Clinical-Scale NK Cells for Cancer Therapy Made from Pluripotent Stem Cells

Dan Kaufman’s laboratory has done it again. The Kaufman laboratory at the University of Minnesota in collaboration with scientists from MD Anderson Cancer Center in Houston, Texas have designed a protocol to make natural killer cells from embryonic stem and induced pluripotent stem cells.

Natural killer cells provide a very important contribution to the innate immune response. These cells produce molecules called cytokines and they also kill virally infected cells and malignant cells. NK cells are unique among the cells of the immune system in that they have the ability to recognize foreign, infected or stressed cells in the absence of antibodies and Major Histocompatibility Complex proteins (the cell surface proteins that act as bar codes used by the immune system uses to determine if a cell is yours or not yours). Therefore, NKs typically work faster than the rest of the immune system.

Natural killer cells or NK cells have been used to treat patients with refractory cancers. Unfortunately, a major problem with using NK cells is growing a sufficient quantity of cells for therapy. Using pluripotent stem cells to make NK cells is an intriguing possibility, but the protocols for differentiating NK cells from embryonic stem cells (ESCs) is tedious and inefficient. However, the Kaufman laboratory has provided a much more efficient and straight-forward way to derive NK cells, thus allowing for the production of clinical scale quantities of NK cells.

The Kaufman lab protocol involves first deriving embryoid bodies from ESCs or induced pluripotent stem cells (iPSCs), which are made from adult cells through genetic engineering techniques that causes the cells to de-differentiate into ESC-like cells known as iPSCs. Embryoid bodies are three-dimensional aggregates of pluripotent stem cells that assume a kind of spherical shape and have a variety of cells differentiating into a wide range of cell types. Embryoid bodies can contain beating heart muscle, neural-type cells, blood progenitors cells, and even muscle or bone cells in their interiors in a haphazard arrangement. Forming embryoid bodies or EBs from cultured ESCs or iPSCs is rather easy, but controlling the differentiation of the cells in the EBs is quite another matter.

embryoid bodies
embryoid bodies

Kaufman and others discovered that if the EBs were incubated with artificial antigen-presenting cells that expressed a surface-bound version of the protein IL21 (interleukin 21) plus a cocktail of cytokines, these pluripotent stem cells could efficiently form NK cells.

Functional assays of the NK cells differentiated from ESCs and iPSCs easily showed that the NK cells for functional in every way and expressed all the cell surface molecules characteristic of NK cells. Furthermore, all ESC and iPSC lines examined were able to make NK cells, but the efficiency with which they made them different rather widely.

In conclusion, Kaufman and others state in their paper, “our ability to now produce large numbers of cytotoxic NK cells means that prospect hESC- and iPSC-derived hematopoietic products for diverse clinical therapies can be realized in the not-too-distant future.” For some cancer patients, that day cannot come soon enough.

A Protein Responsible for Cancer Stem Formation Provides a Drug Target

Eighty-five percent of all tumors are carcinomas, which are tumors that form in layers of cells that line surfaces.  Such cell layers are known as an epithelium. When carcinomas form, they undergo an “epithelial-mesenchymal” transformation” or EMT.  EMT means that cells go from being closely aligned and tightly bound to each other in a an organized layer to cells that have little to do with each other and grow in unorganized clumps.  Is there a molecule that unites the carcinomas and if so is this molecule a potential drug target for cancer treatments?

Mammary Carcinoma
Mammary Carcinoma

Researchers at the University of Texas MD Anderson Cancer Center have identified a protein that seems to play a pivotal role in EMT.  This protein, FOXC2, may lay at the nexus of why some carcinomas resist chemotherapy and grow uncontrollably and spread.  FOXC2 could, conceivably represent a novel drug target for chemotherapy.

Sendurai Mani, assistant professor of Translational Molecular Pathology and co-director of the Metastasis Research Center at MD Anderson, said, “We found that FOXC2 lies at the crossroads of the cellular properties of cancer stem cells and cells that have undergone EMT, a process of cellular change associated with generating cancer stem cells.”

Cancer stem cells are fewer in number than other tumor cells, yet research has tied them to cancer progression and resistance to treatment.  Abnormal activation of EMT can actually create cancer stem cells, according to Mani.

Mani continued, “There are multiple molecular pathways that activate EMT.  We found many of these pathways also activate FOXC2 expression to launch this transition, making FOXC2 a potentially efficient check point to block EMT from occurring. ”  Mani’s research group used experiments with cultured cells and mice to discover these concepts, but the next step will require assessing the levels of FOXC2 expression in human tumors samples.

In the meantime, these new data from Mani’s research team may have profound implication for the treatment of particular types of carcinomas that have proven to be remarkably stubborn.  Breast cancers, for example, are typically carcinomas of the mammary gland ductal system.  A specific group of breasts cancers are very notoriously resistant to treatment, and FOXC2 seems to be at the center of such breast cancers.

The anti-cancer drug sunitinib, which is marketed under the trade name Sutent, has been approved by the US Food and Drug Administration (US FDA) for three different types of cancers.  In this study, sunitinib proved effective against these particularly stubborn types of breast cancer; the so-called “triple-negative, claudin-low” breast cancers.


Mani explained why such breast cancers are so resistant to treatment:  “FOXC2 is a transcription factor, a protein that binds to DNA in the promoter region of genes to activate them.  For a variety of reasons, transcription factors are hard to target with drugs.”

However, sunitinib seems to target these triple-negative breast cancers.  When mice with triple-negative breast cancer were treated with sunitinib, the treated mice had smaller primary tumors, longer survival, and fewer incidences of metastasis.  The cancer cells also showed a marked decreased in their ability to form “mammospheres,” or balls of cancer stem cells (this is an earmark of cancer stem cells).  Thus sunitinib seem to attack cancer stem cells.

As it turns out, FOXC2 activates the expression of the platelet-derived growth factor receptor-beta (PDGFRc-beta).  Activation of PDGFRc-beta drives cell proliferation in FOXC2-positive cells, and sunitinib inhibits PDGFRc-beta and inhibits cells that have active FOXC2 and undergoing EMT.

Triple-negative breast cancer cells lack receptors that are used by the most common anti-cancer drugs.  These deficiencies are responsible for the resistance of these cancers to treatment.  Such cancer cells also tend to under go EMT because they lack the protein claudin, which binds epithelial cells together.  Without claudin, these cancer cells become extremely aggressive.

Since cells undergoing EMT are heavily expressing FOXC2, Mani and his colleagues used a small RNA molecule that makes a short hairpin and inhibits FOXC2 synthesis.  Unfortunately, blocking FOXC2 had no effect on cell growth, but it did alter the physical appearance of the cells and reduced their expression of genes associated with EMT and increased the expression of E-cadherin, a protein necessary for epithelial cell organization.  Breast cancer cells also became less invasive when FOXC2 was inhibited, and they down-regulated CD44 and CD24, which are markers of cancer stem cells..  Additionally, triple-negative breast cancer cells that had FOXC2 inhibited had a reduced ability to make mammospheres.  Thus, FOXC2 expression is elevated in cancer stem cells, and inhibition of FOXC2 decreased the ability of the cancer stem cells to behave as cancer stem cells.


Mani’s group also approached these experiments from another approach by overexpressing FOXC2 in malignant mammary epithelial cells.  This forced FOXC2 expression drove cells to undergo EMT and become much more aggressive and metastatic (the cancer spread to the liver, hind leg, lungs, and brain).  Breast cancer cells without forced FOXC2 overexpression showed no tendency to metastasize.

Finally, Mani’s group examined metastatic mammary tumors that were highly aggressive when implanted into nude mice (mice that cannot reject transplants).  Two of the tumors were claudin-negative and both of these tumors showed elevated FOXC2 expression.  When FOXC2 expression was blocked by Mani’s hairpin RNA, the claudin-negative tumors became less aggressive and grew more as mesenchymal cells.  The cells that underwent EMT also showed high levels of PDGF-RC-beta expression.

Mani said of these data: “We thought PDGF-B might be a drugable target in these FOXC2-expressing cells.”  Mani’s group also showed that suppressing FOXC2 reduced the expression of PDGFRC-Beta.  Thus, this small molecule might be an effective therapeutic strategy for treating these hard-to-treat breast cancers.

MD Anderson has filed a patent application connected to this study.

See Hollier B.G., Tinnirello A.A., Werden S.J., Evans K.W., Taube J.H., Sarkar T.R., Sphyris N., Shariati M., Kumar S.V., Battula V.L., Herschkowitz J.I., Guerra R., Chang J.T., Miura N., Rosen J.M., and Mani S.A.,. FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer. Cancer Research. e-Pub 2/2013.