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Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer.
No full textPatient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer
Validated machine learning tools to distinguish immune checkpoint inhibitor, radiotherapy, COVID-19 and other infective pneumonitis.
Get PDFBACKGROUND: Pneumonitis is a well-described, potentially disabling, or fatal adverse effect associated with both immune checkpoint inhibitors (ICI) and thoracic radiotherapy. Accurate differentiation between checkpoint inhibitor pneumonitis (CIP) radiation pneumonitis (RP), and infective pneumonitis (IP) is crucial for swift, appropriate, and tailored management to achieve optimal patient outcomes. However, correct diagnosis is often challenging, owing to overlapping clinical presentations and radiological patterns. METHODS: In this multi-centre study of 455 patients, we used machine learning with radiomic features extracted from chest CT imaging to develop and validate five models to distinguish CIP and RP from COVID-19, non-COVID-19 infective pneumonitis, and each other. Model performance was compared to that of two radiologists. RESULTS: Models to distinguish RP from COVID-19, CIP from COVID-19 and CIP from non-COVID-19 IP out-performed radiologists (test set AUCs of 0.92 vs 0.8 and 0.8; 0.68 vs 0.43 and 0.4; 0.71 vs 0.55 and 0.63 respectively). Models to distinguish RP from non-COVID-19 IP and CIP from RP were not superior to radiologists but demonstrated modest performance, with test set AUCs of 0.81 and 0.8 respectively. The CIP vs RP model performed less well on patients with prior exposure to both ICI and radiotherapy (AUC 0.54), though the radiologists also had difficulty distinguishing this test cohort (AUC values 0.6 and 0.6). CONCLUSION: Our results demonstrate the potential utility of such tools as a second or concurrent reader to support oncologists, radiologists, and chest physicians in cases of diagnostic uncertainty. Further research is required for patients with exposure to both ICI and thoracic radiotherapy
Winners vs Losers: The glutamate-NMDAR signalling axis in the regulation of cell fitness
No full textCell competition is an evolutionary conserved quality control process that eliminates
suboptimal or potentially dangerous cells. For ‘unfit’ cells to be detected, their competitive status
needs to be compared to the collective fitness of cells within the tissue. Here, I demonstrate that the
vesicular glutamate transporter VGlut, and autocrine glutamate signalling, are required for cell
competition and Myc-driven super-competition in epithelia. I find that autocrine secretion of glutamate
stimulates NMDAR-mediated activation of the CaMKII, PKA and CrebB signalling axis, thereby
suppressing loser status and cell death under competitive settings. Accordingly, upon clonal loss of
autocrine glutamate signalling, cells acquire loser status via the induction of TNF. This in turn drives
autocrine TNFR-mediated activation of JNK that triggers loser cell elimination. Inhibiting caspases or
preventing loser cells from transferring lactate to their neighbours removes the fitness disparity and
nullifies cell competition. Further, in a Drosophila model for premalignant cancer, clones that
overexpress the Myc proto-oncogene co-opt the VGlut>extracellular glutamate>NMDAR>CaMKII,
PKA>CrebB signalling circuit to acquire super-competitor status and subdue their wild-type
neighbours. Targeting glutamate signalling converts Myc ‘super-competitor’ clones into ‘losers’,
highlighting new therapeutic opportunities to restrict the evolution of fitter clones
Identification of Genes with Rare Loss of Function Variants Associated with Aggressive Prostate Cancer and Survival.
No full textBACKGROUND: Prostate cancer (PrCa) is a substantial cause of mortality among men globally. Rare germline mutations in BRCA2 have been validated robustly as increasing risk of aggressive forms with a poorer prognosis; however, evidence remains less definitive for other genes. OBJECTIVE: To detect genes associated with PrCa aggressiveness, through a pooled analysis of rare variant sequencing data from six previously reported studies in the UK Genetic Prostate Cancer Study (UKGPCS). DESIGN, SETTING, AND PARTICIPANTS: We accumulated a cohort of 6805 PrCa cases, in which a set of ten candidate genes had been sequenced in all samples. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We examined the association between rare putative loss of function (pLOF) variants in each gene and aggressive classification (defined as any of death from PrCa, metastatic disease, stage T4, or both stage T3 and Gleason score ≥8). Secondary analyses examined staging phenotypes individually. Cox proportional hazards modelling and Kaplan-Meier survival analyses were used to further examine the relationship between mutation status and survival. RESULTS AND LIMITATIONS: We observed associations between PrCa aggressiveness and pLOF mutations in ATM, BRCA2, MSH2, and NBN (odds ratio = 2.67-18.9). These four genes and MLH1 were additionally associated with one or more secondary analysis phenotype. Carriers of germline mutations in these genes experienced shorter PrCa-specific survival (hazard ratio = 2.15, 95% confidence interval 1.79-2.59, p = 4 × 10-16) than noncarriers. CONCLUSIONS: This study provides further support that rare pLOF variants in specific genes are likely to increase aggressive PrCa risk and may help define the panel of informative genes for screening and treatment considerations. PATIENT SUMMARY: By combining data from several previous studies, we have been able to enhance knowledge regarding genes in which inherited mutations would be expected to increase the risk of more aggressive PrCa. This may, in the future, aid in the identification of men at an elevated risk of dying from PrCa
Predictability of B cell clonal persistence and immunosurveillance in breast cancer.
No full textB cells and T cells are important components of the adaptive immune system and mediate anticancer immunity. The T cell landscape in cancer is well characterized, but the contribution of B cells to anticancer immunosurveillance is less well explored. Here we show an integrative analysis of the B cell and T cell receptor repertoire from individuals with metastatic breast cancer and individuals with early breast cancer during neoadjuvant therapy. Using immune receptor, RNA and whole-exome sequencing, we show that both B cell and T cell responses seem to coevolve with the metastatic cancer genomes and mirror tumor mutational and neoantigen architecture. B cell clones associated with metastatic immunosurveillance and temporal persistence were more expanded and distinct from site-specific clones. B cell clonal immunosurveillance and temporal persistence are predictable from the clonal structure, with higher-centrality B cell antigen receptors more likely to be detected across multiple metastases or across time. This predictability was generalizable across other immune-mediated disorders. This work lays a foundation for prioritizing antibody sequences for therapeutic targeting in cancer
A RIPK1-specific PROTAC degrader achieves potent antitumor activity by enhancing immunogenic cell death.
No full textReceptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies
A comparative analysis of risk factor associations with interval and screen-detected breast cancers: A large UK prospective study.
No full textThe associations of certain factors, such as age and menopausal hormone therapy, with breast cancer risk are known to differ for interval and screen-detected cancers. However, the extent to which associations of other established breast cancer risk factors differ by mode of detection is unclear. We investigated associations of a wide range of risk factors using data from a large UK cohort with linkage to the National Health Service Breast Screening Programme, cancer registration, and other health records. We used Cox regression to estimate adjusted relative risks (RRs) and 95% confidence intervals (CIs) for associations between risk factors and breast cancer risk. A total of 9421 screen-detected and 5166 interval cancers were diagnosed in 517,555 women who were followed for an average of 9.72 years. We observed the following differences in risk factor associations by mode of detection: greater body mass index (BMI) was associated with a smaller increased risk of interval (RR per 5 unit increase 1.07, 95% CI 1.03-1.11) than screen-detected cancer (RR 1.27, 1.23-1.30); having a first-degree family history was associated with a greater increased risk of interval (RR 1.81, 1.68-1.95) than screen-detected cancer (RR 1.52, 1.43-1.61); and having had previous breast surgery was associated with a greater increased risk of interval (RR 1.85, 1.72-1.99) than screen-detected cancer (RR 1.34, 1.26-1.42). As these differences in associations were relatively unchanged after adjustment for tumour grade, and are in line with the effects of these factors on mammographic density, they are likely to reflect the effects of these risk factors on screening sensitivity
Whole genome sequencing refines stratification and therapy of patients with clear cell renal cell carcinoma.
No full textClear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer, but a comprehensive description of its genomic landscape is lacking. We report the whole genome sequencing of 778 ccRCC patients enrolled in the 100,000 Genomes Project, providing for a detailed description of the somatic mutational landscape of ccRCC. We identify candidate driver genes, which as well as emphasising the major role of epigenetic regulation in ccRCC highlight additional biological pathways extending opportunities for therapeutic interventions. Genomic characterisation identified patients with divergent clinical outcome; higher number of structural copy number alterations associated with poorer prognosis, whereas VHL mutations were independently associated with a better prognosis. The observations that higher T-cell infiltration is associated with better overall survival and that genetically predicted immune evasion is not common supports the rationale for immunotherapy. These findings should inform personalised surveillance and treatment strategies for ccRCC patients
The Emerging Role of LPA as an Oncometabolite.
No full textLysophosphatidic acid (LPA) is a phospholipid that displays potent signalling activities that are regulated in both an autocrine and paracrine manner. It can be found both extra- and intracellularly, where it interacts with different receptors to activate signalling pathways that regulate a plethora of cellular processes, including mitosis, proliferation and migration. LPA metabolism is complex, and its biosynthesis and catabolism are under tight control to ensure proper LPA levels in the body. In cancer patient specimens, LPA levels are frequently higher compared to those of healthy individuals and often correlate with poor responses and more aggressive disease. Accordingly, LPA, through promoting cancer cell migration and invasion, enhances the metastasis and dissemination of tumour cells. In this review, we summarise the role of LPA in the regulation of critical aspects of tumour biology and further discuss the available pre-clinical and clinical evidence regarding the feasibility and efficacy of targeting LPA metabolism for effective anticancer therapy
Observational and genetic associations between cardiorespiratory fitness and cancer: a UK Biobank and international consortia study
No full textAbstract
Background
The association of fitness with cancer risk is not clear.
Methods
We used Cox proportional hazards models to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for risk of lung, colorectal, endometrial, breast, and prostate cancer in a subset of UK Biobank participants who completed a submaximal fitness test in 2009-12 (
N
= 72,572). We also investigated relationships using two-sample Mendelian randomisation (MR), odds ratios (ORs) were estimated using the inverse-variance weighted method.
Results
After a median of 11 years of follow-up, 4290 cancers of interest were diagnosed. A 3.5 ml O
2
⋅min
−1
⋅kg
−1
total-body mass increase in fitness (equivalent to 1 metabolic equivalent of task (MET), approximately 0.5 standard deviation (SD)) was associated with lower risks of endometrial (HR = 0.81, 95% CI: 0.73–0.89), colorectal (0.94, 0.90–0.99), and breast cancer (0.96, 0.92–0.99). In MR analyses, a 0.5 SD increase in genetically predicted O
2
⋅min
−1
⋅kg
−1
fat-free mass was associated with a lower risk of breast cancer (OR = 0.92, 95% CI: 0.86–0.98). After adjusting for adiposity, both the observational and genetic associations were attenuated.
Discussion
Higher fitness levels may reduce risks of endometrial, colorectal, and breast cancer, though relationships with adiposity are complex and may mediate these relationships. Increasing fitness, including via changes in body composition, may be an effective strategy for cancer prevention.
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