DNA-nanorobot-guided thrombin-inducing tumor infarction: raising new potential clinical concerns.

DNA-nanorobot-guided thrombin-inducing tumor infarction (DNA NanorobotTh-ITI) is emerging as a powerful therapeutic strategy for treatment of solid cancers. The technology represents a major advance in the application of DNA nanotechnology for anticancer therapy. More importantly, the technology is being translated from preclinical studies to the clinic owing to its promising anticancer effects with fewer toxicities demonstrated in preclinical settings. However, despite these beneficial effects of the technology, it is important to point out that some important potential clinical concerns remain to be addressed. Here, we raise these clinical concerns along with these beneficial effects of the technology. Hopefully, these newly raised potential clinical concerns could drive forward research in this field to expedite its clinical translation.

Elusive Neurotoxicity in T Cell-Boosting Anticancer Therapies.

Several T cell-boosting anticancer therapies (including anti-CD19 CAR-T cells and bi-specific T cell engagers, BiTEs) have been approved by the FDA for specific clinical indications. Their potency has been demonstrated in various clinical trials, but some life-threatening complications such as neurotoxicity remain poorly understood. Thus, by conducting multifaceted investigations, a better understanding of T cell immunotherapy-associated neurotoxicity to bridge the gap between basic research and clinical practice is urgently needed.

Approved CAR T cell therapies: ice bucket challenges on glaring safety risks and long-term impacts.

Two autologous chimeric antigen receptor (CAR) T cell therapies (Kymriah™ and Yescarta™) were recently approved by the FDA. Kymriah™ is for the treatment of pediatric patients and young adults with refractory or relapse (R/R) B cell precursor acute lymphoblastic leukemia and Yescarta™ is for the treatment of adult patients with R/R large B cell lymphoma. In common, both are CD19-specific CAR T cell therapies lysing CD19-positive targets. Their dramatic efficacy in the short term has been highlighted by many media reports. By contrast, their glaring safety gaps behind the miracles remain much less addressed. Here, we focus on addressing the crucial challenges in relation to the gaps.

Breakthroughs in modern cancer therapy and elusive cardiotoxicity: Critical research-practice gaps, challenges, and insights.

To date, five cancer treatment modalities have been defined. The three traditional modalities of cancer treatment are surgery, radiotherapy, and conventional chemotherapy, and the two modern modalities include molecularly targeted therapy (the fourth modality) and immunotherapy (the fifth modality). The cardiotoxicity associated with conventional chemotherapy and radiotherapy is well known. Similar adverse cardiac events are resurging with the fourth modality. Aside from the conventional and newer targeted agents, even the most newly developed, immune-based therapeutic modalities of anticancer treatment (the fifth modality), e.g., immune checkpoint inhibitors and chimeric antigen receptor (CAR) T-cell therapy, have unfortunately led to potentially lethal cardiotoxicity in patients. Cardiac complications represent unresolved and potentially life-threatening conditions in cancer survivors, while effective clinical management remains quite challenging. As a consequence, morbidity and mortality related to cardiac complications now threaten to offset some favorable benefits of modern cancer treatments in cancer-related survival, regardless of the oncologic prognosis. This review focuses on identifying critical research-practice gaps, addressing real-world challenges and pinpointing real-time insights in general terms under the context of clinical cardiotoxicity induced by the fourth and fifth modalities of cancer treatment. The information ranges from basic science to clinical management in the field of cardio-oncology and crosses the interface between oncology and onco-pharmacology. The complexity of the ongoing clinical problem is addressed at different levels. A better understanding of these research-practice gaps may advance research initiatives on the development of mechanism-based diagnoses and treatments for the effective clinical management of cardiotoxicity.

Activation of CECR1 in M2-like TAMs promotes paracrine stimulation-mediated glial tumor progression.

Background: The majority of glioma-associated microglia/macrophages have been identified as M2-type macrophages with immune suppressive and tumor supportive action. Recently, the extracellular adenosine deaminase protein Cat Eye Syndrome Critical Region Protein 1 (CECR1) was shown to regulate macrophage maturation. In this study, we investigate the role of CECR1 in the regulation of the glioma-associated macrophage response. Methods: Expression of CECR1 was assessed in human glioma samples. CECR1-mediated macrophage response was studied in vitro, using donor derived CD14+ monocytes and the THP-1 monocytic cell line. The response of the human glioma cell line U87 to conditioned medium of macrophages preconditioned with recombinant human CECR1 or CECR1 silencing was also assessed. Results: CECR1 was strongly expressed in high-grade gliomas (P < .001) and correlated positively with the M2 phenotype markers in tumor-associated microglia/macrophages (TAMs) (overall, P < .05). In vitro studies confirmed the presence of a significantly higher level of CECR1 expression in M2-like macrophages exposed to U87 conditioned medium (P < .001). CECR1 knockdown or stimulation of macrophages affected differentiation toward the M2-like phenotype. Stimulation of U87 cells with conditioned medium of CECR1 knockdown or stimulated macrophages affected tumor cell proliferation and migration, coinciding with altered intracellular signaling of mitogen-activated protein kinase (MAPK). In glioma tissue samples, CECR1 expression correlated with Ki67 and MAPK signaling protein. Conclusions: CECR1 is a potent regulator of TAM polarization and is consistently highly expressed by M2-type TAMs, particularly in high-grade glioma. Paracrine effects induced by CECR1 in M2-like TAMs activate MAPK signaling and stimulate the proliferation and migration of glioma cells.

Expression site of P2RY12 in residential microglial cells in astrocytomas correlates with M1 and M2 marker expression and tumor grade.

The role of resident microglial cells in the pathogenesis and progression of glial tumors is still obscure mainly due to a lack of specific markers. Recently P2RY12, a P2 purinergic receptor, was introduced as a specific marker for microglial cells under normal and pathologic conditions. Here we analyzed the expression of P2RY12 in astrocytomas of various malignancy grades in relation to markers for M1 and M2 macrophage activation profiles by using two web-based glioma datasets and confocal immunohistochemistry to 28 astrocytoma samples grades II-IV. In the gliomas, P2RY12 immunoreactivity delineated CD68 negative cells with otherwise microglial features from CD68 positive tumor associated macrophages (TAMs). The presence of P2RY12 positive cells correlated positively with overall survival. P2RY12 mRNA levels and membrane-bound localization of P2RY12 were inversely correlated with increasing malignancy grade, and the expression site of P2RY12 shifted from cytoplasmic in low-grade gliomas, to nuclear in high-grade tumors. The cytoplasmic expression of P2RY12 was associated with the expression of M1 markers, characteristic of the pro-inflammatory macrophage response. In contrast, the nuclear localization of P2RY12 was predominant in the higher graded tumors and associated with the expression of the M2 marker CD163.We conclude that P2RY12 is a specific marker for resident microglia in glioma and its expression and localization correspond to tumor grade and predominant stage of M1/M2 immune response.

Pregnancy Zone Protein is Increased in the Alzheimer's Disease Brain and Associates with Senile Plaques.

Increased levels of pregnancy zone protein (PZP) were found in the serum of persons who later developed Alzheimer's disease (AD) in comparison to controls who remained dementia free. We suggested that this increase is due to brain derived PZP entering the blood stream during the early phase of the disease. Here we investigate the possible involvement of PZP in human AD pathogenesis. We observed increased PZP immunoreactivity in AD postmortem brain cortex compared to non-demented controls. In the AD cortex, PZP immunoreactivity localized to microglial cells that interacted with senile plaques and was occasionally observed in neurons. Our data link the finding of elevated serum PZP levels with the characteristic AD pathology and identify PZP as a novel component in AD.

Circulating glioma biomarkers.

Validated biomarkers for patients suffering from gliomas are urgently needed for standardizing measurements of the effects of treatment in daily clinical practice and trials. Circulating body fluids offer easily accessible sources for such markers. This review highlights various categories of tumor-associated circulating biomarkers identified in blood and cerebrospinal fluid of glioma patients, including circulating tumor cells, exosomes, nucleic acids, proteins, and oncometabolites. The validation and potential clinical utility of these biomarkers is briefly discussed. Although many candidate circulating protein biomarkers were reported, none of these have reached the required validation to be introduced for clinical practice. Recent developments in tracing circulating tumor cells and their derivatives as exosomes and circulating nuclear acids may become more successful in providing useful biomarkers. It is to be expected that current technical developments will contribute to the finding and validation of circulating biomarkers.

Fast tracking of co-localization of multiple markers by using the nanozoomer slide scanner and NDPViewer.

The detection of co-localization of immunohistochemical markers in tissues or cells requires the application of the confocal laser scanning microscope (CLSM) to multiple immunofluorescence (MIF) stainings. CLSM is operationally sophisticated but requires time-consuming procedures of imaging and reconstruction, and a professional operator is required for manipulation of the microscopic system. Therefore, this technique is less suitable for the examination of many samples in a short time. Moreover, the technique only allows imaging of selected areas of a sample at one time and is not practical for fast panoramic mapping and tracking of whole tissue sections. Here we show a powerful high-throughput and operationally simple histological approach using the Hamamatsu NDP slide scanner (Hamamatsu Nanozoomer 2.0HT) and its viewing platform (NDP.Viewer). The approach not only enables fast mapping and tracking of overlapping spots or regions stained with multiple markers, but also offers panoramic screening of whole tissue sections with fully electronic manipulation for the visualization and analysis of any individual regions.

Intratumoral, not circulating, endothelial progenitor cells share genetic aberrations with glial tumor cells.

Literature data indicate that glioma stem cells may give rise to both tumor cells and endothelial progenitor cells (EPCs). Malignant glioma patients usually have increased levels of circulating (EPCs) and these cells are known to contribute to the glioma neovasculature. In this study we compared the intratumoral and circulating EPCs of glioma patients for a set of common glioma genotypical aberrations (amplification of EGFR; deletion of PTEN and aneusomy of chromosomes 7 and 10). We found that the EPCs present in the tumor tissues, not the circulating EPCs, share genetic aberrations with the tumor cells. EPCs with EGFR amplification were found in 46% and with PTEN deletion in 36% of the cases. EPCs with polysomy 7 and monosomy 10 were detected in 56% and 38% of the cases while centrosomal abnormalities in EPCs were found in 68% of the cases. The presence of genetic aberrations of glioma cells in intratumoral EPCs may point to transdifferentiation of glioma stem cells into EPCs. However, the tissue specific CD133 splice variant of blood EPCs was detected in the glioma tissues but not in control brains, suggestive of a blood origin of at least part of the intratumoral EPCs. The findings highlight the complexity of the cellular constituents of glioma neovascularization which should be taken into account when developing anti-angiogenic strategies for gliomas.

Isocitrate dehydrogenase 1R132H mutation in microglia/macrophages in gliomas: indication of a significant role of microglia/macrophages in glial tumorigenesis.

Somatic mutation of Isocitrate dehydrogenase 1 (IDH1) at the locus of R132 (IDH1 (R132H)) occurs in > 70% of WHO grade II-III gliomas and secondary glioblastomas. To date it remains unknown whether the mutation is restricted to glial tumor cells. Microglial cells are the resident macrophages in the central nervous system. Tumor-infiltrating microglial cells/macrophages are major stromal cellular components of malignant gliomas and substantially contribute to the tumor mass. Differential identification of the IDH1 (R132H) mutant cellular components is of particular importance for understanding of the mutation-associated tumor biology. Here we discovered that a significant portion of CD68(+), Iba1(+), CX3CR1(+) microglial cells/macrophages also harbor the IDH1R132H mutation. The findings provide novel insights for understanding the mutation-associated tumor biology relevant to clinical applications as a predictive and/or prognostic marker or therapeutic target.

HeNe laser (633 nm)-coupled confocal microscope allows simulating magnetic resonance imaging/computed tomography scan of the brain and eye: a noninvasive optical approach applicable to small laboratory animals.

Magnetic resonance imaging (MRI) and computed tomography (CT) are noninvasive medical imaging techniques used for the detailed visualization of internal organs of the human body. Because CT uses X-rays for imaging, there is a risk of radiation exposure. In contrast, MRI uses radiowaves and magnetic fields for imaging; thus, there are no reported biological hazards. However, neither MRI nor CT is suitable as a noninvasive imaging tool applicable in small laboratory animals such as zebrafish embryos or larvae. The recently established micro-CT scanner is only suitable for scanning adult fish and a staining procedure is required for imaging. In addition, CT-based scanning is generally more suitable for skeletal imaging but not for visualization of soft tissues because of its lower contrast. In this study, we evaluated whether 633 nm HeNe laser-coupled confocal microscope allows simulating MRI/CT scan and imaging soft tissues such as brain and eye in zebrafish embryos/larvae. We show that the 633 nm HeNe laser can penetrate well into intact brain and eye of zebrafish. It represents a noninvasive imaging method with high resolution while not requiring contrast agents, enabling the detection of differential signals from normal and pathological organs such as brain and eye.

Glut1/SLC2A1 is crucial for the development of the blood-brain barrier in vivo.

OBJECTIVE: The overall permeability of the blood-brain barrier (BBB) is regulated by specialized cerebral endothelial cells and their junctional complexes, consisting of adherens junctions (AJs) and tight junctions (TJs). Among the members of the glucose transporters (Glut), Glut1 is a unique molecule expressed in the cerebral endothelial cells. Glut1 and the junctional proteins are concomitantly downregulated in situations in which breakdown of the BBB has taken place. We hypothesized that the expression of Glut1 may play a significant role in the development of the cerebral microvasculature with BBB properties. To date, there is no information on the role of Glut1 during the development of BBB. In the present study, the in vivo effects of Glut1 knockdown on the cerebral vascular development were investigated. METHODS: Zebrafish was used as a model organism. We confirmed that the structure of the zebrafish homologue of Glut1 is highly similar to the human Glut1 and that the function of the Glut1-mediated cerebral uptake of glucose is evolutionally conserved. RESULTS: In the Glut1 knockdown model, we observed loss of the cerebral endothelial cells, with concomitant downregulation of the junctional proteins important for intactness of the AJs/TJs and impaired cerebral circulation. The resulting leaky BBB caused vasogenic brain edema. INTERPRETATION: The data suggest a crucial role of Glut1 in the development of the cerebral endothelial cells with BBB properties in vivo. The findings suggest that modulation of Glut1 expression and function may open new directions of research for therapeutic strategies to prevent vasogenic brain edema.

Expression sites of colligin 2 in glioma blood vessels.

In a previous study using state-of-the-art proteomic techniques, we identified colligin 2 (HSP47) as a glioma blood vessel-specific protein. In the present study we precisely localized the expression of colligin 2 in the blood vessels of diffusely infiltrating gliomas and relate the expression to the distinct cellular components of the vessels by using multiple immunolabeling and confocal microscopy. We grouped the glioma blood vessels into morphological categories ranging from normal looking capillaries to vessels with hypertrophic and sclerotic changes. The expression patterns of various markers of endothelial and pericytic differentiation were correlated with the position of the cells in the vessels and the expression of colligin 2. We found that colligin 2 is expressed in all categories of glioma blood vessels in cells with endothelial and pericytic lineage. Expression of colligin 2 was also found in cells scattered around blood vessels and in few glial fibrillary acidic protein-positive cells within the blood vessels. There is overlap in the expression of colligin 2 and the collagens type I and IV for which colligin 2 is a chaperon. We conclude that colligin 2 is expressed in all cellular components of glioma blood vessels and may serve as a general marker for active angiogenesis.