RESUMEN
Defining how interactions between tumor subpopulations contribute to invasion is essential for understanding how tumors metastasize. Here, we find that the heterogeneous expression of the transcription factor ΔNp63 confers distinct proliferative and invasive epithelial-to-mesenchymal transition (EMT) states in subpopulations that establish a leader-follower relationship to collectively invade. A ΔNp63-high EMT program coupled the ability to proliferate with an IL1α- and miR-205-dependent suppression of cellular protrusions that are required to initiate collective invasion. An alternative ΔNp63-low EMT program conferred cells with the ability to initiate and lead collective invasion. However, this ΔNp63-low EMT state triggered a collateral loss of fitness. Importantly, rare growth-suppressed ΔNp63-low EMT cells influenced tumor progression by leading the invasion of proliferative ΔNp63-high EMT cells in heterogeneous primary tumors. Thus, heterogeneous activation of distinct EMT programs promotes a mode of collective invasion that overcomes cell intrinsic phenotypic deficiencies to induce the dissemination of proliferative tumor cells. SIGNIFICANCE: These findings reveal how an interaction between cells in different EMT states confers properties that are not induced by either EMT program alone.
Asunto(s)
Transición Epitelial-Mesenquimal/fisiología , Invasividad Neoplásica/fisiopatología , Proteínas de Neoplasias/metabolismo , Factores de Transcripción/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Proteínas Supresoras de Tumor/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Extensiones de la Superficie Celular , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/genética , Matriz Extracelular/patología , Femenino , Humanos , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , ARN Interferente Pequeño/metabolismo , Esferoides Celulares , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Neoplasias de la Mama Triple Negativas/genética , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells. Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.
Asunto(s)
Técnicas de Cocultivo/métodos , Linfocitos T Citotóxicos/citología , Comunicación Celular , Línea Celular Tumoral , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Humanos , Invasividad Neoplásica , Esferoides Celulares/patologíaRESUMEN
Base excision repair (BER) is one of the most frequently used cellular DNA repair mechanisms and modulates many human pathophysiological conditions related to DNA damage. Through live cell and in vitro reconstitution experiments, we have discovered a major sub-pathway of conventional long-patch BER that involves formation of a 9-nucleotide gap 5' to the lesion. This new sub-pathway is mediated by RECQ1 DNA helicase and ERCC1-XPF endonuclease in cooperation with PARP1 poly(ADP-ribose) polymerase and RPA The novel gap formation step is employed during repair of a variety of DNA lesions, including oxidative and alkylation damage. Moreover, RECQ1 regulates PARP1 auto-(ADP-ribosyl)ation and the choice between long-patch and single-nucleotide BER, thereby modulating cellular sensitivity to DNA damage. Based on these results, we propose a revised model of long-patch BER and a new key regulation point for pathway choice in BER.
