Tumor Microenvironment Modifications Induced by Afatinib in Squamous Cell Carcinoma of the Head and Neck: A Window-of-Opportunity Study (EORTC-90111-24111).

PURPOSE: The EORTC-90111-24111 phase II window study evaluated afatinib versus no preoperative treatment in patients with primary squamous cell carcinoma of the head and neck (HNSCC). We investigated afatinib-induced tumor and microenvironment modifications by comparing pre- and posttreatment tumor biopsies. PATIENTS AND METHODS: Thirty treatment-naïve patients with primary HNSCC were randomized. Twenty-five patients received afatinib for 14 days before surgery (40 mg 1×/day) and 5 patients were attributed to the control arm. Biopsies were taken at work-up and during surgery. Good quality RNA samples were used for omics analyses. The control arm was enlarged by samples coming from our previous similar window study. RESULTS: IHC analyses of afatinib-treated tumor biopsies showed a decrease in pEGFR (P ≤ 0.05) and pERK (P ≤ 0.05); and an increase in CD3+ (P ≤ 0.01) and CD8+ (P ≤ 0.01) T-cell infiltration, and in CD3+ (P ≤ 0.05) T-cell density. RNA sequencing analyses of afatinib-treated tumor samples showed upregulation of inflammatory genes and increased expression scores of signatures predictive of response to programmed cell death protein 1 blockade (P ≤ 0.05). In posttreatment biopsies of afatinib-treated patients, two clusters were observed. Cluster 1 showed a higher expression of markers and gene sets implicated in epithelial-to-mesenchymal transition (EMT) and activation of cancer-associated fibroblasts (CAF) compared with cluster 2 and controls. CONCLUSIONS: Short-term treatment with afatinib in primary HNSCC induces CD3+ and CD8+ tumor infiltration and, in some patients, EMT and CAF activation. These results open perspectives to overcome resistance mechanisms to anti-HER therapy and to potentiate the activity of immune checkpoint inhibitors.

Liquid biopsy for mutational profiling of locoregional recurrent and/or metastatic head and neck squamous cell carcinoma.

OBJECTIVES: The molecular landscape of head and neck squamous cell carcinoma (HNSCC) harbors potentially actionable genomic alterations. We aimed to study the utility of liquid biopsy to (i) characterize the mutational landscape of recurrent/metastatic HNSCC using a comprehensive gene panel and (ii) estimate the concordance between DNA mutations identified from circulating tumor DNA (ctDNA) and matched tumor tissues. MATERIALS AND METHODS: Targeted next-generation sequencing (NGS) was performed on cell-free DNA (cfDNA) of 39 patients with locoregional recurrent (n = 19) and/or metastatic (n = 20) HNSCC. Tumor biopsy (n = 18) was sequenced using the same technique. RESULTS: ctDNA was detected in 51% of patients (20/39) with a higher probability of detection in metastatic than locoregional recurrent disease (70% versus 30%, p = 0.025). 81% and 58% of the tissue tumor variants were not detected in plasma when considering all patients and only metastatic patients with detectable ctDNA, respectively. In a multivariate analysis, the likelihood of detecting the tissue tumor variant in plasma was related to metastatic status (p = 0.012), tumor variant allele frequency (p < 0.001) and ctDNA quantity (p < 0.001). 26% of the variants were detected only in liquid and not in the solid biopsy. Three patients without an available tumor sample had plasma containing three different potentially actionable PIK3CA mutations. CONCLUSION: CtDNA detection and characterization using targeted NGS is feasible in metastatic HNSCC. Liquid biopsies do not reflect the complete mutation profile of the tumor but have the potential to identify actionable mutations when tumor biopsies are not available as well as variants not found in matched tumor tissue.

Metabolic Imaging Using Hyperpolarized Pyruvate-Lactate Exchange Assesses Response or Resistance to the EGFR Inhibitor Cetuximab in Patient-Derived HNSCC Xenografts.

PURPOSE: Optimal head and neck squamous cell carcinoma (HNSCC) patient selection for anti-EGFR-based therapy remains an unmet need since only a minority of patients derive long-term benefit from cetuximab treatment. We assessed the ability of state-of-the-art noninvasive in vivo metabolic imaging to probe metabolic shift in cetuximab-sensitive and -resistant HNSCC patient-derived tumor xenografts (PDTXs). EXPERIMENTAL DESIGN: Three models selected based on their known sensitivity to cetuximab in patients (cetuximab-sensitive or acquired-resistant HNC007 PDTXs, cetuximab-naïve UCLHN4 PDTXs, and cetuximab-resistant HNC010 PDTXs) were inoculated in athymic nude mice. RESULTS: Cetuximab induced tumor size stabilization in mice for 4 weeks in cetuximab-sensitive and -naïve models treated with weekly injections (30 mg/kg) of cetuximab. Hyperpolarized 13C-pyruvate-13C-lactate exchange was significantly decreased in vivo in cetuximab-sensitive xenograft models 8 days after treatment initiation, whereas it was not modified in cetuximab-resistant xenografts. Ex vivo analysis of sensitive tumors resected at day 8 after treatment highlighted specific metabolic changes, likely to participate in the decrease in the lactate to pyruvate ratio in vivo. Diffusion MRI showed a decrease in tumor cellularity in the HNC007-sensitive tumors, but failed to show sensitivity to cetuximab in the UCLHN4 model. CONCLUSIONS: This study constitutes the first in vivo demonstration of cetuximab-induced metabolic changes in cetuximab-sensitive HNSCC PDTXs that were not present in resistant tumors. Using metabolic imaging, we were able to identify hyperpolarized 13C-pyruvate as a potential marker for response and resistance to the EGFR inhibitor in HNSCC.

2'-deoxy-2'-[18F] fluoro-D-glucose positron emission tomography, diffusion-weighted magnetic resonance imaging, and choline spectroscopy to predict the activity of cetuximab in tumor xenografts derived from patients with squamous cell carcinoma of the head and neck.

We investigated changes on 2'-deoxy-2'-[18F]fluoro-D-glucose positron emission tomography (18FDG-PET), diffusion-weighted magnetic resonance imaging (DW-MRI), and choline spectroscopy as early markers of cetuximab activity in squamous cell carcinoma of the head and neck (SCCHN). SCCHN patient-derived tumor xenografts models were selected based on their cetuximab sensitivity. Three models were resistant to cetuximab and two were sensitive (one was highly sensitive and the other one was moderately sensitive). Cetuximab was infused on day 0 and 7. Maximal standardized uptake values (SUVmax), apparent diffusion coefficient (ADC), and total choline pool were measured at baseline and at day 8. To investigate the possible clinical relevance of our pre-clinical findings, we also studied the SUVmax and ADC modifications induced by cetuximab in five patients. Cetuximab induced a significant decrease in SUVmax and an increase in ADC at day 8 compared to baseline in the most cetuximab-sensitive model but not in the other models. At day 8, in one resistant model, SUVmax was decreased compared to baseline and was significantly lower than the controls. Choline spectroscopy was not able to predict cetuximab activity. The five patients treated with cetuximab had a 18FDG-PET partial response. One patient had a partial response according to RECISTv1.1. Interestingly, this last had also an increase in ADC value above 25%. Our preclinical data support the use of PDTX to investigate imaging techniques to detect early treatment response. Our pre-clinical and clinical data suggest that DW-MRI and 18FDG-PET should be further investigated to predict cetuximab activity.

Improvement of pig islet function by in vivo pancreatic tissue remodeling: a "human-like" pig islet structure with streptozotocin treatment.

Pig islets demonstrate significantly lower insulin secretion after glucose stimulation than human islets (stimulation index of ∼12 vs. 2 for glucose 1 and 15 mM, respectively) due to a major difference in ß- and α-cell composition in islets (60% and 25% in humans and 90% and 8% in pigs, respectively). This leads to a lower rise in 3',5'-cyclic adenosine monophosphate (cAMP) in pig ß-cells. Since glucagon is the major hormonal effector of cAMP in ß-cells, we modified pig islet structure in vivo to increase the proportion of α-cells per islet and to improve insulin secretion. Selected doses (0, 30, 50, 75, and 100 mg/kg) of streptozotocin (STZ) were intravenously injected in 32 young pigs to assess pancreatic (insulin and glucagon) hormone levels, islet remodeling (histomorphometry for α- and ß-cell proportions), and insulin and glucagon secretion in isolated islets. Endocrine structure and hormonal content of pig islets were compared with those of human islets. The dose of STZ was significantly correlated with reductions in pancreatic insulin content (p< 0.05, r(2) = 0.77) and the proportion of ß-cells (p < 0.05, r(2) = 0.88). A maximum of 50 mg/kg STZ was required for optimal structure remodeling, with an increased proportion of α-cells per islet (26% vs. 48% α-cells per islet for STZ <50 mg/kg vs. >75 mg/kg; p < 0.05) without ß-cell dysfunction. Three months after STZ treatment (30/50 mg/kg STZ), pig islets were isolated and compared with isolated control islets (0 mg/kg STZ). Isolated islets from STZ-treated (30/50 mg/kg) pigs had a higher proportion of α-cells than those from control animals (32.0% vs. 9.6%, respectively, p < 0.05). After in vitro stimulation, isolated islets from STZ-treated pigs demonstrated significantly higher glucagon content (65.4 vs. 21.0 ng/ml, p < 0.05) and insulin release (144 µU/ml) than nontreated islets (59 µU/ml, p < 0.05), respectively. Low-dose STZ (<50 mg/kg) can modify the structure of pig islets in vivo and improve insulin secretion after isolation.

Beta-5 score to evaluate pig islet graft function in a primate pre-clinical model.

BACKGROUND: We developed a composite scoring system to accurately assess pig islet function in pre-clinical primate studies. METHODS: Two scoring methods that have been clinically validated in human islet allotransplantation were tested in six non-diabetic and nine streptozotocin (STZ)-induced diabetic primates: (i) SUITO index=[1500 × fasting C-peptide (ng/ml)]/[fasting blood glucose (FBG, mg/dl) - 63] and (ii) CP/G ratio =[fasting C-peptide (ng/ml) × 100]/FBG (mg/dl). Both scores were analysed as a function of the ß-cell mass of the native primate pancreas. Next, a proposed ß5 score based on FBG values, daily glycosuria, post-prandial glycosuria, polydipsia, and polyuria was validated on the same primates. Ranges of normal and pathologic values for each parameter were assessed during 5 months in non-diabetic and diabetic primates, respectively. Finally, scores were tested on the nine STZ-induced diabetic primates, four of which were transplanted with microencapsulated pig islets and five with macroencapsulated pig islets. All parameters required for each score were measured prior to transplantation and up to 12 weeks post-transplantation. For the CP/G ratio after transplantation, primate C-peptide was replaced by porcine C-peptide. RESULTS: The Suito index was not correlated with the pancreatic ß-cell mass in contrast to the CP/G ratio (R(2) = 0.17, P = 0.645 vs. R(2) = 0.76, P = 0.003; respectively). The internal consistency of the parameters implied by the ß5 score was confirmed by a Cronbach's alpha test of 0.97. Diabetes was confirmed by a significant decrease in the CP/G ratio and the ß5 score before and after diabetes induction, respectively. After transplantation, a significant correlation was found between the CP/G ratio and the ß5 score, which reflected the functionality of pig islet xenografts and diabetes control. In addition, the CP/G ratio and ß5 score were correlated with the glycosylated hemoglobin course after transplantation and diabetes correction with macroencapsulated pig islets. CONCLUSION: The proposed ß5 score provides a valid tool to accurately assess islet transplantation in a primate pre-clinical model.

Alginate macroencapsulation of pig islets allows correction of streptozotocin-induced diabetes in primates up to 6 months without immunosuppression.

BACKGROUND: This study assessed the capacity of alginate-encapsulated islets to reverse diabetes in a pig-to-primate model. METHODS: Adult pig islets were encapsulated in microcapsules implanted under the kidney capsule (n=4) or in a subcutaneous macrodevice (n=5) in diabetic primates. Fasting blood glucose (FBG), insulin, porcine C-peptide, glycosylated hemoglobin (HbA1C), and cellular and humoral responses were followed. RESULTS: Nonencapsulated pig islets were rejected within 7 days. A transient decrease of FBG was observed only during the 2 weeks after microencapsulated pig islet implantation under the kidney capsule. After subcutaneous transplantation of a macrodevice, diabetes was corrected up to a maximum of 6 months in five animals: FBG less than 107 mg/dL and HbA1C at 8% ± 1.4%. Two of the five animals received a new macrodevice between 25 and 35 weeks after the first graft dysfunction (HbA1C ≥ 13), and diabetes was controlled for an additional 18 weeks in these animals. Although a strong humoral response was elicited after transplantation of encapsulated islets, a total impermeability of alginate 3% wt/vol to IgG was demonstrated before and up to 20 weeks after transplantation of the subcutaneous macrodevice. CONCLUSIONS: Pig islets encapsulated in a subcutaneous macrodevice can control diabetes up to 6 months without immunosuppression.

In vivo selection of biocompatible alginates for islet encapsulation and subcutaneous transplantation.

Islet encapsulation requires several properties including (1) biocompatibility, (2) immunoprotection, and (3) oxygen diffusion for islet survival and diabetes correction. New chemical alginates were tested in vivo and compared with traditional high-mannuronate and -guluronate alginates. New alginates with coupled peptide sequence (sterile lyophilized high mannuronate [SLM]-RGD3% and sterile lyophilized high guluronate [SLG]-RGD3%), to improve encapsulated cell adherence in the matrix, and alginates with a very low viscosity (VLDM7% and VLDG7%), to reduce implant size by loading a higher number of islets per volume of polymer, were implanted subcutaneously in 70 Wistar rats for comparison with alginates of high viscosity and high content of mannuronic (SLM3%) or guluronic acids (SLG3%). Permeability of alginates to 36-, 75-, and 150-kDa lectins coupled to fluorescein isothiocynate was quantified before implantation and at 2, 4, and 12 weeks after implantation. Biocompatibility (fibrosis, graft stability, immunologic infiltration by CD3/CD68 cells, and neovascularization) was assessed at each explantation time. Permeability to small molecules was found for all alginates. Impermeability to 150-kDa molecules, such as IgG, was observed only for SLM3% before implantation and was maintained up to 12 weeks after implantation. SLM3% and SLG3% demonstrated better graft stability with lower CD3/CD68 recruitment and fibrosis than the other alginates. SLM3% induced a significantly higher angiogenesis and maintained oxygen pressure at approximately 40 mm Hg for up to 4 weeks after implantation as measured by in vivo electronic paramagnetic resonance oximetry. SLM-encapsulated pig islets implanted subcutaneously in rats demonstrated no inflammatory/immunologic reactions and islets functioned for up to 60 days without immunosuppression. A traditional alginate made of high mannuronic content (SLM3%) is an adapted material to immunoprotect islets in subcutaneous tissue. No improvement was found with lower viscosity and use of GRGDSP-peptide sequence.

Native pancreatic alpha-cell adaptation in streptozotocin-induced diabetic primates: importance for pig islet xenotransplantation.

BACKGROUND: Metabolic compatibility between donor and recipient species is an important matter for pig islet xenotransplantation. Glucagon is a key hormone for the function of pig islets as well as control of hypoglycemia in the recipients of the islets. Because a discrepancy exists in the composition of glucagon cells of pig and human/primate islets, the present study was designed to determine the role of native recipient glucagon cells in the treatment of diabetes by islet transplantation in a "pig-to-primate" model. METHODS: Streptozotocin-treated (50 mg/kg) monkeys (n = 12, follow-up of 6 to 231 days) were compared with non-diabetic animals (n = 5; follow-up, 180 days). Metabolic [fasting and intravenous glucose tolerance tests (IVGTTs) for serum levels of glucose, insulin, glucagon] and morphologic (endocrine volume density and cell mass for insulin and glucagon) were compared between non-diabetic and diabetic animals. Six additional diabetic primates were given transplants of 15 000 adult pig islet equivalents without immunosuppression to monitor glucose, glucagon, insulin, and porcine C-peptide levels until 48 h after transplantation. RESULTS: Elevated fasting blood glucose, pathologic IVGTT, destruction of 95% of beta-cell mass, and glycosylated hemoglobin (>13%) were assessed in diabetic monkeys. The serum glucagon levels and glucagon cell mass correlated significantly with diabetes time course of diabetes (R = 0.940, p = 0.005; R = 0.663, p = 0.019, respectively). A mean increase of 89% in glucagon cell mass was observed for primates suffering from diabetes >53 days. No response of glucagon secretion was observed for diabetic animals during IVGTT, because no increase of serum insulin levels followed glucose loading. Blood glucose levels dropped after pig islet xenografts in diabetic primates. This reduction was maintained by an insulin level >20 microU/ml over the period of time of xenograft function (porcine C-peptide >0.1 ng/ml). A total restoration of native primate glucagon sensitivity to insulin was found after pig islets xenotransplantation as revealed by a reduction of 80% of the glucagon level. When graft dysfunction (>24 h post-transplantation), the insulin level dropped and glucagon levels rose again (>50 pg/ml). CONCLUSIONS: Native glucagon cells provide morphologic and functional plasticity to diabetes. Adult pig islet xenotransplantation can restore the sensitivity of primate glucagon to insulin but cannot protect the diabetic recipient against hypoglycemia.

Regeneration of abdominal wall musculofascial defects by a human acellular collagen matrix.

This work studied the reconstruction of an abdominal wall defect by a human acellular collagen matrix. The abdominal wall defect was cured in 40 rats by implanting (i) polypropylene (Pro), (ii) polyester (Mers) meshes, and (iii) human acellular collagen matrix with two orientations: fibres in parallel (fascia lata longitudinal [FLL]) or perpendicular (fascia lata transversal [FLT]) to native rats' abdominal walls. Hernia recurrence, adhesions, and histology (for inflammation and remodelling) were assessed at 4 and 8 weeks after implantation. Two large abdominal eventrations were cured by a human acellular matrix in human patients. A higher hernia recurrence rate was observed for rats transplanted with FLL than with FLT/Pro/Mers at 4 and 8 weeks after implantation. A lower adhesion rate was achieved for FLL/FLT than for Pro/Mers meshes (p<0.05). A decrease in immunologic cell infiltrations in FLL/FLT was observed between day 30 and day 60 (p<0.05). Collagen, elastin, and muscular tissues were found only in FLL/FLT matrix; a weaker muscular cell infiltration for FLL occurred at 8 weeks. Human abdominal eventrations were totally cured by using FLT as confirmed by computed tomography scanning at 12 and 16 months after implantation. In conclusion, human acellular collagen matrix, placed in an FLT position, can induce an abdominal wall reconstitution without adhesions and hernia recurrence.

Parameters favouring successful adult pig islet isolations for xenotransplantation in pig-to-primate models.

BACKGROUND: In the near future, adult porcine islets of Langerhans appear as an unlimited source of insulin-producing cells which could play a major role for treating diabetes mellitus. There is, however, an obvious lack of pre-clinical results and data in the pig-to-primate model. One of the main hurdles of this model is certainly related to the difficulty of reproducing regularly successful porcine islet isolation. This experimental work was designed to provide guidelines applicable in pig pancreas procurement and islet isolation for successful islet xenotransplantation into primates. METHODS: Pancreases were harvested from adult Belgium Landrace pigs (n = 79) in a single centre. The impact on islet yield of (1) pancreas procurement (blood exsanguination and warm ischaemia time (WIT)), (2) cold storage solutions (classic UW and modified UW (without hydroxyethyl starch and inverse K+/Na+ concentration)), (3) a dynamic or static method of pancreas digestion, and (4) the endotoxin content and enzymatic activity from five different batches of Liberase PI was studied. In addition, pancreatic biopsies (n = 18), performed before isolation, were retrospectively analyzed to study the impact of histomorphometry on porcine islet yield. Finally, two diabetic cynomolgus monkeys were transplanted without immunosuppression with 15,000 pig islet equivalents/kg body weight of recipient to assess in vivo the function of freshly isolated islets. Univariate and multivariate analyses were performed. RESULTS: By multiple linear regression, the most significant variables that significantly improved islet yield were, firstly, the presence of <30 EU (endotoxin units) of endotoxin in Liberase batches, followed by a WIT under 10 min and the use of blood exsanguination before pancreas harvesting (P < 0.005). In contrast, isolation method (dynamic vs. static) and the solution used for storage (short-term) (UW vs. modified UW) did not significantly influence islet yield. The correlation of retrospective histomorphometry analysis of native pancreas and extemporaneous biopsy before isolation clearly determined a positive relationship between isolated islet number and the number of islets/cm2 (r = 0.708, P < 0.01) or with the percentage of large islets (r = 0.680, P < 0.01) found in pancreas biopsies. Pig pancreases containing more than 82 islets/cm2 and more than 42% of large islets (>100 microm) thus enabled more than 120,000 islet equivalents to be obtained in 90% of the cases, which is an ideal amount of islets to transplant into a primate of 4 to 5 kg. In vivo, a reduction of blood glucose (<200 mg/dl), associated with porcine C-peptide production, was observed in two primates after transplantation with adult pig islets. At day 7 post-transplantation, however, loss of islet function was associated with graft destruction and immune reaction. CONCLUSIONS: Morphological screening of the pig pancreas before isolation, optimal blood exsanguination, WIT <10 min, and an endotoxin content <30 EU/mg in Liberase PI batches determine successful pig islet isolation for xenotransplantation in primates.

Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: proof of concept.

BACKGROUND: Pig islets xenotransplantation remains associated with a strong humoral and cellular xenogeneic immune responses. The aim of this study was to assess the long-term biocompatibility of alginate encapsulated pig islets after transplantation in primates. METHODS: Adult pig islets encapsulated in alginate under optimal conditions (n=7) or not (n=5) were transplanted under the kidney capsule of nondiabetic Cynomolgus maccacus. Additional primates received empty capsules (n=1) and nonencapsulated pig islets (n=2) as controls. Capsule integrity, cellular overgrowth, pig islet survival, porcine C-peptide and anti-pig IgM/IgG antibodies were examined up to 6 months after implantation. RESULTS: Nonencapsulated islets and islets encapsulated in nonoptimal capsules were rapidly destroyed. In seven primates receiving perfectly encapsulated pig islets, part of the islets survived up to 6 months after implantation without immunosuppression. Porcine C-peptide was detected after 1 month in 71% of the animals. The majority of grafts (86%) were intact and completely free of cellular overgrowth or capsule fibrosis. Explanted capsules, after 135 (n=2/2) and 180 (n=2/3) days, demonstrated residual insulin content and responses to glucose challenge (stimulation index of 2.2). Partial islet survival was obtained despite an elicited anti-pig IgG humoral response. CONCLUSIONS: Optimal alginate encapsulation significantly prolonged adult pig islet survival into primates for up to 6 months, even in the presence of antibody response.

The influence of implantation site on the biocompatibility and survival of alginate encapsulated pig islets in rats.

This work investigated the impact of implantation sites on the biocompatibility of alginate encapsulated pig islets. Non-diabetic rats were implanted with adult pig islets encapsulated in alginate either intraperitoneally (IP; n=25), subcutaneously (SC; n=37) or under the kidney capsule (KC; n=34). Capsule biocompatibility (retrieval rate, capsule diameter, degree of capsule broken and cellular overgrowth, CD68/CD3 staining) as well as islets viability and functionality were assessed until 30 days after transplantation. Implantation site did not significantly influence the biocompatibility of empty alginate capsules after transplantation (n=48). Most of the empty capsules (>90%) were retrieved after harvesting and were free of cellular overgrowth until day 30 post-transplantation. Three days after implantation, no significant difference for encapsulated pig islets was observed in terms of capsule biocompatibility and islet functionality in peritoneum, KC or subcutaneously. However, between days 5 and 30 after transplantation, explanted capsules from IP demonstrated a higher degree of broken capsules (>13%) and capsules with severe cellular overgrowth (>50%, CD68+ infiltration) than capsules removed from SC and KC (p<0.05). This was associated with a significant reduction of islet viability, insulin content and insulin secretion. In rats, the peritoneum site seems not appropriate for promoting the engraftment of encapsulated pig islets. Kidney subcapsular and subcutaneous spaces represent an interesting alternative.

Streptozotocin-induced diabetes in large animals (pigs/primates): role of GLUT2 transporter and beta-cell plasticity.

BACKGROUND: To induce irreversible diabetes in large animals, the efficiency of streptozotocin (STZ) was evaluated in pigs, primates and compared to the gold standard model in rats. METHODS: Low (50 mg/kg) and high (150 mg/kg) doses of STZ were tested. Hepatic/renal function, glucose metabolism (intravenous glucose tolerance tests, fasting blood glucose) and histomorphometry were evaluated prior to, 1, and 4 weeks after STZ treatment. RESULTS: In rats and primates, expressing a high level of GLUT2 expression on beta cells, a dose of 50 mg/kg STZ induced irreversible diabetes (due to the 97% destruction of beta cell mass) without provoking liver or renal failure. In pigs, despite the use of high STZ dose, partial correction of hyperglycaemia was observed four weeks after STZ injection (decreased fasting blood glucose and intravenous glucose tolerance tests; increased insulin production). The correction of hyperglycaemia was associated with significant hypertrophy of immature pig beta-cell clusters (+30%, P<0.05), whereas no hypertrophy was observed in rats/primates. CONCLUSION: These results demonstrated that STZ might be used to induce irreversible diabetes in rats and primates. In contrast, the low STZ sensitivity in pigs related to a low expression of GLUT2, higher number of immature beta cells and compensatory beta-cell hypertrophy, renders STZ-induced diabetes inappropriate for studying islet allografts in swine.

A simple method using a polymethylpenten chamber for isolation of human pancreatic islets.

OBJECTIVES: Isolation of large numbers of intact and functional human islets remains difficult and expensive. We describe a novel method using a polymethylpenten chamber (PMPC) and compare its efficacy to the classic method using a stainless steel chamber (SSC). METHODS: Five pancreases obtained from cadaveric donors were processed with the SSC method, and the islets were purified with a Cobe cell separator. The next 15 pancreases (similar donor characteristics) were distended with Liberase HI, minced, and digested in a PMPC whose thermic properties did not require continuous heating to maintain temperature of the prewarmed medium at 37 degrees C. The digestion was done in 2 phases to avoid damaging the first freed islets. Digested tissue was filtered on a column of 6-mm glass beads and 500-microm mesh screen, so that tissue volume was small enough to permit purification on discontinuous Ficoll gradients in tubes. RESULTS: With the PMPC method, the extent of digestion (+/-70%), yield (approximately 5000 IEQ/g), and final purity (73%) and viability (84%) of the islets was similar to those with the SSC, but the proportion of large islets (>150 microm in diameter) was higher. Cell composition (beta vs. non-beta cells) of isolated islets was not different from that of islets in situ in the same pancreas. Islet function, assessed by perifusion, showed an excellent average stimulation index of approximately 13-fold (15 vs. 1 mmol/L glucose, without cAMP-raising agent). CONCLUSIONS: This new method for isolation of human islets uses simple, low-cost, and potentially disposable material and requires a team of only 2 persons. The technique is as efficient as the classic SSC method and provides islets with excellent integrity and insulin-secreting capacity.