Daughter Cells and Erythroid Cells Budding from PGCCs and Their Clinicopathological Significances in Colorectal Cancer

Abstract

Purpose: We previously reported that polyploid giant cancer cells (PGCCs) induced by cobalt chloride (CoCl₂) exhibit cancer stem cell properties. Daughter cells generated by PGCCs possess epithelial mesenchymal transition (EMT) phenotype changes and EMT plays an important role in cancer development and progression. This study investigated the characteristics of PGCCs from LoVo and HCT116 induced by CoCl₂ and the clinicopathological significances of PGCCs in colorectal cancer (CRC). Materials and Methods: Western blotting and immunocytochemical staining were used to compare the expression levels of EMT-related proteins between PGCCs with budding daughter cells and the control cells. In addition, tissue samples were collected from 159 patients with CRC for analysis of PGCCs, vasculogenic mimicry (VM), and single stromal PGCCs with budding, as well as immunohistochemical staining for cathepsin B, vimentin, and hemoglobin A. Results: Single PGCCs induced by CoCl₂ formed spheroids in vitro. Poorly differentiated CRCs showed the highest numbers of PGCCs and VM, and expression of cathepsin B. There was greater expression of EMT-related proteins in PGCCs with budding daughter cells than in control cells. The expression of vimentin located in PGCC nuclei. Single stomal PGCCs with budding were detected in 27.45% of well differentiated, 50% of moderately differentiated, and 90.20% of poorly differentiated CRC samples. PGCCs can generate erythroid cells that express delta-hemoglobin to form VM. Erythroid cells generated by PGCCs were positive for hemoglobin A immunocytochemical staining. Conclusion: PGCCs from LoVo and HCT116 treated by CoCl₂ exhibited cancer stem cell properties. The number of PGCCs and VM were associated with CRC differentiation and daughter cells budded from PGCCs may promote the lymph node metastasis via expression of EMT-related proteins. PGCCs and their newly generated erythroid cells form VM structures.

Keywords: Cancer stem cells.; Colorectal cancer; Epithelial-mesenchymal transition; Polyploid giant cancer cells; Vasculogenic mimicry.

Figure 1

A. Morphologic characteristics of regular LoVo and PGCCs. (a) Morphologic characteristics of regular LoVo cells cultured in complete RPMI-1640 (10×). (b) LoVo PGCCs survived from the treatment of 300 µM CoCl₂ for 48 h (black arrow heads, 10×). (c) Single PGCCs from LoVo (black arrow heads) with their budding daughter cells (red arrow heads, 10×). (d) Formation of cancer spheroids from LoVo PGCCs (black arrow heads, 10×). B. Morphologic characteristics of regular HCT116 and PGCCs. (a) Morphologic characteristics of regular HCT116 cells cultured in complete RPMI-1640 (10×). (b) HCT116 PGCCs survived from the treatment of 300 µM CoCl₂ for 72 h (Black arrow heads, 10×). (c) Single PGCCs from HCT116 (black arrow heads) with their budding daughter cells (red arrow heads, 10×). (d) Formation of cancer spheroids from HCT116 PGCCs (black arrow heads, 10×). C. Single LoVo PGCCs generated small daughter cells via budding over a 9-day-period continuous observation (From a to f). D. Presence of PGCCs in CRC tissue specimens and their association with cathepsin B expression. (a) PGCCs in well differentiated CRCs (black arrow heads, H&E, 20×). (b) PGCCs in moderately differentiated CRCs (black arrow heads, H&E, 20×). (c) PGCCs in poorly differentiated CRCs (black arrow heads, H&E, 20×). (d) PGCCs were positive for cathepsin B IHC staining (black arrow heads, IHC, 20×).

Figure 2

A. ICC staining of vimentin, E-cadherin and N-cadherin. (a) Vimentin staining of control LoVo cells (ICC, 20×). (b) Positive vimentin staining located both in the cytoplasm (black arrow heads) and the nuclei of PGCCs (red arrow heads) (ICC, 20×). (c) Positive E-cadherin staining in control LoVo cells (ICC, 20×). (d) E-cadherin staining in LoVo cells after CoCl₂ treatment (ICC, 20×). (e) N-cadherin staining in control LoVo cells (ICC, 20×). (f) Positive N-cadherin staining in LoVo cells after CoCl₂ treatment (ICC, 20×). B. Western blot analysis showed the expression of EMT related proteins including Twist, Slug, and Snail expression in LoVo and HCT116 before and after CoCl₂ treatment. C. Vimentin expression in CRCs with different differentiation statuses. (a) Stromal cells were positive for vimentin IHC staining in well differentiated CRCs (black arrow heads, IHC, 20×). (b) Stromal cells were positive for vimentin IHC staining in moderately differentiated CRCs (black arrow heads, IHC, 20×). (c) The positive vimentin IHC staining in the nuclei of cancer cells (black arrow heads) and the cytoplasm of stromal cells (red arrow heads) in moderately differentiated CRCs (IHC, 20×). (d) The positive vimentin IHC staining located in the nuclei of PGCC (black arrow heads) and the cytoplasm of stromal cells (red arrow heads) in poorly differentiated CRCs (IHC, 20×). (e) PGCC nuclei were positive for vimentin IHC staining in poorly differentiated CRCs (black arrow heads, IHC, 20×). (f) The positive vimentin IHC staining in both the nuclei (black arrow heads) and cytoplasm (red arrow heads) of PGCCs in poorly differentiated CRCs (IHC, 20×). D. Budding PGCCs were associated with tumor cell invasion. (a) Black arrows indicate daughter cells budded from PGCCs (black arrow heads, H&E, 20×). (b) PGCCs located at the invasion front (black arrow heads, H&E, 20×). (c) Single PGCCs located in the stroma (black arrow heads, H&E, 20×). (d) PGCCs and budded daughter cells formed tumor emboli (black arrow head, H&E, 20×). (e) PGCCs at the invasion front were positive for cathepsin B IHC staining (black arrow heads, IHC, 20×). (f) Single stromal PGCCs were positive for cathepsin B IHC staining (black arrow heads, IHC, 20×).

Figure 3

A. Generation of erythroid cells by PGCCs from LoVo and HCT116 cancer cell lines. (a) PGCCs from LoVo were observed in paraffin-embedded spheroids (black arrow heads, H&E, 20×). (b) Erythroid cells appeared in the cytoplasm of LoVo PGCCs (black arrow heads, H&E, 20×). (c) Erythroid cells generated from LoVo cell block after CoCl₂ treatment (black arrow heads, H&E, 20×). (d) Erythroid cells in (c) were positive for anti-delta hemoglobin (Black arrow heads, IHC 20×). (e) PGCCs from HCT116 were observed in paraffin-embedded spheroids (black arrow heads, H&E, 20×). (f) Erythroid cells appeared in HCT116 PGCCs cytoplasm (black arrow heads, H&E, 20×). (g) Erythroid cells generated from HCT116 cell block after CoCl₂ treatment (black arrow heads, H&E, 20×). (h) Erythroid cells in (g) were positive for anti-delta hemoglobin (Black arrow heads, IHC, 20×). B. PGCCs produced daughter tumor cells and erythroid cells to form VM. (a) Erythroid cells (black arrow heads) adhere to PGCC surfaces (H&E, 20×). (b) Erythroid cells located in the cytoplasm of PGCCs (black arrow heads, H&E, 20×). (c) Erythroid cells adhered to PGCC surfaces were positive for hemoglobin A (black arrow heads) and red blood cells in EVs (red arrow heads) were negative for hemoglobin A (IHC, 20×). (d) Erythroid cells in the cytoplasm of PGCCs were positive for hemoglobin A (black arrow heads, IHC, 20×). (e) Erythroid cells budding from PGCCs (black arrow heads) and tumor cells formed VM (red arrow heads) (H&E, 20×). C. Presence of VM in CRCs. (a) VM without a PAS-positive basement membrane (black arrowheads, H&E, 20×). (b) VM without a basement membrane (red arrow heads) and EVs (black arrow heads) that were stained with both CD34 and PAS (double-staining, 20×). (c) VM structure with a PAS-positive basement membrane (black arrow heads, H&E, 20×). (d) VM with a basement membrane was positive for PAS staining and negative for CD34 staining (black arrow heads, double staining, 20×). D. The relationship between tumor differentiation and cathepsin B expression levels and the number of PGCCs and VM in CRCs.