Characterization of Cancer Stem Cells in Colon Adenocarcinoma Metastasis to the Liver

Background Fifty percent of colorectal cancer (CRC) patients develop liver metastasis. This study identified and characterized cancer stem cells (CSCs) within colon adenocarcinoma metastasis to the liver (CAML). Methods 3,3-Diaminobenzidine immunohistochemical (IHC) staining was performed on nine CAML samples for embryonic stem cell (ESC) markers OCT4, SOX2, NANOG, c-Myc, and KLF4. Immunofluorescence (IF) IHC staining was performed to investigate coexpression of two markers. NanoString mRNA expression analysis and colorimetric in situ hybridization (CISH) were performed on four snap-frozen CAML tissue samples for transcript expression of these ESC markers. Cells stained positively and negatively for each marker by IHC and CISH staining were counted and analyzed. Results 3,3-Diaminobenzidine IHC staining, and NanoString and CISH mRNA analyses demonstrated the expression of OCT4, SOX2, NANOG, c-Myc, and KLF4 within in all nine CAML samples, except for SOX2 which was below detectable levels on NanoString mRNA analysis. IF IHC staining showed the presence of a SOX2+/NANOG+/KLF4+/c-Myc+/OCT− CSC subpopulation within the tumor nests, and a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4− CSC subpopulation and a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4+ CSC subpopulation within the peritumoral stroma. Conclusion The novel finding of three CSC subpopulations within CAML provides insights into the biology of CRC.

pathway (CIMP) (2,5,6). Of these, CIP appears to be the most relevant in the pathogenesis of CRC (2,5,6). The liver is a common metastatic site for CRC. Approximately 20% of CRC patients present with liver metastasis at the time of diagnosis (7) and overall 50% of CRC patients develop liver metastasis during the course of their disease, accounting for 2/3 of the disease-related deaths (7,8).
The survival time for untreated CRC patients with liver metastasis remains at 5-20 months (8). Liver resection is the mainstay treatment for CRC metastatic to the liver (CRCML) and it is the only intervention that significantly increases life expectancy, with a 5-year survival rate of up to 58% (7,8). However, only 20% of patients with CRCML are operable at time of diagnosis (7). Chemotherapy plays a palliative role if used as a primary treatment, so recent focus has been on neoadjuvant chemotherapy to downstage the metastatic liver disease to increase operability (7,8).
Stem cells are essential to the growth and development as well as the maintenance of human tissues, with the key abilities of self-renewal and maintenance of pluripotency, and differentiation through asymmetrical division (9)(10)(11).
The cancer stem cell (CSC) concept of cancer proposes that a subpopulation of cells within the cancer possess properties of embryonic stem cells (ESCs) and are thought to be the quintessential drivers of carcinogenesis due to their ability to promote angiogenesis, invasion, metastasis, and resistance to apoptosis as well as hijacking stem cell properties (12,13).
OCT4 ensures the survival of ESCs through maintenance of the pluripotent state and self-renewal capabilities (11,13,14). SOX2 and OCT4 act in partnership and indispensably together to regulate the expression of genes specific for maintaining stem cell pluripotency and self-renewal (11,15). NANOG also contributes to the transcriptional factor regulatory network involved in maintaining the key properties of stem cells, facilitated through control of genes maintaining pluripotency (16). c-Myc is a protooncogene with a significant role in fundamental cellular process and is involved in generation of induced pluripotent cells through activation of downstream targets which enhance proliferation and transformation of cells (16,17). KLF4 is involved in regulation of the cell cycle, somatic cell reprogramming, and pluripotent characteristics (18).
A number of other stem cell markers associated with CRC CSCs have also been reported in the literature. CD44 is a cell surface glycoprotein with a number of roles in regulating cellular structure and cell-cell interactions (19,20). Overexpression of CD44 has been implicated in early colorectal carcinogenesis, with CD44 knockdown preventing this process (19,20). Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a marker specific to crypt base columnar cells (CBCs) which are undifferentiated stem cells that give rise to the colonic mucosa (19). LGR5 knockdown results in tumor regression (21). Epithelial cell adhesion molecule (EpCAM) is a marker unique to epithelial cells and consequently tumors of epithelial morphology, with functions in cell signaling, and regulating processes such as proliferation, differentiation and metastasis (19). EpCAM + cells have also been extensively characterized as CRC CSCs (19).

Tissue samples
Positive human control tissues used for the primary antibodies were seminoma for OCT4 and NANOG (23), skin for SOX2 (24), breast cancer for KLF4 (25) and prostate for c-Myc (26). A negative CAML control sample was prepared for DAB IHC staining using an IgG isotype control (ready-to-use; cat#IR600, Dako, Santa Clara, CA, USA). Negative controls for IF IHC staining was performed using a section of glioblastoma tissue with the combined use of primary isotype mouse (ready-to-use; cat# IR750, Dako, Copenhagen, Denmark) and rabbit (read-to-use; cat# IR600, Dako) antibodies.

image analysis
3,3-Diaminobenzidine IHC-stained slides were viewed and images were captured using the Olympus BX53 microscope fitted with an Olympus DP21 digital camera (Olympus, Tokyo, Japan). IF IHC-stained slides were viewed and imaged using the Olympus FV1200 biological confocal laser-scanning microscope and processed with cellSens Dimension 1.11 software using 2D deconvolution algorithm (Olympus).

nanostring mrna expression analysis
RNA was extracted from six snap-frozen samples of CAML from the same cohort of nine patients used for DAB IHC staining, was analyzed using NanoString nCounter™ Gene Expression Assay (NanoString Technologies, Seattle, WA, USA). Total RNA was extracted using the MagJET RNA kit (Thermo Fisher Scientific) with the protocol adapted for tissue, and run on a KingFisher Duo machine (Thermo Fisher Scientific). The RNA samples were then quantitated on a Qubit ® 2.0 fluorometer (Invitrogen, Life Technologies) and were subject to RNA integrity analysis via the 2100 Bioanalyzer Instrument (Agilent Technologies). The samples then underwent NanoString nCounter gene expression assay performed by New Zealand Genomics (Dunedin, New Zealand) according to the manufacturer's protocol. Probes for the genes encoding OCT4 (POU5F1, NM_002701.4), SOX2 (NM_003106.2), NANOG (NM_024865.2), KLF4 (NM_004235.4), c-Myc (NM_002467.3), and the housekeeping gene GUSB (NM_000181.1) were designed and synthesized by NanoString Technologies. Raw data were analyzed using nSolver™ software (NanoString Technologies) using standard settings and normalized against the housekeeping gene.

colormetric In Situ hybridization
The 4μm-thick formalin-fixed paraffin-embedded CAML sections of six patients from the original cohort of nine patients used for DAB IHC staining were used for CISH staining using the Leica Bond Rx autostainer and detected using the ViewRNA eZ Detection kit (Affymetrix, CA, USA) as previously described (27). Probes for the stem cell markers c-Myc (NM_002467), KLF4 (NM_004235), NANOG (NM_024865), SOX2 (NM_003106), and OCT4 (POU5F1, NM_002701) were obtained from Affymetrix. Positive controls were demonstrated on human tissues: breast cancer for KLF4, normal prostate for c-Myc, skin for SOX2, and seminoma for OCT4 and NANOG. Negative control was demonstrated on a sample of Bacillus (NM_L38424).

cell counting
Counting of cells with nucleus or cytoplasm that stained positively for OCT4, SOX2, NANOG, KLF4, and c-Myc was each performed in six fields of view at 400× magnification representative of both tumor nests (TNs) and the peritumoral stroma for each of the original nine samples of CAML used for DAB IHC staining. In each field of view, cells within the TNs and those within the peritumoral stroma were counted separately and hence images were taken to incorporate areas of tissue containing cells both within the TNs and the peritumoral stroma.
Cell counting for CISH-stained slides was performed at 1,000× magnification which meant that cells within the TNs and those within the peritumoral stroma could not be incorporated in a single image and hence images were taken separately for cells within the TNs and those within the peritumoral stroma.

statistical analyses
Statistical analysis for comparison of markers demonstrated by DAB IHC and CISH staining was performed to determine significance. Total counts of cells that stained positively and those that stained negatively for each ESC marker within the TNs and the peritumoral stroma were collated. The proportion of positive staining cells was calculated for each marker in both the TNs and peritumoral stroma. χ 2 tests were then carried out using the Quantpsy (www.quantpsy.org) software to determine statistical significance with regards to the proportional expression of each marker, and related t-tests were used to compare the proportional expression of each ESC marker within the TNs against those within the peritumoral stroma using SPSS V.24.
To determine the expression of c-Myc (Figures 1D-F,  green), we performed dual staining with SOX2 ( Figure 1D, red), NANOG (Figure 1E, red), and KLF4 (Figure 1F, red). c-Myc was expressed by cells within the TNs (Figures 1D-F, green,  arrows) and cells within the peritumoral stroma (Figures 1D-F  arrowheads). Intriguingly the c-Myc + population within the peritumoral stroma also expressed SOX2 (Figure 1D, red), NANOG ( Figure 1E, red), and KLF4 (Figure 1F, red). Split images of all the stains presented in Figure 1 are shown in Figure S3 in Supplementary Material. Minimal staining was present on the negative control ( Figure S3M in Supplementary Material), confirming the specificity of the primary antibodies used.

nanostring mrna expression analysis
NanoString mRNA analysis confirmed expression of mRNA transcripts for OCT4, NANOG, KLF4, and c-Myc in all six CAML samples relative to the housekeeping gene GUSB (Figure 2). SOX2 mRNA transcript expression was below detectable levels in all CAML samples. RNA integrity analysis confirmed that two of the four samples had RIN > 7.  Figure S5B in Supplementary Material, pink, arrows), seminoma for NANOG ( Figure S5C in Supplementary Material, pink, arrows), breast cancer for KLF4 ( Figure S5D in Supplementary Material, pink, arrows), and normal prostate for c-Myc ( Figure  S5E in Supplementary Material, pink, arrows). The negative control was a sample of Bacillus (NM_L38424) ( Figure S5F in Supplementary Material).

cell counting and statistical analyses
Statistical analyses of the results of cell counting of DAB-stained slides showed that there was statistically significant difference between the mean proportions of OCT4 + , SOX2 + , NANOG + , and KLF4 + cells in the peritumoral stroma compared to those within the TNs (p < 0.05), but there were no significant differences for c-Myc (p > 0.05) (Figure 3A). When comparing the total proportional of positively stained cells within the TNs and the peritumoral stroma for each marker, χ 2 statistical analysis demonstrated a hierarchy of expression of these markers with increasing abundance: NANOG > SOX2 > KLF4 > c-Myc > OCT4. All differences were statistically significant (p < 0.01).
The means for the cell counts on CISH-stained slides ( Figure 3B) analyzed by χ 2 statistical analysis demonstrated the following hierarchy: c-Myc > KLF4 > NANOG > OCT4 > SOX2. All comparisons were highly statistically significant between markers (p < 0.0001) except for the comparison between KLF4 and NANOG which was not statistically significant (p = 0.884).
Interestingly the presence of SOX2 was demonstrated by IHC staining and CISH, but was not detectable by NanoString mRNA analysis. This discrepancy may be due to an observed  50% degradation of the mRNA during the process of NanoString mRNA analysis. The relative abundance of the CSC markers used in this study may reflect the presence of a hierarchy of CSCs within this tumor.
The presence of three putative CSC subpopulations is intriguing. OCT was expressed only by cells within the peritumoral stroma, but not those within the TNs. We speculate these OCT4 + cells within the peritumoral stroma may represent the most primitive CSC subpopulation within CAML. This OCT4 + CSC subpopulation may give rise to the downstream OCT4 − subpopulations within the peritumoral stroma and those within the TNs (Figure 4A), although this putative presence of a CSC hierarchy remains the topic of further investigation.
Recent literature has reported on the heterogeneity of CSC (30,31) and suggests a putative CSC hierarchy and the upregulation of OCT4 as potentially identifying the most primitive phenotype in the CSC population (32,33).
It would be of great interest to carry out analysis of tissue samples of the primary CRCs that gave rise the liver metastases that we have studied, to determine the CSC phenotype. Unfortunately, this was not available from the cohort of patients included in this study, but will a subject of future investigation.
Epithelial-mesenchymal transition (EMT) is a pathway in which epithelial tumor cells deregulate the cytoskeletal structure to acquire mesenchymal morphology, with pathological implications in cancer metastasis and tumor invasion of the surrounding tissues (34,35). The WNT/β catenin signaling pathway is activated in CRC, with growing evidence that this pathway interacts with the transcription and expression of embryonic transcription factors to modulate EMT (34,35). The mesenchymal-epithelial transition (MET) pathway facilitates metastasis of tumor cells, which once integrated into a host tissue, their mesenchymal morphology transformed into epithelial phenotype (34,35). Again, WNT/β catenin pathway is implicated in this morphological change, as inhibition of this pathway blocks MET (3,4,35).
It is exciting to speculate the presence of an OCT4 + CSC subpopulation within a primary CRC that undergoes an EMT process (36). The EMT process proposes that the epithelial CRC CSC acquire a mesenchymal phenotype which enables it to detach and metastasize to the liver via the portal circulation, in a similar way that circulating cancer cells are proposed to originate (37,38). We speculate that these OCT4 + CSCs, upon "lodgement" in the liver then subsequently undergo MET (39), OCT4 − CSC subpopulation within the peritumoral stroma to complete the metastatic process. This latter CSC subpopulation then potentially gives rise to the OCT4 − CSCs within the TNs ( Figure 4B). Alternatively, the "parent" metastatic OCT4 + CSCs may be seeded into the liver to form the TNs, which then gives rise to a gradual succession of primitive progenies ( Figure 4B) with the OCT4 + being the ultimate end-product of this process. However, it is equally likely that the OCT4 + cells are not derived from the CRCLM, but instead are from the surrounding tissues and this clearly requires further investigation.
Recent studies have demonstrated a role for granulocyte macrophage colony-stimulating factor in inducing EMT in CRC, primarily being secreted by the colon epithelial cells (40,41). Based on this study, it is exciting to speculate that the OCT4 + cells are a result of the process of EMT, although this is the topic of further investigation.
The finding of three putative subpopulations of CSC within CALM provides novel insight and warrants further investigation toward a better understanding of the biology of this common cancer.