Breaking the metastasis cycle: Is ROS-activated β-globin expression required for CTC survival?

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Reactive oxygen species (ROS) are chemical molecules containing a highly reactive oxygen molecule. These molecules are formed as by-products of cellular metabolism, but also serve intracellularly as signaling molecules. ROS examples include peroxides, superoxide, hydroxyl radical and singlet oxygen. When present within cells in significant amounts, ROS can be indicators of oxidative cellular stress, and their persistence at elevated levels can result in cellular damage and potentially cell death.

Cancer exists in three developmental stages. First, as a primary tumor, second as circulating tumor cells (CTCs) and third, as secondary tumors (metastasis). The ability to inhibit or destroy either primary tumors or CTCs would provide an effective control or possible cure for cancer. A recent paper by Zheng, Y. et al. (2017) suggests ROS may play a signaling role for up-regulation of β-globin (HBB) in circulating tumor cells (CTCs), which results in initiation of unique anti-apoptotic properties that increase CTC survival in the circulatory system (1). CTCs are a transient, but crucial intermediary state in the development of cancer metastasis. Originating from primary tumor cells, these cells undergo epithelial-to-mesenchymal transition (EMT) during which time they experience differential gene expression and develop alternative phenotypes. Such changes result in the loss of matrix adhesion as well as acquisition of stem cell-like cellular plasticity properties that contribute to their formation. As CTCs become independent of the parent tumor, they can migrate into the bloodstream and travel to distant locations in the body, with the potential to form secondary tumors.

Within the bloodstream, CTCs are subjected to multiple stress types that differ from the protective nurturing environment of the primary tumor. As single cells, CTCs experience stress from the loss of cell adhesion, changing nutritional environments, and sheer stress from circulation. ROS production is associated with the above conditions and can result in cellular damage and death.

The Zheng et al. study examined the effect of oxidative stress on survivability of CTCs in the bloodstream by isolating breast, prostate, and lung cancer single CTCs and analyzing their individual RNA-seq profiles. Unexpectedly, their findings revealed significant overexpression of β-globin (HBB) in individual CTCs as compared to their primary tumors of origin, and this was without an observed corresponding α-globin (HBA) induction. HHB induction was also observed when H727 (lung carcinoid) and MGH134 (NSCLC) cell cultures, which grow under adherent conditions and have low levels of endogenous HBB expression, were either treated directly with H2O2 (a known ROS) or through induced loss of adherence to the matrix, an action which is known to trigger increased intracellular ROS. Live cell staining of CTCs and blood leucocytes using H2DCFDA and MitoSOX red dyes confirmed accumulated ROS in CTCs only, suggesting β-globin induction in CTCs may be associated with oxidative stress. Transcription factor screening and chromatin-IP studies led to the identification of KLF4 as a possible HHB transcription regulator candidate, which was supported by rapid binding of KLF4 to the HBB promoter. The influence of β-globin on CTC survivability was demonstrated by knockdown experiments of endogenous HBB in cultured breast cancer CTCs, which resulted in reduced proliferation rates in anchorage-independent suspension culture. CTCs could be rescued and survival increased, however, if pre-treated with the anti-oxidant NAC. Finally, using a mouse prostate cancer model, single cell transcriptome profile analyses of all three cancer stages, primary tumor cells, CTCs, and secondary tumor cells, confirmed low HBB in primary tumor cells, substantial HBB induction in CTCs and reduced HBB expression in secondary tumor cells. Thus, this study demonstrated that CTC expression profiles are unique from those of their attached primary tumor of origin and secondary tumors to which they give rise. In particular, high HHB expression levels were driven by ROS signaling activation of anti-apoptotic pathways, resulting in increased survivability of this cancer stage. It should also be noted that Zheng et al. additionally performed tail vein injection of breast CTCs (BRx50 and HBB-depleted BRx50 cultures) into immunosuppressed NSG mice. Multiple lung metastases developed in mice injected with BRx50 CTCs, but mice injected with HBB-depleted CTCs demonstrated significantly impaired potential to generate metastatic lesions.

The key to curing cancer may reside in the ability to break the cycle between primary and secondary tumor formation through prevention of CTC dissemination and survivability in the circulatory system. Research to better understand the uniqueness of individual CTCs requires the development of new technologies that allow both concentration of CTCs from blood samples (CTCs constitute an extremely low percentage of total cells in the bloodstream; 1 CTC per 5,000,000 white blood cells) as well as the facilitation of single cell isolation for analysis. A few technologies exist today which address these needs.

One recently developed method for concentrating and selecting single CTCs for study is described in an application note by Kläver, R. et al. (2017) (2). In this study, an alternative application of the AdnaTest ProstateCancerSelect Kit was used for enriching individual prostate cancer LnCAP95 CTCs from blood using the magnetic beads that are coated with highly specific antibodies for prostate cancer cell surface antigens (both epithelial and tumor antigens), provided with the kit. Beads and captured CTCs are then transferred to a microraft array for isolation of CTCs using the QIAscout instrument. The QIAscout instrument fits onto the objective of an inverted microscope to provide easy visual microscopic single cell isolation of various cell types, including adherent, suspension and fluorescent cells as well asprimary cells and cell lines. The associated 12,000 microraft array supports a suitable environment for single cell viability and growth. AdnaTest magnetic beads andattached prostate cancer CTCs were transferred to the QIAscout array which had been pretreated with the adhesive coating CellTak (Corning) and contained 2 ml cell culture medium. After 4 hours, microrafts were then screened for the presence of settled CTCs. The microrafts containing individual CTCs were dislodged by the instrument’s release needle and were transferred to reaction tubes using a magnetic wand, without cross-contamination, for subsequent mRNA processing and PCR analysis. Learn more about AdnaTest ProstateCancerSelect and QIAscout.

In summary, the mechanisms of cancer metastasis are likely much more intricate than we’ve imagined. Continued single cell analysis of CTCs will contribute significantly to a better understanding of CTC cellular regulation and their role in cancer metastasis.

 

References

  1. 1. Zheng, Y. et al. (2017) Expression of β-globin by cancer cells promotes cell survival during blood-borne dissemination. Nat. Commun.8, doi: 10.1038/ncomms14344. Link

2. Kläver, R. et al. (2017) QIAscout CTC single cell isolation after AdnaTest CancerSelect pre-enrichment. QIAGEN Application Note 1106792, 1–6. Link

 

 

 

 

Joby Chesnick

Dr. Joby Chesnick is a Senior Global Marketing Manager in Demand Generation at QIAGEN. She received her Ph.D. in Biology, with doctoral research specializing in unicellular algal symbioses at Texas A&M University. Afterwards, she was awarded an NSF Post-Doctoral Fellowship in Plant Biology and investigated organelle acquisition by primitive eukaryotes at the University of Washington in Seattle. In 2007, Joby additionally received her M.B.A. from the University of Wisconsin-Madison. Her work experience spans 18 years in the biotech industry, and includes positions in bioinformatics support, technical support, intellectual property, product management, and sales prior to joining QIAGEN in January 2017.

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