A novel method for efficient generation of antigen-specific effector T-cells using dendritic cells transduced with recombinant adeno-associated virus and p38 kinase blockade.

BACKGROUND: The inefficacy of standard therapeutic strategies for ovarian cancer is reflected by the enduring poor prognosis of this malignancy. Due to the potential for exquisite specificity, sensitivity and long-term memory, immunotherapy offers an alternative modality for durable control of the disease, provided appropriate antigens can be identified and presented in the right context. METHODS: We tested a novel dendritic cell vaccine formulation to reprogram autologous antigen-specific T-cells in vitro, in vivo in a murine model of ovarian cancer, and ex vivo using human cells from patients. RESULTS: We show that dendritic cells (DCs) treated with a p38 MAPK inhibitor and transduced with a recombinant adenovirus associated vector (AAV) expressing Sperm protein (Sp) 17 are highly effective in generating antigen-specific T-cell cytotoxic response against ovarian cancer cells. Additionally, these DCs enhanced the differentiation of effector T-cells while reducing the frequency of Foxp3+ T-reg cells in vitro. CONCLUSIONS: This work provides a rationale for translation of pharmacologically reprogrammed DCs into clinical trials for prevention of tumor recurrence and progression in high-risk ovarian cancer patients.

Computer-aided assessment of the extra-cellular matrix during pancreatic carcinogenesis: a pilot study.

BACKGROUND: A hallmark of pancreatic ductal adenocarcinoma is the desmoplastic reaction, but its impact on the tumor behavior remains controversial. Our aim was to introduce a computer -aided method to precisely quantify the amount of pancreatic collagenic extra-cellular matrix, its spatial distribution pattern, and the degradation process. METHODS: A series of normal, inflammatory and neoplastic pancreatic ductal adenocarcinoma formalin-fixed and paraffin-embedded Sirius red stained sections were automatically digitized and analyzed using a computer-aided method. RESULTS: We found a progressive increase of pancreatic collagenic extra-cellular matrix from normal to the inflammatory and pancreatic ductal adenocarcinoma. The two-dimensional fractal dimension showed a significant difference in the collagenic extra-cellular matrix spatial complexity between normal versus inflammatory and pancreatic ductal adenocarcinoma. A significant difference when comparing the number of cycles necessary to degrade the pancreatic collagenic extra-cellular matrix in normal versus inflammatory and pancreatic ductal adenocarcinoma was also found. The difference between inflammatory and pancreatic ductal adenocarcinoma was also significant. Furthermore, the mean velocity of collagenic extra-cellular matrix degradation was found to be faster in inflammatory and pancreatic ductal adenocarcinoma than in normal. CONCLUSION: These findings demonstrate that inflammatory and pancreatic ductal adenocarcinomas are characterized by an increased amount of pancreatic collagenic extra-cellular matrix and by changes in their spatial complexity and degradation. Our study defines new features about the pancreatic collagenic extra-cellular matrix, and represents a basis for further investigations into the clinical behavior of pancreatic ductal adenocarcinoma and the development of therapeutic strategies.

Development of a M cell-targeted microparticulate platform, BSK02™, for oral immunization against the ovarian cancer antigen, sperm protein 17.

Although it only accounts for approximately 5% of all female cancer cases, ovarian cancer (OC) ranks as the fifth leading cause of death due to cancer in women. We have evaluated the potential of an orally administered microparticulate vaccine incorporating an immunodominant epitope peptide derived from the cancer/testis antigen sperm protein 17 (SP17) aberrantly expressed in OC, to retard the progression of the disease. The peptide antigen and the immune-stimulatory toll-like receptor 9 ligand CpG oligonucleotide were incorporated into spray dried microparticles composed of enteric and sustained release polymers together with the Aleuria aurantia lectin targeting microfold cells present in the gut-associated lymphoid tissue. These particles were administered via oral route to mice challenged week prior with SP17-expressing ID8 OC cells. Analysis of splenocytes harvested from vaccinated mice revealed strong activation of IFN-γ+/CD8+ lymphocytes in response to re-stimulation with the SP17 antigen. Moreover, vaccinated animals showed significant retardation of ascites/tumor volume in comparison to placebo-treated animals four weeks after the tumor challenge (p = 0.005). Taken together, our results suggest that vaccination against SP17 using orally administered microparticles could potentially be used as an effective consolidation strategy for OC patients with residual tumor or high probability for relapse following first-line treatments. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 29-36, 2019.

Cancer Cells Exploit Notch Signaling to Redefine a Supportive Cytokine Milieu.

Notch signaling is a well-known key player in the communication between adjacent cells during organ development, when it controls several processes involved in cell differentiation. Notch-mediated communication may occur through the interaction of Notch receptors with ligands on adjacent cells or by a paracrine/endocrine fashion, through soluble molecules that can mediate the communication between cells at distant sites. Dysregulation of Notch pathway causes a number of disorders, including cancer. Notch hyperactivation may be caused by mutations of Notch-related genes, dysregulated upstream pathways, or microenvironment signals. Cancer cells may exploit this aberrant signaling to "educate" the surrounding microenvironment cells toward a pro-tumoral behavior. This may occur because of key cytokines secreted by tumor cells or it may involve the microenvironment through the activation of Notch signaling in stromal cells, an event mediated by a direct cell-to-cell contact and resulting in the increased secretion of several pro-tumorigenic cytokines. Up to now, review articles were mainly focused on Notch contribution in a specific tumor context or immune cell populations. Here, we provide a comprehensive overview on the outcomes of Notch-mediated pathological interactions in different tumor settings and on the molecular and cellular mediators involved in this process. We describe how Notch dysregulation in cancer may alter the cytokine network and its outcomes on tumor progression and antitumor immune response.

Role of MSC-derived galectin 3 in the AML microenvironment.

In acute myeloid leukemia (AML), high Galectin 3 (LGALS3) expression is associated with poor prognosis. The role of LGALS3 derived from mesenchymal stromal cells (MSC) in the AML microenvironment is unclear; however, we have recently found high LGALS3 expression in MSC derived from AML patients is associated with relapse. In this study, we used reverse phase protein analysis (RPPA) to correlate LGALS3 expression in AML MSC with 119 other proteins including variants of these proteins such as phosphorylated forms or cleaved forms to identify biologically relevant pathways. RPPA revealed that LGALS3 protein was positively correlated with expression of thirteen proteins including MYC, phosphorylated beta-Catenin (p-CTNNB1), and AKT2 and negatively correlated with expression of six proteins including integrin beta 3 (ITGB3). String analysis revealed that proteins positively correlated with LGALS3 showed strong interconnectivity. Consistent with the RPPA results, LGALS3 suppression by shRNA in MSC resulted in decreased MYC and AKT expression while ITGB3 was induced. In co-culture, the ability of AML cell to adhere to MSC LGALS3 shRNA transductants was reduced compared to AML cell adhesion to MSC control shRNA transductants. Finally, use of novel specific LGALS3 inhibitor CBP.001 in co-culture of AML cells with MSC reduced viable leukemia cell populations with induced apoptosis and augmented the chemotherapeutic effect of AraC. In summary, the current study demonstrates that MSC-derived LGALS3 may be critical for important biological pathways for MSC homeostasis and for regulating AML cell localization and survival in the leukemia microenvironmental niche.

The cancer-testis antigen, sperm protein 17, a new biomarker and immunological target in head and neck squamous cell carcinoma.

Head and Neck Squamous Cell Carcinoma is a deadly and locally aggressive malignancy that frequently portends a poor prognosis. Since current treatment modalities including surgery, chemotherapy and radiation are heavily debilitating and often result in recurrence intense efforts have been put into the development of novel less toxic and more lasting treatment strategies. Recently, immunotherapy has been proposed as a promising alternative that could potentially meet these requirements. SP17 is a validated cancer-testis antigen in multiple myeloma, ovarian cancer and non-small cell lung cancer. We aim at studying SP17 expression in HNSCC and its immunogenicity as a possible future target for HNSCC therapeutic vaccines. SP17 expression was evaluated in tissue specimens of HNSCC patients and controls. Moreover, SP17 immunogenicity was studied by generating autologous dendritic cells in vitro from the peripheral blood mononucleated cells of HNSCC patients and testing their ability to induce SP17 specific cytotoxic lymphocytes capable of killing autologous tumor cells in vitro. SP17specific immune responses were also evaluated in HNSCC patients as circulating anti-SP17 autoantibodies. SP17 was expressed in HNSCC tissues of HNSCC patients. Autologous dendritic cells pulsed with SP17 antigen induced powerful SP17 MHC class-I restricted, perforin-dependent, cytotoxic T-cells capable of efficiently killing autologous tumor cells in vitro. SP17-specific autoantibodies were detectable in the serum of HNSCC patients irrespective of tumor site or TNM stage. In conclusion, SP17 is an ideal immunotherapeutic target for HNSCC and a potential serological biomarker of the disease.

Cancer testis antigen Sperm Protein 17 as a new target for triple negative breast cancer immunotherapy.

Breast carcinoma is a major health issue for millions of women. Current therapies have serious side effects, and are only partially effective in patients with metastatic tumors. Thus, the need for novel and less toxic therapies is urgent. Moreover, hormonal and antibody therapies effective in other subtypes are not effective in Triple Negative Breast Cancer (TNBC). Immunotherapeutic strategies directed against specific tumor-associated antigens (TAAs) and mediated by specific cytotoxic T lymphocytes (CTL) have been largely underexplored in this disease. Cancer-testis antigens (CTA) are a group of TAAs displaying the ideal characteristics of promising vaccine targets, i.e. strong immunogenicity and cancer specificity. The CTA, Sperm Protein 17 (SP17), has been found to be aberrantly expressed in different neoplasms, including ovarian and esophageal cancers, nervous system tumors and multiple myeloma, and has been suggested as a candidate target for immunotherapy. Here, we evaluated SP17 expression levels in breast cancer cell lines, invasive ductal breast carcinoma, including patients with TNBC, and adjacent non-neoplastic breast tissue, and determined whether SP17 was capable of generating SP17-specific cytotoxic T lymphocytes in vitro. We showed that SP17 is expressed in breast cancer cell lines and primary breast tumors and importantly in TNBC subtype, but not in adjacent non-tumoral breast tissue or unaffected tissues, except in male germinal cells. Furthermore, we detected specific anti-SP17 antibodies in patients' sera and we generated SP17-specific, HLA class I-restricted, cytotoxic T lymphocytes capable of efficiently killing breast cancer cells.

PI3K/AKT signaling inhibits NOTCH1 lysosome-mediated degradation.

The pathways of NOTCH and PI3K/AKT are dysregulated in about 60% and 48% of T-cell acute lymphoblastic leukemia (T-ALL) patients, respectively. In this context, they interact and cooperate in controlling tumor cell biology. Here, we propose a novel mechanism by which the PI3K/AKT pathway regulates NOTCH1 in T-ALL, starting from the evidence that the inhibition of PI3K/AKT signaling induced by treatment with LY294002 or transient transfection with a dominant negative AKT mutant downregulates NOTCH1 protein levels and activity, without affecting NOTCH1 transcription. We showed that the withdrawal of PI3K/AKT signaling was associated to NOTCH1 phosphorylation in tyrosine residues and monoubiquitination of NOTCH1 detected by Ubiquitin capture assay. Co-immunoprecipitation assay and colocalization analysis further showed that the E3 ubiquitin ligase c-Cbl interacts and monoubiquitinates NOTCH1, activating its lysosomal degradation. These results suggest that the degradation of NOTCH1 could represent a mechanism of control by which NOTCH1 receptors are actively removed from the cell surface. This mechanism is finely regulated by the PI3K/AKT pathway in physiological conditions. In pathological conditions characterized by PI3K/AKT hyperactivation, such as T-ALL, the excessive AKT signaling could lead to NOTCH1 signaling dysregulation. Therefore, a therapeutic strategy directed to PI3K/AKT in T-ALL could contemporaneously inhibit the dysregulated NOTCH1 signaling. © 2015 Wiley Periodicals, Inc.

Sex-driven differences in immunological responses: challenges and opportunities for the immunotherapies of the third millennium.

PURPOSE OF THE STUDY: Male-based studies, both at the biochemical and at the pre-clinical/clinical trial levels, still predominate in the scientific community. Many studies are based on the wrong assumption that both sexes are fundamentally identical in their response to treatments. As a result, findings obtained mainly in males are applied to females, resulting in negative consequences female patients. In cancer immunotherapy, there is still a scarce focus on this topic. Here we review the main differences in immune modulation and immune system biology between males and females with a particular focus on how these differences affect cancer immunotherapy and cancer vaccines. METHODS: We reviewed articles published on PubMed from 1999 to 2014, using the keywords: sex hormones, immune response, estrogen, immunotherapy, testosterone, cancer vaccines, sex-based medicine. We also present new data wherein the expression of the cancer testis antigen, Ropporin-1, was determined in patients with multiple myeloma, showing that the expression of Ropporin-1 was influenced by sex. RESULTS: Male and female immune systems display radical differences mainly due to the immune regulatory effects of sex hormones. These differences might have a dramatic impact on the immunological treatment of cancer. Moreover, the expression of tumor antigens that can be targeted by anti-cancer vaccines is associated with sex. CONCLUSION: Future clinical trials focusing on cancer immunotherapy will need to take into account the differences in the immune response and in the frequency of target antigen expression between male and females, in order to optimize these anti-cancer immunotherapies of the third millennium.

Targeting Tumor Initiating Cells through Inhibition of Cancer Testis Antigens and Notch Signaling: A Hypothesis.

Tumor initiating cells (TICs) differ from normal stem cells (SCs) in their ability to initiate tumorigenesis, invasive growth, metastasis and the acquisition of chemo and/or radio-resistance. Over the past years, several studies have indicated the potential role of the Notch system as a key regulator of cellular stemness and tumor development. Furthermore, the expression of cancer testis antigens (CTA) in TICs, and their role in SC differentiation and biology, has become an important area of investigation. Here, we propose a model in which CTA expression and Notch signaling interacts to maintain the sustainability of self-replicating tumor populations, ultimately leading to the development of metastasis, drug resistance and cancer progression. We hypothesize that Notch-CTA interactions in TICs offer a novel opportunity for meaningful therapeutic interventions in cancer.

Chimeric antigen receptor engineering: a right step in the evolution of adoptive cellular immunotherapy.

Cancer immunotherapy comprises different therapeutic strategies that exploit the use of distinct components of the immune system, with the common goal of specifically targeting and eradicating neoplastic cells. These varied approaches include the use of specific monoclonal antibodies, checkpoint inhibitors, cytokines, therapeutic cancer vaccines and cellular anticancer strategies such as activated dendritic cell (DC) vaccines, tumor-infiltrating lymphocytes (TILs) and, more recently, genetically engineered T cells. Each one of these approaches has demonstrated promise, but their generalized success has been hindered by the paucity of specific tumor targets resulting in suboptimal tumor responses and unpredictable toxicities. This review will concentrate on recent advances on the use of engineered T cells for adoptive cellular immunotherapy (ACI) in cancer.

Novel antigens in non-small cell lung cancer: SP17, AKAP4, and PTTG1 are potential immunotherapeutic targets.

Lung cancer is the leading cause of cancer deaths in both genders worldwide, with an incidence only second to prostate cancer in men and breast cancer in women. The lethality of the disease highlights the urgent need for innovative therapeutic options. Immunotherapy can afford efficient and specific targeting of tumor cells, improving efficacy and reducing the side effects of current therapies. We have previously reported the aberrant expression of cancer/testis antigens (CTAs) in tumors of unrelated histological origin. In this study we investigated the expression and immunogenicity of the CTAs, Sperm Protein 17 (SP17), A-kinase anchor protein 4 (AKAP4) and Pituitary Tumor Transforming Gene 1 (PTTG1) in human non-small cell lung cancer (NSCLC) cell lines and primary tumors. We found that SP17, AKAP4 and PTTG1 are aberrantly expressed in cancer samples, compared to normal lung cell lines and tissues. We established the immunogenicity of these CTAs by measuring CTA-specific autoantibodies in patients' sera and generating CTA-specific autologous cytotoxic lymphocytes from patients' peripheral blood mononuclear cells. Our results provide proof of principle that the CTAs SP17/AKAP4/PTTG1 are expressed in both human NSCLC cell lines and primary tumors and can elicit an immunogenic response in lung cancer patients.

Galectins as therapeutic targets for hematological malignancies: a hopeful sweetness.

Galectins are family of galactose-binding proteins known to play critical roles in inflammation and neoplastic progression. Galectins facilitate the growth and survival of neoplastic cells by regulating their cross-talk with the extracellular microenvironment and hampering anti-neoplastic immunity. Here, we review the role of galectins in the biology of hematological malignancies and their promise as potential therapeutic agents in these diseases.

Galectins in cancer: carcinogenesis, diagnosis and therapy.

A major breakthrough in the field of medical oncology has been the discovery of galectins and their role in cancer development, progression and metastasis. In this review article we have condensed the results of a number of studies published over the past decade in an effort to shed some light on the unique role played by the galectin family of proteins in neoplasia, and how this knowledge may alter the approach to cancer diagnosis as well as therapy in the future. In this review we have also emphasized the potential use of galectin inhibitors or modulators in the treatment of cancer and how this novel treatment modality may affect patient outcomes in the future. Based on current pre-clinical models we believe the use of galectin inhibitors/modulators will play a significant role in cancer treatment in the future. Early clinical studies are underway to evaluate the utility of these promising agents in cancer patients.

Galectin-3 inhibition suppresses drug resistance, motility, invasion and angiogenic potential in ovarian cancer.

OBJECTIVE: Ovarian cancer is the most deadly gynecologic malignancy worldwide. Since the pathogenesis of ovarian cancer is incompletely understood, and there are no available screening techniques for early detection, most patients are diagnosed with advanced, incurable disease. In an effort to develop innovative and effective therapies for ovarian cancer, we tested the effectiveness of Galecti-3C in vitro. This is a truncated, dominant negative form of Galectin-3, which is thought to act by blocking endogenous Galectin-3. METHODS: We produced a truncated, dominant-negative form of Galectin-3, namely Galetic-3C. Ovarian cancer cell lines and primary cells from ovarian cancer patients were treated with Galectin-3C, and growth, drug sensitivity, and angiogenesis were tested. RESULT: We show, for the first time, that Galectin-3C significantly reduces the growth, motility, invasion, and angiogenic potential of cultured OC cell lines and primary cells established from OC patients. CONCLUSIONS: Our findings indicate that Galectin-3C is a promising new compound for the treatment of ovarian cancer.

Notch signaling drives multiple myeloma induced osteoclastogenesis.

Multiple myeloma (MM) is closely associated with bone destruction. Once migrated to the bone marrow, MM cells unbalance bone formation and resorption via the recruitment and maturation of osteoclast precursors. The Notch pathway plays a key role in different types of cancer and drives several biological processes relevant in MM, including cell localization within the bone marrow, proliferation, survival and pharmacological resistance. Here we present evidences that MM can efficiently drive osteoclastogenesis by contemporaneously activating Notch signaling on tumor cells and osteoclasts through the aberrant expression of Notch ligands belonging to the Jagged family. Active Notch signaling in MM cells induces the secretion of the key osteoclastogenic factor, RANKL, which can be boosted in the presence of stromal cells. In turn, MM cells-derived RANKL causes the upregulation of its receptor, RANK, and Notch2 in pre-osteoclasts. Notch2 stimulates osteoclast differentiation by promoting autocrine RANKL signaling. Finally, MM cells through Jagged ligands expression can also activate Notch signaling in pre-osteoclast by direct contact. Such synergism between tumor cells and pre-osteoclasts in MM-induced osteoclastogenesis can be disrupted by silencing tumor-derived Jagged1 and 2. These results make the Jagged ligands new promising therapeutic targets in MM to contrast bone disease and the associated co-morbidities.

Selective expression and immunogenicity of the cancer/testis antigens SP17, AKAP4 and PTTG1 in non-small cell lung cancer: new candidates for active immunotherapy.

ABSTRACT BACKGROUND. Lung cancer is the leading cause of cancer deaths in both genders worldwide, with an incidence only second to prostate cancer in men and breast cancer in women. The lethality of the disease highlights the urgent need for innovative therapeutic options. Immunotherapy can afford efficient and specific targeting of tumor cells, improving efficacy and reducing the side effects of current therapies. We have previously reported the aberrant expression of cancer/testis antigens (CTAs) in tumors of unrelated histological origin. In this study we investigated the expression and immunogenicity of the cancer/testis antigens (CTAs) Sperm Protein 17 (SP17), A-kinase anchor protein 4 (AKAP4) and Pituitary Tumor Transforming Gene 1 (PTTG1) in human non-small cell lung cancer (NSCLC) cell lines and primary tumors. METHODS. We used RT-PCR, immunofluorescence, flow cytometry, ELISA and cytotoxicity assays to determine the expression levels and immunogenicity of SP17, AKAP4 and PTTG1 in human NSCLC cell lines and primary tumors. RESULTS. We found that SP17, AKAP4 and PTTG1 are aberrantly expressed in NSCLC cancer cell lines and primary tumor tissues from patients, compared to normal lung cell lines and tissues. We established the immunogenicity of these CTAs by measuring CTA-specific autoantibodies in patients' sera and generating CTA-specific autologous cytotoxic lymphocytes (CTLs) from patients' peripheral blood mononuclear cells (PBMCs). CONCLUSIONS. Our results provide proof of principle that the CTAs SP17/AKAP4/PTTG1 are expressed in both human NSCLC cell lines and primary tumors and can elicit an immunogenic response in NSCLC patients. Based on our findings, further studies are warranted to explore the feasibility of developing CTA-specific immunotherapeutic strategies for NSCLC patients.

The role of human papilloma virus (HPV) infection in non-anogenital cancer and the promise of immunotherapy: a review.

Over the past 30 years, human papilloma virus (HPV) has been shown to play a role in the development of various cancers. Most notably, HPV has been linked to malignant progression in neoplasms of the anogenital region. However, high-risk HPV has also been suggested to play a significant role in the development of cancers in other anatomic locations, such as the head and neck, lung, breast and bladder. In 2006, the first vaccine for HPV, Gardasil, was approved for the prevention of subtypes 6, 11, 16 and 18. A few years later, Cevarix was approved for the prevention of subtypes 16 and 18, the HPV subtypes most frequently implicated in malignant progression. Although increased awareness and vaccination could drastically decrease the incidence of HPV-positive cancers, these approaches do not benefit patients who have already contracted HPV and developed cancer as a result. For this reason, researchers need to continue developing treatment modalities, such as targeted immunotherapies, for HPV-positive lesions. Here, we review the potential evidence linking HPV infection with the development of non-anogenital cancers and the potential role of immunotherapy in the prevention and eradication of HPV infection and its oncogenic sequela.

Anti-galectin-3 therapy: a new chance for multiple myeloma and ovarian cancer?

Here we review the role of Galectins in the molecular pathogenesis of multiple myeloma and ovarian cancer, with a special focus on Glectin-3. Multiple myeloma is the second most common hematologic malignancy worldwide. Because the pathogenesis of multiple myeloma is still incompletely understood, there is no ultimately effective cure, and this cancer results fatal. Ovarian cancer is the most lethal gynecologic malignancy worldwide. Due to the lack of screening techniques for early detection, patients are mostly diagnosed with advanced disease, which results ultimately fatal. Multiple myeloma and ovarian cancer have different biologies, but they share a strong dependence on adhesion with extracellular matrix and other cells. Galectin-3 plays a key role in regulating such adhesive abilities of tumor cells. Here we discuss the outcomes and possible mechanism of action of a truncated, dominant negative form of Galectin-3, Galectin-3C, in these malignancies. Overall, we report that Galectin-3C is a promising new compound for effective adjuvant therapies in advanced, refractory multiple myeloma and ovarian cancer.