Orit Rozenblatt-Rosen, Aviv Regev, Philipp Oberdoerffer, Tal Nawy, Anna Hupalowska, Jennifer E Rood, Orr Ashenberg, Ethan Cerami, Robert J Coffey, Emek Demir, Li Ding, Edward D Esplin, James M Ford, Jeremy Goecks, Sharmistha Ghosh, Joe W Gray, Justin Guinney, Sean E Hanlon, Shannon K Hughes, Shelley E Hwang, Christine A Iacobuzio-Donahue, Judit Jané-Valbuena, Bruce E Johnson, Ken S Lau, Tracy Lively, Sarah A Mazzilli, Dana Pe'er, Sandro Santagata, Alex K Shalek, Denis Schapiro, Michael P Snyder, Peter K Sorger, Avrum E Spira, Sudhir Srivastava, Kai Tan, Robert B West, and Elizabeth H Williams. 2020. “The Human Tumor Atlas Network: Charting Tumor Transitions across Space and Time at Single-Cell Resolution.” Cell, 181, 2, Pp. 236-249.Abstract
Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.
Dale A Sandercock, Mark W Barnett, Jennifer E Coe, Alison C Downing, Ajit J Nirmal, Pierpaolo Di Giminiani, Sandra A Edwards, and Tom C Freeman. 2019. “Transcriptomics Analysis of Porcine Caudal Dorsal Root Ganglia in Tail Amputated Pigs Shows Long-Term Effects on Many Pain-Associated Genes.” Front Vet Sci, 6, Pp. 314.Abstract
Tail amputation by tail docking or as an extreme consequence of tail biting in commercial pig production potentially has serious implications for animal welfare. Tail amputation causes peripheral nerve injury that might be associated with lasting chronic pain. The aim of this study was to investigate the short- and long-term effects of tail amputation in pigs on caudal DRG gene expression at different stages of development, particularly in relation to genes associated with nociception and pain. Microarrays were used to analyse whole DRG transcriptomes from tail amputated and sham-treated pigs 1, 8, and 16 weeks following tail treatment at either 3 or 63 days of age (8 pigs/treatment/age/time after treatment; = 96). Tail amputation induced marked changes in gene expression (up and down) compared to sham-treated intact controls for all treatment ages and time points after tail treatment. Sustained changes in gene expression in tail amputated pigs were still evident 4 months after tail injury. Gene correlation network analysis revealed two co-expression clusters associated with amputation: Cluster A (759 down-regulated) and Cluster B (273 up-regulated) genes. Gene ontology (GO) enrichment analysis identified 124 genes in Cluster A and 61 genes in Cluster B associated with both "inflammatory pain" and "neuropathic pain." In Cluster A, gene family members of ion channels e.g., voltage-gated potassium channels (VGPC) and receptors e.g., GABA receptors, were significantly down-regulated compared to shams, both of which are linked to increased peripheral nerve excitability after axotomy. Up-regulated gene families in Cluster B were linked to transcriptional regulation, inflammation, tissue remodeling, and regulatory neuropeptide activity. These findings, demonstrate that tail amputation causes sustained transcriptomic expression changes in caudal DRG cells involved in inflammatory and neuropathic pain pathways.
Sze Sing Lee, Jaichandran Sivalingam, Ajit J Nirmal, Wai Har Ng, Irene Kee, In Chin Song, Chin Yong Kiong, Kristoffer A Gales, Frederic Chua, Edgar M Pena, Bryan E Ogden, and Oi Lian Kon. 2018. “Durable engraftment of genetically modified FVIII-secreting autologous bone marrow stromal cells in the intramedullary microenvironment.” J Cell Mol Med, 22, 7, Pp. 3698-3702.Abstract
Genetically modified FVIII-expressing autologous bone marrow-derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture-expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could support durable engraftment of FVIII-secreting BMSC in vivo. A zinc finger nuclease integrated human FVIII transgene into PPP1R12C (intron 1) of culture-expanded primary canine BMSCs. FVIII-secretory capacity of implanted BMSCs in each dog was expressed as an individualized therapy index (number of viable BMSCs implanted × FVIII activity secreted/million BMSCs/24 hours). Plasma samples before and after implantation were assayed for transgenic FVIII protein using an anti-human FVIII antibody having negligible cross-reactivity with canine FVIII. Plasma transgenic FVIII persisted for at least 48 weeks after implantation in the intramedullary cavity. Transgenic FVIII protein levels were low after intramuscular implantation and undetectable after both intravenous infusion and subcutaneous implantation. All plasma samples were negative for anti-human FVIII antibodies. Plasma concentrations and durability of transgenic FVIII secretion showed no correlation with the therapy index. Thus, the implantation site microenvironment is crucial. The intramedullary microenvironment, but not extramedullary tissues, supported durable engraftment of genetically modified autologous FVIII-secreting BMSCs.
Ajit J Nirmal, Tim Regan, Barbara B Shih, David A Hume, Andrew H Sims, and Tom C Freeman. 2018. “Immune Cell Gene Signatures for Profiling the Microenvironment of Solid Tumors.” Cancer Immunol Res, 6, 11, Pp. 1388-1400.Abstract
The immune composition of the tumor microenvironment regulates processes including angiogenesis, metastasis, and the response to drugs or immunotherapy. To facilitate the characterization of the immune component of tumors from transcriptomics data, a number of immune cell transcriptome signatures have been reported that are made up of lists of marker genes indicative of the presence a given immune cell population. The majority of these gene signatures have been defined through analysis of isolated blood cells. However, blood cells do not reflect the differentiation or activation state of similar cells within tissues, including tumors, and consequently markers derived from blood cells do not necessarily transfer well to tissues. To address this issue, we generated a set of immune gene signatures derived directly from tissue transcriptomics data using a network-based deconvolution approach. We define markers for seven immune cell types, collectively named , and demonstrate how these markers can be used for the quantitative estimation of the immune cell content of tumor and nontumor tissue samples. The utility of is demonstrated through the stratification of melanoma patients into subgroups of prognostic significance and the identification of immune cells with the use of single-cell RNA-sequencing data derived from tumors. Use of is facilitated by an R package (imsig). .
Clare Pridans, Kristin A Sauter, Katharine M Irvine, Gemma M Davis, Lucas Lefevre, Anna Raper, Rocio Rojo, Ajit J Nirmal, Philippa Beard, Michael Cheeseman, and David A Hume. 2018. “Macrophage colony-stimulating factor increases hepatic macrophage content, liver growth, and lipid accumulation in neonatal rats.” Am J Physiol Gastrointest Liver Physiol, 314, 3, Pp. G388-G398.Abstract
Signaling via the colony-stimulating factor 1 receptor (CSF1R) controls the survival, differentiation, and proliferation of macrophages. Mutations in CSF1 or CSF1R in mice and rats have pleiotropic effects on postnatal somatic growth. We tested the possible application of pig CSF1-Fc fusion protein as a therapy for low birth weight (LBW) at term, using a model based on maternal dexamethasone treatment in rats. Neonatal CSF1-Fc treatment did not alter somatic growth and did not increase the blood monocyte count. Instead, there was a substantial increase in the size of liver in both control and LBW rats, and the treatment greatly exacerbated lipid droplet accumulation seen in the dexamethasone LBW model. These effects were reversed upon cessation of treatment. Transcriptional profiling of the livers supported histochemical evidence of a large increase in macrophages with a resident Kupffer cell phenotype and revealed increased expression of many genes implicated in lipid droplet formation. There was no further increase in hepatocyte proliferation over the already high rates in neonatal liver. In conclusion, treatment of neonatal rats with CSF1-Fc caused an increase in liver size and hepatic lipid accumulation, due to Kupffer cell expansion and/or activation rather than hepatocyte proliferation. Increased liver macrophage numbers and expression of endocytic receptors could mitigate defective clearance functions in neonates. NEW & NOTEWORTHY This study is based on extensive studies in mice and pigs of the role of CSF1/CSF1R in macrophage development and postnatal growth. We extended the study to neonatal rats as a possible therapy for low birth weight. Unlike our previous studies in mice and pigs, there was no increase in hepatocyte proliferation and no increase in monocyte numbers. Instead, neonatal rats treated with CSF1 displayed reversible hepatic steatosis and Kupffer cell expansion.
Barbara B Shih, Ajit J Nirmal, Denis J Headon, Arne N Akbar, Neil A Mabbott, and Tom C Freeman. 2017. “Derivation of marker gene signatures from human skin and their use in the interpretation of the transcriptional changes associated with dermatological disorders.” J Pathol, 241, 5, Pp. 600-613.Abstract
Numerous studies have explored the altered transcriptional landscape associated with skin diseases to understand the nature of these disorders. However, data interpretation represents a significant challenge due to a lack of good maker sets for many of the specialized cell types that make up this tissue, whose composition may fundamentally alter during disease. Here we have sought to derive expression signatures that define the various cell types and structures that make up human skin, and demonstrate how they can be used to aid the interpretation of transcriptomic data derived from this organ. Two large normal skin transcriptomic datasets were identified, one RNA-seq (n = 578), the other microarray (n = 165), quality controlled and subjected separately to network-based analyses to identify clusters of robustly co-expressed genes. The biological significance of these clusters was then assigned using a combination of bioinformatics analyses, literature, and expert review. After cross comparison between analyses, 20 gene signatures were defined. These included expression signatures for hair follicles, glands (sebaceous, sweat, apocrine), keratinocytes, melanocytes, endothelia, muscle, adipocytes, immune cells, and a number of pathway systems. Collectively, we have named this resource SkinSig. SkinSig was then used in the analysis of transcriptomic datasets for 18 skin conditions, providing in-context interpretation of these data. For instance, conventional analysis has shown there to be a decrease in keratinization and fatty metabolism with age; we more accurately define these changes to be due to loss of hair follicles and sebaceous glands. SkinSig also highlighted the over-/under-representation of various cell types in skin diseases, reflecting an influx in immune cells in inflammatory disorders and a relative reduction in other cell types. Overall, our analyses demonstrate the value of this new resource in defining the functional profile of skin cell types and appendages, and in improving the interpretation of disease data. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Jaichandran Sivalingam, Dimitar Kenanov, Hao Han, Ajit Johnson Nirmal, Wai Har Ng, Sze Sing Lee, Jeyakumar Masilamani, Toan Thang Phan, Sebastian Maurer-Stroh, and Oi Lian Kon. 2016. “Multidimensional Genome-wide Analyses Show Accurate FVIII Integration by ZFN in Primary Human Cells.” Mol Ther, 24, 3, Pp. 607-19.Abstract
Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. We also showed AAVS1 ZFN activity and durable FVIII transgene secretion in primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. Our study suggests that, with close attention to the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious.
Anand Krishnan, Vivek R Gopinath, Ajit Johnson, Asha S Nair, and Radhakrishna M Pillai. 2013. “Cell-cycle analysis and micronuclei frequency reveals G0/G1 blockers as weak micronuclei inducers.” Drug Chem Toxicol, 36, 2, Pp. 249-54.Abstract
Micronuclei (MN) formation is generally attributed to error in DNA synthesis or mitosis, which are represented by the S or G(2)/M phase respectively, in the cell-cycle histogram. Interestingly, many of the known anticancer drugs target these cell-cycle phases to elicit cytotoxicity. Here, we attempted to identify whether any correlation exists between the cell-cycle effect and MN induction potential using various treatments. In addition, we tracked down MN in cycling cells to assess its final fate. We treated SiHa cells with various known drugs and correlated their effects on cell-cycle and MN frequency. MN-tracking studies were performed in peripheral mononuclear and siHa cells upon staining with Giemsa and ethidium bromide respectively. We observed MN induction by all the tested drugs irrespective of their basic effect on cell cycle. However, MN induction was more with drugs which interfere with the S or G(2)/M than the G(0)/G(1) phase. Our results indicate G(0)/G(1) blockers to be comparatively safer drugs. Additionally, our results show that expulsion out of cells may be one of the main fates of drug-induced MN.
Aleksandra Bartnik, Ajit Johnson Nirmal, and Shi-Yu Yang. 2012. “Peptide Vaccine Therapy in Colorectal Cancer.” Vaccines (Basel), 1, 1, Pp. 1-16.Abstract
Colorectal cancer is the third most common cause of cancer-related deaths and the second most prevalent (after breast cancer) in the western world. High metastatic relapse rates and severe side effects associated with the adjuvant treatment have urged oncologists and clinicians to find a novel, less toxic therapeutic strategy. Considering the limited success of the past clinical trials involving peptide vaccine therapy to treat colorectal cancer, it is necessary to revise our knowledge of the immune system and its potential use in tackling cancer. This review presents the efforts of the scientific community in the development of peptide vaccine therapy for colorectal cancer. We review recent clinical trials and the strategies for immunologic monitoring of responses to peptide vaccine therapy. We also discuss the mechanisms underlying the therapy and potential molecular targets in colon cancer.