Bioengineering Approaches Research Articles

Emerging Bioengineering Approaches for Cancer and Tumor Therapy

Cancer has become a significant socioeconomic burden globally, with millions of new cases and deaths each year. The promising field of bioengineering has recently undergone significant advancements, providing new methods for combating cancer. Increasing attention has been directed toward understanding the molecular mechanisms of human diseases, supported by the availability of various genetic tools and rapid technological advancements. These developments have enabled the use of the latest gene therapy techniques for cancer treatment, including gene editing, gene deletion, and correcting defective genes through methods such as TALENs, Zinc fingers, RNAi, CRISPR, site-directed mutagenesis (SDM), and enzyme therapy to modulate catalytic activity. In addition, bioengineering vaccines like mRNA vaccines, bioinformatics, computational tools, artificial intelligence (AI), nanotechnology, and chemotherapy are emerging as significant cancer treatment strategies. Among these, gene editing and gene therapy have gained particular attention in recent years and are often used in combination with other therapeutic approaches. The engineering of enzymes and advancements in nanotechnology have also significantly progressed. AI and bioinformatics have contributed to more precise diagnosis, prediction, and prognosis, enabling tailored treatment of cancer and tumors. Imaging and radiotherapy, enhanced by AI, have improved surgical outcomes, even from remote locations. Precision oncology has emerged, using bacteria and viruses to target tumors directly. In this review, we discuss recent advancements and challenges in various cancer therapies.

An Enabling Peptide Ligation Induced by Thiol-Salicylaldehyde Ester for Chemical Protein Synthesis.

Chemical protein synthesis by amide-forming ligation of two unprotected peptide segments offers an effective strategy for the preparation of protein derivatives that are not accessible through bioengineering approaches. Herein, an unprecedented chemical ligation between peptides with C-terminal 2-mercaptobenzaldehyde (thiol-salicylaldehyde, TSAL) esters and peptides bearing N-terminal cysteine/penicillamine is reported. Reactive peptide TSAL esters can be obtained from peptide hydrazides in an operationally simple and highly effective manner. This chemoselective peptide ligation enables the rapid production of N,S-benzylidene acetal intermediates, which can readily be converted into native amide bonds even at sterically hindered junctions. In addition, the current method can be applied compatibly in concert with other types of ligations and subsequent desulfurization chemistry, thereby facilitating convergent protein synthesis. The effectiveness of this new method is also showcased by the total synthesis of proteins ubiquitin and hyalomin-3 (Hyal-3), the efficient synthesis of protein ubiquitin-fold modifier 1 (UFM1) via a C-to-N sequential TSAL ester-induced ligation strategy, and the chemical synthesis of protein Mtb CM through a combined strategy of Ser/Thr ligation and TSAL ester-induced ligations.

Organoid Models in Cancer Immunotherapy: Bioengineering Approach for Personalized Treatment

Organoid Models in Cancer Immunotherapy: Bioengineering Approach for Personalized Treatment

Brain organoids: A new tool for modelling of neurodevelopmental disorders.

Neurodevelopmental disorders are mostly studied using mice as models. However, the mouse brain lacks similar cell types and structures as those of the human brain. In recent years, emergence of three-dimensional brain organoids derived from human embryonic stem cells or induced pluripotent stem cells allows for controlled monitoring and evaluation of early neurodevelopmental processes and has opened a window for studying various aspects of human brain development. However, such organoids lack original anatomical structure of the brain during maturation, and neurodevelopmental maturation processes that rely on unique cellular interactions and neural network connections are limited. Consequently, organoids are difficult to be used extensively and effectively while modelling later stages of human brain development and disease progression. To address this problem, several methods and technologies have emerged that aim to enhance the sophisticated regulation of brain organoids developmental processes through bioengineering approaches, which may alleviate some of the current limitations. This review discusses recent advances and application areas of human brain organoid culture methods, aiming to generalize optimization strategies for organoid systems, improve the ability to mimic human brain development, and enhance the application value of organoids.

Revitalizing Bacillus Calmette-Guérin Immunotherapy for Bladder Cancer: Nanotechnology and Bioengineering Approaches.

Bacillus Calmette-Guérin (BCG) immunotherapy has been a cornerstone treatment for non-muscle-invasive bladder cancer for decades and still faces challenges, such as severe immune adverse reactions, which reduce its use as a first-line treatment. This review examines BCG therapy's history, mechanisms, and current status, highlighting how nanotechnology and bioengineering are revitalizing its application. We discuss novel nanocarrier systems aimed at enhancing BCG's efficacy while mitigating specific side effects. These approaches promise improved tumor targeting, better drug loading, and an enhanced stimulation of anti-tumor immune responses. Key strategies involve using materials such as liposomes, polymers, and magnetic particles to encapsulate BCG or functional BCG cell wall components. Additionally, co-delivering BCG with chemotherapeutics enhances drug targeting and tumor-killing effects while reducing drug toxicity, with some studies even achieving synergistic effects. While most studies remain experimental, this research direction offers hope for overcoming BCG's limitations and advancing bladder cancer immunotherapy. Further elucidation of BCG's mechanisms and rigorous safety evaluations of new delivery systems will be crucial for translating these innovations into clinical practice.

A Multipronged Bioengineering, Spectroscopic and Theoretical Approach in Unravelling the Excited-State Dynamics of the Archetype Mycosporine Amino Acid.

Mycosporine glycine (MyG) was produced by the fermentation of a purposely engineered bacterial strain and isolated from this sustainable source. The ultrafast spectroscopy of MyG was then investigated in its native, zwitterionic form (MyGzwitter), via femtosecond transient electronic absorption spectroscopy. Complementary nonadiabatic (NAD) simulations suggest that, upon photoexcitation to the lowest excited singlet state (S1), MyGzwitter undergoes efficient nonradiative decay to repopulate the electronic ground state (S0). We propose an initial ultrafast ring-twisting mechanism toward an S1/S0 conical intersection, followed by internal conversion to S0 and subsequent vibrational cooling. This study illuminates the workings of the archetype mycosporine, providing photoprotection, in the UV-B range, to organisms such as corals, macroalgae, and cyanobacteria. This study also contributes to our growing understanding of the photoprotection mechanisms of life.

Molecular Mechanisms and Enhancement of Piezoelectricity in the M13 Virus

AbstractUnderstanding the structure and function of bioelectric materials is challenging due to the complex nature of biomaterials and a lack of appropriate tools. The precisely defined structures and genetic tunability of viruses provide an excellent model system to investigate bioelectrical behavior in biomaterials. This study presents the molecular mechanisms of piezoelectricity in the M13 bacteriophage (phage) under various mechanical stresses for bio‐piezoelectric generation. A computational approach is used to calculate the piezoelectric tensors of the M13 phage and quantify its direction‐dependent dipole moments. By computationally designing negatively charged residues on the phage surface, the surface charge density is enhanced to 16.7 µC cm−2. Using genetic engineering, phages are experimentally designed with different charges and tail structures to create model phage nanostructures, including individual phages, vertically standing phage films, and horizontally aligned phage films. Their vertical, horizontal, and shear‐mode piezoelectric properties are then measured using scanning probe microscopy techniques. The resulting phage‐based piezoelectric energy generators exhibit an effective piezoelectric coefficient of 15.4 pm V−1 and a power density of 4.2 µW cm−2. This phage‐based bioengineering approach provides a versatile platform for investigating fundamental mechanisms of bioelectricity and designing bioelectric materials for applications in energy harvesting, biomemory, and biosensors.

P-792 Human endometrial organoids embedded in hybrid endometrial-derived hydrogels as a biocompatible treatment for endometrial pathologies in preclinical models

Abstract Study question Are human endometrial organoids (hEOs) labelled with Molday-ION Rhodamine B (MIRB) within hybrid endometrial-derived hydrogels a biocompatible approach to treat endometrial pathologies in immunocompetent mice? Summary answer The direct intrauterine injection of hEOs labelled with MIRB within hybrid endometrial-derived hydrogels demonstrated xenogeneic biocompatibility in preclinical models over a 2-week period. What is known already The lack of effective treatments for endometrial pathologies emphasizes the urgent need for novel bioengineering approaches. In this context, hEOs are presented as promising candidates for tissue regeneration, since they offer a native and partially undifferentiated cellular source with high proliferative capacity, which is fostered after its culture within hybrid endometrial-derived hydrogels (Gómez-Álvarez et al. 2023). Their concurrent injection aims to enhance proliferation and differentiation through its rich biochemical composition. No prior study has successfully labelled and injected hEOs in immunocompetent models, which is crucial to discern whether the therapeutic effect operates at paracrine level or involves direct tissue regeneration. Study design, size, duration This study was approved by an ethics committee. Decellularized porcine endometrium was used to create endometrial-derived hydrogels as previously described and then mixed with PuraMatrix to obtain the hybrid endometrial-derived hydrogel. After 7 days of culture, hEOs (passages 6-7) were labelled with MIRB for final tracking. Finally, hEOs were mixed with the hybrid hydrogel and injected into the uterine horns in immunocompetent mice (n = 3). After two weeks, biocompatibility was evaluated through blood and macroscopical analyses. Participants/materials, setting, methods After 7 days, hEOs were collected and labelled with MIRB (50 µg/ml) for their final tracking in the murine endometrium. Endometrial-derived hydrogel (8 mg/ml) was mixed with PuraMatrix 1% (50:50 ratio) to generate the hybrid hydrogel. After its characterization and in vitro validation (Gómez-Álvarez et al. 2023), it was used as the bioactive carrier. Each animal received a single intrauterine injection (50 µl, ∼100.000 cells). Blood, spleen, and uterus were collected after 2-week period. Main results and the role of chance Hybrid endometrial-derived hydrogels demonstrated biocompatibility and in vivo stability in mice over a 2-week period (Gómez-Álvarez et al. 2023). In this study, we aim to use this hybrid hydrogel with hEOs as a biocompatible treatment in preclinical models. After 7 days of culture, hEOs were successfully labelled with MIRB (∼85% efficacy), which was confirmed under fluorescence microscopy. The single intrauterine injection (50 µl, ∼100.000 cells) did not cause apparent adverse immune reactions in mice (n = 3) during the 2-week period. All mice exhibited no signs of distress and their behavior remained within normal parameters. Furthermore, the macroscopic examination of uterine horns revealed normal appearance, characterized by the absence of anomalies and tumors. At day 14 post-injection, haematocrit analyses showed normal white blood cell count: 0.89 ± 0.53 · 109/L neutrophils; 7.82 ± 2.23 · 109/L lymphocytes; 0.21 ± 0.13 · 109/L monocytes; 0.04 ± 0.04 · 109/L eosinophils; 0.00 ± 0.00 · 109/L basophils. In addition, no significant changes in red blood cells (9.31 ± 0.54 · 1012/L) and platelets (1063.00 ± 318.58 · 109/L) were observed. Histological analyses will be perform to assess the presence of hEOs labelled with MIRB in the murine endometrium and dilucidate their mechanism of action. Limitations, reasons for caution This study has been carried out in healthy C57BL/6 mice. To confirm the scope of this technique, the next step is to explore the regenerative potential of hEOs labelled with MIRB within hybrid endometrial-derived hydrogels in murine models with endometrial damage. Wider implications of the findings These findings demonstrate the biocompatibility of hEOs labelled with MIRB within hybrid endometrial-derived hydrogels in immunocompetent C57BL/6 mice. This bioengineering approach holds the potential to improve the treatment landscape for specific endometrial pathologies. Trial registration number Not applicable

A bio-engineering approach to generate bioinspired (spider) silk protein-based materials

A bio-engineering approach to generate bioinspired (spider) silk protein-based materials

Flexible nano-liposomes-encapsulated recombinant UL8-siRNA (r/si-UL8) based on bioengineering strategy inhibits herpes simplex virus-1 infection

Herpes simplex virus-1 (HSV-1) infection can cause various diseases and the current therapeutics have limited efficacy. Small interfering RNA (siRNA) therapeutics are a promising approach against infectious diseases by targeting the viral mRNAs directly. Recently, we employed a novel tRNA scaffold to produce recombinant siRNA agents with few natural posttranscriptional modifications. In this study, we aimed to develop a specific prodrug against HSV-1 infection based on siRNA therapeutics by bioengineering technology. We screened and found that UL8 of the HSV-1 genome was an ideal antiviral target based on RNAi. Next, we used a novel bio-engineering approach to manufacture recombinant UL8-siRNA (r/si-UL8) in Escherichia coli with high purity and activity. The r/si-UL8 was selectively processed to mature si-UL8 and significantly reduced the number of infectious virions in human cells. r/si-UL8 delivered by flexible nano-liposomes significantly decreased the viral load in the skin and improved the survival rate in the preventive mouse zosteriform model. Furthermore, r/si-UL8 also effectively inhibited HSV-1 infection in a 3D human epidermal skin model. Taken together, our results highlight that the novel siRNA bioengineering technology is a unique addition to the conventional approach for siRNA therapeutics and r/si-UL8 may be a promising prodrug for curing HSV-1 infection.

Reclamation of an eroded lakeshore slope using small vegetation islands and terraces

Bioengineering approaches to slope stabilization involve biological and mechanical methods with low impact on the environment and landscape. Slope stabilization using vegetation patches or islands can greatly decrease runoff and soil erosion on an eroded lake shore. While vegetation islands showed promise for forest and grassland reclamation, they have rarely been used on eroded lake shores. A study was conducted to determine whether bioengineering using native vegetation islands and terraces could successfully revegetate and stabilize a severely eroded lake shore slope in the central aspen parkland, Alberta, Canada. Three gullies and three slopes were terraced using pressure treated planks, then transplanted with non-dormant (mid-August) and dormant (mid-October) 20 cm diameter vegetation islands in a soil amended with 25% (by volume) compost. Volumetric water content was significantly less in lower slope positions than upper slope positions, and sediment transport to lower slope positions was much reduced. After one year, both non-dormant and dormant transplanted islands survived and established on the terraced slope, under relatively inhospitable growing conditions. Although non-dormant islands had significantly lower vegetation mortality (10% or less) than dormant islands on both slopes and gullies, no differences in health and vigour were found by the end of the study. Overall plant species number on the site increased during the study. Graminoid species densities increased, while those of forbs, shrub and tree species declined in the transplanted vegetation islands. Non-dormant islands had greater graminoid density than dormant islands, where Poa pratensis was the dominant species in the islands. Bioengineering with vegetation islands and terraces can assist in trapping runoff and sediment, and accelerate establishment of a diversity of plant species and groups relative to traditional revegetation methods.

Angiogenesis is uncoupled from osteogenesis during calvarial bone regeneration

Bone regeneration requires a well-orchestrated cellular and molecular response including robust vascularization and recruitment of mesenchymal and osteogenic cells. In femoral fractures, angiogenesis and osteogenesis are closely coupled during the complex healing process. Here, we show with advanced longitudinal intravital multiphoton microscopy that early vascular sprouting is not directly coupled to osteoprogenitor invasion during calvarial bone regeneration. Early osteoprogenitors emerging from the periosteum give rise to bone-forming osteoblasts at the injured calvarial bone edge. Microvessels growing inside the lesions are not associated with osteoprogenitors. Subsequently, osteogenic cells collectively invade the vascularized and perfused lesion as a multicellular layer, thereby advancing regenerative ossification. Vascular sprouting and remodeling result in dynamic blood flow alterations to accommodate the growing bone. Single cell profiling of injured calvarial bones demonstrates mesenchymal stromal cell heterogeneity comparable to femoral fractures with increase in cell types promoting bone regeneration. Expression of angiogenesis and hypoxia-related genes are slightly elevated reflecting ossification of a vascularized lesion site. Endothelial Notch and VEGF signaling alter vascular growth in calvarial bone repair without affecting the ossification progress. Our findings may have clinical implications for bone regeneration and bioengineering approaches.

Nature-based solution using a bioengineering strategy for soil erosion control in tropical climate with intense rainfall patterns

BackgroundIn the face of escalating soil erosion exacerbated by intense rainfall in tropical climates, the quest for sustainable and effective erosion control techniques is paramount. Nature-based solutions (NbS) using bioengineering strategies have emerged as a potential avenue, yet their efficacy and adaptability in such environmental conditions are not fully explored. ObjectiveThis study seeks to bridge the research gap by evaluating the effectiveness of a bioengineering approach, which involved a strategic plantation of Pigeonpea, Sesame, and Cowpea, as an NbS for soil erosion control under the challenging conditions of tropical climates. It emphasizes the rhizospheric characteristics of these species and their potential to offer a sustainable and environmentally friendly solution to soil erosion. MethodsEmploying a random block experimental design, the study compared the performance of Pigeonpea, Sesame, and Cowpea against conventional biosynthetic erosion control methods across various plots. Over 25 rainfall events, comprehensive data collection was undertaken, encompassing 150 measures of precipitation, surface runoff, and soil loss mass. Results: The findings reveal bioengineering solution's comparable, if not superior, effectiveness to conventional techniques, marked by minimal soil loss and enhanced water infiltration capabilities. Analysis of Covariance showcased a very large effect size, significantly underscoring bioengineering's efficacy in soil loss control. Notably, bioengineering's contribution extends beyond erosion control, fostering aquifer recharge and preventing sedimentation in water bodies. ConclusionsThis strategic plantation of Pigeonpea, Sesame, and Cowpea stands out as a promising NbS for soil erosion control in tropical climates, characterized by its sustainable approach and minimal environmental footprint. The study advocates for the broader integration of Pigeonpea, Sesame, and Cowpea in soil conservation practices, highlighting its potential to transform erosion control strategies towards greater environmental sustainability and effectiveness.

Cationic Glucan Dendrimer Gel-Mediated Local Delivery of Anti-OC-STAMP-siRNA for Treatment of Pathogenic Bone Resorption.

Osteoclast stimulatory transmembrane protein (OC-STAMP) plays a pivotal role in the promotion of cell fusion during osteoclast differentiation (osteoclastogenesis) in the context of pathogenic bone resorption. Thus, it is plausible that the suppression of OC-STAMP through a bioengineering approach could lead to the development of an effective treatment for inflammatory bone resorptive diseases with minimum side effects. Here, we synthesized two types of spermine-bearing (Spe) cationic glucan dendrimer (GD) gels (with or without C12) as carriers of short interfering RNA (siRNA) to silence OC-STAMP. The results showed that amphiphilic C12-GD-Spe gel was more efficient in silencing OC-STAMP than GD-Spe gel and that the mixture of anti-OC-STAMP siRNA/C12-GD-Spe significantly downregulated RANKL-induced osteoclastogenesis. Also, local injection of anti-OC-STAMP-siRNA/C12-GD-Spe could attenuate bone resorption induced in a mouse model of periodontitis. These results suggest that OC-STAMP is a promising target for the development of a novel bone regenerative therapy and that C12-GD-Spe gel provides a new nanocarrier platform of gene therapies for osteolytic disease.

#1967 Towards the design optimisation of catheters for paediatric dialysis using a bioengineering approach

Abstract Background and Aims Central venous lines (CVLs) used for haemodialysis (HD) in children are associated with a high complication rate, leading to inadequate dialysis and need for a line replacement in nearly 45% of children on HD. This high complication rate may be due to poor CVL configurations, designed without considering specific anatomical differences between children and adults. Using a bioengineering approach, we aim to provide a fluid dynamics characterization of different CVL models commonly used in children to establish an association between CVL design and clinical complications, and to improve CVL performance by optimising their design. Method Computational fluid dynamics (CFD) using modelling and simulations is a powerful engineering tool that use computers to study complex systems, applying mathematics, physics and computer science. Four models of CVLs of varying designs and sizes (6.5Fr, 8Fr, 10Fr and 14Fr) were scanned by microCT to reconstruct computational models. CFD analyses were set up to simulate blood flow within the CVLs under both ideal and realistic models of superior vena cava. A variety of flow rates, routinely used in clinical practice, were applied to study the whole range of working conditions. Haemodynamic features that cannot be measured in vivo such as high shear stresses and large areas of stagnation, were analysed. CFD findings were compared to clinical outcomes (n = 26 patients with 57 lines) and to the blood clots distribution of CVLs removed from patients (n = 8). Fluid dynamics characteristics of the CVLs studied were used to set up a preliminary design optimisation process of a 6.5F CVL model. Using sampling methods, a variety of CVL designs were created starting from the original model and results were compared. Results In all the simulated CVLs, the arterial lumens showed the highest stagnation and recirculation areas. The role of arterial tip configuration was negligible for the blood flows and the proximal side holes played a major role for blood aspiration being also subject to the highest levels of shear stress, regardless of the design. In all the anatomical models, blood velocity increased after catheter insertion (Fig. 1a) together with wall shear stresses. CFD results were in accordance with the clinical data which showed a higher recurrence rate of thrombosis for the 8Fr and 10Fr CVLs. Also, microCT of the CVLs removed due to complications confirmed presence of thrombosis at the catheter tips. Results suggest that shear stress is the parameter most strongly related to clinical complications. The preliminary design optimisation study was used to find the parameters which have the highest influence on the CVL performance. Preliminarily optimised designs of the 6.5F model showed a better haemodynamic performance, reducing stagnation areas (Fig. 1b). Conclusion Characterisation of the fluid dynamics of commercially available CVLs explains some of the clinical outcomes experienced by patients. Identification of potential design-related complications in CVLs has allowed to set up the optimisation process which will lead to new CVL designs with improved blood flow distribution and minimal damage to blood cells. Funding Kidney Research UK, PhD fellowship and MedTech grants.

Multifunctional Nanofibrous Hollow Microspheres for Enhanced Periodontal Bone Regeneration.

Destructive periodontitis destroys alveolar bone and eventually leads to tooth loss. While guided bone regeneration, which is based on creating a physical barrier to hinder the infiltration of epithelial and connective tissues into defect sites, has been widely used for alveolar bone regeneration, its outcomes remain variable. In this work, a multifunctional nanofibrous hollow microsphere (NFHMS) is developed for enhanced alveolar bone regeneration. The NFHMS is first prepared via combining a double emulsification and a thermally induced phase separation process. Next, E7, a short peptide with high specific affinity to bone marrow-derived stem cells (BMSCs), is conjugated onto the surface of NFHMS. After that, bone forming peptide (BFP), a short peptide derived from bone morphology protein 7 isloaded in calcium phosphate (CaP) nanoparticles, which are further encapsulated in the hollow space of the NFHMS-E7 to form NFHMS-E7-CaP/BFP. The NFHMS-E7-CaP/BFP selectively promoted the adhesion of BMSCs and expelled the adhesion of fibroblasts and epithelial cells. In addition, the BFP is sustainedly released from the NFHMS-E7-CaP/BFP to enhance the osteogenesis of BMSCs. A rat challenging fenestration defect model showed that the NFHMS-E7-CaP/BFP significantly enhanced alveolar bone tissue regeneration. This work provides a novel bioengineering approach for guided bone regeneration.

Crosstalking with Dendritic Cells: A Path to Engineer Advanced T Cell Immunotherapy.

Crosstalk between dendritic cells (DCs) and T cells plays a crucial role in modulating immune responses in natural and pathological conditions. DC-T cell crosstalk is achieved through contact-dependent (i.e., immunological synapse) and contact-independent mechanisms (i.e., cytokines). Activated DCs upregulate co-stimulatory signals and secrete proinflammatory cytokines to orchestrate T cell activation and differentiation. Conversely, activated T helper cells "license" DCs towards maturation, while regulatory T cells (Tregs) silence DCs to elicit tolerogenic immunity. Strategies to efficiently modulate the DC-T cell crosstalk can be harnessed to promote immune activation for cancer immunotherapy or immune tolerance for the treatment of autoimmune diseases. Here, we review the natural crosstalk mechanisms between DC and T cells. We highlight bioengineering approaches to modulate DC-T cell crosstalk, including conventional vaccines, synthetic vaccines, and DC-mimics, and key seminal studies leveraging these approaches to steer immune response for the treatment of cancer and autoimmune diseases.

Towards Greener and More Cost-efficient Biosynthesis of Pharmaceuticals and Fragrance Molecules.

Enzymes are natural catalysts which are gaining momentum in chemical synthesis due to their exquisiteselectivity and their biodegradability. However, the cost-efficiency and the sustainability of the overall biocatalytic process must be enhanced to unlock completely the potential of enzymes for industrial applications. To reach this goal, enzyme immobilization and the integration into continuous flow reactors have been the cornerstone of our research. We showed key examples of the advantages of those tools for the biosynthesis of antivirals, anticancer drugs, and valuable fragrance molecules. By combining new strategies to immobilize biocatalysts, innovative bioengineering approaches, and process development, the performance of the reactions could be boosted up to 100-fold.

Innovations in bio-engineering and cell-based approaches to address immunological challenges in islet transplantation.

Human allogeneic pancreatic islet transplantation is a life-changing treatment for patients with severe Type 1 Diabetes (T1D) who suffer from hypoglycemia unawareness and high risk of severe hypoglycemia. However, intensive immunosuppression is required to prevent immune rejection of the graft, that may in turn lead to undesirable side effects such as toxicity to the islet cells, kidney toxicity, occurrence of opportunistic infections, and malignancies. The shortage of cadaveric human islet donors further limits islet transplantation as a treatment option for widespread adoption. Alternatively, porcine islets have been considered as another source of insulin-secreting cells for transplantation in T1D patients, though xeno-transplants raise concerns over the risk of endogenous retrovirus transmission and immunological incompatibility. As a result, technological advancements have been made to protect transplanted islets from immune rejection and inflammation, ideally in the absence of chronic immunosuppression, to improve the outcomes and accessibility of allogeneic islet cell replacement therapies. These include the use of microencapsulation or macroencapsulation devices designed to provide an immunoprotective environment using a cell-impermeable layer, preventing immune cell attack of the transplanted cells. Other up and coming advancements are based on the use of stem cells as the starting source material for generating islet cells 'on-demand'. These starting stem cell sources include human induced pluripotent stem cells (hiPSCs) that have been genetically engineered to avoid the host immune response, curated HLA-selected donor hiPSCs that can be matched with recipients within a given population, and multipotent stem cells with natural immune privilege properties. These strategies are developed to provide an immune-evasive cell resource for allogeneic cell therapy. This review will summarize the immunological challenges facing islet transplantation and highlight recent bio-engineering and cell-based approaches aimed at avoiding immune rejection, to improve the accessibility of islet cell therapy and enhance treatment outcomes. Better understanding of the different approaches and their limitations can guide future research endeavors towards developing more comprehensive and targeted strategies for creating a more tolerogenic microenvironment, and improve the effectiveness and sustainability of islet transplantation to benefit more patients.

Abstract 5768: Proximity labeling with molecular signal sensing of oncogenic PIK3CA

Abstract PIK3CA mutations are major oncogenic drivers for oncogenesis. Mutant PIK3CA continuously activates the PI3K pathway, transmitting oncogenic signals within the transformed cells. Despite decades of efforts focused on targeting oncogenic PIK3CA for cancer therapy, the precise molecular and functional machinery remains incomplete due to the molecular complexity of the PI3K pathway. Mapping of proximity proteins in the oncogenic PI3K pathway and identifying mutant signals will signify a deep understanding of oncogenesis and a broad range of therapeutic applications. In this study, we hypothesize that PIK3CA mutant cancer cells establish a unique molecular program networking with distinct proteins, which differs from that of cancer cells with wild-type (WT) PIK3CA. To test this hypothesis, we have developed a novel bioengineering approach to decode protein-protein interactions of oncogenic PIK3CA and identify key molecular partners, potentially providing therapeutic vulnerabilities in cancer cells. First, we applied the proximity labeling technique, based on the fusion of a promiscuous protein ligase to a targeting protein of interest (POI), featuring proximity-dependent biotinylation of interacting and neighboring proteins. The promiscuous biotin ligases, BioID and BioID2, were utilized to integrate WT PIK3CA or gain-of-function hotspot mutations in at E545K or H1047R residues. To optimize the labeling protocol and biotinylation conditions, the BioID and BioID2 plasmids were fused with each POI, incorporating multiple combinations of protein tags, including myc, HA, and red fluorescence protein. Lamin A was used as the positive control, and a total of 15 plasmids were constructed in pcDNA3.1 mammalian expression vector. The sizes of the plasmids and the gene sequences were validated by PCR amplification and Sanger sequencing. Next, we employed a programmable nuclease, the prokaryotic Argonaute (pAgo), assisted by fluorescence resonance energy transfer technology, to detect oncogenic signals derived from mutant PIK3CA in cell-free conditions. To test our methods, we engineered two cancer cell lines (DHSA-1426 and COSB) derived from naturally occurring hemangiosarcoma in dogs, to induce PIK3CA H1047R mutations using CRISPR/Cas9. Our technical approach enables temporal and spatial molecular sensing of oncogenic PIK3CA and its functional interactome in living cancer cells by integrating high-resolution molecular mapping with a pAgo-based detection system. Our ongoing work involves the standardization and optimization of proximity labeling for oncogenic PIK3CA across ontogenetically distinct cancer cells. Furthermore, we will determine the sensitivity and specificity of the integrated approach to achieve proximity labeling with programmable detection of molecular signals from oncogenic PIK3CA. Citation Format: Rong Li, Donghee Lee, Md Abdullah, Jong Hyuk Kim. Proximity labeling with molecular signal sensing of oncogenic PIK3CA [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 5768.