Dual-color imaging of nuclear-cytoplasmic dynamics, viability, and proliferation of cancer cells in the portal vein area

We used dual-color in vivo cellular imaging to visualize trafficking, nuclear-cytoplasmic dynamics, and the viability of cancer cells after their injection into the portal vein of mice. For these studies, we used dual-color fluorescent cancer cells that express green fluorescent protein (GFP) linked to histone H2B in the nucleus and retroviral red fluorescent protein (RFP) in the cytoplasm. Human HCT-116-GFP-RFP colon cancer and mouse mammary tumor (MMT) cells were HCT-116-GFP-RFP in the portal vein of nude mice. The cells were observed intravitally in the liver at the single-cell level using the Olympus OV100 whole-mouse imaging system. Most HCT-116-GFP-RFP cells remained in sinusoids near peripheral portal veins. Only a small fraction of the cancer cells invaded the lobular area. Extensive clasmocytosis (destruction of the cytoplasm) of the HCT-116-GFP-RFP cells occurred within 6 hours. The number of apoptotic cells rapidly increased within the portal vein within 12 hours of injection. Apoptosis was readily visualized in the dual-color cells by their altered nuclear morphology. The data suggest rapid death of HCT-116-GFP-RFP cells in the portal vein. In contrast, dual-color MMT-GFP-RFP cells injected into the portal vein mostly survived in the liver of nude mice 24 hours after injection. Many surviving MMT-GFP-RFP cells showed invasive figures with cytoplasmic protrusions. The cells grew aggressively and formed colonies in the liver. However, when the host mice were pretreated with cyclophosphamide, the HCT-116-GFP-RFP cells also survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells.     

Orthotopic Transplant Mouse Models with Green Fluorescent Protein-expressing Cancer Cells to Visualize Metastasis and Angiogenesis

There have been major efforts in metastasis research in recent years, especially on the role of angiogenesis in the metastatic process. Much of the information in this area has been obtained from model systems that are not representative of clinical cancer. The technique of surgical orthotopic implantation (SOI) has allowed the development of clinically relevant metastatic models of human cancer in immunodeficient rodents such as the nude and SCID mouse. In order to allow direct visualization of the metastatic process, we took advantage of the green fluorescent protein (GFP) of the jellyfish, Aequorea victoria. A series of cancer cell lines have been stably transfected with vectors containing humanized GFP cDNA. To utilize GFP expression for metastasis studies, fragments of subcutaneously growing tumor, which were comprised of GFP-expressing cells, were implanted by SOI in nude mice. Subsequent metastases were visualized in systemic organs by GFP fluorescence in the lung, liver, bones, brain and other organs down to the single-cell level. With this fluorescent tool, we detected and visualized for the first time tumor cells at the microscopic level in fresh viable tissue in their normal host organs even in the live animal. Angiogenesis is readily visualized in the transplanted GFP-expressing tumors in real time in situ in the live animal using simple laparotomy and fluorescent techniques. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance to visualize and study cancer metastasis and the role of angiogenesis and other factors in the metastatic process. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tumor imaging with multicolor fluorescent protein expression

Imaging with fluorescent proteins has been revolutionary and has led to the new field of in vivo cell biology. Many new applications of this technology have been developed. Green fluorescent protein (GFP)-labeled or red fluorescent protein (RFP)-labeled HT-1080 human fibrosarcoma cells were used to determine clonality of metastasis by imaging of metastatic colonies after mixed implantation of the red and green fluorescent cells. Resulting pure red or pure green colonies were scored as clonal, whereas mixed yellow colonies were scored as nonclonal. Dual-color fluorescent cancer cells expressing GFP in the nucleus and RFP in the cytoplasm were engineered. The dual-color cancer cells enable real-time nuclear–cytoplasmic dynamics to be visualized in living cells in vivo, including mitosis and apoptosis. The nuclear and cytoplasmic behavior of dual-color cancer cells in real time in blood vessels was observed as they trafficked by various means or extravasated in an abdominal skin flap. Dual-color cancer cells were also visualized trafficking through lymphatic vessels where they were imaged via a skin flap. Seeding and arresting of single dual-color cancer cells in the lung, accumulation of cancer-cell emboli, cancer-cell viability, and metastatic colony formation were imaged in real time in an open-chest nude mouse model using assisted ventilation. Novel treatment was evaluated in these imageable models. UVC irradiation killed approximately 70% of the dual-color cancer cells in a nude mouse model. An RFP-expressing glioma was transplanted to the spinal cord of transgenic nude mice expressing nestin-driven green fluorescent protein (ND-GFP). In ND-GFP mice, GFP is expressed in nascent blood vessels and neural stem cells. ND-GFP cells staining positively for neuronal class III-β-tubulin or CD31 surrounded the tumor, suggesting that the tumor stimulated both neurogenesis and angiogenesis. The tumor caused paralysis and also metastasized to the brain. The Salmonella typhimurium A1-R tumor-targeting bacterial strain was administered in the orthotopic spinal cord glioma model. The treated animals had a significant increase in survival and decrease in paralysis. S. typhimurium A1-R was effective against primary bone tumor and lung metastasis expressing RFP in a nude mouse model. S. typhimurium A1-R was effective against both axillary lymph and popliteal lymph node metastases of human dual-color pancreatic cancer and fibrosarcoma cells, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. Imaging with fluorescent proteins will reveal mechanisms of cancer progression and provide visual targets for novel therapeutics.     

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.     

Transgenic nude mouse with ubiquitous green fluorescent protein expression as a host for human tumors

We report here the development of the transgenic green fluorescent protein (GFP) nude mouse with ubiquitous GFP expression. The GFP nude mouse was obtained by crossing nontransgenic nude mice with the transgenic C57/B6 mouse in which the beta-actin promoter drives GFP expression in essentially all tissues. In crosses between nu/nu GFP male mice and nu/+ GFP female mice, the embryos fluoresced green. Approximately 50% of the offspring of these mice were GFP nude mice. Newborn mice and adult mice fluoresced very bright green and could be detected with a simple blue-light-emitting diode flashlight with a central peak of 470 nm and a bypass emission filter. In the adult mice, the organs all brightly expressed GFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, and duodenum. The following systems were dissected out and shown to have brilliant GFP fluorescence: the entire digestive system from tongue to anus; 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 GFP. Pancreatic islets showed GFP fluorescence. The spleen cells were also GFP positive. Red fluorescent protein (RFP)-expressing human cancer cell lines, including PC-3-RFP prostate cancer, HCT-116-RFP colon cancer, MDA-MB-435-RFP breast cancer, and HT1080-RFP fibrosarcoma were transplanted to the transgenic GFP nude mice. All of these human tumors grew extensively in the transgenic GFP nude mouse. Dual-color fluorescence imaging enabled visualization of human tumor-host interaction by whole-body imaging and at the cellular level in fresh and frozen tissues. The GFP mouse model should greatly expand our knowledge of human tumor-host interaction.     

The camptothecin derivative CPT-11 inhibits angiogenesis in a dual-color imageable orthotopic metastatic nude mouse model of human colon cancer

Recent studies have shown the expression of a stem cell marker protein, nestin, in nascent blood vessels in nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice. In the present study, we visualized tumor angiogenesis and evaluated the antiangiogenic efficacy of CPT-11 in ND-GFP nude mice using dual-color fluorescence imaging. We orthotopically implanted ND-GFP nude mice with the human cancer cell line HCT-116 expressing red fluorescent protein (RFP). The mice were treated with CPT-11 at 40 mg/kg on days 7, 10, 14. Tumor angiogenesis was imaged and visualized by dual-color fluorescence imaging on day 17, three days after the last CPT-11 treatment. Tumor volume and the mean nascent blood vessel density were determined and compared to the control mice. The growing tumor had high expressions of nestin in the nascent blood vessels. The nascent blood vessels showed co-localization of the endothelial-cell-specific marker CD-31 under immunohistochemical staining. The nascent blood vessels were highly visible and their density was determined. ND-GFP nude mice that were administered CPT-11 showed significant reduction in the mean nascent blood vessel density and tumor volume. The dual-color model of ND-GFP transgenic nude mice orthotopically implanted with HCT-116 expressing RFP proved to be effective in visualizing and quantitating tumor growth and tumor angiogenesis. The results showed that CPT-11 is an effective inhibitor of angiogenesis and provided strong implications for wider clinical application of CPT-11 for colon cancer.     

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.     

In vivo tumor delivery of the green fluorescent protein gene to report future occurrence of metastasis

The green fluorescent protein (GFP) gene was administered to intraperitoneally (i.p.) growing human stomach cancer in nude mice to visualize future regional and distant metastases. GFP retroviral supernatants were injected i.p. from day 4 to day 10 after i.p. implantation of the cancer cells. Tumor and metastasis fluorescence was visualized every other week with the use of fluorescence optics via a laparotomy on the tumor-bearing animals. At 2 weeks after retroviral GFP delivery, GFP-expressing tumor cells were observed in gonadal fat, greater omentum, and intestine, indicating that these primary i.p. growing tumors were efficiently transduced by the GFP gene and could be visualized by its expression. At the second and third laparotomies, GFP-expressing tumor cells were observed to have spread to lymph nodes in the mesentery and other regional sites. At the fourth laparotomy, widespread tumor growth was visualized by GFP expression, inducing liver metastasis. No normal tissues were found to be transduced by the GFP retrovirus. Thus, reporter gene transduction of the primary tumor enabled detection of its subsequent metastasis. This gene therapy model could be applied to primary tumors before resection or other treatment to have a fluorescent early detection system for metastasis and recurrence.     

Circulating human prostate cancer cells from an orthotopic mouse model rapidly captured by immunomagnetic beads and imaged by GFP expression

Circulating tumor cells (CTCs) are potential precursors of metastasis. They are also of use in diagnosing malignancy and for prognostic purposes. Our laboratory has previously isolated CTCs from orthotopic nude mouse models of human prostate cancer cells where the PC-3 cancer cells express green fluorescent protein (GFP). It was found that orthotopic tumors produced CTCs and not subcutaneous tumors, which may explain why orthotopic tumors metastasize and subcutaneous tumors do not. However, in this previous study, CTCs were observed only after culture. In the present study, using the GFP-expressing PC-3 orthotopic model and immunomagnetic beads coated with anti-epithelial cell adhesion molecule (EpCAM) and anti-prostate specific membrane antigen (PSMA), GFP-expressing CTC were isolated within 15 minutes and were readily visualized by GFP fluorescence. It was possible to immediately place the immunomagnetic-bead-captured GFP-expressing PC-3 CTCs in 3-dimensional sponge cell culture, where they proliferated. The combination of GFP expression and the use of immunomagnetic beads is a very powerful method to obtain CTCs for either immediate analysis or for biological characterization in vivo or in 3-dimensional culture.     

过表达IL-18 抑制人结直肠癌细胞HCT-116的增殖

目的：研究过表达IL-18 基因对人结直肠癌（colorectal cancer,CRC）HCT-116 细胞体内外增殖的影响及其可能的机制。方法：构建含人IL-18 基因片段的载体,采用慢病毒转染法获得稳定过表达人IL-18 的CRC HCT-116 细胞株HCT-116/IL-18,CCK-8 法检测HCT-116/IL-18 细胞与野生型HCT-116 细胞的增殖,Western blotting 检测两组细胞内IL-18、Cyclin D1、增殖细胞核抗原（PCNA）、DNA损伤修复酶（PARP）蛋白的表达。将HCT-116、HCT-116/IL-18 细胞分别接种于裸鼠左右两侧腋下,观察成瘤性及移植瘤的生长情况,免疫组化法检测移植瘤组织中IL-18 及PCNA的表达。结果：IL-18 基因在HCT-116 细胞内过表达,可延缓HCT-116 的增殖（P<0.05 或P<0.01）;与HCT-116 细胞相比,HCT-116/IL-18 细胞内PARP表达明显增强,PCNA、Cyclin D1 表达减弱（P<0.01）。HCT-116/IL-18 细胞系在裸鼠体内成瘤率明显降低,成瘤率为43%,与对照组比较其移植瘤成瘤时间晚、生长慢、肿块小,且HCT-116 / IL-18 异种移植瘤PCNA蛋白表达下调（P<0.01）。结论：过表达IL-18 基因对HCT-116 细胞生长增殖具有抑制作用,其机制可能与IL-18调控细胞周期和促进DNA损伤有关。     

Color-coded real-time subcellular fluorescence imaging of the interaction between cancer and host cells in live mice

Stromal cells are essential for tumor growth. Stromal cells interact with cancer cells during tumor growth and progression. We report here the development of a tri-color imageable mouse model to visualize the interaction between host cells and cancer cells. To observe subcellular cancer cell dynamics in vivo, HT-1080 human fibrosarcoma cells were labeled in the nucleus with histone H2B-green fluorescent protein (GFP) and with retroviral red fluorescent protein (RFP) in the cytoplasm. HT-1080-GFP-RFP cells were sprinkled over a skin-flap in transgenic GFP immunocompetent mice. After 24 h, the mice were imaged with an Olympus IV100 laser scanning microscope. HT-1080-GFP-RFP cells were visualized surrounded by host-derived lymphocytes and macrophages both expressing GFP. It was possible to observe host GFP macrophages contacting, engulfing, and digesting dual-color HT-1080-GFP-RFP cells in real time. The dual-color cancer cells were readily visible after being engulfed in the GFP macrophages. Other cancer cells were visualized being killed by lymphocytes. The results of this study show that differentially labeling cells with spectrally-distinct fluorescent protein can allow subcellular-resolution imaging of cell-cell interactions between host and cancer cells.     

Lymph node metastasis of oral cancer visualized in live tissue by green fluorescent protein expression

Here, we report the establishment of a stably transfected cell line which expresses high levels of green fluorescent protein (GFP), thus permitting the detection and visualization of developing tumors and lymph node metastases after injection into nude mice. Cells of the human oral squamous carcinoma cell line (SAS-L1) were transfected with an expression vector containing a cDNA encoding humanized GFP and the neomycin resistance gene. A clone with stable high-level expression of GFP was selected in vitro using G418. To study metastasis formation, GFP-expressing cells were injected orthotopically into the tongue of nude mice. The resultant tumor growth in the tongue and micrometastases in the lymph nodes could be visualized by GFP fluorescence. Therefore a useful model has been developed for the study of oral cancer, firstly to understand the metastatic process and secondly for the evaluation of potential treatments.     

Non-invasive Fluorescent-protein Imaging of Orthotopic Pancreatic-cancer-patient Tumorgraft Progression in Nude Mice

In order to individualize and therefore have more effective treatment for pancreatic cancer, we have developed a multicolor, imageable, orthotopic mouse model for individual patients with pancreatic cancer by passaging their tumors through transgenic nude mice expressing green fluorescent protein (GFP) and red fluorescent protein (RFP). The tumors acquired brightly fluorescent stroma from the transgenic host mice, which was stably associated with the tumors through multiple passages. In the present study, pancreatic cancer patient tumor specimens were initially established in NOD.CB17-Prkdc(scid)/NcrCrl (NOD/SCID) mice. The tumors were then passaged orthotopically into transgenic nude mice ubiquitously expressing GFP and subsequently to nude mice ubiquitously expressing RFP. The tumors, with very bright GFP and RFP stroma, were then orthotopically passaged to non-transgenic nude mice. It was possible to image the brightly fluorescent tumors non-invasively longitudinally as they progressed in the non-transgenic nude mice. This non-invasive imageable tumorgraft model will be valuable to screen for effective treatment options for individual patients with pancreatic cancer, as well as for the discovery of improved agents for this treatment-resistant disease.     

一种改良的使用micro-CT扫描的肿瘤终末期小鼠血管造影方法

目的:探讨裸鼠移植瘤模型中血管的分布和密度,试图改进造影剂注射方法来提高肿瘤部位造影剂灌注效率。方法:每只鼠麻醉后,于右侧皮下注射HCT-116细胞悬液,成瘤后定期测量肿瘤长径和短径。待裸鼠肿瘤负荷达到生命终结标准,肿瘤重量超过体重的10%或者出现恶液质时,使用不同方法注射造影剂,应用微计算机断层扫描技术（micro-CT）进行造影,探究肿瘤的血管分布,比较不同注射方法的造影效率。结果:经比较,麻醉后,经下腔静脉注射造影剂的裸鼠造影效率显著高于尾静脉注射与腹腔注射,并且与尾静脉注射相比,对操作者的要求明显降低。结论:经下腔静脉注射造影剂后使用micro-CT造影可成为研究肿瘤终末期小鼠血供的有效方法,为观察肿瘤细胞对血管的调控提供证据。     

Chimeric Mouse model to track the migration of bone marrow derived cells in glioblastoma following anti-angiogenic treatments

Bone marrow derived cells (BMDCs) have been shown to contribute in the tumor development. In vivo animal models to investigate the role of BMDCs in tumor development are poorly explored. We established a novel chimeric mouse model using as low as 5 × 10⁶ GFP+ BM cells in athymic nude mice, which resulted in >70% engraftment within 14 d. In addition, chimera was established in NOD-SCID mice, which displayed >70% with in 28 d. Since anti-angiogenic therapies (AAT) were used as an adjuvant against VEGF-VEGFR pathway to normalize blood vessels in glioblastoma (GBM), which resulted into marked hypoxia and recruited BMDCs to the tumor microenvironment (TME). We exploited chimeric mice in athymic nude background to develop orthotopic U251 tumor and tested receptor tyrosine kinase inhibitors and CXCR4 antagonist against GBM. We were able to track GFP+ BMDCs in the tumor brain using highly sensitive multispectral optical imaging instrument. Increased tumor growth associated with the infiltration of GFP+ BMDCs acquiring suppressive myeloid and endothelial phenotypes was seen in TME following treatments. Immunofluorescence study showed GFP+ cells accumulated at the site of VEGF, SDF1 and PDGF expression, and at the periphery of the tumors following treatments. In conclusion, we developed a preclinical chimeric model of GBM and phenotypes of tumor infiltrated BMDCs were investigated in context of AATs. Chimeric mouse model could be used to study detailed cellular and molecular mechanisms of interaction of BMDCs and TME in cancer.     

利用荧光素酶标记的肿瘤细胞建立活体成像动物模型

目的建立稳定表达荧光素酶的人乳腺癌细胞株并构建适用于小动物活体成像系统观察的裸鼠皮下移植瘤模型。方法采用脂质体将携带荧光素酶基因的质粒转染到人乳腺癌细胞株MCF-7中,G418筛选出稳定表达荧光素酶的单克隆细胞株。扩增后接种于裸鼠,建立裸鼠皮下移植瘤模型,通过活体动物成像系统监测肿瘤的生长过程。结果获得了高水平稳定表达荧光素酶的乳腺癌单克隆细胞株,该单克隆细胞株与母细胞系MCF-7具有相似的生长特性。将稳定表达荧光素酶的克隆接种于裸鼠皮下可成瘤,小动物活体成像系统能准确监测肿瘤细胞在体内的生长过程。结论成功建立了稳定表达荧光素酶的乳腺癌单克隆细胞株。采用活体动物成像系统构建的裸鼠皮下移植瘤模型是拓展肿瘤体内生长、转移及治疗相关研究的理想模型。     

Growth factor-mediated interaction between tumor cells and stromal fibroblasts in an experimental model of human small-cell lung cancer

Malignant tumors induce development of their own stromal tissues during the processes of growth, progression and metastasis. Since the vascular architecture among the various stromal elements is well known to facilitate tumor growth and has been a target of therapy, the importance of stromal fibroblasts has recently been established. To elucidate the interaction between the tumor and its stromal fibroblasts, the present study took advantage of a unique experimental model consisting of a human small-cell lung cancer cell line, WA-ht, and its mouse stromal fibroblast cell line, WA-mFib, both originally derived from a xenograft tumor in a mouse subcutis. Co-culture with the WA-mFib cells significantly augmented the plating efficiency of WA-hT cells in vitro, and their co-inoculation in nude mice shortened latency and tumor doubling time. Histochemical detection of beta-gal, transfected into WA-mFib cells, demonstrated their contribution to the nude mouse xenograft tumor formation as its tumor stroma. Elevated hepatocyte growth factor (HGF) from fibroblasts followed by elevated production of vascular endothelial growth factor (VEGF) from both tumor cells and fibroblasts were demonstrated by ELISA in supernatants of their co-culture, accompanied by enhanced colonogenicity of the tumor cells; these enhanced features were not observed in their respective monocultures. Antisense oligonucleotides to HGF cancelled these augmentation effects with co-culture. The findings highlight the substantial roles of tumor stromal fibroblasts, interacting with soluble growth factors, in promoting the malignant propensity of the tumor.     

Imageable fluorescent metastasis resulting in transgenic GFP mice orthotopically implanted with human-patient primary pancreatic cancer specimens

Tumors from pancreatic cancer patients were established in NOD/SCID mice immediately after surgery and subsequently passaged orthotopically in transgenic nude mice ubiquitously expressing green fluorescent protein (GFP). The primary patient tumors acquired GFP-expressing stroma. Subsequent liver metastases, and disseminated peritoneal metastases maintained the stroma from the primary tumor, and possibly recruited additional GFP-expressing stroma, resulting in their very bright fluorescence. The GFP-expressing stroma included cancer-associated fibroblasts and tumor-associated macrophages in both the primary and metastatic tumors. This imageable model of metastasis from a patient-tumor is an important advance over patient "tumorgraft" models currently in use, which are implanted subcutaneously, do not metastasize and are not imageable. The new imageable model of patient pancreatic cancer metastasis provides unique opportunities to identify current and novel antimetastatic therapeutics for individual patients.     

经小动物内窥镜行肠黏膜下注射肿瘤细胞建立原位结直肠癌模型的技术探讨

目的:探讨使用活体小动物内窥镜进行原位注射人源性结肠癌细胞系建立原位结直肠癌模型的技术方法。方法:6~8周龄BALB/c雌性裸鼠20只,异氟醚气体持续麻醉,通过小动物结肠镜的引导,使用30G注射针将人结肠癌HCT-116细胞注射到裸鼠肠黏膜下,在肿瘤细胞注射3、7、10、15天进行小鼠内窥镜检查,观察裸鼠结直肠的成瘤情况。结果:利用小动物内窥镜行黏膜注射肿瘤细胞建立结直肠癌模型的技术成功率达100%。在注射肿瘤细胞时未出现穿孔,在小鼠内窥镜第7天随访时有1例裸鼠在肠镜通过肿瘤时出现肠壁穿孔。本研究中,没有出现因内窥镜注射导致的裸鼠死亡,所有19例存活裸鼠在第15天随访时均形成原位结直肠肿瘤,使用该方法建立裸鼠原位结直肠癌的建模成功率为95%。结论:通过小鼠内窥镜引导行肠黏膜下注射肿瘤细胞建立结直肠癌原位模型是一项快速而且有效的建模技术,该模型可更好地用于药物检测、基因功能评估和肿瘤转移。     

Expression of secretory phospholipase A2 in colon tumor cells potentiates tumor growth

Secretory phospholipase A2 (sPLA2-IIA) has been shown to attenuate intestinal tumorigenesis in Apc(Min) mice, demonstrating that it is a tumor modifier. To further explore the actions of sPLA2-IIA in tumorigenesis, sPLA2-IIA was overexpressed in two cell lines where it is normally absent, the murine colon tumor cell line AJ02nm0, and human colon carcinoma cell line HCT-116. Two allelic variants of sPLA2-IIA were tested in this study; sPLA2-IIA(AKR) and sPLA2-IIA(SWR), which are derived from AKR/J and SWR/J mice, respectively, and differ by a single amino acid at position 63 in the calcium- and receptor-binding domain. There was no change in cell-doubling time for either allele when compared to vector controls. Furthermore, sodium butyrate and arachidonic acid (AA)-induced cell death were unchanged in control and transfected cells. Addition of the sPLA2 substrate, palmitoyl-arachidonoyl-phosphatidic acid (PAPA), to AJ02nm0 cells resulted in a modest (12%-24%), but significant (P < 0.01), inhibition of growth that was dependent on sPLA2-IIA expression. However, when AJ02nm0 and HCT-116 cells were injected subcutaneously (sc) into nude mice, Pla2g2a expression resulted in a 2.5-fold increase in tumor size. In addition, sPLA2-IIA expressing HCT-116 tumors were found to be more infiltrative than controls. We conclude that the ability of sPLA2-IIA to slow tumor cell growth is dependent upon the availability of substrate, and that in some instances sPLA2-IIA may actually enhance tumor growth. Mechanisms that may account for differences between the tumor explant model versus the Apc(Min) model of intestinal cancer are discussed.