Integrative cancer biology-on-chip: Blood science and angiogenesis-enabled Ovarian TME-Chips determine platelet pathophysiology in antiangiogenic escape
Introduction
Platelets are the first responders in vascular pathobiology of cancer and play both structural and functional roles as reporters, messengers, and active transporters within the tumor microenvironment (TME). But what remains a major unmet need is a detailed knowledge of mechanisms that result in platelet trafficking into the TME. Relatedly, there is little understanding that informs how platelets influence tumor angiogenesis, as well as conferring resistance against antiangiogenic and immune therapy, partly because the TME comprises complex interactions between platelets, endothelium, and cancer cells. This need is driven, in part, by the lack of reductionist models to study this complex biology. Here, we have engineered a tumor microenvironment-chip that is capable of recapitulation and dissection of angiogenesis due to complex and combinatorial signaling arising from platelets, endothelial and ovarian cancer cells (aTME-Chip). With this platform, we have reproduced the spatiotemporal dynamics of endothelial sprouting within a TME composed of several human ovarian cancer cell types. We have then proceeded to incrementally and systematically include the influence of circulating and extravasating human platelets on ovarian tumor angiogenesis and resistance against anti-angiogenic therapy within the aTME-Chips, using both normal and patient cells.
Methods
The aTME-Chip consists of microfluidic device with two straight channels on the side and a gel channel in the center made of PDMS soft lithography. Cancer cells were encapsulated in the central gel channel while primary endothelial cells (EC) were seeded in a straight channel to form confluent lumen on one side and culture media was flowed through the straight channel on other side to induce angiogenesis. The study was performed for a period of seven days with ovarian cancer cell lines. Platelets from whole blood were perfused in the device through the EC lumen undergoing angiogenesis in the aTME-Chip. The contribution of platelets in evading the effect of anti-angiogenic drug was performed by exposing the aTME-Chip to Bevacizumab, an anti-VEGF therapeutic agent for 48h. Images were captured and analyzed every 48h over the period of 7 days.
Results
Our results show that upon the co-culture of ovarian cancer cells and ECs in the aTME-Chip, systematic barrier disruption, sprouting, and angiogenesis occurs over time in the presence or absence of moving platelets. By including three distinct human ovarian cancer cell-types that may induce angiogenesis differentially, the aTME-Chip systematically and quantitatively several outcomes– firstly, introduction of platelets significantly increases the proangiogenic activity within TME. Secondly, we find that the temporal dynamics of platelet-led angiogenic signaling are dependent on ovarian cancer cell type and seeding density. Further, platelets either activated exogenously by cancer cells or derived clinically from an ovarian cancer patient exert a proangiogenic signaling within the TME. Finally, on exposing the aTME-Chips to anti-VEGF bevacizumab treatment, we observed significant retrogression of angiogenic sprouts in aTME-Chip in absence of platelets while the angiogenesis was found to be transiently arrested in aTME-Chip with platelets. Upon withdrawal of anti-angiogenic therapy after 48 h, angiogenesis rebound aggressively in aTME-Chip when platelets were present. Therefore, the presence of platelets within the tumor microenvironment confers chemoresistance thereby evading the action of anti-VEGF drug to control the disease spread.
Conclusion
Our aTME-Chip enables recapitulation and dissection of angiogenesis due to complex and combinatorial signaling arising from platelets as well as endothelial and ovarian cancer cells. This aTME-Chip may be deployed to derive novel anti-angiogenic targets that could work alone or in combination with antiplatelet treatments, ultimately preventing the growth and therapeutic resistance of cancers.