Raouf, A. | bioRxiv Science

bioRxiv2026

Architecture Matters: Design Rules for Multigene IDO1/PD L1 Cassettes in Human Skin Cells

Allogeneic cell therapies require the coordinated expression of multiple immunomodulatory genes, yet multigene circuits that function in permissive cell lines often fail in differentiated human tissues for unclear reasons. Here, we systematically dissect how transcriptional architecture governs functional immunoregulation in engineered human keratinocyte and fibroblast lines. Using site-specific large-cargo integration (eePASSIGE) as an enabling tool, we determined that genomic insertion efficiency was not the limiting factor for phenotype; rather, promoter arrangement and gene order dictated expression hierarchy. A single-promoter EF1-IDO1-T2A-GFP design that expressed robustly in HEK293T cells was nearly silent in skin-derived cells, preventing reporter-based enrichment. In dual- and tri-modular cassettes, we observed severe transcriptional interference: a downstream CMV promoter driving GFP or PD-L1/iCasp9 (via EMCV-IRES) markedly suppressed the upstream EF1-IDO1 unit, despite intact integration (resulting in [~]175-625-fold attenuation), demonstrating strong promoter interference within the circuit. Functionally, co-culture assays revealed a hierarchical immunomodulatory logic: high IDO1 expression proved to be a requisite threshold for T-cell suppression, whereas PD-L1 provided measurable benefit only against highly activated, PD-1+ T cells in vitro. Collectively, these data establish a site-specific framework for generating immune-tuned skin cells and define essential design rules for avoiding promoter interference in next-generation translational skin substitutes.

bioengineering↗

bioRxiv2024

A robust protocol for the systematic collection and expansion of cells from ER+ breast cancertumors and their matching tumor-adjacent tissues

Therapy resistance and tumor recurrence are major challenges in the clinical management of breast cancer. Current data indicates that the breast tumor microenvironment (TME) and the tumor immune microenvironment (TIME) are important modulators of breast cancer cell response to chemotherapies and the development of therapy resistance. To this end, the ability to recreate the tumor microenvironment in the laboratory using autologous primary cells that make up the breast TME has become an indispensable tool for cancer researchers as it allows the study of tumor immunobiology in the context of therapy resistance. Moreover, the clinical relevance of data obtained from single cell transcriptomics and proteomics platforms would be greatly improved if primary autologous tumor cells were used. In this article, we report a robust and efficient workflow to obtain autologous cancer cells, cancer-associated fibroblasts, and tumor-infiltrating immune cells from primary human breast cancer tumors obtained from mastectomy procedures. As well, we show that this protocol can be used to obtain normal-like epithelial cells, fibroblasts, and immune cells from the matching tumor-adjacent breast tissue samples. Also, a robust methodology to expand each of these primary cell types in vitro is presented that allows the maintenance of the primary tumor cell phenotype. The availability of a large number of autologous primary human breast tumor cells and their matching tumor-adjacent tissues will facilitate the study of differential and cancer cell-specific gene expression patterns that will further our understanding of how the TME and TIME influence therapy resistance in the breast tumor context.

cancer biology↗