Role of Myeloid-Derived Suppressor Cells in High-Dose-Irradiated TRAMP-C1 Tumors: A Therapeutic Target and an Index for Assessing Tumor Microenvironment.

PURPOSE: To investigate the temporal and spatial infiltration of TRAMP-C1 tumors by myeloid-derived suppressor cells (MDSCs) after high-dose radiation therapy (RT), and to explore their effect on tumor growth. METHODS AND MATERIALS: TRAMP-C1 intramuscularly tumors were irradiated with a single dose of 8 Gy or 25 Gy. The dynamics of infiltrated MDSCs and their intratumoral spatial distribution were assessed by immunohistochemistry and flow cytometry. Cytokine levels in the blood and tumor were analyzed by multiplex immunoassay. Mice were injected with anti-Gr-1 antibody to determine whether MDSCs affect tumor growth after RT. RESULTS: CD11b+Gr-1+ MDSCs infiltrated TRAMP-C1 tumors irradiated with 25 Gy, but not 8 Gy, within 4 hours and recruitment persisted for at least 2 weeks. Both CD11b+Ly6G+Ly6C+ polymorphonuclear-MDSCs (PMN-MDSCs) and CD11b+Ly6G-Ly6Chi monocytic-MDSCs (M-MDSCs) were involved. Tumor RT also increased the representation of both MDSC subpopulations in the spleen and peripheral blood. Levels of multiple cytokines were increased in the tumors at 2 weeks, including GM-CSF, G-CSF, CCL-3, CCL-5, CXCL-5, IL-6, IL-17α, and VEGF-a; while G-CSF, IL-6, and TNF-α levels increased in the blood. PMN-MDSCs aggregated in the central necrotic region of the irradiated tumors over time, where they were associated with avascular hypoxia (CD31-PIMO+). MDSCs expressed the proangiogenic factor, matrix metalloproteinase-9, and, within the necrotic area, high levels of arginase-1 and indoleamine 2,3-dioxygenase. Depletion of PMN-MDSCs by Gr-1 antibody increased the efficacy of high-dose RT. CONCLUSIONS: PMN-MDSCs infiltrate TRAMP-C1 tumors after high-dose RT. Their spatial distribution suggests they are involved in the evolution of an intratumoral state of necrosis associated with avascular hypoxia, and their phenotype is consistent with them being immunosuppressive. They appear to promote tumor growth after RT, making them a prime therapeutic target for therapeutic intervention. Assessment of MDSCs and cytokine levels in blood could be an index of the need for such an intervention.

Sunitinib Treatment-elicited Distinct Tumor Microenvironment Dramatically Compensated the Reduction of Myeloid-derived Suppressor Cells.

BACKGROUND/AIM: The clinical response rate of prostate cancer to tyrosine kinase inhibitor (TKI) monotherapy is low. The mechanisms of resistance to TKI are unclear. This study aimed to examine if the tumor microenvironment (TME) is involved in the resistance. MATERIALS AND METHODS: The anti-vascular effect of Sutent was examined by immunofluorescent staining in TRAMP-C1 tumor. The percentage of CD11b+ population were analyzed by flow cytometry. The level of cytokines and chemokines were measured by multiplex immunoassay. RESULTS: The Sutent monotherapy caused 1.5 days of tumor growth delay, chronic hypoxia, and more mature vasculature. Sutent monotherapy increased the percentage of polymorphonuclear myeloid-derived suppressor cells (MDSCs) in peripheral blood. The evolved TME triggered the re-distribution of myeloid cells in chronically hypoxic areas. The multiplex immunoassay indicated higher levels of several cytokines and chemokines both in tumors and the blood. CONCLUSION: Sunitinib treatment induced a distinct tumor microenvironment that impaired the efficient reduction of MDSCs by TKI.

Radiosensitization of ultrasmall GNP-PEG-cRGDfK in ALTS1C1 exposed to therapeutic protons and kilovoltage and megavoltage photons.

PURPOSE: One of the promising radiosensitizers is the ultrasmall gold nanoparticle (GNP) with a hydrodynamic diameter <3 nm. We studied functionalized ultrasmall GNPs (1.8 nm diameter) coated by polyethylene glycol (PEG) and conjugated with cyclic RGDfK (2.6 nm hydrodynamic diameter) for targeting of alpha(v) beta(3) integrin (αvß3) in the murine ALTS1C1 glioma cell line. MATERIALS AND METHODS: We investigated the uptake, toxicity and radiosensitivity of GNP-PEG-cRGDfKs in ALTS1C1 cells exposed to protons, kilovoltage photons and megavoltage photons. The in vitro uptake and toxicity of GNPs in the hepatocytes and Kupffer cells were assessed for murine AML12 hepatocyte and RAW 264.7 macrophage cell lines. The in vivo biodistribution of GNPs in the ALTS1C1 tumor model was tested using the inductively coupled plasma mass spectrometry. RESULTS: Results indicated GNPs accumulated in the cytoplasm with negligible toxicity for a moderate concentration of GNPs. Observed sensitizer enhancement ratios and dose enhancement factors are 1.21-1.66 and 1.14-1.33, respectively, for all radiations. CONCLUSION: Ultrasmall GNP-PEG-cRGD can be considered as a radiosensitizer. For radiotherapy applications, the delivery method should be developed to increase the GNP uptake in the tumor and decrease the uptakes in undesirable organs.

Decline of Tumor Vascular Function as Assessed by Dynamic Contrast-Enhanced Magnetic Resonance Imaging Is Associated With Poor Responses to Radiation Therapy and Chemotherapy.

PURPOSE: To investigate whether changes in the volume transfer coefficient (K(trans)) in a growing tumor could be used as a surrogate marker for predicting tumor responses to radiation therapy (RT) and chemotherapy (CT). METHODS AND MATERIALS: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was consecutively performed on tumor-bearing mice, and temporal and spatial changes of K(trans) values were measured along with tumor growth. Tumor responses to RT and CT were studied before and after observed changes in K(trans) values with time. RESULTS: Dynamic changes with an initial increase and subsequent decline in K(trans) values were found to be associated with tumor growth. When each tumor was divided into core and peripheral regions, the K(trans) decline was greater in core, although neither vascular structure or necrosis could be linked to this spatial difference. Tumor responses to RT were worse if applied after the decline of K(trans), and there was less drug distribution and cell death in the tumor core after CT. CONCLUSION: The K(trans) value in growing tumors, reflecting the changes of tumor microenvironment and vascular function, is strongly associated with tumor responses to RT and CT and could be a potential surrogate marker for predicting the tumor response to these treatments.

Combination of vessel-targeting agents and fractionated radiation therapy: the role of the SDF-1/CXCR4 pathway.

PURPOSE: To investigate vascular responses during fractionated radiation therapy (F-RT) and the effects of targeting pericytes or bone marrow-derived cells (BMDCs) on the efficacy of F-RT. METHODS AND MATERIALS: Murine prostate TRAMP-C1 tumors were grown in control mice or mice transplanted with green fluorescent protein-tagged bone marrow (GFP-BM), and irradiated with 60 Gy in 15 fractions. Mice were also treated with gefitinib (an epidermal growth factor receptor inhibitor) or AMD3100 (a CXCR4 antagonist) to examine the effects of combination treatment. The responses of tumor vasculatures to these treatments and changes of tumor microenvironment were assessed. RESULTS: After F-RT, the tumor microvascular density (MVD) was reduced; however, the surviving vessels were dilated, incorporated with GFP-positive cells, tightly adhered to pericytes, and well perfused with Hoechst 33342, suggesting a more mature structure formed primarily via vasculogenesis. Although the gefitinib+F-RT combination affected the vascular structure by dissociating pericytes from the vascular wall, it did not further delay tumor growth. These tumors had higher MVD and better vascular perfusion function, leading to less hypoxia and tumor necrosis. By contrast, the AMD3100+F-RT combination significantly enhanced tumor growth delay more than F-RT alone, and these tumors had lower MVD and poorer vascular perfusion function, resulting in increased hypoxia. These tumor vessels were rarely covered by pericytes and free of GFP-positive cells. CONCLUSIONS: Vasculogenesis is a major mechanism for tumor vessel survival during F-RT. Complex interactions occur between vessel-targeting agents and F-RT, and a synergistic effect may not always exist. To enhance F-RT, using CXCR4 inhibitor to block BM cell influx and the vasculogenesis process is a better strategy than targeting pericytes by epidermal growth factor receptor inhibitor.

A preclinical study to explore vasculature differences between primary and recurrent tumors using ultrasound Doppler imaging.

The purpose of this preclinical study was to perform a longitudinal investigation of the function and morphology of the vasculatures of primary and recurrent tumors, because recurrent tumors have lower curability. Thus, elucidating differences in the features of the vasculatures of primary and recurrent tumors could help to improve tumor therapies. The transgenic adenocarcinoma of the mouse prostate tumors were transplanted in nonirradiated and with 25 Gy of preirradiation normal tissues to produce the primary and recurrent tumor models, respectively. The perfusion and branching index of tumor vasculatures were characterized to reveal the function and morphology information, respectively. The blood vessels were more dilated and continuous in recurrent tumors than in primary tumors. During tumor progression, the perfusion increased in primary tumors but did not change significantly in recurrent tumors. The tumor perfusion was lower in recurrent tumors than in primary tumors, whereas branching index in 2-D ultrasound images did not differ between the two tumor models. Furthermore, the introducing 3-D volumetric power Doppler image may have the potential for accurately revealing the morphologic features within tumors. The results of this study suggest that power Doppler imaging is an easily applied and rapid method for noninvasively assessing the vascular features of primary and recurrent tumors and for exploring differences between their vasculature pathways.

Characterization of tumor vasculature distributions in central and peripheral regions based on Doppler ultrasound.

PURPOSE: Tumor heterogeneity is a major obstacle to therapy, and thus, how to achieve the maximal therapeutic gain in tumor suppression is an important issue. To accomplish this goal, assessing changes in tumor behaviors before treatment is helpful for physicians to adjust treatment schedules. In this study, the authors longitudinally and spatially investigated tumor perfusion and vascular density by power Doppler imaging and immunohistochemical analysis, respectively. Moreover, the authors developed a method to describe quantitatively the spatial distribution of the vasculature within the central and peripheral regions of tumors. METHODS: Tumor perfusion was estimated by power Doppler images at an operating frequency of 25 MHz. To avoid the attenuation effect of such high-frequency ultrasound, murine tumors were subcutaneously transplanted into the thighs of mice and then monitored for 11 days. The tumors were removed at various time intervals for immunohistochemical analysis of their vascular density using CD31 staining. The spatial characteristics of the tumor vasculature were quantified by a γ value, which characterizes the rate at which vascular signals increase with the fractional sizes of the peripheral area within the tumor. RESULTS: During tumor progression, the volume of tumor perfusion in the power Doppler images was strongly correlated with the vascular density determined by immunohistochemical analysis. In addition, the γ value significantly decreased with increased tumor size in the power Doppler images but not in the immunohistochemical analysis. CONCLUSIONS: Although the tumor perfusion and vascular density estimates showed good temporal correlations during tumor progression, they did not show good spatial correlations due to tumor perfusion patterns changing from homogeneous to heterogeneous. In contrast to the perfusion patterns, the vascular density of the tumor remained uniformly distributed. In the present study, no necrosis regions were found in the tumor experiments. Furthermore, the measurement of γ value is a simple method for assessing the vasculatures of spatial distribution within tumors.

Irradiation promotes an m2 macrophage phenotype in tumor hypoxia.

Macrophages display different phenotypes with distinct functions and can rapidly respond to environmental changes. Previous studies on TRAMP-C1 tumor model have shown that irradiation has a strong impact on tumor microenvironments. The major changes include the decrease of microvascular density, the increase of avascular hypoxia, and the aggregation of tumor-associated macrophages in avascular hypoxic regions. Similar changes were observed no matter the irradiation was given to tissue bed before tumor implantation (pre-IR tumors), or to established tumors (IR tumors). Recent results on three murine tumors, TRAMP-C1 prostate adenocarcinoma, ALTS1C1 astrocytoma, and GL261 glioma, further demonstrate that different phenotypes of inflammatory cells are spatially distributed into different microenvironments in both IR and pre-IR tumors. Regions with avascular hypoxia and central necrosis have CD11b(high)/Gr-1+ neutrophils in the center of the necrotic area. Next to them are CD11b(low)/F4/80+ macrophages that sit at the junctions between central necrotic and surrounding hypoxic regions. The majority of cells in the hypoxic regions are CD11b(low)/CD68+ macrophages. These inflammatory cell populations express different levels of Arg I. This distribution pattern, except for neutrophils, is not observed in tumors receiving chemotherapy or an anti-angiogenesis agent which also lead to avascular hypoxia. This unique distribution pattern of inflammatory cells in IR tumor sites is interfered with by targeting the expression of a chemokine protein, SDF-1α, by tumor cells, and this also increases radiation-induced tumor growth delay. This indicates that irradiated-hypoxia tissues have distinct tumor microenvironments that favor the development of M2 macrophages and that is affected by the levels of tumor-secreted SDF-1α.

In vivo imaging of radiation-induced tissue apoptosis by (99m)Tc(I)-his (6)-annexin A5.

OBJECTIVE: A recombinant annexin A5 with the N-terminal extension of six histidine residues was labeled with (99m)Tc(I)-tricarbonyl ion to produce the (99m)Tc-labeled annexin A5, referred to (99m)Tc(I)-his(6)-annexin A5. We have explored the agent as an effective imaging probe for in vivo detecting the apoptosis of internal tissue subjected with high radiation doses in a γ-irradiated mouse model. METHODS: [(99m)Tc(CO)(3)(OH(2))(3)](+) was prepared and taken to directly label his(6)-annexin A5. The radiochemical purity of (99m)Tc(I)-his(6)-annexin A5 after size-exclusion separation was measured by HPLC. The binding affinity of (99m)Tc(I)-his(6)-annexin A5 to apoptotic cells was assessed using 20 Gy-irradiated Jurkat T cells. The effectiveness of (99m)Tc(I)-his(6)-annexin A5 as an imaging probe to detect the internal tissue apoptosis was assessed by biodistribution study and nanoSPECT/CT using the animal model of C57BL/6J mice conducted with 10 Gy γ irradiation. RESULTS: The radiochemical purity of (99m)Tc(I)-his(6)-annexin A5 could attain ≥95%. The binding affinity of (99m)Tc(I)-his(6)-annexin A5 to the 20 Gy-irradiated Jurkat cells was found to be ca. 20-fold higher than that to the sham-irradiated cells. In the animal imaging study, the splenic uptake of (99m)Tc(I)-his(6)-annexin A5 for the 10 Gy-irradiated mice showed from ca. 3-fold to 5-fold higher than those of the sham-irradiated mice from 45 to 165 min postinjection. The corresponding intestinal uptake showed from ca. 2-fold to 3-fold higher during the same period of time postinjection. The biodistribution study demonstrated the organ uptakes comparable with the imaging results. The apoptotic extents of the spleen and the intestine from the SPECT/CT imaging were correlated with an immunohistochemical staining assay for caspase 3 active form fragment. CONCLUSION: This work is the first study to demonstrate that (99m)Tc(I)-his(6)-annexin A5 is a potential clinical imaging agent for detecting radiation-induced tissue apoptosis in an animal model.

Vasculatures in tumors growing from preirradiated tissues: formed by vasculogenesis and resistant to radiation and antiangiogenic therapy.

PURPOSE: To investigate vasculatures and microenvironment in tumors growing from preirradiated tissues (pre-IR tumors) and study the vascular responses of pre-IR tumors to radiation and antiangiogenic therapy. METHODS AND MATERIALS: Transgenic adenocarcinoma of the mouse prostate C1 tumors were implanted into unirradiated or preirradiated tissues and examined for vascularity, hypoxia, and tumor-associated macrophage (TAM) infiltrates by immunohistochemistry. The origin of tumor endothelial cells was studied by green fluorescent protein-tagged bone marrow (GFP-BM) transplantation. The response of tumor endothelial cells to radiation and antiangiogenic agent was evaluated by apoptotic assay. RESULTS: The pre-IR tumors had obvious tumor bed effects (TBE), with slower growth rate, lower microvascular density (MVD), and more necrotic and hypoxic fraction compared with control tumors. The vessels were dilated, tightly adhered with pericytes, and incorporated with transplanted GFP-BM cells. In addition, hypoxic regions became aggregated with TAM. As pre-IR tumors developed, the TBE was overcome at the tumor edge where the MVD increased, TAM did not aggregate, and the GFP-BM cells did not incorporate into the vessels. The vessels at tumor edge were more sensitive to the following ionizing radiation and antiangiogenic agent than those in the central low MVD regions. CONCLUSIONS: This study demonstrates that vasculatures in regions with TBE are mainly formed by vasculogenesis and resistant to radiation and antiangiogenic therapy. Tumor bed effects could be overcome at the edge of larger tumors, but where vasculatures are formed by angiogenesis and sensitive to both treatments. Vasculatures formed by vasculogenesis should be the crucial target for the treatment of recurrent tumors after radiotherapy.