Assessing the impact of epigenetic inhibitors on reversion of the chromatin landscape from metastatic to non-metastatic organoids

Colorectal cancer, a leading cause of cancer-related mortality due to distant metastases, is driven by activating mutations in the Wnt and MAPK pathways. Understanding the interplay between these crucial pathways during metastatic progression is essential for developing effective treatments. Here we developed an immunocompetent mouse model of metastatic colorectal cancer using in vivo orthotopic passaging. We demonstrate that highly metastatic tumor cells show increased accessibility of AP-1 TF motifs and less accessibility at TCF/LEF, suggesting higher MAPK and less WNT activity, respectively. Overall design: We have developed an immuno-competent mouse model of metastatic colorectal cancer by sequentially passaging small intestinal organoids with mutations in Apc, Kras, Tp53 and Smad4 in vivo. Organoids from either Passage 1 (P1, m4) or Passage 5 (P5, m484) were orthotopically injected into the colon of C57BL/6N mice. 5 weeks post orthotopic injection colon tumors were harvested, digested into single cells and live Epcam+ cells sorted. P1 tumors: n=4; P5 tumors: n=4

结直肠癌（colorectal cancer）是因远处转移导致癌症相关死亡的主要病因，其发生由Wnt通路（Wnt pathway）与MAPK通路（MAPK pathway）的激活性突变驱动。阐明上述关键通路在转移进展过程中的相互作用，对于开发有效治疗手段至关重要。本研究通过体内原位传代（in vivo orthotopic passaging）技术，构建了转移性结直肠癌的免疫健全小鼠模型（immunocompetent mouse model）。研究发现，高转移性肿瘤细胞的AP-1转录因子（AP-1 TF）基序开放程度更高，而TCF/LEF结合位点的开放程度更低，这分别提示其MAPK通路活性更高、Wnt通路活性更低。整体实验设计：本研究通过体内连续传代携带Apc、Kras、Tp53及Smad4突变的小肠类器官，构建了转移性结直肠癌的免疫健全小鼠模型。将第1代（P1，m4）与第5代（P5，m484）的类器官原位注射至C57BL/6N小鼠的结肠内。原位注射5周后，收获结肠肿瘤组织，消化为单细胞悬液并分选活的上皮细胞黏附分子阳性（Epcam+）细胞。P1组肿瘤样本量n=4；P5组肿瘤样本量n=4