Biology subjects

Johnson, J. O.

Publications and source records attributed to Johnson, J. O..

8 recordsLinked to original sources

bioRxiv2026

Spatially resolved multimodal hallmarks of response to neoadjuvant immunotherapies in the melanoma ecosystem in 2D and 3D

Neoadjuvant immunotherapy has transformed cancer treatment, yet the spatial molecular architecture governing response and resistance across distinct immune checkpoint blockade (ICB) regimens remains poorly defined. We assembled the largest neoadjuvant ICB (NICB) spatial multi-omics cohort to date, profiling over 112 million cells at single-cell resolution across three melanoma ICB regimens using MERFISH spatial transcriptomics, multiplexed immunofluorescence, and scRNA-sequencing. These analyses revealed the full multicellular spatial architecture of the NICB tumor microenvironment, including mature TLS with germinal centers, TCF7+ stem-like T cells, myeloid cells organized into spatially distinct cellular neighborhoods with unique intercellular signaling circuits, and CCL19/CCL21-expressing fibroblasts as a previously unrecognized stromal scaffold sustaining these immune hubs. We developed three purpose-built computational tools that together enabled comprehensive quantification of this microenvironment for the first time: SCIRA for whole-slide single-cell receptor-ligand quantification, GCSCAN for molecularly grounded TLS and germinal center structural delineation, and PathNet-TLS for automated TLS detection on H&E images. Applying these tools across the cohort, we defined the immune and stromal composition and cellular neighborhood organization distinguishing responders from non-responders. We also quantified cell-cell interactions and regimen-specific immune architectures, including a markedly stronger mature TLS/germinal center response with IPI-NIVO than NIVO-RELA. Importantly, GCSCAN-quantified TLS and germinal center density each stratified disease-free survival, with responders that lack germinal centers having an elevated risk of relapse. Open-top light-sheet imaging and CODA-based 3D reconstruction further uncovered interconnected germinal center-TLS tunnels invisible to standard 2D histopathology. These findings establish a discovery-to-tool paradigm linking single-cell tumor microenvironment interrogation to clinically deployable computational pathology for biomarker-driven NICB assessment across cancer types.

cancer biology↗

bioRxiv2026

BCL11B targeting in tumor CD8+ T cells amplifies anti-tumor response by blocking exhaustion while promoting stemness and cytotoxicity

Tumor infiltrating CD8+ T cells (TILs) progress to a state of terminal exhaustion (Ttex) which have impaired functionality and are nonrenewable. However their precursors (Tpex) are renewable and can generate efficient effector cells. We started from the observation that melanoma patients undergoing therapy with checkpoint inhibitors show increased survival when their T cells have low BCL11B mRNA. In line with this, ablation of Bcl11b in CD8+ TILs conferred a superior anti-tumor response in murine melanoma and ovarian cancer models. Bcl11b KO TILs failed to progress to the Ttex state and retained elevated stemness. Bcl11b exerted its role by repressing expression of essential transcription factors (TF) controlling stemness, and conversely by promoting expression of exhaustion-associated TFs and inhibitory receptor genes, through complex epigenetic control. In addition, Bcl11b KO CD8+ T cells showed increased Ag-specific cytolytic activity and elevated Gzmb and Prf1 proteins, but no increase in their mRNAs, however presented higher expression of genes with role in translation. Furthermore, CRISPR-CAS9-mediated deletion of BCL11B in human TILs from a patient with poor response to adoptive cell therapy with autologous TILs, improved their cytolytic activity and promoted expression of the stemness-associated TF TCF1, underlying its potential therapeutic use. HIGHLIGHTS- Adoptive transfer of Bcl11b KO CD8+ TILs surpasses WT in tumor burden reduction - Bcl11b ablation reprograms TILs and impairs the progression to Ttex state - Bcl11b KO CD8+ T cells have elevated cytotoxicity and kill only Ag-MHCI targets - BCL11B deletion in nonresponder ACT-TIL improves cytolytic activity and elevates TCF1 GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/742578v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@c3b965org.highwire.dtl.DTLVardef@1c09d75org.highwire.dtl.DTLVardef@a19748org.highwire.dtl.DTLVardef@d2fd6b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Collagen remodeling promotes a GPCR-mediated mechanosensory immune checkpoint in ADGRG1+ CD8+ T cells and serves as a spatial biomarker of response to immunotherapy

Accessibility of immune cells to the tumor microenvironment (TME) in many solid tumors can be influenced by extracellular matrix (ECM) deposition, organization, and remodeling within tumor stroma. Specifically, lysyl hydroxylase-2 (LH2)-catalyzed lysine hydroxylation in type-I collagen telopeptides leads to formation of intermolecular collagen cross-links creating a stiffened and proteolytically resistant, stable ECM. Advanced head and neck squamous cell carcinomas (HNSCC) frequently exhibit desmoplasia with elevated LH2 expression, and Immune-checkpoint (ICI) therapy is effective in only <20% patients, suggesting that ECM remodeling mechanistically governs the composition and function of TME infiltrates. We show that elevated LH2 and collagen alignment promotes stromal accumulation of CD8 T-cells, and poor response to ICI in HPV-HNSCCs. Integration of clinical biopsies, transcriptomic datasets, and an immunocompetent syngeneic mouse model revealed that aligned collagen spatially restricts adhesion G protein-coupled receptor positive (ADGRG1) CD8 T-cells to activate a non-canonical GPCR-mediated mechanosensory program that drives dysfunction and exhaustion. Statement of significanceDesmoplasia is common in solid tumors, and immune checkpoint inhibitors benefit only some patients. Current biomarkers like PD-L1 and TMB have limited value. Our findings reveal a previously unrecognized collagen-ADGRG1 mechanosensory immune checkpoint, offering a clinically tractable, spatially resolved biomarker to better stratify patients for immunotherapy.

cancer biology↗

bioRxiv2024

Eco-evolutionary Guided Pathomic Analysis to Predict DCIS Upstaging

Cancers evolve in a dynamic ecosystem. Thus, characterizing cancers ecological dynamics is crucial to understanding cancer evolution and can lead to discovering novel biomarkers to predict disease progression. Ductal carcinoma in situ (DCIS) is an early-stage breast cancer characterized by abnormal epithelial cell growth confined within the milk ducts. In this study, we show that ecological analysis of hypoxia and acidosis biomarkers can significantly improve prediction of DCIS upstaging. First, we developed a novel eco-evolutionary designed approach to define habitats in the tumor intraductal microenvironment based on oxygen diffusion distance. Then, we identified cancer cells with metabolic phenotypes attributed to their habitats, including CA9 for hypoxia responding phenotype, and LAMP2b for acid adapted phenotype. Traditionally these markers have shown limited predictive capabilities for DCIS progression, if any. However, when analyzed from an ecological perspective, their power to differentiate between non-upstaged and upstaged DCIS increased significantly. Second, we discovered distinct niches with spatial patterns of these biomarkers and used the distribution of such niches to predict patient upstaging. The niches were characterized by pattern analysis of both cellular and spatial features. With a 5-fold validation on the biopsy cohort, we trained a random forest classifier to achieve the area under curve (AUC) of 0.74. Our results affirm the importance of tumor ecological features in eco-evolutionary-designed approaches for novel biomarkers discovery. SignificanceOur results affirm the importance of spatial and ecological features in eco-evolutionary-designed biomarkers discovery studies in the era of digital pathology.

cancer biology↗

bioRxiv2024

Histology-guided mathematical model of tumor oxygenation: sensitivity analysis of physical and computational parameters

A hybrid off-lattice agent-based model has been developed to reconstruct the tumor tissue oxygenation landscape based on histology images and simulated interactions between vasculature and cells with microenvironment metabolites. Here, we performed a robustness sensitivity analysis of that models physical and computational parameters. We found that changes in the domain boundary conditions, the initial conditions, and the Michaelis constant are negligible and, thus, do not affect the model outputs. The model is also not sensitive to small perturbations of the vascular influx or the maximum consumption rate of oxygen. However, the model is sensitive to large perturbations of these parameters and changes in the tissue boundary condition, emphasizing an imperative aim to measure these parameters experimentally.

systems biology↗

bioRxiv2020

Targeting hypoxic habitats with hypoxia pro-drug evofosfamide in preclinical models of sarcoma

Hypoxic regions (habitats) within tumors are heterogeneously distributed and can be widely variant. Hypoxic habitats are generally pan-therapy resistant. For this reason, hypoxia-activated prodrugs (HAPs) have been developed to target these resistant volumes. The HAP evofosfamide (TH-302) has shown promise in preclinical and early clinical trials of sarcoma. However, in a phase III clinical trial, TH-302 did not improve survival in combination with doxorubicin (dox), most likely due to a lack of patient stratification based on hypoxic status. Herein, our goal was to develop deep-learning (DL) models to identify hypoxic habitats, using multiparametric (mp) MRI and co-registered histology, and to non-invasively monitor response to TH-302 in a patient-derived xenograft (PDX) of rhabdomyosarcoma and a syngeneic model of fibrosarcoma (RIF-1). A DL convolutional neural network showed strong correlations (>0.81) between the true hypoxic portion in histology and the predicted hypoxic portion in multiparametric MRI. TH-302 monotherapy or in combination with Dox delayed tumor growth and increased survival in the hypoxic PDX model (p<0.05), but not in the RIF-1 model, which had lower volume of hypoxic habitats. Control studies showed that RIF-1 resistance was due to hypoxia and not to other causes. Notably, PDX tumors developed resistance to TH-302 under prolonged treatment. In conclusion, response to TH-302 can be attributed to differences in hypoxia status prior therapy. Development of non-invasive MR imaging to assess hypoxia is crucial in determining the effectiveness of TH-302 therapy and to follow response. In further studies, our approach can be used to better plan therapeutic schedules to avoid resistance. One Sentence SummaryDevelopment of non-invasive MR imaging to assess hypoxia is crucial in determining the effectiveness of TH-302 therapy and to follow response.

cancer biology↗

bioRxiv2020

Establishing a Living Biobank of Patient-Derived Organoids of Intraductal Papillary Mucinous Neoplasms of the Pancreas

Pancreatic cancer (PaCa) is the third leading cause of cancer-related deaths in the United States. There is an unmet need to develop strategies to detect PaCa at an early, operable stage and prevent its progression. Intraductal papillary mucinous neoplasms (IPMNs) are cystic PaCa precursors that comprise nearly 50% of pancreatic cysts detected incidentally via cross-sectional imaging. Since IPMNs can progress from low- and moderate-grade dysplasia to high-grade dysplasia and invasion, the study of these lesions offers a prime opportunity to develop early detection and prevention strategies. Organoids are an ideal preclinical platform to study IPMNs, and the objective of the current investigation was to establish a living biobank of patient-derived organoids (PDO) from IPMNs. IPMN tumors and adjacent normal pancreatic tissues were successfully harvested from 15 patients with IPMNs undergoing pancreatic surgical resection at Moffitt Cancer Center & Research Institute (Tampa, FL) between May of 2017 and March of 2019. Organoid cultures were also generated from cryopreserved tissues. Organoid count and size were determined over time by both Image-Pro Premier 3D Version 9.1 digital platform and Matlab application of a Circular Hough Transform algorithm, and histologic and genomic characterization of a subset of the organoids was performed using immunohistochemistry and targeted sequencing, respectively. The success rates for organoid generation from IPMN tumor and adjacent normal pancreatic tissues were 81% and 87%, respectively. IPMN organoids derived from different epithelial subtypes showed different morphologies in vitro, and organoids recapitulated histologic and genomic characteristics of the parental IPMN tumor. In summary, this pre-clinical model has the potential to provide new opportunities to unveil mechanisms of IPMN progression to invasion and to shed insight into novel biomarkers for early detection and targets for chemoprevention.

cancer biology↗

bioRxiv2019

Mutations in the SPTLC1 gene are a cause of amyotrophic lateral sclerosis that may be amenable to serine supplementation

Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of childhood motor disorder with a heterogeneous clinical presentation. The underlying causes of this condition are poorly understood, hindering the development of effective therapies. In a whole-exome sequencing trio-family study of three unrelated juvenile patients diagnosed with ALS and failure to thrive, we identified de-novo mutations in SPTLC1 (p.Ala20Ser in two patients and p.Ser331Tyr) not present in their healthy parents or siblings. SPTLC1 encodes a subunit of the serine palmitoyltransferase complex, a key enzyme in sphingolipid biosynthesis. Mutations in this gene are known to cause hereditary sensory autonomic neuropathy, type 1A, with a characteristic increase in plasma levels of neurotoxic deoxymethyl-sphinganine. We found an increase of this metabolite in one of our patients carrying the p.Ala20Ser mutation. Treatment of one of the patients with high dose, oral L-serine led to an increase in body weight, suggesting that serine supplementation may be beneficial among patients carrying mutations in this gene.

genetics↗