課題番号 ：S-15-NM-0046
利用形態 ：機器利用
利用課題名（日本語） ：
Program Title (English) ：Effect of the extracellular matrix on cellular aggregate invasion
利用者名（日本語） ：
Username (English) ：Nada Khalifat1), Pr. Françoise Brochard-Wyart2)
所属名（日本語） ：
Affiliation (English) ：1) Pierre et Marie Curie University, Paris France, 2) Institut Curie, Paris France

１．概要（Summary ）
Cellular aggregates have been used in experimental cancer research for several decades and serve as in vitro models of tumor micro regions to study the mechanisms involved in the control of proliferation, invasion and metastasis. They have a surface tension, and they form spheroids to minimize their surface energy. They spread like drops of a simple viscous liquid, where the parameters controlling the wetting properties (adhesion, viscosity and friction coefficient) depend upon cellular activity. In this project, our aim is to extend our previous studies regarding the spreading of sarcoma 180 cell line, to cells secreting extracellular matrix: the highly metastatic colon carcinoma cells CT26 line.
２．実験（Experimental）
Cell culture and aggregates preparation: NIMS MMSPF’S facility
CT26 cells are cultured at 37°C under 95% air / 5% CO2 atmosphere in culture medium consisted of Dulbecco’s Modified Eagle Medium (DMEM) enriched with 10% calf serum Upon reaching confluence, cells are prepared for aggregation following a procedure similar to that of by the pendant drop technique (fig.1)

Preparation of polyacrylamide substrates: NIMS MANA
Flexible polyacrylamide (PAA) is prepared by allowing polyacrylamide solutions to polymerize between two chemically modified glass cover slips and summarized on Fig. 2.The cover slip with the attached polyacrylamide gel is place at the bottom of a chamber for the spreading experiments.

Figure 2: Substrate preparation for spreading of cell aggregates.
Microscopy and Image analysis: NIMS MMSPF’S facility and Institut Curie Nikon Microscopy Center
Aggregates were placed on a fibronectin coated glass coverslip (or a polyacrylamide gel) that formed the bottom of a cylindrical experimental chamber filled with CO2-equilibrated culture medium maintained at 37°C using a heating platform. To prevent evaporation, the open surface was sealed with mineral oil.
Long time spreading is observed using an inverted microscope (Leica) equipped with x10 and x20 air objectives. And, recording at an acquisition rate of 1 frame every 10 min. The spreading experiment consists in observing the aggregate from below to determine if a precursor film (monolayer of cells) expands around the aggregate. The presence of a precursor film is the signature of spreading coefficient S>0. The spreading dynamics is characterized by measuring the contact and the precursor film areas by tracing the contour of the spreading aggregate, using ImageJ software (National Institutes of Health) and taking the enclosed area.
Immunofluorescence Staining and Imaging: NIMS MMSPF’S facility
Immunostaining performed on the aggregate after spreading. The aggregate were then incubated for 45 min at RT in PBS complemented with 3% FCS and stored overnight at 4 °C with primary antibody diluted in the same buffer. Secondary fluorescent antibodies were added for 45 min. We used Life Act-GFP stable line or WT CT26 cells. Imaging was performed using Leica confocal microscope with x 63 oil objective.
３．結果と考察（Results and Discussion）
The analogy between tissues and liquids has been very fruitful to describe the spreading of tissues, using the physics of capillarity and wetting. Tissue spreading results from a competition between cell-cell adhesion (Wcc) and cell-substrate adhesion (Wcs) controlled by integrins binding to fibronectin. We can tune Wcs by varying substrate rigidity. The sign of the spreading parameter: S = Wcs – Wcc controls the wetting properties. If S < 0, cell-cell adhesion is dominant and the aggregate does not spread: “partial wetting”. If S > 0, “complete wetting” occurs, and a “precursor film” made of a cellular monolayer flows outwards from the edge of the aggregate (Fig.3).

Figure 3: Illustration of the two regimes of wetting aggregates.
We characterize the spreading by measuring D (E) = Rc V*, where the dependence of the diffusion coefficient D on the substrate elastic modulus E is proportional to the radius of the aggregate Rc and to the velocity V*(Fig.4).

Figure 4: Dependence of the velocity V* upon substrate rigidity. D(E) = Rc V*.
We observe complete wetting with decreasing the substrate elastic modulus from 16.7 to 0.2kPa, and a slowdown of velocity when it passes by a maximum at around 8kPa. In addition, the cellular morphology appeared heterogeneous and depending on substrates rigidity, they exhibit protrusions with diverse morphology and dynamic Fig.5.

Figure 4: Immunofluorescence confocal Image of the precursor film of CT26 on soft substrate exhibiting protrusions (Green: Actin in green & Laminin network in pink).
４．その他・特記事項（Others）
The project is still running.
５．論文・学会発表（Publication/Presentation）
The paper is in preparation.
Role of the extracellular matrix in the spreading of cellular aggregates
Nada Khalifat, Grégory Beaune, Guillaume Duclos, Tomita Vasilica Stirbat, Françoise M. Winnik, and Françoise Brochard-Wyart

６．関連特許（Patent）
なし。