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. 2009 Nov 15;69(22):8555-62.
doi: 10.1158/0008-5472.CAN-08-4673. Epub 2009 Nov 3.

Lin-Sca-1+CD49fhigh stem/progenitors are tumor-initiating cells in the Pten-null prostate cancer model

David J Mulholland  1 , Li Xin, Ashkan Morim, Devon Lawson, Owen Witte, Hong Wu
Affiliations

Lin-Sca-1+CD49fhigh stem/progenitors are tumor-initiating cells in the Pten-null prostate cancer model

David J Mulholland et al. Cancer Res. .

Abstract

We have shown previously that Pten deletion leads to the expansion of subset of prostate cancer cells positive for CK5 and p63. Although this subpopulation may be involved in tumor initiation or progression, studies to date have not functionally validated this hypothesis. Using in vitro sphere-forming assay and in vivo prostate reconstitution assay, we show here the presence of a tumor-initiating subpopulation in the Pten prostate cancer mouse model. Specifically, we show that the Lin(-)Sca-1(+)CD49f(high) (LSC) subpopulation overlaps with CK5(+);p63(+) cells and is significantly increased during prostate cancer initiation and progression and after castration. Mutant spheres mimic the structural organization of the epithelial compartment in the Pten-null primary tumor. Sorted LSC cells from either Pten-null spheres or primary tumors are able to regenerate prostate epithelial structure with cancerous morphology, closely mimicking that of primary cancers. Therefore, the LSC subpopulation is capable of initiating a cancerous phenotype that recapitulates the pathology seen in the primary lesions of the Pten mutant prostate model.

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Figures

Figure 1
Figure 1. LSC content correlates with Pten mutant prostate cancer progression and is enhanced upon androgen ablation
(A) Increased LSC+ subpopulation during prostate cancer initiation (PIN, 7 weeks) and progression (cancer, 20 weeks) in Pten mutants prostate (n=3) as compared to controls (n=3). (B) Castration enhances the percentage LSC+ cells in Pten mutants (n=3) relative to intact mutants (n=3) at 10 wks. This trend is also observed in control mice; however, the absolute LSC content is significantly lower than mutant mice. (C) The majority of mutant and control LSC+ cells are positive for the basal cell markers, p63 and CK5 (p63+/CK5+).
Figure 2
Figure 2. Prostate spheres derived from Pten mutants are larger and more heterogeneous than control spheres
(A) CD49fhigh cells isolated from Pten mutant mice contain most sphere forming activity (n=3). While LSC+ cells formed spheres that were predominantly >50 μm in diameter, LSC- cells formed small, non-spheroid structures that were exclusively <50 μm in diameter (right). (B) Prostate spheres formed from ROSA26-LacZ;Pb-Cre+;PtenL/L mice provide genetic validation that Pten deletion (LacZ+) leads to enhanced sphere diameter as compared to WT (LacZ-) spheres. (C) Castration of Pten mutant mice enhances sphere forming potential (p<0.005); however, exogenous androgen (R1881) supplement to mutant sphere cultures from intact mice does not significantly enhance sphere forming activity (p>0.05).
Figure 3
Figure 3. Deletion of Pten promotes progenitor expansion in prostate spheres
(A) Immunocytochemical analysis showing that mutant spheres (middle two panels) have a multilayered structure with increased p63+ and Ki67+ cells, similar to the primary cancer (right panels). Bars: left two panels = 100 μm; insets = 40 μm; right panel = 75 μm. (B) Pten-null spheres (LacZ+) display progenitor expansion and increased cell proliferation index (Fig 3B, right). Similar trends can also be observed in the absence of the LacZ marker in spheres generated from total unsorted cells (left). (C) Spheres from sorted LSChigh cells can be serially passed. Conversely, LSClow fractions yielded very inefficient sphere forming activities both for control and mutant cultures and could not be serial passaged.
Figure 4
Figure 4. Cells derived from the mutant spheres are capable of generating cancerous structure upon in vivo reconstitution
(A) Upper, a schematic illustration of experimental procedure. LSC cells from either control or Pten mutant prostates (6-8 wks) were plated in sphere forming conditions, expanded by one passage (P1), dissociated to single cells and combined with cells from UGSM in Matrigel/PrEGM for grafting. While cells from control spheres formed single layered prostate acini, sphere cells from mutant mice propagated multilayered AR-positive neoplastic structures (lower panels); bar = 75 μm (B) Grafts generated from mutant spheres (upper panels) recapitulate phenotypes associated with primary cancer (lower panels), including expansion of p63+/CK5+ cells and activation of the PTEN downstream effector, P-AKT; bar = 50 μm.
Figure 5
Figure 5. LSC cells from Pten mutant mice can initiate prostate cancer development
(A) Total (Lin-), LSClow or LSChigh sorted cells from Pten mutant prostates (6-8 wks) were each recombined with UGSM cells. Tissue regenerations revealed that while total and LSChigh cells could reconstitute prostate cancer structure, LSClow cells generated little detectable glandular structure. Bars, low mag = 500 μm; high mag =200 μm. (B) Prostate regenerations from mutant LSChigh cells recapitulate primary cancer morphology with AR and P-AKT positive cells in addition to increased p63+ and Ki67+ index (upper panels). Control LSChigh grafts mimic primary normal prostate structure containing low levels of P-AKT and p63+ cells (lower panels); bar = 100 μm.
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