上皮间质转化和肿瘤干细胞

最近,两个新的概念出现在肿瘤生物学中：上皮间质转化（epitheilal-mesenchymal transition,EMT）和肿瘤干细胞（cancer stem cells,CSCs）。EMT不仅赋予细胞迁移和侵袭特征,还可使肿瘤细胞获得自我更新能力而具有干细胞的特性,从而促进CSCs的产生。本文讨论EMT和CSCs之间的联系、EMT获得干细胞特征促进CSCs产生、EMT是形成CSCs的重要环节及二者在干细胞巢中的转化,并分析影响EMT和CSCs的因素。研究EMT在肿瘤发生中的作用,与CSCs理论相融合,从而可能发现新的肿瘤靶向治疗。     

Emodin exerts antitumor effects in ovarian cancer cell lines by preventing the development of cancer stem cells via epithelial mesenchymal transition

Ovarian cancer has the worst prognosis among all types of gynecological malignancies and patients are often diagnosed at an advanced stage with distant metastasis. In the present study, it was found that emodin, a small molecular chemical drug derived from natural plants, has antitumor effects on ovarian cancer cells. Emodin induced cytotoxicity and inhibited proliferation in the ovarian cancer cell lines, SK-OV-3, A2780 and PA-1. In addition, emodin inhibited the migration and invasion abilities of the ovarian cancer cells by inhibiting epithelial-mesenchymal transition (EMT), which was evidenced by the downregulation of N-cadherin and vimentin, and the upregulation of E-cadherin protein expression levels. When a subcutaneous xenograft SK-OV-3 tumor mouse model was used, emodin notably reduced the tumor growth rate and inhibited tumor cell proliferation. Furthermore, mechanical analysis revealed that emodin markedly inhibited EMT and reduced the stemness of tumor cells, which was evidenced by the decrease in the protein expression of CD133 and Oct4. Pulmonary metastasis of the ovarian cancer cells was significantly suppressed in the tumor mouse model by the administration of emodin. In addition, flow cytometry analysis indicated that emodin significantly reduced the proportion of ovarian cancer stem-like cells in metastatic lung tissues. In conclusion, emodin, a potent inhibitor of EMT, could serve as a potential candidate for ovarian cancer therapy.     

上皮间质转化和肿瘤干细胞

最近,两个新的概念出现在肿瘤生物学中:上皮间质转化(epitheilal-mesenchymal transition,EMT)和肿瘤干细胞(cancer stem cells,CSCs)。EMT不仅赋予细胞迁移和侵袭特征,还可使肿瘤细胞获得自我更新能力而具有干细胞的特性,从而促进CSCs的产生。本文讨论EMT和CSCs之间的联系、EMT获得干细胞特征促进CSCs产生、EMT是形成CSCs的重要环节及二者在干细胞巢中的转化,并分析影响EMT和CSCs的因素。研究EMT在肿瘤发生中的作用,与CSCs理论相融合,从而可能发现新的肿瘤靶向治疗。     

结直肠癌干细胞及靶向治疗研究进展

肿瘤干细胞是一类未分化的原始肿瘤细胞,是肿瘤转移,复发和耐药的根源。研究结直肠癌干细胞及标志物对于寻求针对肿瘤干细胞的治疗措施将是肿瘤治疗研究的新方向,针对性的靶向治疗将成为肿瘤治疗的研究热点。     

阿司匹林对结肠炎相关结直肠癌小鼠模型肿瘤细胞凋亡的影响

目的探讨阿司匹林对结肠炎相关结直肠癌（CAC）小鼠模型的肿瘤形成、肿瘤细胞凋亡以及肠道炎症的影响。方法 将60只雄性Balb／c小鼠随机分为模     

The roles of microRNAs and epithelial-mesenchymal transition in colorectal cancer metastasis

Colorectal cancer (CRC) is the second most common cause of cancer death worldwide. Distant metastasis is the major cause of death in patients with CRC. During progression to metastasis in which maligna...     

Oxidized high mobility group B‐1 enhances metastability of colorectal cancer via modification of mesenchymal stem/stromal cells

High mobility group box‐1 (HMGB1) is known to be a chemotactic factor for mesenchymal stem/stromal cells (MSCs), but the effect of post‐translational modification on its function is not clear. In this study, we hypothesized that differences in the oxidation state of HMGB1 would lead to differences in the function of MSCs in cancer. In human colorectal cancer, MSCs infiltrating into the stroma were correlated with liver metastasis and serum HMGB1. In animal models, oxidized HMGB1 mobilized three‐fold fewer MSCs to subcutaneous tumors compared with reduced HMGB1. Reduced HMGB1 inhibited the proliferation of mouse bone marrow MSCs (BM‐MSCs) and induced differentiation into osteoblasts and vascular pericytes, whereas oxidized HMGB1 promoted proliferation and increased stemness, and no differentiation was observed. When BM‐MSCs pretreated with oxidized HMGB1 were co‐cultured with syngeneic cancer cells, cell proliferation and stemness of cancer cells were increased, and tumorigenesis and drug resistance were promoted. In contrast, co‐culture with reduced HMGB1‐pretreated BM‐MSCs did not enhance stemness. In an animal orthotopic transplantation colorectal cancer model, oxidized HMGB1, but not reduced HMGB1, promoted liver metastasis with intratumoral MSC chemotaxis. Therefore, oxidized HMGB1 reprograms MSCs and promotes cancer malignancy. The oxidized HMGB1–MSC axis may be an important target for cancer therapy.     

BRACHYURY confers cancer stem cell characteristics on colorectal cancer cells

Cancer stem cells (CSCs) are initiating cells in colorectal cancer (CRC). Colorectal tumours undergo epithelial to mesenchymal transition (EMT)-like processes at the invasive front, enabling invasion and metastasis, and recent studies have linked this process to the acquisition of stem cell-like properties. It is of fundamental importance to understand the molecular events leading to the establishment of cancer initiating cells and how these mechanisms relate to cellular transitions during tumourigenesis. We use an in vitro system to recapitulate changes in CRC cells at the invasive front (mesenchymal-like cells) and central mass (epithelial-like cells) of tumours. We show that the mesoderm inducer BRACHYURY is expressed in a subpopulation of CRC cells that resemble invasive front mesenchymal-like cells, where it acts to impose characteristics of CSCs in a fully reversible manner, suggesting reversible formation and modulation of such cells. BRACHYURY, itself regulated by the oncogene β-catenin, influences NANOG and other 'stemness' markers including a panel of markers defining CRC-CSC whose presence has been linked to poor patient prognosis. Similar regulation of NANOG through BRACHYURY was observed in other cells lines, suggesting this might be a pathway common to cancer cells undergoing mesenchymal transition. We suggest that BRACHYURY may regulate NANOG in mesenchymal-like CRC cells to impose a 'plastic-state', allowing competence of cells to respond to signals prompting invasion or metastasis.     

siRNA沉默Twist基因对结直肠癌细胞顺铂化疗敏感性的影响

目的 结直肠癌(colorectal cancer,CRC)是世界范围内最常见的恶性肿瘤之一,本研究探讨应用小分子干扰RNA (siRNA)沉默Twist信号通路对结直肠癌顺铂化疗敏感性的影响及其可能机制.方法 0、0.0625、0.125、0.25、0.5和1μg/mL的顺铂作用于SW480和LoVo这种结直肠癌细胞株48 h.Twist-siRNA转染这2种结直肠癌细胞株.CCK-8法计算转染前后顺铂对于两者的抑制率.EdU法检测癌细胞增殖能力.Hoechest 3344荧光染色检测细胞形态.蛋白印迹法检测不同药物作用下的Twist及上皮间质化相关标志物的表达变化.结果 LoVo和SW480细胞组中Twist表达量分别为0.824±0.124和0.191±0.039 9;Twist在LoVo中高表达,在SW480中低表达,差异有统计学意义,P＜0.05.SW480和LoVo对于顺铂的IC50值分别为(3.394±0.083)和(6.737±0.796) μg/mL,P＜0.05.wist siRNA干扰后SW480和LoVo对于顺铂的IC50值分别为(0.113 7±0.008 8)和(0.459 7±0.025 0) μg/mL,与单用顺铂组相比,差异均有统计学意义,均P＜0.05.Hoechest3344荧光染色结果表明,Twist转染组比未转染组出现了更加明显的细胞凋亡形态变化.CCK-8检测结果显示,采用Twist siRNA干扰后SW480和LoVo对于顺铂的IC50值分别为(0.113 7±0.008 8)和(0.459 7±0.025 0)μg/mL,与单用顺铂组相比,差异有统计学意义,P＜0.05.EdU法检测结果显示,Twist siRNA+顺铂组、顺铂组和正常组SW480细胞增殖率分别为(14.63±1.33)％、(25.55±1.73)％和(51.23±3.31)％;LoVo细胞增殖率分别为(22.35±2.39)％、(29.27±1.85)％和(64.62±3.61)％;与正常组和单用顺铂组相比,Twist siRNA+顺铂组的细胞增殖率明显降低,差异均有统计学意义,均P＜0.05.蛋白印迹法结果表明,Twist siRNA作用的同时伴随E-cadherin表达升高,而Vimentin表达降低,差异有统计学意义,P＜0.05.结论 应用siRNA降低Twist信号通路活性可通过抑制上皮间质化过程而增加结直肠癌对于顺铂的化疗敏感性.     

MicroRNA和EMT在结肠癌转移中的作用(英文)

Colorectal cancer （CRC） is the second most common cause of cancer death worldwide. Distant metastasis is the major cause of death in patients with CRC. During progression to metastasis in which malignant cells disseminate from the primary tumor to seeding other organs, a multistep process is involved. Cancer cells proliferate, invade microenvironment, en- ter into the blood circulation, then survive and colonize into distant organs. MicroRNAs （miRNAs） and epithelial-mesenchymal transition （EMT） are key regulators and mechanism in tumorigenesis and cancer metastasis. We review the roles of EMT and microRNAs, especially EMT related microRNAs in the metastatic pathway of CRC. MicroRNAs provide us a set of potential therapeutic applications and molecular target for CRC.     

Mouse induced pluripotent stem cell microenvironment generates epithelial-mesenchymal transition in mouse Lewis lung cancer cells

Induced pluripotent stem (iPS) cells may be a powerful tool in regenerative medicine, but their potential tumorigenicity is a significant challenge for the clinical use of iPS cells. Previously, we succeeded in converting miPS cells into cancer stem cells (CSCs) under the conditions of tumor microenvironment. Both stem cells and tumor cells are profoundly influenced by bi-directional communication with their respective microenvironment, which dictates cell fate determination and behavior. The microenvironment derived from iPS cells has not been well studied. In this paper, we have investigated the effects of secreted factors from Nanog-mouse iPS (miPS) cells on mouse Lewis lung cancer (LLC) cells that are found in the conditioned media. The results demonstrated that miPS cells secrete factors that can convert the epithelia phenotype of LLC cells to a mesenchymal phenotype, and that can promote tumorigenisity, migration and invasion. Furthermore, LLC cells that have been exposed to miPS conditioned medium became resistant to apoptosis. These various biological effects suggest that the miPS microenvironment contain factors that can promote an epithelial-mesenchymal transition (EMT) through an active Snail-MMP axis or by suppressing differentiation in LLC cells.     

大肠癌细胞HCT-15肿瘤干细胞样细胞分离及成瘤性比较

目的:依据人大肠癌细胞HCT-15的表面标志物,分离其中的干细胞亚群。建立裸鼠体内原代大肠癌荷瘤模型,比较各亚群肿瘤体积和质量。方法:利用免疫磁珠分选技术,分离其中CD133/CD44干细胞亚群。分选得到CD133+CD44+、CD133+CD44-亚群分别接种于裸鼠体内,并观察肿瘤大小和质量。结果:CD133+CD44+和CD133+CD44-细胞亚群成功的从HCT-15细胞中分离出来。接种CD133+CD44+细胞的裸鼠形成的肿瘤体积［（2.76±0.22）cm3］和质量［（5.2±0.21）g］明显高于接种CD133+CD44-细胞裸鼠的肿瘤体积与质量［（1.56±0.34）cm3,（3.4±0.18）g］（P＜0.05）。结论:CD44阳性的大肠癌肿瘤干细胞成瘤能力明显高于CD44阴性的大肠癌肿瘤干细胞。     

Chemoresistant colorectal cancer cells and cancer stem cells mediate growth and survival of bystander cells

Background:

Recent studies suggest that cancer stem cells (CSCs) mediate chemoresistance, but interestingly, only a small percentage of cells in a resistant tumour are CSCs; this suggests that non-CSCs survive by other means. We hypothesised that chemoresistant colorectal cancer (CRC) cells generate soluble factors that enhance survival of chemonaive tumour cells.

Methods:

Chemoresistant CRC cells were generated by serial passage in oxaliplatin (Ox cells). Conditioned media (CM) was collected from parental and oxaliplatin-resistant (OxR) cells. CRC cells were treated with CM and growth and survival were assessed. Tumour growth rates were determined in nude mice after cells were treated with CM. Mass spectrometry (MS) identified proteins in CM. Reverse phase protein microarray assays determined signalling effects of CM in parental cells.

Results:

Oxaliplatin-resistant CM increased survival of chemo-naive cells. CSC CM also increased growth of parental cells. Parental and OxR mixed tumours grew larger than tumours composed of parental or OxR cells alone. Mass spectrometry detected unique survival-promoting factors in OxR CM compared with parental CM. Cells treated with OxR CM demonstrated early phosphorylation of EGFR and MEK1, with later upregulation of total Akt .We identified progranulin as a potential mediator of chemoresistance.

Conclusion:

Chemoresistant tumour cells and CSCs may promote resistance through soluble factors that mediate survival in otherwise chemosensitive tumour cells.     

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。     

Epithelial mesenchymal transition and tumor budding in aggressive colorectal cancer: tumor budding as oncotarget

Epithelial mesenchymal transition (EMT) is proposed as a critical mechanism for the acquisition of malignant phenotypes by epithelial cells. In colorectal cancer, tumor cells having undergone EMT are histologically represented by the presence of tumor buds defined as single cells or small clusters of de-differentiated tumor cells at the invasive front. Tumor budding is not a static, histological feature rather it represents a snap-shot of a dynamic process undertaken by an aggressive tumor with the potential to disseminate and metastasize. Strong, consistent evidence shows that tumor budding is a predictor of lymph node metastasis, distant metastatic disease, local recurrence, worse overall and disease-free survival time and an independent prognostic factor. Moreover, the International Union against Cancer (UICC) recognizes tumor budding as a highly relevant, additional prognostic parameter. The aim of this review is to summarize the evidence supporting the implementation of tumor budding into diagnostic pathology and patient management and additionally to illustrate its worthiness as a potential therapeutic target.     

Mesenchymal stem cells in the colorectal tumor microenvironment: recent progress and implications

Mesenchymal stem cells (MSCs) are nonhematopoietic multipotent adult stem cells. They have been shown to have a natural tropism for many tumors types, including colorectal, and are capable of escaping host immune surveillance. MSCs are known to engraft at tumors and integrate into their architecture, potentially as carcinoma-associated fibroblasts. In contrast with other malignancies, our understanding of the interactions between colorectal cancer cells and MSCs remains limited. Considering the established importance of inflammation in the colorectal cancer primary tumor microenvironment and the role of stromal cells in this process, there is a potential wealth of information to be gleaned from further investigation of interactions between these cell populations. Epithelial-mesenchymal transition is central to colorectal cancer progression and MSCs have also been implicated in this process. This review explores the current knowledge (both in vitro and in vivo) of interactions between colorectal cancer cells and MSCs. It highlights potential effects of cell source, number and ratio on outcome of in vivo studies and explores strategies to more accurately explore their role in the primary tumor microenvironment. As our understanding of the underlying molecular processes in colorectal cancer develops, elucidation of these interactions will be central to development of novel therapeutic strategies for this prevalent disease.     

The requirement for freshly isolated human colorectal cancer (CRC) cells in isolating CRC stem cells

Background:

Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs.

Methods:

Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment.

Results:

None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not.

Conclusions:

PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs.     

CD133-positive tumor cell content is a predictor of early recurrence in colorectal cancer

Background

The aims of this study were to demonstrate the tumorigenicity of CD133+ colon cancer cells in vitro, analyze the correlations between spheroid formation and clinicopathologic variables, and screen for overexpressed genes in CD133+ colon cancer stem cells. Moreover, the aim of this study was to establish a living tumor tissue bank using surgically resected specimens.

Methods

Using LoVo cell line, we isolated CD133+ cells and performed clonogenic assay and animal experiment to test tumorigenicity of CD133+ cells. Twenty-nine surgical samples were freshly collected from 27 patients who received curative or palliative surgery, and the samples were mechanically and enzymatically dissociated into single cells.

Results

We confirmed the enhanced tumorigenicity of CD133+ cells isolated from LoVo cell line both in vitro and in vivo. Of these 29 samples, 8 (28%) contained >3% CD133+ cells. Sphere formation was significantly higher in samples from patients with lymphatic invasion than in those without lymphatic invasion [54.5% (6/11) vs. 12.5% (2/16); P=0.033] and in samples containing >3% of CD133+ cells than in those containing ≤3% of CD133+ cells [36.4% (4/11) vs. 0% (0/16); P=0.019].

Conclusions

These findings indicate that CD133 is a valid marker for identifying cancer stem cells from fresh surgically resected colorectal cancer tissues. Furthermore, we successfully established a living tumor tissue bank using surgically resected colorectal tissues with a viability of >70%.     

Genistein suppresses FLT4 and inhibits human colorectal cancer metastasis

Dietary consumption of genistein, found in soy, has been associated with a potentially protective role in colorectal cancer (CRC) development and progression. Herein we demonstrate that genistein will inhibit human CRC cell invasion and migration, that it does so at non-cytotoxic concentrations and we demonstrate this in multiple human CRC cell lines. After orthotopic implantation of human CRC tumors into mice, oral genistein did not inhibit tumor growth, but did inhibit distant metastasis formation, and was non-toxic to mice. Using a qPCR array, we screened for genistein-induced changes in gene expression, followed by Western blot confirmation, demonstrating that genistein downregulated matrix metalloproteinase 2 and Fms-Related Tyrosine Kinase 4 (FLT4; vascular endothelial growth factor receptor 3). After demonstrating that genistein suppressed neo-angiogenesis in mouse tumors, we examined FLT4 expression in primary CRC and adjacent normal colonic tissue from 60 human subjects, demonstrating that increased FLT4 significantly correlates with increased stage and decreased survival. In summary, we demonstrate for the first time that genistein inhibits human CRC metastasis at dietary, non-toxic, doses. FLT4 is identified as a marker of metastatic disease, and as a response marker for small molecule therapeutics that inhibit CRC metastasis.