Proposals submitted to the FDA for MSC-based products are undergoing a rapid expansion that is characterized by increased variability in donor and tissue sources, manufacturing processes, proposed ...functional mechanisms, and characterization methods. Here we discuss the diversity in MSC-based clinical trial product proposals and highlight potential challenges for clinical translation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Pyruvate kinase M2 (PKM2) is an alternatively spliced variant, which mediates the conversion of glucose to lactate in cancer cells under normoxic conditions, known as the Warburg effect. Previously, ...we demonstrated that PKM2 is one of 97 genes that are overexpressed in non-small-cell lung cancer (NSCLC) cell lines. Herein, we demonstrate a novel role of subcellular PKM2 expression as a biomarker of therapeutic response after targeting this gene by shRNA or small molecule inhibitor (SMI) of PKM2 enzyme activity in vitro and in vivo. We examined two established lung cancer cell lines, nine patients derived NSCLC and three normal lung fibroblast cell lines for PKM2 mRNA, protein and enzyme activity by RT-qPCR, immunocytochemistry (ICC), and Western blot analysis. All eleven NSCLC cell lines showed upregulated PKM2 enzymatic activity and protein expression mainly in their cytoplasm. Targeting PKM2 by shRNA or SMI, NSCLC cells showed significantly reduced mRNA, enzyme activity, cell viability, and colony formation, which also downregulated cytosolic PKM2 and upregulated nuclear enzyme activities. Normal lung fibroblast cell lines did not express PKM2, which served as negative controls. PKM2 targeting by SMI slowed tumor growth while gene-silencing significantly reduced growth of human NSCLC xenografts. Tumor sections from responding mice showed >70% reduction in cytoplasmic PKM2 with low or undetectable nuclear staining by immunohistochemistry (IHC). In sharp contrast, non-responding tumors showed a >38% increase in PKM2 nuclear staining with low or undetectable cytoplasmic staining. In conclusion, these results confirmed PKM2 as a target for cancer therapy and an unique function of subcellular PKM2, which may characterize therapeutic response to anti-PKM2 therapy in NSCLC.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Th2 cytokines, interleukin (IL)-4 and -13, are structurally and functionally related. They regulate immune responses and the immune microenvironment, not only under normal physiological ...conditions, but also in cancer. Both cytokines bind to their high-affinity receptors and form various configurations of receptor subtypes. We and others have reported that IL-4 and IL-13 bind to IL-4Rα and IL-13Rα1 chains, forming functional receptors in cancer cells. IL-13 also binds with high affinity to a private chain IL-13Rα2. After forming ligand-receptor complexes, both cytokines initiate signal transduction and mediate biological effects, such as tumor proliferation, cell survival, cell adhesion and metastasis. In certain cancers, the presence of these cytokine receptors may serve as biomarkers of cancer aggressiveness.
In a series of studies, we reported that overexpression of IL-4 and IL-13 receptors on cancer cells provides targets for therapeutic agents for cancer therapy. In addition, both of these cytokines and their receptors have been shown to play important roles in modulating the immune system for tumor growth. IL-4, IL-13 and their receptors seem to play a role in cancer stem cells and provide unique targets to eradicate these cells. In this review article, we summarize some of the important attributes of IL-4 and IL-13 receptors in cancer biology and discuss pre-clinical and clinical studies pertaining to recombinant immunotoxins designed to target these receptors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. A variety of targeted agents are being tested in the clinic including cancer vaccines, immunotoxins, antibodies and T ...cell immunotherapy for GBM. We have previously reported that IL-13 receptor subunits α1 and α2 of IL-13R complex are overexpressed in GBM. We are investigating the significance of
IL-13Rα1
and
α2
expression in GBM tumors. In order to elucidate a possible relationship between
IL-13Rα1
and
α2
expression with severity and prognoses of subjects with GBM, we analyzed gene expression (by microarray) and clinical data available at the public The Cancer Genome Atlas (TCGA) database (Currently known as Global Data Commons). More than 40% of GBM samples were highly positive for
IL-13Rα2
mRNA (Log2 ≥ 2) while only less than 16% samples were highly positive for
IL-13Rα1
mRNA. Subjects with high
IL-13Rα1
and
α2
mRNA expressing tumors were associated with a significantly lower survival rate irrespective of their treatment compared to subjects with
IL-13Rα1
and
α2
mRNA negative tumors. We further observed that
IL-13Rα2
gene expression is associated with GBM resistance to temozolomide (TMZ) chemotherapy. The expression of
IL-13Rα2
gene did not seem to correlate with the expression of genes for other chains involved in the formation of IL-13R complex (
IL-13Rα1
or
IL-4Rα
) in GBM. However, a positive correlation was observed between
IL-4Rα
and
IL-13Rα1
gene expression. The microarray data of
IL-13Rα2
gene expression was verified by RNA-Seq data. In depth analysis of TCGA data revealed that immunosuppressive genes (such as
FMOD, CCL2, OSM
, etc.) were highly expressed in
IL-13Rα2
positive tumors, but not in
IL-13Rα2
negative tumors. These results indicate a direct correlation between high level of
IL-13R
mRNA expression and poor patient prognosis and that immunosuppressive genes associated with
IL-13Rα2
may play a role in tumor progression. These findings have important implications in understanding the role of IL-13R in the pathogenesis of GBM and potentially other cancers.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Previously, we have demonstrated that a variety of human cancers including the ovarian cancer express IL‐13Rα2, a high affinity receptor for IL‐13. Herein, we have examined if IL‐13 regulates ...invasion and metastasis of ovarian cancer through IL‐13Rα2 in vitro and in vivo in animal models of human ovarian cancer. We tested cell invasion and protease activity in IL‐13Rα2‐overexpressing and IL‐13Rα2‐negative ovarian tumor cell lines. IL‐13 treatment significantly augmented both cell invasion and enzyme activities in only IL‐13Rα2‐positive cells but not in IL‐13Rα2‐negative cells in vitro. Mechanistically, IL‐13 enhanced ERK1/2, AP‐1 and MMP activities only in IL‐13Rα2‐positive cells but not in IL‐13Rα2‐negative cells. In contrast, other signaling pathways such as IRS1/2, PI3K and AKT do not seem to be involved in IL‐13 induced signaling in ovarian cancer cell lines. Highly specific inhibitors for MMP and AP‐1 efficiently inhibited both invasion and protease activities without impacting the basal level invasion and protease activities in vitro. In orthotopic animal model of human ovarian cancer, IL‐13Rα2‐positive tumors metastasized to lymph nodes and peritoneum earlier than IL‐13Rα2‐negative tumors. Interestingly, the IL‐13Rα2‐positive tumor bearing mice died earlier than mice with IL‐13Rα2‐negative tumor. Intraperitoneal injection of IL‐13 further shortened survival of IL‐13Rα2‐positive tumor bearing mice compared to IL‐13Rα2‐negative tumor mice. IL‐13Rα2‐positive tumors and lymph node metastasis expressed higher levels of MMPs and higher ERK1/2 activation compared to IL‐13Rα2‐negative tumors. Taken together, IL‐13Rα2 is involved in cancer metastasis through activation of ERK/AP‐1 and that targeting IL‐13Rα2 might not only directly kill primary tumors but also prevent cancer metastasis.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Adrenocortical carcinoma (ACC) is a rare but aggressive endocrine malignancy that usually results in a fatal outcome. To allow the better clinical management and reduce mortality, we searched for ...clinical and molecular markers that are reliable predictor of disease severity and clinical outcome in ACC patients. We determined a correlation between the overexpression of IL-13Rα2 and the clinical outcome in ACC patients using comprehensive data available in The Cancer Genome Atlas (TCGA) database. The dataset of 79 ACC subjects were divided into groups of low, medium, or high expression of IL-13Rα2 as determined by RNA-seq. These patients were also stratified by length of survival, overall survival, incidence of a new tumor event, incidence of metastasis, and production of excess hormones. We report a correlation between IL-13Rα2 expression and survival of subjects with ACC. High expression of IL-13Rα2 in ACC tumors was significantly associated with a lower patient survival rate and period of survival compared to low expression (p = 0.0084). In addition, high IL-13Rα2 expression was significantly associated with a higher incidence of new tumor events and excess hormone production compared to low or medium IL-13Rα2 expression. Within the cohort of patients that produced excess hormone, elevated IL-13Rα2 expression was significantly associated with a lower survival rate. Additionally, IL-13Rα1 had a potential relationship between transcript level and ACC survival. Our results and promising antitumor activity in preclinical models and trials indicate that IL-13Rα2 expression is an important prognostic biomarker of ACC disease outcome and a promising target for therapeutic treatment of ACC.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Current evidence suggests that initiation, growth, and invasion of cancer are driven by a small population of cancer stem cells (CSC). Previous studies have identified CD44+ cells as cancer stem ...cells in head and neck squamous cell carcinoma (HNSCC). However, CD44 is widely expressed in most cells in HNSCC tumor samples and several cell lines tested. We previously identified a small population of CD24+/CD44+ cells in HNSCC. In this study, we examined whether this population of cells may represent CSC in HNSCC.
CD24+/CD44+ cells from HNSCC cell lines were sorted by flow cytometry, and their phenotype was confirmed by qRT-PCR. Their self-renewal and differentiation properties, clonogenicity in collagen gels, and response to anticancer drugs were tested in vitro. The tumorigenicity potential of CD24+/CD44+ cells was tested in athymic nude mice in vivo.
Our results show that CD24+/CD44+ cells possessed stemness characteristics of self-renewal and differentiation. CD24+/CD44+ cells showed higher cell invasion in vitro and made higher number of colonies in collagen gels compared to CD24-/CD44+ HNSCC cells. In addition, the CD24+/CD44+ cells were more chemo-resistant to gemcitabine and cisplatin compared to CD24-/CD44+ cells. In vivo, CD24+/CD44+ cells showed a tendency to generate larger tumors in nude mice compared to CD24-/CD44+ cell population.
Our study clearly demonstrates that a distinct small population of CD24+/CD44+ cells is present in HNSCC that shows stem cell-like properties. This distinct small population of cells should be further characterized and may provide an opportunity to target HNSCC CSC for therapy.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Previously, we have demonstrated that Interleukin 13 receptor alpha 2 (IL-13Rα2) is overexpressed in approximate 78% Glioblastoma multiforme (GBM) samples. We have also demonstrated that IL-13Rα2 can ...serve as a target for cancer immunotherapy in several pre-clinical and clinical studies. However, the significance of overexpression of IL-13Rα2 in GBM and astrocytoma and signaling through these receptors is not known. IL-13 can signal through IL-13R via JAK/STAT and AP-1 pathways in certain cell lines including some tumor cell lines. Herein, we have investigated a role of IL-13/IL-13Rα2 axis in signaling through AP-1 transcription factors in human glioma samples in situ.
We examined the activation of AP-1 family of transcription factors (c-Jun, Fra-1, Jun-D, c-Fos, and Jun-B) after treating U251, A172 (IL-13Rα2 +ve) and T98G (IL-13Rα2 -ve) glioma cell lines with IL-13 by RT-qPCR, and immunocytochemistry (ICC). We also performed colorimetric ELISA based assay to determine AP-1 transcription factor activation in glioma cell lines. Furthermore, we examined the expression of AP-1 transcription factors in situ in GBM and astrocytoma specimens by multiplex-immunohistochemistry (IHC). Student t test and ANOVA were used for statistical analysis of the results.
We have demonstrated up-regulation of two AP-1 transcription factors (c-Jun and Fra-1) at mRNA and protein levels upon treatment with IL-13 in IL-13Rα2 positive but not in IL-13Rα2 negative glioma cell lines. Both transcription factors were also overexpressed in patient derived GBM specimens, however, in contrast to GBM cell lines, c-Fos is also overexpressed in patient derived specimens. Astrocytoma specimens showed lesser extent of immunostaining for IL-13Rα2 and three AP-1 factors compared to GBM specimens. By transcription factor activation assay, we demonstrated that AP-1 transcription factors (C-Jun and Fra-1) were activated upon treatment of IL-13Rα2 + GBM cell lines but not IL-13Rα2 - GBM cell line with IL-13. Our results demonstrate functional activity of AP-1 transcription factor in GBM cell lines in response to IL-13.
These results indicate that IL-13/IL-13Rα2 axis can mediate signal transduction in situ via AP-1 pathway in GBM and astrocytoma and may serve as a new target for GBM immunotherapy.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Human bone marrow‐derived multipotent mesenchymal stromal cells, often referred to as mesenchymal stem cells (MSCs), represent an attractive cell source for many regenerative medicine applications ...due to their potential for multi‐lineage differentiation, immunomodulation, and paracrine factor secretion. A major complication for current MSC‐based therapies is the lack of well‐defined characterization methods that can robustly predict how they will perform in a particular in vitro or in vivo setting. Significant advances have been made with identifying molecular markers of MSC quality and potency using multivariate genomic and proteomic approaches, and more recently with advanced techniques incorporating high content imaging to assess high‐dimensional single cell morphological data. We sought to expand upon current methods of high dimensional morphological analysis by investigating whether short term cell and nuclear morphological profiles of MSCs from multiple donors (at multiple passages) correlated with long term mineralization upon osteogenic induction. Using the combined power of automated high content imaging followed by automated image analysis, we demonstrated that MSC morphology after 3 days was highly correlated with 35 day mineralization and comparable to other methods of MSC osteogenesis assessment (such as alkaline phosphatase activity). We then expanded on this initial morphological characterization and identified morphological features that were highly predictive of mineralization capacities (>90% accuracy) of MSCs from additional donors and different manufacturing techniques using linear discriminant analysis. Together, this work thoroughly demonstrates the predictive power of MSC morphology for mineralization capacity and motivates further studies into MSC morphology as a predictive marker for additional in vitro and in vivo responses. Stem Cells 2016;34:935–947
High dimensional early morphological signatures extracted from single cells using high content image analysis can be used to predict mineralization capacity of culture‐expanded human mesenchymal stem cells. The top left panel shows day 3 images for cellular and nuclear morphology analysis. Principal component analysis and linear discriminant analysis are used to develop morphology‐based models that accurately (>90%) predict mineralization.
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