A transgenic red fluorescent protein‐expressing nude mouse for color‐coded imaging of the tumor microenvironment

The tumor microenvironment (TME) is critical for tumor growth and progression. We have previously developed color‐coded imaging of the TME using a green fluorescent protein (GFP) transgenic nude mouse as a host. However, most donor sources of cell types appropriate for study in the TME are from mice expressing GFP. Therefore, a nude mouse expressing red fluorescent protein (RFP) would be an appropriate host for transplantation of GFP‐expressing stromal cells as well as double‐labeled cancer cells expressing GFP in the nucleus and RFP in the cytoplasm, thereby creating a three‐color imaging model of the TME. The RFP nude mouse was obtained by crossing non‐transgenic nude mice with the transgenic C57/B6 mouse in which the β‐actin promoter drives RFP (DsRed2) expression in essentially all tissues. In crosses between nu/nu RFP male mice and nu/+ RFP female mice, the embryos fluoresced red. Approximately 50% of the offspring of these mice were RFP nude mice. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, duodenum, the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart, and major arteries and veins. The skinned skeleton highly expressed RFP. The bone marrow and spleen cells were also RFP positive. GFP‐expressing human cancer cell lines, including HCT‐116‐GFP colon cancer and MDA‐MB‐435‐GFP breast cancer were orthotopically transplanted to the transgenic RFP nude mice. These human tumors grew extensively in the transgenic RFP nude mouse. Dual‐color fluorescence imaging enabled visualization of human tumor–host interaction. The RFP nude mouse model should greatly expand our knowledge of the TME. J. Cell. Biochem. 106: 279–284, 2009. © 2008 Wiley‐Liss, Inc.     

Single Unpurified Breast Tumor-Initiating Cells from Multiple Mouse Models Efficiently Elicit Tumors in Immune-Competent Hosts

The tumor-initiating cell (TIC) frequency of bulk tumor cell populations is one of the criteria used to distinguish malignancies that follow the cancer stem cell model from those that do not. However, tumor-initiating cell frequencies may be influenced by experimental conditions and the extent to which tumors have progressed, parameters that are not always addressed in studies of these cells. We employed limiting dilution cell transplantation of minimally manipulated tumor cells from mammary tumors of several transgenic mouse models to determine their tumor-initiating cell frequency. We determined whether the tumors that formed following tumor cell transplantation phenocopied the primary tumors from which they were isolated and whether they could be serially transplanted. Finally we investigated whether propagating primary tumor cells in different tissue culture conditions affected their resident tumor-initiating cell frequency. We found that tumor-initiating cells comprised between 15% and 50% of the bulk tumor cell population in multiple independent mammary tumors from three different transgenic mouse models of breast cancer. Culture of primary mammary tumor cells in chemically-defined, serum-free medium as non-adherent tumorspheres preserved TIC frequency to levels similar to that of the primary tumors from which they were established. By contrast, propagating the primary tumor cells in serum-containing medium as adherent populations resulted in a several thousand-fold reduction in their tumor-initiating cell fraction. Our findings suggest that experimental conditions, including the sensitivity of the transplantation assay, can dramatically affect estimates of tumor initiating cell frequency. Moreover, conditional on cell culture conditions, the tumor-initiating cell fraction of bulk mouse mammary tumor cell preparations can either be maintained at high or low frequency in vitro thus permitting comparative studies of tumorigenic and non-tumorigenic cancer cells.     

绿色荧光裸鼠的建立和验证

目的建立系统性表达绿色荧光蛋白的裸鼠,接种人源肺癌细胞验证该模型是否具有免疫缺陷性,并观察双色荧光的成像效果。方法利用系统性表达绿色荧光蛋白的C57BL/6J小鼠与BALB/C裸小鼠多代杂交和互交,建立稳定表达绿色荧光蛋白的裸鼠。大体解剖观察胸腺生长情况,整体和器官荧光成像验证绿色荧光蛋白的表达情况。以2×106/只的剂量对其皮下腋下接种表达红色荧光蛋白的人类A549肺癌细胞（RFP-A549）,通过观测肿瘤生长来验证模型的免疫缺陷性。同时,利用红色荧光标记的肿瘤和绿色宿主鼠,对双色的整体成像效果进行观测。结果构建出系统性表达绿色荧光蛋白的裸鼠,大体解剖可见胸腺缺失。在激发光的激发下,绿色荧光裸鼠全身发出清晰的绿色荧光,脑、心脏、肺脏、肝脏、肾脏,肠胃及胰腺等主要器官可见明显绿色荧光。接种RFP-A549细胞后,成瘤率达到100%,整体动物荧光成像表现出清晰的双色。结论本研究构建出的绿色荧光裸鼠,动物整体可以清晰地表达绿色荧光并具有免疫缺陷性     

Development of a Clinically-Precise Mouse Model of Rectal Cancer

Currently-used rodent tumor models, including transgenic tumor models, or subcutaneously growing tumors in mice, do not sufficiently represent clinical cancer. We report here development of methods to obtain a highly clinically-accurate rectal cancer model. This model was established by intrarectal transplantation of mouse rectal cancer cells, stably expressing green fluorescent protein (GFP), followed by disrupting the epithelial cell layer of the rectal mucosa by instilling an acetic acid solution. Early-stage tumor was detected in the rectal mucosa by 6 days after transplantation. The tumor then became invasive into the submucosal tissue. The tumor incidence was 100% and mean volume (±SD) was 1232.4 ± 994.7 mm³ at 4 weeks after transplantation detected by fluorescence imaging. Spontaneous lymph node metastasis and lung metastasis were also found approximately 4 weeks after transplantation in over 90% of mice. This rectal tumor model precisely mimics the natural history of rectal cancer and can be used to study early tumor development, metastasis, and discovery and evaluation of novel therapeutics for this treatment-resistant disease.     

荧光标记技术在肿瘤模型研究中的应用

目的利用绿色荧光小鼠和红色荧光蛋白标记肿瘤细胞,建立荧光标记的小鼠肿瘤模型,并建立活体荧光成像和荧光显微镜成像在整体和细胞水平直接观察肿瘤的技术。方法将小鼠B16黑色素瘤细胞接种到绿色荧光蛋白转基因小鼠皮下,建立GFP小鼠肿瘤模型。以红色荧光蛋白作为标记基因导入小鼠黑色素瘤细胞B16细胞,建立稳定表达红色荧光蛋白的细胞株。将表达红色荧光蛋白B16细胞接种到绿色荧光转基因小鼠皮下,建立双荧光小鼠肿瘤模型。用荧光显微镜和活体荧光成像系统检测小鼠肿瘤的发生发展。结果分别建立了GFP小鼠肿瘤模型和双色荧光小鼠肿瘤模型。利用活体荧光影像仪可以观察双色荧光小鼠模型中受体绿色荧光组织和红色荧光移植肿瘤相互融合。利用荧光显微镜,可以观察到肿瘤内绿色荧光标记的来源于受体小鼠的血管和免疫细胞。经香菇多糖刺激的GFP小鼠肿瘤模型的移植瘤组织中,来源于受体小鼠绿色荧光标记的免疫细胞明显多于经生理盐水刺激的对照小鼠。结论利用绿色荧光小鼠和红色荧光RFP标记肿瘤细胞建立荧光标记的小鼠肿瘤模型,采用活体荧光成像仪和荧光显微镜可在整体和细胞水平直接观察肿瘤的生长以及肿瘤与宿主的相互作用。     

Studying primary tumor-associated fibroblast involvement in cancer metastasis in mice

Stromal cells have been studied extensively in the primary tumor microenvironment. In addition, mesenchymal stromal cells may participate in several steps of the metastatic cascade. Studying this interaction requires methods to distinguish and target stromal cells originating from the primary tumor versus their counterparts in the metastatic site. Here we illustrate a model of human tumor stromal cell-mouse cancer cell coimplantation. This model can be used to selectively deplete human stromal cells (using diphtheria toxin, DT) without affecting mouse cancer cells or host-derived stromal cells. Establishment of novel genetic models (e.g., transgenic expression of the DT receptor in specific cells) may eventually allow analogous models using syngeneic cells. Studying the role of stromal cells in metastasis using the model outlined above may take 8 weeks.     

实验小鼠在癌症研究中的应用及其进展

小鼠是生物医学研究中使用数量最多的哺乳类实验动物。人类利用小鼠模型进行癌症研究已有100多年的历史,小鼠大量的遗传变异可作为研究人类癌症的借鉴。特别是近年来,培育成功的转基因、基因敲除等遗传工程小鼠模型,使我们对人类癌症发生有了深刻的认识,为评估癌症的诊断方法,革新预防和治疗方案提供了一个很有价值的平台。本文着重介绍了癌症研究中常用的小鼠模型、GEM模型及取得的最新进展等,分析了小鼠肿瘤模型的局限性,并对其发展趋势进行展望。     

Color-coded intravital imaging demonstrates a transforming growth factor-β (TGF-β) antagonist selectively targets stromal cells in a human pancreatic-cancer orthotopic mouse model

Pancreatic cancer is a recalcitrant malignancy, partly due to desmoplastic stroma which stimulates tumor growth, invasion, and metastasis, and inhibits chemotherapeutic drug delivery. Transforming growth factor-β (TGF-β) has an important role in the formation of stromal desmoplasia. The present study describes the ability of color-coded intravital imaging to demonstrate the efficacy of a TGF-β inhibitor to target stroma in an orthotopic mouse model of pancreatic cancer. The BxPC-3 human pancreatic adenocarcinoma cell line expressing green fluorescent protein (GFP), which also has a high TGF-β expression level, was used in an orthotopic model in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). Fourteen mice were randomized into a control group (n = 7, vehicle, i.p., weekly, for 3 weeks) and a treated group (n = 7, SB431542 [TGF-β receptor type I inhibitor] 0.3 mg, i.p., weekly, for 3 weeks). Stromal cells expressing RFP and cancer cells expressing GFP were observed weekly for 3 weeks by real-time color-coded intravital imaging. The RFP fluorescence area from the stromal cells, relative to the GFP fluorescence area of the cancer cells, was significantly decreased in the TGF-β-inhibitor-treatment group compared to the control group. The present study demonstrated color-coded imaging in an orthotopic pancreatic-cancer cell-line mouse model can readily detect the selective anti-stromal-cell targeting of a TGF-β inhibitor.     

造血干细胞全标记红色和绿色荧光转基因小鼠的筛选

目的对五种荧光转基因小鼠造血干细胞中的荧光标记细胞进行分析,筛选造血干细胞全标记红色和绿色荧光转基因小鼠,为造血干细胞分化机制体内示踪研究提供理想的动物模型。方法采用活体荧光影像系统对两种红色荧光转基因小鼠品系C57BL/6J-TgN（CAG-DsRed-1和CAG-DsRed-2）ZLFILAS和三种绿色荧光转基因小鼠品系C57BL/6J-TgN（CAG-EGFP-1、CAG-EGFP-2和CAG-EGFP-3）ZLFILAS的荧光标记进行比较;采用流式细胞术检测各转基因小鼠的骨髓lin（-）c-kit（＋）Sca-1＋（LSK）造血干细胞荧光标记细胞比率,根据标记比率筛选造血干细胞全标记红色和绿色荧光转基因小鼠。结果活体荧光影像分析表明转基因小鼠均系统性表达红色或绿色荧光。流式细胞术检测表明LSK造血干细胞中高度表达红色和绿色荧光,其中,C57BL/6J-TgN（CAG-DsRed-1）ZLFILAS和C57BL/6J-TgN（CAG-EGFP-1）ZLFILAS的造血干细胞全部为荧光标记细胞。结论筛选获得在造血干细胞中全标记的红色和绿色荧光转基因小鼠,可为造血干细胞体内研究提供有效示踪工具。     

A transgenic mouse model genetically tags all activated CD8 T cells

Identifying and characterizing Ag-specific CD8+ T cells are central to the study of immunological memory. Although powerful strategies such as MHC tetramers and peptide-induced cytokine production assays exist for identifying Ag-specific CD8+ T cells, alternate strategies that are not dependent upon a priori knowledge of the immunodominant and subdominant antigenic epitopes, as well as the MHC background of the animal are of obvious utility. In this study, we present a transgenic mouse model that uses Cre-loxP recombination to permanently mark all activated CD8+ T cells with beta-galactosidase. We used the lymphocytic choriomeningitis virus infection model to track the dynamics of the antiviral CD8+ T cell responses. We show that in this transgenic mouse model system, all of the antiviral effector and memory CD8+ T cells are contained within the beta-gal-marked CD8+ T cell population.     

Reduced plasma APOA1 level is associated with Gastric Tumor Growth in MKN45 mouse xenograft model

There is no suitable diagnostic and prognostic biomarker for gastric cancer. The biggest hurdles in biomarker discovery are (i) the low abundance of cancer cell-specific proteins that limits their detection and (ii) complex inter-patient variations that complicate the discovery process. To circumvent these issues, we conducted proteomics on the plasma of gastric cancer mouse xenograft and attempted to identify proteins released by cancer cells. MKN45 gastric cancer cells were subcutaneously implanted into immune-incompetent nude mice. Plasma samples collected from mice with different tumor sizes (low, mid and high tumor loads) were subjected to iTRAQ and mass spectrometric analyses. Detection of human APOA1 in mouse plasma was verified and its expression level was shown to be lower in mice with large tumors compared to those with small tumors. Studies on a panel of about 14 gastric cancer cell lines supported the notion that APOA1 in mouse plasma was of human gastric cancer cell origin. While the clinical utility of APOA1 remains to be ascertained with a larger scale study, the current work supported the feasibility of using mouse xenograft model for gastric cancer biomarker discovery.     

A transient parabiosis skin transplantation model in mice

Parabiosis-conjoined surgery to provide a shared circulation between two mice-has been previously developed to study the hematopoietic system. This protocol describes the use of parabiosis for efficient transplantation of skin from a transgenic to a wild-type mouse. It can be used to study the role of stromal cells in a spontaneous model of distant cancer dissemination (metastasis). We have recently shown that primary tumor-derived stromal cells may facilitate metastasis by providing a provisional stroma at the secondary site. Studying the role of primary tumor-derived stroma cells requires methods for distinguishing and targeting stromal cells originating from the primary tumor versus their counterparts in the metastatic site. Parabiosis may also be used, taking advantage of the shared circulation between the parabiosed mice, to study tumor metastasis from one parabiont to another, or to investigate the role of circulating inflammatory cells or stem cells. Studying the role of stromal cells in metastasis using this model typically takes up to 11 weeks.     

2-Methoxyestradiol induces G2/M arrest and apoptosis in prostate cancer

Few therapeutic treatment options are available for patients suffering from metastatic androgen-independent prostate cancer. We investigated the ability of the estrogen metabolite 2-methoxyestradiol to inhibit the proliferation of a variety of human prostate cancer cell lines in vitro and to inhibit the growth of androgen-independent prostate cancer in a transgenic mouse model in vivo. Our results showed that 2-methoxyestradiol is a powerful growth inhibitor of LNCaP, DU 145, PC-3, and ALVA-31 prostate cancer cells. Cell flow cytometry of 2-methoxyestradiol-treated DU 145 cells showed a marked accumulation of cells in the G2/M phase of the cell cycle and an increase in the sub-G1 fraction (apoptotic). In addition, staining for annexin V, changes in nuclear morphology, and inhibition of caspase activity support a role for apoptosis. More importantly, we showed that 2-methoxyestradiol inhibits prostate tumor progression in the Ggamma/T-15 transgenic mouse model of androgen-independent prostate cancer without toxic side effects. These results in cell culture and an animal model support investigations into the clinical use of 2-methoxyestradiol in patients with androgen-independent prostate cancer.     

Development of the transgenic cyan fluorescent protein (CFP)‐expressing nude mouse for “Technicolor” cancer imaging

A major goal for in vivo biology is to develop models which can express multiple colors of fluorescent proteins in order to image many processes simultaneously in real time. Towards this goal, the cyan fluorescent protein (CFP) nude mouse was developed by crossing non‐transgenic nude mice with the transgenic CK/ECFP mouse in which the β‐actin promoter drives expression of CFP in almost all tissues. In crosses between nu/nu CFP male mice and nu/+ CFP female mice, approximately 50% of the embryos fluoresced blue. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest fluorescent signals of all internal organs which vary in intensity. Orthotopic implantation of XPA‐1 human pancreatic cancer cells expressing red fluorescent protein (RFP); or green fluorescent protein (GFP) in the nucleus and RFP in the cytoplasm, was performed in female nude CFP mice. Color‐coded fluorescence imaging of these human pancreatic cancer cells implanted into the bright blue fluorescent pancreas of the CFP nude mouse afforded novel insight into the interaction of the pancreatic tumor and the normal pancreas, in particular the strong desmoplastic reaction of the tumor. The naturally enhanced blue fluorescence of the pancreas in the CFP mouse serves as an ideal background for color‐coded imaging of the interaction of implanted cancer cells and the host. The CFP nude mouse will provide unique understanding of the critical interplay between the cancer cells and their microenvironment. J. Cell. Biochem. 107: 328–334, 2009. © 2009 Wiley‐Liss, Inc.     

Transformation of diatom Phaeodactylum tricornutum by electroporation and establishment of inducible selection marker

Antibodies are important drugs for treating cancer and there is strong rationale for using multiple antibodies to improve outcomes. We labeled two breast cancer binding antibodies, anti-ErbB2 and anti-EpCAM, with infrared fluorescence dyes of different wavelengths and determined their in vivo distribution in a breast cancer xenograft model using a near-infrared (NIR) fluorescence imaging system. Our data show that these two antibodies can be readily assessed simultaneously in mouse xenograft model. This will help guide design of dosing strategies for multiple antibodies and identify potential interaction that could affect pharmacokinetics and possible side effects.     

Establishment of heparan sulphate deficient primary endothelial cells from EXT-1<Superscript>flox/flox</Superscript> mouse lungs and sprouting aortas

Angiogenesis is a hallmark of expanding tissue e.g. during embryogenesis and wound healing in physiology as well as in diseases such as cancer and atherosclerosis. Key steps of the angiogenic process involve growth factor-mediated stimulation of endothelial cell sprouting and tube formation. Heparan sulphate proteoglycans (HSPGs) have been implicated as important co-receptors of several pro-angiogenic proteins. The importance of HSPGs in physiology was underscored by the finding that knockout of the gene encoding HS polymerase, EXT-1, resulted in early embryonic lethality. Here, we describe the establishment of HS-deficient endothelial cells from sprouting aortas as well as from the lungs of EXT-1^flox/flox mice. Recombination of the loxP-flanked EXT-1 locus by Cre-expressing adenovirus was demonstrated at the mRNA level. Moreover, depletion of HS polysaccharides was verified by flow cytometry and fluorescence microscopy methodology using phage display-derived anti-HS antibodies. In summary, we provide a genetic model to unravel the functional role of HSPGs specifically in primary endothelial cells during early steps of angiogenesis. Our studies are applicable to most loxP-based transgenic mouse strains, and may thus be of general importance in the angiogenesis field.     

Integrated Proteomic Analysis of Human Cancer Cells and Plasma from Tumor Bearing Mice for Ovarian Cancer Biomarker Discovery

Background

The complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery.

Methodology/Principal Findings

We investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease.

Conclusions/Significance

Integrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers.     

Direct discovery and validation of a peptide/MHC epitope expressed in primary human breast cancer cells using a TCRm monoclonal antibody with profound antitumor properties

The identification and validation of new cancer-specific T cell epitopes continues to be a major area of research interest. Nevertheless, challenges remain to develop strategies that can easily discover and validate epitopes expressed in primary cancer cells. Regarded as targets for T cells, peptides presented in the context of the major histocompatibility complex (MHC) are recognized by monoclonal antibodies (mAbs). These mAbs are of special importance as they lend themselves to the detection of epitopes expressed in primary tumor cells. Here, we use an approach that has been successfully utilized in two different infectious disease applications (WNV and influenza). A direct peptide-epitope discovery strategy involving mass spectrometric analysis led to the identification of peptide YLLPAIVHI in the context of MHC A*02 allele (YLL/A2) from human breast carcinoma cell lines. We then generated and characterized an anti-YLL/A2 mAb designated as RL6A TCRm. Subsequently, the TCRm mAb was used to directly validate YLL/A2 epitope expression in human breast cancer tissue, but not in normal control breast tissue. Moreover, mice implanted with human breast cancer cells grew tumors, yet when treated with RL6A TCRm showed a marked reduction in tumor size. These data demonstrate for the first time a coordinated direct discovery and validation strategy that identified a peptide/MHC complex on primary tumor cells for antibody targeting and provide a novel approach to cancer immunotherapy.