Tumor‐Initiating Label‐Retaining Cancer Cells in Human Gastrointestinal Cancers Undergo Asymmetric Cell Division

Label‐retaining cells (LRCs) have been proposed to represent adult tissue stem cells. LRCs are hypothesized to result from either slow cycling or asymmetric cell division (ACD). However, the stem cell nature and whether LRC undergo ACD remain controversial. Here, we demonstrate label‐retaining cancer cells (LRCCs) in several gastrointestinal (GI) cancers including fresh surgical specimens. Using a novel method for isolation of live LRCC, we demonstrate that a subpopulation of LRCC is actively dividing and exhibits stem cells and pluripotency gene expression profiles. Using real‐time confocal microscopic cinematography, we show live LRCC undergoing asymmetric nonrandom chromosomal cosegregation LRC division. Importantly, LRCCs have greater tumor‐initiating capacity than non‐LRCCs. Based on our data and that cancers develop in tissues that harbor normal‐LRC, we propose that LRCC might represent a novel population of GI stem‐like cancer cells. LRCC may provide novel mechanistic insights into the biology of cancer and regenerative medicine and present novel targets for cancer treatment. STEM CELLS 2012; 30:591–598

The concept of ACD-NRCC was introduced by Cairns [11]. It is one possible method by which stem cells divide asymmetrically and self-renew. ACD-NRCC suggests that each chromosome in some stem cells contains one DNA strand that is conserved throughout multiple ACDs (Fig. 1A). By maintaining these DNA template strands within the daughter stem cell, stem cells could avoid accumulation of mutations from replication errors. It is a potential mechanism by which replication errors are preferentially segregated into the daughter cell destined to differentiate and eventually be eliminated like most mature epithelial cells [1, 2, 4-6, 12-16, 19]. However, other investigators could not confirm the existence of ACD-NRCC or LRC [17,[20][21][22][23]. The question whether LRCs are generated by ACD-NRCC versus slow cycling remains highly controversial [18].
To test the cancer stem cells hypothesis [24][25][26][27][28][29][30][31][32], we developed a novel methodology that enables us to isolate live label-retaining cancer cells (LRCCs). Based on the fact that solid organ cancers are derived from tissues that contain LRC, and that LRCs are thought to be adult tissue stem cells, we tested human cancer cell lines and fresh surgical cancer specimens for the existence of LRCC. We tested whether LRCC possess stem-like properties, and the mechanism by which LRCCs are generated. Finally, based on the cancer stem cell hypothesis, we tested their tumor-initiating capacity in immunocompromised mice.
Here, we show the existence of LRCC in gastrointestinal (GI) cancers. LRCC express proliferation markers, cell cycle checkpoint genes, and a mitotic marker suggesting that LRCCs are not quiescent but rather undergo active cell division. For the first time, to our knowledge, we demonstrate live LRCC undergoing label-retaining ACD with NRCC. Finally, we demonstrate that LRCCs have greater tumorinitiating capacity than non-LRC generating tumors with only 10 cells, and a stem cells gene expression profile. Taken together, these findings suggest that LRCC represent a novel class of common GI cancer stem cells, and as such may provide new insights into the biology of cancer and the stem cell origins of cancer.

Fresh Primary Human Cancer Cells and Cancer Cell Lines
Fresh tissue was obtained on National Cancer Institute protocol 09-C-0079. Tumors were harvested and processed into spheroids, transplanted into nude mice once, harvested, and used in this study (Supporting Information Materials and Methods and Table  S1).

Isolation of Live LRCC
Isolation of live LRCC was done as described (Supporting Information Fig. S1A, S1B and Materials and Methods).

Real-Time Confocal Cinematography for the Detection of LRCC Undergoing ACD-NRCC
Cancer cells with Cy-5-labeled DNA were isolated and plated onto collagen-IV-coated slide chambers (Supporting Information Fig. S2 and Materials and Methods). Confocal cinematography imaging was performed on a Zeiss LSM 710 NLO confocal equipped with an environmental chamber (Carl Zeiss, Deutschland, Germany, http://www.zeiss.de).

Statistics
All data are presented as the means 6 SEM. Statistical differences were evaluated as follows: (a) the statistical significance of observing ACD-NRCC was calculated with the two-tailed p value by the exact binomial test. (b) Fisher's exact test was used to test for significance of tumor-initiating capacity (Supporting Information Materials and Methods).

A Subpopulation of LRCCs Is Not Quiescent and Undergoes Active Cell Division
We developed a novel method that allowed for the isolation of live LRC (Materials and Methods). To test whether LRCC undergo active cell division, we isolated live LRCC and non-LRCC ( Fig. 1B) from three HCC cell lines and three surgical specimens (three colon cancers, Supporting Information Materials and Methods). The relative percentages of LRCC ranged from 1.3% to 2.0% (n ¼ 6).
To validate these findings, we tested the cell cycle duration of LRCC and the non-LRCC. The cell cycle duration of LRCC was 34.9 6 8.8 hours, and the cell cycle duration of the non-LRCC was 36 6 9.2 (n ¼ 18, p ¼ .95, Fig. 1F). Finally, we tested and compared LRCC versus non-LRCC for the expression of key cell cycle checkpoint genes. Using qRT-PCR cell cycle array, we show that there is no statistical difference in the expression of all tested genes (cyclin A2, CCNA2; D1, CCND1; D2, CCND2; D3, CCND3; E1, CCNE1; cell division control protein 2, CDC2; cyclin-dependent kinase 2, CDK2; 4, CDK4; and 6, CDK6) between LRCC and non-LRCC (Fig. 1G, n ¼ 18). Interestingly, CCND2, a gene expressed during the mid-G1 and exit from G0-G1 phase, was expressed 4.2 6 0.2-fold higher in the LRCC than in the non-LRCC. Exit from G0 into the G1 phase is thought to herald stem cells activation.
In summary, using five layers of evidence, we show that a subpopulation of LRCC is actively dividing, mitigating the quiescence/slow-cycling hypothesis in LRCC. These findings suggest that LRCC could undergo ACD-NRCC.

LRCC Undergo ACD with NRCC
To test the alternative hypothesis asking the question whether LRCC undergo ACD-NRCC, we developed a novel method to detect live LRCC ( Fig. 2A, Materials and Methods). Cells were grown for one cell cycle in serum-free media and underwent a double-thymidine arrest to increase the probability of cells being synchronously in G1-S phase at the inception of the experiment. Subsequently, we added complete media and allowed DNA synthesis to occur in an environment rich with the DNA nucleotide analog Cy5-dUTP (2 0 -deoxyuridine 5 0 -triphosphate), as described in Materials and Methods. After incorporation of Cy5-dUTP into the DNA, cells were grown for one more cell cycle in culture. Using FACS, we sorted only Cy5-dUTP-high positive cancer cells with >99% purity. Cy5-dUTP-positive cancer cells were then placed on collagen-IV-coated chamber slides, and their nuclei were labeled with the vital stain Cyto9. Subsequently, we initiated continuous confocal microscopic cinematography of live cells undergoing cell divisions. In Figure 2B, 2C, we show one such representative ACD-NRCC. The still pictures in Figure 2B, 2C, were taken from a continuous video where at time t ¼ 0 minute, one can see a single cell with a single nucleus containing DNA labeled with Cy5-dUTP (Fig. 2B, green). Following the same cell, at time t ¼ 210 minutes, one can observe one cell with two nuclei during mitosis; however, here, only one of the nuclei contains Cy5-dUTP-labeled DNA ( Fig. 2C and Supporting Information Video S1). At time t ¼ 600 minutes, one can observe two cells: one with Cy5-dUTP-labeled DNA (Fig. 2B, green and Supporting Information Video S1) and the other with unlabeled DNA (Fig. 2B, blue and Supplemental Video S1). To ascertain that these are not two cells over each other, we used confocal microscopic cinematography to deconstruct the layers (Z stacking) confirming one cell dividing into two. To fully appreciate this phenomenon, we attached a video of live LRCC undergoing ACD-NRCC in real time (Supporting Information Video S1). As far as we know, this is the first time, to our knowledge, that ACD-NRCC is recorded in live cells and in real time. In the first set of experiments, we observed 104 cell divisions in three different experiments, 2/104 of these cells underwent ACD-NRCC. In subsequent experiments (n ¼ 16), the relative proportion of cells undergoing ACD-NRCC was 1.9%-2.7%. LRCC undergoing ACD-NRCC is a rare but statistically significant phenomenon (p ¼ .001, statistics in Materials and Methods).

LRCC Exhibit Greater Tumor-Initiating Capacity than Non-LRCC
To further understand the biological implications and the potential stem cell nature of LRCC, we tested the tumor-initiating capacity of LRCC (Materials and Methods). We isolated live LRCC and non-LRCC from one HCC cell line (PLC/PRF/5) and fresh cancer cells from a surgical specimen (CSCL-04-Ke derived from colorectal cancer). All in vivo experiments were done in a blinded fashion. Moreover, mice were scrambled blindly within and among cages, and we used coded electronic transponders to track the mice. All experiments were terminated at 16 weeks. The sealed envelope containing the blinding code was opened in the presence of all involved. We transplanted 10 cells into 20 Nude/SCID mice per each group (LRCC group and non-LRCC group). We found that LRCC exhibited superior tumor-initiating capacity when compared with non-LRCC: 14/20 versus 2/20 of the mice generated tumors (p ¼ .0005, Fisher's exact test). The LRCC generated faster and larger tumors than the non-LRCC, 8 weeks versus 14 weeks (Fig. 3).

Stem Cells and Pluripotency Gene Expression Profiling of LRCC
To gain further understanding of the potential stem cell nature of LRCC, we isolated live LRCC and non-LRCC and compared their gene expression profiles (Materials and Methods). We performed qRT-PCR SuperArray analysis: WNT (84 genes), stem cells (84 genes), and pluripotency (11 genes). We analyzed three HCC cell lines and three freshly isolated colon cancers from surgical specimens (n ¼ 18).
Taken together, our data suggest that LRCCs have gene expression profile consistent with stem-like phenotype. To integrate these results, we used the Ingenuity pathway analysis software to generate a stem cell pathway map for LRCC (Fig. 5).

DISCUSSION
Although, several investigators using membrane dyes demonstrated the existence of quiescent non-DNA LRCs in rodents, little evidence exists to suggest the existence of nonquiescent DNA LRCs in human cancers (LRCC) [12]. Demonstrating nonquiescent DNA LRCCs in GI cancers is shown here for the first time, to our knowledge. It gives further credence to the hypothesis that cancers, similarly to adult tissue, might be driven by stem-like cells.
The existence of ACD-NRCC has been questioned. Among several reasons for the controversy was the inability to reproducibly test live LRC or LRCC. Here, we show live LRCC undergoing ACD-NRCC in real time. Potentially, the existence of LRCC undergoing ACD-NRCC may pave the way for novel strategies to target cancer via targeting the mechanisms underlying LRCC. Furthermore, using similar studies, it may provide novel understanding into adult tissue stem cells (LRC) and regenerative medicine.
Isolation of cancer stem cells has been based mostly on cell-surface markers or the side population. More recently Pine et al. demonstrated the existence of cells that undergo ACD in lung cancer [12]. However, their observation was done on fixed cells, and thus they were unable to test whether these cells function like stem cells. Our method uses the ability for stem cells to retain DNA labels. Since LRCCs were identified in diverse GI cancers and most of GI cancers develop in tissues known to harbor LRC, it is conceivable that the property of DNA label retaining could be used to study a potential common stem-like cancer cells in GI cancers. This is an exciting prospect as it may provide a common platform for the comparison of malignant transformation among diverse stem-like cells. This can provide invaluable insights for the development of novel cancer therapeutics against LRCC. Additionally, it may provide a common platform to study the differences between normal adult tissue stem cells (LRC) and stemlike cancer cells (LRCC), providing further insights into carcinogenesis and potentially adult tissue regeneration.
In view of the fact that LRCC undergo active cell division, the question of relative resistance to chemotherapy must be explained differently. Clearly, quiescence can explain resistance of cancer stem cells to chemotherapy. However, if LRCCs undergo active cell division there must be other mechanisms to protect them from chemotherapy. Further investigation of such mechanisms could be the base for a novel approach for anticancer drug developments.
Here we show, for the first time, to our knowledge, that putative HCC and colorectal cancer stem cells, that is, LRCC can generate tumors with only 10 cells. The LRCCs have superior tumor-initiating capacity than the non-LRCC (p ¼ .0005). Previous studies using different cancer stem cells' markers (side population, CD133, CD44/CD24/EpCam) demonstrated tumorinitiating capacity consistently always with more than 10 cells. However, there is fundamental variability among studies in terms of conditions. The LRCC from established cell lines and fresh tumors generated large tumors within 8 weeks using only 10 cells (FACS technique). Thus, based on their ability to generate tumors, LRCC should be considered as putative novel stem-like cancer cells or tumor-initiating cells.
A potential drawback of our methodology is the introduction of modified nucleotides into the cells. However, since the non-LRCCs are derived from cells that underwent introduction of modified nucleotides, like the LRCC, but did not retain DNA label, it is unlikely that the introduction of modified nucleotides was the source of LRCC behaving like stem cells.
In conclusion, using multiple lines of evidence, we demonstrated that LRCC possess stem cells' traits. We showed that LRCC undergo ACD-NRCC, a property suggested previously only to stem cells. We demonstrated that LRCCs have exquisite ability to initiate tumors with only 10 cells, a property associated with stem-like cancer cells. We demonstrate that LRCCs when compared with non-LRCCs have stem cells' gene expression profile. In particular, LRCCs express all six human genes used to generate induced pluripotent stem (iPS) cells (OCT3/4, SOX2, NANOG, c-MYC, LIN28 and KLF4). Expression of these six genes with highly unusual upregulation of SOX2 (38.9 6 13.1-fold, p ¼ .035) and LIN28 (107.5 6 4.4-fold, p ¼ .0089) is highly suggestive of stem cell gene expression profile. We propose that the LRCC could be common subpopulation of Figure 5. Stem cell pathways analysis for label-retaining cancer cell (LRCC). Using our gene expression data and the ingenuity pathway analysis platform, we show that LRCC upregulates known stem cells, pluripotency, and Wnt pathway genes and downregulates genes associated with gastrointestinal differentiation and apoptosis suggesting a stem cell gene expression profile. Abbreviations: AES, amino-terminal enhancer of split; AKT, v-akt murine thymoma viral oncogene homolog; BMP, bone morphogenetic protein; CCND, cyclin D; CSNK, casein kinase; CXCL, chemokine (C-X-C motif) ligand; CYP, cytochrome P450; DTX, deltex homolog; DVL, dishevelled; FGF, fibroblast growth factor; FOXN, forkhead box N; GSK, glycogen synthase kinase; KLF, Kruppel-like factor; KRT, keratin; LEF, lymphoid enhancer-binding factor; LGR, leucinerich repeat containing G protein-coupled receptor; LIN28, lin-28 homolog; MEK-ERK, mitogen-activated protein kinase kinase-MAP kinase; MME, membrane metallo-endopeptidase; MYC, v-myc myelocytomatosis viral oncogene homolog; NANOG, nanog homeobox; NEUROG, neurogenin; NOGGIN, noggin; OCT, octamer-binding protein; RB, retinoblastoma; SMAD, mothers against DPP homolog; SOX, SRY (sex determining region Y)-box; TCF, T-cell specific transcription factor; UTF, undifferentiated embryonic cell transcription factor; WNT, Wingless-type MMTV integration site family. novel stem-like cancer cells or tumor-initiating cells. Finally, the ability to isolate live LRCC and LRC has implications beyond cancer; it may provide novel insights into normal adult tissue stem cells, tissue regeneration, and tissue degeneration into cancer.

SUMMARY
LRCs are thought to be tissue stem cells. GI cancers are derived from tissues containing LRCs. We show, in live cells, that various GI cancers contain LRCCs undergoing ACD and exhibit stem cells and pluripotency gene expression profiles. Importantly, LRCCs have greater tumor-initiating capacity than non-LRCC. We propose that LRCC might represent a novel population of stem-like cancer cells.

ACKNOWLEDGMENT
This study was supported by the intramural grant provided by the NIH/National Cancer Institute.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors declare no potential conflicts of interests.