P. T. White1, C. Subramanian1, P. T. Grogan1,2, S. Cai3, M. L. Forrest3, M. S. Cohen1 1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2University Of Kansas,Department Of Pharmacology, Toxicology And Therapeutics,Kansas City, KS, USA 3University Of Kansas,Department Of Pharmaceutical Sciences,Lawrence, KS, USA
Introduction: As the second leading cause of cancer-related death in US women, breast cancer recurrence, metastatic spread, and drug resistance remain a significant challenge despite current therapeutic advances. If only a small subpopulation of tumor cells called progenitor or stem cells survive treatment, they have the ability to transform and recreate a tumor or metastasize. To date no therapeutic specifically targets this cancer stem cell (CSC) population mainly due to the heterogeneity of its surface markers, although CD44 positivity is a commonality among most CSCs. We have developed a CD44-targeted hyaluronic acid nanoparticle drug-delivery platform and hypothesize that this targeting when conjugated to standard chemotherapy drugs will selectively target breast CSCs leading to an improved, more durable therapeutic response.
Methods: A panel of validated human breast cancer cell lines (SUM159,MDA-MB-468LN,T47D,SK-BR-3) were evaluated for CD44 expression levels by flow cytometry(FC). Cy5-labeled HA-drug conjugates (cisplatin, doxorubicin, and docetaxel) were evaluated by FC and fluorescent microscopy for cellular uptake and CD44 targeting. Breast CSCs were identified in the cell lines by CD44,ALDH1,CD133 marker positivity. Drug efficacy, toxicity and survival was performed using our prior published MDA-468LN triple neg model in female Nu/Nu mice via breast fat pad injection(10^6 cells). Animals were treated with either 50% or 75% MTD cis, dox, docetaxel(ACT) combination therapy weekly x 3wks vs. same doses conjugated to 35kDa HA nanoparticles vs. control then followed for survival, relapse, and tumor CD44 expression by Western Blot (WB).
Results: Receptor-mediated active drug uptake was CD44 dependent with SUM159 and MDA468LN having 80-90% uptake and >90% expression compared to 1-5% expression and uptake with CD44-low SK-BR-3 and T47D cells (p<0.001). Uptake is time dependent with a peak at 19hrs. Drug uptake was abrogated by 70-80% via CD44 saturation with excess free HA or CD44 antibody vs. unsaturated(p<0.01). 100% of HA-ACT therapy mice(75%MTD) had a complete clinical response during treatment without observed toxicity vs. 40% std ACT group(p<0.01). 85% of the HA-ACT mice(75% MTD) had a complete durable pathologic response 8 weeks post end of treatment vs. 0% survival in the std ACT and control arms by end of study(p<0.001). Tumor lysates of the HA-triple therapy mice had almost complete inhibition of CD44 expression by WB even 8 weeks after completion of treatment compared to no inhibition with the std. ACT and control groups.
Conclusions: HA-conjugation of chemotherapeutics is a novel approach to selectively target CD44 expressing breast tumor cells which includes the vast majority of breast CSCs. This in combination with improved tumoral drug delivery lead to significant improvement in survival and durability of response compared to standard combination therapy supporting further translation toward clinical applications.