Sequencing cancer

Cancer forms highly heterogeneous tissues at several molecular levels, genomic, proteomic, transcriptomic and other epigenetic traits. The level of complexity is further augmented by the dynamic nature of tumor progression with cancer cell populations evolving in a clonal manner. The clonal evolution of tumors is shaped by selective pressure exerted by endogenous factors such as intra tumor dynamics between different clones and exogenous factors such as microenvironment and therapeutics. The technical advances in next generation sequencing has accelerated and facilitated a massive acquisition of genomic and transcriptional data from different cancers during the last decade. Despite the increased knowledge in the transcriptional and genomic landscape of tumors, many questions have still not been fully addressed and one of the explanations lies in the heterogenetic nature of cancer tissues that has to be desiccated into its fundamental parts - the single cancer cells.

In this thesis the heterogenetic nature of normal- and cancer tissues and the implications to single-cell based methods are discussed. In order to study the transcriptome of single-cells delicate molecular tools needs to be developed. In the two first papers we describe a single-cell RNA sequencing method that is highly sensitive and can produce full gene expression profiles of hundreds of single-cells per sequencing experiment.

Several models have been proposed for tumor evolution and one fundamental question is the hierarchical organization of tumor propagating cells. In paper III we studied the tumor progression in myelodysplastic syndromes (MDS), a clonal hematological disorder in which multiple haematological lineages are affected. We found that the putative MDS-stem cell (SC) population is functionally and molecularly distinct from its downstream progenitors and that in MDS, self-renewal and that acquisition of somatic mutations was restricted to the MDS-stem cell population. By targeted sequencing of purified populations and single-cell derived stem cell clones we could track all somatic mutations found in the bone marrow of MDS patients to the distinct MDS-stem cells providing definitive evidence for the existence of rare human MDSSCs in vivo.

Another aspect of heterogeneity in solid tumors is the genetic heterogeneity between primary tumor and the metastatic lesions. In paper IV we addressed the heterogeneity in metastatic breast cancer by comparing the genetic profiles of ten pairs of primary tumor to their metastatic lesion obtained from exome sequencing. We found a marked heterogeneity in number of somatic mutations as well as the extent of chromosomal aberrations in the metastatic lesions. We also found a number of mutated genes to be significantly enriched in the metastases. The clinical implications to metastatic heterogeneity is supported by altered receptor status and drug resistance in metastatic lesions and suggest that extended characterization of the metastases is of great importance.

In summary, heterogeneity is the main characteristic of both normal and cancer tissues. To resolve the mixed patterns of gene regulation and genomic aberrations, single-cell based approaches needs to be applied and will be a powerful tool to shed light on questions in tumor biology such as transcription dynamics and genetic selection.

List of scientific papers

I. Saiful Islam, Una Kjällquist, Annalena Moliner, Pawel Zajac Jian-Bing Fan, Peter Lönnerberg and Sten Linnarsson. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Research. (2011) 21:1160-7.
https://doi.org/10.1101/gr.110882.110

II. Saiful Islam, Una Kjällquist, Annalena Moliner, Pawel Zajac, Jian-Bing Fan, Peter Lönnerberg and Sten Linnarsson. Highly multiplexed and strand-specific single-cell RNA 5' end sequencing. Nature Protocols. (2012) 7:813–28.
https://doi.org/10.1038/nprot.2012.022

III. Petter S. Woll, Una Kjällquist, Onima Chowdhury, Helen Doolittle, David C. Wedge, Supat Thongjuea, Rikard Erlandsson, Mtakai Ngara, Kristina Anderson, Qiaolin Deng, Adam J. Mead, Laura Stenson, Alice Giustacchini, Sara Duarte, Eleni Giannoulatou, Stephen Taylor, Mohsen Karimi, Christian Scharenberg, Teresa Mortera-Blanco, Iain C. Macaulay, Sally-Ann Clark, Ingunn Dybedal, Dag Josefsen, Pierre Fenaux, Peter Hokland, Mette S Holm, Mario Cazzola, Luca Malcovati, Sudhir Tauro, David Bowen, Jacqueline Boultwood, Andrea Pellagatti, John E. Pimanda, Ashwin Unnikrishnan, Paresh Vyas, Gudrun Göhring, Brigitte Schlegelberger, Magnus Tobiasson, Gunnar Kvalheim, Stefan N. Constantinescu, Claus Nerlov, Lars Nilsson, Peter Campbell, Rickard Sandberg, Elli Papaemmanuil, Eva Hellström-Lindberg, Sten Linnarsson, Sten Eirik W. Jacobsen. Myelodysplastic syndromes are propagated by rare and distinct human cancer stem cells in vivo. Cancer Cell. (2014) [Accepted]
https://pubmed.ncbi.nlm.nih.gov/24835589

IV. Una Kjällquist, Nicholas P. Tobin, Rikard Erlandsson, Eva Karlsson, Linda S.Lindström, Siker Kimbung, Anna Danielsson, Ingrid Hedenfalk, Thomas Hatschek, Jonas Bergh and Sten Linnarsson. Heterogeneity and clonal evolution revealed by exome sequencing of primary breast cancers with paired metastatic lesions. [Manuscript]