Cell lines are key tools for preclinical cancer research, but it remains unclear how well they represent patient tumor samples. Direct comparisons of tumor and cell line transcriptional profiles are ...complicated by several factors, including the variable presence of normal cells in tumor samples. We thus develop an unsupervised alignment method (Celligner) and apply it to integrate several large-scale cell line and tumor RNA-Seq datasets. Although our method aligns the majority of cell lines with tumor samples of the same cancer type, it also reveals large differences in tumor similarity across cell lines. Using this approach, we identify several hundred cell lines from diverse lineages that present a more mesenchymal and undifferentiated transcriptional state and that exhibit distinct chemical and genetic dependencies. Celligner could be used to guide the selection of cell lines that more closely resemble patient tumors and improve the clinical translation of insights gained from cell lines.
The development of metastases is an extended and inefficient process involving multiple steps. The last of these involves the growth of micrometastases into macroscopic tumors. We show here that ...intravenously injected, nonmetastatic cancer cells cease proliferating after extravasating into the parenchyma of the lungs; this response is attributable to the cells inability to trigger adhesion-related signaling events when they are scattered sparsely within the extracellular matrix (ECM) of the parenchyma. We recapitulate this situation by culturing these nonmetastatic cells at low seeding density in ECM-derived gels in vitro, in which they undergo cell-cycle arrest resulting, in part, from insufficient activation of focal adhesion kinase (FAK). Metastatic cancer cells, in contrast, show sufficient FAK activation to enable their proliferation within ECM gels in vitro and continue cell-cycle progression within the lung parenchyma in vivo. Activation of FAK in these metastatic cells depends on expression of the β₁ subunit of integrins, and proliferation of these cells after extravasation in the lungs is diminished by knocking down the expression of either FAK or integrin β₁. These results demonstrate the critical role of integrin β₁-FAK signaling axis in controlling the initial proliferation of micrometastatic cancer cells disseminated in the lungs.
Assays to study cancer cell responses to pharmacologic or genetic perturbations are typically restricted to using simple phenotypic readouts such as proliferation rate. Information-rich assays, such ...as gene-expression profiling, have generally not permitted efficient profiling of a given perturbation across multiple cellular contexts. Here, we develop MIX-Seq, a method for multiplexed transcriptional profiling of post-perturbation responses across a mixture of samples with single-cell resolution, using SNP-based computational demultiplexing of single-cell RNA-sequencing data. We show that MIX-Seq can be used to profile responses to chemical or genetic perturbations across pools of 100 or more cancer cell lines. We combine it with Cell Hashing to further multiplex additional experimental conditions, such as post-treatment time points or drug doses. Analyzing the high-content readout of scRNA-seq reveals both shared and context-specific transcriptional response components that can identify drug mechanism of action and enable prediction of long-term cell viability from short-term transcriptional responses to treatment.
Regulatory networks orchestrated by key transcription factors (TFs) have been proposed to play a central role in the determination of stem cell states. However, the master transcriptional regulators ...of adult stem cells are poorly understood. We have identified two TFs, Slug and Sox9, that act cooperatively to determine the mammary stem cell (MaSC) state. Inhibition of either Slug or Sox9 blocks MaSC activity in primary mammary epithelial cells. Conversely, transient coexpression of exogenous Slug and Sox9 suffices to convert differentiated luminal cells into MaSCs with long-term mammary gland-reconstituting ability. Slug and Sox9 induce MaSCs by activating distinct autoregulatory gene expression programs. We also show that coexpression of Slug and Sox9 promotes the tumorigenic and metastasis-seeding abilities of human breast cancer cells and is associated with poor patient survival, providing direct evidence that human breast cancer stem cells are controlled by key regulators similar to those operating in normal murine MaSCs.
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► Slug and Sox9 suffice to convert differentiated mammary epithelial cells to stem cells ► Maintenance of naturally arising mammary stem cells (MaSCs) requires Slug and Sox9 ► These transcription factors activate autoregulatory genetic programs to induce MaSCs ► Slug and Sox9 promote breast cancer tumor-initiating and metastatic abilities
Activation of two transcription factors, Slug and Sox9, is sufficient to convert differentiated mammary epithelial cells into stem cells and to promote metastasis in breast cancer cells.
Genetic and chemical perturbations impact diverse cellular phenotypes, including multiple indicators of cell health. These readouts reveal toxicity and antitumorigenic effects relevant to drug ...discovery and personalized medicine. We developed two customized microscopy assays, one using four targeted reagents and the other three targeted reagents, to collectively measure 70 specific cell health phenotypes including proliferation, apoptosis, reactive oxygen species, DNA damage, and cell cycle stage. We then tested an approach to predict multiple cell health phenotypes using Cell Painting, an inexpensive and scalable image-based morphology assay. In matched CRISPR perturbations of three cancer cell lines, we collected both Cell Painting and cell health data. We found that simple machine learning algorithms can predict many cell health readouts directly from Cell Painting images, at less than half the cost. We hypothesized that these models can be applied to accurately predict cell health assay outcomes for any future or existing Cell Painting dataset. For Cell Painting images from a set of 1500+ compound perturbations across multiple doses, we validated predictions by orthogonal assay readouts. We provide a web app to browse predictions: http://broad.io/cell-health-app. Our approach can be used to add cell health annotations to Cell Painting datasets.
Abstract Disseminated tumor cells must negotiate multiple situations that challenge their viability and/or proliferative capacity before they can successfully colonize distant organ sites. Thus, the ...shear stress caused by the blood flow may physically damage tumor cells during their translocation from primary tumors to distant organs via the circulation. In addition, the tissue microenvironment of distant organs is generally unfamiliar to tumor cells, limiting their proliferation within the parenchyma of these organs. Each of these situations involves various types of interactions between tumor cells and host components, which either support or inhibit the establishment and subsequent progression of metastases. The initial formation of micrometastases, as well as their subsequent growth – often termed colonization – therefore require complex adaptations by tumor cells to various host components, most of which are never encountered by these cells during their growth within primary tumor sites. These difficulties explain why the colonization of distant organs by disseminated tumor cells is an extraordinarily demanding task and thus inefficient, and suggests a number of potential targets that might be used in the future to interfere therapeutically with this process. Studying the details of tumor–host interactions at each of the steps leading up to successful metastatic colonization may therefore pave the way for designing therapeutic strategies to counteract the metastatic spread of malignant tumors.
Disseminated cancer cells that have extravasated into the tissue parenchyma must interact productively with its extracellular matrix components to survive, proliferate, and form macroscopic ...metastases. The biochemical and cell biologic mechanisms enabling this interaction remain poorly understood. We find that the formation of elongated integrin β(1)-containing adhesion plaques by cancer cells that have extravasated into the lung parenchyma enables the proliferation of these cells via activation of focal adhesion kinase. These plaques originate in and appear only after the formation of filopodium-like protrusions (FLP) that harbor integrin β(1) along their shafts. The cytoskeleton-regulating proteins Rif and mDia2 contribute critically to the formation of these protrusions and thereby enable the proliferation of extravasated cancer cells. Hence, the formation of FLPs represents a critical rate-limiting step for the subsequent development of macroscopic metastases.
Although the mechanisms of metastatic dissemination have begun to be uncovered, those involved in the establishment of extravasated cancer cells in foreign tissue microenvironments remained largely obscure. We have studied the behavior of recently extravasated cancer cells in the lungs and identified a series of cell biologic processes involving the formation of filopodium-like protrusions and the subsequent development of elongated, mature adhesion plaques, which contribute critically to the rapid proliferation of the micrometastatic cells and thus are prerequisites to the eventual lung colonization by these cells.
A critical function of tumor suppressor p53 is the induction of apoptosis in cells exposed to noxious stresses. We report a previously unidentified pro-apoptotic gene, Noxa. Expression of Noxa ...induction in primary mouse cells exposed to x-ray irradiation was dependent on p53. Noxa encodes a Bcl-2 homology 3 (BH3)-only member of the Bcl-2 family of proteins; this member contains the BH3 region but not other BH domains. When ectopically expressed, Noxa underwent BH3 motif-dependent localization to mitochondria and interacted with anti-apoptotic Bcl-2 family members, resulting in the activation of caspase-9. We also demonstrate that blocking the endogenous Noxa induction results in the suppression of apoptosis. Noxa may thus represent a mediator of p53-dependent apoptosis.
The success of anticancer therapy is usually limited by the development of drug resistance. Such acquired resistance is driven, in part, by intratumoural heterogeneity - that is, the phenotypic ...diversity of cancer cells co-inhabiting a single tumour mass. The introduction of the cancer stem cell (CSC) concept, which posits the presence of minor subpopulations of CSCs that are uniquely capable of seeding new tumours, has provided a framework for understanding one dimension of intratumoural heterogeneity. This concept, taken together with the identification of the epithelial-to-mesenchymal transition (EMT) programme as a critical regulator of the CSC phenotype, offers an opportunity to investigate the nature of intratumoural heterogeneity and a possible mechanistic basis for anticancer drug resistance. In fact, accumulating evidence indicates that conventional therapies often fail to eradicate carcinoma cells that have entered the CSC state via activation of the EMT programme, thereby permitting CSC-mediated clinical relapse. In this Review, we summarize our current understanding of the link between the EMT programme and the CSC state, and also discuss how this knowledge can contribute to improvements in clinical practice.
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