PLoS One. 2016 Jan 8;11(1):e0146052. doi: 10.1371/journal.pone.0146052.

What Do We Learn from Spheroid Culture Systems? Insights from Tumorspheres Derived from Primary Colon Cancer Tissue

Komal Qureshi-Baig1, Pit Ullmann1, Fabien Rodriguez1, Sónia Frasquilho2, Petr V. Nazarov3, Serge Haan1, Elisabeth Letellier1

1 Molecular Disease Mechanisms Group, Life Sciences Research Unit, University of Luxembourg, 6 Avenue du Swing, L-4367, Campus Belval, Luxembourg, 2 Integrated Biobank of Luxembourg, 6 rue Nicolas Ernest Barblé, L-1210, Luxembourg, Luxembourg, 3 Luxembourg Institute of Health, Genomics Research Unit, 84 Val Fleuri, L-1526, Luxembourg, Luxembourg

Citation: Qureshi-Baig K, Ullmann P, Rodriguez F, Frasquilho S, Nazarov PV, Haan S, et al. (2016) What Do We Learn from Spheroid Culture Systems? Insights from Tumorspheres Derived from Primary Colon Cancer Tissue. PLoS ONE 11(1): e0146052. doi:10.1371/journal.pone.0146052



Due to their self-renewal and tumorigenic properties, tumor-initiating cells (TICs) have been hypothesized to be important targets for colorectal cancer (CRC). However the study of TICs is hampered by the fact that the identification and culturing of TICs is still a subject of extensive debate. Floating three-dimensional spheroid cultures (SC) that grow in serum free medium supplemented with growth factors are supposed to be enriched in TICs. We generated SC from fresh clinical tumor specimens and compared them to SC isolated from CRC cell-lines as well as to adherent differentiated counterparts. Patient-derived SC display self-renewal capacity and can induce serial transplantable tumors in immuno-deficient mice, which phenotypically resemble the tumor of origin. In addition, the original tumor tissue and established SC retain several similar CRC-relevant mutations. Primary SC express key stemness proteins such as SOX2, OCT4, NANOG and LGR5 and importantly show increased chemoresistance ability compared to their adherent differentiated counterparts and to cell line-derived SC. Strikingly, cells derived from spheroid or adherent differentiating culture conditions displayed similar self-renewal capacity and equally formed tumors in immune-deficient mice, suggesting that self-renewal and tumor-initiation capacity of TICs is not restricted to phenotypically immature spheroid cells, which we describe to be highly plastic and able to reacquire stem-cell traits even after long differentiation processes. Finally, we identified two genes among a sphere gene expression signature that predict disease relapse in CRC patients. Here we propose that SC derived from fresh patient tumor tissue present interesting phenotypic features that may have clinical relevance for chemoresistance and disease relapse and therefore represent a valuable tool to test for new CRC therapies that overcome drug resistance.

PMID: 26745821



Colorectal carcinoma (CRC) is the third most frequently diagnosed cancer type for both men and women and the second most common cause of cancer mortality in Western countries [1]. Despite great progress made during the last decades, a lot of controversy still remains over the backgrounds of cancer onset, metastasis and CRC progression. A rare subset of cells, the so-called tumor-initiating cells (TICs) also known as cancer stem cells (CSCs) are thought to be responsible for tumor evolution [2]. TICs have first been described in the frame of hematopoietic malignancies [3]. A few years later, TICs were also identified in a wide array of solid tumors such as, breast, skin, brain, pancreas, lung and colon [3]. Despite significant advances in colon TIC research, the isolation, identification and characterization of colon TICs remains incompletely established.

We wondered if SC derived from freshly resected colon tumor tissue display TIC properties, such as tumor initiation ability in vivo, and if they harbor chemoresistance traits. Interestingly, we observed that primary SC displayed an important tumor-initiation capacity. Mice transplanted with 100 cells of tumor tissue derived from patients T6, T18 and T20 all initiated tumor growth. This result nicely shows the tumorigenic potential of SC derived from primary tumor tissue (Fig 1a). Furthermore, the resulting primary and secondary xenografts resembled the original primary tumor found in the patients (Fig 1b). Thus, we concluded that SC isolated from primary human tumor samples do not only possess tumor-initiating capacity, but they also recapitulate the phenotype of the primary tumor.



In a next step we asked the question how the adherent differentiated counterparts of SC would behave in vivo. Cells derived from SC had a much higher expression of pluripotency markers such as NANOG, SOX2 and OCT4, compared to cells derived from differentiated adherent cultures. We then investigated the tumor-initiation capacity of the adherent differentiated counterparts and compared them to SC by injecting 100 cells of each on both flanks of immunocompromised mice. We followed tumor formation over time and found out that SC and adherent differentiated counterparts display similar tumor-initiation capacity. This finding surprisingly hints towards the fact that tumor-initiating capacity of TICs is not restricted to phenotypically immature spheroid cells (Fig 1c). Regarding this observation, we suggest that our SC are highly plastic and may be influenced by their microenvironment. In this line, our results evoke that SC are able to reacquire stem-cell traits even after long differentiation processes.

Once we had established primary SC and characterized them in regard to TIC properties, we asked ourselves whether these SC displayed chemoresistance abilities. To investigate this hypothesis, we applied 5-FU at various concentrations to SC and compared them to their respective adherent differentiated counterpart. We demonstrated that primary SC displayed higher resistance to 5-FU treatment compared to their differentiated counterparts (Fig 2). The increased sensitivity to 5-FU is consistent with the high proliferation rate of the adherent cultures, as chemotherapeutics are known to mostly target fast-proliferating cells.

Taking into account our findings, we can assume that SC from primary tumor tissue might represent an interesting tool for in vitro studies of new CRC therapies as these culture conditions favor the enrichment of cells that are highly resistant to conventional therapies and that need to be targeted to circumvent disease relapse. 





We demonstrate that SC derived from primary colon tumor tissue seem to represent a superior model to adherent differentiated culture systems to screen for new therapeutic targets mostly due to their increased resistance to chemotherapeutics. Finally, our findings underline the clinical pertinence of our SC, and in future, they may represent an interesting tool to test for new CRC therapies that may overcome drug resistance.



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  3. Kreso A, Dick JE (2014) Evolution of the cancer stem cell model. Cell Stem Cell 14:275–291. doi: 10.1016/j.stem.2014.02.006



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