Identification of Cancer Stem Cell Subpopulations in Head and Neck Metastatic Malignant Melanoma

Abstract

Cancer stem cells (CSCs) have been identified in many cancer types. This study identified and characterized CSCs in head and neck metastatic malignant melanoma (HNmMM) to regional lymph nodes using induced pluripotent stem cell (iPSC) markers. Immunohistochemical (IHC) staining performed on 20 HNmMM tissue samples demonstrated expression of iPSC markers OCT4, SOX2, KLF4, and c-MYC in all samples, while NANOG was expressed at low levels in two samples. Immunofluorescence (IF) staining demonstrated an OCT4+/SOX2+/KLF4+/c-MYC+ CSC subpopulation within the tumor nests (TNs) and another within the peritumoral stroma (PTS) of HNmMM tissues. IF also showed expression of NANOG by some OCT4+/SOX2+/KLF4+/c-MYC+ cells within the TNs in an HNmMM tissue sample that expressed NANOG on IHC staining. In situ hybridization (n = 6) and reverse-transcription quantitative polymerase chain reaction (n = 5) on the HNmMM samples confirmed expression of all five iPSC markers. Western blotting of primary cell lines derived from four of the 20 HNmMM tissue samples showed expression of SOX2, KLF4, and c-MYC but not OCT4 and NANOG, and three of these cell lines formed tumorspheres in vitro. We demonstrate the presence of two putative CSC subpopulations within HNmMM, which may be a novel therapeutic target in the treatment of this aggressive cancer.

Keywords: cancer stem cell; head and neck cancer; induced pluripotent stem cell; malignant melanoma; melanoma metastasis.

Figure 1

Expression of OCT4, SOX2, KLF4, c-MYC, and NANOG in head and neck metastatic malignant melanoma (HNmMM). Representative hematoxylin and eosin stained section (A) and immunohistochemical stained sections (B–F) of HNmMM demonstrating the tumor organized into tumor nests (TNs, arrows) surrounded by the peritumoral stroma (PTS, arrowheads). OCT4 (B, brown) was expressed on the nucleus and cytoplasm of the cells within the TNs and the cells within the PTS. SOX2 (C, brown) was predominantly expressed in the nucleus of the cells within the TNs with some cytoplasmic expression in the cells within the TNs and the cells within the PTS. KLF4 (D, brown) was expressed predominantly in the cytoplasm of the cells within the TNs with weak staining in the cells within the PTS. Moderate cytoplasmic and focal nuclear expression of c-MYC (E, brown) was present in the cells within the TNs and the cells within the PTS. NANOG (F, brown) was expressed in the cytoplasm of the cells within the TNs in two of the 20 tissue samples. Nuclei were counterstained with hematoxylin (B–F, blue). Original magnification 400×; n = 20.

Figure 2

Graph demonstrating mean percentage positive expression of induced pluripotent stem cell markers NANOG, OCT4, KLF4, c-MYC, and SOX2 by cells within the tumor nests and the peritumoral stroma on immunohistochemical sections of head and neck metastatic malignant melanoma. Error bars represent 95% confidence intervals of the mean. Three replicates from each of the 19 patient tissue samples were used for an Analysis of Variance (ANOVA), thus giving a sample size of 57 for each of the following markers: OCTs, SOX2, KLF4, and c-MYC (n = 57). Similarly, for NANOG, three replicates from each of the two patient tissue samples were used for an ANOVA, thus giving a sample size of 6 (n = 6). ***, p < 0.0005; *, p < 0.05.

Figure 3

Immunofluorescence stains of head and neck metastatic malignant melanoma tissue samples demonstrating expression of induced pluripotent stem cell markers. Representative sections showing expression of OCT4 (A–C, green), SOX2 (B, red), and KLF4 (C, red) were expressed on the cells within both the tumor nests (TNs, thick arrows) and the peritumoral stroma (PTS, arrowheads). The c-MYC+ [(D–F), green] cells within the TNs and the PTS also expressed SOX2 (E, red) and KLF (F, red). NANOG (A, red) was expressed on the OCT4+ (A, green) cells within both the TNs (thick arrows) and the PTS (arrowheads), and some cells (thick arrows) and not the others (thin arrows) within the TNs. All slides were counterstained with 4′,6′-diamidino-2-phenylindole (blue). Original magnification 400×; n = 2. The insets show enlarged views of the corresponding images.

Figure 4

Representative in situ hybridization images of head and neck metastatic malignant melanoma tissue samples demonstrating mRNA transcript expression (brown) of induced pluripotent stem cell markers OCT4 (A), NANOG (B), SOX2 (C), KLF4 (D), and c-MYC (E). Nuclei were counterstained with hematoxylin (A–E, blue). Original magnification 1000×; n = 6.

Figure 5

Graph demonstrating mean percentage positive expression of induced pluripotent stem cell markers KLF4, SOX2, OCT4, NANOG, and c-MYC by cells within the tumor nests and the peritumoral stroma on in situ hybridization-stained sections of head and neck metastatic malignant melanoma. Error bars represent 95% confidence intervals of the mean. Six replicates from each of the six patient tissue samples were used for ANOVA, thus giving a sample size of 36 for each marker (n = 36). ***, p < 0.0005; *, p < 0.05; ns, not statistically significant.

Figure 6

Quantitative transcriptional profiling of induced pluripotent stem cell (iPSC) markers c-MYC, KLF4, OCT4, NANOG, and SOX2. Graphs demonstrating average fold change (2^∆∆Ct) values of triplicate RT-qPCR runs carried out on five snap-frozen head and neck metastatic malignant melanoma (HNmMM) tissue samples (B) four HNmMM -derived primary cell lines (A) amplifying mRNA transcripts for these iPSC markers. CT values of the iPSC markers were normalized to the housekeeping genes GAPDH and PSMB4 to calculate ΔCT, and expression compared relative to that of UHR (y = 1). Error bars represent 95% confidence intervals of the geometric mean.

Figure 7

Representative Western blot images of total protein extracted from four head and neck metastatic malignant melanoma-derived primary cell lines probed for induced pluripotent stem cell markers. Blots were probed for OCT4 (A), NANOG (B), SOX2 (C), KLF4 (D), and c-MYC (E) and detected with HRP conjugated goat anti-rabbit antibody. α-Tubulin was used as the loading control (F) and detected using HRP conjugated mouse IgGĸ binding protein. Arrows indicate the presence of the proteins with their expected band sizes.

Figure 8

Representative high-magnification z-stack (40×) images of tumorsphere formation in suspension culture of the three head and neck metastatic malignant melanoma (HNmMM)-derived primary cell lines, averaging 62.7 µm in diameter (A–C). A representative high magnification (40×) image of one HNmMM-derived primary cell line that did not form spheres, as defined by the tumorsphere criteria in the Methods section; instead, they averaged a diameter of 21 µm (D).