Sharmeela Kaushal

Half-Antibody Functionalized Lipid−Polymer Hybrid Nanoparticles for Targeted Drug Delivery to Carcinoembryonic Antigen Presenting Pancreatic Cancer Cells

Current chemotherapy regimens against pancreatic cancer are met with little success as poor tumor vascularization significantly limits the delivery of oncological drugs. High-dose targeted drug delivery, through which a drug delivery vehicle releases a large payload upon tumor localization, is thus a promising alternative strategy against this lethal disease. Herein, we synthesize anti-carcinoembryonic antigen (CEA) half-antibody conjugated lipid-polymer hybrid nanoparticles and characterize their ligand conjugation yields, physicochemical properties, and targeting ability against pancreatic cancer cells. Under the same drug loading, the half-antibody targeted nanoparticles show enhanced cancer killing effect compared to the corresponding nontargeted nanoparticles.

Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model.

The aim of this study was to evaluate a new fluorescently labeled chimeric anti‐CEA antibody for improved detection and resection of colon cancer.

PLGA nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses

The peptide vaccine clinical trials encountered limited success because of difficulties associated with stability and delivery, resulting in inefficient antigen presentation and low response rates in patients with cancer. The purpose of this study was to develop a novel delivery approach for tumor antigenic peptides in order to elicit enhanced immune responses using poly(DL-lactide-co-glycolide) nanoparticles (PLGA-NPs) encapsulating tumor antigenic peptides. PLGA-NPs were made using the double emulsion-solvent evaporation method. Artificial antigen-presenting cells were generated by human dendritic cells (DCs) loaded with PLGA-NPs encapsulating tumor antigenic peptide(s). The efficiency of the antigen presentation was measured by interferon-γ ELISpot assay (Vector Laboratories, Burlingame, CA). Antigen-specific cytotoxic T lymphocytes (CTLs) were generated and evaluated by CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Fitchburg, WI). The efficiency of the peptide delivery was compared between the methods of emulsification in incomplete Freund’s adjuvant and encapsulation in PLGA-NPs. Our results showed that most of the PLGA-NPs were from 150 nm to 500 nm in diameter, and were negatively charged at pH 7.4 with a mean zeta potential of −15.53 ± 0.71 mV; the PLGA-NPs could be colocalized in human DCs in 30 minutes of incubation. Human DCs loaded with PLGA-NPs encapsulating peptide induced significantly stronger CTL cytotoxicity than those pulsed with free peptide, while human DCs loaded with PLGA-NPs encapsulating a three-peptide cocktail induced a significantly greater CTL response than those encapsulating a two-peptide cocktail. Most importantly, the peptide dose encapsulated in PLGA-NPs was 63 times less than that emulsified in incomplete Freund’s adjuvant, but it induced a more powerful CTL response in vivo. These results demonstrate that the delivery of peptides encapsulated in PLGA-NPs is a promising approach to induce effective antitumor CTL responses in vivo.

KRas Induces a Src/PEAK1/ErbB2 Kinase Amplification Loop That Drives Metastatic Growth and Therapy Resistance in Pancreatic Cancer

Early biomarkers and effective therapeutic strategies are desperately needed to treat pancreatic ductal adenocarcinoma (PDAC), which has a dismal 5-year patient survival rate. Here, we report that the novel tyrosine kinase PEAK1 is upregulated in human malignancies, including human PDACs and pancreatic intraepithelial neoplasia (PanIN). Oncogenic KRas induced a PEAK1-dependent kinase amplification loop between Src, PEAK1, and ErbB2 to drive PDAC tumor growth and metastasis in vivo. Surprisingly, blockade of ErbB2 expression increased Src-dependent PEAK1 expression, PEAK1-dependent Src activation, and tumor growth in vivo, suggesting a mechanism for the observed resistance of patients with PDACs to therapeutic intervention. Importantly, PEAK1 inactivation sensitized PDAC cells to trastuzumab and gemcitabine therapy. Our findings, therefore, suggest that PEAK1 is a novel biomarker, critical signaling hub, and new therapeutic target in PDACs.

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression

Regulation of the actin-myosin cytoskeleton plays a central role in cell migration and cancer progression. Here, we report the discovery of a cytoskeleton-associated kinase, pseudopodium-enriched atypical kinase 1 (PEAK1). PEAK1 is a 190-kDa nonreceptor tyrosine kinase that localizes to actin filaments and focal adhesions. PEAK1 undergoes Src-induced tyrosine phosphorylation, regulates the p130Cas-Crk-paxillin and Erk signaling pathways, and operates downstream of integrin and epidermal growth factor receptors (EGFR) to control cell spreading, migration, and proliferation. Perturbation of PEAK1 levels in cancer cells alters anchorage-independent growth and tumor progression in mice. Notably, primary and metastatic samples from colon cancer patients display amplified PEAK1 levels in 81% of the cases. Our findings indicate that PEAK1 is an important cytoskeletal regulatory kinase and possible target for anticancer therapy.

Fluorescence-Guided Surgery Allows for More Complete Resection of Pancreatic Cancer, Resulting in Longer Disease-Free Survival Compared with Standard Surgery in Orthotopic Mouse Models

BACKGROUND Negative surgical margins are vital to achieve cure and prolong survival in patients with pancreatic cancer. We inquired if fluorescence-guided surgery (FGS) could improve surgical outcomes and reduce recurrence rates in orthotopic mouse models of human pancreatic cancer. STUDY DESIGN A randomized active-control preclinical trial comparing bright light surgery (BLS) to FGS was used. Orthotopic mouse models of human pancreatic cancer were established using the BxPC-3 pancreatic cancer cell line expressing red fluorescent protein (RFP). Two weeks after orthotopic implantation, tumors were resected with BLS or FGS. Pre- and postoperative images were obtained with the OV-100 Small Animal Imaging System to assess completeness of surgical resection in real time. Postoperatively, noninvasive whole body imaging was done to assess recurrence and follow tumor progression. Six weeks postoperatively, mice were sacrificed to evaluate primary pancreatic and metastatic tumor burden at autopsy. RESULTS A more complete resection of pancreatic cancer was achieved using FGS compared with BLS: 98.9% vs 77.1%, p = 0.005. The majority of mice undergoing BLS (63.2%) had evidence of gross disease with no complete resections; 20% of mice undergoing FGS had complete resection and an additional 75% had only minimal residual disease (p = 0.0001). The mean postoperative tumor burden was significantly less with FGS compared with BLS: 0.08 ± 0.06 mm(2) vs 2.64 ± 0.63 mm(2), p = 0.001. The primary tumor burden at termination was significantly less with FGS compared with BLS: 19.3 ± 5.3 mm(2) vs 6.2 ± 3.6 mm(2), p = 0.048. FGS resulted in significantly longer disease-free survival than BLS (p = 0.02, hazard ratio = 0.39, 95% CI 0.17, 0.88). CONCLUSIONS Surgical outcomes were improved in pancreatic cancer using fluorescence-guidance. This novel approach has significant potential to improve surgical treatment of cancer.

Knockdown of the β1 integrin subunit reduces primary tumor growth and inhibits pancreatic cancer metastasis

To address the role of β1 integrins in pancreatic cancer progression, we stably knocked down β1 integrin subunit expression in human FG‐RFP pancreatic cancer cells using lentiviral‐based RNA interference. We then examined the effects of β1 integrin subunit knockdown on pancreatic cancer cell adhesion, migration and proliferation on tumor microenvironment‐specific extracellular matrix proteins in vitro and on tumor progression in vivo using a clinically relevant fluorescent orthotopic mouse model of pancreatic cancer. Knockdown of the β1 integrin subunit inhibited cell adhesion, migration and proliferation on types I and IV collagen, fibronectin and laminin in vitro. In vivo, knockdown of the β1 integrin subunit reduced primary tumor growth by 50% and completely inhibited spontaneously occurring metastasis. These observations indicate a critical role for the β1 integrin subunit in pancreatic cancer progression and metastasis in particular. Our results suggest the β1 integrin subunit as a therapeutic target for the treatment of pancreatic cancer, especially in the adjuvant setting to prevent metastasis of this highly aggressive cancer.

Metronomic Gemcitabine in Combination with Sunitinib Inhibits Multisite Metastasis and Increases Survival in an Orthotopic Model of Pancreatic Cancer

Metronomic chemotherapy suppresses growth of primary tumors and established metastases. However, its effect on metastatic progression is essentially unknown. We report the treatment of a metastatically competent model of pancreatic cancer with metronomic gemcitabine and sunitinib. Mice with orthotopic, red fluorescent protein-expressing, pancreatic cancer tumorgrafts were treated with gemcitabine on a metronomic (1 mg/kg daily, METG) or maximum tolerated dose (150 mg/kg twice weekly, MTDG) schedule with or without sunitinib (SU). Rates of primary tumor growth, metastasis, ascites, and survival were calculated. Gemcitabine at a daily dose of 2 mg or greater led to toxicity within 1 month in mice without tumors but METG at 1 mg/kg/d was well tolerated. Mice with pancreatic cancer tumorgrafts died with metastatic disease at a median of 25 days. METG/SU significantly prolonged median overall survival (44 days) compared with control or either regimen alone (P < 0.05). Primary tumor growth was inhibited by METG/SU (P = 0.03) but neither METG nor sunitinib alone. In contrast, treatment with METG suppressed metastasis at multiple sites, an effect enhanced by sunitinib. MTDG with or without sunitinib had the most favorable effect on primary tumor growth and survival, but its antimetastatic efficacy was similar to that of METG/SU. von Willebrand factor expression was inhibited by METG. Antimetastatic activity approaching that of MTDG is achieved with a total dose reduced 42 times using METG and is further enhanced by sunitinib. Our results suggest the potential of this therapeutic paradigm against pancreatic cancer in the adjuvant and maintenance settings. Mol Cancer Ther; 9(7); 2068–78. ©2010 AACR.

Fluorescent LYVE-1 antibody to image dynamically lymphatic trafficking of cancer cells in vivo.

BACKGROUND The lymphatic system is a major route for cancer cell dissemination, and a potential target for antitumor therapy. Despite ongoing interest in this area of research, the real-time behavior of cancer cells trafficking in the lymphatic system is poorly understood due to lack of appropriate tools to image this process. MATERIALS AND METHODS We have used monoclonal-antibody and fluorescence technology to color-code lymphatic vessels and the cancer cells inside them in a living animal. Monoclonal anti-mouse LYVE-1 antibody was conjugated to a green fluorophore and delivered to the lymphatic system of a nude mouse, allowing imaging of mouse lymphatics. Tumor cells engineered to express red fluorescent protein were then imaged traveling within the labeled lymphatics in real time. RESULTS AlexaFluor-labeled monoclonal anti-mouse LYVE-1 created a durable signal with clear delineation of lymphatic architecture. The duration of fluorescent signal after conjugated LYVE-1 delivery was far superior to that of fluorescein isothiocyanate-dextran or control fluorophore-conjugated IgG. Tumor cells engineered to express red fluorescent protein delivered to the inguinal lymph node enabled real-time tracking of tumor cell movement within the green fluorescent-labeled lymphatic vessels. CONCLUSIONS This technology offers a powerful tool for the in vivo study of real-time trafficking of tumor cells within lymphatic vessels, for the deposition of the tumor cells in lymph nodes, as well as for screening of potential antitumor lymphatic therapies.

Fluorescence-guided surgery of human colon cancer increases complete resection resulting in cures in an orthotopic nude mouse model

BACKGROUND We inquired if fluorescence-guided surgery (FGS) could improve surgical outcomes in fluorescent orthotopic nude mouse models of human colon cancer. METHODS We established fluorescent orthotopic mouse models of human colon cancer expressing a fluorescent protein. Tumors were resected under bright light surgery (BLS) or FGS. Pre- and post-operative images with the OV-100 Small Animal Imaging System (Olympus Corp, Tokyo Japan) were obtained to assess the extent of surgical resection. RESULTS All mice with primary tumor that had undergone FGS had complete resection compared with 58% of mice in the BLS group (P = 0.001). FGS resulted in decreased recurrence compared with BLS (33% versus 62%, P = 0.049) and lengthened disease-free median survival from 9 to >36 wk. The median overall survival increased from 16 wk in the BLS group to 31 weeks in the FGS group. FGS resulted in a cure in 67% of mice (alive without evidence of tumor at >6 mo after surgery) compared with only 37% of mice that underwent BLS (P = 0.049). CONCLUSIONS Surgical outcomes in orthotopic nude mouse models of human colon cancer were significantly improved with FGS. The present study can be translated to the clinic by various effective methods of fluorescently labeling tumors.