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PDF - 480K, CX3CL1/fractalkine gene and protein expression in healthy colon mucosa, CRC and established cell lines.

PDF file, 3314K, Representative IHC analysis of ALDH1A1 expression in large tissues prostate sections.

PDF file, 46K, Analysis of the expression of ALDH specific isoforms in BPH and PCa surgical specimens.

PDF file, 39K, Characteristics of PCa patients.

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 3H-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, IL1β 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. Clin Cancer Res; 20(12); 3094–106. ©2014 AACR.

PDF - 216K, Notch Box plot of histoscore values in CRC TMA upon staining with GM-CSF and M-CSF specific reagents.

PDF file, 1824K, Differential expression and localization of ALDH1A1 and ALDH7A1 proteins in BPH.

PDF file, 41K.

Purpose: High aldehyde dehydrogenase (ALDH) has been suggested to selectively mark cells with high tumorigenic potential in established prostate cancer cell lines. However, the existence of cells with high ALDH activity (ALDHbright) in primary prostate cancer specimens has not been shown so far. We investigated the presence, phenotype, and clinical significance of ALDHbright populations in clinical prostate cancer specimens.Experimental Design: We used ALDEFLUOR technology and fluorescence-activated cell-sorting (FACS) staining to identify and characterize ALDHbright populations in cells freshly isolated from clinical prostate cancer specimens. Expression of genes encoding ALDH-specific isoforms was evaluated by quantitative real-time PCR in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer tissues. ALDH1A1-specific expression and prognostic significance were assessed by staining two tissue microarrays that included more than 500 samples of BPH, prostatic intraepithelial neoplasia (PIN), and multistage prostate cancer.Results: ALDHbright cells were detectable in freshly excised prostate cancer specimens (n = 39) and were mainly included within the EpCAM(+) and Trop2(+) cell populations. Although several ALDH isoforms were expressed to high extents in prostate cancer, only ALDH1A1 gene expression significantly correlated with ALDH activity (P < 0.01) and was increased in cancers with high Gleason scores (P = 0.03). Most importantly, ALDH1A1 protein was expressed significantly more frequently and at higher levels in advanced-stage than in low-stage prostate cancer and BPH. Notably, ALDH1A1 positivity was associated with poor survival (P = 0.02) in hormone-naïve patients.Conclusions: Our data indicate that ALDH contributes to the identification of subsets of prostate cancer cells of potentially high clinical relevance. Clin Cancer Res; 19(19); 5361–71. ©2013 AACR.

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.

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.

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.

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 naive CD8(+) T cells. We have generated a replication incompetent CD40 ligand-expressing recombinant vaccinia virus (rVV40L) to promote the differentiation of human naive 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 naive 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.

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.

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.

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

Purpose: High aldehyde dehydrogenase (ALDH) has been suggested to selectively mark cells with high tumorigenic potential in established prostate cancer cell lines. However, the existence of cells with high ALDH activity (ALDHbright) in primary prostate cancer specimens has not been shown so far. We investigated the presence, phenotype, and clinical significance of ALDHbright populations in clinical prostate cancer specimens. Experimental Design: We used ALDEFLUOR technology and fluorescence-activated cell-sorting (FACS) staining to identify and characterize ALDHbright populations in cells freshly isolated from clinical prostate cancer specimens. Expression of genes encoding ALDH-specific isoforms was evaluated by quantitative real-time PCR in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer tissues. ALDH1A1-specific expression and prognostic significance were assessed by staining two tissue microarrays that included more than 500 samples of BPH, prostatic intraepithelial neoplasia (PIN), and multistage prostate cancer. Results: ALDHbright cells were detectable in freshly excised prostate cancer specimens (n = 39) and were mainly included within the EpCAM(+) and Trop2(+) cell populations. Although several ALDH isoforms were expressed to high extents in prostate cancer, only ALDH1A1 gene expression significantly correlated with ALDH activity (P < 0.01) and was increased in cancers with high Gleason scores (P = 0.03). Most importantly, ALDH1A1 protein was expressed significantly more frequently and at higher levels in advanced-stage than in low-stage prostate cancer and BPH. Notably, ALDH1A1 positivity was associated with poor survival (P = 0.02) in hormone-naïve patients. Conclusions: Our data indicate that ALDH contributes to the identification of subsets of prostate cancer cells of potentially high clinical relevance. Clin Cancer Res; 19(19); 5361–71. ©2013 AACR.

BACKGROUND: Cancer initiation and progression might be driven by small populations of cells endowed with stem cell-like properties. Here we comparatively addressed the expression of genes encoding putative stemness regulators including c-Myc, Klf4, Nanog, Oct4A and Sox2 genes in benign prostatic hyperplasia (BPH) and prostate cancer (PCA). METHODS: Fifty-eight PCA and thirty-nine BPH tissues samples were used for gene expression analysis, as evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of specific Klf4 isoforms was tested by conventional PCR. Klf4 specific antibodies were used for protein detection in a tissue microarray including 404 prostate samples. RESULTS: Nanog, Oct4A and Sox2 genes were comparably expressed in BPH and PCA samples, whereas c-Myc and Klf4 genes were expressed to significantly higher extents in PCA than in BPH specimens. Immunohistochemical studies revealed that Klf4 protein is detectable in a large majority of epithelial prostatic cells, irrespective of malignant transformation. However, in PCA, a predominantly cytoplasmic location was observed, consistent with the expression of a differentially spliced Klf4alpha isoform. CONCLUSION: Klf4 is highly expressed at gene and protein level in BPH and PCA tissues but a cytoplasmic location of the specific gene product is predominantly detectable in malignant cells. Klf4 location might be of critical relevance to steer its functions during oncogenesis.

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.

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 β1 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.

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.

© 2015 Elsevier Ltd. 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.

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.

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 3H-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, IL1β 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. Clin Cancer Res; 20(12); 3094–106. ©2014 AACR.

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.

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 χ2 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.

The possibility of using the immune system of patients to control tumor outgrowth in a therapeutic setting has always been highly appealing to both clinicians and researchers. However, although cancer cells express tumor-associated antigens that can be targeted by T-cells, clinical trials suggest that the induction of specific immune responses per se may be insufficient to achieve clinical goals. Based on these trial data, in addition to experimental data revealing the complexity of mechanisms controlling immune responsiveness, a reassessment of immunotherapy procedures is underway. As a result, a second generation of antitumor treatments that includes reagents of potential pharmaceutical relevance is being developed. In this review, the most recent literature addressing issues related to immunotherapy for solid tumors is discussed.

In cancer immunotherapy, induction of tumor-reactive CD8+ T cells displaying phenotypic and functional profile of central memory T cells (TCM) is associated with favorable prognosis. A key element in the generation of TCM CD8+ cells is represented by the help provided by CD4+ T cells during the priming of naïve CD8+ T cells. In particular CD40 ligand (CD40L) expressed and/or secreted by activated CD4+ T cells triggers CD40 receptor expressed on antigen presenting cells (APCs) thereby enhancing their antigen presentation capacity. In order to bypass the requirement of CD4+ T cells we generated a non-replicating recombinant vaccinia virus encoding human CD40L (rVV40L) and compared its ability to shape CD8-mediated immune response to soluble CD40L recombinant protein (sCD40L). In this regard, our data clearly underline the different biological properties of membrane-bound CD40L, as provided by rVV40L infection, as compared to its soluble form, in CD14+ APCs activation. Notably, considering expression of IL-12p40, IFN-a and -b genes, cytokines of proven relevance for memory T cell induction, rVV40L-infection was much more potent than s40L-treatment alone or combine with WT infection. In parallel, s40L-stimulation induced a much more significant expression of IL-10 and indoleamine-2, 3-dioxygenase (IDO) genes encoding immunosuppressive factors. Considering a panel of molecules involved in the generation of the immunological synapse with T cells on CD14+ cells, rVV40L appeared to promote a less intense up regulation of CD80 co-stimulatory molecules but, most importantly, only a minor increase of programmed death ligand-1 (PD-L1). Therefore, gene expression and phenotypic profiles suggested that rVV40L-infected CD14+ cells might be highly effective APC in the induction of TCM CD8+ cells. Indeed, a single in vitro stimulation of naïve CD8+ T cells by rVV40L-infected CD14+ cells, in the absence of CD4+ T cells, was able to promote the rapid generation of central memory TAA (MAGE and MART-1) specific CD8+ T cells. These TCM were characterized by the typical central memory phenotype, as indicated by co-expression of CD45RO, CD62L and IL-7Ra, and by the high proliferative potential upon antigen recognition. Collectively our data indicate that rVV40L efficiently modulates the quality of different APC signals delivered during the formation of the immunological synapse with CD8+ T cells. These in-vitro observations validate the strong clinical potential of our recombinant vaccinia virus constructs co-expressing CD40L for cancer immunotherapies. Citation Format: Emanuele Trella, Evangelos Panopoulos, Nermin Raafat, Chantal Mengus, Emmanuel Traunecker, Swantje Heidtmann, Michael Heberer, Daniel Oertli, Giulio Cesare Spagnoli, Paul Zajac. CD40 ligand expressing recombinant vaccinia virus (rVV40L) modulation of central memory CD8-mediated immune response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2490. doi:10.1158/1538-7445.AM2015-2490

Assessment of the effectiveness of chemotherapeutic compounds in bidimensional cell cultures poorly mimics resistance development and the tumor-microenvironment complexity in vivo. In this study we addressed the suitability of a perfused bioreactor to sustain colorectal cancer cell growth and to test established treatment regimens in an in-vitro setting as compared to normal cell culture or xenografts of the same cell line. By culturing the HT29 colorectal cancer cell line in 3D on collagen scaffolds tissue-like structures characterized by a heterogeneous pattern of proliferating and apoptotic cells closely resembling xenografts from the same cell line as generated in immunodeficient mice could be successfully established. CDX2, a colorectal cancer tumor-marker , was expressed in 3D static and perfused cultures, but not in 2D cultures. Upon perfusion, homogeneous seeding on scaffolds could be obtained and significantly higher numbers of tumor cells were recovered, as compared to static cultures (13.7 fold increase, p After 48 hours treatment with clinically relevant concentrations of 5-FU no effect on numbers of cells cultured in 3D under perfusionand as xenografts was observed, as compared to a 55.8% inhibition of 2D cultures. The fraction of Ki67 negative cells (G0 cell phase), was increased after a 48 hours treatment in the 3D perfused and xenografts, but it reached again levels similar to untreated cells after 96 hours. In contrast,cells surviving n 2D cell cultures remained largely positive for Ki67. Importantly, in perfused cultures we could only observe a marginal effect on the expression of BCL-2, TRAF1, FLIP apoptosis resistance genes, as compared to a significant down regulation of their expression in 2D cell cultures and 3D static conditions. In the same line, a combination of ABT-199, a new clinically approved BCL-2 inhibitor, and 5-FU induced additional cytostatic and cytotoxic effects in 3D perfused but not in 2D cell cultures (83.3% inhibition vs. 39.8% p=0.003). Notably, we found that BCL-2 expression in tissues from neoadjuvant treated patients with resistance to treatment was higher than in stage and grade matched colorectal cancers from untreated patients or patients responding to treatment. Cultures in perfused bioreactors offer the possibility of generating tumour tissue-like structures over a short time period. These structures are characterized by sensitivity to chemotherapeutic treatments similar to that of xenografted tumors and significantly different from standard 2D cellular assays.In particular, our data suggest that inhibition of BCL-2 in combination with 5-FU treatment in colorectal cancer could be endowed with therapeutic potential. Citation Format: Christian Hirt, Adam Papadimitropoulos, Evangelos Panopoulos, Valentina Mele, Manuele Muraro, Eleonora Cremonesi, Raoul Droeser, Chantal Mengus, Michael Heberer, Daniel Oertli, Giandomenica Iezzi, Paul Zajac, Serenella Eppenberger-Castori, Luigi Tornillo, Luigi Terracciano, Ivan Martin, Giulio Spagnoli. Resistance of colorectal cancer cells to 5-FU treatment in three dimensional cell culture models under perfusion involves BCL-2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2024. doi:10.1158/1538-7445.AM2014-2024

Recombinant vaccinia virus (rVV) is one of the most immunogenic viral vectors. Its immunogenicity can be further modulated by the insertion of transgenes or deletion of viral genes which are involved in immune responses or specific cellular pathways. We generated a rVV encoding human CD154 (CD40L) and confirmed an efficient licensing of antigen presenting cells (APC), upon infection with a non-replicating (psoralen UV-inactivated) CD40L-rVV, as demonstrated by increased expression of (i) surface ligand/receptors (MHC I/II, CD80), (ii) Th1 cytokines (IL-12, IL-15, TNFa) and most importantly (iii) an increased capacity to stimulate TAA specific CTL proliferation. The hereby presented results are further characterizing some aspects of the pleiotropic roles in immuno-oncology of CD40 triggering via CD40L expressing recombinant vaccinia virus. One of the immuno-modulatory functions of CD40L-CD40 is to play a key role during CD8+ priming for the generation of memory responses. Using sorted naive CD8+ Tcells (CD45RA+CD62L+) primed with allogeneic CD14+ infected with rVV 40L we observed an increased central memory phenotype (CD45RO+ CD62L+) as compared to culture primed with wild type virus infected cells. Moreover, rVV-CD40L stimulation of CD14+ cells also led to the inhibition of co-cultured tumor cells’ proliferation, correlating with increased TNFα expression. Finally, apoptosis of CD40+ tumor cell lines was induced upon direct infection with this CD40L non-replicating recombinant virus. These data are confirming the potent capacities of recombinant vaccinia viruses expressing CD40L to efficiently trigger multiple CD40 mediated anti-tumor pathways. Not only this vector enhances the generation of tumor specific CD8+ T cells effector and memory responses, but it also promotes anti-tumoral functions of innate immune cells and is even able to directly induce apoptosis in CD40+ tumor cells. These in-vitro observations underline the strong clinical potential of recombinant vaccinia virus expressing CD40L in cancer viro- and immunotherapies. Citation Format: Evangelos Panopoulos, Emanuele Trella, Nermin Raafat, Chantal Mengus, Giulio Spagnoli, Paul Zajac. Recombinant Vaccinia virus expressing CD40L: a multipotent antitumor immunogenic reagent. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2843. doi:10.1158/1538-7445.AM2013-2843

Background: Polyomavirus BK (BKV) large tumor antigen (L-Tag) contributes to oncogenesis by regulating crucial pathways of human cell cycle when non-permissive cells are infected. Therefore, L-Tag has been identified as an important target of immune surveillance in L-Tag expressing tumors. The role of L-Tag as a target of tumor immune surveillance in prostate cancer (PCa) patients has not been investigated so far. In this study we thus analyzed the immunological profile exerted by L-Tag peptide-specific induction in newly diagnosed PCa patients by means of BKV L-Tag serology and local molecular testing, and compared it to age-matched benign prostatic hyperplasia (BPH) patients. Methods: We enrolled 60 newly diagnosed PCa patients and 50 age-matched BPH. Detection of BKV L-Tag DNA was carried out in random punches from tumor areas of surgically excised specimens. BKV L-Tag seroprevalence was determined by enzyme immunoassay (EIA). Systemic immune-regulatory activity was tested upon BKV L-Tag peptide-pool induction and analyzed using cytokine gene expressions (qrt-PCR), antigen-specific regulatory T cell (Treg) characterization (flow cytometry) and immune-regulatory protein release (multiplex fluorescent bead immunoassay). Data were correlated to 5-year follow-up clinical information. Five-year follow-up after surgery was completed on 48/60 PCa patients. Biochemical recurrence (BR) with early censoring was defined as the first PSA ≤ 0.2ng/ml. Results: In peripheral blood mononuclear cells (PBMCs) from PCa patients with evidence of biochemical recurrence (BR) and BKV L-Tag positive lesions, BKV L-Tag peptide-pool, but not the Epstein Barr/Influenza virus/Cytomegalovirus (CEF) peptide-pool, induced a particular gene profile, which was characterized by high expression of IL-10 and TGFβ-1 and a low expression of IFN-γ and TNF-α genes. This signature was confirmed by protein secretion data and correlated with levels of anti-BKV L-Tag IgG activity. Furthermore, in these PCa patients the L-Tag peptide pool significantly boosted IL-10 secreting CD4+CD25+(high)/FoxP3+(high)/CD103+ T cells exerting suppressive functions on autologous effectors (CD4+, CD25− and CD8+ T cell fractions) by reducing cell proliferation and IFN-γ production. Conclusion: Significant associations between immune regulatory activity exerted by BKV L-Tag induction, BKV L-Tag DNA positive tumor lesions and IgG activity against L-Tag were observed in PCa patients. In detail, BR+ PCa patients shared features such as functional BKV L-Tag specific immune regulatory profile with marked BKV L-Tag positive lesion and strong L-Tag-specific IgG activity, thereby hinting at a potential BKV involvement in prostate cancer progression. Our findings are thus consistent with a systemic BKV L-Tag-related tolerogenic environment governing prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 556. doi:1538-7445.AM2012-556

OBJECTIVE: Tumor initiation and progression might be driven by rare populations of cells endowed with stem-properties, and therefore defined as cancer stem cells (CSC). High Aldehyde dehydrogenase (ALDH) activity has been suggested to selectively identify CSC in several tumor types including breast cancer, and, possibly, prostate cancer (PCA). In this study, we investigated presence and potential CSC characteristics of cells with high ALDH activity (ALDH bright) in PCA cell lines, fresh surgical specimens, and primary cultures. MATERIALS AND METHODS: PC3, Du145, VCaP, and LNCaP PCA cell lines were evaluated. Surgical specimens, including Benign Prostate Hyperplasia (BPH) and PCA, were used directly for gene expression studies. Surgical samples were also enzymatically digested for functional analysis and the establishment of primary cultures. ALDH activity was tested using ALDEFLUOR® technology. Cells were sorted from individual cell lines by flow cytometry and evaluated for CSC properties, including spheroid formation ability, clonogenicity, stemness-related gene expression, ALDH specific isoforms expression, and tumorigenicity upon injection in NOD/SCID mice. RESULTS: PCA cell lines and primary cultures displayed heterogeneous ALDH activity and expression of specific ALDH isoforms. Despite a higher expression of Oct4A and Klf4 stemness associated genes, ALDH bright cells isolated from Du145 and PC3 did not show improved spheroid formation or clonogenic capacity, as compared to their dim counterparts. Interestingly, however, ALDH bright cells were associated with a significantly higher tumorigenic capacity in vivo as compared to ALDH low cells. Nevertheless, ALDH bright cells lost their higher tumorigenic capacity following serial “in vivo” passages. Most importantly, a well defined ALDH bright population could also be detected in cells isolated from PCA specimens (n>20). ALDH activity was consistent with a strong expression of several ALDH specific isoforms in PCA tissues. Notably, a significant increase of defined ALDH isoforms such as ALDH1A3 (p=0.001) was detectable in tissues obtained from patients with PCA as compared to BPH or normal specimens. CONCLUSIONS: ALDH bright subsets can be detected in cells isolated from fresh PCA tissue samples, PCA cell lines and primary cultures. These populations appear to be associated with increased “in vivo” tumorigenicity rather than enhanced “in vitro” stem properties in the cell lines investigated. Importantly, expression of ALDH specific isoforms appears to be increased in clinical PCA as compared to BPH and “normal” samples, thereby suggesting a putative role of ALDH in PCA tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3384. doi:1538-7445.AM2012-3384

Prostate cancer (PCA) is a leading cause of cancer death in men. Underlying immunosuppressive mechanisms in benign prostate hyperplasia (BPH) and PCA patients are not fully clarified. We analyzed homeostatic proliferation of CD8+ T cells upon stimulation with common receptor γ chain IL-2, IL-7 and IL-15 cytokines in PCA as compared to BPH patients. CD8+ T cells exhaustion was assessed by evaluating Programmed Death-1 (PD-1) receptor and its ligand PD-L1 expression in PBMC and tissue infiltrating CD8+ T cells from PCA and BPH patients. 57 BPH and 76 PCA patients were enrolled. Gene expression was quantified by Real-Time PCR. T cell proliferation was evaluated by CFSE dilution. CD132, CD122, PD-1 and PD-Ll expression were assessed by flow cytometry. IL-2, IL-7 and IL-15 gene expression were quantified in PCA (n=57) and BPH (n=32) tissues. IL-7 and IL-15 gene expression were significantly increased in PCA tissues as compared to BPH (p=0.024; p=0.031). No significant differences were observed for IL-2 gene expression. CD8+ cells from BPH (n=17) and PCA (n=21) patients showed a significantly decreased responsiveness to IL-7 and IL-15 (p=0.021; p=0.015) as compared to healthy donors (n=9). Response to IL-2 was similar. A trend towards a lower response to IL-15 in PCA as compared to BPH patients was detectable. Percentage of CD8+ T cells expressing the common γ chain (CD132) of homeostatic cytokines receptor is significantly decreased in PCA (n=13) and BPH (n=16) patients as compared to healthy donors (n=8) (p= 0.024; p Taken together these data indicate that both BPH and PCA patients display a decreased responsiveness to IL-7 and IL-15 homeostatic cytokines. Interestingly, high percentages of peripheral blood CD8+ T cells express PD-1 in BPH and PCA patients. Notably, PD-1 and its ligand are highly expressed in tissue infiltrating CD8+ T cells in BPH and PCA patients, thus raising the issue of the role of T cells exhaustion in PCA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4782.

Recombinant poxviruses expressing tumor associated antigens (TAAs) are evaluated since 20 years as immunogenic vaccine vector in clinical trials. Possible limitation to recombinant viral vector is due to either prior systemic immunity to poxviruses or immunodominance of viral antigens which may reduce the induction of immune response against weaker tumor antigens. To address this issue, we developed a recombinant Vaccinia virus expressing HSV type I protein ICP47. This protein down-regulates MHC class-I antigen presentation by blocking the transporter associated with antigen processing (TAP), which translocates peptides, generated by proteasomal protein degradation, into the endoplasmic reticulum for loading onto MHC class I molecule. Herpes simplex virus (HSV) US12 gene, coding for infected cell protein 47 (ICP47) was introduced into wild type vaccinia virus and into a r.VV expressing MART-1/Melan-A27–35 HLA-A201 endoplasmic reticulum (ER)-targeted epitope. Following infection with non-replicating recombinant virus, effect of ICP47 expression on cell surface MHC-class-I, MHC class-II and co-stimulatory molecules was characterized by antibody staining and FACS analysis. Human T-lymphocytes were stimulated in vitro with autologous CD14+ cells infected with r.VV-US12, r.VV-Mart-US12 or control virus. Responsiveness of specific CD8+ and CD4+ to viral proteins and recombinant epitopes were monitored by MHC-multimer staining and interferon gamma (IFNg) expression analysis. Cells infected with HSV-US12-r.VV, demonstrated a decreased ability of presenting MHC class-I antigens to CD8+ T cells whereas MHC-class-II restricted presentation to CD4+ T cells remained unaffected. Co-expression of ER-Melan-A/Mart-126–35 appeared to partially compensate for the ICP47 related surface MHC class-I molecule down-regulation and preserve a strong capacity to induce CTL response against the TAA derived peptide. Thus, viral vectors expressing ICP47 confirmed a diminished TAP-dependant processing of endogenous class-I restricted epitopes while the immunogenic potential of recombinant epitopes directly targeted to the ER was enhanced. Such reagents could become of high relevance especially in multiple-boost vaccine protocol required in cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1500.

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.