The evaluation of angiogenesis inhibitors requires the analysis of the precise structure and function of tumor vessels. The anti-angiogenic agents lenvatinib and sorafenib are multi-target tyrosine ...kinase inhibitors that have been approved for the treatment of hepatocellular carcinoma (HCC). However, the different effects on tumor vasculature between lenvatinib and sorafenib are not well understood. In this study, we analyzed the effects of both drugs on vascular structure and function, including vascular normalization, and investigated whether the normalization had a positive effect on a combination therapy with the drugs and radiation using micro X-ray computed tomography with gold nanoparticles as a contrast agent, as well as immunohistochemical analysis and interstitial fluid pressure (IFP) measurement. In mice subcutaneously transplanted with mouse HCC cells, treatment with lenvatinib or sorafenib for 14 days inhibited tumor growth and reduced the tumor vessel volume density. However, analysis of integrated data on vessel density, rates of pericyte-covering and perfused vessels, tumor hypoxia, and IFP measured 4 days after drug treatment showed that treatment with 3 mg/kg of lenvatinib significantly reduced the microvessel density and normalized tumor vessels compared to treatment with 50 mg/kg of sorafenib. These results showed that lenvatinib induced vascular normalization and improved the intratumoral microenvironment in HCC tumors earlier and more effectively than sorafenib. Moreover, such changes increased the radiosensitivity of tumors and enhanced the effect of lenvatinib and radiation combination therapy, suggesting that this combination therapy is a powerful potential application against HCC.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
In typical super-resolution microscopy, high temporal resolution is sacrificed to achieve a spatial resolution beyond the diffraction limit. In this study, we have developed a new structured ...illumination microscopy (SIM) system using two Pockels cells, achieving a temporal resolution of 1 ms. The introduction of the two Pockels cells enables control of the phases of the illumination pattern along the X- and Y-axes in microseconds. We obtained five images consecutively with different illumination patterns and reconstructed a super-resolution image. We observed fluorescent nanoparticles in living cells for 8 s with super resolution and a frame rate of 1,000 fps.
Anticancer drug efficacy varies because the delivery of drugs within tumors and tumor responses are heterogeneous; however, these features are often more homogenous in vitro. This difference makes it ...difficult to accurately determine drug efficacy. Therefore, it is important to use living tumor tissues in preclinical trials to observe the heterogeneity in drug distribution and cell characteristics in tumors. In the present study, to accurately evaluate the efficacy of an antibody-drug conjugate (ADC) containing a microtubule inhibitor, we established a cell line that expresses a fusion of end-binding protein 1 and enhanced green fluorescent protein that serves as a microtubule plus-end-tracking protein allowing the visualization of microtubule dynamics. This cell line was xenografted into mice to create a model of living tumor tissue. The tumor cells possessed a greater number of microtubules with plus-ends, a greater number of meandering microtubules, and a slower rate of microtubule polymerization than the in vitro cells. In tumor tissues treated with fluorescent dye-labeled ADCs, heterogeneity was observed in the delivery of the drug to tumor cells, and microtubule dynamics were inhibited in a concentration-dependent manner. Moreover, a difference in drug sensitivity was observed between in vitro cells and tumor cells; compared with in vitro cells, tumor cells were more sensitive to changes in the concentration of the ADC. This study is the first to simultaneously evaluate the delivery and intracellular efficacy of ADCs in living tumor tissue. Accurate evaluation of the efficacy of ADCs is important for the development of effective anticancer drugs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In addition to genomic signaling, Estrogen receptor alpha (ERα) is associated with cell proliferation and survival through extranuclear signaling contributing to endocrine therapy (ET) resistance. ...However, the relationship between extranuclear ERα and ET resistance has not been extensively studied. We sought to measure extranuclear ERα expression by immunohistochemistry using phosphor-integrated dots (IHC-PIDs) and to assess its predictive value for ET resistance. After quantitative detection of ERα by IHC-PIDs in vitro, we developed "the nearest-neighbor method" to calculate the extranuclear ERα. Furthermore, tissue sections from 65 patients with HR+/HER2- BC were examined by IHC-PIDs, and the total ERα, nuclear ERα, extranuclear ERα PIDs score, and ratio of extranuclear-to-nuclear ERα (ENR) were measured using the novel method. We demonstrate that quantification of ERα using IHC-PIDs exhibited strong correlations to real-time qRT-PCR (
= 0.94) and flow cytometry (
= 0.98). High ERα ENR was significantly associated with poor overall survival (
= 0.048) and disease-free survival (DFS) (
= 0.007). Multivariate analysis revealed that the ERα ENR was an independent prognostic factor for DFS hazard ratio, 3.8; 95% CI, 1.4-11.8;
= 0.006. Our automated measurement has high accuracy to localize and assess extranuclear ERα. A high ERα ENR in HR
/HER2
BC indicates decreased likelihood of benefiting from ET.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Previous studies showed that gold nanoparticles (AuNPs) are useful radiosensitizers which optimize radiation therapy under low-dose radiation. However, the mechanisms of AuNP radiosensitization, ...including the amount and localization of the AuNPs interacting with cancer cells, has not yet been quantified. To answer these questions, we prepared AuNPs conjugated with anti-human epidermal growth factor receptor type 2 (HER2) antibody via polyethylene glycol (PEG) chains (AuNP-PEG-HER2ab). AuNP-PEG-HER2ab specifically bound to the HER2-expressing cancer cells and entered the cells via endocytosis. Whether endocytosis of AuNP-PEG-HER2ab occurred had no effect on radiosensitization efficacy by AuNP-PEG-HER2ab in vitro. The radiosensitization efficacy in vitro depended on dose of AuNP-PEG-HER2ab or dose of X-ray. Moreover, AuNP-PEG-HER2ab administrated into tumor-bearing mice was localized to both the periphery of the tumor tissue and near the nuclei in cancer cells in tumor deep tissue. The localization of AuNP-PEG-HER2ab in tumor tissues was important factors for in vivo powerful radiosensitization efficacy.
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•The gold nanoparticles targeting cancer cells (AuNP-PEG-HER2ab) were synthesized.•The AuNP-PEG-HER2ab was specifically bound to HER2-expressing cancer cells.•The number of AuNPs interacting to tumor cells affected radiosensitization efficacy.•Whether endocytosis of AuNPs occurred had no effect on radiosensitization efficacy.•The localization of AuNPs in tumor tissues might affect radiosensitization efficacy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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•Short-term synthesis of 2 nm gold nanoparticles (sAu/GSH) with excretory capacity.•Synthesis of X-ray contrast agents using sAu/GSH and lactoferrin complex (sAu/GSH-LF).•Improved ...blood retention time of sAu/GSH by complexing sAu/GSH and LF.•CT imaging of morphological changes in tumor vessels with time using sAu/GSH-LF.
The technology to accurately image the morphology of tumor vessels with X-ray contrast agents is important to clarify mechanisms underlying tumor progression and evaluate the efficacy of chemotherapy. However, in clinical practice, iodine-based contrast agents present problems such as short blood retention owing to a high clearance ability and insufficient X-ray absorption capacity when compared with other high atomic number elements. To resolve these issues, gold nanoparticles (AuNPs), with a high atomic number, have attracted a great deal of attention as contrast agents for angiography, and have been employed in small animal models. Herein, we developed novel contrast agents using AuNPs and captured changes in tumor vessel morphology with time using X-ray computed tomography (CT). First, glutathione-supported single nanometer-sized AuNPs (sAu/GSH) (diameter, 2.2 nm) were fabricated using tetrakis(hydroxymethyl)phosphonium chloride as a reducing agent. The sAu/GSH particles were intravenously injected into mice, remained in vessels for a few minutes, and were then excreted by the kidneys after 24 h, similar to the commercial contrast agent iopamidol. Next, the Au/GSH and lactoferrin (sAu/GSH-LF) (long axis size, 17.3 nm) complex was produced by adding lactoferrin to the sAu/GSH solution under the influence of a condensing agent. On intravenously administering sAu/GSH-LF to mice, the blood retention time was 1−3 h, which was considerably longer than that observed with iopamidol and sAu/GSH. Moreover, we succeeded in imaging morphological changes in identical tumor vessels for several days using X-ray CT with sAu/GSH-LF.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Immune checkpoint inhibitor therapy has been attracting attention as a new cancer treatment and is likely to be widely used in combination with radiotherapy. Therefore, examination of the effects of ...X-ray irradiation on sentinel lymph nodes and lymphatic vessels, which are involved in antigen presentation, is important for therapy. The hindlimbs of mice were irradiated with X-rays (total radiation doses: 2, 10, and 30 Gy), and X-ray computed tomography (CT) imaging was performed using 15-nm or 2-nm gold nanoparticles (AuNPs) as contrast agents on days 7, 14, and 28 after irradiation to evaluate the diameter of the collecting lymph vessels and lymph flow within the irradiated area. X-ray CT imaging data using 15-nm AuNPs on day 28 after irradiation showed that the diameter of the collecting lymph vessels was significantly larger in all irradiated groups compared to the control group (p ≤ 0.01). CT imaging with 2-nm AuNPs showed that lymphatic drainage was significantly reduced in the lymph nodes irradiated with 10 Gy and 30 Gy compared to the lymph nodes irradiated with 2 Gy (p ≤ 0.05). Additionally, immunohistochemical analyses were conducted to evaluate the area density and morphology of high endothelial venules (HEVs) in the lymph nodes, which are important vessels for naive T cells to enter the lymph nodes. The expression level of MECA-79, which specifically localized to HEVs, was significantly decreased in the 10 Gy and 30 Gy irradiation groups compared to the control group (p ≤ 0.05). There was a significant decrease in normal HEV morphology (p ≤ 0.05) and a significant increase in abnormal HEV morphology (p ≤ 0.05) in all irradiated groups. These results also showed that X-ray irradiation induced a time- and radiation dose-dependent increase in the diameter of the collecting lymph vessels, stagnation of intralymphatic lymph flow, and a reduction in the area density of HEVs and their abnormal morphology, demonstrating that X-ray irradiation affected the immune responses. Therefore, these findings suggest that X-ray irradiation to lymph nodes may impair the opportunity for antigen presentation in the lymph nodes, which is the key to cancer immunity, and that for this reason, it is important to carefully plan irradiation of sentinel lymph nodes and develop treatment strategies according to future treatment options.
•X-ray irradiation caused dilation of collecting lymphatic vessels.•X-ray irradiation caused stagnation of lymphatic flow within the lymph nodes.•X-ray irradiation reduced the high endothelial venule (HEV) area in lymph nodes.•X-ray irradiation induced morphological abnormalities in HEVs in lymph nodes.•X-ray irradiation affects the immune response in the lymphatic network.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Fluorescence imaging is a very useful method for visualizing molecules and cells, but when tissues are measured", decrease in resolution due to increased scattering and absorption of light in ...proportion to tissue thickness (problem 1)" and "decrease in signal to noise(S/N)ratio of positive signal due to tissue autofluorescence(problem 2)"are problems to be solved. In this paper, to develop a technology to improve the analysis accuracy of drug efficacy mechanisms in preclinical trial of drug discovery, we performed development of a supporting technology for drug discovery of antibody drug conjugates by imaging living tumor tissues, while solving problem 1. This technology is expected to lead to an improvement in the success rate of clinical trials. Next, to develop a diagnostic method to predict the response to neoadjuvant chemotherapy with antibody drugs for breast cancer, we performed development of fluorescence imaging of pathological tissues using fluorescent nanoparticles with ultra-high brightness, while solving problem 2. This diagnostic technology makes it possible to evaluate the expression level of the target protein of antibody drug with high quantitative and wide range sensitivity. This improved the accuracy of drug efficacy prediction. Therefore, patients who are expected to have a low drug efficacy will be able to select anticancer drugs with different mechanisms of action. These results of this study showed the reduction of drug discovery costs and improvement of individualized medicine. Thus, this study will greatly contribute to the development of precision medicine.
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