The story of SUM-44 PE
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After working with over 40 breast cancer specimens that had come to my laboratory, we decided that we needed to focus our attention on pleural effusion derived specimens for a specific reason. We had learned from our earlier experiences with primary tumor-derived cells that it is sometimes difficult to tell the difference between proliferating normal human mammary epithelial cells and breast cancer cells that emerge in primary cancer-derived cultures, particularly when growth factor enriched media were used. Indeed, we were fooled by some specimens in which normal the HMEC cells that emerged in these cultures had different characteristics (particularly proliferative lifespan) than normal cells derived from reduction mammoplasty specimens. As a result, we knew we needed to work with some breast cancer specimens in which we could be sure there were no normal HMEC cells present so that we could convince ourselves that we actually could grow breast cancer cells.
We received a pleural effusion specimen from a patient of Dr. Weber’s (fittingly enough) in mid-May of 1991. This patient had a lobular breast cancer that was estrogen receptor positive and she had failed hormonal therapy and chemotherapy. Being derived from a lobular breast cancer, these cells have an E-cadherin point mutation. Thus, much like the patient who provided the cells that became the MCF-7 cell line, SUM-44 was derived from a patient with ER positive, metastatic breast cancer. This particular specimen was the most highly enriched in breast cancer cells that we had received to that point. Many pleural effusion specimens from breast cancer patients are cytology negative and thus it is not possible to culture breast cancer cells from such specimens. However, SUM-44 was not one of those. It was quite clear after the initial purification of the cells from this specimen that it was highly enriched with breast cancer cells (not good news for the patient), and this allowed us to not only start primary cultures from the specimen, but to directly freeze several tubes of cells before they ever saw a culture dish. This was important because at this time, we were still attempting to use the culture medium that we had just developed that was specifically designed to support the culture of luminal mammary epithelial cells to grow breast cancer cells. To this point, we had become confident that we had the ability to culture normal luminal cells, and the hypothesis at that time was that, since most breast cancer cells were keratin-19 positive and hence luminal (that was the belief at the time), media that only supported the growth of basal-like cells would not support the growth of breast cancer cells. So, armed with this culture medium, and now with a pleural effusion specimen that we knew contained millions of bone fide breast cancer cells, we tested this medium on the SUM-44 specimen. And, the cells failed to grow! This was our first direct evidence that enriched, growth factor supplemented media that supported the growth of normal cells of many lineages would not necessarily support the growth of breast cancer cells. So, armed with that knowledge, we thawed frozen vials of the SUM-44 cells and started growing the cells in media in which specific factors were systematically removed from the media, and that was immediately rewarding. We found that SUM-44 cells would grow continuously and could be serially passaged in a medium containing 5% serum, plus insulin and hydrocortisone. The addition of other factors, namely EGF, of cyclic AMP agonists, or even collagen coating the plates (all things that normal cells responded well to) all had strong negative effects on the growth of these cells. Furthermore, we then found that SUM-44 cells grew perfectly well in our serum-free medium supplemented with insulin and hydrocortisone.
As indicated above, the SUM-44 cell line was developed from the outset in our Ham’s F12 medium supplemented with 5% FBS, insulin and hydrocortisone, and in this medium the cells could be serially passaged. However, they could not be serially passaged in a manner that we and others had gotten used to from with other cell lines. Indeed, we learned two additional very important lessons from our work with SUM-44; first, that true breast cancer cells grow quite slowly in culture and have doubling times of approximately 200 hours! And, second, that these breast cancer cells are often density dependent for their growth. Thus, in order to successfully propagate these cells, patience was required to allow the cells to come to a high level of confluence and the cells have to be split at low split ratios. Thus, the culture conditions for SUM-44, which we still use, calls for a 1:3 split every two weeks!
The lessons learned from SUM-44 began to shed light on why so many labs, including our own, had failed in their efforts to culture primary breast cancer cells. We failed because we were making assumptions about the biology of breast cancer cells that were incorrect: namely, that breast cancer cells grow fast (wrong), and that breast cancer cells can grow at clonal density (often wrong). This problem was compounded by the fact that normal HMEC cells, when provided with the right cocktail of hormones and growth factors grow very fast, and this makes it difficult to impossible to culture breast cancer cells from primary tumors using enriched media, and this is as lesson that our lab and a few others learned the hard way. Thus, from this one specimen, we learned that to successfully culture human breast cancer cells, culture conditions needed to be used that allowed the cancer cells to seed the culture dish at high density, and a medium needed to be used that would support the growth of the breast cancer cells and not the growth of normal HMEC cells, or fibroblasts, or mesothelial cells, in the case of pleural effusions. We hypothesized that if we could provide these conditions, our success rate for developing breast cancer cell lines would increase significantly, and this turned out to be correct.
