Page 321 - 2014 Printable Abstract Book
P. 321
pathways: endocytosis (chlorpromazine), early endosomal entrapment (bafilomycin), late endosomal
persistence (lactacystin), and endolysosomal fusion (chloroquine). Transmission electron microscopy
(TEM) identified subcellular compartmental localization and intra-compartmental geometry of
internalized cGNRs. Radiosensitization was evaluated by clonogenic assays and correlated with
measurement of reactive oxygen species (ROS), γH2AX-foci and micronuclei. Intracellular gold content
was quantified by ICP-MS, and the effect of cytoplasmic cGNR distribution was evaluated using Monte
Carlo simulations. Results: As expected, blockade of cGNR internalization with chlorpromazine abrogated
the cGNR-mediated radiosensitization. Surprisingly, bafilomycin, lactacystin and chloroquine treatment
resulted in greater radiosensitization by cGNRs. Radiosensitization was associated with increases in ROS
levels, number of micronuclei in binucleated cells, and γH2AX-foci post-radiation. ICP-MS results
confirmed that intracellular gold content was comparable in all instances besides treatment with
chlorpromazine. TEM images confirmed that treatment with bafilomycin, lactacystin and chloroquine
resulted in disaggregation of cGNRs within endosomes. Monte Carlo results suggest that the yield of
secondary electrons is 26% higher with the disaggregated (solitary) geometry than the clustered
geometry. The electrons escaping clusters of nanoparticles have energy below 500eV (average range:
2.1um), possibly due to shielding and self-attenuation within clusters. Conversely, the disaggregated
distribution of nanoparticles generated a majority of electrons (59%) with energy above 7keV (average
range: 8.4um). Conclusion: Collectively these results suggest that disaggregation of nanoparticles within
endosomes by pharmacologic interventions has the potential to substantially improve radiosensitization
via increased density of ionizations, increased ROS, and more DNA damage.
1
(PS5-59) HIF-1 in myeloid cells for angiogenesis and tumor response to radiotherapy. G-One Ahn ; Chan-
2
2
1
1
1
1
1
Ju Lee ; Beom-Ju Hong ; Seoyeon Bok ; Hoibin Jeong ; Young-Eun Kim ; Hak Jae Kim ; Il Han Kim ; Irving L.
Weissman ; and J. Martin Brown , Pohang University of Science and Technology, Pohang, Korea, Republic
3
3
1
2
of ; Seoul National University College of Medicine, Seoul, Korea, Republic of ; and Stanford University,
3
Stanford, CA
Strong evidence now suggest that myeloid cells (cells that give rise to monocytes and
macrophages) promote tumor growth by secreting various angiogenic factors including vascular
endothelial growth factor (VEGF) and Bv8. We and others have also reported that myeloid cells
significantly contribute to tumor recurrence after radiotherapy. Blocking myeloid recruitment to tumors
genetically, by neutralizing antibodies, or pharmacologically significantly inhibited tumor regrowth
following irradiation, all of which further confirm that myeloid cells are a major therapeutic target for
radiotherapy. Then what regulates angiogenic properties in myeloid cells? Recently, we have
demonstrated that hypoxia-inducible factor-1 (HIF-1) in myeloid cells is a critical regulator for
angiogenesis by generating a novel strain of myeloid-specific knockout (KO) mice targeting HIF pathways.
We observed enhanced angiogenic phenotype, including erythema, increased VEGF production in the
bone marrow lysates, and increased neovascularization into the subcutaneously implanted matrigel, in
mice inactivated for pVHL (von Hippel Lindau tumor suppressor), and a negative regulator for HIF, in
myeloid cells. All of these effects were completely abrogated when HIF-1 is genetically or
pharmacologically inactivated, suggesting that it is a HIF-1 dependent effect. We further found that
monocytes, among the myeloid cell lineage are the major effector mediating angiogenic phenotypes in
these mice, by producing VEGF and S100A8. To investigate the role of HIF in myeloid cells in influencing
tumor radiosensitivity, we implanted Lewis lung carcinoma in the myeloid-specific HIF-1α or HIF-2α KO
319 | P a g e
persistence (lactacystin), and endolysosomal fusion (chloroquine). Transmission electron microscopy
(TEM) identified subcellular compartmental localization and intra-compartmental geometry of
internalized cGNRs. Radiosensitization was evaluated by clonogenic assays and correlated with
measurement of reactive oxygen species (ROS), γH2AX-foci and micronuclei. Intracellular gold content
was quantified by ICP-MS, and the effect of cytoplasmic cGNR distribution was evaluated using Monte
Carlo simulations. Results: As expected, blockade of cGNR internalization with chlorpromazine abrogated
the cGNR-mediated radiosensitization. Surprisingly, bafilomycin, lactacystin and chloroquine treatment
resulted in greater radiosensitization by cGNRs. Radiosensitization was associated with increases in ROS
levels, number of micronuclei in binucleated cells, and γH2AX-foci post-radiation. ICP-MS results
confirmed that intracellular gold content was comparable in all instances besides treatment with
chlorpromazine. TEM images confirmed that treatment with bafilomycin, lactacystin and chloroquine
resulted in disaggregation of cGNRs within endosomes. Monte Carlo results suggest that the yield of
secondary electrons is 26% higher with the disaggregated (solitary) geometry than the clustered
geometry. The electrons escaping clusters of nanoparticles have energy below 500eV (average range:
2.1um), possibly due to shielding and self-attenuation within clusters. Conversely, the disaggregated
distribution of nanoparticles generated a majority of electrons (59%) with energy above 7keV (average
range: 8.4um). Conclusion: Collectively these results suggest that disaggregation of nanoparticles within
endosomes by pharmacologic interventions has the potential to substantially improve radiosensitization
via increased density of ionizations, increased ROS, and more DNA damage.
1
(PS5-59) HIF-1 in myeloid cells for angiogenesis and tumor response to radiotherapy. G-One Ahn ; Chan-
2
2
1
1
1
1
1
Ju Lee ; Beom-Ju Hong ; Seoyeon Bok ; Hoibin Jeong ; Young-Eun Kim ; Hak Jae Kim ; Il Han Kim ; Irving L.
Weissman ; and J. Martin Brown , Pohang University of Science and Technology, Pohang, Korea, Republic
3
3
1
2
of ; Seoul National University College of Medicine, Seoul, Korea, Republic of ; and Stanford University,
3
Stanford, CA
Strong evidence now suggest that myeloid cells (cells that give rise to monocytes and
macrophages) promote tumor growth by secreting various angiogenic factors including vascular
endothelial growth factor (VEGF) and Bv8. We and others have also reported that myeloid cells
significantly contribute to tumor recurrence after radiotherapy. Blocking myeloid recruitment to tumors
genetically, by neutralizing antibodies, or pharmacologically significantly inhibited tumor regrowth
following irradiation, all of which further confirm that myeloid cells are a major therapeutic target for
radiotherapy. Then what regulates angiogenic properties in myeloid cells? Recently, we have
demonstrated that hypoxia-inducible factor-1 (HIF-1) in myeloid cells is a critical regulator for
angiogenesis by generating a novel strain of myeloid-specific knockout (KO) mice targeting HIF pathways.
We observed enhanced angiogenic phenotype, including erythema, increased VEGF production in the
bone marrow lysates, and increased neovascularization into the subcutaneously implanted matrigel, in
mice inactivated for pVHL (von Hippel Lindau tumor suppressor), and a negative regulator for HIF, in
myeloid cells. All of these effects were completely abrogated when HIF-1 is genetically or
pharmacologically inactivated, suggesting that it is a HIF-1 dependent effect. We further found that
monocytes, among the myeloid cell lineage are the major effector mediating angiogenic phenotypes in
these mice, by producing VEGF and S100A8. To investigate the role of HIF in myeloid cells in influencing
tumor radiosensitivity, we implanted Lewis lung carcinoma in the myeloid-specific HIF-1α or HIF-2α KO
319 | P a g e
