Steven A. Curley

A recellularized human colon model identifies cancer driver genes

Refined cancer models are needed to bridge the gaps between cell line, animal and clinical research. Here we describe the engineering of an organotypic colon cancer model by recellularization of a native human matrix that contains cell-populated mucosa and an intact muscularis mucosa layer. This ex vivo system recapitulates the pathophysiological progression from APC-mutant neoplasia to submucosal invasive tumor. We used it to perform a Sleeping Beauty transposon mutagenesis screen to identify genes that cooperate with mutant APC in driving invasive neoplasia. We identified 38 candidate invasion-driver genes, 17 of which, including TCF7L2, TWIST2, MSH2, DCC, EPHB1 and EPHB2 have been previously implicated in colorectal cancer progression. Six invasion-driver genes that have not, to our knowledge, been previously described were validated in vitro using cell proliferation, migration and invasion assays and ex vivo using recellularized human colon. These results demonstrate the utility of our organoid model for studying cancer biology.

Generation of an in vitro 3D PDAC stroma rich spheroid model

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic/stromal reaction, which contributes to the poor clinical outcome of this disease. Therefore, greater understanding of the stroma development and tumor-stroma interactions is highly required. Pancreatic stellate cells (PSC) are myofibroblast-like cells located in exocrine areas of the pancreas, which as a result of inflammation produced by PDAC migrate and accumulate in the tumor mass, secreting extracellular matrix components and producing the dense PDAC stroma. Currently, only a few orthotopic or ectopic animal tumor models, where PDAC cells are injected into the pancreas or subcutaneous tissue layer, or genetically engineered animals offer tumors that encompass some stromal component. Herein, we report generation of a simple 3D PDAC inxa0vitro micro-tumor model without an addition of external extracellular matrix, which encompasses a rich, dense and active stromal compartment. We have achieved this inxa0vitro model by incorporating PSCs into 3D PDAC cell culture using a modified hanging drop method. It is now known that PSCs are the principal source of fibrosis in the stroma and interact closely with cancer cells to create a tumor facilitatory environment that stimulates local and distant tumor growth. The 3D micro-stroma models are highly reproducible with excellent uniformity, which can be used for PDAC-stroma interaction analysis and high throughput automated drug-screening assays. Additionally, the increased expression of collagenous regions means that molecular based perfusion and cytostaticity of gemcitabine is decreased in our Pancreatic adenocarcinoma stroma spheroids (PDAC-SS) model when compared to spheroids grown without PSCs. We believe this model will allow an improved knowledge of PDAC biology and has the potential to provide an insight into pathways that may be therapeutically targeted to inhibit PSC activation, thereby inhibiting the development of fibrosis in PDAC and interrupting PSC-PDAC cell interactions so as to inhibit cancer progression.

The Effects of Non-Invasive Radiofrequency Treatment and Hyperthermia on Malignant and Nonmalignant Cells

Background: Exposure of biological subjects to electromagnetic fields with a high frequency is associated with temperature elevation. In our recent studies, we reported that non-invasive radiofrequency (RF) treatment at 13.56 MHz with the field ranging from 1 KeV to 20 KeV/m2 inhibits tumor progression in animals with abdominal tumor xenografts and enhances the anticancer effect of chemotherapy. The RF treatment was followed by temperature elevation in tumors to approximately 46 °C during 10 min of exposure. In contrast, the temperature of normal tissues remained within a normal range at approximately 37 °C. Whether all biological effects of RF treatment are limited to its hyperthermic property remains unclear. Here, we compared how RF and hyperthermia (HT) treatments change the proliferation rate, oxygen consumption and autophagy in malignant and nonmalignant cells. Methods: In the current study, cancer and nonmalignant cells of pancreatic origin were exposed to the RF field or to conventional HT at 46 °C, which was chosen based on our previous in vivo studies of the tumor-specific RF-induced hyperthermia. Results: Only RF treatment caused declines in cancer cell viability and proliferation. RF treatment also affected mitochondrial function in cancer cells more than HT treatment did and, unlike HT treatment, was followed by the elevation of autophagosomes in the cytoplasm of cancer cells. Importantly, the effects of RF treatment were negligible in nonmalignant cells. Conclusion: The obtained data indicate that the effects of RF treatment are specific to cancer cells and are not limited to its hyperthermic property.

Radiofrequency treatment alters cancer cell phenotype

The importance of evaluating physical cues in cancer research is gradually being realized. Assessment of cancer cell physical appearance, or phenotype, may provide information on changes in cellular behavior, including migratory or communicative changes. These characteristics are intrinsically different between malignant and non-malignant cells and change in response to therapy or in the progression of the disease. Here, we report that pancreatic cancer cell phenotype was altered in response to a physical method for cancer therapy, a non-invasive radiofrequency (RF) treatment, which is currently being developed for human trials. We provide a battery of tests to explore these phenotype characteristics. Our data show that cell topography, morphology, motility, adhesion and division change as a result of the treatment. These may have consequences for tissue architecture, for diffusion of anti-cancer therapeutics and cancer cell susceptibility within the tumor. Clear phenotypical differences were observed between cancerous and normal cells in both their untreated states and in their response to RF therapy. We also report, for the first time, a transfer of microsized particles through tunneling nanotubes, which were produced by cancer cells in response to RF therapy. Additionally, we provide evidence that various sub-populations of cancer cells heterogeneously respond to RF treatment.

Development and Validation of Insulin-like Growth Factor-1 Score to Assess Hepatic Reserve in Hepatocellular Carcinoma

Background Child-Turcotte-Pugh (CTP) score is the standard tool to assess hepatic reserve in hepatocellular carcinoma (HCC), and CTP-A is the classic group for active therapy. However, CTP stratification accuracy has been questioned. We hypothesized that plasma insulin-like growth factor 1 (IGF-1) is a valid surrogate for hepatic reserve to replace the subjective parameters in CTP score to improve its prognostic accuracy. Methods We retrospectively tested plasma IGF-1 levels in the training set (n = 310) from MD Anderson Cancer Center. Recursive partitioning identified three optimal IGF-1 ranges that correlated with overall survival (OS): greater than 50ng/mL = 1 point; 26 to 50ng/mL = 2 points; and less than 26ng/mL = 3 points. We modified the CTP score by replacing ascites and encephalopathy grading with plasma IGF-1 value (IGF-CTP) and subjected both scores to log-rank analysis. Harrell’s C-index and U-statistics were used to compare the prognostic performance of both scores in both the training and validation cohorts (n = 155). All statistical tests were two-sided. Results Patients’ stratification was statistically significantly stronger for IGF-CTP than CTP score for the training (P = .003) and the validation cohort (P = .005). Patients reclassified by IGF-CTP relative to their original CTP score were better stratified by their new risk groups. Most important, patients classified as A by CTP but B by IGF-CTP had statistically significantly worse OS than those who remained under class A by IGF-CTP in both cohorts (P = .03 and P < .001, respectively, from Cox regression models). AB patients had a worse OS than AA patients in both the training and validation set (hazard ratio [HR] = 1.45, 95% confidence interval [CI] = 1.03 to 2.04, P = .03; HR = 2.83, 95% CI = 1.65 to 4.85, P < .001, respectively). Conclusions The IGF-CTP score is simple, blood-based, and cost-effective, stratified HCC better than CTP score, and validated well on two independent cohorts. International validation studies are warranted.

Theory and Experimental Validation of a Spatio-temporal Model of Chemotherapy Transport to Enhance Tumor Cell Kill

It has been hypothesized that continuously releasing drug molecules into the tumor over an extended period of time may significantly improve the chemotherapeutic efficacy by overcoming physical transport limitations of conventional bolus drug treatment. In this paper, we present a generalized space- and time-dependent mathematical model of drug transport and drug-cell interactions to quantitatively formulate this hypothesis. Model parameters describe: perfusion and tissue architecture (blood volume fraction and blood vessel radius); diffusion penetration distance of drug (i.e., a function of tissue compactness and drug uptake rates by tumor cells); and cell death rates (as function of history of drug uptake). We performed preliminary testing and validation of the mathematical model using in vivo experiments with different drug delivery methods on a breast cancer mouse model. Experimental data demonstrated a 3-fold increase in response using nano-vectored drug vs. free drug delivery, in excellent quantitative agreement with the model predictions. Our model results implicate that therapeutically targeting blood volume fraction, e.g., through vascular normalization, would achieve a better outcome due to enhanced drug delivery. Author Summary Cancer treatment efficacy can be significantly enhanced through the elution of drug from nano-carriers that can temporarily stay in the tumor vasculature. Here we present a relatively simple yet powerful mathematical model that accounts for both spatial and temporal heterogeneities of drug dosing to help explain, examine, and prove this concept. We find that the delivery of systemic chemotherapy through a certain form of nano-carriers would have enhanced tumor kill by a factor of 2 to 4 over the standard therapy that the patients actually received. We also find that targeting blood volume fraction (a parameter of the model) through vascular normalization can achieve more effective drug delivery and tumor kill. More importantly, this model only requires a limited number of parameters which can all be readily assessed from standard clinical diagnostic measurements (e.g., histopathology and CT). This addresses an important challenge in current translational research and justifies further development of the model towards clinical translation.

Pancreatic adenocarcinoma response to chemotherapy enhanced with non-invasive radio frequency evaluated via an integrated experimental/computational approach

Although chemotherapy combined with radiofrequency exposure has shown promise in cancer treatment by coupling drug cytotoxicity with thermal ablation or thermally-induced cytotoxicity, limited access of the drugxa0to tumor loci in hypo-vascularized lesions has hampered clinical application. We recently showed that high-intensity short-wave capacitively coupled radiofrequency (RF) electric-fields may reach inaccessible targetsxa0in vivo. This non-invasive RF combined with gemcitabine (Gem) chemotherapy enhanced drug uptake and effect in pancreatic adenocarcinoma (PDAC), notorious for having poor response and limited therapeutic options, but without inducing thermal injury. We hypothesize that the enhanced cytotoxicity derives from RF-facilitated drug transport in the tumor microenvironment. We propose an integrated experimental/computational approach to evaluate chemotherapeutic response combined with RF-induced phenotypic changes in tissue with impaired transport. Results show that RF facilitates diffusive transport in 3D cell cultures representing hypo-vascularized lesions, enhancing drug uptake and effect. Computational modeling evaluates drug vascular extravasation and diffusive transport as key RF-modulated parameters, with transport being dominant. Assessment of hypothetical schedules following current clinical protocol for Stage-IV PDAC suggests that unresponsive lesions may be growth-restrained when exposed to Gem plus RF. Comparison of these projections to experiments inxa0vivo indicates that synergy may result from RF-induced cell phenotypic changes enhancing drug transport and cytotoxicity, thus providing a potential baseline for clinically-focused evaluation.

Biotransport kinetics and intratumoral biodistribution of malonodiserinolamide-derivatized [60]fullerene in a murine model of breast adenocarcinoma

[60]Fullerene is a highly versatile nanoparticle (NP) platform for drug delivery to sites of pathology owing to its small size and both ease and versatility of chemical functionalization, facilitating multisite drug conjugation, drug targeting, and modulation of its physicochemical properties. The prominent and well-characterized role of the enhanced permeation and retention (EPR) effect in facilitating NP delivery to tumors motivated us to explore vascular transport kinetics of a water-soluble [60]fullerene derivatives using intravital microscopy in an immune competent murine model of breast adenocarcinoma. Herein, we present a novel local and global image analysis of vascular transport kinetics at the level of individual tumor blood vessels on the micron scale and across whole images, respectively. Similar to larger nanomaterials, [60]fullerenes displayed rapid extravasation from tumor vasculature, distinct from that in normal microvasculature. Temporal heterogeneity in fullerene delivery to tumors was observed, demonstrating the issue of nonuniform delivery beyond spatial dimensions. Trends in local region analysis of fullerene biokinetics by fluorescence quantification were in agreement with global image analysis. Further analysis of intratumoral vascular clearance rates suggested a possible enhanced penetration and retention effect of the fullerene compared to a 70 kDa vascular tracer. Overall, this study demonstrates the feasibility of tracking and quantifying the delivery kinetics and intratumoral biodistribution of fullerene-based drug delivery platforms, consistent with the EPR effect on short timescales and passive transport to tumors.

PO-515 Novel water-solube [60]fullerene nanotherapeutic agent for pancreatic cancer treatment

Introduction The rapid development of nanotechnology is of great interest to researchers focused on translational medicine and novel targeted cancer treatment. This novel ‘nano’ approach has been used by medicinal chemist to design potential drugs which could easily broke almost any biological barriers. It has been published earlier, that engineered nanoparticles, including carbon nanomaterials, could enter tumours via their leaky vessels system and retain due to a weak drainage in the tumour microenvironment. Here we prestent the synthesis of water-soluble d-glucosamine derivatives used as drug delivery tools and selective FYN and LCK kinase inhibitor for pancreatic cancer treatment. Material and methods Human PDAC line PANC-1, ASCP-1 and PAN-O2 was obtained from American Type Culture Collection (ATCC, USA) or from National Cancer Institute. Nuclear magnetic resonance spectra were measured on a Bruker 400 MHz NMR Spectrometer with TMS as an internal standard. MS datasets were collected using Autoflex II MALDI-TOF mass spectrometer, and MS electrospray ionisation time-of-flight (ESI-microTOF) mass spectrometer, both instruments from Bruker Daltonics Inc (Fremont, CA). High-resolution spectra were performed using Shimadzu IT-ToF LC-MS System and flash chromatography was performed using Agilent 971-FP Flash Purification System. Results and discussions To date we have designed and synthesised three glucosamine-based, water-soluble [60]fullerene derivatives with high wate solubility up to 400u2009mg/ml. It was observed that all fullerenes form two aggregate fraction 20–30u2009nm and 400–500u2009nm. Initial dark cytotoxicity studies on pancreatic cancer cell line PANC1 have been carried out using flow cytometry and propidium iodide (PI) apoptosis staining. It has been shown that all 3 glycofullerenes are non-toxic even in high concentrations (up to 1u2009mg/ml, incubation 3 and 24u2009hours). Moreover, synthesised [60]fullerene derivative localises preferentially in the nucleus of PSC cells, with some localization in the cell cytoplasm. Conclusion Our results show predominant cellular nuclear internalisation of our novel hexakis glucosamine C60 derivative. In addition, synthesised nanotherapeutic is inherently non-toxic up to concentrations of 1u2009mg/ml, and displays strong photodynamic cytotoxic behaviour, when illuminated with both blue and green light. Moreover designed nanotherapeutic is showing selective inhibition to two kinases FYN A and LCK. MS thanks National Science Centre (Poland) for the support (grant UMO- 2016/23/D/NZ7/00912)

Induction of VX2 para-renal carcinoma in rabbits: generation of animal model for loco-regional treatments of solid tumors.

BackgroundAnimal models of para-renal cancer can provide useful information for the evaluation of tumor response to loco-regional therapy experiments in solid tumors. The aim of our study was to establish a rabbit para-renal cancer model using locally implanted VX2 tumors.MethodsIn order to generate a rabbit model of para-renal cancer, we established four hind limb donor rabbits by using frozen VX2 tumor samples. Following inoculation, rabbits were monitored for appetite and signs of pain. Viable tumors appeared as palpable nodules within 2xa0weeks of inoculation. Tumor growth was confirmed in all rabbits by high-resolution ultrasound analysis and histology. Once tumor growth was established, hind limb tumors extraction was used for tumor line propagation and para-renal tumor creation. Twenty-one rabbit models bearing para-renal cancer were established by implanting VX2 tumor into the para-renal capsula. Tumors developed into discreet 2–3xa0cm nodules within 1–3 weeks of implantation. Serial renal ultrasonography follow-up, starting 1xa0week after tumor implantation, was performed. Two weeks after tumor implantation, rabbits were euthanized and tumors and other organs were collected for histopathology.ResultsTumor growth after VX2 tumor fragment implantation was confirmed in all rabbits by high-resolution ultrasound (US) imaging examinations of the para-renal regions and was measured with digital caliper. The para-renal injection of VX2 tumor fragments, achieved tumor growth in 100% of cases. All data were confirmed by histological analysis.ConclusionsWe generated for the first time, a model of para-renal cancer by surgical tumor implantation of VX2 frozen tumor fragments into rabbit’s para-renal region. This method minimizes the development of metastases and the use of non-necrotic tumors and will optimize the evaluation of tumor response to loco-regional therapy experiments.