Human Explants Research Articles

Abstract 6732: Immunomodulatory and anti-tumor effect of a CCR2-targeted STING agonist iADC in human lung cancer

Abstract Introduction: Antibody drug conjugates (ADCs) targeting cancer-cell antigens can improve therapeutic index and show prominent clinical impact. Limited studies have used ADCs to target antigens present in non-malignant cells in the tumor microenvironment or to deliver immunomodulatory agents. We developed an immune-cell directed antibody drug conjugate (iADC) TAK-500 that selectively activates STING in C-C chemokine receptor type 2 (CCR2) expressing cells to achieve enhanced efficacy over non-targeted STING agonists. Here, we spatially mapped CCR2 protein in human NSCLC and studied the immunomodulatory role and anti-tumor effect of the novel iADC TAK-500 on human lung cancer tumor explants ex vivo. Methods: Using multiplexed quantitative immunofluorescence (mQIF) panels we measured the levels of CCR2 protein, myeloid cell populations and tumor infiltrating lymphocyte (TIL) subsets in 411 primary NSCLC carcinomas from treatment-naïve patients in 2 independent cohorts and determined their clinical significance. In addition, we longitudinally studied CCR2 levels, myeloid cells (DAPI/CCR2/CD68/CD11b), TILs (DAPI/CK/CD4/CD8/CD20), T-cell functional markers (DAPI/CK/CD3/Ki-67/GZMB [Granzyme B]) and cancer-cell death/proliferation markers (DAPI/CK/TFR1/Ki-67/CC3 [Cleaved Caspase 3]) in 314 primary cultured tissue fragments from paired tumor and non-tumor specimens resected from 11 lung cancer patients at Yale New Haven Hospital between 2021-2023 (8 adenocarcinomas, 2 small cell carcinomas, and 1 squamous cell carcinoma). Each biopsy was divided into 4-mm2 fragments, cultured in extracellular matrix for 5 days with or without TAK-500 treatment. Samples were processed into FFPE tissue and stained with mQIF panels using a tissue microarray format. Results: CCR2 protein was detected in 386 of 411 (94%) NSCLCs from 2 independent cohorts predominantly localized in CD11b+ intratumor myeloid cells and CD68+ tumor-associated macrophages (TAMs). Elevated CCR2 was associated with adenocarcinoma histology, increased PD-L1, activating KRAS mutations and longer survival. Treatment with TAK-500 for 5 days reduced CCR2 levels in myeloid cells and CD68+ TAMs, without significant changes in TIL or T-cell GZMB/Ki-67 levels. TAK-500 also significantly reduced tumor proliferation (Ki-67 expression) and increased tumor cell apoptosis (CC3 levels) after 5 days of treatment. Notably, these TAK-500-induced changes were observed only in tumor specimens with high baseline CCR2 levels above the median in retrospective NSCLC cohorts. No significant effect was observed in the paired non-tumor fragments. Conclusion: Our results reveal prominent myeloid CCR2 expression in a subset of human NSCLCs with distinct clinicopathologic/molecular characteristics. Using a novel lung cancer explant ex vivo system, we demonstrate a CCR2-dependent immunomodulatory and anti-tumor effect of TAK-500 iADC. Citation Format: Angelo Porciuncula, Vicky A. Appleman, Alex Parent, Jeff Raizer, Richard C. Gregory, Neil Lineberry, Adnan O. Abu-Yousif, Kurt A. Schalper. Immunomodulatory and anti-tumor effect of a CCR2-targeted STING agonist iADC in human lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6732.

Abstract 4071: The first-in-class PD1-IL18 conjugate BPT567 induces potent anti-tumor immunity by preferentially activating PD1+IL18R+ intratumoral effector T cells in cis

Abstract Antibody-cytokine conjugates leverage orthogonal mechanisms of action (MoA) in one molecule to induce potent antitumor immune responses. PD-1-targeting conjugates are of particular interest since they preferentially target antigen-experienced PD-1+ CD8+ T cells enriched in the tumor microenvironment (TME) while simultaneously blocking the PD-(L)1 pathway and inducing potent cytokine receptor stimulation in the same CD8+ T cell in cis (cis-signaling). Interleukin 18 (IL-18) is a proinflammatory cytokine able to integrate both innate and adaptive immunity resulting in profound anti-tumor immune responses mediated by T effector and NK cells. BPT567 has been generated via chemical conjugation of the anti-PD-1 Ab LipustobartTM to an enhanced human IL-18 variant of increased potency and reduced sensitivity to IL-18 binding protein (IL-18BP). BPT567 is designed to specifically target and activate a subset of tumor-infiltrating PD-1+ IL18R+ CD8+ T effector cells recently described to exhibit superior cytotoxic and proliferative activity (Codarri Deak et al., Nature 2022). Due to its ability to signal in cis, BPT567 shows enhanced potency as well as increased resistance to IL-18BP when analyzing IFNγ release in vitro in PD-1+ cells. Subsequent PD-1 receptor occupancy analyses showed that it is fully sufficient for BPT567 to engage a low number of PD-1 receptors to induce maximum IFNγ release in NK92 cells expressing human PD-1. Strikingly, despite addressing overlapping epitopes in PD-1, even a 200-fold excess of pembrolizumab has no impact on the in vitro potency of BPT567. In vivo, BPT567 exhibits strong anti-tumor efficacy at significantly lower IL-18 doses compared to the combination of an untargeted antibody-IL-18 conjugate and an anti-PD1 Ab further corroborating the importance of cis-signaling as a unique MoA of this PD1-IL18 conjugate. Detailed analyses of tumor-infiltrating immune leukocytes (TIL) revealed that BPT567 triggers a preferential expansion of CD8+ T effector memory cells within the TME. In line with this finding, activity in the MC38 tumor model is dependent on the presence of CD8+ T cells while depletion of other immune cell subsets (e.g. NK cells, CD4+ T cells, macrophages) had no impact on efficacy. Although not required for anti-tumor efficacy, CD4+ T cells are essential for proficient formation of an immunological memory. In an effort to confirm findings generated in murine models in relevant human ex vivo systems, we analyzed the release of cytokines and chemokines in dissociated cells isolated from primary human tumor explants. Also in this human model, BPT567 is able to induce significantly stronger IFNγ release compared to either single agents or the combination of untargeted IL-18 and an anti-PD1 Ab, thus confirming the unique MoA of the PD1-IL18 conjugate triggering superior TIL activation in cis. Citation Format: Kea Martin, Thuy Luu, Jean-Philippe Carralot, Lilian Gremlich, Petra Herzig, Caoimhe Herr, Roy Meoded, Philipp Moosmann, Arnaud Goepfert, Alfred Zippelius, Vijaya Pattabiraman, Bertolt Kreft. The first-in-class PD1-IL18 conjugate BPT567 induces potent anti-tumor immunity by preferentially activating PD1+IL18R+ intratumoral effector T cells in cis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4071.

Hepatitis E virus infects human testicular tissue and Sertoli cells

Globally, hepatitis E virus (HEV) infections are prevalent. The finding of high viral loads and persistent viral shedding in ejaculate suggests that HEV replicates within the human male genital tract, but its target organ is unknown and appropriate models are lacking. We aimed to determine the HEV tropism in human testis and its potential influence on male reproductive health. We conducted ex vivo culture of human testis explants and in vitro culture of primary human Sertoli cells. Clinically derived HEV genotype 1 (HEV1) and HEV3 virions, as well as rat derived HEV-C1, were used for inoculation. Transcriptomic analysis was performed on testis tissues collected from tacrolimus-treated rabbits with chronic HEV3 infection. Our findings reveal that HEV3, but not HEV1 or HEV-C1, can replicate in human testis explants and primary human Sertoli cells. Tacrolimus treatment significantly enhanced the replication efficiency of HEV3 in testis explants and enabled successful HEV1 infection in Sertoli cells. HEV3 infection disrupted the secretion of several soluble factors and altered the cytokine microenvironment within primary human Sertoli cells. Finally, intratesticular transcriptomic analysis of immunocompromised rabbits with chronic HEV infection indicated a downregulation of genes associated with spermatogenesis. HEV can infect the human testicular tissues and Sertoli cells, with increased replication efficiency when exposed to tacrolimus treatment. These findings shed light on how HEV may persist in ejaculate of patients with chronic hepatitis E and provide valuable ex vivo tool for studying countermeasures.

Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies.

Organotypic culture of human brain explants as a preclinical model for AI-driven antiviral studies

Viral neuroinfections represent a major health burden for which the development of antivirals is needed. Antiviral compounds that target the consequences of a brain infection (symptomatic treatment) rather than the cause (direct-acting antivirals) constitute a promising mitigation strategy that requires to be investigated in relevant models. However, physiological surrogates mimicking an adult human cortex are lacking, limiting our understanding of the mechanisms associated with viro-induced neurological disorders. Here, we optimized the Organotypic culture of Post-mortem Adult human cortical Brain explants (OPAB) as a preclinical platform for Artificial Intelligence (AI)-driven antiviral studies. OPAB shows robust viability over weeks, well-preserved 3D cytoarchitecture, viral permissiveness, and spontaneous local field potential (LFP). Using LFP as a surrogate for neurohealth, we developed a machine learning framework to predict with high confidence the infection status of OPAB. As a proof-of-concept, we showed that antiviral-treated OPAB could partially restore LFP-based electrical activity of infected OPAB in a donor-dependent manner. Together, we propose OPAB as a physiologically relevant and versatile model to study neuroinfections and beyond, providing a platform for preclinical drug discovery.

Growth modulatory effects of fenretinide encompass keratinocyte terminal differentiation: A favorable outcome for oral squamous cell carcinoma chemoprevention.

Oral squamous cell carcinoma (OSCC) is world-wide health problem associated with high morbidity and mortality. From both the patient and socio-economic perspectives, prevention of progression of premalignant oral intraepithelial neoplasia (OIN) to OSCC is clearly the preferable outcome. Optimal OSCC chemopreventives possess a variety of attributes including high-tolerability, bioavailability, efficacy and preservation of an intact surface epithelium. Terminal differentiation, which directs oral keratinocytes leave the proliferative pool to form protective cornified envelopes, preserves the protective epithelial barrier while concurrently eliminating growth-aberrant keratinocytes. This study employed human premalignant oral keratinocytes and an OSCC cell line to evaluate the differentiation-inducing capacity of the synthetic retinoid, fenretinide (4HPR). Full thickness oral mucosal explants were evaluated for proof of concept differentiation studies. Results of this study characterize the ability of 4HPR to fulfill all requisite components for keratinocyte differentiation i.e. nuclear import via binding to CRABP-II (molecular modeling), binding to and subsequent activation of retinoic acid nuclear receptors (receptor activation assays), increased expression and translation of genes associated with keratinocyte differentiation (RT-PCR, immunoblotting) upregulation of a transglutaminase enzyme essential for cornified envelope formation (TGM3, functional assay) and augmentation of terminal differentiation in human oral epithelial explants (image-analyses quantified corneocyte desquamation). These data build upon the chemoprevention repertoire of 4HPR that includes function as a small molecule kinase inhibitor and inhibition of essential mechanisms necessary for basement membrane invasion. An upcoming clinical trial, which will assess whether a 4HPR-releasing mucoadhesive patch induces histologic, clinical and molecular regression in OIN lesions, will provide essential clinical insights.

Abstract B017: Understanding the impact of transporter heterogeneity on the efficacy of PARP inhibitors

Abstract B017: Understanding the impact of transporter heterogeneity on the efficacy of PARP inhibitors

Mechanical loading rescues mechanoresponsiveness in a human osteoarthritis explant model despite Wnt activation

ObjectivesOptimizing rehabilitation strategies for osteoarthritis necessitates a comprehensive understanding of chondrocytes’ mechanoresponse in both health and disease, especially in the context of the interplay between loading and key pathways involved in osteoarthritis development, like canonical Wnt signaling. This study aims to elucidate the role of Wnt signaling in the mechanoresponsiveness of healthy and osteoarthritic human cartilage. MethodsWe used an ex-vivo model involving short-term physiological mechanical loading of human cartilage explants. First, the loading protocol for subsequent experiments was determined. Next, loading was applied to non-OA explants with or without Wnt activation with CHIR99021. Molecular read-outs of anabolic, pericellular matrix and matrix remodeling markers were used to assess the effect of Wnt on cartilage mechanoresponse. Finally, the same set-up was used to study the effect of loading in cartilage from patients with established OA. ResultsOur results confirm that physiological loading maintains expression of anabolic genes in non-OA cartilage, and indicate a deleterious effect of Wnt activation in the chondrocyte mechanoresponsiveness. This suggests that loading-induced regulation of chondrocyte markers occurs downstream of canonical Wnt signaling. Interestingly, our study highlighted contrasting mechanoresponsiveness in the model of Wnt activation and the established OA samples, with established OA cartilage maintaining its mechanoresponsiveness, and mechanical loading rescuing the chondrogenic phenotype. ConclusionThis study provides insights into the mechanoresponsiveness of human cartilage in both non-OA and OA conditions. These findings hold the potential to contribute to the development of strategies that optimize the effect of dynamic compression by correcting OA pathological cell signaling.

PPARγ antagonists induce aromatase transcription in adipose tissue cultures

Aromatase is the rate-limiting enzyme in the biosynthesis of estrogens and a key risk factor for hormone receptor-positive breast cancer. In postmenopausal women, estrogens synthesized in adipose tissue promotes the growth of estrogen receptor positive breast cancers. Activation of peroxisome proliferator-activated receptor gamma (PPARγ) in adipose stromal cells (ASCs) leads to decreased expression of aromatase and differentiation of ASCs into adipocytes. Environmental chemicals can act as antagonists of PPARγ and disrupt its function. This study aimed to test the hypothesis that PPARγ antagonists can promote breast cancer by stimulating aromatase expression in human adipose tissue.Primary cells and explants from human adipose tissue as well as A41hWAT, C3H10T1/2, and H295R cell lines were used to investigate PPARγ antagonist-stimulated effects on adipogenesis, aromatase expression, and estrogen biosynthesis. Selected antagonists inhibited adipocyte differentiation, preventing the adipogenesis-associated downregulation of aromatase. NMR spectroscopy confirmed direct interaction between the potent antagonist DEHPA and PPARγ, inhibiting agonist binding. Short-term exposure of ASCs to PPARγ antagonists upregulated aromatase only in differentiated cells, and a similar effect could be observed in human breast adipose tissue explants. Overexpression of PPARG with or without agonist treatment reduced aromatase expression in ASCs.The data suggest that environmental PPARγ antagonists regulate aromatase expression in adipose tissue through two mechanisms. The first is indirect and involves inhibition of adipogenesis, while the second occurs more acutely.

ILT2 and ILT4 Drive Myeloid Suppression via Both Overlapping and Distinct Mechanisms.

Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.

A host-microbial metabolite interaction gut-on-a-chip model of the adult human intestine demonstrates beneficial effects upon inulin treatment of gut microbiome.

Background: The gut and its microbiome have a major impact on many aspects of health and are therefore also an attractive target for drug- or food-based therapies. Here, we report on the added value of combining a microbiome screening model, the i-screen, with fresh intestinal tissue explants in a microfluidic gut-on-a-chip model, the Intestinal Explant Barrier Chip (IEBC). Methods: Adult human gut microbiome (fecal pool of 6 healthy donors) was cultured anaerobically in the i-screen platform for 24 h, without and with exposure to 4 mg/mL inulin. The i-screen cell-free culture supernatant was subsequently applied to the luminal side of adult human colon tissue explants (n = 3 donors), fixed in the IEBC, for 24 h and effects were evaluated. Results: The supplementation of the media with inulin promoted the growth of Anaerostipes, Bifidobacterium, Blautia, and Collinsella in the in vitro i-screen, and triggered an elevated production of butyrate by the microbiota. Human colon tissue exposed to inulin-treated i-screen cell-free culture supernatant or control i-screen cell-free culture supernatant with added short-chain fatty acids (SCFAs) showed improved tissue barrier integrity measured by a 28.2%-34.2% reduction in FITC-dextran 4000 (FD4) leakage and 1.3 times lower transport of antipyrine. Furthermore, the release of pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α was reduced under these circumstances. Gene expression profiles confirmed these findings, but showed more profound effects for inulin-treated supernatant compared to SCFA-supplemented supernatant. Conclusion: The combination of i-screen and IEBC facilitates the study of complex intestinal processes such as host-microbial metabolite interaction and gut health.

Targeting an inflammation-amplifying cell population can attenuate osteoarthritis-associated pain

BackgroundUnderstanding of pain in osteoarthritis, its genesis, and perception is still in its early stages. Identification of precise ligand-receptor pairs that transduce pain and the cells and tissues in which they reside will elucidate new therapeutic approaches for pain management. Our recent studies had identified an inflammation-amplifying (Inf-A) cell population that is expanded in human OA cartilage and is distinctive in the expression of both IL1R1 and TNF-R2 receptors and active Jnk signaling cascade.MethodsIn this study, we have tested the function of the cartilage-resident IL1R1+TNF-R2+ Inf-A cells in OA. We have identified that the IL1R1+TNF-R2+ Inf-A cells expand in aged mice as well as after anterior cruciate ligament tear upon tibia loading and OA initiation in mice. We targeted and modulated the Jnk signaling cascade in InfA through competitive inhibition of Jnk signaling in mice and human OA explants and tested the effects on joint structure and gait in mice.ResultsModulation of Jnk signaling led to attenuation of inflammatory cytokines CCL2 and CCL7 without showing any structural improvements in the joint architecture. Interestingly, Jnk inhibition and lowered CCL2 and 7 are sufficient to significantly improve the gait parameters in treated PTOA mice demonstrating reduced OA-associated pain. Consistent with the mice data, treatment with JNK inhibitor did not improve human OA cartilage explants.ConclusionThese studies demonstrate that Inf-A, an articular-cartilage resident cell population, contributes to pain in OA via secretion of CCL2 and 7 and can be targeted via inhibition of Jnk signaling.

Intracellular Pseudomonas aeruginosa within the Airway Epithelium of Cystic Fibrosis Lung Tissues.

Rationale: Pseudomonas aeruginosa is the major bacterial pathogen colonizing the airways of adult patients with cystic fibrosis (CF) and causes chronic infections that persist despite antibiotic therapy. Intracellular bacteria may represent an unrecognized reservoir of bacteria that evade the immune system and antibiotic therapy. Although the ability of P. aeruginosa to invade and survive within epithelial cells has been described invitro in different epithelial cell models, evidence of this intracellular lifestyle in human lung tissues is currently lacking. Objectives: To detect and characterize intracellular P. aeruginosa in CF airway epithelium from human lung explant tissues. Methods: We sampled lung explant tissues from patients with CF undergoing lung transplantation and non-CF lung donor control tissue. We analyzed lung tissue sections for the presence of intracellular P. aeruginosa using quantitative culture and microscopy, in parallel to histopathology and airway morphometry. Measurements and Main Results: P. aeruginosa was isolated from the lungs of seven patients with CF undergoing lung transplantation. Microscopic assessment revealed the presence of intracellular P. aeruginosa within airway epithelial cells in three of the seven patients analyzed at a varying but low frequency. We observed those events occurring in lung regions with high bacterial burden. Conclusions: This is the first study describing the presence of intracellular P. aeruginosa in CF lung tissues. Although intracellular P. aeruginosa in airway epithelial cells is likely relatively rare, our findings highlight the plausible occurrence of this intracellular bacterial reservoir in chronic CF infections.

Tissue-engineered and autologous pericardium in congenital heart surgery: comparative histopathological study of human vascular explants.

The goal of this histological study was to assess the biocompatibility of vascular patches used in the repair of congenital heart defects. We examined tissue-engineered bovine (n = 7) and equine (n = 7) patches and autologous human pericardium (n = 7), all explanted due to functional issues or follow-up procedures. Techniques like Movat-Verhoeff, von Kossa and immunohistochemical staining were used to analyse tissue composition, detect calcifications and identify immune cells. A semi-quantitative scoring system was implemented to evaluate the biocompatibility aspects, thrombus formation, extent of pannus, inflammation of pannus, cellular response to patch material, patch degradation, calcification and neoadventitial inflammation. We observed distinct material degradation patterns among types of patches. Bovine patches showed collagen disintegration and exudate accumulation, whereas equine patches displayed edematous swelling and material dissolution. Biocompatibility scores were lower in terms of cellular response, degradation and overall score for human autologous pericardial patches compared to tissue-engineered types. The extent of pannus formation was not influenced by the type of patch. Bovine patches had notable calcifications causing tissue hardening, and foreign body giant cells were more frequently seen in equine patches. Plasma cells were frequently detected in the neointimal tissue of engineered patches. Our results confirm the superior biocompatibility of human autologous patches and highlight discernible variations in the changes of patch material and the cellular response to patch material between bovine and equine patches. Our approach implements the semi-quantitative scoring of various aspects of biocompatibility, facilitating a comparative quantitative analysis across all types of patches, despite their inherent differences.

P626 JNJ-77242113, an oral peptide selectively targeting the IL-23 receptor, demonstrates pharmacodynamic activity in rat and human colon tissue explants following oral dosing

Abstract Background The interleukin (IL)-23 pathway is a pathogenic driver in psoriasis, psoriatic arthritis, and inflammatory bowel disease (IBD). There are currently no approved oral therapies selectively targeting the IL-23 pathway. JNJ-77242113 (JNJ-2113) is a targeted oral peptide that binds the IL-23 receptor with high affinity, and potently and selectively inhibits IL-23 proximal signaling and downstream cytokine production. Oral JNJ-2113 showed dose-dependent inhibition of IL-23–induced IL-17A production in rat blood and attenuated signs of disease in a rat colitis model. A Phase 1 study showed inhibition of IL-23–induced IFN-γ production in blood from participants dosed with JNJ-2113 (Fourie et al. ISID. 2023). Methods We evaluated IL-23–induced IL-22 expression in colon tissue after exposure to JNJ-2113 or JNJ-2100 (a tool molecule with similar affinity and potency). IL-22 mRNA was measured after IL-23 stimulation in rat colon biopsies treated with JNJ-2100 in vitro or in colon tissue from rats 6 h after oral JNJ-2100 dosing. Human colon explants from donors were treated in vitro with JNJ-2100 or JNJ-2113, stimulated with IL-23, and IL-22 mRNA was measured. Colon and rectum biopsies from healthy participants dosed for 10 days with oral JNJ-2113 or placebo in a Phase 1 study were obtained, stimulated with IL-23 ex vivo, and IL-22 mRNA was evaluated. Results Overall, inhibition of IL-23–induced IL-22 expression was observed in colon tissue exposed to JNJ-2100 or JNJ-2113. Dose-dependent inhibition of IL-22 expression was observed in rat colon biopsies exposed to JNJ-2100 in vitro. Colon explants from rats dosed with 0.3, 3, and 30 mg/kg oral JNJ-2100 showed dose-dependent inhibition of IL-22 expression (52%, 88% and 96%, respectively) compared with explants from rats treated with vehicle. Human colon explants exposed to JNJ-2100 or JNJ-2113 in vitro also showed dose-dependent inhibition of IL-23–induced IL-22 expression, with >90% inhibition at the highest doses. Importantly, in colon biopsies from Phase 1 participants dosed with oral JNJ-2113 versus placebo, significant inhibition of IL-23–induced IL-22 expression was seen (P<0.05), with an average of 65% and 66% inhibition observed in sigmoid colon and rectum biopsies, respectively. Conclusion These results demonstrate pharmacodynamic activity of JNJ-2100 in vitro and ex vivo in rat colon tissue, of JNJ-2100 and JNJ-2113 in vitro in human colon explants, and of JNJ-2113 in colon biopsies from healthy human volunteers. These data support development of JNJ-2113 for the treatment of IBD. Of note, a Phase 2b dose-ranging study has demonstrated clinical activity of JNJ-2113 in moderate-to-severe plaque psoriasis (NCT05223868).

Fibronectin mediates activin A-promoted human trophoblast migration and acquisition of endothelial-like phenotype

BackgroundDuring human early placentation, a proportion of extravillous trophoblasts (EVTs) migrate to the maternal decidua, differentiating into endovascular EVTs to remodel spiral arteries and ensure the establishment of blood circulation at the maternal-fetal interface. Inadequate EVT migration and endovascular differentiation are closely associated with adverse pregnancy outcomes such as miscarriage. Activin A and fibronectin are both secretory molecules abundantly expressed at the maternal-fetal interface. Activin A has been reported to regulate EVT biological functions. However, whether fibronectin mediates activin A-promoted EVT migration and acquisition of endothelial-like phenotype as well as the underlying molecular mechanisms remain unknown. Additionally, the role of fibronectin in pregnancy establishment and maintenance warrants further investigation.MethodsPrimary and immortalized (HTR8/SVneo) human EVTs were used as in vitro study models. Cultured human first-trimester chorionic villous explants were utilized for ex vivo validation. A local fibronectin knockdown model in ICR mouse uteri, achieved by nonviral in vivo transfection with small interfering RNA (siRNA) targeting fibronectin 1 (si-Fn1), was employed to explore the roles of fibronectin in the establishment and maintenance of early pregnancy.ResultsOur results showed that activin A treatment significantly induced fibronectin 1 (FN1) mRNA expression and fibronectin protein production, which is essential for human trophoblast migration and endothelial-like tube formation. Both basal and activin A-upregulated fibronectin expression were abolished by the TGF-β type I receptor inhibitor SB431542 or siRNA-mediated knockdown of activin receptor-like kinase (ALK4) or SMAD4. Moreover, activin A-increased trophoblast migration and endothelial-like tube formation were attenuated following the depletion of fibronectin. Fibronectin knockdown via intrauterine siRNA administration reduced CD31 and cytokeratin 8 (CK8) expression at the maternal-fetal interface, resulting in a decrease in the number of implantation sites and embryos.ConclusionsOur study demonstrates that activin A promotes trophoblast cell migration and acquisition of endothelial-like phenotype via ALK4-SMAD2/3-SMAD4-mediated fibronectin upregulation. Furthermore, through a local fibronectin knockdown model in mouse uteri, we found that the absence of fibronectin at the maternal-fetal interface impedes endovascular migration of trophoblasts and decidual vascularization, thereby interfering with early embryo implantation and the maintenance of pregnancy. These findings provide novel insights into placental development during early pregnancy establishment and contribute to the advancement of therapeutic approaches for managing pregnancy complications related to trophoblast dysfunction.

Loratadine as an Anti-inflammatory Agent Against Clostridium difficile Toxin B.

Clostridium difficile infection (CDI) is a debilitating nosocomial infection. C. difficile produces toxins A and B, which cause inflammation. Existing therapies have issues with recurrence, cost, and safety. We aim to discover a safe, effective, and economical nonmicrobiological therapeutic approach against CDI. We included human primary peripheral blood mononuclear cells (PBMCs), fresh human colonic explants, and humanized HuCD34-NCG mice. Toxin A+B+ VPI 10463 and A-B+ ribotype 017 C. difficile strains were used. We used single-cell RNA profiling and high-throughput screening to find actionable toxin B-dependent pathways in PBMCs. Histamine 1 receptor-related drugs were found among the hit compounds that reversed toxin-mediated macrophage inflammatory protein (MIP) 1α expression in PBMCs. We identified loratadine as the safest representative antihistamine for therapeutic development. Loratadine inhibited toxin B-induced MIP-1α secretion in fresh human colonic tissues. Oral loratadine (10 mg/kg/d) maintained survival, inhibited intestinal CCl3 messenger RNA expression, and prevented vancomycin-associated recurrence in the VPI 10463-infected mice and ribotype 017-infected hamsters. Splenocytes from loratadine-treated mice conferred anti-inflammatory effects to the VPI 10463-infected T/B-cell--deficient Rag-/- mice. Oral loratadine suppressed human MIP-1α expression in monocytes/macrophages in toxin B-expressing ribotype 017-infected humanized HuCD34-NCG mice. Loratadine may be repurposed to optimize existing therapies against CDI.

Abstract B062: CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to therapy. Stromal cancer-associated fibroblasts (CAFs) play a key role in promoting tumor progression and chemoresistance, by creating a fibrotic microenvironment that impedes drug access and feeds PDAC cells nutrients and pro-tumor signals. CRO-67 (Patent PCT/AU2023/050505) is a novel drug developed with Noxopharm Ltd using a rational medicinal chemistry design to improve bioavailability of chromans which have potent anti-cancer activity. Using our patient-derived PDAC tumor in a dish model (explants; maintain multicellular architecture and fibrosis) we previously showed that CRO-67 has both anti-tumor and CAF reprogramming capacity in 4 patient explants [Cancer Res (2022) 82 (22_Supplement):C073]. Aims: 1) To expand evaluation of CRO-67 in additional patient-derived PDAC tumor explants given PDAC heterogeneity. 2) Assess the effect of CRO-67 on PDAC cell and CAF function. 3) Validate the therapeutic potential of CRO-67 on PDAC growth in vivo. Methods: 1) PDAC tumor samples collected from 7 patients undergoing pancreatic resection. Tumor explants (1–2mm diameter) were cultured on gelatin sponges, treated with CRO-67 (0–50μg/mL) every 3 days and fixed on day 12. Therapeutic response assessed by immunohistochemistry for cytokeratin (PDAC cells), α-smooth muscle actin (CAFs), bromodeoxyuridine (proliferation) and TUNEL (cell death). 2) PDAC cells (MiaPaCa-2) and patient-derived CAFs were treated with CRO-67 (1.5μM) for 48h before proliferation analysis (IncuCYTE S3) and for 24h before apoptosis (Annexin V/DAPI staining; flow cytometry) and cell cycle (DAPI staining; flow cytometry) analysis. 3) Subcutaneous PDAC (BxPC3 human cells) tumors in mice were treated with CRO-67 (2.5mg/kg Intraperitoneally, twice a day) for 21 days and tumor volume measured (calipers). Results: 1) CRO-67 treatment decreased tumor and CAF cell frequency in 6/6 (no quantifiable tumor in patient 7 explants) and 7/7 patient PDAC explants, respectively. It also decreased explant cell proliferation and increased cell death versus controls. 2) CRO-67 completely abolished proliferation of MiaPaCa-2 and reduced the average growth rate of CAFs by 81.3±8.8% (p=0.0025; n=5), increased apoptosis in MiaPaCa-2 by 455.7±21.4% (p=0.003; n=3) and in CAFs by 172.6±13.1% (p=0.0067; n=5) and increased the fraction of cells in G2/M cell cycle phase by 163.8±12.8% (p<0.0001; n=3) in MiaPaCa-2 and by 74.6±8.6% (p=0.0015; n=5) in CAFs versus controls. 3) CRO-67 reduced PDAC tumors in vivo by 56.7±6.6% (p=0.0013; n=9 mice/group) versus controls. Conclusions: CRO-67: 1) reduced PDAC cells and CAFs in human PDAC explants via reduced proliferation and increased death; 2) reduced proliferation of PDAC cells and CAFs in vitro, increased apoptosis and disrupted cell cycle progression through G2/M phase; 3) reduced PDAC tumor growth in vivo. Implication: We predict CRO-67 is a potential ‘dual cell’ therapy with direct anti-tumor effects and CAF reprogramming capacity, warranting further investigation in vivo. Citation Format: Keilah Garcia Netto, Shannon Chiang, John Kokkinos, Koroush S. Haghighi, Aparna Raina, Omali Pitiyarachchi, Janet Youkhana, Quach Truong, Daniel Wenholz, Xiang Li, Olivier Laczka, Naresh Kumar, John Wilkinson, David Goldstein, George Sharbeen, Phoebe A. Phillips. CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B062.

Abstract B101: Tumor-selective inhibition of active RAS in pancreatic ductal adenocarcinoma

Abstract RM-042 is a broad-spectrum inhibitor of the active (GTP-bound) form of KRAS, HRAS, and NRAS, with affinity for both mutant and wild type (WT) variants (RASMULTI(ON)). Given that more than 90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, we assessed the therapeutic potential of RM-042 in a comprehensive range of PDAC models, including human and murine cell lines, human patient-derived organoids, human PDAC explants, subcutaneous and orthotopic cell-line or patient derived xenografts, syngeneic allografts, and genetically engineered mouse models. We observed broad and remarkable anti-tumor activity across these models following direct RAS inhibition by RM-042 at doses and concentrations that were tolerable in vivo. Analyses of tumor and normal tissues following RM-042 treatment revealed divergent cellular responses to RAS inhibition. While we observed anti-proliferative effects in both normal and tumor tissues, RAS-dependent tumors alone exhibited a susceptibility to cell death via apoptosis within hours of dosing. Finally, using single cell regulatory network analysis, we found that different subtypes of PDAC malignant epithelial cells responded in distinct ways to RAS inhibition by RM-042, resulting in an enrichment of the more differentiated gastro-intestinal lineage state (GLS) cellular subtype. Together, these data have significant implications for the clinical development of RAS inhibitors in the setting of PDAC. A related inhibitor, RMC-6236, is currently in early clinical evaluation (NCT05379985). Citation Format: Urszula N. Wasko, Jingjing Jiang, Lorenzo Tomassoni, Yingyun Wang, Bianca Lee, Margo Orlen, Kristina Drizyte-Miller, Marie Menard, Julien Dilly, Steven A. Sastra, Carmine F. Palermo, Stephanie Chang, Andrew J. Aguirre, Channing J. Der, Robert H. Vonderheide, Ben Z. Stanger, Andrea Califano, Mallika Singh, Kenneth P. Olive. Tumor-selective inhibition of active RAS in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B101.

Optimal transcorneal electrical stimulation parameters for preserving photoreceptors in a mouse model of retinitis pigmentosa.

JOURNAL/nrgr/04.03/01300535-202419110-00034/figure1/v/2024-03-08T184507Z/r/image-tiff Retinitis pigmentosa is a hereditary retinal disease that affects rod and cone photoreceptors, leading to progressive photoreceptor loss. Previous research supports the beneficial effect of electrical stimulation on photoreceptor survival. This study aims to identify the most effective electrical stimulation parameters and functional advantages of transcorneal electrical stimulation (tcES) in mice affected by inherited retinal degeneration. Additionally, the study seeked to analyze the electric field that reaches the retina in both eyes in mice and post-mortem humans. In this study, we recorded waveforms and voltages directed to the retina during transcorneal electrical stimulation in C57BL/6J mice using an intraocular needle probe with rectangular, sine, and ramp waveforms. To investigate the functional effects of electrical stimulation on photoreceptors, we used human retinal explant cultures and rhodopsin knockout (Rho-/-) mice, demonstrating progressive photoreceptor degeneration with age. Human retinal explants isolated from the donors' eyes were then subjected to electrical stimulation and cultured for 48 hours to simulate the neurodegenerative environment in vitro. Photoreceptor density was evaluated by rhodopsin immunolabeling. In vivo Rho-/- mice were subjected to two 5-day series of daily transcorneal electrical stimulation using rectangular and ramp waveforms. Retinal function and visual perception of mice were evaluated by electroretinography and optomotor response (OMR), respectively. Immunolabeling was used to assess the morphological and biochemical changes of the photoreceptor and bipolar cells in mouse retinas. Oscilloscope recordings indicated effective delivery of rectangular, sine, and ramp waveforms to the retina by transcorneal electrical stimulation, of which the ramp waveform required the lowest voltage. Evaluation of the total conductive resistance of the post-mortem human compared to the mouse eyes indicated higher cornea-to-retina resistance in human eyes. The temperature recordings during and after electrical stimulation indicated no significant temperature change in vivo and only a subtle temperature increase in vitro (~0.5-1.5°C). Electrical stimulation increased photoreceptor survival in human retinal explant cultures, particularly at the ramp waveform. Transcorneal electrical stimulation (rectangular + ramp) waveforms significantly improved the survival and function of S and M-cones and enhanced visual acuity based on the optomotor response results. Histology and immunolabeling demonstrated increased photoreceptor survival, improved outer nuclear layer thickness, and increased bipolar cell sprouting in Rho-/- mice. These results indicate that transcorneal electrical stimulation effectively delivers the electrical field to the retina, improves photoreceptor survival in both human and mouse retinas, and increases visual function in Rho-/- mice. Combined rectangular and ramp waveform stimulation can promote photoreceptor survival in a minimally invasive fashion.