A pan-cancer organoid platform for precision medicine.

Patient-derived tumor organoids (TOs) are emerging as high-fidelity models to study cancer biology and develop novel precision medicine therapeutics. However, utilizing TOs for systems-biology-based approaches has been limited by a lack of scalable and reproducible methods to develop and profile these models. We describe a robust pan-cancer TO platform with chemically defined media optimized on cultures acquired from over 1,000 patients. Crucially, we demonstrate tumor genetic and transcriptomic concordance utilizing this approach and further optimize defined minimal media for organoid initiation and propagation. Additionally, we demonstrate a neural-network-based high-throughput approach for label-free, light-microscopy-based drug assays capable of predicting patient-specific heterogeneity in drug responses with applicability across solid cancers. The pan-cancer platform, molecular data, and neural-network-based drug assay serve as resources to accelerate the broad implementation of organoid models in precision medicine research and personalized therapeutic profiling programs.

Allele-specific open chromatin in human iPSC neurons elucidates functional disease variants.

Most neuropsychiatric disease risk variants are in noncoding sequences and lack functional interpretation. Because regulatory sequences often reside in open chromatin, we reasoned that neuropsychiatric disease risk variants may affect chromatin accessibility during neurodevelopment. Using human induced pluripotent stem cell (iPSC)-derived neurons that model developing brains, we identified thousands of genetic variants exhibiting allele-specific open chromatin (ASoC). These neuronal ASoCs were partially driven by altered transcription factor binding, overrepresented in brain gene enhancers and expression quantitative trait loci, and frequently associated with distal genes through chromatin contacts. ASoCs were enriched for genetic variants associated with brain disorders, enabling identification of functional schizophrenia risk variants and their cis-target genes. This study highlights ASoC as a functional mechanism of noncoding neuropsychiatric risk variants, providing a powerful framework for identifying disease causal variants and genes.

Organizing a COVID-19 triage unit: a Swiss perspective.

Background: With the rapid global spread of the acute respiratory syndrome coronavirus 2, urgent health-care measures have been implemented. We describe the organizational process in setting up a coronavirus disease 2019 triage unit in a Swiss tertiary care hospital. Methods: Our triage unit was set-up outside of the main hospital building and consists of three areas: 1. Pre-triage, 2. Triage, and 3. Triage plus. The Pre-triage check-points identify any potential COVID-19-infected patients and re-direct them to the main Triage area where trained medical staff screen which patients undergo diagnostic testing. If testing is indicated, nasopharyngeal swabs are performed. If patients require further investigations, they are referred to Triage plus. At this stage, patients are then discharged home after additional testing or admitted to the hospital for management. Observations: A total of 1265 patients were screened between 10 March 2020 and 12 April 2020 at our Triage unit. Of these, 112 (8.9%) tested positive. 73 (65%) of the positively-tested patients were female and 39 (35%) were male. The mean age for all patients was 43.8 years (SD 16.3 years). Distinguishing between genders, mean age for females was 41.1 (SD 16.5) and mean age for males was 48.6 (SD 14.9), with females being significantly younger than males (p < 0.001). Conclusion: Our triage unit was set-up as part of a large-scale restructuring process. Current challenges include low sensitivity for test results as well as limited staff and resources. We hope that our experience will help other health care institutions develop similar triage systems.

The Genetic Relevance of Human Induced Pluripotent Stem Cell-Derived Microglia to Alzheimer's Disease and Major Neuropsychiatric Disorders.

Microglia are the primary innate immune cell type in the brain that have been implicated in the pathogenesis of several neurodegenerative and neuropsychiatric disorders, most notably Alzheimer's disease (AD) and schizophrenia. Microglia generated from human induced pluripotent stem cells (hiPSCs) represent a promising in vitro cellular model for studying the neuroimmune interactions involved in these disorders. Among several methods of generating -hiPSC-derived microglia (iMG) - varying in duration and resultant purity - a recent protocol by Brownjohn et al. [Stem Cell Reports. 2018 Apr;10(4):1294-307] is particularly simple and efficient. However, the replicability of this method, transcriptomic similarity of these iMG to primary adult microglia, and their genetic relevance to disease (i.e., enrichment of disease risk loci in genes preferentially expressed in these cells) remains unclear. Using two hiPSC lines, we demonstrated that Brownjohn's protocol can rapidly generate iMG that morphologically and functionally resembled microglia. The iMG cells we generated were found to be transcriptionally similar to previously reported iMG, as well as fetal and adult microglia. Furthermore, by using cell type-specific gene expression to partition disease heritability, we showed that iMG cells are genetically relevant to AD but found no significant enrichments of risk loci of Parkinson's disease, schizophrenia, major depressive disorder, bipolar disorder, autism spectrum disorder, or body mass index. Across a range of neuronal and immune cell types, we found only iMG, primary microglia, and microglia-like cell types exhibited a significant enrichment for AD heritability. Our results thus support the use of iMG as a human cellular model for understanding AD biology and underlying genetic factors, as well as for developing and efficiently screening new therapeutics.

Safety and activity of ibrutinib in combination with nivolumab in patients with relapsed non-Hodgkin lymphoma or chronic lymphocytic leukaemia: a phase 1/2a study.

BACKGROUND: Preclinical studies have shown synergistic antitumour effects between ibrutinib and immune-checkpoint blockade. The aim of this study was to assess the safety and activity of ibrutinib in combination with nivolumab in patients with relapsed or refractory B-cell malignant diseases. METHODS: We did a two-part, open-label, phase 1/2a study at 21 hospitals in Australia, Israel, Poland, Spain, Turkey, and the USA. The primary objective of part A (dose escalation) was to assess the safety of daily oral ibrutinib (420 mg or 560 mg) in combination with intravenous nivolumab (3 mg/kg every 2 weeks) to ascertain a recommended phase 2 dose in patients with relapsed or refractory high-risk chronic lymphocytic leukaemia or small lymphocytic lymphoma (del17p or del11q), follicular lymphoma, or diffuse large B-cell lymphoma. Dose optimisation was investigated using a modified toxicity probability interval design. The primary objective of the part B expansion phase was to establish the preliminary activity (the proportion of patients who achieved an overall response) of the combination of ibrutinib and nivolumab in four cohorts: relapsed or refractory high-risk chronic lymphocytic leukaemia or small lymphocytic lymphoma (del17p or del11q), follicular lymphoma, diffuse large B-cell lymphoma, and Richter's transformation. All participants who received at least one dose of treatment were included in the primary analysis and analyses were done by disease cohort. This trial is registered with ClinicalTrials.gov, number NCT02329847. The trial is ongoing. FINDINGS: Between March 12, 2015, and April 11, 2017, 144 patients were enrolled in the study. Three patients died before receiving study treatment; thus, 141 patients were included in the analysis, 14 in part A and 127 in part B. One dose-limiting toxicity (grade 3 hyperbilirubinaemia) was reported at the 420 mg dose in the diffuse large B-cell lymphoma cohort, which resolved after 5 days. The combination of ibrutinib and nivolumab led to overall responses in 22 (61%) of 36 patients with high-risk chronic lymphocytic leukaemia or small lymphocytic lymphoma, 13 (33%) of 40 patients with follicular lymphoma, 16 (36%) of 45 patients with diffuse large B-cell lymphoma, and 13 (65%) of 20 patients with Richter's transformation. The most common all-grade adverse events were diarrhoea (47 [33%] of 141 patients), neutropenia (44 [31%]), and fatigue (37 [26%]). 11 (8%) of 141 patients had adverse events leading to death; none were reported as drug-related. The most common grade 3-4 adverse events were neutropenia (40 [28%] of 141 patients) and anaemia (32 [23%]). The incidence of grade 3-4 neutropenia ranged from eight (18%) of 45 patients with diffuse large B-cell lymphoma to 19 (53%) of 36 patients with chronic lymphocytic leukaemia or small lymphocytic lymphoma; incidence of grade 3-4 anaemia ranged from five (13%) of 40 patients with follicular lymphoma to seven (35%) of 20 patients with Richter's transformation. The most common serious adverse events included anaemia (six [4%] of 141 patients) and pneumonia (five [4%]). The most common grade 3-4 immune-related adverse events were rash (11 [8%] of 141 patients) and increased alanine aminotransferase (three [2%]). INTERPRETATION: The combination of ibrutinib and nivolumab had an acceptable safety profile and preliminary activity was similar to that reported with single-agent ibrutinib in chronic lymphocytic leukaemia or small lymphocytic lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma. The clinical response in patients with Richter's transformation was promising and supports further clinical assessment. FUNDING: Janssen R&D.

Thrombospondin-2 overexpression in the skin of transgenic mice reduces the susceptibility to chemically induced multistep skin carcinogenesis.

BACKGROUND: We have previously reported stromal upregulation of the endogenous angiogenesis inhibitor thrombospondin-2 (TSP-2) during multistep carcinogenesis, and we found accelerated and enhanced skin angiogenesis and carcinogenesis in TSP-2 deficient mice. GOALS: To investigate whether enhanced levels of TSP-2 might protect from skin cancer development. METHODS: We established transgenic mice with targeted overexpression of TSP-2 in the skin and subjected hemizygous TSP-2 transgenic mice and their wild-type littermates to a chemical skin carcinogenesis regimen. RESULTS: TSP-2 transgenic mice showed a significantly delayed onset of tumor formation compared to wild-type mice, whereas the ratio of malignant conversion to squamous cell carcinomas was comparable in both genotypes. Computer-assisted morphometric analysis of blood vessels revealed pronounced tumor angiogenesis already in the early stages of carcinogenesis in wild type mice. TSP-2 overexpression significantly reduced tumor blood vessel density in transgenic mice but had no overt effect on LYVE-1 positive lymphatic vessels. The percentage of desmin surrounded, mature tumor-associated blood vessels and the degree of epithelial differentiation remained unaffected. The antiangiogenic effect of transgenic TSP-2 was accompanied by a significantly increased number of apoptotic tumor cells in transgenic mice. CONCLUSION: Our results demonstrate that enhanced levels of TSP-2 in the skin result in reduced susceptibility to chemically-induced skin carcinogenesis and identify TSP-2 as a new target for the prevention of skin cancer.

Dabrafenib in patients with Val600Glu or Val600Lys BRAF-mutant melanoma metastatic to the brain (BREAK-MB): a multicentre, open-label, phase 2 trial.

BACKGROUND: Brain metastases are common in patients with metastatic melanoma and median overall survival from their diagnosis is typically 17-22 weeks. We assessed dabrafenib in patients with Val600Glu or Val600Lys BRAF-mutant melanoma metastatic to the brain. METHODS: We undertook a multicentre, open-label, phase 2 trial in 24 centres in six countries. We enrolled patients with histologically confirmed Val600Glu or Val600Lys BRAF-mutant melanoma and at least one asymptomatic brain metastasis (≥5 mm and ≤40 mm in diameter). Eligible patients were aged 18 years or older, had an Eastern Cooperative Oncology Group performance status of 0 or 1, and had adequate organ function. Patients were split into two cohorts: those in cohort A had not received previous local treatment for brain metastases and those in cohort B had progressive brain metastases after previous local treatments. Patients received 150 mg oral dabrafenib twice a day until disease progression, death, or unacceptable adverse events. The primary endpoint was the proportion of patients with Val600Glu BRAF-mutant melanoma who achieved an overall intracranial response, which was defined as a complete response or partial response assessed with a modified form of Response Evaluation Criteria in Solid Tumors (RECIST 1.1). We included patients who received at least one dose of dabrafenib in efficacy and safety analyses. This study is registered with ClinicalTrials.gov, number NCT01266967. FINDINGS: Between Feb 2, 2011, and Aug 5, 2011, we enrolled 172 patients: 89 (52%) in cohort A and 83 (48%) in cohort B. 139 (81%) had Val600Glu BRAF-mutant melanoma. 29 (39·2%, 95% CI 28·0-51·2) of 74 patients with Val600Glu BRAF-mutant melanoma in cohort A achieved an overall intracranial response, as did 20 (30·8%, 19·9-43·4) of 65 in cohort B. One (6·7%, 0·2-31·9) of 15 patients with Val600Lys BRAF-mutant melanoma achieved an overall intracranial response in cohort A, as did four (22·2%, 6·4-47·6) of 18 such patients in cohort B. Treatment-related adverse events of grade 3 or worse occurred in 38 (22%) patients. Eleven (6%) patients developed squamous-cell carcinoma (five [6%] patients in cohort A, of whom one also had keratoacanthoma; six [7%] in cohort B). Four grade 4 treatment-related adverse events occurred in cohort A: one blood amylase increase, one convulsion, one lipase increase, and one neutropenia. Two grade 4 events occurred in cohort B: one agranulocytosis and one intracranial haemorrhage. 51 (30%) patients had a serious adverse event. The three most frequent serious adverse events were pyrexia (ten [6%] patients), intracranial haemorrhage (ten [6%]; one treatment-related), and squamous-cell carcinoma (11 [6%]). INTERPRETATION: Dabrafenib has activity and an acceptable safety profile in patients with Val600Glu BRAF-mutant melanoma and brain metastases irrespective of whether they are untreated or have been previously treated but have progressed. FUNDING: GlaxoSmithKline.

The impact of infections on critically ill acute heart failure patients: an observational study.

BACKGROUND: Hospitalised patients with acute heart failure (AHF) suffer from a high morbidity and mortality, which might, at least partly, be influenced by concomitant infections. The aim of this observational study was to investigate the impact of infections on the clinical course of critically ill patients with AHF, both present on intensive care unit (ICU) admission and acquired during the ICU stay. METHODS: From 178 consecutive AHF patients, 76 were treated medically and 21 required emergency cardiac surgery. The remaining 81 patients, who underwent elective cardiac surgery, were excluded from the assessment of infections on ICU admission, but were included in the analysis of nosocomial infections during the ICU stay. RESULTS: A total of 16% of patients (16/97) had infections on ICU admission. These patients had longer ICU (6 vs. 3 days, p = 0.04) and hospital (19 vs. 11 days, p = 0.04) stays than patients without infections. Although not statistically significant, there was a trend for increased mortality at 30 days (44% vs. 24%, p = 0.13) and 6 months (57% vs. 31%, p = 0.13) in AHF patients with infections on ICU admission. Infection complications during the ICU stay occurred in 17% (30/178) of AHF patients and significantly increased their mortality at 30 days (33% vs. 14%, p = 0.02) and 6 months (41% vs. 18%, p = 0.02). CONCLUSIONS: In this observational study, infections present on ICU admission or occurring during the ICU stay had a negative impact on the morbidity and mortality of critically ill patients with AHF. Future studies are needed to gain a better understanding of the interactions between heart failure and infections, as a better knowledge of this field may have an important therapeutic potential.

Successful pregnancy in pulmonary arterial hypertension associated with systemic lupus erythematosus: a case report.

INTRODUCTION: Pulmonary arterial hypertension is a complication of systemic lupus erythematosus. Mortality in pregnant patients with pulmonary arterial hypertension related to connective tissue disease is as high as 56%. The authors report the first case of a successful maternal-fetal outcome in a pregnant patient with systemic lupus erythematosus-associated pulmonary arterial hypertension treated with sildenafil and inhaled iloprost during pregnancy and until several weeks after caesarean section. CASE PRESENTATION: The case presented is of a 29-year-old woman with systemic lupus erythematosus and associated severe pulmonary arterial hypertension. Vasodilator therapy with bosentan and sildenafil, immunosuppressive therapy with prednisone, hydroxychloroquine and azathioprine and oral anticoagulation (phenprocoumon) had normalized her right ventricular over right atrial pressure when she was diagnosed in her 5th week of pregnancy. The teratogenic drugs bosentan and phenprocoumon were stopped, the latter replaced by low molecular weight heparin. During the 35th week, a slight increase in pulmonary pressure was found. Therapy with inhaled iloprost was established. A caesarean section was performed in the 37th week and a healthy baby was delivered. The patient remained stable until 11 weeks after delivery, when an increase in right ventricular over right atrial pressure was noted. Bosentan was reintroduced and prednisone and azathioprine doses were increased. The patient has remained stable until the present time. CONCLUSION: Pulmonary arterial hypertension has been considered a contraindication for pregnancy. Novel vasodilator therapy, combined with immunosuppressants in this patient with systemic lupus erythematosus, may "cure" pulmonary arterial hypertension and permit pregnancy with successful outcome. However, postpartum exacerbation of systemic lupus erythematosus and pulmonary arterial hypertension have to be considered.

Presentation and outcome of critically ill medical and cardiac-surgery patients with acute heart failure.

BACKGROUND: The aim of the study was to investigate presentation and outcome of consecutive acute heart failure (AHF) patients admitted to the intensive care unit (ICU) including also patients undergoing cardiac surgery, thereby providing comparative information on all critically ill AHF subgroups. METHODS: The prospective observational study with 6-month follow up was performed in the cardio-thoracic and the medical ICU of a university hospital. AHF was defined according to the European Society of Cardiology guidelines. Univariate Cox regression was used to calculate hazard ratio (HR) and 95% confidence intervals (CI) for risk factors. RESULTS: A total of 192 patients fulfilled the AHF criteria, of whom 86 and 24 underwent elective and emergency cardiac surgery, respectively. The remaining 82 medical patients had no surgical interventions. Cardiogenic shock was diagnosed in 32% of all patients and was the most common AHF presentation. Medical, elective surgery and emergency surgery AHF patients had a mortality at 30 days of 31%, 4.7% and 22% (<0.05) and at 180 days of 42%, 6.1% and 23% (<0.05), respectively. While the presence of cardiogenic shock was associated with a poor outcome (HR 1.8, CI 1.0-3.0; p=0.04), post-operative cardiac stunning had a good prognosis (HR 0.06, CI 0.01-0.47; p<0.01). Mortality worsened when infections (HR 2.8, CI 1.5-5.7; p<0.01) or renal dysfunction (HR 4.4, CI 2.2-8.4, p<0.01) were present on ICU admission. CONCLUSIONS: Medical patients, patients undergoing elective cardiac surgery and patients requiring emergency cardiac surgery are three distinct AHF-subpopulations. Co-morbidities and surgical treatment options affect long-term outcome.

Cytochrome P450 enzyme-mediated drug metabolism at exposure to acute hypoxia (corresponding to an altitude of 4,500 m).

OBJECTIVE: To investigate the effect of acute hypoxia and concomitant changes in portal blood flow on the disposition of drugs mainly metabolized by the cytochrome P(450) enzymes (CYP) 3A4 (verapamil) and CYP1A2 (theophylline). METHODS: Twenty healthy male participants were studied on two 14-h study days in a normobaric hypoxic chamber and were allocated randomly to one of two groups receiving short infusions of either theophylline (6 mg kg (-1) body weight) or verapamil (5 mg) intravenously. According to a randomized, cross-over design, participants were once exposed to normoxia and once to hypoxia (12% oxygen corresponding to the ambient( P)O(2) at an altitude of 4,500 m above sea level). The concentrations of theophylline, 1,3-dimethyluric acid, verapamil, and norverapamil were determined in serial blood samples by means of liquid chromatography-mass spectrometry (LC/MS/MS). Portal blood flow was assessed by transabdominal duplex ultrasonography. RESULTS: Acute hypoxia did not alter the pharmacokinetics of theophylline [half-life+/-SD: 9.29+/-1.77 versus 9.39+/-1.40 (hypoxia)], 1,3-dimethyluric acid (12.9+/-4.72 versus 15.1+/-8.59), verapamil (2.00+/-0.98 versus 1.79+/-0.58), or norverapamil (7.98+/-2.94 versus 9.91+/-6.40). Individual changes of elimination half-life and changes in capillary oxygen saturation,( P)O(2), or portal vein flow were not correlated. Portal vein flow was unaffected by hypoxia. CONCLUSIONS: Acute hypoxia corresponding to hypoxia at altitudes of 4,500 m does not impair the metabolism mediated by CYP1A2 or CYP3A4. At rapid ascent to and short-term stay at altitudes up to 4,500 m, the doses of drugs metabolized by these CYPs do therefore not require dose modification, and major changes in the disposition of already administered drugs are not to be expected.

Expression of the type-1 repeats of thrombospondin-1 inhibits tumor growth through activation of transforming growth factor-beta.

In the present study, the type-1 repeats of thrombospondin-1 (TSP-1) were transfected into A431 cells. Expression of all three type-1 repeats (3TSR) and expression of just the second type-1 repeat containing the transforming growth factor (TGF)-beta activating sequence KRFK (TSR2 + KRFK) significantly inhibited in vivo tumor angiogenesis and growth in nude mice. These tumors expressed increased levels of both active and total TGF-beta. A431 cells expressing the second type-1 repeat without the KRFK sequence (TSR2 - KRFK) produced tumors that were slightly larger than the 3TSR and TSR2 + KRFK tumors. These tumors expressed elevated levels of active TGF-beta but levels of total TGF-beta were not different from control tumors. Injection of the peptide, LSKL, which blocks TSP-1 activation of TGF-beta, reversed the growth inhibition observed with cells expressing TSR2 + KRFK to a level comparable to controls. Various residues in the WSHWSPW region and the VTCG sequence of both TSR2+/- KRFK were mutated. Although mutation of the VTCG sequence had no significant effect on tumor growth, mutation of the WSHWSPW sequence reduced inhibition of tumor growth. These findings suggest that the inhibition of tumor angiogenesis and growth by endogenous TSP-1 involves regulation of both active and total TGF-beta and the sequences KRFK and WSHWSPW in the second type-1 repeat.

An N-terminal 80 kDa recombinant fragment of human thrombospondin-2 inhibits vascular endothelial growth factor induced endothelial cell migration in vitro and tumor growth and angiogenesis in vivo.

We have previously shown that stable overexpression of the thrombospondin-2 (TSP-2) gene inhibited the tumor growth and angiogenesis of human squamous cell carcinoma xenotransplants. To investigate the potential antitumoral efficacy of systemic TSP-2 therapy, we expressed a recombinant 80 kDa fragment of human TSP-2 (TSP-2/NTF), encompassing the N-terminal globular region through the three type 1 repeats, in human kidney 293 EBNA cells, using a modified pCEP4 expression vector. Daily intraperitoneal injections of TSP-2/NTF resulted in a significant inhibition of the growth of human A431 squamous cell carcinomas in vivo and in reduced tumor vascularization. To further investigate possible mechanisms of the antiangiogenic activity of TSP-2/NTF, several in vitro angiogenesis assays were performed in human dermal microvascular endothelial cells. TSP-2/NTF inhibited vascular endothelial growth factor induced migration of human dermal microvascular endothelial cells and inhibited tube formation on Matrigel in vitro. TSP-2/NTF also inhibited vascular endothelial growth factor induced angiogenesis in an in vivo Matrigel assay. Moreover, TSP-2/NTF potently induced human dermal microvascular endothelial cell apoptosis in vitro but did not affect A431 tumor cell proliferation or apoptosis. These findings identify TSP-2/NTF as a potent systemic inhibitor of tumor growth and angiogenesis, acting by direct inhibition of several endothelial cell functions involved in neovascularization.

Angiogenesis, lymphangiogenesis, and melanoma metastasis.

The induction of angiogenesis is a critical point in the development of most human tumors - including melanomas. Some of the earliest studies in the field of tumor angiogenesis showed that transplantation of human melanoma fragments into the hamster cheek pouch stimulated blood vessel growth. Since then, numerous studies have demonstrated that human melanomas also induce angiogenesis. The prognostic importance of the degree of melanoma vascularization, however, has remained controversial. Elevated expression of several angiogenic factors, including vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8, has been detected in primary cutaneous melanomas, and the importance of these mediators in promoting melanoma angiogenesis and metastasis has been confirmed in tumor xenotransplant models. Based upon these findings, several clinical trials of angiogenesis inhibitors have been initiated in human melanoma patients and are currently underway. Recent experimental evidence indicates that tumor-associated lymphangiogenesis also plays an important role in mediating tumor spread to regional lymph nodes. These observations have important implications for prognosis and treatment of human melanomas.

Thrombospondin-1 selectively inhibits early-stage carcinogenesis and angiogenesis but not tumor lymphangiogenesis and lymphatic metastasis in transgenic mice.

The roles played by the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in the early stages of multi-step carcinogenesis and in the control of hematogenous versus lymphatic metastasis are unknown. To investigate these issues we compared tumor development in normal mice and in transgenic mice with targeted overexpression of TSP-1 in the epidermis following a standard two-step chemical skin carcinogenesis regimen. Overexpression of TSP-1 resulted in delayed and reduced development of premalignant epithelial hyperplasias, but did not inhibit the malignant conversion to squamous cell carcinomas. TSP-1 overexpression also suppressed tumor angiogenesis and distant organ metastasis, but failed to inhibit tumor-associated lymphangiogenesis or lymphatic tumor spread to regional lymph nodes. Concomitant with these results, we found that the endothelial TSP-1 receptor CD36 was mostly absent from cutaneous lymphatic vessels. Our findings indicate the potential use of TSP-1 for the prevention of premalignant stages of tumorigenesis and are likely to have implications for the further development of anti-angiogenic cancer therapies.

Systemic inhibition of tumor growth and angiogenesis by thrombospondin-2 using cell-based antiangiogenic gene therapy.

Recent studies indicate that continuous administration improves the antitumoral efficacy of angiogenesis inhibitors, as compared with intermittent dosing, suggesting a potential role of gene therapy in antiangiogenic tumor therapy. We established a tissue-engineered implant system for the continuous in vivo production of thrombospondin-2 (TSP-2), a potent endogenous inhibitor of tumor growth and angiogenesis. Fibroblasts were retrovirally transduced to overexpress TSP-2 and were seeded onto biodegradable polymer scaffolds. After transplantation into the peritoneal cavity of nude mice, bioimplants maintained high levels of TSP-2 secretion over extended time periods, resulting in increased levels of circulating TSP-2. Bioimplant-generated TSP-2 potently inhibited tumor growth and angiogenesis of human squamous cell carcinomas, malignant melanomas, and Lewis lung carcinomas that were implanted at a distant site. These results provide the first proof-of-principle for the feasibility and therapeutic efficiency of systemic, cell-based antiangiogenic gene therapy using biodegradable polymer grafts for the treatment of cancer.

Activation of the tie2 receptor by angiopoietin-1 enhances tumor vessel maturation and impairs squamous cell carcinoma growth.

The distinct roles of angiopoietin (Ang)-1 and Ang2, counteracting ligands for the endothelium-specific Tie2 receptor, in tumor development and progression have remained poorly understood. We investigated the expression of Ang1 and Ang2 during multistep mouse skin carcinogenesis and in human squamous cell carcinoma (SCC) xenografts. Expression of Ang2, but not of Ang1, was up-regulated in angiogenic tumor vessels already in early stages of skin carcinogenesis and was also strongly increased in SCCs. Stable overexpression of Ang1 in human A431 SCCs resulted in a more than 70% inhibition of tumor growth, associated with enhanced Tie2 phosphorylation levels, as compared with low levels in control transfected tumors. No major changes in the vascular density, vascular endothelial growth factor mRNA and protein expression, and vascular endothelial growth factor receptor-2 phosphorylation levels were observed in Ang1-expressing tumors. However, the fraction of tumor blood vessels with coverage by alpha-smooth muscle actin-positive periendothelial cells was significantly increased, indicative of an increased vascular maturation status. These findings identify an inhibitory role of Ang1/Tie2 receptor-mediated vessel maturation in SCC growth and suggest that up-regulation of its antagonist, Ang2, during early-stage epithelial tumorigenesis contributes to the angiogenic switch by counteracting specific vessel-stabilizing effects of Ang1.

The alpha(1)beta(1) and alpha(2)beta(1) integrins provide critical support for vascular endothelial growth factor signaling, endothelial cell migration, and tumor angiogenesis.

Angiogenesis is a complex process, involving functional cooperativity between cytokines and endothelial cell (EC) surface integrins. In this study, we investigated the mechanisms through which the alpha(1)beta(1) and alpha(2)beta(1) integrins support angiogenesis driven by vascular endothelial growth factor (VEGF). Dermal microvascular EC attachment through either alpha(1)beta(1) or alpha(2)beta(1) supported robust VEGF activation of the Erk1/Erk2 (p44/42) mitogen-activated protein kinase signal transduction pathway that drives EC proliferation. Haptotactic EC migration toward collagen I was dependent on alpha(1)beta(1) and alpha(2)beta(1) as was VEGF-stimulated chemotaxis of ECs in a uniform collagen matrix. Consistent with the functions of alpha(1)beta(1) and alpha(2)beta(1) in supporting signal transduction and EC migration, antibody antagonism of either integrin resulted in potent inhibition of VEGF-driven angiogenesis in mouse skin. Moreover, combined antagonism of alpha(1)beta(1) and alpha(2)beta(1) substantially reduced tumor growth and angiogenesis of human squamous cell carcinoma xenografts. Collectively, these studies identify critical collaborative functions for the alpha(1)beta(1) and alpha(2)beta(1) integrins in supporting VEGF signal transduction, EC migration, and tumor angiogenesis.