Cancer-stromal cell interactions in breast cancer brain metastases induce glycocalyx-mediated resistance to HER2-targeting therapies.

Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases.

Sub-Tip-Radius Near-Field Interactions in Nano-FTIR Vibrational Spectroscopy on Single Proteins.

Tip-enhanced vibrational spectroscopy has advanced to routinely attain nanoscale spatial resolution, with tip-enhanced Raman spectroscopy even achieving atomic-scale and submolecular sensitivity. Tip-enhanced infrared spectroscopy techniques, such as nano-FTIR and AFM-IR spectroscopy, have also enabled the nanoscale chemical analysis of molecular monolayers, inorganic nanoparticles, and protein complexes. However, fundamental limits of infrared nanospectroscopy in terms of spatial resolution and sensitivity have remained elusive, calling for a quantitative understanding of the near-field interactions in infrared nanocavities. Here, we demonstrate the application of nano-FTIR spectroscopy to probe the amide-I vibration of a single protein consisting of ∼500 amino acid residues. Detection with higher tip tapping demodulation harmonics up to the seventh order leads to pronounced enhancement in the peak amplitude of the vibrational resonance, originating from sub-tip-radius geometrical effects beyond dipole approximations. This quantitative characterization of single-nanometer near-field interactions opens the path toward employing infrared vibrational spectroscopy at the subnanoscale and single-molecule levels.

N-2,6-Di(isopropyl)phenyl-2-azaphenalenyl radical cations.

N-2,6-Di(isopropyl)phenyl-2-azaphenalenyl radical cations were obtained as a dark brown air-sensitive crystalline compound. The high HOMA values and the ACID calculation indicate relatively high aromatic character of a 5,8-di-tert-butyl derivative, and clean generation of a derivative without tert-butyl groups indicates that the di(isopropyl)phenyl group is sufficient for hampering the formation of the σ-dimer.

Tenosynovitis With Psammomatous Calcification Preoperatively Diagnosed as Intra-Articular Free Body of the Young Male Wrist: A Case Report.

Tenosynovitis with psammomatous calcifications is a rare condition primarily affecting female patients in the distal extremities. This case report presents a unique instance of tenosynovitis with psammomatous calcification in a 31-year-old man presenting with wrist pain. Initial misdiagnosis and unsuccessful steroid injections prompted further investigation, leading to the discovery of an extra-articular calcified mass. Arthroscopic resection was attempted but found to be unnecessary because the lesion was located outside the joint. Histopathological examination confirmed the diagnosis of tenosynovitis with psammomatous calcification. After mass removal, the patient experienced relief from wrist pain and resumed work within a month. Subsequent follow-ups at 9 months showed no recurrence of pain, with full range of wrist motion and no grip power weakness. This case highlights the importance of differentiating tenosynovitis with psammomatous calcification from intra-articular lesions, particularly in atypical presentations, and demonstrates the effectiveness of surgical intervention in resolving symptoms.

Peripheral blood TCR clonotype diversity as an age-associated marker of breast cancer progression.

Immune escape is a prerequisite for tumor growth. We previously described a decline in intratumor activated cytotoxic T cells and T cell receptor (TCR) clonotype diversity in invasive breast carcinomas compared to ductal carcinoma in situ (DCIS), implying a central role of decreasing T cell responses in tumor progression. To determine potential associations between peripheral immunity and breast tumor progression, here, we assessed the peripheral blood TCR clonotype of 485 breast cancer patients diagnosed with either DCIS or de novo stage IV disease at younger (<45) or older (≥45) age. TCR clonotype diversity was significantly lower in older compared to younger breast cancer patients regardless of tumor stage at diagnosis. In the younger age group, TCR-α clonotype diversity was lower in patients diagnosed with de novo stage IV breast cancer compared to those diagnosed with DCIS. In the older age group, DCIS patients with higher TCR-α clonotype diversity were more likely to have a recurrence compared to those with lower diversity. Whole blood transcriptome profiles were distinct depending on the TCR-α Chao1 diversity score. There were more CD8+ T cells and a more active immune environment in DCIS tumors of young patients with higher peripheral blood TCR-α Chao1 diversity than in those with lower diversity. These results provide insights into the role that host immunity plays in breast cancer development across different age groups.

Antenna-coupled infrared nanospectroscopy of intramolecular vibrational interaction.

Many photonic and electronic molecular properties, as well as chemical and biochemical reactivities are controlled by fast intramolecular vibrational energy redistribution (IVR). This fundamental ultrafast process limits coherence time in applications from photochemistry to single quantum level control. While time-resolved multidimensional IR-spectroscopy can resolve the underlying vibrational interaction dynamics, as a nonlinear optical technique it has been challenging to extend its sensitivity to probe small molecular ensembles, achieve nanoscale spatial resolution, and control intramolecular dynamics. Here, we demonstrate a concept how mode-selective coupling of vibrational resonances to IR nanoantennas can reveal intramolecular vibrational energy transfer. In time-resolved infrared vibrational nanospectroscopy, we measure the Purcell-enhanced decrease of vibrational lifetimes of molecular vibrations while tuning the IR nanoantenna across coupled vibrations. At the example of a Re-carbonyl complex monolayer, we derive an IVR rate of (25±8) cm-1 corresponding to (450±150) fs, as is typical for the fast initial equilibration between symmetric and antisymmetric carbonyl vibrations. We model the enhancement of the cross-vibrational relaxation based on intrinsic intramolecular coupling and extrinsic antenna-enhanced vibrational energy relaxation. The model further suggests an anti-Purcell effect based on antenna and laser-field-driven vibrational mode interference which can counteract IVR-induced relaxation. Nanooptical spectroscopy of antenna-coupled vibrational dynamics thus provides for an approach to probe intramolecular vibrational dynamics with a perspective for vibrational coherent control of small molecular ensembles.

Biological reconstruction for massive bone defect following resection of tumor in Japan.

BACKGROUND: Several procedures of biological reconstruction for massive bone defect are available following tumor resection. Since the 1980s, allografting has been advanced mainly in the United States. However, allogeneic bone grafting has not been sufficiently developed in Japan for socioreligious reasons, and many other biological reconstructive methods have been developed. STATUS OF BIOLOGICAL RECONSTRUCTION: Bone lengthening, recycled and vascularized bone grafting have yielded favorable outcomes. Once bone union is achieved, reoperation is scarcely performed, with lower rate of infection than that observed with a prosthesis. However, there are disadvantages, such as complicated surgical procedures and relatively common postoperative complications. However, if sufficient donors are available, allogeneic bone grafting can be a good alternative. PROSPECTS OF THE JAPANESE ORTHOPAEDIC ASSOCIATION: Regenerative medicine with iPS cells, etc., which is under investigation, is expected to be employed for defect reconstruction. However, several biological reconstructive procedures should be further developed. These procedures are not inferior to prosthetic and allograft reconstructions in the short term, but rather are superior in the long term. Favorable outcomes are being obtained by combining recycled bone reconstruction and vascularized bone grafting, suggesting possible improvement in the future. Data should be accumulated to develop biological reconstruction in Japan. Although Japan has the challenge in terms of the ability to convey its message, the disadvantages of the procedures should be minimized. CONCLUSION: The construction of an allogeneic bone grafting system should be promoted, while biological reconstruction methods developed in Japan should be further developed and convey our message clearly and logically.

9,9-Bis[4-(N-aryl)phenyl]methylidene-xanthylidene Derivatives Displaying Mechano-, Crystallo-, and Thermochromism.

Tetraphenylethylene (TPE) derivatives bearing a xanthene moiety are of interest because they have novel optical properties. 9,9-Bis[4-(N,N-diphenylamino)phenyl] and 9,9-bis[4-(9-carbazolyl)-phenyl]methylidene-xanthylidenes 3 and 4 were synthesized using Suzuki-Miyaura coupling of 9,9-dibromomethylidene-xanthylidene with the corresponding boronic acids. Diphenylamino derivative 3 exhibits mechanochromism and mechanofluorochromism (MC and MFC) reflected in absorption and fluorescence color changes. In contrast, carbazolyl derivative 4 displays thermo- and crystallo-chromism in addition to MC and MFC in the solid state. Powder X-ray diffraction and single crystal X-ray crystallographic analysis reveal that the solid state photophysical properties of these substances are governed by conformational changes rather by the creation of planar π-conjugation extended geometries.

Nanoscale heterogeneity of ultrafast many-body carrier dynamics in triple cation perovskites.

In high fluence applications of lead halide perovskites for light-emitting diodes and lasers, multi-polaron interactions and associated Auger recombination limit the device performance. However, the relationship of the ultrafast and strongly lattice coupled carrier dynamics to nanoscale heterogeneities has remained elusive. Here, in ultrafast visible-pump infrared-probe nano-imaging of the photoinduced carrier dynamics in triple cation perovskite films, a ~20 % variation in sub-ns relaxation dynamics with spatial disorder on tens to hundreds of nanometer is resolved. We attribute the non-uniform relaxation dynamics to the heterogeneous evolution of polaron delocalization and increasing scattering time. The initial high-density excitation results in faster relaxation due to strong many-body interactions, followed by extended carrier lifetimes at lower densities. These results point towards the missing link between the optoelectronic heterogeneity and associated carrier dynamics to guide synthesis and device engineering for improved perovskites device performance.

Unique Glycoform-Dependent Monoclonal Antibodies for Mouse Mucin 21.

Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.

An in vivo orthotopic serial passaging model for a metastatic renal cancer study.

We developed an in vivo serial passaging model for renal cancer with orthotopic renal subcapsular inoculation. We detail the procedures for renal subcapsular inoculation of cancer cells in mice, followed by in vivo and exvivo bioluminescence imaging, tumor-bearing kidney dissociation, and in vivo passaging. This protocol is capable of reproducing the coevolution between cancer cells and the primary tumor microenvironment. It enables us to unveil the systemic dynamics of metastasis and develop a therapeutic strategy for metastatic renal cancer. For complete details on the use and execution of this protocol, please refer to Nishida et al. (2020).

Semi-empirical quantum optics for mid-infrared molecular nanophotonics.

Nanoscale infrared (IR) resonators with sub-diffraction limited mode volumes and open geometries have emerged as new platforms for implementing cavity quantum electrodynamics at room temperature. The use of IR nanoantennas and tip nanoprobes to study strong light-matter coupling of molecular vibrations with the vacuum field can be exploited for IR quantum control with nanometer spatial and femtosecond temporal resolution. In order to advance the development of molecule-based quantum nanophotonics in the mid-IR, we propose a generally applicable semi-empirical methodology based on quantum optics to describe light-matter interaction in systems driven by mid-IR femtosecond laser pulses. The theory is shown to reproduce recent experiments on the acceleration of the vibrational relaxation rate in infrared nanostructures. It also provides physical insights on the implementation of coherent phase rotations of the near-field using broadband nanotips. We then apply the quantum framework to develop general tip-design rules for the experimental manipulation of vibrational strong coupling and Fano interference effects in open infrared resonators. We finally propose the possibility of transferring the natural anharmonicity of molecular vibrational levels to the resonator near-field in the weak coupling regime to implement intensity-dependent phase shifts of the coupled system response with strong pulses and develop a vibrational chirping model to understand the effect. The semi-empirical quantum theory is equivalent to first-principles techniques based on Maxwell's equations, but its lower computational cost suggests its use as a rapid design tool for the development of strongly coupled infrared nanophotonic hardware for applications ranging from quantum control of materials to quantum information processing.

Ultrafast infrared nano-imaging of far-from-equilibrium carrier and vibrational dynamics.

Ultrafast infrared nano-imaging has demonstrated access to ultrafast carrier dynamics on the nanoscale in semiconductor, correlated-electron, or polaritonic materials. However, mostly limited to short-lived transient states, the contrast obtained has remained insufficient to probe important long-lived excitations, which arise from many-body interactions induced by strong perturbation among carriers, lattice phonons, or molecular vibrations. Here, we demonstrate ultrafast infrared nano-imaging based on excitation modulation and sideband detection to characterize electron and vibration dynamics with nano- to micro-second lifetimes. As an exemplary application to quantum materials, in phase-resolved ultrafast nano-imaging of the photoinduced insulator-to-metal transition in vanadium dioxide, a distinct transient nano-domain behavior is quantified. In another application to lead halide perovskites, transient vibrational nano-FTIR spatially resolves the excited-state polaron-cation coupling underlying the photovoltaic response. These examples show how heterodyne pump-probe nano-spectroscopy with low-repetition excitation extends ultrafast infrared nano-imaging to probe elementary processes in quantum and molecular materials in space and time.

Genome-wide analysis of DNA methylation identifies the apoptosis-related gene UQCRH as a tumor suppressor in renal cancer.

DNA hypermethylation is frequently observed in clear cell renal cell carcinoma (ccRCC) and correlates with poor clinical outcomes. However, the detailed function of DNA hypermethylation in ccRCC has not been fully uncovered. Here, we show the role of DNA methylation in ccRCC progression through the identification of a target(s) of DNA methyltransferases (DNMT). Our preclinical model of ccRCC using the serial orthotopic inoculation model showed the upregulation of DNMT3B in advanced ccRCC. Pretreatment of advanced ccRCC cells with 5-aza-deoxycytidine, a DNMT inhibitor, attenuated the formation of primary tumors through the induction of apoptosis. DNA methylated sites were analyzed genome-wide using methylation array in reference to RNA-sequencing data. The gene encoding ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissues was lower than normal adjacent tissues. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo, implying the tumor-suppressive role of UQCRH. Moreover, 5-aza-deoxycytidine enhanced the therapeutic efficiency of mammalian target of rapamycin inhibitor everolimus in vivo. These findings suggested that the DNMT3B-induced methylation of UQCRH may contribute to renal cancer progression and implicated clinical significance of DNMT inhibitor as a therapeutic option for ccRCC.

Risk factors for preoperative nasal carriage of methicillin-resistant bacteria in total hip and knee arthroplasty patients.

BACKGROUND: The present study aimed to identify risk factors for preoperative nasal carriage of resistant bacteria - MRSA methicillin-resistant Staphylococcus (S.) aureus, MRSE (methicillin-resistant Staphylococcus epidermidis), and MRCNS (methicillin-resistant coagulase negative staphylococci) in total hip and knee arthroplasty (THA and TKA) patients. METHODS: Nasal cultures were obtained from 538 patients before THA (262 primary and 26 revision) and TKA (241 primary and 9 revision). These were classified either as methicillin-resistant bacteria (group MR) or methicillin-susceptible bacteria (including culture-negative) (group MS). Patient characteristics were compared between these groups using logistic regression models. RESULTS: The resistant bacteria were preoperatively present in 33.1% (178 patients) among all patients. MRSE, MRCNS, and MRSA were detected in 27.5% (148 patients), 3.7% (20 patients), and 1.9% (10 patients). In the unadjusted comparisons of the patient characteristics between the groups MR and MS, a significant difference was found in the percentages of diabetic patients (15.2% vs. 9.2%, P = 0.04); the association remained after the multivariable adjustment for possible risk factors (P < 0.001). In addition, the diabetic patients in the group MR showed a higher percentage of receiving insulin injection than those in the group MS (25.9% vs. 6.1%, P = 0.063), and their mean levels of HbA1c were significantly higher in the group MR than the MS (6.8% vs. 6.4%, P = 0.03). CONCLUSIONS: We identified diabetes as a risk factor for the preoperative nasal carriage of resistant bacteria. Our results suggest that, in order to prevent a surgical site infection (SSI), extra care should be taken in performing joint arthroplasties for diabetic patients, especially using insulin and with high HbA1c levels (≥6.6%) prior to the surgical procedures.

Multidimensional Nano-Imaging of Structure, Coupling, and Disorder in Molecular Materials.

A hierarchy of intramolecular and intermolecular interactions controls the properties of biomedical, photophysical, and novel energy materials. However, multiscale heterogeneities often obfuscate the relationship between microscopic structure and emergent function, and they are generally difficult to access with conventional optical and electron microscopy techniques. Here, we combine vibrational exciton nanoimaging in variable-temperature near-field optical microscopy (IR s-SNOM) with four-dimensional scanning transmission electron microscopy (4D-STEM), and vibrational exciton modeling based on density functional theory (DFT), to link local microscopic molecular interactions to macroscopic three-dimensional order. In the application to poly(tetrafluoroethylene) (PTFE), large spatio-spectral heterogeneities with C-F vibrational energy shifts ranging from sub-cm-1 to ≳25 cm-1 serve as a molecular ruler of the degree of local crystallinity and disorder. Spatio-spectral-structural correlations reveal a previously invisible degree of highly variable local disorder in molecular coupling as the possible missing link between nanoscale morphology and associated electronic, photonic, and other functional properties of molecular materials.

2D Vibrational Exciton Nanoimaging of Domain Formation in Self-Assembled Monolayers.

Order, disorder, and domains affect many of the functional properties in self-assembled monolayers (SAMs). However, carrier transport, wettability, and chemical reactivity are often associated with collective effects, where conventional imaging techniques have limited sensitivity to the underlying intermolecular coupling. Here we demonstrate vibrational excitons as a molecular ruler of intermolecular wave function delocalization and nanodomain size in SAMs. In the model system of a 4-nitrothiophenol (4-NTP) SAM on gold, we resolve coupling-induced peak shifts of the nitro symmetric stretch mode with full spatio-spectral infrared scattering scanning near-field optical microscopy. From modeling of the underlying 2D Hamiltonian, we infer domain sizes and their distribution ranging from 3 to 12 nm across a field of view on the micrometer scale. This approach of vibrational exciton nanoimaging is generally applicable to study structural phases and domains in SAMs and other molecular interfaces.

Heterogenous expression of endoglin marks advanced renal cancer with distinct tumor microenvironment fitness.

Intratumoral heterogeneity, including in clear cell renal cell carcinoma, is a potential cause of drug resistance and metastatic cancer progression. We specified the heterogeneous population marked by endoglin (also known as CD105) in a preclinical model of clear cell renal cell carcinoma progression. Highly malignant derivatives of human clear cell renal cell carcinoma OS-RC-2 cells were established as OS5Ks by serial orthotopic inoculation in our previous study. Expression of both ENG (encoding endoglin) mRNA and protein were heterogeneously upregulated in OS5Ks, and the endoglin-positive (ENG+ ) population exhibited growth dependency on endoglin in anchorage-independent cultures. Despite the function of endoglin as a type III receptor, transforming growth factor ß and bone morphogenetic protein-9 signaling were unlikely to contribute to the proliferative phenotype. Although endoglin has been proposed as a marker for renal cancer-initiating cells, the OS5K-3 ENG+ population did not enrich other reported cancer-initiating cell markers or differentiate into the ENG- population. Mouse tumor inoculation models revealed that the tumor-forming capabilities of OS5K-3 ENG+ and ENG- cells in vivo were highly dependent on the microenvironment, with the renal microenvironment most preferable to ENG+ cells. In conclusion, the renal microenvironment, rather than the hypothesized ENG+ cell-centered hierarchy, maintains cellular heterogeneity in clear cell renal cell carcinoma. Therefore, the effect of the microenvironment should be considered when evaluating the proliferative capability of renal cancer cells in the experimental settings.

Does intraoperative mechanical prophylaxis prevent venous thromboembolism in total knee arthroplasty? - effectiveness of passive-assisted ankle motion in surgical/non-surgical side.

BACKGROUND: Gradual compression stocking (GCS) and intermittent pneumatic compression device (IPCD) are used for intraoperative mechanical prophylaxis against venous thromboembolism (VTE) during total knee arthroplasty (TKA). In this study, we applied a passive-assisted ankle motion in combination with GCS and IPCD during TKA and evaluated its effectiveness in preventing postoperative VTE. METHODS: We included 77 patients who underwent primary unilateral TKA. Patients were divided into group A (53 patients who underwent GCS and IPCD on their non-surgical side limb) and group B (24 patients who underwent passive ankle dorsiflexion motion in addition to GCS and IPCD on their non-surgical side limb). Deep vein thrombosis (DVT) was assessed using lower extremity ultrasonography (US). The incidence of VTE in each affected limb was compared between the two groups. RESULTS: US was performed 4 days after surgery on average. The incidence of DVT in groups A and B was 47.2 and 70.8 %, respectively. In group A, 22.6 % of DVTs were found only on the surgical side, 11.3 % on the non-surgical side, and 13.2 % on both sides. On the other hand, in group B, 41.7 % of DVTs were found only on the surgical side, 4.2 % on the non-surgical side, and 25.0 % on both sides. No significant difference in the incidence of VTE was noted between the 2 groups. CONCLUSIONS: The intraoperative application of passive ankle motion plus GCS and IPCD might not further reduce the incidence of postoperative DVT in TKA patients.