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, 36 (34), 4887-4900

Dedifferentiation Into Blastomere-Like Cancer Stem Cells via Formation of Polyploid Giant Cancer Cells

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Dedifferentiation Into Blastomere-Like Cancer Stem Cells via Formation of Polyploid Giant Cancer Cells

N Niu et al. Oncogene.

Abstract

Our recent perplexing findings that polyploid giant cancer cells (PGCCs) acquired embryonic-like stemness and were capable of tumor initiation raised two important unanswered questions: how do PGCCs acquire such stemness, and to which stage of normal development do PGCCs correspond. Intriguingly, formation of giant cells due to failed mitosis/cytokinesis is common in the blastomere stage of the preimplantation embryo. However, the relationship between PGCCs and giant blastomeres has never been studied. Here, we tracked the fate of single PGCCs following paclitaxel-induced mitotic failure. Morphologically, early spheroids derived from PGCCs were indistinguishable from human embryos at the blastomere, polyploid blastomere, compaction, morula and blastocyst-like stages by light, scanning electron or three-dimensional confocal scanning microscopy. Formation of PGCCs was associated with activation of senescence, while budding of daughter cells was associated with senescence escape. PGCCs showed time- and space-dependent activation of expression of the embryonic stem cell markers OCT4, NANOG, SOX2 and SSEA1 and lacked expression of Xist. PGCCs acquired mesenchymal phenotype and were capable of differentiation into all three germ layers in vitro. The embryonic-like stemness of PGCCs was associated with nuclear accumulation of YAP, a key mediator of the Hippo pathway. Spheroids derived from single PGCCs grew into a wide spectrum of human neoplasms, including germ cell tumors, high-grade and low-grade carcinomas and benign tissues. Daughter cells derived from PGCCs showed attenuated capacity for invasion and increased resistance to paclitaxel. We also observed formation of PGCCs and dedifferentiation in ovarian cancer specimens from patients treated with chemotherapy. Taken together, our findings demonstrate that PGCCs represent somatic equivalents of blastomeres, the most primitive cancer stem cells reported to date. Thus, our studies reveal an evolutionarily conserved archaic embryonic program in somatic cells that can be de-repressed for oncogenesis. Our work offers a new paradigm for cancer origin and disease relapse.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
PGCCs exhibited a developmental pattern similar to that of blastomeres. (A) Experimental design. D, day; SEM, scanning electron microscopy; WB, western blotting; IF, immunofluorescence; IHC, immunohistochemistry; sD, scanning day. (B) Number (mean±s.d.) of regular cancer-cell-derived spheroids and PGCC-derived spheroids per 5000 cells at sD7. Regular cancer-cell-derived spheroids: Hey, 7±1.7; SKOV3, 2.3±1.2; MDA-HGSC-1, 50.3±5.9. PGCC-derived spheroids: Hey, 46.7±7.8; SKOV3, 11±2; MDA-HGSC-1, 174.0±8.8. (C) Diameter (mean±s.d.) of regular cancer-cell-derived spheroids (Hey, 363.0±94.5; SKOV3, 182.5±32.9; MDA-HGSC-1, 482.5±91.5) and PGCC-derived spheroids (Hey, 104.2±62.0; SKOV3, 84.0±37.0; MDA-HGSC-1, 92.5±22.5) at sD7. (D) Light microscopic images of control regular Hey cells, Hey-derived PGCCs and resulting spheroids at sD7. Scale bars, 50 μm. (E) Light microscopic images of 'spheroid' formed from a single regular Hey cell (20 μm in diameter) in serum-free stem cell medium. Scale bars, 50 μm. (F) Scanning electron microscopy images of regular Hey cell under mitosis (a) and spheroid derived from regular Hey cell (b). FL, filopodium; RF, ruffle. Scale bars, 10 μm. (G) Light microscopic images of spheroid formation from a single Hey-derived PGCC. Vacuole in the cytoplasm of PGCC is indicated with white arrowhead in panel sD2. The vacuolation is probably due to accumulation of autophagosomes. Scale bars, 50 μm. (H) Light microscopic nuclear morphology and size of PGCC and PGCC-derived spheroid with budding. Hoechst 33342 stain. Scale bars, 50 μm. (I) Scanning electron microscopy images of Hey-derived PGCCs. (a and b) PGCC mimicking single polyploid blastomere. (c–j) PGCC cleavage division mimicking blastomere division, including two-cell (c), three-cell (d), four-cell (e), six-cell (f), compaction-like (g), PGCCs with multiple small cleaved daughter cells resembling a morula (h–j). Scale bars, 10 μm. (J) 3D confocal scanning images of Hey-derived PGCC (a and b) and spheroid derived from Hey-derived PGCC (c and d). (a and b) Blastocyst-like PGCC with budding (a) and cross-sectional view of panel a showing multiple nuclei in a single PGCC (b). Scale bars, 100 μm. (c and d) front (c) and back (d) views of PGCC-derived spheroid. Scale bars, 10 μm.
Figure 2
Figure 2
Generation of embryonic-like stemness of PGCCs. (a) Immunofluorescence photos of OCT4, SSEA1, SOX2 and NANOG in spheroids derived from Hey-derived PGCCs cultured in stem cell medium and reattached on coverslips at different time points. sD, spheroid day. Scale bars, 50 μm. (b) Hematoxylin–eosin (H&E) staining of spheroids from regular Hey cells and PGCCs at sD7 in stem cell medium. Bottom panels are enlarged views of the boxed areas. Black arrow, anaplastic nuclei in a cyst; orange arrow, erythrocyte-like cell in cyst; blue arrow, macrophage-like cell. Scale bars, 50 μm. (c) IHC (colored by DAB, brown) showed the expression of cytokeratin (CK), fibronectin and OCT3/4 in spheroids derived from regular Hey cells and Hey-derived PGCCs. Black arrow indicates rare positive OCT3/4 cells in the middle of spheroids. Scale bars, 50 μm. (d) Immunofluorescence photos of OCT4, NANOG, SSEA1, SOX4 and SOX2 in regular MDA-HGSC-1 cells and spheroids derived from MDA-HGSC-1-derived PGCCs on recovery day 7 (sD1) in stem cell medium. Scale bars, 50 μm. (e) Immunofluorescence photos of Xist in regular Hey and MDA-HGSC-1 cells, PGCCs derived from these cells, and PGCC-derived daughter cells. Boxed regions in the two daughter panels are daughter cells with high magnification, indicated by white arrowheads. Bars, 20 μm. (f) Quantification of OCT4, NANOG and DAZL by qRT-PCR analysis in Hey and SKOV3 cells allowed recovering in regular medium at different times after paclitaxel treatment. (g) Expression of OCT4, NANOG and SOX2 in Hey- and SKOV3-derived PGCCs at different times after paclitaxel treatment, detected by western blotting.
Figure 3
Figure 3
Differentiation of PGCCs in vitro. (A) Differentiation of PGCC to cells of different morphology at 2 months of culture in regular medium. (a) Regular Hey cells. (b–f) Daughter cells of different morphology, including epithelium-like (b), fibroblast-like (c), mixed epithelial and mesenchymal cells (d), neuron-like (e) and small spore-like cells (f). (B) Normalized mRNA expression of proteins associated with three germ layers in Hey-derived and SKOV3-derived PGCCs over 28 days of recovery in regular medium. AFP, SMA and PAX6 are markers for endoderm, mesoderm and ectoderm, respectively. (C) Expression of proteins associated with three germ layers in regular Hey and SKOV3 cells, PGCCs derived from these cells and PGCC-derived daughter cells. AFP, SMA and β3-tubulin are markers for endoderm, mesoderm and ectoderm, respectively. Ctrl, regular cancer cell (day 0); PG, PGCC at sD1; Dau, daughter cell. (DF) Adipocyte (D), chondrocyte (E) and osteocyte (F) differentiation of regular Hey cells and reattached spheroids derived from PGCCs cultured in specific differentiation medium for 14 days. Black arrow in D, oil red–positive vesicle. Blue arrow in D, negative cell (internal reference). Black arrows in E, blue signals of sulfate chondroitin. (G) Immunofluorescence images of three-germ-layer differentiation of regular Hey cells and reattached spheroids from PGCCs. (H) 3D culture of regular Hey cells and PGCCs (at sD1) in the Alvetex scaffold system for 7 days. Regular Hey cells penetrated into the scaffold membrane, while PGCCs mainly grew on the surface of the membrane and formed a monolayer epithelial-like structure (hematoxylin–eosin (H&E) staining). Human-specific cytokeratin (CK) positivity confirmed the epithelium source.
Figure 4
Figure 4
Subcellular localization of YAP and differentiation of PGCCs to endoderm and ectoderm (astrocytes). (a) Expression and distribution of YAP in test cells. In regular cells and PGCC-derived daughter cells, YAP was mainly located in the cytoplasm, but in PGCCs, YAP was predominantly located in the nucleus. Translocation of YAP to the nucleus in PGCCs could be inhibited by dobutamine (Dobu), leading to continued endoreplication and budding failure and death. (b) Proportion of YAP in nucleus in regular cells, PGCCs and PGCC-derived daughter cells. The proportion of YAP in the nucleus was significantly higher in PGCCs (Hey, 73.0±3.0% SKOV3, 72.6±0.6%) and daughter cells (Hey, 9.7±0.6% SKOV3, 8.6±0.6%) than in regular cells (Hey, 0.7±0.5% SKOV3, 0.4±0.5%). (c and d) Endoderm differentiation (marked by AFP, c-Kit and CXCR4) and astrocyte differentiation (marked by GFAP and CD133) were inhibited by dobutamine (Dobu). Scale bars, 50 μm.
Figure 5
Figure 5
Formation of germ cell tumors and carcinomas of different grades from PGCC-derived spheroids. (A) Hematoxylin–eosin (H&E)-stained images from xenografts formed by control Hey cells and PGCC-derived spheroids. (a) control Hey cells; (b) low-power view of multiple foci of dysgerminoma in a background of carcinoma; (c) high-power view of dysgerminoma showing vesicular nuclei and clear cytoplasm of tumor cells characteristic of primordial germ cells; (d) dysgerminoma with skeletal muscle differentiation; (e) mixed dysgerminoma and embryonic carcinoma; (f) dysgerminoma with mesenchymal morphology; (g) high-grade carcinoma; (h) mixed high- and low-grade tumor with high-power view; (i) benign squamous cells with immunohistochemical staining against cytokeratin. (B) H&E staining and IHC for SALL4, cytokeratin (CK) and OCT4 on continuous sections of xenografts formed by regular Hey cells (Control) and malignant germ cell tumors generated from spheroids derived from PGCCs. In control xenografts formed by regular Hey cells, cancer cells were positive for human-specific CK, but not for OCT4 and SALL4. In xenografts formed by PGCC-derived spheroids, all of the cancer cells were positive for human-specific CK, and there were clusters of cells positive for OCT4 and SALL4. Gray circles show the same subpopulations. Black cycle indicates cytokeratin positive cells. Scale bars, 50 μm.
Figure 6
Figure 6
Acquisition of drug resistance and increased expression of epithelial and mesenchymal markers in PGCC-derived daughter cells. (a) Percentage of polyploid cells (blue dots in gate P2) among regular Hey and SKOV3 cells and PGCC-derived daughter cells. (b) Percentage of apoptotic cells (Q2+Q4) among regular Hey and SKOV3 cells and PGCC-derived daughter cells after exposure to 100 nm PTX and recovery for 48 h. (c) Sensitivity of PGCC-derived daughter cells compared with corresponding regular cancer cells to PTX, vincristine, carboplatin, olaparib and topotecan (24 h treatment). Results are presented as the ratio of the IC50 value for daughter cells to the IC50 value for regular cancer cells. The IC50 values are indicated in Supplementary Table 2. (d and e) Invasion ability of regular Hey and SKOV3 cells and PGCC-derived daughter cells. In matrigel-transwell invading experiments, the number of invaded regular Hey cells (72.6±11.8/cm2) and SKOV3 cells (63.8±8.7/cm2) was higher than the number of invaded daughter cells (Hey daughter, 32.7±3.8/cm2; SKOV3 daughter, 28.6±3.4/cm2). (f) Expression of epithelium-related and mesenchyme-related protein in tested cell lines, detected by western blotting. Reg, regular cancer cells; PG, PGCCs at day 7 after paclitaxel treatment; Dau, daughter cells. (g) Acquisition of mesenchymal phenotype in xenografts generated by PGCC-derived daughter cells. Left panel, tumor nodules of regular control and daughter cell groups are indicated with blue and black arrows, respectively. Right upper panel, nodule size of test groups. Right lower panels, H&E staining of xenografts. Similar-sized cancer cells in control are indicated with black arrows, and heterogeneous-size cancer cells in the daughter group are indicated with blue (epithelial-like) and green (mesenchymal-like) arrows.
Figure 7
Figure 7
PGCCs and differentiation in ovarian cancer specimens. (ac) Hematoxylin–eosin (H&E) staining shows the PGCC populations before and after chemotherapy in three paired cases. In the first two cases (A), cells in the pre-chemotherapy specimen were homogeneous in size and morphology with small round nuclei; the PGCCs (black arrows) present after chemotherapy had large bizarre nuclei and rich cytoplasm. In the third case (B), some large cancer cells were present before chemotherapy (a; blue arrows), but PGCCs present after chemotherapy (b) were larger. Some small daughter cells (black arrows) budded off from the PGCCs (c, higher magnification of b) and some erythrocyte-like vacuoles formed in the cytoplasm of PGCCs (d, higher magnification of b) and released out (blue arrows). In case 4 (C), there was prominent budding from PGCCs after chemotherapy (indicated by black arrows). (D) Nuclear area of cancer cells before chemotherapy and PGCCs and daughter cells after chemotherapy. (E) Expression of OCT4, NANOG, SOX2 and YAP in cancer cells before chemotherapy and PGCCs after chemotherapy. (F) RNA in situ hybridization analysis of Xist. Before chemotherapy, Xist was widely positive in most nuclei of cancer cells and stromal cells; after chemotherapy, there were fewer positive spots in the nuclei of PGCCs.
Figure 8
Figure 8
Schematic of blastomere model for cancer origin and disease relapse. (A) Blastomere-mediated embryogenesis during differentiation (normal development) and subsequent tumor formation associated with maturation arrest of stem cells in different organs during adulthood. Following gamete fusion (sex), zygote will develop into blastomeres, compaction, morula and blastocyst/ICM. Inner cell mass will develop into primordial germ cells, which will proceed with spermatogenesis (male) or oogenesis (female); while three germ layers will develop into somatic tissues containing stem cells of primitive multipotency (a), intermediate potency (b) or late oligo-potency (c) or mature stable cells (d). Maturation arrest at different developmental hierarchies leads to formation of germ cell tumors (GCTs) or germ-layer-specific tumor with different grades of malignancy: high-grade tumors (HG), low-grade tumors (LG), benign tumors (BN) or metaplasia (MP). BM, blastomere; Endo, endoderm; Ecto, ectoderm; Meso, mesoderm; GCs, germ cells; ICM, inner cell mass. M' and M represent different genetic/epigenetic mutations acquired in germ cells and somatic cells respectively during embryonic or in adult development. a, b, c, and d represent different levels of hierarchy in normal development. (B) Induced blastomere-like-mediated oncogenesis via dedifferentiation (reprogramming) of somatic cells. Following mitotic failure induced by paclitaxel (PTX), somatic cells initiate endoreplication to generate PGCCs, which grow into blastomere-like (BM-like), compaction-like and morula-like cell masses (self-renewal) and then differentiate into structures morphologically similar to the blastocyst with inner cell mass and then into the three germ layers and germ cell lineage (termination). The entire process mimics development of preimplantation embryo via the giant cell cycle (preimplantation-like). The germ cells and primitive stem cells arrest at different developmental hierarchies to generate germ cell tumors (GCTs) and somatic tumors with different levels of malignancy. a′, b′, c′ and d′ correspond to levels of developmental hierarchy following stress-induced reprogramming.

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