BME PhD Candidate, Cindy Farino Reyes, will be defending her dissertation.
An Engineered Hydrogel Platform for Understanding Breast Cancer Dormancy, Reactivation, and Chemoresistance for Drug Development
When: Tuesday January 11, 2022 @ 10 AM EST
NO IN-PERSON ATTENDANCE
VIRTUAL: https://udel.zoom.us/j/93342333532 Password: Farino
Committee Chair: John Slater, PhD
Committee: Kelvin Lee, PhD; Jennifer Sims-Mourtada, PhD; Elise Corbin, PhD
Metastasis remains the leading cause of cancer-associated death worldwide. A latency period ranging from months to decades is often observed prior to relapse as disseminated tumor cells (DTCs) can undergo dormancy upon infiltration of secondary organs. While eliminating these dormant cells can prevent recurrence, current chemotherapeutics fail to effectively target and eliminate dormant populations. Mechanistic understanding of dormancy-associated chemoresistance could lead to development of targeted therapeutic strategies. In the effort towards developing new therapeutic strategies to eliminate dormant cells, we developed an in vitro, hydrogel platform to mimic generic premetastatic niches during initial DTC infiltration of secondary tissues.
In particular, we investigated the role of hydrogel properties (matrix adhesivity and degradability) in regulating the phenotypic fate of breast cancer using five breast cancer cell lines: the triple negative MDA‑MB‑231 (231) parental line, three organotropic sublines derived from the parental 231 line: BoM-1833 (bone‑tropic), LM2-4175 (lung‑tropic), and BrM2a‑831 (brain-tropic), and the estrogen receptor positive (ER+) MCF7 cell line. Each cell line was individually encapsulated and cultured for 15 days in three distinct, poly(ethylene glycol) (PEG)-based hydrogel formulations composed of proteolytically degradable PEG (PEG-PQ), integrin‑ligating RGDS, and the non‑degradable crosslinker N‑vinyl pyrrolidone (NVP). We varied concentrations of RGDS and NVP to mimic favorable and unfavorable premetastatic niches including a permissive niche with high levels of adhesivity and degradability (Gel 1: ++ adhesivity, ++ degradability), a non-permissive niche with moderate levels of adhesivity and degradability (Gel 2: + adhesivity, + degradability), and a non-permissive niche with no adhesivity but high degradability (Gel 3: – adhesivity, ++ degradability). Dormancy-associated metrics including temporal changes in viable cell density, proliferation, metabolism, apoptosis, phosphorylated‑ERK (p-ERK) and -p38 (p-p38), and morphological characteristics were quantified. A multimetric classification approach was implemented to categorize each hydrogel-induced phenotype as: (1) growth, (2) balanced tumor dormancy, (3) balanced cellular dormancy, or (4) restricted survival, cellular dormancy. Hydrogels with high adhesivity and degradability (permissive gel 1) promoted growth in all five cell lines tested. Hydrogels with no adhesivity, but high degradability (non-permissive gel 3), induced restricted survival, cellular dormancy in the parental 231 and MCF7 lines and balanced cellular dormancy in the organotropic lines demonstrating the role of cell adhesion in inducing dormancy while also highlighting the enhanced survival capabilities of organotropic sublines. Hydrogels with moderate adhesivity and degradability (non-permissive gel 2) induced balanced cellular dormancy in the parental 231 and lung‑tropic lines and balanced tumor mass dormancy in bone‑ and brain‑tropic lines and MCF7s, demonstrating that matrix confinement can induce dormancy. The ability to induce escape from dormancy, reactivation, via “on-demand” dynamic incorporation of RGDS into a highly degradable hydrogel was also demonstrated.
With an established dormancy and reactivation platform, we implemented this system to quantify dormancy-associated chemoresistance. The cellular responses to doxorubicin (DOX), paclitaxel (PAC), and 5-fluorouracil (5-FU) in the growth formulation (permissive gel 1), and dormant formulations (non-permissive gels 2 and 3) in parental 231s were quantified via measurement of half maximal inhibitory concentration (IC50) and half maximal effective concentration (EC50) values. Under PAC and 5-FU treatment, parental 231s residing in either dormant state exhibited increased chemoresistance compared to 231s in the growth state in permissive gel 1. DOX was further tested on growing, dormant, and reactivated parental 231s, organotropic sublines, and MCF7s. Parental 231s undergoing dormancy demonstrated increased chemoresistance with a 1.4 to 1.8-fold increase in EC50 and 1.3 to 1.8-fold increase in IC50 compared to cells in the growth state. Furthermore, reactivated parental 231s were also chemoresistant and displayed a ~2.5 fold increase in the EC50. Similar results were observed for organotropic sublines and MCF7s. To mechanistically investigate the role of dormancy in conferring DOX resistance, cytoplasmic and nuclear accumulation of DOX was measured. The results indicated statistically significantly higher DOX accumulation rates and nuclear localization, determined by nuclear to cytoplasmic mean intensity ratio (NC ratio), in cells in the growth state (permissive gel 1) compared to the two dormant (non-permissive gels 2 and 3) and reactivated states. These results further validated the utility of implementing engineered hydrogels as an in vitro platform to induce breast cancer dormancy for the development of anti-dormancy therapeutic strategies and provided valuable insight into the mechanisms involved in dormant and reactivated cell chemoresistance.
Based on our findings that chemoresistant dormant and reactivated cells exhibit decreased DOX intracellular accumulation rates and subsequent localization to the nucleus, we investigated the role of three ABC-efflux pumps (P-gp, MRP1, and BCRP) which are known to be overexpressed in chemoresistant cancers. Immunofluorescence analysis revealed that chemoresistant dormant and reactivated cells expressed significantly greater levels of P-gp, MRP1, and BCRP (excluding MCF7s which had similar levels of BCRP expression in all states), compared to more chemosensitive cells residing in the growth state induced by permissive gel 1. To determine if differential efflux pump expression played a role in chemoresistance, efflux pumps were inhibited using a P-gp inhibitor (10 nM tariquidar), MRP1 inhibitor (50 µM MK571), BCRP inhibitor (10 µM Ko 143), or a combination of all three inhibitors on parental 231s, brain-tropic 231s, and MCF7s. Results showed that efflux pump inhibition significantly increased chemosensitivity, indicated by significant decreases in EC50 values, in all three cell lines. For instance, P-gp+MRP1+BCRP inhibition decreased EC50 values to 4.2 ± 1.1 µM (permissive gel 1, ~11-fold decrease), 0.4 ± 0.1 µM (non-permissive gel 2, ~170-fold decrease), 1.1 ± 0.5 µM (non-permissive gel 3, ~81-fold decrease), and 1.4 ± 0.3 µM (reactivated, ~81-fold decrease) compared to no inhibition, with significantly lower EC50 values in dormant and reactivated parental 231s than cells in permissive gel 1. Increased chemoresistance was accompanied by significant increases in the DOX accumulation rate (~3-11-fold increase) and NC ratio (~1.2-3.2-fold increase) for all three cell lines. Furthermore, efflux pump inhibition led to significantly greater accumulation rates and, in most cases, greater NC ratios in dormant and reactivated cells compared to growing cells in permissive gel 1. These results provide novel insights in the role of efflux pumps in dormancy and reactivated cell chemoresistance and proposes a therapeutic strategy to target and eliminate dormant and reactivated cells in vivo.
Taken together this work provides a well-characterized hydrogel platform to induce breast cancer dormancy and reactivation as demonstrated in five breast cancer lines, implements the platform to quantify dormant and reactivated cell chemoresistance, provides novel insight regarding DOX accumulation and localization in dormant and reactivated cells, provides insight into the role of efflux pumps in chemoresistance and DOX transport, and demonstrates the ability to increase DOX efficacy in treatment of highly chemoresistant dormant and reactivated cancer via efflux pump inhibition in vitro. Future implementation of these results could be used to model dormancy in other cancers and investigate mechanisms involved in dormancy and reactivation. Furthermore, we show that this platform can be used in drug development and plan future studies to test our treatment strategy in vivo, with the ultimate goal to eliminate dormant cells and aid in preventing metastatic relapse.