Maintain Cell Viability Research Articles

Preparation of thermo-responsive polymer encapsulated exosomes and its role as a therapeutic agent for blood clot lysis

Tissue plasminogen activators induce enzymatic activation of plasminogen to plasmin that cleaves fibrin strands in blood clots. In the present study, extracellular vesicles such as exosomes from fibrosarcoma cell line HT1080 were utilized as clot-busting agents. These exosomes were being used for clot lysis of whole blood which showed 28% lysis within 10 h, which was comparable to that of the streptokinase (commercial plasmin activator) with no significant difference. These exosomes were able to facilitate the migration of endothelial cells in a scratch wound assay where normalized wound area remaining was 7.5% at 18 h. Also, exosomes aided in attenuation of oxidative stress generated on the cells, thereby maintaining cell viability. These exosomes were further encapsulated in a thermo-responsive polymer for better localized delivery that showed no cytotoxic effects, and sustained delivery was achieved up to a concentration of 117 µg/mL in 25 days, which corresponds to around 65% of the total amount of exosomes added. When a combination of exosomes and thermo–responsive polymer was utilized, the clot lysis activity reached to around 22% in 72 h. Thus, it proves the potential of this combinatorial approach which can be effectively used for thrombus degradation and healing of endothelium lining in damaged blood vessels.

Alginate functionalized biomimetic 3D scaffold improves cell culture and cryopreservation for cellular therapy

The clinical translation of cellular therapy is hampered by the scarcity of reliable and consistent cell sources. In this study, we developed an exquisite scaffold featuring the hierarchical structure and biofunctions of silkworm cocoons (CryoSiCo), for boosting cell manufacture and cryopreservation. CryoSiCo was constructed by a creative bottom-up fabrication technique integrating electrospinning, in situ surface functionalization and freeze-shaping, generating a 3D cocoon-mimicking fibrous scaffold composed of graphene oxide-incorporated polylactic acid/gelatin inner fiber core and alginate outer fiber shell. CryoSiCo provided rapid and uniform rewarming for cryopreserved cells, and maximally maintained cell viability and proliferation capability, allowing for effective cryopreservation. Importantly, CryoSiCo could cryopreserve stem cell-scaffold constructs with high cell survival and functions, which can be directly implanted to restore tissue defects. Thus, CryoSiCo represents an appealing biomimetic strategy for storing precious cells and tissue engineered constructs, showing a broad application for fundamental research and applied medicine.

Cytocompatible asymmetrical coating for Janus carrier synthesis through capillary wetting and ascending

Despite the successful implementation of elegant strategies for the fabrication of Janus microstructures, two critical factors have limited the applicability of most techniques for the partial modification of living cell surfaces: harsh conditions that could disintegrate cells, and the lack of an effective route to accomplish a mild modification for living cells. In this study, an expeditious synthesis, named lower-half occupation by capillary ascended liquids (LOCAL), is proposed for the fabrication of asymmetrical structures surrounding not only microbeads but also both living adherent and buoyant mammalian cells. The microbeads or living cells are safely supported and trapped on the apical sides of a micropillar array, which prevents them from contacting the bottom substrate. As the coating agents further transfer and contact the trapped particles through interpillar capillary flow, the autonomous capillary ascending coats the free bottom surfaces of the target particles within 2 min, with significantly small quantities of coating agents. The self-assembled architectures of the cells demonstrate thoroughly maintained cell viability, highlighting the potential of the LOCAL method as a desirable alternative to the widely applied state-of-art methods for developing Janus beads and Janus cells.

Ferroptosis Regulation by the NGLY1/NFE2L1 Pathway

Cells are equipped with various mechanisms to maintain cell viability upon exposure to stress. The Cap ‘n’ Collar (CNC) family includes five vertebrate stress‐responsive transcription factors. The CNC member nuclear factor erythroid 2 like 2 (NFE2L2) is considered the master regulator of the cellular oxidative stress response. Fellow CNC member nuclear erythroid 2 like 1 (NFE2L1) is better known for its role in responding to proteasome dysfunction. We hypothesized that NFE2L1 also responds to oxidative stress. We demonstrate that genetic disruption of NFE2L1 sensitizes cancer cells to death by a non‐apoptotic, oxidative form of cell death termed ferroptosis. We also show that genetic disruption of N‐glycanase 1 (NGLY1), which catalyzes NFE2L1 deglycosylation and sequence editing, sensitizes cancer cells to ferroptosis through an NFE2L1‐dependent mechanism. Expression of a sequence‐edited NFE2L1 mutant, which mimics NGLY1‐processed NFE2L1, rescues cellular resistance to ferroptosis in NFE2L1 gene‐disrupted cells. Finally, we provide evidence that the ferroptosis protection conferred by NFE2L1 is mediated by the expression of glutathione peroxidase 4 (GPX4). All together, our results highlight the role of NFE2L1 in ferroptosis resistance and implicate NFE2L1 and NGLY1 as potential targets for cancer therapy.

Donation after circulatory death and lung transplantation.

ABSTRACTLung transplantation is the most effective modality for the treatment of patients with end-stage lung diseases. Unfortunately, many people cannot benefit from this therapy due to insufficient donor availability. In this review and update article, we discuss donation after circulatory death (DCD), which is undoubtedly essential among the strategies developed to increase the donor pool. However, there are ethical and legislative considerations in the DCD process that are different from those of donation after brain death (DBD). Among others, the critical aspects of DCD are the concept of the end of life, cessation of futile treatments, and withdrawal of life-sustaining therapy. In addition, this review describes a rationale for using lungs from DCD donors and provides some important definitions, highlighting the key differences between DCD and DBD, including physiological aspects pertinent to each category. The unique ability of lungs to maintain cell viability without circulation, assuming that oxygen is supplied to the alveoli-an essential aspect of DCD-is also discussed. Furthermore, an updated review of the clinical experience with DCD for lung transplantation across international centers, recent advances in DCD, and some ethical dilemmas that deserve attention are also reported.

Evaluation of a hyaluronic acid hydrogel (Restylane Lyft) as a scaffold for dental pulp regeneration in a regenerative endodontic organotype model

Scaffolds are crucial elements for dental pulp regeneration. Most of the currently used scaffolds in regenerative endodontic procedures (REPs) are unsuitable for chairside clinical use. This study aimed to evaluate the effect of an injectable synthetic scaffold (Restylane Lyft) on human bone marrow mesenchymal stem cell (hBMSC) viability, proliferation, and osteo/dentinogenic differentiation in a regenerative endodontic organotype model (REM). hBMSC were loaded in an REM either alone (hBMSC group) or mixed with the Restylane Lyft scaffold (Restylane/hBMSC group) and cultured in basal culture medium (n = 9/group). hMSC on culture plates served as controls. Cell viability and proliferation were measured using AlamarBlue assay. The loaded REM was cultured in an osteogenic differentiation medium to measure alkaline phosphatase activity (ALP) and examine the expression of the osteo/dentinogenic markers using real-time reverse transcriptase polymerase chain reaction. Cell viability in all groups increased significantly over 5days. The Restylane/hBMSC group showed significantly higher ALP activity and dentin sialophosphoprotein, osteocalcin, and bone sialoprotein genes expression than the hBMSC and the control groups. Restylane Lyft, a hyaluronic acid (HA) injectable, FDA-approved hydrogel, maintained cell viability and proliferation and promoted osteo/dentinogenic differentiation of hBMSC when cultured in an REM. Henceforth, it could be a promising chairside scaffold material for REPs.

TLR4 and SARM1 modulate survival and chemoresistance in an HPV-positive cervical cancer cell line

Human Papillomavirus is responsible for a wide range of mucosal lesions and tumors. The immune system participate in tumorigenesis in different ways. For example, signaling pathways triggered by Toll-like receptors (TLR) play a role in chemotherapy resistance in several tumor types and are candidates for contributing to the development of HPV-induced tumors. Here, we studied the receptor TLR4 and the adaptor molecule SARM1 in HeLa cells, an HPV-positive cervical cancer cell line. Knocking out of these genes individually proved to be important for maintaining cell viability and proliferation. TLR4 knock out cells were more sensitive to cisplatin treatment, which was illustrated by an increased frequency of apoptotic cells. Furthermore, TLR4 and SARM1 modulated ROS production, which was induced by cell death in response to cisplatin. In conclusion, TLR4 and SARM1 are important for therapy resistance and cervical cancer cell viability and may be relevant clinical targets.

Fabrication of hydrogels with adjustable mechanical properties through 3D cell-laden printing technology

Biocompatible hydrogels have drawn enormous interest for their essential applications in tissue engineering. However, the preparation of hydrogels that meet the requirements of both moldability and biocompatibility is still a challenge. In this study, the hydrogel with adjustable mechanical properties was prepared for cell-laden printing by the combination of photo-crosslinking and ion crosslinking. A mixture of gelatin methacrylate (GelMA), sodium alginate (Alg), chondroitin 4-sulfate sodium salt (CS), and sodium carboxymethyl cellulose (NaCMC) was used as the hydrogel material. Systematical rheological analysis revealed that the formability of this hydrogel were tuned by adjusting the ratio of GelMA to NaCMC. Furthermore, this composite hydrogel could promote cell proliferation, which could be significantly influenced by the content of NaCMC and CS (101.2% cell viability over 7 days of culture by the developed hydrogel extract). The survival rate of cultured cells in hydrogel extracts with NaCMC and CS was 25.9% higher than that without NaCMC and CS. This hydrogel allowed L929 fibroblast cells to maintain cell viability after printing, demonstrating that the hydrogel was suitable for cell-laden printing. Overall, this work provided practical and fundamental insights into developing the biocompatible hydrogel for cell proliferation and migration.

Effect of Calcium Sulphate Pre-crosslinking on Rheological Parameters of Alginate Based Bio-Inks and on Human Corneal Stromal Fibroblast Survival in 3D Bio-Printed Constructs

The principle of three-dimensional (3D) bio-printing involves integration of biomaterials, live cells and controlled motor systems for creating complex biomimetic constructs. Bio-ink is one of the most important components in the process of 3D bio-printing and needs to be sufficiently viscous to be dispensed as a free-standing filament but be biocompatible to maintain cell viability and function. Alginate has been used widely for 3D bio-printing due to its biocompatibility, tunable properties, rapid gelation, low cost, and ability to be functionalized to direct cell behavior. By tuning the physiochemical parameters of alginate-based bio-inks, such as viscosity, improvements in print resolution, fidelity and growth characteristics of encapsulated cells can be achieved. This study aimed to improve the printability of low concentration alginate bio-inks by utilizing calcium sulphate (CaSO4) pre-crosslinking. A variety of alginates, differing in their viscosity, molecular weight and b-D-mannuronate and α-L-guluronate residues were investigated by wet spinning and bio-printing. Rheological and structural properties of pre-crosslinked alginates were characterized with the aim of mitigating the resolution problems associated with the use of low percentage alginate bio-inks, more favorable for maintaining cell viability. Pre-crosslinking produced a significant effect on viscosity of biomaterials improving their suitability for the bio-printing process and influencing the final resolution of the printed structure. Medium viscosity high b-D-mannuronate containing alginate (MVM) showed the highest degree of viscosity change compared to the control (p < 0.0001; n = 6), assessed by single value viscometry analysis and shear rheology, after pre-crosslinking and was subsequently used in experiments with cells. The survival of human corneal stromal fibroblasts (CSFs) was assessed using CellTiterGlo metabolic assay and confirmed with Calcein acetoxymethyl and Ethidium homodimer -1 live/dead staining in pre-crosslinked alginate fibers and bio-printed lattices. Encapsulation of CSFs in pre-crosslinked alginate-based bio-inks did not have a detrimental effect on CSF viability compared to the non-pre-crosslinked control over 7 days under standard cell culture conditions (p > 0.05, n = 3). Overall, printability of low percentage alginate bio-inks was improved by pre-crosslinking without affecting the biocompatibility of the bio-inks.

Effects of SRC and IKKβ Kinase Inhibition in Ischemic Factors Modeling In Vitro and In Vivo

The search for new molecular targets whose modulation can reduce nerve cell dysfunction and neuronal death during ischemic damage is one of the most significant issues in both fundamental and clinical neurobiology. Various kinase enzymes are often considered to be such promising targets since they are involved in key molecular cascades that regulate cell adaptation to stress factors. Our work is devoted to the study of the role of two kinases—SRC and IKKβ—in maintaining the neural networks’ functional activity under a hypoxic condition in vivo and in vitro. SRC kinase is a cytoplasmic non-receptor protein tyrosine kinase. It is involved in the regulation of cell proliferation and differentiation; its expression in nerve cells changes during hypoxia. IKKβ kinase is involved in the regulation of the activity of the transcription factor NF-κB, which is a pleiotropic regulator of many cellular signaling pathways. We have shown that blockade of SRC and IKKβ kinases by selective inhibitors maintains cell viability in modeling hypoxic damage in vitro but does not allow for the preservation of the bioelectrical activity of neurons. Studies in vivo have shown the neuroprotective effect of SRC but not IKKβ kinase inhibition in the modeling of cerebral ischemia in mice.

Effect of corn stigma extract on physical and antioxidant properties of biodegradable and edible gelatin and corn starch films

The development of bio-based food packaging with antioxidant properties is an important research topic and has gained prominence these days. In this study, bioactive films were developed based gelatin-corn starch (GCS) incorporated with corn stigma extract (CSE) at different concentrations (15% and 25%; w/v). In preliminary tests, the extract maintained cell viability above 90% indicating that it is safe for application as an active ingredient. Insertion of the extract did not influence the thickness of the films but caused a slight change in optical properties. Scanning electron microscopy (SEM) analysis revealed interactions between the extract's bioactive compounds with gelatin and corn starch compounds, which may have improved the mechanical properties (elongation at break, Young's modulus). The addition of 25% corn stigma extract increased the contact angle, giving the film a hydrophobic character. Furthermore, at this concentration, a 15% reduction in water vapor permeability was observed. The elaborated films showed complete biodegradability before the tenth day of the study. It can be inferred that the films with corn stigma extract have good antioxidant properties, indicating that they can be used as an ingredient for food packaging.

Design of experiments approach to developing a robust ink for bioprinting

Despite advancements in tissue engineering, the methods used to generate three-dimensional (3D) in vitro models for rapid screening and characterization studies remain time and labor intensive. Bioprinting offers an opportunity to offset these limitations by providing a scalable, high-throughput method with precise control over biomaterial scaffold and cellular deposition. However, the process of formulating bioinks can be complex in terms of balancing the mechanical integrity of a bioscaffold and viability of cells. One key factor, especially in alginate-based bioinks, is the rate of bioscaffold dissolution. It must allow cells to replace the bioscaffold with extracellular matrix (ECM), yet remain durable during extended tissue culture. This study uses a Design of Experiments (DoE) approach to understand the dependencies of multiple variables involved in the formulation and processing of an alginate-based bioink. The focus of the DoE was to understand the effects of hydrogel composition on bioink durability while maintaining cell viability. Three ingredients were varied in all: alginate, nanocellulose, and fibrinogen. Their effects on the bioink were then measured with respect to extrudability, strength, and stiffness as determined by dynamic mechanical analysis (DMA). The DoE demonstrated that mechanical integrity increased with increasing alginate concentration. In contrast, fibrinogen and nanofibril concentration had no statistically significant effect. The optimized ink containing fibroblasts was printable using multiple nozzle sizes while also supporting fibroblast cell viability. DMA characterization further showed that the composition of the cell culture medium did not modulate the degradation rate of the hydrogel. Ultimately, the study outlines a methodology for formulating a bioink that will result in robust bioscaffolds for in vitro model development.

Cyclin-Dependent Kinase Synthetic Lethality Partners in DNA Damage Response.

Cyclin-dependent kinases (CDKs) are pivotal mediators and effectors of the DNA damage response (DDR) that regulate both the pathway components and proteins involved in repair processes. Synthetic lethality (SL) describes a situation in which two genes are linked in such a way that the lack of functioning of just one maintains cell viability, while depletion of both triggers cell death. Synthetic lethal interactions involving CDKs are now emerging, and this can be used to selectively target tumor cells with DNA repair defects. In this review, SL interactions of CDKs with protooncogene products MYC, poly (ADP-ribose) polymerase (PARP-1), and cellular tumor antigen p53 (TP53) are discussed. The individual roles of each of the SL partners in DDR are described.

Therapy resistance: opportunities created by adaptive responses to targeted therapies in cancer.

Normal cells explore multiple states to survive stresses encountered during development and self-renewal as well as environmental stresses such as starvation, DNA damage, toxins or infection. Cancer cells co-opt normal stress mitigation pathways to survive stresses that accompany tumour initiation, progression, metastasis and immune evasion. Cancer therapies accentuate cancer cell stresses and invoke rapid non-genomic stress mitigation processes that maintain cell viability and thus represent key targetable resistance mechanisms. In this Review, we describe mechanisms by which tumour ecosystems, including cancer cells, immune cells and stroma, adapt to therapeutic stresses and describe three different approaches to exploit stress mitigation processes: (1) interdict stress mitigation to induce cell death; (2) increase stress to induce cellular catastrophe; and (3) exploit emergent vulnerabilities in cancer cells and cells of the tumour microenvironment. We review challenges associated with tumour heterogeneity, prioritizing actionable adaptive responses for optimal therapeutic outcomes, and development of an integrative framework to identify and target vulnerabilities that arise from adaptive responses and engagement of stress mitigation pathways. Finally, we discuss the need to monitor adaptive responses across multiple scales and translation of combination therapies designed to take advantage of adaptive responses and stress mitigation pathways to the clinic.

Inositol depletion regulates phospholipid metabolism and activates stress signaling in HEK293T cells

Inositol plays a significant role in cellular function and signaling. Studies in yeast have demonstrated an “inositol-less death” phenotype, suggesting that inositol is an essential metabolite. In yeast, inositol synthesis is highly regulated, and inositol levels have been shown to be a major metabolic regulator, with its abundance affecting the expression of hundreds of genes. Abnormalities in inositol metabolism have been associated with several human disorders. Despite its importance, very little is known about the regulation of inositol synthesis and the pathways regulated by inositol in human cells. The current study aimed to address this knowledge gap. Knockout of ISYNA1 (encoding myo-inositol-3-P synthase 1) in HEK293T cells generated a human cell line that is deficient in de novo inositol synthesis. ISYNA1-KO cells exhibited inositol-less death when deprived of inositol. Lipidomic analysis identified inositol deprivation as a global regulator of phospholipid levels in human cells, including downregulation of phosphatidylinositol (PI) and upregulation of the phosphatidylglycerol (PG)/cardiolipin (CL) branch of phospholipid metabolism. RNA-Seq analysis revealed that inositol deprivation induced substantial changes in the expression of genes involved in cell signaling, including extracellular signal-regulated kinase (ERK), and genes controlling amino acid transport and protein processing in the endoplasmic reticulum (ER). This study provides the first in-depth characterization of the effects of inositol deprivation on phospholipid metabolism and gene expression in human cells, establishing an essential role for inositol in maintaining cell viability and regulating cell signaling and metabolism.

Multifunctional polyphenol-based silk hydrogel alleviates oxidative stress and enhances endogenous regeneration of osteochondral defects

In osteochondral defects, oxidative stress caused by elevated levels of reactive oxygen species (ROS) can disrupt the normal endogenous repair process. In this study, a multifunctional hydrogel composed of silk fibroin (SF) and tannic acid (TA), the FDA-approved ingredients, was developed to alleviate oxidative stress and enhance osteochondral regeneration. In this proposed hydrogel, SF first interacts with TA to form a hydrogen-bonded supramolecular structure, which is subsequently enzymatically crosslinked to form a stable hydrogel. Furthermore, TA had multiple phenolic hydroxyl groups that formed interactions with the therapeutic molecule E7 peptide for controlled drug delivery. In vitro investigations showed that SF-TA and SF-TA-E7 hydrogels exhibited a multitude of biological effects including scavenging of ROS, maintaining cell viability, and promoting the proliferation of bone marrow mesenchymal stem cells (BMSCs) against oxidative stress. The proteomic analysis indicated that SF-TA and SF-TA-E7 hydrogels suppressed oxidative stress, which in turn improved cell proliferation in multiple proliferation and apoptosis-related pathways. In rabbit osteochondral defect model, SF-TA and SF-TA-E7 hydrogels promoted enhanced regeneration of both cartilage and subchondral bone as compared to hydrogel without TA incorporation. These findings indicated that the multifunctional SF-TA hydrogel provided a microenvironment suitable for the endogenous regeneration of osteochondral defects.

Conductive ionic liquid/chitosan hydrogels for neuronal cell differentiation

To regulate cell behaviors and promote nerve function recovery, three-dimensional (3D) conductive hydrogel can transmit intercellular electrical signals, and effectively provide the cell survival environment. However, producing hydrogels with simultaneous high conductivity, favorable biocompatibility, and tissue-matching properties remains a challenge for spinal cord injury (SCI) treatment. Here, a conductive, multifunctional, and biocompatible VPImBF4 ionic liquid (IL) with photosensitive chitosan-based hydrogel ([email protected]) is developed. The [email protected] hydrogel exhibits a 3D microporous structure that could maintain cell viability and improve cell growth. Elastic modulus, conductivity, and biodegradability of the [email protected] hydrogels are investigated with tissue-matching mechanical properties. The [email protected] conductive hydrogels synergistically enhance neuronal cell proliferation and promote neuronal cells differentiation via upregulates synapse gene (Tubulin β3, GAP43, Synaptophysin) expression. Furthermore, in vivo studies of the [email protected] conductive hydrogels as implants demonstrate low-inflammation and neovascularize promotion and appropriate biodegradable properties. The developed [email protected] conductive hydrogel is a promising therapeutic scaffold biomaterial for SCI repair.

Proline-based solution maintains cell viability and stemness of canine adipose-derived mesenchymal stem cells after hypothermic storage.

Transportation of mesenchymal stem cells (MSCs) under hypothermic conditions in 0.9% normal saline solution (NSS) might increase cell death and alter the stemness of MSCs. The present study aimed to evaluate the effect of proline-based solution (PL-BS) on cell viability and the stemness of newly established canine adipose-derived mesenchymal stem cells (cAD-MSCs) under hypothermic conditions. Characterized cAD-MSCs were stored in 1, 10, and 100 mM PL-BS or NSS at 4°C for 6, 9, and 12 hours prior to an evaluation. The results demonstrated that storage in 1 mM PL-BS for 6 hours decreased cell apoptosis and proliferation ability, but improved cell viability and mitochondrial membrane potential. cAD-MSCs maintained their high expression of CD44 and CD90, but had a low expression of CD34 and MHC class II. Trilineage differentiation ability of cAD-MSCs was not affected by storage in 1 mM PL-BS. Gene expression analysis demonstrated that immunomodulatory genes, including IDO, HGF, PGE-2, and IL-6, were upregulated in cAD-MSCs stored in 1 mM PL-BS. In conclusion, PL-BS can be effectively applied for storing cAD-MSCs under hypothermic conditions. These findings provide a new solution for effective handling of cAD-MSCs which might be promising for clinical applications.

From Rest to Growth: Life Collisions of Gordonia polyisoprenivorans 135.

In the process of evolution, living organisms develop mechanisms for population preservation to survive in unfavorable conditions. Spores and cysts are the most obvious examples of dormant forms in microorganisms. Non-spore-forming bacteria are also capable of surviving in unfavorable conditions, but the patterns of their behavior and adaptive reactions have been studied in less detail compared to spore-forming organisms. The purpose of this work was to study the features of transition from dormancy to active vegetative growth in one of the non-spore-forming bacteria, Gordonia polisoprenivorans 135, which is known as a destructor of such aromatic compounds as benzoate, 3-chlorobenzoate, and phenol. It was shown that G. polyisoprenivorans 135 under unfavorable conditions forms cyst-like cells with increased thermal resistance. Storage for two years does not lead to complete cell death. When the cells were transferred to fresh nutrient medium, visible growth was observed after 3 h. Immobilized cells stored at 4 °C for at least 10 months regenerated their metabolic activity after only 30 min of aeration. A study of the ultrathin organization of resting cells by transmission electron microscopy combined with X-ray microanalysis revealed intracytoplasmic electron-dense spherical membrane ultrastructures with significant similarity to previously described acidocalcisomas. The ability of some resting G. polyisoprenivorans 135 cells in the population to secrete acidocalcisome-like ultrastructures into the extracellular space was also detected. These structures contain predominantly calcium (Ca) and, to a lesser extent, phosphorus (P), and are likely to serve as depots of vital macronutrients to maintain cell viability during resting and provide a quick transition to a metabolically active state under favorable conditions. The study revealed the features of transitions from active growth to dormant state and vice versa of non-spore-forming bacteria G. polyisoprenivorans 135 and the possibility to use them as the basis of biopreparations with a long shelf life.

Abstract P5-10-05: HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers

Abstract HER2 is amplified in about 20% of breast cancers. HER3 is as essential as HER2 for maintaining cell viability in HER2+ breast cancer cells. Inhibition of HER2 tyrosine kinase activity results in upregulation of HER3 transcription and phosphorylation. We sought to identify HER3 binding partners upon pharmacological inhibition of HER2 using the irreversible pan HER inhibitor neratinib. We immunoaffinity-purified HER3 from HER2+ BT474 cells treated ± neratinib. Following immunoprecipitation using a HER3 antibody, binding partners were released under reducing conditions. Mass spectrometry experiments identified non-muscle myosin IIA (NMIIA) increased upon inhibition of HER2 with neratinib and decreased under DMSO control treatment from HER3 immunoprecipitates. To validate the presence of NMIIA, we performed immunoprecipitation experiments in HER2+ BT474 and MDA-MB-453 cells using a HER3 antibody. Immunoblots showed increased NMIIA levels upon treatment with 200nM neratinib for 24 hours in both cell lines. Myosin heavy chain 9 (MYH9) gene encodes the protein NMIIA. NMIIA localizes to actin stress fibers and has been implicated in regulation of cell contractility and stress fiber organization.To test if there is an interaction between HER3 and NMIIA, we immunoprecipitated NMIIA from BT474 and MDA-MB-453 cells using an NMIIA antibody. The products were analyzed using immunoblots. The results indicated that HER3 levels were increased upon inhibition of HER2 with 200 nM neratinib for 24 hours.We next examined long term overall survival of primary breast cancer patients who have high and low levels of gene expression for MYH9 from the METABRIC cohort. We observed that patients with high levels of MYH9 have a statistically significant worse overall survival versus patients who express low levels of MYH9.We next sought to examine overall levels of HER3 and MYH9 mRNA and protein upon treatment with neratinib in HER2+ breast cancer whole cell lysates. We observed that mRNA and protein levels of both HER3 and MYH9 increased upon HER2 inhibition with 24 hours of neratinib treatment. To further investigate the role of NMIIA, we knocked out NMIIA in BT474 and MDA-MB-453 using inducible lentiviral shRNA targeting MYH9. This system utilizes the Tet-On 3G induction system and is a highly controlled system that consists of an inducible RNA polymerase II promoter. In the presence of doxycycline, the TRE3G promoter is bound and activated by the constitutively expressed Tet-On 3G trans-activator protein. Non-targeting shRNA was used as control and cells were selected in 1-2 ug/ml puromycin. Western blot analysis demonstrated that cells infected with lentiviral shRNA targeting MYH9 treated with doxycycline had reduced protein expression of NMIIA. BT474 and MDA-MB-453 transduced with shMYH9 were used to perform growth, migration, and invasion assays. The results indicated that MYH9 knockdown + neratinib treatment suppresses BT474 and MDA-MB-453 growth, migration, and invasion. Currently, in vivo xenograft experiments examining BT474 and MDA-MB-453 tumor growth ±NMIIA shRNA in the presence or absence of neratinib treatment are underway. In conclusion, we have identified that NMIIA mRNA and protein levels are increased in the presence of neratinib treatment. Our data reveals that HER2 inhibition increases NMIIA to promote HER2+ breast cancer growth. Citation Format: Samar M Alanazi, Rosalin Mishra, Hima Patel, Long Yuan, Mary Kate Kilroy, Joan T Garrett. HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-05.