cancer stem cell hypothesis markers

The cancer stem cell hypothesis: In search of definitions, markers, and relevance

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A Guide to Cancer Stem Cell Markers

Cancer stem cells (CSCs) are cancer cells that initiate tumor formation, growth, and metastasis. These cells contribute to these processes by remaining undifferentiated and self-renewing — features that are largely specified by the expression of select transcription factors. In addition to transcription factors, cancer stem cells also upregulate certain surface markers, which can be used to distinguish cancer stem cells from other cancer cells. Still, more markers of stemness — the capability of a cell for self-renewal and differentiation — can also be found in the cytoplasm of cancer cells. Some markers are common to a wide variety of tumor types (“inclusive markers”), whereas others are more specific (“exclusive markers”). This article summarizes the most commonly described cancer stem cell markers in each of these categories.

Inclusive Cancer Stem Cell Markers

There are no universal cancer stem cell markers that can identify cancer stem cells and only cancer stem cells of every cancer. That is because the few stemness markers that are shared by all cancer types are also shared by non-cancer cells (including normal stem cells and various differentiated cell types). The Yamanaka (or pluripotency) factors OCT4, MYC, KLF4, and SOX2 are prime examples of markers that denote stemness, cancer or not. Together, these four transcription factors induce pluripotency by regulating a variety of developmental signaling pathways such as the Notch and Wnt signaling pathways. Notch and Wnt signaling can also serve as markers of stemness in multiple cancers, including breast and colorectal cancers. NANOG and SALL4 are further examples of a pluripotency factor also found in cancer stem cells.

Figure image: Cancer stem cell markers (CSCs) are often shared by other cell types, including other stem cells (stemness markers), differentiated cells, and tissue-specific cancer cells.

Cancer stem cells also share markers with differentiated (non-stem) cell types. For example, the lymphocyte markers CD19, CD24, CD38, and CD44 have also been detected in the stem cell populations of several cancers. In fact, the glycoprotein CD44 has been detected on the stem cells of most cancers, including breast, pancreatic, prostate, colorectal, ovarian, lung, liver, head/neck, blood, bladder, gastric, brain, bone, and cervical cancers.

Other such markers include CD24 and CD90 (THY1), both of which are expressed in neurons and lymphocytes as well as blood, liver, breast, brain, lung, head/neck, and gastric cancer stem cells; and the epithelial cell markers CD133 (PROM1) and EPCAM, which are also expressed by brain, colorectal, lung, liver, prostate, pancreatic, breast, ovarian, and gastric cancer stem cells.

Exclusive Cancer Stem Cell Markers

Exclusive cancer stem cell markers are those that label the stem cells of only one or two cancers. Exclusive refers to expression among different cancer stem cell populations, as some of these markers may also be expressed by analogous normal stem cell populations. These markers are outlined below by cancer type.

Bladder cancer.  CD47 is a marker of bladder cancer stem cells, although it can be found on virtually all bladder cells, albeit at lower expression levels. This marker has been shown to promote bladder cancer engraftment, and its silencing promotes macrophage phagocytosis of bladder cancer cells. Bladder cancer stem cells are also CD66c lo  (CEACAM6 lo ).

Blood cancers.  The first cancer stem cell markers to be confirmed  in vivo  were of the blood cancer acute myeloid leukemia, where CD34 + CD38 -  hematopoietic stem cells were discovered to promote AML tumorigenesis. Despite being mostly exclusive to blood cancers, these markers are also expressed by noncancerous hematopoietic stem cells. IL3RA is another blood cancer stem cell marker that labels noncancerous hematopoietic stem cells. HAVCR2 (TIM-3), however, marks cancerous hematopoietic stem cells but not noncancerous ones, enabling their differentiation.

Brain cancers.  Initially discovered in neural embryonic progenitors, FUT4 is a gene that encodes the enzyme responsible for synthesizing the Lewis X (CD15) carbohydrate. As such, both markers can be used to identify brain cancer stem cells.

Breast cancer.  Stem cells in breast cancer share many markers with the stem cells of other cancer types, such as ITGA6 (CD49f) and CD90. ITGB3 (CD61), however, is a breast cancer-specific stem cell marker.

Colorectal cancer.  While no marker uniquely labels colorectal cancer stem cells, the glycoprotein dipeptidyl-peptidase 4 (CD26), encoded by DPP4, is perhaps the most exclusive of stemness in colorectal cancer, where it promotes metastasis. Gastric cancer.  Exclusive stem cell markers of gastric cancers include LINGO2, LETM1, and MSI2. LINGO2 and LETM1 regulate the Akt signaling and contribute to cancer processes like angiogenesis, metastasis, and tumorigenicity. MSI2 regulates similar processes. Silencing LINGO2 also silences the pluripotency factor and inclusive cancer stem cell marker OCT4. The brain cancer stem cell marker Lewis X/CD15 is also associated with gastric cancer stem cells.

Head & neck cancer.  The primary exclusive cancer stem cell marker of head and neck cancer is NGFR (CD271). LGR5 (GPR49), while also present on the same cells, is expressed by colorectal, gastric, and breast cancer stem cells.

Liver cancer.  The oncofetal marker (one that is expressed in embryonic and cancerous but not adult tissues) AFP can be used to identify liver cancer stem cells. This marker is a secreted biomarker of malignant liver cancer that can be detected in blood.

Lung cancer.  Like blood cancer stem cells, lung cancer stem cells express CD34. PLAUR (CD87) can be used to distinguish blood cancer stem cells from lung cancer stem cells.

Melanoma.  In addition to marking head & neck cancer stem cells, NGFR (CD271) is a well-established cancer stem cell marker that maintains stemness in melanoma cells. The keratinocyte marker ABCB5 and the lymphocyte marker CD20 (MS4A1) are additional markers of stemness in melanoma.

Ovarian and cervical cancers.  Another normal stem cell marker, ENG (CD105) identifies both ovarian and cervical cancer stem cells. The marker is also associated with angiogenesis, especially in and around tumors.

Prostate cancer.  Prostate cancer stem cells are CD44 + CD133 + HNRNPA2B1 hi , mirroring their highly proliferative yet noncancerous CD133 + HNRNPA2B1 hi  epithelial cousins in the prostate. Although CD44 and CD133 are found in many cancer stem cell populations, HNRNPA2B1 is limited to those of prostate cancer. CD151 is another marker exclusively expressed by prostate cancer stem cells.

Table of cancer stem cell markers

The table below lists human and mouse cancer stem cells markers as described in recent literature. The majority of proteins listed are either glycoprotein markers or transcription factors, but other defining markers, such as receptors, binding proteins, and even signaling pathways, are also included. Accompanying each marker are links to relevant antibodies and ELISA kits, as these immunodetection tools are routinely used in cell characterization studies via flow cytometry and immunostaining. The associated products are offered by a variety of manufacturers and can serve as a useful reference for cancer stem cell identification and profiling.

Note: *This marker is a carbohydrate antigen. Information on Protein Type, Localization, and Size (kDa) obtained from UniProt.org (for human genes only). 

1. Murar M, Vaidya A. Cancer stem cell markers: premises and prospects. Biomark Med. 2015;9(12):1331-1342. doi:10.2217/bmm.15.85

2. Hadjimichael C, Chanoumidou K, Papadopoulou N, Arampatzi P, Papamatheakis J, Kretsovali A. Common stemness regulators of embryonic and cancer stem cells. World J Stem Cells. 2015;7(9):1150-1184. doi:10.4252/wjsc.v7.i9.1150

3. Zhao W, Li Y, Zhang X. Stemness-Related Markers in Cancer. Cancer Transl Med. 2017;3(3):87-95. doi:10.4103/ctm.ctm_69_16

4. Gopalan V, Islam F, Lam AK. Surface Markers for the Identification of Cancer Stem Cells. Methods Mol Biol. 2018;1692:17-29. doi:10.1007/978-1-4939-7401-6_2

5. Walcher L, Kistenmacher AK, Suo H, et al. Cancer Stem Cells-Origins and Biomarkers: Perspectives for Targeted Personalized Therapies. Front Immunol. 2020;11:1280. Published 2020 Aug 7. doi:10.3389/fimmu.2020.01280

6. Atashzar MR, Baharlou R, Karami J, et al. Cancer stem cells: A review from origin to therapeutic implications. J Cell Physiol. 2020;235(2):790-803. doi:10.1002/jcp.29044

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• Research article
• Open access
• Published: 23 November 2011

Cancer stem cell markers in breast cancer: pathological, clinical and prognostic significance

• H Raza Ali 1 , 2 ,
• Sarah-Jane Dawson 1 , 2 , 3 ,
• Fiona M Blows 5 ,
• Elena Provenzano 2 , 3 , 4 , 6 ,
• Paul D Pharoah 1 , 4 , 5 &
• Carlos Caldas 1 , 2 , 3 , 4  

Breast Cancer Research volume  13 , Article number:  R118 ( 2011 ) Cite this article

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Introduction

The cancer stem cell (CSC) hypothesis states that tumours consist of a cellular hierarchy with CSCs at the apex driving tumour recurrence and metastasis. Hence, CSCs are potentially of profound clinical importance. We set out to establish the clinical relevance of breast CSC markers by profiling a large cohort of breast tumours in tissue microarrays (TMAs) using immunohistochemistry (IHC).

We included 4, 125 patients enrolled in the SEARCH population-based study with tumours represented in TMAs and classified into molecular subtype according to a validated IHC-based five-marker scheme. IHC was used to detect CD44/CD24, ALDH1A1, aldehyde dehydrogenase family 1 member A3 (ALDH1A3) and integrin alpha-6 (ITGA6). A 'Total CSC' score representing expression of all four CSC markers was also investigated. Association with breast cancer specific survival (BCSS) at 10 years was assessed using a Cox proportional-hazards model. This study was complied with REMARK criteria.

In ER negative cases, multivariate analysis showed that ITGA6 was an independent prognostic factor with a time-dependent effect restricted to the first two years of follow-up (hazard ratio (HR) for 0 to 2 years follow-up, 2.4; 95% confidence interval (95% CI), 1.2 to 4.8; P = 0.009). The composite 'Total CSC' score carried independent prognostic significance in ER negative cases for the first four years of follow-up (HR for 0 to 4 years follow-up, 1.3; 95% CI, 1.1 to 1.6; P = 0.006).

Conclusions

Breast CSC markers do not identify identical subpopulations in primary tumours. Both ITGA6 and a composite Total CSC score show independent prognostic significance in ER negative disease. The use of multiple markers to identify tumours enriched for CSCs has the greatest prognostic value. In the absence of more specific markers, we propose that the effective translation of the CSC hypothesis into patient benefit will necessitate the use of a panel of markers to robustly identify tumours enriched for CSCs.

The existence of tumour initiating cells also called cancer stem cells (CSCs) in breast cancer has been demonstrated by several studies [ 1 – 3 ]. It has been shown that xenotransplanted cell subpopulations enriched for CSCs can generate tumours in non-obese severe-combined immunodeficient (NOD/SCID) mice from a fraction of the number of unselected cells required to form tumours. In addition, tumours resulting from the implantation of small numbers of CSCs recapitulate the molecular heterogeneity of the original mixed population. The CSC hypothesis holds that since this subpopulation of cells is exclusively able to form tumours they underpin both disease recurrence and metastasis [ 4 ]. Therefore, CSCs are potentially of major clinical significance.

In order to demonstrate the functional characteristics which define a CSC, it is necessary to isolate candidate CSCs. This has been achieved by use of cell-surface markers and by tagging cells which exhibit characteristics associated with stemness. The combination of CD44 and CD24 first enabled Al-Hajj et al . to prospectively isolate a CSC subpopulation of from eight of nine patients with breast cancer [ 1 ]. After excluding non-epithelial cells (lineage - ), CD44 + CD24 -/low cells were enriched by flow cytometry and subsequently implanted into NOD/SCID mice. The CD44 + CD24 -/low cells were able to form tumours in NOD/SCID mice from fewer cells than the mixed population with 10- to 50-fold enrichment for this ability. The resulting xenografts were found to exhibit the same phenotypic diversity as the original tumours [ 1 ].

A similar paradigm for experimentation was used to show that cell subpopulations with high aldehyde dehydrogenase (ALDH) activity were enriched for CSCs [ 3 ]. The ALDEFLUOR assay uses a biochemical reaction to tag cells with high ALDH activity with cytoplasmic fluorescence, permitting their enrichment by flow cytometry. Ginestier et al . found that ALDEFLUOR-positive normal mammary epithelial cells from reduction mammoplasties were enriched for sphere-forming ability and in vivo outgrowth potential, forming 10-fold more ducts in NOD/SCID mice. Similarly, ALDEFLUOR-positive cells from xenografts of human breast carcinomas were able to form tumours in NOD/SCID mice from as few as 500 cells, whereas ALDEFLUOR-negative cells inconsistently formed tumours and required 50, 000 cells to do so. Again, the tumours resulting from the implantation of ALDEFLUOR-positive cells contained both ALDEFLUOR-positive and negative cells in proportions similar to the original mixed population. The clinical relevance of this finding was investigated by using immunohistochemistry (IHC) to stain for aldehyde dehydrogenase family 1 member A1 (ALDH1A1) in 481 primary breast carcinomas. ALDH1A1 retained independent prognostic significance in a multivariate analysis [ 3 ]. The ALDEFLUOR assay is designed to detect expression of ALDH1A1 (STEMCELL Technologies SARL, Grenoble, France) and, consistent with this, Ginestier et al . found that ALDH1A1 expression was restricted to the ALDEFLUOR-positive subpopulation from normal mammary epithelial cells. However, the identity of the aldehyde dehydrogenase isoform(s) responsible for ALDEFLUOR-positivity in malignant breast epithelial cells has been questioned. Marcato et al . sought to establish whether ALDEFLUOR-positivity in primary breast tumours and breast cancer cell-lines related to a particular isoform(s) of ALDH or a global increase in ALDH activity [ 5 ]. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) not ALDH1A1, was found to correlate most strongly with ALDEFLUOR-positivity and, using immunofluorescence (IF) in primary tumours, was also found to correlate with metastasis and tumour grade. Moreover, the knockdown of ALDH1A3 in three breast cancer cell lines abrogated ALDEFLUOR activity [ 5 ].

An alternative approach to the CSC problem was used by Pece et al. , who, by exploiting the quiescent nature of normal mammary stem cells, isolated sufficient numbers to derive a gene expression signature [ 2 ]. The lipophilic dye PKH26 was used to isolate the most mitotically inactive fraction of self-renewing epithelial cells from reduction mammoplasties. The resulting gene signature was found to correlate with the grade of breast tumours. This correlation was established directly by comparison with published datasets and, indirectly, both by the prospective isolation of primary breast cancer cells using a subset of high-ranking markers from the gene signature and by IHC of breast tumours. By IHC and IF it was shown that grade 3 breast tumours contained a three- to four-fold greater proportion of cells expressing these high-ranking markers compared to grade 1 tumours. The authors argue that the grade of breast tumours is a function of their CSC content [ 2 ]. Prominent amongst the markers of the normal mammary stem cell-derived signature was CD49f or alpha-6 integrin (ITGA6). ITGA6 is a cell-surface protein which has been shown to identify adult mouse mammary stem cells [ 6 ] and a tumorigenic subpopulation in the MCF-7 breast cancer cell line [ 7 ] as well as regulating CSCs in glioblastoma [ 8 ].

Although CD44 + CD24 -/low , ALDH1A1, ALDH1A3 and ITGA6 appear to enrich for CSCs it is important to note that this is not always the case. For example, the CD44 + CD24 -/low phenotype was not successful in identifying CSCs in one of the nine patient specimens originally reported [ 1 ]. Similarly, Hwang-Verslues et al . found that the expression of stem cell markers, including CD44 + CD24 -/low and ALDH1A1, varied between breast cancer cell lines and between primary tumours, and that these markers did not universally enrich for CSCs [ 9 ]. Heterogeneity amongst the phenotype of CSCs and the existence of multiple clones of cells acting as CSCs are well-established concepts in the haematological malignancies [ 10 ]. It has been proposed that breast CSCs may exhibit heterogeneity between the subtypes of breast cancer in a manner analogous to the haematological malignancies [ 11 ].

Although several studies have profiled CSC markers in primary breast tumours [ 12 – 17 ], they have reached different conclusions and their precise clinical significance remains uncertain. We set out to establish the clinical relevance of the CSC hypothesis in breast cancer by profiling a large cohort of primary breast carcinomas using IHC and tissue microarrays (TMAs). We hypothesised that the significance of CSC markers may not be universal amongst breast cancers but may be subtype specific. In order to assess the relationship between subtype and CSC markers, we have divided tumours into molecular subtypes according to a validated panel of IHC markers and stratified all analyses by oestrogen receptor status (ER).

Materials and methods

Study population.

The SEARCH breast study was used for this work. SEARCH is a large prospective population-based study of women diagnosed with breast cancer identified through the East Anglia Cancer Registry. It includes prevalent cases diagnosed before the age of 55 during 1991 to 1996 and still alive in 1996 and incident cases consisting of women under the age of 70 diagnosed after 1996; details of this study have been published previously [ 18 ]. A total of 4, 125 patients were included. Data on age at diagnosis, vital status including breast cancer-specific mortality, follow-up time, time between diagnosis and study entry, lymph node status, histological grade, tumour size, detection by mammographic screening, hormone therapy and chemotherapy were available. Details of the characteristics of the cohort are provided in Table 1 . The SEARCH (Studies of Epidemiology and Risk Factors in Cancer Heredity) study is approved by the Cambridgeshire 4 Research Ethics Committee; all study participants provided written informed consent.

Immunohistochemistry and scoring

Paraffin embedded tissue blocks containing primary breast carcinoma were constructed as tissue microarrays (TMAs) as previously described [ 19 ]. Each tumour was represented by a 0.6 mm tissue core. Staining patterns in histologically normal breast tissue were assessed from one block. IHC was used to assay for the expression of cancer stem-cell related and other relevant proteins as detailed in Additional file 1 . Briefly, 3 to 4 μm paraffin sections were dewaxed in xylene and rehydrated through graded alcohols. IHC was conducted using a BondMaX auto-immunostainer (Leica, Bucks, UK). Bound primary antibody was detected using a polymer-conjugated secondary antibody and staining was developed with 3-3'-diaminobenzidine (DAB). Double-immunostaining for detection of the CD44 + CD24 -/low phenotype was done in sequence, with detection of bound mouse anti-CD24 antibody with a biotinylated secondary antibody developed with DAB and detection of bound rabbit anti-CD44 with a polymer-conjugated secondary antibody developed using alkaline phosphatase with fast-red as a chromogen. Stained TMAs were viewed following digitisation using the Ariol platform (Genetix Limited, Hampshire, UK). The extent of staining was assessed blinded to all patient and tumour characteristics. Only membranous CD44 expression was scored while cytoplasmic and apical staining of lumens was scored for CD24. For ALDH1A1 and ALDH1A3 only cytoplasmic staining was considered and expression by stromal cells was assessed separately. All CSC markers were scored by a pathologist (HRA) using an Allred scoring system accounting for both the intensity of staining (0 = none, 1 = weak, 2 = moderate, 3 = strong) and the proportion of stained cells (0 = 0%, 1 = < 1%, 2 = 1 to 10%, 3 = 11 to 33%, 4 = 34 to 66%, 5 = > 66%) producing a sum score of the two values (intensity + proportion = 0 to 8). The scoring system was chosen in consultation between HRA and a senior pathologist (EP). HRA has extensive experience in interpreting IHC in breast cancer TMAs, with Kappa agreement statistics of 0.81, 0.88 0.65 and 0.85 for the markers aurora kinase a (AURKA), Trans-acting T-cell-specific transcription factor GATA-3 (GATA3), mast/stem cell growth factor receptor kit (c-Kit) and DNA replication licensing factor MCM2 (MCM2) respectively. The cut-offs for scoring systems used for each antigen are detailed in Additional file 1 . In order to address whether the combination of these markers offered superior prognostic value than their use separately, a "Total CSCs" variable was also created by adding the four dichotomised scores together to produce five categories. However, since only five cases were positive for all four markers, the four-marker-positive and three-marker-positive categories were merged leaving four categories (0 to 3).

Definition of molecular subtype

Tumours were classified into six molecular subtypes using a validated IHC-based surrogate classifier according to the expression of ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (CK5/6) and epidermal growth factor receptor (EGFR) [ 20 ]. Molecular subtypes were defined as: luminal 1a (ER+ or PR+, HER2-, CK5/6- and EGFR-), luminal 1b (ER+ or PR+, HER2-, CK5/6+ or EGFR+), luminal 2 (ER+ or PR+, HER2+), HER2 (ER- and PR-, HER2+), core basal phenotype (CBP) (ER- and PR-, HER2-, CK5/6+ or EGFR+) and 5-marker negative phenotype (5NP) (ER-, PR-, HER2-, CK5/6-, EGFR-).

Statistical analyses

All analyses were stratified by ER status since ER expression defines fundamentally distinct diseases within breast cancer [ 20 , 21 ]. Correlations between ordinal variables were assessed using Spearman's rank correlation. Associations between categorical variables were assessed using Pearson's chi-square test or Fisher's exact test as appropriate. Associations with age were assessed using a Wilcoxon rank-sum test. A log-rank test was used to compare survival between groups in Kaplan-Meier survival plots. A Cox proportional-hazards model was used to investigate association with breast cancer-specific survival (BCSS) at 10 years follow-up, providing a hazard ratio (HR) and 95% confidence interval (95% CI) for each variable. Although the date of diagnosis was used to calculate time-to-event, since SEARCH is an ongoing study the date of study entry was used to determine time under observation in order to adjust for the bias of prevalent cases in a prospectively recruiting study (left-truncation) [ 22 ]. Likelihood ratios from univariate analyses were used to decide whether to model markers as continuous or dichotomised variables. Cut-points for dichotomisation were informed by comparing strata with non-expressing cases against BCSS in a Cox-proportional hazards model where there was no trend to hazard ratios, a pre-determined cut-point of > 2 was applied. Analyses exploring associations with clinical, molecular and survival data were also conducted using zero as a cut-point for dichotomisation of CSC markers in order to determine the extent to which patterns were dependent on different cut-points. Multivariate analyses were conducted for CSC markers significantly associated with BCSS on univariate analysis. Multivariate models were modified in a backward stepwise manner until the most parsimonious fit was attained. Covariates in the initial model included age (> 55 years), lymph node status, grade, tumour size (< 2 cm, 2 to 4.9 cm, ≥5 cm), endocrine therapy, adjuvant chemotherapy, PR and HER2 status. Grade, tumour size and 'Total CSCs' were modelled as continuous variables. Standard log-log plots were used to explore compliance with the Cox proportional-hazards assumption. For variables which violated the assumption, the Cox model was extended to include a coefficient which varied as a function of log-time, where if the HR decreases with time the log of the coefficient is < 1 and, conversely, > 1 if the HR increases with time. The P -value of the time-varying coefficient was also used to determine whether it was reasonable to model a variable as time-dependent in different subgroups. This work complied with reporting recommendations for tumour marker prognostic studies (REMARK) criteria [ 23 ]. All analyses were conducted using Intercooled Stata version 11.1 (StataCorp, College Station, TX, USA). All Stata command lines used to produce reported analyses can be made available on request. Heatmaps and dendograms for a single randomly selected imputed dataset using Allred scores were produced using Cluster [ 24 ] and Java TreeView as previously described [ 25 ].

Missing data

The technical limitations of TMAs inevitably result in missing data. Tumour characteristics, such as size and morphology, tend to be correlated with the missingness of TMA data. Hence analyses, which exclude cases with missing data (complete case analysis (CCA)), can be biased [ 26 ]. In order to adjust for this source of bias we used multiple imputation (MI). MI is a method for handling missing data which has recently been validated for use in molecular pathology studies and been shown to produce more precise, less biased HRs compared to CCA [ 27 ]. MI generates a specified number of datasets wherein instances of missing data are resolved by randomly generated values which have been inferred under a model which takes account of the rest of the data. Subsequent analyses are performed on each imputed dataset and the results combined in a manner which accounts for the variability between imputed values. We used the ice command in Stata (StataCorp) to perform multiple imputation by chained equations [ 28 , 29 ] for 50 datasets across all IHC markers and relevant clinical variables including an outcome indicator (Nelson-Aalen estimator) to avoid inappropriate attenuation of associations [ 30 ]. Imputed data were then analysed using the mi commands. Results of survival analyses for both CCA and MI are presented for comparison.

CSC markers have distinct expression patterns in normal and neoplastic breast tissue

CSC markers showed distinct patterns of staining in normal breast tissue (Figure 1 ). Double-immunostaining for CD44 + CD24 -/low revealed membranous CD44 expression primarily in myoepithelial cells, although there was also some expression by luminal cells. CD24 localised to the apical surface of luminal cells and also stained intra-luminal secretions. These patterns are consistent with those previously reported [ 14 ]. In keeping with the observations of Ginestier et al . [ 3 ], strong ALDH1A1 expression was seen in isolated luminal cells in terminal-ductal lobular units (TDLUs). However, in some TDLUs ALDH1A1 expression was observed more frequently, including occasional TDLUs where almost all cells were positive for ALDH1A1, again in keeping with staining patterns previously reported [ 14 ]. Myoepithelial cells were also observed to express ALDH1A1 both in ducts and, less often, in TDLUs. Nearly all stromal cells expressed ALDH1A1. ALDH1A3 was expressed very weakly in the cytoplasm of all mammary epithelial cells and stromal cells. For ITGA6, membranous staining of myoepithelial cells was predominant while staining of luminal cells was seen less frequently.

Photomicrographs of CSC marker expression in normal breast tissue . A . Double immunostaining for CD44 (red) and CD24 (brown) reveals membranous CD44 expression of myoepithelial cells and some luminal cells. CD24 stains luminal apical membranes and secretions. B . IHC for ALDH1A1 showed different patterns, including staining of single luminal cells (right panel), of whole lobules (left panel) and stromal cells. C . IHC for ALDH1A3 (left panel) shows weak cytoplasmic of most epithelial and mesenchymal cells. IHC for ITGA6 (right panel) shows membranous staining of both myoepithelial and luminal cells.

CSC markers were expressed at different levels in primary breast carcinomas (Figure 2 and Table 2 ). ALDH1A1 expression was least frequent amongst the CSC markers, with 59% of cases having an Allred score of 0 compared to ALDH1A3 expression where 43% of cases were scored as 0. There were more tumours with a maximum Allred score of 8 for the CD44 + CD24 -/low phenotype than the other CSC markers (4% for CD44 + CD24 -/low and ≤1% for the other CSC markers). There was a gradation of staining for all markers, ranging from single isolated cells to small clusters of cells to rare cases where all cells were strongly stained (Figure 2 ).

Photomicrographs of CSC marker expression in invasive breast carcinoma . A . Membranous CD44 (red) staining and cytoplasmic CD24 (brown) staining of carcinoma cells. Tumours contained different proportions of positive cells, including cases dominated by CD44 + CD24 -/low cells (left panel) and others composed of CD24 expressing cells exclusively (right panel). B . Examples of low (left panel) and high (right panel) ALDH1A1 expression. C . Examples of high ALDH1A3 (left panel) and high ITGA6 (right panel) expression.

The correlations between CSC markers were stronger in ER- than ER+ disease (Additional file 2 ). In ER+ disease, ITGA6 was the only marker significantly correlated with all other CSC markers; it was most strongly correlated with ALDH1A3 with a Spearman's rho of 0.16, P < 0.0001. CD44 + CD24 -/low was significantly correlated with ITGA6 only (Spearman's rho = 0.09, P = 0.0006). ALDH1A1 and ALDH1A3 were only weakly correlated in ER+ disease (Spearman's rho = 0.07, P = 0.0035). By contrast, in ER- disease all CSC markers were significantly positively correlated. The correlations between markers were also generally stronger in ER- cases. ITGA6 and CD44 + CD24 -/low were the most strongly correlated markers (Spearman's rho = 0.29, P < 0.0001) while the weakest correlations were between ALDH1A1 and CD44 + CD24 -/low (Spearmans's rho = 0.11, P = 0.0141) and between ALDH1A1 and ITGA6 (Spearmans's rho = 0.11, P = 0.0176).

Association with clinical and molecular characteristics

CD44 + CD24 -/low and ALDH1A1 expression were significantly associated with clinical features in analyses stratified by ER status (Table 3 ). In ER+ disease, CD44 + CD24 -/low was associated with favourable clinical parameters. Of CD44 + CD24 -/low positive tumours, 33% were grade 1 whereas only 23% of CD44 + CD24 -/low negative tumours were grade 1 ( P = 0.006). Similarly, 68% of CD44 + CD24 -/low positive tumours were node negative, compared to 60% of CD44 + CD24 -/low negative cases ( P = 0.008). In ER+ disease CD44 + CD24 -/low positive tumours were associated with ductal morphology with 81% of CD44 + CD24 -/low positive cases being ductal compared to 73% of CD44 + CD24 -/low negative cases ( P = 0.012). ADLH1A1 positivity was significantly associated with high tumour grade in ER- disease only, with 43% of ALDH1A1 positive tumours being grade 3 compared to 20% of ALDH1A1 negative tumours ( P = 0.012). In contrast to ER+ disease, the CD44 + CD24 -/low phenotype was associated with higher tumour grade in ER- cases with 76% of positive tumours being grade 3 compared to 66% of negative cases ( P = 0.020). However, as observed in ER+ disease, CD44 + CD24 -/low positive tumours were more often node-negative in ER- cases also, with 64% of CD44 + CD24 -/low positive tumours being node-negative compared to 51% of CD44 + CD24 -/low negative cases ( P = 0.012). In accordance with a putative CSC-marker, ALDH1A1 was significantly associated with positive lymph node status in ER- disease with 59% of ALDH1A1 positive cases being node positive compared to 43% of negative cases ( P = 0.036). Notably, for analyses stratified by ER status, both ALDH1A3 and ITGA6 were not significantly associated with any clinical features.

All CSC markers were significantly associated with negative ER and PR status (Additional file 3 ). ITGA6 positive tumours showed the strongest association with 63% of cases being ER-, compared to 22% of ITGA6 negative cases ( P < 0.0001). Both ALDH1A1 and ALDH1A3 were associated with positive HER2 status where 26% of tumours positive for either marker were HER2 positive and 11% of negative cases were HER2 positive ( P < 0.0001). By contrast, CD44 + CD24 -/low positive cases were significantly associated with negative HER2 status ( P = 0.025). These relationships were reflected in the pattern of association with molecular subtype (Table 4 ). The distribution of all CSC markers by molecular subtype is illustrated as a heatmap in Figure 3 . Both CD44 + CD24 -/low and ALDH1A3 were negatively associated with the luminal 1a subtype in both ER+ and ER- disease. The luminal subtypes in the ER- subgroup are ER-, PR+ tumours, of which there were 128. In ER- disease ALDH1A1 was also negatively associated with the luminal 1a subtype. Within ER- disease, we also found CD44 + CD24 -/low to be associated with the CBP (basal) subtype consistent with previous reports [ 12 ]. There was a strong association between ALDH1A1 positivity and the HER2 subtype in ER- disease with 38% of ALDH1A1 positive tumours being of the HER2 subtype compared to 18% of ALDH1A1 negative cases ( P = 0.001).

Heatmap of CSC marker expression across breast cancer molecular subtypes . Heatmap illustrating the unclustered distribution of cases from a single randomly selected imputed dataset across molecular subtypes defined by a five-marker IHC classifier. CSC markers arranged by average linkage clustering.

CSC markers were significantly associated with higher proliferation measured by Ki67 labelling (Table 4 ). ALDH1A3 positivity was associated with high Ki67 expression in ER+ disease, with 39% of ALDH1A3 positive cases having a Ki67 fraction of > 10% compared to 22% of ALDH1A3 negative cases ( P = 0.002). In ER- disease, all CSC markers except ALDH1A3 were significantly associated with > 10% Ki67 staining. This relationship was strongest amongst ALDH1A1 positive tumours where 73% of positive cases were also Ki67 positive whereas just 51% of negative cases were Ki67 positive ( P = 0.003). Associations with clinical and molecular characteristics for non-CSC markers (CD44 - CD24 + , CD44 + CD24 + , stromal ALDH1A1, stromal ALDH1A3) are detailed in Additional files 4 and 5 .

CSC markers predict poor outcome in ER- disease

There were 1, 127 cases with complete data for all relevant clinical variables and all IHC markers of a potential 4, 125 (27%). The median follow-up time was 8.54 years with a total of 740 deaths of which 563 were deaths from breast cancer. There were 507 deaths from breast cancer when follow-up was restricted to 10 years. Further details of the characteristics of the study cohort can be found in Table 1 .

On univariate analysis, CSC markers showed distinct associations with survival and were more often associated with outcome in ER- disease (Additional files 6 and 7 ). The CD44 + CD24 -/low phenotype was not significantly associated with survival. Although ALDH1A1 was associated with poor outcome in both ER+ (HR 2.5, 95% CI 1.1 to 5.6, P = 0.027) and ER- disease (HR 2.4, 95% CI 1.4 to 4.1, P = 0.002) when complete data were analysed, analysis of imputed data only reproduced the association within ER- disease (HR 1.9, 95% CI 1.1 to 3.2, P = 0.022) and not in ER+ cases (HR 1.6, 95% CI 0.73 to 3.6, P = 0.233). ALDH1A3 was significantly associated with survival within the ER- subgroup in both complete (HR 1.8, 95% CI 1.1 to 3.1, P = 0.026) and imputed (HR 1.7, 95% CI 1.1 to 2.9, P = 0.032) datasets. Similarly, ITGA6 was associated with poorer survival in ER- disease only. This association was time-dependent in both the complete and imputed data with the extended Cox-model showing that the hazard associated with ITGA6 positivity fell over time. The Total CSC score, representing a composite measure of all four CSC markers, also showed an association with poorer survival restricted to ER- disease and in the imputed dataset this effect was time-dependent with a reduction in hazard over time.

On multivariate analysis both ITGA6 and the Total CSC composite score retained independent prognostic value in ER- disease (Table 5 and Figure 4 ). Multivariate analyses were restricted to CSC markers, which were associated with outcome on univariate analysis. ALDH1A1 showed independent prognostic value in ER- disease in CCA only. This effect was not reproduced when imputed data were analysed. ALDH1A3 was not significantly associated with outcome on multivariate analysis. As observed in univariate analyses, ITGA6 showed a time-dependent prognostic effect in both complete (HR 7.5, 95% CI 2.6 to 21.6, P < 0.001; T 0.18 95% CI 0.06 to 0.54, P = 0.002) and imputed (HR 2.8, 95% CI 1.2 to 6.3, P = 0.013; T 0.50, 95% CI 0.24 to 1.0, P = 0.055) datasets (CCA five-year BCSS adjusted for tumour size, grade and node status: ITGA6 negative = 87%; ITGA6 positive = 77%). In CCA the Total CSC variable was best modelled by not allowing for time-dependence. The Total CSCs composite score showed independent prognostic significance in complete data for ER- disease, conferring a 70% increased relative risk of event. In imputed data, the Total CSCs score also retained a significant association with survival for ER- disease and in a model allowing for time-dependence this effect diminished with time (HR 1.8, 95% CI 1.2 to 2.6, P = 0.002; T 0.71, 95% CI 0.51 to 0.99, P = 0.042). The five-year BCSS estimates for complete data adjusted for tumour size, grade and node status were Total CSCs = 0, 88%; Total CSCs = 1, 77%; Total CSCs = 2, 84%; Total CSCs = 3, 11%. Although for complete data the adjusted five-year survival is higher for a Total CSC score of 2 compared to 1, this was not reproduced in imputed data according to hazard ratios from a Cox proportional-hazards model where estimates of hazard increased successively with higher Total CSCs scores (data not shown). In order to investigate the relationship with survival time for ITGA6 and the Total CSCs score, follow-up time was divided into four periods (Table 6 ). Period-specific survival analyses showed that for ITGA6 adverse outcome associated with positivity was restricted to the first two years of follow-up, after which ITGA6 expression was not significantly associated with survival. Similarly, for the Total CSCs score unfavourable prognosis was restricted to the first four years after which there was no significant association with survival.

Kaplan-Meier survival plots of ITGA6 and Total CSC expression in ER- cases for BCSS . A . ITGA6 expression as a dichotomised variable (zero- to two-year follow-up, log-rank P = 0.0016; n (events): ITGA6- = 309 (14), ITGA6+ = 52 (8)). B . Total CSC composite score (zero- to four-year follow-up, log-rank P = 0.0173; n (events): CSC 0 = 164 (25), CSC 1 = 84 (22), CSC 2 = 19 (5), CSC 3 to 4 = 7 (4)).

The CSC hypothesis holds that CSCs are solely responsible for tumour recurrence and metastasis [ 4 ]. The existence of CSCs in solid tumours was first demonstrated in breast cancer in 2003; since then other studies have also shown that a CSC population can be isolated from primary breast tumours [ 1 – 3 ]. The idea that CSCs are resistant to chemo- and radiotherapy has also been supported by some studies [ 31 – 33 ]. These findings are potentially of profound clinical importance and many attempts to understand their clinical relevance have been made. However, despite these efforts, the significance of CSCs remains uncertain and many questions persist. We have attempted to establish the clinical relevance of CSCs in breast cancer by using IHC to assay for putative CSC markers in a large cohort of primary breast tumours in TMAs. We find that CSC markers show distinct patterns of expression and association with clinical and molecular features. We also show that the prognostic significance of CSC markers is largely restricted to ER- disease and that the most robust predictor of outcome is a composite score representing expression of all four markers investigated. We show that this score is the most powerful predictor of outcome and an independent prognostic factor in ER- disease.

Our study has some potential limitations. First, since putative CSCs were originally identified using flow cytometry we have assumed that this assay can be reasonably translated into an IHC based equivalent. Although we would expect these modalities to identify a population with a high degree of overlap, it is probable that there will be some discordance. Second, we have used TMAs to detect a subpopulation of cells of reputed scarcity and as a result there is likely to be some sampling error. However, we have attempted to mitigate this effect by using a very large study cohort which has also enabled us to address important questions, especially those related to subtype, with statistical robustness. Finally, our analyses should be considered exploratory. Validation studies using identical methodology in independent cohorts are necessary before definitive conclusions can be drawn. Analyses of associations with clinical, molecular and outcome data where zero was used as a cut-point for dichotomisation are presented in Additional files 8 , 9 , 10 and 11 . Most reported analyses are reproduced in these data, including the independent prognostic value of the 'Total CSC' score in the ER- subgroup. Although we find a small reduction in the hazard associated with CSC-positive cases treated with adjuvant chemotherapy compared to those who did not receive chemotherapy (data not shown), questions relating to the chemo-resistance of CSC-enriched tumours are best addressed in the context of randomised clinical trials.

The CD44 + CD24 -/low phenotype was the first marker described to enrich for breast CSCs [ 1 ]. This prompted several attempts to characterise CD44 + CD24 -/low cells in primary breast carcinomas. The prevalence of CD44 + CD24 -/low cells has been shown to be associated with the basal-like subtype [ 12 , 14 ], to favour distant metastasis [ 34 ] and to be inversely associated with lymph node status [ 13 ]. An association with survival has been demonstrated by one study [ 15 ] and gene signatures derived from CD44+ primary breast cancer cells and CD44 + CD24 - breast cancer cells (from xenografts or pleural effusions) have also been shown to correlate with outcome [ 35 , 36 ]. We also found that tumours enriched for the CD44 + CD24 -/low phenotype were associated with the basal-like subtype and with negative lymph node status. In addition, we found that CD44 + CD24 -/low tumours were associated with the luminal 1b subtype which, like basal-like tumours, is a subtype defined by basal cytokeratin expression. However, we did not find an association with survival.

Utilising the ALDEFLUOR assay, Ginestier et al . were able to use high aldehyde dehydrogenase activity as a basis for the enrichment of breast CSCs [ 3 ]. The group also found ALDH1A1 to be an independent prognostic factor when detected by IHC in primary breast carcinomas [ 3 ]. However, subsequent studies have not upheld the prognostic significance of ALDH1A1 [ 16 , 17 ]. Despite this and in keeping with the CSC hypothesis, ALDH1A1 has been found to predict response to chemotherapy [ 37 ]. We found that ALDH1A1 was an independent prognostic factor in ER- disease by CCA but that this finding was not reproduced when imputed data were analysed. Since missingness of data tends to be correlated across variables, estimates from CCA can be biased [ 26 , 27 ]. MI adjusts for this form of selection bias; hence, we consider estimates derived from MI more reliable than those from CCA. Our findings, coupled with those of other studies, imply that ALDH1A1 alone may not be a robust prognostic factor in breast cancer.

The assumption that ALDEFLUOR positivity of tumour cells correlates with ALDH1A1 expression by IHC has been questioned. Marcato et al . investigated which isoform of the aldehyde dehydrogenase family was most responsible for ALDEFLUOR positivity and found ALDH1A3 rather than ALDH1A1 to be the basis of ALDEFLUOR activity [ 5 ].

Although we found ALDH1A1 and ALDH1A3 to be positively correlated, the relationship was not strong (ER- cases, Spearman's rho = 0.19, P < 0.0001) and many cases showed discordant expression. We found ALDH1A3 to be significantly associated with survival in ER- disease on univariate analysis but this association was lost after adjustment for known prognostic factors in multivariate analysis.

ITGA6 expression has been linked to mammary stem cell biology in different ways. It has been used as a marker of murine mammary stem cells [ 5 , 6 ] and of tumorigenic cells of the MCF-7 breast cancer cell-line [ 7 ]. Pece et al . found ITGA6 to be highly expressed by normal human mammary stem cells and also showed that ITGA6 expression correlated with tumour grade [ 2 ]. Although we did not find a significant association between ITGA6 expression and higher tumour grade, there is a trend towards this in the ER- subgroup. ITGA6 expression has previously been shown to predict poor outcome in breast cancer [ 38 ]. We found ITGA6 to be an independent prognostic factor in ER- disease albeit restricted to the first two years of follow-up, after which ITGA6 expression was not associated with survival.

There is no highly specific marker for breast CSCs, rather the markers investigated in this study enrich tumour cell subpopulations for CSCs. We found a weak to moderate correlation between CSC markers, implying that different populations defined by these markers have some overlap but that most cells do not express these markers concurrently.

The idea of combining markers to increase the purity of subpopulations for CSCs was utilised by Ginestier et al . who showed that the combination of CD44 + CD24 -/low and ALDEFLUOR activity enabled the isolation of cells able to form tumours in NOD/SCID mice from as few as 20 cells, compared to 500 cells when sorted by ALDEFLUOR activity alone [ 3 ]. Based on this finding, Neumeister et al . set out to establish the significance of combined CSC marker expression by investigating the expression of CD44 and ALDH1A1 in a cohort of 639 primary breast tumours [ 17 ]. They found no association with survival when they analysed the markers separately, but found the combination of the two markers to be an independent predictor of outcome [ 17 ]. Along these lines, we generated a score representing the sum of the dichotomised scores for all four markers. We found that this score was an independent prognostic factor in ER- disease.

In summary, we have investigated the expression of putative CSC markers in a large cohort of primary breast carcinomas, treating ER+ and ER- tumours as distinct entities. We found that the patterns of association with clinical and molecular characteristics are different between CSC markers but that all markers were strongly associated with negative ER status. CSC markers did not carry significant prognostic value in ER+ tumours; therefore, additional markers enabling IHC quantification of CSCs in ER+ tumours (75 to 80% of all breast cancers) are required. In ER- disease, although only ITGA6 retained independent prognostic significance, a composite score representing expression of all four markers was the most powerful predictor of outcome. Based on our findings and in the absence of more specific CSC markers, we propose that it may be necessary to utilise a panel of CSC markers in order to effectively translate knowledge of CSCs into patient benefit.

Abbreviations

retinal dehydrogenase 1

aldehyde dehydrogenase family 1 member A3

aurora kinase a

breast cancer-specific survival

core-basal phenotype

complete case analysis

confidence interval

cytokeratin 5/6

cancer stem cell

3-3'-diaminobenzidine

epidermal growth factor receptor

estrogen receptor

human epidermal growth factor receptor 2

hazard ratio

immunofluorescence

immunohistochemistry

integrin alpha-6

multiple imputation

non-obese severe combined immunodeficiency

progesterone receptor

Study of Epidemiology and Risk Factors in Cancer Heredity

terminal-ductal lobular unit

tissue microarray.

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Acknowledgements

We are very grateful to the participants of the SEARCH breast study who permitted the use of their tissue for research and to the many individuals who have made this work possible. In particular, we acknowledge: the SEARCH team, the Eastern Cancer Registration and Information Centre, and Leigh-Anne McDuffus and Dr Will Howat at the Histopathology Core Facility at the CRUK Cambridge Research Institute for immunohistochemical staining.

HRA is supported by a fellowship funded by the Addenbrooke's Charitable Trust and the NIHR Cambridge Biomedical Research Centre. SJD is supported by a fellowship funded by the Commonwealth Scholarship and Fellowship program and Cancer Research UK. SEARCH is funded by programme grants from Cancer Research UK (C490/A11019 and C490/A11024).

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Department of Oncology, University of Cambridge, Cambridge, CB1 9RN, UK

H Raza Ali, Sarah-Jane Dawson, Paul D Pharoah & Carlos Caldas

Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 ORE, UK

H Raza Ali, Sarah-Jane Dawson, Elena Provenzano & Carlos Caldas

Cambridge Breast Unit, Addenbrooke's Hospital, Cambridge University Hospital NHS Foundation Trust and NIHR Cambridge Biomedical Research Centre, Cambridge, CB2 2QQ, UK

Sarah-Jane Dawson, Elena Provenzano & Carlos Caldas

Cambridge Experimental Cancer Medicine Centre (ECMC), Cancer Research UK Cambridge Research Institute, Li Ka-Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK

Elena Provenzano, Paul D Pharoah & Carlos Caldas

Strangeways Research Laboratories, University of Cambridge, Cambridge, CB1 9RN, UK

Fiona M Blows & Paul D Pharoah

Department of Histopathology, Addenbrooke's Hospital, Cambridge University Hospital NHS Foundation Trust, Cambridge, CB2 2QQ, UK

Elena Provenzano

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Correspondence to H Raza Ali .

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The authors declare that they have no competing interests.

Authors' contributions

HRA, EP, PDP and CC designed the study. HRA scored TMAs for CSC markers and conducted statistical analyses. SJD and EP scored TMAs for non-CSC markers. FMB constructed TMAs and compiled clinical data. PDP and CC are the project leaders for molecular pathology studies in SEARCH. All authors read and approved the final manuscript.

Electronic supplementary material

Additional file 1: reagents and protocols for immunohistochemistry. (pdf 115 kb), additional file 2: contingency table for csc markers by spearman's rank correlation. (pdf 92 kb), additional file 3: csc marker associations with er, pr and her2. (pdf 102 kb), additional file 4: non-csc marker associations with clinical characteristics. (pdf 192 kb), additional file 5: non-csc marker associations with molecular characteristics. (pdf 137 kb), additional file 6: univariate survival analyses for all clinical and molecular markers (cca). (pdf 137 kb), additional file 7: univariate survival analyses for all clinical and molecular markers (mi). (pdf 124 kb), 13058_2011_2861_moesm8_esm.pdf.

Additional file 8: CSC marker associations with clinical characteristics using zero as a cut-point for dichotomisation. (PDF 120 KB)

13058_2011_2861_MOESM9_ESM.PDF

Additional file 9: CSC marker associations with molecular characteristics using zero as a cut-point for dichotomisation. (PDF 115 KB)

13058_2011_2861_MOESM10_ESM.PDF

Additional file 10: Univariate survival analyses of CSC markers using zero as a cut-point for dichotomisation. (PDF 99 KB)

13058_2011_2861_MOESM11_ESM.PDF

Additional file 11: Multivariate survival analyses for 'Total CSCs' using zero as a cut-point for dichotomisation of constituent CSC markers. (PDF 102 KB)

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Ali, H.R., Dawson, SJ., Blows, F.M. et al. Cancer stem cell markers in breast cancer: pathological, clinical and prognostic significance. Breast Cancer Res 13 , R118 (2011). https://doi.org/10.1186/bcr3061

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Received : 16 August 2011

Revised : 21 November 2011

Accepted : 23 November 2011

Published : 23 November 2011

DOI : https://doi.org/10.1186/bcr3061

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The Cancer Stem Cell Hypothesis: Failures and Pitfalls

Rahman, Maryam MD * ; Deleyrolle, Loic PhD * ; Vedam-Mai, Vinata PhD * ; Azari, Hassan PhD * ; Abd-El-Barr, Muhammad MD, PhD *† ; Reynolds, Brent A PhD *

*Department of Neurosurgery, University of Florida, Gainesville, Florida; †Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

Received, January 15, 2010.

Accepted, June 5, 2010.

Correspondence: Brent Reynolds, PhD, or Maryam Rahman, MD, Box 100265, Department of Neurosurgery, University of Florida, Gainesville, FL 32610. E-mail: [email protected] or [email protected]

Based on the clonal evolution model and the assumption that the vast majority of tumor cells are able to propagate and drive tumor growth, the goal of cancer treatment has traditionally been to kill all cancerous cells. This theory has been challenged recently by the cancer stem cell (CSC) hypothesis, that a rare population of tumor cells, with stem cell characteristics, is responsible for tumor growth, resistance, and recurrence. Evidence for putative CSCs has been described in blood, breast, lung, prostate, colon, liver, pancreas, and brain. This new hypothesis would propose that indiscriminate killing of cancer cells would not be as effective as selective targeting of the cells that are driving long-term growth (ie, the CSCs) and that treatment failure is often the result of CSCs escaping traditional therapies.

The CSC hypothesis has gained a great deal of attention because of the identification of a new target that may be responsible for poor outcomes of many aggressive cancers, including malignant glioma. As attractive as this hypothesis sounds, especially when applied to tumors that respond poorly to current treatments, we will argue in this article that the proposal of a stemlike cell that initiates and drives solid tissue cancer growth and is responsible for therapeutic failure is far from proven. We will present the point of view that for most advanced solid tissue cancers such as glioblastoma multiforme, targeting a putative rare CSC population will have little effect on patient outcomes. This review will cover problems with the CSC hypothesis, including applicability of the hierarchical model, inconsistencies with xenotransplantation data, and nonspecificity of CSC markers.

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The cancer stem cell hypothesis: a work in progress

Affiliation.

• 1 Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5324, USA. [email protected]
• PMID: 17075578
• DOI: 10.1038/labinvest.3700488

There is a growing body of evidence that supports the idea that malignant tumors are initiated and maintained by a population of tumor cells that share similar biologic properties to normal adult stem cells. This model, the cancer stem cell (CSC) hypothesis, is based on the observation that tumors, like adult tissues, arise from cells that exhibit the ability to self-renew as well as give rise to differentiated tissue cells. Although the concept of the CSC is not entirely new, advances made over the past two decades in our understanding of normal stem cell biology in conjunction with the recent application of these concepts to experimentally define CSCs have resulted in the identification of CSCs in several human malignancies.

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Understanding pancreatic cancer stem cells and their role in carcinogenesis: a narrative review

The purpose of this review article is to describe the pathogenesis of pancreatic cancer and to better understand the role of abnormal stem cells in the development of pancreatic cancer.

Pancreatic cancer is a highly fatal disease that is caused by the uncontrolled proliferation of pancreatic exocrine or neuroendocrine glands. It is believed that pancreatic cancers arise from a small population of abnormal cancer stem cells (CSCs) that promote tumorigenesis, tumor metastasis and therapeutic resistance. The molecular markers CD133, CXCR4, DCLK1, c-MET, ABCG2 and Lgr5 are routinely used to detected and observe the behaviours of pancreatic cancer stem cells (PCSCs).

A comprehensive search was performed on PubMed, Google Scholar, Scopus, Clinicaltrials.gov and Web of Science using related keywords. Articles focusing on PCSCs and pancreatic cancer pathogenesis, biochemistry and clinical trials were selected.

Conclusions

Although very little is known about the exact cause of pancreatic cancer, PCSCs seem to play an important role in carcinogenesis. Mutated biochemical cascades include Sonic Hedgehog, K-RAS-JNK, DLL4/Notch and Nodal/Activin. Several clinical trials are trying to determine if the transplantation of hematopoietic stem cell or peripheral stem cells could be useful for the treatment of such an aggressive tumor.

Introduction and background

Pancreatic cancers are aggressive tumors which can be subdivided into two large categories: exocrine pancreatic cancers (e.g., adenocarcinoma) and neuroendocrine pancreatic cancers ( 1 ). Pancreatic adenocarcinomas represent approximately 85% of all pancreatic cancer cases and arise from pancreatic exocrine glands, whereas pancreatic neuroendocrine cancers represent slightly less than 5% of all pancreatic cancer cases and originate from endocrine tissues of pancreas ( 1 ).

According to GLOBOCAN 2018, pancreatic cancer is the 11 th most common malignancy across the globe ( 2 ). It remains the seventh and third leading cause of cancer death worldwide and in the United States of America, respectively ( 2 , 3 ). A 2021 Surveillance, Epidemiology, and End Results (SEER) analysis estimated that there were 40,430 new cases of pancreatic cancer (79% men and 31% women) and 48,220 pancreas cancer-related deaths in the United-States ( 4 ). This disease has an overall poor 5-year survival rate (10%) and has a slightly higher mortality rate in men (12.7 per 100,000) than in women (9.6 per 100,000) ( 4 ).

The exact cause of pancreatic remains poorly understood, but involves a plethora of risk factors such as smoking status, genetic predispositions, advanced age, diabetes mellitus, obesity and a family history of chronic pancreatitis ( 5 ). The signs and symptoms of pancreatic cancer are mainly dependent on two factors: (I) size/location of the tumor and (II) affected organs. Based on these parameters, pancreatic tumors can be assigned different numerical stages to describe their progression. If a pancreatic cancer is ≤2 cm (Ia) or >2cm (Ib) and only found in the pancreas, it is classified as a stage I neoplasm ( 5 ). Stage II cancers often grown nearby organs but have not infiltrated lymph nodes ( 5 ). A diagnosis of stage III pancreatic cancer indicates that the initial tumor is large and has invaded the lymphatic system but has not spread to distant sites ( 5 ). Stage IV pancreatic tumors are characterized by their ability to metastasize to distant organs (e.g., lungs or liver) ( 5 ). Unfortunately, a significant portion of patients remain asymptomatic until the advanced stage. Signs and symptoms include unintentional weight loss, nausea/vomiting, anorexia, and abdominal/back discomfort. The presence of jaundice, pale stools, dark urine and pruritus are signs that the underlying tumor might have metastasized or is exerting mass effect ( 5 ).

Pancreatic cancer remains a disease that is very hard to treat despite the availability of various treatment options. It is assumed that this neoplasm arises from a small population of irregular stem cells that divide uncontrollably. The purpose of this narrative review is to shed some light on the genomic complexity of pancreatic cancer and to better understand the role of pancreatic cancer stem cells (PCSCs) in tumor development. We present the following article in accordance with the Narrative Review reporting checklist (available at https://dx.doi.org/10.21037/sci-2021-067 ).

A thorough investigation was performed using the following search engines: PubMed, Google Scholar, Scopus, Clinicaltrials.gov and Web of Science. The most recent query was performed in December 2021 while reviewing the article. The selected articles were either basic research, clinical research, or translational research papers. Keywords such as “pancreatic cancer”, “pancreatic cancer stem cells”, “stem cells”, “pancreatic cancer biochemical cascades”, “pathogenesis” and “pancreatic cancer clinical trials” were used. A paper was only included if it followed one or more of the following criteria: (I) written in English; (II) published the data of in vitro or in vivo studies that discussed the behaviour of cancer stem cells (CSCs) in pancreatic cancer; (III) published data on drugs targeting PCSCs. A total of 48 articles were used for the creation of this literature review.

PDAC subtyping

Molecular subtypes.

Bailey et al . postulated that pancreatic adenocarcinomas (PDACs) can be subdivided into four different categories based on their expression patterns ( 6 ). The squamous subtype was associated with a poor prognosis because it harbored mutations in several important tumor suppressor genes (e.g., TP53 and KDM6A ) which led to a complete loss of endodermal identity ( 6 ). The pancreatic progenitor subtype was characterized by its preferential expression of PDX1 , MNX1 and FOXA2/3 ( 6 ). These genes play an important role in the early development of the pancreas via the regulation of glycosylation, fatty acid oxidation and steroid hormone/drug metabolism ( 7 ). Although the immunogenic subtype is similar to the pancreatic progenitor subtype in terms of genetic expression patterns, it defined by remarkable immune cell infiltration due to aberrancies in B-cell signalling pathway, antigen presentation and Toll-like receptor (TLR) signalling pathways ( 6 ). The aberrantly differentiated endocrine exocrine ( ADEX ) subtype was caused by genes involved in exocrine function (e.g., NR5A2 and RBPJL ), endocrine differentiation (e.g., NEUROD1 and NKX2-2 ) and mutant KRAS activation ( 6 ).

Immune subtypes

According to Liu et al. , PDACs can be separated into three different immune clusters (C1-3) ( 8 ). Subtype C1 pancreatic cancers are viewed as immune-cold because they lacked several key immune modulators such as INF-γ and TGF-β ( 8 ). In contrast, subtype C2 cancers have an immune-suppressive phenotype as they are associated with elevated TGF-β enrichment scores and low lymphocyte enrichment scores ( 8 ). Subtype C3 pancreatic tumors are immune-hot cancers because they display an abundance of inflammatory markers and are linked with severe immune cell infiltration ( 8 ).

Cancer stems cells and pancreatic cancer

Cancer stem cell hypothesis and tumorigenesis.

It is hypothesized that all cancers originate from a very small population of abnormal pluripotent stem cells also known as CSCs or tumour-initiating cells ( 9 ). The ability of CSCs to self-renew indefinitely and differentiate into various cells is an important feature in tumor formation, progression, metastasis, and therapy resistance ( 10 - 12 ). Although scientists are still trying to determine the exact origin of CSCs, it is thought that CSCs arise from non-malignant progenitor cells that have acquired various somatic mutations ( 9 ). Another hypothesis is that CSCs are the product of differentiated cells re-acquiring stem-cell like properties via the epithelial-to-mesenchymal transition (EMT) ( 13 ). Markopoulos et al . have noticed that inflammatory cytokines (e.g., TNFα, TGFβ, IL-1 and IL-6) may accelerate this phenomenon by activating master transcription factors (TFs) such as Smads, STAT3 and NF-kB and EMT-inducing TF families such as Snail, Twist and Zeb ( 14 ).

CSCs increase in number by undergoing symmetric cell division which generates two identical pluripotent daughter cells ( 15 ). CSCs can also self-renew through asymmetric cell division, but this process produces a tumor progenitor cell (TPC) and a daughter cell possessing stem-cell properties ( 15 ). The delicate balance between symmetric and asymmetric cellular division is tightly controlled by various oncogenes that employ the Hedgehog, Notch and Wnt signalling mediators ( 16 , 17 ).

In 2007, Li et al. first defined PCSCs by observing the behaviour of human PDAC cells transplanted into immunosuppressed mice ( 18 ). There was a small subpopulation of PCSCs, which represented 0.2-0.8% of all the tumor cells, which simultaneously expressed the CD44, CD24 and ESA/EpCAM (epithelial specific antigen) surface markers ( 18 ). These neoplastic cells displayed a 100-fold increase in tumorigenic potential compared to their CD44 - CD24 - ESA - counterparts and were able to generate the development signal molecule sonic hedgehog (SHH) and undergo symmetric as well as asymmetric cellular division ( 18 ). Other known PCSC markers include CD133, ALDH1, DCLK1, CXCR4, ABCG2, c-Met, and Lgr5 ( 19 - 24 ).

Also known as prominin-1, CD133 is a pentaspan transmembrane glycoprotein that serves as a biological marker for stem cells and CSCs ( 25 ). Although the exact role of CD133 in the progression of cancer remains elusive, a study by Hermann et al . revealed that a population of CD133 + /CXCR + CSCs were able to sustain pancreatic tumor growth and were essential for metastasis ( 26 ). CD133 is thought to confer a metastatic phenotype by upregulating the expression of N-cadherin via the Src signalling pathway, which plays a critical role in the EMT regulatory loop ( 27 , 28 ). Moreover, the expression of CD133 in PDACs was associated with tumorigenesis and resistance to chemotherapy ( 26 ). The chemokine receptor CXCR4, once bound to its primary ligand CXCL12, has also been implicated in pancreatic cancer tumorigenesis, infiltration, and metastasis ( 29 ). Billadeau et al. proposed that this process, in part, involves the activation of ERK-mediated biochemical cascades which control the expression of different angiogenesis-related genes such as VEGF, CD44, HIF1α and IL8 ( 30 ). As part of the ALDH super-family, ALDH1 is a cytosolic enzyme that plays a vital role in the detoxification of exogenous and endogenous aldehydes ( 31 ). This protein marker was first described by Ginestier et al. in 2007 and is now used as a functional marker for normal stem cells ( 32 ). Rasheed et al . noticed that increased expression of ALDH1A1 in pancreatic cancer cells was associated with a worse prognosis ( 20 ). Furthermore, ALDH1 has been shown to regulate the proliferation of PDACs and provide them with gemcitabine and cyclophosphamide resistance ( 33 ). DCLK1 is a serine/threonine-protein kinase belonging to the doublecortin (DCX) family that plays an important role in PCSC biology. Several studies noticed that DCLK1 was overexpressed in PCSCs displaying invasive and metastatic properties ( 21 ). This tumor marker accelerates the development of malignant features in CSCs by robustly upregulating genes (e.g., SNAI 2 , CDH2 and VIM ) that modulate EMT ( 34 ). Furthermore, it is postulated that DCLK1 overexpression might sustain neoplastic growth as a study by Westphalen et al. discovered that DCLK1+ cells were necessary for the regeneration of pancreatic tissue following injury ( 35 ). The hepatocyte growth factor (HGF)/c-MET axis is one of many signalling pathways that is necessary for the expression of DCLK1 in tumor cells. It facilitates pancreatic cancer progression by mediating the interaction between PCSCs and stromal pancreatic stellate cells (PSCs) ( 36 ). Although ABCG2 and Lgr5 have also been implicated in the development of PDACs, very little is known about their precise role in development of pancreatic cancer tumorigenesis ( 37 , 38 ).

Aberrant biochemical pathways causing therapy resistance

Tumors are composed of a heterogenous mix of active tumor cell lines and clonal stem cells; the latter being subdivided into CSCs and tumor initiating cells (TICs). While CSCs tend to remain quiescent in the periphery, TICs are responsible for the continued proliferation of the tumor progeny ( 16 , 17 ). Experiments by Hermann et al. found that PCSCs have profound resistance to very high concentrations of gemcitabine (up to 100 micrograms/mL) ( 26 ). These same concentrations were able to induce apoptosis in virtually all other tumor cell lines. In fact, gemcitabine notably led to selective pressure of CD133+ PCSCs, leading to the chief hypothesis that pancreatic treatments may expand the tumorigenic cell population by producing a relapsed progeny of resistant tumor cell types. Early studies of hematopoietic stem cells by Goodell et al. in 1996 suggested that stem cell resistance to therapy is likely linked to their quiescent nature, enhanced repair of DNA damage, and anti-apoptotic mechanisms such as efflux membrane transporters, specifically, ABC transporters ( 39 ).

Since the discovery of PCSCs as potential drivers of therapeutic resistance, several studies ( Table 1 ) have researched the proposed molecular pathways. Hedgehog signaling through the SHH pathway was identified as a mediator of tumorigenesis by Thayer et al. in 2003 ( 40 ). Hedgehog signaling is essential in pancreatic embryonic pathways and its misexpression was shown to lead to the development of precursor lesions and the subsequent development of mutations in K-RAS and HER-2/neu ( 40 ). Several studies evaluating the inhibition of this pathway suggest that PCSCs may be induced to undergo apoptosis, thus serving as an adjuvant to chemotherapy ( 41 - 43 ).

CSC, cancer stem cell; PCSC, pancreatic cancer stem cell.

The Notch signaling pathway is responsible for stem cell renewal, differentiation, and survival and is an important driver of pancreatic embryonic development. Overexpression of Notch proteins by CD133+ PCSCs has been shown to promote self-renewal through vascular development and is key in therapeutic resistance ( 44 ). Hoey et al. had previously shown that targeting delta like ligand 4 (DLL4), an important component of Notch signaling, in colon and breast cancer reduced the frequency of TICs ( 45 ). They expanded this knowledge to pancreatic cancer in 2012 and found that combining anti-human DLL4 and anti-murine DLL4 had pronounced reductions in TICs, likely by the induction of dysfunctional vasculature within the tumor microenvironment ( 44 ).

Nodal/Activin are components of the TGF-beta superfamily and are chiefly responsible for the regulation of embryonic stem cells. Lonardo et al . found that nodal/activin were highly expressed in PCSCs and the inhibition of their activin-like (Alk) 4/7 receptor reduced or eliminated the capacity for their self-renewal ( 46 ). This effect was enhanced by co-blockade with SHH inhibitors and ultimately showed reversal of chemoresistance of PCSCs. The c-Jun NH2-terminal kinase (JNKs) is a sub-group of the mitogen-activated protein kinases, which are often dysregulated in many cancer types. JNKs have been shown to be crucial to stem cell self-renewal in human glioblastoma, which led to their evaluation by Okada et al . in PCSCs ( 47 , 48 ). Inhibition of the JNK axis deprived the PCSCs of their ability to sustain tumor growth. Additionally, K-ras mutations were shown to contribute to the maintenance of the PCSCs, and combination therapies which targeted K-Ras-JNK significantly reduced TICs and tumor bulk growth ( 48 ).

Ongoing clinical trials and future directives

Most clinical trials currently investigating PCSCs are largely focused on hematopoietic stem cell transplant (HSCT) or the transplantation of peripheral stem cells ( Table 2 ). A phase I, single-arm trial evaluating metastatic pancreatic adenocarcinoma with BRCA 1 or 2 mutation is treating patients with a drug combination of melphalan, BCNU (carmustine), and vitamins in association with autologous HSCT. The primary outcomes of this study are the evaluation of toxicity and adverse events. The medical college of Wisconsin (through collaboration with Massachusetts Institute of Technology) is evaluating whether the antibiotic doxycycline can kill a significant fraction of metakaryotic (PCSCs) cells in pre-treated pancreatic adenocarcinoma. This phase 2 trial will administer doxycycline during radiation treatment and following neoadjuvant chemotherapy. Patients will then undergo surgical resection.

Researchers are currently studying the feasibility and safety profile of PSCA-specific CAR-T cells (BPX-601) with concurrent administration of rimiducid in PSCA-positive advanced solid tumors. This is a Phase I/II dose escalation and expansion trial which will evaluate the safety and efficacy in metastatic pancreatic and prostate cancer. A Chinese study sponsored by Shenzhen University General Hospital is currently recruiting for a Phase I trial studying the efficacy and safety of CD276-targeted CAR-T cells in refractory pancreatic cancer. This biologic is a member of the B7 co-stimulatory family and has been shown to be overexpressed in many cancer types and is associated with a poorer prognosis.

Pancreatic tumor heterogeneity is driven by sub-groups and functional differences within sub-group clones. PCSCs and their counterparts, tumor-initiating cells, are largely responsible for this diverse tumor microenvironment and often play crucial roles in the recurrence and chemoresistance seen in advanced pancreatic cancer. Many of the molecular pathways involved in the self-renewal and function of stem cells have been identified and their co-inhibition coupled with standard therapeutic regimens may improve progression, recurrence, and overall survival.

Acknowledgments

Funding : None.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/ .

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://dx.doi.org/10.21037/sci-2021-067

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/sci-2021-067 ). The authors have no conflicts of interest to declare.

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What Are Cancer Stem Cells?

They influence how a tumor grows

Role in Cancer Growth

Resistance to therapy, importance of cancer stem cell research.

Cancer stem cells are a small subpopulation of cells found within tumors that are tumorigenic, meaning they can create a cancerous tumor. Self-renewal and the ability to differentiate into diverse cell types are hallmark features of cancer stem cells. They can reproduce themselves and sustain cancer in the body. They are therefore hypothesized to be the primary driver of cancer growth and metastasis . This is called the stem cell theory of cancer. Effective cancer treatment then must target and attack these cells. Doing so can improve the chances of cancer remission.

Cancer stem cells have been identified in brain , breast , colon , ovarian , pancreatic , and prostate tumors, as well as in melanoma , multiple myeloma , nonmelanoma skin cancer , and leukemia .

Cancer stem cell research is ongoing, and new studies are emerging frequently.

What Are Stem Cells?

Stem cells are undifferentiated (or only partly differentiated) human cells that can turn into different types of cells in the body, from nerve cells (neurons) to brain cells. They can also fix damaged tissues. They must possess two major qualities: self-renewal and the capacity to differentiate. Stem cell-based therapies are also being studied to treat serious illnesses such as paralysis and Alzheimer's disease .

There are two types of stem cells: embryonic and adult stem cells. Embryonic stem cells come from unused embryos and are created from an in vitro fertilization process. They are pluripotent, meaning they can turn into more than one cell type. Within adult stem cells, there are two different types: one type comes from fully developed tissues such as the brain, skin, and bone marrow, and the other is induced pluripotent stem cells, which have been changed in the lab to be more like embryonic stem cells.

luismmolina / Getty Images

The stem cell theory of cancer hypothesizes that cancer stem cells are thought to drive tumor initiation and may be responsible for therapeutic resistance and cancer recurrence.

Like many areas of biomedical research, cancer stem cells are an evolving field of study. Multiple studies have indicated that insufficient evidence exists to confirm the existence of cancer stem cells. A review of 1,000 Web of Science publications revealed that only 49% supported the cancer stem cell hypothesis.

Cell surface markers can be used to identify cancer stem cells, as has been done in research that supports the hypothesis that these stem cells do not respond to traditional therapies such as chemotherapy . This research also supports the idea that cancer stem cells are the source of cancer metastasis.

Like all stem cells, cancer cells must have the following characteristics:

• Self-renewal: When stem cells divide into more stem cells, this process is referred to as cell renewal.
• Cell differentiation: Cell differentiation is when a cell changes from a less differentiated to a more differentiated cell type.

Cancer stem cells use specific signaling pathways. It is hypothesized that cancer stem cells can also act as a reservoir of cancer cells, which may cause a relapse after surgery, radiation, or chemotherapy has eliminated all observable signs of cancer. Targeting these cells would thus highly improve the chances of a patient's remission if cancer stem cells are the origin of the tumor.

Cancer stem cells have the capacity to change into more specialized cell types, so they can potentially lead to tumor cell heterogeneity. Due to this quality, they are cited as a major factor of chemoresistance. Their highly resistant nature can lead to tumors metastasizing and tumor regrowth. As such, the developing research on cancer stem cells could dramatically change the prognosis of multiple cancer types.

Also, many new anticancer therapies are evaluated based on their ability to shrink tumors, but if the therapies are not killing the cancer stem cells, the tumor will soon grow back, often with resistance to the previously used therapy.

Cancer stem cell research is critical because it addresses the potential root cause of cancer proliferation and can lead to the development of more effective and safer treatments. Treatments targeting cancer stem cells will likely have fewer side effects compared with existing options because they will leave other kinds of cells untouched.  

Understanding these cells can also help modify current treatments for maximum effect. Research has shown that cancer stem cells are resistant to the ionizing radiation used to treat cancer. Understanding this resistance may in the future help researchers find compounds that undermine this process and make cancer stem cells vulnerable to radiation damage.  

A Word From Verywell

Cancer stem cell research offers promising hope for the continually evolving field of cancer therapeutics, but more research needs to be done to confirm the stem cell theory of cancer. Cancer stem cell research has the potential to generate better treatments for cancer with fewer side effects, as well as to improve the efficacy of current treatment options. If the theory is proven, therapies targeting cancer stem cells may even be able to lower the rate of cancer recurrence. While its existence is still up for debate, it represents an exciting opportunity to advance cancer care and improve cancer survival.

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Tumor microenvironment acidosis favors pancreatic cancer stem cell properties and in vivo metastasis

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The acidic tumor microenvironment favors cancer aggressiveness via incompletely understood pathways. Here, we asked whether acidic environments select for cancer stem cell (CSC) properties. Bulk RNA-seq of Panc-1 human pancreatic cancer cells adapted to extracellular pH 6.5 revealed upregulation of CSC markers including CD44, EpCam, Nestin and aldehyde dehydrogenases, and CSC pathway enrichment. We therefore assessed CSC characteristics of acid-adapted (AA) and non-adapted (Ctrl) PaTu8988s and MiaPaca-2 pancreatic cancer cells. Compared to Ctrl, AA cells exhibited increased ALDH- and β-catenin activity and pancreatosphere-forming efficiency, classical CSC characteristics. Panc-1, PaTu8988s and MiaPaCa-2 AA cells differed in CSC marker expression, and AA cells did not exhibit typical flow cytometric CSC populations. However, single-nucleus sequencing identified the acid adaptation-induced emergence of a population with clear CSC characteristics. Finally, in an orthotopic mouse model, AA Panc-1 cells drove strongly increased aggressiveness and liver metastasis compared to Ctrl cells.

We conclude that acid-adaptation of pancreatic cancer cells leads to enrichment of a CSC phenotype with unusual traits, providing new insight into how acidic tumor microenvironments favor cancer aggressiveness.

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