(68d) Dormant Brain Metastatic Breast Cancer Spheroids in Biomimetic Hyaluronic Acid Hydrogels Exhibit Resistance to Therapy Via p38 Signaling

Breast cancer is a leading cause of mortality among women worldwide, posing significant challenges due to its propensity for metastasis to secondary organs. Brain metastatic breast cancer (BMBC) poses a considerable challenge to patient prognosis, necessitating a deeper understanding of its underlying mechanisms. One crucial aspect is the dormancy phase, where disseminated tumor cells remain quiescent, evading treatment and potentially leading to relapse. These dormant cells, impervious to therapy, represent a formidable obstacle to effective treatment strategies. Therefore, development of experimental model systems that model dormancy and the associated resistance to therapy is imperative for developing innovative therapeutic interventions. In this study, we leveraged a biomimetic hyaluronic acid (HA) hydrogel platform that has been previously shown by our group to induce dormancy and a scaffold-free environment (i.e., suspension culture) to stimulate cell proliferation, to delineate the response of dormant versus proliferating BMBC spheroids to both chemo (Paclitaxel) and targeted (Lapatinib) therapies.

In our study, we utilized two distinct cell lines representing BMBC: triple-negative MDA-MB-231Br cells and human epidermal growth factor receptor 2 positive BT474Br3 cells. Spheroids derived from these cell lines were subjected to Paclitaxel or Lapatinib treatment over a 72 h period in HA hydrogel or suspension cultures. We found a marked reduction in proliferation in treated proliferating spheroids, as evidenced by Ki67 and EdU staining, while displaying a contrasting pattern in apoptosis, as indicated by Annexin-V staining. Interestingly, dormant spheroids exhibited negligible changes in these proliferation and apoptosis markers post-treatment, highlighting their intrinsic resistance to therapy. Additionally, we found a significant decrease in the % extracellular signal-regulated kinase 1/2 (p-ERK) to % phosphorylated p38 (p-p38) ratio in proliferating spheroids post treatment, implying their increased susceptibility to therapy compared to dormant spheroids, which maintained similar ratios. Consequently, inhibition of p38 in dormant spheroids cultured on HA hydrogels resulted in a marked increase in proliferation, and subsequent response to therapy, further highlighting the intricate interplay between dormancy-associated signaling pathways and therapeutic response. Finally, we demonstrated that the observed resistance to therapy was also reversible as the transfer of dormant spheroids cultured on HA hydrogel for 5 days to suspension cultures resulted in proliferation and response to therapy at the end of day 13. Overall, such biomimetic hydrogel-based culture platforms provide useful tools to study dormancy associated drug resistance in BMBC.