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Supplementary Figure 1 from Three-Dimensional Culture of Mouse Renal Carcinoma Cells in Agarose Macrobeads Selects for a Subpopulation of Cells with Cancer Stem Cell or Cancer Progenitor Properties

Supplementary Table 4 from Three-Dimensional Culture of Mouse Renal Carcinoma Cells in Agarose Macrobeads Selects for a Subpopulation of Cells with Cancer Stem Cell or Cancer Progenitor Properties

Supplementary Movie 1 from Hydrophilic Agarose Macrobead Cultures Select for Outgrowth of Carcinoma Cell Populations That Can Restrict Tumor Growth

Supplementary Table 2 from Three-Dimensional Culture of Mouse Renal Carcinoma Cells in Agarose Macrobeads Selects for a Subpopulation of Cells with Cancer Stem Cell or Cancer Progenitor Properties

Supplementary Table 3 from Three-Dimensional Culture of Mouse Renal Carcinoma Cells in Agarose Macrobeads Selects for a Subpopulation of Cells with Cancer Stem Cell or Cancer Progenitor Properties

Cancer cells and their associated tumors have long been considered to exhibit unregulated proliferation or growth. However, a substantial body of evidence indicates that tumor growth is subject to both positive and negative regulatory controls. Here, we describe a novel property of tumor growth regulation that is neither species nor tumor-type specific. This property, functionally a type of feedback control, is triggered by the encapsulation of neoplastic cells in a growth-restricting hydrogel composed of an agarose matrix with a second coating of agarose to form 6- to 8-mm diameter macrobeads. In a mouse cell model of renal adenocarcinoma (RENCA cells), this process resulted in selection for a stem cell–like subpopulation which together with at least one other cell subpopulation drove colony formation in the macrobeads. Cells in these colonies produced diffusible substances that markedly inhibited in vitro and in vivo proliferation of epithelial-derived tumor cells outside the macrobeads. RENCA cells in monolayer culture that were exposed to RENCA macrobead-conditioned media exhibited cell-cycle accumulation in S phase due to activation of a G2/M checkpoint. At least 10 proteins with known tumor suppression functions were identified by analysis of RENCA macrobead-conditioned media, the properties of which offer opportunities to further dissect the molecular basis for tumor growth control. More generally, macrobead culture may permit the isolation of cancer stem cells and other cells of the stem cell niche, perhaps providing strategies to define more effective biologically based clinical approaches to treat neoplastic disease. Cancer Res; 71(3); 725–35. ©2011 AACR.

Supplementary Table 1 from Three-Dimensional Culture of Mouse Renal Carcinoma Cells in Agarose Macrobeads Selects for a Subpopulation of Cells with Cancer Stem Cell or Cancer Progenitor Properties

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Although human islet transplantation has proven to provide clinical benefits, especially the near complete amelioration of hypoglycemia, the supply of human islets is limited and insufficient to meet the needs of all people that could benefit from islet transplantation. Porcine islets, secreting insulin nearly identical to that of human insulin, have been proposed as a viable supply of unlimited islets. Further, encapsulation of the porcine islets has been shown to reduce or eliminate the use of immunosuppressive therapy that would be required to prevent rejection of the foreign islet tissue. The goal of the current study was to determine the long-term safety and efficacy of agarose encapsulated porcine islets (macrobeads) in diabetic cynomolgus macaques, in a study emulating a proposed IND trial in which daily exogenous insulin therapy would be reduced by 50% with no loss of glucose regulation. Four of six animals implanted with macrobeads demonstrated ≥ 30% reduction in insulin requirements in year 1 of follow-up. Animals were followed for 2, 3.5, and 7.4 years with no serious adverse events, mortality or evidence of pathogen transmission. This study supports the continued pursuit of encapsulated porcine islet therapy as a promising treatment option for diabetes mellitus.

It has long been recognized that defects in cell cycle checkpoint and DNA repair pathways give rise to genomic instability, tumor heterogeneity, and metastasis. Despite this knowledge, the transcription factor-mediated gene expression programs that enable survival and proliferation in the face of enormous replication stress and DNA damage have remained elusive. Using robust omics data from two independent studies, we provide evidence that a large cohort of lung adenocarcinomas exhibit significant genome instability and overexpress the DNA damage responsive transcription factor MYB proto-oncogene like 2 (MYBL2). Across two studies, elevated MYBL2 expression was a robust marker of poor overall survival and disease-free survival outcomes, regardless of disease stage. Clinically, elevated MYBL2 expression identified patients with aggressive early onset disease, increased lymph node involvement, and increased incidence of distant metastases. Analysis of genomic sequencing data demonstrated that MYBL2 High lung adenocarcinomas had elevated somatic mutation burden, widespread chromosomal alterations, and alterations in single-strand DNA break repair pathways. In this study, we provide evidence that impaired single-strand break repair, combined with a loss of cell cycle regulators TP53 and RB1, give rise to MYBL2-mediated transcriptional programs. Omics data supports a model wherein tumors with significant genomic instability upregulate MYBL2 to drive genes that control replication stress responses, promote error-prone DNA repair, and antagonize faithful homologous recombination repair. Our study supports the use of checkpoint kinase 1 (CHK1) pharmacological inhibitors, in targeted MYBL2 High patient cohorts, as a future therapy to improve lung adenocarcinoma patient outcomes.

Objective The complexity, heterogeneity and capacity of malignant neoplastic cells and tumors for rapid change and evolution suggest that living-cell-based biological-systems approaches to cancer treatment are merited. Testing this hypothesis, the tumor marker, metabolic activity, and overall survival (OS) responses, to the use of one such system, implantable macrobeads [RENCA macrobeads (RMBs)], in phase I and IIa clinical trials in advanced, treatment-resistant metastatic colorectal cancer (mCRC) are described here. Methods Forty-eight mCRC patients (30 females; 18 males), who had failed all available, approved treatments, underwent RMB implantation (8 RMB/kg body weight) up to 4 times in phase I and phase IIa open-label trials. Physicals, labs [tumor and inflammation markers, lactate dehydrogenase (LDH)] and positron emission tomography-computed tomography (PET-CT) imaging to measure number/volume and metabolic activity of the tumors were performed pre- and 3-month-post-implantation to evaluate safety and initial efficacy (as defined by biological responses). PET-CT maximum standard uptake value (SUVmax) (baseline and d 90; SUVmax ≥2.5), LDH, and carcinoembryonic antigen (CEA) and/or cancer antigen 19-9 (CA 19-9) response (baseline, d 30 and/or d 60) were assessed and compared to OS. Results Responses after implantation were characterized by an at least 20% decrease in CEA and/or CA 19-9 in 75% of patients. Fluorodeoxyglucose (FDG)-positive lesions (phase I, 39; 2a, 82) were detected in 37/48 evaluable patients, with 35% stable volume and stable or decreased SUV (10) plus four with necrosis; 10, increased tumor volume, SUV. LDH levels remained stable and low in Responders (R) (d 0-60, 290.4-333.9), but increased steadily in Non-responders (NR) (d 0-60, 382.8-1,278.5) (d 60, P=0.050). Responders to RMBs, indicated by the changes in the above markers, correlated with OS (R mean OS=10.76 months; NR mean OS=4.9 months; P=0.0006). Conclusions The correlations of the tumor marker, tumor volume and SUV changes on PET-CT, and LDH levels themselves, and with OS, support the concept of a biological response to RMB implantation and the validity of the biological-systems approach to mCRC. A phase III clinical trial is planned.

Background The use of porcine islets to replace insulin-producing islet β-cells, destroyed during the diabetogenic disease process, presents distinct challenges if this option is to become a therapeutic reality for the treatment of type 1 diabetes. These challenges include a thorough evaluation of the microbiological safety of the islets. In this study, we describe a robust porcine islet-screening program that provides a high level of confidence in the microbiological safety of porcine islets suitable for clinical trials. Methods A four-checkpoint program systematically screens the donor herd (Large White – Yorkshire × Landrace F1 hybrid animals), individual sentinel and pancreas donor animals and, critically, the islet macrobeads themselves. Molecular assays screen for more than 30 known viruses, while electron microscopy and in vitro studies are employed to screen for potential new or divergent (emergent) viruses. Results Of 1207 monthly samples taken from random animals over a 2-year period, only a single positive result for Transmissible gastroenteritis virus was observed, demonstrating the high level of biosecurity maintained in the source herd. Given the lack of clinical signs, positive antibody titers for Porcine reproductive and respiratory syndrome virus, Porcine parvovirus, and Influenza A confirm the efficacy of the herd vaccination program. Porcine respiratory coronavirus was found to be present in the herd, as expected for domestic swine. Tissue homogenate samples from six sentinel and 11 donor animals, over the same 2-year period, were negative for the presence of viruses when co-cultured with six different cell lines from four species. The absence of adventitious viruses in separate islet macrobead preparations produced from 12 individual pancreas donor animals was confirmed using validated molecular (n = 32 viruses), in vitro culture (cells from four species), and transmission electron microscopy assays (200 cell profiles per donor animal) over the same 2-year period. There has been no evidence of viral transmission following the implantation of these same encapsulated and functional porcine islets into non-immunosuppressed diabetic cynomolgus macaques for up to 4 years. Isolated peripheral blood mononuclear cells from all time points were negative for PCV (Type 2), PLHV, PRRSV, PCMV, and PERV-A, PERV-B, and PERV-C by PCR analysis in all six recipient animals. Conclusion The four-checkpoint program is a robust and reliable method for characterization of the microbiological safety of encapsulated porcine islets intended for clinical trials.

This work assesses the potential capability of the next generation of high-precision Radial Velocity (RV) instruments for Earth-twin exoplanet detection. From the perspective of the importance of data sampling, the Terra Hunting Experiment aims to do this through an intense series of nightly RV observations over a long baseline on a carefully selected target list, via the brand-new instrument HARPS3. This paper describes an end-to-end simulation of generating and processing such data to help us better understand the impact of uncharacterised stellar noise in the recovery of Earth-mass planets with orbital periods of the order of many months. We consider full Keplerian systems, realistic simulated stellar noise, instrument white noise, and location-specific weather patterns for our observation schedules. We use Bayesian statistics to assess various planetary models fitted to the synthetic data, and compare the successful planet recovery of the Terra Hunting Experiment schedule with a typical reference survey. We find that the Terra Hunting Experiment can detect Earth-twins in the habitable zones of solar-type stars, in single and multi-planet systems, and in the presence of stellar signals. Also that it out-performs a typical reference survey on accuracy of recovered parameters, and that it performs comparably to an uninterrupted space-based schedule., Comment: 22 pages, 13 figures

The encapsulation of porcine islets is an attractive methodology for the treatment of Type I diabetes. In the current study, the use of pravastatin as a mild anti-inflammatory agent was investigated in pancreatectomized diabetic canines transplanted with porcine islets encapsulated in agarose-agarose macrobeads and given 80 mg/day of pravastatin (n=3) while control animals did not receive pravastatin (n=3). Control animals reached preimplant insulin requirements on days 18, 19, and 32. Pravastatin-treated animals reached preimplant insulin requirements on days 22, 27, and 50. Two animals from each group received a second macrobead implant: control animals remained insulin-free for 15 and 21 days (AUC = 3003 and 5078 mg/dL/24 hr days 1 to 15) and reached preimplant insulin requirements on days 62 and 131. Pravastatin treated animals remained insulin-free for 21 and 34 days (AUC = 1559 and 1903 mg/dL/24 hr days 1 to 15) and reached preimplant insulin requirements on days 38 and 192. Total incidence (83.3% versus 64.3%) and total severity (22.7 versus 18.3) of inflammation on tissue surfaces were higher in the control group at necropsy. These findings support pravastatin therapy in conjunction with the transplantation of encapsulated xenogeneic islets for the treatment of diabetes mellitus.

We present a description of a new instrument development, HARPS3, planned to be installed on an upgraded and roboticized Isaac Newton Telescope by end-2018. HARPS3 will be a high resolution (R = 115,000) echelle spectrograph with a wavelength range from 380-690 nm. It is being built as part of the Terra Hunting Experiment - a future 10 year radial velocity measurement programme to discover Earth-like exoplanets. The instrument design is based on the successful HARPS spectrograph on the 3.6m ESO telescope and HARPS-N on the TNG telescope. The main changes to the design in HARPS3 will be: a customised fibre adapter at the Cassegrain focus providing a stabilised beam feed and on-sky fibre diameter ~ 1.4 arcsec, the implementation of a new continuous flow cryostat to keep the CCD temperature very stable, detailed characterisation of the HARPS3 CCD to map the effective pixel positions and thus provide an improved accuracy wavelength solution, an optimised integrated polarimeter and the instrument integrated into a robotic operation. The robotic operation will optimise our programme which requires our target stars to be measured on a nightly basis. We present an overview of the entire project, including a description of our anticipated robotic operation., Comment: 13 pages, 8 figures, SPIE conference proceedings

We present preliminary results from an experiment designed to measure the effective pixel positions of a CCD to sub-pixel precision. This technique will be used to characterise the 4k x 4k CCD destined for the HARPS-3 spectrograph. The principle of coherent beam interference is used to create intensity fringes along one axis of the CCD. By sweeping the physical parameters of the experiment, the geometry of the fringes can be altered which is used to probe the pixel structure. We also present the limitations of the current experimental set-up and suggest what will be implemented in the future to vastly improve the precision of the measurements.

The culture of tumor cell lines in three-dimensional scaffolds is considered to more closely replicate the in vivo tumor microenvironment than the standard method of two-dimensional cell culture. We hypothesized that our method of encapsulating and maintaining viable and functional pancreatic islets in agarose–agarose macrobeads (diameter 6–8 mm) might provide a novel method for the culture of tumor cell lines. In this report we describe and characterize tumor colonies that form within macrobeads seeded with mouse renal adenocarcinoma cells. Approximately 1% of seeded tumor cells survive in the macrobead and over several months form discrete elliptical colonies appearing as tumor cell niches with increasing metabolic activity in parallel to colony size. The tumor colonies demonstrate ongoing cell turnover as shown by BrdU incorporation and activated caspase-3 and TUNEL staining. Genes upregulated in the tumor colonies of the macrobead are likely adaptations to this novel environment, as well as an amplification of G1/S cell-cycle checkpoints. The data presented, including SCA-1 and Oct4 positivity and the upregulation of stem cell–like genes such as those associated with the Wnt pathway, support the notion that the macrobead selects for a subpopulation of cells with cancer stem cell or cancer progenitor properties. Cancer Res; 71(3); 716–24. ©2011 AACR.

Cancer cells and their associated tumors have long been considered to exhibit unregulated proliferation or growth. However, a substantial body of evidence indicates that tumor growth is subject to both positive and negative regulatory controls. Here, we describe a novel property of tumor growth regulation that is neither species nor tumor-type specific. This property, functionally a type of feedback control, is triggered by the encapsulation of neoplastic cells in a growth-restricting hydrogel composed of an agarose matrix with a second coating of agarose to form 6- to 8-mm diameter macrobeads. In a mouse cell model of renal adenocarcinoma (RENCA cells), this process resulted in selection for a stem cell–like subpopulation which together with at least one other cell subpopulation drove colony formation in the macrobeads. Cells in these colonies produced diffusible substances that markedly inhibited in vitro and in vivo proliferation of epithelial-derived tumor cells outside the macrobeads. RENCA cells in monolayer culture that were exposed to RENCA macrobead-conditioned media exhibited cell-cycle accumulation in S phase due to activation of a G2/M checkpoint. At least 10 proteins with known tumor suppression functions were identified by analysis of RENCA macrobead-conditioned media, the properties of which offer opportunities to further dissect the molecular basis for tumor growth control. More generally, macrobead culture may permit the isolation of cancer stem cells and other cells of the stem cell niche, perhaps providing strategies to define more effective biologically based clinical approaches to treat neoplastic disease. Cancer Res; 71(3); 725–35. ©2011 AACR.

The information presented here is generally known to dc motor manufacturers but is not common knowledge to dc motor users. As there is a necessity for the equipment to perform beyond its original specifications and adjustment, commutation issues may arise that could increase maintenance requirements or downtime. Understanding the commutation implications of reducing the motor field may direct a user on a course of action, should commutation deteriorate and maintenance costs increase unacceptably. Some applications will tolerate this speed increase and others may not. In some cases, it may be possible to shift neutral slightly in the direction of motor rotation to improve commutation at the higher loads in weak field, with a possible sacrifice in performance at lower loads or with full field. The motor may be readjusted by the blackband method to improve commutation at the weak field settings at all loads, with some sacrifice of performance at base speed. The motor could be returned to the OEM for black-band adjustment with the reduced main field excitation or sent to a motor shop with this capability. Otherwise, it may be necessary to consider the other options of obtaining a higher speed motor, using a different gearbox or using a lower base speed motor and eliminating the gearbox.

The ability to culture porcine islets for extended times allows for both their functional assessment and the assurance of their microbiological safety prior to transplantation. We have previously shown that agarose-encapsulated porcine islets can be cultured for at least 24 weeks. In the current study, porcine islet agarose macrobeads cultured for up to 67 weeks were assessed for their ability to restore normoglycemia, respond to an intraperitoneal glucose challenge, maintain spontaneously diabetic BB rats free of insulin therapy for more than 6 months, and for their biocompatibility. Porcine islets were encapsulated in agarose macrobeads and subjected to weekly static perifusion assays for the assessment of insulin production. After in vitro culture for either 9, 40, or 67 weeks, 56–60 macrobeads were transplanted to each spontaneously diabetic BB rat. Transplanted rats were monitored daily for blood glucose levels. Glucose tolerance tests and assessments for porcine C-peptide were conducted at various intervals throughout the study. Normoglycemia (100–200 mg/dl) was initially restored in all islet transplanted rats. Moderate hyperglycemia (200–400 mg/dl) developed at around 30 days posttransplantation and continued throughout the study period of 201–202 days. Importantly, all rats that received encapsulated porcine islets continued to gain weight and were free of exogenous insulin therapy for the entire study. Porcine C-peptide (0.2–0.9 ng/ml) was detected in the serum of islet recipients throughout the study period. No differences were detected between recipient animals receiving islet macrobeads of various ages. These results demonstrate that the encapsulation of porcine islets in agarose macrobeads allows for extended culture periods and is an appropriate strategy for functional and microbiological assessment prior to clinical use.

We have previously described the use of a double coated agarose-agarose porcine islet macrobead for the treatment of type I diabetes mellitus. In the current study, the long-term viral safety of macrobead implantation into pancreatectomized diabetic dogs treated with pravastatin (n=3) was assessed while 2 dogs served as nonimplanted controls. A more gradual return to preimplant insulin requirements occurred after a 2nd implant procedure (days 148, 189, and >652) when compared to a first macrobead implantation (days 9, 21, and 21) in all macrobead implanted animals. In all three implanted dogs, porcine C-peptide was detected in the blood for at least 10 days following the first implant and for at least 26 days following the second implant. C-peptide was also present in the peritoneal fluid of all three implanted dogs at 6 months after 2nd implant and in 2 of 3 dogs at necropsy. Prescreening results of islet macrobeads and culture media prior to transplantation were negative for 13 viruses. No evidence of PERV or other viral transmission was found throughout the study. This study demonstrates that the long-term (2.4 years) implantation of agarose-agarose encapsulated porcine islets is a safe procedure in a large animal model of type I diabetes mellitus.

The transplantation of porcine islets of Langerhans to treat type 1 diabetes may provide a solution to the demand for insulin-producing cells. Porcine islets encapsulated in agarose–agarose macrobeads have been shown to function in nonimmunosuppressed xenogeneic models of both streptozotocin-induced and autoimmune type 1 diabetes. One advantage of agarose encapsulation is the ability to culture macrobeads for extended periods, permitting microbiological and functional assessment. Herein we describe optimization of the agarose matrix that results in improved islet function. Porcine islets (500 IEQs) from retired breeding sows were encapsulated in 1.5% SeaKem Gold (SG), 0.8% SG, or 0.8% Litex (Li) agarose, followed by an outer capsule of 5% SG agarose. Insulin production by the encapsulated islets exhibited an agarose-specific effect with 20% (0.8% SG) to 50% (0.8% Li) higher initial insulin production relative to 1.5% SG macrobeads. Insulin production was further increased by 40–50% from week 2 to week 12 in both agarose types at the 0.8% concentration, whereas islets encapsulated in 1.5% SG agarose increased insulin production by approximately 20%. Correspondingly, fewer macrobeads were required to restore normoglycemia in streptozotocin-induced diabetic female CD(SD) rats that received 0.8% Li (15 macrobeads) or 0.8% SG (17 macrobeads) as compared to 1.5% SG (19 macrobeads). Islet cell proliferation was also observed during the first 2 months postencapsulation, peaking at 4 weeks, where approximately 50% of islets contained proliferative cells, including β-cells, regardless of agarose type. These results illustrate the importance of optimizing the microenvironment of encapsulated islets to improve islet performance and advance the potential of islet xenotransplantation for the treatment of type 1 diabetes.

Streptozotocin (STZ), a nitrosourea with DNA alkylating properties, has been widely used to induce hyperglycemia by specifically destroying the insulin-producing β-cells of the islets of Langerhans in experimental models of Type I diabetes. STZ's known carcinogenic properties, however, raise concerns about its suitability for long-term studies. We conducted a formal study of STZ's carcinogenic effects in long-term surviving diabetic Wistar-Furth rats. To determine if insulin therapy or islet transplantation exacerbated tumorigenesis, rats were randomly assigned to one of four experimental groups: normal animals with no treatment (Group 1, n=12); normal animals that underwent peritoneal implantation of porcine islets encapsulated in a double layer of agarose to form islet macrobeads (normal + islets; group 2, n=12); STZ treatment followed by daily exogenous insulin (STZ + insulin; group 3, n=18) and STZ treatment followed by the intraperitoneal implantation of porcine islet macrobeads (STZ + islets; group 4, n=14). At 215 days post-STZ induction, no renal proliferative lesions were observed in animals that did not receive STZ (group 1 and group 2) whereas adenoma incidences of 57% for group 3 and 34% for group 4 were observed. By terminal necropsy at day 351, the incidence and severity of renal proliferative lesions increased with tubular carcinoma observed in 67% of group 3 and 60% of group 4 animals. We conclude that the STZ-induced diabetic rat model is not suitable for long-term studies because of progressive renal tumorigenesis. Our experiments also demonstrate the safety and effectiveness of porcine islet macrobeads for the treatment of diabetes.

Carbon brushes are commonly used on DC or AC applications such as commutators and slip rings. The commutator application has been studied extensively and there are many trouble shooting tools to assist the technician at resolving motor or generator problems. Slip ring applications have been widely used on high speed turbine generators, slower speed synchronous motor/generators and wound rotor motors. In recent years, with the aid of modern electronics, the doubly fed asynchronous generator and wound rotor motor have increased in popularity. Troubleshooting tools for the slip ring application used on this type of equipment are limited. This paper will address the modes of failure for carbon brushes and slip rings and the root cause analysis for these failures. The information for this paper was generated from motor/generator inspections over a large range of applications. The intent of this paper is to assist the technician with the evaluation of slip rings and carbon brushes in order to reduce maintenance cost and increase uptime.

DC motors have been the workhorse of variable speed drives in the paper industry for many years. DC motor speed is easily controlled by varying the supplied armature voltage and/or excitation (field) current. Many mills have inquired about operating motors beyond the original motor rating. With armature voltage limited by the drive, motor speed may be increased by reducing the shunt field current, but not without consideration of vibration, machine adjustment, brush performance, and possible commutation issues because the machine was not designed and tested at those conditions. This paper examines and presents test/field data displaying the effects of field weakening on DC machine performance and maintenance.

Signaling pathways controlling cellular proliferation are major targets of cancer therapeutics, however, accurate assessment of activation status of targetable signaling cascades remains a challenge. We have developed proximity ligation assay (PLA)-based methodologies to detect signaling-associated complexes for a variety of receptor tyrosine kinases, intracellular adaptors and phosphorylation events in formalin-fixed, paraffin-embedded tissues. By assessing the degree of functional association of oncogenic kinases (such as EGFR and ALK) with intracellular adaptor molecules, it is possible to characterize the activation status of targetable kinase activity and monitor variations across tissue types, within tissues and in response to perturbations. Here, we present data for a panel of PLAs using cell lines, patient specimens and patient-derived xenografts models. We show that EGFR-GRB2 PLA reflects erlotinib pharmacodynamics and has predictive capacity for response to both erlotinib and cetuximab. We demonstrate that PLA can identify alterations in multiple bypass signaling cascades associated with therapeutic escape using both in vitro models of acquired resistance and patient specimens. We observe striking intratumoral variation in abundance of signaling-associated complexes, likely reflecting influences from the tumor microenvironment and phenotypic heterogeneity. We are currently expanding our PLA panel to include emerging therapeutic targets (MET and FGFR1) and evaluating performance of these assays to characterize signaling complexes in circulating tumor cells obtained from advanced stage non-small cell lung cancer patients. Collectively, our data suggests that PLA-based methodologies can be harnessed to explore the cancer interactome and can be important tools to enable precision medicine. Citation Format: Matthew A. Smith, Leah Clark, Richard Hall, Kate Fisher, Takeshi Yoshida, Vincent Vuaroqueaux, Heinz-Herbert Fiebig, Ann Chen, Eric B. Haura. Characterizing pathway vulnerabilities and tumor heterogeneity through signaling-associated complexes. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B54.

For clinical xenogenic islet transplantation to be successful, several requirements must be met. Among them is a sizeable and reliable source of fully functional and microbiologically safe islets. The inherent variability among porcine pancreases, with respect to islet yield, prompted us to develop a Biopsy Score technique to determine the suitability of each pancreas for islet isolation processing. The Biopsy Score consists of an assessment of five variables: warm ischemia time, pancreas color, fat content, islet size, and islet demarcation, each of which is assigned a value of −1 or +1, depending on whether or not the established criteria is met. For determination of islet size and demarcation, fresh biopsies of porcine pancreases are stained with dithizone (DTZ) solution and examined under a dissecting microscope. Based on the scoring of such biopsies in pancreases from 26—56-month-old sows, we report here that the presence of large (>100 μm diameter), well-demarcated islets in the pancreas biopsy is a reliable predictor of isolation success. Encapsulation of the isolated porcine islets within the inner layer of a 1.5% agarose and an outer layer of 5.0% agarose macrobead, containing 500 equivalent islet number (EIN), provides for extended in vitro functional viability (>6 months of insulin production in response to glucose), as well as for comprehensive microbiological testing and at least partial isolation of the xenogeneic islets from the host immune system. All microbiological testing to date has been negative, except for the presence of porcine endogenous retrovirus (PERV). Taken together, we believe that the Biopsy Score enhancement of our islet isolation technique and our agarose-agarose macroencapsulation methodology bring us significantly closer to realizing clinical porcine islet xenotransplantation for the treatment of insulin-dependent diabetic patients.

In the era of precision medicine, matching patients to targeted therapies is a critical goal and novel biomarker strategies must be implemented as new therapeutic options are introduced into the clinic. However, most traditional biomarkers fail to capture the actual signaling activity occurring within a patient's tumor. Proximity ligation assays (PLA) represent a novel approach to quantify receptor tyrosine kinase activity in clinical specimens, yielding a fluorescent readout of in situ signaling complexes. We have developed PLA assays for multiple receptor tyrosine kinase-adaptor pairs, including EGFR in complex with its major adaptor protein, GRB2. Here, we demonstrate robust performance of these assays in both cell lines and FFPE tissue specimens and provide evidence of predictive biomarker capacity for tyrosine kinase inhibitor response in lung cancer patients. For EGFR-GRB2, we demonstrate PLA is highly sensitive and specific, reflecting kinase activity and not merely expression levels of EGFR or GRB2. In non-small cell lung cancer (NSCLC) cell lines, high PLA signal is associated with EGFR mutation status and levels of phospho-EGFR. PLA also correlates with results from co-immunoprecipitation experiments and is abrogated by erlotinib in a dose-dependent manner. In multiple cell line xenograft models, PLA signal is observed in cytokeratin-staining regions and reveals dissociation of EGFR-GRB2 signaling complexes in response to erlotinib. In patient-derived lung adenocarcinoma xenografts, significantly higher PLA signal is observed in EGFR mutant xenografts relative to wildtype xenografts and is strongly reduced with erlotinib treatment. We have analyzed three cohorts of mixed histology NSCLC tumor specimens (N =354 patients) and scored these on a 0-3+ scale, finding good agreement of incidence of ≥ 2 PLA score across the three cohorts. EGFR protein expression levels as determined by Automated Quantitative Analysis (AQUA) correlate with EGFR-GRB2 PLA signal, but clear instances are defined where PLA signals are discordant, indicating “value-added” over EGFR expression analysis. High PLA score was not limited to adenocarcinomas and was observed in multiple histologies, with significant enrichment in metastatic brain lesions (P Collectively, our data indicate that PLA assays measuring EGFR:GRB2 signaling complexes can serve as biomarkers for druggable kinase activity. We believe this novel proteome-level approach has the potential to be applied in a predictive capacity in multiple malignancies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B27. Citation Format: Matthew A. Smith, Richard Hall, Kate Fisher, Ann Chen, Eric B. Haura. Proximity ligation assays as biomarkers for receptor tyrosine kinase activity in lung tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B27.

Predictive biomarkers are critical to optimize the use of receptor tyrosine kinase (RTK) inhibitors for cancer therapy. Because multi-protein complexes are critical to maintain oncogenic signaling, we hypothesized that assays that measure protein-protein interactions could identify patients with RTK activity and predict RTK inhibitor response. We developed in situ proximity ligation assays (PLA) to assess the degree of active EGFR signaling in both non-small cell lung cancer (NSCLC) cell lines and formalin-fixed paraffin-embedded (FFPE) patient specimens with the goal of establishing PLA assays as biomarkers of erlotinib sensitivity. Using in situ PLA, we evaluated EGFR association with its signaling adaptors, GRB2 and SHC1. By confocal microscopy we observed increased PLA signals in EGFR mutant NSCLC cell lines (PC9, H1650, HCC4006, HCC827) relative to EGFR wildtype (H23, H1299, H322, A549), indicative of constitutively active signaling complexes in mutant cell lines. PLA signal intensity was independent of EGFR, GRB2 and SHC1 protein expression levels, but did correlate with co-IP and pEGFR immunoblot results. Erlotinib dose-dependently reduced PLA signals in PC9 cells. H1650 xenografts exhibited high PLA signals that were reduced by erlotinib, corresponding with tumor shrinkage. Conversely, low PLA signals were observed in xenografts from EGFR wildtype cell lines (H322, H157). In human xenografts, PLA signals were significantly higher in EGFR mutant xenografts and reduced by erlotinib. In human FFPE specimens, PLA signals were absent in normal non-epithelial tissues and low in epithelial portions. FFPE lung tumor specimens (N = 221) revealed a wide range of PLA signal intensities (scored as 0+ to 3+), which localized to cytokeratin-positive regions. High PLA signals were found in approximately 25% of specimens across multiple histological classifications, including adenocarcinoma, squamous and large cell carcinoma. Furthermore, specimens from metastatic brain lesions were highly enriched for high PLA signal (p2+) in specimens with relatively low EGFR protein expression and low PLA signal ( Citation Format: Matthew A. Smith, Richard Hall, Scott Haake, Jiannong Li, Eric B. Haura. Proximity ligation assays reveal protein-protein interactions associated with oncogenic signaling and drug sensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3502. doi:10.1158/1538-7445.AM2013-3502