Transfected Tumor Cells Research Articles

INT-1B3, an LNP formulated miR-193a-3p mimic, promotes anti-tumor immunity by enhancing T cell mediated immune responses via modulation of the tumor microenvironment and induction of immunogenic cell death.

microRNAs (miRNAs) are small, non-coding RNAs that regulate expression of multiple genes. MiR-193a-3p functions as a tumor suppressor in many cancer types, but its effect on inducing specific anti-tumor immune responses is unclear. Therefore, we examined the effect of our lipid nanoparticle (LNP) formulated, chemically modified, synthetic miR-193a-3p mimic (INT-1B3) on anti-tumor immunity. INT-1B3 inhibited distant tumor metastasis and significantly prolonged survival. INT-1B3-treated animals were fully protected against challenge with autologous tumor cells even in absence of treatment indicating long-term immunization. Protection against autologous tumor cell challenge was hampered upon T cell depletion and adoptive T cell transfer abrogated tumor growth. Transfection of tumor cells with our miR-193a-3p mimic (1B3) resulted in tumor cell death and apoptosis accompanied by increased expression of DAMPs. Co-culture of 1B3-transfected tumor cells and immature DC led to DC maturation and these mature DC were able to stimulate production of type 1 cytokines by CD4+ and CD8+ T cells. CD4-CD8- T cells also produced type 1 cytokines, even in response to 1B3-transfected tumor cells directly. Live cell imaging demonstrated PBMC-mediated cytotoxicity against 1B3-transfected tumor cells. These data demonstrate for the first time that miR-193a-3p induces long-term immunity against tumor development via modulation of the tumor microenvironment and induction of immunogenic cell death.

A novel molecular imaging probe [99mTc]Tc-HYNIC-FAPI targeting cancer-associated fibroblasts

Fibroblast activation protein (FAP) is higher expressed on cancer-associated fibroblasts (CAFs) in most malignant epithelial neoplasms, which is lower expressed in normal tissues. As a promising small molecular probe, FAP inhibitor (FAPI) shows the specific binding to FAP. This study aimed to explore a novel molecular probe [99mTc]Tc-HYNIC-FAPI targeting CAFs. The in vitro characteristics of the probe were also evaluated. The FAPI targeting FAP was designed, synthesized and conjugated with the chelator 6-hydrazinylnicotinic acid (HYNIC) for radiolabeling with 99mTc. The radiolabeling yield, radiochemical purity and stability were evaluated by Instant thin-layer chromatography (ITLC) and High performance liquid chromatography (HPLC). Lipophilicity was performed by the distribution coefficient test. The binding and migration ability of the probe was assessed using the FAP transfected tumor cell line. The radiolabeling yield of [99mTc]Tc-HYNIC-FAPI was (97.29 ± 0.46) %. The radiochemical purity was more than 90% and kept stable until 6 h. The radioligand was shown as lower lipophilicity, of which logD7.4 value was − 2.38 pm 0.13. In vitro experiments, the results indicated that the probe showed binding properties, and inhibited the migration of tumor cells. The novel [99mTc]Tc-HYNIC-FAPI probe was successfully radiosynthesized and exhibited good radiochemical purity, stability and in vitro binding ability to tumor cells. The [99mTc]Tc-HYNIC-FAPI will be a promising SPECT/CT imaging probe.

PIL-12 delivered by polymer based nanovector for anti-tumor genetherapy

Finding more effective and safe non-viral vectors to transfer genes into cancer cells has become the key of immune gene therapy for cancer. Herein a triblock compound MPEG2000–PDLLA4000–MPEG2000 modified by cationic liposome DOTAP was used as a non-viral vector DOTAP/MPEG2000–PDLLA4000–MPEG2000 (DMPM) to effectively transfer interleukin (IL)-12 plasmid (pIL-12) into tumor tissue. IL-12 produced by transfected tumor cells successfully inducing lymphocyte proliferation and promoting interferon-γ (IFN-γ) secretion, which resulted in tumor cells death. The ability of DMPM to transfer pIL-12 and the immune effect induced by IL-12 in cells had been explored. The anti-tumor effect, mechanism and safety of pIL-12/DMPM in mice cancer model were investigated in this study. Our results showed that the pIL-12 transferred by DMPM was highly expressed both in CT26 cells and B16-F10 cells. IL-12 expressed in the culture supernatant of transfected tumor cells stimulated lymphocyte proliferation and promoted IFN-γ secretion. The experimental result confirmed that pIL-12/DMPM therapy significantly reduced tumor growth in mice model. We designed the nanocomposite DMPM to deliver pIL-12 for cancer treatment and explored its therapeutic efficacy and the underlying anti-tumor mechanism. Our study suggested pIL-12 loaded by DMPM complex would be an effective strategy for cancer treatment.

Abstract 5199: Tumor-specific antibody response induced by novel immunomodulator, IFx-Hu2.0, reflects extent of epitope spreading to multiple tumor epitopes

Abstract While the role of T cells in cancer immunotherapy has been well studied and is relatively clear and well defined, there are few studies elucidating the more complicated involvement of B cells in anti-cancer processes. Preclinical data showed that IFx-Hu2.0 primes both innate and adaptive immune responses through the expression of Emm55, a bacterial protein. Recognition by antigen presenting cells (APC) is followed by interantigenic epitope spreading to tumor antigens expressed in transfected tumor cells. The current study was designed to determine the extent of epitope spreading through quantitating the IgM and IgG responses to known melanoma antigens. Plasma samples (pre-and post-intralesional injection of IFx-Hu2.0) from seven patients with unresectable stage III-IV cutaneous melanoma enrolled in a first-in-human, phase 1 open-label trial (NCT03655756) were analyzed and compared using PEPperCHIP® Melanoma Antigen Microarrays with 21 melanoma-associated antigens converted into linear 15 amino acid peptides with a peptide-peptide overlap of 13 amino acids. The resulting array contained 4,125 different peptides printed in duplicate along with multiple control peptides. The arrays were incubated with plasma samples [collected immediately prior to and four weeks (N=6) or two weeks (N=1) post treatment and three weeks post injection #2 (N=1)] at three dilutions, followed by staining with secondary and control antibodies. Readout with a LI-COR Odyssey Imaging System was followed by quantification of spot intensities and peptide annotation with PepSlide® Analyzer. For generation of IgM/IgG intensity ratios, a minimal baseline intensity of 200 fluorescence units was set. Changes in both IgM and IgG epitope recognition to melanoma-associated antigens was detected in all patients. Baseline and IFx-Hu2.0-induced antibody response profiles for all individuals were heterogeneous, with no dominant common antibody response, pointing to the unpredictability of specific epitope recognition and a relative independence from MHC haplotype requirements due to epitope spreading. Notwithstanding the signals from adjacent peptides, high numbers of newly recognized melanoma epitopes were observed for all patients and all plasma samples. These data suggest enhanced crosstalk between innate and adaptive immune cells, epitope presentation by APC to T and B cells and epitope spreading to multiple previously unrecognized tumor epitopes. IgM and IgG levels were maintained at four weeks post IFx-Hu2.0 administration suggesting continued primary recognition and maturation of effector immune response. B cells represent an important proportion of infiltrating lymphocytes in melanoma and are associated with good prognosis in most cancer types. Studies are underway to determine the relative contribution of B cell activity to anti-tumor effects seen with IFx-Hu2.0. Citation Format: Patricia Lawman, Michael Shamblott, Ashraf Dehlawi, James Bianco. Tumor-specific antibody response induced by novel immunomodulator, IFx-Hu2.0, reflects extent of epitope spreading to multiple tumor epitopes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5199.

Platelet Pharmacytes for the Hierarchical Amplification of Antitumor Immunity in Response to Self-Generated Immune Signals.

Systemic immunosuppression mediated by tumor-derived exosomes is an important cause for the resistance of immune checkpoint blockade (ICB) therapy. Herein, self-adaptive platelet (PLT) pharmacytes are engineered to mediate cascaded delivery of exosome-inhibiting siRNA and anti-PD-L1 (aPDL1) toward synergized antitumor immunity. In the pharmacytes, polycationic nanocomplexes (NCs) assembled from Rab27 siRNA (siRab) and a membrane-penetrating polypeptide are encapsulated inside the open canalicular system of PLTs, and cytotoxic T lymphocytes (CTLs)-responsive aPDL1 nanogels (NGs) are covalently backpacked on the PLT surface. Upon systemic administration, the pharmacytes enable prolonged blood circulation and active accumulation to tumors, wherein PLTs are activated to liberate siRab NCs, which efficiently transfect tumor cells, silence Rab27a, and inhibit exosome secretion. The immunosuppression is thus relieved, leading to the activation, proliferation, and tumoral infiltration of cytotoxic T cells, which trigger latent aPDL1 release. As such, the competitive aPDL1 exhaustion by PD-L1-expressing exosomes is minimized to sensitize ICB. Synergistically, siRab and aPDL1 induce strong antitumor immunological response and memory against syngeneic murine melanoma. This study reports a bioinspired mechanism to resolve the blood circulation/cell internalization contradiction of polycationic siRNA delivery systems, and renders an enlightened approach for the spatiotemporal enhancement of antitumor immunity.

Blockade of HIF-1α and STAT3 by hyaluronate-conjugated TAT-chitosan-SPION nanoparticles loaded with siRNA molecules prevents tumor growth

HIF-1α and STAT3 are two of the critical factors in the growth, proliferation, and metastasis of cancer cells and play a crucial role in inhibiting anti-cancer immune responses. Therefore, we used superparamagnetic iron oxide (SPION) nanoparticles (NPs) coated with thiolated chitosan (ChT) and trimethyl chitosan (TMC) and functionalized with hyaluronate (H) and TAT peptide for delivery of siRNA molecules against STAT3 and HIF-1α to cancer cells both in vivo and in vitro. The results indicated that tumor cell transfection with siRNA-encapsulated NPs robustly inhibited proliferation and migration and induced apoptosis in tumor cells. Furthermore, simultaneous silencing of HIF-1α and STAT3 significantly repressed cancer development in two different tumor types (4T1 breast cancer and CT26 colon cancer) which were associated with upregulation of cytotoxic T lymphocytes and IFN-γ secretion. The findings suggest inhibiting the HIF-1α/STAT3 axis by SPION-TMC-ChT-TAT-H NPs as an effective way to treat cancer.

The Role of MicroRNA Expression for Proliferation and Apoptosis of Tumor Cells: Impact of Hypoxia-Related Acidosis.

The metabolic microenvironment in tumors is characterized by hypoxia and acidosis. Extracellular pH sometimes decreases to even below 6.0. Previous experiments showed that tissue pH has an impact on tumor cell proliferation and apoptosis. However, the mechanism of how cell cycle progression is affected by decreased pH is not fully understood yet. One possible mechanism includes changes in the expression of miRNAs. The aim of this study was to analyze the impact of pH-regulated miRNAs (miR-183 and miR-215) on proliferation, apoptosis, and necrosis of tumor cells. Therefore, AT1 prostate and Walker-256 mammary carcinoma cells were transfected with the miRNAs or with the respective antagomirs and incubated at pH7.4 and 6.6 for 24h. AT1 cells underwent a G0/G1 cell cycle arrest under acidic conditions and showed a marked reduction of the number of actively DNA-synthesizing cells. In Walker-256 cells, acidosis induced a reduction of apoptosis and additionally a significant increase in necrotic cell death. Transfection of tumor cells with miR-183 or miR-215, which were significantly downregulated under acidic conditions, had no impact on cell death of AT1 or Walker-256 cells. Overexpression of miR-183, which is also downregulated by acidosis, intensified G0/G1 cell cycle arrest in AT1 cells. Previous studies revealed that hypoxia-related tumor acidosis affects the expression of different small noncoding RNAs. However, not all of these acidosis-regulated miRNAs seem to have an impact on proliferation, apoptosis, and necrosis of tumor cells. While miR-215 had no influence, miR-183 seems to be an interesting candidate that could amplify the impact of extracellular acidosis on malignant behavior of tumor cells.

Abstract 953: Herpes simplex virus thymidine kinase/ganciclovir-mediated apoptotic tumor cells induce anti-tumor immunity

Abstract Immune cells can recognize tumor antigen and elicit an adaptive immune response against tumor cells. It has been reported that apoptotic or necrotic death of tumor cells releases tumor antigen stimulating anti-tumor immunity. However, the immune responses against apoptotic tumor cells in vivo are poorly understood. To investigate whether apoptotic tumor cells affect anti-tumor immunity, local immune responses in tumor were evaluated by induction of apoptotic tumor cells with herpes simplex virus thymidine kinase (HSVtk)/ ganciclovir (GCV) system. HSVtk gene transduced tumor cells were resulted in apoptosis in vitro. Administration of GCV triggered apoptosis in HSVtk transfected tumor cells in vivo and regressed the tumor. Comprehensive gene expression analysis of the bulk tumor showed that IFNγ and IL-12 were upregulated. CD8+ T cells were increased in the treatment group compared to the control group. The tumor regression by induction of GCV-triggered apoptosis was inhibited by anti-CD8 depleting antibody. These findings indicate that apoptotic tumor cells induce CD8+ T cell-dependent anti-tumor immunity. Citation Format: Sho Umegaki, Hidekazu Shirota, Chikashi Ishioka. Herpes simplex virus thymidine kinase/ganciclovir-mediated apoptotic tumor cells induce anti-tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 953.

C-X-C Motif Chemokine Ligand 14 is a Unique Multifunctional Regulator of Tumor Progression.

Cancer is a leading cause of death and disease worldwide, with a tremendous financial impact. Thus, the development of cost-effective novel approaches for suppressing tumor growth and progression is essential. In an attempt to identify the mechanisms responsible for tumor suppression, we screened for molecules downregulated in a cancer progression model and found that the chemokine CXCL14, also called BRAK, was the most significantly downregulated. Increasing the production of CXCL14 protein by transfecting tumor cells with a CXCL14 expression vector and transplanting the cells into the back skin of immunodeficient mice suppressed tumor cell growth compared with that of parental tumor cells, suggesting that CXCL14 suppressed tumor growth in vivo. However, some studies have reported that over-expression of CXCL14, especially in stromal cells, stimulated the progression of tumor formation. Transgenic mice expressing 10-fold more CXCL14 protein than wild-type C57BL/6 mice showed reduced rates of chemical carcinogenesis, transplanted tumor growth, and metastasis without apparent side effects. CXCL14 also acts as an antimicrobial molecule. In this review, we highlight recent studies involving the identification and characterization of CXCL14 in cancer progression and discuss the reasons for the context-dependent effects of CXCL14 on tumor formation.

Development of an Orthotopic Intrasplenic Xenograft Mouse Model of Canine Histiocytic Sarcoma and Its Use in Evaluating the Efficacy of Treatment with Dasatinib.

Canine histiocytic sarcoma is a highly aggressive and metastatic hematopoietic neoplasm that responds poorly to currently available treatment regimens. Our goal was to establish a clinically relevant xenograft mouse model to assess the preclinical efficacy of novel cancer treatment protocols for histiocytic sarcoma. We developed an intrasplenic xenograft mouse model characterized by consistent tumor growth and development of metastasis to the liver and other abdominal organs. This model represents the metastatic or disseminated form of canine histiocytic sarcoma, which is considered the most clinically challenging form of the disease. Transfection of tumor cells with a luciferase vector supported the use of in vivo bioluminescence imaging to track tumor progression over time and to assess the response of this murine model to novel chemotherapeutic agents. Dasatinib treatment of the mice with intrasplenic xenografts decreased tumor growth and increased survival times, compared with mice treated with vehicle only. Our findings indicate the potential of dasatinib for the treatment of histiocytic sarcoma in dogs and for similar diseases in humans. These results warrant additional studies to clinically test the efficacy of dasatinib in dogs with histiocytic sarcoma.

Immune evasion by TGFβ-induced miR-183 repression of MICA/B expression in human lung tumor cells

ABSTRACTImmune escape is a hallmark of cancer. In human lung cancer, we have identified a unique microRNA (miR)-based pathway employed by tumor cells to repress detection by immune cells via the NKG2D-MICA/B receptor-ligand system. MICA/B is readily induced by cell transformation and serves as a danger signal and ligand to alert NK and activated CD8+ T cells. However, immunohistochemical analysis indicated that human lung adenocarcinoma and squamous cell carcinoma specimens express little MICA/B while high levels of miR-183 were detected in both tumor types in a TCGA database. Human lung tumor cell lines confirmed the reverse relationship in expression of MICA/B and miR-183. Importantly, a miR-183 binding site was identified on the 3ʹuntranslated region (UTR) of both MICA and MICB, suggesting its role in MICA/B regulation. Luciferase reporter constructs bearing the 3ʹUTR of MICA or MICB in 293 cells supported the function of miR-183 in repressing MICA/B expression. Additionally, anti-sense miR-183 transfection into H1355 or H1299 tumor cells caused the upregulation of MICA/B. Abundant miR-183 expression in tumor cells was traced to transforming growth factor-beta (TGFβ), as evidenced by antisense TGFβ transfection into H1355 or H1299 tumor cells which subsequently lost miR-183 expression accompanied by MICA/B upregulation. Most significantly, anti-sense miR-183 transfected tumor cells became more sensitive to lysis by activated CD8+ T cells that express high levels of NKG2D. Thus, high miR-183 triggered by TGFβ expressed in lung tumor cells can target MICA/B expression to circumvent detection by NKG2D on immune cells.

Abstract 3803: Immune escape from NK cells and activated CD8 T cells by miR-183 repression of MICA/B expression in human lung cancer

Abstract Immune escape is a hallmark of cancer. In human lung cancer, we have identified a unique microRNA (miR)-based pathway employed by tumor cells to repress detection by immune cells via the NKG2D-MICA/B receptor-ligand system. MICA/B is readily induced by cell transformation and serves as a danger signal and ligand to alert NK and activated CD8+ T cells. However, immunohistochemical analysis indicated that human lung adenocarcinoma and squamous cell carcinoma specimens express little MICA/B while high levels of miR-183 were detected in both tumor types in a TCGA database. Human lung tumor cell lines confirmed the reverse relationship in expression of MICA/B and miR-183. Importantly, a miR-183 binding site was identified on the 3' untranslated region (UTR) of both MICA and MICB, suggesting its role in MICA/B regulation. Luciferase reporter constructs bearing the 3'UTR of MICA or MICB in 293 cells supported the function of miR-183 in repressing MICA/B expression. Additionally, anti-sense miR-183 transfection into H1355 or H1299 tumor cells caused the upregulation of MICA/B. Abundant miR-183 expression in tumor cells was traced to transforming growth factor-beta (TGFβ), as evidenced by anti-sense TGFβ transfection into H1355 or H1299 tumor cells, which subsequently lost miR-183 expression accompanied by MICA/B upregulation. Most significantly, anti-sense miR-183 transfected tumor cells became more sensitive to lysis by activated CD8+ T cells that express high levels of NKG2D. Thus, high miR-183 triggered by TGFβ expressed in lung tumor cells can target MICA/B expression to circumvent detection by NKG2D on immune cells. Citation Format: Nhan Tu, Thu Le Trinh, Sarah S. Donatelli, Melba M. Tejera, Danielle L. Gilvary, Jun-Min Zhou, Erika A. Egsioglu, Sheng Wei, Domenico Coppola, Julie Y. Djeu. Immune escape from NK cells and activated CD8 T cells by miR-183 repression of MICA/B expression in human lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3803.

Potential of Protein-based Anti-metastatic Therapy with Serpins and Inter α-Trypsin Inhibitors.

In this review we summarize the principles of anti-metastatic therapy with selected serpin family proteins, such as pigment epithelial-derived factor (PEDF) and maspin, as well as inter α-trypsin inhibitor (IαIs) light chains (bikunin) and heavy chains (ITIHs). Case-by-case, antimetastatic activity may be dependent or independent of the protease-inhibitory activity of the corresponding proteins. We discuss the incidence of target deregulation in different tumor entities, mechanisms of deregulation, context-dependent functional issues as well as in vitro and in vivo target validation studies with transfected tumor cells or recombinant protein as anti-metastatic agents. Finally, we comment on possible clinical evaluation of these proteins in adjuvant therapy.

The Role of MiR-197 in Regulating Gastric Cancer Radiation Sensitivity

The purpose of this study was to define the roles of miR-197 in determining sensitivity of gastric cancer to radiation therapy, to explore the underlying mechanism and to evaluate the potential of miR-197 as a biomarker for predicting radio-sensitivity. Tumor specimens from 8 patients with a histological diagnosis of gastric adenocarcinoma (stage IIIB/IIIC) which was unrescectable under original evaluation were used in the micro-RNA profiling and comparison. And these specimens were from our phase II study (NCT02024217) which had been completed. Human gastric cancer cell lines, MGC-803 and MKN28, were used in vitro (cell culture) studies. Transfection of tumor cells with the mimic or inhibitor of miR-197, and reporter gene assay, were performed to investigate the role of miR-197 in determining radio-sensitivity and the target gene. Higher expression of miR-197 was observed in human gastric cancer specimens and cell lines that were insensitive to radiation therapy, as compared to sensitive cancer specimens and the cell lines. We also found that miR-197 negatively regulated the expression of PTPN9. The role of miR-197 in conferring cellular resistance to radiation treatment was validated in cell culture models and animal experiment. The effect of miR-197 on radio-resistance was mediated through targeting the 3'-UTR of PTPN9 gene. The expression level of miR-197 in gastric cancer may serve as a biomarker for sensitivity to radiation therapy, and targeting miR-197 may mean a new approach to sensitizing gastric cancer to radiation treatment.

Abstract 4977: Isolation and characterization of a PRAME-specific TCR with high avidity, potent antitumor efficacy and a favorable preclinical safety profile

Abstract Some of the challenges regarding adoptive T cell transfer as an immunotherapeutic approach for the treatment of cancer patients include the initial induction, isolation and later characterization of suitable TCR candidates. In addition, the selection of appropriate target antigens is important and complex as such antigens need to meet some critical demands. Published data as well as in depth in silico analyses revealed the tumor associated antigen PRAME a suitable candidate. Classified as cancer/testis antigen, PRAME is highly expressed in tumors with only limited or no expression in normal tissues, other than testis. Here we describe the induction of PRAME-specific, HLA-A*02:01-restricted CD8-positive T cells by using an in vitro priming approach. Mature dendritic cells transfected with ivtRNA encoding full-length PRAME were cocultured with autologous CD8-enriched PBL from a healthy, HLA-A*02:01-positive blood donor. Initially primed T cells were expanded for 2 weeks and T cells bearing TCRs specific for PRAME-derived epitopes were identified by multimer staining and FACS analysis. Single cells were sorted into 96 well plates and after an expansion period of another 2 weeks, growing clones were screened for specificity. TCR sequences of specific clones were identified by using NGS and reconstructed in a retroviral vector system enabling stable transduction of effector cells. The resulting TCR-modified T cells were thoroughly analyzed and characterized using a dedicated set of assays to evaluate multiple parameters concerning safety and efficacy. This characterization revealed TCR T4.8-1-29 to be highly specific for a PRAME-derived epitope presented on HLA-A*02:01. Only epitope-positive target cells expressing the appropriate HLA-restriction element induced cytokine secretion by T4.8-1-29 expressing effector cells, leading to recognition as well as specific lysis of these target cells. Recognition and lysis, not just of peptide-loaded T2 cells, but also of PRAME-transfected APC, transfected tumor cells as well as endogenously PRAME-positive tumor cells was mediated by this TCR. T4.8-1-29-expressing effectors show a high natural avidity for the target epitope without the need of further manipulation, e.g. functional efficacy enhancement by affinity maturation. In addition, the toxicity assessment using various in vitro and in silico tools revealed a favorable preclinical safety profile for this TCR. In summary, by using an in vitro priming approach utilizing cells from healthy donors, it was possible to generate a TCR recognizing a PRAME-derived epitope with high natural avidity and specificity. The TCR shows a favorable safety profile, demonstrating that this approach is useful to generate TCRs potentially qualifying for evaluation in clinical trials. Citation Format: Manon Weis, Carina Wehner, Christian Ellinger, Susanne Wilde, Dolores J. Schendel. Isolation and characterization of a PRAME-specific TCR with high avidity, potent antitumor efficacy and a favorable preclinical safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4977. doi:10.1158/1538-7445.AM2017-4977

Abstract 1462: Exosome-derived miR-429 contributes to prostate cancer chemoresistance

Abstract Prostate cancer (PCa) is the most common cancer in men and the 2nd leading cause of cancer-related death in the United States. Certain miRNAs, act as tumor promoting or inhibiting factors in tumor development, function primarily by targeting specific mRNAs for degradation or inhibition of translation of their targeted proteins. Emerging evidence indicates that exosomes play a key role in tumor-host crosstalk, and exosome secretion, composition, and functional capacity are altered as tumors progress to aggressive phenotypes. From our prior report, we have constructed a potential network of 29 deregulated miRNAs, including 19 up and 10 down, in exosome samples derived from two kinds of paclitaxel resistant PCa cells (PC3-TXR and DU145-TXR) compared with their parental cells (PC3 and DU145). In this study, to further determine the roles of certain miRNAs derived from exosomes in chemoresistant cells, we firstly confirmed the lower expression level of miR-429 miRNA in the exosomes derived from the paclitaxel resistant PCa cells compared to their parental cells. In addition, when the miR-429 was over-expressed in PC3-TXR and DU145-TXR cells by stable transfection, epithelial-to-mesenchymal transition (EMT) markers significantly altered together with morphological changes. Overexpression of miR-429 decreased the tumor cell migration and invasion that associated with inactivation of PI-3K/AKT and Notch signaling pathways since the pathway specific inhibitors significantly diminished the transfected tumor cell migration and invasion. Finally, we found that overexpression of miRNA-429 significantly enhanced chemo-sensitivity of the chemoresistant PCa cells. These data suggested that exosome-derived miR-429 contributes to PCa chemoresistance and miR-429 may become a potential therapeutic target for PCa patients. This work was supported by Natural Science Foundation of China (NSFC) Key Project (81130046); NSFC (81171993; 81272415); Guangxi Projects of China (2013GXNSFEA053004; 2012GXNSFCB053004; 2013GXNSFBA019177; 1355004-5; 201201ZD004; GZPT13-35; 14122008-22; 11-031-05-K2; KY2015YB057; 14-045-12-K2). Note: This abstract was not presented at the meeting. Citation Format: Yang Liu, Liye Xie, Jing Li, Atsushi Mizokami, Evan T. Keller, Yi Lu, Jian Zhang. Exosome-derived miR-429 contributes to prostate cancer chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1462. doi:10.1158/1538-7445.AM2017-1462

Abstract 2559: FoxF1 is a potential oncogene in prostate cancer

Abstract Background: FoxF1 belongs to the family of forkhead box transcription factors. Many forkhead box proteins are linked to cancer development and progression. However, the regulation and exact function of FoxF1 remains unclear. The aim of this study was to elucidate how the transcriptional activity of FoxF1 is controlled and to assess the role of FoxF1 within prostate cancer. Design: For assessment of FoxF1 functions tumor cell lines were stably transfected with wild type FoxF1 or mutant FoxF1 constructs lacking putative phosphorylation sites. Protein expression and localization of FoxF1 were analyzed by immunprecipitation and Western Blot. Using tissue microarrays of prostate cancer cohorts we analyzed the protein expression and intracellular localization of FoxF1 in tumors compared to benign tissue. Results: Primary tumors and distant metastases exhibited a significantly higher FoxF1 expression compared to benign prostate tissue. In tumors and metastases nuclear localized FoxF1 was more abundant compared to benign tissue. Transfection of tumor cell lines with FoxF1 followed by immunprecipitation and Western Blot showed that nuclear FoxF1 but not cytoplasmic FoxF1 is phosphorylated in tyrosine residues, conceivably tyrosine-39, as suggested by literature data. We further transfected tumor cell lines with different amino acid exchange mutants of putative phosphorylation sites and analyzed the transcription of the known FoxF1 target gene Pecam-1, revealing a complex pattern of regulation, dependent on the site of phosphorylation. Overexpression of FoxF1 in tumor cell lines resulted in epithelial-mesenchymal transition, as observed by loss of E-cadherin expression, gain of Vimentin expression, the up-regulation of Snail and Twist and changes to a spindle-like morphology. Furthermore, overexpression of FoxF1 led to anchorage-independent growth in soft agar as well as increased migration rates in vitro without affecting the proliferation rate. Transfection of tumor cell lines with FoxF1 followed by immunprecipitation and Western Blot showed that nuclear FoxF1 but not cytoplasmic FoxF1 is phosphorylated in tyrosine residues, conceivably tyrosine-39, as suggested by literature data. We further transfected tumor cell lines with different amino acid exchange mutants of putative phosphorylation sites and analyzed the transcription of the known FoxF1 target gene Pecam-1, revealing a complex pattern of regulation. Conclusion: In summary, our results point to a role of FoxF1 as a potential oncogene in prostate cancer whose nuclear localization and activity is regulated by phosphorylation. Citation Format: Carolin Bossmann, Constanze Merz, Yuri Tolkach, Jessica Carlsson, Sven Perner, Ove Andrén, Glen Kristiansen, Michael Nowak. FoxF1 is a potential oncogene in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2559. doi:10.1158/1538-7445.AM2017-2559

Antibody Distance from the Cell Membrane Regulates Antibody Effector Mechanisms.

Immunotherapy using mAbs, such as rituximab, is an established means of treating hematological malignancies. Abs can elicit a number of mechanisms to delete target cells, including complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity, and Ab-dependent cellular phagocytosis. The inherent properties of the target molecule help to define which of these mechanisms are more important for efficacy. However, it is often unclear why mAb binding to different epitopes within the same target elicits different levels of therapeutic activity. To specifically address whether distance from the target cell membrane influences the aforementioned effector mechanisms, a panel of fusion proteins consisting of a CD20 or CD52 epitope attached to various CD137 scaffold molecules was generated. The CD137 scaffold was modified through the removal or addition of cysteine-rich extracellular domains to produce a panel of chimeric molecules that held the target epitope at different distances along the protein. It was shown that complement-dependent cytotoxicity and Ab-dependent cellular cytotoxicity favored a membrane-proximal epitope, whereas Ab-dependent cellular phagocytosis favored an epitope positioned further away. These findings were confirmed using reagents targeting the membrane-proximal or -distal domains of CD137 itself before investigating these properties in vivo, where a clear difference in the splenic clearance of transfected tumor cells was observed. Together, this work demonstrates how altering the position of the Ab epitope is able to change the effector mechanisms engaged and facilitates the selection of mAbs designed to delete target cells through specific effector mechanisms and provide more effective therapeutic agents.

Dual-Targeting Nanoparticles for In Vivo Delivery of Suicide Genes to Chemotherapy-Resistant Ovarian Cancer Cells.

Ovarian cancer is the most lethal gynecologic cancer. Claudin-3 and -4, the receptors for Clostridium perfringens enterotoxin (CPE), are overexpressed in more than 70% of these tumors. Here, we synthesized and characterized poly(lactic-co-glycolic-acid) (PLGA) nanoparticles (NPs) modified with the carboxy-terminal-binding domain of CPE (c-CPE-NP) for the delivery of suicide gene therapy to chemotherapy-resistant ovarian cancer cells. As a therapeutic payload, we generated a plasmid encoding for the diphtheria toxin subunit-A (DT-A) under the transcriptional control of the p16 promoter, a gene highly differentially expressed in ovarian cancer cells. Flow cytometry and immunofluorescence demonstrated that c-CPE-NPs encapsulating the cytomegalovirus (CMV) GFP plasmid (CMV GFP c-CPE-NP) were significantly more efficient than control NPs modified with a scrambled peptide (CMV GFP scr-NP) in transfecting primary chemotherapy-resistant ovarian tumor cell lines in vitro (P = 0.03). Importantly, c-CPE-NPs encapsulating the p16 DT-A vector (p16 DT-A c-CPE-NP) were significantly more effective than control p16 DT-A scr-NP in inducing ovarian cancer cell death in vitro (% cytotoxicity: mean ± SD = 32.9 ± 0.15 and 7.45 ± 7.93, respectively, P = 0.03). In vivo biodistribution studies demonstrated efficient transfection of tumor cells within 12 hours after intraperitoneal injection of CMV GFP c-CPE-NP in mice harboring chemotherapy-resistant ovarian cancer xenografts. Finally, multiple intraperitoneal injections of p16 DT-A c-CPE-NP resulted in a significant inhibition of tumor growth compared with control NP in chemotherapy-resistant tumor-bearing mice (P = 0.041). p16 DT-A c-CPE-NP may represent a novel dual-targeting therapeutic approach for the selective delivery of gene therapy to chemotherapy-resistant ovarian cancer cells. Mol Cancer Ther; 16(2); 323-33. ©2016 AACR.

Recovery HLA-A2 and Beta2-microglobulin Expression in Tumor Cells Using Viral Vectors

Background: Tumor elimination and the success of cancer immunotherapy depend on the proper expression of HLA class I complex (HLA-I) required for the presentation of tumor-associated peptides to cytotoxic T-lymphocytes. Tumors escape immune attack by losing HLA-I expression, often due to irreversible genetic/chromosomal alterations, including mutations in beta2-microglobulin (B2M) or lack of HLA-A2 allele due to a haplotype loss. The introduction of these genes and re-expression of the missing HLA-I specificity on the tumor cell surface is an attractive strategy to induce tumor rejection by T-lymphocytes. Methods: Using genomic HLA-I typing and gene sequencing we determined HLA-I phenotypes and alterations in different human tumor cell lines previously characterized in our laboratory. We used adeno- and adeno-associated viruses to reconstitute/up-regulate HLA-A2 or/and B2M expression in these cells in vitro. Using flow cytometry and immunocytochemistry we evaluated levels and patterns of HLA-I expression in these cells. Methods: Using genomic HLA-I typing and gene sequencing we determined HLA-I phenotypes and alterations in different human tumor cell lines previously characterized in our laboratory. We used adeno- and adeno-associated viruses to reconstitute/up-regulate HLA-A2 or/and B2M expression in these cells in vitro. Using flow cytometry and immunocytochemistry we evaluated levels and patterns of HLA-I expression in these cells. Conclusion: Using gene therapy, it is possible to recover normal B2M and HLA-A2 gene expression caused by structural “hard” alteration and to induce co-expression of both genes in cells naturally lacking HLA-A2 allele. In addition, we demonstrated that transfected tumor cells ae able to express seven HLA-I alleles. The recovery of the missing HLA-I molecules frequently observed in tumor cells using adeno and adeno-associated viruses can be a useful strategy to circumvent cancer immune escape and increase tumor rejection.