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Additional file 1: Supplementary Fig. S1. Validation of PAK2 as an essential kinase for CMS4 cell lines. A, PAK1–3 mRNA expression levels in a panel of 28 CRC cell lines, also including those used for the drop-out screen, as determined by quantitative PCR. Of note: diamond for PAK3 located on x-axis indicates no mRNA could be detected in this sample. B, C, 2Log mRNA expression levels of PAK4–6 in CRC cell lines (B) and tumors (C), determined by microarray or RNA sequencing. D, Western blot for PAK1 protein expression in HT55 & SW948 (CMS2) and HuTu-80 & MDST8 (CMS4). 2,2,2-Trichloroethanol (2,2,2TCE) signal (excerpt taken around 60 kDa region) indicates amount of protein loaded per cell line. Numbers on the left represent molecular weight in kDa according to a stained protein ladder loaded in the gel. Representative blot of N = 3 is displayed. E, F, PAK2 (E) and PAK1 (F) gene expression levels in cells expressing non-silencing (NS) or PAK2 targeting shRNA (PAK2-KD), as determined by qPCR. Expression was normalized to NS condition, N = 3, mean + S.D. is plotted. G, PAK2 gene expression levels in cells expressing non-silencing (NS) or PAK2 targeting shRNA (PAK2-KD), as determined by qPCR in CMS4 cell lines not included in the initial screen (CaR-1, LS123) and a CMS4 line in which PAK2 was not a significant drop-out in the initial screen (Colo320-HSR). H, Relative contribution of PAK2 KD cells to co-culture in competition assay set-up, over time, in additional CMS4 cell lines. Contribution of shRNA-turboRFP expressing cells was first normalized to contribution on day 0 post sort, and subsequently calculated relative to contribution of NS shRNA-turboRFP expressing cells at each individual time-point. N ≥ 2, mean + s.e.m. is plotted. Supplementary Fig. S2. PAK2 loss alters cell morphology, not induction of apoptosis, of CMS4 cell lines. A, Validation of successful PAK2 knock-out (KO) in OUMS-23 CRISPR-Cas9-edited single cell knock-out clones. WT = wildtype. Numbers on the left represent molecular weight in kDa according to a stained protein ladder loaded in the gel. B, Phase contrast images of OUMS-23 WT and KO cells 7 days post-plating. Region of interest (ROI) selected for lower panels is indicated in white dashed box. C, D, Outgrowth of WT and PAK2-KO cells plated on plastic (C) or Matrigel (D) relative to day 0, as measured 4 days post-plating by CellTiter Glo. Representative experiment of N = 3 is shown. Data plotted is mean + S.D. N.S. = not significant, ** = P < 0.005, *** = P < 0.001, **** = P < 0.0001 in two-tailed, Welch-corrected Student’s T-test. E, F, G, Relative outgrowth of cells after 3 days of treatment with different concentrations of Oxaliplatin (E), 5-FU (F) and Paclitaxel (G) measured using CellTiter Glo and normalized to DMSO treated control relative to a day 0 baseline measurement. Data plotted is mean ± S.D. Representative experiment of N = 3 is shown. H, Percentage of apoptotic cells present 48 hours post plating as assessed by Nicoletti assay. Bar graphs depict mean + s.e.m., N = 3, n.s. = not significant as calculated by two-tailed, Welch-corrected Student’s T-test. I, Phase contrast images of CMS2 (HT55) and CMS4 (HuTu-80, MDST8) WT and KO cells 5 days post-plating. Scale bar represents 50 μm. Supplementary Fig. S3. PAK2 deficiency affects actin cytoskeleton turnover and focal adhesion localization. A, ARHGEF7 (βPIX) gene expression levels in cells expressing non-silencing (NS) or ARHGEF7 (βPIX) targeting shRNAs (Sh-1, Sh-2), as determined by qPCR. Expression was normalized to NS condition, N = 3, mean + s.d. is plotted. B, C, Region of interest stills (Fig. 4D) from time-lapse TIRF microscopy at indicated time points (min = minutes) of cell lines transduced with Lifeact-GFP (green) and paxillin-mCherry (magenta). Scale bars indicate 5 μm. See corresponding Supplementary Movies 5and 6, available online, for the full time-lapse recording. D, E, Heatmaps show actin cytoskeleton dynamics over 60 minutes for HuTu-80 (D) and MDST8 (E) in a unique color per time frame. Note the rapid emergence and disappearance of filopodia in the WT condition and relative stability of filopodia over time in PAK2-KO cells. Supplementary Fig. S4. Metastatic colonization and secondary spread is strongly reduced by PAK2 loss. A, PAK2 gene expression levels in primary cultures expressing non-silencing (NS) or PAK2 targeting shRNA (PAK2-KD), as determined by qPCR. B, Relative outgrowth of NS or PAK2-KD primary cell lines plated on low-adherent culture plates relative to day 0, as measured 4 days post-plating by CellTiter Glo.. Data plotted is mean ± S.D. Representative experiment of N = 3 is shown. C, D, Quantification (C) and representative images (D) of Migration Transwell experiments. Bar plots display relative area percentage covered by cells (purple) in Transwell image. Data plotted is mean ± S.D. Values representing N = 2 are plotted. E, Representative images of liver and lung sections stained for human Ku80 (DAB, indicated in brown), derived from two mice per condition. Along with the other Ku80-stained sections, these images were the source for quantification as included in Fig. 5G, I. F, Macroscopic images of tumor burden in full livers and lungs of mice injected with a 50–50% mixture of HuTu-80 WT-Dendra (green) and PAK2-KO-tdTomato cells (magenta). Pictures were taken shortly after mice were sacrificed.

Animal models are 78% accurate in determining whether drugs will alter contractility of the human heart. To evaluate the suitability of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for predictive safety pharmacology, we quantified changes in contractility, voltage, and/or Ca2+ handling in 2D monolayers or 3D engineered heart tissues (EHTs). Protocols were unified via a drug training set, allowing subsequent blinded multicenter evaluation of drugs with known positive, negative, or neutral inotropic effects. Accuracy ranged from 44% to 85% across the platform-cell configurations, indicating the need to refine test conditions. This was achieved by adopting approaches to reduce signal-to-noise ratio, reduce spontaneous beat rate to ≤ 1 Hz or enable chronic testing, improving accuracy to 85% for monolayers and 93% for EHTs. Contraction amplitude was a good predictor of negative inotropes across all the platform-cell configurations and of positive inotropes in the 3D EHTs. Although contraction- and relaxation-time provided confirmatory readouts forpositive inotropes in 3D EHTs, these parameters typically served as the primary source of predictivity in 2D. The reliance of these “secondary” parameters to inotropy in the 2D systems was not automatically intuitive and may be a quirk of hiPSC-CMs, hence require adaptations in interpreting the data from this model system. Of the platform-cell configurations, responses in EHTs aligned most closely to the free therapeutic plasma concentration. This study adds to the notion that hiPSC-CMs could add value to drug safety evaluation.

Competitive cell-interactions play a crucial role in quality control during development and homeostasis. Here we show that cancer cells use such interactions to actively eliminate wild-type intestine cells in enteroid monolayers and organoids. This apoptosis-dependent process boosts proliferation of intestinal cancer cells. The remaining wild-type population activates markers of primitive epithelia and transits to a fetal-like state. Prevention of this cell fate transition avoids elimination of wild-type cells and, importantly, limits the proliferation of cancer cells. JNK signalling is activated in competing cells and is required for cell fate change and elimination of wild-type cells. Thus, cell competition drives growth of cancer cells by active out-competition of wild-type cells through forced cell death and cell fate change in a JNK dependent manner.

Summary Competitive cell interactions play a crucial role in quality control during development and homeostasis. Here, we show that cancer cells use such interactions to actively eliminate wild-type intestine cells in enteroid monolayers and organoids. This apoptosis-dependent process boosts proliferation of intestinal cancer cells. The remaining wild-type population activates markers of primitive epithelia and transits to a fetal-like state. Prevention of this cell-state transition avoids elimination of wild-type cells and, importantly, limits the proliferation of cancer cells. Jun N-terminal kinase (JNK) signaling is activated in competing cells and is required for cell-state change and elimination of wild-type cells. Thus, cell competition drives growth of cancer cells by active out-competition of wild-type cells through forced cell death and cell-state change in a JNK-dependent manner., Graphical abstract, Highlights • In organoids, wild-type intestine cells are actively eliminated by cancer cells • Cell competition boosts proliferation of intestinal cancer cells • Remaining wild-type cells adopt a fetal-like state • JNK signaling in wild-type cells drives cell competition, Interactions between cells helps coordinated decision making in tissues. Krotenberg Garcia et al. show that intestinal cancer cells use competitive cell interactions to actively eliminate surrounding wild-type cells. These interactions drive oncogenic growth in organoids.

Integrin α3β1 plays a crucial role in tumor formation in the two-stage chemical carcinogenesis model (DMBA and TPA treatment). However, the mechanisms whereby the expression of α3β1 influences key oncogenic drivers of this established model are not known yet. Using an in vivo mouse model with epidermal deletion of α3β1 and in vitro Matrigel cultures of transformed keratinocytes, we demonstrate the central role of α3β1 in promoting the activation of several protumorigenic signaling pathways during the initiation of DMBA/TPA‒driven tumorigenesis. In transformed keratinocytes, α3β1-mediated focal adhesion kinase/Src activation leads to in vitro growth of spheroids and to strong Akt and STAT 3 activation when the α3β1-binding partner tetraspanin CD151 is present to stabilize cell‒cell adhesion and promote Smad2 phosphorylation. Remarkably, α3β1 and CD151 can support Akt and STAT 3 activity independently of α3β1 ligation by laminin-332 and as such control the essential survival signals required for suprabasal keratin-10 expression during keratinocyte differentiation. These data demonstrate that α3β1 together with CD151 regulate the signaling pathways that control the survival of differentiating keratinocytes and provide a mechanistic understanding of the essential role of α3β1 in early stages of skin cancer development.

Although hair bulge stem cells are not the cancer cells-of-origin, they contribute to two-stage DMBA/TPA skin carcinogenesis in an α3β1-dependent manner., Epidermal-specific deletion of integrin α3β1 almost completely prevents the formation of papillomas during 7,12-Dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) two-stage skin carcinogenesis. This dramatic decrease in tumorigenesis was thought to be due to an egress and premature differentiation of α3β1-depleted hair bulge (HB) stem cells (SCs), previously considered to be the cancer cells-of-origin in the DMBA/TPA model. Using a reporter mouse line with inducible deletion of α3β1 in HBs, we show that HB SCs remain confined to their niche regardless of the presence of α3β1 and are largely absent from skin tumors. However, tumor formation was significantly decreased in mice deficient for α3β1 in HB SCs. RNA sequencing of HB SCs isolated from short-term DMBA/TPA–treated skin showed α3β1-dependent expression of the matricellular protein connective tissue growth factor (CCN2), which was confirmed in vitro, where CCN2 promoted colony formation and 3D growth of transformed keratinocytes. Together, these findings show that HBs contribute to skin tumorigenesis in an α3β1-dependent manner and suggest a role of HB SCs in creating a permissive environment for tumor growth through the modulation of CCN2 secretion.