Human Microtumors Generated in 3D: Novel Tools for Integrated In Situ Studies of Cancer Immunotherapies.

Cellular immunotherapy targeting human tumor antigens is a promising strategy to treat solid tumors. Yet clinical results of cellular immunotherapy are disappointing. Moreover, the currently available in vitro human tumor models are not designed to study the optimization of T-cell therapies of solid tumors. Here, we describe a novel assay for multiparametric in situ analysis of therapeutic effects on individual human three-dimensional (3D) tumors. In this assay, tumors of several millimeter diameter are generated from human cancer cell lines of different tumor entities in a collagen type I microenvironment. A newly developed approach for efficient morphological analysis reveals that these in vitro tumors resemble many characteristics of the corresponding clinical cancers such as histological features, immunohistochemical staining patterns, distinct tumor growth compartments and heterogeneous protein expression. To assess the response to therapy with tumor antigen specific T-cells, standardized protocols are described to determine T-cell infiltration and tumor destruction by monitoring soluble factors and tumor growth. Human tumors engineered in 3D collagen scaffolds are excellent in vitro surrogates for avascular tumor stages allowing integrated analyses of the antitumor efficacy of cancer specific immunotherapy in situ.

AT1-receptor blockade, but not renin inhibition, reduces aneurysm growth and cardiac failure in fibulin-4 mice.

AIMS: Increasing evidence supports a role for the angiotensin II-AT1-receptor axis in aneurysm development. Here, we studied whether counteracting this axis via stimulation of AT2 receptors is beneficial. Such stimulation occurs naturally during AT1-receptor blockade with losartan, but not during renin inhibition with aliskiren. METHODS AND RESULTS: Aneurysmal homozygous fibulin-4 mice, displaying a four-fold reduced fibulin-4 expression, were treated with placebo, losartan, aliskiren, or the ß-blocker propranolol from day 35 to 100. Their phenotype includes cystic media degeneration, aortic regurgitation, left ventricular dilation, reduced ejection fraction, and fractional shortening. Although losartan and aliskiren reduced hemodynamic stress and increased renin similarly, only losartan increased survival. Propranolol had no effect. No drug rescued elastic fiber fragmentation in established aneurysms, although losartan did reduce aneurysm size. Losartan also increased ejection fraction, decreased LV diameter, and reduced cardiac pSmad2 signaling. None of these effects were seen with aliskiren or propranolol. Longitudinal micro-CT measurements, a novel method in which each mouse serves as its own control, revealed that losartan reduced LV growth more than aneurysm growth, presumably because the heart profits both from the local (cardiac) effects of losartan and its effects on aortic root remodeling. CONCLUSION: Losartan, but not aliskiren or propranolol, improved survival in fibulin-4 mice. This most likely relates to its capacity to improve structure and function of both aorta and heart. The absence of this effect during aliskiren treatment, despite a similar degree of blood pressure reduction and renin-angiotensin system blockade, suggests that it might be because of AT2-receptor stimulation.

Stratification based on methylation of TBX2 and TBX3 into three molecular grades predicts progression in patients with pTa-bladder cancer.

The potential risk of recurrence and progression in patients with non-muscle-invasive bladder cancer necessitates followup by cystoscopy. The risk of progression to muscle-invasive bladder cancer is estimated based on the European Organisation of Research and Treatment of Cancer score, a combination of several clinicopathological variables. However, pathological assessment is not objective and reproducibility is insufficient. The use of molecular markers could contribute to the estimation of tumor aggressiveness. We recently demonstrated that methylation of GATA2, TBX2, TBX3, and ZIC4 genes could predict progression in Ta tumors. In this study, we aimed to validate the markers in a large patient set using DNA from formalin-fixed and paraffin-embedded tissue. PALGA: the Dutch Pathology Registry was used for patient selection. We included 192 patients with pTaG1/2 bladder cancer of whom 77 experienced progression. Methylation analysis was performed and log-rank analysis was used to calculate the predictive value of each methylation marker for developing progression over time. This analysis showed better progression-free survival in patients with low methylation rates compared with the patients with high methylation rates for all markers (P<0.001) during a followup of ten-years. The combined predictive effect of the methylation markers was analyzed with the Cox-regression method. In this analysis, TBX2, TBX3, and ZIC4 were independent predictors of progression. On the basis of methylation status of TBX2 and TBX3, patients were divided into three new molecular grade groups. Survival analysis showed that only 8% of patients in the low molecular grade group progressed within 5 years. This was 29 and 63% for the intermediate- and high-molecular grade groups. In conclusion, this new molecular-grade based on the combination of TBX2 and TBX3 methylation is an excellent marker for predicting progression to muscle-invasive bladder cancer in patients with primary pTaG1/2 bladder cancer.

Extracellular matrix defects in aneurysmal Fibulin-4 mice predispose to lung emphysema.

BACKGROUND: In this study we set out to investigate the clinically observed relationship between chronic obstructive pulmonary disease (COPD) and aortic aneurysms. We tested the hypothesis that an inherited deficiency of connective tissue might play a role in the combined development of pulmonary emphysema and vascular disease. METHODS: We first determined the prevalence of chronic obstructive pulmonary disease in a clinical cohort of aortic aneurysms patients and arterial occlusive disease patients. Subsequently, we used a combined approach comprising pathological, functional, molecular imaging, immunological and gene expression analysis to reveal the sequence of events that culminates in pulmonary emphysema in aneurysmal Fibulin-4 deficient (Fibulin-4(R)) mice. RESULTS: Here we show that COPD is significantly more prevalent in aneurysm patients compared to arterial occlusive disease patients, independent of smoking, other clinical risk factors and inflammation. In addition, we demonstrate that aneurysmal Fibulin-4(R/R) mice display severe developmental lung emphysema, whereas Fibulin-4(+/R) mice acquire alveolar breakdown with age and upon infectious stress. This vicious circle is further exacerbated by the diminished antiprotease capacity of the lungs and ultimately results in the development of pulmonary emphysema. CONCLUSIONS: Our experimental data identify genetic susceptibility to extracellular matrix degradation and secondary inflammation as the common mechanisms in both COPD and aneurysm formation.

Telomerase reverse transcriptase promoter mutations in bladder cancer: high frequency across stages, detection in urine, and lack of association with outcome.

BACKGROUND: Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. OBJECTIVES: To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). DESIGN, SETTING, AND PARTICIPANTS: A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription-quantitative polymerase chain reaction. The two most frequent mutations were investigated, using a SNaPshot assay, in an independent set of 184 non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo and 21 mo, respectively). Voided urine from patients with suspicion of incident UBC (n=174), or under surveillance after diagnosis of non-muscle-invasive UBC (n=194), was tested using a SNaPshot assay. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease-specific survival, and overall survival. RESULTS AND LIMITATIONS: In the two series, 78 of 111 (70%) and 283 of 357 (79%) tumors harbored TERT mutations, C228T being the most frequent substitution (83% for both series). TERT mutations were not associated with clinical or pathologic parameters, but were more frequent among FGFR3 mutant tumors (p=0.0002). There was no association between TERT mutations and mRNA expression (p=0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC patients. The sensitivity was 62% in incident and 42% in recurrent UBC. A limitation of the study is its retrospective nature. CONCLUSIONS: Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may help to detect UBC recurrences.

SOCS2 mediates the cross talk between androgen and growth hormone signaling in prostate cancer.

UNLABELLED: Anabolic signals such as androgens and the growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis play an essential role in the normal development of the prostate but also in its malignant transformation. In this study, we investigated the role of suppressor of cytokine signaling 2 (SOCS2) as mediator of the cross talk between androgens and GH signals in the prostate and its potential role as tumor suppressor in prostate cancer (PCa). We observed that SOCS2 protein levels assayed by immunohistochemistry are elevated in hormone therapy-naive localized prostatic adenocarcinoma in comparison with benign tissue. In contrast, however, castration-resistant bone metastases exhibit reduced levels of SOCS2 in comparison with localized or hormone naive, untreated metastatic tumors. In PCa cells, SOCS2 expression is induced by androgens through a mechanism that requires signal transducer and activator of transcription 5 protein (STAT5) and androgen receptor-dependent transcription. Consequentially, SOCS2 inhibits GH activation of Janus kinase 2, Src and STAT5 as well as both cell invasion and cell proliferation in vitro. In vivo, SOCS2 limits proliferation and production of IGF-1 in the prostate in response to GH. Our results suggest that the use of GH-signaling inhibitors could be of value as a complementary treatment for castration-resistant PCa. SUMMARY: Androgen induced SOCS2 ubiquitin ligase expression and inhibited GH signaling as well as cell proliferation and invasion in PCa, whereas reduced SOCS2 was present in castration-resistant cases. GH-signaling inhibitors might be a complementary therapeutic option for advanced PCa.

Hypermethylation of the polycomb group target gene PCDH7 in bladder tumors from patients of all ages.

PURPOSE: Bladder tumors in patients younger than 20 years show a low incidence of the genetic and epigenetic aberrations typically found in older patients. One of the most common epigenetic aberrations in human malignancies is DNA hypermethylation. Polycomb group complexes have an important role during lineage choices in embryogenesis and their target genes are 12 times more likely to be methylated than nonpolycomb group target genes. We hypothesized that methylation of polycomb group target genes is an early event in urothelial carcinogenesis and thus might be observed in young patients. MATERIALS AND METHODS: We stratified 167 patients by age into 4 groups, including age less than 20 years in 14, 20 to 40 in 48, 40 to 60 in 47 and greater than 60 in 58. Five previously identified polycomb group target genes (MEIS1, ONECUT2, OTX1, PCDH7 and SOX21) were selected for methylation analysis. Methylation ratios were calculated by using the unmethylated and methylated signal. The outcome represented the fraction of methylated cells within one tumor. Genes with similar methylation ratios in all age groups were considered as potential bladder cancer initiating candidates. RESULTS: Three genes showed higher methylation ratios in tumors from older patients, including ONECUT2, SOX21 and OTX1 (each p <0.001). MEIS1 showed a similar methylation ratio in all groups but the median methylation ratio was low. PCDH7 showed a similar median methylation percent in all age categories, ie 54% at less than 20, 59% at 20 to 40, 59% at 40 to 60 and 67% at greater than 60 years (p = 0.1). CONCLUSIONS: Tumors from young patients showed less methylation for most markers. PCDH7 showed high methylation ratios in all age categories. Therefore, it could have an important role in early urothelial carcinogenesis.

The microtubule plus-end tracking protein CLASP2 is required for hematopoiesis and hematopoietic stem cell maintenance.

Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators of microtubule behavior has been studied mainly in cultured cells. We show here that absence of murine CLASP2 in vivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization of microtubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.

Age-related neuronal degeneration: complementary roles of nucleotide excision repair and transcription-coupled repair in preventing neuropathology.

Neuronal degeneration is a hallmark of many DNA repair syndromes. Yet, how DNA damage causes neuronal degeneration and whether defects in different repair systems affect the brain differently is largely unknown. Here, we performed a systematic detailed analysis of neurodegenerative changes in mouse models deficient in nucleotide excision repair (NER) and transcription-coupled repair (TCR), two partially overlapping DNA repair systems that remove helix-distorting and transcription-blocking lesions, respectively, and that are associated with the UV-sensitive syndromes xeroderma pigmentosum (XP) and Cockayne syndrome (CS). TCR-deficient Csa(-/-) and Csb(-/-) CS mice showed activated microglia cells surrounding oligodendrocytes in regions with myelinated axons throughout the nervous system. This white matter microglia activation was not observed in NER-deficient Xpa(-/-) and Xpc(-/-) XP mice, but also occurred in Xpd(XPCS) mice carrying a point mutation (G602D) in the Xpd gene that is associated with a combined XPCS disorder and causes a partial NER and TCR defect. The white matter abnormalities in TCR-deficient mice are compatible with focal dysmyelination in CS patients. Both TCR-deficient and NER-deficient mice showed no evidence for neuronal degeneration apart from p53 activation in sporadic (Csa(-/-), Csb(-/-)) or highly sporadic (Xpa(-/-), Xpc(-/-)) neurons and astrocytes. To examine to what extent overlap occurs between both repair systems, we generated TCR-deficient mice with selective inactivation of NER in postnatal neurons. These mice develop dramatic age-related cumulative neuronal loss indicating DNA damage substrate overlap and synergism between TCR and NER pathways in neurons, and they uncover the occurrence of spontaneous DNA injury that may trigger neuronal degeneration. We propose that, while Csa(-/-) and Csb(-/-) TCR-deficient mice represent powerful animal models to study the mechanisms underlying myelin abnormalities in CS, neuron-specific inactivation of NER in TCR-deficient mice represents a valuable model for the role of NER in neuronal maintenance and survival.

Impaired vascular contractility and aortic wall degeneration in fibulin-4 deficient mice: effect of angiotensin II type 1 (AT1) receptor blockade.

Medial degeneration is a key feature of aneurysm disease and aortic dissection. In a murine aneurysm model we investigated the structural and functional characteristics of aortic wall degeneration in adult fibulin-4 deficient mice and the potential therapeutic role of the angiotensin (Ang) II type 1 (AT(1)) receptor antagonist losartan in preventing aortic media degeneration. Adult mice with 2-fold (heterozygous Fibulin-4(+/R)) and 4-fold (homozygous Fibulin-4(R/R)) reduced expression of fibulin-4 displayed the histological features of cystic media degeneration as found in patients with aneurysm or dissection, including elastin fiber fragmentation, loss of smooth muscle cells, and deposition of ground substance in the extracellular matrix of the aortic media. The aortic contractile capacity, determined by isometric force measurements, was diminished, and was associated with dysregulation of contractile genes as shown by aortic transcriptome analysis. These structural and functional alterations were accompanied by upregulation of TGF-ß signaling in aortas from fibulin-4 deficient mice, as identified by genome-scaled network analysis as well as by immunohistochemical staining for phosphorylated Smad2, an intracellular mediator of TGF-ß. Tissue levels of Ang II, a regulator of TGF-ß signaling, were increased. Prenatal treatment with the AT(1) receptor antagonist losartan, which blunts TGF-ß signaling, prevented elastic fiber fragmentation in the aortic media of newborn Fibulin-4(R/R) mice. Postnatal losartan treatment reduced haemodynamic stress and improved lifespan of homozygous knockdown fibulin-4 animals, but did not affect aortic vessel wall structure. In conclusion, the AT(1) receptor blocker losartan can prevent aortic media degeneration in a non-Marfan syndrome aneurysm mouse model. In established aortic aneurysms, losartan does not affect aortic architecture, but does improve survival. These findings may extend the potential therapeutic application of inhibitors of the renin-angiotensin system to the preventive treatment of aneurysm disease.

Nephrotoxicity in mice after repeated imaging using 111In-labeled peptides.

UNLABELLED: We determined the renal radiation dose of a series of (111)In-labeled peptides using animal SPECT. Because the animals' health deteriorated, renal toxicity was assessed. METHODS: Wild-type and megalin-deficient mice were imaged repeatedly at 3- to 6-wk intervals to quantify renal retention after injection of 40-50 MBq of (111)In-diethylenetriaminepentaacetic acid-labeled peptides (octreotide, exendin, octreotate, neurotensin, and minigastrin analogs), and the absorbed kidney radiation doses were estimated. Body weight, renal function parameters, and renal histology were determined at 16-20 wk after the first scan and compared with those in naive animals. RESULTS: Because of high renal retention, (111)In-diethylenetriaminepentaacetic acid-exendin-4 scans resulted in a 70-Gy kidney radiation dose in wild-type mice. Megalin-deficient kidneys received 20-40 Gy. The other peptides resulted in much lower renal doses. Kidney function monitoring indicated renal damage in imaged animals. CONCLUSION: Micro-SPECT enables longitudinal studies in 1 animal. However, long-term nephrotoxic effects may be induced after high renal radiation doses, even with (111)In-labeled radiotracers.

In-depth investigation of the molecular pathogenesis of bladder cancer in a unique 26-year old patient with extensive multifocal disease: a case report.

BACKGROUND: The molecular characteristics and the clinical disease course of bladder cancer (BC) in young patients remain largely unresolved. All patients are monitored according to an intensive surveillance protocol and we aim to gain more insight into the molecular pathways of bladder tumors in young patients that could ultimately contribute to patient stratification, improve patient quality of life and reduce associated costs. We also determined whether a biomarker-based surveillance could be feasible. CASE PRESENTATION: We report a unique case of a 26-year-old Caucasian male with recurrent non-muscle invasive bladder tumors occurring at a high frequency and analyzed multiple tumors (maximal pTaG2) and urine samples of this patient. Analysis included FGFR3 mutation detection, FGFR3 and TP53 immunohistochemistry, mircosatellite analysis of markers on chromosomes 8, 9, 10, 11 and 17 and a genome wide single nucleotide polymorphism-array (SNP). All analyzed tumors contained a mutation in FGFR3 and were associated with FGFR3 overexpression. None of the tumors showed overexpression of TP53. We found a deletion on chromosome 9 in the primary tumor and this was confirmed by the SNP-array that showed regions of loss on chromosome 9. Detection of all recurrences was possible by urinary FGFR3 mutation analysis. CONCLUSIONS: Our findings would suggest that the BC disease course is determined by not only a patient's age, but also by the molecular characteristics of a tumor. This young patient contained typical genetic changes found in tumors of older patients and implies a clinical disease course comparable to older patients. We demonstrate that FGFR3 mutation analysis on voided urine is a simple non-invasive method and could serve as a feasible follow-up approach for this young patient presenting with an FGFR3 mutant tumor.

Stimulated neovascularization, inflammation resolution and collagen maturation in healing rat cutaneous wounds by a heparan sulfate glycosaminoglycan mimetic, OTR4120.

Heparan sulfate glycosaminoglycans (HS-GAGs) are not only the structural elements of tissue architecture but also regulate the bioavailability and transduction pathways of heparan sulfate-bound polypeptides released by cells or the extracellular matrix. Heparan sulfate-bound polypeptides include inflammatory mediators, chemokines, angiogenic factors, morphogens, and growth-promoting factors that induce cell migration, proliferation, and differentiation in wound healing. OTR4120, a polymer engineered to mimic the properties of HS-GAGs, is used to replace the natural HS-GAGs that are degraded during wound repair, and enhance the tissue regeneration by preserving the cellular microenvironment and the endogenous signals needed for tissue regeneration. We previously demonstrated that OTR4120 treatment had a long-term effect on increasing breaking strength and vasodilation in healing rat full-thickness excisional wounds. The present study investigates the underlying mechanisms of the effects of OTR4120 treatment in improving the quality of cutaneous wound repair. We found that OTR4120 treatment stimulated inflammation resolution and increased neovascularization. OTR4120 treatment also promoted epidermal migration and proliferation during reepithelialization. Moreover, the granulation tissue formation and collagen maturation were improved in OTR4120-treated wounds. Three months after wounding, the effects of OTR4120 treatment on vascularization and inflammation resolution were normalized, except for an improved neodermis. We conclude that OTR4120 is a potential matrix therapeutic agent that ensures the quality of normal cutaneous wound repair and may restore impaired wound healing characterized by deficient angiogenesis and prolonged inflammation.

Changes in the penile arteries of the rat after fractionated irradiation of the prostate: a pilot study.

INTRODUCTION: External beam radiotherapy for prostate cancer leads to erectile dysfunction in 36%-43% of patients. The underlying mechanism is largely unknown, although some clinical studies suggest that the arterial supply to the corpora cavernosa is responsible. Two animal experimental studies reported on the effects of a single fraction of prostate irradiation on the penile structures. However, irradiation in multiple fractions is more representative of the actual clinical treatment. AIM: The present prospective, controlled study was initiated to investigate the effect of fractionated prostate irradiation on the arteries of the corpora cavernosa. MAIN OUTCOME MEASURES: Histological evaluation of the penile tissue in comparison with control rats at 2, 4, and 9 weeks after irradiation. METHODS: The prostate of twelve rats was treated with external beam radiation in 5 daily fractions of 7.4 gray. Three control rats were treated with sham irradiation. Prostatic and penile tissue was evaluated for general histology (hematoxylin-eosin). The penile tissue was further evaluated after combined staining for collagen (resorcin fuchsin) and alpha-smooth muscle actin (SMA) (Biogenex). RESULTS: The prostate showed adequate irradiation with fibrosis occurring at 9 weeks after irradiation. The corpora cavernosa showed arteries that had developed loss of smooth muscle cells expressing SMA, thickening of the intima, and occlusions. All the control rats maintained normal anatomy. CONCLUSION: This is the first animal experimental study that demonstrates changes in the arteries of the corpora cavernosa after fractionated irradiation to the prostatic area. The preliminary data suggests that erectile dysfunction after radiotherapy might be caused by radiation damage to the arterial supply of the corpora cavernosa.

The development of multiple bladder tumour recurrences in relation to the FGFR3 mutation status of the primary tumour.

Non-muscle invasive bladder cancers (NMI-BCs) represent 75% of bladder cancers upon presentation. After removal of the primary tumour by transurethral resection, multiple recurrences continue to develop in 70% of patients. Consequently, prolonged and costly surveillance by cystoscopy is required. Mutations in the FGFR3 oncogene are common in NMI-BCs and are associated with a lower chance of progression to muscle-invasive disease. Here we analysed the consistency of FGFR3 mutations in primary and recurrent tumours. This knowledge is of crucial importance if FGFR3 mutation analysis on urinary cells is to be used as an alternative for cystoscopical surveillance. To this end, we monitored the disease process and FGFR3 mutation status of primary and recurrent tumours in 118 patients with NMI-BC. During median follow-up of 8.8 years, these patients underwent 2133 cystoscopies and 80 patients developed 414 recurrences. FGFR3 mutations were equally prevalent in primary and recurrent tumours (63%). Patients can have different types of FGFR3 mutations in different tumours. Recurrence risk was not significantly different for patients with a mutant or wild-type primary tumour. Recurrence rates varied widely between patients but were constant for a patient and were unrelated to FGFR3 status. In the mutant patient group, in contrast to the wild-type group, recurrences continued to develop after 10 years. In 81% of the recurrences of patients with a mutant primary tumour, a mutation was found. Moreover, recurrences in this patient group were of lower stage and grade than those of patients with a wild-type primary tumour (p < 0.001). These results suggest that surveillance by FGFR3 mutation analysis on voided urine in combination with a reduced cystoscopy frequency of patients presenting with an FGFR3 mutant tumour is worth investigating.

Targeting a single mismatched minor histocompatibility antigen with tumor-restricted expression eradicates human solid tumors.

Regressions of metastatic solid tumors after allogeneic human leukocyte antigen (HLA)-matched stem cell transplantation (SCT) are often associated with detrimental graft-versus-host disease (GVHD). The graft-versus-host reaction of the HLA-matched donor is directed mainly against the multiple mismatched minor histocompatibility antigens (mHags) of the patient. mHags are strong HLA-restricted alloantigens with differential tissue distribution. Ubiquitously expressed mHags are the prime in situ targets of GVHD. The mHag HA-1 is hematopoiesis restricted, but displays additionally an aberrant expression on solid tumors. Thus, HA-1 might be an excellent target to boost the anti-solid tumor effect of allogeneic SCT without inducing severe GVHD. Here, we show that cytotoxic T lymphocytes (CTLs) solely targeting the human mHag HA-1 are capable of eradicating 3-dimensional human solid tumors in a highly mHag-specific manner in vitro, accompanied by interferon-gamma release. In vivo, HA-1-specific CTLs distribute systemically and prevent human breast cancer metastases in immunodeficient mice. Moreover, HA-1-specific CTLs infiltrate and inhibit the progression of fully established metastases. Our study provides the first proof for the efficacy of a clinically applicable concept to exploit single mismatched mHags with hematopoiesis- and solid tumor-restricted expression for boosting the anti-solid tumor effect of allogeneic SCT.

Sox2 is important for two crucial processes in lung development: branching morphogenesis and epithelial cell differentiation.

The primary lung bud originates from the foregut and develops into the bronchial tree by repetitive branching and outgrowing of the airway. The Sry related HMG box protein Sox2 is expressed in a cyclic manner during initiation and branching morphogenesis of the lung. It is highly expressed in non-branching regions and absent from branching regions, suggesting that downregulation of Sox2 is mandatory for airway epithelium to respond to branch inducing signals. Therefore, we developed transgenic mice that express a doxycycline inducible Sox2 in the airway epithelium. Continuous expression of Sox2 hampers the branching process resulting in a severe reduction of the number of airways. In addition, the bronchioli transiently go over into enlarged, alveolar-like airspaces, a pathology described as bronchiolization of alveoli. Furthermore, a substantial increase was observed of cGRP positive neuroendocrine cells and Delta Np63 isoform expressing (pre-) basal cells, which are both committed precursor-like cells. Thus, Sox2 prevents airways from branching and prematurely drives cells into committed progenitors, apparently rendering these committed progenitors unresponsive to branch inducing signals. However, Sox2 overexpression does not lead to a complete abrogation of the epithelial differentiation program.

RGTA OTR 4120, a heparan sulfate proteoglycan mimetic, increases wound breaking strength and vasodilatory capability in healing rat full-thickness excisional wounds.

ReGeneraTing Agents (RGTAs), a family of polymers engineered to protect and stabilize heparin-binding growth factors, have been shown to promote tissue repair and regeneration. In this study, the effects of one of these polymers, RGTA OTR4120, on healing of full-thickness excisional wounds in rats were investigated. Two 1.5 cm diameter circular full-thickness excisional wounds were created on the dorsum of a rat. After creation of the wounds, RGTA OTR4120 was applied. The progress of healing was assessed quantitatively by evaluating the wound closure rate, vasodilatory capability, and wound breaking strength. The results showed a triple increase of the local vascular response to heat provocation in the RGTA OTR4120-treated wounds as compared with vehicle-treated wounds. On days 14 and 79 after surgery, the wounds treated with RGTA OTR4120 gained skin strength 12% and 48% of the unwounded skin, respectively, and displayed a significantly increased gain in skin strength when compared with control animals. These results raise the possibility of efficacy of RGTA OTR4120 in accelerating surgically cutaneous wound healing by enhancing the wound breaking strength and improving the microcirculation.

Long-term toxicity of [(177)Lu-DOTA (0),Tyr (3)]octreotate in rats.

PURPOSE AND METHODS: Studies on peptide receptor radionuclide therapy (PRRT) using radiolabelled somatostatin analogues have shown promising results with regard to tumour control. The efficacy of PRRT is limited by uptake and retention in the proximal tubules of the kidney, which might lead to radiation nephropathy. We investigated the long-term renal toxicity after different doses of [(177)Lu-DOTA(0),Tyr(3)]octreotate and the effects of dose fractionation and lysine co-injection in two tumour-bearing rat models. RESULTS: Significant renal toxicity was detected beyond 100 days after start of treatment as shown by elevated serum creatinine and proteinuria. Microscopically, tubules were strongly dilated with flat epithelium, containing protein cylinders. Creatinine levels rose significantly after 555 MBq [(177)Lu-DOTA(0),Tyr(3)]octreotate, but were significantly lower after 278 MBq (single injection) or two weekly doses of 278 MBq. Renal damage scores were maximal after 555 MBq and significantly lower in the 278 and 2x278 MBq groups. Three doses of 185 MBq [(177)Lu-DOTA(0),Tyr(3)]octreotate with intervals of a day, a week or a month significantly influenced serum creatinine (469+/-18, 134+/-70 and 65+/-15 micromol/l, respectively; p<0.001). Renal histological damage scores were not significantly influenced by dose fractionation. Lysine co-administration with three weekly treatments of 185 MBq significantly lowered serum creatinine and proteinuria. CONCLUSION: Injection of high doses of [(177)Lu-DOTA(0),Tyr(3)]octreotate resulted in severe renal damage in rats as indicated by proteinuria, elevated serum creatinine and histological damage. This damage was dose dependent and became overt between 100 and 200 days after treatment. Dose fractionation had significant beneficial effects on kidney function. Also, lysine co-injection successfully prevented functional damage.

Amifostine protects rat kidneys during peptide receptor radionuclide therapy with [177Lu-DOTA0,Tyr3]octreotate.

PURPOSE: In peptide receptor radionuclide therapy (PRRT) using radiolabelled somatostatin analogues, the kidneys are the major dose-limiting organs, because of tubular reabsorption and retention of radioactivity. Preventing renal uptake or toxicity will allow for higher tumour radiation doses. We tested the cytoprotective drug amifostine, which selectively protects healthy tissue during chemo- and radiotherapy, for its renoprotective capacities after PRRT with high-dose [(177)Lu-DOTA(0),Tyr(3)]octreotate. METHODS: Male Lewis rats were injected with 278 or 555 MBq [(177)Lu-DOTA(0),Tyr(3)]octreotate to create renal damage and were followed up for 130 days. For renoprotection, rats received either amifostine or co-injection with lysine. Kidneys, blood and urine were collected for toxicity measurements. At 130 days after PRRT, a single-photon emission computed tomography (SPECT) scan was performed to quantify tubular uptake of (99m)Tc-dimercaptosuccinic acid (DMSA), a measure of tubular function. RESULTS: Treatment with 555 MBq [(177)Lu-DOTA(0),Tyr(3)]octreotate resulted in body weight loss, elevated creatinine and proteinuria. Amifostine and lysine treatment significantly prevented this rise in creatinine and the level of proteinuria, but did not improve the histological damage. In contrast, after 278 MBq [(177)Lu-DOTA(0),Tyr(3)]octreotate, creatinine values were slightly, but not significantly, elevated compared with the control rats. Proteinuria and histological damage were different from controls and were significantly improved by amifostine treatment. Quantification of (99m)Tc-DMSA SPECT scintigrams at 130 days after [(177)Lu-DOTA(0),Tyr(3)]octreotate therapy correlated well with 1/creatinine (r(2)=0.772, p<0.001). CONCLUSION: Amifostine and lysine effectively decreased functional renal damage caused by high-dose [(177)Lu-DOTA(0),Tyr(3)]octreotate. Besides lysine, amifostine might be used in clinical PRRT as well as to maximise anti-tumour efficacy.