One of the last trends in the battle between cancer stem cell and clonal evolution proponents and opponents is that “they are not mutually exclusive”. Peace. Is problem solved now? We are coming to realizing the great cancer complexity in which applying just one universal theory of tumor progression sounds elusive. On top of this complexity, a new interesting theory has appeared – the phenoypic plasticity of cancer cells.
There was some evidence coming from a few labs that cancer stem cells (CSC) could change their surface phenotype, genotype or signaling pathways during tumor progression. One of the most convincing proofs of cancer phenotypic plasticity and heterogeneity came up in recent papers by both Sean Morrison and Martin Carroll groups. In order to illustrate the concept of cancer stem cell phenotypic plasticity, I’ll focus on these two studies.
First of all I’ll summarize the paper from Morrison’s group:
…recent studies carried out in cancer cell lines have suggested that some phenotypic and functional attributes of tumorigenic cells can reversibly turn on and off (Mani et al., 2008,Pinner et al., 2009,Roesch et al., 2010,Sharma et al., 2010). This raises the question of whether reversible changes are observed in primary cancers from patients and whether many or few cells in these cancers can undergo such changes. If most cells in a cancer can reversibly gain and lose competence to form a tumor, then this is a transient state rather than a hierarchically determined attribute possessed only by rare cancer stem cells. These studies also raise the separate question of whether phenotypic heterogeneity in patient tumors is driven mainly by irreversible or reversible phenotypic changes.
1. Very high frequencies (30%) of tumorigenic cells were observed in total unselected melanoma cells. It was irrespective of source (obtained directly from patients or xenografted) and stage of disease (primary cutaneous melanomas (I-II) or metastastatic (III-IV).
2. There was no correlation between tumorigenic cell frequency and tumor growth rate / aggressiveness. Slow growing tumors can be tumorigenic and vice versa.
3. High tumorigenic rate was maintained indefinitely in a serial transplant assay
4. Surface markers (as many as 22) do not distinguish a difference between tumorigenic versus nontumorigenic melanoma cells
5. Metastatic potential is irrespective of surface markers expression (checked on CD271)
6. Melanoma cells with different phenotype can recapitulate tumor heterogeneity. So the competence to form a tumor is reversible.
Very important part – assays battle:
Our results with CD271 are different from the results reported by Boiko et al. (2010) even though both studies used the same anti-CD271 antibody, both studied a similar spectrum of melanoma stages (mainly stage III), and both studied a combination of xenografted tumors and tumors obtained directly from patients. The most obvious potential explanation for the difference in results lies in the different assays used in the two studies: different enzymatic dissociation conditions (25 min in our study versus up to 3 hr in their study), different injection sites (subcutaneous versus intradermal), and different recipient mice (NSG versus Rag−/−IL2Rγ−/−).
Thus, the assay we used appears to be 10,000-fold more sensitive than the assay used by Boiko et al. (2010). Using this more sensitive assay, we find that CD271− cells have at least as much ability to form tumors as CD271+ cells. It will now be critical for other labs to independently assess whether they also observe tumor formation by CD271− melanoma cells.
As Sean Morrison noted in his talks this year, they are getting pretty much the same data for human glioblastoma. To complete the picture read this interview with Mark Shackleton.
Martin Carroll’s group used very sensitive xenotransplantation model (NSG) and confirmed phenotypic heterogeneity in acute myelogenous leukemic (AML) stem cells. But most importantly they found that AML stem cell can not be defined by known surface markers and therefore possess phenotypic heterogeneity. The brief summary:
1. Unlike melanoma, AML stem cells are rare, irrespective of xenotransplant model used.
2. All sorted cell fractions of AML samples (carrying different mutations) were able to engraft in NSG mice irrespective of surface markers expression and recapitulate disease. They checked: Lin- versus Lin+(dim), CD34+ versus CD34-, CD38+ versus CD38-, CD45RA+ versus CD45RA- and combinations. Thus, AML stem cells are widely distributed throughout different phenotypes. Absolute distribution of AML stem cells does not correlate with immunophenotype.
3. Any sorted cell fraction of AML sample recapitulated phenotypic diversity of original disease phenotype in primary and secondary recipients.
We report here that for each sample evaluated SL-ICs are consistently found in both Lin– and Lin(dim) fractions, indicating that the transition to lineage marker expression is not necessarily associated with a loss of capacity to initiate AML. Remarkably, we found that none of the cell surface markers (Lin, CD34, CD38, CD45RA, and CD123) used to identify hematopoietic stem cells and myeloid progenitors in normal BM could be used to consistently segregate AML-initiating and self-renewing cells.
This study does not address the important question relating to the nature of the cell responsible for initiating AML. The phenotypic heterogeneity of SL-ICs and, in particular, our characterization of SL-ICs with myeloid progenitor-like phenotypes strengthen the concept that AML-initiating cells are not necessarily derived from hematopoietic stem cells as initially hypothesized.
The data described here present a fundamental challenge to the LSC model as it was originally proposed. The original model described a binary model of tumor heterogeneity in which there were cells capable of tumor reconstitution in a NOD/SCID mouse (stem cells) and cells that had no capacity for tumor reconstitution. Furthermore, LSCs were proposed to reside only in the immature compartment of hematopoietic cells, suggesting that limited differentiation within tumor cells recapitulated normal hematopoietic differentiation. In contrast, our results demonstrate that cells with features resembling a more mature hematopoietic cell can reconstitute a leukemia within the more sensitive NSG mouse model.
I think the consequences of this study is very impactful for the field.
First of all, more than a decade-old dogma about the restriction of leukemic stem cells to a particular phenotype finally crashed. Secondly, if AML stem cell can not be defined by known surface markers and possess phenotypic heterogeneity, the validity of many studies involving sorting of AML stem cells could be questioned. A few groups sorted AML cells based on expression of CD34 and CD38, called them “leukemic stem cells” and studied gene expression signature, metabolism and other quaities in order to find new potential druggable targets. One of the very recent studies like that just came out of press.
I’m pretty sure we will get more and more data about irrelevance of surface markers for cancer stem cell function.
Phenotypic plasticity is one of the nails in the coffin of simplistic models of cancer development, such as clonal evolution and cancer stem cell. You can add here genetic and epigenetic plasticity and you can get a sense how complex it is. Even functional characteristics of tumorigenic cells can be reversibly switched on and off. For example, chemotherapy resistance can be acquired stochastically and reflect the reversible state of cancer cells. Modeling and understanding of cancer stem cell phenotypic plasticity is the way to go in order to develop new therapies.