A complete understanding of the biological mechanisms regulating devastating disease such as cancer remains elusive. Pancreatic and brain cancers are primary among the cancer types with poor prognosis. Molecular biomarkers have emerged as group of proteins that are preferentially overexpressed in cancers and with a key role in driving disease progression and resistance to chemotherapy. The epidermal growth factor receptor (EGFR), a cell proliferative biomarker is particularly highly expressed in most cancers including brain and pancreatic cancers. The ability of EGFR to sustain prolong cell proliferation is augmented by biomarkers such as Bax, Bcl-XL and Bcl-2, proteins regulating the apoptotic process. To better understand the role and effect of the microenvironment on these biomarkers in pancreatic cancer (PaCa); we analysed two pancreatic tumor lines (AsPc-1 and MiaPaCa-2) in 2D, 3D in-vitro cultures and in orthotopic tumors at different growth stages. We also investigated in patient derived glioblastoma (GBM) tumor cultures, the ability to utilize the EGFR expression to specifically deliver photosensitizer to the cells for photodynamic therapy. Overall, our results suggest that (1) microenvironment changes affect biomarker expression; thereby it is critical to understand these effects prior to designing combination therapies and (2) EGFR expression in tumor cells indeed could serve as a reliable and a robust biomarker that could be used to design targeted and image-guided photodynamic therapy.
The purpose of this study is to strategically combine two clinical-relevant, nanotechnology-based therapies to facilitate rapid clinical translation and immediately improve on the dismal statistics of pancreatic cancer (PanCa) patients. We hypothesized that benzoporphyrin derivative (BPD)-based photodynamic therapy (PDT) (Phase I/II study, solid PanCa) destroys tumor efflux transporters, which may help maintain high intracellular concentrations of Irinotecan (CPT-11) (Phase III study, metastatic PanCa) to reduce tumor burden and prolong survival. We test our hypothesis in orthotopic PanCa models.
Two types of liposomes were fabricated by adapting procedures from literature. They are: (i) Liposome with BPD in lipid bilayer (LBPD) and (ii) Liposome encapsulating CPT-11 in aqueous core (LCPT-11). Lipids (DPPC, DOTAP, Cholesterol, DSPE-mPEG at a molar ratio of 2:0.2:1.0:0.2) were mixed in chloroform (for LBPD, dissolve with 0.2 mM BPD), and the chloroform was evaporated. Lipid films were rehydrated for 2h in an aqueous solution (for LCPT-11, contain 7 mM CPT-11) with freeze thaw cycles. The resulting dispersion was extruded through polycarbonate membranes (100 nm pore size) to form unilamellar vesicles. Liposome size and polydispersity were measured by dynamic light scattering. BPD (or CPT-11) concentration was determined by UV-Vis spectroscopy. Human pancreatic cancer cells (MIA PaCa-2 or AsPC-1, ATCC) were implanted orthotopically in Swiss Nu/Nu mice (4-6 weeks old, ~25 mg) on day 0. Animals were anesthetized with Ketamine/Xylazine and the pancreas was exteriorized. Cells (1 x 106 in 50 μL of Matrigel-containing media) were injected into the pancreas using a 301/2-gauge needle, and the incision was closed with 4-0 sutures. Treatments were initiated when the tumors reach ~35 mm3 on day 9 (determined by ultrasound imaging). Tumor bearing mice were intravenously (tail vein) injected with LBPD (0.25 mg/kg) and LCPT-11 (20 mg/kg) 1h before light administration. Interstitial PDT (690 nm laser, 100mW/cm2, 75 J/cm2) was performed on the exteriorized pancreas of the anesthetized animal, and then followed by wound closure with sutures.
We have delivered reproducible LBPDs (137±9 nm) and LCPT-11s (122±5 nm), with a polydispersity index less than 0.05, were found stable for up to 3 weeks of storage. The BPD and CPT-11 loading efficiency in liposomes were found to be ~75% and ~50%, respectively. The longitudinal ultrasound monitoring of orthotopic tumor volume in response to combination LBPD-PDT and LCPT- 11 was carried out with appropriate controls. We observed that LBPD-PDT significantly enhances the tumoricidal efficacy of LCPT-11 and significantly inhibited tumor growth up to at least 3 weeks post-treatment (p < 0.05). Tumor volumes for the combination group on day 30 were ~3 fold and ~5 fold less than single treatments and no treatment groups, respectively.Photodynamic therapy (and Irinotecan chemotherapy) alone has already shown promise in treatment of PanCa in clinical studies. This study recognizes that the genetic complexity and heterogeneity of PanCa make it extremely difficult for any single treatment to impact outcome. To overcome therapy resistance, it is critical to understand the limitations of single treatment and develop new combination regimens based on interactive mechanisms. We performed pilot studies in orthotopic PanCa models that demonstrated LBPD-PDT could improve the efficacy of liposomal Irinotecan treatment. We anticipate the findings of this study, based on two clinically relevant treatments, will form the basis for rapid translation of a novel combination regimen for PanCa patients.
Glioblastoma (GBM) is an aggressive cancer with dismal survival rates and few new treatment options. Fluorescence guided resection of GBM followed by photodynamic therapy (PDT) has shown promise in several chemo- or radiotherapy non-responsive GBM treatments clinically. PDT is an emerging light and photosensitizer (PS) mediated cytotoxic method. However, as with other therapeutic modalities, the outcomes are variable largely due to the nonpersonalization of dose parameters. The variability can be attributed to the differences in heterogeneous photosensitizer accumulation in tumors. Building upon our previous findings on utilizing PS fluorescence for designing tumor-specific PDT dose, we explore the use of photoacoustic imaging, a technique that provides contrast based on the tissue optical absorption properties, to obtain 3D information on the tumoral photosensitizer accumulation. The findings of this study will form the basis for customized photodynamic therapy for glioblastoma and have the potential to serve as a platform for treatment of other cancers.