Biology subjects

Forward, S.

Publications and source records attributed to Forward, S..

6 recordsLinked to original sources

bioRxiv2026

HER2 mutation-derived neoantigens in NSCLC as actionable targets for TCR therapy

HER2 mutations are oncogenic drivers in 1-6% of non-small cell lung cancers (NSCLC), but therapeutic resistance limits the durability of current HER2-targeted treatments. Here, we identify T-cell receptors (TCRs) targeting recurrent HER2 hotspot mutations as a potential immunotherapeutic strategy for HER2-mutant NSCLC. Using neoepitope prediction and antigen-specific T-cell enrichment, we isolated HLA-A*02:01restricted TCRs recognizing HER2 A775insYVMA, S310F, and G776delinsVC mutations, collectively covering approximately 60% of HER2-mutant NSCLC. These TCRs selectively recognized mutant HER2 epitopes without detectable wild-type reactivity and some displayed cross-recognition of related hotspot variants, expanding the spectrum of targetable tumors. The G776delinsVC-specific TCR also exhibited co-receptorindependent activity showcased by its ability to activate CD4+ T cells. Importantly, timelapse single-cell flow cytometry analyses demonstrated that TCR-engineered T cells repeatedly reacquired activated polyfunctional states following serial antigen stimulation, while serial tumor rechallenge assays confirmed sustained cytotoxic activity across multiple rounds of tumor killing. These findings identify recurrent HER2 mutations as shared immunotherapeutic targets and provide a foundation for the development of TCR-based therapies for HER2-mutant NSCLC.

immunology↗

bioRxiv2025

Differentiation stage-specific use of cap-independent and cap-dependent translation initiation in hematopoiesis

Cell stress can increase the use of m7G-cap-independent, IRES-mediated translation initiation relative to cap-dependent translation (IRES/Cap). Reporters that quantify IRES/Cap have demonstrated differential activity across cultured cell types and stress conditions. By generating an IRES/Cap reporter mouse, we were able to systematically evaluate IRES/Cap across distinct tissues and cell types during physiological stresses and lineage commitment. Caloric stress invoked the expected boost in IRES/Cap translation regardless of differentiation state, but unexpectedly IRES/Cap progressively increased during hematopoietic and epithelial (hair follicle) differentiation under normal, homeostatic conditions. This was independent of total protein output or cell cycle. Even within cells of a given differentiation state, cells with lower relative-IRES utilization had markedly higher multipotent capability in vivo. The RNA processing protein PTBP1 is a mediator of this translation initiation preference. Therefore, low IRES/Cap is a signature of high stemness and suggests modulation of translation initiation participates in cell differentiation state.

developmental biology↗

bioRxiv2024

Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi-pass acquisition

The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins, and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular fluorescent proteins and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cell analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.

bioengineering↗

bioRxiv2022

Laser particle barcoding for multi-pass high-dimensional flow cytometry

Flow cytometry is a standard technology in life science and clinical laboratories used to characterize the phenotypes and functional status of cells, especially immune cells. Recent advances in immunology and immuno-oncology as well as drug and vaccine discovery have increased the demand to measure more parameters. However, the overlap of fluorophore emission spectra and one-time measurement nature of flow cytometry are major barriers to meeting the need. Here, we present multi-pass flow cytometry, in which cells are tracked and measured repeatedly through barcoding with infrared laser-emitting microparticles. We demonstrate the benefits of this approach on several pertinent assays with human peripheral blood mononuclear cells (PBMCs). First, we demonstrate unprecedented time-resolved flow characterization of T cells before and after stimulation. Second, we show 33-marker deep immunophenotyping of PBMCs, analyzing the same cells in 3 back-to-back cycles. This workflow allowed us to use only 10-13 fluorophores in each cycle, significantly reducing spectral spillover and simplifying panel design. Our results open a new avenue in multi-dimensional single-cell analysis based on optical barcoding of individual cells.

bioengineering↗

bioRxiv2022

Highly efficient, all-organic bioluminescence-photosensitizer conjugate eradicates early-stagetumors and prevents metastasis in mice

Photodynamic therapy (PDT) is an established treatment modality using light-activatable drugs. Despite its unique cytotoxic mechanism, the shallow penetration of light has been a serious drawback limiting the applications of PDT. Here, we report bioluminescence-activated PDT (BL-PDT) using efficient bioluminescence resonance energy transfer (BRET) conjugates of clinically approved photosensitizers, Ce6, and luciferase proteins. A high photon-to-Ce6 conversion efficiency (80%), along with intracellular delivery by membrane-fusion liposomes, enabled effective cancer cell killing in vitro. In a syngeneic mouse model of aggressive 4T1 triple-negative breast cancer, as well as a xenograft model of MDA-MB-231 tumors, BL-PDT resulted in complete tumor remission and prevention of metastasis, as well as neo-adjuvant effects. Our result shows the promise of molecularly activable, clinically viable, depth-unlimited phototherapy.

cancer biology↗

bioRxiv2018

Wavelength-encoded laser particles for massively-multiplexed cell tagging

Large-scale single-cell analyses have become increasingly important given the role of cellular heterogeneity in complex biological systems. However, no current techniques enable optical imaging of uniquely-tagged individual cells. Fluorescence-based approaches can only distinguish a handful of distinct cells or cell groups at a time because of spectral crosstalk between conventional fluorophores. Here we show a novel class of imaging probes emitting coherent laser light, called laser particles. Made of silica-coated semiconductor microcavities, these laser particles have single-mode emission over a broad range from 1170 to 1580 nm with sub-nm linewidths, enabling massive spectral multiplexing. We demonstrate the stability and biocompatibility of these probes in vitro and their utility for wavelength-multiplexed cell tagging and imaging. We demonstrate real-time tracking of thousands of individual cells in a 3D tumor model for several days showing different behavioral phenotypes. We expect laser particles will enable new approaches for single-cell analyses.

biophysics↗