CD40 ligand-expressing recombinant vaccinia virus promotes the generation of CD8(+) central memory T cells.

Central memory CD8(+) T cells (TCM ) play key roles in the protective immunity against infectious agents, cancer immunotherapy, and adoptive treatments of malignant and viral diseases. CD8(+) TCM cells are characterized by specific phenotypes, homing, and proliferative capacities. However, CD8(+) TCM -cell generation is challenging, and usually requires CD4(+) CD40L(+) T-cell "help" during the priming of naïve CD8(+) T cells. We have generated a replication incompetent CD40 ligand-expressing recombinant vaccinia virus (rVV40L) to promote the differentiation of human naïve CD8(+) T cells into TCM specific for viral and tumor-associated antigens. Soluble CD40 ligand recombinant protein (sCD40L), and vaccinia virus wild-type (VV WT), alone or in combination, were used as controls. Here, we show that, in the absence of CD4(+) T cells, a single "in vitro" stimulation of naïve CD8(+) T cells by rVV40L-infected nonprofessional CD14(+) antigen presenting cells promotes the rapid generation of viral or tumor associated antigen-specific CD8(+) T cells displaying TCM phenotypic and functional properties. These observations demonstrate the high ability of rVV40L to fine tune CD8(+) mediated immune responses, and strongly support the use of similar reagents for clinical immunization and adoptive immunotherapy purposes.

MAGE-A10 cancer/testis antigen is highly expressed in high-grade non-muscle-invasive bladder carcinomas.

Bladder cancer is a common urinary malignancy and a prevalent cause of cancer-related death. Current therapies of early stage non-muscle-invasive bladder cancer (NMIBC) are frequently associated with undesirable toxicities and recurrence. Active antigen-specific immunotherapy may provide a valid therapeutic option for patients with NMIBC. Cancer-testis antigens (CTA) expressed in various tumour types and in a limited range of healthy tissues may represent potential targets for specific immunotherapy. MAGE-A10 is probably the most immunogenic antigen of the MAGE-A family. We evaluated the expression of MAGE-A10 in NMIBC. Seventy-nine patients undergoing surgical treatment for NMIBC were enrolled in the study. MAGE-A10 gene expression was assessed by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed on paraffin-embedded sections. MAGE-A10 gene was specifically expressed in one-third of NMIBC (n = 24: 32.43%). Gene expression was correlated with high tumour grade. MAGE-A10 protein was exclusively detectable in nuclei of tumour cells. More importantly, MAGE-A10 protein was also more frequently detectable in high-grade tumours (p = 0.0001) and in stage T1 tumours invading subepithelial tissue or lamina propria (p = 0.01). A strong correlation between MAGE-A10 staining score and tumour grade and stage could accordingly be observed. These data indicate that MAGE-A10 expression is a feature of aggressive NMIBC and might be used as a novel target for specific immunotherapy of these cancers.

Differential patterns of large tumor antigen-specific immune responsiveness in patients with BK polyomavirus-positive prostate cancer or benign prostatic hyperplasia.

The role of the polyomavirus BK (BKV) large tumor antigen (L-Tag) as a target of immune response in patients with prostate cancer (PCa) has not been investigated thus far. In this study, we comparatively analyzed humoral and cellular L-Tag-specific responsiveness in age-matched patients bearing PCa or benign prostatic hyperplasia, expressing or not expressing BKV L-Tag-specific sequences in their tissue specimens, and in non-age-matched healthy individuals. Furthermore, results from patients with PCa were correlated to 5-year follow-up clinical data focusing on evidence of biochemical recurrence (BR) after surgery (prostate specific antigen level of ≥0.2 ng/ml). In peripheral blood mononuclear cells (PBMC) from patients with PCa with evidence of BR and BKV L-Tag-positive tumors, stimulation with peptides derived from the BKV L-Tag but not those derived from Epstein-Barr virus, influenza virus, or cytomegalovirus induced a peculiar cytokine gene expression profile, characterized by high expression of interleukin-10 (IL-10) and transforming growth factor ß1 and low expression of gamma interferon genes. This pattern was confirmed by protein secretion data and correlated with high levels of anti-BKV L-Tag IgG. Furthermore, in PBMC from these PCa-bearing patients, L-Tag-derived peptides significantly expanded an IL-10-secreting CD4(+) CD25(+(high)) CD127(-) FoxP3(+) T cell population with an effector memory phenotype (CD103(+)) capable of inhibiting proliferation of autologous anti-CD3/CD28-triggered CD4(+) CD25(-) T cells. Collectively, our findings indicate that potentially tolerogenic features of L-Tag-specific immune response are significantly associated with tumor progression in patients with BKV(+) PCa.

Immunohistochemical analysis of the expression of MAGE-A and NY-ESO-1 cancer/testis antigens in diffuse large B-cell testicular lymphoma.

BACKGROUND: Primary testicular lymphoma (PTL) is a rare and lethal disease. The most common histological subtype is diffuse large B-cell lymphoma (DLBCL). Standard treatments are frequently ineffective. Thus, the development of novel forms of therapy is urgently required. Specific immunotherapy generating immune responses directed against antigen predominantly expressed by cancer cells such as cancer-testis antigens (CTA) may provide a valid alternative treatment for patients bearing PTL, alone or in combination with current therapies. METHODS: Three monoclonal antibodies (mAbs), 77B recognizing MAGE-A1, 57B recognizing an epitope shared by multiple MAGE-A CTA (multi-MAGE-A specific) and D8.38 recognizing NY-ESO-1/LAGE-1 were used for immunohistochemical staining of 27 PTL, including 24 DLBCL. RESULTS: Expression of MAGE-A1 was infrequently detectable in DLBCL specimens (12.50%), whereas multi-MAGE-A and NY-ESO-1/LAGE-1 specific reagents stained the cytoplasms of tumor cells in DLBCL specimens with higher frequencies (54.17% and 37.50%, respectively) with different expression levels. CONCLUSIONS: These results suggest that MAGE-A and NY-ESO-1/LAGE-1, possibly in combination with other CTA, might be used as targets for specific immunotherapy in DLBCL.

Preventing vaccinia virus class-I epitopes presentation by HSV-ICP47 enhances the immunogenicity of a TAP-independent cancer vaccine epitope.

Herpes simplex virus protein ICP47, encoded by US12 gene, strongly downregulates major histocompatibility complex (MHC) class-I antigen restricted presentation by blocking transporter associated with antigen processing (TAP) protein. To decrease viral vector antigenic immunodominance and MHC class-I driven clearance, we engineered recombinant vaccinia viruses (rVV) expressing ICP47 alone (rVV-US12) or together with endoplasmic reticulum (ER)-targeted Melan-A/MART-1(27-35) model tumor epitope (rVV-MUS12). In this study, we show that antigen presenting cells (APC), infected with rVV-US12, display a decreased ability to present TAP dependent MHC class-I restricted viral antigens to CD8+ T-cells. While HLA class-I cell surface expression is strongly downregulated, other important immune related molecules such as CD80, CD44 and, most importantly, MHC class-II are unaffected. Characterization of rVV-MUS12 infected cells demonstrates that over-expression of a TAP-independent peptide, partially compensates for ICP47 induced surface MHC class-I downregulation (30% vs. 70% respectively). Most importantly, in conditions where clearance of infected APC by virus-specific CTL represents a limiting factor, a significant enhancement of CTL responses to the tumor epitope can be detected in cultures stimulated with rVV-MUS12, as compared to those stimulated by rVV-MART alone. Such reagents could become of high relevance in multiple boost protocols required for cancer immunotherapy, to limit vector-specific responsiveness.

VCP/p97 cofactor UBXN1/SAKS1 regulates mitophagy by modulating MFN2 removal from mitochondria.

Initiation of PINK1- and PRKN-dependent mitophagy is a highly regulated process involving the activity of the AAA-ATPase VCP/p97, a cofactor-guided multifunctional protein central to handling ubiquitinated client proteins. Removal of ubiquitinated substrates such as the mitofusin MFN2 from the outer mitochondrial membrane by VCP is critical for PRKN accumulation on mitochondria, which drives mitophagy. Here we characterize the role of the UBA and UBX-domain containing VCP cofactor UBXN1/SAKS1 during mitophagy. Following mitochondrial depolarization and depending on PRKN, UBXN1 translocated alongside VCP to mitochondria. Prior to mitophagy, loss of UBXN1 led to mitochondrial fragmentation, diminished ATP production, and impaired ER-mitochondrial apposition. When mitophagy was induced in cells lacking UBXN1, mitochondrial translocation of VCP and PRKN was impaired, diminishing mitophagic flux. In addition, UBXN1 physically interacted with PRKN in a UBX-domain depending manner. Interestingly, ectopic expression of the pro-mitophagic VCP cofactor UBXN6/UBXD1 fully reversed impaired PRKN recruitment in UBXN1-/- cells. Mechanistically, UBXN1 acted downstream of PINK1 by facilitating MFN2 removal from mitochondria. In UBXN1-/- cells exposed to mitochondrial stress, MFN2 formed para-mitochondrial blobs likely representing blocked intermediates of the MFN2 removal process partly reversible by expression of UBXN6. Presence of these MFN2 blobs strongly correlated with impaired PRKN translocation to depolarized mitochondria. Our observations connect the VCP cofactor UBXN1 to the initiation and maintenance phase of PRKN-dependent mitophagy, and indicate that, upon mitochondrial stress induction, MFN2 removal from mitochondria occurs through a specialized process.

Tumor infiltration by FcγRIII (CD16)+ myeloid cells is associated with improved survival in patients with colorectal carcinoma.

The prognostic significance of macrophage and natural killer (NK) cell infiltration in colorectal carcinoma (CRC) microenvironment is unclear. We investigated the CRC innate inflammatory infiltrate in over 1,600 CRC using two independent tissue microarrays and immunohistochemistry. Survival time was assessed using the Kaplan-Meier method and Cox proportional hazards regression analysis in a multivariable setting. Spearman's rank correlation tested the association between macrophage and lymphocyte infiltration. The Basel study included over 1,400 CRCs. The level of CD16+ cell infiltration correlated with that of CD3+ and CD8+ lymphocytes but not with NK cell infiltration. Patients with high CD16+ cell infiltration (score 2) survived longer than patients with low (score 1) infiltration (p = 0.008), while no survival difference between patients with score 1 or 2 for CD56+ (p = 0.264) or CD57+ cell (p = 0.583) infiltration was detected. CD16+ infiltrate was associated with improved survival even after adjusting for known prognostic factors including pT, pN, grade, vascular invasion, tumor growth and age [(p = 0.001: HR (95% CI) = 0.71 (0.6-0.9)]. These effects were independent from CD8+ lymphocyte infiltration [(p = 0.036: HR (95% CI) = 0.81 (0.7-0.9)] and presence of metastases [(p = 0.002: HR (95% CI) = 0.43 (0.3-0.7)]. Phenotypic studies identified CD16+ as CD45+CD33+CD11b+CD11c+ but CD64- HLA-DR-myeloid cells. Beneficial effects of CD16+ cell infiltration were independently validated by a study carried out at the University of Athens confirming that patients with CD16 score 2 survived longer than patients with score 1 CRCs (p = 0.011). Thus, CD16+ cell infiltration represents a novel favorable prognostic factor in CRC.

Elevated levels of circulating IL-7 and IL-15 in patients with early stage prostate cancer.

BACKGROUND: Chronic inflammation has been suggested to favour prostate cancer (PCA) development. Interleukins (IL) represent essential inflammation mediators. IL-2, IL-7, IL-15 and IL-21, sharing a common receptor γ chain (c-γ), control T lymphocyte homeostasis and proliferation and play major roles in regulating cancer-immune system interactions. We evaluated local IL-2, IL-7, IL-15 and IL-21 gene expression in prostate tissues from patients with early stage PCA or benign prostatic hyperplasia (BPH). As control, we used IL-6 gene, encoding an IL involved in PCA progression. IL-6, IL-7 and IL-15 titres were also measured in patients' sera. METHODS: Eighty patients with BPH and 79 with early (1 to 2c) stage PCA were enrolled. Gene expression in prostate tissues was analyzed by quantitative real-time PCR (qRT-PCR). Serum IL concentrations and acute phase protein titres were evaluated by ELISA. Mann-Whitney, Wilcoxon and χ(2) tests were used to compare IL gene expression and serum titers in the two groups of patients. Receiver operating characteristic (ROC) curves were constructed to evaluate the possibility to distinguish sera from different groups of patients based on IL titers. RESULTS: IL-2 and IL-21 gene expression was comparably detectable, with low frequency and at low extents, in PCA and BPH tissues. In contrast, IL-6, IL-7 and IL-15 genes were expressed more frequently (p < 0.0001, p = 0.0047 and p = 0.0085, respectively) and to significantly higher extents (p = 0.0051, p = 0.0310 and p = 0.0205, respectively) in early stage PCA than in BPH tissues. Corresponding proteins could be detected to significantly higher amounts in sera from patients with localized PCA, than in those from patients with BPH (p = 0.0153, p = 0.0174 and p = 0.0064, respectively). Analysis of ROC curves indicates that IL-7 (p = 0.0039), but not IL-6 (p = 0.2938) or IL-15 (p = 0.1804) titres were able to distinguish sera from patients with malignancy from those from patients with benign disease. Serum titres of C reactive (CRP), high mobility group B1 (HMGB1) and serum amyloid A (SAA) acute phase proteins were similar in both groups of patients. CONCLUSIONS: Expression IL-7 and IL-15 genes in prostate tissues and corresponding serum titres are significantly increased in patients with early stage PCA as compared with patients with BPH.

New dimensions in tumor immunology: what does 3D culture reveal?

Experimental models indicate that tumor cells in suspension, unlike solid tumor fragments, might be unable to produce life-threatening cancer outgrowth when transferred to animal models, irrespective of the number of cells transferred, although they induce specific immune responses. Human tumor cells cultured in three dimensions display increased pro-angiogenic capacities and resistance to interferons, chemotherapeutic agents or irradiation, as compared with cells cultured in two-dimensional (2D) monolayers. Tumor cells cultured in three dimensions were also shown to be characterized by defective immune recognition by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens (TAAs) and by a capacity to inhibit CTL proliferation and dendritic cell (DC) functions. Downregulation of human leukocyte antigen (HLA) or TAA expression and high production of lactic acid might play a role in the elicitation of these effects. Here, we propose that growth in 3D architectures might provide new insights into tumor immunology and could represent an integral missing component in pathophysiological tumor immune escape mechanisms.

Whole blood assessment of antigen specific cellular immune response by real time quantitative PCR: a versatile monitoring and discovery tool.

BACKGROUND: Monitoring of cellular immune responses is indispensable in a number of clinical research areas, including microbiology, virology, oncology and autoimmunity. Purification and culture of peripheral blood mononuclear cells and rapid access to specialized equipment are usually required. We developed a whole blood (WB) technique monitoring antigen specific cellular immune response in vaccinated or naturally sensitized individuals. METHODS: WB (300 microl) was incubated at 37 degrees C with specific antigens, in the form of peptides or commercial vaccines for 5-16 hours. Following RNAlater addition to stabilize RNA, the mixture could be stored over one week at room temperature or at 4 degrees C. Total RNA was then extracted, reverse transcribed and amplified in quantitative real-time PCR (qRT-PCR) assays with primers and probes specific for cytokine and/or chemokine genes. RESULTS: Spiking experiments demonstrated that this technique could detect antigen specific cytokine gene expression from 50 cytotoxic T lymphocytes (CTL) diluted in 300 microl WB. Furthermore, the high sensitivity of this method could be confirmed ex-vivo by the successful detection of CD8+ T cell responses against HCMV, EBV and influenza virus derived HLA-A0201 restricted epitopes, which was significantly correlated with specific multimer staining. Importantly, a highly significant (p = 0.000009) correlation between hepatitis B surface antigen (HBsAg) stimulated IL-2 gene expression, as detectable in WB, and specific antibody titers was observed in donors vaccinated against hepatitis B virus (HBV) between six months and twenty years before the tests. To identify additional markers of potential clinical relevance, expression of chemokine genes was also evaluated. Indeed, HBsAg stimulated expression of MIP-1beta (CCL4) gene was highly significantly (p = 0.0006) correlated with specific antibody titers. Moreover, a longitudinal study on response to influenza vaccine demonstrated a significant increase of antigen specific IFN-gamma gene expression two weeks after immunization, declining thereafter, whereas increased IL-2 gene expression was still detectable four months after vaccination. CONCLUSION: This method, easily amenable to automation, might qualify as technology of choice for high throughput screening of immune responses to large panels of antigens from cohorts of donors. Although analysis of cytokine gene expression requires adequate laboratory infrastructure, initial antigen stimulation and storage of test probes can be performed with minimal equipment and time requirements. This might prove important in "field" studies with difficult access to laboratory facilities.

In Vitro Modeling of Tumor-Immune System Interaction.

Immunotherapy has emerged during the past two decades as an innovative and successful form of cancer treatment. However, frequently, mechanisms of actions are still unclear, predictive markers are insufficiently characterized, and preclinical assays for innovative treatments are poorly reliable. In this context, the analysis of tumor/immune system interaction plays key roles, but may be unreliably mirrored by in vivo experimental models and standard bidimensional culture systems. Tridimensional cultures of tumor cells have been developed to bridge the gap between in vitro and in vivo systems. Interestingly, defined aspects of the interaction of cells from adaptive and innate immune systems and tumor cells may also be mirrored by 3D cultures. Here we review in vitro models of cancer/immune cell interaction and we propose that updated technologies might help develop innovative treatments, identify biologicals of potential clinical relevance, and select patients eligible for immunotherapy treatments.

Expression of Indoleamine 2,3-Dioxygenase Induced by IFN-γ and TNF-α as Potential Biomarker of Prostate Cancer Progression.

Inflammation has been suggested to play an important role in onset and progression of prostate cancer (PCa). Histological analysis of prostatectomy specimens has revealed focal inflammation in early stage lesions of this malignancy. We addressed the role of inflammatory stimuli in the release of PCa-specific, tumor-derived soluble factors (PCa-TDSFs) already reported to be mediators of PCa morbidity, such as indoleamine 2,3-dioxygenase (IDO) and interleukin (IL)-6. Inflammation-driven production and functions of PCa-TDFSs were tested "in vitro" by stimulating established cell lines (CA-HPV-10 and PC3) with IFN-γ or TNF-α. Expression of genes encoding IDO, IL-6, IFN-γ, TNF-α, and their receptors was investigated in tumor tissues of PCa patients undergoing radical prostatectomy, in comparison with benign prostatic hyperplasia (BPH) specimens. IFN-γ and TNF-α-treatment resulted in the induction of IDO and IL-6 gene expression and release in established cell lines, suggesting that the elicitation of PCa-TDSFs by these cytokines might contribute to progression of cancer into an untreatable phenotype. An analysis based on timing of biochemical recurrence revealed the prognostic value of IDO but not IL-6 gene expression in predicting recurrence-free survival in patients (RFS) with PCa. In addition, a urine-based mRNA biomarker study revealed the diagnostic potential of IDO gene expression in urines of men at risk of PCa development.

Use of multicellular tumor spheroids to dissect endothelial cell-tumor cell interactions: a role for T-cadherin in tumor angiogenesis.

This study addresses establishment of an "in vitro" melanoma angiogenesis model using multicellular tumor spheroids (MCTS) of differentiated (HBL) or undifferentiated (NA8) melanoma cell lines. DNA microarray assay and qRT-PCR indicated upregulation of pro-angiogenic factors IL-8, VEGF, Ephrin A1 and ANGPTL4 in NA8-MCTSs (vs. monolayers) whereas these were absent in MCTS and monolayer cultures of HBL. Upon co-culture with endothelial cell line HMEC-1 NA8-MCTS attract, whereas HBL-MCTS repulse, HMEC-1. Overexpression of T-cadherin in HMEC-1 leads to their increased invasion and network formation within NA8-MCTS. Given an appropriate angiogenic tumor microenvironment, T-cadherin upregulation on endothelial cells may potentiate intratumoral angiogenesis.

Expression of Indoleamine 2,3-Dioxygenase Gene Is a Feature of Poorly Differentiated Non-muscle-invasive Urothelial Cell Bladder Carcinomas.

AIM: To evaluate indoleamine 2,3-dioxygenase (IDO) gene expression in non-muscle-invasive urothelial cell bladder carcinoma (NMIBC). PATIENTS AND METHODS: Seventy-four patients undergoing surgical treatment for NMIBC were enrolled in the study. IDO gene expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: IDO gene expression was detectable significantly more frequently (48/74, 64.86% vs. 5/21, 23.81%, p<0.001) and to significantly higher extents (p=0.01) in cancer tissues than in normal bladder mucosa. IDO gene expression was observed significantly more frequently in large (p=0.02), high-grade (p=0.05) and stage T1 (p=0.03) than in small, low-grade and stage Ta tumors. Expression levels were also significantly higher in large, high-grade and stage T1 tumors (p<0.01, p=0.05 and p=0.03, respectively). A direct positive correlation between IDO gene expression in tumor tissues and tumor size (R=0.24, p=0.04), grade (R=0.23, p=0.05) and stage (R=0.25, p=0.03) was detected. Multivariate analysis suggested a trend (p=0.08) towards longer overall survival in patients bearing tumors that did not express IDO gene. CONCLUSION: These data indicate that IDO gene expression is a feature of aggressive NMIBC, suggesting a potential immunosuppressive role of IDO.

MAGE-A Antigens and Cancer Immunotherapy.

MAGE-A antigens are expressed in a variety of cancers of diverse histological origin and germinal cells. Due to their relatively high tumor specificity, they represent attractive targets for active specific and adoptive cancer immunotherapies. Here, we (i) review past and ongoing clinical studies targeting these antigens, (ii) analyze advantages and disadvantages of different therapeutic approaches, and (iii) discuss possible improvements in MAGE-A-specific immunotherapies.

T-cadherin in prostate cancer: relationship with cancer progression, differentiation and drug resistance.

Prostate cancer represents the second leading cause of cancer-related death in men. T-cadherin (CDH13) is an atypical GPI-anchored member of the cadherin family of adhesion molecules. Its gene was reported to be downregulated in a small series of prostate tumours. T-cadherin protein expression/localisation in prostate tissue has never been investigated. The purpose of our study was to analyse CDH13 gene and protein levels in large sets of healthy and cancer prostate tissue specimens and evaluate CDH13 effects on the sensitivity of prostate cancer cells to chemotherapy. Analysis of CDH13 gene expression in the TCGA RNAseq dataset for prostate adenocarcinoma (N = 550) and in tissue samples (N = 101) by qPCR revealed weak positive correlation with the Gleason score in cancer and no difference between benign and malignant specimens. Immunohistochemical analysis of tissue sections (N = 12) and microarrays (N = 128 specimens) demonstrated the presence of CDH13 on the apical surface and at intercellular contacts of cytokeratin 8-positive luminal cells and cells double-positive for cytokeratin 8 and basal marker p63. T-cadherin protein expression was markedly upregulated in cancer as compared to benign prostate hyperplasia, the increase being more prominent in organ-confined than in advanced hormone-resistant tumours, and correlated negatively with the Gleason pattern. T-cadherin protein level correlated strongly with cytokeratin 8 and with an abnormal diffuse/membrane localisation pattern of p63. Ectopic expression of CDH13 in metastatic prostate cancer cell line DU145 reduced cell growth in the presence of doxorubicin. We conclude that CDH13 protein, but not its gene expression, is strongly upregulated in early prostate cancer, correlates with changes in luminal/basal differentiation and p63 localisation, and promotes sensitivity of cancer cells to doxorubicin. These data identify CDH13 as a novel molecule relevant for prostate cancer progression and response to therapy.

Nonreplicating recombinant vaccinia virus expressing CD40 ligand enhances APC capacity to stimulate specific CD4+ and CD8+ T cell responses.

Recombinant poxviruses expressing immunomodulatory molecules together with specific antigens represent powerful vaccines for cancer immunotherapy. Recently, we and others have demonstrated, in vitro and in vivo, that coexpression of CD80 and CD86 costimulatory molecules enhances the immunogenic capacity of a recombinant vaccinia virus (rVV) encoding different tumor-associated antigens. To further investigate the capacity of these vectors to provide ligands for different costimulatory pathways relevant in the generation of T cell responses, we constructed a recombinant virus (rVV) expressing CD40 ligand or CD154 (CD154rVV). Upon binding the CD40 receptor expressed on antigen presenting cells (APC), this molecule, physiologically expressed on activated CD4+ T cells, increases their antigen presentation and immunostimulatory capacities. Therefore, we evaluated the effects of CD154rVV infection on APC activation and its consequences on T cell stimulation. CD154rVV infection of autologous fibroblasts, monocytes, or iDC promoted the expression of a number of cytokines, including GM-CSF, TNF-alpha, and IL-15 in iDC. Most importantly, IL-12 p40 gene expression and protein secretion were induced by CD154rVV but not by wild-type VV (WT VV) in either CD14+ cells or iDC, and these effects could be blocked by anti-CD40 monoclonal antibodies. Furthermore, phenotypic characterization of CD154rVV infected iDC revealed enhanced expression of CD83 and CD86 surface markers as compared with wild-type vaccinia virus infection. As expected, VV infection triggered cytokines gene expression in cultures including APC and T cells from VV immune donors. However, cytokine genes typically expressed by T cell receptor triggered T cells such as those encoding IL-2 and IFN-gamma, or T cell proliferation, were detectable to a significantly higher extent in CD154rVV infected cultures, as compared with WT VV. Activation of specific CD8+ T cells was then investigated using MART-1/Melan-A(27-35) epitope as the model of tumor-associated antigen (TAA). In the presence of CD154rVV activated APCs, significantly higher numbers of specific cytotoxic CD8+ T cells were detected, as compared with cultures performed in the presence of WT VV or in the absence of virus. Taken together, these data indicate that functional CD154 expression from rVV infected cells promotes APC activation, thereby enhancing antigen-specific T cell generation. Such a recombinant vector might help bypass the requirement for activated helper cells during CTL priming, thus qualifying as a potentially relevant vector in the generation of CD8+ T cell responses in cancer immunotherapy.

Bioreactor-engineered cancer tissue-like structures mimic phenotypes, gene expression profiles and drug resistance patterns observed "in vivo".

Anticancer compound screening on 2D cell cultures poorly predicts "in vivo" performance, while conventional 3D culture systems are usually characterized by limited cell proliferation, failing to produce tissue-like-structures (TLS) suitable for drug testing. We addressed engineering of TLS by culturing cancer cells in porous scaffolds under perfusion flow. Colorectal cancer (CRC) HT-29 cells were cultured in 2D, on collagen sponges in static conditions or in perfused bioreactors, or injected subcutaneously in immunodeficient mice. Perfused 3D (p3D) cultures resulted in significantly higher (p < 0.0001) cell proliferation than static 3D (s3D) cultures and yielded more homogeneous TLS, with morphology and phenotypes similar to xenografts. Transcriptome analysis revealed a high correlation between xenografts and p3D cultures, particularly for gene clusters regulating apoptotic processes and response to hypoxia. Treatment with 5-Fluorouracil (5-FU), a frequently used but often clinically ineffective chemotherapy drug, induced apoptosis, down-regulation of anti-apoptotic genes (BCL-2, TRAF1, and c-FLIP) and decreased cell numbers in 2D, but only "nucleolar stress" in p3D and xenografts. Conversely, BCL-2 inhibitor ABT-199 induced cytotoxic effects in p3D but not in 2D cultures. Our findings advocate the importance of perfusion flow in 3D cultures of tumor cells to efficiently mimic functional features observed "in vivo" and to test anticancer compounds.

"In vitro" 3D models of tumor-immune system interaction.

Interaction between cancer cells and immune system critically affects development, progression and treatment of human malignancies. Experimental animal models and conventional "in vitro" studies have provided a wealth of information on this interaction, currently used to develop immune-mediated therapies. Studies utilizing three-dimensional culture technologies have emphasized that tumor architecture dramatically influences cancer cell-immune system interaction by steering cytokine production and regulating differentiation patterns of myeloid cells, and decreasing the sensitivity of tumor cells to lymphocyte effector functions. Hypoxia and increased production of lactic acid by tumor cells cultured in 3D architectures appear to be mechanistically involved. 3D culture systems could be further developed to (i) include additional cell partners potentially influencing cancer cell-immune system interaction, (ii) enable improved control of hypoxia, and (iii) allow the use of freshly derived clinical cancer specimens. Such advanced models will represent new tools for cancer immunobiology studies and for pre-clinical assessment of innovative treatments.

GM-CSF Production by Tumor Cells Is Associated with Improved Survival in Colorectal Cancer.

PURPOSE: Colorectal cancer infiltration by CD16(+) myeloid cells correlates with improved prognosis. We addressed mechanistic clues and gene and protein expression of cytokines potentially associated with macrophage polarization. EXPERIMENTAL DESIGN: GM-CSF or M-CSF-stimulated peripheral blood CD14(+) cells from healthy donors were cocultured with colorectal cancer cells. Tumor cell proliferation was assessed by (3)H-thymidine incorporation. Expression of cytokine genes in colorectal cancer and autologous healthy mucosa was tested by quantitative, real-time PCR. A tumor microarray (TMA) including >1,200 colorectal cancer specimens was stained with GM-CSF- and M-CSF-specific antibodies. Clinicopathological features and overall survival were analyzed. RESULTS: GM-CSF induced CD16 expression in 66% ± 8% of monocytes, as compared with 28% ± 1% in cells stimulated by M-CSF (P = 0.011). GM-CSF but not M-CSF-stimulated macrophages significantly (P < 0.02) inhibited colorectal cancer cell proliferation. GM-CSF gene was expressed to significantly (n = 45, P < 0.0001) higher extents in colorectal cancer than in healthy mucosa, whereas M-CSF gene expression was similar in healthy mucosa and colorectal cancer. Accordingly, IL1ß and IL23 genes, typically expressed by M1 macrophages, were expressed to significantly (P < 0.001) higher extents in colorectal cancer than in healthy mucosa. TMA staining revealed that GM-CSF production by tumor cells is associated with lower T stage (P = 0.02), "pushing" growth pattern (P = 0.004) and significantly (P = 0.0002) longer survival in mismatch-repair proficient colorectal cancer. Favorable prognostic effect of GM-CSF production by colorectal cancer cells was confirmed by multivariate analysis and was independent from CD16(+) and CD8(+) cell colorectal cancer infiltration. M-CSF expression had no significant prognostic relevance. CONCLUSIONS: GM-CSF production by tumor cells is an independent favorable prognostic factor in colorectal cancer.