Jonathan G. Sham

Porous chitosan-hyaluronic acid scaffolds as a mimic of glioblastoma microenvironment ECM

Cancer therapeutics are developed through extensive screening; however, many therapeutics evaluated with 2D in vitro cultures during pre-clinical trials suffer from lower efficacy in patients. Replicating the in vivo tumor microenvironment in vitro with three-dimensional (3D) porous scaffolds offers the possibility of generating more predictive pre-clinical models to enhance cancer treatment efficacy. We developed a chitosan and hyaluronic acid (HA) polyelectrolyte complex 3D porous scaffold and evaluated its physical properties. Chitosan-HA (C-HA) scaffolds had a highly porous network. C-HA scaffolds were compared to 2D surfaces for in vitro culture of U-118 MG human glioblastoma (GBM) cells. C-HA scaffold cultures promoted tumor spheroid formation and increased stem-like properties of GBM cells as evidenced by the upregulation of CD44, Nestin, Musashi-1, GFAP, and HIF-1α as compared with 2D cultures. Additionally, the invasiveness of GBM cells cultured in C-HA scaffolds was significantly enhanced compared to those grown in 2D cultures. C-HA scaffold cultures were also more resistant to chemotherapy drugs, which corresponded to the increased expression of ABCG2 drug efflux transporter. These findings suggest that C-HA scaffolds offer promise as an in vitro GBM platform for study and screening of novel cancer therapeutics.

Pancreatic ductal adenocarcinoma contains an effector and regulatory immune cell infiltrate that is altered by multimodal neoadjuvant treatment

Objective The immune response to pancreatic ductal adenocarcinoma (PDA) may play a role in defining its uniquely aggressive biology; therefore, we sought to clearly define the adaptive immune infiltrate in PDA. Design We used immunohistochemistry and flow cytometry to characterize the immune infiltrate in human PDA and compared our findings to the patients’ peripheral blood. Results In contrast to the myeloid cell predominant infiltrate seen in murine models, T cells comprised the majority of the hematopoietic cell component of the tumor stroma in human PDA. Most intratumoral CD8+ T cells exhibited an antigen-experienced effector memory cell phenotype and were capable of producing IFN-γ. CD4+ regulatory T cells (Treg) and IL-17 producing T helper cells were significantly more prevalent in tumor than in blood. Consistent with the association with reduced survival in previous studies, we observed higher frequencies of both myeloid cells and Treg in poorly differentiated tumors. The majority of intratumoral T cells expressed the co-inhibitory receptor programmed death-1 (PD-1), suggesting one potential mechanism through which PDA may evade antitumor immunity. Successful multimodal neoadjuvant therapy altered the immunoregulatory balance and was associated with reduced infiltration of both myeloid cells and Treg. Conclusion Our data show that human PDA contains a complex mixture of inflammatory and regulatory immune cells, and that neoadjuvant therapy attenuates the infiltration of intratumoral cells associated with immunosuppression and worsened survival.

Squamous cell carcinoma

Cutaneous squamous cell carcinoma (SCC) is a common cancer arising from malignant proliferation of epidermal keratinocytes. SCC represents a broad range of disease, from superficially invasive to aggressive, highly lethal tumors. This chapter will review the epidemiology, risk factors, pathogenesis, diagnosis, and treatment of cutaneous SCC, with a particular focus on disease in the lower extremity.

Iron-Oxide-Based Nanovector for Tumor Targeted siRNA Delivery in an Orthotopic Hepatocellular Carcinoma Xenograft Mouse Model

Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Small interfering RNA (siRNA) holds promise as a new class of therapeutics for HCC, as it can achieve sequence-specific gene knockdown with low cytotoxicity. However, the main challenge in the clinical application of siRNA lies in the lack of effective delivery approaches that need to be highly specific and thus incur low or no systemic toxicity. Here, a nonviral nanoparticle-based gene carrier is presented that can specifically deliver siRNA to HCC. The nanovector (NP-siRNA-GPC3 Ab) is made of an iron oxide core coated with chitosan-polyethylene glycol (PEG) grafted polyethyleneimine copolymer, which is further functionalized with siRNA and conjugated with a monoclonal antibody (Ab) against human glypican-3 (GPC3) receptor highly expressed in HCC. A rat RH7777 HCC cell line that coexpresses human GPC3 and firefly luciferase (Luc) is established to evaluate the nanovector. The nanoparticle-mediated delivery of siRNA against Luc effectively suppresses Luc expression in vitro without notable cytotoxicity. Significantly, NP-siLuc-GPC3 Ab administered intravenously in an orthotopic model of HCC is able to specifically bound to tumor and induce remarkable inhibition of Luc expression. The findings demonstrate the potential of using this nanovector for targeted delivery of therapeutic siRNA to HCC.

Nanoparticle mediated silencing of DNA repair sensitizes pediatric brain tumor cells to γ‐irradiation

Medulloblastoma (MB) and ependymoma (EP) are the most common pediatric brain tumors, afflicting 3000 children annually. Radiotherapy (RT) is an integral component in the treatment of these tumors; however, the improvement in survival is often accompanied by radiation‐induced adverse developmental and psychosocial sequelae. Therefore, there is an urgent need to develop strategies that can increase the sensitivity of brain tumors cells to RT while sparing adjacent healthy brain tissue. Apurinic endonuclease 1 (Ape1), an enzyme in the base excision repair pathway, has been implicated in radiation resistance in cancer. Pharmacological and specificity limitations inherent to small molecule inhibitors of Ape1 have hindered their clinical development. Here we report on a nanoparticle (NP) based siRNA delivery vehicle for knocking down Ape1 expression and sensitizing pediatric brain tumor cells to RT. The NP comprises a superparamagnetic iron oxide core coated with a biocompatible, biodegradable coating of chitosan, polyethylene glycol (PEG), and polyethyleneimine (PEI) that is able to bind and protect siRNA from degradation and to deliver siRNA to the perinuclear region of target cells. NPs loaded with siRNA against Ape1 (NP:siApe1) knocked down Ape1 expression over 75% in MB and EP cells, and reduced Ape1 activity by 80%. This reduction in Ape1 activity correlated with increased DNA damage post‐irradiation, which resulted in decreased cell survival in clonogenic assays. The sensitization was specific to therapies generating abasic lesions as evidenced by NP:siRNA not increasing sensitivity to paclitaxel, a microtubule disrupting agent. Our results indicate NP‐mediated delivery of siApe1 is a promising strategy for circumventing pediatric brain tumor resistance to RT.

Glypican-3–Targeting F(ab′)2 for 89Zr PET of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is an increasingly lethal malignancy for which management is critically dependent on accurate imaging. Glypican-3 (GPC3) is a cell surface receptor overexpressed in most HCCs and provides a unique target for molecular diagnostics. The use of monoclonal antibodies (mAbs) that target GPC3 (αGPC3) in PET imaging has shown promise but comes with inherent limitations associated with mAbs such as long circulation times. This study used 89Zr-conjugated F(ab′)2 fragments directed against GPC3 (89Zr-αGPC3-F(ab′)2) to evaluate the feasibility of the fragments as a diagnostic immuno-PET imaging probe. Methods: Immobilized ficin was used to digest αGPC3, creating αGPC3-F(ab′)2 fragments subsequently conjugated to 89Zr. In vivo biodistribution and PET studies were performed on GPC3-expressing HepG2 and GPC3-nonexpressing RH7777 orthotopic xenografts. Results: Reliable αGPC3-F(ab′)2 production via immobilized ficin digestion was verified by high-performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. 89Zr-αGPC3-F(ab′)2 demonstrated F(ab′)2-dependent, antigen-specific cell binding. HepG2 tumor uptake was higher than any other tissue, peaking at 100 ± 21 percentage injected dose per gram (%ID/g) 24 h after injection, a value 33- to 38-fold higher than GPC3-nonexpressing RH7777 tumors. The blood half-life of the 89Zr-αGPC3-F(ab′)2 conjugate was approximately 11 h, compared with approximately 115 h for historic mAb controls. This shorter half-life enabled clear tumor visualization on PET 4 h after administration, with a resultant peak tumor-to-liver contrast ratio of 23.3. Blocking antigen-expressing tumors with an excess of nonradiolabeled αGPC3 resulted in decreased tumor uptake similar to native liver. The kidneys exhibited high tissue uptake, peaking at 24 h with 83 ± 12 %ID/g. HepG2 tumors ranging from 1.5 to 7 mm were clearly visible on PET, whereas larger RH7777 tumors displayed signal lower than background liver tissue. Conclusion: This study demonstrates the feasibility of using 89Zr-αGPC3-F(ab′)2 for intrahepatic tumor localization with small-animal PET. Faster blood clearance and lower background liver uptake enable excellent signal-to-noise ratios at early time points. Increased renal uptake is similar to that as has been seen with clinical radioactive peptide imaging. 89Zr-αGPC3-F(ab′)2 addresses some of the shortcomings of whole-antibody immuno-PET probes. Further optimization is warranted to maximize probe sensitivity and specificity in the process of clinical translation.

Glypican-3–Targeted 89Zr PET Imaging of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a devastating malignancy in which imperfect imaging plays a primary role in diagnosis. Glypican-3 (GPC3) is an HCC-specific cell surface proteoglycan overexpressed in most HCCs. This paper presents the use of 89Zr-conjugated monoclonal antibody against GPC3 (89Zr-αGPC3) for intrahepatic tumor localization using PET. Methods: Polymerase chain reaction confirmed relative GPC3 expression in cell lines. In vitro binding, in vivo biodistribution, and small-animal PET studies were performed on GPC3-expressing HepG2 and non–GPC3-expressing HLF and RH7777 cells and orthotopic xenografts. Results: 89Zr-αGPC3 demonstrated antibody-dependent, antigen-specific tumor binding. HepG2 liver tumors exhibited high peak uptake (836.6 ± 86.6 percentage injected dose [%ID]/g) compared with background liver (27.5 ± 1.6 %ID/g). Tumor-to-liver contrast ratio was high and peaked at 32.5. The smallest HepG2 tumor (<1 mm) showed lower peak uptake (42.5 ± 6.4 %ID/g) and tumor-to-liver contrast (1.57) but was still clearly visible on PET. Day 7 tissue activity was still substantial in HepG2 tumors (466.4 ± 87.6 %ID/g) compared with control RH7777 tumors (3.9 ± 1.3 %ID/g, P < 0.01), indicating antigen specificity by 89Zr-αGPC3. HepG2 tumor treated with unlabeled αGPC3 or heat-denatured 89Zr-αGPC3 demonstrated tumor activity (2.1 %ID/g) comparable to that of control xenografts, confirming antibody dependency. Conclusion: This study demonstrated the feasibility of using 89Zr-αGPC3 to image HCC in the liver, as well as the qualitative determination of GPC3 expression via small-animal PET. The ability to clarify the identity of small liver lesions with an HCC-specific PET probe would provide clinicians with vital information that could significantly alter patient management, warranting further investigation for clinical translation.

Evaluating the mechanisms of improved glucose homeostasis after bariatric surgery in Ossabaw miniature swine

Background. Roux-en-Y gastric bypass (RYGB) is the most common bariatric operation; however, the mechanism underlying the profound weight-independent effects on glucose homeostasis remains unclear. Large animal models of naturally occurring insulin resistance (IR), which have been lacking, would provide opportunities to elucidate such mechanisms. Ossabaw miniature swine naturally exhibit many features that may be useful in evaluating the anti diabetic effects of bariatric surgery. Methods. Glucose homeostasis was studied in 53 Ossabaw swine. Thirty-two received an obesogenic diet and were randomized to RYGB, gastrojejunostomy (GJ), gastrojejunostomy with duodenal exclusion (GJD), or Sham operations. Intravenous glucose tolerance tests and standardized meal tolerance tests were performed prior to, 1, 2, and 8 weeks after surgery and at a single time-point for regular diet control pigs. Results. High-calorie-fed Ossabaws weighed more and had greater IR than regular diet controls, though only 70% developed IR. All operations caused weight-loss-independent improvement in IR, though only in pigs with high baseline IR. Only RYGB induced weight loss and decreased IR in the majority of pigs, as well as increasing AUCinsulin/AUCglucose. Conclusions. Similar to humans, Ossabaw swine exhibit both obesity-dependent and obesity-independent IR. RYGB promoted weight loss, IR improvement, and increased AUCinsulin/AUCglucose, compared to the smaller changes following GJ and GJD, suggesting a combination of upper and lower gut mechanisms in improving glucose homeostasis.

Nanoparticle Biokinetics in Mice and Nonhuman Primates

Despite the preponderance of iron oxide nanoparticles (NPs) designed for theranostic applications, widespread clinical translation of these NPs lags behind. A better understanding of how NP pharmacokinetics vary between small and large animal models is needed to rapidly customize NPs for optimal performance in humans. Here we use noninvasive magnetic resonance imaging (MRI) to track iron oxide NPs through a large number of organ systems in vivo to investigate NP biokinetics in both mice and nonhuman primates. We demonstrate that pharmacokinetics are similar between mice and macaques in the blood, liver, spleen, and muscle, but differ in the kidneys, brain, and bone marrow. Our study also demonstrates that full-body MRI is practical, rapid, and cost-effective for tracking NPs noninvasively with high spatiotemporal resolution. Our techniques using a nonhuman primate model may provide a platform for testing a range of NP formulations.

A Large Animal Survival Model to Evaluate Bariatric Surgery Mechanisms

Background The impact of Roux-en-Y gastric bypass (RYGB) on type 2 diabetes mellitus is thought to result from upper and/or lower gut hormone alterations. Evidence supporting these mechanisms is incomplete, in part because of limitations in relevant bariatric-surgery animal models, specifically the lack of naturally insulin-resistant large animals. With overfeeding, Ossabaw swine develop a robust metabolic syndrome, and may be suitable for studying post-surgical physiology. Whether bariatric surgery is feasible in these animals with acceptable survival is unknown. Methods Thirty-two Ossabaws were fed a high-fat, high-cholesterol diet to induce obesity and insulin resistance. These animals were assigned to RYGB (n = 8), RYGB with vagotomy (RYGB-V, n = 5), gastrojejunostomy (GJ, n = 10), GJ with duodenal exclusion (GJD, n = 7), or sham operation (n = 2) and were euthanized 60 days post-operatively. Post-operative changes in weight and food intake are reported. Results Survival to scheduled necropsy among surgical groups was 77%, living an average of 57 days post-operatively. Cardiac arrest under anesthesia occurred in 4 pigs. Greatest weight loss (18.0% ± 6%) and food intake decrease (57.0% ± 20%) occurred following RYGB while animals undergoing RYGB-V showed only 6.6% ± 3% weight loss despite 50.8% ± 25% food intake decrease. GJ (12.7% ± 4%) and GJD (1.2% ± 1%) pigs gained weight, but less than sham controls (13.4% ± 10%). Conclusions A survival model of metabolic surgical procedures is feasible, leads to significant weight loss, and provides the opportunity to evaluate new interventions and subtle variations in surgical technique (e.g. vagus nerve sparing) that may provide new mechanistic insights.