Cell competition promotes metastatic intestinal cancer through a multistage process.

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.

Subtype-specific kinase dependency regulates growth and metastasis of poor-prognosis mesenchymal colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) can be divided into four consensus molecular subtypes (CMS), each with distinct biological features. CMS4 is associated with epithelial-mesenchymal transition and stromal infiltration (Guinney et al., Nat Med 21:1350-6, 2015; Linnekamp et al., Cell Death Differ 25:616-33, 2018), whereas clinically it is characterized by lower responses to adjuvant therapy, higher incidence of metastatic spreading and hence dismal prognosis (Buikhuisen et al., Oncogenesis 9:66, 2020). METHODS: To understand the biology of the mesenchymal subtype and unveil specific vulnerabilities, a large CRISPR-Cas9 drop-out screen was performed on 14 subtyped CRC cell lines to uncover essential kinases in all CMSs. Dependency of CMS4 cells on p21-activated kinase 2 (PAK2) was validated in independent 2D and 3D in vitro cultures and in vivo models assessing primary and metastatic outgrowth in liver and peritoneum. TIRF microscopy was used to uncover actin cytoskeleton dynamics and focal adhesion localization upon PAK2 loss. Subsequent functional assays were performed to determine altered growth and invasion patterns. RESULTS: PAK2 was identified as a key kinase uniquely required for growth of the mesenchymal subtype CMS4, both in vitro and in vivo. PAK2 plays an important role in cellular attachment and cytoskeletal rearrangements (Coniglio et al., Mol Cell Biol 28:4162-72, 2008; Grebenova et al., Sci Rep 9:17171, 2019). In agreement, deletion or inhibition of PAK2 impaired actin cytoskeleton dynamics in CMS4 cells and, as a consequence, significantly reduced invasive capacity, while it was dispensable for CMS2 cells. Clinical relevance of these findings was supported by the observation that deletion of PAK2 from CMS4 cells prevented metastatic spreading in vivo. Moreover, growth in a model for peritoneal metastasis was hampered when CMS4 tumor cells were deficient for PAK2. CONCLUSION: Our data reveal a unique dependency of mesenchymal CRC and provide a rationale for PAK2 inhibition to target this aggressive subgroup of colorectal cancer.

Generation of mixed murine organoids to model cellular interactions.

Cell competition is a mechanism of interaction that dictates cell selection based on differences in cellular fitness. We designed a protocol to generate mixed murine organoids and enteroid monolayers used to study such complex cellular interactions in a mammalian system. This protocol is dedicated to follow the behavior of different cell populations over time, using (time-lapse) microscopy or transcriptome/proteome analysis. For complete details on the use and execution of this protocol, please refer to Krotenberg Garcia et al. (2021).

Active elimination of intestinal cells drives oncogenic growth in organoids.

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.

Integrin α3ß1 Is a Key Regulator of Several Protumorigenic Pathways during Skin Carcinogenesis.

Integrin α3ß1 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 α3ß1 influences key oncogenic drivers of this established model are not known yet. Using an in vivo mouse model with epidermal deletion of α3ß1 and in vitro Matrigel cultures of transformed keratinocytes, we demonstrate the central role of α3ß1 in promoting the activation of several protumorigenic signaling pathways during the initiation of DMBA/TPA‒driven tumorigenesis. In transformed keratinocytes, α3ß1-mediated focal adhesion kinase/Src activation leads to in vitro growth of spheroids and to strong Akt and STAT 3 activation when the α3ß1-binding partner tetraspanin CD151 is present to stabilize cell‒cell adhesion and promote Smad2 phosphorylation. Remarkably, α3ß1 and CD151 can support Akt and STAT 3 activity independently of α3ß1 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 α3ß1 together with CD151 regulate the signaling pathways that control the survival of differentiating keratinocytes and provide a mechanistic understanding of the essential role of α3ß1 in early stages of skin cancer development.

Integrin α3ß1 in hair bulge stem cells modulates CCN2 expression and promotes skin tumorigenesis.

Epidermal-specific deletion of integrin α3ß1 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 α3ß1-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 α3ß1 in HBs, we show that HB SCs remain confined to their niche regardless of the presence of α3ß1 and are largely absent from skin tumors. However, tumor formation was significantly decreased in mice deficient for α3ß1 in HB SCs. RNA sequencing of HB SCs isolated from short-term DMBA/TPA-treated skin showed α3ß1-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 α3ß1-dependent manner and suggest a role of HB SCs in creating a permissive environment for tumor growth through the modulation of CCN2 secretion.

Human-iPSC-Derived Cardiac Stromal Cells Enhance Maturation in 3D Cardiac Microtissues and Reveal Non-cardiomyocyte Contributions to Heart Disease.

Cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) are functionally immature, but this is improved by incorporation into engineered tissues or forced contraction. Here, we showed that tri-cellular combinations of hiPSC-derived CMs, cardiac fibroblasts (CFs), and cardiac endothelial cells also enhance maturation in easily constructed, scaffold-free, three-dimensional microtissues (MTs). hiPSC-CMs in MTs with CFs showed improved sarcomeric structures with T-tubules, enhanced contractility, and mitochondrial respiration and were electrophysiologically more mature than MTs without CFs. Interactions mediating maturation included coupling between hiPSC-CMs and CFs through connexin 43 (CX43) gap junctions and increased intracellular cyclic AMP (cAMP). Scaled production of thousands of hiPSC-MTs was highly reproducible across lines and differentiated cell batches. MTs containing healthy-control hiPSC-CMs but hiPSC-CFs from patients with arrhythmogenic cardiomyopathy strikingly recapitulated features of the disease. Our MT model is thus a simple and versatile platform for modeling multicellular cardiac diseases that will facilitate industry and academic engagement in high-throughput molecular screening.