(W1230-01-06) Peptide Conjugated Hypoxia-Responsive Nanoparticles to Decrease Triple-Negative Breast Cancer Stemness in a Patient-Derived Xenograft Mice Model

Purpose: TNBC (triple-negative breast cancer) tumors are diagnosed in about 10-15% of patients. Aggressive tumors have few treatment options because of a lack of expression of the hormone receptors estrogen, progesterone, and HER2. TNBC has an unfavorable prognosis, with high chances of relapse and metastasis. Moreover, uncontrolled cell growth and inadequate blood supply result in low oxygen levels or hypoxia in the tumors. Hypoxia enhances cancer cell survival through increased aggressiveness, metastasis, and resistance to chemotherapy, leading to poor outcomes. We observed increased neuropilin-1 (NRP-1) receptor expression in patient-derived TNBC xenograft tumors. Hence, we decided to prepare targeted hypoxia-responsive polymersomes (Ps) containing the stemness inhibitor Napabucasin (NAPA) and the anticancer drug doxorubicin (DOX) separately. We incorporated an iRGD peptide-conjugated polymer in the polymersomes for active targeting and subsequent solid tumor penetration, which can also assist in reducing the cancer stemness marker with patient-derived xenograft in vivo and in vitro models.

Methods: We observed a pronounced synergy between NAPA and DOX in regulating TNBC cell proliferation under normoxia (21% oxygen) and hypoxia (0.2% oxygen). Subsequently, we prepared targeted, iRGD peptide-conjugated, hypoxia-responsive polymersomes (Ps) containing NAPA and DOX. We performed the immunofluorescence imaging of the neuropilin-1 (NRP-1) receptor on MDA-MB-231 and patient-derived (PDX) TNBC cells in normoxia and hypoxia. For in vitro studies, we developed a TNBC PDX mouse model using tumor samples from patients at Sanford Broadway Clinic, Fargo, ND, with IRB approval. For in vitro studies, we developed tumors in different stages of F1-F5 generations and utilized F5 generation PDX tumor-bearing female NSG. The mice were given polymersome-encapsulated drug via intravenous injection twice weekly for 4 weeks and observed for an additional week. The percentage of tumor volume growth was calculated and compared to the control group. After the treatment, tumors from all groups were dissected and fixed in 10% formalin for histological evaluation. We analyzed the tumor core necrosis and the average area of necrosis between the groups by staining the tumors with hematoxylin and eosin. Also, we examined the solid TNBC PDX tumors by immunostaining them with a cancer stemness marker.

Results: We observed a synergistic effect of NAPA and DOX in regulating cell proliferation under normoxia (21% oxygen) and hypoxia (1 % oxygen). Interestingly, the synergistic effect was higher in hypoxia compared to normoxia. We observed increased NRP-1 receptor expression on MDA-MB-231 and PDX-TNBC cells under hypoxia. Also, we observed a reduction in the cancer stemness marker in 2D monolayer and 3D spheroids with targeted combination drug therapy compared to the control. TNBC PDX mice treated with DOX and NAPA alone and a combination of encapsulated DOX with NAPA (non-targeted and targeted) showed significant weight loss compared to the control (over 4 weeks). In the initial analysis, we observed higher tumor necrosis for the targeted encapsulated polymersome group compared to the control. The tumor size and cancer stemness markers decreased significantly with targeted combination therapy compared to the control group.

Conclusion: The combination of NAPA and DOX has shown promising results in controlling the growth of MD-MB-231 cells. Encapsulation of NAPA and DOX enhances the release of drugs in hypoxic conditions, thereby killing the cancer cells and reducing cancer stemness markers.


Increased NRP-1 expression in MDA-MB-231 and PDX cells in hypoxia. AF-388 (blue) shows DAPI, AF-569 (green) shows phalloidin, and AF-647 (red) shows the NRP-1 expression. Data is represented as mean ± SEM from n = 3.


Effect of NAPA and DOX combination on PDX Spheroids Using Automated Lionheart FX live microscopy. The reduction in spheroid size was measured using MATLAB software. Data shows significantly reduced spheroids’ size (n = 4).


Effect of encapsulated NAPA and DOX combination treatment on PDX-TNBC mouse model. Data shows that combining encapsulated NAPA and DOX significantly reduces tumor volume (n = 3-4).