- Top of page
- MATERIALS AND METHODS
- Author contributions
- Supporting Information
Many cancers appear to be driven by stem-like cells that self-renew, differentiate to yield heterogeneous progeny and survive adverse microenvironments (Dalerba et al, 2007). That these may also initiate metastasis has driven efforts to identify and characterize them. Tumour-initiating stem cell (T-ISC)-enriched populations have been identified in various cancers by discrete surface markers and by their ability to generate tumour spheres and xenograft tumours with high frequency (Frank et al, 2010; Magee et al, 2012; O'Brien et al, 2009). Elegant lineage tracing experiments recently provided compelling evidence for the cancer stem cell model (Chen et al, 2012; Driessens et al, 2012; Schepers et al, 2012). In primary breast cancers, CD44+CD24neg/low ESA+ cells were enriched for xenograft formation compared to bulk tumour cells (Al Hajj et al, 2003). Aldehyde dehydrogenase 1 (ALDH1) activity also marks breast cancer cells enriched for stem cell properties and those with both ALDH1+ and CD44+CD24neg/low are most tumourigenic, but infrequent (<1%; Ginestier et al, 2007).
For cancers of breast, pancreas, prostate, head and neck and colon, the T-ISC phenotype consistently includes surface CD44+ expression (Al Hajj et al, 2003; Hurt et al, 2008; Li et al, 2007; O'Brien et al, 2007; Prince et al, 2007). CD44 expression has been associated with poor prognosis and metastasis, supporting the idea that stem-like cells generate metastases (Liu et al, 2010; Shipitsin et al, 2007; Yang et al, 2008). In contrast, the relationship between surface CD24 expression and stemness differs between solid tumours. Overexpression of this glycosylated surface protein increases cancer cell proliferation and migration (Aigner et al, 1998). While surface CD24 is observed in subsets of T-ISC from liver (Lee et al, ), colon (Yeung et al, 2010) and pancreas (Li et al, 2007), the T-ISC phenotype described for primary breast cancers show negative or low level surface CD24 (CD44+CD24neg/low; Al Hajj et al, 2003). This contrasts with normal mammary progenitors cells, which express CD24 (Pece et al, 2010; Spike et al, 2012). It is noteworthy that CD24+ cells are increased in metastatic compared to primary breast cancers (Shipitsin et al, 2007).
Populations surviving chemotherapy and radiation appear to be enriched for T-ISC (Calcagno et al, 2010; Li et al, 2008; Tanei et al, 2009), possibly due to membrane transporters, greater quiescence and enhanced DNA repair, permitting T-ISC regeneration (Frank et al, 2010; O'Brien et al, 2009). Thus, identification and interdiction of T-ISC specific pathways may permit greater anti-cancer treatment efficacy. To date, the difficulty of isolating viable T-ISC from solid tumours in sufficient quantity to permit their molecular characterization has limited development of T-ISC-directed therapies that circumvent drug resistance or induce differentiation. Breast cancer cell lines have been shown to contain T-ISC analogous to those in primary breast cancers, permitting isolation of the large numbers of T-ISC required for functional characterization (Charafe-Jauffret et al, 2009; Fillmore & Kuperwasser, 2008).
Stem-like cell subsets within a cancer may vary not only in their self-renewal potential, but also in their ability to successfully engage different metastatic niches. While T-ISC or a sub-population thereof have been broadly posited as giving rise to metastasis, relatively few experimental models have addressed this directly. ALDH1+CD44+CD24neg subpopulations in breast cancer lines yielded more xenograft metastasis than ALDH1-CD44low/−CD24+ (Croker et al, 2008), but metastatic potential was not limited to the very low minority ALDH1+ population. While increasing data indicate the presence of functional heterogeneity within T-ISC enriched populations in other tumours (Hermann et al, 2007; Lee et al, ; Pang et al, 2010; Yang et al, 2008), identification and characterization of mammary T-ISC subsets that consistently metastasize or that mediate therapy resistance presents a challenge.
The present study was undertaken to identify discrete subsets among T-ISC of the most deadly form of breast cancer: that lacking estrogen and progesterone receptors and HER2 amplification (so called triple negative—hereafter TNBC). We postulated that, as for normal stem cells, primary TNBC-derived cultures and immortal lines might exhibit an aberrant T-ISC hierarchy with precursor/progeny populations that differ in molecular pathways conferring self-renewal, tumourigenicity and metastatic potential.
Here we demonstrate a hierarchical relationship between distinct subsets within CD44+CD24neg/low subpopulations from a TNBC line and from two TNBC patient-derived dissociated tumours (DTs). The minority CD44+CD24low+ population shows greater sphere formation and gives rise to CD44+CD24neg progeny. In contrast, cells arising from CD44+CD24neg are exclusively CD44+CD24neg both in 2D culture and in spheres. CD44+CD24low+ are enriched for embryonic stem cell (ES) and metastatic gene expression signatures, tumour sphere, soft agar colony forming and tumour forming cells compared to CD44+CD24neg, and in the MDA-MB-231 model show greater metastatic potential. In CD44+CD24low+ cells, Notch1 was shown to directly transactivate SOX2 to drive self-renewal. Although Notch has been previously implicated in breast cancer stem cell self-renewal (Harrison et al, 2010; McGowan et al, 2011; Sansone et al, 2007) the CD44+CD24neg T-ISC sub-population was unaffected by Notch inhibition in 2D culture, sphere and xenograft assays, revealing a heretofore unappreciated heterogeneity in GSI responsiveness in T-ISC.
- Top of page
- MATERIALS AND METHODS
- Author contributions
- Supporting Information
While many solid tumours appear to be driven by T-ISCs, emerging data suggest that phenotypically distinct subsets exist within T-ISC populations (Meyer et al, 2010; Patel et al, 2012; Visvader & Lindeman, 2012) and thus targeting of this population therapeutically may prove more challenging than previously appreciated. Earlier work showed primary human CD44+CD24neg/lowESA+ breast cancer cells were enriched for T-ISC, with <1/200 generating tumours in immune compromised hosts (Al Hajj et al, 2003). Fillmore et al. showed CD44+CD24neg/lowESA+ in both ER positive and ER negative breast cancer cell lines were enriched in stem cell properties and T-ISC, while the strongly surface CD24 positive CD44+ cells such as we observe in ER positive cancer lines were not (Fillmore & Kuperwasser, 2008). Aldefluor activity also marks stem-like properties and its combined enrichment together with surface CD44+CD24neg/low yields a highly tumourigenic population (Ginestier et al, 2007). The CD44+CD24neg/low primary human breast cancer population is enriched by taxane chemotherapy (Li et al, 2008), and exhibits distinct gene expression profiles, prognostic of poor patient outcomes (Creighton et al, 2009; Liu et al, 2007; Shipitsin et al, 2007; Tsunoda et al, 2011).
Here we show that cells with low level surface CD24 expression (CD24low+) and CD24 negative (CD24neg) cells comprise distinct phenotypes in the T-ISC-enriched CD44+CD24neg/low population of TNBC lines and primary dissociated tumour cultures. The CD44+ population with low surface CD24 positivity (CD44+CD24low+) represents 11–23% of CD44+ cells, has greater sphere forming potential, increased clonogenicity and appears to give rise to CD24neg progeny. CD44+CD24neg cells show fewer sphere-generating cells (a proxy for stem cell function in vitro) that decline with serial passage, and generate only CD44+CD24neg progeny in 2D and 3D culture. ESA+ and ALDH1+ cells were only seen in the CD44+CD24low+ population, revealing further heterogeneity within this phenotype. Both populations generate orthotopic xenografts, but the proportion of T-ISC in the CD44+CD24low+ population is higher (1 in 72 T-ISC frequency in CD44+CD24low+ compared to 1 in 44,936 for CD44+CD24neg in DT-22). Notably, CD44+CD24low+ cells preferentially express ES genes and metastatic gene signatures, show greater motility and invasion, and in the MDA-MB-231 model, yield tumours that metastasize while CD44+CD24neg did not. The CD24neg subpopulation within CD44+CD24neg/low cells showed no Notch1 activation and was GSI insensitive both in 3D (spheres and soft agar colonies) and in xenografts, highlighting important therapeutic implications of heterogeneity in cancer stem cell populations.
The difficulty of isolating sufficient T-ISCs from primary cancers to permit their molecular characterization has hampered efforts to define pathways critical for T-ISC self-renewal, therapy resistance and metastasis. The present study used not only TNBC lines, but also confirmed findings in human breast cancer cultures at early passage. Early passage patient tumour cultures—while an imperfect substitute for primary tumour analysis—permitted molecular assays in T-ISC subsets requiring large cell numbers that are not feasible in primary cancers. It is worth noting that not all TNBC lines and DTs assayed exhibit the dominant CD44/CD24 marker pattern evaluated herein. Present work addressed only the CD44+CD24neg/low subset of TNBC. Further investigation is required to determine its relevance to T-ISC in other breast cancer types.
In all three models assayed, CD44+CD24low+ cells gave rise to both CD44+CD24low+ and CD44+CD24neg phenotypes in 2D culture with asymmetric growth kinetics. Spheres seeded by single CD44+CD24low+ cells comprised progeny – both phenotypes, while spheres generated by single CD44+CD24neg cells yielded only CD44+CD24neg, suggesting that CD24neg arise from CD24low and are restricted to produce only like progeny. Notably, while tumours generated from sorted CD44+CD24low+ DT-22 cells comprised both phenotypes, tumours arising from sorted CD44+CD24neg cells were almost entirely CD44+CD24neg. The 3–4% CD44+CD24low+ cells present in all of 18 orthotopic tumours generated from CD44+CD24neg-enriched cells may reflect the lack of purity in the initial sorted cells injected (4% CD24low present in the CD24neg enriched population) or could represent phenotypic conversion within a cancer population from a lower to a greater self-renewal potential (discussed further below). These reproducible patterns seen not only in MDA-MB-231, but also in two independent primary TNBC tumour-derived cultures, support a lineage relationship rather than random T-ISC heterogeneity.
The genetic plasticity and aberrant differentiation within cancer cell populations would permit clonal evolution within T-ISC populations, but do not preclude the existence of abortive developmental hierarchies that could contribute to heterogeneity. While other explanations are possible, our data are compatible with a hierarchy such as what has been observed in human leukaemia stem cell subsets (Goardon et al, 2011), colon (Bu et al, 2013) and liver cancer T-ISC (Lee et al, ). Further evidence for the existence of stem-like cancer cells giving rise to clonally related cells with reduced self-renewal capacity was recently provided by lineage tracing in squamous skin papilloma (Driessens et al, 2012). Present data support the notion that precursor–progeny relationships can exist in T-ISC populations, yielding subsets that differ in self-renewal and, potentially, in the ability to establish metastasis (Dalerba et al, 2007).
CD24 may be a marker of metastatic potential. Its expression is increased in breast and bladder cancer metastasis compared to primaries and confers poor prognosis (Athanassiadou et al, 2009; Bircan et al, 2006; Overdevest et al, 2011; Shipitsin et al, 2007) and CD24 knockdown abrogates metastasis in bladder cancer (Overdevest et al, 2011). Present data link low level surface CD24 to metastatic potential in TNBC. In the MDA-MB-231, as few as 100 CD44+CD24low+ cells yielded orthotopic tumours that generated multi-organ metastasis and lung micrometastasis, while tumours from up to a half million CD44+CD24neg cells failed to metastasize. Moreover, CD44+CD24low+ cells in both MDA-MB-231 and DT-22 showed greater motility and invasion, and preferential expression of gene profiles observed in breast cancers metastatic to lung (Minn et al, 2005) or brain (Bos et al, 2009) and that characterize bone metastasis (Kang et al, 2003). A T-ISC hierarchy with progressive loss of both CD24 and metastatic potential was also observed in a hepatocellular cancer, in which a highly metastatic CD24+ T-ISC population gave rise to CD24neg cells that failed to metastasize (Lee et al, ). While present work provides novel evidence for a putative metastatic hierarchy within breast cancer T-ISC in the MDA-MB-231 line, this remains to be confirmed in additional models since DT-22 does not form tumours that metastasize.
Present data provide not only an example of heterogeneity in T-ISC of TNBC, but also in molecular pathways and susceptibility to therapeutic targeting. Notch family members play complex and critical roles in fate determination in mammogenesis (Bouras et al, 2008; Raouf et al, 2008; Sale et al, 2013) and are implicated in breast cancer stem cell self-renewal (Harrison et al, 2010; Sansone et al, 2007; Wang et al, 2009). NOTCH1 and NOTCH4 pro-viral integration sites mediate mammary tumour formation (Dievart et al, 1999; Gallahan & Callahan, 1987), Notch4 is upregulated in T-ISC in primary non-invasive (Farnie et al, 2007) and invasive ER positive breast cancers (Harrison et al, 2010) and Notch1 overexpression in breast cancer correlates with worse prognosis (Reedijk et al, 2005; Stylianou et al, 2006).
Here we observed higher NOTCH 1, 2 and 4 gene expression in CD44+CD24low+ cells in MDA-MB-231 and higher levels of N1-ICD, N2-ICD and N4-ICD in both DT-22 and MDA-MB-231 models. Expression profiling showed that Notch driven genes, ES signature genes (Assou et al, 2007), genes targeted by Nanog, Oct4 and Sox2 (NOS targets) and a subset of these with transcriptional function (NOS-TFs; Boyer et al, 2005) were preferentially expressed by CD24low+ compared to CD24neg cells. Moreover, Sox2 and Nanog proteins were higher in the Notch-activated CD24low+ cells, leading us to investigate the link between Notch and Sox2. Sox2 is a driver of ES self-renewal and may play a role in human cancers (Leis et al, 2011; Nakatsugawa et al, 2011; Sarkar & Hochedlinger, 2013; Xiang et al, 2011). Notch inhibition reduced Sox2, sphere and colony formation, and in vivo tumour growth exclusively in the CD44+CD24low+ population, without affecting global cell proliferation. Of the four different Notch genes, only N1-ICD overexpression significantly induced SOX2 in our mammary cell models, and N1-ICD bound the SOX2 promoter in both. Moreover, N1-ICD overexpressing MDA-MB-231 showed a Sox2-dependent increase in the proportions of ALDH1+ and of CD44+CD24low+ cells, and in sphere formation, suggesting that Notch1 critically activates SOX2 to drive T-ISC self-renewal in these ER negative breast cancer models.
Heterogeneity in driving pathways and in the phenotypes of T-ISC has become increasingly apparent (Patel et al, 2012; Schober & Fuchs, 2011; Visvader & Lindeman, 2012). T-ISC of different surface phenotypes and metastatic ability have been shown to co-exist in pancreatic (Hermann et al, 2007), colon (Pang et al, 2010) and liver cancer models (Lee et al, ; Yang et al, 2008). Recent work by Kim et al showed that tumour initiating cells in breast cancers of either basal or luminal phenotypes co-exist, with the latter generating more invasive tumours. They also provide evidence that basal-like cancer cells with stem cell traits may give rise to progeny cells with luminal phenotype but not vice versa and cells with luminal markers can initiate tumours (Kim et al, 2012).
The present study provides evidence for two phenotypically distinct populations within the TNBC CD44+CD24neg/low, in which CD44+CD24neg arise from a CD44+CD24low+ precursor population. We also show evidence for phenotypic heterogeneity within the CD44+CD24low+ population which comprises subsets of ESA+ and ESA− and ALDH1+ and ALDH1− cells with different levels of self-renewal as evidenced by limiting dilution sphere assays. Whether precursor–progeny relationships exist within the ESA and ALDH1-based subgroups is yet unknown. ALDH1 activity is very infrequent in CD44+CD24neg/low breast cancer cells (Charafe-Jauffret et al, 2009; Croker et al, 2008; Ginestier et al, 2007), and populations enriched for both aldefluor activity and CD44+CD24neg/low show a high T-ISC frequency (Ginestier et al, 2007). It is noteworthy that our ALDH1+ CD44+CD24low+ cells showed the greatest abundance of sphere forming cells and also the greatest responsiveness to Notch pathway inhibition.
Several groups have presented evidence for potential phenotypic conversion within marker-defined populations (Meyer et al, 2009). In tert-immortalized and oncogene transformed HMEC models, CD44 negative cells have been shown to generate CD44 positive progeny suggesting that more “differentiated cells” can revert to a more primordial stem-like precursor (Chaffer et al, 2011). Different subsets within ER negative breast cancer lines sorted by CD44, CD24 and ESA status appear to undergo transitions compatible with a model of stochastic phenotypic conversion (Gupta et al, 2011). Here we show that N1-ICD overexpression not only increased CD24low+ cells, suggesting an increase in self-renewal/symmetric division, but also led to a low rate of conversion of CD44+CD24neg cell to CD44+CD24low+, an event we have not observed spontaneously in culture over several years. Interestingly, xenografts generated from sorted CD44+CD24neg DT-22 cells consistently contained 2–8% CD44+CD24low cells in all of 18 tumours, raising the possibility of a niche-induced inter-conversion in vivo. Taken together, these observations raise the intriguing possibility that the tumour microenvironment may regulate the phenotypic heterogeneity present within tumour initiating stem cells as observed in other systems (Lu et al, 2013).
An important implication of heterogeneity within malignant stem cell populations is that more primitive stem cells may have not only a greater metastatic propensity, they may also differ from their bulk progeny and escape therapy to re-populate. The importance of the Notch pathway to mammogenesis and mammary cancer has led to the clinical development of Notch inhibitors (Morgan et al, 2013), several of which are in clinical trials. Notch inhibitors have been shown to augment pre-clinical efficacy of estrogen receptor blockade (Rizzo et al, 2008), trastuzumab (Osipo et al, 2008) and radiation (Phillips et al, 2006), and enhance chemotherapy response through depletion of stem cells in preclinical models (Qiu et al, 2013) and clinical trials (Schott et al, 2013). They may also have particular efficacy in TNBC (Clementz et al, 2011; O'Toole et al, 2013). Loss of experimental brain metastasis following Notch1 knockdown in xenograft models lends support for Notch-targeted therapies (McGowan et al, 2011).
T-ISC heterogeneity may limit the efficacy of therapies that oppose Notch signalling. The two TNBC T-ISC populations characterized herein, differed notably in their responses to GSI. RO4929097, a GSI in clinical trials for cancer, had no effect on tumours derived from CD44+CD24neg cells, which comprise a majority of the population in TNBC lines. Only CD44+CD24low+-generated tumours responded to RO4929097. Even in the N1-ICD-expressing CD44+CD24low+ population, sphere formation and tumour growth were not fully abrogated by GSI treatment. This could reflect incomplete Notch inhibition or the presence of a Notch-independent CD44+CD24low+ subpopulation. While Notch pathway inhibitors hold promise for preferentially targeting the more malignant T-ISC, Notch1/Sox2 dependence may be restricted within T-ISC subsets. The presence of functionally discrete T-ISC subpopulations may also limit tumour responses to other targeted therapies. Present findings support further phenotypic and molecular characterization of distinct T-ISC subpopulations, since they may illuminate limitations of current therapy and open new avenues for more effective cancer treatment.