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3D Flipwell Engineering for Developing Asynchronous Systems for Toxicologic and Immunomodulatory Therapies in Bacterial, Gut, and Immune Cells

To study the roles of bacterial secretome in immune cell polarization and gut mucosal homeostasis, we developed a 3-dimensional co-culture model using ...

Case Western Reserve University School of Medicine, Case Comprehensive Cancer Center

Gut mucosa consists of stratified layers of microbes, semi-permeable mucus, epithelium and stroma abundant in immune cells. Although tightly regulated, interactions between gut commensals and immune cells play indispensable roles in homeostasis and cancer pathogenesis in the body. Thus, there is a critical need to develop a robust model for the gut mucosal microenvironment. Here, we report our novel co-culture utilizing 3D Flipwell system for establishing the stratified layers of discrete mucosal components. This method allows for analyzing synchronous effects of test stimuli on gut bacteria, mucus, epithelium and immune cells, as well as their crosstalks. In the present report, we tested the immuno-stimulatory effects of sepiapterin (SEP, the precursor of the cofactor of nitric oxide synthase (NOS)-BH) on the gut mucosal community. We previously reported that SEP effectively reprogrammed tumor-associated macrophages and inhibited breast tumor cell growth. In our co-cultures, SEP largely promoted mucus integrity, bacterial binding, and M1-like polarization of macrophages. Conversely, these phenomena were absent in control-treated cultures. Our results demonstrate that this novel co-culture may serve as a robust in vitro system to recapitulate the effects of pharmacological agents on the gut mucosal microenvironment, and could potentially be expanded to test the effects outside the gut.

Novel 3D Flipwell system that models gut mucosal microenvironment for studying interactions between gut microbiota, epithelia and immunity.

HER2+ breast tumors have abundant immune-suppressive cells, including M2-type tumor-associated macrophages (TAMs). Although TAMs consist of the immune-stimulatory M1 type and immune-suppressive M2 type, the M1/M2-TAM ratio is reduced in immune-suppressive tumors, contributing to their immunotherapy refractoriness. M1- versus M2-TAM formation depends on differential arginine metabolism, where M1-TAMs convert arginine to nitric oxide (NO) and M2-TAMs convert arginine to polyamines (PAs). We hypothesize that such distinct arginine metabolism in M1- versus M2-TAMs is attributed to different availability of BH (NO synthase cofactor) and that its replenishment would reprogram M2-TAMs to M1-TAMs. Recently, we reported that sepiapterin (SEP), the endogenous BH precursor, elevates the expression of M1-TAM markers within HER2+ tumors. Here, we show that SEP restores BH levels in M2-like macrophages, which then redirects arginine metabolism to NO synthesis and converts M2 type to M1 type. The reprogrammed macrophages exhibit full-fledged capabilities of antigen presentation and induction of effector T cells to trigger immunogenic cell death of HER2+ cancer cells. This study substantiates the utility of SEP in the metabolic shift of the HER2+ breast tumor microenvironment as a novel immunotherapeutic strategy.

Reprogramming of breast tumor-associated macrophages with modulation of arginine metabolism.

Transforming Growth Factor β (TGFβ) is a pleiotropic cytokine closely linked to tumors. Previously, we pharmacologically inhibited basal nitric oxide (NO) production in healthy mammary glands and found that this induced precancerous progression accompanied by upregulation of TGFβ and desmoplasia. In the present study, we tested whether NO directly S-nitrosylates (forms an NO-adduct at a cysteine residue) TGFβ for inhibition, whereas reduction of NO denitrosylates TGFβ for de-repression. We introduced mutations to 3 C-terminal cysteines of TGFβ1 which were predicted to be S-nitrosylated. We found that these mutations indeed impaired S-nitrosylation of TGFβ1 and shifted the binding affinity towards the receptor from the latent complex. Furthermore, in silico structural analyses predicted that these S-nitrosylation-defective mutations strengthen the dimerization of mature protein, whereas S-nitrosylation-mimetic mutations weaken the dimerization. Such differences in dimerization dynamics of TGFβ1 by denitrosylation/S-nitrosylation likely account for the shift of the binding affinities toward the receptor versus latent complex. Our findings, for the first time, unravel a novel mode of TGFβ regulation based on S-nitrosylation or denitrosylation of the protein.

Reduced S-nitrosylation of TGFβ1 elevates its binding affinity toward the receptor and promotes fibrogenic signaling in the breast.

Arginine metabolism in tumors is often shunted into the pathway producing pro-tumor and immune suppressive polyamines (PAs), while downmodulating the alternative nitric oxide (NO) synthesis pathway. Aiming to correct arginine metabolism in tumors, arginine deprivation therapy and inhibitors of PA synthesis have been developed. Despite some therapeutic advantages, these approaches have often yielded severe side effects, making it necessary to explore an alternative strategy. We previously reported that supplementing sepiapterin (SEP), the endogenous precursor of tetrahydrobiopterin (BH the essential NO synthase cofactor), could correct arginine metabolism in tumor cells and tumor-associated macrophages (TAMs) and induce their metabolic and phenotypic reprogramming. We saw that oral SEP treatment effectively suppressed the growth of HER2-positive mammary tumors in animals. SEP also has no reported dose-dependent toxicity in clinical trials for metabolic disorders. In the present study, we tested our hypothesis that a long-term administration of SEP to individuals susceptible to HER2-positive mammary tumor would protect them against tumor occurrence.

Article	Year
Novel 3D Flipwell system that models gut mucosal microenvironment for studying interactions between gut microbiota, epithelia and immunity. Scientific reports\| PubMed ID: 36650266	2023
Reprogramming of breast tumor-associated macrophages with modulation of arginine metabolism. Life science alliance\| PubMed ID: 39191486	2024
Reduced S-nitrosylation of TGFβ1 elevates its binding affinity toward the receptor and promotes fibrogenic signaling in the breast. The Journal of biological chemistry\| PubMed ID: 39571651	2024
Immunogenic shift of arginine metabolism triggers systemic metabolic and immunological reprogramming to suppress HER2 + breast cancer. Cancer & metabolism\| PubMed ID: 40114277	2025

Saori Furuta

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