Near-Infrared Fluorescence Axillary Reverse Mapping (ARM) Procedure in Invasive Breast Cancer: Relationship between Fluorescence Signal in ARM Lymph Nodes and Clinical Outcomes.

The near-infrared (NIR) fluorescence axillary reverse mapping (ARM) procedure is a promising tool to identify and preserve arm lymphatic drainage during axillary lymph node dissection (ALND). The ARMONIC clinical trial was conducted to validate the technique on a large cohort of patients and to analyze the predictive clinical factors for ARM lymph node metastasis. For the first time, the fluorescence signal intensity from the ARM lymph nodes was measured and correlated with clinical findings. A total of 109 patients with invasive breast cancer and indications of mastectomy and ALND underwent the NIR fluorescence ARM procedure. Indocyanine green was administered by intradermal injection followed by intraoperative identification and resection of the ARM lymph nodes with NIR fluorescence camera guidance. The fluorescence signal intensity and signal distribution were then measured ex vivo and compared with clinical outcomes. ARM lymph nodes were successfully identified by fluorescence in 94.5% of cases. The mean normalized fluorescence signal intensity value was 0.47 with no significant signal difference between metastatic and non-metastatic ARM lymph nodes (p = 0.3728). At the microscopic level, the fluorescence signal distribution was focally intense in lymphoid tissue areas. Only the preoperative diagnosis of metastasis in the axillary nodes of patients was significantly associated with a higher ARM node fluorescence signal intensity (p = 0.0253), though it was not significantly associated with the pathological nodal (pN) status (p = 0.8081). Based on an optimal cut-off fluorescence value, the final sensitivity and specificity of the NIR fluorescence ARM procedure for ARM lymph node metastatic involvement were 64.7% and 47.3%, respectively. Although our preliminary results did not show that fluorescence signal intensity is a reliable diagnostic tool, the NIR fluorescence ARM procedure may be useful for ARM lymph node identification. Clinical trial registration: NCT02994225.

Endothelial and hematopoietic hPSCs differentiation via a hematoendothelial progenitor.

BACKGROUND: hPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages. METHODS: Two hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144+-embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144+, respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other's (e.g. hPSC-ECs vs. hPSC-EB-CD144+) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations. RESULTS: A hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144+ contributed to the generation of CD45+ human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144+. Endothelial differentiation of hPSC-EB-CD144+ yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144+ generated an intermediate population of > 90% CD43+ hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144+, hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144+CD45+, a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors. CONCLUSION: The present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering.

Cord blood-endothelial colony forming cells are immunotolerated and participate at post-ischemic angiogenesis in an original dorsal chamber immunocompetent mouse model.

BACKGROUND: Cardiovascular diseases are the main cause of morbidity and mortality worldwide. Restoring blood supply to ischemic tissues is an essential goal for the successful treatment of these diseases. Growth factor or gene therapy efficacy remains controversial, but stem cell transplantation is emerging as an interesting approach to stimulate angiogenesis. Among the different stem cell populations, cord blood-endothelial progenitor cells (CB-EPCs) and more particularly cord blood-endothelial progenitor cell-derived endothelial colony forming cells (CB-ECFCs) have a great proliferative potential without exhibiting signs of senescence. Even if it was already described that CB-ECFCs were able to restore blood perfusion in hind-limb ischemia in an immunodeficient mouse model, until now, the immunogenic potential of allogenic CB-ECFCs remains controversial. Therefore, our objectives were to evaluate the immune tolerance potency of CB-ECFCs and their capacity to restore a functional vascular network under ischemic condition in immunocompetent mice. METHODS: In vitro, the expression and secretion of immunoregulatory markers (HLA-G, IL-10, and TGF-ß1) were evaluated on CB-ECFCs. Moreover, CB-ECFCs were co-cultured with activated peripheral blood mononuclear cells (PBMCs) for 6 days. PBMC proliferation was evaluated by [3H]-thymidine incorporation on the last 18 h. In vivo, CB-ECFCs were administered in the spleen and muscle of immunocompetent mice. Tissues were collected at day 14 after surgery. Finally, CB-ECFCs were injected intradermally in C57BL/6JRj mice close to ischemic macrovessel induced by thermal cauterization. Mice recovered until day 5 and were imaged, twice a week until day 30. RESULTS: Firstly, we demonstrated that CB-ECFCs expressed HLA-G, IL-10, and TGF-ß1 and secreted IL-10 and TGF-ß1 and that they could display immunosuppressive properties in vitro. Secondly, we showed that CB-ECFCs could be tolerated until 14 days in immunocompetent mice. Thirdly, we revealed in an original ischemic model of dorsal chamber that CB-ECFCs were integrated in a new functional vascular network. CONCLUSION: These results open up new perspectives about using CB-ECFCs as an allogeneic cell therapy product and gives new impulse to the treatment of cardiovascular diseases.

Multimodal imaging for tumour characterization from micro- to macroscopic level using a newly developed dorsal chamber designed for long-term follow-up.

Optical imaging of living animals is a unique method of studying the dynamics of physiological and pathological processes at a subcellular level. One-shot acquisitions at high resolution can be achieved on exteriorized organs before animal euthanasia. For longitudinal follow-up, intravital imaging can be used and involves imaging windows implanted in cranial, thoracic or dorsal regions. Several imaging window models exist, but none have proven to be applicable for long-term monitoring and most biological processes take place over several weeks. Moreover, none are compatible with multiple imaging modalities, meaning that different biological parameters cannot be assessed in an individual animal. We developed a new dorsal chamber that was well tolerated by mice (over several months) and allowed individual and collective cell tracking and behaviour analysis by optical imaging, ultrasound and magnetic resonance tomography. This new model broadens potential applications to areas requiring study of long-term biological processes, as in cancer research.

Establishment and characterization of in vivo orthotopic bioluminescent xenograft models from human osteosarcoma cell lines in Swiss nude and NSG mice.

Osteosarcoma is one of the most common primary bone tumors in childhood and adolescence. Metastases occurrence at diagnosis or during disease evolution is the main therapeutic challenge. New drug evaluation to improve patient survival requires the development of various preclinical models mimicking at best the complexity of the disease and its metastatic potential. We describe here the development and characteristics of two orthotopic bioluminescent (Luc/mKate2) cell-derived xenograft (CDX) models, Saos-2-B-Luc/mKate2-CDX and HOS-Luc/mKate2-CDX, in different immune (nude and NSG mouse strains) and bone (intratibial and paratibial with periosteum activation) contexts. IVIS SpectrumCT system allowed both longitudinal computed tomography (CT) and bioluminescence real-time follow-up of primary tumor growth and metastatic spread, which was confirmed by histology. The murine immune context influenced tumor engraftment, primary tumor growth, and metastatic spread to lungs, bone, and spleen (an unusual localization in humans). Engraftment in NSG mice was found superior to that found in nude mice and intratibial bone environment more favorable to engraftment compared to paratibial injection. The genetic background of the two CDX models also led to distinct primary tumor behavior observed on CT scan. Saos-2-B-Luc/mKate2-CDX showed osteocondensed, HOS-Luc/mKate2-CDX osteolytic morphology. Bioluminescence defined a faster growth of the primary tumor and metastases in Saos-2-B-Luc/mKate2-CDX than in HOS-Luc/mKate2-CDX. The early detection of primary tumor growth and metastatic spread by bioluminescence allows an improved exploration of osteosarcoma disease at tumor progression, and metastatic spread, as well as the evaluations of anticancer treatments. Our orthotopic models with metastatic spread bring complementary information to other types of existing osteosarcoma models.

Establishment and characterization of new orthotopic and metastatic neuroblastoma models.

BACKGROUND/AIM: Treatment of metastatic neuroblastoma remains a challenge in pediatric oncology. Relevant preclinical models may improve exploration of oncogenesis and new therapies. We developed new orthotopic and metastatic models derived from stage 4 neuroblastoma. MATERIAL AND METHODS: Orthotopic and systemic models were established in BalbC Rag2(-/-)gammaC(-/-) mice following adrenal and intravenous injection of luciferase-transfected IMR-32 and IGR-N91 cells, respectively. RESULTS: All four models exhibited 100% tumor take rate. Metastatic spread of orthotopic IMR-32-Luc cells was observed mainly to the lung, liver and bone; that of IGR-N91-Luc cells to liver, spleen and adrenals. Interestingly, systemic IMR-32-Luc cells metastasized rather to the lung, liver and bone, and IGR-N91-Luc to liver, lung, spleen and adrenals. Feasibility of non-invasive, real-time antitumor response evaluation was validated in the systemic models. CONCLUSION: These neuroblastoma models with distinct patterns of metastatic spread represent relevant tools for exploring local and metastatic tumor cell tropism, mechanisms of spread and evaluating new cancer therapeutics.

Reversing resistance to vascular-disrupting agents by blocking late mobilization of circulating endothelial progenitor cells.

UNLABELLED: The prevailing concept is that immediate mobilization of bone marrow-derived circulating endothelial progenitor cells (CEP) is a key mechanism mediating tumor resistance to vascular-disrupting agents (VDA). Here, we show that administration of VDA to tumor-bearing mice induces 2 distinct peaks in CEPs: an early, unspecific CEP efflux followed by a late yet more dramatic tumor-specific CEP burst that infiltrates tumors and is recruited to vessels. Combination with antiangiogenic drugs could not disrupt the early peak but completely abrogated the late VDA-induced CEP burst, blunted bone marrow-derived cell recruitment to tumors, and resulted in striking antitumor efficacy, indicating that the late CEP burst might be crucial to tumor recovery after VDA therapy. CEP and circulating endothelial cell kinetics in VDA-treated patients with cancer were remarkably consistent with our preclinical data. These findings expand the current understanding of vasculogenic "rebounds" that may be targeted to improve VDA-based strategies. SIGNIFICANCE: Our findings suggest that resistance to VDA therapy may be strongly mediated by late, rather than early, tumor-specific recruitment of CEPs, the suppression of which resulted in increased VDA-mediated antitumor efficacy. VDA-based therapy might thus be significantly enhanced by combination strategies targeting late CEP mobilization.

Combining functional imaging and interstitial pressure measurements to evaluate two anti-angiogenic treatments.

BACKGROUND: Interstitial hypertension is responsible for poor capillary blood flow and hampered drug delivery. The efficacy of combined sorafenib/bevacizumab treatment given according to different administration schedules has been evaluated by measuring both interstitial pressure (IP) and quantitative dynamic contrast-enhanced ultrasonography (DCE-US) parameters in melanoma-bearing mice. MATERIAL AND METHODS: [corrected] Sixty mice were xenografted with B16F10 melanoma. Animals received a daily administration over 4 days (D0 to D3) of either sorafenib at 30 mg/kg, bevacizumab at 2.5 mg/kg alone, or different schedules of combined treatments. Perfusion parameters determined using an Aplio® sonograph (Toshiba) with SonoVue® contrast agent (Bracco) were compared to IP measurements using fiberoptic probes (Samba®) at D0, D2, D4, D8. RESULTS: The mean baseline IP values ranged between 6.55 and 31.29 mmHg in all the groups. A transient IP decrease occurred at D2 in all treated groups, and especially in the concomitant group which exhibited a significant IP reduction compared to D0. A significant decrease in both the peak intensity and the area under the curve was observed at D4 in the group with concomitant administration of both molecules which yielded maximal inhibition of the tumor volume and the number of vessels. No correlation was found between IP values and volume or perfusion parameters, indicating complex relationships between IP and vascularization. No IP gradients were found between the center and the periphery but IP values in these two regions were significantly correlated (R = 0.93). CONCLUSION: The results suggest that IP variations could be predictive of vascular changes and that one single IP measurement is sufficient to fully characterize the whole tumor.

The anti-angiogenic activity of IL-12 is increased in iNOS-/- mice and involves NK cells.

We have previously reported that the in vivo transfer of murine interleukin-12 (IL-12) gene using a Semliki Forest virus vector induced tumor regression through inhibition of tumor blood vessel formation. To examine whether IL-12 anti-angiogenic activity interferes with the NO pathway, we used inducible nitric oxide synthase-deficient mice (iNOS-/-) and demonstrated that the anti-tumor effect of IL-12 is more pronounced in these mice. In addition, despite the increased level of intratumoral VEGF in iNOS-/- mice, IL-12 induced a stronger inhibition of blood vessel formation. Histological analysis of SFV-IL-12-treated tumors showed an increase in natural killer (NK) perivascular infiltration in iNOS-/- as compared to control mice. In vitro IL-12-stimulated murine splenic NK cells displayed significant killing activity towards established murine endothelial cells used as targets. These studies indicate that the anti-angiogenic activity of IL-12 interferes with iNOS pathway and involves NK cell recruitment.

Early quantitative evaluation of a tumor vasculature disruptive agent AVE8062 using dynamic contrast-enhanced ultrasonography.

OBJECTIVES: To evaluate the early tumor vasculature disrupting effects of the AVE8062 molecule and the feasibility of dynamic contrast-enhanced ultrasonography (DCE-US) in the quantitative assessment of these effects. MATERIAL AND METHODS: AVE8062 was administered at a single dose (41 mg/kg) to 40 melanoma-bearing nude mice, which were all imaged before and after drug administration (5 + 15 minutes, 1, 6, and 24 hours). Using an ultrasound scanner (Aplio, Toshiba), intratumor vessels were counted in power Doppler mode and tumor microvasculature was assessed in a specific harmonic mode associated with a perfusion and quantification software for contrast-uptake quantification (Sonovue, Bracco). The peak intensity (PI), time-to-PI (T PI), and full-width at half maximum (FWHM) were extracted from the time-intensity curves expressed as linear raw data. Histologic analysis evaluated microvessel density (MVD) and necrosis at each time point studied. Statistical significance was estimated (paired sum rank and Mann-Whitney tests) to evaluate drug activity and to compare its efficacy at the different time points. RESULTS: In power Doppler mode, intratumoral vessels depletion started 15 minutes postinjection (32%, P = 0.004) and the decrease was maximal at 6 hours (51%, P = 0.002). PI decreased by 3.5- and 45.7-fold at 1 and 6 hours, respectively, compared with preinjection values (P = 0.016 and P = 0.008). The decrease at 6 hours was significantly different from the variation at 1 hour (P = 0.0012) and at 24 hours (P = 0.0008). T PI and FWHM showed a significant increase exclusively at 6 hours (P = 0.0034, P = 0.0039). Histology revealed significantly decreased MVD and increased necrosis at 24 hours (P < 0.01). CONCLUSION: DCE-US allowed quantitative in vivo evaluation of the functional effects of AVE8062, which was found most effective on tumoral microvasculature 6 hours after its administration. A clinical phase-1 study of AVE8062 is ongoing using the same ultrasonography methodology before and 6 and 24 hours postadministration.

Acoustic characterization of a new trisacryl contrast agent. Part II: Flow phantom study and in vivo quantification.

The biocompatible trisacryl particles (TMP) are made of a cross-linked acrylic copolymer. Their inherent acoustic properties, studied for a contrast agent application, have been previously demonstrated in a in vitro Couette device. To measure their acoustic behaviour under circulating blood conditions, the TMP backscatter enhancement was further evaluated on a home-made flow phantom at different TMP doses (0.12-15.6 mg/ml) suspended in aqueous and blood media, and in nude mice (aorta and B16 grafted melanoma). Integrated backscatter (IB) was measured by spectral analysis of the Doppler signals recorded from an ultrasound system (Aplio) combined with a 12-MHz probe. Doppler phantom experiments revealed a maximal IB of 17+/-0.88 dB and 7.5+/-0.7 dB in aqueous and blood media, respectively. IB measured on mice aorta, in pulsed Doppler mode, confirmed a constant maximal value of 7.29+/-1.72 dB over the first minutes after injection of a 7.8 mg/ml TMP suspension. Following the injection, a 60% enhancement of intratumoral vascularization detection was observed in power Doppler mode. A preliminary histological study revealed inert presence of some TMP in lungs 8 and 16 days after injection. Doppler phantom experiments on whole blood allowed to anticipate the in vivo acoustic behaviour. Both protocols demonstrated TMP effectiveness in significantly increasing Doppler signal intensity and intratumoral vascularization detection. However, it was also shown that blood conditions seemed to shadow the TMP contrast effect, as compared to in vitro observations. These results encourage further investigations on the specific TMP targeting and on their bio-distribution in the different tissues.

Combination of HIFU therapy with contrast-enhanced sonography for quantitative assessment of therapeutic efficiency on tumor grafted mice.

The objective was to evaluate treatment efficiency of a new high-intensity focused ultrasound (HIFU) prototype combining a therapeutic transducer with a sonographic probe. The optimal HIFU sequence was defined on ex vivo samples before in vivo evaluation of tumor ablation was performed by perfusion quantification after contrast agent injection. The original feature of this prototype is a 9-MHz sonographic probe in a HIFU device and connected to an Aplio (Toshiba) sonograph. Acoustical power and treatment time were determined on ex vivo livers to generate 1-cm-long lesions. Lesion reproducibility was assessed for the power and treatment time selected. The gap between lesions and HIFU displacement shot procedures were optimized to ablate a 1-cm3 volume. The optimized protocol was applied to five murine tumors in vivo. Tumor ablation was quantified according to (1) contrast uptake (CU) after HIFU using perfusion software (Toshiba) in "vascular recognition imaging" mode and Sonovue (Bracco) contrast agent, and (2) the percentage of necrosis quantified on histologic slides. Ex vivo results: optimized settings, at 442 W/cm2 applied during three cycles (3 s on/5 s off) generated 10 identical elementary lesions measuring 9.78 (+/-0.66) * 2.11 (+/-0.33) mm2. A 4-mm gap between adjacent lesions and a 2-min pause between shot lines were found optimal. In vivo results: 60 % (+/-22) mean reduction in CU after HIFU and tumor necrosis histologically estimated at 58 % (+/-5.7) were quantified for the five animals. The therapeutic potential of this HIFU prototype was demonstrated in vivo through objective quantification of tumor ablation based on CU.

Angiogenesis and tumor growth inhibition by a matrix metalloproteinase inhibitor targeting radiation-induced invasion.

In this study, we have evaluated the interactions between ionizing radiation and a matrix metalloproteinase (MMP) inhibitor. Using Matrigel invasion assays, we show that ionizing radiation induced a dose-dependent increase in the invasive phenotype of cultured B16 melanoma cells and that conditioned medium from these irradiated B16 cells promoted endothelial cell [human microvascular endothelial cells (HMEC)] invasiveness. To determine whether the radiation-induced changes in invasive phenotype could be due to changes in MMP activation, we have tested the ability of the MMP inhibitor Metastat to modulate the ionizing radiation-induced invasive phenotype using both an in vitro melanoma model and a mouse s.c. tumor model. In these studies, Metastat inhibited the ionizing radiation-induced invasive phenotype in cultured B16 cells and similarly inhibited the increase in HMEC invasion induced by conditioned medium from irradiated B16 cells. Conversely, ionizing radiation increased B16 MMP-2 activity and the conditioned medium from irradiated B16 induced HMEC MMP-2 activity. To further investigate the interaction between ionizing radiation and MMP activation, we then studied the effects of ionizing radiation on downstream effectors of the MMP system. We found that ionizing radiation induced vascular endothelial growth factor (VEGF) secretion by B16 melanoma cells and that this secretion was inhibited by Metastat. Similarly, conditioned medium from irradiated B16 was also able to increase VEGF secretion in HMECs. Moreover, ionizing radiation-induced melanoma cell invasiveness was partially inhibited by an anti-VEGF monoclonal antibody. In vivo, ionizing radiation plus concomitant Metastat yielded the greatest growth inhibition of melanoma s.c. tumors and this effect correlated with inhibition of angiogenesis as measured by both Doppler ultrasonography and platelet/endothelial cell adhesion molecule-1 staining. Finally, ionizing radiation modulated MMP-2, VEGF, and VEGF receptor expression in these tumor samples using immunohistochemistry. Taken together, these results suggest that there is an ionizing radiation-induced tumor survival pathway and a possible paracrine ionizing radiation-induced stimulatory pathway emanating from tumor cells toward the endothelial bed that is impeded when Metastat is given simultaneously. This model could provide in vivo evidence of the antitumor efficacy of combining a MMP inhibitor with ionizing radiation to target radiation-induced invasion and angiogenesis.

In vitro echogenicity characterization of poly[lactide-coglycolide] (plga) microparticles and preliminary in vivo ultrasound enhancement study for ultrasound contrast agent application.

OBJECTIVES: This work includes (1) the characterization of a reproducible poly[lactide-coglycolide] (PLGA) microparticle preparation with an optimial mean diameter and size distribution and (2) the preliminary in vivo ultrasonographic investigation of PLGA microparticles. METHODS: A first series of PLGA microparticle preparations (1 to 15 mum) was acoustically characterized on a hydrodynamic device to select the most appropriate for ultrasound contrast agent application. Preparations of 3-microm microparticles were selected, characterized at different doses, and then injected into 20 melanoma grafted mice for contrast-enhanced power Doppler ultrasonography evaluation. RESULTS: The 3-microm microparticles (3.26-microm mean diameter with 0.41-microm standard deviation) led to in vitro enhancement of 18.3 dB at 0.62 mg/mL. In vivo experiments showed 47% enhancement of intratumoral vascularization detection after PLGA injection, significantly correlated (P < 0.0001) with preinjection intravascularization and tumoral volume. No toxicity was histologically observed. CONCLUSION: The 3-microm PLGA microparticles provided significant enhancement in vitro and in vivo without any toxicity.

Canstatin acts on endothelial and tumor cells via mitochondrial damage initiated through interaction with alphavbeta3 and alphavbeta5 integrins.

Canstatin, the noncollagenous domain of collagen type IV alpha-chains, belongs to a series of collagen-derived angiogenic inhibitors. We have elucidated the functional receptors and intracellular signaling induced by canstatin that explain its strong antitumor efficacy in vivo. For this purpose, we generated a canstatin-human serum albumin (CanHSA) fusion protein, employing the HSA moiety as an expression tag. We show that CanHSA triggers a crucial mitochondrial apoptotic mechanism through procaspase-9 cleavage in both endothelial and tumor cells, which is mediated through cross-talk between alphavbeta3- and alphavbeta5-integrin receptors. As a point of reference, we employed the first three kringle domains of angiostatin (K1-3), fused with HSA, which, in contrast to CanHSA, act only on endothelial cells through alphavbeta3-integrin receptor-mediated activation of caspase-8 alone, without ensuing mitochondrial damage. Taken together, these results provide insights into how canstatin might exert its strong anticancer effect.

In vivo echographic evidence of tumoral vascularization and microenvironment interactions in metastatic orthotopic human neuroblastoma xenografts.

Human neuroblastoma (NB) is the second most frequent solid tumor of childhood and represents a highly heterogeneous disease at clinical and biologic levels. Little progress has been made to improve the poor prognosis of patients with high-stage NB. Tumor progression and metastatic dissemination still represent major obstacles to the successful treatment of advanced stage disease. In order to develop and evaluate new, targeted, therapeutic strategies, fully defined and biologically relevant in vivo models of NB are strongly needed. We have developed an orthotopic model of metastatic human NB in the nude mouse, using 2 well-characterized NB cell lines. Tumor growth, vascular properties and metastatic patterns were investigated using a sensitive and newly developed in vivo echographic technology in addition to immunohistochemistry and PCR analyses. Results show that implantation of low numbers of NB cells directly into the adrenal gland of nude mice resulted in rapid and homogeneous tumor growth without tumor morbidity. Nude mice were shown to rapidly develop highly vascularized adrenal tumors that selectively metastasized to the liver and bone marrow. In addition, the newly formed mouse vessels in orthotopic but not in heterotopic tumors, were found to express the highly angiogenic alphavbeta3 integrin marker, indicating the development of a truly malignant neovasculature in orthotopic conditions only. This observation confirms the impact of the regional microenvironment on tumor biology and suggests the existence of cross-talk with the tumor cells. In conclusion, such model faithfully reproduces the growth, vascular and metastatic patterns as observed in patients. It therefore represents a powerful and biologically relevant tool to improve our understanding of the biology of NB and to develop and assess new antiangiogenic and metastasis-targeted therapies.

[Interest of color Doppler ultrasonography to evaluate a new anti-angiogenic treatment with thalidomide in metastatic renal cell carcinoma]. / Intérêt de l'échographie-Doppler pour évaluer l'efficacité d'un traitement anti-angiogénique par thalidomide dans le cancer du rein métastatique.

PURPOSE: To assess the effectiveness of Doppler Ultrasonography (DUS) in evaluating the anti-angiogenic effect of thalidomide. MATERIALS AND METHODS: [corrected] Forty patients with metastases of cancer of the kidney were included in a phase II therapeutic trial. Twenty eight had tumor targets which could be evaluated by DUS. Before treatment, the volume was studied in 59 tumors and the vascularity in 54 tumors. During treatment, comparative DUS examinations were performed every 6 weeks. RESULTS: Before treatment, the mean tumor volume was 41 cm3, more than 10 vessels were visible in 57% of them. A correlation was noted between the size of the tumors and the degree of vascularity. A stabilisation of tumor volume correlated with a decrease in the number of vessels and an increase in vascularity preceding an increase in tumor volume were also observed. CONCLUSION: DUS is effective in assessing tumour size and vascularity.

Validation of a new method for quantifying in vivo murine tumor necrosis by sonography.

RATIONALE AND OBJECTIVES: At present, the gold standard to evaluate tumor necrosis is histology. We described here a new method to quantify the degree of tumor necrosis by ultrasonography. This technique combines ultrasound exploration of tissue and post-treatment of the numerical sequences using a dedicated software to evaluate backscattered power within the tumor. MATERIALS AND METHODS: In order to establish that the backscattered power could be considered as a relevant marker of tumor necrosis, we performed (1) intra- and interoperator reproducibility in estimation of tumor dimensions obtained on sonographic scans; and (2) intra- and interoperator reproducibility in quantification of backscattered power in postprocessing using the HDILab software. The third part of the study consisted of correlating the degree of tumor necrosis estimated by histology and the ultrasound backscattered power, both obtained on xenografted melanomas at different days after tumor transplantation. RESULTS: Results concerning tumor size estimations and quantification of echogenicity were reproducible (coefficient of variation < 4.33%). The degree of necrosis measured in histology and echogenicity were significantly negatively correlated (P < 0.003). CONCLUSION: In conclusion, backscattered power could be considered as a relevant parameter to quantify tumor necrosis in vivo.

High-frequency sonography and color Doppler in the management of pigmented skin lesions.

We aimed to evaluate high-frequency sonography (HFS) coupled with color Doppler in the management of pigmented skin lesions (PSL). HFS examination was performed in 111 patients with 130 PSL. A color Doppler study was conducted in 107 lesions, to assess intralesional vascularization. Imaging findings were compared with histologic diagnosis. In the case of melanoma, sonographic and histologic maximum thickness measurements were compared. HFS showed 114 of the 130 lesions. Among the detected lesions, HFS alone provided 100% sensitivity and 100% specificity in the distinction of melanoma/nevi from other lesions, and 100% sensitivity and 32% specificity in the distinction of melanomas from nonmelanoma lesions. Sonographic and histologic measurement of melanoma thickness strongly correlated (r > 0.96, p < 0.001). Color Doppler detection of intralesional vessels had a 100% specificity and 34% sensitivity in the distinction of melanomas from other PSL. HFS coupled with color Doppler is a simple, reliable tool for PSL management.