Undoubtedly, monitoring the crosstalk between cancer cells and CAFs in vitro will provide great help for the research of tumor microenvironment and the screening of related anticancer drugs. In this research, we built a model in vitro, which can reflect the crosstalk between cancer cells and fibroblasts via cellular aggregates. To compare with the traditional methods that researching cancer cells and CAFs separately, this co-culture enables researchers to research cancer cells and CAFs as a whole, which could be a very interesting research perspective. In addition, to compare with the methods like subcutaneous injection of mice, co-culture in soft agar culture medium enables researchers to monitor cellular aggregates’ growth and progress so that they can gain more detailed information about cancer process. In practice, the co-culture method is simple, convenient and timesaving, which can reduce the cost for researchers.
In the aggregate formation experiment, we speculate that the reason why the TS/A-ME-iLX-2 aggregates presented the above area trend (Fig. 2c) is that the nutrients are gradually consumed, or ME-iLX-2 was inferior in the nutritional competition between TS/A and ME-iLX-2 which may cause the decreasing of promoting aggregates formation. The decrease of area on the sixth day may be mainly due to the lack of nutrients in the system. We added DMEM on the sixth day, which can explain the increasing from the sixth day to the seventh day. According to above-mentioned information, we conjectured that the nutrient requirement of TS/A-ME-iLX-2 aggregates is more than of TS/A-LX-2 aggregates. Moreover, the areas of TS/A-ME-iLX-2 aggregates are larger than the areas of TS/A-LX-2 aggregates on the same day. In conclusion, we speculate that ME-iLX-2 provide a better environment to aggregate formation.
In eugenol inhibition of aggregates, considering the differences of the quantity, area and morphology of cell aggregates, we believe that this inhibition may occur in two ways: 1. Eugenol inhibits the cellular aggregation which can decrease the number of cellular aggregates. 2. Eugenol inhibits the growth of cellular aggregates after its formation. The differences between control group and experimental group demonstrated eugenol’s inhibition of the aggressive structure of cancer in vitro as well as this soft agar co-culture model’s function of anticancer drug screening.
As for the mechanism of the aggregate’s formation, Sharma et al pointed out that CD44 may be the medium between CAFs and cancer cells. Yamaguchi et al. pointed out that this behavior may be related to the activation of intracellular signaling pathways that regulate actomyosin contractility. Based on the research by Sharma et al., we found that CD44 expression is significantly higher in TS/A-ME-iLX-2 aggregates than in TS/A-LX-2 aggregates, which not only confirms perspective of Sharma et al, but also can be used to refer that, as CAFs, ME-iLX-2 enhance its expression of CD44 to improve its ability to attract TS/A together, which can be a reason why the amount and area of TS/A-ME-iLX-2 aggregates are higher(Fig. 5). In addition, it is also a possible way to increase the amount and area of TS/A-ME-iLX-2 aggregates that CAFs promote the propagation of cancer cells in many ways.
Actually, the composition of the tumor microenvironment is quite complex. Not only does it includes CAFs which is researched in this research, but also includes endothelial cells, mesenchymal stem cells (MSCs), immune cells and even vascular, lymphatic network, etc.. Though the soft-agar co-culture can simulate the crosstalk between cancer cells and CAFs, the capability of its simulated tumor microenvironment needs to be further studied. Considering that MSCs have the ability to recruit and differentiate into CAFs, and CAFs can also recruit MSCs, we speculate that MSCs can promote cellular communication in this soft-agar model. What’s more, biophysical interactions in tumor microenvironment also promote the metastasis of cancer cells. It is also need to be further studied that how to simulate the chemical and mechanical signals better.
In this research, we counted aggregates’ amount and area to reflect their formation and growth, because there were great morphological differences among the cell aggregates, which is particularly significant between the co-culture system and separated fibroblasts. However, it also leads to the problem that the amount and area are always inevitably different when we make statistics. There is still a lack of more scientific indicators and algorithms to make a more accurate assessment of cellular aggregates.