Biological Behaviors And Metastatic Spread Of Colorectal Cancer Cells In Mouse Models

[Background]Colorectal cancer（CRC）is a leading cause of cancer deaths and metastatic spread is the major factor in CRC mortality.Heterogeneity in cellular phenotypes among a collection of primary tumor subclones that might be present in varying abundance in multiple distinct metastatic lesions in a single organ site or among other different organ sites may underlie CRC progression,recurrence and resistance to therapy.However,data on the extent and basis of cancer cell heterogeneity in metastatic lesions are limited.To study the clonal properties of metastatic lesions and dynamic behaviors of metastatic colorectal cancer cells,we carried out multi-color lineage tracing to address how metastatic CRC cells contribute to intra-tumoral heterogeneity of metastases and explore biological factors contributing to dynamic metastatic behaviors.[Methods]We have used a murine CRC cell line with Apc,Kras,and Trp53 gene defects as well as CRC engraftment models that generate lung and liver metastases,including spontaneous metastasis models.We carried out multi-color lineage tracing to address how metastatic CRC cells contribute to intra-tumoral heterogeneity of metastases.CRC cell line20390 was generated from the tumor-derived organoid from a CDX2P-Cre ER^T2Apc^flox/+Kras^LSL-G12D/+Trp53^flox/flox（AKP）mouse,which was used for generating mouse engraftment models that generate lung and liver metastases,including spontaneous metastasis models in this study.We can visualize the clonality by color compositions.[Results]（1）Target DNA sequencing showed the cells overexpressing m Cherry or EYFP had very similar genomic profiles.（2）Polyclonal metastases can be generated by polyclonal seeding and/or multiple waves of reseeding in CRC mouse models.（3）Cancer cells in an existing metastatic lesion can escape and seed a new site.Metastatic cancer cells reseed existing lung metastatic lesions and primary tumors.Intermixing of metastatic clones contributes to the intra-tumoral heterogeneity of primary tumors and metastases.（4）The clonal composition of CRC metastases varies depending on the organ location of cancer cells（5）The site of neutrophil infiltration in metastases varies depending on the organ colonized by the cancer cells.[Conclusions]Dynamic behaviors of CRC metastatic cells like recurrent seeding and reseeding contribute to intra-lesion heterogeneities.Polyclonal seeding is also a possible mechanism forming polyclonal metastases.Physical treatment for human CRC should pay attention to the elimination of metastatic lesions since metastatic cancer cells can escape and generate a new metastatic lesion.