Growth pattern of the de novo small clusters of colorectal cancer is regulated by Notch signaling at detachment

Cancer cell clusters have a higher capacity for metastasis formation than single cells. This suggests that cancer cell clusters, as the smallest unit of cancer, have different biological properties than single cells. The nature of the de novo cancer cell clusters newly formed from the tumor mass is largely unknown. We have developed a model using organoid technology to generate small cell clusters from larger cancer cell masses. We used it to follow the growth pattern of each colon cancer cell from a single cell to a four-cell cluster. Growth patterns were classified into three subgroups: actively growing spheroids (AG), poorly growing spheroids (PG), and dead cells. As the initial number of cells in the cluster increased, AG spheroids increased and dead cells decreased. The other group was PG spheroids, which stopped growing after a short period of growth; the ratio was independent of initial cell number. Under growth permissive conditions, most of the PG spheroids did not grow, but a few did. Comparison of AG and PG spheroids by RNA sequencing showed that Notch signaling was enriched in PG spheroids. Notch signaling was robustly activated in small clusters immediately after dissociation, but not in single-cells. Inhibition of Notch signaling dramatically increased the percentage of PG spheroids, with growth characteristics similar to those of naturally induced PG spheroids. To analyze the rare PG spheroids which grew under growth permissive conditions, we have developed a method to easily and gently prepare numerous small clusters. PG spheroids induced by notch inhibition also contained a small population that grew under growth permissive conditions. In contrast to the low frequency of growth of PG spheroids in vitro, when injected into mice, xenograft tumors derived from spheroids treated with Notch inhibitors showed growth rates comparable to those of non-treated spheroids. Thus, PG spheroids have a stable phenotype but have a strong ability to change to an AG phenotype under certain microenvironments.