Frequency of Cancer Stem Cells Higher than Predicted

Co-injection of 786-O, a ccRCC cell line, with human serum (UFS) and cancer-associated fibroblasts (CAF) leads to greater cancer transplantation success rate, a higher estimate of the frequency of cancer stem cells and a more clinically relevant clear cell differentiation in the resulting tumors. A. 786-O cells alone. B. 786-O cells + UFS + CAF. Scale bars = 50μm. Image Source: Modified from [1].

With the help of BioCision’s CoolCell® freezing containers, scientists re-examine a popular hypothesis for the spread of cancer – and find that tumorigenic cancer stem cells occur much more often than previously thought.

Cancer is perhaps the most feared disease in the developed world today. In the US, more than 1.5 million new cases of cancer are predicted to occur in 2016, with about 600,000 expected deaths. Despite much progress in our fight against this disease, including promising immunotherapeutic approaches, the disease continues to threaten many lives. Most, if not all, cancers can spread from their original location in the human body to a new one. This ability to metastasize is one reason why cancers are so difficult to eradicate.

How do these metastases form? One theory is based on the low-rate existence of distinct tumorigenic cancer stem cells (CSC), which can self-renew, differentiate and proliferate. The theory presumes that CSCs are the only tumor cells that can initiate and build full metastases. Science can functionally identify the frequency of such “tumorigenic” cancer stem cells. You inoculate a suspension of a defined number of tumor cells into an animal model of choice and subsequently correlate the number of inoculated cells with the incidence of successful tumor development in the animal. If you succeed to generate a tumor when you inoculate 105 cells, but not when you inoculate only 104 cells, then the frequency of cancer stem cells is lower than 10-4 but higher than 10-5. Right?

Well. No. Not true. Scientists now found out that previous estimates of cancer stem cell frequencies may have severely underestimated their actual occurrence. This startling discovery was recently published in Scientific Reports. [1]

The researchers were puzzled by a stark discrepancy between their success rates during clear cell renal cell carcinoma (ccRCC) tumor transplantation into their animal model. Transplantation of small unprocessed tumor fragments generated tumors much more frequently than ccRCC cell suspensions, even though both approaches transferred similar number of tumor cells. The scientists wondered why.

It’s not that they were not careful. To minimize adverse effects during cryopreservation, the team used BioCision’s CoolCell® containers, which ensured maximal cell survival and slow freezing of the cell suspensions at the optimal rate of -1°C / min. Flow cytometry nevertheless showed that the frequency of tumor cells declined from 66% to 33% when the tumor tissue cells were put in suspension. In addition to this selective destruction of tumor cells, the necessary digestion and washing steps during cell suspension apparently removed components that were beneficial for tumor cell engraftment. To counteract this, the researchers provided human serum and cancer-associated fibroblasts. Et voilà – tumor cell engraftment efficiency promptly increased a whopping 10fold.

Another often overlooked factor in tumor transplantation is anoikis, a programmed cell death due to a sudden lack of cell-cell and cell-extracellular matrix communication channels. When the scientists obstructed anoikis with an inhibitor of a specific kinase, the tumorigenic potential of both ex vivo ccRCC cells and in vitro cell lines increased even further. Finally, the scientists also showed that the standard trypan blue cell viability assay overestimates the number of living cells by 4 – 7fold.

So what is the upshot of these findings? The scientists argue that the standard processing steps performed in labs worldwide may give rise to a substantial underestimate of the frequency of cancer stem cells in human cancers. Tumorigenic cells may therefore not be rare at all in ccRCC or other cancers.

This certainly would lead to changes in our approach to fight metastases. Knowing your enemy is half the battle. Learning that you still have to do more homework on that half may seem painful. But it may ultimately help to win the fight.


[1] Gedye, C. et al. Cancer stem cells are underestimated by standard experimental methods in clear cell renal cell carcinoma. Sci Rep 6:25220. doi: 10.1038/srep25220. 2016.