Specific Target Cells Research Articles

Folate-modified liposomes mediate the co-delivery of cisplatin with miR-219a-5p for the targeted treatment of cisplatin-resistant lung cancer

Cisplatin (DDP) resistance, often leading to first-line chemotherapy failure in non-small cell lung cancer (NSCLC), poses a significant challenge. MiR-219a-5p has been reported to enhance the sensitivity of human NSCLC to DDP. However, free miR-219a-5p is prone to degradation by nucleases in the bloodstream, rendering it unstable. In light of this, our study developed an efficient nanodrug delivery system that achieved targeted delivery of DDP and miR-219a-5p by modifying liposomes with folate (FA). Based on the results of material characterization, we successfully constructed a well-dispersed and uniformly sized (approximately 135.8 nm) Lipo@DDP@miR-219a-5p@FA nanodrug. Agarose gel electrophoresis experiments demonstrated that Lipo@DDP@miR-219a-5p@FA exhibited good stability in serum, effectively protecting miR-219a-5p from degradation. Immunofluorescence and flow cytometry experiments revealed that, due to FA modification, Lipo@DDP@miR-219a-5p@FA could specifically bind to FA receptors on the surface of tumor cells (A549), thus enhancing drug internalization efficiency. Safety evaluations conducted in vitro demonstrated that Lipo@DDP@miR-219a-5p@FA exhibited no significant toxicity to non-cancer cells (BEAS-2B) and displayed excellent blood compatibility. Cellular functional experiments, apoptosis assays, and western blot demonstrated that Lipo@DDP@miR-219a-5p@FA effectively reversed DDP resistance in A549 cells, inhibited cell proliferation and migration, and further promoted apoptosis. In summary, the Lipo@DDP@miR-219a-5p@FA nanodrug, through specific targeting of cancer cells and reducing their resistance to DDP, significantly enhanced the anti-NSCLC effects of DDP in vitro, providing a promising therapeutic option for the clinical treatment of NSCLC.

450 Developing Methods for High-Resolution Characterization of Plasma Cells

OBJECTIVES/GOALS: Antibodies play an important role in the pathogenesis of a wide range of diseases, including cancer, autoimmune diseases, and infections. There are currently no reliable methods to isolate and study specific plasma cell subpopulations as antibody production sources. We aim to develop methods to study plasma cells in high resolution. METHODS/STUDY POPULATION: We will use molecular cloning to engineer fusion proteins that would bind plasma cell proteins to study these cells based on their surface features. The first phase of our study consists of assessing the efficacy of this plasma cell isolation method in established cell lines (e.g., RPMI 8226) and also antibody-secreting cell lines that we are establishing as a part of this study. In the second phase of the study, we will assess the efficacy of this method by studying antigen-specific plasma cell populations in the bone marrow aspiration samples of 20 healthy volunteers using various assays, including ELISPOT, flow cytometry, and fluorescent microscopy. RESULTS/ANTICIPATED RESULTS: We have designed the constructs and have completed the cloning. The final plasmids have been verified using various restriction enzymes and Sanger sequencing. Following the transfection of Freestyle HEK 293F cells and isolation of respective proteins, we expect to be able to utilize these engineered proteins to differentiate various antibody-secreting plasma cells. We will use cell lines for proof-of-concept experiments and will subsequently move this method to human bone marrow samples. We expect to be able to visualize multiple specific antibody-secreting plasma cell populations using fluorescent microscopy and utilize this method to isolate them by cell sorting via flow cytometry. DISCUSSION/SIGNIFICANCE: We expect to be able to use this method to target specific plasma cell clones in the advancement of precision medicine regarding the treatment of plasma cell disorders (e.g., multiple myeloma) and also expand its use in other areas, such as antibody discovery and the assessment of the humoral immune responses in infectious diseases.

Application of phytochemicals in photodynamic therapy of cancer

SignificanceHerbal medicine is an important part of the health care system in many countries as they exhibit chemopreventive and chemotherapeutic effects. The combination of phytochemicals with Photodynamic therapy is a novel approach to enhancing therapeutic efficacy. ApproachWe studied the effect of various plant extracts, phytochemicals, photosensitizers and their combinations to induce cell death in the lung, breast, colon, cervical and skin cancer cells. The efficacy of the drug moieties was evaluated using viability, proliferation, and cytotoxicity studies. Detailed cell death mechanism was studied using protein expression profiling using immunofluorescence, flow cytometry, ELISA and western blotting. ResultsThe results revealed that PDT and combination therapy groups showed significant cell death in breast and lung cancer cells. Conjugation of photosensitizers with nanoparticles and phytochemicals enhanced the drug delivery and treatment outcome. ConclusionsCombination of PDT with other existing treatments or replacing the synthetic PSs with natural ones is a promising approach in the fight against cancer with improved specific cancer cell targeting.

The miR-183/96/182 cluster regulates sensory innervation, resident myeloid cells and functions of the cornea through cell type-specific target genes

The conserved miR-183/96/182 cluster (miR-183C) is expressed in both corneal resident myeloid cells (CRMCs) and sensory nerves (CSN) and modulates corneal immune/inflammatory responses. To uncover cell type-specific roles of miR-183C in CRMC and CSN and their contributions to corneal physiology, myeloid-specific miR-183C conditional knockout (MS-CKO), and sensory nerve-specific CKO (SNS-CKO) mice were produced and characterized in comparison to the conventional miR-183C KO. Immunofluorescence and confocal microscopy of flatmount corneas, corneal sensitivity, and tear volume assays were performed in young adult naïve mice; 3′ RNA sequencing (Seq) and proteomics in the trigeminal ganglion (TG), cornea and CRMCs. Our results showed that, similar to conventional KO mice, the numbers of CRMCs were increased in both MS-CKO and SNS-CKO vs age- and sex-matched WT control littermates, suggesting intrinsic and extrinsic regulations of miR-183C on CRMCs. The number of CRMCs was increased in male vs female MS-CKO mice, suggesting sex-dependent regulation of miR-183C on CRMCs. In the miR-183C KO and SNS-CKO, but not the MS-CKO mice, CSN density was decreased in the epithelial layer of the cornea, but not the stromal layer. Functionally, corneal sensitivity and basal tear volume were reduced in the KO and SNS-CKO, but not the MS-CKO mice. Tear volume in males is consistently higher than female WT mice. Bioinformatic analyses of the transcriptomes revealed a series of cell-type specific target genes of miR-183C in TG sensory neurons and CRMCs. Our data elucidate that miR-183C imposes intrinsic and extrinsic regulation on the establishment and function of CSN and CRMCs by cell-specific target genes. miR-183C modulates corneal sensitivity and tear production through its regulation of corneal sensory innervation.

Single-cell transcriptomics reveals comprehensive microenvironment and highlights the dysfuntional state of NK cells in endometrioid carcinoma.

Endometrioid endometrial cancer (EEC) is one of the most common gynecologic malignancies. The interaction between cancer cells and the cells in the tumor microenvironment (TME) plays a crucial role in determining disease progression and response to treatment. To better understand the diversity in the TME of ECC, we conducted a comprehensive analysis using single-cell RNA sequencing across 21 samples, including 16 ECC and 5 adjacent normal tissues. We primarily focused on tumor-infiltrating natural killer (NK) cells and their cell-cell interactions with other immune cell types. We identified a CD56dim_DNAJB1 NK cells subset, which had low cytotoxic capability and high stress levels, suggesting a dysfunctional state. This subset showed strong interactions with tumor-associated macrophages through several ligand-receptor pairs. Additionally, we observed that tumor-infiltrating LAMP3+ dendritic cells may inhibit CD8+ T cells or attract regulatory T cells to the tumor area. These dendritic cells also had impaired activation effects on NK cells within the TME. Our study provides valuable insights into the role of NK cells in cancer immunity and highlights the potential of targeting specific NK cell subsets for therapeutic purposes.

Self-Signal-Triggered Drug Delivery System for Tumor Therapy Using Cancer Cell Membrane-Coated Biocompatible Mn3O4 Nanocomposites.

In anti-cancer metastasis treatment, precise drug delivery to cancer cells remains a challenge. Innovative nanocomposites are developed to tackle these issues effectively. The approach involves the creation of manganese oxide (Mn3O4) nanoparticles (NPs) and their functionalization using trisodium citrate to yield functionalized Mn3O4 NPs (F-Mn3O4 NPs), with enhanced water solubility, stability, and biocompatibility. Subsequently, the chemotherapeutic drug doxorubicin (DOX) is encapsulated with Mn3O4 NPs, resulting in DOX/Mn3O4 NPs. To achieve cell-specific targeting, These NPs are coated with HeLa cell membranes (HCM), forming HCM/DOX/Mn3O4. For further refinement, a transferrin (Tf) receptor is integrated with cracked HCM to create Tf-HCM/DOX/Mn3O4 nanocomposites (NC) with specific cell membrane targeting capabilities. The resulting Tf-HCM/DOX/Mn3O4 NC exhibits excellent drug encapsulation efficiency (97.5%) and displays triggered drug release when exposed to NIR laser irradiation in the tumor's environment (pH 5.0 and 6.5). Furthermore, these nanocomposites show resistance to macrophage uptake and demonstrate homotypic cancer cell targeting specificity, even in the presence of other tumor cells. In vitro toxicity tests show that Tf-HCM/DOX/Mn3O4 NC achieves significant anticancer activity against HeLa and BT20 cancer cells, with percentages of 76.46% and 71.36%, respectively. These results indicate the potential of Tf-HCM/DOX/Mn3O4 NC as an effective nanoplatform for chemo-photothermal therapy.

Lipid-Based Nanoparticle Functionalization with Coiled-Coil Peptides for In Vitro and In Vivo Drug Delivery.

For the delivery of drugs, different nanosized drug carriers (e.g., liposomes, lipid nanoparticles, and micelles) have been developed in order to treat diseases that afflict society. Frequently, these vehicles are formed by the self-assembly of small molecules to encapsulate the therapeutic cargo of interest. Over decades, nanoparticles have been optimized to make them more efficient and specific to fulfill tailor-made tasks, such as specific cell targeting or enhanced cellular uptake. In recent years, lipid-based nanoparticles in particular have taken center stage; however, off-targeting side effects and poor endosomal escape remain major challenges since therapies require high efficacy and acceptable toxicity.To overcome these issues, many different approaches have been explored to make drug delivery more specific, resulting in reduced side effects, to achieve an optimal therapeutic effect and a lower required dose. The fate of nanoparticles is largely dependent on size, shape, and surface charge. A common approach to designing drug carriers with targeting capability is surface modification. Different approaches to functionalize nanoparticles have been investigated since the attachment of targeting moieties plays a significant role in whether they can later interact with surface-exposed receptors of cells. To this end, various strategies have been used involving different classes of biomolecules, such as small molecules, nucleic acids, antibodies, aptamers, and peptides.Peptides in particular are often used since there are many receptors overexpressed in different specific cell types. Furthermore, peptides can be produced and modified at a low cost, enabling high therapeutic screening. Cell-penetrating peptides (CPPs) and cell-targeting peptides (CTPs) are frequently used for this purpose. Less studied in this context are fusogenic coiled-coil peptides. Lipid-based nanoparticles functionalized with these peptides are able to avoid the endolysosomal pathway; instead such particles can be taken up by membrane fusion, resulting in increased delivery of payload. Furthermore, they can be used for targeting cells/organs but are not directed at surface-exposed receptors. Instead, they recognize complementary peptide sequences, facilitating their uptake into cells.In this Account, we will discuss peptides as moieties for enhanced cytosolic delivery, targeted uptake, and how they can be attached to lipid-based nanoparticles to alter their properties. We will discuss the properties imparted to the particles by peptides, surface modification approaches, and recent examples showing the power of peptides for in vitro and in vivo drug delivery. The main focus will be on the functionalization of lipid-based nanoparticles by fusogenic coiled-coil peptides, highlighting the relevance of this concept for the development of future therapeutics.

Hydrogen-bonded cytosine-endowed supramolecular polymeric nanogels: Highly efficient cancer cell targeting and enhanced therapeutic efficacy

We demonstrate that cytosine moieties within physically cross-linked supramolecular polymers not only manipulate drug delivery and release, but also confer specific targeting of cancer cells to effectively enhance the safety and efficacy of chemotherapy—and thus hold significant potential as a new perspective for development of drug delivery systems. Herein, we successfully developed physically cross-linked supramolecular polymers (PECH-PEG-Cy) comprised of hydrogen-bonding cytosine pendant groups, hydrophilic poly(ethylene glycol) side chains, and a hydrophobic poly(epichlorohydrin) main chain. The polymers spontaneously self-assemble into a reversibly hydrogen-bonded network structure induced by cytosine and directly form spherical nanogels in aqueous solution. Nanogels with a high hydrogen-bond network density (i.e., a higher content of cytosine moieties) exhibit outstanding long-term structural stability in cell culture substrates containing serum, whereas nanogels with a relatively low hydrogen-bond network density cannot preserve their structural integrity. The nanogels also exhibit numerous unique physicochemical characteristics in aqueous solution, such as a desirable spherical size, high biocompatibility with normal and cancer cells, excellent drug encapsulation capacity, and controlled pH-responsive drug release properties. More importantly, in vitro experiments conclusively indicate the drug-loaded PECH-PEG-Cy nanogels can selectively induce cancer cell-specific apoptosis and cell death via cytosine receptor-mediated endocytosis, without significantly harming normal cells. In contrast, control drug-loaded PECH-PEG nanogels, which lack cytosine moieties in their structure, can only induce cell death in cancer cells through non-specific pathways, which significantly inhibits the induction of apoptosis. This work clearly demonstrates that the cytosine moieties in PECH-PEG-Cy nanogels confer selective affinity for the surface of cancer cells, which enhances their targeted cellular uptake, cytotoxicity, and subsequent induction of programmed cell death in cancer cells.

Abstract 3205: MicroRNA-peptide polyelectrolyte complex nanoparticle-hydrogel composite attenuates mesothelioma growth

Abstract Diffuse pleural mesothelioma (DPM) is a biologically unusual, aggressive asbestos-associated cancer affecting the entire pleura surface. DPM defies current standard multimodal therapies due to intrinsic chemoresistance and inability to achieve negative-margin resection of complex surface anatomy. Hence, current non-specific drug regimens ultimately fail because the biology of this tumor is poorly understood and even if surgically resected, tumor inevitably recurs from residual microscopic tumor foci. These various clinical challenges led us to develop a therapeutic platform delivering microRNA (miRNA), a novel anti-cancer agent therapeutic regimen, with the goal of eradicating residual microscopic tumor foci thereby improving the efficacy of multimodal therapy. We engineered a peptide-based biodegradable hydrogel that can be injected or sprayed directly to coat anatomic surfaces (surface-fill hydrogel; SFH), functioning as a therapeutic depot. Properties of SFH allow us to specify critical composition parameters such as biopersistence time, therapeutic payload, cell-specific targeting etc. In the first step of SFH formulation, miRNA complexed with different amphiphilic cationic peptides varying in length (19-26 amino acids) and amines (9-17), to form stable nanoparticles. Flash nanocomplexation (FNC) were used to produce highly scalable and kinetically controlled assembly of miRNA-peptide polyelectrolyte complex (PEC) nanoparticles (NPs), which utilizes a confined impinging jet (CIJ) mixer to achieve turbulent micromixing of the NPs components, yielding highly uniform and stable NPs. Next, NPs were encapsulated into another peptide hydrogel, formed via self-assembly of amphiphilic cationic peptide HLT2, thus forming a surface-fill hydrogel NPs composite. The SFH-NPs composite was not toxic to DPM cells and demonstrated efficient release of miRNA-peptide NPs from the SFH matrix in-vitro. The uptake of FAM-labeled miRNA-peptide NPs was confirmed by flowcytometry. Biodistribution analysis of NPs in mice bearing luciferase-positive DPM xenografts treated locally with Cy3-miRNA-SFH composite showed that Cy3-miRNA was concentrated in tumor cells but not in other organs. Furthermore, miR-215 exhibiting potent anti-tumor activity in DPM was used to study the therapeutic efficacy of SFH miRNA-nanoparticle composites. Subcutaneous DPM xenografts treated locally with miR-215 SFH composite shrank tumor significantly. Thus, delivery of specific miRNA-peptide PEC nanoparticles via SFH therapeutic platform in preclinical models of DPM displayed potent anti-cancer effects. Together, this new generation microRNA-peptide polyelectrolyte complex nanoparticles-SFH composite is a very promising delivery platform that could be extended for a diverse array of locoregional therapies. Citation Format: Anand Singh, Anderson Caleb, Joel P. Schneider, Chuong D. Hoang. MicroRNA-peptide polyelectrolyte complex nanoparticle-hydrogel composite attenuates mesothelioma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3205.

Abstract 480: Nanotherapeutic strategies to improve targeted radionuclide therapy

Abstract Cancer is a leading cause of death worldwide. Several multidisciplinary approaches exist for cancer treatment, including radiotherapy and chemotherapy. Radiotherapy uses high-energy radiation to kill cancer cells; chemotherapy inhibits cancer cell proliferation and often kills cells by targeting the cell cycle. Resistance to radiotherapy and chemotherapy is a key determining factor in the outcome of therapeutic efficacy. Conventional nontargeted radiotherapy also affects distant nonirradiated cells, leading to DNA damage and changes in the cell cycle that are often linked to secondary carcinogenesis in patients. In recent years, targeted radionuclide therapy (TRT) has emerged as a promising personalized treatment strategy that delivers cytotoxic levels of radiation directly and specifically to cancer cells. Among radioisotopes,225Ac exhibits desirable properties for TRT: multiple α-particle emission and high cytotoxicity. One of the challenges with 225Ac is the nonspecific toxicity caused by the release and relocation of its decay daughters. Different approaches have been proposed to prevent the relocation of decay daughters, including nanoparticles. Nanoparticles have been pursued as a promising delivery vehicle of α-emitting radioisotopes to the tumor site. Here, we show specific targeting of Her2-positive breast cancer cells with 225Ac-radiolabeled lanthanum orthovanadate (LaVO4) nanoparticles. Nanoparticles’ surfaces were functionalized to improve biocompatibility and conjugated to target cancer cells, respectively. Cellular uptake and localization of these engineered nanoparticles were analyzed by a Confocal microscope and IncuCyte live cell analysis system. Results confirm its localization in the nucleus following the endolysosomal path, enhancing the nanoparticles’ effectiveness as a delivery vehicle for α-emitting radioisotopes with the potential of increased treatment efficacy. Upon addition of cancer-targeting ligands, these nanoparticles can achieve higher efficiency by delivering the radioisotope to the tumor cells with a potential of safe encapsulation of all α-emitters in the decay chain. The anticancer efficiency of 225Ac-radiolabeled nanoparticles was demonstrated by a dose-dependent effective killing of 3D breast cancer spheroids using both IncuCyte live imaging and the cell survival assays. Ultimately, the results of this study will be crucial in determining the future use of targeted radiolabeled nanoparticle-based delivery systems as an approach for more efficacious cancer treatment. Citation Format: Debjani Pal, Miguel Toro Gonzáleza, Amber N. Bibleb, Brian Sanders, Anna Plechaty, Owee Kirpekar, Mircea Podar, Sandra M. Davern. Nanotherapeutic strategies to improve targeted radionuclide therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 480.

Abstract 6569: KLIPP: Precision targeting of cancer with CRISPR

Abstract Current cancer therapeutic treatments are typically associated with significant collateral damage to normal tissues and the emergence of treatment resistance. Thus, there is an unmet need to develop therapies that are uniquely cancer specific and unhindered by the emergence of resistance. We have developed an RNA therapy approach based on CRISPR technology, known as “KLIPP”, that targets cancer-specific structural variant junctions (SVJs), a so far untapped cancer “Achilles heel”, that are highly abundant across cancer types. KLIPP uses a “split enzyme” system consisting of dCas9 conjugated to an endonuclease, Fok1, that requires dimerization for its activity. By designing sgRNAs that bind to sequences flanking the cancer-specific SVJs, Fok1-dCas9 dimers are formed, leading to the induction of DNA double strand breaks (DSBs) specifically at SVJs in cancer cells. We verify induction of DSBs by γH2AX foci staining in the nucleus of KLIPP-treated cells and resulting loss of clonogenic survival and induction of apoptosis, while cells lacking the targeted SVJs are unaffected. Importantly, we show elimination of orthotopic bladder cancer tumors in vivo after turning on expression of the KLIPP reagents by doxycycline in the drinking water. Finally, we show successful lipid nanoparticle delivery of KLIPP reagents to orthotopic bladder tumors in vivo using catheter-mediated delivery, leading to specific targeting of bladder cancer cells. Independently of the cancer-driving mutations, the omnipresence of cancer-specific SVJs is exploited by KLIPP to force cancer cells into apoptosis while leaving normal cells unperturbed. We believe that the successful implementation of precision KLIPP therapy could be transformative for the treatment of cancer patients. Citation Format: Mats Ljungman, Huibin Yang, Radhika Suhas Hulbatte, Natalie Gratsch, Alan Kelher, John Raupp, Yin Wang, Kristen Hong Dorsey, Troy Halseth, Anna Schwendeman, Philip Palmbos. KLIPP: Precision targeting of cancer with CRISPR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6569.

Abstract 1341: SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status

Abstract Background: Monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) are frontline therapies for numerous EGFR-dependent epithelial tumors. However, their efficacy faces challenges due to on-target skin toxicity and intrinsic or acquired resistance. Within the tumor microenvironment, innate immune cells, including macrophages and natural killer (NK) cells, play a significant role but are hindered by the lack of specific cancer cell targeting and a suppressive microenvironment. To enhance innate immune cell cytotoxicity, we have developed SM2235, a humanized tetravalent bispecific VHH antibody. SM2235 partially blocks EGFR and EGF interaction while conditionally activating innate immune cells through CD16A upon engagement with cancer cells. Methods: SM2235 was engineered by combining a partially blocking EGFR single-domain antibody with a high-affinity CD16A-activating single-domain antibody on an inactivated human IgG1 Fc backbone. The binding affinity and specificity were confirmed using bio-layer interferometry and ELISA with recombinant proteins, followed by flow cytometry with EGFR+ human cancer cell lines and CD16A+ primary NK cells. SM2235's efficacy in blocking EGFR-EGF interaction and downstream signaling was assessed through competitive ELISA and proliferation assays in both EGFR-dependent and independent cancer cell lines. Its ability to induce target-dependent NK cell and macrophage activation was evaluated through activation markers as well as cytotoxicity and phagocytosis assays. In vivo efficacy of SM2235 against EGFR+ human tumors was measured using xenograft assays in NSG mice, covering cetuximab-sensitive and resistant cancer cell lines. Preliminary pharmacokinetics and safety studies were conducted in cynomolgus monkeys. Results: SM2235 exhibits sub-nanomolar affinity for EGFR+ human cancer cell lines and CD16A+ primary human NK cells. It partially inhibits EGFR-dependent cancer cell proliferation. Compared to cetuximab, SM2235 significantly enhances target-dependent CD16A-mediated NK cell and macrophage activation and cytotoxicity against EGFR+ cancer cells in ADCC and ADCP assays in vitro. SM2235 effectively inhibits EGFR+ xenograft tumors in vivo, irrespective of KRAS mutation status. In cynomolgus monkeys, SM2235 demonstrates excellent tolerance with a 20-hour serum half-life of approximately 20 hours and no dose-dependent skin toxicity. Conclusions: Preclinical characterization of SM2235 shows promising in vivo efficacy against EGFR+ human cancers, regardless of KRAS mutation, with a favorable safety profile marked by minimal skin toxicity. Citation Format: Huiqin Geng, Wenjing Song, Xiaodan Liu, Shihao Lu, Hong Zhou, Simin Yang, Siyao Xie, Yi Wei, Xing Zhang, Sheila Zhou, Yanbin Liang. SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1341.

Abstract 7254: Evaluation of scFv and diabody pseudotyped Sindbis engineered retrovectors for targeted cancer therapy

Abstract Engineering viral vectors to target specific cells and organs after intravenous delivery remains a challenge. Transcriptional targeting with cell-specific promoters is a commonly used approach but does not change vector biodistribution. We have developed a novel gene delivery system targeted to tumor cells by pseudotyping the vector with Sindbis Virus (SB) envelope bearing an antigen-binding domain. Prototype vectors were generated by inserting a universal IgG binding domain from Protein A into the SB envelope (SB-ZZ). Antibodies (Abs) against various cell-surface targets (HLA, HER2, CD47, EGFR, Nectin-4), were conjugated to SB-ZZ vectors delivering a luciferase (Luc) reporter gene. Targeted transduction was confirmed on cells expressing the corresponding antigens by significantly higher Luc activity compared to SB-ZZ vectors without Abs. SB envelopes were further engineered by adding or removing furin cleavage sites and linkers, transduction-enhancing point mutations, and direct incorporation of single-chain variable fragment (scFv) in different VH and VL orientations, or diabody (DB) sequences, against the same targets. The specificity of optimized HER2-targeting vectors delivering the Luc reporter was evaluated in SKBR3 (HER2 over-expressing) and MDA-MB-468 (HER2-null) cells. Robust Luc activity was observed in SKBR3 cells, but only negligible activity in MDA-MB-468 cells. In MDA-MB-468 cells overexpressing HER2, significantly higher transduction was observed compared to the wild-type cells. When comparing HER2-targeting vectors with scFv vs. DB sequences based on the same antibody, transduction capability was higher with the DB motif in HER2-positive cell lines. Targeting vectors delivering HSV-eTK were also constructed, and ganciclovir (GCV)-mediated cell killing was evaluated in vitro. SKBR3 cells transduced with HER2-targeting vectors showed significantly more cell killing after GCV treatment than after transduction with an amphotropic reference vector. Our results indicate a method for directing gene therapy vectors to specific populations of tumor cells, and provide a framework for highly modular targeted gene therapy and immunotherapeutics. Experiments are underway to examine in vivo delivery of targeted vs amphotropic vectors. Citation Format: Makoto Sato, Alexander Cunha, Sara Collins, Noriyuki Kasahara, Cecilia Roh, Robert G. Johnson. Evaluation of scFv and diabody pseudotyped Sindbis engineered retrovectors for targeted cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7254.

Distinctive whole-brain cell types predict tissue damage patterns in thirteen neurodegenerative conditions.

For over a century, brain research narrative has mainly centered on neuron cells. Accordingly, most neurodegenerative studies focus on neuronal dysfunction and their selective vulnerability, while we lack comprehensive analyses of other major cell types' contribution. By unifying spatial gene expression, structural MRI, and cell deconvolution, here we describe how the human brain distribution of canonical cell types extensively predicts tissue damage in 13 neurodegenerative conditions, including early- and late-onset Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, amyotrophic lateral sclerosis, mutations in presenilin-1, and 3 clinical variants of frontotemporal lobar degeneration (behavioral variant, semantic and non-fluent primary progressive aphasia) along with associated three-repeat and four-repeat tauopathies and TDP43 proteinopathies types A and C. We reconstructed comprehensive whole-brain reference maps of cellular abundance for six major cell types and identified characteristic axes of spatial overlapping with atrophy. Our results support the strong mediating role of non-neuronal cells, primarily microglia and astrocytes, in spatial vulnerability to tissue loss in neurodegeneration, with distinct and shared across-disorder pathomechanisms. These observations provide critical insights into the multicellular pathophysiology underlying spatiotemporal advance in neurodegeneration. Notably, they also emphasize the need to exceed the current neuro-centric view of brain diseases, supporting the imperative for cell-specific therapeutic targets in neurodegeneration.

Distinctive whole-brain cell types predict tissue damage patterns in thirteen neurodegenerative conditions

For over a century, brain research narrative has mainly centered on neuron cells. Accordingly, most neurodegenerative studies focus on neuronal dysfunction and their selective vulnerability, while we lack comprehensive analyses of other major cell types’ contribution. By unifying spatial gene expression, structural MRI, and cell deconvolution, here we describe how the human brain distribution of canonical cell types extensively predicts tissue damage in 13 neurodegenerative conditions, including early- and late-onset Alzheimer’s disease, Parkinson’s disease, dementia with Lewy bodies, amyotrophic lateral sclerosis, mutations in presenilin-1, and 3 clinical variants of frontotemporal lobar degeneration (behavioral variant, semantic and non-fluent primary progressive aphasia) along with associated three-repeat and four-repeat tauopathies and TDP43 proteinopathies types A and C. We reconstructed comprehensive whole-brain reference maps of cellular abundance for six major cell types and identified characteristic axes of spatial overlapping with atrophy. Our results support the strong mediating role of non-neuronal cells, primarily microglia and astrocytes, in spatial vulnerability to tissue loss in neurodegeneration, with distinct and shared across-disorder pathomechanisms. These observations provide critical insights into the multicellular pathophysiology underlying spatiotemporal advance in neurodegeneration. Notably, they also emphasize the need to exceed the current neuro-centric view of brain diseases, supporting the imperative for cell-specific therapeutic targets in neurodegeneration.

Heating ability of amine functionalized Fe3O4 as a function of field amplitude and frequency for hyperthermia application

The functionalization of magnetic nanoparticles (MNPs) is crucial for their biomedical applications such as magnetic hyperthermia, which attracted attention in recent years as a promising alternative or complementary therapy to traditional cancer treatments. Here, we investigate a synthetic approach based on the hydrothermal process for the amine-functionalized magnetite (Fe3O4-NH2) MNPs for possible use in hyperthermia. The characterization of coated MNPs by FTIR confirms the functionalization with the amine group, while the XRD shows how the magnetite phase was formed. Magnetic measurements of both coated and uncoated MNPs at room temperature revealed their ferrimagnetic behavior. It was found from magnetic hyperthermia measurements under an alternating magnetic field (AMF) that functionalized with amine MNPs reach hyperthermia temperatures (42 °C)) in relatively short times (around 10 min.) with a adequate value of sample absorption rate (SAR)in the range between 33 to 45 W g–1. The (NH2) groups on the surface of the (Fe3O4) nanoparticles prepared by hydrothermal process make them amenable to further functionalization with various biomolecules. This enables the nanoparticles to be tailored for specific applications, such as drug delivery or targeting specific cells or tissues. Moreover, the appropriate crystallinity combined with the relatively adequate heating ability and adequate saturation suggest that the functionalized MNPs are promising for hyperthermia application.

Molecular Determinants Involved in the Docking and Uptake of Tumor-Derived Extracellular Vesicles: Implications in Cancer.

Extracellular vesicles produced by tumor cells (TEVs) influence all stages of cancer development and spread, including tumorigenesis, cancer progression, and metastasis. TEVs can trigger profound phenotypic and functional changes in target cells through three main general mechanisms: (i) docking of TEVs on target cells and triggering of intra-cellular signaling; (ii) fusion of TEVs and target cell membranes with release of TEVs molecular cargo in the cytoplasm of recipient cell; and (iii) uptake of TEVs by recipient cells. Though the overall tumor-promoting effects of TEVs as well as the general mechanisms involved in TEVs interactions with, and uptake by, recipient cells are relatively well established, current knowledge about the molecular determinants that mediate the docking and uptake of tumor-derived EVs by specific target cells is still rather deficient. These molecular determinants dictate the cell and organ tropism of TEVs and ultimately control the specificity of TEVs-promoted metastases. Here, we will review current knowledge on selected specific molecules that mediate the tropism of TEVs towards specific target cells and organs, including the integrins, ICAM-1 Inter-Cellular Adhesion Molecule), ALCAM (Activated Leukocyte Cell Adhesion Molecule), CD44, the metalloproteinases ADAM17 (A Disintegrin And Metalloproteinase member 17) and ADAM10 (A Disintegrin And Metalloproteinase member 10), and the tetraspanin CD9.

Nanoemulsions of Phoenix dactylifera L. (Decaffeinated) and Coffea arabica L. Extracts as a Novel Approach for the Treatment of Carbon Tetrachloride-Mediated Liver Fibrosis.

Liver fibrosis is a condition characterized by the excessive buildup of scar tissue in the liver. This scarring occurs as a result of chronic liver damage, often caused by conditions such as hepatitis, alcohol abuse, certain metabolic disorders, genetic abnormalities, autoimmunity, and noninfectious diseases such as fatty liver which leads to liver fibrosis. Nanoparticles have gained attention in recent years as potential therapeutic agents for liver fibrosis. They offer unique advantages due to their small size, large surface area, and ability to carry drugs or target specific cells or tissues. Studies have suggested that nanoemulsions may enhance drug delivery systems, enabling targeted drug delivery to specific sites in the liver and improving therapeutic outcomes. In this study, we explore the protective and therapeutic values with phytochemical profiling of the used agro-wastes decaffeinated palm date seeds (Phoenix dactylifera L., PSC) coffee and caffeinated Arabic coffee seeds (Coffea arabica L.; ACS). Both ACS and PSC extracts were converted into nanoemulsion (NE) forms using the oleic acid/Tween 80 system, which was recruited for the purpose of treating a rat model with liver fibrosis. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to record the sizes, morphologies, hydrodynamic diameters, and ζ-potentials of the prepared NE-ACSE and NE-PSCE. Accordingly, the NE-ACSE and NE-PSCE imaged via TEM and their ζ-potentials were recorded at 20.7, 23.3 nm and -41.4, -28.0 mV, respectively. The antioxidant properties were determined with a DPPH scavenging assay. The synthesized NE-PSCE and NE-ACSE were employed to treat a rat model with CCl4-induced liver fibrosis, to estimate the role of each emulsion-based extract in the treatment of liver fibrosis through recording inflammatory parameters, liver functions, antioxidant enzymes, and histopathological analysis results. The nanoemulsion forms of both ACSE and PSCE provided significant increases in antioxidant enzymes, reducing inflammatory parameters, compared to other groups, where liver functions were decreased with values close to those of the control group. In conclusion, both nanoemulsions, ACSE and PSCE, provided a new avenue as therapeutic approaches for liver diseases, and further studies are encouraged to obtain maximum efficiency of treatment via the combination of both extracts.

Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines.

Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate-TraD and TraD-T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.

A magnetic separation method for isolating and characterizing the biomolecular corona of lipid nanoparticles

Lipid nanoparticle (LNP) formulations are a proven method for the delivery of nucleic acids for gene therapy as exemplified by the worldwide rollout of LNP-based RNAi therapeutics and mRNA vaccines. However, targeting specific tissues or cells is still a major challenge. After LNP administration, LNPs interact with biological fluids (i.e., blood), components of which adsorb onto the LNP surface forming a layer of biomolecules termed the "biomolecular corona (BMC)" which affects LNP stability, biodistribution, and tissue tropism. The mechanisms by which the BMC influences tissue- and cell-specific targeting remains largely unknown, due to the technical challenges in isolating LNPs and their corona from complex biological media. In this study, we present a new technique that utilizes magnetic LNPs to isolate LNP-corona complexes from unbound proteins present in human serum. First, we developed a magnetic LNP formulation, containing >40 superparamagnetic iron oxide nanoparticles (IONPs)/LNP, the resulting LNPs containing iron oxide nanoparticles (IOLNPs) displayed a similar particle size and morphology as LNPs loaded with nucleic acids. We further demonstrated the isolation of the IOLNPs and their corresponding BMC from unbound proteins using a magnetic separation (MS) system. The BMC profile of LNP from the MS system was compared to size exclusion column chromatography and further analyzed via mass spectrometry, revealing differences in protein abundances. This new approach enabled a mild and versatile isolation of LNPs and its corona, while maintaining its structural integrity. The identification of the BMC associated with an intact LNP provides further insight into LNP interactions with biological fluids.