Sable, S. B. | Search bioRxiv

bioRxiv2025

Identification and characterization of host-directed therapeutics for tuberculosis using a versatile human 3D tuberculoma bioplatform

Host-directed therapies (HDT) represent a pivotal strategy in combating both drug-susceptible and drug-resistant tuberculosis (TB). Current evaluations, however, show limited success in animal models and clinical settings, emphasizing the need for more effective HDT candidates. With few druggable targets validated within granulomas, it is essential to verify the effectiveness of HDT candidates identified through traditional macrophage cultures in the context of the tuberculous granuloma milieus. Bioengineering a scalable, high-throughput screening (HTS) platform that replicates the physiological microenvironments in the hallmark tubercular lesions could significantly improve the identification of relevant HDT candidates and new treatment strategies. Here, we developed a facile, HTS-compatible bioplatform that generates tuberculoma-emulating structures, following three-dimensional (3D) co-cultures of human cells and pathogenic mycobacteria. Employing high-content imaging alongside immunological and transcriptomic approaches, we demonstrated that these 3D structures exhibit classic tuberculoma attributes and develop crucial transformations. Utilizing this system, we screened antibody biosimilars and potential HDT compounds. Our findings demonstrate the systems versatility in discovering antimicrobials and HDT candidates that effectively reduce mycobacterial burdens and granuloma lesions, while elucidating their immune mechanisms within 3D milieus. Many compounds effective in two-dimensional (2D) cultures were ineffective once granulomas formed in our 3D model. Notably, several promising compounds were found to induce rapid autophagy flux, and we validated the effectiveness of one such compound, the multi-kinase inhibitor AT9283, in a mouse model. Our findings highlight several HDT candidates for potential repurposing in TB treatment, offering a robust tool for accelerating therapeutic discoveries and advancing translational research for TB and other granulomatous diseases. SummarySafer and shorter treatment regimens for tuberculosis (TB) are urgently needed. Host-directed therapies (HDT) are being explored to enhance antibiotic regimens and address antimicrobial resistance. To expedite the discovery of HDT candidates and the development of new treatment strategies, scalable in vitro systems are needed that can replicate the critical features and microenvironments of TB lesions in a format compatible with high-throughput screening (HTS). We developed an HTS-compatible bioplatform using 3D co-cultures of human cells and fluorescent mycobacteria, creating tuberculoma-like structures with classic solid, necrotic, and cavitary transformations that exhibit crucial microenvironments. This facile system enabled us to screen antibody biosimilars and HDT compounds in 3D human in vitro tuberculomas, identifying candidates that inhibited mycobacterial growth and granuloma lesions while revealing potential innate immune mechanisms. The study revealed several promising HDT candidates that could be repurposed for TB treatment, introducing a versatile tool for screening therapeutic libraries. Additionally, it presents a framework for enhancing human in-vitro granuloma models, as advances in tissue-like systems emerge to recapitulate the architecture and multilineage differentiation in the lungs.

immunology↗

bioRxiv2025

Methodology for a freshly engineered or cryo-preserved 3D tuberculoma bioplatform for studying tuberculosis biology and high-content screening of therapeutics

Tuberculomas are the conglomeration of tuberculous granulomas into structurally organized three-dimensional (3D) masses that result from Mycobacterium tuberculosis infection and represent one of the more severe morphological forms of tuberculosis (TB). Several in vitro models that mimic human TB granulomas have been reported to decipher complex host-pathogen interactions and discover new prophylactic and therapeutic interventions. They serve as ethical bridge approaches to human studies. However, these models need improvements in generating well-organized granuloma lesions, classic tuberculoma structures, and relevant microenvironments. They are impractical for screening extensive chemical and genetic libraries owing to their low throughput, limited scalability, batch-to-batch variability, and high costs. Here, we describe a mycobacteria-in-spheroid co-culture workflow in a standard 96-well plate format that generates a robust 3D cell culture model. This model reproduces key attributes and microenvironments in human tuberculomas and can be scaled up as a high-throughput screening (HTS)-compatible bioplatform. The tuberculoma-like structures generated encompass organized, florid granulomatous lesions and exhibit solid, necrotic, and cavitary morphologies. This model can be developed using freshly isolated human primary cells or a monocytic cell line with virulent mycobacteria. The platform combines the entire workflow from generation to imaging of tuberculoma-like structures in situ. It permits the serial quantitation of drug efficacy and monitoring of lesion resolution over several days to weeks following a single treatment. Additionally, we outline a methodology for adopting this workflow for cryo-preservation, enhancing its potential for commercial application. The ease of generation, pliability, cryo-shelf stability, and reproducibility of the bioplatform make it ideal for HTS applications and implementation in the discovery programs of TB and other granulomatous diseases.

bioengineering↗