Cd44-positive Cancer Stem Cells Expressing Cellular Prion Protein Contribute to Metastatic Capacity in Colorectal Cancer

Cancer stem cells are implicated in tumor progression, metastasis, and recurrence, although the exact mechanisms remain poorly understood. Here, we show that the expression of cellular prion protein (PrPc, PRNP) is positively correlated with an increased risk of metastasis in colorectal cancer. PrPc defines a subpopulation of CD44-positive cancer stem cells that contributes to metastatic capacity. PrPc(+)CD44(+) colorectal cancer stem cells displayed high liver metastatic capability, unlike PrPc(-)CD44(+) stem cells, that was inhibited by RNAi-mediated attenuation of PrPc. Notably, administration of PrPc monoclonal antibodies significantly inhibited tumorigenicity and metastasis of colorectal cancer stem cells in mouse models of orthotopic metastasis. PrPc promoted epithelial to mesenchymal transition (EMT) via the ERK2 (MAPK1) pathway, thereby conferring high metastatic capacity. Our findings reveal the function of PrPc in regulating EMT in cancer stem cells, and they identify PrPc as candidate therapeutic target in metastatic colorectal cancer.


Introduction
Cancer is a major cause of mortality, and most human cancer deaths are related to metastasis (1,2).The difficulty in diagnosing and therapeutically eliminating metastasis is related to our limited understanding of the molecules specifically expressed on the surface of metastatic cancer cells.Accumulating evidence suggests that a subset of cancer cells, operationally termed cancer stem cells (CSCs) or tumor-initiating cells, possesses enhanced ability to regenerate tumors and survive anti-cancer therapies (3).
CSCs were first identified in the hematopoietic system (4), and have now been reported in most human solid tumors on the basis of their functional properties (5)(6)(7).Colorectal cancer stem cells (CCSCs) have been distinguished using CD44 or CD133 either alone (8)(9)(10), or combined with other markers, such as EpCAM, CD166, CD29, CD24, LGR5 and aldehyde dehydrogenase 1 (ALDH1) (11)(12)(13).Previous studies by ourselves and other authors have shown that as few as 100 CD44 + cancer cells isolated from patients were able to develop into a heterogeneous tumor, and that spheroids derived from a single CD44 + cancer cell could recapitulate the heterogeneous hierarchy of tumor cells (8,14).These studies indicate that CCSCs may trigger the formation of colorectal cancer (CRC), and that CD44 is a functionally important surface biomarker of CCSCs.
Importantly, an antibody specific to CD44 selectively eradicated leukemic stem cells in vivo by altering the fate of leukemic stem cells (15).Monoclonal antibodies targeting surface antigens specifically expressed in CSCs offer a potentially powerful strategy to eliminate CSCs with minimal toxicity.Besides CD44, monoclonal antibodies targeting CD123 and CD47 have been reported to eradicate leukemia stem cells (16).
Recent evidence suggests that CSCs may be endowed with enhanced disseminating Research.
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Cellular prion protein (PrPc) is a highly-conserved glycoprotein present in all vertebrates and has the same protein sequence as the scrapie prion protein (PrPsc), a pathogenic factor in scrapie in sheep， bovine spongiform encephalopathy in cattle, and kuru in human beings (25,26).In addition to the central involvement of PrPc in prion diseases, several lines of evidence have intriguingly implicated PrPc in human tumors including glioblastoma, breast, prostate, gastric and colorectal cancers (CRCs) (27).PrPc interacts homophilically with itself or heterotypically with components of the extracellular matrix (ECM) such as laminin and vitronectin, to mediate cell adhesion (28).PrPc expression is required for the self-renewal of CD34 + hematopoietic stem cells (29,30).However, despite tantalizing evidence linking PrPc to human cancers, its physiological function and exact role in cancer development remain elusive.Here we report that high levels of PrPc expression (in PrPc + cells) identify a functionally distinct subpopulation of CD44 + CCSCs, which contributes to CRC initiation and metastasis.Our findings thus reveal hitherto-unknown roles of PrPc in CCSCs and EMT, and suggest that PrPc could provide a potential therapeutic target in metastatic CRCs.

Cells, animals, and samples
Fresh colon samples from CRC patients and paired normal subjects were collected from the tumor bank of Beijing Cancer Hospital (Beijing, China), as approved by the Research Ethics Board at the Beijing Institute for Cancer Research.A tissue microarray (TMA) was constructed as described previously (31,32).The NCI-60 human tumor cell line panel was kindly provided by Dr. Jacque Grassi from the National Cancer Institute (Betheda, MD, USA).SW480 and MDA-MB-231 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA), authenticated using polyphasic (genotypic and phenotypic) testing every 6 months, cultured according to the Manufacturer's instructions.
Human primary CRC cells were isolated from patients and cultured in with serum free medium.All CCSCs were subcultured for fewer than 20 passages.4-week-old female nude mice (BALB/c-nu /nu) and 5-week-old female NOD/SCID mice were purchased from Chinese Academy of Medical Sciences, and all experiments were performed under standard conditions in accordance with the national and institutional regulations.

Tumorigenesis and metastasis in orthotopic xenograft models
NOD/SCID mice were anesthetized with pentobarbital and the cecums were exteriorized by laparotomy.Various numbers of CD44 + PrPc + , CD44 + PrPc -, CD44 -PrPc + , and CD44 -PrPc -subpopulations freshly sorted from patients, or 50,000 DsRed labeled PrPc over-expressing or knockdown CCSCs were injected into the cecal wall.All the animals were monitored for up to 80-100 days or until tumor-associated death occurred.Primary tumors and metastasis were evaluated by a whole-body fluorescence imaging system (In-Vivo FX PRO, Carestream).

Results
The PrPc expression level is correlated with clinical and pathological features and outcomes in CRC patients.
On the basis of our previous observations that CD44 is a robust marker for CCSCs (14), we hypothesized that additional factors may exist that are co-expressed with CD44 in the promotion of migration and metastasis of CSCs.We therefore compared gene expression in colorectal cancer, liver metastases and normal tissues by cDNA array performed in our lab.In addition, we conducted a CD44 co-expression analysis from Oncomine datasets (Ki_Colon, Koinuma_Colon, and Smith_ Colorectal_2 datasets; [33][34][35].Interestingly, we found that several genes were in fact co-expressed with CD44 and up-regulated in metastases; one of these genes was PRNP (data not shown), which encodes PrPc.
To find out whether PrPc and CD44 are associated with the clinical and pathological features, we carried out IHC staining in a tissue microarray (TMA) containing 309 colorectal tumor specimens.CD44 and PrPc were found to be co-expressed in most of the tumors, especially in those that were poorly differentiated (correlation coefficient = 0.73) (Fig. 1A).The PrPc expression level was significantly higher in poorly-differentiated than in moderately-and well-differentiated samples (P < 0.001; Fig. 1A and Supplementary Fig. S1A), whereas the level of CD44 was not associated with clinical stage (P = 0.143) (Fig. 1A).This observation was supported by examining multiple Oncomine cDNA microarray datasets, which revealed that the PRNP mRNA expression levels were significantly elevated in high-grade and advanced-stage CRCs (Supplementary Fig. S1B).Importantly, the CRC patients with the highest PrPc expression were also found to have the poorest prognosis, with a median survival of only 12 months (Fig. 1B), which was significantly (P < 0.0001) shorter than that of patients whose primary tumor was PrPc-negative (median survival >5 years).
CRC-related death is usually attributable to recurrence and distant metastasis.Out of 309 specimens, there were 228 primary CRCs without metastasis versus 81 cases with concurrent distant metastasis.Strikingly, we found that PrPc was highly expressed in the primary tumors of the patients with liver or lung metastasis (P = 0.0007) (Fig. 1C).
Oncomine cDNA microarray data analysis also showed a close correlation of the PrPc level with disease recurrence and early patient death (Supplementary Fig. S1C).We also observed that PrPc expression was most prominent at the interface and invasion fronts of tumor nodules (identified by cytokeratin 20 staining; Fig. 1D), which may constitute a reservoir of mobile CSCs (36).In the lymph nodes that had been invaded by CRC cells, PrPc + cells were also observed in vascular invasions (Fig. 1E and Supplementary Fig. S1D).In addition, high levels of PrPc were present in CRC cells and could be seen invading the tumor stroma (Fig. 1F).Collectively, these data indicate that high PrPc expression is associated with advanced clinical stages, increased metastatic risk, and reduced patient survival.

A subpopulation of PrPc + CRC cells possesses CSC properties
We next examined PrPc and CD44 co-expression patterns at the cellular level, and determined the biological and tumorigenic properties of PrPc-expressing cells in CRC.
We next asked whether PrPc is associated with CD44 in contributing to CSC properties.
To evaluate their tumorigenic potential, the CD44 + PrPc + , CD44 + PrPc -, CD44 -PrPc + , and CD44 -PrPc -subpopulations isolated from 9 patients (i.e., cases 22-30, Supplementary Table 1) were subcutaneously injected into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice.As predicted by their clonogenicity data, xenograft tumors were generated from the CD44 + PrPc + , CD44 + PrPc -and CD44 -PrPc + cells, but not from the CD44 -PrPc -cells in 6 out of 9 cases (Supplementary Table 2).In limited-dilution assays, the CD44 than the other subpopulations (P < 0.001) (Supplementary Table 2).We frequently observed that tumor cells had invaded the underlying muscle tissue in tumors regenerated from CD44 + PrPc + (but not in those regenerated from CD44 + PrPc -) cells (Fig. 2E).To further test their self-renewal capabilities in vivo, 100 CD44 + PrPc + cancer cells purified from a first-generation xenograft tumor were transplanted into NOD/SCID mice.Our findings indicated that 8 out of 9 transplantations grew secondary xenograft tumors that recapitulated the phenotype of the original tumor, and even higher tumorigenic capacities were detected in the third transplantation (Supplementary Fig. S2D and data not shown).
Similar tumor-initiating capacities were also observed in nude mice.We isolated the four subpopulations pre-purified from CD45 -tumor cells, and injected them subcutaneously into nude mice (cases 1-11, Supplementary Table 1).Tumor development was observed in 8 cases (Supplementary Fig. S2E).Again, the CD44 + PrPc + CRC cells exhibited the greatest tumor-initiating capacity (P < 0.05) (Supplementary Table 2).As shown in Supplementary Figure S2E, the reconstituted tumors were histologically heterogeneous.
PrPc and CD44 co-expression was detected in only a minor subset of cells in regenerated tumors (Supplementary Fig. S2F-G), also suggesting that the CD44 + PrPc + cells were capable of self-renewal.
As in leukemic stem cells (15), knocking down CD44 in CRC cells significantly inhibited tumor regeneration (14).To investigate the role of PrPc in tumor initiation, we isolated primary CRC cells from patients and cultured them in ultra-low attachment dishes without serum.Remarkably, PrPc knockdown in the CRC cells also retarded tumor growth in nude mice, although the inhibitory effect was slightly less apparent than with CD44 knockdown (Fig. 2F).However, combined knockdown of both CD44 and PrPc completely abolished tumor development (Fig. 2F).Taken together, these data suggest that the CD44 + PrPc + subpopulation of CRC cells does possess CSC properties.

PrPc + CSC is more metastatic
Since PrPc is highly expressed in metastatic tissues, we attempted to find out whether it contributes to metastasis in CRC.In order to recapitulate human cancers with greater fidelity, we transplanted 500, 2,000 and 10,000 cells from each of the CD44 + PrPc + , CD44 + PrPc -, CD44 -PrPc + and CD44 -PrPc -subpopulations isolated from 7 patients (i.e., case 24-30, Supplementary Table 1), into the cecal wall of NOD/SCID mice (38).As shown in Table 1, 6 out of the 7 patient-derived CRC cells produced cecal tumors, which was consistent with the results obtained from subcutaneously-implanted tumor cells (Supplementary Table 2).The CD44 + PrPc + cells clearly formed tumors with the greatest efficiency (P <0.01) (Table 1 and Supplementary Table 2).Remarkably, macroscopic and microscopic analysis revealed that liver metastases were almost exclusively observed in the mice implanted with PrPc + cells (i.e., CD44 + PrPc + and CD44 -PrPc + ) (Fig. 3A-C; Table 1).Furthermore, semi-quantitative measurement of the metastatic area and the metastatic index confirmed that the tumors derived from PrPc + CRC cells were highly metastatic (Fig. 3D-E).
Most of the migrating cells (84.25±3.14%)were found to be PrPc-positive, versus only 1.25±0.22% of the non-migrant cells (Fig. 3G).Collectively, these observations imply that PrPc is crucial for CCSC migration in vitro and for metastasis in vivo.

PrPc is functionally important for cell migration and cancer metastasis
Since CSC is generally rare and it is difficult to label and trace its growth in animals, we generated a cell line of CD44 + CCSCs isolated from a CRC patient.These cells have all the characteristics of CSCs including constitutive expression of CD44, and the capacity to form spheres and generate xenograft tumors (14).The CCSCs were grown in ultra-low attachment dishes without serum, and were subcultured once a week for fewer than 20 passages.To investigate the unique role of PrPc in metastasis, we used this cell line pre-labeled with DsRed and in which PrPc had either been knocked down by shRNAs or was over-expressed via the pBABE-PURO retrovirus system.As shown in Figure 4A-C, PrPc knockdown strongly inhibited tumor growth and liver metastasis as assessed by whole-body fluorescence imaging and H&E staining, suggesting that PrPc play a central role in CSC metastasis.In contrast both tumor growth and liver metastasis were promoted by PrPc over-expression.Moreover, the level of PrPc expression was correlated with the metastatic index (Fig. 4D).Similar results were observed using the spleen metastasis model (Supplementary Fig. S4A-C).In addition, transwell migration assays revealed that the levels of PrPc correlated with the migratory capacity of CCSCs (Fig. 4E).It is noteworthy that the PrPc levels did not alter the cell-cycle profiles (Supplementary Fig. S4D).Using CRC cells with CSC properties, we were able to demonstrate that PrPc is functionally required for the migration of CCSCs and contributes to CRC metastasis.

PrPc antibodies inhibit migration and metastasis
Surface markers unique to CSCs would provide ideal therapeutic targets on these cells, and indeed monoclonal antibodies have demonstrated therapeutic efficacy in both preclinical tumor models and cancer patients (41).We were therefore interested in addressing the question of whether PrPc could be used as a therapeutic target, in a manner similar to CD44 (15).We generated several monoclonal antibodies against PrPc, out of which we selected the IgG1 antibody.The specificity of the antibodies was verified by ELISA, Western blotting (WB), and IHC staining (Supplementary Fig. S4E-G).The migration of CD44 + CCSCs in vitro was strongly inhibited after they had been treated with 1 μg/ml anti-PrPc antibodies.The inhibitory effect obtained was greater than that associated with the anti-CD44 antibody (~1.7 fold).A combination of 1 μg/ml anti-CD44 and 1 μg/ml anti-PrPc antibodies was able to inhibit migration by up to ~80% (Fig. 4F); whereas PrPc antibodies did not show any effect on cell migration in PrPc knockdown cells (data not shown).
It was essential to find out whether PrPc monoclonal antibodies can prevent CRC metastases in vivo.NOD/SCID mice bearing orthotopic implantations of CCSCs were administered 200 μg antibodies intraperitoneally (i.p) on a weekly basis.ELISA analysis revealed that the concentration of anti-PrPc antibody in the serum of tumor-bearing animals was maintained at 0.87 -1 μg/ml (data not shown).The orthotopic and metastatic tumors were monitored by whole-body fluorescence imaging every week and after 8-10 weeks the mice were sacrificed and underwent pathology examinations.The presence of anti-PrPc antibodies dramatically decreased tumorigenicity and liver metastases (Fig. 4G), whereas their bodyweight did not decrease (not shown).CCSCs pre-coated with anti-PrPc antibodies before orthotopic implantation produced a similar inhibitory effect (Supplementary Fig. S4H).
To make sure that the observed metastasis-inhibiting effects of anti-PrPc antibodies were not due to inhibition of primary tumor growth, we pre-inoculated the CCSCs orthotopically into the cecal wall for 3 weeks.Mice with equivalent tumors were then grouped and injected with 100 or 200 μg antibodies weekly.The results clearly showed that the anti-PrPc antibody inhibited tumor growth and metastasis at the lower dosage (100 μg/week) and eliminated tumor growth at higher dosage (200 μg/week) (Supplementary Fig. S4I).

PrPc promotes EMT in CCSCs
We consistently observed that the PrPc-overexpressing CCSCs exhibited an elongated mesenchymal-like morphology, whereas the PrPc knockdown cells were flatter (Fig. 5A; Supplementary Fig. S4G).Also, PrPc + CRC cells generated branching colonies with loose cell-cell interaction (see Fig. 2D), characteristic of the EMT phenotype.This prompted us to investigate whether PrPc promotes epithelial-mesenchymal transition (EMT), a known mechanism of CSC migration and invasion.Cells undergoing EMT retain stem cell functionality and form invasive front colonies (42).We first used quantitative RT-PCR (primers shown in Supplementary Table 3) to measure several EMT-related genes by manipulating PrPc levels.In PrPc over-expressing cells, mesenchymal genes such as N-cadherin and Twist were significantly up-regulated, whereas E-cadherin, an epithelial marker, was down-regulated (Supplementary Fig. S5A).
In addition, there were increased levels of matrix metalloproteinase 2 (MMP2) and MMP11 (Supplementary Fig. S5A), which might have contributed to the metastasis observed in PrPc over-expressing cells.In contrast, knock-down of PrPc resulted in a reversed-expression pattern (Supplementary Fig. S5A).Western blot analysis and double IF staining further confirmed the correlation of PrPc with the expression of EMT-related molecules (Fig. 5B-C; Supplementary Fig. S5B).
Twist is a key transcription factor regulating mesenchymal cell fate, differentiation and morphogenesis (43).Since PrPc expression had been found to influence the expression of Twist (Supplementary Fig. S5A), we reasoned that PrPc might regulate EMT via Twist.
To test this, Twist was ectopically expressed in CCSCs in which PrPc was stably knocked down by shRNA.We observed that constitutively overexpressed Twist was indeed able to induce EMT in PrPc-knocked down CCSCs, leading to increased levels of Snail and N-cadherin (Fig. 5D).However, overexpression of PrPc in the Twist RNAi cells was unable to restore EMT, probably because Snail and N-cadherin expression was lower than in the parental cells (Fig. 5D).Overall, we conclude that PrPc regulates the EMT phenotype by modulating Twist.
Next, we used qRT-PCR to measure the mRNA levels of Twist, N-cadherin, and E-cadherin in CD44 + PrPc + , CD44 + PrPc -, CD44 -PrPc + , and CD44 -PrPc -subpopulations of CRC cells freshly purified from 6 patients.The results showed that both Twist and N-cadherin were expressed at significantly higher levels in the PrPc-positive (i.e.CD44 + PrPc + and CD44 -PrPc + ) CRC cells than in the PrPc -subpopulations (Fig. 5E).
Furthermore, ~82% of N-cadherin + cells were also PrPc +  cells were PrPc -in CD45 -EpCAM + CRC cells as analyzed by FCM (Fig. 5F).These findings suggest that PrPc is positively correlated with the mesenchymal properties of cells from CRC patients.

Evidence that PrPc-induced EMT is ERK2 dependent
EMT is regulated by the TGF-and ERK signaling pathways (44).To understand the molecular mechanisms of PrPc-induced EMT, we first assessed the activation of these two pathways (45).Knocking down PrPc appeared to result in a reduction of both Phospho-smad2 and ERK1/2; however, over-expression of PrPc activated ERK1/2 but not smad2, suggesting that ERK signaling may act downstream of PrPc (Fig. 6A).In support of this, we found that U0126, an inhibitor of ERK signaling, suppressed the expression of several EMT-associated molecules (Fig. 6B) as well as EMT itself, especially when PrPc was over-expressed.
Laminin is a PrPc ligand (46), which, we reasoned, could activate events occurring downstream of PrPc, such as EMT.Laminin did enhance both ERK2 phosphorylation and the EMT phenotype (Fig. 6C-D).Interestingly, PrPc antibody blocked the laminin-induced ERK activation, EMT protein expression and EMT morphology in a dose-dependent manner, (Fig. 6C-D), probably via interference with the interaction between laminin and its receptor PrPc.IF staining confirmed that the anti-PrPc antibody attenuated the laminin-induced N-cadherin expression (Fig. 6E).In addition, transwell migration assays revealed that U0126 blocked the PrPc overexpression-induced migration, and that the anti-PrPc antibody also attenuated laminin-enhanced migration (Fig. 6F).Our findings thus suggest that ERK2 signaling pathway is involved in PrPc-induced EMT.

Discussion
It has been demonstrated that CSCs contribute to both tumor initiation and cancer metastasis in many tumor systems.However, in most cases, the underlying mechanisms are not well understood.In this study, we have identified a distinct PrPc + subpopulation in CD44 + CCSCs that displays enhanced capacity to regenerate tumors.These results emphasize the functionalities of PrPc as an "enrichment" factor for CCSC.Our data demonstrate that PrPc is a novel CCSC biomarker, which, by itself, functionally contributes to both CRC tumorigenicity and metastasis.Interestingly, the PrPc + CRC cells tend to localize at the invasion fronts in patient tumors, are more mesenchymal, and behave like other cancer cells undergoing EMT, properties that have probably given these cells a greater capacity to disseminate and metastasize.These observations indicate PrPc's role in regulating CCSC likely occurring through its ability to promote EMT and the exact mechanism warrants future investigations.In terms of the mechanism involved, our work implies that PrPc may regulate EMT through ERK2-and Twist-dependent signaling pathways.Importantly, the interaction of PrPc with its ligand, laminin, directly activates ERK2 signaling and promotes EMT.Therefore, conceptually, interference with PrPc expression/functions, the PrPc-laminin interaction or its downstream signaling events, may be a promising avenue in developing new therapies targeting PrPc + CRC cell-mediated metastasis.
Previous work has shown that PrP1, a PrPc ortholog in the zebra fish, regulates the stability of E-cadherin mediated cell adhesion (47), suggesting that PrPc expression may endow the cell with properties of EMT, which is a developmentally-conserved regulatory mechanism.In addition, PrP c -null (PRNP 0/0 ) cells display less migratory activity than PRNP +/+ cells (48).The more aggressive phenotype of PRNP +/+ cells is consistent with our observations that PrPc contributes to CCSC migration.Recently, CD26 + CRC cells have also been found to possess CSC properties, with greatly increased metastatic capacity, and, intriguingly, like PrPc, CD26 also functions as an adhesion molecule that binds to ECM (24).These findings, together with our present study, strongly suggest that the enhanced tumorigenic and metastatic capacities of CCSCs are probably determined by the CSC-intrinsic expression of functional molecules such as PrPc and CD26, which in turn mediate the interactions of CSCs with their microenvironment.Future work will attempt to elucidate the potential relationship between PrPc + and CD26 + CCSCs.
Previous studies have shown that the expression of PrPc is positively correlated with invasiveness and drug resistance in both gastric and breast cancers (27,49).In addition, Weinberg and colleagues have reported that endoglin and PrPc are enriched in mammosphere-initiating cells (50).Although PrPc has been reported to be highly expressed in some CRCs (51), as far as we are aware this is the first report linking PrPc to CCSCs and our data help to explain how PrPc is associated with cancer metastasis.
The level of PrPc was inversely correlated with patient survival, suggesting a potential prognostic value in CRC.It has recently emerged that monoclonal antibodies targeting surface molecules expressed in CSCs demonstrate powerful therapeutic efficacy by eliminating CSCs and leading to tumor resolution (16).Our findings therefore have significant clinical implications, since PrPc + CSCs may provide important new targets for therapy in metastatic CRC.Indeed, PrPc monoclonal antibodies display strong inhibitory effects on CCSC tumorigenesis and metastasis in orthotopic xenograft models, suggesting possible therapeutic applications in the clinic.Overall, our findings throw new light on the mechanisms underlying CRC cell metastasis, and are of clinical importance for the design of therapeutic strategies for treating CRC.Table 1 ：Tumor-initiating incidence and liver metastasis in the orthotopic model *Indicated numbers (500, 2,000 and 10,000) of CD44 + PrPc + , CD44 + PrPc -, CD44 -PrPc + , and CD44 -PrPc -subpopulations freshly isolated from CD45 -EpCAM + CRC cells in 7 patients (i.e., case 24-30, Table S1) were transplanted into cecal wall of NOD/SCID mice.In 6 out of 7 cases, xenograft tumors were regenerated (day 100).
The incidence of tumor regeneration at the cecum and the incidence of metastasis to the liver are shown.

Figure 1 .
Figure 1.PrPc is associated with high grade tumors and poor prognosis in patients.A: Heatmap of CD44 and PrPc protein expression in CRCs (n = 309 patients).Data are grouped by the following tumor grades: Grade 1, well-differentiated; Grade 2, moderately-differentiated; Grade 3, poorly differentiated.CD44 and PrPc expression are semi-quantitatively described as 0: negative; 1: low; 2: moderate; and 3: high.B: Kaplan-Meier plot showing the survival percentage of 109 CRC patients (> 5 years after surgery) grouped by PrPc expression level.The numbers of patients (n) in each group are indicated in parentheses.C: The PrPc expression level is positively correlated with metastasis.Patients with a concurrent metastasis (M, red) and those without a metastasis (Non-M, blue) are grouped by PrPc expression levels.Y axis: number of patients.** P < 0.001.D: Double IF staining of cytokeratin 20 (green) and PrPc (red) in CRC showing preferential localization of PrPc-positive cells in the invasive fronts of tumor islets (indicated by dotted white lines).Scale bar, 200 μm.E: Representative IF images of PrPc expression in invasive vasculatures in the draining lymph node (white dotted outline).PrPc + /cytokeratin 20 + tumor cells (yellow) are magnified and indicated (arrows).Scale bar, 200 μm.F: IHC staining shows PrPc + CRC cell invasion into stromal cells (red arrows).Scale bar, 100 μm.

Figure 2 . 0001 Figure 3 .
Figure 2. The CD44 + PrPc + subpopulation of CRC cells possesses CSC properties A: Double IF staining of CD44 (green) and PrPc (red) in a representative CRC cryosection.The section was counterstained with DAPI (blue).Scale bar, 100 μm.B: A representative 4-color flow cytometric analysis of a CRC.Cells were gated by CD45

Figure 5 .
Figure 5. PrPc modulates EMT in CCSCs A: Phase-contrast images of CCSCs in which PrPc was either over-expressed or knocked down.Note the morphological changes after the PrPc expression level was modulated.Scale bar, 50 μm.B: Western blot analysis of N-cadherin and E-cadherin in CCSCs in which PrPc was either over-expressed or knocked down.Monoglycosylated (30 kDa) and unglycosylated (28 kDa) PrPc expressed on CCSCs were detected by anti-PrPc (Clone5) antibody.C: Double IF staining of N-cadherin (green) and E-cadherin (red) in CCSCs in which PrPc was either over-expressed or knocked down.Scale bar, 50 μm.D: Quantitative RT-PCR analysis of EMT related genes in Twist or PrPc manipulated CCSCs.The mRNA expression levels were normalized to that of parental CCSCs.Purple bars, knock-in of twist in PrPc RNAi CCSCs.Yellow bars, knock-in of PrPc in Twist RNAi cells.**P < 0.001, ***P < 0.0001.E: Relative mRNA expression levels of N-cadherin, E-cadherin and Twist in the

Figure 6 .
Figure 6.PrPc-regulated EMT is ERK2 dependent A: Western blot analysis of the proteins indicated in CCSCs in which PrPc was either over-expressed or knocked down.B: Parental CCSCs and those over-expressing PrPc were treated with the ERK2 inhibitor U0126 for 24 h, and a whole-cell lysate was used for WB analysis of the molecules indicated.C: CCSCs were treated with 5 μg/ml anti-PrPc antibody (Ab), 2 μg/ml laminin (LN), and a combination of Ab and LN for 24 h, after which cells were harvested for WB analysis.D: Phase-contrast images of CCSCs maintained under the conditions indicated.Note that the anti-PrPc antibodies rescued the morphological changes in the presence of LN.Scale bar, 100 μm.E: Double IF staining of N-cadherin (green) and E-cadherin (red) in CCSCs in the presence of 10 μM U0126, 2 μg/ml LN, and indicated dose of anti-PrPc antibodies (μg/ml).Scale bar, 20 μm.F: Migration of CCSCs in the presence of U0126, 2 μg/ml LN, and indicated dose of anti-PrPc antibodies (μg/ml).
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on February 15, 2013; DOI: 10.1158/0008-5472.
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