Diversity of Extracellular Vesicles (EV) in Plasma of Cancer Patients

Extracellular vesicles (EVs) are produced by all cells and are found in all body fluids. They function as intercellular messengers that carry and deliver signals regulating cellular interactions in health and disease. EVs are emerging as potential biomarkers of diseases and responses to therapies, and much attention is being devoted to understanding their role in physiological as well as pathological events. EVs are heterogenous in their origin, size, molecular characteristics, genetic content and functions. Isolation of EV subsets from plasma and characterization of their distinct properties have been a limiting factor in ongoing efforts to understand their biological importance. Here, we discuss the immunoaffinity-based strategies that are available for isolating distinct subsets of EVs from plasma and provide a road-map to their successful immunocapture and molecular profiling, with special attention to tumor-derived EVs or TEX

Lost in Translation: Obstacles to Translational Medicine

When we launched the Journal of Translational Medicine a few months ago, we were interested primarily in exploring scientific consideration of this discipline. However, as editors of JTM, we have been contacted almost daily to discuss the problems faced by scientists and clinicians around the world who are challenging the traditional boundaries of science and medicine. Through these conversations, we have learned that translational medicine is in fact "lost in translation," inspiring much angst, many promises and some Federal appropriations. However, little has been done to substantively promote this important field. Authoritative reviews on the subject are available to the interested reader [1-7]. In this article, we will address JTM's "constituency" to report what we've learned about the obstacles to translational medicine from the myriad of phone conversations and e-mail interactions

STAT1 contributes to HLA class I upregulation and CTL reactivity after anti-EGFR mAb cetuximab therapy in head and neck cancer patients

Squamous cell carcinoma of head and neck (HNSCC) cells express low HLA class I and antigen processing machinery (APM) components, such as transporter TAP-1/2, which is associated with the reduced sensitivity to cytotoxic T lymphocyte (CTL) mediated lysis. Epidermal growth factor receptor (EGFR) is overexpressed in HNSCC and is associated with the poor prognosis. FDA approved anti-EGFR blockade mAb cetuximab inhibits HNSCC proliferation, and induces EGFR-specific CTL. However, the molecular mechanism(s) underlying the EGFR-specific CTL recognition of HNSCC in the therapeutic efficacy of anti-EGFR mAb is still emerging. We show that cetuximab or EGFR knockdown enhanced expression of HLA class I antigens, which is associated with the EGFR expression level on HNSCC. These findings were validated in a prospective trial of neoadjuvant cetuximab therapy. Interestingly, upregulation of HLA-B/C alleles were more pronounced than HLA-A alleles after cetuximab or EGFR knockdown treatment. EGFR signaling blockade or EGFR depletion also enhanced IFN gamma receptor (IFNAR) on HNSCC and augmented induction of HLA class I and TAP-1/2 caused by IFN gamma treatment. Cetuximab or EGFR knockdown enhanced the level of HLA class I, STAT-1, TAP-1/2 in a STAT-1+/+ cell line but not in STAT-1-/- cell line, documenting the STAT-1 dependence of this effect. We also found that Src homology domain-containing phosphatase 2 (SHP-2), which is downstream of EGFR and also overexpressed in SCCHN, can suppress the immunostimulatory effect of cetuximab treatment on HLA class I/STAT-1 upregulation, and dual targeting of EGFR and SHP-2 co-operates in the most efficient reversal of immune escape phenotype. In addition, cetuximab-based EGFR inhibition and SHP-2 depletion enhanced the recognition of HNSCC cells by EGFR 853-861 specific CTL, and enhanced surface presentation of non-EGFR TA, such as MAGE-3 271-279 , indicating that a broad tumor antigen repertoire is processed and presented by HLA/APM upregulation. These findings elucidate a novel immune escape mechanism associated with EGFR signaling through STAT1 suppression and the reversal with cetuximab, which may provide additional targets for on-going mAb-based immunotherapy

EGFR-mediated tumor immunoescape: The imbalance between phosphorylated STAT1 and phosphorylated STAT3

The epidermal growth factor receptor (EGFR) supports the escape of malignant cells from immunosurveillance by inhibiting the activation of signal transducer and activator of transcription 1 (STAT1) while promoting that of STAT3. We have recently demonstrated that protein tyrosine phosphatase, non-receptor type 11 (PTNP11, best known as SHP2), a phosphatase that operates downstream of EGFR, is responsible for the dephosphorylation of active STAT1 and for the inhibition of the antigen-processing machinery (APM), hence favoring tumor immunoescape. Thus, EGFR signaling may skew the tumor microenvironment to suppress cellular immune responses

507. Chondroitin Sulfate Proteoglycan 4 (CSPG4)-Redirected T Cells Eliminate Glioblastoma-Derived Neurospheres

Adoptive therapy with chimeric antigen receptor-redirected T cells (CAR-Ts) remains challenging for the treatment of glioblastoma (GBM) because of the heterogeneous expression of targetable tumor antigens, which leads to the selection of antigen-loss variants. In addition, the emerging role of GBM-derived neurospheres (GBM-NS) as a critical cell subset in causing GBM recurrence highlights the need to eradicate these cells to achieve sustained responses. By exploiting a well-established culture system, we generated and expanded GBM-NS from 23 surgical samples, and tested them using flow cytometry for the expression of CSPG4, a membrane bound tumor antigen found to be overexpressed in GBM by mRNA profiling. We observed that 70% of GBM-NS displayed high expression of CSPG4 (from 71% to 99%), 17% moderate-high expression (from 51% to 70%), and 13% moderate-low expression (<50%). Based on these results, we hypothesized that CSPG4-specific CAR-Ts would represent a broadly applicable strategy for the treatment of GBM. We generated CSPG4. CAR-Ts, encoding the 4-1BB endodomain, from 6 healthy donors and tested them against 19 of the 23 generated GBM-NS that robustly grow in vitro. CSPG4.CAR-Ts efficiently eliminated all GBM-NS, with high to moderate-low CSPG4 expression, in co-culture experiments at E:T ratios ranging from 2:5 to 1:5 (0.2±0.5% and 0.6±0.9% residual GBM-NS, respectively). By contrast, GBM-NS continued to grow in the presence of control T cells (60.7±17.6% residual GBM-NS). CSPG4.CAR-Ts, but not control T cells, also rapidly proliferated in response to GBM-NS as evaluated by the CFSE assay. CSPG4. CAR-Ts showed a Th1 cytokine profile in response to GBM-NS, releasing significantly more IFN-γ (3593.8±1718.1 pg/ml/2×10^5 cells) and IL-2 (258.8±153.3 pg/ml/2×10^5 cells) than control T cells (1.8±2.5 and 0.9±1.2 pg/ml/2×10^5 cells, respectively). For the in vivo experiments we compared CSPG4.CAR-Ts encoding CD28, 4-1BB, or CD28-4-1BB co-stimulatory endodomains. Two GBM-NS with moderate-low and high CSPG4 expression, respectively were selected and transduced to express the FFluciferase gene to monitor the tumor growth by in vivo bioluminescence imaging. Both GBM-NS and T cells were intracranially injected in 5 wks old female nude mice. CSPG4.CAR-Ts were efficient in controlling tumor growth of both moderate-low and high CSPG4-expressing GBM-NS. We observed an early eradication of the tumor mass in high-CSPG4 expressing GBM-NS, and a significant improved survival in both mice bearing high or moderate-low CSPG4-expressing GBM-NS. CAR-Ts encoding 4-1BB were significantly more efficient than those encoding CD28 or CD28-4-1BB in prolonging tumor free survival (p=0.04). Our data suggest that CSPG4 is an attractive target for CAR-Ts in GBM and that the strategy we have shown to be effective in mice has the potential to be translated to a clinical setting

Adoptively transferred human lung tumor specific cytotoxic T cells can control autologous tumor growth and shape tumor phenotype in a SCID mouse xenograft model

<p>Abstract</p> <p>Background</p> <p>The anti-tumor efficacy of human immune effector cells, such as cytolytic T lymphocytes (CTLs), has been difficult to study in lung cancer patients in the clinical setting. Improved experimental models for the study of lung tumor-immune cell interaction as well as for evaluating the efficacy of adoptive transfer of immune effector cells are needed.</p> <p>Methods</p> <p>To address questions related to the <it>in vivo </it>interaction of human lung tumor cells and immune effector cells, we obtained an HLA class I ⁺lung tumor cell line from a fresh surgical specimen, and using the infiltrating immune cells, isolated and characterized tumor antigen-specific, CD8⁺CTLs. We then established a SCID mouse-human tumor xenograft model with the tumor cell line and used it to study the function of the autologous CTLs provided via adoptive transfer.</p> <p>Results</p> <p>The tumor antigen specific CTLs isolated from the tumor were found to have an activated memory phenotype and able to kill tumor cells in an antigen specific manner <it>in vitro</it>. Additionally, the tumor antigen-specific CTLs were fully capable of homing to and killing autologous tumors <it>in vivo</it>, and expressing IFN-γ, each in an antigen-dependent manner. A single injection of these CTLs was able to provide significant but temporary control of the growth of autologous tumors <it>in vivo </it>without the need for IL-2. The timing of injection of CTLs played an essential role in the outcome of tumor growth control. Moreover, immunohistochemical analysis of surviving tumor cells following CTL treatment indicated that the surviving tumor cells expressed reduced MHC class I antigens on their surface.</p> <p>Conclusion</p> <p>These studies confirm and extend previous studies and provide additional information regarding the characteristics of CTLs which can be found within a patient's tumor. Moreover, the <it>in vivo </it>model described here provides a unique window for observing events that may also occur in patients undergoing adoptive cellular immunotherapy as effector cells seek and destroy areas of tumor growth and for testing strategies to improve clinical effectiveness.</p

Polyclonality of BRAF Mutations in Acquired Melanocytic Nevi

Melanocytic nevi are thought to be senescent clones of melanocytes that have acquired an oncogenic BRAF mutation. BRAF mutation is considered to be a crucial step in the initiation of melanocyte transformation. However, using immunomagnetic separation or laser-capture microdissection, we examined BRAF mutations in sets of approximately 50 single cells isolated from acquired melanocytic nevi from 13 patients and found a substantial number of nevus cells that contained wild-type BRAF mixed with nevus cells that contained BRAF(V600E). Furthermore, we simultaneously amplified BRAF exon 15 and a neighboring single nucleotide polymorphism (SNP), rs7801086, from nevus cell samples obtained from four patients who were heterozygous for this SNP. Subcloning and sequencing of the polymerase chain reaction products showed that both SNP alleles harbored the BRAF(V600E) mutation, indicating that the same BRAF(V600E) mutation originated from different cells. The polyclonality of BRAF mutations in acquired melanocytic nevi suggests that mutation of BRAF may not be an initial event in melanocyte transformation.ArticleJOURNAL OF THE NATIONAL CANCER INSTITUTE. 101(20):1423-1427 (2009)journal articl