Prostate cancer is notoriously heterogeneous even when diagnosed initially as localized disease, being composed of phenotypically diverse malignant cell populations. Indeed, this tumor cell heterogeneity is the basis for the Gleason Grading system which combines the scores of the degree of morphological abnormalities of the most common, as well as the second most common, population of malignant cells within the primary prostate cancer lesion. Besides morphological heterogeneity, individual prostate cancer sites are also characteristically heterogeneous for the cellular expression of a series of differentiation marker [i.e., androgen receptor (AR), prostate specific antigen (PSA), prostate specific membrane antigen, and lineage specific cytokeratins]. While it is clear that prostate cancer is derived from the glandular epithelial compartment, this compartment is likewise composed of a heterogeneous mixture of morphological distinct cells types, including basal, intermediate, and luminal-secretory cells [1
While the normal prostatic epithelial compartment is phenotypically heterogeneous, these cells are genetically identical being derived from the hierarchical expansion of a series of progenitors derived from a normal parental epithelial stem cell [1
]. These stem cells reside in a tissue specific microenvironmental stem cell niche which allows them to maintain self-renewal ability and also generate a hierarchically expanding cascade of phenotypically diverse progeny having only limited self-renewal ability [1
]. Based upon a growing knowledge of the role of normal prostate stem cells in both tissue renewal and the development of normal phenotypic heterogeneity, the characteristic tumor cell heterogeneity is consistent with the lethality of prostate cancers being the result of the hierarchical expansion from a minor population of cancer cells with an unlimited self-renewal capacity, termed cancer initiating cells (CICs). Recent experimental studies have documented that even though CICs are a minor population of cancer cells, they drive both the lethality and heterogeneity of the prostate cancer [3
]. This is because these CICs have unlimited self-renewal ability while also giving rise to a hierarchically expanding cascade of phenotypically diverse malignant progeny which have only a limited proliferative ability even though they share the malignant genotype inherited from their CICs parents [3
During prostate carcinogenesis, AR is transformed from a growth suppressive into a ligand dependent oncogenic protein directly stimulating the growth of prostate cancer cells [3
]. This is because AR is a ligand dependent transcription factor for the expression of malignancy associated genes, like ETS fusion genes [12
]. In addition, AR protein also gains the ability to be a licensing factor for DNA replication in prostate cancer cells [13
]. Initially, this malignant growth stimulation requires a physiological level of androgen [i.e., testosterone and dihydrotestosterone (DHT)] providing the rationale for why androgen ablation (i.e., castration) is standard therapy for metastatic prostate cancer [3
]. Unfortunately, after a variable period of response, there is progression to a castrate resistant state which despite secondary approaches to further lower androgen, eventually kills the patient [15
]. Thus, prostate cancer will kill 30,000 US males this year alone [16
]. The progression to this castration resistant state is due to CICs acquiring the novel ability to generate survival and proliferation signaling without requiring full occupancy of the AR by its normal cognate ligands [17
]. Defining the mechanism for how CICs capture AR as a ligand dependent oncogenic protein during prostate carcinogenesis and how it eventually acquires ligand independent signaling during malignant progression is required to develop successful therapies for both the prevention and treatment of this devastating disease. Thus, isolating and propagating human prostate CICs from the full spectrum of clinical stages is more than an academic exercise; it is mandatory so that these cells can be used for such mechanistic studies.
Attempts to develop methods to isolate and propagate CICs have relied upon using established human prostate cancer cell lines since these clearly contain CICs based upon their lethal growth as xenografts in immune-deficient mice. Presently, prostate cancer cell lines have been established from only one primary prostate cancer from a patient with localized disease (i.e., E006AA), with the remaining established lines being derived from one primary prostate cancer (i.e., CWR-22) and from distant sites from <10 patients with metastatic disease. Thus, there is an urgent need for obtaining additional CIC containing lines particularly derived from primary prostate cancers. The major limitation for establishing new CIC driven human prostate cancer cell lines is not in dissociating these cells from patient specimens in a viable form. Instead the limitations are: (i) inability to prevent overgrowth of normal prostatic epithelial and mesenchymal cells also present within the starting clinical sample, and (ii) the inability to propagate the self-renewing CICs, as opposed to their more differentiated progeny which have only limited proliferative potential. Thus more refined methods are needed to propagate prostate CICs from a large series of prostate cancers patients.
One such refinement is based upon the fact that in serum free-growth factor defined (SFD) media containing high Ca2+
(i.e., >1 mM), normal human prostate epithelial and mesenchymal cells attach to standard tissue culture plastic [18
], but CICs from human prostate cancer cell lines and from early passage cultures derived from chemotherapy/radiation recurrent prostate patients do not and instead self-associate in suspension and grow as non-adherent spheroids, termed prostaspheres [9
]. Growth of such non-adherent prostaspheres documents that the CICs have lost contact inhibition which is a fundamental characteristic of cancer versus normal cells. In the present study, CICs from seven additional human prostate cancer lines were documented to form prostaspheres when plated onto standard tissue culture flask in high Ca2+
/SFD-media supplemented with androgen. Based upon these facts, single cell suspensions derived from a large series (n = 72) of radical prostatectomy specimen were plated onto standard tissue culture flask in high Ca2+
/SFD-media supplemented with androgen in order to fractionate normal contaminating host cells from human cancer cells by allowing the latter to form prostaspheres. These prostaspheres were then tested to determine whether they contained self-renewing CICs as documented by their ability to be continuously propagated in vitro.