Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

A computational method to aid the design and analysis of single cell RNA-seq experiments for cell type identification.

BACKGROUND: The advent of single cell RNA sequencing (scRNA-seq) enabled researchers to study transcriptomic activity within individual cells and identify inherent cell types in the sample. Although numerous computational tools have been developed to analyze single cell transcriptomes, there are no published studies and analytical packages available to guide experimental design and to devise suitable analysis procedure for cell type identification. RESULTS: We have developed an empirical methodology to address this important gap in single cell experimental design and analysis into an easy-to-use tool called SCEED (Single Cell Empirical Experimental Design and analysis). With SCEED, user can choose a variety of combinations of tools for analysis, conduct performance analysis of analytical procedures and choose the best procedure, and estimate sample size (number of cells to be profiled) required for a given analytical procedure at varying levels of cell type rarity and other experimental parameters. Using SCEED, we examined 3 single cell algorithms using 48 simulated single cell datasets that were generated for varying number of cell types and their proportions, number of genes expressed per cell, number of marker genes and their fold change, and number of single cells successfully profiled in the experiment. CONCLUSIONS: Based on our study, we found that when marker genes are expressed at fold change of 4 or more, either Seurat or SIMLR algorithm can be used to analyze single cell dataset for any number of single cells isolated (minimum 1000 single cells were tested). However, when marker genes are expected to be only up to fold change of 2, choice of the single cell algorithm is dependent on the number of single cells isolated and rarity of cell types to be identified. In conclusion, our work allows the assessment of various single cell methods and also aids in the design of single cell experiments.

Initial results of hypoxia imaging using 1-alpha-D: -(5-deoxy-5-[18F]-fluoroarabinofuranosyl)-2-nitroimidazole ( 18F-FAZA).

PURPOSE: Tumour hypoxia is thought to play a significant role in the outcome of solid tumour therapy. Positron emission tomography (PET) is the best-validated noninvasive technique able to demonstrate the presence of hypoxia in vivo. The locally developed PET tracer for imaging hypoxia, 1-alpha-D: -(5-deoxy-5-[(18)F]-fluoroarabinofuranosyl)-2-nitroimidazole ((18)F-FAZA), has been shown to accumulate in experimental models of tumour hypoxia and to clear rapidly from the circulation and nonhypoxic tissues. The safety and general biodistribution patterns of this radiopharmaceutical in patients with squamous cell carcinoma of the head and neck (HNSCC), small-cell lung cancer (SCLC) or non-small-cell lung cancer (NSCLC), malignant lymphoma, and high-grade gliomas, were demonstrated in this study. METHODS: Patients with known primary or suspected metastatic HNSCC, SCLC or NSCLC, malignant lymphoma or high-grade gliomas were dosed with 5.2 MBq/kg of (18)F-FAZA, then scanned 2-3 h after injection using a PET or PET/CT scanner. Images were interpreted by three experienced nuclear medicine physicians. The location and relative uptake scores (graded 0 to 4) of normal and abnormal (18)F-FAZA biodistribution patterns, the calculated tumour-to-background (T/B) ratio, and the maximum standardized uptake value were recorded. RESULTS: Included in the study were 50 patients (32 men, 18 women). All seven patients with high-grade gliomas showed very high uptake of (18)F-FAZA in the primary tumour. In six out of nine patients with HNSCC, clear uptake of (18)F-FAZA was observed in the primary tumour and/or the lymph nodes in the neck. Of the 21 lymphoma patients (15 with non-Hodgkin's lymphoma and 6 with Hodgkin's disease), 3 demonstrated moderate lymphoma-related uptake. Of the 13 lung cancer patients (12 NSCLC, 1 SCLC), 7 had increased (18)F-FAZA uptake in the primary lung tumour. No side effects of the administration of (18)F-FAZA were observed. CONCLUSION: This study suggests that (18)F-FAZA may be a very useful radiopharmaceutical to image hypoxia in the tumour types selected. Especially the high uptake by gliomas was encouraging. Given the good imaging properties, including acceptable T/B ratios in the tumour categories studied, (18)F-FAZA could be considered as a very promising agent for assessing the hypoxic fraction of these tumour types.

Synthesis and evaluation of novel 4-amino-4,6-androstadiene-3,17-dione: an analog of formestane.

Synthesis of 4-amino-4,6-androstadiene-3,17-dione 7, an analog of formestane used in breast cancer therapy as an aromatase inhibitor, from 4-acetoxy-4-androstene-3,17-dione 2 is described. This is the first report of a 4-amino diene (4,6) system in this series of molecules. The new (7) and reported molecules were screened by the National Cancer Institute (NCI, Bethesda, USA) for in vitro antitumor activity against 60 human cancer cell lines. Molecule 7 showed best activity against breast cancer cell line (MCF-7).

Optimization of a two-step desolvation method for preparing gelatin nanoparticles and cell uptake studies in 143B osteosarcoma cancer cells.

PURPOSE: To establish a matrix of parameters to synthesize nanoparticles of different sizes and to investigate the cellular uptake of these nanoparticles by osteosarcoma cancer cells in order to investigate their potential as therapeutic drugdelivery carriers. METHODS: Gelatin A and B were used to synthesize nanoparticles by a two-step desolvation process. Different parameters were investigated, including temperature, pH, concentration of glutaraldehyde, type of desolvating agent and nature of gelatin. For cell uptake studies, Texas Red labeled nanoparticles were incubated with 143B osteosarcoma cells and then evaluated using confocal laser scanning microscopy (CLSM). RESULTS: The systematic investigation of the synthesis parameters showed that it is possible to prepare gelatin-based nanoparticles with different particle sizes and a narrow size distribution. Temperature and nature of the gelatin were the most important synthesis factors. Bioimaging using CLSM showed uptake of the nanoparticles by 143B osteosarcoma cancer cells. CONCLUSIONS: Osteosarcoma cancer cells take up gelatin nanoparticles. This might improve the clinical effectiveness of anti-cancer treatments if nanoparticles are used as a drug delivery system and has important implications for future cancer treatment strategies.

Synthesis and biodistribution of 8-iodo-11-(4-methylpiperazino)-5H-dibenzo[b,e][1,4]-diazepine: Iozapine.

8-Iodo-11-(4-methylpiperazino)-5H-dibenzo[b,e][1,4]-diazepine: Iozapine, a potential D(4)-receptor ligand was synthesized using oxidative iodo-destannylation reaction. The preliminary biodistribution studies of radioiodinated iozapine have shown that the compound is taken up in the brains of mice and rabbits.