Tumors and tumor-derived cell lines contain polyploid giant cells with significantly elevated genomic content, often with multiple nuclei. The frequency of giant cells can increase markedly following anticancer treatment. Although giant cells enter a dormant phase and therefore do not form macroscopic colonies (aggregates of ≥50 cells) in the conventional in vitro colony formation assay, they remain viable and metabolically active. The purpose of this commentary is to underscore the potential importance of polyploid/multinucleated giant cells in metastasis and cancer recurrence following exposure to anticancer agents. We also discuss the possibility that most preclinical (cell-based and animal model) drug discovery approaches might not account for delayed responses that are associated with dormant giant cells.

Roles of Polyploid/Multinucleated Giant Cancer Cells in Metastasis and Disease Relapse Following Anticancer Treatment

Recently, the development of multifunctional theranostic nanoplatforms to realize tumor-specific imaging and enhanced cancer therapy via responding or modulating the tumor microenvironment (TME) has attracted tremendous interests in the field of nanomedicine. Herein, tungsten disulfide (WS2 ) nanoflakes with their surface adsorbed with iron oxide nanoparticles (IONPs) via self-assembly are coated with silica and then subsequently with manganese dioxide (MnO2 ), on to which polyethylene glycol (PEG) is attached. The obtained WS2 -IO/S@MO-PEG appears to be highly sensitive to pH, enabling tumor pH-responsive magnetic resonance imaging with IONPs as the pH-inert T2 contrast probe and MnO2 as the pH-sensitive T1 contrast probe. Meanwhile, synergistic combination tumor therapy is realized with such WS2 -IO/S@MO-PEG, by utilizing the strong near-infrared light and X-ray absorbance of WS2 for photothermal therapy (PTT) and enhanced cancer radiotherapy (RT), respectively, as well as the ability of MnO2 to decompose tumor endogenous H2 O2 and relieve tumor hypoxia to further overcome hypoxia-associated radiotherapy resistance. The combination of PTT and RT with WS2 -IO/S@MO-PEG results in a remarkable synergistic effect to destruct tumors. This work highlights the promise of developing multifunction nanocomposites for TME-specific imaging and TME modulation, aiming at precision cancer synergistic treatment.

Manganese Dioxide Coated WS2@Fe3O4/sSiO2 Nanocomposites for pH-Responsive MR Imaging and Oxygen-Elevated Synergetic Therapy

Tumour microenvironment responsive nanoconstructs for cancer theranostic

Pancreatic ductal adenocarcinoma (PDAC) tumor growth is enhanced by tumor-associated macrophages (TAMs), yet the mechanisms by which tumor cells and TAMs communicate are not fully understood. Here we show that exosomes secreted by PDAC cell lines differed in their surface proteins, lipid composition, and efficiency of fusing with THP-1-derived macrophages in vitro. Exosomes from AsPC-1, an ascites-derived human PDAC cell line, were enriched in ICAM-1, which mediated their docking to macrophages through interactions with surface-exposed CD11c on macrophages. AsPC-1 exosomes also contained much higher levels of arachidonic acid (AA), and they fused at a higher rate with THP-1-derived macrophages than did exosomes from other PDAC cell lines or from an immortalized normal pancreatic ductal epithelial cell line (HPDE) H6c7. Phospholipase A2 enzymatic cleavage of arachidonic acid from AsPC-1 exosomes reduced fusion efficiency. PGE2 secretion was elevated in macrophages treated with AsPC-1 exosomes but not in macrophages treated with exosomes from other cell lines, suggesting a functional role for the AsPC-1 exosome-delivered arachidonic acid in macrophages. Non-polarized (M0) macrophages treated with AsPC-1 exosomes had increased levels of surface markers indicative of polarization to an immunosuppressive M2-like phenotype (CD14hi CD163hi CD206hi). Furthermore, macrophages treated with AsPC-1 exosomes had significantly increased secretion of pro-tumoral, bioactive molecules including VEGF, MCP-1, IL-6, IL-1β, MMP-9, and TNFα. Together, these results demonstrate that compared to exosomes from other primary tumor-derived PDAC cell lines, AsPC-1 exosomes alter THP-1-derived macrophage phenotype and function. AsPC-1 exosomes mediate communication between tumor cells and TAMs that contributes to tumor progression.

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Tumor-promoting effects of pancreatic cancer cell exosomes on THP-1-derived macrophages.

Polyploid giant cancer cells (PGCC) constitute a dangerous subpopulation of cancer cells and are a driving force in cancer recurrence. These unique cells arise from diploid tumor cells in response to stress encountered in the tumor microenvironment or during cancer therapy. PGCC are greatly dedifferentiated, acquire pluripotency, and are able to replicate through a form of asymmetric division called neosis, which results in new populations that are themselves able to differentiate into new cell types or to re-establish tumors. Progeny tend to be more genetically unstable than the founding population due to the dysregulation required to transition through a PGCC state. Therefore, cancers that escape stressors through this mechanism tend to re-emerge with a more aggressive phenotype that is therapy resistant. This review focuses on the clinical significance of PGCC, the need for standardized nomenclature and molecular markers, as well as possible avenues to develop therapies aimed at PGCC and the process of neosis. The biology underlying the development of PGCC including cell cycle checkpoint dysregulation, stress responses, dedifferentiation, stemness and epithelial-mesenchymal transition is discussed.

Giants and monsters: Unexpected characters in the story of cancer recurrence.

Background/Aim: Fusion of cancer cells has been studied for over half a century. However, the steps involved after initial fusion between cells, such as heterokaryon formation and nuclear fusion, have been difficult to observe in real time. In order to be able to visualize these steps, we have established cancer-cell sublines from the human HT-1080 fibrosarcoma, one expressing green fluorescent protein (GFP) linked to histone H2B in the nucleus and a red fluorescent protein (RFP) in the cytoplasm and the other subline expressing RFP in the nucleus (mCherry) linked to histone H2B and GFP in the cytoplasm. Materials and Methods: The two reciprocal color-coded sublines of HT-1080 cells were fused using the Sendai virus. The fused cells were cultured on plastic and observed using an Olympus FV1000 confocal microscope. Results: Multi-nucleate (heterokaryotic) cancer cells, in addition to hybrid cancer cells with single-or multiple-fused nuclei, including fused mitotic nuclei, were observed among the fused cells. Heterokaryons with red, green, orange and yellow nuclei were observed by confocal imaging, even in single hybrid cells. The orange and yellow nuclei indicate nuclear fusion. Red and green nuclei remained unfused. Cell fusion with heterokaryon formation and subsequent nuclear fusion resulting in hybridization may be an important natural phenomenon between cancer cells that may make them more malignant. Conclusion: The ability to image the complex processes following cell fusion using reciprocal color-coded cancer cells will allow greater understanding of the genetic basis of malignancy.

Color-coded Live Imaging of Heterokaryon Formation and Nuclear Fusion of Hybridizing Cancer Cells.

Background/aim The interaction between pancreatic-cancer cells and stromal cells in the tumor microenvironment (TME) is of particular importance in cancer progression and metastasis. The present report demonstrates the role of cancer-associated fibroblasts (CAFs) and multinucleate pancreatic-cancer cells in peritoneal metastasis. Materials and methods An orthotopic mouse model of pancreatic cancer was established with the human pancreatic cancer cell line BxPC3, which stably expresses green fluorescent protein (GFP). Results BxPC3-GFP cells formed peritoneal metastases by week 18 after orthotopic implantation. Using an Olympus FV1000 confocal microscope, multi-nucleated cancer cells were frequently observed in the peritoneal metastases. The primary pancreatic tumor and peritoneal-metastases were harvested, cultured and then transplanted subcutaneously. Subcutaneous tumors established from peritoneal-metastatic cells were larger than subcutaneous tumors established from primary-tumor cells. Subcutaneous tumors of each type were subsequently cultured in vitro. CAFs were observed growing out from the tumors established from peritoneal-metastatic cells, but not the tumors established from the primary cancer. Conclusion The results of the present study suggest that multi-nucleated cancer cells and CAFs were related to peritoneal metastasis of pancreatic cancer.

Imaging the Role of Multinucleate Pancreatic Cancer Cells and Cancer-Associated Fibroblasts in Peritoneal Metastasis in Mouse Models.

Background/aim Fatty liver disease is increasing in the developed and developing world. Liver metastasis from malignant lymphoma in the fatty liver is poorly understood. In a previous report, we developed color-coded imaging of the tumor microenvironment (TME) of the murine EL4-RFP malignant lymphoma during metastasis, including the lung. In the present report, we investigated the potential and microenvironment of the fatty liver induced by a choline-deficient diet as a metastatic site in this mouse lymphoma model. Materials and methods C57BL/6-GFP transgenic mice were fed with a choline-deficient diet in order to establish a fatty liver model. EL4-RFP cells were injected in the spleen of normal mice and fatty-liver mice. Metastases in mice with fatty liver or normal liver were imaged with the Olympus SZX7 microscope and the Olympus FV1000 confocal microscope. Results Metastases of EL4-RFP were observed in the liver, ascites and bone marrow. Primary tumors were imaged in the spleen at the injection site. The fewest metastases were observed in the fatty liver. In addition, the fewest cancer-associated fibroblasts (CAFs) were observed in the fatty liver. Conclusion The relative metastatic resistance of the fatty liver may be due to the reduced number of CAFs in the fatty livers. The mechanism of the effect of the choline-deficient diet is discussed.

Choline-Deficient-Diet-Induced Fatty Liver Is a Metastasis-Resistant Microenvironment

Background: The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. In a previous study, EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were established and injected into the tail vein of C57/BL6 green fluorescent protein (GFP) transgenic mice. Metastasis was observed at multiple sites which were also enriched with host GFP-expressing stromal cells. In the present study, our aim was to establish an orthotopic model of EL4-RFP. Materials and Methods: In the present study, EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice as an orthotopic model of lymphoma. Resultant primary tumor and metastases were imaged with the Olympus FV1000 scanning laser confocal microscope. Results: EL4-RFP metastasis was observed 21 days later. EL4-RFP tumors in the spleen (primary injection site), liver, supra-mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized by color-coded imaging. EL4-RFP metastases in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Small tumors were observed in the spleen, which were rich in host stromal cells. In the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture. Conclusion: Color-coded imaging of the lymphoma microenvironment suggests an important role of stromal cells in lymphoma progression and metastasis.

Color-Coded Imaging of Syngeneic Orthotopic Malignant Lymphoma Interacting with Host Stromal Cells During Metastasis

Background/aim Our laboratory pioneered color-coded imaging of the tumor microenvironment (TME). We observed recruitment of cancer and stromal cells to the TME and recombination between cancer and stromal cells. The aim of the present study was to observe the dynamics of the TME by color-coded imaging during metastasis and in the formation of a pre-metastatic niche. Materials and methods Red-fluorescent protein (RFP-expressing) mouse colon-cancer 26 cells were initially injected subcutaneously in green-fluorescent protein (GFP) nude mice. The resulting subcutaneous tumors were harvested and cultured. The cultured subcutaneous tumors contained RFP colon cancer cells, GFP stromal cells and recombinant cancer-stromal cells expressing yellow fluorescence. After 14 days culture, the cells were injected into the spleen. Results After splenic injection, colon-cancer 26 metastases were observed in the liver, ascites, and bone marrow. Using the Olympus FV1000 confocal microscope, the cells cultured from tumors and metastasis in each site were visualized. RFP colon-cancer cells, GFP stromal cells derived from host GFP nude mice, and recombinant yellow-fluorescent cells were observed in each organ. In addition, in the liver, areas with only GFP stromal cells were observed and assumed to be a pre-metastatic niche. Conclusion Color-coded imaging demonstrated the dynamics of colon cancer and stromal cells at different metastatic sites including the formation of recombinant cancer-stromal cells.

Kosuke Hasegawa

Papers

Color-coded Live Imaging of Heterokaryon Formation and Nuclear Fusion of Hybridizing Cancer Cells.

Imaging the Role of Multinucleate Pancreatic Cancer Cells and Cancer-Associated Fibroblasts in Peritoneal Metastasis in Mouse Models.

Choline-Deficient-Diet-Induced Fatty Liver Is a Metastasis-Resistant Microenvironment

Color-Coded Imaging of Syngeneic Orthotopic Malignant Lymphoma Interacting with Host Stromal Cells During Metastasis

Color-coded Imaging Distinguishes Cancer Cells, Stromal Cells, and Recombinant Cancer-stromal Cells in the Tumor Microenvironment During Metastasis.