Berbamine targets cancer stem cells and reverses cabazitaxel resistance via inhibiting IGF2BP1 and p-STAT3 in prostate cancer.

BACKGROUND: Cancer stem cells (CSCs) are a small subpopulation of tumor cells with the capability of self-renewal and drug resistance, leading to tumor progression and disease relapse. Our study aimed to investigate the antitumor effect of berbamine, extracted from berberis amurensis, on prostate CSCs. METHODS: Sphere formation was used to collect prostate CSCs. The viability, proliferation, invasion, migration, and apoptosis assays were used to evaluate the antitumor effect of berbamine on prostate CSCs. Prostate CSC markers were analyzed by flow cytometry and qRT-PCR. Small RNA sequencing analysis was conducted to analyse miRNAs. Exosomes were extracted using the ExoQuick-TC kit and verified by testing exosomal markers using western blot. RESULTS: Berbamine targets prostate CSCs. Additionally, berbamine enhanced the antitumor effect of cabazitaxel, a second-line chemotherapeutic drug for advanced prostate cancer, and re-sensitized Cabazitaxel-resistant PCa cells (CabaR-DU145) to cabazitaxel by inhibiting ABCG2, CXCR4, IGF2BP1, and p-STAT3. Berbamine enhanced the expression of let-7 miRNA family and miR-26b and influenced the downstream targets IGF2BP1 and p-STAT3, respectively. Silencing CXCR4 and ABCG2 downregulated the expression of IGF2BP1 and p-STAT3, respectively. Importantly, berbamine enhanced also levels of exosomal let-7 family and miR-26b, suggesting that berbamine possibly influences the expression of let-7 family and miR-26b through exosome delivery. Exosomes derived from berbamine-treated CabaR-DU145 cells re-sensitized the cells to cabazitaxel. CONCLUSION: Berbamine enhanced the toxic activity of cabazitaxel and reversed cabazitaxel resistance potentially through CXCR4/exosomal let-7/IGF2BP1 and ABCG2/exosomal miR-26b/p-STAT3 axes.

Fluorescent Polyvinylphosphonate Bioconjugates for Selective Cellular Delivery.

To date, many poly(ethylene glycol) (PEG) and poly(N-isopropylacrylamide) (PNIPAAm) biomolecule conjugates have been described, but they often show long response times, are not bio-inert, or lose function in biological fluids. Herein, we present a modular synthetic approach to generate polyvinylphosphonate biomolecule conjugates. These conjugates exhibit a sharp phase transition temperature even under physiological conditions where few other examples with this property have been described to date. Furthermore, it was feasible to add biological functions to the polymers via the conjugation step. The polyvinylphosphonate cholesterol constructs are attached to the cellular membrane and the folic acid anchored polymers are shuttled into the cells. This is an exceptional finding through a straightforward synthetic approach.

The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation.

Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four ß-strands and an α-helix which form a parallel dimer by interaction of two ß-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.

Prospective evaluation of immunological, molecular-genetic, image-based and microbial analyses to characterize tumor response and control in patients with unresectable stage III NSCLC treated with concurrent chemoradiotherapy followed by consolidation therapy with durvalumab (PRECISION): protocol for a prospective longitudinal biomarker study.

Background: Concurrent platinum-based chemoradiotherapy (CRT) followed by durvalumab maintenance treatment represents the new standard of care in unresectable stage III non-small cell lung cancer (NSCLC). In this prospective hypothesis-generating single-center study, we aim to identify a framework of prognostic and predictive biomarkers by longitudinal characterization of tumor- and patient (host)-related parameters over all phases of multimodal treatment. Methods: This study will enroll 40 patients (≥18 years, Eastern Cooperative Oncology Group performance status (ECOG PS) 0-2, with a diagnosis of PD-L1 positive (≥1%), inoperable stage III NSCLC) with an indication for CRT followed by maintenance treatment with durvalumab according to European Medicines Agency (EMA) approval. Comprehensive analysis will include peripheral blood cellular and humoral immunophenotyping and circulating tumor DNA as well as gut/saliva microbiota analyses. Additional morphological analysis with 18F-FDG-PET/computed tomography (CT) before, 6 weeks, 6 and 12 months after the end of CRT is included. Statistical analysis using multiple testing will be used to examine the impact of different parameters on progression-free survival (PFS) and overall survival (OS) as well as tumor response and response duration. Discussion: This protocol describes the methodology of a comprehensive biomarker study in order to identify a framework of prognostic and predictive markers for unresectable stage III NSCLC in a real-world setting. Trial Registration: ClinicalTrials.gov identifier (NCT05027165), data registered on August 2021.

Generation of Epstein-Barr Virus Antigen-Specific T Cell Receptors Recognizing Immunodominant Epitopes of LMP1, LMP2A, and EBNA3C for Immunotherapy.

Epstein-Barr virus (EBV) infections in healthy individuals are usually cleared by immune cells, wherein CD8+ T lymphocytes play the most important role. However, in some immunocompromised individuals, EBV infections can lead to the development of cancer in B, T, natural killer (NK) cells and epithelial cells. Most EBV-associated cancers express a limited number of virus-specific antigens such as latent membrane proteins (LMP1 and LMP2) and nuclear proteins (EBNA1, -2, EBNA3A, -B, -C, and EBNA-LP). These antigens represent true tumor-specific antigens and can be considered useful targets for T cell receptor (TCR) gene therapy to treat EBV-associated diseases. We used a TCR isolation platform based on a single major histocompatibility complex class I (MHC I) K562 cell library for the detection, isolation, and re-expression of TCRs targeting immunodominant peptide MHC (pMHC). Mature dendritic cells (mDCs) were pulsed with in vitro-transcribed (ivt) RNA encoding for the selected antigen to stimulate autologous T cells. The procedure allowed the mDCs to select an immunogenic epitope of the antigen for processing and presentation on the cell surface in combination with the most suitable MHC I molecule. We isolated eight EBV-specific TCRs. They recognize various pMHCs of EBV antigens LMP1, LMP2A, and EBNA3C, some of them described previously and some newly identified in this study. The TCR genes were molecularly cloned into retroviral vectors and the resultant TCR-engineered T cells secreted interferon-γ after antigen contact and were able to lyse tumor cells. The EBV-specific TCRs can be used as a basis for the generation of a TCR library, which provides a valuable source of TCRs for the production of EBV-specific T cells to treat EBV-associated diseases in patients with different MHC I types.

Reduced frequency of perforin-positive CD8+ T cells in menstrual effluent of endometriosis patients.

Endometriosis is a widespread disease and commonly reduces the life quality of those affected. Scientific literature indicates different underlying immunological changes. Frequently examined tissues are peripheral blood, endometrial tissue and peritoneal fluid. Yet, knowledge on immunological differences in menstrual effluent (ME) is scarce. In this study, between January 2018 and August 2019, 12 women with endometriosis (rASRM classification: stages I-IV) and 11 healthy controls were included. ME was collected using menstrual cups and venous blood samples (PB) were taken. Mononuclear cells were obtained from ME (MMC) and PB (PBMC) and analyzed using flow cytometry. Concentrations of cell adhesion molecules (ICAM-I and VCAM-I) and cytokines (IL-6, IL-8 and TNF-α) were measured using ELISA. CD8 + T cells obtained from ME were significantly less often perforin-positive in women with endometriosis compared to healthy controls. A comparison between MMC and PBMC revealed that MMC contained significantly less T cells and more B cells. The CD4/CD8 ratio was significantly higher in MMC, and Tregs were significantly less frequently in MMC. In ME, T cells and NK cells expressed significantly more CD69. NK cells obtained from ME were predominantly CD56bright/CD16dim and had a lower frequency of perforin + cells compared to PBMC NK cells. Moreover, ICAM-1 plasma levels were significantly reduced in women with endometriosis compared to healthy controls. In conclusion, CD8 + T cells obtained from the ME were significantly less perforin-positive in endometriosis patients indicating a reduced cytotoxic potential. MMC are distinctively different from PBMC and, thus, seem to be of endometrial origin.

Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells.

Background: Treatment of B-cell malignancies with CD19-directed chimeric antigen receptor (CAR) T-cells marked a new era in immunotherapy, which yet has to be successfully adopted to solid cancers. Epigenetic inhibitors of DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) can induce broad changes in gene expression of malignant cells, thus making these inhibitors interesting combination partners for immunotherapeutic approaches. Methods: Urothelial carcinoma cell lines (UCC) and benign uroepithelial HBLAK cells pretreated with the DNMTi decitabine or the HDACi romidepsin were co-incubated with CAR T-cells directed against EGFR or CD44v6, and subsequent cytotoxicity assays were performed. Effects on T-cell cytotoxicity and surface antigen expression on UCC were determined by flow cytometry. We also performed next-generation mRNA sequencing of inhibitor-treated UCC and siRNA-mediated knockdown of potential regulators of CAR T-cell killing. Results: Exposure to decitabine but not romidepsin enhanced CAR T-cell cytotoxicity towards all UCC lines, but not towards the benign HBLAK cells. Increased killing could neither be attributed to enhanced target antigen expression (EGFR and CD44v6) nor fully explained by changes in the T-cell ligands PD-L1, PD-L2, ICAM-1, or CD95. Instead, gene expression analysis suggested that regulators of cell survival and apoptosis were differentially induced by the treatment. Decitabine altered the balance between survival and apoptosis factors towards an apoptosis-sensitive state associated with increased CAR T-cell killing, while romidepsin, at least partially, tilted this balance in the opposite direction. Knockdown experiments with siRNA in UCC confirmed BID and BCL2L1/BCLX as two key factors for the altered susceptibility of the UCC. Conclusion: Our data suggest that the combination of decitabine with CAR T-cell therapy is an attractive novel therapeutic approach to enhance tumor-specific killing of bladder cancer. Since BID and BCL2L1 are essential determinants for the susceptibility of a wide variety of malignant cells, their targeting might be additionally suitable for combination with immunotherapies, e.g., CAR T-cells or checkpoint inhibitors in other malignancies.

Antitumor effector functions of T cells are dependent on in vivo priming and restricted T-cell receptor expression.

Tumor-specific T cells are crucial for immunologic control of malignant disease. T cells can be induced in vivo by vaccination or adoptively transferred after activation ex vivo. We investigated the requirements for generating T cells with optimal antitumor effector functions in a murine lymphoma model. Using adoptive transfer, we show that in vivo efficacy of T cells cannot be predicted by tumor reactivity in vitro. A restricted T-cell receptor beta chain repertoire of T-cell populations stimulated ex vivo against tumor cells was necessary but not sufficient for tumor protectivity. Tumor elimination furthermore required vaccination of donor mice, hence in vivo priming. The in vivo priming step may allow tumor-specific T cells to accumulate in vitro more rapidly and to survive for longer periods after withdrawal of the antigenic stimulus and adoptive transfer. A possible survival benefit of in vivo induced T cells may be ascribed to the responsiveness to homeostatic cytokines and to unique cytokine milieus encountered in vivo. Most importantly, monoclonal T cells cannot inhibit tumor growth. A prerequisite of tumor rejection was the expression of at least 2 T-cell receptor beta chains by transferred T-cell populations. This finding has implications for designing adoptive transfer strategies for the clinic.

Dendritic cell-based immunogens for B-cell chronic lymphocytic leukemia.

Hybrids generated from tumor cells and dendritic cells (DC) have been proposed as tools for treating malignant disease. Here, we study the underlying principles and the feasibility for the adjuvant therapy of human B cell chronic-lymphocytic leukemia (B-CLL). CLL cells and allogeneic DC were only mixed or additionally fused. Using a combination of FACS and fluorescence microscopic analyses, we show that DC-CLL hybrids can be successfully generated. However, fusion frequencies have to be critically evaluated because the number of fused cells is overestimated when based on FACS analyses alone. The capability of activating patients' PBMC was examined by measuring cytokine secretion in co-culture assays. We made a systematic comparison of the immunostimulatory capacities of different stimulator cell populations, including DC-CLL fusion samples, unfused mixtures of DC and CLL cells as well as DC or tumor cells alone. Surprisingly, even unfused mixtures had a pronounced tumor-directed immunostimulatory effect. This could be explained by the capture of antigens from surrounding leukemia cells by DC during co-cultivation. Although fusion frequencies were low, PBMC stimulation was significantly more effective when the mixtures were subjected to cell fusion. The most potent stimulus was provided by DC-CLL fusion samples derived from mature DC, probably due to their enhanced costimulatory capacity. In summary, DC-tumor cell hybrids might be feasible in the treatment of B-CLL. It should be considered that FACS analysis is not sufficient to assess fusion frequencies and that interactions between unfused DC and CLL cells also result in PBMC activation.

Vaccination against B-cell chronic lymphocytic leukemia with trioma cells: preclinical evaluation.

PURPOSE: Trioma cells are lymphoma cells that have been fused to a hybridoma and have thereby been modified to express an immunoglobulin directed against surface receptors of antigen-presenting cells. Trioma cells that potentially include all lymphoma-derived antigens will be targeted to professional antigen-presenting cells in vivo. This allows uptake, processing, and presentation of tumor-derived antigens to T lymphocytes. In a mouse model, vaccination with trioma cells conferred long-lasting, T cell-dependent tumor immunity and was even able to eradicate established lymphomas. Here, we investigated whether this potent approach is effective in the human system. EXPERIMENTAL DESIGN: Malignant cells from 11 patients with B cell chronic-lymphocytic leukemia (B-CLL) were fused to an anti-Fc receptor hybridoma. The resulting trioma cells were extensively characterized with respect to their clonal origin. The induction of autologous tumor-specific T lymphocytes in the presence of trioma and antigen-presenting cells was examined in vitro by determining cytokine secretion in coculture assays. RESULTS: In seven cases, trioma cells could successfully be generated from B-CLL cells. Stimulation of autologous lymphocytes with trioma cells induced a leukemia-specific T-cell response. Immunostimulatory trioma cells were also obtained from two patients with solid B-cell lymphoma. CONCLUSIONS: Trioma-mediated immunization may be a promising adjuvant treatment of human malignancies of the B-cell lineage, particularly of B-CLL, which has still a very poor prognosis. Our in vitro results pave the way for clinical application.

Harnessing innate and adaptive immunity for adoptive cell therapy of renal cell carcinoma.

The development of immunotherapies for renal cell carcinoma (RCC) has been the subject of research for several decades. In addition to cytokine therapy, the benefit of various adoptive cell therapies has again come into focus in the past several years. Nevertheless, success in fighting this immunogenic tumor is still disappointing. RCC can attract a multitude of different effector cells of both the innate and adaptive immune system, including natural killer (NK) cells, gammadelta T cells, NK-like T cells, peptide-specific T cells, dendritic cells (DC), and regulatory T cells (Tregs). Based on intensive research on the biology and function of different immune cells, we now understand that individual cell types do not act in isolation but function within a complex network of intercellular interactions. These interactions play a pivotal role in the efficient activation and function of effector cells, which is a prerequisite for successful tumor elimination. This review provides a current overview of the diversity of effector cells having the capacity to recognize RCC. Aspects of the functions and anti-tumor properties that make them attractive candidates for adoptive cell therapies, as well as experience in clinical application are discussed. Improved knowledge of the biology of this immune network may help us to effectively harness various effector cells, placing us in a better position to develop new therapeutic strategies to successfully fight RCC.

A polyvalent cellular vaccine induces T-cell responses against specific self-antigens overexpressed in chronic lymphocytic B-cell leukemia.

B cell-derived chronic lymphocytic leukemia (CLL) is an incurable disease that requires innovative therapeutic regimens. Experimental approaches of immunotherapy aiming at induction of systemic T-cell responses have been developed. Trioma cells provide a potent vaccine derived from malignant B cells that allows multiple antigens (Ags) from the parental tumor to be ingested by Ag-presenting cells. Like other strategies using modified whole tumor cells, this approach induces polyvalent responses. Using trioma cell-pulsed dendritic cells (DCs) for T-cell activation in vitro, we asked whether specific Ags overexpressed by CLL can be identified as target structures of such responses and what is the nature of these Ags. Expression levels of several genes in CLL samples were quantitated by reverse transcriptase-polymerase chain reaction. T lymphocytes were polyvalently stimulated by trioma-pulsed DCs and specificities were tested by determining cytokine secretion in the presence of target cells transfected with RNA coding for those Ags that were found to be overexpressed. We demonstrate that DCs pulsed with the modified tumor cells efficiently activate T lymphocytes against CLL and that overexpressed Ags related to leukemogenesis, such as BCL-2, MDM2, and ETV5, serve as targets for those T cells. Immune escape by Ag loss or mutation is less likely to occur if immunity is directed against altered self-proteins that are involved in malignant transformation. Therefore, vaccines based on modified tumor cells such as triomas hold promise for immunotherapy of CLL and other malignancies. Polyvalent vaccines originally designed as individualized therapeutics may be more broadly applicable, at least in patients showing similar Ag patterns.

Allorestricted T cells with specificity for the FMNL1-derived peptide PP2 have potent antitumor activity against hematologic and other malignancies.

Cell-based immunotherapy in settings of allogeneic stem cell transplantation or donor leukocyte infusion has curative potential, especially in hematologic malignancies. However, this approach is severely restricted due to graft-versus-host disease (GvHD). This limitation may be overcome if target antigens are molecularly defined and effector cells are specifically selected. We chose formin-related protein in leukocytes 1 (FMNL1) as a target antigen after intensive investigation of its expression profile at the mRNA and protein levels. Here, we confirm restricted expression in peripheral blood mononuclear cells (PBMCs) from healthy donors but also observe overexpression in different leukemias and aberrant expression in transformed cell lines derived from solid tumors. We isolated allorestricted T-cell clones expressing a single defined TCR recognizing a particular HLA-A2-presented peptide derived from FMNL1. This T-cell clone showed potent antitumor activity against lymphoma and renal cell carcinoma cell lines, Epstein-Barr virus (EBV)-transformed B cells, and primary tumor samples derived from patients with chronic lymphocytic leukemia (CLL), whereas nontransformed cells with the exception of activated B cells were only marginally recognized. Allorestricted TCRs with specificity for naturally presented FMNL1-derived epitopes may represent promising reagents for the development of adoptive therapies in lymphoma and other malignant diseases.