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.

A Pan-Cancer Landscape of ABCG2 across Human Cancers: Friend or Foe?

Emerging evidence from research or clinical studies reported that ABCG2 (ATP-binding cassette sub-family G member 2) interrelates with multidrug resistance (MDR) development in cancers. However, no comprehensive pan-cancer analysis is available at present. Therefore, we explore multiple databases, such as TCGA to investigate the potential therapeutic roles of ABCG2 across 33 different tumors. ABCG2 is expressed on a lower level in most cancers and shows a protective effect. For example, a lower expression level of ABCG2 was detrimental to the survival of adrenocortical carcinoma (TCGA-ACC), glioblastoma multiforme (GBM), and kidney renal clear cell carcinoma (KIRC) patients. Distinct associations exist between ABCG2 expression and stemness scores, microenvironmental scores, microsatellite instability (MSI), and tumor mutational burden (TMB) of tumor patients. We observed a significant positive correlation between the ABCG2 mutation site and prognosis in uterine corpus endometrial carcinoma (UCEC) patients. Moreover, transmembrane transporter activity and hormone biosynthetic-associated functions were found to be involved in the functionality of ABCG2 and its related genes. The cDNAs of cancer cell lines were collected to detect exon mutation sequences and to analyze ABCG2 mRNA expression. The mRNA expression level of ABCG2 showed a significant difference among spheres and drug-resistant cancer cell lines compared with their corresponding adherent cancer cell lines in six types of cancer. This pan-cancer study provides, for the first time, a comprehensive understanding of the multifunctionality of ABCG2 and unveils further details of the potential therapeutic role of ABCG2 in pan-cancer.

Induction of porcine-specific regulatory T cells with high specificity and expression of IL-10 and TGF-ß1 using baboon-derived tolerogenic dendritic cells.

BACKGROUND: Regulatory T cells (Treg) play an important role in maintenance of homeostasis in vivo. Treg application to alleviate allo-organ rejection is being studied extensively. However, natural Treg (nTreg) expansion in vitro is laborious and expensive. Antigen-specific Treg are more effective and require lower cell numbers than use of nTreg for immune control. The baboon, as a non-human primate experimental animal model, is widely used in xenotransplantation research. An effective method to generate baboon xeno-specific Treg would benefit research on immune tolerance in xenotransplantation using this model system. METHOD: Baboon tolerogenic dendritic cells (tolDC) were generated in 3 days from monocytes isolated from baboon peripheral blood mononuclear cells in medium supplemented with anti-inflammatory cytokines. After loading with porcine-specific (PS) in vitro-transcribed RNA (ivtRNA), tolDC were used to induce CD4+ T cells to become porcine-specific Treg (PSTreg) in cocultures supplemented with IL-2 and rapamycin for 10 days. Anti-inflammatory and inflammatory cytokine expression was evaluated at the mRNA and protein levels in both baboon tolDC and PSTreg. Functional assays, suppression of activation markers on porcine-specific effector T cells (PSTeff) and inhibition of PSTeff proliferation, were used to test PSTreg specificity. RESULTS: TolDC generated with this method exhibited a tolerogenic phenotype, expressed CCR7 and produced high levels of IL-10 and TGF-ß1, whereas IL-12p40 and IFN-γ were not expressed. PSTreg were successfully generated in cocultures of CD4+ T cells and PS ivtRNA-loaded tolDC. They exhibited a CD3+  CD4+  CD25+  CD127low/-  CD45RAlow  Foxp3+ phenotype and were characterized by high expression of IL-10 and TGF-ß1 mRNA and protein. They showed upregulated expression of EBI3 and GARP mRNA. PSTreg exhibited highly suppressive effects toward PSTeff, secreting high amounts of IL-10 and TGF-ß1 cytokine upon interaction with PSTeff and suppressing IFN-γ expression on PSTeff. CONCLUSION: In this study, a fast 3-day method to generate baboon-derived tolDC is provided that allows subsequent induction of PSTreg displaying high porcine-antigen specificity and expression of IL-10 and TGF-ß1. Porcine-specific baboon Treg can be used in porcine solid organ or cell xenotransplantation studies through adoptive cell transfer into host baboons.

Pre-clinical heterotopic intrathoracic heart xenotransplantation: a possibly useful clinical technique.

BACKGROUND: As a step towards clinical cardiac xenotransplantation, our experimental heterotopic intrathoracic xenotransplantation model offers a beating and ejecting donor heart while retaining the recipient's native organ as a backup in case of graft failure. Clinically applicable immunosuppressive regimens (IS) were investigated first, then treatments known to be effective in hypersensitized patients or those with recalcitrant rejection reactions. METHODS: Consecutive experiments were carried out between 2009 and 2013. Twenty-one genetically modified pigs (GGTA1-knockout/hCD46/± thrombomodulin, in one case HLA-E instead) were used as donors. In all experiments, two cycles of immunoabsorption reduced preformed antibodies. Recipient baboons were divided into two groups according to IS regimen: In group one (n = 10), pre-treatment started either one (anti-CD20) or four weeks (anti-CD20 plus the proteasome inhibitor bortezomib) prior to transplantation. The extended conventional (as for allotransplantation) immunosuppressive maintenance regimen included anti-thymocyte globuline, tacrolimus, mycophenolate mofetil, methylprednisolone and weekly anti-CD20. In group two (n = 11), myeloablative pre-treatment as in multiple myeloma patients (long and short regimens) was added to extended conventional IS; postoperative total thoracic and abdominal lymphoid irradiation (TLI; single dose of 600 cGY) was used to further reduce antibody-producing cells. RESULTS: In the perioperative course, the surgical technique was safely applied: 19 baboons were weaned off extracorporeal circulation and 17 extubated. Nine animals were lost in the early postoperative course due to causes unrelated to surgical technique or IS regimen. Excluding these early failures, median graft survival times of group 1 and 2 were 18.5 (12-50) days and 16 (7-35) days. Necropsy examination of group 1 donor organs revealed hypertrophy of the left ventricular wall in the six longer-lasting grafts; myocardial histology confirmed pre-clinical suspicion of humoral rejection, which was not inhibited by the extended conventional IS including intensified treatments, and signs of thrombotic microangiopathy. Grafts of group 2 presented with only mild-to-moderate features of humoral rejection and thrombotic microangiopathy, except in one case of delayed rejection on day 17. The other experiments in this group were terminated because of untreatable pulmonary oedema, recurring ventricular fibrillation, Aspergillus sepsis, as well as a combination of a large donor organ and late toxic side effects due to TLI. CONCLUSIONS: Longer-term results were difficult to achieve in this model due to the IS regimens used. However, we conclude that heterotopic intrathoracic heart transplantation may be an option for clinical xenotransplantation.

Expression and prognostic significance of a comprehensive epithelial-mesenchymal transition gene set in renal cell carcinoma.

PURPOSE: Epithelial-mesenchymal transition enhances tumor cell motility and has a critical role in invasion and metastasis in a number of carcinomas. A set of transcription factors acts as a master regulator of the epithelial-mesenchymal transition process. To our knowledge it is unknown whether epithelial-mesenchymal transition is important for clear cell renal cell carcinoma progression. Therefore, we comprehensively assessed mRNA levels of epithelial-mesenchymal transition associated genes in renal cell carcinoma as well as their prognostic relevance. MATERIALS AND METHODS: We determined the expression of a set of 46 epithelial-mesenchymal transition related genes by oligonucleotide microarray and gene set enrichment analyses using RNA from 14 samples each of normal kidneys, and G1 and G3 primary renal cell carcinomas. Expression of select epithelial-mesenchymal transition genes was validated by real-time polymerase chain reaction in normal kidneys, primary renal cell carcinomas and metastases in an independent cohort of 112 patients. Results were combined with followup data for survival analysis. RESULTS: The epithelial-mesenchymal transition gene set was preferentially expressed in primary renal cell carcinoma compared to normal tissue (false discovery rate 0.01). No difference was found between G1 and G3 tumors. Quantitative reverse transcriptase-polymerase chain reaction revealed down-regulation of critical epithelial-mesenchymal transition genes such as CDH2 and ZEB1 in metastases, suggesting epithelial-mesenchymal transition reversal during metastasis. Kaplan-Meier analysis demonstrated a better outcome in patients with low CXCR4, vimentin, fibronectin and TWIST1 mRNA levels. Multivariate analyses revealed that CXCR4 and VIM up-regulation represents an independent prognostic marker for poor cancer specific survival in patients with renal cell carcinoma. CONCLUSIONS: Taken together, our data provide strong evidence that epithelial-mesenchymal transition occurs in renal cell carcinoma. Thus, interference with epithelial-mesenchymal transition in renal cell carcinoma might represent a future therapeutic option.

CXC chemokine receptor 4 is essential for maintenance of renal cell carcinoma-initiating cells and predicts metastasis.

In many solid tumors, cancer stem cells (CSC) represent a population with tumor-initiating, self-renewal, and differentiation potential, which can be identified by surface protein markers. No generally applicable markers are yet known for renal cell carcinoma (RCC). Two RCC cell lines (RCC-26, RCC-53) were found to differ widely in their capacity to form spheres in vitro and to establish tumors in mice, potentially reflecting differences in CSC content. A subpopulation expressing the CXC chemokine receptor 4 (CXCR4) was present only in the more tumorigenic cell line RCC-53. When grown as spheres, most of the RCC-53 cells were CXCR4-positive, expressed stem cell-associated transcription factor genes at elevated levels, and were more resistant toward the tyrosine kinase inhibitors sunitinib, sorafenib, and pazopanib. Sorted CXCR4-positive cells exhibited greater capacity for sphere formation and tumor growth-inducing potential in vivo than CXCR4-negative cells. Significantly, higher CXCR4 mRNA levels in primary RCC tumors from patients with localized but not disseminated disease predicted shorter survival. Downregulation of CXCR4 expression by small interfering RNA (siRNA) or pharmacological inhibition by AMD3100 compromised tumor sphere formation, viability of CXCR4-positive cells, and increased their responsiveness toward tyrosine kinase inhibitors. In conclusion, CXCR4 identifies a subpopulation of tumor-initiating cells in RCC cell lines and plays a role in their maintenance. The relative insensitivity of such cells to tyrosine kinase inhibitors might contribute to the development of therapy resistance in RCC patients. Future therapies therefore could combine blockade of the CXCR4 signaling pathway with standard therapies for more effective treatments of metastatic RCC.

High immune response rates and decreased frequencies of regulatory T cells in metastatic renal cell carcinoma patients after tumor cell vaccination.

Our previously reported phase I clinical trial with the allogeneic gene-modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (T(H)1/T(H)2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.

Identification of a novel combination treatment strategy in clear cell renal cell carcinoma stem cells with shikonin and ipilimumab.

Background: Management of clear cell renal cell carcinoma (ccRCC) has changed rapidly in recent years with the advent of immune checkpoint inhibitors (ICIs). However, only a limited number of patients can sustainably respond to immune checkpoint inhibitors and many patients develop resistance to therapy, creating an additional need for therapeutic strategies to improve the efficacy of systemic therapies. Methods: Binding probability and target genes prediction using online databases, invasion, migration, and apoptosis assays as well as the inhibition of cancer stem cells (CSCs) markers in ccRCC cell lines were used to select the most promising phytochemicals (PTCs). Mixed lymphocyte tumor cell culture (MLTC) system and flow cytometry were performed to confirm the potential combination strategy. The potential immunotherapeutic targets and novel CSC markers were identified via the NanoString analysis. The mRNA and protein expression, immune signatures as well as survival characteristics of the marker in ccRCC were analyzed via bioinformation analysis. Results: Shikonin was selected as the most promising beneficial combination partner among 11 PTCs for ipilimumab for the treatment of ccRCC patients due to its strong inhibitory effect on CSCs, the significant reduction of FoxP3+ Treg cells in peripheral blood mononuclear cells (PBMCs) of patients and activation of the endogenous effector CD3+CD8+ and CD3+CD4+ T cells in response to the recognition of tumor specific antigens. Based on NanoString analysis VCAM1, CXCL1 and IL8 were explored as potential immunotherapeutic targets and novel CSC markers in ccRCC. The expression of VCAM1 was higher in the tumor tissue both at mRNA and protein levels in ccRCC compared with normal tissue, and was significantly positively correlated with immune signatures and survival characteristics in ccRCC patients. Conclusion: We propose that a combination of shikonin and ipilimumab could be a promising treatment strategy and VCAM1 a novel immunotherapeutic target for the treatment of ccRCC.

Modified vaccinia virus Ankara delivers a robust surrogate marker for immune monitoring to sarcoma cells even if cells are being exposed to chemotherapy and heat treatment.

PURPOSE: Adding hyperthermia to chemotherapy improved the clinical outcome of patients with high risk soft tissue sarcoma. Further improvement might be possible if combined with vaccination strategies. As no sarcoma-associated antigens are known, the ectopic expression of a surrogate marker for which immune monitoring tools are available, is envisaged. We tested surrogate marker transfer into sarcoma cells in vitro using modified vaccinia virus Ankara (MVA), which has well established clinical safety. We examined its robustness against standard sarcoma treatment modalities, such as ifosfamide and hyperthermia. MATERIALS AND METHODS: We transduced sarcoma cell lines and primary tumour cells from sarcoma patients with MVA encoding the human tyrosinase gene (MVA-hTyr). Kinetics of tyrosinase expression and the potency to activate tyrosinase-specific cytotoxic T cells were assessed. In addition cells were exposed to chemotherapy and heat, imitating the clinical setting. RESULTS: Tyrosinase was ectopically expressed in sarcoma cells. Infected cells presented tyrosinase epitopes for T cell recognition even if exposed to ifosfamide/heat. CONCLUSIONS: As sarcoma patients receive surgery up front or after neoadjuvant systemic chemotherapy/hyperthermia, tumour material is generally available. Our data document that primary sarcoma cells can be infected with MVA-hTyr in vitro and antigen presentation is not affected by ifosfamide or heat treatment. Infected cells can serve as a source for vaccine preparation. MVA-hTyr infection of tumour cells lacking defined antigens is a feasible system to introduce a robust surrogate marker to provide an immune monitoring marker for assessing the induction of antigen-specific T cell activation.

The role of Shikonin in improving 5-aminolevulinic acid-based photodynamic therapy and chemotherapy on glioblastoma stem cells.

Glioblastoma multiforme is a malignant neoplasia with a median survival of less than two years and without satisfactory therapeutic options. The so-called glioblastoma stem cells escape the established radio- and chemotherapies and lead to tumor recurrence in most cases. The alkaloid Shikonin with its various anti stem cell properties and the interstitial photodynamic therapy with 5-aminolevulinic acid seem to be promising new options in the therapy of glioblastoma. In this study, in vitro investigations were performed to observe the influence of Shikonin on viability, proliferation, induction of apoptosis and the capability of forming tumor spheres in U-87 MG and the primary glioblastoma cell line GB14. The combined effect with the chemotherapeutic temozolomide and photodynamic treatment on the mRNA expression of glioma specific stem cell markers and further examined intracellular protoporphyrin IX accumulation under Shikonin treatment was analyzed. Shikonin effectively inhibited the capability of forming tumor spheres and enhanced temozolomide effectiveness in the reduction of proliferation and in the induction of apoptosis. Additionally, Shikonin increased the mRNA expression of the tumor suppressing Neurofibromatosis type 1 (NF1) gene and showed modulating effects on intracellular protoporphyrin IX.

Identification of EZH2 as Cancer Stem Cell Marker in Clear Cell Renal Cell Carcinoma and the Anti-Tumor Effect of Epigallocatechin-3-Gallate (EGCG).

The aim of the study was to develop a new therapeutic strategy to target cancer stem cells (CSCs) in clear cell renal cell carcinoma (ccRCC) and to identify typical CSC markers to improve therapy effectiveness. It was found that the corrected-mRNA expression-based stemness index was upregulated in kidney renal clear cell carcinoma (KIRC) tissues compared to non-tumor tissue and increased with higher tumor stage and grade. EZH2 was identified as a CSC marker and prognosis factor for KIRC patients. The expression of EZH2 was associated with several activated tumor-infiltrating immune cells. High expression of EZH2 was enriched in immune-related pathways, low expression was related to several metabolic pathways. Epigallocatechin-3-gallate (EGCG) was identified as the most potent suppressor of EZH2, was able to inhibit viability, migration, and invasion, and to increase the apoptosis rate of ccRCC CSCs. KIF11, VEGF, and MMP2 were identified as predictive EGCG target genes, suggesting a potential mechanism of how EZH2 might regulate invasiveness and migration. The percentages of FoxP3+ Treg cells in the peripheral blood mononuclear cells of ccRCC patients decreased significantly when cultured with spheres pretreated with EGCG plus sunitinib compared to spheres without treatment. Our findings provide new insights into the treatment options of ccRCC based on targeting CSCs.

Dendritic cells pulsed with RNA encoding allogeneic MHC and antigen induce T cells with superior antitumor activity and higher TCR functional avidity.

Adoptive transfer of T cells expressing transgenic T-cell receptors (TCRs) with antitumor function is a hopeful new therapy for patients with advanced tumors; however, there is a critical bottleneck in identifying high-affinity TCR specificities needed to treat different malignancies. We have developed a strategy using autologous dendritic cells cotransfected with RNA encoding an allogeneic major histocompatibility complex molecule and a tumor-associated antigen to obtain allo-restricted peptide-specific T cells having superior capacity to recognize tumor cells and higher functional avidity. This approach provides maximum flexibility because any major histocompatibility complex molecule and any tumor-associated antigen can be combined in the dendritic cells used for priming of autologous T cells. TCRs of allo-restricted T cells, when expressed as transgenes in activated peripheral blood lymphocytes, transferred superior function compared with self-restricted TCR. This approach allows high-avidity T cells and TCR specific for tumor-associated self-peptides to be easily obtained for direct adoptive T-cell therapy or for isolation of therapeutic transgenic TCR sequences.

Diagnostic and Prognostic Role of miR-192 in Different Cancers: A Systematic Review and Meta-Analysis.

INTRODUCTION: It has been shown that miR-192 is abnormally expressed in a variety of cancer types and participates in different kinds of signaling pathways. The role of miR-192 in the diagnosis and prognosis of cancer has not been verified. This article is aimed at exploring the diagnostic and prognostic value of miR-192 through a systematic review and meta-analysis. METHODS: A systematic search was performed through PubMed, Embase, Web of Science, and Cochrane Library databases up to June 16, 2020. A total of 16 studies were enrolled in the meta-analyses, of which 11 articles were used for diagnostic meta-analysis and 5 articles were used for prognostic meta-analysis. The values of sensitivity and specificity using miR-192 expression as a diagnostic tool were pooled in the diagnostic meta-analysis. The hazard ratios (HRs) of overall survival (OS) with 95 confidence intervals (CIs) were extracted from the studies, and pooled HRs were evaluated in the prognostic meta-analysis. Eleven studies including 667 cancer patients and 514 controls met the eligibility criteria for the diagnostic meta-analysis. Five studies including 166 patients with high miR-192 expression and 236 patients with low miR-192 expression met the eligibility criteria for the prognostic meta-analysis. RESULTS: The overall diagnostic accuracy was as follows: sensitivity 0.79 (95%CI = 0.75-0.82), specificity 0.74 (95%CI = 0.64-0.82), positive likelihood ratio 3.03 (95%CI = 2.11-4.34), negative likelihood ratio 0.29 (95%CI = 0.23-0.37), diagnostic odds ratio 10.50 (95%CI = 5.89-18.73), and area under the curve ratio (AUC) 0.82 (95%CI = 0.78-0.85). The overall prognostic analysis showed that high expression of miR-192 in patients was associated with positive survival (HR = 0.62, 95%CI : 0.41-0.93, p = 0.020). CONCLUSION: Our results revealed that miR-192 was a potential biomarker with good sensitivity and specificity in cancers. Moreover, highly expressed miR-192 predicted a good prognosis for patients.

Shikonin enhances the antitumor effect of cabazitaxel in prostate cancer stem cells and reverses cabazitaxel resistance by inhibiting ABCG2 and ALDH3A1.

Cancer stem cells (CSCs) are a small population among cancer cells, defined as capable of self-renewal, and driving tumor growth, metastasis, and therapeutic relapse. The development of therapeutic strategies to target CSCs is of great importance to prevent tumor metastasis and relapse. Increasing evidence shows that shikonin has inhibiting effects on CSCs. This study was to determine the effect of shikonin on prostate CSCs, and on drug resistant cells. Sphere formation assay was used to enrich prostate CSCs. The effect of shikonin on viability, proliferation, migration, and invasion was studied. Typical CSCs markers were analyzed by flow cytometry and RT-qPCR. The cytotoxic mechanism of shikonin was analyzed by staining for annexin V, reactive oxygen species (ROS) and mitochondrial membrane potential. To study the effect of shikonin on drug-resistant cells a cabazitaxel resistant cell line was established. Shikonin inhibited the viability, proliferation, migration, and invasion of prostate CSCs. Shikonin enhanced the antitumor effect of cabazitaxel, which is a second-line chemotherapeutic drug in advanced prostate cancer. Shikonin induced apoptosis through generating ROS and disrupting the mitochondrial membrane potential. Furthermore, shikonin suppressed the expression of ALDH3A1 and ABCG2 in prostate CSCs, which are two markers related to drug-resistance. When inhibiting the expression of ABCG2 and ALDH3A1, the cabazitaxel resistant cells acquired more sensibility to cabazitaxel. Shikonin enhances the cytotoxic activity of cabazitaxel in prostate CSCs and reverses the cabazitaxel-resistant state.

MOP-dependent enhancement of methadone on the effectiveness of ALA-PDT for A172 cells by upregulating phosphorylated JNK and BCL2.

BACKGROUND: Methadone, as a long-acting opioid analgesic, shows an ability to sensitize the treatment of ALA-PDT for glioblastoma cells (A172) in vitro by promoting apoptosis. However, the mechanisms how methadone enhances the effectiveness of ALA-PDT for tumor cells remains to be clarified. METHODS: The expression of mu opioid receptor (MOP), apoptosis, phosphorylated c-Jun N-terminal kinase (JNK) and phosphorylated apoptosis regulator B cell lymphoma 2 (BCL2) were measured by flow cytometry. Cytotoxicity was determined using Cell Counting Kit-8 (CCK-8). A MOP antagonist, naloxone, was used to evaluate the role of MOP in the above process. RESULTS: It was found that A172 cells show the expression of MOP and that naloxone inhibits the enhancement of the methadone effect on apoptosis following ALA-PDT (p < 0.05). Phosphorylated JNK and BCL2 induced by ALA-PDT were promoted in the presence of methadone (p < 0.05). These methadone effects were also inhibited by naloxone (p < 0.05). CONCLUSIONS: The results suggest that apoptosis induced by ALA-PDT is enhanced by methadone, mostly MOP-mediated, through the upregulation of accumulation of phosphorylated JNK and BCL2, leading to a promotion of cytotoxicity of ALA-PDT for A172 cells.

Methadone enhances the effectiveness of 5-aminolevulinic acid-based photodynamic therapy for squamous cell carcinoma and glioblastoma in vitro.

Although having shown promising clinical outcomes, the effectiveness of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) for squamous cell carcinoma (SCC) and glioblastoma remains to be improved. The analgesic drug methadone is able to sensitize various tumors to chemotherapy. In this in vitro study, the influence of methadone to the effectiveness of ALA-PDT for SCC (FADU) and glioblastoma (A172) was investigated on the protoporphyrin IX (PpIX) fluorescence, survival rates, apoptosis, and cell cycle phase, each with or without the presence of methadone. The production of PpIX was increased by methadone in FADU cells while it was decreased in A172 cells. The survival rates of both cell lines treated by ALA-PDT were significantly reduced by the combination with methadone (P < .05). Methadone also significantly increased the percentage of apoptotic cells and improved the effect of ALA-PDT on the cell cycle phase arrest in the G0/G1 phase (P < .05). This study demonstrates the potential of methadone to influence the cytotoxic effect of ALA-PDT for both SCC and glioblastoma cell lines.

The intracellular detection of MIP-1beta enhances the capacity to detect IFN-gamma mediated HIV-1-specific CD8 T-cell responses in a flow cytometric setting providing a sensitive alternative to the ELISPOT.

BACKGROUND: T-cell mediated immunity likely plays an important role in controlling HIV-1 infection and progression to AIDS. Several candidate vaccines against HIV-1 aim at stimulating cellular immune responses, either alone or together with the induction of neutralizing antibodies, and assays able to measure CD8 and CD4 T-cell responses need to be implemented. At present, the IFN-gamma-based ELISPOT assay is considered the gold standard and it is broadly preferred as primary assay for detection of antigen-specific T-cell responses in vaccine trials. However, in spite of its high sensitivity, the measurement of the sole IFN-gamma production provides limited information on the quality of the immune response. On the other hand, the introduction of polychromatic flow-cytometry-based assays such as the intracellular cytokine staining (ICS) strongly improved the capacity to detect several markers on a single cell level. RESULTS: The cumulative analysis of 275 samples from 31 different HIV-1 infected individuals using an ICS staining procedure optimized by our laboratories revealed that, following antigenic stimulation, IFN-gamma producing T-cells were also producing MIP-1beta whereas T-cells characterized by the sole production of IFN-gamma were rare. Since the analysis of the combination of two functions decreases the background and the measurement of the IFN-gamma+ MIP-1beta+ T-cells was equivalent to the measurement of the total IFN-gamma+ T-cells, we adopted the IFN-gamma+ MIP-1beta+ data analysis system to evaluate IFN-gamma-based, antigen-specific T-cell responses. Comparison of our ICS assay with ELISPOT assays performed in two different experienced laboratories demonstrated that the IFN-gamma+ MIP-1beta+ data analysis system increased the sensitivity of the ICS up to levels comparable to the sensitivity of the ELISPOT assay. CONCLUSION: The IFN-gamma+ MIP-1beta+ data evaluation system provides a clear advantage for the detection of low magnitude HIV-1-specific responses. These results are important to guide the choice for suitable highly sensitive immune assays and to build reagent panels able to accurately characterize the phenotype and function of responding T-cells. More importantly, the ICS assay can be used as primary assay to evaluate HIV-1-specific responses without losing sensitivity in comparison to the ELISPOT assay.

Generation of clinical grade dendritic cells with capacity to produce biologically active IL-12p70.

BACKGROUND: For optimal T cell activation it is desirable that dendritic cells (DCs) display peptides within MHC molecules as signal 1, costimulatory molecules as signal 2 and, in addition, produce IL-12p70 as signal 3. IL-12p70 polarizes T cell responses towards CD4+ T helper 1 cells, which then support the development of CD8+ cytotoxic T lymphocytes. We therefore developed new maturation cocktails allowing DCs to produce biologically active IL-12p70 for large-scale cancer vaccine development. METHODS: After elutriation of leukapheresis products in a closed bag system, enriched monocytes were cultured with GM-CSF and IL-4 for six days to generate immature DCs that were then matured with cocktails, containing cytokines, interferon-gamma, prostaglandin E2, and a ligand for Toll-like receptor 8, with or without poly (I:C). RESULTS: Mature DCs expressed appropriate maturation markers and the lymph node homing chemokine receptor, CCR7. They retained full maturity after culture for two days without maturation cocktails and following cryopreservation. TLR ligand stimulation induced DCs capable of secreting IL-12p70 in primary cultures and after one day of coculture with CD40L-expressing fibroblasts, mimicking an encounter with T cells. DCs matured with our new cocktails containing TLR8 ligand, with or without poly (I:C), induced alloresponses and stimulated virus-specific T cells after peptide-pulsing. DCs matured in cocktails containing TLR8 ligand without poly (I:C) could also be loaded with RNA as a source of antigen, whereas DCs matured in cocktails containing poly (I:C) were unable to express proteins following RNA transfer by electroporation. CONCLUSION: Our new maturation cocktails allowed easy DC harvesting, stable maturation and substantial recoveries of mature DCs after cryopreservation. Our procedure for generating DCs is easily adaptable for GMP-compliance and yields IL-12p70-secreting DCs suitable for development of cancer vaccines using peptides or RNA as sources of immunizing antigens.

Adjuvant therapeutic vaccination in patients with non-small cell lung cancer made lymphopenic and reconstituted with autologous PBMC: first clinical experience and evidence of an immune response.

BACKGROUND: Given the considerable toxicity and modest benefit of adjuvant chemotherapy for non-small cell lung cancer (NSCLC), there is clearly a need for new treatment modalities in the adjuvant setting. Active specific immunotherapy may represent such an option. However, clinical responses have been rare so far. Manipulating the host by inducing lymphopenia before vaccination resulted in a magnification of the immune response in the preclinical setting. To evaluate feasibility and safety of an irradiated, autologous tumor cell vaccine given following induction of lymphopenia by chemotherapy and reinfusion of autologous peripheral blood mononuclear cells (PBMC), we are currently conducting a pilot-phase I clinical trial in patients with NSCLC following surgical resection. This paper reports on the first clinical experience and evidence of an immune response in patients suffering from NSCLC. METHODS: NSCLC patients stages I-IIIA are recruited. Vaccines are generated from their resected lung specimens. Patients undergo leukapheresis to harvest their PBMC prior to or following the surgical procedure. Furthermore, patients receive preparative chemotherapy (cyclophosphamide 350 mg/m2 and fludarabine 20 mg/m2 on 3 consecutive days) for induction of lymphopenia followed by reconstitution with their autologous PBMC. Vaccines are administered intradermally on day 1 following reconstitution and every two weeks for a total of up to five vaccinations. Granulocyte-macrophage-colony-stimulating-factor (GM-CSF) is given continuously (at a rate of 50 microg/24 h) at the site of vaccination via minipump for six consecutive days after each vaccination. RESULTS: To date, vaccines were successfully manufactured for 4 of 4 patients. The most common toxicities were local injection-site reactions and mild constitutional symptoms. Immune responses to chemotherapy, reconstitution and vaccination are measured by vaccine site and delayed type hypersensitivity (DTH) skin reactions. One patient developed positive DTH skin tests so far. Immunohistochemical assessment of punch biopsies taken at the local vaccine site reaction revealed a dense lymphocyte infiltrate. Further immunohistochemical differentiation showed that CD1a+ cells had been attracted to the vaccine site as well as predominantly CD4+ lymphocytes. The 3-day combination chemotherapy consisting of cyclophosphamide and fludarabine induced a profound lymphopenia in all patients. Sequential FACS analysis revealed that different T cell subsets (CD4, CD8, CD4CD25) as well as granulocytes, B cells and NK cells were significantly reduced. Here, we report on clinical safety and feasibility of this vaccination approach during lymphoid recovery and demonstrate a patient example. CONCLUSION: Thus far, all vaccines were well tolerated. The overall trial design seems safe and feasible. Vaccine site reactions associated with infusion of GM-CSF via mini-pump are consistent with the postulated mechanism of action. More detailed immune-monitoring is required to evaluate a potential systemic immune response. Further studies to exploit homeostasis-driven T cell proliferation for the induction of a specific anti-tumor immune response in this clinical setting are warranted.

Cytotoxic markers and frequency predict functional capacity of natural killer cells infiltrating renal cell carcinoma.

PURPOSE: Renal cell carcinoma harbors high numbers of infiltrating lymphocytes with apparent limited efficacy in tumor control. This study focused on the natural killer (NK) cells infiltrating renal cell carcinoma. EXPERIMENTAL DESIGN: Tumor-infiltrating lymphocytes (TIL) were isolated from renal cell carcinoma and analyzed for NK cell frequency and phenotype (n = 34). NK cells were enriched and tested for effector function. RESULTS: Two renal cell carcinoma subtypes were identified, one containing high (>20% of the lymphocyte population, n = 14), the other low (<20%, n = 20), NK cell numbers. NK cells of both groups were noncytolytic ex vivo but differed in CD16 and cytotoxic effector molecule expression as well as in their capacity to acquire cytotoxic activity: The majority of NK cells from tumors with high NK cell content (high NK-TIL) were CD16(bright), whereas few CD16bright NK cells were found in tumors with low NK cell frequencies (low NK-TIL). The CD16 dichotomy correlated with different capacities to develop cytotoxicity after short-term activation with interleukin-2 ex vivo: Low NK-TIL remained noncytolytic against K562 and unresponsive to signals via the activating receptor NKp46 despite expression of receptor and adaptor molecules. In contrast, high NK-TIL acquired cytotoxic function. As described for peripheral CD16bright NK cells, NK cells from high-NK tumors showed high per cell expression of granzyme A, granzyme B, and perforin. NK cells from low NK-TIL resembled CD16(neg/dim) peripheral NK cells with few cytotoxin+ cells and lower expression of perforin. CONCLUSION: The extent of NK cell infiltration and the expression of markers (CD16 and cytotoxins) predict the functional capacity of NK cells infiltrating renal cell carcinoma and can be used to characterize subgroups of renal cell carcinoma.