Biology subjects

Lee, P. P.

Publications and source records attributed to Lee, P. P..

18 recordsLinked to original sources

bioRxiv2025

Modelling Immune Dynamics in Locally Advanced MSI-H/dMMR Colorectal Cancer with Neoadjuvant Pembrolizumab Treatment: From Differential Equations to an Agent-Based Framework

Colorectal cancer (CRC) is the third most common malignancy worldwide, and accounts for approximately 10% of all cancers and an estimated 850,000 deaths annually. Within CRC, MSI-H/dMMR tumours are highly immunogenic due to their high mutational burden and neoantigen load, yet can evade immunosurveillance via PD-1/PD-L1-mediated signalling. Pembrolizumab, an anti-PD-1 antibody approved for unresectable or metastatic MSI-H/dMMR CRC, is emerging as a promising neoadjuvant option in the locally advanced setting, inducing rapid, deep and durable immune responses. In this work, we construct a minimal model of neoadjuvant pembrolizumab therapy in locally advanced MSI-H/dMMR CRC (laMCRC) using ordinary differential equations (ODEs), providing a highly extensible model that captures the main immune dynamics involved. On the other hand, agent-based models (ABMs) naturally capture stochasticity, interactions at an individual level, and discrete events that lie beyond the scope of differential-equation formulations. As such, we also convert our ODE model, with parameters calibrated to experimental data, to an ABM, preserving its dynamics while providing a flexible platform for future mechanistic investigation and modelling.

cancer biology↗

bioRxiv2025

Optimisation of pembrolizumab therapy for de novo metastatic MSI-H/dMMR colorectal cancer using data-driven delay integro-differential equations

Colorectal cancer (CRC), the third most commonly diagnosed cancer worldwide, presents a growing public health concern, with 20% of new diagnoses involving de novo metastatic disease and up to 80% of these patients presenting with unresectable metastatic lesions. Microsatel-lite instability-high (MSI-H) CRC and deficient mismatch repair (dMMR) CRC constitute 15% of all CRC, and 4% of metastatic CRC, and, while less responsive to conventional chemotherapy, exhibit notable sensitivity to immunotherapy, especially programmed cell death protein 1 (PD-1) checkpoint inhibitors such as pembrolizumab. Despite this, there is a significant need to optimise immunotherapeutic regimens to maximise clinical efficacy and patient quality of life whilst minimising financial burden. In this work, we adapt our mechanistic model for locally advanced MSI-H/dMMR CRC to de novo metastatic MSI-H/dMMR CRC (dnmMCRC), deriving model parameters from pharmacokinetic, bioanalytical, and radiographic studies, as well as bulk RNA-sequencing data deconvolution from the TCGA COADREAD and GSE26571 datasets. We finally optimised treatment with pembrolizumab to balance efficacy, efficiency, and toxicity in dnmMCRC, comparing against currently FDA-approved regimens, analysing factors influencing treatment success and comparing immune dynamics to those in locally advanced disease.

cancer biology↗

bioRxiv2025

Lymphocytes and monocytes undergo swift suppression of IL-10R, IL-6R, and IL-2Rβγ signaling under high concentrations of different cytokines

The JAK-STAT signaling pathway is fundamental for immune system regulation. It involves phosphorylation of several types of STAT proteins in response to binding of cytokines to immune cell receptors. Traditionally, the immune signaling studies focus on measuring the levels of phosphorylated STATs (pSTATs) following individual cytokine application. We developed an experimental approach, based on multiparametric flow cytometry, to simultaneously measure the levels of five pSTATs after 15 minutes of cell treatment with high doses of individual cytokines and their paired combinations. Analysis of our experimental data involving peripheral blood mononuclear cells from healthy donors reveals systematic suppression of IL-10R, IL-6R, and IL-2R{beta}{gamma} signaling in T cells, B cells, NK cells, and monocytes. This suppression is mediated by at least all tested cytokines that do not induce relevant pSTATs by themselves. Remarkably, the cytokines with negligible own signaling do act as prominent selective signaling suppressors. In contrast, the signaling of IFNAR, IFNGR, IL-4R, and IL-2R{beta}{gamma} remains largely unaffected by co-application of other cytokines. We propose that this pattern of signaling suppression represents an evolutionary developed mechanism enhancing the promptness, specialization, and efficiency of the immune response, while increased concentration of cytokines serves as a danger signal of inefficient response. We hypothesize that selective signaling suppression arises from the differential sensitivity of conformations of cytokine-receptor complexes to the increase of cell surface tension and stiffness, which is caused by effects following the binding of cytokines to membrane-associated molecules, including glycocalyx elements. While the rewiring of immune cell signaling should represent a powerful evolutionary tool for augmentation of adaptive response, it should also lead to the prolonged suppression of counteracting signaling pathways, culminating in cytokine release syndrome and contributing to autoimmune diseases.

immunology↗

bioRxiv2025

Ligand Discrimination in Immune Cells: Signal Processing Insights into Immune Dysfunction in ER+ Breast Cancer

Prior studies have shown that approximately 40% of estrogen receptor positive (ER+) breast cancer (BC) patients harbor immune signaling defects in their blood at diagnosis, and the presence of these defects predicts overall survival. Therefore, it is of interest to quantitatively characterize and measure signaling errors in immune signaling systems in these patients. Here we propose a novel approach combining communication theory and signal processing concepts to model ligand discrimination in immune cells in the peripheral blood. We use the model to measure the specificity of ligand discrimination in the presence of molecular noise by estimating the probability of error, which is the probability of making a wrong ligand identification. We apply our model to the JAK/STAT signaling pathway using high dimensional spectral flow cytometry measurements of transcription factors, including phosphorylated STATs and SMADs, in immune cells stimulated with several cytokines (IFN{gamma}, IL-2, IL-6, IL-4, and IL-10) from 19 ER+ breast cancer patients and 32 healthy controls. In addition, we apply our model to 10 healthy donor samples treated with a clinically approved JAK1/2 inhibitor. Our results show reduced ligand identification accuracy and higher levels of molecular noise in BC patients as compared to healthy controls, which may indicate altered immune signaling and the potential for immune cell dysfunction in these patients. Moreover, the inhibition of JAK1/2 produces ligand misidentification and molecular noise rates similar to, or even greater than, those observed in breast cancer. These results suggest a means to improve the use of signaling kinase inhibitor therapies by identifying patients with favorable ligand discrimination specificity profiles in their immune cells. One Sentence SummaryWe use a communication model to measure ligand discrimination errors in immune cells and molecular noise in cytokine signaling in ER+ breast cancer patients as compared to healthy controls.

cancer biology↗

bioRxiv2023

Systems profiling reveals recurrently dysregulated cytokine signaling responses in ER+ breast cancer patients' blood

Cytokines mediate cell-to-cell communication across the immune system and therefore are critical to immunosurveillance in cancer and other diseases. Several cytokines show dysregulated abundance or signaling responses in breast cancer, associated with the disease and differences in survival and progression. Cytokines operate in a coordinated manner to affect immune surveillance and regulate one another, necessitating a systems approach for a complete picture of this dysregulation. Here, we profiled cytokine signaling responses of peripheral immune cells from breast cancer patients as compared to healthy controls in a multidimensional manner across ligands, cell populations, and responsive pathways. We find alterations in cytokine responsiveness across pathways and cell types that are best defined by integrated signatures across dimensions. Alterations in the abundance of a cytokines cognate receptor do not explain differences in responsiveness. Rather, alterations in baseline signaling and receptor abundance suggesting immune cell reprogramming are associated with altered responses. These integrated features suggest a global reprogramming of immune cell communication in breast cancer. Significance StatementWhile individual cytokine responses have previously been observed to be altered in breast cancer, cytokine signaling responses are tightly interconnected in a way that has not been previously characterized. Here, we profile cytokine signaling responses and find alterations that are shared across both pathways and cell types. The signatures across these measurements better define the alterations and point to a broad immunosuppression response. HighlightsO_LIBaseline and post-stimulation cytokine signaling profiles differ between healthy donors and breast cancer patients. C_LIO_LIChanges in cytokine response are not explained by differences in abundance of the cognate receptor C_LIO_LIFeatures of signaling response and receptor abundance dysregulation are coordinated across patients C_LIO_LIIntegrated patterns of dysregulation in breast cancer patients share features of Th17 like-response as well as regulatory-like B and CD8+ T cells C_LI

cancer biology↗

bioRxiv2022

PD-L1 blockade restores CAR T cell activity through IFNγ-regulation of CD163+ macrophages

BackgroundThe immune suppressive tumor microenvironment (TME) that inhibits T cell infiltration, survival, and anti-tumor activity has posed a major challenge for developing effective immunotherapies for solid tumors. Chimeric antigen receptor (CAR)-engineered T cell therapy has shown unprecedented clinical response in treating patients with hematological malignancies, and intense investigation is underway to achieve similar responses with solid tumors. Immunologically cold tumors, including prostate cancers, are often infiltrated with abundant tumor-associated macrophages (TAMs), and infiltration of CD163+ M2 macrophages correlates with tumor progression and poor responses to immunotherapy. However, the impact of TAMs on CAR T cell activity alone and in combination with TME immunomodulators is unclear. MethodsTo model this in vitro, we utilized a novel co-culture system with tumor cells, CAR T cells, and polarized M1 or M2 macrophages from CD14+ PBMCs collected from healthy human donors. Tumor cell killing, T cell activation and proliferation, and macrophage phenotypes were evaluated by flow cytometry, cytokine production, RNA sequencing, and functional blockade of signaling pathways using antibodies and small molecule inhibitors. We also evaluated the TME in humanized mice following CAR T cell therapy for validation of our in vitro findings. ResultsWe observed inhibition of CAR T cell activity with the presence of M2 macrophages, but not M1 macrophages, coinciding with a robust induction of PD-L1 in M2 macrophages. We observed similar PD-L1 expression in TAMs following CAR T cell therapy in the TME of humanized mice. PD-L1, but not PD-1, blockade in combination with CAR T cell therapy altered phenotypes to more M1-like subsets and led to loss of CD163+ M2 macrophages via IFN{gamma} signaling, resulting in improved anti-tumor activity of CAR T cells. ConclusionThis study reveals an alternative mechanism by which the combination of CAR T cells and immune checkpoint blockade modulates the immune landscape of solid tumors to enhance therapeutic efficacy of CAR T cells.

immunology↗

bioRxiv2021

Control-theoretic immune tradeoffs explain SARS-CoV-2 virulence and transmission variation

Dramatic variation in SARS-CoV-2 virulence and transmission between hosts has driven the COVID-19 pandemic. The complexity and dynamics of the immune response present a challenge to understanding variation in SARS-CoV-2 infections. To address this challenge, we apply control theory, a framework used to study complex feedback systems, to establish rigorous mathematical bounds on immune responses. Two mechanisms of SARS-CoV-2 biology are sufficient to create extreme variation between hosts: (1) a sparsely expressed host receptor and (2) potent, but not unique, suppression of interferon. The resulting model unifies disparate and unexplained features of the SARS-CoV-2 pandemic, predicts features of future viruses that threaten to cause pandemics, and identifies potential interventions.

systems biology↗

bioRxiv2020

A minimal model of T cell avidity may identify subtherapeutic vaccine schedules

T cells protect the body from cancer by recognising tumour-associated antigens. Recognising these antigens depends on multiple factors, one of which is T cell avidity, i.e., the total interaction strength between a T cell and a cancer cell. While both high- and low-avidity T cells can kill cancer cells, durable anti-cancer immune responses require the selection of high-avidity T cells. Previous experimentation with anti-cancer vaccines, however, has shown that most vaccines elicit low-avidity T cells. Optimising vaccine schedules may remedy this by preferentially selecting high-avidity T cells. Here, we use mathematical modelling to develop a simple, phenomenological model of avidity selection that may identify vaccine schedules that disproportionately favour low-avidity T cells. We calibrate our model to our prior, more complex model, and then validate it against several experimental data sets. We find that the sensitivity of the models parameters change with vaccine dosage, which allows us to use a patients data and clinical history to screen for suitable vaccine strategies.

immunology↗

bioRxiv2020

Ivermectin converts cold tumors hot and synergies with immune checkpoint blockade for treatment of breast cancer

We show that treatment with the FDA-approved anti-parasitic drug ivermectin induces immunogenic cancer cell death (ICD) and robust T cell infiltration into breast tumors. As an allosteric modulator of the ATP/P2x4/P2x7 axis which operates in both cancer and immune cells, ivermectin also selectively targets immunosuppressive populations including myeloid cells and Tregs, resulting in enhanced Teff/Tregs ratio. While neither agent alone showed efficacy in vivo, combination therapy with ivermectin and checkpoint inhibitor anti-PD1 antibody achieved synergy in limiting tumor growth (p=0.03) and promoted complete responses (p<0.01), also leading to immunity against contralateral re-challenge with demonstrated anti-tumor immune responses. Going beyond primary tumors, this combination achieved significant reduction in relapse after neoadjuvant (p=0.03) and adjuvant treatment (p<0.001), and potential cures in metastatic disease (p<0.001). Statistical modeling confirmed bona fide synergistic activity in both the adjuvant (p=0.007) and metastatic settings (p<0.001). Ivermectin has dual immunomodulatory and ICD-inducing effects in breast cancer, converting cold tumors hot, thus represents a rational mechanistic partner with checkpoint blockade.

immunology↗

bioRxiv2020

NLRP3 inflammasome contributes to host defense against Talaromyces marneffei infection

Talaromyces marneffei is an important thermally dimorphic pathogen causing disseminated mycoses in immunocompromised individuals in southeast Asia. Previous study has suggested that NLRP3 inflammasome plays a critical role in antifungal immunity. However, the mechanism underlying the role of NLRP3 inflammasome activation in host defense against T. marneffei remains unclear. We show that T. marneffei yeasts but not conidia induce potent IL-1{beta} response, which is differentially regulated in discrete immune cell types. Dectin-1/Syk signaling pathway mediates pro-IL-1{beta} production, and NLRP3 inflammasome is activated to trigger the processing of pro-IL-1{beta} into IL-1{beta}. The activated NLRP3 inflammasome partially promotes Th1 and Th17 immune responses against T. marneffei yeasts. In vivo, mice with NLRP3 or caspase-1 deficiency exhibit higher mortality rate and fungal load compared to wild-type mice. Herein, our study provides the first evidence that NLRP3 inflammasome contributes to host defense against T. marneffei infection, which may have implications for future antifungal therapeutic designs.

microbiology↗

bioRxiv2020

Immature dendritic cells promote high avidity tuning of vaccine T cell response

Therapeutic vaccines can elicit tumor-specific cytotoxic T lymphocytes (CTLs), but durable reductions in tumor burden require vaccines that stimulate high-avidity CTLs. Recent advances in immunotherapy responses have led to renewed interest in vaccine approaches, including dendritic cell vaccine strategies. However, dendritic cell requirements for vaccines that generate potent anti-tumor T-cell responses are unclear. Here we use mathematical modeling to show that counterintuitively, increasing levels of immature dendritic cells may lead to selective expansion of high-avidity CTLs. This finding contrasts with traditional dendritic cell vaccine approaches that have sought to harness ex vivo generated mature dendritic cells. We show that the injection of vaccine antigens in the context of increased numbers of immature dendritic cells results in a decreased overall peptide:MHC complex load that favors high-avidity CTL activation and expansion. Overall, our results provide a firm basis for further development of this approach, both alone and in combination with other immunotherapies such as checkpoint blockade.

immunology↗

bioRxiv2020

Dissecting Response to Cancer Immunotherapy by Applying Bayesian Network Analysis to Flow Cytometry Data

Cancer immunotherapy, specifically immune checkpoint blockade therapy, has been found to be effective in the treatment of metastatic cancers. However, only a subset of patients achieve clinical responses. Consequently, elucidating immune system-related pre-treatment biomarkers that are predictive with respect to sustained clinical response is a major research priority. Another research priority is evaluating changes in the immune system before and after treatment in responders and non-responders. Our group has been studying immune signaling networks as an accurate reflection of the global immune state. Flow cytometry (FACS, Fluorescence-activated cell sorting) data characterizing immune signaling in peripheral blood mononuclear cells (PBMC) from gastroesophageal adenocarcinoma (GEA) patients were used to analyze changes in immune signaling networks in this setting. Here, we describe a novel computational pipeline developed by us to perform secondary analyses of FACS data using systems biology / machine learning techniques and concepts. It is centered around comparative Bayesian network analyses of immune signaling networks and is capable of detecting true positive signals that conventional methods (such as FlowJo manual gating) might miss. Future studies are planned to validate and follow up immune markers (and combinations / interactions thereof) associated with clinical responses that were identified by this computational pipeline.

systems biology↗

bioRxiv2020

Arhgap25 deficiency leads to decreased numbers of peripheral blood B cells and defective germinal center reactions

Rho family GTPases are critical for normal B cell development and function and their activity is regulated by a large and complex network of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, the role of GAPs in B cell development is poorly understood. Here we show that the novel Rac-GAP ARHGAP25 is important for B cell development in mice in a CXCR4-dependent manner. We show that Arhgap25 deficiency leads to a significant decrease in peripheral blood B cell numbers, as well as defects in mature B cell differentiation. Arhgap25-/- B cells respond to antigen stimulation in vitro and in vivo but have impaired germinal center formation and decreased IgG1 class switching. Additionally, Arhgap25-/- B cells exhibit increased chemotaxis to CXCL12. Taken together, these studies demonstrate an important role for Arhgap25 in peripheral B cell development and antigen response.

immunology↗

bioRxiv2019

Selective Ablation of Cancer Cells with Low Intensity Pulsed Ultrasound

Ultrasound can be focused into deep tissues with millimeter precision to perform non-invasive ablative therapy for diseases such as cancer. In most cases, this ablation uses high intensity ultrasound to deposit non-selective thermal or mechanical energy at the ultrasound focus, damaging both healthy bystander tissue and cancer cells. Here we describe an alternative low intensity pulsed ultrasound approach that leverages the distinct mechanical properties of neoplastic cells to achieve inherent cancer selectivity. We show that when applied at a specific frequency and pulse duration, focused ultrasound selectively disrupts a panel of breast, colon, and leukemia cancer cell models in suspension without significantly damaging healthy immune or red blood cells. Mechanistic experiments reveal that the formation of acoustic standing waves and the emergence of cell-seeded cavitation lead to cytoskeletal disruption, expression of apoptotic markers, and cell death. The inherent selectivity of this low intensity pulsed ultrasound approach offers a potentially safer and thus more broadly applicable alternative to non-selective high intensity ultrasound ablation.

bioengineering↗

bioRxiv2019

Phenotypic Switching of Naïve T cells to Immune-suppressive Treg-like Cells by Mutant KRAS

Oncogenic (mutant) Ras protein Kirsten rat sarcoma viral oncogene homolog (KRAS) promotes uncontrolled proliferation, altered metabolism, and loss of genome integrity in a cell-intrinsic manner. Here, we demonstrate that CD4+ T cells when incubated with tumor-derived exosomes from mutant (MT) KRAS non-small cell lung cancer (NSCLC) cells, patient sera, or a mouse xenograft model, induce phenotypic conversion to FOXP3+ Treg-like cells that are immune-suppressive. Furthermore, transfecting T cells with MT KRAS cDNA alone induced phenotypic switching and mathematical modeling supported this conclusion. Single-cell sequencing identified the interferon pathway as the mechanism underlying the phenotypic switch. These observations highlight a novel cytokine-independent, cell-extrinsic role for KRAS in T cell phenotypic switching. Thus, targeting this new class of Tregs represents a unique therapeutic approach for NSCLC. Since KRAS is the most frequently mutated oncogene in a wide variety of cancers, the findings of this investigation are likely to be of broad interest and have a large scientific impact.

cancer biology↗

bioRxiv2019

Fractal dimension, occupancy and hotspot analyses of B cell spatial distribution predict clinical outcome in breast cancer

While the density of tumor-infiltrating lymphocytes (TILs) is now well known to correlate with clinical outcome, the clinical significance of spatial distribution of TILs is not well characterized. We have developed novel statistical techniques (including fractal dimension differences, a hotspot analysis, a box counting method that we call occupancy and a way to normalize cell density that we call thinning) to analyze the spatial distribution (at different length scales) of various types of TILs in triple negative breast tumors. Consistent with prior reports, the density of CD20+ B cells within tumors is not correlated with clinical outcome. However, we found that their spatial distribution differs significantly between good clinical outcome (no recurrence within at least 5 years of diagnosis) and poor clinical outcome (recurrence with 3 years of diagnosis). Furthermore, CD20+ B cells are more spatially dispersed in good outcome tumors and are more likely to infiltrate into cancer cell islands. Lastly, we found significant correlation between the spatial distributions of CD20+ B cells and CD8+ (cytotoxic) T cells (as well as CD3+ T cells), regardless of outcome. These results highlight the significance of the spatial distribution of TILs, especially B cells, within tumors.\n\nSignificance StatementImmune cells can fight cancer. For example, a patient has a good prognosis when a high density of killer T cells, a type of immune cell that can kill cancer cells, infiltrates into a tumor. However, there is no clear association between prognosis and the density of B cells, another type of immune cell, in a tumor. We developed several statistical techniques to go beyond cell density and look at the spatial distribution, i.e., the pattern or arrangement of immune cells, in tumors that have been removed from patients with triple negative breast cancer. We find that B cells and killer T cells tend to be more spread out in the tumors of patients whose cancer did not recur.

cancer biology↗

bioRxiv2019

Cell non-autonomous interactions during non-immune stromal progression in the breast tumor microenvironment

SummaryThe breast tumor microenvironment of primary and metastatic sites is a complex milieu of differing cell populations, consisting of tumor cells and the surrounding stroma. Despite recent progress in delineating the immune component of the stroma, the genomic expression landscape of the non-immune stroma (NIS) population and their role in mediating cancer progression and informing effective therapies are not well understood. Here we obtained 52 cell-sorted NIS and epithelial tissue samples across 37 patients from i) normal breast, ii) normal breast adjacent to primary tumor, iii) primary tumor, and iv) metastatic tumor sites. Deep RNA-seq revealed diverging gene expression profiles as the NIS evolves from normal to metastatic tumor tissue, with intra-patient normal-primary variation comparable to inter-patient variation. Significant expression changes between normal and adjacent normal tissue support the notion of a cancer field effect, but extended out to the NIS. Most differentially expressed protein-coding genes and lncRNAs were found to be associated with pattern formation, embryogenesis, and the epithelial-mesenchymal transition. We validated the protein expression changes of a novel candidate gene, C2orf88, by immunohistochemistry staining of representative tissues. Significant mutual information between epithelial ligand and NIS receptor gene expression, across primary and metastatic tissue, suggests a unidirectional model of molecular signaling between the two tissues. Furthermore, survival analyses of 827 luminal breast tumor samples demonstrated the predictive power of the NIS gene expression to inform clinical outcomes. Together, these results highlight the evolution of NIS gene expression in breast tumors and suggest novel therapeutic strategies targeting the microenvironment.

cancer biology↗

bioRxiv2017

On the mechanism of long-range alignment order of fibroblasts

Long-range alignment ordering of fibroblasts have been observed in the vicinity of cancerous tumors and can be recapitulated with in vitro experiments. However, the mechanisms driving their ordering are not understood. Here we show that local collision-driven nematic alignment interactions among fibroblasts are insufficient to explain observed long-range alignment. One possibility is that there exists another orientation field co-evolving with the cells and reinforcing their alignment. We propose that this field reflects the mechanical cross-talk between the fibroblasts and the underlying fibrous material on which they move. We demonstrate that this new long-range interaction can give rise to high nematic order and to the observed patterning of the cancer microenvironment.\n\nSignificance StatementLong-range alignment patterns of fibroblasts have been observed both in vivo and in vitro. However, there has not been much understanding of the underlying mechanism. In this work, we demonstrate that these patterns cannot be simply explained by their steric interaction with one another during collisions. Instead, we propose that fibroblasts may collectively align through non-local interactions arising from their modification of an underlying extracellular matrix. The proposed mechanism explains the observed co-alignment between fibroblasts and collagen fibers around tumors and can be be tested in future experiments that can image the dynamics of this pattern formation in vivo or in vitro

biophysics↗