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Evaluation of efficacy of anti-xCT vaccination on TUBO or 4T1 derived lung metastases

Soft agar colony formation assay of cells transfected with xCT

Analysis of the cellular response induced by anti-xCT vaccination

qPCR analysis of xCT expression in tumorspheres

Effect of immunotherapy with non CSC-specific targets

Cellular localization and correlation with breast cancer patient clinical outcome (CO) of genes differentially expressed in TUBO cells and tumorspheres

Resistance to therapy and lack of curative treatments for metastatic breast cancer suggest that current therapies may be missing the subpopulation of chemoresistant and radioresistant cancer stem cells (CSC). The ultimate success of any treatment may well rest on CSC eradication, but specific anti-CSC therapies are still limited. A comparison of the transcriptional profiles of murine Her2+ breast tumor TUBO cells and their derived CSC-enriched tumorspheres has identified xCT, the functional subunit of the cystine/glutamate antiporter system xc−, as a surface protein that is upregulated specifically in tumorspheres. We validated this finding by cytofluorimetric analysis and immunofluorescence in TUBO-derived tumorspheres and in a panel of mouse and human triple negative breast cancer cell-derived tumorspheres. We further show that downregulation of xCT impaired tumorsphere generation and altered CSC intracellular redox balance in vitro, suggesting that xCT plays a functional role in CSC biology. DNA vaccination based immunotargeting of xCT in mice challenged with syngeneic tumorsphere-derived cells delayed established subcutaneous tumor growth and strongly impaired pulmonary metastasis formation by generating anti-xCT antibodies able to alter CSC self-renewal and redox balance. Finally, anti-xCT vaccination increased CSC chemosensitivity to doxorubicin in vivo, indicating that xCT immunotargeting may be an effective adjuvant to chemotherapy. Cancer Res; 76(1); 62–72. ©2015 AACR.

Immunofluorescence analysis of xCT expression in human tissues

Hydrogels that provide mechanical support and sustainably release therapeutics have been used to treat tendon injuries. However, most hydrogels are insufficiently tough, release drugs in bursts, and require cell infiltration or suturing to integrate with surrounding tissue. Here we report that a hydrogel serving as a high-capacity drug depot and combining a dissipative tough matrix on one side and a chitosan adhesive surface on the other side supports tendon gliding and strong adhesion (larger than 1,000 J msup-2/sup) to tendon on opposite surfaces of the hydrogel, as we show with porcine and human tendon preparations during cyclic-friction loadings. The hydrogel is biocompatible, strongly adheres to patellar, supraspinatus and Achilles tendons of live rats, boosted healing and reduced scar formation in a rat model of Achilles-tendon rupture, and sustainably released the corticosteroid triamcinolone acetonide in a rat model of patellar tendon injury, reducing inflammation, modulating chemokine secretion, recruiting tendon stem and progenitor cells, and promoting macrophage polarization to the M2 phenotype. Hydrogels with 'Janus' surfaces and sustained-drug-release functionality could be designed for a range of biomedical applications.

A key to enhance the low translatability of preclinical drug discovery are in vitro human three-dimensional (3D) microphysiological systems (MPS). Here, we show a new method for automated engineering of 3D human skeletal muscle models in microplates and functional compound screening to address the lack of muscle wasting disease medication. To this end, we adapted our recently described 24-well plate 3D bioprinting platform with a printhead cooling system to allow microvalve-based drop-on-demand printing of cell-laden Matrigel containing primary human muscle precursor cells. Mini skeletal muscle models develop within a week exhibiting contractile, striated myofibers aligned between two attachment posts. As an in vitro exercise model, repeated high impact stimulation of contractions for 3 h by a custom-made electrical pulse stimulation (EPS) system for 24-well plates induced interleukin-6 myokine expression and Akt hypertrophy pathway activation. Furthermore, the known muscle stimulators caffeine and Tirasemtiv acutely increase EPS-induced contractile force of the models. This validated new human muscle MPS will benefit development of drugs against muscle wasting diseases. Moreover, our Matrigel 3D bioprinting platform will allow engineering of non-self-organizing complex human 3D MPS., Alave-Furrer et al adapted their recently-developed 3D bioprinting platform to allow microvalve-based drop-on-demand printing of cell-laden Matrigel containing primary human muscle precursor cells. Their bioprinting platform recapitulated aspects of exercise and pharmacological responses and thus could aid the engineering of more complex 3D microphysiological systems.

Effects of repeated injection of monosodium urate (MSU) crystals, in combination with lipopolysaccharide (LPS), into rat knee joints every two weeks for a maximum of five administrations were investigated. Joint swelling, nociception and hard/soft tissue changes were assessed longitudinally by non-invasive imaging. MSU crystals induced joint swelling, synovial membrane thickening, fibrosis of the infrapatellar fat pad, tidemark breaching, and cartilage invasion by inflammatory cells. Several inflammatory proteins were present in synovial fluid. A higher sensitivity to mechanical stimulus was detected in paws of limbs receiving MSU/LPS compared to saline-injected limbs. In MSU/LPS-challenged joints, magnetic resonance imaging (MRI) revealed increased synovial fluid volume in the posterior region of the joint, alterations in the infrapatellar fat pad reflecting a progressive decrease of fat volume and fibrosis formation, and a significant increase in the relaxation time T2 in femoral cartilage, consistent with a reduction of proteoglycan content. MRI also showed cyst formation in the tibia, femur remodeling, and T2 reductions in extensor muscles consistent with fibrosis development. Repeated intra-articular MSU/LPS injections in the rat knee joint induced pathology in multiple tissues and may be a useful means to investigate the relationship between urate crystal deposition and the development of degenerative joint disease.

Assessment of myelin integrity in peripheral nerve injuries and pathologies has largely been limited to post-mortem analysis owing to the difficulty in obtaining biopsies without affecting nerve function. This is further encumbered by the small size of the tissue and its location. Therefore, the development of robust, non-invasive methods is highly attractive. In this study, we used magnetic resonance imaging (MRI) techniques, including magnetization transfer ratio (MTR), to longitudinally and non-invasively characterize both the sciatic nerve crush and lysolecithin (LCP) demyelination models of peripheral nerve injury in rodents. Electrophysiological, gene expression and histological assessments complemented the extensive MRI analyses in young and aged animals. In the nerve crush model, MTR analysis indicated a slower recovery in regions distal to the site of injury in aged animals, as well as incomplete recovery at six weeks post-crush when analyzing across the entire nerve surface. Similar regional impairments were also found in the LCP demyelination model. This research underlines the power of MTR for the study of peripheral nerve injury in small tissues such as the sciatic nerve of rodents and contributes new knowledge to the effect of aging on recovery after injury. A particular advantage of the approach is the translational potential to human neuropathies.

Hydrogels are under active development for controlled drug delivery, but their clinical translation has been limited by low drug loading capacity, deficiencies in mechanical toughness and storage stability, and poor control over the drug release that often results in burst release and short release duration. This work reports a design of composite clay hydrogels, which simultaneously achieve a spectrum of mechanical, storage, and drug loading/releasing properties to address the critical needs from translational perspectives. The clay nanoparticles provide large surface areas to adsorb biological drugs, and assemble into microparticles that are physically trapped within and toughen hydrogel networks. The composite hydrogels demonstrate feasibility of storage, and extended release of large quantities of an insulin-like growth factor-1 mimetic protein (8 mg/mL) over four weeks. The release rate was primarily governed by ionic exchange and could be upregulated by low pH, which is typical for injured tissues. A rodent model of Achilles tendon injury was used to demonstrate that the composite hydrogels allow for highly extended and localized release of biological drugs in vivo, while demonstrating biodegradation and biocompatibility. These attributes make the composite hydrogel a promising system for drug delivery and regenerative medicine. [46,47,48]

Objective Colony-stimulating factor 1 receptor (CSF-1R) essentially modulates monocyte proliferation, migration, and activation, which are considered important for the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine CSF-1R expression in human RA as well as the efficacy of a specific anti–CSF-1R monoclonal antibody (AFS98) in 2 different animal models of RA. Methods CSF-1R expression was examined in blood, synovium, and bone samples from RA patients, osteoarthritis (OA) patients, and healthy subjects. The efficacy of AFS98 was examined by clinical assessment, histology, and bone histomorphometry in collagen-induced arthritis (CIA) and serum-transfer arthritis. Results CSF-1R expression was increased in the synovium of RA patients compared to OA patients and healthy controls in fibroblast-like synoviocytes, follicular dendritic cells, macrophages, and osteoclasts. Circulating RA monocytes and neutrophils but not lymphocytes were CSF-1R+. In mice, blockade of CSF-1R abrogated cartilage damage, bone erosion, and systemic bone loss, and this was associated with the depletion of osteoclasts in both models. While blockade of CSF-1R did not affect inflammation in passive serum-transfer arthritis, it significantly reduced inflammation in CIA, and this was associated with the absence of synovial macrophages and reduced splenic CD11b+Gr-1− monocytes. Conclusion CSF-1R was broadly expressed in human RA. Blockade of CSF-1R protected against bone and cartilage destruction in both mouse models and also showed significant antiinflammatory effects in the CIA model. These data provide evidence for CSF-1R as a therapeutic target in RA.

Organ-specific cell types are maintained by tissue homeostasis and may vary in nature and/or frequency in pathological situations. Moreover, within a cell lineage, some sub-populations, defined by combinations of cell-surface markers, may have specific functions. Dendritic cells are the epitome of such a population as they may be subdivided into discrete sub-groups with defined functions in specific compartments of various organs. Technically, to study the distribution of DC sub-populations, it involves performing multiparametric immunofluorescence on well-conserved organ structures. However, immunodetection may be impacted by protein cross-linking and antigenic epitope masking by the use of 10% neutral-buffered formalin. To circumvent this and to preserve a good morphological tissue structure, we evaluated alternative fixatives such as Periodate Lysine Paraformaldehyde or Tris Zinc fixatives in combination with other embedding techniques. The cryosection protocols were adapted for optimal antigen detection but offered a poor morphological preservation. We therefore developed a new methodology based on Tris Zinc fixative, gelatin-sucrose embedding and freezing. Using multiple DC markers, we demonstrate that this treatment is an optimal protocol for cell-surface marker detection on high-quality tissue sections.

Ankylosing spondylitis is a common rheumatic disease involving both inflammatory erosive osteopenia and bony overgrowth. Main disease features are recapitulated in small rodents challenged with complete Freund’s adjuvant. MRI was used to follow longitudinally in vivo changes induced in the rat spine and micro-CT as terminal assessment of bone damage. Histochemistry methods were used to validate these imaging modalities in view of preclinical drug testing and translational applications of spine imaging. Animals were examined using a 3D fat-suppressed gradient-echo sequence, following the injection of gadolinium. At the end of the study, spines were excised for micro-CT and histological examination. Signals reflecting inflammation were detected at levels L5-L6 of the lumbar spine throughout the experimental period, peaking at day 27 after adjuvant. At day 14 the inflammatory response occurred along ligaments but it expanded to nearby soft tissues at later time points. From day 27 onwards inflammation was also detected within the bone, in areas where erosion occurred, and bone-like structures were formed. Micro-CT showed bone remodeling. Histology of isolated spines confirmed the inflammation and bone remodeling observed in vivo. The present study including three complementary approaches clearly demonstrates the potential of imaging for longitudinal assessments of changes in the spine in this animal model in view of preclinical pharmacological studies. The excellent correlation seen between the in vivo images and the histology underlines its fundamental role in the validation of non-invasive imaging readouts.

Resistance to therapy and lack of curative treatments for metastatic breast cancer suggest that current therapies may be missing the subpopulation of chemoresistant and radioresistant cancer stem cells (CSC). The ultimate success of any treatment may well rest on CSC eradication, but specific anti-CSC therapies are still limited. A comparison of the transcriptional profiles of murine Her2+ breast tumor TUBO cells and their derived CSC-enriched tumorspheres has identified xCT, the functional subunit of the cystine/glutamate antiporter system xc−, as a surface protein that is upregulated specifically in tumorspheres. We validated this finding by cytofluorimetric analysis and immunofluorescence in TUBO-derived tumorspheres and in a panel of mouse and human triple negative breast cancer cell-derived tumorspheres. We further show that downregulation of xCT impaired tumorsphere generation and altered CSC intracellular redox balance in vitro, suggesting that xCT plays a functional role in CSC biology. DNA vaccination based immunotargeting of xCT in mice challenged with syngeneic tumorsphere-derived cells delayed established subcutaneous tumor growth and strongly impaired pulmonary metastasis formation by generating anti-xCT antibodies able to alter CSC self-renewal and redox balance. Finally, anti-xCT vaccination increased CSC chemosensitivity to doxorubicin in vivo, indicating that xCT immunotargeting may be an effective adjuvant to chemotherapy. Cancer Res; 76(1); 62–72. ©2015 AACR.

Oncolytic virotherapy is an emergent promising therapeutic approach for the treatment of cancer. We have constructed a vaccinia virus (WR strain) deleted for thymidine kinase (TK) and ribonucleotide reductase (RR) genes that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. We evaluated this construct (VV-FCU1) in the orthotopic model of renal carcinoma (RenCa). Systemic administration of VV-FCU1 resulted in orthotopic tumor growth inhibition, despite temporary expression of viral proteins. VV-FCU1 treatment was associated with an infiltration of tumors by CD8+ T lymphocytes and a decrease in the proportion of infiltrating Tregs, thus modifying the ratio of CD8+/CD4+ Treg in favor of CD8+cytotoxic T cells. We demonstrated that VV-FCU1 treatment prolonged survival of animals implanted with RenCa cells in kidney. Depletion of CD8+ T cells abolished the therapeutic effect of VV-FCU1 while depletion of CD4+ T cells enhanced its protective activity. Administration of the prodrug 5-fluorocytosine (5-FC) resulted in a sustained control of tumor growth but did not extend survival. This study shows the importance of CD4+ and CD8+ T cells in vaccinia virus-mediated oncolytic virotherapy and suggests that this approach may be evaluated for the treatment of human renal cell carcinoma.

Colony-stimulating factor 1 receptor (CSF-1R) essentially modulates monocyte proliferation, migration, and activation, which are considered important for the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine CSF-1R expression in human RA as well as the efficacy of a specific anti-CSF-1R monoclonal antibody (AFS98) in 2 different animal models of RA.CSF-1R expression was examined in blood, synovium, and bone samples from RA patients, osteoarthritis (OA) patients, and healthy subjects. The efficacy of AFS98 was examined by clinical assessment, histology, and bone histomorphometry in collagen-induced arthritis (CIA) and serum-transfer arthritis.CSF-1R expression was increased in the synovium of RA patients compared to OA patients and healthy controls in fibroblast-like synoviocytes, follicular dendritic cells, macrophages, and osteoclasts. Circulating RA monocytes and neutrophils but not lymphocytes were CSF-1R+. In mice, blockade of CSF-1R abrogated cartilage damage, bone erosion, and systemic bone loss, and this was associated with the depletion of osteoclasts in both models. While blockade of CSF-1R did not affect inflammation in passive serum-transfer arthritis, it significantly reduced inflammation in CIA, and this was associated with the absence of synovial macrophages and reduced splenic CD11b+Gr-1- monocytes.CSF-1R was broadly expressed in human RA. Blockade of CSF-1R protected against bone and cartilage destruction in both mouse models and also showed significant antiinflammatory effects in the CIA model. These data provide evidence for CSF-1R as a therapeutic target in RA.

The aim of this study was to monitor in vivo with low field MRI growth of a murine orthotopic glioma model following a suicide gene therapy.The gene therapy consisted in the stereotactic injection in the mice brain of a modified vaccinia virus Ankara (MVA) vector encoding for a suicide gene (FCU1) that transforms a non toxic prodrug 5-fluorocytosine (5-FC) to its highly cytotoxic derivatives 5-fluorouracil (5-FU) and 5'-fluorouridine-5'monophosphate (5'-FUMP). Using a warmed-up imaging cell, sequential 3D T1 and T2 0.1T MRI brain examinations were performed on 16 Swiss female nu/nu mice bearing orthotopic human glioblastoma (U87-MG cells). The 6-week in vivo MRI follow-up consisted in a weekly measurement of the intracerebral tumor volume leading to a total of 65 examinations. Mice were divided in four groups: sham group (n=4), sham group treated with 5-FC only (n=4), sham group with injection of MVA-FCU1 vector only (n=4), therapy group administered with MVA-FCU1 vector and 5-FC (n=4). Measurements of tumor volumes were obtained after manual segmentation of T1- and T2-weighted images.Intra-observer and inter-observer tumor volume measurements show no significant differences. No differences were found between T1 and T2 volume tumor doubling times between the three sham groups. A significant statistical difference (p0.05) in T1 and T2 volume tumor doubling times between the three sham groups and the animals treated with the intratumoral injection of MVA-FCU1 vector in combination with 2 weeks per os 5-FC administration was demonstrated.Preclinical low field MRI was able to monitor efficacy of suicide gene therapy in delaying the tumor growth in an in vivo mouse model of orthotopic glioblastoma.

To redress the immune imbalances created by pathologies such as cancer, it would be beneficial to create novel cytokine molecules, which combine desired cytokine activities with reduced toxicities. Due to their divergent but complementary activities, it is of interest to combine interleukin-2 (IL-2) and IL-18 into one recombinant molecule for immunotherapy. Evaluation of a fusokine protein that combines murine IL-2/IL-18 shows that it is stable, maintains IL-2 and IL-18 bioactivities, has notably reduced IL-2 associated toxicities, and has a novel lymphocyte-stimulating activity. An adeno-viral expression system was used to explore the biology of this “fusokine”. Inclusion of the IL-18 prosequence (proIL-18) increases the expression, secretion, and potency of this fusokine. In vivo gene transfer experiments show that Ad-IL-2/proIL-18 dramatically outdoes Ad-IL-2, Ad-proIL-18, or the combination of both, by inducing high rates of tumor rejection in several murine models. Both innate and adaptive effector mechanisms are required for this antitumor activity.

Ad CFTR, a replication-deficient adenovirus expressing the human cystic fibrosis transmembrane conductance regulator (CFTR), was administered by aerosolization in a single escalating dose to three pairs (cohorts) of cystic fibrosis (CF) patients. Buffer only was administered to the nose and lungs 9-14 days before nasal instillation of virus followed the day after by aerosolization of Ad CFTR to the lung. Nasal doses (defined in terms of viral plaque forming units, pfu) were 10(5), 10(7), and 4 x 10(8), whereas aerosolized doses were 10(7), 10(8), 5.4 x 10(8) for each cohort, respectively. No acute toxic effects were observed in the first 4 weeks after virus treatment. Shedding of infectious Ad CFTR was never detected, whereas detection of vector DNA sequences and CFTR expression demonstrated DNA transfer to the nose and airways of patients. No significant deviations in immunological and inflammatory parameters were observed in serum and in bronchoalveolar lavage (BAL). Importantly, for all patients, the serum anti-adenovirus antibody levels did not change significantly from baseline and no antibodies against adenovirus were found in BAL.

The chicken chorioallantoic membrane (CAM) assay represents one of the most widely used in vivo screening assay for genes with angiogenic (blood vessel-inducing) or angiostatic (inhibition of vessel formation or their destruction) activities. Here we show that adenovirus gene transfer vectors infect cells in the CAM and lead to expression of the viral transgene. Furthermore, infection with an adenovirus vector containing the human vascular endothelial growth factor gene induced the formation of new blood vessels. This improved method saves a considerable amount of time in the identification of genes that can influence blood vessel formation because the expensive and time-consuming production and purification of recombinant protein can be omitted.

Background Primary cutaneous lymphomas (CLs), characterized by an accumulation of clonal T or B lymphocytes preferentially localized in the skin, have been successfully treated with interferons (IFNs) which counterbalance the Th2-immunosuppressive state associated with this pathology. In a phase I/II clinical trial, we correlated the local immune infiltrate and the anti-tumor effects of repeated intralesional administrations of an adenovirus vector expressing human interferon-gamma (IFN-g) termed TG1042, in patients with advanced primary cutaneous T-cell lymphomas (CTCL) or multilesional cutaneous B-cell lymphomas (CBCL). Methods For each patient, variation in time of specific lymphocyte populations, defined by immunohistochemical stainings, was assessed in biopsies of injected lesions. For each patient, the change in local immune response was associated with the patient’s objective response at the end of the study. Results Immunohistochemical analyses of biopsies indicate that infiltration of CD8+ T lymphocytes and of TIA-1+ cytotoxic T-cells in lesions injected with TG1042 correlates with clinical benefit. Conclusions These data suggest for the first time that a CD8+ cytotoxic infiltrate, induced by local expression of IFN-g correlates with a clinical response. Trial registration The phase I step (TG1042.01) does not have a registration number. The phase II step (TG1042.06) registration number was NCT00394693.