Fig 1.
Co-culture of ACT1 cell cluster with NTECs.
ACT1 cells were cultured for 5 days until they formed small cell clusters of approximately 500 μm in diameter. Then, NTECs were added to establish co-cultures. (A) ACT1 cluster in monoculture at Day 7. (B) ACT1 cluster with NTECs two days after co-culture. NTECs were gradually crowded around ACT1 cell clusters. The bar in (A) indicates 100 μm.
Fig 2.
Suppression of ACT1 cell growth by competition with NTECs.
ACT1 cells were cultured for 5 days until they formed small cell clusters, and then GFP-NTECs were added and cultured for 3 more days. ACT1 cluster sizes at Day 8 were measured in ATC1 monocultures (A) and in co-cultures (B), and their sizes were compared (C) as described in Materials and Methods. ACT1 clusters and NTECs were fixed and stained with anti-53BP1 antibody (red fluorescence). The bar indicates 100 μm.
Fig 3.
Time-lapse analysis demonstrates NTEC-specific cell death.
ACT1 cells labeled with Qtracker 655 were incubated for 3 days before NTECs were added. Time-lapse imaging was started 48 hours after co-culture (A). Representative images of co-cultures at 18 h (B) and 24 h (C) thereafter. Spatiotemporal occurrence of dead NTECs, which appered as small round-shaped cells within 100 μm distance from the ACT1 clusters, was recorded and summed every 5 hours. Total number of dead cells indicates the summed number of dead cells obtained every 5 hours (D). The bar indicates 40 μm.
Fig 4.
Time-lapse analysis demonstrates NTEC-specific apoptosis.
ACT1 cells labeled with Qtracker 655 (red fluorescence) were incubated for 3 days before NTECs were added. Time-lapse imaging was started 48 hours after co-culture at which time point CellEvent Caspase-3/7 Green detection reagent was added and incubated for 30 min (A). The freeform shape indicated by red lined corresponds to ATC1 cluster borders. Representative images of cocultures with CellEvent Caspase-3/7 Green detection reagent incubated for further 6 hours (B), 12 hours (C) and 16 hours (D). The bar indicates 50 μm.
Fig 5.
Detection of phosphorylation of ERK1/2.
ACT1 cell clusters were co-cultured with GFP-NTECs for 5 days, fixed with formaldehyde, and stained with anti-phosphorylated ERK1/2 antibody (red) and anti-Ki-67 antibody (green). (A) Merged image. (B) Red fluorescence image showing regional phosphorylation of ERK1/2 in NTECs. The bar indicates 100 μm.
Fig 6.
Analysis of region-specific phosphorylation of ERK1/2.
ACT1 cell clusters were co-cultured with GFP-NTECs for 5 days, fixed with formaldehyde, and stained with anti-phosphorylated ERK1/2 antibody. (A) Merged image. (B) Green fluorescence image showing GFP-NTECs. Area designated with a white dashed line indicates ACT1 cell cluster. (C) Red fluorescence image showing phosphorylation of ERK1/2. (D) NTECs with phosphorylated ERK1/2 signals zoned according to their distance from the ACT1 cluster. Red cells are phosphorylated ERK1/2-positive within 100 pixels from the cluster, orange 100–200 pixels, yellow 200–300 pixels, green 300–400 pixels, cyan 400–500 pixels. Percentage of NTECs with phosphorylated ERK1/2 is indicated in (E). The bar in (A) indicates 100 μm.
Fig 7.
Western blot analysis of RB and CKIs.
Total protein was extracted from both NTECs and ACT1 cells cultured either alone (NTEC or ACT1 only, respectively) or from cell co-cultures (NTEC or ACT1 competed, respectively) and subjected to western blot analysis with indicated antibodies.
Fig 8.
Western blot analysis of cell cycle regulators and Akt.
Total protein was extracted from both NTECs and ACT1 cells cultured either alone (NTEC or ACT1 only, respectively) or from cell co-cultures (NTEC or ACT1 competed, respectively) and subjected to western blot analysis with indicated antibodies.
Fig 9.
Western blot analysis of MAP-kinase pathway.
Total protein was extracted from both NTECs and ACT1 cells cultured either alone (NTEC or ACT1 only, respectively) or from cell co-cultures (NTEC or ACT1 competed, respectively) and subjected to western blot analysis with indicated antibodies.
Fig 10.
Reduced cell competition between terminally arrested NTECs and ACT1 cell cluster.
ACT1 cells were cultured for 5 days until they formed small cell clusters and then GFP-NTECs irradiated with 10 Gy of γ-rays were added and cultured for 3 more days. ACT1 cluster sizes were measured 3 days later (on Day 8). ACT1 clusters and GFP-NTECs were fixed and stained with anti-53BP1 antibody (red fluorescence). (A) ACT1 cluster in monoculture. (B) ACT1 cluster co-cultured with GFP-NTECs at Day 8. (C) ACT1 cluster with 10 Gy-irradiated GFP-NTECs at Day 8. (D) Comparison of the ACT1 cluster sizes. Statistical difference was evaluated by Mann-Whitney test. The bar indicates 100 μm.
Fig 11.
Summary of reciprocal stress response.
Cell competition between anaplastic thyroid cancer cells and normal thyroid follicular epithelial cells provoked reciprocal stress response. In competed ACT1 cell clusters, activation of Akt resulted in dephosphorylation of RB, which gave rise to reduced growth. In contrast, competed NTECs, especially those neighboring ACT1 clusters, were forced to die by apoptosis via unscheduled activation of ERK1/2. Our results demonstrate that cell competition is a bi-directional phenomenon, thourgh which the initiation, propagation, and metastasis of tumors are hindered.