Cu-ATSM Imaging for Cancer Stem Cell-rich Regions: In Vivo and In Vitro characterization

ntroduction: Cancer stem cells are recently noticed to contribute to tumor malignant behaviors, such as resistance to therapy and metastasis ability in tumors. On the other hand, it has been also known that tumor hypoxia is associated with such tumor malignancy. This indicates that tumor hypoxia might be a specific environment to keep up cancer stem cells within tumors. In this study, we examined relationships between existence of cancer stem cells and accumulation of hypoxia imaging agent 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM) in vivo and conducted in vitro characterization. Methods: Double-tracer autoradiography and immunohistchemistry was performed with mouse colon carcinoma (Colon-26) tumor-bearing mice. In autoradiography, mixture of 74 MBq of 18FDG and 0.37 MBq of 64Cu-ATSM was intravenously injected. The distribution of radio-labeled tracers was compared with the immunohistochemical staining for CD133 expression, which reflected the characteristic of cancer stem cells in Colon-26 cells. Additionally, 64Cu-ATSM uptake and survival of CD133+ cells under hypoxia was also examined with Colon-26 cells in vitro. Results: In Colon-26 tumors, 64Cu-ATSM localizes preferentially in regions with a high density of CD133+ cells. Density of CD133+ cells was highest in regions of high 64Cu-ATSM uptake and lowest in regions of high uptake of 18FDG. In addition, we found that in vitro culturing of Colon-26 cells under hypoxia increased both 64Cu-ATSM uptake and the proportion of CD133+ cells present. Conclusion: Our findings show that, in Colon-26 tumors, 64Cu-ATSM accumulates in CD133+ cell-rich regions and that these cells would be resistant to hypoxic environment. Therefore, 64Cu-ATSM could be a potential imaging agent for cancer stem cell-rich regions within tumors.World Molecular Imaging Congres

Effect of Multimerization of a Linear Arg-Gly-Asp Peptide on Integrin Binding Affinity and Specificity

Multivalent interactions are frequently used to enhance ligand-receptor binding affinity. In this study,mono-, di- and trimeric Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Tyr (AVTGRGDSY) peptides, labeled with 125I orCy5.5, were compared in vitro and in vivo. Using human embryonic kidney HEK293 (naturally aV-positive andb 3-negative), HEK293(b 1) (b 1-transfected and aVb 3-negative), HEK293(b 3) (b 3-transfected and strongly aVb 3-positive), and human glioblastoma U87MG (naturally aVb 3-positive) cell lines we evaluated their binding affinityand specificity. In vitro, the monomeric AVTGRGDSY showed specific binding to both HEK293(b 1) andHEK293(b 3) cells. Multimerization resulted in no change toward HEK293 cells, diminished binding withHEK293(b 1) cells, but substantially enhanced binding with aVb 3-positive HEK293(b 3) and U87MG cells. Moreover,multimeric AVTGRGDSY peptides were found to be nearly comparable to the same molar concentration ofa well-known aVb 3-specific cyclo(RGDfV) (c(RGDfV)) peptide in specificity and affinity for targeting aVb 3 integrin.Non-invasive in vivo optical imaging demonstrated that as compared to its monomeric analogue, the Cy5.5-labeled dimeric AVTGRGDSY peptide produced markedly enhanced tumor-to-background contrast inHEK293(b 3) tumor-bearing mice than in HEK293(b 1) tumor-bearing mice. In conclusion, the present studyshowed the difference of monomeric and multimeric linear Arg-Gly-Asp (RGD)-containing compound in integrinselectivity and affinity. Our data provide useful information for the design of novel RGD peptides

3D tumor spheroids constructed with nano-sized pattern scaffoldings: a novel model for molecular imaging research

3D multicellular spheroid (MCS) of tumor cells is expected to be a useful in vitro model reflecting characteristics of in vivo tumors, in tumor research. However, conventional methods to form 3D MCSs have problems in convenience and cell viability. Here we propose a method to acquire 3D MCSs, which are constructed from spontaneous migration and gathering of tumor cells and sustain high proliferation and viability, by simply culturing cells on the plates with nano-sized pattern scaffoldings on surface of the bottom (nano-pattern culture). In this study, we examined detailed characteristics of 3D MCSs constructed with nano-pattern culture to evaluate its utility in molecular imaging research. Morphology and formation process of the MCSs were examined with an inverted light microscope and SEM and with time-lapse analysis, respectively. Proliferation, viability, gene expression profile and response to hypoxia of the MCSs were also investigated. Transcription of hypoxia-inducible factor 1 (HIF-1) was examined with GFP reporter assay. Our observations showed that tumor cells elongated foots to grasp nano-sized pattern scaffoldings, migrated and gathered each other, which results in formation of adherent MCSs. On the other hand, gene expression profile of the cells on nano-sized pattern scaffoldings was mostly similar to that of 2D monolayer cells in cell adhesion and motility, which indicates that nano-pattern culture can liberate latent potential of tumor cells in cell adhesion and motility rather than 2D culture. Additionally, the mature MCSs constructed with nano-pattern culture were up-regulated in gene expression of multicellular organismal development and response to hypoxia. Transcription of HIF-1 and gene expression of HIF-1 target genes were also activated and there were hypoxic regions detected by pimonidazol binding, in the MCSs from this culture. These facts demonstrate that 3D culture system with nano-sized pattern scaffoldings can provide a model for cell migration, construction of organizational structure and hypoxia in 3D formation, which are linked with characteristics of in vivo tumors. Therefore, the nano-pattern culture system could be useful in development of molecular imaging probes.World Molecular Imaging Congress 201

Tumor uptake of radiolabeled acetate reflects the expression of cytosolic acetyl-CoA synthetase: implications for the mechanism of acetate PET.

INTRODUCTION: [1-(11)C]Acetate positron emission tomography (PET) is used for myocardial studies. In the myocardium, mitochondrial acetyl-CoA synthetase (ACSS1) mainly contributes to the radiopharmaceutical uptake. [1-(11)C]Acetate PET is also used for tumor diagnosis; however, the uptake mechanism of radiolabeled acetate in tumors remains unclear. Our previous study reported that cytosolic acetyl-CoA synthetase (ACSS2) was expressed in tumor cells and up-regulated under hypoxia, whereas expression of ACSS1 was negligible regardless of the oxygen conditions. We also indicated that ACSS2 is a bidirectional enzyme that controls acetyl-CoA/acetate metabolism in tumor cells. In this study, to elucidate the basic mechanism of tumor acetate uptake, we focused on ACSS2 and investigated the role of ACSS2 in the uptake of radiolabeled acetate in tumor cells. METHODS: [1-(14)C]Acetate uptake and ACSS2 expression were examined in four tumor cell lines under normoxia or hypoxia. An ACSS2 knockdown study was also performed. RESULTS: [1-(14)C]Acetate uptake was increased in the tumor cells under hypoxia. This pattern followed that of ACSS2 expression. The incorporated (14)C was mostly distributed in the lipid-soluble fractions, and this tendency increased under hypoxia. ACSS2 knockdown led to a corresponding reduction in [1-(14)C]acetate uptake in all tumor cell lines examined under normoxia and hypoxia. CONCLUSIONS: ACSS2 plays an important role in the uptake of radiolabeled acetate in tumor cells, which is different from that in the myocardium, which mainly involves ACSS1. The uptake of radiolabeled acetate in tumors increased under hypoxia along with up-regulation of ACSS2 expression. This suggests a possible mechanism for acetate PET for tumors

Cu-ATSM Imaging for Cancer Stem Cell-rich Regions: In Vivo and In Vitro characterization

ntroduction: Cancer stem cells are recently noticed to contribute to tumor malignant behaviors, such as resistance to therapy and metastasis ability in tumors. On the other hand, it has been also known that tumor hypoxia is associated with such tumor malignancy. This indicates that tumor hypoxia might be a specific environment to keep up cancer stem cells within tumors. In this study, we examined relationships between existence of cancer stem cells and accumulation of hypoxia imaging agent 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM) in vivo and conducted in vitro characterization. Methods: Double-tracer autoradiography and immunohistchemistry was performed with mouse colon carcinoma (Colon-26) tumor-bearing mice. In autoradiography, mixture of 74 MBq of 18FDG and 0.37 MBq of 64Cu-ATSM was intravenously injected. The distribution of radio-labeled tracers was compared with the immunohistochemical staining for CD133 expression, which reflected the characteristic of cancer stem cells in Colon-26 cells. Additionally, 64Cu-ATSM uptake and survival of CD133+ cells under hypoxia was also examined with Colon-26 cells in vitro. Results: In Colon-26 tumors, 64Cu-ATSM localizes preferentially in regions with a high density of CD133+ cells. Density of CD133+ cells was highest in regions of high 64Cu-ATSM uptake and lowest in regions of high uptake of 18FDG. In addition, we found that in vitro culturing of Colon-26 cells under hypoxia increased both 64Cu-ATSM uptake and the proportion of CD133+ cells present. Conclusion: Our findings show that, in Colon-26 tumors, 64Cu-ATSM accumulates in CD133+ cell-rich regions and that these cells would be resistant to hypoxic environment. Therefore, 64Cu-ATSM could be a potential imaging agent for cancer stem cell-rich regions within tumors.World Molecular Imaging Congres

Comparison of in vitro characteristics of a monomeric, dimeric and trimeric fibronectin-derived linear

Multivalent interactions are frequently used to enhance ligand-receptor binding affinity. In this study a mono-, di- and trimeric AVTGRGDSY peptide derived from fibronectin were compared concerning the integrin receptor binding affinity and/or specificity. Methods: AVTGRGDSY monomer, dimer and trimer were synthesized, labeled with 125I or Cy5.5. Using human embryonic kidney cells HEK293 (naturally alphaV-positive and beta3-negative), HEK293(beta1) (beta1-transfected and alphaVbeta3-negative), HEK293(beta3)( beta3-transfected and strongly alphaVbeta3-positive), and human glioblastoma cells U87MG (naturally alphaVbeta3-positive),cell-binding assay, competitive inhibition assay, cell adhesion assay and confocal laser scanning microscopic study were performed to determine the bioactivities of these peptides. Results: The monomeric AVTGRGDSY showed specific binding to both HEK293(beta1) and HEK293(beta3) cells. Multimerization resulted in no change with HEK293 cells, diminished binding for HEK293(beta1) cells, but substantially enhanced binding for alphaVbeta3-positive HEK293(beta3) and U87MG cells in the presence of Mn2+. Moreover, the multimeric AVTGRGDSY peptides were found to be nearly comparable with th alphaVbeta3-specific cyclo(RGDfV) peptide in specificity and affinity for targeting alphaVbeta3 integrin. Conclusion: The multimeric AVTGRGDSY peptide might be an efficient alphaVbeta3-targeting molecule. The present study would be useful for better understanding of the molecular basis of the interaction between RGD ligands and integrin receptors.World Molecular Imaging Congres