Biology subjects

Munn, L. L.

Publications and source records attributed to Munn, L. L..

14 recordsLinked to original sources

bioRxiv2026

Immortalized smooth muscle cells enhance in vitro vasculogenesis

De novo vessel formation (vasculogenesis) in vitro is a key step in tissue engineering to preserve tissue viability for long-term assays and testing therapeutic agents. However, in vitro vasculogenesis is often unreliable due to differences in vascular-supporting cells, including endothelial cells and stromal cells such as smooth muscle cells (SMCs) and fibroblasts. Here, we developed a robust co-culture system of HUVECs and SMCs to generate stable vascular networks capable of maintaining tissue viability over extended periods. Given that SMC plasticity is a major limitation in supporting endothelial network formation, we systematically evaluated the effects of passage number, confluency, and freezing on primary SMC function. To overcome this limitation, we generated immortalized supportive SMCs, which preserved their vasculogenic gene program and functional capacity even at high passage. In addition, we identified and validated key genes associated with endothelial support, including CD248, C3, and FBLN1, all essential for vasculogenesis. Immortalized SMCs consistently maintained expression of these genes and supported robust vessel formation under variable culture conditions. Collectively, this study demonstrates that immortalized SMCs provide a stable, reproducible platform for endothelial-SMC co-cultures, enabling long-term vascularized tumor models suitable for functional studies and therapeutic screening.

developmental biology↗

bioRxiv2026

Volumetric mechanoplasticity couples melanoma drug tolerance to susceptibility to CD8+ T cell killing

Cell swelling has been reported in physiological tumor contexts, but whether sustained cell volume expansion leaves a durable state that reshapes drug and immune responses in melanoma is unknown. Using controlled hypotonic dilution as an experimental handle, we compare two priming regimens with comparable hypotonic exposure but different magnitude and persistence of volume expansion, enabling us to test which post-recovery phenotypes track with sustained enlargement beyond hypotonic exposure alone. Sustained enlargement produces a measurable size imprint after return to isotonic conditions, accompanied by nuclear lamina and chromatin remodeling with p53 pathway engagement and suppression of replication and growth programs. After recovery, primed cells reinforce F-actin organization, migrate faster in 3D collagen, and induce antioxidant and anti-ferroptosis defenses consistent with improved stress survival. A gene expression signature derived from this response is associated with poorer outcome in TCGA skin cutaneous melanoma. Unexpectedly, the same volume history also increases IFN-{gamma} response, elevates MHC-I, reduces sialylation, and increases susceptibility to CD8+ T cell killing. These findings indicate that persistent volume history can couple drug tolerance programs to an exploitable increase in immune visibility.

biophysics↗

bioRxiv2025

Efficient calculation of fluid transport in porous media with moving boundaries

A novel hybrid model combining the lattice Boltzmann (LB) and finite difference (FD) methods is proposed to simulate transport in through a junction of actively contracting lymphatic vessels, while also handling flow of interstitial liquid in the surrounding porous tissue. Details of the dynamically flexing walls and valves in the lymphatic vessel and its near vicinity are modeled using a high-resolution LB method, whereas overall efficiency was significantly improved by using low-resolution FD in the larger tissue domain distant from the vessel. Pressure and velocity conditions at the interface between subdomains of the two numerical methods are matched by imposing a partial bounce-back ratio in LB corresponding to the permeability coefficient{kappa} in Darcys law for flow through porous media. Parameters governing the match between the algorithms at their interface can be estimated from the Kozeny-Carman relationship for porous media and further refined with a simpler, parallel flow geometry that also serves to validate the method. Test calculations show that the hybrid method is roughly four times faster than the LB method and permits computation over significantly larger domains. This method should be applicable to a large range of problems involving fluid flow in porous media with embedded conduits that have non-stationary boundaries. Author summaryIt is generally acknowledged that the finite difference method (FDM) is faster and requires less memory than the lattice Boltzmann method (LBM) for comparable domain sizes. However, LBM performs better for simulating fluid flow near complex, deformable, or moving boundaries. For this reason, it can be beneficial to create hybrid models that combine FDM and LBM. In this work, we use such a hybrid model to simulate a contracting lymphatic bifurcation in fluid. Our goal is to demonstrate the models robustness and high efficiency.

bioengineering↗

bioRxiv2025

A Hybrid Multiscale Model for Predicting CAR-T Therapy Outcomes in SolidTumors

T cell distribution within tumors ("tumor hotness") critically determines immunotherapy success. However, despite numerous strategies to enhance intratumoral T cell accumulation--such as multi-target CAR-Ts and combinatorial approaches--limited mechanistic understanding of T cell-microenvironment interactions has constrained progress. To address this, we developed a physiological mechanistic model of the 3D tumor microenvironment (TME) to evaluate CAR-T performance under environmental fluctuations and different infusion strategies. The model integrates key vascular (rolling, adhesion, endothelial suppression) and interstitial (ECM density, metabolic competition, chemokine sensitivity) barriers. Our simulations reveal that collagen density and metabolic competition dominate CAR-T efficacy. Enhancing vascular adhesion improves infiltration but remains limited by collagen and metabolism. Endothelial suppression markedly reduces tumor hotness, while its alleviation enhances response. Systemic infusion yields higher tumor hotness than intratumoral delivery, but combined routes or reduced collagen restore efficacy even in dense tumors. This mechanistic framework enables rational optimization of CAR-T strategies. Significance StatementThe success of immunotherapies such as CAR-T cells depends on their ability to infiltrate and persist within solid tumors, yet the mechanisms that govern this process remain poorly understood. Using a mechanistic 3D model of the tumor microenvironment, we quantitatively dissected how vascular and interstitial barriers--including endothelial suppression, collagen density, metabolic competition, and chemokine cues--shape CAR-T distribution ("tumor hotness"). Our results reveal that stromal and metabolic constraints, rather than vascular adhesion alone, dominate CAR-T efficacy. This framework bridges molecular, cellular, and tissue-scale mechanisms, providing a quantitative foundation for optimizing CAR-T design and delivery strategies to overcome resistance in solid tumors.

immunology↗

bioRxiv2025

Optimizing Cancer Vaccinations Using a Physiologically Based Pharmacokinetic (PBPK) Model

Antigen-based tumor vaccines rely on adjuvants to stimulate local inflammation, recruit antigen-presenting cells (APCs), and enhance immune activation. However, the complex interplay between antigen transport, lymphatic drainage, and immune cell dynamics across organs remains poorly understood, limiting the rational design of vaccination strategies. Here, we present a multiscale compartmental Physiologically Based Pharmacokinetic (PBPK) model of antigen vaccination that integrates systemic circulation, lymphatic connectivity, and immune cell activation at the whole-body level. The model incorporates arterial, venous, and lymphatic flows, organ-specific interstitium and lymph node (LN) networks, and a superficial skin network. The model reproduces spatiotemporal distributions of antigen and suppressive factors, APC activation, and nT priming across activation sites, including LNs and spleen. Our results show that the sensitivity of vaccination-induced immunity is highly related to antigen and suppressive factor production by the tumor, and that early-stage vaccination, enhances immunity. Since the model is able to identify optimal vaccination administration over the course of tumor growth for each patient with certain levels of antigen and immune suppressive factors, it can serve as the foundation for digital twins of patients to help inform anti-cancer vaccination strategies. TeaserA PBPK model links body-wide immune transport to optimize cancer vaccine design and delivery.

systems biology↗

bioRxiv2025

Optimizing T-cell-mediated cancer killing: An agent-based model

Immunotherapies for cancer aim to improve immune cell function by helping immune cells recognize and kill tumors. A group of immunotherapies target T-cells, aiming to improve the patients prognosis. However, these therapies are not always successful, and the underlying mechanisms of T-cell elimination of tumors remain poorly understood. We developed a three-dimensional agent-based model to explore this behavior that probabilistically models cell growth, migration and interaction parameters. Through this project, we explore the influence of cytokines like IL-2 and CCL2 on T-cell behavior and optimize the interactions between T-cells and cancer cells to most efficiently kill tumors using modeling. How individual parameters such as T-cell killing rate, proliferation, infiltration and cytotoxicity is still largely unexplored, and our study aims to fill that gap by stochastically simulating T-cells as they attack a cancer spheroid in order to identify new potential strategies to enhance T-cell killing of tumors. Our study found that, from greatest to least effect, increasing the killing, proliferation, and infiltration rate of T-cells increased the clearance rate of the tumor. A 17% increase in killing rate was enough to control and shrink the tumor, while a 15% increase in their proliferation rate was sufficient to control the tumor without regressing it; a 30% increase in their proliferation rate was sufficient to shrink the tumor. Increasing the infiltration was only able to control the tumor, not cause it to regress.

cancer biology↗

bioRxiv2025

The anti-virus T cell response dominates the anti-cancer response in oncolytic virus therapy

Oncolytic viruses have strong potential as immunotherapies. By causing cancer cells to die and relieve antigens, these viruses can stimulate robust, systemic immune responses that may eliminate disseminated disease and prevent recurrence. Unfortunately, clinical trials using oncolytic viruses have not induced clearance of metastasis or protection from recurrence. Likewise, the combination of the only FDA-approved oncolytic virus-- Talimogene laherparapvec--with immune checkpoint blockade did not improve progression-free or overall survival. Because of these disappointing clinical trials, we sought to measure the ability of oncolytic viruses to induce cancer antigen presentation and the elicitation of cancer antigen-specific immune responses. Our data revealed that despite improved antigen presentation by dendritic cells, priming of cancer antigen-specific T cells was limited. However, viral antigen-specific T cells did develop and were in a phenotypic state to induce an effective response against virally infected cells. These preclinical results were mirrored in human peripheral blood samples. Overall, these data show that oncolytic virus treatment induces a response against the virus itself, but not cancer antigen, explaining the lack of response in metastatic disease. These interesting findings identify a critical mechanism that needs to be overcome to increase the efficacy of oncolytic virus therapy.

immunology↗

bioRxiv2025

Overcoming impaired antigen presentation in tumor draining lymph nodes facilitates immunotherapy

Immunotherapies have revolutionized cancer care in recent decades, but approved therapies often fail and currently only target specific steps in the generation of anti-cancer immune responses. Notably, the majority of approved immunotherapies do not target antigen processing and presentation, which are key steps in the development of immune responses and harbor potential as targets to improve immunotherapy. Here, we demonstrate that breast tumors induce locoregional lymph node impairment in antigen presentation--but not in antigen processing--which limits anti-cancer antigen- specific T cell responses. Inhibition of the locoregional T cell response was due to a tumor-mediated reduction of the cytokine IL1{beta} in tumor draining lymph nodes, which impaired antigen presentation. Further, we tested the ability of dendritic cells in lymph nodes at various distances from the primary tumor to be activated utilizing an antigen- agnostic adjuvant delivery strategy. We observed improved anti-tumor T cell responses when the adjuvant was delivered to cancer antigen-positive lymph nodes distant from the tumor, suggesting these lymph nodes can be targeted to improve anti-cancer immune responses. When combined with immune checkpoint blockade, delivery of the adjuvant to distant lymph nodes led to long-term survival and protection from recurrence. Antigen presentation and T cell responses could also be recovered by exogenous delivery of IL1{beta} via intratumoral injection, with improved survival when combined with immune checkpoint blockade. Our study demonstrates that tumor- induced locoregional impairment of antigen presentation can be overcome by the appropriate introduction of immunological adjuvant to tumor-distant lymph nodes or by restoring IL1{beta} to the tumor-draining lymph node. These strategies can induce high- quality, durable immune responses and have clinical implications for expanding the efficacy of immunotherapies. SummaryBreast tumors induce locoregional lymph node impairments in antigen presentation, which can be remedied via either IL1{beta} delivery or antigen-agnostic adjuvant therapies to distant lymph nodes, facilitating better immunotherapy responses.

cancer biology↗

bioRxiv2025

Physiologically-based pharmacokinetic model for CAR-T cells delivery and efficacy in solid tumors

Abnormal blood vessels limit the delivery and function of endogenous T cells as well as adoptively transferred Chimeric Antigen Receptor (CAR)-T cells in the tumor microenvironment (TME). We recently showed that vascular normalization using anti-VEGF therapy can overcome these challenges and improve the outcome of CAR-T therapy in glioblastoma models in mice. Here, we developed a physiologically based pharmacokinetic model to simulate the dynamics of both adoptively transferred CAR-T cells and endogenous immune cells in solid tumors following vascular normalization. Similar to our data, our model simulations show that vascular normalization reprograms the TME from immunosuppressive to immunosupportive--enhancing infiltration of endogenous CD8 T cells and CAR-T cells, increasing M1 macrophages, and reducing M2 macrophages and regulatory T cells--thereby improving efficacy. Strikingly, vascular normalization reduces the number of infused CAR-T cells needed for tumor control by an order of magnitude. Moreover, synchronizing a second CAR-T infusion at their peak proliferative phase maximizes antitumor function. Furthermore, the efficacy of CAR-T cells engineered to secrete anti-VEGF antibody depends on the ability of CAR-T cells to induce vascular normalization. Additionally, combining vascular and stromal normalization can improve the efficacy of anti-VEGF antibody-producing FAP-CAR-T cells for the treatment of desmoplastic tumors such as pancreatic ductal adenocarcinoma. Finally, the model predicts that local CAR-T delivery can sustain high concentrations within the TME and induce recruitment of other antitumor immune cells, improving outcomes. Our model provides a versatile framework to optimize dosing strategies, treatment sequencing, and delivery routes for improving CAR-T therapies for solid tumors. Significance Statement Preclinical studies and early clinical trials of CAR-T therapy show encouraging responses in glioblastoma, diffuse midline gliomas, and neuroblastoma, yet substantial obstacles remain for effective CAR-T therapy for solid tumors. Building on our discovery that judicious VEGF blockade normalizes tumor vessels and enhances CD8T-cell infiltration, we developed a mathematical model to optimize CAR-T therapy for solid tumors. Simulations predict that vascular normalization can render the TME immunosupportive and decrease CAR-T doses tenfold. In desmoplastic tumors, FAP-CAR-T efficacy is improved by combining anti-VEGF and stromal normalizing agents. Optimal scheduling and direct intratumoral delivery can mitigate T-cell exhaustion and improve tumor control further. Thus, our model serves as a strategic roadmap for optimal CAR-T deployment in solid tumors.

cancer biology↗

bioRxiv2025

Lymph nodes link sex-biased immune aging to compromised antigen recognition

The numerical abundance of diverse naive CD8 T cell clones is essential to provide broad protection against infection and cancer. Here, we uncover a sex-biased mechanism of immune aging in which an early, male-biased depletion of naive CD8 T cells driven by accelerated, antigen-agnostic differentiation into virtual memory cells is combined with age-related thymic involution that limits naive CD8 T cell replenishment. These mechanisms lead to contraction of lymph nodes and reduced local naive T cell clone availability, limiting cancer antigen recognition capacity. Therapeutic thymus regeneration via androgen ablation repopulates naive CD8 T cells in lymph nodes, which reinvigorates cancer-specific T cell responses and enhances responsiveness to immune checkpoint blockade. These findings reveal the crucial impact of sex and age on naive T cell clone abundance in lymph nodes and suggest strategies to restore immune competence in middle-aged males.

immunology↗

bioRxiv2024

A Multiresolution Approach with Method-Informed Statistical Analysis for Quantifying Lymphatic Pumping Dynamics

Despite significant strides in lymphatic system imaging, the timely diagnosis of lymphatic disorders remains elusive. One main cause for this is the absence of standardized, quantitative methods for real-time analysis of lymphatic contractility. Here, we address this unmet need by combining near-infrared lymphangiography imaging with an innovative analytical workflow. We combined data acquisition, signal processing, and statistical analysis to integrate traditional peak and-valley with advanced wavelet time-frequency analyses. Decision theory was used to evaluate the primary drivers of attributable variance in lymphangiography measurements to generate a strategy for optimizing the number of repeat measurements needed per subject to increase measurement reliability. This approach not only offers detailed insights into lymphatic pumping behaviors across species, sex and age, but also significantly boosts the reliability of these measurements by incorporating multiple regions of interest and evaluating the lymphatic system under various gravitational loads. By addressing the critical need for improved imaging and quantification methods, our study offers a new standard approach for the imaging and analysis of lymphatic function that can improve our understanding, diagnosis, and treatment of lymphatic diseases. The results highlight the importance of comprehensive data acquisition strategies to fully capture the dynamic behavior of the lymphatic system.

physiology↗

bioRxiv2023

Lymphatic muscle cells are unique cells that undergo aging induced changes

Lymphatic muscle cells (LMCs) within the wall of collecting lymphatic vessels exhibit tonic and autonomous phasic contractions, which drive active lymph transport to maintain tissue-fluid homeostasis and support immune surveillance. Damage to LMCs disrupts lymphatic function and is related to various diseases. Despite their importance, knowledge of the transcriptional signatures in LMCs and how they relate to lymphatic function in normal and disease contexts is largely missing. We have generated a comprehensive transcriptional single-cell atlas--including LMCs--of collecting lymphatic vessels in mouse dermis at various ages. We identified genes that distinguish LMCs from other types of muscle cells, characterized the phenotypical and transcriptomic changes in LMCs in aged vessels, and uncovered a pro-inflammatory microenvironment that suppresses the contractile apparatus in advanced-aged LMCs. Our findings provide a valuable resource to accelerate future research for the identification of potential drug targets on LMCs to preserve lymphatic vessel function as well as supporting studies to identify genetic causes of primary lymphedema currently with unknown molecular explanation.

cell biology↗

bioRxiv2023

Cancer immunotherapy responses persist after lymph node resection

Lymphatic transport facilitates the presentation of cancer antigens in tumor-draining lymph nodes (tdLNs), leading to T cell activation and the generation of systemic anti-cancer immune surveillance. Surgical removal of tdLNs to control cancer progression is routine in clinical practice. However, whether removing tdLNs impairs immune checkpoint blockade (ICB) is still controversial. Our analysis demonstrates that melanoma patients remain responsive to PD-1 checkpoint blockade after regional LN dissection. We were able to recapitulate the persistent response to ICB after regional LN resection in murine melanoma and mammary carcinoma models. Mechanistically, soluble antigen is diverted to distant LNs after tdLN dissection. Consistently, robust ICB responses in patients with head and neck cancer after primary tumor and tdLN resection correlated with the presence of reactive LNs in distant sites. These findings indicate that distant LNs sufficiently compensate for the removal of direct tdLNs and sustain the response to ICB.

cancer biology↗

bioRxiv2023

Reprogramming Intrahepatic Cholangiocarcinoma Immune Microenvironment by Chemotherapy and CTLA-4 Blockade Enhances Anti-PD1 Therapy

Intrahepatic cholangiocarcinoma (ICC) has limited therapeutic options and a dismal prognosis. Anti-PD-L1 immunotherapy combined with gemcitabine/cisplatin chemotherapy has recently shown efficacy in biliary tract cancers, but responses are seen only in a minority of patients. Here, we studied the roles of anti-PD1 and anti-CTLA-4 immune checkpoint blockade (ICB) therapies when combined with gemcitabine/cisplatin and the mechanisms of treatment benefit in orthotopic murine ICC models. We evaluated the effects of the combined treatments on ICC vasculature and immune microenvironment using flow cytometry analysis, immunofluorescence, imaging mass cytometry, RNA-sequencing, qPCR, and in vivo T-cell depletion and CD8+ T-cell transfer using orthotopic ICC models and transgenic mice. Combining gemcitabine/cisplatin with anti-PD1 and anti-CTLA-4 antibodies led to substantial survival benefits and reduction of morbidity in two aggressive ICC models, which were ICB-resistant. Gemcitabine/cisplatin treatment increased the frequency of tumor-infiltrating lymphocytes and normalized the ICC vessels, and when combined with dual CTLA-4/PD1 blockade, increased the number of activated CD8+Cxcr3+IFN-{gamma}+ T-cells. Depletion of CD8+ but not CD4+ T-cells compromised efficacy. Conversely, CD8+ T-cell transfer from Cxcr3-/- versus Cxcr3+/+ mice into Rag1-/- immunodeficient mice restored the anti-tumor effect of gemcitabine/cisplatin/ICB combination therapy. Finally, rational scheduling of the ICBs (anti-CTLA-4 "priming") with chemotherapy and anti-PD1 therapy achieved equivalent efficacy with continuous dosing while reducing overall drug exposure. In summary, gemcitabine/cisplatin chemotherapy normalizes vessel structure, increases activated T-cell infiltration, and enhances anti-PD1/CTLA-4 immunotherapy efficacy in aggressive murine ICC. This combination approach should be clinically tested to overcome resistance to current therapies in ICC patients. One Sentence SummaryImmune microenvironment reprogramming by chemotherapy and priming using CTLA-4 blockade render ICCs responsive to anti-PD-1 immunotherapy.

cancer biology↗