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Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy., Competing Interests: Declaration of interests D.A.M. holds ownership interest in iOncologi, Inc. E.J.S is a paid consultant for Siren Biotechnology. The manuscript discusses patented technologies from H.R.M.-G., P.C., S.Q., J.M., A.P.G., J.H., W.G.S., M.R., D.A.M., and E.J.S. Patented technologies are under option to license by iOncologi, Inc., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable success as an immunotherapy for hematological malignancies, and its potential for treating solid tumors is an active area of research. However, limited trafficking and mobility of T cells within the tumor microenvironment (TME) present challenges for CAR T cell therapy in solid tumors. To gain a better understanding of CAR T cell function in solid tumors, we subjected CD70-specific CAR T cells to a challenge by evaluating their immune trafficking and infiltration through a confined 3D microchannel network in a bio-conjugated liquid-like solid (LLS) medium. Our results demonstrated successful CAR T cell migration and anti-tumor activity against CD70-expressing glioblastoma and osteosarcoma tumors. Through comprehensive analysis of cytokines and chemokines, combined with in situ imaging, we elucidated that immune recruitment occurred via chemotaxis, and the effector-to-target ratio plays an important role in overall antitumor function. Furthermore, through single-cell collection and transcriptomic profiling, we identified differential gene expression among the immune subpopulations. Our findings provide valuable insights into the complex dynamics of CAR T cell function in solid tumors, informing future research and development in this promising cancer treatment approach. STATEMENT OF SIGNIFICANCE: The use of specialized immune cells named CAR T cells to combat cancers has demonstrated remarkable success against blood cancers. However, this success is not replicated in solid tumors, such as brain or bone cancers, mainly due to the physical barriers of these solid tumors. Currently, preclinical technologies do not allow for reliable evaluation of tumor-immune cell interactions. To better study these specialized CAR T cells, we have developed an innovative in vitro three-dimensional model that promises to dissect the interactions between tumors and CAR T cells at the single-cell level. Our findings provide valuable insights into the complex dynamics of CAR T cell function in solid tumors, informing future research and development in this promising cancer treatment approach., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

To prospectively determine whether brain tumors will respond to immune checkpoint inhibitors (ICIs), we developed a novel mRNA vaccine as a viral mimic to elucidate cytokine release from brain cancer cells in vitro. Our results indicate that cytokine signatures following mRNA challenge differ substantially from ICI responsive versus non-responsive murine tumors. These findings allow for creation of a diagnostic assay to quickly assess brain tumor immunogenicity, allowing for informed treatment with ICI or lack thereof in poorly immunogenic settings.

Messenger RNA (mRNA) has emerged as a remarkable tool for COVID-19 prevention but its use for induction of therapeutic cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Herein, we develop a facile approach for substantially enhancing immunogenicity of tumor-derived mRNA in lipid-particle (LP) delivery systems. By using mRNA as a molecular bridge with ultrapure liposomes and foregoing helper lipids, we promote the formation of 'onion-like' multi-lamellar RNA-LP aggregates (LPA). Intravenous administration of RNA-LPAs mimics infectious emboli and elicits massive DC/T cell mobilization into lymphoid tissues provoking cancer immunogenicity and mediating rejection of both early and late-stage murine tumor models. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for toll-like receptor engagement, RNA-LPAs stimulate intracellular pathogen recognition receptors (RIG-I) and reprogram the TME thus enabling therapeutic T cell activity. RNA-LPAs were safe in acute/chronic murine GLP toxicology studies and immunologically active in client-owned canines with terminal gliomas. In an early phase first-in-human trial for patients with glioblastoma, we show that RNA-LPAs encoding for tumor-associated antigens elicit rapid induction of pro-inflammatory cytokines, mobilization/activation of monocytes and lymphocytes, and expansion of antigen-specific T cell immunity. These data support the use of RNA-LPAs as novel tools to elicit and sustain immune responses against poorly immunogenic tumors.

Introduction: Brain tumors remain especially challenging to treat due to the presence of the blood-brain barrier. The unique biophysical properties of nanomaterials enable access to the tumor environment with minimally invasive injection methods such as intranasal and systemic delivery., Methods: In this review, we will discuss approaches taken in NP delivery to brain tumors in preclinical neuro-oncology studies and ongoing clinical studies., Results: Despite recent development of many promising nanoparticle systems to modulate immunologic function in the preclinical realm, clinical work with nanoparticles in malignant brain tumors has largely focused on imaging, chemotherapy, thermotherapy and radiation., Conclusion: Review of early preclinical studies and clinical trials provides foundational safety, feasibility and toxicology data that can usher a new wave of nanotherapeutics in application of immunotherapy and translational oncology for patients with brain tumors.

Objective: Non-invasive prenatal testing (NIPT) based on fetal cells in maternal blood has the advantage over NIPT based on circulating cell-free fetal DNA in that there is no contamination with maternal DNA. This will most likely result in better detection of chromosomal aberrations including subchromosomal defects. The objective of this study was to test whether fetal cells enriched from maternal blood can be used for cell-based NIPT., Methods: We present a method for enriching fetal cells from maternal blood, subsequent amplification of the fetal genome and detection of chromosomal and subchromosomal variations in the genome., Results: An average of 12.8 fetal cells from 30 mL of maternal blood were recovered using our method. Subsequently, whole genome amplification on fetal cells resulted in amplified fetal DNA in amounts and quality high enough to generate array comparative genomic hybridization as well as next-generation sequencing profiles. From one to two fetal cells, we were able to demonstrate copy number differences of whole chromosomes (21, X-, and Y) as well as subchromosomal aberrations (ring X)., Conclusion: Intact fetal cells can be isolated from every maternal blood sample. Amplified DNA from isolated fetal cells enabled genetic analysis by array comparative genomic hybridization and next-generation sequencing. © 2016 John Wiley & Sons, Ltd., (© 2016 John Wiley & Sons, Ltd.)

Objective: The goal was to develop methods for detection of chromosomal and subchromosomal abnormalities in fetal cells in the mother's circulation at 10-16 weeks' gestation using analysis by array comparative genomic hybridization (CGH) and/or next-generation sequencing (NGS)., Method: Nucleated cells from 30 mL of blood collected at 10-16 weeks' gestation were separated from red cells by density fractionation and then immunostained to identify cytokeratin positive and CD45 negative trophoblasts. Individual cells were picked and subjected to whole genome amplification, genotyping, and analysis by array CGH and NGS., Results: Fetal cells were recovered from most samples as documented by Y chromosome PCR, short tandem repeat analysis, array CGH, and NGS including over 30 normal male cells, one 47,XXY cell from an affected fetus, one trisomy 18 cell from an affected fetus, nine cells from a trisomy 21 case, three normal cells and one trisomy 13 cell from a case with confined placental mosaicism, and two chromosome 15 deletion cells from a case known by CVS to have a 2.7 Mb de novo deletion., Conclusion: We believe that this is the first report of using array CGH and NGS whole genome sequencing to detect chromosomal abnormalities in fetal trophoblastic cells from maternal blood. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd., (© 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.)

Objective: Detection of genomic copy number abnormalities in a single cell using array comparative genomic hybridization (CGH) offers a promising non-invasive alternative for prenatal diagnosis. Our objective was to compare three commercially available whole-genome amplification (WGA) kits for their capacity to produce high quality DNA from single cells that is suitable for both molecular genotyping and array CGH., Methods: We examined kit performance on unfixed, fixed and fixed/permeabilized lymphoblastoid cells. Molecular genotyping methods were used to evaluate the fidelity of amplified DNA for genomic profiling, while array CGH was used to assess copy number from single cells harboring trisomy 21, a DiGeorge syndrome deletion, a CMT1A duplication or a MECP2 duplication., Results: Molecular genotyping was achieved from single cells but performance varied between WGA kits. Furthermore, we consistently detected a dosage difference in sex chromosomes for gender mismatched hybridizations and for chromosome 21 in trisomy 21 cells. The 2.5 Mb DiGeorge syndrome deletion was also detected using all three WGA platforms, whereas the 1.3 Mb CMT1A and the 0.6 Mb MECP2 duplications were not consistently detected., Conclusion: These data suggest that single cell molecular genotyping and copy number analysis can be accomplished when WGA conditions are optimized. © 2016 John Wiley & Sons, Ltd., (© 2016 John Wiley & Sons, Ltd.)

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the United States. Metaphase-based cytogenetic tests, such as G-Band karyotyping, are among the most effective to detect CLL and provide significant prognostic information. However, the use of metaphase cytogenetics is currently problematic due to the low mitotic index of most CLL cells in vitro cultures. Even when metaphases can be generated in the presence of traditional B-cell mitogen LPS, the quality is often poor and aberrations escape detection., Purpose: We hypothesized that immuno-stimulatory interleukin-2(IL-2) plus cytosine-phosphodiester-guanine oligodeoxynucleotide (CpG ODN) can work as a novel B-cell mitogen to stimulate bone marrow cultures which result in a higher mitotic index than regular standard bone marrow cultures stimulated with LPS. This will increase the clonal chromosomal aberration detection rate in patients with CLL., Methods: Bone marrow samples from CLL patients were divided and parallel cultures were set up using LPS and CpG Oligonucleotide/ IL-2 (IL-2/CpG) as mitogens, respectively. Mitotic index was read under the microscope blindly by three different readers (SQ, LV, RM). G-banding, and Spectral Karyotyping (SKY) were performed to confirm and compare abnormalities., Results: The readings showed that mitotic index in IL-2/CpG stimulated bone marrow cultures was seven times higher than that of standard LPS bone marrow cultures with an average standard deviation of "0.92'" and CI of 95%, p less than 0.05. G-Banding and Spectral Karyotyping (SKY) showed the same abnormalities in IL-2/CpG found in LPS Bone marrow cultures., Conclusion: According to the results, IL-2/CpG cultures should be used in the cytogenetic lab for chromosomal analysis instead of LPS due to the higher mitotic index that helps in reducing false negative results. Further research should be done in order to lower false negative CLL detection results.