Blockade of CCR5 and CXCR3 attenuates murine acute graft-versus-host disease through modulating donor-derived T-cell distribution and function.

Lymphocyte trafficking via chemokine receptors such as C-C chemokine receptor 5 (CCR5) and CXCR3 plays a critical role in the pathogenesis of acute graft-versus-host disease (aGVHD). Our previous studies showed that the addition of CCR5 or CXCR3 antagonists could only slightly alleviate the development of aGVHD. Given the specificity of T lymphocytes bearing CXCR3 and CCR5, we investigated whether combined CCR5 and CXCR3 blockade could further attenuate murine aGVHD. A mouse model of aGVHD was established to assess the efficacy of CCR5 and/or CXCR3 blockade on the development of aGVHD. The distribution of lymphocytes was calculated by quantification of immunostaining cells. The immunomodulatory effect on T cells was assessed by evaluating T-cell proliferation, viability, and differentiation. Using the murine allogeneic hematopoietic stem cell transplantation model, we demonstrated that blockade of both CCR5 and CXCR3 could efficiently alleviate the development of aGVHD. Further investigation on the immune mechanisms for this prophylactic effect showed that more T cells were detained into secondary lymphoid organs (SLOs), which may lead to reduced infiltration of T cells into GVHD target organs. Our study also showed that T cells detained in SLOs dampened the activation, suppressed the polarization toward T helper type 1 (Th1) and T cytotoxic type 1 (Tc1) cells, and induced the production of Treg cells. These data suggest that concurrent blockade of CCR5 and CXCR3 attenuates murine aGVHD through modulating donor-derived T-cell distribution and function, and this might be applicable for aGVHD prophylaxis in clinical settings.

Near-Infrared II Hyperspectral Imaging Improves the Accuracy of Pathological Sampling of Multiple Cancer Types.

Pathological histology is the "gold standard" for clinical diagnosis of cancer. Incomplete or excessive sampling of the formalin-fixed excised cancer specimen will result in inaccurate histologic assessment or excessive workload. Conventionally, pathologists perform specimen sampling relying on naked-eye observation, which is subjective and limited by human perception. Precise identification of cancer tissue, size, and margin is challenging, especially for lesions with inconspicuous tumors. To overcome the limits of human eye perception (visible: 400-700 nm) and improve the sampling efficiency, in this study, we propose using a second near-infrared window (NIR-II: 900-1700 nm) hyperspectral imaging (HSI) system to assist specimen sampling on the strength of the verified deep anatomical penetration and low scattering characteristics of the NIR-II optical window. We used selected NIR-II HSI narrow bands to synthesize color images for human eye observation and also applied a machine learning-based algorithm on the complete NIR-II HSI data for automatic tissue classification to assist pathologists in specimen sampling. A total of 92 tumor samples were collected, including 7 types. Sixty-two (62/92) samples were used as the validation set. Five experienced pathologists marked the contour of the cancer tissue on conventional color images by using different methods, and compared it with the "gold standard," showing that NIR-II HSI-assisted methods had significant improvements in determining cancer tissue compared with conventional methods (conventional color image with or without X-ray). The proposed system can be easily integrated into the current workflow, with high imaging efficiency and no ionizing radiation. It may also find applications in intraoperative detection of residual lesions and identification of different tissues.

High Dynamic Range Dual-Modal White Light Imaging Improves the Accuracy of Tumor Bed Sampling After Neoadjuvant Therapy for Breast Cancer.

OBJECTIVES: Accurate evaluation of residual cancer burden remains challenging because of the lack of appropriate techniques for tumor bed sampling. This study evaluated the application of a white light imaging system to help pathologists differentiate the components and location of tumor bed in specimens. METHODS: The high dynamic range dual-mode white light imaging (HDR-DWI) system was developed to capture antiglare reflection and multiexposure HDR transmission images. It was tested in 60 specimens of modified radical mastectomy after neoadjuvant therapy. We observed the differential transmittance among tumor tissue, fibrosis tissue, and adipose tissue. RESULTS: The sensitivity and specificity of HDR-DWI were compared with x-ray or visual examination to determine whether HDR-DWI was superior in identifying tumor beds. We found that tumor tissue had lower transmittance (0.12 ± 0.03) than fibers (0.15 ± 0.04) and fats (0.27 ± 0.07) (P < .01). CONCLUSIONS: HDR-DWI was more sensitive in identifying fiber and tumor tissues than cabinet x-ray and visual observation (P < .01). In addition, HDR-DWI could identify more fibrosis areas than the currently used whole slide imaging did in 12 samples (12/60). We have determined that HDR-DWI can provide more in-depth tumor bed information than x-ray and visual examination do, which will help prevent diagnostic errors in tumor bed sampling.

Lymph Node Fibroblastic Reticular Cells Attenuate Immune Responses Through Induction of Tolerogenic Macrophages at Early Stage of Transplantation.

BACKGROUND: Fibroblastic reticular cells (FRCs) are a type of stromal cells located in the T zone in secondary lymphoid organs. Previous studies showed that FRCs possess the potential to promote myeloid differentiation. We aim to investigate whether FRCs in lymph nodes (LNs) could induce tolerogenic macrophage generation and further influence T-cell immunity at an early stage of allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: LNs were assayed to confirm the existence of proliferating macrophages after allo-HSCT. Ex vivo-expanded FRCs and bone marrow cells were cocultured to verify the generation of macrophages. Real-time quantitative PCR and ELISA assays were performed to observe the cytokines expressed by FRC. Transcriptome sequencing was performed to compare the difference between FRC-induced macrophages (FMs) and conventional macrophages. Mixed lymphocyte reaction and the utilization of FMs in acute graft-versus-host disease (aGVHD) mice were used to test the inhibitory function of FMs in T-cell immunity in vitro and in vivo. RESULTS: We found a large number of proliferating macrophages near FRCs in LNs with tolerogenic phenotype under allo-HSCT conditions. Neutralizing anti-macrophage colony-stimulating factor receptor antibody abolished FMs generation in vitro. Phenotypic analysis and transcriptome sequencing suggested FMs possessed immunoinhibitory function. Mixed lymphocyte reaction proved that FMs could inhibit T-cell activation and differentiation toward Th1/Tc1 cells. Injection of FMs in aGVHD mice effectively attenuated aGVHD severity and mortality. CONCLUSIONS: This study has revealed a novel mechanism of immune regulation through the generation of FRC-induced tolerogenic macrophages in LNs at an early stage of allo-HSCT.

Reconstructing virtual large slides can improve the accuracy and consistency of tumor bed evaluation for breast cancer after neoadjuvant therapy.

BACKGROUND: To explore whether the "WSI Stitcher", a program we developed for reconstructing virtual large slide through whole slide imaging fragments stitching, can improve the efficiency and consistency of pathologists in evaluating the tumor bed after neoadjuvant treatment of breast cancer compared with the conventional methods through stack splicing of physical slides. METHODS: This study analyzed the advantages of using software-assisted methods to evaluate the tumor bed after neoadjuvant treatment of breast cancer. This new method is to use "WSI Stitcher" to stitch all the WSI fragments together to reconstruct a virtual large slide and evaluate the tumor bed with the help of the built-in ruler and tumor proportion calculation functions. RESULTS: Compared with the conventional method, the evaluation time of the software-assisted method was shortened by 35%(P < 0.001). In the process of tumor bed assessment after neoadjuvant treatment of breast cancer, the software-assisted method has higher intraclass correlation coefficient when measuring the length (0.994 versus 0.934), width (0.992 versus 0.927), percentage of residual tumor cells (0.947 versus 0.878), percentage of carcinoma in situ (0.983 versus 0.881) and RCB index(0.997 versus 0.772). The software-assisted method has higher kappa values when evaluating tumor staging(0.901 versus 0.687) and RCB grading (0.963 versus 0.857). CONCLUSION: The "WSI Stitcher" is an effective tool to help pathologists with the assessment of breast cancer after neoadjuvant treatment.

Automatic Brain Midline Surface Delineation on 3D CT Images With Intracranial Hemorrhage.

Brain midline delineation plays an important role in guiding intracranial hemorrhage surgery, which still remains a challenging task since hemorrhage shifts the normal brain configuration. Most previous studies detected brain midline on 2D plane and did not handle hemorrhage cases well. We propose a novel and efficient hemisphere-segmentation framework (HSF) for 3D brain midline surface delineation. Specifically, we formulate the brain midline delineation as a 3D hemisphere segmentation task, and employ an edge detector and a smooth regularization loss to generate the midline surface. We also introduce a distance-weighted map to keep the attention on the midline. Furthermore, we adopt rectification learning to handle various head poses. Finally, considering the complex situation of ventricle break-in for hemorrhages in bilateral intraventricular (B-IVH) cases, we identify those cases via a classification model and design a midline correction strategy to locally adjust the midline. To our best knowledge, it is the first study focusing on delineating the brain midline surface on 3D CT images of hemorrhage patients and handling the situation of ventricle break-in. Extensive validation on our large in-house datasets (519 patients) and the public CQ500 dataset (491 patients), demonstrates that our method outperforms state-of-the-art methods on brain midline delineation.

LYG1 Deficiency Attenuates the Severity of Acute Graft-Versus-Host Disease via Skewing Allogeneic T Cells Polarization Towards Treg Cells.

Acute graft-versus-host disease (aGVHD) is a lethal complication after allogeneic hematopoietic stem cell transplantation. The mechanism involves the recognition of host antigens by donor-derived T cells which induces augmented response of alloreactive T cells. In this study, we characterized the role of a previously identified novel classical secretory protein with antitumor function-LYG1 (Lysozyme G-like 1), in aGVHD. LYG1 deficiency reduced the activation of CD4+ T cells and Th1 ratio, but increased Treg ratio in vitro by MLR assay. By using major MHC mismatched aGVHD model, LYG1 deficiency in donor T cells or CD4+ T cells attenuated aGVHD severity, inhibited CD4+ T cells activation and IFN-γ expression, promoted FoxP3 expression, suppressed CXCL9 and CXCL10 expression, restrained allogeneic CD4+ T cells infiltrating in target organs. The function of LYG1 in aGVHD was also confirmed using haploidentical transplant model. Furthermore, administration of recombinant human LYG1 protein intraperitoneally aggravated aGVHD by promoting IFN-γ production and inhibiting FoxP3 expression. The effect of rhLYG1 could partially be abrogated with the absence of IFN-γ. Furthermore, LYG1 deficiency in donor T cells preserved graft-versus-tumor response. In summary, our results indicate LYG1 regulates aGVHD by the alloreactivity of CD4+ T cells and the balance of Th1 and Treg differentiation of allogeneic CD4+ T cells, targeting LYG1 maybe a novel therapeutic strategy for preventing aGVHD.

The Potential Genes Mediate the Pathogenicity of Allogeneic CD4+T Cell in aGVHD Mouse Model.

Acute graft-versus-host disease (aGVHD) remains a significant and severe complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Due to the occurrence of aGVHD, allo-HSCT significantly increases the mortality rate compared with autologous hematopoietic stem cell transplantation (auto-HSCT). In this study, auto-HSCT and allo-HSCT aGVHD mouse models were built to detect the difference in CD4+ lymphocyte in different tissues based on ribonucleic acid sequencing (RNA-Seq) analysis. Clustering analysis, functional annotation, and pathway enrichment analysis were performed on differentially expressed genes (DEGs). The protein-protein interaction (PPI) network was used to find hub genes. CD4+T cells were activated by MLR and cytokine stimulation. Cells were sorted out by a flow cell sorter. The selected genes were verified by qRT-PCR, histology, and immunofluorescence staining. The GSE126518 GEO dataset was used to verify the hub genes. Enrichment analysis revealed four immune-related pathways that play an important role in aGVHD, including immunoregulatory interactions between a lymphoid and a nonlymphoid cell, chemokine receptors binding chemokines, cytokine and cytokine receptor interaction, and the chemokine signaling pathway. At the same time, with the PPI network, 11 novel hub genes that were most likely to participate in immunoregulation in aGVHD were identified, which were further validated by qRT-PCR and the GSE126518 dataset. Besides, the protein expression level of Cxcl7 was consistent with the sequencing results. In summary, this study revealed that immunoregulation-related DEGs and pathways played a vital role in the onset of aGVHD. These findings may provide some new clues for probing the pathogenesis and treatment of aGVHD.

In Vitro Immunological Effects of CXCR3 Inhibitor AMG487 on Dendritic Cells.

AMG 487 is the targeted blocker of chemokine receptor CXCR3 and improves inflammatory symptoms by blocking the inflammatory cycle. Here we investigated whether AMG 487 affects dendritic cell (DC) biology and function. The expression of co-stimulatory markers on DCs was reduced, indicating the semi-mature state of DC when AMG 487 was added throughout the in vitro differentiation period. Additionally, when added solely during the final lipopolysaccharide-induced activation step, AMG 487 inhibited DC activation, as demonstrated by a decreased expression of activation markers. AMG487 also promoted the expression of PD-L2 and impaired the ability to induce antigen-specific T cell responses. Our results demonstrated that AMG 487 significantly affects DC maturity in vitro and function leading to impaired T cell activation, inducing DCs to have characteristics similar to tolerogenic DCs. AMG 487 may directly play an immunomodulatory role during DC development and functional shaping.

Deeply-Supervised Networks With Threshold Loss for Cancer Detection in Automated Breast Ultrasound.

ABUS, or Automated breast ultrasound, is an innovative and promising method of screening for breast examination. Comparing to common B-mode 2D ultrasound, ABUS attains operator-independent image acquisition and also provides 3D views of the whole breast. Nonetheless, reviewing ABUS images is particularly time-intensive and errors by oversight might occur. For this study, we offer an innovative 3D convolutional network, which is used for ABUS for automated cancer detection, in order to accelerate reviewing and meanwhile to obtain high detection sensitivity with low false positives (FPs). Specifically, we offer a densely deep supervision method in order to augment the detection sensitivity greatly by effectively using multi-layer features. Furthermore, we suggest a threshold loss in order to present voxel-level adaptive threshold for discerning cancer vs. non-cancer, which can attain high sensitivity with low false positives. The efficacy of our network is verified from a collected dataset of 219 patients with 614 ABUS volumes, including 745 cancer regions, and 144 healthy women with a total of 900 volumes, without abnormal findings. Extensive experiments demonstrate our method attains a sensitivity of 95% with 0.84 FP per volume. The proposed network provides an effective cancer detection scheme for breast examination using ABUS by sustaining high sensitivity with low false positives. The code is publicly available at https://github.com/nawang0226/abus_code.

3D Inception U-net with Asymmetric Loss for Cancer Detection in Automated Breast Ultrasound.

PURPOSE: Breast cancer is the most common cancer and the leading cause of cancer-related deaths for women all over the world. Recently, automated breast ultrasound (ABUS) has become a new and promising screening modality for whole breast examination. However, reviewing volumetric ABUS is time-consuming and lesions could be missed during the examination. Therefore, computer-aided cancer detection in ABUS volume is extremely expected to help clinician for the breast cancer screening. METHODS: We develop a novel end-to-end 3D convolutional network for automated cancer detection in ABUS volume, in order to accelerate reviewing and meanwhile to provide high detection sensitivity with low false positives (FPs). Specifically, an efficient 3D Inception Unet-style architecture with fusion deep supervision mechanism is proposed to attain decent detection performance. In addition, a novel asymmetric loss is designed to help the network balancing false positive and false negative regions, thus improving detection sensitivity for small cancerous lesions. RESULTS: The efficacy of our network was extensively validated on a dataset including 196 patients with 661 cancer regions. Our network obtained a detection sensitivity of 95.1% with 3.0 FPs per ABUS volume. Furthermore, the average inference time of the network was 0.1 second per volume, which largely shortens the conventional reviewing time. CONCLUSIONS: The proposed network provides efficient and accurate cancer detection scheme using ABUS volume, and may assist clinicians for more efficient breast cancer screening.

CXCR3 blockade combined with cyclosporine A alleviates acute graft-versus-host disease by inhibiting alloreactive donor T cell responses in a murine model.

Chemotaxis of T cells to acute graft-versus-host disease (aGvHD) target tissues directed by chemokines and their receptors plays a key role in the pathogenesis of aGvHD. Blockade of lymphocyte migration by targeting chemokine receptors may be a viable strategy for the prevention and treatment of aGvHD, which is quite distinguishable from typical efforts to use immunosuppressive medications that have been associated with some side effects. CXCR3 and its ligands have been reported to be correlated with aGvHD pathogenesis. Using the small-molecule CXCR3 antagonist AMG487, we demonstrated that AMG487 combined with cyclosporine A (CsA) effectively alleviated aGvHD with a prolonged mean survival time and significantly inhibited the infiltration of inflammatory cells in aGvHD target tissues in a murine aGvHD model. In addition, AMG487 combined with CsA inhibited the activation, proliferation and differentiation of donor-derived T cells in the spleens. Further results showed that the concentrations of Th1 cells associated with pro-inflammatory cytokines such as IFN-γ and TNFα in serum were decreased. In addition, AMG487 treatment did not alter CXCR3 and CCR5 expression in donor-derived T cells but elevated the serum CXCL9 and CXCL10 levels. This novel and effective approach has the potential to develop a new clinical method to prevent and treat aGvHD.

Covalent-reaction-induced interfacial assembly to transform doxorubicin into nanophotomedicine with highly enhanced anticancer efficiency.

Herein, we show that a molecular assembly offers tremendous opportunities of affording existing building units with new physicochemical properties, holding promise in wide applications. Herein, we develop a facile covalent assembly using a natural occurring linker, genipin, to efficiently transform a traditional chemo drug, doxorubicin, into a nanophotomedicine. A possible mechanism is proposed, in which doxorubicin reacts with genipin through covalent bonding to produce poorly soluble units, which further form nuclei and mediate the interfacial assembly to generate uniform nanoparticles. Such assembled nanophotomedicine shows remarkably enhanced singlet oxygen generation ability (about 1000 folds), leading to a much higher photodynamic activity. Moreover, this self-carried nanodrug exhibits adjustable size, excellent colloidal stability, high capacity and preferable endocytosis. These favorable features lead to greatly improved anticancer efficiency under light at the same dosage, compared to that of pure doxorubin. We believe this study brings a new dimension to develop advanced drug delivery systems by molecular assembly.

Biomimetic membrane-conjugated graphene nanoarchitecture for light-manipulating combined cancer treatment in vitro.

We report that through facile lipid self-assembly, biomimetic membrane-conjugated mesoporous silica-coated graphene oxide is constructed as targeting nanocarrier toward efficient combination of photothermal therapy and chemotherapy. Impressively, the simple surface modification with folate-contained lipid bilayer allows the graphene-based nanoarchitecture above to be selectively internalized by tumor cells overexpressing relevant receptors. Compared to pure drug, 7-fold doxorubicin is delivered into tumor cells by the nanoarchitecture. After cellular internalization, upon near infrared light illumination, graphene oxide in the nanoarchitecture can convert light energy into heat to kill cancer cells partially. Simultaneously, hyperthermia will drive rapid release of doxorubicin from the nanoarchitecture above to further cause the death of more cancer cells. Thus, integrated cancer treatment with higher efficacy is achieved in vitro compared to that of individual therapy.

Rational assembly of a biointerfaced core@shell nanocomplex towards selective and highly efficient synergistic photothermal/photodynamic therapy.

To optimize synergistic cancer therapy, we rationally assemble an inorganic-organic nanocomplex using a folate-modified lipid bilayer spread on photosensitizer-entrapped mesoporous silica nanoparticle (MSN) coated gold nanorods (AuNRs). In this hybrid bioconjugate, the large specific surface area and pore size of AuNR@MSN guarantee a high loading capacity of small photosensitive molecules. The modification with selective mixed liposomes on the surface of AuNR@MSN enables faster cellular internalization and enhancement of endocytosis. Under one-time NIR two-photon illumination, AuNR-mediated hyperthermia can kill cancer cells directly. Meanwhile, the loaded photosensitizer, hypocrellin B, generates two kinds of reactive oxygen species (ROS) to induce cell apoptosis. Remarkably, hyperthermia can improve the yield of ROS. After intravenous injection of this bioconjugate into female BALB/c nude mice followed by laser irradiation (808 nm, 1.3 W cm(-2), 6 min), the tumor growth is suppressed completely. The tumors are not recurrent within the observation time (19 days), and the normal or main organs are not obviously pathological. Thus, such a simplified and selective cancer treatment, combining photothermal and photodynamic therapy in a synergistic manner, provides outstanding efficiency in vivo. This nanocomplex with well-defined core@shell nanostructures integrated with a two-photon technique holds great promise to improve cancer phototherapy with a high efficiency in the clinic.