Glioblastoma (GBM) remains the most devastating primary central nervous system malignancy with a ... more Glioblastoma (GBM) remains the most devastating primary central nervous system malignancy with a median survival of around 15 months. The past decades of research have not yielded significant advancements in the treatment of GBM. In that same time, a novel class of molecules, long non-coding RNAs (lncRNAs), has been found to play a multitude of roles in cancer and normal biology. The increased accessibility of next generation sequencing technologies and the advent of lncRNA-specific microarrays have facilitated the study of lncRNA etiology. Molecular and computational methods can be applied to predict lncRNA function. LncRNAs can serve as molecular decoys, scaffolds, super-enhancers, or repressors. These molecules can serve as phenotypic switches for GBM cells at the expression and/or epigenetic levels. LncRNAs can affect stemness/differentiation, proliferation, invasion, survival, DNA damage response, and chromatin dynamics. Aberrant expression of these transcripts may facilitate t...
Brain metastases (BM) from breast cancer (BC) are associated with significant morbidity and morta... more Brain metastases (BM) from breast cancer (BC) are associated with significant morbidity and mortality. However, the mechanism of how BC cells initiate and propagate BM tumors remains elusive. In this study, we established a set of patient-derived models of circulating tumor cells (CTCs) from either hormone receptor positive (HR+) or negative (HR-) cells, and subsequent BM cells in vivo. Our brain-primed BC derivative lines exhibited a trend to form metastatic tumors in the brain more frequently than in the bones or lungs. In contrast, the naĂ¯ve BC cells rarely formed BM tumors. Micro-MRI of mouse brains after cardiac injection of brain-primed BC derivatives detected the persistent attachment and subsequent colonization of these circuiting cells, whereas naĂ¯ve BC cells, when injected into arterial circulation, were quickly eradicated from the brain. Transcriptomic expression analysis showed that ALDH isoforms including ALDH1A3 are highly upregulated in BM cells in contrast to BC cells. Silencing of ALDH1A3 by lentiviral shRNA infection specifically attenuated in vivo BM formation from the BC derivatives through arterial circulation, while it did not affect formation of the metastases in the lungs and bones. RNA sequencing detected that the expression of tissue transglutaminase (TG2), constitutive activator of transcription factor-kB (NF-kB), is higher in BM but not in lung and bone metastasis and its expression was attenuated by reducing ALDH1A3. Clinically, matched pairs of human primary BC and BM tissue samples showed the elevation of ALDH1A3 expression in BM tumors. In addition, ALDH1A3 expression was significantly correlated with poor prognosis. To develop therapeutics for ALDH1A3, we designed and synthesized a small molecule ALDH1A3 inhibitor, termed GA11. Systemic treatment of the CTC-BM mouse models with GA11 yielded a prominent inhibitory effect on BM formation. Taken together, our results suggest that ALDH1A3 might be a promising therapeutic target for BM tumors.
Glioblastoma (GBM), or grade IV astrocytoma, is a deadly disease due in part to the high degree o... more Glioblastoma (GBM), or grade IV astrocytoma, is a deadly disease due in part to the high degree of intratumoral heterogeneity that contributes to treatment failures. One cellular subset enriched for the capacity to propagate tumor growth and promote therapeutic resistance is brain tumor initiating cells (BTICs). The aim of the study was to define the roles of GTP cyclohydrolase 1 (GCH1) in regulation of BTIC phenotypes via reactive species. We examined GCH1 mRNA and protein expression in patient-derived xenografts, clinical samples and glioma gene expression datasets. GCH1 levels were modulated using lentiviral expression systems, and effects on cell growth, self-renewal, reactive species production and in vitro animal survival were determined. We found that GCH1 RNA and protein expression were increased in BTICs in comparison to non-BTICs, but that GCH1 was not exclusive to the BTIC fraction. Indeed, GCH1 was elevated in GBM in comparison to normal brain. Overexpression of GCH1 in GBM cells increased cell growth in vitro and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 knockdown with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal models. Mechanistically, GCH1 increased CD44 expression and was critical for controlling reactive species balance, including suppressing reactive oxygen species production. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in BTICs promotes tumor maintenance and is a key regulator of reactive oxygen species in GBM.
Accurate patient-derived models of cancer are needed for profiling the disease and for testing th... more Accurate patient-derived models of cancer are needed for profiling the disease and for testing therapeutics. These models must not only be accurate, but also suitable for high-throughput screening and analysis. Here we compare two derivative cancer models, microtumors and spheroids, to the gold standard model of patient-derived orthotopic xenografts (PDX) in glioblastoma multiforme (GBM). To compare these models, we constructed a custom NanoString panel of 350 genes relevant to GBM biology. This custom assay includes 16 GBM-specific gene signatures including a novel GBM subtyping signature. We profiled 11 GBM-PDX with matched orthotopic cells, derived microtumors, and derived spheroids using the custom NanoString assay. In parallel, these derivative models underwent drug sensitivity screening. We found that expression of certain genes were dependent on the cancer model while others were model-independent. These model-independent genes can be used in profiling tumor-specific biology ...
JNCI: Journal of the National Cancer Institute, 1977
A combination of two cell separation methods was utilized for the isolation of thymus-derived lym... more A combination of two cell separation methods was utilized for the isolation of thymus-derived lymphocytes (TL) from enzymatically disaggregated tumors. Passage through Sephadex G-10-glass bead columns to remove adherent cell types followed by exposure to IgG-coated sheep red blood cell monolayers for removal of Fc receptor-bearing inflammatory cells provided functional TL suspensions of high purity with good percentage recovery.
Publisher Summary This chapter reviews the application of a single technique, that is, velocity s... more Publisher Summary This chapter reviews the application of a single technique, that is, velocity sedimentation at unit gravity toward the objective of separating tumor cell suspensions into functionally analyzable cell populations. This technique relies primarily on size differences of cells in tumor cell suspensions and in some instances, these differences have permitted nearly quantitative recovery of the various subpopulations. The success of this separation method is highly dependent on the quality of the single cell suspension to be fractionated. The maximum number of cells that can be separated effectively is determined by the level of resolution required to provide sufficiently separated subpopulations and by a phenomenon known as the streaming limit. Resolution is improved by layering a very narrow band of cells on an undisturbed gradient. Unit gravity velocity sedimentation has been used to separate and analyze the subpopulations of cells found in a number of tumors that grow as a single-cell suspension in the peritoneal cavity.
Somatomedin C, also called insulin-like growth factor I (Sm-C/IGF-I), is a highly conserved polyp... more Somatomedin C, also called insulin-like growth factor I (Sm-C/IGF-I), is a highly conserved polypeptide required for the proliferation of many cell types. Since several attempts in our laboratory to recover monoclonal antibody-secreting hybrids to this peptide by the direct fusion of hyperimmunized splenocytes with myeloma cells had been unsuccessful, we modified our approach by coculturing hyperimmunized BALB/c splenocytes and a small amount of the antigen for 5 days prior to fusion with the P3X63Ag.8.653 myeloma cell line. Of 88 microcultures at risk, specific antibody was detected in 24. Two clones were expanded in ascites fluid and characterized as to isotype, affinity, and specificity. Both were IgG1,kappa and bound human Sm-C/IGF-I with affinity constants of 1.09 and 1.01 X 10(10) liter/mol, respectively. Both clones were quite specific for Sm-C/IGF-I with inconsequential binding to insulin-like growth factor II, multiplication-stimulating activity, any of the chymotryptic fragments of Sm-C/IGF-I, insulin preparations, hGH, hTSH, mEGF, or mouse albumin. In vitro boosting after primary in vivo immunization appears to provide monoclones of an IgG isotype in contrast to primary in vitro immunization, which reportedly favors an IgM isotype. The antibodies produced in this study have proved to be extraordinarily useful in defining the physiologic role of Sm-I/IGF-I with immunoneutralization techniques and in the purification of human Sm-C/IGF-I by affinity chromatography.
Clinical cancer research : an official journal of the American Association for Cancer Research, 1999
Malignant gliomas remain incurable with current interventions. Encouraging investigational approa... more Malignant gliomas remain incurable with current interventions. Encouraging investigational approaches include the use of genetically modified herpes simplex-1 (HSV-1) viruses as direct cytotoxic agents. Combining attenuated HSV-1 with standard therapy, human U-87 malignant glioma xenografts grown in the hind limb or intracranially in athymic nude mice were exposed to ionizing radiation, inoculated with genetically modified HSV R3616, or received both virus and radiation. The combination of virus with fractionated ionizing radiation suggests a synergistic action and results in reduced tumor volumes and longer survivals when compared with treatment with either modality alone.
Glioblastoma (GBM) remains the most devastating primary central nervous system malignancy with a ... more Glioblastoma (GBM) remains the most devastating primary central nervous system malignancy with a median survival of around 15 months. The past decades of research have not yielded significant advancements in the treatment of GBM. In that same time, a novel class of molecules, long non-coding RNAs (lncRNAs), has been found to play a multitude of roles in cancer and normal biology. The increased accessibility of next generation sequencing technologies and the advent of lncRNA-specific microarrays have facilitated the study of lncRNA etiology. Molecular and computational methods can be applied to predict lncRNA function. LncRNAs can serve as molecular decoys, scaffolds, super-enhancers, or repressors. These molecules can serve as phenotypic switches for GBM cells at the expression and/or epigenetic levels. LncRNAs can affect stemness/differentiation, proliferation, invasion, survival, DNA damage response, and chromatin dynamics. Aberrant expression of these transcripts may facilitate t...
Brain metastases (BM) from breast cancer (BC) are associated with significant morbidity and morta... more Brain metastases (BM) from breast cancer (BC) are associated with significant morbidity and mortality. However, the mechanism of how BC cells initiate and propagate BM tumors remains elusive. In this study, we established a set of patient-derived models of circulating tumor cells (CTCs) from either hormone receptor positive (HR+) or negative (HR-) cells, and subsequent BM cells in vivo. Our brain-primed BC derivative lines exhibited a trend to form metastatic tumors in the brain more frequently than in the bones or lungs. In contrast, the naĂ¯ve BC cells rarely formed BM tumors. Micro-MRI of mouse brains after cardiac injection of brain-primed BC derivatives detected the persistent attachment and subsequent colonization of these circuiting cells, whereas naĂ¯ve BC cells, when injected into arterial circulation, were quickly eradicated from the brain. Transcriptomic expression analysis showed that ALDH isoforms including ALDH1A3 are highly upregulated in BM cells in contrast to BC cells. Silencing of ALDH1A3 by lentiviral shRNA infection specifically attenuated in vivo BM formation from the BC derivatives through arterial circulation, while it did not affect formation of the metastases in the lungs and bones. RNA sequencing detected that the expression of tissue transglutaminase (TG2), constitutive activator of transcription factor-kB (NF-kB), is higher in BM but not in lung and bone metastasis and its expression was attenuated by reducing ALDH1A3. Clinically, matched pairs of human primary BC and BM tissue samples showed the elevation of ALDH1A3 expression in BM tumors. In addition, ALDH1A3 expression was significantly correlated with poor prognosis. To develop therapeutics for ALDH1A3, we designed and synthesized a small molecule ALDH1A3 inhibitor, termed GA11. Systemic treatment of the CTC-BM mouse models with GA11 yielded a prominent inhibitory effect on BM formation. Taken together, our results suggest that ALDH1A3 might be a promising therapeutic target for BM tumors.
Glioblastoma (GBM), or grade IV astrocytoma, is a deadly disease due in part to the high degree o... more Glioblastoma (GBM), or grade IV astrocytoma, is a deadly disease due in part to the high degree of intratumoral heterogeneity that contributes to treatment failures. One cellular subset enriched for the capacity to propagate tumor growth and promote therapeutic resistance is brain tumor initiating cells (BTICs). The aim of the study was to define the roles of GTP cyclohydrolase 1 (GCH1) in regulation of BTIC phenotypes via reactive species. We examined GCH1 mRNA and protein expression in patient-derived xenografts, clinical samples and glioma gene expression datasets. GCH1 levels were modulated using lentiviral expression systems, and effects on cell growth, self-renewal, reactive species production and in vitro animal survival were determined. We found that GCH1 RNA and protein expression were increased in BTICs in comparison to non-BTICs, but that GCH1 was not exclusive to the BTIC fraction. Indeed, GCH1 was elevated in GBM in comparison to normal brain. Overexpression of GCH1 in GBM cells increased cell growth in vitro and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 knockdown with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal models. Mechanistically, GCH1 increased CD44 expression and was critical for controlling reactive species balance, including suppressing reactive oxygen species production. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in BTICs promotes tumor maintenance and is a key regulator of reactive oxygen species in GBM.
Accurate patient-derived models of cancer are needed for profiling the disease and for testing th... more Accurate patient-derived models of cancer are needed for profiling the disease and for testing therapeutics. These models must not only be accurate, but also suitable for high-throughput screening and analysis. Here we compare two derivative cancer models, microtumors and spheroids, to the gold standard model of patient-derived orthotopic xenografts (PDX) in glioblastoma multiforme (GBM). To compare these models, we constructed a custom NanoString panel of 350 genes relevant to GBM biology. This custom assay includes 16 GBM-specific gene signatures including a novel GBM subtyping signature. We profiled 11 GBM-PDX with matched orthotopic cells, derived microtumors, and derived spheroids using the custom NanoString assay. In parallel, these derivative models underwent drug sensitivity screening. We found that expression of certain genes were dependent on the cancer model while others were model-independent. These model-independent genes can be used in profiling tumor-specific biology ...
JNCI: Journal of the National Cancer Institute, 1977
A combination of two cell separation methods was utilized for the isolation of thymus-derived lym... more A combination of two cell separation methods was utilized for the isolation of thymus-derived lymphocytes (TL) from enzymatically disaggregated tumors. Passage through Sephadex G-10-glass bead columns to remove adherent cell types followed by exposure to IgG-coated sheep red blood cell monolayers for removal of Fc receptor-bearing inflammatory cells provided functional TL suspensions of high purity with good percentage recovery.
Publisher Summary This chapter reviews the application of a single technique, that is, velocity s... more Publisher Summary This chapter reviews the application of a single technique, that is, velocity sedimentation at unit gravity toward the objective of separating tumor cell suspensions into functionally analyzable cell populations. This technique relies primarily on size differences of cells in tumor cell suspensions and in some instances, these differences have permitted nearly quantitative recovery of the various subpopulations. The success of this separation method is highly dependent on the quality of the single cell suspension to be fractionated. The maximum number of cells that can be separated effectively is determined by the level of resolution required to provide sufficiently separated subpopulations and by a phenomenon known as the streaming limit. Resolution is improved by layering a very narrow band of cells on an undisturbed gradient. Unit gravity velocity sedimentation has been used to separate and analyze the subpopulations of cells found in a number of tumors that grow as a single-cell suspension in the peritoneal cavity.
Somatomedin C, also called insulin-like growth factor I (Sm-C/IGF-I), is a highly conserved polyp... more Somatomedin C, also called insulin-like growth factor I (Sm-C/IGF-I), is a highly conserved polypeptide required for the proliferation of many cell types. Since several attempts in our laboratory to recover monoclonal antibody-secreting hybrids to this peptide by the direct fusion of hyperimmunized splenocytes with myeloma cells had been unsuccessful, we modified our approach by coculturing hyperimmunized BALB/c splenocytes and a small amount of the antigen for 5 days prior to fusion with the P3X63Ag.8.653 myeloma cell line. Of 88 microcultures at risk, specific antibody was detected in 24. Two clones were expanded in ascites fluid and characterized as to isotype, affinity, and specificity. Both were IgG1,kappa and bound human Sm-C/IGF-I with affinity constants of 1.09 and 1.01 X 10(10) liter/mol, respectively. Both clones were quite specific for Sm-C/IGF-I with inconsequential binding to insulin-like growth factor II, multiplication-stimulating activity, any of the chymotryptic fragments of Sm-C/IGF-I, insulin preparations, hGH, hTSH, mEGF, or mouse albumin. In vitro boosting after primary in vivo immunization appears to provide monoclones of an IgG isotype in contrast to primary in vitro immunization, which reportedly favors an IgM isotype. The antibodies produced in this study have proved to be extraordinarily useful in defining the physiologic role of Sm-I/IGF-I with immunoneutralization techniques and in the purification of human Sm-C/IGF-I by affinity chromatography.
Clinical cancer research : an official journal of the American Association for Cancer Research, 1999
Malignant gliomas remain incurable with current interventions. Encouraging investigational approa... more Malignant gliomas remain incurable with current interventions. Encouraging investigational approaches include the use of genetically modified herpes simplex-1 (HSV-1) viruses as direct cytotoxic agents. Combining attenuated HSV-1 with standard therapy, human U-87 malignant glioma xenografts grown in the hind limb or intracranially in athymic nude mice were exposed to ionizing radiation, inoculated with genetically modified HSV R3616, or received both virus and radiation. The combination of virus with fractionated ionizing radiation suggests a synergistic action and results in reduced tumor volumes and longer survivals when compared with treatment with either modality alone.
Uploads
Papers by Yancey Gillespie