Cell competition promotes metastatic intestinal cancer through a multistage process

Summary Cell competition plays an instrumental role in quality control during tissue development and homeostasis. Nevertheless, cancer cells can exploit this process for their own proliferative advantage. In our study, we generated mixed murine organoids and microtissues to explore the impact of cell competition on liver metastasis. Unlike competition at the primary site, the initial effect on liver progenitor cells does not involve the induction of apoptosis. Instead, metastatic competition manifests as a multistage process. Initially, liver progenitors undergo compaction, which is followed by cell-cycle arrest, ultimately forcing differentiation. Subsequently, the newly differentiated liver cells exhibit reduced cellular fitness, rendering them more susceptible to outcompetition by intestinal cancer cells. Notably, cancer cells leverage different interactions with different epithelial populations in the liver, using them as scaffolds to facilitate their growth. Consequently, tissue-specific mechanisms of cell competition are fundamental in driving metastatic intestinal cancer.

Displays the cancer cells in pure (dark green) and mixed (light green) that were followed during ;me-lapse imaging.The number of cancer cells, normalized to T=0, is ploCed against ;me.Each line represents an organoid.

Table S1
Related to Figure S6 Overview of all oligonucleo;de sequences used for qPCR experiments in Figure S6.

Figure S2 :
Figure S2: Increased expansion of compe?ng cancer cells Related to Figure 2

Figure S3 :
Figure S3: Cancer induces compac?on and cell cycle arrest of wild-type liver cells Related to Figure 3 (A) Quan;fica;on of the inter-nuclear distance of cancer cells in pure and mixed organoids; each dot represents one organoid (mean ± SEM; Ordinary one-way ANOVA, Sidak's mul;ple comparisons test; p<0.4681; n= 26 and 64 organoids).(B-C) Quan;fica;on of the percentage of EdU-/pH3-wild-type cells a\er 1.5 h (B) or 24 h (C) of EdU treatment; each dot represents one organoid (mean ± SEM; Ordinary one-way ANOVA, Sidak's mul;ple comparisons test; p<0.0001; n= 55 and 47 organoids in B; p<0.0001; n=37 and 48 organoids in C). (D) Z projec;on confocal images of pure wild-type (D and D') and Mixed (D'' and D''') FUCCI2 organoids fixed three days a\er plan;ng.White doCed line indicates wild-type cells.Scale bars represent 50 µm.

Figure S6 -
Figure S6 -WT liver acts as a scaffold for tumor growth during compe??onRelated to Figure6(A-E) Display the expression of hepatocyte marker genes Albumin (A), HNF4 (B) and Cyp3a (C) and liver progenitor marker genes Krt19 (D) and Tbx3 (E) rela;ve to the housekeeping gene GAPDH, in liver progenitor organoids cultured for 3 days in isola;on and expansion medium, in liver progenitor organoids at the day 15 of the differen;a;on protocol and in micro;ssues 3 days a\er pla;ng.(F) Representa;ve 3D-reconstructed s;tched confocal images of pure (le\) and mixed (right) micro;ssues formed from APC -/-(F) and wild-type small intes;ne (F') organoids together with hepatocyte-like WT cells (Magenta).The insets display a 6x magnifica;on of the area in the white box.(G) Quan;fica;on of the percentage of intes;ne cells in the mixed AKP, APC -/-and wild-type micro;ssues.The dashed line indicates the expected contribu;on based on the star;ng ra;o (Mean; Ordinary one-way ANOVA, mul;ple comparisons; Tukey´s mul;ple comparison test; AKP vs APC, p= 0.0049, n= 17 and n= 5; AKP vs SI WT, p<0.0001, n= 17 and n= 7; APC vs SI WT p= 0.4293, n= 5 and n= 7).(H) Representa;ve 3D-reconstructed s;tched confocal images of mixed micro;ssues formed from AKP cancer (green) and hepatocyte-like WT cells (magenta), treated with vehicle control (DMSO) or pancaspase inhibitor (ZVAD).The insets display a 2.5x magnifica;on of the area in the white boxes.Scale bar in s;tched overview image represent 500 µm and in the magnifica;on 50 µm.