RNA-seq of 4T1 mouse breast cancer cells cultured on soft vs stiff polyacrylamide hydrogels for 24 hours
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ABSTRACT: The tumour microenvironment is a critical element involved in tumour progression and responsiveness to therapies. Using functionalized tunable stiffness hydrogel, mimicking the mechanical properties of healthy and tumour tissues, we explore how the stiffness of the microenvironment can influence cancer cells by generating RNA-seq transcriptional profiles of 4T1 mouse breast cancer cells cultured on soft vs stiff polyacrylamide hydrogels for 24 hours.
Project description:Human bone marrow mesenchymal stem cells (MSCs) were co-cultured for 7 days with endothelial cells, where they participated in the formation of microcapillaries. MSCs that were exposed to the microcapillaries or kept as monocultures were isolated by FACS and analyzed by RNAseq.
Project description:Background Colorectal cancer (CRC), a malignancy with high incidence and mortality rates, presents particularly severe prognoses at the stages of invasion and metastasis despite significant advancements in diagnostic and therapeutic technologies. The tumour microenvironment, especially the extracellular matrix (ECM) stiffness, has garnered considerable attention for its impact on CRC progression and metastasis. However, the precise relationship and molecular mechanisms between ECM stiffness and CRC prognosis remain unclear. Methods This study included 107 CRC patients. Tumor stiffness was assessed using magnetic resonance elastography (MRE), and collagen content was analysed with Masson staining. CRC cell lines SW480 and HCT116 were cultured on matrices of varying stiffness, followed by transcriptome sequencing to identify stiffness-related differential genes and investigate their functions and signalling pathways. Additionally, an HSF4 knockout CRC cell model was constructed to evaluate the effects of HSF4 on cell proliferation, migration, and invasion through both in vitro and in vivo experiments, exploring its role in tumour growth and metastasis. Results The study found that CRC tumour stiffness was significantly higher than normal tissue and positively correlated with collagen content and TNM staging. Transcriptome analysis revealed that high-stiffness matrices significantly regulated cell functions and signalling pathways. High expression of HSF4 was strongly associated with tumour stiffness and poor prognosis. HSF4 expression increased with higher TNM stages in CRC tissues, and its knockout significantly inhibited cell proliferation, migration, and invasion, particularly on high-stiffness matrices. In vivo experiments further confirmed that HSF4 promoted tumour growth and metastasis, independent of collagen protein increase. Conclusion This study reveals a close relationship between tumour stiffness and CRC prognosis, identifying HSF4 as a critical player in CRC progression. HSF4 promotes tumour proliferation and metastasis by regulating EMT-related signalling pathways, with its high expression closely linked to tumour stiffness. Although LOXL1 increased collagen content and a-SMA expression, HSF4's role in promoting tumour growth and metastasis was independent of collagen protein. HSF4 holds potential as a target for CRC prognosis evaluation and treatment. Future research should further explore the regulatory mechanisms of HSF4 and its potential for clinical application.
Project description:Extracellular matrix (ECM) elasticity is perceived by cells via focal adhesion structures, which transduce mechanical cues into chemical signaling to conform cell behavior. Although the contribution of ECM compliance to the control of cell migration or division has been extensively studied, little has been reported regarding whole proteome adaptation to changes of mechanical forces. Uropathogenic E. coli (UPEC) are responsible of 70-95% of the Urinary Tract Infections (UTIs), comprising cystitis, prostatitis and pyelonephritis. These are recurrent or chronic infections causing significant costs in public health. UPEC produce the Cytotoxic Necrotizing Factor1 (CNF1) toxin, targeting one of the major host mechano-sensor and -adaptor, namely the actin cytoskeleton. We have discovered that the CNF1 and integrin-dependent invasion of cells by UPEC is highly dependent on ECM stiffness. Here we aim to analyze primary human cells treated or not with CNF1 and cultured on a fibronectin ECM of variable stiffness by label free shotgun proteomics, to bring new concepts on the manipulation of host tissue mechanics by pathogens.
Project description:Background: the transcription of tumor mutations from DNA into RNA has implications for biology, epigenetics and clinical practice. It is not clear if mutations are in general transcribed and, if so, at what proportion to the wild-type allele. Here, we examined the correlation between DNA mutation allele frequency and RNA mutation allele frequency. Methods: we sequenced the exome and transcriptome of tumor cell lines with large copy number variations, identified heterozygous single nucleotide mutations and absolute DNA copy number, and determined the corresponding DNA and RNA mutation allele fraction. Results: we found that 99% of the DNA mutations in expressed genes are expressed as RNA. Moreover, we found a high correlation between the DNA and RNA mutation allele frequency. Exceptions are mutations that cause premature termination codons and therefore activate nonsense-mediated decay. Beyond this, we did not find evidence of any wide-scale mechanism, such as allele-specific epigenetic silencing, preferentially promoting mutated or wild-type alleles. Conclusion: taken together, our data strongly suggest that genes are equally transcribed from all alleles, mutated and wild-type, and thus transcribed in proportion to their DNA allele frequency.
Project description:In this project, we cultured rat hippocampal neurons in mechanically different environments and studied electrical maturation. We compared WT neurons with two different Piezo1 knockdown conditions. These two knockdown conditions were generated in two independent CRISPR-Cas9 KD assays. Each assay is based on four different CRISPR-Cas9 guides targeting the Piezo1 gene. RNA sequencing was used to analyse the pathway leading to the stiffness dependent maturation behaviour.
Project description:We report that macrophage elasticity plays a dominant role in bacterial phagocytosis, release of TNF-alpha, and production of reactive oxygen species. We show that macrophage elasticity is modulated by mechanical factors including substrate rigidity and substrate stretch. Changes in macrophage elasticity are dependent upon the degree of actin polymerization, and mediated in part through small rhoGTPase activity. Moreover, the functional effects of macrophage elasticity are not predicted by gene expression profiles. Murine RAW 267.4 macrophages were separately grown on 2 matrix stiffness levels (1200, 150000 Pascals) for 0, 2, 6, 18 hours with 3 replicate sample experiments per condition. Total RNA extracted from the cells and profiled by microarrays. Keywords: Murine RAW 267.4 macrophage, matrix stiffness, phagocytosis, cell elasticity.
Project description:Cancers evade the immune system in order to grow or metastasise through the process of cancer immunoediting. While checkpoint inhibitor therapy has been effective for reactivating tumour immunity in some cancers, many solid cancers, including breast cancer, remain largely non-responsive. Understanding the way non-responsive cancers evolve to evade immunity, what resistance pathways are activated and whether this occurs at the clonal level will improve immunotherapeutic design. We tracked cancer cell clones during the immunoediting process and determined clonal transcriptional profiles that allow immune evasion in murine mammary tumour growth in response to immunotherapy with anti-PD1 and anti-CTLA4. Clonal diversity was significantly restricted by immunotherapy treatment at both the primary and metastatic sites. These findings demonstrate that immunoediting selects for pre-existing breast cancer cell populations, that immunoediting is not a static process and is ongoing during metastasis and immunotherapy treatment. Isolation of immunotherapy resistant clones revealed unique and overlapping transcriptional signatures. The overlapping gene signature was predictive of poor survival in basal-like breast cancer patient cohorts. Some of these overlapping genes have existing small molecules which can be used to potentially improve immunotherapy response.
Project description:Cells interact with their mechanical environment and respond in consequence. Mechanical cues can have a wide range of influences on cell behaviour, ranging from guidance of differentiation and cell fate to immune activation. The impact of substrate stiffness on primary macrophages - a key player in innate immunity and inflammation - had not been previously studied. We prepared bone marrow-derived macrophage cultures from adult rat hematopoietic stem cells exposed to M-CSF, and cultured these on polyacrylamide substrates of controlled stiffness (ranging from 50 to 0.1 kPa shear modulus, covering the range found in physiological tissues) for 3 days. The RNA from these cells was then extracted and sequenced.
Project description:Mesenchymal stromal cells were cultured in 3D PEG hydrogels for 7 days in the presence of serum-free media or conditioned media from a panel of breast cancer cells (MCF-7, MDA-MB-231, MDA-MB-231 lung-tropic, MDA-MB-231 brain-tropic, MDA-MB-231 bone-tropic). In all cases, the secretomes were collected after cancer cells were in serum-free media for 24h.
Project description:Wood stiffness is the most important wood quality trait of forest trees for structural timber production. We investigated genes differentially transcribed in radiate pine trees with distinct wood stiffness using bulked segregant analysis (BSA) and cDNA microarrays. Transcript accumulation in earlywood (EW) and latewood (LW) of high (HS) and low stiffness (LS) trees in two progeny trials was compared. Radiata pine trees used for microarray experiment were selected from two progeny trials planted at Flynn and Kromelite, Australia. Based on the IML-based MOE measurement, five families with highest and lowest MOE each were selected from each trial, which represented two segregant populations with contrasting wood stiffness. Two individuals from each selected family were further sampled. Developing xylem tissues of selected trees in Flynn trial were sampled in spring (October) and autumn (April), representing earlywood (EW) and latewood (LW) of juvenile aged trees, respectively. Collection of xylem tissues from Kromelite trial was arranged in summer (late November) when latewood (LW) was formed. The xylem tissues were scraped at breast height with a sharp chisel after the bark was removed. In Flynn trial EW and LW tissues were collected from the same sampled trees on opposite sides of the trunk. Transcript accumulation was compared in trees with highest (HS) and lowest stiffness (LS) using xylem samples from Flynn collected in spring (EW) and autumn (LW), as well as Kromelite in summer (LW), respectively. Bulked segregant analysis (BSA) was used for the experiment design. Total RNA samples extracted from the five trees with HS were pooled at equal amount, and compared to the bulked five individuals with LS. This pooling strategy can partly minimize the genetic variation among different genotypes. Dye swaps were applied in each biological replicate.