Biomimetic Isolation of Highly Tumorigenic and Metastatic Circulating Tumor Cells

Introduction:: Metastasis is the most lethal and advanced stage of cancer¹. Metastatic tumors may result from circulating tumor cells (CTCs). During the metastatic cascade, most CTCs die due to anoikis, shear stress, and immune surveillance, whereas only a small fraction of CTCs can survive, settle in a distant site, and initiate metastasis². These robust CTCs possess stem cell-like characteristics to initiate tumors in distant organs³. Thus, it is vital to isolate the stem cell-like CTCs to characterize these cells and screen strategies for the prevention of metastasis. Conventional isolation methods for CTCs are surface biomarker-based, however, there are no definitive markers for all heterogeneous stem cell-like CTCs⁴. Moreover, no studies have been reported that demonstrate the isolation of tumorigenic and metastatic stem-like CTCs. Here, we advanced a bioinspired “one cell in one core-shell microcapsule” (1C1C) culture⁵ to isolate highly tumorigenic and metastatic stem-like CTCs. We hypothesize that non stem cell-like CTCs undergo programmed cell death due to the lack of appropriate extracellular matrix (ECM) and neighboring cells (i.e., anoikis) in 1C1C culture, whereas CTCs with stem cell-like features can survive and proliferate from one cell. The CTC clones isolated with the 1C1C approach show high stemness and tumorigenic/metastatic abilities. This method can be invaluable for the discovery of targeted therapies against metastasis.

Materials and Methods:: Core-shell microcapsules with one patient-derived CTC per microcapsule were generated by using a non-planar microfluidic device, as illustrated in Fig. 1a. One, single CTC is encapsulated in a hyaluronic acid (HA)/alginate hydrogel core with a semipermeable alginate hydrogel shell. After ~21 days of 1C1C culture, the resultant clone-forming cells (CFCs) were stained with putative cancer stem cell markers (CD44, CD133, ALDH1A1, and KLF4). The expressions of these markers and pluripotent markers (NANOG, SOX2, SSEA4, and OCT4) were quantified with flow cytometry. CTCs from conventional adherent culture (2D) and suspension culture (3Dsus) were used as controls in vitro and in vivo. Five hundred cells from each group were injected into the fat pad of NOD-SCID mice to study tumorigenesis. Tumor weight of mice sacrificed on day 120 was measured. For the metastasis study, 10,000 cells were intravenously (i.v.) injected into NOD-SCID mice and lung tissue of the mice sacrificed on day 210 were analyzed for metastatic nodules.


Results, Conclusions, and Discussions:: The biomimetic 1C1C culture is able to isolate and expand highly tumorigenic and metastatic stem cell-like CTCs. The semi-permeable hydrogel can allow one stem cell-like CTC to recreate its own microenvironment within a nanoliter-scaled hydrogel for survival. CTCs from ~1% of one-cell microcapsules can form clones. These cells are defined as clone-forming cells (CFCs). Immunofluorescence images of CFCs display four different expression phenotypes, suggesting that these cells are heterogeneous in the serum-containing culture medium. Moreover, high expression of KLF4 and ALDH1A1 is shown over the entire CFC population. These observations are confirmed by quantitative analysis of the markers with flow cytometry. The CFCs from 1C1C culture show ~1.5-fold (for CD44, CD133, NANOG, and SOX2), 2-fold (for SSEA4, and OCT4), 2.8-fold (for ALDH1A1), and 8-fold (for KLF4) higher expression than cells from the 2D control group (Fig. 1b). In vivo studies demonstrate that 1C1C clone cells have much higher tumorigenic and metastatic abilities than cells from the two conventional cultures (Fig. 1c). Collectively, the 1C1C culture is able to isolate stem cell-like CTCs with high tumorigenic and metastatic potential. It may be invaluable for screening therapies against the highly tumorigenic and metastatic CFCs for combating cancer metastasis, the major cause of cancer-related mortality.


Acknowledgements (Optional): : This research was supported by the Intramural Research Program of the National Institutes of Health, the National Cancer Institute.

References (Optional): : ¹Gerstberger et al. (2023). Cell 186, 1564-1579. ²Micalizzi et al. (2017). Genes Dev 31, 1827-1840. ³Oskarsson et al. (2014). Cell Stem Cell 14, 306-32. ⁴Woo et al. (2018). Transl Res 201, 128-135. ⁵Wang et al. (2020). Adv Sci 7, 2000259.