Nicholas Saguibo and Dr. Marc Hansen, Physiology and Developmental Biology

Introduction

The TGF-β signaling pathway has been researched extensively over the past few years, and has been shown to be active in the majority of metastatic tumors. Interestingly, tumors expressing TGF-β activity are positively correlated with poorer prognosis in patients, making it a logical target for cancer therapeutics. Furthermore, research conducted in our lab in recent years has demonstrated that the stiffness of the substrate upon which metastatic cancer cells are adhered plays a significant role in the rate of metastasis. To summarize: the harder the substrate, the higher the rate of metastasis. This same research has also demonstrated that softer, more pliable substrates inhibit tumor migration and increase cell apoptosis (programmed cell death).

In line with these findings, we set out to develop a novel screening technology that utilizes the TGF-β pathway to identify chemical compounds that “trick” the tumor into thinking it is adhered to a softer substrate. Instead of undergoing migration, cells are triggered to undergo apoptosis. This approach to anti-cancer drug development is unique in that it alters the communication between the tumor and its microenvironment, instead of targeting the tumor itself. Furthermore, it provides an alluring way for researchers to approach cancer drug discovery.

Methodology

Cell culture

An array of immortalized cancer cell lines were cultured using appropriate growth medium supplemented with 10% Fetal Bovine Serum and 1% 1000X PSK. Cell cultures were incubated between experiments at 37°C and 5.0% CO₂. Growth medium primarily used for cell culture was RPMI or lowglucose DMEM, depending on the cell line. TGFβ was purchased through Antigenix America.

BIS-acrylamide Substrate Formation

To generate flexible substrates, we adapted the protocol suggested by Wang and Pelham [1]. The glass well bottoms of a 96-well plate were etched with sodium hydroxide and silanized with 3- aminopropyltrimethoxysilane. The wells were subsequently treated with 0.5% (v/v) glutaraldehyde to activate the glass for covalent linkage to polyacrylamide gels. Flexible polyacrylamide gels were prepared using 8% acrylamide and 0.06% BIS and polymerized to the activated, silanized surface of the 96-well plate. In order for cells to grow on this flexible BIS-acrylamide substrate, we had to activate the substrate for covalent linkage with an extracellular matrix. This was accomplished by adding 1 mM sulfo-SANPAH to each well and exposing the well to UV light for 5 minutes. The sulfo-SANPAH was then washed, and the gel substrates were incubated overnight with collagen I. Finally, surfaces were washed with PBS and proper growth medium was added to wells prior to seeding with cells.

Results

Although countless hours were spent by our team working to perfect the substrate screening technology we had proposed, we were unable to produce a marketable drug screen that accurately predicted anti-tumor efficacy in small molecule compounds. This is not to say we have abandoned any hopes of eventually perfecting our idea; on the contrary, our screening technology is still being developed by research assistants in the Hansen Lab. Furthermore, although we certainly desired to have a working anti-cancer drug screen developed within a year, we found difficulty adapting the above-described substrate formation protocol to a 96-well format. Although there have been reports of the successful formation of soft substrates in 96-well and 384-well formats [2], we did not have the resources available at the time to recreate the methods by which these are produced. Because our screening technology is dependent on our ability to efficiently screen thousands of compounds in a short amount of time, it was crucial for us to develop our screen using either a 96- or 384-well format. Because of the difficulties we experienced in adapting our soft substrate formation protocol to multiwell plates, however, producing a workable screen in such a format merits further investigation and development.

Discussion

Although there has been much progress made in the fight against cancer, there is still a dire need for the discovery of new, targeted anti-cancer drugs. Developing a screening technology that determines the anti-cancer efficacy of investigational small molecule compounds, therefore, is a logical route for discovering new therapeutics. We set out to develop such a screen using a novel approach to drug discovery; to date, no one, to our knowledge, has designed an anti-cancer screening technology that purposely aims to alter the intimate relationship between a cancer cell and its immediate surroundings, also known as the tumor microenvironment. We hypothesized that by developing a screen to detect small molecule compounds that disrupt this cell-substrate relationship, we would be able to isolate potentially promising anti-cancer drugs. As previously described, however, the methods and principles behind this screen require further investigation and study in order for it to be a viable way of detecting novel anticancer therapeutics.

Conclusion

Although were unable to perfect our drug screen, we were successful in laying the groundwork for future research assistants within the Hansen lab to develop this technology and further utilize it to study the associations between cell-substrate interactions. It is our hope that academic and industrial institutions will one day be able to utilize this screening technology as an efficient tool to provide new leads for small molecule anti-cancer compounds.

References

i. YL Wang, RJ Pelham Jr., Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells. Methods Enzymol. 298. (1998) 489-496.

ii. JD Mih, AS Sharif, et al, A Multiwell Platform for Studying Stiffness-Dependent Cell Biology. PLoS One. 6 (5). (2011).