Anti bacterial function of secreted human FABP3.

FABP3 belongs to a large family of cytoplasmic fatty acid binding proteins that are expressed in a tissue-specific manner. It is predominantly expressed in breast, muscle and heart. During our exploratory studies on the role of FABP3 in tumorigenesis and our consequent attempts to study the molecular mechanism responsible for the oncogenic potential of FABP3, we came across an unexpected role of FABP3 as an anti-bacterial protein. Presence of the protein was detected in culture media of cell lines stably over-expressing human FABP3. Conditioned medium from these FABP3 over-expressing cells exerted a distinct anti-bacterial activity against E. coli. Our results indicate that binding of FABP3 to the bacterial cell surface contributes to its anti-bacterial activity. Incubation of E. coli bacterial cells with FABP3 protein led to disruption of the physical integrity of bacterial cell membrane causing leakage of cellular components. Further, in silico analysis predicted strong binding of FABP3 to the antibiotic binding sites on the bacterial ribosome. Interestingly, we found that FABP3 is a naturally occurring secretory protein present in milk in abundance as confirmed by western blot and ELISA. Thus, our experimental data together with in silico analysis suggests that FABP3 is secreted in milk, has an anti-bacterial function, shows activity against E. coli by disrupting bacterial membrane and targeting the ribosome, and may play a protective role against bacterial infection in newborns.

Establishment of a three­dimensional triculture model on the novel AXTEX­4D™ platform.

Cancer can be fatal if it is not treated in a timely manner; therefore, there is a high demand for more specific oncology drugs. Unfortunately, drugs showing positive responses on a two­dimensional (2D) culture platform do not often show the same effect in clinical trials. Therefore, three­dimensional (3D) culture platforms are garnering attention since they more closely mimic the tumor microenvironment (TME). The TME stimulates metastasis and drug resistance, and serves an essential role in tumor formation. An accurate understanding of tumor­stroma interactions is undoubtedly required to improve the response of patients to therapeutic strategies, and cancer therapeutic strategies that do not account for the stroma are considered inadequate. It should be noted that 3D monoculture systems do not completely mimic the TME since other cells in the 3D culture are missing, such as fibroblast or endothelial cells, which are essential components of the stroma; therefore, it is essential to develop advanced 3D culture systems. The present study aimed to develop a versatile triculture model that mimics the native TME; therefore, it could aid in high­throughput screening of chemotherapeutic drugs against cancer by evaluating their effects on tumor progression and cell cytotoxicity. The present study demonstrated the use of the AXTEX­4D™ platform in developing triculture tissueoids composed of MCF­7, human umbilical vein endothelial cells and MRC5 cells, and compared it with a 3D monoculture model (MCF­7) and a 2D culture model. The triculture model was validated for proliferation, ECM markers and T­cell infiltration by confocal microscopy. Alamar Blue assay demonstrated that triculture tissueoids exhibited higher drug resistance than the other two models, thus demonstrating their use in the screening of oncology drugs.

Recapitulating Tumor Microenvironment Using AXTEX-4DTM for Accelerating Cancer Research and Drug Screening.

OBJECTIVE: The formation of three-dimensional spheroid tumor model using the scaffold-based platforms has been demonstrated over many years now. 3D tumor models are generated mainly in non-scalable culture systems, using synthetic and biological scaffolds. Many of these models fail to reflect the complex tumor microenvironment and do not allow long-term monitoring of tumor progression. This has resulted in inconsistent data in drug testing assays during preclinical and clinical studies. METHODS: To overcome these limitations, we have developed 3D tissueoids model by using novel AXTEX-4D platform. RESULTS: Cancer 3D tissueoids demonstrated the basic features of 3D cell culture with rapid attachment, proliferation, and longevity with contiguous cytoskeleton and hypoxic core. This study also demonstrated greater drug resistance in 3D-MCF-7 tissueoids in comparison to 2D monolayer cell culture. CONCLUSION: In conclusion, 3D-tissueoids are more responsive than 2D-cultured cells in simulating important tumor characteristics, anti-apoptotic features, and their resulting drug resistance.

AXTEX-4D: A Three-Dimensional Ex Vivo Platform for Preclinical Investigations of Immunotherapy Agents.

The latest advancements in oncology are majorly focused on immuno-oncology (I-O) therapies. However, only ∼7% of drugs are being approved from the preclinical discovery phase to phase 1. The most challenging issues in I-O are the development of active and efficient drugs in an economically feasible way and in a comparatively short time for testing and validation. This mandates an urgent need for the upgradation of preclinical screening models that closely mimic the in vivo tumor microenvironment (TME). The established and most common methods for investigating the tumoricidal activity of I-O drugs are either two-dimensional systems or primary tumor cells in standard tissue culture vessels. Unfortunately, they do not mimic the TME. Consequently, the more in vivo-like three-dimensional (3D) multicellular tumor spheroids are quickly becoming the favored model to examine immune cell-mediated responses in reaction to the administration of I-O drugs. Despite many advantages of multicellular spheroids, challenges (e.g., incompatibility of quantitative assays with spheroid platforms) are still involved in the tedious procedures required for the spheroid culture that is holding back the biological community from adapting the well-recognized spheroid tissue models for studying drug delivery more widely. To this end, we have demonstrated the utility of the 3D ex vivo oncology model, developed on our novel AXTEX-4D™ platform to assess therapeutic efficacies of I-O drugs by investigating immune cell proliferation, migration, infiltration, cytokine profiling, and cytotoxicity of tumor tissueoids. The platform eliminates the need for additional biomolecules such as hydrogels and instead relies on the cancer cells themselves to create their own gradients and microenvironmental factors. In effect, the more comprehensive and ex vivo-like immune-oncology model developed on AXTEX-4D platform can be utilized for high-throughput screening of immunotherapeutic drugs.