With improving biofabrication technology, 3D bioprinted constructs increasingly resemble real tissues. However, the fundamental principles describing how cell-generated forces within these constructs ...drive deformations, mechanical instabilities, and structural failures have not been established, even for basic biofabricated building blocks. Here we investigate mechanical behaviours of 3D printed microbeams made from living cells and extracellular matrix, bioprinting these simple structural elements into a 3D culture medium made from packed microgels, creating a mechanically controlled environment that allows the beams to evolve under cell-generated forces. By varying the properties of the beams and the surrounding microgel medium, we explore the mechanical behaviours exhibited by these structures. We observe buckling, axial contraction, failure, and total static stability, and we develop mechanical models of cell-ECM microbeam mechanics. We envision these models and their generalizations to other fundamental 3D shapes to facilitate the predictable design of biofabricated structures using simple building blocks in the future.
Tumor Treating Fields (TTFields), an approved therapy for glioblastoma (GBM) and malignant mesothelioma, employ noninvasive application of low-intensity, intermediate-frequency, alternating electric ...fields to disrupt the mitotic spindle, leading to chromosome missegregation and apoptosis. Emerging evidence suggests that TTFields may also induce inflammation. However, the mechanism underlying this property and whether it can be harnessed therapeutically are unclear. Here, we report that TTFields induced focal disruption of the nuclear envelope, leading to cytosolic release of large micronuclei clusters that intensely recruited and activated 2 major DNA sensors - cyclic GMP-AMP synthase (cGAS) and absent in melanoma 2 (AIM2) - and their cognate cGAS/stimulator of interferon genes (STING) and AIM2/caspase 1 inflammasomes to produce proinflammatory cytokines, type 1 interferons (T1IFNs), and T1IFN-responsive genes. In syngeneic murine GBM models, TTFields-treated GBM cells induced antitumor memory immunity and a cure rate of 42% to 66% in a STING- and AIM2-dependent manner. Using single-cell and bulk RNA sequencing of peripheral blood mononuclear cells, we detected robust post-TTFields activation of adaptive immunity in patients with GBM via a T1IFN-based trajectory and identified a gene panel signature of TTFields effects on T cell activation and clonal expansion. Collectively, these studies defined a therapeutic strategy using TTFields as cancer immunotherapy in GBM and potentially other solid tumors.
BackgroundAlthough cancer immunotherapy has revolutionized treatment for many solid tumors, their efficacy is limited in GBM due in large part to the ‘cold’ tumor microenvironment (TME) of the GBM ...tumor that lacks critical immune cells like dendritic cells (DC) and T cells but instead is enriched in immunosuppressive cells.1 2 New approaches are necessary to overcome these challenges. The lack of intratumor DCs may be partly due to circulating DCs unable to traverse physical barriers in non-immunogenic tumors, the blood-brain barrier in GBM.3 4 We propose a shift in the scientific and treatment paradigm of cancer immunotherapy, calling for tumor cells to take on a central, active role in initiating their own immunity right in the TME.MethodsUsing NETZEN, an integrated deep-learning, and gene network-based ranking computational platform, we identified cell fate determinants (CFDs) to convert GBM cells directly into induced dendritic cells (iDC) inside the TME. CFDs were delivered using a viral vector, and conversion was assessed by immunophenotyping, scRNA-seq. iDCs were functionally validated by their ability to efficiently cross-present exogenous antigens to naive CD8 T cells, a critical hallmark of natural DCs, and activate tumor-specific T cells to kill GBM cells in a cytotoxicity assay.ResultsA four-CFD subnetwork anchored by PU.1 was sufficient to convert mouse GBM cells to iDCs (CD45+MHCII+CD80/86+MHCI+). iDCs are growth arrested, exhibit 3-fold higher phagocytic activity, and upregulate the canonical antigen processing and presenting machinery by 10–40-fold, resulting in 100-fold greater efficiency at processing ovalbumin and cross-presenting SIINFEKL to naive OTI-CD8+ cytotoxic T lymphocytes (CTL). In addition, iDCs efficiently cross-present exogenous antigens to naïve CD8+ CTLs and elicit >30-fold higher activation and cytotoxicity in tumor-specific T cells than in native GBM cells, confirming their DC-like properties. Lastly, intratumoral GBM-DC conversion in a syngeneic orthotopic GBM model resulted in a significant reduction in tumor burden and an increase in survival compared to controls (Log-rank, p=0.01) with synergistic efficacy with classical DC-based tumor vaccination and potentially with ICIs. We have also successfully converted human GBM cells to iDCs with similar properties using a 2-CFD combination anchored by PU.1.ConclusionsGBM-derived iDCs acquired DC-like functions and could elicit robust intratumor immune activation, thereby overcoming many current limitations of immunotherapy in GBM. We aim to develop a low-cost, off-the-shelf gene therapy-based fate conversion tumor immunotherapy.AcknowledgementsR01CA238387, R42CA228875, 6BC04, K08CA160824, Lacerta, SareptaReferencesKorman AJ, Garrett-Thomson SC, Lonberg N. The foundations of immune checkpoint blockade and the ipilimumab approval decennial. Nature Reviews Drug Discovery 2022;21:509–528, doi:10.1038/s41573-021-00345-8.Yu P, et al. Priming of naive T cells inside tumors leads to eradication of established tumors. Nat Immunol 2004;5:141–149. doi:10.1038/ni1029.Ochoa de Olza M, Navarro Rodrigo B, Zimmermann S, Coukos G. Turning up the heat on non-immunoreactive tumours: opportunities for clinical development. The Lancet Oncology 2020;21:e419-e430. doi:10.1016/S1470-2045(20)30234-5.Hegde S, et al. Dendritic cell paucity leads to dysfunctional immune surveillance in pancreatic cancer. Cancer Cell 2020;37:289–307.e289, doi:https://doi.org/10.1016/j.ccell.2020.02.008.
BackgroundDendritic cell (DC) plays crucial role in eliciting anti-tumor immune response through both innate and adaptive immune system. Our group has developed an adoptive cellular therapy (ACT) ...against high grade gliomas, which significantly improves survival in murine models of CNS malignancies. Our studies have shown that the efficacy is associated with the observed increase in intratumoral dendritic cells (DC) which arise from transferred hematopoietic stem cells (HSC).1 However, tumors develop diverse mechanisms to restrict DC function to evade immune surveillance. In this study, we will address the various DC dysfunctional mechanism in both primary and ACT escaped tumors.MethodsACT treatment was administered to mice bearing the KR158B glioblastoma cell line to get ACT escaped tumor. The mice received 9 Gy of irradiation prior to the transfer of hematopoietic stem cells and tumor-reactive T cells, followed by three doses of BMDC vaccine. Tumor associated DCs sorted from primary and ACT escaped tumors were subjected to assess DC function or gene expression profiling. T cell proliferation, T cell activation markers, and IFNγ secretion were measured by flow cytometry and ELISA as a readout for DC-T cell co-culture functional assays. Brain tumor slices were prepared using a vibratome, and the conditioned medium from the slice culture was used to pre-treat DCs to determine the impact of brain tumor secretion on DCs.ResultsFunctional evidence demonstrated that DCs from ACT-escaped tumors is impaired in activating tumor-reactive T cells. By comparing the gene expressing profiles between tumor associated DCs in primary and ACT escaped tumors,we found a significant decrease in cDC marker genes, antigen assembly genes, and MHC molecules in ACT-escaped tumor DCs, compared to primary tumor DCs, suggesting a decreased cDC population and impaired antigen presentation in ACT-escaped tumor DCs. To determine whether secreted factors from tumor microenvironment that drives DC dysfunction, conditioned medium(CM) from tumor brain slices were tested in inducing DC dysfunction in vitro. CM from primary tumor brain slices suppressed DC function compared to healthy brain slice CM. Further results showed that non-tumor cell-derived secretion from the slices drives DC dysfunction in primary tumors. Strikingly, unlike primary tumor brain CM, ACT-escaped tumor brain slice CM did not decrease DC function capacity, suggesting different tumor-driven DC dysfunctional mechanisms in primary and ACT-escaped tumors.ConclusionsThe primary glioma tumor drives DC dysfunction through non-tumor cell-derived secretion, whereas the ACT-escaped tumor employs a different mechanism involving cDC exclusion and impaired MHC expression and antigen loading.ReferencesWildes TJ, et al. Cross-talk between T Cells and Hematopoietic Stem Cells during Adoptive Cellular Therapy for Malignant Glioma. Clin Cancer Res, 2018; 24(16): 3955–3966.Ethics ApprovalThe study is approved by the University of Florida Institutional Animal Care and Use Committee (IACUC) and are covered under protocol number IACUC202100000053. All participants were given informed consent before taking part.
The postexercise ankle–brachial index (ABI) is recommended in patients with normal resting ABI when peripheral artery disease (PAD) is suspected. The aims of this study were to determine the ...comparative diagnostic accuracy of the resting and postexercise ABI for detecting PAD, and, the effect of the presence of diabetes on these. Three methods of interpretation currently in use were also investigated: a reduction in postexercise ABI by >20% compared to resting ABI, an ABI value of ≤0.90 postexercise, or a reduction in systolic ankle pressure of >30 mmHg postexercise. This retrospective study used colour duplex ultrasound (CDU) as the reference standard. In 278 limbs (whole group), the resting ABI had an overall area under the curve (AUC) of 0.71, with the postexercise ABI yielding a similar diagnostic accuracy of AUC 0.72. In the non-diabetes group (n=171), the resting ABI had an overall AUC of 0.74 and the postexercise ABI had a similar AUC of 0.76. In the diabetes group (n=107), overall accuracy was reduced compared to the non-diabetes group, with the resting ABI having an overall AUC of 0.65 and the postexercise ABI yielding a similar accuracy with an AUC of 0.64. The overall diagnostic accuracy of the postexercise ABI for diagnosing PAD was not greatly improved compared to resting ABI. Given the lower overall diagnostic accuracy in the diabetes group, both the resting and the postexercise ABI results in diabetes populations should be interpreted with caution. There is a risk of undiagnosed disease if relying on these results alone to determine lower limb vascular status.
We report a novel technique to synthesize fluorescent carbon dots decorated graphene oxide (CDs-GO) sheets by microwave irradiation using citric acid as the precursor and ethylenediamine as the ...passivation agent. The samples are characterized by X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) and ultra violet-visible spectroscopy (UV-Vis). Fluorescence studies reveal strong excitation wavelength dependent fluorescence behaviour for these nanocomposites dispersed in water. Third order nonlinear optical properties of these composites are studied using Z scan technique with Q-switched nanosecond pulsed Nd: YAG laser and the measured highest nonlinear optical susceptibility is 4.624x10-10esu. The optical limiting studies of these nanocomposites yield a lower limiting threshold of 104MW/cm2 enabling them as a strong candidate for optical limiting applications.
Background
The resting systolic toe pressure (TP) is a measure of small arterial function in the periphery. TP is used in addition to the ankle‐brachial index when screening for peripheral arterial ...disease (PAD) of the lower limb in those with diabetes, particularly in the presence of lower limb medial arterial calcification. It may be used as an adjunct assessment of lower limb vascular function and as a predictor of wound healing. The aim of this study was to determine the diagnostic accuracy of TP for detecting PAD in people with and without diabetes.
Methods
This was a retrospective case‐control study. Two researchers extracted information from consecutive patient records, including TP measurements, colour Duplex ultrasound results, demographic information, and medical history. Measures of diagnostic accuracy were determined by receiver operating curve (ROC) analysis, and calculation of sensitivity, specificity, and positive and negative likelihood ratios.
Results
Three hundred and nintey‐four participants with suspected PAD were included. In the diabetes group (n = 176), ROC analysis of TP for detecting PAD was 0.78 (95%CI: 0.69 to 0.84). In the control group (n = 218), the ROC of TP was 0.73 (95%CI: 0.70 to 0.80). TP had highest sensitivity when anatomical distribution of disease was both proximal and distal (diabetes group: 79.49%, the control group: 82.61%). TP yielded highest sensitivity in mild disease (50–75% stenosis) in diabetes group, (81.82%) and moderate disease (>75% stenosis) in control group (80.77%).
Conclusions
Our findings indicate that TPs are useful to assist in diagnosing PAD in clinical practice, however, results should be interpreted with caution due to the small probability of PAD being present with a negative test.
BackgroundGlioma-induced immune dysregulation of the hematopoietic system has been described in a limited number of studies. In this study, our group further demonstrates that gliomas interrupt the ...cellular differentiation programming and outcomes of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. HSPCs from glioma-bearing mice are reprogrammed and driven towards expansion of myeloid lineage precursors and myeloid-derived suppressor cells (MDSCs) in secondary lymphoid organs. However, we found this expansion is reversed by immunotherapy. Adoptive cellular therapy (ACT) has been demonstrably efficacious in multiple preclinical models of central nervous system (CNS) malignancies, and here we describe how glioma-induced dysfunction is reversed by this immunotherapeutic platform.MethodsThe impact of orthotopic KR158B-luc glioma on HSPCs was evaluated in an unbiased fashion using single cell RNAseq (scRNAseq) of lineage− cells and phenotypically using flow cytometry. Mature myeloid cell frequencies and function were also evaluated using flow cytometry. Finally, ACT containing total body irradiation, tumor RNA-pulsed dendritic cells, tumor-reactive T cells and HSPCs isolated from glioma-bearing or non-tumor-bearing mice were used to evaluate cell fate differentiation and survival.ResultsUsing scRNAseq, we observed an altered HSPC landscape in glioma-bearing versus non-tumor-bearing mice . In addition, an expansion of myeloid lineage subsets, including granulocyte macrophage precursors (GMPs) and MDSCs, were observed in glioma-bearing mice relative to non-tumor-bearing controls. Furthermore, MDSCs from glioma-bearing mice demonstrated increased suppressive capacity toward tumor-specific T cells as compared with MDSCs from non-tumor-bearing hosts. Interestingly, treatment with ACT overcame these suppressive properties. When HSPCs from glioma-bearing mice were transferred in the context of ACT, we observed significant survival benefit and long-term cures in orthotopic glioma models compared with mice treated with ACT using non-glioma-bearing HSPCs.
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