Reinforced antimyeloma therapy via dual-lymphoid activation mediated by a panel of antibodies armed with bridging-BiTE.

Immunotherapy using bispecific antibodies including bispecific T-cell engager (BiTE) has the potential to enhance the efficacy of treatment for relapsed/refractory multiple myeloma. However, myeloma may still recur after treatment because of downregulation of a target antigen and/or myeloma cell heterogeneity. To strengthen immunotherapy for myeloma while overcoming its characteristics, we have newly developed a BiTE-based modality, referred to as bridging-BiTE (B-BiTE). B-BiTE was able to bind to both a human immunoglobulin G-Fc domain and the CD3 molecule. Clinically available monoclonal antibodies (mAbs) were bound with B-BiTE before administration, and the mAb/B-BiTE complex induced antitumor T-cell responses successfully while preserving and supporting natural killer cell reactivity, resulting in enhanced antimyeloma effects via dual-lymphoid activation. In contrast, any unwanted off-target immune-cell reactivity mediated by mAb/B-BiTE complexes or B-BiTE itself appeared not to be observed in vitro and in vivo. Importantly, sequential immunotherapy using 2 different mAb/B-BiTE complexes appeared to circumvent myeloma cell antigen escape, and further augmented immune responses to myeloma relative to those induced by mAb/B-BiTE monotherapy or sequential therapy with 2 mAbs in the absence of B-BiTE. Therefore, this modality facilitates easy and prompt generation of a broad panel of bispecific antibodies that can induce deep and durable antitumor responses in the presence of clinically available mAbs, supporting further advancement of reinforced immunotherapy for multiple myeloma and other refractory hematologic malignancies.

Reduced dynamic loads due to hip dislocation induce acetabular cartilage degeneration by IL-6 and MMP3 via the STAT3/periostin/NF-κB axis.

Developmental dysplasia of the hip (DDH) is characterized by anatomical abnormalities of the hip joint, ranging from mild acetabular dysplasia to hip subluxation and eventually dislocation. The mechanism underlying the cartilage degeneration of the hip joints exposed to reduced dynamic loads due to hip dislocation remains unknown. We established a rodent hip dislocation (disarticulation; DA) model of DDH (DA-DDH rats and mice) by swaddling. Expression levels of periostin (Postn) and catabolic factors, such as interleukin-6 (IL-6) and matrix metalloproteinase 3 (Mmp3), increased and those of chondrogenic markers decreased in the acetabular cartilage of the DA-DDH models. Postn induced IL-6 and Mmp3 expression in chondrocytes through integrin αVß3, focal adhesion kinase, Src, and nuclear factor-κB (NF-κB) signaling. The microgravity environment created by a random positioning machine induced Postn expression in chondrocytes through signal transducer and activator of transcription 3 (STAT3) signaling. IL-6 stimulated Postn expression via STAT3 signaling. Furthermore, cartilage degeneration was suppressed in the acetabulum of Postn-/- DA-DDH mice compared with that in the acetabulum of wild type DA-DDH mice. In summary, reduced dynamic loads due to hip dislocation induced acetabular cartilage degeneration via IL-6 and MMP3 through STAT3/periostin/NF-κB signaling in the rodent DA-DDH models.

Computer-Aided Detection of Quantitative Signatures for Breast Fibroepithelial Tumors Using Label-Free Multi-Photon Imaging.

Fibroadenomas (FAs) and phyllodes tumors (PTs) are major benign breast tumors, pathologically classified as fibroepithelial tumors. Although the clinical management of PTs differs from FAs, distinction by core needle biopsy diagnoses is still challenging. Here, a combined technique of label-free imaging with multi-photon microscopy and artificial intelligence was applied to detect quantitative signatures that differentiate fibroepithelial lesions. Multi-photon excited autofluorescence and second harmonic generation (SHG) signals were detected in tissue sections. A pixel-wise semantic segmentation method using a deep learning framework was used to separate epithelial and stromal regions automatically. The epithelial to stromal area ratio and the collagen SHG signal strength were investigated for their ability to distinguish fibroepithelial lesions. An image segmentation analysis with a pixel-wise semantic segmentation framework using a deep convolutional neural network showed the accurate separation of epithelial and stromal regions. A further investigation, to determine if scoring the epithelial to stromal area ratio and the SHG signal strength within the stromal area could be a marker for differentiating fibroepithelial tumors, showed accurate classification. Therefore, molecular and morphological changes, detected through the assistance of computational and label-free multi-photon imaging techniques, enable us to propose quantitative signatures for epithelial and stromal alterations in breast tissues.

Therapeutic effects of the PKR inhibitor C16 suppressing tumor proliferation and angiogenesis in hepatocellular carcinoma in vitro and in vivo.

The therapeutic effects of C16, which is an inhibitor of RNA-dependent protein kinase (PKR), on growth of hepatocellular carcinoma (HCC) cells and tumor progression in vitro and in vivo were evaluated. Huh7 cells, a human HCC cell line, were used. The effects of C16 on cell viability were evaluated with the MTT assay, and real-time RT-PCR was performed. Huh7 cells were grafted into immunodeficient mice, and the in vivo effects of C16 on tumorigenesis were examined. C16 suppressed proliferation of HCC cells in a dose-dependent manner in vitro. Mouse models with xenograft transplantation showed that the inhibitor suppressed the growth of HCC cells in vivo. Moreover, C16 decreased angiogenesis in HCC tissue in the xenograft model. Consistent with these results in mice, transcript levels of vascular endothelial growth factor-A and factor-B, platelet-derived growth factor-A and factor-B, fibroblast growth factor-2, epidermal growth factor, and hepatocyte growth factor, which are angiogenesis-related growth factors, were significantly decreased by C16 in vitro. In conclusion, the PKR inhibitor C16 blocked tumor cell growth and angiogenesis via a decrease in mRNA levels of several growth factors. C16 may be useful in the treatment of HCC.

Visualization of epithelial-mesenchymal transition in an inflammatory microenvironment-colorectal cancer network.

Epithelial-mesenchymal transition (EMT) is a biological process by which epithelial cells acquire mesenchymal characteristics. In malignant tumors, EMT is crucial for acquisition of a mesenchymal phenotype with invasive and metastatic properties, leading to tumor progression. An inflammatory microenvironment is thought to be responsible for the development and progression of colorectal cancer (CRC); however, the precise role of inflammatory microenvironments in EMT-related CRC progression remains unclear. Here, we show the spatiotemporal visualization of CRC cells undergoing EMT using a fluorescence-guided EMT imaging system in which the mesenchymal vimentin promoter drives red fluorescent protein (RFP) expression. An inflammatory microenvironment including TNF-α, IL-1ß, and cytokine-secreting inflammatory macrophages induced RFP expression in association with the EMT phenotype in CRC cells. In vivo experiments further demonstrated the distribution of RFP-positive CRC cells in rectal and metastatic tumors. Our data suggest that the EMT imaging system described here is a powerful tool for monitoring EMT in inflammatory microenvironment-CRC networks.

Direct comparison of target-reactivity and cross-reactivity induced by CAR- and BiTE-redirected T cells for the development of antibody-based T-cell therapy.

The development of chimeric antigen receptor (CAR) and bispecific T-cell engager (BiTE) has led to the successful application of cancer immunotherapy. The potential reactivity mediated by CAR- and BiTE-redirected T cells needs to be assessed to facilitate the application of these treatment options to a broader range of patients. Here, we have generated CAR and BiTE possessing the same single chain fragment variable (scFv) specific for the HLA-A2/NY-ESO-1157-165 complex (A2/NY-ESO-1157). Using HLA-A2+NY-ESO-1+ myeloma cells and peptides presented by HLA-A2 molecules as a model, both sets of redirected T cells recognized and killed HLA-A2+NY-ESO-1+ myeloma cells in an A2/NY-ESO-1157-specific manner in vitro. Moreover, CAR- and BiTE-activated T cells showed similar functional avidity, as assessed by cytokine production and killing activity, both displaying antitumor reactivity against HLA-A2+NY-ESO-1+ myeloma cells in vivo. Interestingly, cross-reactivity for homologous peptides presented by HLA-A*02:01 and NY-ESO-1157 peptide presented by HLA-A2 alleles was not identical between CAR- and BiTE-redirected T cells, probably due to structural differences of modified antibodies. These results have demonstrated that both antitumor CAR- and BiTE-activated T cells have comparable potential to recognize tumors, while paying attention to unknown off-target reactivity that would differ for each antibody-based modality even if the same scFv was employed.

In vivo optical imaging of cancer cell function and tumor microenvironment.

In vivo optical imaging using fluorescence and bioluminescence is superior to other methods in terms of spatiotemporal resolution and specificity, and represents a new technology for comprehensively studying living organisms in a less invasive way. Nowadays, it is an indispensable technology for studying many aspects of cancer biology, including dynamic invasion and metastasis. In observations of fluorescence or bioluminescence signals in a living body, various problems were caused by optical characteristics such as absorption and scattering and, therefore, observation of deep tissue was difficult. Recent developments in techniques for observation of the deep tissues of living animals overcame this difficulty by improving bioluminescent proteins, fluorescent proteins, and fluorescent dyes, as well as detection technologies such as two-photon excitation microscopy. In the present review, we introduce these technological developments and in vivo application of bioluminescence and fluorescence imaging, and discuss future perspectives on the use of in vivo optical imaging technology in cancer research.

The HIV co-receptor CCR5 regulates osteoclast function.

C-C chemokine receptor 5 (CCR5) is a co-receptor of HIV. Epidemiological findings suggest that the functional loss of CCR5 is correlated with a lower incidence of bone-destructive diseases as well as of HIV transmission. However, it is not clear whether CCR5 is involved in regulation of the function of bone cells, in addition to that of immune cells. Here we show that blockade of CCR5 using specific antibodies impairs human osteoclast function in vitro. Ccr5-deficient (Ccr5 -/- ) mice presented with dysfunctional osteoclasts and were resistant to osteoporosis induced by receptor activator of nuclear factor kappa-B ligand (RANKL), which triggers osteoporosis independently of inflammatory and immunomodulatory pathways. Furthermore, Ccr5 deficiency impairs the cellular locomotion and bone-resorption activity of osteoclasts, which is associated with the disarrangement of podosomes and adhesion complex molecules including Pyk2. Overall, the data provides evidence that CCR5 has an essential role in bone-destructive conditions through the functional regulation of osteoclasts.

Impact of immediate breast reconstruction on postoperative adjuvant chemotherapy: a single center study.

BACKGROUND: Various studies have indicated a worldwide increase in the number of immediate breast reconstruction surgeries. However, breast reconstruction should not delay or prevent postoperative cancer therapeutics such as adjuvant chemotherapy and radiotherapy. In response to these developments, our team researched the impact of immediate breast reconstruction on postoperative adjuvant chemotherapy. METHODS: From April 2006 to March 2011, 116 patients at Saitama Cancer Center underwent postoperative adjuvant chemotherapy following mastectomy with or without immediate breast reconstruction. Fifty patients received postoperative adjuvant chemotherapy following mastectomy with immediate breast reconstruction (IBR group), and 66 patients received the same treatment but without immediate breast reconstruction (non-IBR group). The outcomes were studied retrospectively by chart review. Patients' average age, body mass index, postoperative complication rate, and days to adjuvant chemotherapy were calculated. RESULTS: Mean age and body mass index of patients were 47.0 ± 9.0 years, 22.2 ± 3.0 kg/m(2) and 55.5 ± 10.1 years, 23.0 ± 3.6 kg/m(2) in IBR group and non-IBR group, respectively. Postoperative complication rate was 10.0 % in IBR group and 6.1 % in non-IBR group. Days to adjuvant chemotherapy was 61.0 ± 10.5 days in IBR group and 58.0 ± 12.3 days in non-IBR group. CONCLUSIONS: Although complication rate and days to adjuvant chemotherapy were slightly increased in IBR group, the delay was not critical to the initiation of adjuvant chemotherapy in these patient groups.

Tongue pressure in patients with tongue cancer resection and reconstruction.

OBJECTIVE: Assessment of tongue function following tongue reconstruction is important to evaluate patient status. To assess tongue function in patients who had undergone tongue reconstruction, the surgical team used a simple, hand-held tongue pressure measurement device to measure tongue power. METHODS: Tongue power of 30 patients (25 males, 5 females; average age: 53.6±15.0 years) was calculated using a hand-held tongue pressure measurement device, six months postoperation. The defects were classified into minimal glossectomy (MG) (n=8), near-half partial glossectomy of the mobile tongue (PG) (n=5), hemi-glossectomy (HG) (n=4), more than half partial glossectomy of the mobile tongue (SG-MT) (n=7), and subtotal glossectomy (SG) (n=6). As seen in other tongue assessments, a simple articulatory test, food evaluation, and speech intelligibility assessment were also performed; resulting correlations were statistically calculated using tongue pressure values. RESULTS: The tongue pressure values were 94.0±14.5% in MG, 48.5±13.2(a) % in PG, 40.4±18.7(a) % in HG, 19.3±7.7(a,b) % in SG-MT, and 15.3±5.6(a,b) % in SG (a: <0.05 vs. MG, b: <0.05 vs. PG). The Pearson r was 0.77, 0.67, and 0.74 when correlated with simple articulatory test, food evaluation, and speech intelligibility assessment, respectively. CONCLUSION: Tongue pressure measurement in patients with tongue cancer resection and reconstruction facilitated determination of patients' tongue function status.

Roles of raft-anchored adaptor Cbp/PAG1 in spatial regulation of c-Src kinase.

The tyrosine kinase c-Src is upregulated in numerous human cancers, implying a role for c-Src in cancer progression. Previously, we have shown that sequestration of activated c-Src into lipid rafts via a transmembrane adaptor, Cbp/PAG1, efficiently suppresses c-Src-induced cell transformation in Csk-deficient cells, suggesting that the transforming activity of c-Src is spatially regulated via Cbp in lipid rafts. To dissect the molecular mechanisms of the Cbp-mediated regulation of c-Src, a combined analysis was performed that included mathematical modeling and in vitro experiments in a c-Src- or Cbp-inducible system. c-Src activity was first determined as a function of c-Src or Cbp levels, using focal adhesion kinase (FAK) as a crucial c-Src substrate. Based on these experimental data, two mathematical models were constructed, the sequestration model and the ternary model. The computational analysis showed that both models supported our proposal that raft localization of Cbp is crucial for the suppression of c-Src function, but the ternary model, which includes a ternary complex consisting of Cbp, c-Src, and FAK, also predicted that c-Src function is dependent on the lipid-raft volume. Experimental analysis revealed that c-Src activity is elevated when lipid rafts are disrupted and the ternary complex forms in non-raft membranes, indicating that the ternary model accurately represents the system. Moreover, the ternary model predicted that, if Cbp enhances the interaction between c-Src and FAK, Cbp could promote c-Src function when lipid rafts are disrupted. These findings underscore the crucial role of lipid rafts in the Cbp-mediated negative regulation of c-Src-transforming activity, and explain the positive role of Cbp in c-Src regulation under particular conditions where lipid rafts are perturbed.

Complex lower face reconstruction using a combined technique of Estlander flap and subscapular artery system free flaps.

When advanced mandibular carcinoma is resected, the defect may include lip and oral commissure. Free flap insertion is commonly used to reconstruct the lip defect. Although improvements in the oral reconstructive method via free flap use have been reported, functional and aesthetic results of the oral sphincter remain limited. This case report describes two individuals presenting with massive lower face defects, including a lower lip defect and a mandibular bone defect. Reconstruction was accomplished using the Estlander flap and free subscapular system of flaps. In both cases, the free subscapular artery system flap was elevated from the mandibular bone defect and other mucosal defect. The lower lip and oral commissure defect was reconstructed via Estlander flap. Free flaps survived 100% and both cases healed without complication. Patients regained good oral sphincter function with no reports of drooling. Thus, in cases involving massive lower face resection, including that of the lower lip and mandibular bone, this method of reconstruction when combined with lip-switch flap and subscapular artery system flap can prove to be useful.

A new nasal cavity and maxilla reconstruction method using jejunum flap with non-vascularised bone.

Reconstruction of the midface is still challenging for reconstructive surgeons because of its complex structure and the need for an aesthetic result. We used a free jejunum flap for the nasal cavity and non-vascularised bone covered by the jejunal seromuscular patch for the facial bone structure. One patient who had a midface defect received reconstructive surgery using free jejunum flap with non-vascularised bone. The nasal cavity reconstruction using jejunal mucosa was moisturised and had less crust formation. The nasal cavity space was very large and patients could breathe easily via the nose. The non-vascularised bone covered by the jejunal seromuscular patch did not dry out or become less absorbent. The reconstruction of the nasal cavity and maxilla using free jejunum flap with non-vascularised bone is novel and useful in some surgical cases.

Lower lip reconstruction using a combined technique of the webster and johanson methods.

Oral reconstruction, especially with lower lip defects greater than 80% of the lip, is still challenging for plastic surgeons. Webster technique is mostly used for lower lip defects greater than 80% of the lip; however, resulting scars in the chin area (Schuchardt flap, a half-circle scar) are relatively conspicuous in Asian populations because of the trapdoor deformity. On the other hand, Johanson staircase flap technique, which is used to reconstruct lower lip defects of up to two thirds of the lip, results in relatively inconspicuous scarring and prevents trapdoor deformity. Thus, instead of Schuchardt flaps, we designed staircase flaps with a Webster technique. Two patients with lower lip carcinoma were operated on using this new technique. Large triangles of the skin and subcutaneous fat were removed from the nasolabial folds, and small staircases were removed from the lower lip to allow medial movement of the cheek tissues. The cosmetic and functional results were improved from the original Webster technique.

Usefulness of Harmonic Focus during anterolateral thigh flap elevation.

Meticulous hemostasis and careful ligation of branches are necessary for pedicle dissection during flap elevation. The aim of this study was to evaluate the effectiveness of the Harmonic Focus handpiece (Ethicon Endo-Surgery, Inc., Blue Ash, Cincinnati, OH, USA) in reducing operation time, bleeding volume, and volume of postoperative drainage during anterolateral thigh flap elevation. Ten patients requiring flap elevation were divided into two groups: (1) Harmonic Focus group (three men, two women), and (2) control group (three men, two women). Operating time was found to be lower in the Harmonic Focus group than in the control group. Bleeding volume and postoperative drainage volume were nearly identical in the Harmonic Focus group and the control group, and the number of silk ligatures was significantly lower in the Harmonic Focus group compared with the control group. Although somewhat costly, the Harmonic Scalpel with the Harmonic Focus handpiece is advantageous for flap elevation, and it is likely that Harmonic Scalpel use will increase in plastic surgery.

Control and inhibition analysis of complex formation processes.

BACKGROUND: Proteolytic degradation of the extracellular matrix (ECM) is a key event in tumour metastasis and invasion. Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade most of the components of the ECM. Several broad-spectrum MMP inhibitors (MMPIs) have been developed, but have had little success due to side effects. Thus, it is important to develop mathematical methods to provide new drug treatment strategies. Matrix metalloproteinase 2 (MMP2) activation occurs via a mechanism involving complex formation that consists of membrane type 1 MMP (MT1-MMP), tissue inhibitor of matrix metalloproteinase 2 (TIMP2) and MMP2. Here, we focus on developing a method for analysing the complex formation process. RESULTS: We used control analysis to investigate inhibitor responses in complex formation processes. The essence of the analysis is to define the response coefficient which measures the inhibitory efficiency, a small fractional change of concentration of a targeting molecule in response to a small fractional change of concentration of an inhibitor. First, by using the response coefficient, we investigated models for general classes of complex formation processes: chain reaction systems composed of ordered steps, and chain reaction systems and site-binding reaction systems composed of unordered multi-branched steps. By analysing the ordered step models, we showed that parameter-independent inequalities between the response coefficients held. For the unordered multi-branched step models, we showed that independence of the response coefficients with respect to equilibrium constants held. As an application of our analysis, we discuss a mathematical model for the MMP2 activation process. By putting the experimentally derived parameter values into the model, we were able to conclude that the TIMP2 and MMP2 interaction is the most efficient interaction to consider in selecting inhibitors. CONCLUSIONS: Our result identifies a new drug target in the process of the MMP2 activation. Thus, our analysis will provide new insight into the design of more efficient drug strategies for cancer treatment.

Mathematical modeling of invadopodia formation.

In invasive cancer cells, specialized sub-cellular membrane structures which carry out a pivotal process in cancer invasion, termed invadopodia, are observed. Invadopodia appear irregularly within the cytoplasm and their general shape is small punctuated finger-like protrusions with dimension up to several µm long. They may exist and persist on a timescale between several tens of minutes to one hour. The formation of invadopodia requires the integration of several processes that include actin reorganization, extracellular matrix (ECM) degradation, signaling processes through receptors such as the epidermal growth factor receptor (EGFR) and matrix metalloproteinase (MMP) synthesis and delivery to the location of the invading front. In this paper, we consider a mathematical model investigating the coupling of these fundamental processes, and we investigate how invadopodia appear in this model. We investigate the spatio-temporal dynamics of the model in two spatial dimensions by using numerical computational simulations. We show that in a special parameter region of the model, random fluctuations of ECM degradation and a positive feedback loop regarding the up-regulation of MMPs allow us to reproduce finger-like protrusions which have similar size and lifetime as invadopodia. This study provides a new insight into how invadopodia appear in cancer cells and why space and time scales exist for invadopodia.

[Fundamental evaluation of HCV core antigen method comparison with Cobas Amplicor HCV monitor v2.0 (high range method)].

Quantitative measurement of hepatitis C virus (HCV) has been performed by PCR method. However, PCR method has problems such as a special instrument, a complicated manual skill and a high cost. Recently, simple and highly sensitive HCV core antigen (Ag) method has been developed. We performed fundamental evaluation of HCV core Ag method, and compared HCV core Ag method with HCV PCR high-range method. The intra-assay and inter-assay variation coefficients for HCV core Ag were calculated to be within the ranges of 1.0-11.3% and 0.8-9.3%, respectively. The test of dilution linearity revealed the unstableness in the vicinity of a cut-off level of 50 fmol/L. Based on the result of the high-range method; sensitivity, specificity, positive predictive value, negative predictive value, and agreement rate were 97.0%, 100%, 100%, 82.0%, and 96.5%, respectively. The correlation between the HCV core Ag method and the high-range method was r = 0.87. Cost per sample and time from sample preparation to final report for HCV core Ag were cheaper and shorter than those of HCV PCR method, respectively. We consider that the HCV core Ag method seems to be useful as the quantitative measurement of HCV with respect to rapidness, easiness and low cost.