Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system

With the use of dual-color fluorescent cells and a highly sensitive whole-mouse imaging system with both macro-optics and micro-optics, we report here the development of subcellular real-time imaging of cancer cell trafficking in live mice. To observe cytoplasmic and nuclear dynamics in the living mouse, tumor cells were labeled in the nucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with an Olympus OV100 whole-mouse imaging system with a sensitive CCD camera and five objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. With the dual-color cancer cells and the highly sensitive whole-mouse imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time. This imaging technology will enable further understanding of the critical steps of metastasis and provide visible targets for antimetastasis drug development.     

Comparison of cancer-cell seeding, viability and deformation in the lung, muscle and liver, visualized by subcellular real-time imaging in the live mouse

The comparison of cancer cell seeding, deformation and viability in the lung, muscle and liver of nude mice in real-time is reported here. The mice were intubated to support ventilation with positive end-respiratory pressure (PEEP) for imaging on the lung. Human fibrosarcoma cells with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm (dual-color HT-1080 cells) were injected into the tail vein for lung imaging, the portal vein for liver imaging or the abdominal aorta for muscle imaging which was performed with an Olympus OV100 Small Animal Imaging System. The length of the cytoplasm and nuclei in 20 seeded cancer cells were measured. A large number of cells initially arrested in the lung capillaries and many cells formed aggregates. The cell number decreased rapidly at 6 and 24 h. There was no significant difference in cancer cell survival when immunocompetent C57BL/6 mice were used in place of the nude mice, suggesting that T cell reaction is not very important in the first 24 h after seeding of cancer cells in the lung. In the lung and liver, little cancer cell deformation occurred. In contrast in the muscle, the cytoplasm and nuclei of the seeded cells were highly deformed and many fragmented cells were observed. The rate of cancer cell death was highest in the lung and lowest in the muscle. In each organ, single disseminated cells tended to die earlier than aggregated cells. The results of this study suggest that the early steps of metastasis are different in the lung, liver and muscle.     

Whole-body subcellular multicolor imaging of tumor-host interaction and drug response in real time

To noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment and drug response at the cellular level in live animals in real time, we developed a new imageable three-color animal model. The model consists of green fluorescent protein (GFP)-expressing mice transplanted with dual-color cancer cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm. The Olympus IV100 Laser Scanning Microscope, with ultra-narrow microscope objectives ("stick objectives"), is used for three-color whole-body imaging of the two-color cancer cells interacting with the GFP-expressing stromal cells. In this model, drug response of both cancer and stromal cells in the intact live animal is also imaged in real time. Various in vivo phenomena of tumor-host interaction and cellular dynamics were imaged, including mitotic and apoptotic tumor cells, stromal cells interacting with the tumor cells, tumor vasculature, and tumor blood flow. This new model system enables the first cellular and subcellular images of unperturbed tumors in the live intact animal. New visible real-time targets for novel anticancer agents are provided in this model, including the color-coded interacting cancer and stromal cells, tumor vasculature, and blood flow. This imageable model should lead to many new insights of in vivo cancer cell biology and to novel drug discovery.     

Abrogation of the interaction between osteopontin and alphavbeta3 integrin reduces tumor growth of human lung cancer cells in mice

Osteopontin (OPN) is a multifunctional cytokine involved in cell signaling by interacting with alphavbeta3 integrins. Recent clinical studies have indicated that OPN expression is associated with tumor progression and poor prognosis among patients with lung cancer. However, the biological role of OPN in human lung cancer has not yet been well-defined. The purpose of this study is to investigate and provide evidence for the causal role of OPN regarding tumor growth and angiogenesis in human lung cancer. In this study, we developed a stable OPN transfectant from human lung cancer cell line SBC-3 which does not express the intrinsic OPN mRNA. To reveal the in vivo effect of OPN on tumor growth of human lung cancer, we subcutaneously injected OPN-overexpressing SBC-3 cells (SBC-3/OPN) and control cells (SBC-3/NEO) into the nude mice. Transfection with the OPN gene significantly increased in vivo tumor growth and neovascularization of SBC-3 cells in mice. These in vivo effects of OPN were markedly suppressed with administration of anti-alphavbeta3 integrin monoclonal antibody or anti-angiogenic agent, TNP-470. Furthermore, recombinant OPN protein enhanced human umbilical vein endothelial cell (HUVEC) proliferation in vitro, and this enhancement was significantly inhibited with the addition of anti-alphavbeta3 integrin antibody. Taken together, these results suggest that OPN plays a crucial role for tumor growth and angiogenesis of human lung cancer cells in vivo by interacting with alphavbeta3 integrin. Targeting the interaction between OPN and alphavbeta3 integrin could be effective for future development of anti-angiogenic therapeutic agents for patients with lung cancer.     

New whole-body multimodality imaging of gastric cancer peritoneal metastasis combining fluorescence imaging with ICG-labeled antibody and MRI in mice

Background

Peritoneal metastasis is the most frequent pattern of recurrence after curative surgery for gastric cancer. However, such a recurrence is difficult to detect by conventional computed tomography (CT) and magnetic resonance imaging (MRI) at an early stage. To improve the sensitivity and specificity of diagnostic imaging for peritoneal metastasis, we developed a new type of multimodality imaging combining fluorescence imaging with near-infrared fluorophore (NIR)-labeled antibodies and MRI.

Methods

Dual optical imaging of peritoneal metastasis was carried out using luciferase-tagged gastric cancer cell lines and XenoLight CF750 or indocyanine green (ICG)-labeled anti-human epidermal growth factor receptor (EGFR) or CEA antibody as a probe in mice with Ivis in vivo imaging system.

Results

This whole-body fluorescent imaging system sensitively detected metastatic foci <1 mm in diameter in the peritoneal cavity noninvasively. Fluorescence imaging proved to be specific because the fluorescence signal was abolished by blocking with excess unlabeled antibody. Although this fluorescence imaging had higher sensitivity for detection of small-sized peritoneal metastases than MRI, it proved difficult to accurately determine organ distribution of the metastasis. We thus developed a multimodality imaging system by the fusion of the three-dimensional fluorescence image with the MRI image and demonstrated its improved diagnostic accuracy over either method alone.

Conclusion

The present results suggest that multimodality imaging consisting of fluorescence imaging with NIR-labeled EGFR or CEA antibody and MRI allows sensitive, specific, and anatomically accurate detection of peritoneal metastasis noninvasively at an early stage.     

Metabolic interaction between cancer cells and stromal cells according to breast cancer molecular subtype

Introduction

The aim of this study was to investigate the differential expression of markers related to metabolic, mitochondrial and autophagy status in different molecular subtypes of breast cancer.

Methods

Using tissue microarray sections generated from 740 cases of breast cancer, we performed immunohistochemical staining for Glut-1, CAIX, MCT4, ATP synthase, glutaminase, BNIP3, Beclin-1, LC3A, LC3B and p62. Based on the immunohistochemical expression of estrogen receptor (ER), progesterone (PR), HER2, and Ki-67 labeling index, the cases were classified into luminal A, luminal B, HER2 and triple-negative breast cancer (TNBC). We further classified metabolic phenotypes of tumors according to glycolytic status by assessing Glut-1 and CAIX expression as follows: Warburg type: tumor (glycolysis type), stroma (nonglycolysis type); reverse Warburg type: tumor (nonglycolysis type), stroma (glycolysis type); mixed type: tumor (glycolysis type), stroma (glycolysis type); and null type: tumor (nonglycolysis type), stroma (nonglycolysis type).

Results

Expression of Glut-1, MCT4 and LC3A was highest in TNBC and lowest in luminal A type (P < 0.001). Tumors were classified into 298 Warburg type (40.3%), 54 reverse Warburg type (7.3%), 62 mixed type (8.4%) and 326 null type (44.0%). The mixed type had a higher histologic grade, ER negativity, PR negativity and Ki-67 index, whereas the null type showed lower histologic grade, ER positivity, PR positivity and Ki-67 index (P < 0.001). TNBC constituted the major portion of Warburg and mixed types, and luminal A consisted mainly of reverse Warburg and null types (P < 0.001).

Conclusion

Breast cancer is heterogeneous in its metabolic status, and therefore it can be classified into various metabolic phenotypes. Specifically, the Warburg and mixed types had strong associations with TNBC, whereas reverse the Warburg and null types had associations with the luminal type, suggesting a correlation between metabolic phenotype and the biology of breast cancer.     

Synergistic interaction between vinorelbine and gamma-linolenic acid in breast cancer cells

It has been suggested that exogenous unsaturated fatty acids (UFAs) may increase the cytotoxic activity of cancer chemotherapeutic agents. We examined how γ-linolenic acid (GLA; 18 : 3n-6), the most promising UFA in the treatment of human tumors, affects the effectiveness of the lipophilic drug vinorelbine (VNR) on human breast carcinoma cell lines. Cells were exposed simultaneously to VNR and GLA or sequentially to GLA followed by VNR. Cell viability was determined by MTT assay. The increase in VNR-induced cell growth inhibition was measured by dividing the IC₅₀ and IC₇₀ values (50 and 70% inhibitory concentrations, respectively) that were obtained when the cells were exposed to VNR alone with those with VNR plus GLA. We found that GLA enhanced in a dose-dependent manner the cell growth inhibitory activity of VNR on MCF-7 cells (up to 9-fold). As GLA by itself showed anti-proliferative effects, possible GLA-VNR interactions at the cellular level were assessed employing the isobologram analysis and the combination index (CI) method of Chou–Talalay. Both methods showed an overall synergism between GLA and VNR in MCF-7 cells. At a high level of cell kill, the synergism was greater when a 24 h GLA pre-exposure or co-exposures were tested. Synergy was likewise observed with the GLA-VNR combination in MDA-MB-231, T47D, and SK-Br3 breast cancer cells. In all cell lines, the synergism was independent of the treatment schedule and the exposure time. Under conditions inhibiting lipid peroxidation using Vitamin E (dl-α-tocopherol), the enhancing effect of GLA (an easily oxidizable UFA) on VNR activity was partially abolished. However, when Vitamin E was used in combination, a similar synergistic increase in growth inhibition was obtained. These latter observations strongly implies that the synergistic effects of GLA with VNR are not mediated through a mechanism involving a generation of lipoperoxides. For comparison, the effects of other UFAs were examined on VNR chemosensitivity: GLA was the most potent at enhancing VNR activity, followed by docosahexaenoic acid (22 : 6n-3), eicosapentaenoic acid (20 : 5n-3) and α-linolenic acid (18 : 3n-3), whereas linoleic acid (18 : 2n-6) and arachidonic acid (20 : 4n-6) did not increase VNR chemosensitivity. Very high concentrations of oleic acid (OA; 18 : 1n-9), an UFA inversely correlated with breast cancer risk, also enhanced VNR effectiveness. Thus, various types of UFAs were not equivalent with respect to their actions on VNR effectiveness. In conclusion, our results give experimental support to the hypothesis that some UFAs can be used as modulators of tumor cell chemosensitivity and provide the rationale for in vivo preclinical investigation.     

Nitrogen movement between host and tumor in mice inoculated with Ehrlich ascitic tumor cells

Tumors function as a nitrogen trap, and they compete with the host for nitrogen compounds. In experiments with whole animals infected with Ehrlich ascitic tumor cells, the glutamine, glutamate, asparagine, and aspartate concentrations were determined for host plasma, ascitic liquid, and tumor cells, throughout the period of tumor growth. Concentration gradients of glutamine or asparagine were created from the host tissues towards the ascitic liquid. The countergradient step from ascitic liquid to tumor cells may be overcome by an active transport process with an apparent Km for glutamine of 3.1 X 10(-4) M. On the other hand, a reverse flux of glutamate and aspartate was seen to take place from cells to plasma. In vitro incubations of tumor cells with near physiological concentrations of glutamine, or asparagine plus glucose, confirmed the host-to-tumor nitrogen movement previously deduced from the relative amino acid concentrations in plasma, ascitic liquid, and tumor cells. The ammonemia detected in tumor-bearing mice at the terminal stage could result from the hydrolysis of glutamine, which was rapidly metabolized by the tumor cells.     

细胞外基质与肿瘤干细胞相互作用的研究进展

细胞外基质(extracellular matrix,ECM)是由蛋白质与糖类等生物大分子在细胞表面或细胞间构成的复杂网络结构.细胞外基质的过量沉积或结构形态异常是肿瘤微环境(tumor microenvironment,TME)的重要特征之一.肿瘤干细胞(cancer stem cells,CSC)指肿瘤内部一类具有...     

Cellular efflux pump and interaction between cisplatin and paclitaxel in ovarian cancer cells

OBJECTIVE: The aim of this study was to evaluate the combination effect of paclitaxel (PTX) and cisplatin (CDDP) and to determine the mechanisms of interaction between these agents. METHODS AND RESULTS: We used human ovarian adenocarcinoma cell lines, namely a parent cell line (KF), a CDDP-resistant cell line (KFr) and a PTX-resistant cell line (KFTx).The combination effect of PTX and CDDP was synergistic on KF and KFTx and additive on KFr. The incidence of anaphase or telophase, evaluated by immunofluorescence microscopy, decreased with PTX and significantly decreased with PTX and CDDP in KF and KFTx. The concentration of PTX, which was measured by high-performance liquid chromatography, was higher in KF and KFTx cells treated with a combination of PTX and CDDP than those treated with PTX alone. Multidrug resistance gene mRNA appeared in KFTx and its expression decreased after exposure to PTX and CDDP. After exposure to CDDP, the expression of multidrug resistance-associated protein (MRP) and the concentration of glutathione increased in KF, but not in KFr or KFTx. MRP expression slightly increased in KF and KFTx after exposure to PTX. In contrast, its expression decreased in KFr. CONCLUSION: The present study suggests that CDDP enhances PTX accumulation and that the interaction of these agents is synergistic in CDDP-sensitive cells.     

Fluorescence lifetime imaging of E-combretastatin uptake and distribution in live mammalian cells

To investigate within live mammalian cells the uptake and disposition of combretastatins, fluorescence lifetime imaging was used with two-photon excitation (2PE). Combretastatin A4 (CA4) and analogues are potential anticancer drugs due to their ability to inhibit angiogenesis. E(trans)-combretastatins are considerably less active than the Z(cis)-combretastatins proposed for clinical use. However the E-combretastatins exhibit stronger intrinsic fluorescence with quantum yields and lifetimes that depend markedly on solvent polarity and viscosity. It is proposed that 2PE in the red and near-infrared tissue window may allow in situ isomerization of E-combretastatins to the more active Z-isomer, offering spatial and temporal control of drug activation and constitute a novel form of photodynamic therapy. In the present work we have characterised 2PE of E-CA4 and have used fluorescence lifetime imaging with 2PE to study uptake and intracellular disposition of E-CA4 and an analogue. The results show that these molecules accumulate rapidly in cells and are located mainly in lipidic environments such as lipid droplets. Within the droplets the local concentrations may be up to two orders of magnitude higher than that of the drug in the surrounding medium.     

In vivo color-coded imaging of the interaction of colon cancer cells and splenocytes in the formation of liver metastases

The role of host cells in tumor progression and metastasis is critical. Intrasplenic injection of tumor cells has long been known as an effective method of developing liver metastases in nude mice, whereas portal vein (PV) injection of tumor cells can result in rapid death of the tumor cells. Host cells were thought to play a role in these phenomena. We report here that after splenic injection of tumor cells, splenocytes cotraffic with the tumor cells to the liver and facilitate metastatic colony formation. Human colon cancer cells that express green fluorescent protein (GFP) linked to histone H2B in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP) were injected in either the PV or spleen of nude mice and imaged at the subcellular level in vivo. Extensive clasmocytosis (destruction of the cytoplasm) of the cancer cells occurred within 6 hours after PV injection and essentially all the cancer cells died. In contrast, splenic injection of these tumor cells resulted in the aggressive formation of liver and distant metastasis. GFP spleen cells were found in the liver metastases that resulted from intrasplenic injection of the tumor cells in transgenic nude mice ubiquitously expressing GFP. When GFP spleen cells and the RFP cancer cells were coinjected in the PV, liver metastasis resulted that contained GFP spleen cells. These results suggest a novel tumor-host interaction that enables efficient formation of liver metastasis via intrasplenic injection.     

Equilibrium between host and cancer caused by effector T cells killing tumor stroma

The growth of solid tumors depends on tumor stroma. A single adoptive transfer of CD8(+) CTLs that recognize tumor antigen-loaded stromal cells, but not the cancer cells because of MHC restriction, caused long-term inhibition of tumor growth. T cells persisted and continuously destroyed CD11b(+) myeloid-derived, F4/80(+) or Gr1(+) stromal cells during homeostasis between host and cancer. Using high-affinity T-cell receptor tetramers, we found that both subpopulations of stromal cells captured tumor antigen from surrounding cancer cells. Epitopes on the captured antigen made these cells targets for antigen-specific T cells. These myeloid stromal cells are immunosuppressive, proangiogenic, and phagocytic. Elimination of these myeloid cells allowed T cells to remain active, prevented neovascularization, and prevented tumor resorption so that tumor size remained stationary. These findings show the effectiveness of adoptive CTL therapy directed against tumor stroma and open a new avenue for cancer treatments.     

肝血管生成:在非转移性结肠直肠癌中肿瘤宿主的相互作用(英文)

Objective  

Angiogenesis is a crucial step for tumor growth and progression. Changes of liver angiogenesis (without metastatic invasion) in response to primary tumors are not known. The aim of the study was to investigate the liver angiogenesis in non-metastatic colorectal cancer (CRC).     

Beneficial bacteria stimulate host immune cells to counteract dietary and genetic predisposition to mammary cancer in mice

Recent studies suggest health benefits including protection from cancer after eating fermented foods such as probiotic yogurt, though the mechanisms are not well understood. Here we tested mechanistic hypotheses using two different animal models: the first model studied development of mammary cancer when eating a Westernized diet, and the second studied animals with a genetic predilection to breast cancer. For the first model, outbred Swiss mice were fed a Westernized chow putting them at increased risk for development of mammary tumors. In this Westernized diet model, mammary carcinogenesis was inhibited by routine exposure to Lactobacillus reuteri ATCC‐PTA‐6475 in drinking water. The second model was FVB strain erbB2 (HER2) mutant mice, genetically susceptible to mammary tumors mimicking breast cancers in humans, being fed a regular (non‐Westernized) chow diet. We found that oral supplement with these purified lactic acid bacteria alone was sufficient to inhibit features of mammary neoplasia in both models. The protective mechanism was determined to be microbially‐triggered CD4+CD25+ lymphocytes. When isolated and transplanted into other subjects, these L. reuteri‐stimulated lymphocytes were sufficient to convey transplantable anti‐cancer protection in the cell recipient animals. These data demonstrate that host immune responses to environmental microbes significantly impact and inhibit cancer progression in distal tissues such as mammary glands, even in genetically susceptible mice. This leads us to conclude that consuming fermentative microbes such as L. reuteri may offer a tractable public health approach to help counteract the accumulated dietary and genetic carcinogenic events integral in the Westernized diet and lifestyle.     

胃癌相关幽门螺杆菌与宿主细胞作用机制的研究

目的:鉴定幽门螺杆菌(Hp)与宿主细胞人类胃上皮细胞(AGS)作用后差异蛋白的改变.方法:应用双向电泳分离菌株26695与AGS细胞相互作用前后的蛋白,应用Image Master 2Dv3.1软件对蛋白图谱进行比较,对目的蛋白进行基质辅助激光解析及电离飞行时间质谱分析,应用Mascot软件进行蛋白搜库.结果:共监测到5个差异蛋白斑点,经质谱分析鉴定为2种蛋白,分别是热体克蛋白60和烷基过氧化还原酶.结论:烷基过氧化还原酶可能进入AGS细胞或结合于AGS细胞膜上,有助于明确Hp与宿主细胞相互作用后的致病过程.     

Simple and rapid determination of homozygous transgenic mice via in vivo fluorescence imaging

Setting up breeding programs for transgenic mouse strains require to distinguish homozygous from the heterozygous transgenic animals. The combinational use of the fluorescence reporter transgene and small animal in-vivo imaging system might allow us to rapidly and visually determine the transgenic mice homozygous for transgene(s) by the in vivo fluorescence imaging. RLG, RCLG or Rm17LG transgenic mice ubiquitously express red fluorescent protein (RFP). To identify homozygous RLG transgenic mice, whole-body fluorescence imaging for all of newborn F2-generation littermates produced by mating of RFP-positive heterozygous transgenic mice (F1-generation) derived from the same transgenic founder was performed. Subsequently, the immediate data analysis of the in vivo fluorescence imaging was carried out, which greatly facilitated us to rapidly and readily distinguish RLG transgenic individual(s) with strong fluorescence from the rest of F2-generation littermates, followed by further determining this/these RLG individual(s) showing strong fluorescence to be homozygous, as strongly confirmed by mouse mating. Additionally, homozygous RCLG or Rm17LG transgenic mice were also rapidly and precisely distinguished by the above-mentioned optical approach. This approach allowed us within the shortest time period to obtain 10, 8 and 2 transgenic mice homozygous for RLG, RCLG and Rm17LG transgene, respectively, as verified by mouse mating, indicating the practicality and reliability of this optical method. Taken together, our findings fully demonstrate that the in vivo fluorescence imaging offers a visual, rapid and reliable alternative method to the traditional approaches (i.e., mouse mating and real-time quantitative PCR) in identifying homozygous transgenic mice harboring fluorescence reporter transgene under the control of a ubiquitous promoter in the situation mentioned in this study.