer cells are terminally differentiated activated CD8 cytotoxic T-EMRA lymphocytes. Exp Hematol 2009;37:616–28. Cappuzzello E, Tosi A, Zanovello P, Sommaggio R, Rosato A. Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies. Oncoimmunology 2016 Aug;5(8):e1199311. The research leading to these results has received funding from Fondazione AIRC under IG 2018 - ID. 21354 project - P.I. Rosato Antonio Disclosure Information A. Dalla Pieta: None. E. Cappuzzello: None. P. Palmerini: None. R. Sommaggio: None. G. Astori: None. K. Chieregato: None. O. Perbellini: None. M. Tisi: None. C. Visco: None. M. Ruggeri: None. A. Rosato: None.

Background Cytokine-Induced Killer (CIK) cells share several functional and phenotypical properties of both T and natural killer (NK) cells, and represent an attractive approach for cell-based immunotherapy as they do not require antigen-specific priming for tumor cell recognition, and can be efficiently and rapidly expanded in vitro. We recently reported that CIK cells have a relevant expression of FcγRIIIa (CD16a), which can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their lytic activity in an antigen-specific manner. Here, we report the assessment and the efficacy of this combined approach against triple negative breast cancer (TNBC), an aggressive tumor that still requires reliable therapeutic options. Materials and methods Different primitive and metastatic TNBC cancer mouse models were established in NSG mice, either by implanting patient-derived TNBC samples or MDA-MB-231 cells subcutaneously or orthotopically into the mammary fat pad, or by injecting MDA-MB-231 cells intravenously. The combined treatment consisted in the repeated intratumoral or intravenous injection of CIK cells and cetuximab, while the mAb alone or CIK cells plus Rituximab served as control treatments. Tumor growth and metastasis were monitored by bioluminescence or immunohistochemistry, and survival was recorded. Results CIK cells plus cetuximab significantly restrained primitive tumor growth in mice, either implanted with TNBC patient-derived tumor xenografts or injected with MDA-MB-231 TNBC cell line. Moreover, in both experimental and spontaneous metastatic models the combined approach almost completely abolished metastasis spreading and dramatically improved survival. The antigen-specific mAb favored tumor and metastasis tissue infiltration by CIK cells, and in particular led to an enrichment of the CD16a+ subset. Conclusions Data highlight the potentiality of a novel immunotherapy approach where a non-specific cytotoxic cell population can be converted into tumor-specific effectors with clinical-grade antibodies, thus providing not only a therapeutic option for TNBC but also a valid alternative to more complex approaches based on chimeric antigen receptor-engineered cells. Disclosure Information R. Sommaggio: None. E. Cappuzzello: None. A. Dalla Pieta: None. P. Palmerini: None. A. Tosi: None. D. Carpanese: None. L. Nicole: None. A. Rosato: None.

TP53 missense mutations leading to the expression of mutant p53 oncoproteins are frequent driver events during tumorigenesis. p53 mutants promote tumor growth, metastasis and chemoresistance by affecting fundamental cellular pathways and functions. Here, we demonstrate that p53 mutants modify structure and function of the Golgi apparatus, culminating in the increased release of a pro-malignant secretome by tumor cells and primary fibroblasts from patients with Li-Fraumeni cancer predisposition syndrome. Mechanistically, interacting with the hypoxia responsive factor HIF1α, mutant p53 induces the expression of miR-30d, which in turn causes tubulo-vesiculation of the Golgi apparatus, leading to enhanced vesicular trafficking and secretion. The mut-p53/HIF1α/miR-30d axis potentiates the release of soluble factors and the deposition and remodeling of the ECM, affecting mechano-signaling and stromal cells activation within the tumor microenvironment, thereby enhancing tumor growth and metastatic colonization., p53 mutants can promote tumorigenesis by affecting fundamental cellular pathways and functions. In this study, the authors demonstrate a novel mutant-p53/HIF1α/miR-30d axis that impacts Golgi structure, trafficking, and secretion of proteins essential for tumor growth and metastasis.

Background & Aim Cytokine-Induced Killer (CIK) cells are a population of effector cells that represent a promising tool for adoptive cell therapy. From a technical and methodological point of view, they are easily expandable ex-vivo and safe, and demonstrated efficacy against hematological malignancies. CIK cells exert cytotoxicity against a broad range of tumor histotypes but not against normal tissues and hematopoietic precursors. We recently reported that they have a relevant expression of FcγRIIIa (CD16a), which can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their cytotoxicity in an antigen-specific manner, to improve their antitumor activity. Indeed, the engagement of CD16a on CIK cells leads to a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian cancer both in vitro and in vivo. Based on this observation, we investigated whether CIK cells could be specifically retargeted against B-cell malignancies by combination with anti-CD20 mAbs, namely Rituximab® and Obinutuzumab®. Methods, Results & Conclusion CIK cells were obtained from peripheral blood mononucleated cells of healthy donors, and stimulated in vitro with IFN-γ, CD3 mAb (OKT3) and IL-2 for 14 days; fresh IL-2 was provided every 3-4 days. CIK cell phenotype was analyzed by multicolor flow cytometry; cytotoxic activity was assessed by calcein AM-release assay against EHEB (CLL), Raji (Burkitt lymphoma), RCK-8, TMB-8, Karpas 422 (DLBCL) tumor cell lines, and primary samples obtained from CLL and DLBCL patients after written informed consent. The combination with either the mAbs significantly increased CIK cell cytotoxicity not only against lymphoma cell lines, but also against the primary tumor samples. Depletion of NK cells from CIK cell bulk cultures did not affect target cell lysis, thus confirming that the antibody-mediated increase of cytotoxicity was mainly accountable to the CIK cell fraction. Taken together, these data indicate that the combination treatment with CIK cells and anti-CD20 mAbs could represent a novel approach for the treatment of hematological malignancies, alternative to the use of chimeric antigen receptor (CAR)-T cells.

In cancer, the tumour suppressor gene TP53 undergoes frequent missense mutations that endow mutant p53 proteins with oncogenic properties. Until now, a universal mutant p53 gain-of-function program has not been defined. By means of multi-omics: proteome, DNA interactome (chromatin immunoprecipitation followed by sequencing) and transcriptome (RNA sequencing/microarray) analyses, we identified the proteasome machinery as a common target of p53 missense mutants. The mutant p53–proteasome axis globally affects protein homeostasis, inhibiting multiple tumour-suppressive pathways, including the anti-oncogenic KSRP–microRNA pathway. In cancer cells, p53 missense mutants cooperate with Nrf2 (NFE2L2) to activate proteasome gene transcription, resulting in resistance to the proteasome inhibitor carfilzomib. Combining the mutant p53-inactivating agent APR-246 (PRIMA-1MET) with the proteasome inhibitor carfilzomib is effective in overcoming chemoresistance in triple-negative breast cancer cells, creating a therapeutic opportunity for treatment of solid tumours and metastasis with mutant p53. Walerych et al. show that p53 missense mutants upregulate the proteasome and increase breast cancer cell resistance to proteasome inhibitors. Combined inhibition of p53 mutants and the proteasome leads to increased therapeutic efficacy.

Triple‐negative breast cancer (TNBC) represents the most aggressive breast tumor subtype. However, the molecular determinants responsible for the metastatic TNBC phenotype are only partially understood. We here show that expression of the mitochondrial calcium uniporter (MCU), the selective channel responsible for mitochondrial Ca2+ uptake, correlates with tumor size and lymph node infiltration, suggesting that mitochondrial Ca2+ uptake might be instrumental for tumor growth and metastatic formation. Accordingly, MCU downregulation hampered cell motility and invasiveness and reduced tumor growth, lymph node infiltration, and lung metastasis in TNBC xenografts. In MCU‐silenced cells, production of mitochondrial reactive oxygen species (mROS) is blunted and expression of the hypoxia‐inducible factor‐1α (HIF‐1α) is reduced, suggesting a signaling role for mROS and HIF‐1α, downstream of mitochondrial Ca2+. Finally, in breast cancer mRNA samples, a positive correlation of MCU expression with HIF‐1α signaling route is present. Our results indicate that MCU plays a central role in TNBC growth and metastasis formation and suggest that mitochondrial Ca2+ uptake is a potential novel therapeutic target for clinical intervention.

Background & Aim Cytokine-Induced Killer (CIK) cells are ex vivo expanded T cells with NK cell phenotype. They express both CD3 and CD56 antigens, and exert a potent antitumor activity against a variety of tumors. Several clinical trials demonstrated the safety and the feasibility of CIK cell therapy, with very low side effects and minimal graft-versus-host toxicity. In this study, we developed a GMP-compliant protocol for robust large-scale expansion of CIK cells using G-Rex® gas-permeable static culture flasks. Methods, Results & Conclusion CIK cells were obtained by stimulating healthy donor PBMCs with GMP-grade IFN-γ, IL-2 and CD3 mAbs, and were cultured in G-Rex6® or G-Rex®6M well plates. CIK cells in G-Rex6® were split only once at day 7 to reduce cell density, whereas the number of CIK cells culterd in G-Rex®6M was not adjusted. In both culture conditions, fresh IL-2 was provided every 3-4 days. We compared these two culture protocols with the culture in standard flasks. Phenotype was analyzed by flow cytometry and cytotoxicity was assessed against several tumor cell lines by calcein-release assay. CIK cells cultured in G-Rex6® well plates showed an outstanding cell expansion compared to G-Rex®6M well plates or standard culture flasks, with a 400-fold expansion and a mean of 109 total cells obtained per single well in 14 days, starting from just 2.5 × 106 cells per well. Moreover, the cultures in G-Rex6® were characterized by an higher percentage of CD3+CD56+ cells, as compared to G-Rex®6M or standard culture flasks (38.6±14.9%, 35.0±15.5%, 24.0±17.8%, respectively). Cells cultured in all devices had a comparable expression of NKG2D, NKp30, NKp44, 2B4 receptors. Importantly, CIK cells expanded in G-Rex®6 were as cytotoxic as cells expanded in standard culture flasks. Conversely, CIK cells cultured in G-Rex®6M showed a remarkable reduction of cytotoxicity against tumor cell targets, thus suggesting that cell density during expansion could affect CIK cell activity. We propose a GMP-compliant protocol for robust large-scale production of CIK cells. G-Rex® system allows to obtain large amounts of CIK cells highly enriched in the CD3+CD56+ subset and endowed with high cytotoxic activity; this can be accomplished with just a single cell culture split at day 7, which dramatically reduces the culture manipulation as compared to the standard culture flasks. Notably, this strategy can be further and easily scalable to produce CIK cells for clinical immunotherapy applications.

Cytokine-Induced killer (CIK) cells are raising growing interest in cellular antitumor therapy, as they can be easily expanded with a straightforward and inexpensive protocol, and are safe requiring only GMP-grade cytokines to obtain very high amounts of cytotoxic cells. CIK cells do not need antigen-specific stimuli to be activated and proliferate, as they recognize and destroy tumor cells in an HLA-independent fashion through the engagement of NKG2D. In several preclinical studies and clinical trials, CIK cells showed a reduced alloreactivity compared to conventional T cells, even when challenged across HLA-barriers; only in a few patients, a mild GVHD occurred after treatment with allogeneic CIK cells. Additionally, their antitumor activity can be redirected and further improved with chimeric antigen receptors, clinical-grade monoclonal antibodies or immune checkpoint inhibitors. The evidence obtained from a growing body of literature support CIK cells as a very promising cell population for adoptive immunotherapy. In this review, all these aspects will be addressed with a particular emphasis on the role of the cytokines involved in CIK cell generation, expansion and functionalization.

The identification of universal tumor-specific antigens shared between multiple patients and/or multiple tumors is of great importance to overcome the practical limitations of personalized cancer immunotherapy. Recent studies support the involvement of DEPDC1 in many aspects of cancer traits, such as cell proliferation, resistance to induction of apoptosis and cell invasion, suggesting that it may play key roles in the oncogenic process. In this study, we report that DEPDC1 expression is upregulated in most types of human tumors, and closely linked to a poorer prognosis; therefore, it might be regarded as a novel universal oncoantigen potentially suitable for targeting many different cancers. In this regard, we report the identification of a HLA-A*0201 allele-restricted immunogenic DEPDC1-derived epitope, which is able to induce cytotoxic T lymphocytes (CTL) exerting a strong and specific functional response in vitro toward not only peptide-loaded cells but also triple negative breast cancer (TNBC) cells endogenously expressing the DEPDC1 protein. Such CTL are also therapeutically active against human TNBC xenografts in vivo upon adoptive transfer in immunodeficient mice. Overall, these data provide evidence that this DEPDC1-derived antigenic epitope can be exploited as a new tool for developing immunotherapeutic strategies for HLA-A*0201 patients with TNBC, and potentially many other cancers.

Gastric carcinoma (GC) is the 2nd most common cause of cancer-related death. Despite advances in conventional treatment and surgical interventions, a high percentage of GC patients still have poor survival. Recently, immunotherapy has become a promising approach to treat GC. Here, we present preclinical and clinical studies encouraging the use of vaccination, adoptive T-cell therapy (ACT), and immune checkpoint inhibitors, such as programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). The ongoing immunotherapy clinical trials have shown promising results in safety and tolerability even in late-stage GC patients. Moreover, we highlight that the combination of ACT with chemotherapy could be the best choice to treat GC.

The YAP and TAZ mediators of the Hippo pathway (hereafter called YAP/TAZ) promote tissue proliferation and organ growth. However, how their biological properties intersect with cellular metabolism remains unexplained. Here, we show that YAP/TAZ activity is controlled by the SREBP/mevalonate pathway. Inhibition of the rate-limiting enzyme of this pathway (HMG-CoA reductase) by statins opposes YAP/TAZ nuclear localization and transcriptional responses. Mechanistically, the geranylgeranyl pyrophosphate produced by the mevalonate cascade is required for activation of Rho GTPases that, in turn, activate YAP/TAZ by inhibiting their phosphorylation and promoting their nuclear accumulation. The mevalonate-YAP/TAZ axis is required for proliferation and self-renewal of breast cancer cells. In Drosophila melanogaster, inhibition of mevalonate biosynthesis and geranylgeranylation blunts the eye overgrowth induced by Yorkie, the YAP/TAZ orthologue. In tumour cells, YAP/TAZ activation is promoted by increased levels of mevalonic acid produced by SREBP transcriptional activity, which is induced by its oncogenic cofactor mutant p53. These findings reveal an additional layer of YAP/TAZ regulation by metabolic cues.

Summary Objective Tissue-based xenografts such as cartilage are rejected within weeks by humoral and cellular mechanisms that preclude its clinical application in regenerative medicine. The problem could be overcome by identifying key molecules triggering rejection and the development of genetic-engineering strategies to counteract them. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage reduces the galactose α1,3-galactose (Gal) antigen and delays rejection. Yet, the role of complement activation in this setting is unknown. Design To determine its contribution, we assessed the effect of inhibiting C5 complement component in α1,3-galactosyltransferase-knockout (Gal KO) mice transplanted with porcine cartilage and studied the effect of human complement on porcine articular chondrocytes (PAC). Results Treatment with an anti-mouse C5 blocking antibody for 5 weeks enhanced graft survival by reducing cellular rejection. Moreover, PAC were highly resistant to complement-mediated lysis and primarily responded to human complement by releasing IL-6 and IL-8. This occurred even in the absence of anti-Gal antibody and was mediated by both C5a and C5b-9. Indeed, C5a directly triggered IL-6 and IL-8 secretion and up-regulated expression of swine leukocyte antigen I (SLA-I) and adhesion molecules on chondrocytes, all processes that enhance cellular rejection. Finally, the use of anti-human C5/C5a antibodies and/or recombinant expression of human complement regulatory molecule CD59 (hCD59) conferred protection in correspondence with their specific functions. Conclusions Our study demonstrates that complement activation contributes to rejection of xenogeneic cartilage and provides valuable information for selecting approaches for complement inhibition.

Inflammation is a significant factor in cancer development, and a molecular understanding of the parameters dictating the impact of inflammation on cancers could significantly improve treatment. The tumor suppressor p53 is frequently mutated in cancer, and p53 missense mutants (mutp53) can acquire oncogenic properties. We report that cancer cells with mutp53 respond to inflammatory cytokines increasing their invasive behavior. Notably, this action is coupled to expression of chemokines that can expose the tumor to host immunity, potentially affecting response to therapy. Mechanistically, mutp53 fuels NF-κB activation while it dampens activation of ASK1/JNK by TNFα, and this action depends on mutp53 binding and inhibiting the tumor suppressor DAB2IP in the cytoplasm. Interfering with such interaction reduced aggressiveness of cancer cells in xenografts. This novel interaction is an unexplored mechanism by which mutant p53 can influence tumor evolution, with implications for our understanding of the complex role of inflammation in cancer.

Cytokine-induced Killer (CIK) cells are a heterogeneous population of ex vivo expanded T lymphocytes capable of MHC-unrestricted antitumor activity, which share phenotypic and functional features with both NK and T cells. Preclinical data and initial clinical studies demonstrated their high tolerability in vivo, supporting CIK cells as a promising cell population for adoptive cell immunotherapy. In this study, we report for the first time that CIK cells display a donor-dependent expression of CD16, which can be engaged by trastuzumab or cetuximab to exert a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian and breast cancer cell lines, leading to an increased lytic activity in vitro, and an enhanced therapeutic efficacy in vivo. Thus, an efficient tumor antigen-specific retargeting can be achieved by a combination therapy with clinical-grade monoclonal antibodies already widely used in cancer therapy, and CIK cell populations that are easily expandable in very large numbers, inexpensive, safe and do not require genetic manipulations. Overall, these data provide a new therapeutic strategy for the treatment of Her2 and EGFR expressing tumors by adoptive cell therapy, which could find wide implementation and application, and could also be expanded to the use of additional therapeutic antibodies.

The aim of the study was to identify immunogenic HLA-A*0201-binding epitopes derived from a number of classical midgut carcinoid-associated proteins. CD8(+) T cells recognizing tumor-associated antigen (TAA) epitopes are of great interest for the establishment of immunotherapy as a novel treatment for this type of malignancy.Midgut carcinoid tumor specimens were microdissected and expression levels of potential TAAs were investigated by quantitative real time PCR. HLA-A*0201-binding motifs were selected using HLA peptide binding prediction algorithms and stabilization of HLA-A*0201 was verified using TAP-deficient T2 cells. Peripheral blood of midgut carcinoid patients was analyzed for peptide epitope recognition and the feasibility of generating peptide-reactive CD8(+) T cells in healthy blood donors was examined by an in vitro stimulation protocol using mature DCs. Activation of patient and healthy donor CD8(+) T cells was analyzed by intracellular flow cytometry staining of interferon gamma.Chromogranin A (CGA), tryptophan hydroxylase 1 (TPH-1), vesicular monoamine transporter 1 (VMAT-1), caudal type homeobox transcription factor 2 (CDX-2), and islet autoantigen 2 (IA-2) are properly expressed by midgut carcinoid tumor cells, with CGA mRNA expressed to greatest level. Midgut carcinoid patients have increased frequencies of peripheral blood CD8(+) T cells recognizing a pool of HLA-A*0201 peptides derived from these proteins compared to healthy age-matched individuals. Activated peptide-specific CD8(+) T cells could also be generated in healthy blood donors by in vitro stimulation.We have identified a number of immunogenic midgut carcinoid-associated peptide epitopes recognized by CD8(+) T cells. We show that midgut carcinoid patients display immune recognition of their tumors. Memory CD8(+) T cells in patient blood are of great interest when pursuing an immunotherapeutic treatment strategy.

Advances in animal transgenesis may allow using xenogeneic chondrocytes in tissue-engineering applications for clinical cartilage repair. Porcine cartilage is rejected by humoral and cellular mechanisms that could be overcome by identifying key molecules triggering rejection and developing effective genetic-engineering strategies. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage protects from galactose α1,3-galactose (Gal)-mediated antibody responses. Now, we studied whether expression of a complement inhibitor provides further protection. First, porcine articular chondrocytes (PAC) were isolated from non-transgenic, single and double transgenic pigs expressing HT and moderate levels of human CD59 (hCD59) and their response to human serum was assessed. High recombinant expression of human complement regulatory molecules hCD59 and hDAF was also attained by retroviral transduction of PAC for further analyses. Complement activation on PAC after exposure to 20 % human serum for 24 hours mainly triggered the release of pro-inflammatory cytokines IL-6 and IL-8. Transgenic expression of HT and hCD59 did not suffice to fully counteract this effect. Nevertheless, the combination of blocking anti-Gal antibodies (or C5a) and high hCD59 levels conferred very high protection. On the contrary, high hDAF expression attained the most dramatic reduction in IL-6/IL-8 secretion by a single strategy, but the additional inhibition of anti-Gal antibodies or C5a did not provide further improvement. Notably, we demonstrate that both hCD59 and hDAF inhibit anaphylatoxin release in this setting. In conclusion, our study identifies genetic-engineering approaches to prevent humoral rejection of xenogeneic chondrocytes for use in cartilage repair.

// Silvia Pegoraro 1 , Gloria Ros 1 , Silvano Piazza 2 , Roberta Sommaggio 3 , Yari Ciani 1,2 , Antonio Rosato 3,4 , Riccardo Sgarra 1 , Giannino Del Sal 1,2 , and Guidalberto Manfioletti 1,* 1 Dipartimento di Scienze della Vita, Universita degli Studi di Trieste, Trieste, Italy 2 Laboratorio Nazionale CIB, (LNCIB), Area Science Park, Trieste, Italy 3 Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy 4 Istituto Oncologico Veneto IRCCS, Padova, Italy Correspondence: Guidalberto Manfioletti, email: // Keywords : Epithelial to mesenchymal transition, stemness, HMGA1, breast cancer, invasion, metastasis, gene-signature Received : June 28, 2013 Accepted : July 7, 2013 Published : July 9, 2013 Abstract Breast cancer is a heterogeneous disease that progresses to the critical hallmark of metastasis. In the present study, we show that the High Mobility Group A1 (HMGA1) protein plays a fundamental role in this process in basal-like breast cancer subtype. HMGA1 knockdown induces the mesenchymal to epithelial transition and dramatically decreases stemness and self-renewal. Notably, HMGA1 depletion in basal-like breast cancer cell lines reduced migration and invasion in vitro and the formation of metastases in vivo . Mechanistically, HMGA1 activated stemness and key migration-associated genes which were linked to the Wnt/beta-catenin, Notch and Pin1/mutant p53 signalling pathways. Moreover, we identified a specific HMGA1 gene expression signature that was activated in a large subset of human primary breast tumours and was associated with poor prognosis. Taken together, these data provide new insights into the role of HMGA1 in the acquisition of aggressive features in breast cancer.

Xenotransplantation of genetically engineered porcine chondrocytes may provide a therapeutic solution for the repair of cartilage defects of various types. However, the mechanisms underlying the humoral and cellular responses that lead to rejection of xenogeneic cartilage are not well understood. In this study, we investigated the interaction between human NK cells and isolated porcine costal chondrocytes (PCC). Our data show that freshly isolated NK cells adhere weakly to PCC. Consequently, PCC were highly resistant to cytolysis mediated by freshly isolated NK cells. However, the presence of human natural Abs in the coculture was often sufficient to trigger cytotoxicity against PCC. Furthermore, IL-2 stimulation of NK cells or activation of PCC with the proinflammatory cytokines TNF-α or IL-1α resulted in increased adhesion, which was paralleled by increased NK cell-mediated lysis of PCC. NK cell adhesion to PCC could be blocked by Abs against human LFA-1 and porcine VCAM-1. NKG2D and NKp44 were involved in triggering cytotoxicity against PCC, which expressed ligands for these activating NK cell receptors. Our data further suggest that NKp30 and NKp46 may contribute to the activation of NK cells by PCC under certain conditions. Finally, comparative studies confirmed that PCC are more resistant than porcine aortic endothelial cells to human NK cell-mediated lysis. Thus, the data demonstrate that human NK cells can kill pig chondrocytes and may therefore contribute to rejection of xenogeneic cartilage. In addition, we identify potential targets for intervention to prevent the NK cell response against pig xenografts.

INTRODUCTION: Our aim was to investigate the immune status of midgut carcinoid patients. Cancer patients generally display suppressed Th1-type immunity that disables mounting of an efficient anti-tumor response. However, little is known about patients with neuroendocrine midgut carcinoids. MATERIAL AND METHODS: Circulating regulatory T cells were determined in patient blood by staining for CD4, CD25 and FoxP3 in flow cytometric analysis. T cell proliferation was measured by Alamar Blue in response to polyclonal activation and the regulatory phenotype of patient CD25+ cells was validated by allogeneic stimulation of CFSE labelled responders. Cytokine levels in patient peripheral blood were measured by ELISA and CBA. Tumor infiltrating T cells were analyzed by immunohistochemistry and immunofluorescence. RESULTS: The results demonstrate that midgut carcinoid patients exhibit increased frequencies of circulating Tregs and patient T cells have a decreased proliferative capacity compared to healthy donors. Systemic Th1-promoting cytokines are reduced. Midgut carcinoid tumors display CD4+ and CD8+ T cell infiltration, always in the presence of regulatory CD4+FoxP3+ cells. DISCUSSION: Midgut carcinoid patients display elevated T regulatory cell numbers and T cell dysfunction. Therapeutic strategies to overcome tumor-induced Th1 immunosuppression are required in combination with anti-tumor vaccinations.

Xenotransplantation of genetically engineered porcine chondrocytes may benefit many patients who suffer cartilage defects. In this work, we sought to elucidate the molecular bases of the cellular response to xenogeneic cartilage. To this end, we isolated pig costal chondrocytes (PCC) and conducted a series of functional studies. First, we determined by flow cytometry the cell surface expression of multiple immunoregulatory proteins in resting conditions or after treatment with human TNF-alpha, IL-1alpha, or IL-1beta, which did not induce apoptosis. TNF-alpha and to a lesser extent IL-1alpha led to a marked upregulation of SLA I, VCAM-1, and ICAM-1 on PCC. SLA II and E-selectin remained undetectable in all the conditions assayed. Notably, CD86 was constitutively expressed at moderate levels, whereas CD80 and CD40 were barely detected. To assess their function, we next studied the interaction of PCC with human monoblastic U937 and Jurkat T cells. U937 cells adhered to resting and in a greater proportion to cytokine-stimulated PCC. Consistent with its expression pattern, pig VCAM-1 was key, mediating the increased adhesion after cytokine stimulation. We also conducted coculture experiments with U937 and PCC and measured the release of pig and human cytokines. Stimulated PCC secreted IL-6 and IL-8, whereas U937 secreted IL-8 in response to PCC. Finally, coculture of PCC with Jurkat in the presence of PHA led to a marked Jurkat activation as determined by the increase in IL-2 secretion. This process was dramatically reduced by blocking pig CD86. In summary, CD86 and VCAM-1 on pig chondrocytes may be important triggers of the xenogeneic cellular immune response. These molecules together with TNF could be considered potential targets for intervention in order to develop xenogeneic therapies for cartilage repair.

Mammary epithelial stem cells are fundamental to maintain tissue integrity. Cancer stem cells (CSCs) are implicated in both treatment resistance and disease relapse, and the molecular bases of their malignant properties are still poorly understood. Here we show that both normal stem cells and CSCs of the breast are controlled by the prolyl-isomerase Pin1. Mechanistically, following interaction with Pin1, Notch1 and Notch4, key regulators of cell fate, escape from proteasomal degradation by their major ubiquitin-ligase Fbxw7α. Functionally, we show that Fbxw7α acts as an essential negative regulator of breast CSCs' expansion by restraining Notch activity, but the establishment of a Notch/Pin1 active circuitry opposes this effect, thus promoting breast CSCs self-renewal, tumor growth and metastasis in vivo. In human breast cancers, despite Fbxw7α expression, high levels of Pin1 sustain Notch signaling, which correlates with poor prognosis. Suppression of Pin1 holds promise in reverting aggressive phenotypes, through CSC exhaustion as well as recovered drug sensitivity carrying relevant implications for therapy of breast cancers. © 2013 The Authors., SCOPUS: ar.j, info:eu-repo/semantics/published

Transplantation may be the best option for the repair of many cartilage lesions including early osteoarthritis. Currently, autologous and allogeneic chondrocytes are grafted into cartilage defects to treat selected patients with moderate clinical success. However, their limited use justifies exploring novel therapies for cartilage repair. Xenotransplantation could become a solution by offering high cell availability, quality and genetic engineering capabilities. The rejection process of xenogeneic cartilage is thus being elucidated in order to develop counteractive strategies. Initial studies determined that pig cartilage xenografts are rejected by a slow process comprising humoral and cellular responses in which the galactose α1,3-galactose antigen participates. Since then, our group has identified key mechanisms of the human response to pig chondrocytes (PCs). In particular, human antibody and complement contribute to PC rejection by inducing a pro-inflammatory milieu. Furthermore, PCs express and up-regulate molecules which are functionally relevant for a variety of cellular immune responses (SLA-I, the potent co-stimulatory molecule CD86, and adhesion molecules VCAM-1 and ICAM-1). These participate by triggering a T cell response, as well as supporting a prominent role of the innate immune responses led by natural killer (NK) cells and monocytes/macrophages. Human NK cells lyse PCs by using selected NK activating receptors, whereas human monocytes are activated by PCs to secrete cytokines and chemokines. All this knowledge sets the bases for the development of genetic engineering approaches designed to avert rejection of xenogeneic chondrocytes and leads the way to developing new clinical applications for cartilage repair.