Neoantigen-targeted CD8+ T cell responses with PD-1 blockade therapy.

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1-14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.

Non-viral precision T cell receptor replacement for personalized cell therapy.

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Amplification of Oncolytic Vaccinia Virus Widespread Tumor Cell Killing by Sunitinib through Multiple Mechanisms.

Oncolytic viruses pose many questions in their use in cancer therapy. In this study, we assessed the potential of mpJX-594 (mouse-prototype JX-594), a replication-competent vaccinia virus administered by intravenous injection, to target the tumor vasculature, produce immune activation and tumor cell killing more widespread than the infection, and suppress invasion and metastasis. These actions were examined in RIP-Tag2 transgenic mice with pancreatic neuroendocrine tumors that developed spontaneously and progressed as in humans. mpJX-594 initially infected tumor vascular endothelial cells, leading to vascular pruning and prolonged leakage in tumors but not in normal organs; parallel effects were observed in U87 gliomas. Viral infection spread to tumor cells, where tumor cell killing was much more widespread than the infection. Widespread tumor cell killing at 5 days was prevented by depletion of CD8+ T lymphocytes and did not require GM-CSF, as mpJX-594 variants that expressed human, mouse, or no GM-CSF produced equivalent amounts of killing. The antivascular, antitumor, and antimetastatic effects of mpJX-594 were amplified by concurrent or sequential administration of sunitinib, a multitargeted receptor tyrosine kinase inhibitor. These effects were not mimicked by selective inhibition of VEGFR2 despite equivalent vascular pruning, but were accompanied by suppression of regulatory T cells and greater influx of activated CD8+ T cells. Together, our results showed that mpJX-594 targets tumor blood vessels, spreads secondarily to tumor cells, and produces widespread CD8+ T-cell-dependent tumor cell killing in primary tumors and metastases, and that these effects can be amplified by coadministration of sunitinib.Significance: These findings reveal multiple unrecognized features of the antitumor properties of oncolytic vaccinia viruses, all of which can be amplified by the multitargeted kinase inhibitor sunitinib. Cancer Res; 78(4); 922-37. ©2017 AACR.

Poxvirus-Based Active Immunotherapy with PD-1 and LAG-3 Dual Immune Checkpoint Inhibition Overcomes Compensatory Immune Regulation, Yielding Complete Tumor Regression in Mice.

Poxvirus-based active immunotherapies mediate anti-tumor efficacy by triggering broad and durable Th1 dominated T cell responses against the tumor. While monotherapy significantly delays tumor growth, it often does not lead to complete tumor regression. It was hypothesized that the induced robust infiltration of IFNγ-producing T cells into the tumor could provoke an adaptive immune evasive response by the tumor through the upregulation of PD-L1 expression. In therapeutic CT26-HER-2 tumor models, MVA-BN-HER2 poxvirus immunotherapy resulted in significant tumor growth delay accompanied by a robust, tumor-infiltrating T cell response that was characterized by low to mid-levels of PD-1 expression on T cells. As hypothesized, this response was countered by significantly increased PD-L1 expression on the tumor and, unexpectedly, also on infiltrating T cells. Synergistic benefit of anti-tumor therapy was observed when MVA-BN-HER2 immunotherapy was combined with PD-1 immune checkpoint blockade. Interestingly, PD-1 blockade stimulated a second immune checkpoint molecule, LAG-3, to be expressed on T cells. Combining MVA-BN-HER2 immunotherapy with dual PD-1 plus LAG-3 blockade resulted in comprehensive tumor regression in all mice treated with the triple combination therapy. Subsequent rejection of tumors lacking the HER-2 antigen by treatment-responsive mice without further therapy six months after the original challenge demonstrated long lasting memory and suggested that effective T cell immunity to novel, non-targeted tumor antigens (antigen spread) had occurred. These data support the clinical investigation of this triple therapy regimen, especially in cancer patients harboring PD-L1neg/low tumors unlikely to benefit from immune checkpoint blockade alone.

Magnetic resonance imaging of stem cell apoptosis in arthritic joints with a caspase activatable contrast agent.

About 43 million individuals in the U.S. encounter cartilage injuries due to trauma or osteoarthritis, leading to joint pain and functional disability. Matrix-associated stem cell implants (MASI) represent a promising approach for repair of cartilage defects. However, limited survival of MASI creates a significant bottleneck for successful cartilage regeneration outcomes and functional reconstitution. We report an approach for noninvasive detection of stem cell apoptosis with magnetic resonance imaging (MRI), based on a caspase-3-sensitive nanoaggregation MRI probe (C-SNAM). C-SNAM self-assembles into nanoparticles after hydrolysis by caspase-3, leading to 90% amplification of (1)H MR signal and prolonged in vivo retention. Following intra-articular injection, C-SNAM causes significant MR signal enhancement in apoptotic MASI compared to viable MASI. Our results indicate that C-SNAM functions as an imaging probe for stem cell apoptosis in MASI. This concept could be applied to a broad range of cell transplants and target sites.

Combination strategy targeting VEGF and HGF/c-met in human renal cell carcinoma models.

Alternative pathways to the VEGF, such as hepatocyte growth factor or HGF/c-met, are emerging as key players in tumor angiogenesis and resistance to anti-VEGF therapies. The aim of this study was to assess the effects of a combination strategy targeting the VEGF and c-met pathways in clear cell renal cell carcinoma (ccRCC) models. Male SCID mice (8/group) were implanted with 786-O tumor pieces and treated with either a selective VEGF receptor tyrosine kinase inhibitor, axitinib (36 mg/kg, 2×/day); a c-met inhibitor, crizotinib (25 mg/kg, 1×/day); or combination. We further tested this drug combination in a human ccRCC patient-derived xenograft, RP-R-01, in both VEGF-targeted therapy-sensitive and -resistant models. To evaluate the resistant phenotype, we established an RP-R-01 sunitinib-resistant model by continuous sunitinib treatment (60 mg/kg, 1×/day) of RP-R-01-bearing mice. Treatment with single-agent crizotinib reduced tumor vascularization but failed to inhibit tumor growth in either model, despite also a significant increase of c-met expression and phosphorylation in the sunitinib-resistant tumors. In contrast, axitinib treatment was effective in inhibiting angiogenesis and tumor growth in both models, with its antitumor effect significantly increased by the combined treatment with crizotinib, independently from c-met expression. Combination treatment also induced prolonged survival and significant tumor growth inhibition in the 786-O human RCC model. Overall, our results support the rationale for the clinical testing of combined VEGF and HGF/c-met pathway blockade in the treatment of ccRCC, both in first- and second-line setting.

Iron administration before stem cell harvest enables MR imaging tracking after transplantation.

PURPOSE: To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants. MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results. RESULTS: In vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling. CONCLUSION: Intravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures.

Inhibition of c-Met reduces lymphatic metastasis in RIP-Tag2 transgenic mice.

Inhibition of VEGF signaling can promote lymph node metastasis in preclinical models, but the mechanism is not fully understood, and successful methods of prevention have not been found. Signaling of hepatocyte growth factor (HGF) and its receptor c-Met can promote the growth of lymphatics and metastasis of some tumors. We sought to explore the contributions of c-Met signaling to lymph node metastasis after inhibition of VEGF signaling. In particular, we examined whether c-Met is upregulated in lymphatics in or near pancreatic neuroendocrine tumors in RIP-Tag2 transgenic mice and whether lymph node metastasis can be reduced by concurrent inhibition of VEGF and c-Met signaling. Inhibition of VEGF signaling by anti-VEGF antibody or sunitinib in mice from the age of 14 to 17 weeks was accompanied by more intratumoral lymphatics, more tumor cells inside lymphatics, and more lymph node metastases. Under these conditions, lymphatic endothelial cells, like tumor cells, had strong immunoreactivity for c-Met and phospho-c-Met. c-Met blockade by the selective inhibitor, PF-04217903, significantly reduced metastasis to local lymph nodes. Together, these results indicate that inhibition of VEGF signaling in RIP-Tag2 mice upregulates c-Met expression in lymphatic endothelial cells, increases the number of intratumoral lymphatics and number of tumor cells within lymphatics, and promotes metastasis to local lymph nodes. Prevention of lymph node metastasis by PF-04217903 in this setting implicates c-Met signaling in tumor cell spread to lymph nodes.

MR imaging features of gadofluorine-labeled matrix-associated stem cell implants in cartilage defects.

OBJECTIVES: The purpose of our study was to assess the chondrogenic potential and the MR signal effects of GadofluorineM-Cy labeled matrix associated stem cell implants (MASI) in pig knee specimen. MATERIALS AND METHODS: Human mesenchymal stem cells (hMSCs) were labeled with the micelle-based contrast agent GadofluorineM-Cy. Ferucarbotran-labeled hMSCs, non-labeled hMSCs and scaffold only served as controls. Chondrogenic differentiation was induced and gene expression and histologic evaluation were performed. The proportions of spindle-shaped vs. round cells of chondrogenic pellets were compared between experimental groups using the Fisher's exact test. Labeled and unlabeled hMSCs and chondrocytes in scaffolds were implanted into cartilage defects of porcine femoral condyles and underwent MR imaging with T1- and T2-weighted SE and GE sequences. Contrast-to-noise ratios (CNR) between implants and adjacent cartilage were determined and analyzed for significant differences between different experimental groups using the Kruskal-Wallis test. Significance was assigned for p<0.017, considering a Bonferroni correction for multiple comparisons. RESULTS: Collagen type II gene expression levels were not significantly different between different groups (p>0.017). However, hMSC differentiation into chondrocytes was superior for unlabeled and GadofluorineM-Cy-labeled cells compared with Ferucarbotran-labeled cells, as evidenced by a significantly higher proportion of spindle cells in chondrogenic pellets (p<0.05). GadofluorineM-Cy-labeled hMSCs and chondrocytes showed a positive signal effect on T1-weighted images and a negative signal effect on T2-weighted images while Ferucarbotran-labeled cells provided a negative signal effect on all sequences. CNR data for both GadofluorineM-Cy-labeled and Ferucarbotran-labeled hMSCs were significantly different compared to unlabeled control cells on T1-weighted SE and T2*-weighted MR images (p<0.017). CONCLUSION: hMSCs can be labeled by simple incubation with GadofluorineM-Cy. The labeled cells provide significant MR signal effects and less impaired chondrogenesis compared to Ferucarbotran-labeled hMSCs. Thus, GadoflurineM-Cy might represent an alternative MR cell marker to Ferucarbotran, which is not distributed any more in Europe or North America.

Controlling escape from angiogenesis inhibitors.

Selective inhibition of vascular endothelial growth factor (VEGF) increases the efficacy of chemotherapy and has beneficial effects on multiple advanced cancers, but response is often limited and the disease eventually progresses. Changes in the tumour microenvironment--hypoxia among them--that result from vascular pruning, suppressed angiogenesis and other consequences of VEGF inhibition can promote escape and tumour progression. New therapeutic approaches that target pathways that are involved in the escape mechanisms add the benefits of blocking tumour progression to those of slowing tumour growth by inhibiting angiogenesis.

Magnetic resonance imaging of ferumoxide-labeled mesenchymal stem cells in cartilage defects: in vitro and in vivo investigations.

The purpose of this study was to (1) compare three different techniques for ferumoxide labeling of mesenchymal stem cells (MSCs), (2) evaluate if ferumoxide labeling allows in vivo tracking of matrix-associated stem cell implants (MASIs) in an animal model, and (3) compare the magnetic resonance imaging (MRI) characteristics of ferumoxide-labeled viable and apoptotic MSCs. MSCs labeled with ferumoxide by simple incubation, protamine transfection, or Lipofectin transfection were evaluated with MRI and histopathology. Ferumoxide-labeled and unlabeled viable and apoptotic MSCs in osteochondral defects of rat knee joints were evaluated over 12 weeks with MRI. Signal to noise ratios (SNRs) of viable and apoptotic labeled MASIs were tested for significant differences using t-tests. A simple incubation labeling protocol demonstrated the best compromise between significant magnetic resonance signal effects and preserved cell viability and potential for immediate clinical translation. Labeled viable and apoptotic MASIs did not show significant differences in SNR. Labeled viable but not apoptotic MSCs demonstrated an increasing area of T2 signal loss over time, which correlated to stem cell proliferation at the transplantation site. Histopathology confirmed successful engraftment of viable MSCs. The engraftment of iron oxide-labeled MASIs by simple incubation can be monitored over several weeks with MRI. Viable and apoptotic MASIs can be distinguished via imaging signs of cell proliferation at the transplantation site.

Suppression of tumor invasion and metastasis by concurrent inhibition of c-Met and VEGF signaling in pancreatic neuroendocrine tumors.

UNLABELLED: Invasion and metastasis increase after the inhibition of VEGF signaling in some preclinical tumor models. In the present study we asked whether selective VEGF inhibition is sufficient to increase invasion and metastasis and whether selective c-Met inhibition is sufficient to block this effect. Treatment of pancreatic neuroendocrine tumors in RIP-Tag2 mice with a neutralizing anti-VEGF antibody reduced tumor burden but increased tumor hypoxia, hypoxia-inducible factor-1α, and c-Met activation and also increased invasion and metastasis. However, invasion and metastasis were reduced by concurrent inhibition of c-Met by PF-04217903 or PF-02341066 (crizotinib). A similar benefit was found in orthotopic Panc-1 pancreatic carcinomas treated with sunitinib plus PF-04217903 and in RIP-Tag2 tumors treated with XL184 (cabozantinib), which simultaneously blocks VEGF and c-Met signaling. These findings document that invasion and metastasis are promoted by selective inhibition of VEGF signaling and can be reduced by the concurrent inhibition of c-Met. SIGNIFICANCE: This report examines the mechanism of increased tumor aggressiveness after anti-VEGF therapy and presents evidence for roles of vascular pruning, hypoxia, and c-Met activation. The results show that simultaneous inhibition of c-Met and VEGF signaling not only slows tumor growth but also reduces invasion and metastasis.

Permeability to macromolecular contrast media quantified by dynamic MRI correlates with tumor tissue assays of vascular endothelial growth factor (VEGF).

PURPOSE: To correlate dynamic MRI assays of macromolecular endothelial permeability with microscopic area-density measurements of vascular endothelial growth factor (VEGF) in tumors. METHODS AND MATERIAL: This study compared tumor xenografts from two different human cancer cell lines, MDA-MB-231 tumors (n=5), and MDA-MB-435 (n=8), reported to express respectively higher and lower levels of VEGF. Dynamic MRI was enhanced by a prototype macromolecular contrast medium (MMCM), albumin-(Gd-DTPA)35. Quantitative estimates of tumor microvascular permeability (K(PS); µl/min × 100 cm(3)), obtained using a two-compartment kinetic model, were correlated with immunohistochemical measurements of VEGF in each tumor. RESULTS: Mean K(PS) was 2.4 times greater in MDA-MB-231 tumors (K(PS)=58 ± 30.9 µl/min × 100 cm(3)) than in MDA-MB-435 tumors (K(PS)=24 ± 8.4 µl/min × 100 cm(3)) (p<0.05). Correspondingly, the area-density of VEGF in MDA-MB-231 tumors was 2.6 times greater (27.3 ± 2.2%, p<0.05) than in MDA-MB-435 cancers (10.5 ± 0.5%, p<0.05). Considering all tumors without regard to cell type, a significant positive correlation (r=0.67, p<0.05) was observed between MRI-estimated endothelial permeability and VEGF immunoreactivity. CONCLUSION: Correlation of MRI assays of endothelial permeability to a MMCM and VEGF immunoreactivity of tumors support the hypothesis that VEGF is a major contributor to increased macromolecular permeability in cancers. When applied clinically, the MMCM-enhanced MRI approach could help to optimize the appropriate application of VEGF-inhibiting therapy on an individual patient basis.

Increased vascular delivery and efficacy of chemotherapy after inhibition of platelet-derived growth factor-B.

Inhibition of platelet-derived growth factor-B (PDGF-B) has multiple effects on tumors, including loss of pericytes, regression of some vessels, normalization of other vessels, and reduction of interstitial pressure. PDGF-B inhibition also increases the efficacy of cancer therapeutics, but the role on tumor vessel efficiency and drug delivery is unclear. We sought to determine whether inhibition of PDGF-B signaling can increase delivery and efficacy of cyclophosphamide in Lewis lung carcinomas or RIP-Tag2 tumors. PDGF-B blockade in Lewis lung carcinoma tumors by the DNA aptamer AX102 for 14 days increased the number of perfused tumor vessels marked by lectin in the bloodstream by 50%. AX102 also increased the width of sleeves of viable tumor cells around blood vessels by 66%, increased tumor cell proliferation by 90%, and increased intratumoral delivery of Hoechst 33342 by 78%. A low dose of cyclophosphamide (20 mg/kg) reduced tumor cell proliferation by 31% when combined with AX102 but not when given alone. Synergy of cyclophosphamide and AX102 on tumor cell proliferation also was found in RIP-Tag2 tumors. Similarly, the PDGF receptor signaling inhibitor imatinib increased delivery of cyclophosphamide and reduced tumor burden in RIP-Tag2 mice, without evidence of tumor cell sensitization to chemotherapy. Together, these findings indicate that inhibition of PDGF-B signaling promotes the delivery and efficacy of chemotherapeutic agents by increasing the efficiency of tumor blood vessels.

VEGF and c-Met blockade amplify angiogenesis inhibition in pancreatic islet cancer.

Angiogenesis inhibitors that block VEGF receptor (VEGFR) signaling slow the growth of many types of tumors, but eventually the disease progresses. Multiple strategies are being explored to improve efficacy by concurrent inhibition of other functionally relevant receptor tyrosine kinases (RTK). XL880 (foretinib, GSK1363089) and XL184 (cabozantinib) are small-molecule inhibitors that potently block multiple RTKs, including VEGFR and the receptor of hepatocyte growth factor c-Met, which can drive tumor invasion and metastasis. This study compared the cellular effects of XL880 and XL184 with those of an RTK inhibitor (XL999) that blocks VEGFR but not c-Met. Treatment of RIP-Tag2 mice with XL999 resulted in 43% reduction in vascularity of spontaneous pancreatic islet tumors over 7 days, but treatment with XL880 or XL184 eliminated approximately 80% of the tumor vasculature, reduced pericytes and empty basement membrane sleeves, caused widespread intratumoral hypoxia and tumor cell apoptosis, and slowed regrowth of the tumor vasculature after drug withdrawal. Importantly, XL880 and XL184 also decreased invasiveness of primary tumors and reduced metastasis. Overall, these findings indicate that inhibition of c-Met and functionally related kinases amplifies the effects of VEGFR blockade and leads to rapid, robust, and progressive regression of tumor vasculature, increased intratumoral hypoxia and apoptosis, and reduced tumor invasiveness and metastasis.

Labeling human mesenchymal stem cells with fluorescent contrast agents: the biological impact.

PURPOSE: This study aims to determine the effect of human mesenchymal stem cell (hMSC) labeling with the fluorescent dye DiD and the iron oxide nanoparticle ferucarbotran on chondrogenesis. PROCEDURES: hMSCs were labeled with DiD alone or with DiD and ferucarbotran (DiD/ferucarbotran). hMSCs underwent confocal microscopy, optical imaging (OI), and magnetic resonance (MR) imaging. Chondrogenesis was induced by transforming growth factor-b and confirmed by histopathology and glycosaminoglycan (GAG) production. Data of labeled and unlabeled hMSCs were compared with a t test. RESULTS: Cellular uptake of DiD and ferucarbotran was confirmed with confocal microscopy. DiD labeling caused a significant fluorescence on OI, and ferucarbotran labeling caused a significant T2* effect on MR images. Compared to nonlabeled controls, progenies of labeled MSCs exhibited similar chondrocyte morphology after chondrogenic differentiation, but the labeled cells demonstrated significantly reduced GAG production (p < 0.05). CONCLUSION: DiD and DiD/ferucarbotran labeling of hMSC does not interfere with cell viability or morphologic differentiation into chondrocytes, but labeled cells exhibit significantly less GAG production compared to unlabeled cells.

MR signal characteristics of viable and apoptotic human mesenchymal stem cells in matrix-associated stem cell implants for treatment of osteoarthritis.

OBJECTIVE: To compare magnetic resonance (MR) signal characteristics of contrast agent-labeled apoptotic and viable human mesenchymal stem cells (hMSCs) in matrix-associated stem cell implants. METHODS: hMSCs were labeled with Food and Drug Administration-approved ferumoxides nanoparticles. One group (A) remained untreated whereas a second group (B) underwent mitomycin C-induced apoptosis induction. Viability of group A and apoptosis of group B was confirmed by caspase-assays and terminal dUTP nick-end labeling (TUNEL) stains. Labeled viable hMSCs, unlabeled viable hMSCs, labeled apoptotic hMSCs, and unlabeled apoptotic hMSCs (n = 7 samples each) in an agarose scaffold were implanted into cartilage defects of porcine patellae specimens and underwent MR imaging at 7 T, using T1-weighted spin-echo sequences, T2-weighted spin-echo sequences, and T2*-weighted gradient-echo sequences. Signal-to-noise ratios (SNR) of the implants were calculated and compared between different experimental groups using linear mixed regression models. RESULTS: Ferumoxides-labeled hMSCs provided a strong negative T2 and T2*-enhancement. Corresponding SNR data of labeled hMSCs were significantly lower compared with unlabeled controls (P < 0.05). Apoptosis induction resulted in a significant signal decline of ferumoxides-labeled hMSC transplants on short echo time T2-weighted spinecho sequences. SNR data of labeled apoptotic hMSCs were significantly lower compared with labeled viable hMSCs (P < 0.05). CONCLUSION: Apoptosis of transplanted ferumoxides-labeled stem cells in cartilage defects can be visualized noninvasively by a significant signal decline on T2-weighted MR images. The described MR signal characteristics may serve as a noninvasive outcome measure for the assessment of matrix-associated stem cell implants in clinical practice. Additional studies are needed to further enhance the observed differences between viable and apoptotic cells, for example, by further optimizing the applied MR pulse sequence parameters or intracellular contrast agent concentration.

The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells.

For in vivo applications of magnetically labeled stem cells, biological effects of the labeling procedure have to be precluded. This study evaluates the effect of different ferucarbotran cell labeling protocols on chondrogenic differentiation of human mesenchymal stem cells (hMSC) as well as their implications for MR imaging. hMSC were labeled with ferucarbotran using various protocols: cells were labeled with 100 microg Fe/ml for 4 and 18 h and additional samples were cultured for 6 or 12 days after the 18 h labeling. Supplementary samples were labeled by transfection with protamine sulfate. Iron uptake was quantified by ICP-spectrometry and labeled cells were investigated by transmission electron microscopy and by immunostaining for ferucarbotran. The differentiation potential of labeled cells was compared with unlabeled controls by staining with Alcian blue and Hematoxylin and Eosin, then quantified by measurements of glucosaminoglycans (GAG). Contrast agent effect at 3 T was investigated on days 1 and 14 of chondrogenic differentiation by measuring signal-to-noise ratios on T(2)-SE and T(2)*-GE sequences. Iron uptake was significant for all labeling protocols (p < 0.05). The uptake was highest after transfection with protamine sulfate (25.65 +/- 3.96 pg/cell) and lowest at an incubation time of 4 h without transfection (3.21 +/- 0.21 pg/cell). While chondrogenic differentiation was decreased using all labeling protocols, the decrease in GAG synthesis was not significant after labeling for 4 h without transfection. After labeling by simple incubation, chondrogenesis was found to be dose-dependent. MR imaging showed markedly lower SNR values of all labeled cells compared with the unlabeled controls. This contrast agent effect persisted for 14 days and the duration of differentiation. Magnetic labeling of hMSC with ferucarbotran inhibits chondrogenesis in a dose-dependent manner when using simple incubation techniques. When decreasing the incubation time to 4 h, inhibition of chondrogenesis was not significant.

Correlative dynamic contrast MRI and microscopic assessments of tumor vascularity in RIP-Tag2 transgenic mice.

The purpose of this study was to define the feasibility of dynamic contrast-enhanced magnetic resonance imaging (MRI) to estimate the vascular density and leakiness of spontaneous islet cell tumors in RIP-Tag2 transgenic mice. Dynamic T(1)-weighted spoiled gradient echo (SPGR) imaging at 2.0 T was performed in 17 RIP-Tag2 mice using a prototype blood pool macromolecular contrast medium (MMCM), albumin-(Gd-DTPA)(35). Kinetic analysis of the dynamic enhancement responses based on a two-compartment model was used to estimate fractional plasma volume (fPV) and the coefficient of endothelial permeability (K(PS)) for each tumor. The MRI estimate of fPV was correlated on a tumor-by-tumor basis with corresponding microscopic measurements of vascular density. The fPV assays by MMCM-enhanced imaging ranged from 2.4%-14.1% of tissue volume. Individual tumor fPV values correlated significantly (r = 0.79, P < 0.001) with the corresponding microscopic estimates of vascularity consisting of the combined area densities of lectin-perfused microvessels plus erythrocyte-stained blood lakes. A biotinylated derivative of the albumin-based MMCM confirmed extravasation of the contrast agent from some tumor blood vessels and accumulation in 25% of blood lakes. The K(PS) values ranged from 0 (no detectable leak) to 0.356 mL/min/100 cm(3). Dynamic MMCM-enhanced MRI is feasible in RIP-Tag2 pancreatic tumors, yielding estimates of vascular permeability and microscopically validated measurements of vascular richness.