Preliminary analysis of cellular sociology of co-cultured glioma initiating cells and macrophages in vitro

doi:10.18679/CN11-6030/R.2016.018

Translational Neuroscience and Clinics

2016, Vol. 2

Issue (2): 77-86 doi: 10.18679/CN11-6030/R.2016.018

Original Articles

Preliminary analysis of cellular sociology of co-cultured glioma initiating cells and macrophages in vitro

Mingxia Zhang¹, Junjie Chen², Lin Wang¹, Xiaoyan Ji¹, Lin Yang¹, Yujing Sheng¹, Hairui Liu¹, Haiyang Wang¹, Aidong Wang¹, Xingliang Dai¹, Xiaonan Li³, Qiang Huang¹, Jun Dong¹

1 Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou 215004, China;
2 Department of Neurology, Xishan People's Hospital, Wuxi 214000, China;
3 Laboratory of Molecular Neuron-oncology, Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX, USA

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Abstract Objective: Real-time monitoring of cytokine secretion at the single immunocyte level, based on the concept of immune cells, sociology has been recently reported. However, the relationships between glioma-initiating cells (GICs) and host immune cells and their mutual interactions in the tumor microenvironment have not been directly observed and remain unclear.
Methods: The dual fluorescence tracing technique was applied to label the co-cultured GICs and host macrophages (Mø), and the interactions between the two types of cells were observed using a live cell imaging system. Fusion cells in the co-culture system were monocloned and proliferated in vitro and their social interactions were observed and recorded.
Results: Using real-time dynamic observation of target cells, 6 types of intercellular conjunction microtubes were found to function in the transfer of intercellular information between GICs and Mø; GICs and host Mø can fuse into hybrid cells after several rounds of mutual interactions, and then these fusion cells fused with each other; Fusion cells generated offspring cells through symmetrical and asymmetrical division or underwent apoptosis. A "cell in cell" phenomenon was observed in the fusion cells, which was often followed by cell release, namely entosis.
Conclusions: Preliminary studies revealed the patterns of cell conjunction via microtubes between GICs and host Mø and the processes of cell fusion, division, and entosis. The results revealed malignant transformation of host Mø, induced by GICs, suggesting complex social relationships among tumor-immune cells in gliomas.

Key words： glioma initiating cells macrophages cellular interactions live cell time-lapse shoot dual fluorescence tracing technique

Received: 20 May 2016 Published: 30 June 2016

Fund: Supported by the National Natural Science Foundation of China (Grant No. 81472739) and the Natural Science Foundation of Jiangsu Province (Grant No. BK20151214).

Corresponding Authors: Jun Dong,E-mail:djdongjun@163.com E-mail: djdongjun@163.com


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	Mingxia Zhang
	Junjie Chen
	Lin Wang
	Xiaoyan Ji
	Lin Yang
	Yujing Sheng
	Hairui Liu
	Haiyang Wang
	Aidong Wang
	Xingliang Dai
	Xiaonan Li
	Qiang Huang
	Jun Dong

Cite this article:

Mingxia Zhang, Junjie Chen, Lin Wang, Xiaoyan Ji, Lin Yang, Yujing Sheng, Hairui Liu, Haiyang Wang, Aidong Wang, Xingliang Dai, Xiaonan Li, Qiang Huang, Jun Dong. Preliminary analysis of cellular sociology of co-cultured glioma initiating cells and macrophages in vitro. Translational Neuroscience and Clinics, 2016, 2(2): 77-86.

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http://tnc.tsinghuajournals.com/10.18679/CN11-6030/R.2016.018 OR http://tnc.tsinghuajournals.com/Y2016/V2/I2/77

Figure 1 Flowchart of the study.

Figure 2 Various types of cell conjunctions. Diameters and lengths of conjunction microtubes varied, with slim (a arrow) and thick tubes
(b, c arrow) observed conjunctions connected nearby cells (a-c) and distant cells (d arrow) according to the positions of relevant cells. These
conjunctions can be also divided into single cell conjunctions (a-d) and multiple cell conjunctions (e, cell ① connected with ②, ③, ④, ⑤
cells, respectively, via conjunction tubes). None of the conjunctions was permanent.

Figure 3 Cell fusion between GICs and Mø. (a–d) GIC actively fused with Mø. A GIC cell extended its pseudopodia to attack several Mø (a). Next, some Mø were fused successfully by the GICs (b), after which another GIC completely fused with Mø and became an RFP/GFP double positive fusion cell. The cell body of the fusion cell gradually became prolonged with a tapered middle section (c). Next, the cell entered prophase of mitosis. Approximately 17 h were required for the cell to divide into two offspring cells (d). (e–l) Mø actively fused GIC. Initially a GIC was enclosed by several Mø (e), and then the Mø (including No. ① cell) further approached and attacked the GIC continuously. It appeared that the cells could not find a breakthrough point, so the No. ① cell changed its attack point (f). After 1,870 min of “fighting”, the shape of Mø (No. ①) changed and had more filamentous pseudopodium and did not maintain close to inserted into the GIC directly (j). Next, the GIC changed from red to light yellow, and several Mø entered the GIC individually, causing the GIC to become dark yellow. Both the RFP and GFP markers were positive (l). (m–o) Re-fusion occurred between the GIC-Mø fusion cells. There were 4 yellow cells in the vision field. The two round cells fused together. Initially, one cell rapidly approached another cell (m). The cells came into contact and began to combine (n). This fusion process took approximately 60 min and the fusion cells were 2× larger than the size of the parent cell (o).

Figure 4 Reproduction of fusion cells. A very large multi-nuclei re-fusion cell was observed (a). After 440 min its body turned into a
sphaerocyst with retracted pseudopodia (b). Another 890 min later, this cell divided into two cells (c); however, after cell division, the
fluorescence of these two cells was initially green and then became yellow after 760 min (d).

Figure 5 Instant apoptosis of fusion cells. One of the fusion cells, the FC-1 cell migrated irregularly before apoptosis. Initially, it moved
close to the No. ① cell (a), but the cells were not in close contact. The migrating cell continued towards the left front and turned back (b).
Next, the FC-1 cell approached to the No. ② cell (c), which extended its pseudopodia, stopped during migration (d) and moved close to
the FC-1 cell (e). Nearly simultaneously, the FC-1 cell collapsed suddenly and its debris was engulfed by the No. ② cell (f). Another fusion
cell, the FC-2 cell, migrated while showing continuous morphologic changes of its cell body. Initially, the cell contacted the surrounding
No. ①–③ Mø cells (g). Only the No. ② cell followed the FC-2 cell closely (h), but they eventually separated (i). The No. ④ cell
approached the FC-2 cell and extended its pseudopodia (j), after which the FC-2 broke into pieces (k), but phagocytosis did not occur. The
FC-2 cell debris approached the No. ⑤ cell (l) gradually, and phagocytosis occurred in No. ⑤ (m–o), while No. ④ had no further
changes (p).

Figure 6 Phenomenon of entosis (scale bar 20 μm). The transient
state in which a live GIC was contained within a Mø (Arrows
marked engulfed cells).

Figure 7 Fate of entotic cells. Entosis occurred in two yellow spherocytes, No. 1 (a) and No. 3 (e). The deep yellow round cell No. 1 released
a slight yellow short spindle cell (b, c), after which the offspring cells rapidly exited. During release, the shape of the parent cell changed
from round to spindle. After release,the shape of the parent cell was restored to its original state (b: No. 1*; d: No. 1*). The No. 3 (e) parent
cell migrated and stretched its small prominences in different directions (f), and then it retracted and released a long spindle pale yellow
offspring (g: No. 3*). The offspring cell moved actively around its parent cell. After 370 min the cell became a spherocyte, similar to its
parent cell (h: No. 3*).

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