Myofibroblasts Enhance Tumor Growth in a Novel Mouse Model of Colorectal Cancer.
Myofibroblasts Enhance Tumor Growth in a Novel Mouse Model of Colorectal Cancer.
复制标题
肌成纤维细胞增强结直肠癌新型小鼠模型中的肿瘤生长。
DOI：
10.1016/j.jss.2019.06.051
复制
发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Yoo,James
中科院分区：
文献类型：
--
作者：
Plummer,Robert;Papageorge,Marianna;Ciomek,Natalie;Liu,Tiegang;Yoo,James
结直肠癌和基质细胞之间的通讯改变了肿瘤微环境，从而调节局部疾病和癌症进展。然而，结肠癌间质细胞的相互作用很难在体内研究。现有动物模型的局限性包括使用免疫功能低下的小鼠、无法对单个器官系统中的细胞群进行遗传修饰或缺乏解剖背景。我们的目标是开发一种新的小鼠模型的结直肠癌，能够研究肿瘤基质细胞的相互作用，在自然的结肠的immune-competent mice.MethodsPrimary小鼠肌成纤维细胞分离C57 BL/6小鼠的结肠，并在细胞培养中生长。基因定义（ApcΔ/Δ; KrasG12D/+;将Trp 53 Δ/Δ）原代小鼠结肠癌细胞以不同浓度（5 × 103至4 × 104个细胞）单独或与同基因肌成纤维细胞（2 × 105个细胞）组合悬浮于无血清培养基（20 μL）中。在异氟烷麻醉后，对免疫活性的8至10周龄C57 BL/6小鼠进行结肠镜检查，利用小动物结肠镜将细胞悬液内窥镜显微注射到结肠壁的粘膜下空间中。监测内镜用于评估肿瘤生长，沿着组织学分析。肿瘤大小是基于肿瘤直径相对于结肠circular.ResultsA共33只小鼠注射的存活率为88%（29/33）的分级系统。结肠直肠癌细胞的内窥镜显微注射导致小鼠远端结肠中的剂量依赖性肿瘤生长，这可以在不处死动物的情况下通过内窥镜进行评估。生长曲线根据注射的结直肠癌细胞浓度而变化，在最低注射细胞浓度（5 × 103个细胞）下无生长，在4周内使用1-2 × 104个细胞进行性生长，而最高结直肠癌细胞浓度（4 × 104个细胞）导致第1周肿瘤变大，随后在4周时间内肿瘤生长稳定下降。联合显微注射2 × 104个结直肠癌细胞和2 × 105个肌成纤维细胞导致更大的肿瘤，持续4周时间，主要由结直肠癌细胞组成。免疫荧光显微镜后共注射结直肠癌细胞与绿色荧光蛋白阳性肌成纤维细胞证实，注射肌成纤维细胞的存在，并保持活力超过4周的时间period.ConclusionsEndoscopic粘膜下显微注射的原代小鼠结直肠癌细胞是可行的，并导致可靠的和可重复的短期增长的结肠肿瘤免疫能力强的小鼠。共注射原代小鼠结直肠癌细胞与同源肌成纤维细胞导致肿瘤生长增强。结直肠癌细胞与同源肌成纤维细胞的共植入为研究免疫活性小鼠天然结肠中肿瘤-基质相互作用提供了一个新的平台。
BackgroundCommunication between colorectal cancer and stromal cells alters the tumor microenvironment to regulate locoregional disease and cancer progression. However, colon cancer–stromal cell interactions are difficult to studyin vivo. Limitations of existing animal models include the use of immunocompromised mice, the inability to genetically modify a cell population in a single organ system, or a lack of anatomic context. Our goal was to develop a novel mouse model of colorectal cancer that is capable of studying tumor-stromal cell interactions in the native colon of immune-competent mice.MethodsPrimary mouse myofibroblasts were isolated from the colon of C57BL/6 mice and were grown in cell culture. Genetically defined (ApcΔ/Δ; KrasG12D/+; Trp53Δ/Δ) primary mouse colon cancer cells were suspended in serum-free media (20 μL) at varying concentrations (5 × 103to 4 × 104cells) either alone or in combination with syngeneic myofibroblasts (2 × 105cells). After isoflurane anesthesia, a colonoscopy was performed on immune-competent 8- to 10-week-old C57BL/6 mice with endoscopic microinjection of the cell suspension into the submucosal space of the colon wall utilizing a small animal colonoscope. Surveillance endoscopy was used to assess for tumor growth, along with histologic analysis. Tumor size is presented on a grading system based on tumor diameter relative to colon circumference.ResultsA total of 33 mice were injected with a survival rate of 88% (29/33). Endoscopic microinjection of colorectal cancer cells resulted in dose-dependent tumor growth in the distal mouse colon that could be assessed endoscopically without animal sacrifice. Growth curves varied depending on the concentration of injected colorectal cancer cells, with no growth at the lowest concentration of injected cells (5 × 103cells), progressive growth over 4 wk using 1-2 × 104cells, while the highest colorectal cancer cell concentration (4 × 104cells) led to larger tumors at week 1 followed by a steady decline in tumor growth over the 4-wk time period. Combined microinjection of 2 × 104colorectal cancer cells with 2 × 105myofibroblasts resulted in much larger tumors that persisted over the 4-wk time period and which were composed primarily of colorectal cancer cells. Immunofluorescence microscopy after coinjection of colorectal cancer cells with green fluorescent protein positive myofibroblasts confirmed that the injected myofibroblasts are present and remain viable over the 4-wk time period.ConclusionsEndoscopic submucosal microinjection of primary mouse colorectal cancer cells is feasible and leads to reliable and reproducible short-term growth of colon tumors in immune-competent mice. Coinjection of primary mouse colorectal cancer cells with syngeneic myofibroblasts leads to enhanced tumor growth. Coimplantation of colorectal cancer cells with syngeneic myofibroblasts provides a novel platform to study tumor-stromal interactions in the native colon of immune-competent mice.