Michele Bianchini

A phase I clinical study of vaccination of melanoma patients with dendritic cells loaded with allogeneic apoptotic/necrotic melanoma cells. Analysis of toxicity and immune response to the vaccine and of IL-10 -1082 promoter genotype as predictor of disease progression

BackgroundSixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression.MethodsPBMC were obtained after leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines each with 5, 10, 15, or 20 × 106 DC/Apo-Nec cell per vaccine, two weeks apart. Immune responses were measured by ELISpot and tetramer analysis. Il-10 genotype was measured by PCR and corroborated by IL-10 production by stimulated PBMC.ResultsImmature DCs efficiently phagocytosed melanoma Apo-Nec cells and matured after phagocytosis as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II, and 75.2 ± 16% reduction in Dextran-FITC endocytosis. CCR7 was also up-regulated upon Apo-Nec uptake in DCs from all patients, and accordingly DC/Apo-Nec cells were able to migrate in vitro toward MIP-3 beta. The vaccine was well tolerated in all patients. The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05). The presence of CD8+T lymphocytes specific to gp100 and Melan A/MART-1 Ags was determined by ELISpot and tetramer analysis in five HLA-A*0201 patients before and after vaccination; one patient had stable elevated levels before and after vaccination; two increased their CD8 + levels, one had stable moderate and one had negligible levels. The analysis of IL-10 promoter -1082 polymorphism in the sixteen patients showed a positive correlation between AA genotype, accompanied by lower in vitro IL-10 production by stimulated PBMC, and faster melanoma progression after lymph nodes surgery (p = 0.04). With a mean follow-up of 49.5 months post-surgery, one stage IIC patient and 7/8 stage III patients remain NED but 7/7 stage IV patients have progressed.ConclusionWe conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags. Patients with a low-producing IL-10 polymorphism appear to have a worst prognosis.Trial registrationClinicaltrials.gov (NHI) NCT00515983

Monocyte-derived dendritic cells loaded with a mixture of apoptotic/necrotic melanoma cells efficiently cross-present gp100 and MART-1 antigens to specific CD8+ T lymphocytes

BackgroundIn the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by γ irradiation (Apo-Nec cells).MethodsWe evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100.ResultsApo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 ± 10.5%) at 12 hs of coculture. By 12–24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC – Dextran uptake (iDC: 81 ± 5%; DC/Apo-Nec 33 ± 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3β. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-γ secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change.ConclusionWe conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3β and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.

Cetuximab-mediated cellular cytotoxicity is inhibited by HLA-E membrane expression in colon cancer cells

Cetuximab, an anti-epidermal growth factor receptor monoclonal antibody, has been shown to increase the median survival of colorectal cancer patients. We previously reported that the expression of HLA-E is significantly increased in primary human colorectal cancer, perhaps contributing to tumour escape from immune surveillance. To establish if HLA-E could be a factor that renders colorectal cancer cells less susceptible to antibody-dependent cellular cytotoxicity (ADCC), in the present study we analysed Cetuximab-mediated cytotoxicity against several colorectal cancer cell lines expressing, or not, HLA-E at the cell surface. We first observed that colorectal cancer cells treated with Cetuximab were killed more efficiently by ADCC. Interestingly, treatment of target cells with recombinant human-β2-microglobulin inhibits Cetuximab-mediated ADCC through HLA-E membrane stabilization. The specific immunosuppressive role of HLA-E was confirmed using an anti-NKG2A monoclonal antibody, that restored the ability of immune cells to kill their target. This result demonstrates that HLA-E at the cell surface can reliably suppress the ADCC effect. On the other hand, Cetuximab induced a direct growth inhibition but only at high concentrations; furthermore, the CDC effect was quite moderate, and we failed to observe a pro-apoptotic effect. Taking into account that our findings suggest that ADCC activity is the main anti-tumour effect observed at clinically achievable concentrations of Cetuximab at the tumour site, we suggest that determination of HLA-E in colorectal cancer could be relevant to predict success of Cetuximab treatment.

Altered phenotype in peripheral blood and tumor-associated NK cells from colorectal cancer patients

Despite NK cells being originally identified because of their ability to kill tumor cells in vitro, only limited information is available on NK cells infiltration of malignant tumors, especially in humans. NK cells infiltrating human colorectal carcinomas (CRCs) were analyzed to identify their potential protective role in an antitumor immune response. The expression and function of relevant molecules were analyzed from different sources, comparing tumor-associated NK cells (TANKs) with autologous peripheral blood NK cells (PB-NKs) from CRC patients—the latter in comparison with PB-NKs from normal donors. TANKs displayed a profound alteration of their phenotype with a drastic reduction of NK cell receptor expression. Co-culture experiments showed that CRC cells produce modulation in NK phenotype and functionality. Moreover, PB-NKs from CRC patients also exhibited an altered phenotype and profound defects in the ability to activate degranulation and IFN-γ production. For the first time, TANK and PB-NK cells from CRC patients have been characterized. It is shown that they are not capable of producing relevant cytokines and degranulate. Taken together, our results suggest that NK cells from CRC patients present alterations of phenotype and function therefore supporting the progression of cancer.

Metallothionein expression in colorectal cancer: Relevance of different isoforms for tumor progression and patient survival

Metallothioneins are a family of small, cysteine-rich proteins with many functions. Immunohistochemical evaluation of all metallothionein 1 + 2 isoforms in colorectal tumors has demonstrated an important down-regulation compared with normal tissue, although its prognostic significance is unclear. Moreover, the contribution of individual isoforms to overall metallothionein down-regulation is not known. To address these important issues, we analyzed the messenger RNA expression levels of all functional metallothionein 1 + 2 isoforms by quantitative reverse transcription polymerase chain reaction in 22 pairs of normal and tumor-microdissected epithelia and correlated these to the overall immunohistochemical protein expression. Our results showed that 5 isoforms (MT1G, 1E, 1F, 1H, and 1M) were lost during the transition from normal mucosa to tumor, whereas MT1X and MT2A were less down-regulated, and their expression was correlated with overall protein positivity. Second, we showed that MT1G hypermethylation occurred in cell lines and in 29% of tumor samples, whereas histone deacetylase inhibitors are able to induce most isoforms. Furthermore, we analyzed by immunohistochemistry 107 normal mucosae, 25 adenomas, 81 carcinomas, and 19 lymph node metastases to evaluate metallothionein expression during different stages of cancer development and to assess its relationship to patient survival. A lower immunohistochemical expression was associated with poorer survival, although it was not an independent predictor. Overall, this study identifies for the first time the relevant metallothionein isoforms for colorectal cancer progression, supports the concept that their loss is associated with worse prognosis, and suggests 2 mechanisms for epigenetic repression of metallothionein expression in colorectal tumors.

Sorting nexin 1 down-regulation promotes colon tumorigenesis

Purpose: Colon cancer is one of the most common human malignancies, yet studies have only begun to identify the multiple mechanisms that underlie the development of this tumor. In this study, we have identified a novel mechanism, dysregulation of endocytic sorting, which promotes colon cancer development. Experimental Design: Immunohistochemical and microarray analyses were done on human colon cancer tissue specimens to determine the levels of one endocytic protein, sorting nexin 1 (SNX1). SW480 cells, a human colon cancer cell line that retains a relatively high level of SNX1 expression, were used to assess the effects of down-regulating this protein by small hairpin RNA. Activation of signal transduction cascades was evaluated in these cells using Western blotting, and multiple functional assays were done. Results: We determined by immunohistochemistry that the level of SNX1 was significantly down-regulated in 75% of human colon cancers. In corroborative studies using microarray analysis, SNX1 message was significantly decreased (log2 ratio less than −1) for 8 of 19 colon carcinomas. Cell lines with reduced SNX1 levels showed increased proliferation, decreased apoptosis, and decreased susceptibility to anoikis. They also showed increased activation of epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 in response to epidermal growth factor. This increased activation was abolished by inhibition of endocytosis. Conclusions: These data suggest that loss of SNX1 may play a significant role in the development and aggressiveness of human colon cancer, at least partially through the mechanism of increased signaling from endosomes. Further, these findings suggest that dysregulation of endocytic proteins may represent a new paradigm in the process of carcinogenesis.

IL-2- or IL-15-activated NK cells enhance Cetuximab-mediated activity against triple-negative breast cancer in xenografts and in breast cancer patients.

Triple-negative breast cancer (TNBC) patients do not benefit from target-specific treatments and is associated with a high relapse rate. Epidermal growth factor receptor is frequently expressed in TNBC and is a candidate for new therapies. In this work, we studied Cetuximab-mediated immune activity by NK cells. Thirteen activating/inhibitory receptors were examined on peripheral blood and tumor infiltrating NK cells. NK-cell functionality was evaluated using as effectors tumor-modulated NK cells and NK cells from patients. We evaluated the treatment with Cetuximab plus IL-2 or IL-15 in vivo in TNBC xenografts. Tumor NK-cells receptor profile showed upregulation of inhibitory receptors and downregulation of activating ones. Tumor-modulated NK cells were less cytotoxic. They could perform antibody-dependent cellular cytotoxicity (ADCC) triggered by Cetuximab, although impaired, it could still be restored by stimulation with IL-2 or IL-15. Patients with advanced disease displayed diminished levels of ADCC compared to healthy volunteers. ADCC was restored and potentiated with both cytokines, which were also effective in enhancing the therapeutic activity of Cetuximab in vivo. The combination of Cetuximab with IL-15 and IL-2 may be considered an attractive therapeutic approach to enhance the clinical efficacy of Cetuximab in TNBC.

Anti-leukemic and anti-GVHD effects of campath-1H in acute lymphoblastic leukemia relapsed after stem-cell transplantation

Despite aggressive approaches, including second transplant, donor lymphocyte infusion and several new agents, the prognosis of acute lymphoid leukemia (ALL) patients relapsing after stem-cell transplantation (SCT) remains poor. Monoclonal-antibodies (moAb) could provide a useful tool in this setting. In particular, anti-CD52 moAb is useful in lymphoid malignancies. We thus treated as compassionate with campath-1H 3 ALL patients relapsed after SCT. In 2 cases we observed a reduction of peripheral blood and/or bone marrow blasts. In 1 case a GVHD grade reduction was observed. Larger trials are required in order to define the role of campath-1H in ALL.

Protein expression changes during human triple negative breast cancer cell line progression to lymph node metastasis in a xenografted model in nude mice

Triple negative breast cancers (TNBC) lacking hormone receptors and HER-2 amplification are very aggressive tumors. Since relevant differences between primary tumors and metastases could arise during tumor progression as evidenced by phenotypic discordances reported for hormonal receptors or HER-2 expression, in this analysis we studied changes that occurred in our TNBC model IIB-BR-G throughout the development of IIB-BR-G-MTS6 metastasis to the lymph nodes (LN) in nude mice, using an antibody-based protein array to characterize their expression profile. We also analyzed their growth kinetics, migration, invasiveness and cytoskeleton structure in vitro and in vivo. In vitro IIB-BR-G-MTS6 cells grew slower but showed higher anchorage independent growth. In vivo IIB-BR-G-MTS6 tumors grew significantly faster and showed a 100% incidence of LN metastasis after s.c. inoculation, although no metastasis was observed for IIB-BR-G. CCL3, IL1β, CXCL1, CSF2, CSF3, IGFBP1, IL1α, IL6, IL8, CCL20, PLAUR, PlGF and VEGF were strongly upregulated in IIB-BR-G-MTS6 while CCL4, ICAM3, CXCL12, TNFRSF18, FIGF were the most downregulated proteins in the metastatic cell line. IIB-BR-G-MTS6 protein expression profile could reflect a higher NFκB activation in these cells. In vitro, IIB-BR-G displayed higher migration but IIB-BR-G-MTS6 had more elevated matrigel invasion ability. In agreement with that observation, IIB-BR-G-MTS6 had an upregulated expression of MMP1, MMP9, MMP13, PLAUR and HGF. IIB-BR-G-MTS6 tumors presented also higher local lymphatic invasion than IIB-BR-G but similar lymphatic vessel densities. VEGFC and VEGFA/B expression were higher both in vitro and in vivo for IIB-BR-G-MTS6. IIB-BR-G-MTS6 expressed more vimentin than IB-BR-G cells, which was mainly localized in the cellular extremities and both cell lines are E-cadherin negative. Our results suggest that IIB-BR-G-MTS6 cells have acquired a pronounced epithelial-to-mesenchymal transition phenotype. Protein expression changes observed between primary tumor-derived IIB-BR-G and metastatic IIB-BR-G-MTS6 TNBC cells suggest potential targets involved in the control of metastasis.

Metallothionein 1G and zinc sensitize human colorectal cancer cells to chemotherapy

Metallothioneins (MT) are a family of low molecular weight proteins that are silenced during colorectal cancer progression, mainly through epigenetic mechanisms, and this loss is associated with poor survival. In this article, we show that overexpression of the MT1G isoform sensitizes colorectal cell lines to the chemotherapeutic agents oxaliplatin (OXA) and 5-fluorouracil (5-FU), in part through enhancing p53 and repressing NF-κB activity. Despite being silenced, MTs can be reinduced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate. In fact, this induction contributes to the cytotoxicity of these agents, given that silencing of MTs by siRNAs reduces their growth-inhibitory activities. Zinc ions also potently enhance MT expression and are cytotoxic to cancer cells. We show for the first time that OXA and 5-FU induce higher levels of intracellular labile zinc, as measured using the fluorescent probe FLUOZIN-3, and that such zinc contributes to the activation of p53 and repression of NF-κB. Addition of zinc enhanced growth inhibition by OXA and 5-FU, and was also capable of resensitizing 5-FU–resistant cell lines to levels comparable with sensitive cell lines. This effect was MT independent because silencing MTs did not affect zinc cytotoxicity. In conclusion, we show that MT induction and zinc administration are novel strategies to sensitize colorectal cancer cells to presently utilized chemotherapeutic agents. Mol Cancer Ther; 13(5); 1369–81. ©2014 AACR.