Metabolic Heterogeneity and Potential Immunotherapeutic Responses Revealed by Single-Cell Transcriptomics of Breast Cancer.

BACKGROUND: Breast cancer (BC) exhibits remarkable heterogeneity. However, the transcriptomic heterogeneity of BC at the single-cell level has not been fully elucidated. METHODS: We acquired BC samples from 14 patients. Single-cell RNA sequencing (scRNA-seq), bioinformatic analyses, along with immunohistochemistry (IHC) and immunofluorescence (IF) assays were carried out. RESULTS: According to the scRNA-seq results, 10 different cell types were identified. We found that Cancer-Associated Fibroblasts (CAFs) exhibited distinct biological functions and may promote resistance to therapy. Metabolic analysis of tumor cells revealed heterogeneity in glycolysis, gluconeogenesis, and fatty acid synthetase reprogramming, which led to chemotherapy resistance. Furthermore, patients with multiple metastases and progression were predicted to benefit from immunotherapy based on a heterogeneity analysis of T cells and tumor cells. CONCLUSIONS: Our findings provide a comprehensive understanding of the heterogeneity of BC, provide comprehensive insight into the correlation between cancer metabolism and chemotherapy resistance, and enable the prediction of immunotherapy responses based on T-cell heterogeneity.

Identifying altered developmental pathways in human globoid cell leukodystrophy iPSCs-derived NSCs using transcriptome profiling.

BACKGROUND: Globoid cell leukodystrophy (GLD) is a devastating neurodegenerative disease characterized by widespread demyelination caused by galactocerebrosidase defects. Changes in GLD pathogenesis occurring at the molecular level have been poorly studied in human-derived neural cells. Patient-derived induced pluripotent stem cells (iPSCs) are a novel disease model for studying disease mechanisms and allow the generation of patient-derived neuronal cells in a dish. RESULTS: In this study, we identified gene-expression changes in iPSCs and iPSC-derived neural stem cells (NSCs) from a patient with GLD (K-iPSCs/NSCs) and normal control (AF-iPSCs/NSCs), in order to investigate the potential mechanism underlying GLD pathogenesis. We identified 194 (K-iPSCs vs. AF-iPSCs) and 702 (K-NSCs vs. AF-NSCs) significantly dysregulated mRNAs when comparing the indicated groups. We also identified dozens of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway terms that were enriched for the differentially expressed genes. Among them, 25 differentially expressed genes identified by RNA-sequencing analysis were validated using real-time quantitative polymerase chain reaction analysis. Dozens of pathways involved in neuroactive ligand-receptor interactions, synaptic vesicle cycle signaling, serotonergic synapse signaling, phosphatidylinositol-protein kinase B signaling, and cyclic AMP signaling were identified as potential contributors to GLD pathogenesis. CONCLUSIONS: Our results correspond to the fact that mutations in the galactosylceramidase gene may disrupt the identified signaling pathways during neural development, suggesting that alterations in signaling pathways contribute to GLD pathogenesis. At the same time, our results demonstrates that the model based on K-iPSCs is a novel tool that can be used to study the underlying molecular basis of GLD.

Gene delivery to breast cancer by incorporated EpCAM targeted DARPins into AAV2.

OBJECTIVE: The aim of this study is to evaluate an AAV vector that can selectively target breast cancer cells and to investigate its specificity and anti-tumor effects on breast cancer cells both in vitro and in vivo, offering a new therapeutic approach for the treatment of EpCAM-positive breast cancer. METHODS: In this study, a modified AAV2 viral vector was used, in which EpCAM-specific DARPin EC1 was fused to the VP2 protein of AAV2, creating a viral vector that can target breast cancer cells. The targeting ability and anti-tumor effects of this viral vector were evaluated through in vitro and in vivo experiments. RESULTS: The experimental results showed that the AAV2MEC1 virus could specifically infect EpCAM-positive breast cancer cells and accurately deliver the suicide gene HSV-TK to tumor tissue in mice, significantly inhibiting tumor growth. Compared to the traditional AAV2 viral vector, the AAV2MEC1 virus exhibited reduced accumulation in liver tissue and had no impact on tumor growth. CONCLUSION: This study demonstrates that AAV2MEC1 is a gene delivery vector capable of targeting breast cancer cells and achieving selective targeting in mice. The findings offer a potential gene delivery system and strategies for gene therapy targeting EpCAM-positive breast cancer and other tumor types.

Generation of the human induced pluripotent stem cell line PUMCi005-A from a patient with Perrault syndrome.

We generated PUMCi005-A, an induced pluripotent stem cell (iPSC) line, from dermal fibroblasts of a 32-year-old female Perrault syndrome patient with double heterozygous (794 G > A and 1181 G > A) mutations in the TWNK gene using Sendai viral delivery of OCT4, SOX2, KLF4, and c-MYC. The PUMCi005-A iPSC line carried the TWNK mutations, displayed typical iPSC morphology, expressed pluripotent stem cell markers, did not have integration of Sendai virus, and exhibited a normal karyotype and differentiation into three germ layers.

rAAV2-Mediated Restoration of GALC in Neural Stem Cells from Krabbe Patient-Derived iPSCs.

Krabbe disease is a rare neurodegenerative fatal disease. It is caused by deficiency of the lysosomal enzyme galactocerebrosidase (GALC), which results in progressive accumulation of galactolipid substrates in myelin-forming cells. However, there is still a lack of appropriate neural models and effective approaches for Krabbe disease. We generated induced pluripotent stem cells (iPSCs) from a Krabbe patient previously. Here, Krabbe patient-derived neural stem cells (K-NSCs) were induced from these iPSCs. By using nine kinds of recombinant adeno-associated virus (rAAV) vectors to infect K-NSCs, we found that the rAAV2 vector has high transduction efficiency for K-NSCs. Most importantly, rAAV2-GALC rescued GALC enzymatic activity in K-NSCs. Our findings not only establish a novel patient NSC model for Krabbe disease, but also firstly indicate the potential of rAAV2-mediated gene therapy for this devastating disease.

Generation and characterization of iPS cell line (CTGUi001-A) from skin fibroblasts of a patient with Fabry disease.

We have generated an iPSCs line (CTGUi001-A) from dermal fibroblasts of a 16-year-old male Fabry disease patient with a novel GLA gene mutation (c.156C > A) using Sendai virus encoding the four Yamanaka factors OCT4, SOX2, KLF4, and c-MYC. The CTGUi001-A iPSC line displayed typical embryonic stem cell-like morphology, carried the GLA gene mutation, expressed several pluripotent stem cell makers, retained normal karyotype (46, XY) and was capable of forming teratomas containing three germ layers.

Single-cell transcriptome sequencing of B-cell heterogeneity and tertiary lymphoid structure predicts breast cancer prognosis and neoadjuvant therapy efficacy.

BACKGROUND: Breast cancer (BC) is a highly heterogeneous disease, and although immunotherapy has recently increased patient survival in a number of solid and hematologic malignancies, most BC subtypes respond poorly to immune checkpoint blockade therapy (ICB). B cells, particularly those that congregate in tertiary lymphoid structures (TLS), play a significant role in antitumour immunity. However, B-cell heterogeneity at single-cell resolution and its clinical significance with TLS in BC need to be explored further. METHODS: Primary tumour lesions and surrounding normal tissues were taken from 14 BC patients, totaling 124,587 cells, for single-cell transcriptome sequencing and bioinformatics analysis. RESULTS: Based on the usual markers, the single-cell transcriptome profiles were classified into various clusters. A thorough single-cell study was conducted with a focus on tumour-infiltrating B cells (TIL-B) and tumour-associated neutrophils (TAN). TIL-B was divided into five clusters, and unusual cell types, such as follicular B cells, which are strongly related to immunotherapy efficacy, were identified. In BC, TAN and TIL-B infiltration are positively correlated, and at the same time, compared with TLS-high, TAN and TIL-B in TLS-low group are significantly positively correlated. CONCLUSIONS: In conclusion, our study highlights the heterogeneity of B cells in BC, explains how B cells and TLS contribute significantly to antitumour immunity at both the single-cell and clinical level, and offers a straightforward marker for TLS called CD23. These results will offer more pertinent information on the applicability and effectiveness of tumour immunotherapy for BC.

Efficacy of Resection of Lateral Wall of Endolymphatic Sac for Treatment of Meniere's Disease.

Background: To explore the long-term efficacy and safety of resection of the lateral wall of the endolymphatic sac for the treatment of intractable Meniere's disease (MD) as an alternative surgical procedure for treating this disorder. Methods: Data from 73 patients who were referred to our hospital and diagnosed with unilateral MD between January 2015 and June 2019 were retrospectively analyzed in this study. Seventy-three patients who had frequent vertigo even after receiving standardized conservative treatment for at least half a year underwent resection of the lateral wall of the endolymphatic sac. Vertigo control and auditory function were assessed. Pure tone audiometry, caloric test, and vestibular evoked myogenic potential were performed to evaluate audiological and vestibular functions. The post-operative follow-up duration was more than 2 years. Results: Among the 73 patients (male 34 cases, female 39 cases; age 20-69 years, average 51.4), vertigo was controlled effectively for 66 cases (90.4%) after 2 years of follow-up; 45 cases (61.6%) were completely controlled, and 21 cases (28.8%) were substantially controlled in this study. The patients of 16.4% had hearing loss with more than 10 dB change based on the four-tone average (0.5, 1, 2 and 3 kHz). No patient had a facial nerve weakness, cerebrospinal fluid leakage, or other complications. Conclusion: Resection of the lateral wall of the endolymphatic sac, which can effectively control vertiginous symptoms in intractable MD patients, represents an effective and safe therapy for this disease. Resection of the lateral wall of the endolymphatic sac is expected to be used as an alternative treatment for MD.

Production and characterization of human induced pluripotent stem cell line (PUMCi002-A) from a Krabbe patient related control to study disease mechanisms associated with GALC mutation.

A KD-control human induced pluripotent stem cells (iPSCs) line (PUMCi002-A) was generated from dermal fibroblasts of a Krabbe patient's father with a c.461C>A mutation in Galactocerebrosidase (GALC) gene. The pluripotency, in vitro differentiation potential and karyotype stability of generated iPSC line were analyzed and confirmed. This cell line can be exploited as a control iPSC line to better understand the mechanisms involved in GALC-associated Krabbe disease and provide plausible new therapeutic directions.

The study of correlation between nomogram prediction of uric acid and different chemotherapy regimens in breast cancer patients.

Background: High levels of serum uric acid (SUA) are associated with a poor survival rate of breast cancer. Meanwhile, a sharp increase in SUA after chemotherapy may lead to tumor lysis syndrome (TLS). We created and validated a nomogram to help doctors better manage the patient's SUA level ahead of time in this study. Methods: From July 2012 to June 2021, 206 patients with breast cancer undergoing chemotherapy participated in the study. They are randomly divided into training set (n=137) and validation set (n=69). Univariate and multivariate logistic regression analysis was used to screen the independent predictors of the risk of elevated uric acid in the whole training set data. The receiver operating characteristic (ROC) curve and decision curve assessed the accuracy and clinical application value of nomogram. Results: We confirmed that body mass index (BMI), age, menopause, EC-T chemotherapy (epirubicin-cyclophosphamide followed by paclitaxel) and THP + C-T (pirarubicin-cyclophosphamide followed by paclitaxel) are independent risk factors for high SUA. We established a nomogram for high SUA risk prediction to help clinicians make individualized choice of chemotherapy regimen. In the training cohort, the area under the ROC curve (AUC) showed statistical accuracy (AUC =0.796). Decision curve analysis proved the clinical value of the nomogram. Conclusions: This nomogram can be used to calculate the specific likelihood of high SUA in patients with breast cancer undergoing chemotherapy with different chemotherapy options.

Long-term efficacy of triple semicircular canal plugging in the treatment of patients with ipsilateral delayed endolymphatic hydrops.

This study aims to explore the long-term efficacy of triple semicircular canal plugging (TSCP) in the treatment of intractable ipsilateral delayed endolymphatic hydrops (DEH), so as to provide an alternative therapy for this disease. Forty-eight patients diagnosed with ipsilateral DEH referred to vertigo clinic of our hospital between Dec. 2010 and Dec. 2017, were included in this study for retrospective analysis. All patients were followed up for 2 years. Vertigo control and auditory functions were measured and analyzed. Pure tone audiometry, caloric test, and vestibular evoked myogenic potential (VEMP) were performed in two-year follow-up. Forty-five patients who accepted intratympanic gentamicin (26.7 mg/mL) twice given one week apart were selected as a control group. The total control rate of vertigo in TSCP group was 97.9% (47/48) in the two-year follow-up, with complete control rate of 83.3% (40/48) and substantial control rate of 14.6% (7/48). The rate of hearing loss was 22.9% (11/48). The total control rate of vertigo in intratympanic gentamicin group was 80.0% (36/45), with complete control rate of 57.8% (26/45) and substantial control rate of 22.2% (10/45), and the rate of hearing loss was 20.0% (9/45). The vertigo control rate of TSCP was significantly higher than that of intratympanic gentamicin (χ2 = 6.01, p < 0.05). There was no significant difference of hearing loss rate between two groups. (χ2 = 0.12, p > 0.05). TSCP, which can reduce vertiginous symptoms in patients with intractable ipsilateral DEH, represents an effective therapy for this disorder.

Imaging Analysis of Patients With Meniere's Disease Treated With Endolymphatic Sac-Mastoid Shunt Surgery.

Objective: Endolymphatic sac surgery is effective in treating intractable Meniere's disease (MD), but the underlying mechanism is still unknown. Our study investigated the mechanism by which endolymphatic sac-mastoid shunt (EMS) surgery is effective in treating MD by means of imaging. Methods: The experiment included 19 patients with intractable MD who underwent 3D-fluid-attenuated inversion recovery (FLAIR) MRI with a 3-Tesla unit 6 h after intravenous administration of gadolinium, before EMS, and 2 years after the surgery. The enhanced perilymphatic space in the bilateral cochlea, vestibule, and canals was visualized and compared with that in the endolymphatic space by quantitatively scoring the scala vestibuli of the cochlea and by measuring the developing area of the vestibules quantitatively. Results: Gadolinium was present in the perilymph of the inner ear in the cochlea, vestibules, and canals of all patients. At the 2-year follow-up, 14 (73.68%) patients had vertigo control. Both before and 2 years after surgery, significant differences were observed in the scala vestibuli scores and the area of vestibular perilymph between the affected and healthy sides. The scala vestibuli scores and the area of vestibular perilymph, however, did not differ when comparing them before and after surgery. Conclusions: According to our results, endolymphatic hydrops was not significantly reduced by surgery. The mechanism by which EMS controls vertigo might be unrelated to the improvement in hydrops.

Intraoperative auditory brainstem response monitoring during semicircular canal plugging surgery in treatment of Meniere's disease.

BACKGROUND: Three semicircular canal plugging (TSCP) is an optimized treatment for intractable Meniere's disease (MD). However, 20-30% of patients experience hearing loss after TSCP, for reasons that remain unclear. OBJECTIVE: To evaluate hearing loss resulting from TSCP. SUBJECTS AND METHODS: This study included 12 patients, which were diagnosed with definite MD and consented to TSCP surgery. Intraoperative auditory brainstem response (ABR) was monitored in each surgical procedure. RESULTS: After opening the mastoid cavity, the ABR threshold increased to 77.08 ± 9.88 dB nHL. The ABR threshold almost recovered to preoperative levels, to 68.33 ± 7.78 dB nHL, after completing TSC outlining. Exposure of three semicircular canal 'blue lines' had little effect on ABR threshold. The most prominent change on hearing loss was observed after mastoid outlining, when 41.67% of patients showed hearing loss ≥10 dB nHL. None of the patients showed a threshold shift ≥10 dB nHL following the last step. CONCLUSIONS: TSCP operation itself caused little hearing damage. SIGNIFICANCE: Ruled out hearing loss as a result of the surgery itself. The reason why 20-30% of patients showed hearing loss in 2-year follow-up visit was not clear, although it may be due to serous fibrous labyrinthitis.

Generation of a human induced pluripotent stem cell line PUMCi001-A from a patient with Krabbe disease.

We have generated PUMCi001-A, an induced pluripotent stem cells (iPSC) line from dermal fibroblasts of a 13-year-old male Krabbe disease patient with two hemizygous (461C > A and 1244G > A) mutations in Galactocerebrosidase (GALC) gene using a Sendai viral delivery of OCT4, SOX2, KLF4, and c-MYC. The PUMCi001-A iPSC line carried the GALC mutations, displayed typical iPSC morphology, expressed pluripotent stem cell makers, exhibited a normal karyotype and differentiation capacity into three germ layers.

Revision surgery after triple semicircular canal plugging and morphologic changes of vestibular organ.

This study aims to investigate the causes of vertigo relapse in patients with Meniere's disease (MD) who had undergone triple semicircular canal plugging (TSCP) and explore the morphologic changes of vestibular organ through revision surgery. Eleven intractable MD patients who underwent TSCP initially and experienced episodic vertigo recurrence later, were enrolled. All patients accepted revision surgery, including seven cases who underwent labyrinthectomy and four cases who underwent repeat TSCP. Pure tone test, caloric test and video-head impulse test (v-HIT) were used to evaluate audiological and vestibular functions. Specimens of canal plugging materials and vestibular end organs were collected from patients who underwent labyrinthectomy during revision surgery. Mineralization and other histological characteristics of canal plugging materials were evaluated by von Kossa staining. Incomplete occlusion or ossification was observed in the semicircular canals (SCs) of all eleven patients, with all three SCs affected in three, the superior SC in five patients, the horizontal SC in two and the posterior SC in one. The results of v-HIT were in accordance with findings discovered intraoperatively. Few mineralized nodules and multiple cavities were found in the von Kossa-stained canal plugging materials. Incomplete occlusion or ossification of SCs was the principal cause of vertigo recurrence in MD patients who underwent TSCP. v-HIT was helpful in determining the responsible SCs.

Long-term outcomes of triple semicircular canal plugging for the treatment of intractable Meniere's disease: A single center experience of 361 cases.

INTRODUCTION: Meniere's disease is a common chronic inner ear disease. Because the definitive pathogenesis is still unknown, there is currently no cure for this disorder. Semicircular canal plugging (SCP), first used to treat patients with intractable benign paroxysmal positional vertigo, has since been applied to patients with intractable peripheral vertigo. This study was aimed to explore the long-term efficacy of triple semicircular canal plugging (TSCP) in the treatment of intractable Meniere's disease (MD) so as to provide a new method in the framework of treatment with MD. METHODS: Three hundred and sixty-one unilateral MD patients, who were treated with TSCP in our hospital between Dec. 2010 and Sep. 2016, were recruited in this study for retrospective analysis. Vertigo control and auditory function were monitored during a period of two-year follow-up. Seventy three patients who were subjected to intratympanic gentamicin were selected as a control group. Pure tone audiometry, caloric test, vestibular evoked myogenic potential (VEMP) were performed in two-year follow-up. RESULTS: The total control rate of vertigo in TSCP group was 97.8% (353/361) in the two-year follow-up, with complete control rate of 80.3% (290/361) and substantial control rate of 17.5% (63/361). The rate of hearing loss was 26.3% (95/361). The total control rate of vertigo in intratympanic gentamicin group was 83.6% (61/73), with complete control rate of 63.0% (46/73) and substantial control rate of 20.5% (15/73). The rate of hearing loss was 24.7% (18/73). The vertigo control rate of TSCP was significantly higher than that of chemical labyrinthectomy(χ2 = 24.798, p <  0.05). There was no significant difference of hearing loss rate between two groups. (χ2 = 0.087, p >  0.05). CONCLUSION: Triple semicircular canal plugging (TSCP), which can reduce vertiginous symptoms in patients with intractable Meniere's disease (MD), represents an effective therapy for this disorder. It might become a new important method in the framework of treatment with MD.

An engineered PD-1-based and MMP-2/9-oriented fusion protein exerts potent antitumor effects against melanoma.

Recent studies showed that the PD-1/PD-L1 checkpoint blockade is a dramatic therapy for melanoma by enhancing antitumor immune activity. Currently, major strategies for the PD-1/PD-L1 blockade have mainly focused on the use of antibodies and compounds. Seeking an alternative approach, others employ endogenous proteins as blocking agents. The extracellular domain of PD-1 (ePD1) includes the binding site with PD-L1. Accordingly, we constructed a PD-1-based recombinantly tailored fusion protein (dFv-ePD1) that consists of bivalent variable fragments (dFv) of an MMP-2/9-targeted antibody and ePD1. The melanoma-binding intensity and antitumor activity were also investigated. We found the intense and selective binding capability of the protein dFv-ePD1 to human melanoma specimens was confirmed by a tissue microarray. In addition, dFv-ePD1 significantly suppressed the migration and invasion of mouse melanoma B16-F1 cells, and displayed cytotoxicity to cancer cells in vitro. Notably, dFv-ePD1 significantly inhibited the growth of mouse melanoma B16-F1 tumor cells in mice and in vivo fluorescence imaging showed that dFv-ePD was gradually accumulated into the B16-F1 tumor. Also the B16-F1 tumor fluorescence intensity at the tumor site was stronger than that of dFv. This study indicates that the recombinant protein dFv-ePD1 has an intensive melanoma-binding capability and exerts potent therapeutic efficacy against melanoma. The novel format of the PD-L1-blocked agent may play an active role in antitumor immunotherapy. [BMB Reports 2018; 51(11): 572-577].

Triple semicircular canal plugging: a novel modality for the treatment of intractable Meniere's disease.

CONCLUSIONS: TSCP, which can reduce vertiginous symptoms in patients with intractable MD, represents an effective therapy for this disorder. OBJECTIVE: To explore the long-term efficacy of triple semicircular canal plugging (TSCP) in the treatment of intractable Meniere's disease (MD). METHODS: Seventy-nine patients diagnosed with unilateral MD referred to a vertigo clinic of the hospital between December 2010 and December 2013 were included in this study for retrospective analysis. TSCP was performed in the affected ear for each patient. Vertigo control and auditory function were measured. Pure tone audiometry, caloric test, and cervical vestibular evoked myogenic potential (cVEMP) were performed in 2-year follow-up. Thirty-six MD patients, who accepted endolymphatic sac decompression (ESD) operation were selected as a comparison group. RESULTS: The total control rate of vertigo in the TSCP group was 98.7% in the 2-year follow-up, with a complete control rate of 81.0% and substantial control rate of 17.7%. The rate of hearing preservation was 70.9%. The total control rate of vertigo in the ESD operation group was 72.2%. The vertigo control rate of TSCP was significantly higher than that of ESD operation. Twenty-four months after treatment, canal paresis was found in the operation side of all patients of TSCP by means of caloric test.

Apoptotic Melanoma B16-F1 Cells Induced by Lidamycin Could Initiate the Antitumor Immune Response in BABL/c Mice.

In the process of tumor cell apoptosis induced by specific regents, calreticulin (CRT) was transferred from endoplasmic reticulum (ER) onto the cell membrane. These tumor cells, when used as the cellular vaccine to immunize experimental animals, could initiate effective antitumor immunoresponse against homologous tumor cells. This is referred to as immunogenic cell death. Lidamycin (LDM) is an enediyne antibiotic, which has extremely potent cytotoxicity to cancer cells. In this study, the mouse melanoma B16-F1 cancer cells were used to investigate the ability of LDM in promoting immunogenic cell death. Our data showed that LDM could induce apoptosis of B16-F1 cancer cells, accompanied by CRT translocation onto the cell membrane. These LDM-treated B16-F1 cells could be recognized and phagocytosed more efficiently by macrophage and dendritic cells. When the LDM-treated apoptotic B16-F1 cells were used as a whole-cell tumor vaccine to immune mice, the mice obtained resistance against rechallenged B16-F1 living cells. At the same time, the specific antitumor immune response was observed in these vaccinated mice. The splenocytes from the mice vaccinated with LDM-treated B16-F1 cells showed significantly enhanced NK lymphocyte activities and also faster growth rate and increased secretion of IFN-γ when encountering the cellular antigens from B16-F1 cells. All these results suggested that LDM could promote immunogenic cell death in B16-F1 cells, and these LDM-treated B16-F1 cells could be used as a sort of cell vaccine to initiate effective antitumor immunoresponse in mice.