(F-236) Neutrophil Extracellular Traps sensing activates directional migration of Brain Metastatic Breast Tumors

Introduction::
Neutrophils are considered as the first line of pathogenic defenders within the innate immune system. Under certain conditions, neutrophils can release DNA into their surroundings, forming protein web structures collectively called Neutrophil Extracellular Traps (NETs). It is known that NETs could enable tumor metatasis¹. However, there is not enough information to sufficiently explain the mechanisms of NETs-mediated metastatic aggression of tumor clusters, which we sought to better comprehend through this work.




Materials and Methods::
Neutrophils were isolated from whole blood of healthy individuals. Brain metastatic subtype of breast cancer cell line MDA-MB-231 (henceforth called BM BrCa) were isolated from mice². BM BrCa spheroids and neutrophils were cultured in a segregated format on our open channel microfluidic device made of Polyacylamide (PAAm), so as to inhibit any initial migratory response of either of the cell types. NETs were labeled with 2.5 µM Sytox Orange, and as required tumor cells were stained with cell tracker (CMFDA - green) and were immnostained for CCDC25 to visualize NETs-induced DNA sensing.




Results, Conclusions, and Discussions::

Segregated neutrophil-tumor spheroid coculture was enabled on our open channel porous PAAm device that allows for biochemical signaling, but at the same time enables cell dynamics only upon on-demand introduction of the Extracellular Matrix (Collagen) (Fig. 1A). Neutrophil response to BM BrCa spheroids was studied through NETosis under static conditions enabled by the segregated co-culture. Significantly greater number of neutrophils NETosed in the presence of BM BrCa compared to parental BrCa, as previously reported². Correspondingly, in the presence of NETs, BM BrCa showed significantly greater CCDC25 activation than parental BrCa, and this was validated with our preliminary qRT-PCR data (Fig. 1B(i)). Introduction of collagen as the extracellular matrix into the segregated co-culture enabled the migration of tumor in response to the NETs (Fig. 1B(ii)). A significant migration of the BM BrCa spheroids, and contrastingly, negligible parental BrCa migration in the presence of NETs was observed. Also, the area of tumor invasion for the BM BrCa was proportional to the NETs density and their proximity, and the alignment of tumor invasion was significantly greater towards NETs (Fig. 1C).

It has been earlier reported that stromal NETs, and not tumor contacted NETs facilitate tumor migration³. Our current results show that NETs sensing of tumors is dependent on their metastatic potential, as observed with the activation of DNA sensor CCDC25 being specific to brain metastatic breast cancer. Taken together, our results suggest that neutrophil reprogramming by the metastatic tumors could be a strategy adopted to generate stromal NETs, which upon being sensed by the tumors, subsequently drive the tumor invasion. This is supported by our results which show that the NETs-tropic migration of metastatic tumors is not just dependent on the presence, but is also controlled by the location and density of NETs.






Acknowledgements (Optional): :

References (Optional): :
[1] Park ,J., Sci Transl Med (2016) vol 8 pp. 361




[2] Safarulla, S., Cancers (2022) Vol. 14 n3 pp. 515




[3] Surendran, V., Biofabrication (2021) vol. 13, n3, 035029