Tracking CSCs

Cancer immunology. Bench to bedside immunotherapy of cancersNima Rezaei (Ed.). Springer-Verlag (2015)

Advances in imaging technology could allow to track the subpopulation of CSCs in a noninvasive manner in order to monitor migration, engraftment, and morphological differentiation and to assess their response to treatment.

Cell tracking can be performed with two molecular imaging approaches:

  1. Direct stem cell labeling by tracers, such as magnetic particles, luminescent nanoparticles, or radionuclides to directly mark cells [244].
  2. Indirect labeling by reporter-gene imaging: cells are transfected with a reporter gene that encodes for molecules that can be detected by imaging after administration of a reporter probe [245].

Both techniques can be applied to integrate existing radiodiagnostics, in order to implement their ability to provide functional information about biodistribution and activity of CSCs. Initially intended for cancer research, these tools have been most extensively tested in baseline research on human physiological stem cells and immune cells, and their use in CSCs tracking is still under investigation at an initial stage.

Bioluminescence imaging (BLI) requires incorporation of a reporter gene, such as ?re?y luciferase (Fluc), in the stem cells: light emission is triggered by interaction with an intravenously administered optical probe, d-luciferin. In a recent experience, breast CSCs were labeled through the expression of optical bifusion reporter genes, to facilitate their visualization in a human xenograft model on NOD/SCID mice [231]. Ethical issues concerning genetic manipulation in vivo represent a severe limitation to this technique.

Fluorescence imaging employs administration of organic fluorophores (such as fluorescein or rhodamine) that emit specific wavelengths following exposition to visible light; another valuable approach that resulted in improved stability of the compound is indirect labeling with genetically encoded fluorescent proteins. Such colorcoding of cancer cells growing in vivo could allow the monitoring of cell–stroma interactions, subcellular processes, and distinction of different cell types with single-cell resolution [246]. Wholebody imaging with fluorescent proteins could represent a powerful technology to follow the dynamics of cancer development and metastasis [247], but low resolution and technical limitations linked to light penetration in depth call for further advancement in technology, like fluorescencemediated molecular tomography [248].

Quantum dots (QDs) are inorganic fluorescent semiconductor nanoparticles with superior optical properties as compared with organic dyes: QDs have been used to study extravasation of intravenously injected, QD-labeled tumor cells in preclinical research [249]. It is still controversial whether their use could affect CD133 expression [250].

In order to increase accuracy, superparamagnetic iron oxide (SPIO, 50–500 nm) nanoparticles, ultrasmall superparamagnetic iron oxide nanoparticles (USPIO, 5 nm) [251], and manganese oxide (MnO) nanoparticles [252] have been tested as contrast agents for MRI [253]. Gadolinium–rhodamine nanoparticles, which provide a stronger positive signal, have been also tested for labeling and tracking cancer cells in vivo in rodents [254].

However, metal nanoparticles are not able to discriminate viable cells. This limitation can be overcome by radionuclides, such as fluorine-19 [255]: it was found that CD34+CD133+CD31+ stem/progenitor cells readily internalized these agent nanoparticles, without the aid of adjunctive labeling techniques, and remained functional in vivo.

Radionuclide imaging can follow the distribution and concentration of radioactive-labeled molecular tracers introduced into a subject. There are two main modalities for radionuclide imaging: positron emission tomography (PET) and single photon emission computed tomography (SPECT). SPECT tracers directly emit a gamma ray in one direction, while PET tracers send two gamma rays in opposite directions, providing higher spatial resolution. Longitudinal tracking is dependent on the specific half-life of decay of the chosen isotope. Disadvantages include leakage of radiotracers from labeled cells and nonspecific uptake by normal tissues. Targeting stem cell surface markers with radiolabeled antibodies could provide information; longitudinal tracking could be performed with appropriate choice of radioisotope, according to its half-life of decay. For example, 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64CuATSM), a PET imaging agent, selectively accumulated in regions of CD133+ high expression in a preclinical model [256].

MicroCT is similar to the conventional CT systems but is capable of achieving a spatial resolution about three orders of magnitude lower (0.3 µm) [257]. Radio-opaque contrast agents, like gold nanoparticles attached to specific ligands, could be useful to target cell population, in order to acquire information on cell topography and behavior [258].

The imaging modalities reviewed in this chapter are characterized by different sensitivity, tissue penetration, and spatial resolution: integration of multiple diagnostic tools in a single imaging session would allow to combine the advantages of each technique.

In summary, CSC-based clinical imaging is a promising goal in the improvement of diagnostic and prognostic tools in cancer therapy. However, current developments do not allow immediate application in the clinical setting; moreover, it is debated whether large-scale use of such techniques would raise ethical issues related to genetic manipulation in patients.