Off-the-Shelf Cord-Blood Mesenchymal Stromal Cells: Production, Quality Control, and Clinical Use.

Background Recently, mesenchymal stromal cells (MSCs) have gained recognition for their clinical utility in transplantation to induce tolerance and to improve/replace pharmacological immunosuppression. Cord blood (CB)-derived MSCs are particularly attractive for their immunological naivety and peculiar anti-inflammatory and anti-apoptotic properties. OBJECTIVES: The objective of this study was to obtain an inventory of CB MSCs able to support large-scale advanced therapy medicinal product (ATMP)-based clinical trials. STUDY DESIGN: We isolated MSCs by plastic adherence in a GMP-compliant culture system. We established a well-characterized master cell bank and expanded a working cell bank to generate batches of finished MSC(CB) products certified for clinical use. The MSC(CB) produced by our facility was used in approved clinical trials or for therapeutic use, following single-patient authorization as an immune-suppressant agent. RESULTS: We show the feasibility of a well-defined MSC manufacturing process and describe the main indications for which the MSCs were employed. We delve into a regulatory framework governing advanced therapy medicinal products (ATMPs), emphasizing the need of stringent quality control and safety assessments. From March 2012 to June 2023, 263 of our Good Manufacturing Practice (GMP)-certified MSC(CB) preparations were administered as ATMPs in 40 subjects affected by Graft-vs.-Host Disease, nephrotic syndrome, or bronco-pulmonary dysplasia of the newborn. There was no infusion-related adverse event. No patient experienced any grade toxicity. Encouraging preliminary outcome results were reported. Clinical response was registered in the majority of patients treated under therapeutic use authorization. CONCLUSIONS: Our 10 years of experience with MSC(CB) described here provides valuable insights into the use of this innovative cell product in immune-mediated diseases.

First clinical application of cord blood mesenchymal stromal cells in children with multi-drug resistant nephrotic syndrome.

BACKGROUND AND OBJECTIVES: Children with multi-drug resistant idiopathic nephrotic syndrome (MDR-INS) usually progress to end-stage kidney disease with a consistent risk of disease recurrence after transplantation. New therapeutic options are needed for these patients. Mesenchymal stromal cells (MSCs) are multipotential non-hematopoietic cells with several immunomodulatory properties and growing clinical applications. Cord blood-derived MSC have peculiar anti-inflammatory and immunosuppressive properties. We aimed at assessing safety and efficacy of cord-blood-derived MSCs (CB-MSCs) in children with MDR-INS. DESIGN, SETTING, PARTICIPANTS: Prospective, open-label, single arm phase I-II pilot study. Pediatric patients with MDR-INS, resistant to at least two lines of therapy, were enrolled. Allogenic CB-MSCs were administered intravenously on days 0, 14, and 21 at a dose of 1.5 × 106 cells/kg. Patients were followed for at least 12 months. The primary outcomes were safety and toxicity. The secondary outcome was remission at 12 months evaluated by urinary protein/urinary creatinine ratio (uPr/uCr). Circulating regulatory T cells (Tregs) were monitored. RESULTS: Eleven pediatric patients with MDR-INS (10 females, median age 13 years) resistant to a median of 3 previous lines of therapy were enrolled. All patients completed the CB-MSC infusion schedule. No patient experienced any infusion-related adverse event or toxicity. Nine patients were assessable for efficacy. At the 12 months follow-up after the treatment, the median uPr/uCr did not change significantly from baseline (8.13 vs. 9.07; p = 0.98), while 3 patients were in partial or complete remission. A lower baseline uPr/uCr was a predictor of remission (2.55 vs. 8.74; p = 0.0238). Tregs count was not associated with CB-MSCs therapy. CONCLUSIONS: CB-MSCs are safe and may have a role in the immunosuppressive therapy of pediatric patients with MDR-INS. This preliminary experience paves the way toward further phase II studies addressing MSC efficacy in immune-mediated kidney diseases.

Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product.

BACKGROUND: A growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). RESULTS: The GMP-compliant protocol defined in this work allows at least 4.11 × 109 Treg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function. CONCLUSIONS: These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases.

Safety and Effectiveness of Cell Therapy in Neurodegenerative Diseases: Take-Home Messages From a Pilot Feasibility Phase I Study of Progressive Supranuclear Palsy.

Mesenchymal stromal cells (MSCs) are multipotent cells with anti-inflammatory properties. Here we tested the safety of MSCs in patients with progressive supranuclear palsy (PSP; ClinicalTrials.gov: NCT01824121; Eudract No. 2011-004051-39). Seven patients were treated. To improve the safety, protocol adjustments were made during the performance of the study. The objectives of our work were: (1) to assess the safety of MSCs and (2) to identify critical issues in cell therapies for neurodegenerative diseases. Autologous MSCs from the bone marrow of PSP patients were administered through the internal carotid arteries. 1-year survival and number of severe adverse events were considered as safety endpoints. Clinical rating scales, neuropsychological assessments, gait and posture analysis, single-photon emission computed tomography, positron emission tomography, and brain magnetic resonance (BMR) were performed at different follow-up times. Peripheral blood levels of inflammatory cytokines were measured before and after cell infusion. Six of the seven treated patients were living 1 year after cell infusion. Asymptomatic spotty lesions were observed at BMR after 24 h in six of the seven treated patients. The last patient in the preliminary cohort (Case 5) exhibited transiently symptomatic BMR ischemic alterations. No severe adverse events were recorded in the last two treated patients. Interleukin-8 serum concentrations decreased in three patients (Case 2, 3, and 4). An adaptive study design, appropriate and up-to-date efficacy measures, adequate sample size estimation, and, possibly, the use of a cellular and/or allogeneic cell sources may help in performing phase II trials in the field.

Chondrogenic and BMP-4 primings confer osteogenesis potential to human cord blood mesenchymal stromal cells delivered with biphasic calcium phosphate ceramics.

Bone marrow mesenchymal stem/stromal cells (BMSCs) show great promise for bone repair, however they are isolated by an invasive bone marrow harvest and their regenerative potential decreases with age. Conversely, cord blood can be collected non-invasively after birth and contains MSCs (CBMSCs) that can be stored for future use. However, whether CBMSCs can replace BMSCs targeting bone repair is unknown. This study evaluates the in vitro osteogenic potential of unprimed, osteogenically primed, or chondrogenically primed CBMSCs and BMSCs and their in vivo bone forming capacity following ectopic implantation on biphasic calcium phosphate ceramics in nude mice. In vitro, alkaline phosphatase (intracellular, extracellular, and gene expression), and secretion of osteogenic cytokines (osteoprotegerin and osteocalcin) was significantly higher in BMSCs compared with CBMSCs, while CBMSCs demonstrated superior chondrogenic differentiation and secretion of interleukins IL-6 and IL-8. BMSCs yielded significantly more cell engraftment and ectopic bone formation compared to CBMSCs. However, priming of CBMSCs with either chondrogenic or BMP-4 supplements led to bone formation by CBMSCs. This study is the first direct quantification of the bone forming abilities of BMSCs and CBMSCs in vivo and, while revealing the innate superiority of BMSCs for bone repair, it provides avenues to induce osteogenesis by CBMSCs.

A circular RNA map for human induced pluripotent stem cells of foetal origin.

BACKGROUND: Adult skin fibroblasts represent the most common starting cell type used to generate human induced pluripotent stem cells (F-hiPSC) for clinical studies. Yet, a foetal source would offer unique advantages, primarily the absence of accumulated somatic mutations. Herein, we generated hiPSC from cord blood multipotent mesenchymal stromal cells (MSC-hiPSC) and compared them with F-hiPSC. Assessment of the full activation of the pluripotency gene regulatory network (PGRN) focused on circular RNA (circRNA), recently proposed to participate in the control of pluripotency. METHODS: Reprogramming was achieved by a footprint-free strategy. Self-renewal and pluripotency of cord blood MSC-hiPSC were investigated in vitro and in vivo, compared to parental MSC, to embryonic stem cells and to F-hiPSC. High-throughput array-based approaches and bioinformatics analyses were applied to address the PGRN. FINDINGS: Cord blood MSC-hiPSC successfully acquired a complete pluripotent identity. Functional comparison with F-hiPSC showed no differences in terms of i) generation of mesenchymal-like derivatives, ii) their subsequent adipogenic, osteogenic and chondrogenic commitment, and iii) their hematopoietic support ability. At the transcriptional level, specific subsets of mRNA, miRNA and circRNA (n = 4,429) were evidenced, casting a further layer of complexity on the PGRN regulatory crosstalk. INTERPRETATION: A circRNA map of transcripts associated to naïve and primed pluripotency is provided for hiPSC of clinical-grade foetal origin, offering insights on still unreported regulatory circuits of the PGRN to consider for the optimization and development of efficient differentiation protocols for clinical translation. FUNDING: This research was funded by Ricerca Corrente 2012-2018 by the Italian Ministry of Health.

Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function.

BACKGROUND: Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. METHODS: We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. RESULTS: Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. CONCLUSIONS: These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation.

Tips and Tricks for Validation of Quality Control Analytical Methods in Good Manufacturing Practice Mesenchymal Stromal Cell Production.

Mesenchymal stromal cells (MSC) for cellular therapy in European Union are classified as advanced therapy medicinal products (ATMPs), and their production must fulfill the requirements of Good Manufacturing Practice (GMP) rules. Despite their classification as medicinal products is already well recognized, there is still a lack of information and indications to validate methods and to adapt the noncompendial and compendial methods to these peculiar biological products with intrinsic characteristics that differentiate them from classic synthetic or biologic drugs. In the present paper, we present the results of the validation studies performed in the context of MSC development as ATMPs for clinical experimental use. Specifically, we describe the validation policies followed for sterility testing, endotoxins, adventitious viruses, cell count, and immunophenotyping. Our work demonstrates that it is possible to fully validate analytical methods also for ATMPs and that a risk-based approach can fill the gap between the prescription of the available guidelines shaped on traditional medicinal products and the peculiar characteristics of these novel and extremely promising new drugs.

Angiogenic and anti-inflammatory properties of mesenchymal stem cells from cord blood: soluble factors and extracellular vesicles for cell regeneration.

In a recent work, our group showed the existence of two distinct mesenchymal stem cell (MSC) subsets within human umbilical cord blood. One less proliferative and short-living (SL-CBMSC), the other with higher growth rate and long-living (LL-CBMSC), and therefore better suited for regenerative medicine applications. We examined whether LL-CBMSC possess peculiar paracrine properties able to affect angiogenesis or inflammatory processes. It was shown for the first time that pro-angiogenic, proliferation-stimulating and tissue repairing factors were released at high level not only as soluble cytokines, but also as mRNA precursors embedded in membrane vesicles. The combination of this primary (proteic factors interacting with surface receptors) and delayed (mRNA transferred and translated via vesicle fusion and cargo release) interaction in endothelial target cells resulted in strong blood vessel induction with the development of capillary-like structures. In addition, LL-CBMSC dynamically modulated their release of pro-angiogenic and anti-inflammatory factors in an in vitro model of damage. In conclusion, LL-CBMSC synthesize and secrete multiple factors that may be attuned in response to the status of the target cell, a crucial requisite when paracrine mechanisms are needed at onset of tissue regeneration.

Finding a new therapeutic approach for no-option Parkinsonisms: mesenchymal stromal cells for progressive supranuclear palsy.

BACKGROUND: The trophic, anti-apoptotic and regenerative effects of bone marrow mesenchymal stromal cells (MSC) may reduce neuronal cell loss in neurodegenerative disorders. METHODS: We used MSC as a novel candidate therapeutic tool in a pilot phase-I study for patients affected by progressive supranuclear palsy (PSP), a rare, severe and no-option form of Parkinsonism. Five patients received the cells by infusion into the cerebral arteries. Effects were assessed using the best available motor function rating scales (UPDRS, Hoehn and Yahr, PSP rating scale), as well as neuropsychological assessments, gait analysis and brain imaging before and after cell administration. RESULTS: One year after cell infusion, all treated patients were alive, except one, who died 9 months after the infusion for reasons not related to cell administration or to disease progression (accidental fall). In all treated patients motor function rating scales remained stable for at least six-months during the one-year follow-up. CONCLUSIONS: We have demonstrated for the first time that MSC administration is feasible in subjects with PSP. In these patients, in whom deterioration of motor function is invariably rapid, we recorded clinical stabilization for at least 6 months. These encouraging results pave the way to the next randomized, placebo-controlled phase-II study that will definitively provide information on the efficacy of this innovative approach. Trial registration ClinicalTrials.gov NCT01824121.

A Chemically Defined Medium-Based Strategy to Efficiently Generate Clinically Relevant Cord Blood Mesenchymal Stromal Colonies.

During the last decade it has been demonstrated that mesenchymal progenitors are present and can be isolated also from cord blood (CB). Recently, we managed to set up a standard protocol allowing the isolation of mesenchymal stromal cells (MSCs) with high proliferative potential and multiple differentiation capabilities, whereas the generation rate of MSC-initiating colonies could still be further improved. Herein, we strikingly succeeded in defining some simple and basic culture conditions based on the use of a chemically defined medium that increased the colony isolation efficiency up to almost 80% of processed CB units. Importantly, this result was achieved irrespective of CB unit white blood cell content and time elapsed from delivery, two limiting parameters involved with processing CB units. Thus, this high efficiency is guaranteed without strict selection of the starting material. In addition, since we are profoundly concerned about how different culture conditions can influence cell behavior, we devoted part of this study to in-depth characterization of the established CB-MSC populations to confirm their stemness features in this novel isolation and culture system. Therefore, an extended study of their immunophenotype, including classical pericytic markers, and a detailed molecular analysis addressing telomere length and also stemness-related microRNA contribution were performed. In summary, we propose a straightforward, extremely efficient, and reliable approach to isolate and expand thoroughly characterized CB-MSCs, even when poor-quality CB units are the only available source, or there is no space for an isolation to fail.

How we make cell therapy in Italy.

In the 21st century scenario, new therapeutic tools are needed to take up the social and medical challenge posed by the more and more frequent degenerative disorders and by the aging of population. The recent category of advanced therapy medicinal products has been created to comprise cellular, gene therapy, and tissue engineered products, as a new class of drugs. Their manufacture requires the same pharmaceutical framework as for conventional drugs and this means that industrial, large-scale manufacturing process has to be adapted to the peculiar characteristics of cell-containing products. Our hospital took up the challenge of this new path in the early 2000s; and herein we describe the approach we followed to set up a pharmaceutical-grade facility in a public hospital context, with the aim to share the solutions we found to make cell therapy compliant with the requirements for the production and the quality control of a high-standard medicinal product.

Extensive Characterization of Platelet Gel Releasate From Cord Blood in Regenerative Medicine.

Platelet gel derived from peripheral blood is widely applied in many clinical fields of surgery as biomaterial containing growth factors with high proliferative properties. In 2010, we studied and patented a platelet gel derived from cord blood. In this study, due to the crucial role of the factors released by the platelet gel, we first extended the characterization of its releasate. Using a wide proteomic array and splitting the two components of the releasate, that is, platelets and plasma, we have been able to study their growth factor content. Interestingly, we discovered high levels of hormones and molecules able to support tissue growth in the cord blood platelet gel releasate and, in addition, higher concentrations of several angiogenic factors if compared with the peripheral blood counterpart. On the contrary, the latter was much richer in inflammatory factors. The second aim of our work was to study the effects on cell culture, immunophenotype, and function of mesenchymal stem cells exposed to these two platelet gel releasates as substitute for the animal serum. Since our findings nicely show that the use of the peripheral versus the cord blood platelet gel releasate can differently influence the mesenchymal stem cell commitment, we can suggest that in addition to its peculiar angiogenic properties cord blood platelet gel releasate shows excellent proliferative properties as cell culture supplement.

Dissection of the cord blood stromal component reveals predictive parameters for culture outcome.

In regenerative medicine, human cord blood-derived multipotent mesenchymal stromal cells (CBMSCs) stand out for their biological peculiarities demonstrated in in vitro and in vivo preclinical studies. Here, we present our 9-year experience for the consistent isolation of CBMSCs. Although nearly one CB unit out of two retains the potential to give rise to MSC colonies, only 46% of them can be cultured till low passages (P≥4), but one-fourth of those reaches even higher passages (P≥8). Subsequent characterization for morphological, clonal, differentiation, and proliferation properties revealed two divergent CBMSC behaviors. In particular, a cumulative population doublings cut-off (CPD=15) was identified that undoubtedly distinguishes two growth curves, and different degrees of commitment toward osteogenesis were observed. These data clearly show the existence of at least two distinct CBMSC subsets: one mainly short-living and less proliferative (SL-CBMSCs), the other long-living, with higher growth rate, and, very importantly, with significantly (P≤0.01) longer telomere (LL-CBMSCs). Moreover, significant differences in the immunoprofile before seeding were found among CB units giving rise to LL-CBMSCs or SL-CBMSCs or showing no colony formation. Finally, all the aforementioned results provided a peculiar and useful set of parameters potentially predictive for CBMSC culture outcome.

Defining the identity of human adipose-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (ADMSCs) are an ideal population for regenerative medical application. Both the isolation procedure and the culturing conditions are crucial steps, since low yield can limit further cell therapies, especially when minimal adipose tissue harvests are available for cell expansion. To date, a standardized procedure encompassing both isolation sites and expansion methods is missing, thus making the choice of the most appropriate conditions for the preparation of ADMSCs controversial, especially in view of the different applications needed. In this study, we compared the effects of three different commercial media (DMEM, aMEM, and EGM2), routinely used for ADMSCs expansion, and two supplements, FBS and human platelet lysate, recently proven to be an effective alternative to prevent xenogeneic antibody transfer and immune alloresponse in the host. Notably, all the conditions resulted in being safe for ADMSCs isolation and expansion with platelet lysate supplementation giving the highest isolation and proliferation rates, together with a commitment for osteogenic lineage. Then, we proved that the high ADMSC hematopoietic supportive potential is performed through a constant and abundant secretion of both GCSF and SCF. In conclusion, this study further expands the knowledge on ADMSCs, defining their identity definition and offers potential options for in vitro protocols for clinical production, especially related to HSC expansion without use of exogenous cytokines or genetic modifications.

Adipogenic potential in human mesenchymal stem cells strictly depends on adult or foetal tissue harvest.

Cell-based therapies promise important developments for regenerative medicine purposes. Adipose tissue and the adipogenic process has become central to an increasing number of translational efforts in addition to plastic and reconstructive surgical applications. In recent experimental clinical trials, human mesenchymal stem cells (MSC) have been proven to be well tolerated because of their low immunoreactivity. MSC are multipotent cells found among mature cells in different tissues and organs with the potentiality to differentiate in many cell types, including osteocytes, chondrocytes and adipocytes, thus being a suitable cell source for tissue engineering strategies. We compared the adipogenic potential of MSC originated from two adult sources as fat pads and bone marrow, and from four foetal sources as umbilical cord blood, Wharton's jelly, amniotic fluid and preterm umbilical cord perivascular cells. Surprisingly, adult MSC displayed higher differentiation capacities confirmed by gene expression analysis on a selected panel of adipogenesis-related genes. Further, an in-depth molecular analysis highlighted the early and vigorous activation of the PPARγ transcription factor-cascade in adipose-derived MSC that resulted to be both delayed and reduced in foetal MSC accounting for their lack of adipogenic potential. Thus, MSC show a different degree of phenotypic plasticity depending on the source tissue, that should be taken into consideration for the selection of the most appropriate MSC type for specific tissue regeneration purposes.

Differential microRNA signature of human mesenchymal stem cells from different sources reveals an "environmental-niche memory" for bone marrow stem cells.

Human mesenchymal stem cells (MSCs) are multipotent cells offering valuable hopes for the treatment of degenerative diseases. MSCs can be found among differentiated cells in many tissues and organs but, unfortunately, their phenotypic similarity hinders a robust cell characterization and discrimination from diverse tissue harvests. MicroRNAs (miRNAs) are crucial managers of gene expression with intriguing and still poorly known roles in stem cell maintenance and differentiation. To identify miRNAs that can discriminate among MSCs, we performed a whole-genome comparative miRNA expression profiling analysis on adipose (AD), bone marrow (BM) and cord blood (CB) derived MSCs, all three considered among the most promising in the field of regenerative medicine. miRNA expression patterns were very similar, meeting their extensive phenotypic and functional overlaps. An in-depth comparison of the few most differentially expressed miRNAs allowed the identification of a highly restricted molecular signature consisting of 5 BMMSC, 11 ADMSC and 11 CBMSC specific miRNAs. Functional analysis of their validated targets allowed the identification of an "environmental-niche memory" for BMMSC and an "epithelial" commitment for ADMSC, providing new insights into the molecular mechanisms discriminating between these MSCs, a crucial element to identify the most appropriate stem cell source for clinical application.

Human CD34+ cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand target both tumor cells and tumor vasculature.

Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 10(5) tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell-derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling-stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.

A computational approach to compare microvessel distributions in tumors following antiangiogenic treatments.

Experimental approaches currently used to quantify the activity of antiangiogenic treatments in cancer therapy do not generally address the importance of spatial distribution of microvessels in target tissues. We report a new computerized method to assess tumor vascularization by quantifying the distribution of functional microvessels as revealed by in vivo staining with sulfosuccinimidyl-6-(biotinamido) hexanoate. Our approach was based on pixel dilation of digital images of blood vessels and addressed the space-filling property of the vessel layouts. This was practically achieved computing the number of dilation cycles (Halo index) needed to permeate a pre-defined amount of each image. Our approach was validated on human tumor xenografts in nonobese diabetic/severe combined immunodeficient mice treated with the antiangiogenic drug sorafenib. For each experimental model, area normalization allowed the unbiased comparison of several hundreds of images showing different amounts of vascular tissue. In two different tumor types, comparison of Halo values showed statistically significant differences between control and sorafenib-treated samples. Conversely, this effect was not observed in samples from an additional xenograft known to resist the antiangiogenic treatment. By separating the analysis of vessel area from the quantification of vessel distributions, our approach can potentially contribute to a better evaluation of the antiangiogenic or vascular-disrupting activity of new drugs or treatments.