Electrophysiology-based stratification of pancreatic tumorigenicity by label-free single-cell impedance cytometry.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer lacking specific biomarkers that can be correlated to disease onset, promotion and progression. To assess whether tumor cell electrophysiology may serve as a marker for PDAC tumorigenicity, we use multi-frequency impedance cytometry at high throughput (∼350 cells/s) to measure the electrical phenotype of single PDAC tumor cells from xenografts, which are derived from primary pancreatic tumors versus those from liver metastases of different patients. A novel phase contrast metric based on variations in the high and low frequency impedance phase responses that is related to electrophysiology of the cell interior is found to be systematically altered as a function of tumorigenicity. PDAC cells of higher tumorigenicity exhibited lowered interior conductivity and enhanced permittivity, which is validated by the dielectrophoresis on the respective cell types. Using genetic analysis, we suggest the role of dysregulated Na+ transport and removal of Ca2+ ions from the cytoplasm on key oncogenic KRAS-driven processes that may be responsible for lowering of the interior cell conductivity. We envision that impedance cytometry can serve as a tool to quantify phenotypic heterogeneity for rapidly stratifying tumorigenicity. It can also aid in protocols for dielectrophoretic isolation of cells with a particular phenotype for prognostic studies on patient survival and to tailor therapy selection to specific patients.

Enrichment of superoxide dismutase 2 in glioblastoma confers to acquisition of temozolomide resistance that is associated with tumor-initiating cell subsets.

BACKGROUND: Intratumor subsets with tumor-initiating features in glioblastoma are likely to survive treatment. Our goal is to identify the key factor in the process by which cells develop temozolomide (TMZ) resistance. METHODS: Resistant cell lines derived from U87MG and A172 were established through long-term co-incubation of TMZ. Primary tumors obtained from patients were maintained as patient-derived xenograft for studies of tumor-initating cell (TIC) features. The cell manifestations were assessed in the gene modulated cells for relevance to drug resistance. RESULTS: Among the mitochondria-related genes in the gene expression databases, superoxide dismutase 2 (SOD2) was a significant factor in resistance and patient survival. SOD2 in the resistant cells functionally determined the cell fate by limiting TMZ-stimulated superoxide reaction and cleavage of caspase-3. Genetic inhibition of the protein led to retrieval of drug effect in mouse study. SOD2 was also associated with the TIC features, which enriched in the resistant cells. The CD133+ specific subsets in the resistant cells exhibited superior superoxide regulation and the SOD2-related caspase-3 reaction. Experiments applying SOD2 modulation showed a positive correlation between the TIC features and the protein expression. Finally, co-treatment with TMZ and the SOD inhibitor sodium diethyldithiocarbamate trihydrate in xenograft mouse models with the TMZ-resistant primary tumor resulted in lower tumor proliferation, longer survival, and less CD133, Bmi-1, and SOD2 expression. CONCLUSION: SOD2 plays crucial roles in the tumor-initiating features that are related to TMZ resistance. Inhibition of the protein is a potential therapeutic strategy that can be used to enhance the effects of chemotherapy.

Molecularly annotation of mouse avatar models derived from patients with colorectal cancer liver metastasis.

Background: Liver is the most common metastatic site in advanced colorectal cancer. Most patients with colorectal cancer liver metastasis (CRLM) do not benefit from current treatment. Patient-derived xenografts (PDXs) with defined molecular signatures are attractive models for preclinical studies. Methods: Successfully established PDXs were evaluated to elucidate their fidelity of patients' biologic characteristics (pathologic, genetic and protein properties, together with chemosensitivity). The genomic variations of PDXs were analyzed by next-generation sequencing to explore the underlying molecular mechanism of metastasis and potential therapeutic targets. Results: CRLM (N=73) showed a significantly higher successful PDX establishment rate than primary specimens (N=26; 76.7% vs. 57.7%). CRLM PDXs recapitulated the pathologic, genetic and protein properties of parental tumors, as well as chemosensitivity. Frequent altered genes in PDXs showed high consistency compared to patients' genomic alterations and were enriched in MAPK, ErbB, cell cycle, focal adhesion pathways for CRLM PDXs, whereas primary tumor-derived PDXs only exhibited genomic variations involving ErbB and cell cycle. The genetic alterations showed high concordance between paired PDXs from primary and metastatic tissues, except for recurrent gene mutations (ARID1A, CDK8, ETV1, STAT5B and WNK3) and common copy number gains in chromosomes 20q (e.g., SRC/AURKA). Several potential drug targets such as KRAS, HER2, and FGFR2 were validated using corresponding inhibitors. Additionally, PDX models could also be used in screening efficient regimens for patients with no druggable alterations. Conclusion: This study has successfully established and validated a large panel of molecularly annotated platforms from patients with CRLM for preclinical studies.

Fat Graft Safety after Oncologic Surgery: Addressing the Contradiction between In Vitro and Clinical Studies.

BACKGROUND: The authors investigate the in vitro and in vivo interaction of human breast cancer cells and human adipose-derived stem cells to address the controversy on the safety of postmastectomy fat grafting. METHODS: The authors co-cultured human adipose-derived stem cells and MDA-MB-231 breast cancer cells in an in vitro cell migration assay to examine the migration of breast cancer cells. In the in vivo arm, the authors injected breast cancer cells (group I), human breast cancer cells plus human adipose-derived stem cells (group II), human breast cancer cells plus human fat graft (group III), and human breast cancer cells plus human fat graft plus human adipose-derived stem cells (group IV) to the mammary fat pads of female nude mice (n = 20). The authors examined the tumors, livers, and lungs histologically after 2 weeks. RESULTS: Migration of breast cancer cells increased significantly when co-cultured with adipose-derived stem cells (p < 0.05). The tumor growth rate in group IV was significantly higher than in groups I and II (p < 0.05). The tumor growth rate in group III was also higher than in groups I and II, but this difference was not statistically significant (p > 0.05). Histologically, there was no liver/lung metastasis at the end of 2 weeks. The vascular density in the tumors from group IV was significantly higher than in other groups (p < 0.01). CONCLUSION: The injection of breast cancer cells, fat graft, and adipose-derived stem cells together increases breast cancer xenograft growth rates significantly.

A Sutureless Vascular Closure Device for Emergent Bovine Xenograft Implantation.

A novel vascular staple (C-staple) was developed that does not enter the vasculature lumen during anastomoses. The objective of this study was to demonstrate C-staple safety when used with a bovine xenograft and compare efficacy of the C-staple procedure with Anastoclip surgical clips or suturing when used with a bovine xenograft. Eight sheep had an acute comparison between suturing and C-staples using both common carotid arteries. Sixteen sheep had xenograft placement in the left carotid artery, eight with C-staples and eight with Anastoclips in a chronic study. Over 6 months, Doppler ultrasound interrogation of the common carotid arteries was performed. After 6 months, arteries were evaluated histopathologically. Cross-clamp and surgical times were longer in the C-staple group than the suture group, and xenograft implantation times were statistically longer with C-staples than with Anastoclips. After 6 months, C-staple biocompatibility was similar to Anastoclips. Patency and hemodynamics of the bovine xenograft were not statistically different between the two groups. C-staples performed as well as the Anastoclips except for implant times, likely due to delivery system differences. Histological findings and clinical outcomes were no different with the two devices. Further refinements of the C-staple delivery system are necessary before proceeding to clinical trials.

A Heterotopic Xenograft Model of Human Airways for Investigating Fibrosis in Asthma.

Limited in vivo models exist to investigate the lung airway epithelial role in repair, regeneration, and pathology of chronic lung diseases. Herein, we introduce a novel animal model in asthma-a xenograft system integrating a differentiating human asthmatic airway epithelium with an actively remodeling rodent mesenchyme in an immunocompromised murine host. Human asthmatic and nonasthmatic airway epithelial cells were seeded into decellularized rat tracheas. Tracheas were ligated to a sterile cassette and implanted subcutaneously in the flanks of nude mice. Grafts were harvested at 2, 4, or 6 weeks for tissue histology, fibrillar collagen, and transforming growth factor-ß activation analysis. We compared immunostaining in these xenografts to human lungs. Grafted epithelial cells generated a differentiated epithelium containing basal, ciliated, and mucus-expressing cells. By 4 weeks postengraftment, asthmatic epithelia showed decreased numbers of ciliated cells and decreased E-cadherin expression compared with nonasthmatic grafts, similar to human lungs. Grafts seeded with asthmatic epithelial cells had three times more fibrillar collagen and induction of transforming growth factor-ß isoforms at 6 weeks postengraftment compared with nonasthmatic grafts. Asthmatic epithelium alone is sufficient to drive aberrant mesenchymal remodeling with fibrillar collagen deposition in asthmatic xenografts. Moreover, this xenograft system represents an advance over current asthma models in that it permits direct assessment of the epithelial-mesenchymal trophic unit.

Evaluation of a Bovine Vascular Graft in Sheep.

The study objective was to determine safety and efficacy of a treated bovine vascular xenograft, in two Good Laboratory Practice compliant studies in sheep following carotid graft implantation. In one study, a 3- to 5-mm diameter xenograft was implanted into the right carotid artery of male sheep and compared to autologous jugular vein and a polymeric grafts similarly implanted. In a second study, a 9.5- to 14-mm diameter xenograft similarly implanted into the right carotid artery was compared to an autologous saphenous vein. Monthly Doppler ultrasound evaluation of implant patency and flow in implants and contralateral control carotid arteries was performed. The small vessel cohort 6 month xenograft patency was equivalent (or better) than animals with polymeric vascular graft or autologous vein implants; the aneurysm incidence was less than that of autologous vein grafts. In the large vessel cohort, all 15 xenografts and 12/15 saphenous vein implants were patent at 6 month follow-up. Tissue histology showed mild inflammatory responses in the xenografts that was slightly greater than suture material. In summary, treated bovine xenograft performance in this small study suggests it may be superior to polymeric autologous vein grafts, and may have a similar failure rate as autologous vein grafts after implantation.

The reverse evolution from multicellularity to unicellularity during carcinogenesis.

Theoretical reasoning suggests that cancer may result from a knockdown of the genetic constraints that evolved for the maintenance of metazoan multicellularity. By characterizing the whole-life history of a xenograft tumour, here we show that metastasis is driven by positive selection for general loss-of-function mutations on multicellularity-related genes. Expression analyses reveal mainly downregulation of multicellularity-related genes and an evolving expression profile towards that of embryonic stem cells, the cell type resembling unicellular life in its capacity of unlimited clonal proliferation. Also, the emergence of metazoan multicellularity ~600 Myr ago is accompanied by an elevated birth rate of cancer genes, and there are more loss-of-function tumour suppressors than activated oncogenes in a typical tumour. These data collectively suggest that cancer represents a loss-of-function-driven reverse evolution back to the unicellular 'ground state'. This cancer evolution model may account for inter-/intratumoural genetic heterogeneity, could explain distant-organ metastases and hold implications for cancer therapy.

The tyrosine phosphatase PTPN14 (Pez) inhibits metastasis by altering protein trafficking.

Factors secreted by tumor cells shape the local microenvironment to promote invasion and metastasis, as well as condition the premetastatic niche to enable secondary-site colonization and growth. In addition to this secretome, tumor cells have increased abundance of growth-promoting receptors at the cell surface. We found that the tyrosine phosphatase PTPN14 (also called Pez, which is mutated in various cancers) suppressed metastasis by reducing intracellular protein trafficking through the secretory pathway. Knocking down PTPN14 in tumor cells or injecting the peritoneum of mice with conditioned medium from PTPN14-deficient cell cultures promoted the growth and metastasis of breast cancer xenografts. Loss of catalytically functional PTPN14 increased the secretion of growth factors and cytokines, such as IL-8 (interleukin-8), and increased the abundance of EGFR (epidermal growth factor receptor) at the cell surface of breast cancer cells and of FLT4 (vascular endothelial growth factor receptor 3) at the cell surface of primary lymphatic endothelial cells. We identified RIN1 (Ras and Rab interactor 1) and PRKCD (protein kinase C-δ) as binding partners and substrates of PTPN14. Similar to cells overexpressing PTPN14, receptor trafficking to the cell surface was inhibited in cells that lacked PRKCD or RIN1 or expressed a nonphosphorylatable RIN1 mutant, and cytokine secretion was decreased in cells treated with PRKCD inhibitors. Invasive breast cancer tissue had decreased expression of PTPN14, and patient survival was worse when tumors had increased expression of the genes encoding RIN1 or PRKCD. Thus, PTPN14 prevents metastasis by restricting the trafficking of both soluble and membrane-bound proteins.

Biomechanical comparison of hamstring tendon fixation devices for anterior cruciate ligament reconstruction: part 1. Five femoral devices.

We conducted a study to biomechanically compare 5 femoral hamstring tendon fixation devices commonly used in anterior cruciate ligament reconstruction. Quadrupled human semitendinosus-gracilis tendon grafts were fixed into porcine femurs using 5 separate fixation devices. For each device, 10 specimens were tested (1500-cycle loading test at 50-200 N). Specimens surviving the cyclic loading then underwent a single load-to-failure (LTF) test. Failure mode, stiffness, ultimate load, and rigidity were recorded. Two of 10 Delta screw (Arthrex), 10 of 10 Bio-TransFix (Arthrex), 10 of 10 Bone Mulch screw (Arthrotek), 10 of 10 EZLoc (Arthrotek), and 10 of 10 Zip Loop (Arthrotek) devices completed the 1500-cycle loading test. Residual displacement was lowest for Bio-TransFix (4.1 mm) followed by Bone Mulch (5.2 mm), EZLoc (6.4 mm), Zip Loop (6.8 mm), and Delta (8.2 mm). Mean stiffness was significantly (P < .001) higher for Bone Mulch (218 N/mm) than for Bio-TransFix (171 N/mm), EZLoc (122 N/mm), Zip Loop (105 N/mm), or Delta (84 N/mm). Mean LTF was significantly ( P < .001) higher for Bone Mulch (867 N) than for Zip Loop (615 N), Bio-TransFix (552 N), EZLoc (476 N), or Delta (410 N). The Bone Mulch screw demonstrated superior strength in the fixation of hamstring grafts in the femur. Bio-TransFix was close behind. The Delta screw demonstrated poor displacement, stiffness, and LTF. When used as the sole femoral fixation device, a device with low LTF, decreased stiffness, and high residual displacement should be used cautiously in patients undergoing aggressive rehabilitation.

Development and histopathological characterization of tumorgraft models of pancreatic ductal adenocarcinoma.

Pancreatic cancer is the one of the deadliest of all malignancies. The five year survival rate for patients with this disease is 3-5%. Thus, there is a compelling need for novel therapeutic strategies to improve the clinical outcome for patients with pancreatic cancer.  Several groups have demonstrated for other types of solid tumors that early passage human tumor xenograft models can be used to define some genetic and molecular characteristics of specific human tumors. Published studies also suggest that murine tumorgraft models (early passage xenografts derived from direct implantation of primary tumor specimens) may be useful in identifying compounds with efficacy against specific tumor types.  Because pancreatic cancer is a fatal disease and few well-characterized model systems are available for translational research, we developed and characterized a panel of pancreatic tumorgraft models for biological evaluation and therapeutic drug testing.  Of the 41 primary tumor specimens implanted subcutaneously into mice, 35 produced viable tumorgraft models.  We document the fidelity of histological and morphological characteristics and of KRAS mutation status among primary (F0), F1, and F2 tumors for the twenty models that have progressed to the F3 generation.  Importantly, our procedures produced a take rate of 85%, higher than any reported in the literature. Primary tumor specimens that failed to produce tumorgrafts were those that either contained <10% tumor cells or that were obtained from significantly smaller primary tumors. In view of the fidelity of characteristics of primary tumor specimens through at least the F2 generation in mice, we propose that these tumorgraft models represent a useful tool for identifying critical characteristics of pancreatic tumors and for evaluating potential therapies. 

Clinicopathological findings in non-human primate recipients of porcine renal xenografts: quantitative and qualitative evaluation of proteinuria.

BACKGROUND: Immunological and histopathological features in pig-to-primate renal xenotransplantation are widely studied. Only limited data have been reported about clinicopathological findings in primate recipients of life-supporting renal xenografts. In human medicine, proteinuria represents a common complication in kidney transplantation and is associated with impaired graft survival. The detection of low molecular weight proteins of tubular origin is considered an early method for predicting potential graft rejection. In this study, the presence and the significance of quantitative and qualitative proteinuria were evaluated in xenotransplanted non-human primates in which kidney function was supported only by the transplanted organ. METHODS: Eight bilaterally nephrectomized cynomolgus monkeys (Macaca fascicularis) were transplanted with a single kidney from α1,3-galactosyltransferase gene-knockout (GTKO) pigs transgenic for human CD39, CD55, CD59, and α1,2-fucosyltransferase. In addition to hematological and biochemical analyses, quantitative and qualitative analysis of proteinuria was evaluated by urinary protein-to-creatinine ratio (UPC ratio) and sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE), respectively. RESULTS: The main hematological and biochemical changes recorded after transplantation were a progressive anemia and a severe and progressive decrease in total proteins. In urine samples, the UPC ratio was low before transplantation and increased after transplantation. Similarly, SDS-AGE was negative before transplantation, but bands consistent with mixed (i.e., tubular and glomerular) proteinuria were observed in all samples collected post-transplantation. CONCLUSIONS: The study of clinicopathological changes in cynomolgus monkey renal xenograft recipients provides a valid help in monitoring the health conditions in the post-transplant period. Moreover, the evaluation of UPC ratio and the use of SDS-AGE technique in urine samples of cynomolgus monkey renal xenograft recipients may be considered a valid, inexpensive, and less time-consuming method than more sophisticated techniques in monitoring proteinuria. Proteinuria and presence of low molecular weight (LMW) proteins were consistently found in urine after transplantation, independent of fluctuations in renal function.

Matrigel alters the pathophysiology of orthotopic human breast adenocarcinoma xenografts with implications for nanomedicine evaluation.

Matrigel, a mouse sarcoma-derived basement membrane protein mixture, is frequently used to facilitate human tumor xenograft growth in rodents. Despite its known effects on tumor growth and metastasis, its impact on tumor pathophysiology and preclinical evaluation of nanomedicines in tumor xenografts has not been reported previously. Herein bilateral MDA435 tumors were established orthotopically with (Mat+) or without (Mat-) co-injection of Matrigel. Tumor perfusion, morphology and nanoparticle retention were evaluated. As compared to Mat- tumors, Mat+tumors exhibited enhanced vascular perfusion and lymphatic flow, greater blood vessel and lymphatic growth within the tumor core, and more deformation and collapse of lymphatics in tumor-associated lymph nodes. These changes were accompanied by reduced nanoparticle retention in Mat+tumors. The results suggest that Matrigel is not a passive medium for tumor growth, but rather significantly alters long-term tumor architecture. These findings have significant implications for the evaluation of therapeutic nanomedicine in xenograft mouse models. FROM THE CLINICAL EDITOR: Matrigel is utilized in facilitating human tumor xenograft growth in rodents. The authors demonstrate that Matrigel is not a passive medium for tumor growth; instead it significantly alters long-term tumor architecture, with major implications in the evaluation of therapeutic nanomedicine in xenograft mouse models.