Performance comparison of high throughput single-cell RNA-Seq platforms in complex tissues.

Single-cell transcriptomics has emerged as the preferred tool to define cell identity through the analysis of gene expression signatures. However, there are limited studies that have comprehensively compared the performance of different scRNAseq systems in complex tissues. Here, we present a systematic comparison of two well-established high throughput 3'-scRNAseq platforms: 10× Chromium and BD Rhapsody, using tumours that present high cell diversity.

Temporal profiling of the breast tumour microenvironment reveals collagen XII as a driver of metastasis.

The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance.

ALTEN: A High-Fidelity Primary Tissue-Engineering Platform to Assess Cellular Responses Ex Vivo.

To fully investigate cellular responses to stimuli and perturbations within tissues, it is essential to replicate the complex molecular interactions within the local microenvironment of cellular niches. Here, the authors introduce Alginate-based tissue engineering (ALTEN), a biomimetic tissue platform that allows ex vivo analysis of explanted tissue biopsies. This method preserves the original characteristics of the source tissue's cellular milieu, allowing multiple and diverse cell types to be maintained over an extended period of time.

Activation of the viral sensor oligoadenylate synthetase 2 (Oas2) prevents pregnancy-driven mammary cancer metastases.

Background: The interferon response can influence the primary and metastatic activity of breast cancers and can interact with checkpoint immunotherapy to modulate its effects. Using N-ethyl-N-nitrosourea mutagenesis, we found a mouse with an activating mutation in oligoadenylate synthetase 2 (Oas2), a sensor of viral double stranded RNA, that resulted in an interferon response and prevented lactation in otherwise healthy mice.

Methods: To determine if sole activation of Oas2 could alter the course of mammary cancer, we combined the Oas2 mutation with the MMTV-PyMT oncogene model of breast cancer and examined disease progression and the effects of checkpoint immunotherapy using Kaplan-Meier survival analysis with immunohistochemistry and flow cytometry.

Advancements in 3D Cell Culture Systems for Personalizing Anti-Cancer Therapies.

Over 90% of potential anti-cancer drug candidates results in translational failures in clinical trials. The main reason for this failure can be attributed to the non-accurate pre-clinical models that are being currently used for drug development and in personalised therapies. To ensure that the assessment of drug efficacy and their mechanism of action have clinical translatability, the complexity of the tumor microenvironment needs to be properly modelled.

Tumor dissociation of highly viable cell suspensions for single-cell omic analyses in mouse models of breast cancer.

Cell preparation with a high rate of viable cells is required to obtain reliable single-cell transcriptomic and epigenomic data. This protocol describes a technique for digestion and single-cell isolation from mouse mammary tumors to achieve ∼90% of viable cells, which can be subsequently processed in a diverse array of high-throughput single-cell "omic platforms," both in an unbiased manner or after selection of a specific cell population. For complete details on the use and execution of this protocol, please refer to Valdes-Mora et al.

Mammary Tumor Organoid Culture in Non-Adhesive Alginate for Luminal Mechanics and High-Throughput Drug Screening.

Mammary tumor organoids have become a promising in vitro model for drug screening and personalized medicine. However, the dependency on the basement membrane extract (BME) as the growth matrices limits their comprehensive application. In this work, mouse mammary tumor organoids are established by encapsulating tumor pieces in non-adhesive alginate.

Generation and analysis of 3D cell culture models for drug discovery.

Successful preclinical drug testing relies in part on data generated using in vitro cell culture models that recapitulate the structure and function of tumours and other tissues in vivo. The growing evidence that 3D cell models can more accurately predict the efficacy of drug responses compared to traditionally utilised 2D cell culture systems has led to continuous scientific and technological advances that enable better physiologically representative in vitro modelling of in vivo tissues. This review will provide an overview of the utility of current 3D cell models from a drug screening perspective and explore the future of 3D cell models for drug discovery applications.

Single-cell transcriptomics reveals involution mimicry during the specification of the basal breast cancer subtype.

Basal breast cancer is associated with younger age, early relapse, and a high mortality rate. Here, we use unbiased droplet-based single-cell RNA sequencing (RNA-seq) to elucidate the cellular basis of tumor progression during the specification of the basal breast cancer subtype from the luminal progenitor population in the MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mammary tumor model. We find that basal-like cancer cells resemble the alveolar lineage that is specified upon pregnancy and encompass the acquisition of an aberrant post-lactation developmental program of involution that triggers remodeling of the tumor microenvironment and metastatic dissemination.

Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer.

Background: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies.

Myeloid-Derived Suppressor Cells as a Therapeutic Target for Cancer.

The emergence of immunotherapy has been an astounding breakthrough in cancer treatments. In particular, immune checkpoint inhibitors, targeting PD-1 and CTLA-4, have shown remarkable therapeutic outcomes. However, response rates from immunotherapy have been reported to be varied, with some having pronounced success and others with minimal to no clinical benefit.

ELF5 modulates the estrogen receptor cistrome in breast cancer.

Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy.

MCL-1 antagonism enhances the anti-invasive effects of dasatinib in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies. It is phenotypically heterogeneous with a highly unstable genome and provides few common therapeutic targets. We found that MCL1, Cofilin1 (CFL1) and SRC mRNA were highly expressed by a wide range of these cancers, suggesting that a strategy of dual MCL-1 and SRC inhibition might be efficacious for many patients.

Single-Cell Transcriptomics in Cancer Immunobiology: The Future of Precision Oncology.

Cancer is a heterogeneous and complex disease. Tumors are formed by cancer cells and a myriad of non-cancerous cell types that together with the extracellular matrix form the tumor microenvironment. These cancer-associated cells and components contribute to shape the progression of cancer and are deeply involved in patient outcome.

Static droplet array for culturing single live adherent cells in an isolated chemical microenvironment.

We present here a new method to easily and reliably generate an array of hundreds of dispersed nanoliter-volume semi-droplets for single-cells culture and analysis. The liquid segmentation step occurs directly in indexed traps by a tweezer-like mechanism and is stabilized by spatial confinement. Unlike common droplet-based techniques, the semi-droplet wets its surrounding trap walls thus supporting the culturing of both adherent and non-adherent cells.

MASTL overexpression promotes chromosome instability and metastasis in breast cancer.

MASTL kinase is essential for correct progression through mitosis, with loss of MASTL causing chromosome segregation errors, mitotic collapse and failure of cytokinesis. However, in cancer MASTL is most commonly amplified and overexpressed. This correlates with increased chromosome instability in breast cancer and poor patient survival in breast, ovarian and lung cancer.

Andy's Algorithms: new automated digital image analysis pipelines for FIJI.

Quantification of cellular antigens and their interactions via antibody-based detection methods are widely used in scientific research. Accurate high-throughput quantitation of these assays using general image analysis software can be time consuming and challenging, particularly when attempted by users with limited image processing and analysis knowledge. To overcome this, we have designed Andy's Algorithms, a series of automated image analysis pipelines for FIJI, that permits rapid, accurate and reproducible batch-processing of 3,3'-diaminobenzidine (DAB) immunohistochemistry, proximity ligation assays (PLAs) and other common assays.

A mutation in the viral sensor 2'-5'-oligoadenylate synthetase 2 causes failure of lactation.

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse.

The innate and adaptive infiltrating immune systems as targets for breast cancer immunotherapy.

A cancer cell-centric view has long dominated the field of cancer biology. Research efforts have focussed on aberrant cancer cell signalling pathways and on changes to cancer cell DNA. Mounting evidence demonstrates that many cancer-associated cell types within the tumour stroma co-evolve and support tumour growth and development, greatly modifying cancer cell behaviour, facilitating invasion and metastasis and controlling dormancy and sensitivity to drug therapy.

MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib.

Background: Metastatic disease is largely resistant to therapy and accounts for almost all cancer deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and chemo-resistance in a wide range of malignancies, and thus its inhibition may prove to be therapeutically useful.

Methods: To examine whether targeting MCL-1 may provide an effective treatment for breast cancer, we constructed inducible models of BIMs2A expression (a specific MCL-1 inhibitor) in MDA-MB-468 (MDA-MB-468-2A) and MDA-MB-231 (MDA-MB-231-2A) cells.

Differential Growth in Periclinal and Anticlinal Walls during Lobe Formation in Arabidopsis Cotyledon Pavement Cells.

Lobe development in the epidermal pavement cells of Arabidopsis thaliana cotyledons and leaves is thought to take place via tip-like growth on the concave side of lobes driven by localized concentrations of actin filaments and associated proteins, with a predicted role for cortical microtubules in establishing the direction of restricted growth at the convex side. We used homologous landmarks fixed to the outer walls of pavement cells and thin-plate spline analysis to demonstrate that lobes form by differential growth of both the anticlinal and periclinal walls. Most lobes formed within the first 24 h of the cotyledons unfurling, during the period of rapid cell expansion.

Chilling to zero degrees disrupts pollen formation but not meiotic microtubule arrays in Triticum aestivum L.

Throughout the wheat-growing regions of Australia, chilling temperatures below 2 °C occur periodically on consecutive nights during the period of floral development in spring wheat (Triticum aestivum L.). In this study, wheat plants showed significant reductions in fertility when exposed to prolonged chilling temperatures in controlled environment experiments.