PROMER technology: A new real-time PCR tool enabling multiplex detection of point mutation with high specificity and sensitivity.

Real-time polymerase chain reaction (real-time PCR) is a powerful tool for the precise quantification of nucleic acids in various applications. In cancer management, the monitoring of circulating tumor DNA (ctDNA) from liquid biopsies can provide valuable information for precision care, including treatment selection and monitoring, prognosis, and early detection. However, the rare and heterogeneous nature of ctDNA has made its precise detection and quantification challenging, particularly for ctDNA containing hotspot mutations. We have developed a new real-time PCR tool, PROMER technology, which enables the precise and sensitive detection of ctDNA containing cancer-driven single-point mutations. The PROMER functions as both a PRObe and priMER, providing enhanced detection specificity. We validated PROMER technology using synthetic templates with known KRAS point mutations and demonstrated its sensitivity and linearity of quantification. Using genomic DNA from human cancer cells with mutant and wild-type KRAS, we confirmed that PROMER PCR can detect mutant DNA. Furthermore, we demonstrated the ability of PROMER technology to efficiently detect mutation-carrying ctDNA from the plasma of mice with human cancers. Our results suggest that PROMER technology represents a promising new tool for the precise detection and quantification of DNA containing point mutations in the presence of a large excess of wild-type counterpart.

hiPSC-derived macrophages improve drug sensitivity and selectivity in a macrophage-incorporating organoid culture model.

Accurate simulation of different cell type interactions is crucial for physiological and precisein vitrodrug testing. Human tissue-resident macrophages are critical for modulating disease conditions and drug-induced injuries in various tissues; however, their limited availability has hindered their use inin vitromodeling. Therefore, this study aimed to create macrophage-containing organoid co-culture models by directly incorporating human-induced pluripotent stem cell (hiPSC)-derived pre-macrophages into organoid and scaffold cell models. The fully differentiated cells in these organoids exhibited functional characteristics of tissue-resident macrophages with enriched pan-macrophage markers and the potential for M1/M2 subtype specialization upon cytokine stimulation. In a hepatic organoid model, the integrated macrophages replicated typical intrinsic properties, including cytokine release, polarization, and phagocytosis, and the co-culture model was more responsive to drug-induced liver injury than a macrophage-free model. Furthermore, alveolar organoid models containing these hiPSC-derived macrophages also showed increased drug and chemical sensitivity to pulmonary toxicants. Moreover, 3D adipocyte scaffold models incorporating macrophages effectively simulated in vivo insulin resistance observed in adipose tissue and showed improved insulin sensitivity on exposure to anti-diabetic drugs. Overall, the findings demonstrated that incorporating hiPSC-derived macrophages into organoid culture models resulted in more physiological and sensitivein vitrodrug evaluation and screening systems.

Single-cell transcriptome analysis reveals subtype-specific clonal evolution and microenvironmental changes in liver metastasis of pancreatic adenocarcinoma and their clinical implications.

BACKGROUND: Intratumoral heterogeneity (ITH) and tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) play important roles in tumor evolution and patient outcomes. However, the precise characterization of diverse cell populations and their crosstalk associated with PDAC progression and metastasis is still challenging. METHODS: We performed single-cell RNA sequencing (scRNA-seq) of treatment-naïve primary PDAC samples with and without paired liver metastasis samples to understand the interplay between ITH and TME in the PDAC evolution and its clinical associations. RESULTS: scRNA-seq analysis revealed that even a small proportion (22%) of basal-like malignant ductal cells could lead to poor chemotherapy response and patient survival and that epithelial-mesenchymal transition programs were largely subtype-specific. The clonal homogeneity significantly increased with more prevalent and pronounced copy number gains of oncogenes, such as KRAS and ETV1, and losses of tumor suppressor genes, such as SMAD2 and MAP2K4, along PDAC progression and metastasis. Moreover, diverse immune cell populations, including naïve SELLhi regulatory T cells (Tregs) and activated TIGIThi Tregs, contributed to shaping immunosuppressive TMEs of PDAC through cellular interactions with malignant ductal cells in PDAC evolution. Importantly, the proportion of basal-like ductal cells negatively correlated with that of immunoreactive cell populations, such as cytotoxic T cells, but positively correlated with that of immunosuppressive cell populations, such as Tregs. CONCLUSION: We uncover that the proportion of basal-like subtype is a key determinant for chemotherapy response and patient outcome, and that PDAC clonally evolves with subtype-specific dosage changes of cancer-associated genes by forming immunosuppressive microenvironments in its progression and metastasis.

Prediction of Acute Hepatotoxicity With Human Pluripotent Stem Cell-Derived Hepatic Organoids.

Recent development of hepatic organoids (HOs) derived from human pluripotent stem cells (hPSCs) provides an alternative in vitro model that can mimic the human liver detoxification pathway for drug safety assessment. By recapitulating the high level of maturity and drug-metabolizing capacity of the liver in a three-dimensional organoid culture, HOs may allow researchers to assess drug toxicity and metabolism more accurately than animal models or hepatocellular carcinoma cells. Although this promising potential has contributed to the development of various protocols, only a few protocols are available to generate functional HOs with guaranteed CYP450 enzymatic activity, the key feature driving toxic responses during drug metabolism. Based on previously published protocols, we describe an optimized culture method that can substantially increase the expression and activity of CYP450s, in particular CYP3A4, CYP2C9, and CYP2C19, in HOs. To generate mass-produced and highly reproducible HOs required as models for toxicity evaluation, we first generated hepatic endodermal organoids (HEOs) from hPSCs capable of in vitro proliferation and cryopreservation. The stepwise protocol includes generating HEOs as well as efficient methods to enhance CYP450 expression and activity in terminally differentiated HOs. Furthermore, we present a simple protocol for the assessment of HO cytotoxicity, one of the hallmarks of drug-induced acute hepatotoxicity. The protocols are relatively straightforward and can be successfully used by laboratories with basic experience in culturing hPSCs. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of hepatic endodermal organoids from human pluripotent stem cells Basic Protocol 2: Expansion and cryopreservation of hepatic endodermal organoids Basic Protocol 3: Differentiation of hepatic organoids from hepatic endodermal organoids Basic Protocol 4: Evaluation of hepatotoxicity using hepatic organoids Support Protocol: Human pluripotent stem cell culture.

Exploring the Anti-Diabetic Potential of Quercetagitrin through Dual Inhibition of PTPN6 and PTPN9.

Protein tyrosine phosphatases (PTPs) are pivotal contributors to the development of type 2 diabetes (T2DM). Hence, directing interventions towards PTPs emerges as a valuable therapeutic approach for managing type 2 diabetes. In particular, PTPN6 and PTPN9 are targets for anti-diabetic effects. Through high-throughput drug screening, quercetagitrin (QG) was recognized as a dual-target inhibitor of PTPN6 and PTPN9. We observed that QG suppressed the catalytic activity of PTPN6 (IC50 = 1 µM) and PTPN9 (IC50 = 1.7 µM) in vitro and enhanced glucose uptake by mature C2C12 myoblasts. Additionally, QG increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and insulin-dependent phosphorylation of Akt in mature C2C12 myoblasts. It further promoted the phosphorylation of Akt in the presence of palmitic acid, suggesting the attenuation of insulin resistance. In summary, our results indicate QG's role as a potent inhibitor targeting both PTPN6 and PTPN9, showcasing its potential as a promising treatment avenue for T2DM.

Predicting disease recurrence in limited disease small cell lung cancer using cell-free DNA-based mutation and fragmentome analyses.

Background: Limited disease (LD) small cell lung cancer (SCLC) treated with definitive concurrent chemoradiotherapy (CCRT) potentially experience disease recurrence. We investigated the feasibility of circulating-tumor DNA (ctDNA)-based genomic and fragmentome analyses to assess the risk of recurrence. Methods: Targeted sequencing was conducted using pre-treatment and on-treatment blood samples from definitive CCRT-treated patients with LD-SCLC (n=50). Based on 12-month recurrence-free survival (RFS), patients were categorized into persistent-response (PeR, n=29) and non-PeR (n=21) groups. Fragmentome analysis was conducted using ctDNA fragments of different lengths: P1 (100-155 bp) and P2 (160-180 bp). Results: Patients with TP53 (n=15) and RB1 (n=11) mutation in on-treatment samples demonstrated significantly shorter RFS than patients with wild-type (WT) (P=0.05, P=0.0014, respectively). Fragmentome analysis of all available on-treatment samples (n=26) revealed that the non-PeR group (n=10) had a significantly higher P1 range (P=0.003) and lower P2 range (P=0.002). The areas under the curves for P1, P2, and the fragmentation ratio (P1/P2) in distinguishing the PeR and non-PeR were 0.850, 0.725, and 0.900, respectively. Using optimal cut-off, longer RFSs were found with the low-fragmentation-ratio group than with the high-fragmentation-ratio group (not reached vs. 7.6 months, P=0.002). Patients with both WT RB1 and a low-fragmentation-ratio (n=10) showed better outcomes than patients with both mutated RB1 and a high-fragmentation-ratio (n=10; hazard ratio, 7.55; 95% confidence interval: 2.14-26.6; P=0.002). Conclusions: RB1 mutations and high fragmentation ratios correlated with early disease recurrence. Analyzing ctDNA could help in predicting early treatment failure and making clinical decisions for high-risk patients.

Establishment of a patient-specific avatar organoid model derived from EUS-guided fine-needle biopsy for timely clinical application in pancreatic ductal adenocarcinoma (with video).

BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) has the worst survival rate among tumors. At the time of diagnosis, more than 80% of PDACs are considered to be surgically unresectable, and there is an unmet need for treatment options in these inoperable PDACs. This study aimed to establish a patient-derived organoid (PDO) platform from EUS-guided fine-needle biopsy (EUS-FNB) collected at diagnosis and to determine its clinical applicability for the timely treatment of unresectable PDAC. METHODS: Patients with suspected PDAC were prospectively enrolled at the Samsung Medical Center from 2015 to 2019. PDAC tissues were acquired by means of EUS-FNB to establish PDAC PDOs, which were comprehensively analyzed for histology, genomic sequencing, and high-throughput screening (HTS) drug sensitivity test. RESULTS: PDAC PDOs were established with a success rate of 83.2% (94/113). It took approximately 3 weeks from acquiring minimal EUS-FNB specimens to generating sufficient PDAC PDOs for the simultaneous HTS drug sensitivity test and genomic sequencing. The high concordance between PDAC tissues and matched PDOs was confirmed, and whole-exome sequencing revealed the increased detection of genetic alterations in PDOs compared with EUS-FNB tissues. The HTS drug sensitivity test showed clinical correlation between the ex vivo PDO response and the actual chemotherapeutic response of the study patients in the real world (13 out of 15 cases). In addition, whole-transcriptome sequencing identified candidate genes associated with nab-paclitaxel resistance, such as ITGB7, ANPEP, and ST3GAL1. CONCLUSIONS: This PDAC PDO platform allows several therapeutic drugs to be tested within a short time window and opens the possibility for timely personalized medicine as a "patient avatar model" in clinical practice.

Clinical characteristics and prognostic factors of non-tuberculous mycobacterial disease in patients with rheumatoid arthritis.

BACKGROUND/AIMS: This study aimed to identify the clinical characteristics of patients with concurrent rheumatoid arthritis (RA) and suspected non-tuberculous mycobacterial (NTM) infections as well as determine their prognostic factors. METHODS: We retrospectively reviewed the medical records of 91 patients with RA whose computed tomography (CT) findings suggested NTM infection. Subsequently, we compared the clinical characteristics between patients with and without clinical or radiological exacerbation of NTM-pulmonary disease (PD) and investigated the risk factors for the exacerbation and associated mortality. RESULTS: The mean age of patients with RA and suspected NTM-PD was 65.0 ± 10.2 years. The nodular/bronchiectatic (NB) form of NTM-PD was the predominant radiographic feature (78.0%). During follow-up, 36 patients (41.9%) experienced a radiological or clinical exacerbation of NTM-PD, whereas 12 patients (13.2%) died. Combined interstitial lung disease (ILD), microbiologically confirmed NTM-PD, and NB with the fibrocavitary (FC) form on chest CT were identified as risk factors for the clinical or radiological exacerbation of NTM-PD. Hydroxychloroquine use was identified as a good prognostic factor. Conversely, history of tuberculosis, ILD, smoking, microbiologically confirmed NTM-PD, and NB with the FC form on chest CT were identified as poor prognostic factors for mortality in suspected NTM-PD. CONCLUSION: ILD and NB with the FC form on chest CT were associated with NTM-PD exacerbation and mortality. Hydroxychloroquine use may lower the risk of NTM-PD exacerbation. Therefore, radiographic features and presence of ILD should be considered when predicting the prognosis of patients with RA and suspected NTM-PD.

Genomic hypomethylation in cell-free DNA predicts responses to checkpoint blockade in lung and breast cancer.

Genomic hypomethylation has recently been identified as a determinant of therapeutic responses to immune checkpoint blockade (ICB). However, it remains unclear whether this approach can be applied to cell-free DNA (cfDNA) and whether it can address the issue of low tumor purity encountered in tissue-based methylation profiling. In this study, we developed an assay named iMethyl, designed to estimate the genomic hypomethylation status from cfDNA. This was achieved through deep targeted sequencing of young LINE-1 elements with > 400,000 reads per sample. iMethyl was applied to a total of 653 ICB samples encompassing lung cancer (cfDNA n = 167; tissue n = 137; cfDNA early during treatment n = 40), breast cancer (cfDNA n = 91; tissue n = 50; PBMC n = 50; cfDNA at progression n = 44), and ovarian cancer (tissue n = 74). iMethyl-liquid predicted ICB responses accurately regardless of the tumor purity of tissue samples. iMethyl-liquid was also able to monitor therapeutic responses early during treatment (3 or 6 weeks after initiation of ICB) and detect progressive hypomethylation accompanying tumor progression. iMethyl-tissue had better predictive power than tumor mutation burden and PD-L1 expression. In conclusion, our iMethyl-liquid method allows for reliable noninvasive prediction, early evaluation, and monitoring of clinical responses to ICB therapy.

Effects of Magnoliae Flos on Atopic Dermatitis-Like Inflammation Evaluated via Extracellular Signal-regulated Kinase or Signal Transducers and Activators of Transcription 1/3 Signalling Pathways.

Atopic dermatitis is a chronic inflammatory skin  disease. Skin is the largest organ and plays a pivotal role in protecting the body. Not only does the skin act as a physical barrier against the external environment, but it also has its own immune system. Atopic dermatitis is caused by prolonged excessive inflammatory responses that worsen under imbalanced cutaneous immune system skin conditions. Although the prevalence and burden of atopic dermatitis is increasing, the standard therapeutic agents remain unclear due to  the complicated pathophysiology of the condition. The objective of this study is to examine the use of Magnoliae flos, the dried flower bud of Magnolia biondii or  related plants. The effects and underlying mechanism of  action of aqueous extract of the buds of Magnoliae flos (MF) were evaluated. Immortalized human keratinocytes (HaCaT) stimulated with tumour necrosis factor-α and interferon-γ mixture and NC/Nga mice stimulated with 2,4-dinitrochlorobenzene were used as atopic dermatitis models, in vitro and in vivo, respectively. The effects of MF were determined by measuring the suppression of pro-inflammatory signalling pathways, such as extracellular signal-regulated kinase or signal transducers and activators of transcription 1/3 and restoring skin barrier molecules. In conclusion, MF is a potential therapeutic alternative for the treatment of atopic dermatitis through repressing inflammatory pathways.

Evaluation of ciliary cleft changes after phacoemulsification using ultrasound biomicroscopy in dogs with cataracts.

Introduction: Glaucoma is one of the most serious complications that causes irreversible blindness after phacoemulsification in dogs; however, a clear mechanism has not been elucidated. This study aimed to analyse the possible anatomical factors associated with glaucoma after phacoemulsification using parameters that reflect the anatomical characteristics of dogs. Materials and methods: A total of 69 eyes of 48 dogs were included in this study. The patients were divided into three groups: normal eye (n = 18), cataract (n = 39), and post-phacoemulsification for at least 2 months after surgery (post-phaco, n = 12). For further analysis, the dogs were subdivided into two groups according to cataract stage: phacoemulsification non-candidate and candidate groups. Non-cataracts and incipient cataracts were categorized into the non-candidate group, whereas immature and mature cataracts were categorized into the candidate group. Measurements of the ciliary cleft parameters, including the area of the ciliary cleft (CCA), length of the ciliary cleft (CCL), width of the ciliary cleft (CCW), iridocorneal angle, and angle opening distance, were obtained using ultrasound biomicroscopy. Results: CCA, CCL, and CCW were significantly higher in the candidate group than in the non-candidate group. CCA, CCL, and CCW were significantly reduced in the post-phaco group compared to those in the cataract group. Based on these results, we found that the ciliary cleft expanded in cataract-affected eyes and narrowed after phacoemulsification. This may indicate that the space between the trabecular meshworks became narrower, potentially leading to an increase in the resistance of the aqueous humor. Conclusion: A narrowed ciliary cleft after phacoemulsification may be an anatomical factor associated with glaucoma.

Genetic assessment of pathogenic germline alterations in lysosomal genes among Asian patients with pancreatic ductal adenocarcinoma.

BACKGROUND: Lysosomes are closely linked to autophagic activity, which plays a vital role in pancreatic ductal adenocarcinoma (PDAC) biology. The survival of PDAC patients is still poor, and the identification of novel genetic factors for prognosis and treatment is highly required to prevent PDAC-related deaths. This study investigated the germline variants related to lysosomal dysfunction in patients with PDAC and to analyze whether they contribute to the development of PDAC. METHODS: The germline putative pathogenic variants (PPV) in genes involved in lysosomal storage disease (LSD) was compared between patients with PDAC (n = 418) and healthy controls (n = 845) using targeted panel and whole-exome sequencing. Furthermore, pancreatic organoids from wild-type and KrasG12D mice were used to evaluate the effect of lysosomal dysfunction on PDAC development. RNA sequencing (RNA-seq) analysis was performed with established PDAC patient-derived organoids (PDOs) according to the PPV status. RESULTS: The PPV in LSD-related genes was higher in patients with PDAC than in healthy controls (8.13 vs. 4.26%, Log2 OR = 1.65, P = 3.08 × 10-3). The PPV carriers of LSD-related genes with PDAC were significantly younger than the non-carriers (mean age 61.5 vs. 65.3 years, P = 0.031). We further studied a variant of the lysosomal enzyme, galactosylceramidase (GALC), which was the most frequently detected LSD variant in our cohort. Autophagolysosomal activity was hampered when GALC was downregulated, which was accompanied by paradoxically elevated autophagic flux. Furthermore, the number of proliferating Ki-67+ cells increased significantly in pancreatic organoids derived from Galc knockout KrasG12D mice. Moreover, GALC PPV carriers tended to show drug resistance in both PDAC cell line and PDAC PDO, and RNA-seq analysis revealed that various metabolism and gene repair pathways were upregulated in PDAC PDOs harboring a GALC variant. CONCLUSIONS: Genetically defined lysosomal dysfunction is frequently observed in patients with young-onset PDAC. This might contribute to PDAC development by altering metabolism and impairing autophagolysosomal activity, which could be potentially implicated in therapeutic applications for PDAC.

Anti-atopic dermatitis effect of fish collagen on house dust mite-induced mice and HaCaT keratinocytes.

Collagen, a major structural protein in mammalian tissues, is effective against skin wounds and osteoarthritis. Although bovine and porcine collagens have mainly been used, several potential risks of mammalian collagen have led to the use of fish collagen (FC) as an alternative. FC and its peptides are used as common cosmeceutical products because of their antihypertensive, anti-bacterial, and antioxidant activities. Despite the effects of FC on wrinkle reduction, UV-protection, and wound healing, the relationship between FC and atopic dermatitis (AD) has not yet been reported. Therefore, we investigated the anti-AD effects of FC against house dust mite (Dermatophagoides farinae, HDM)-induced AD in NC/Nga mice and TNF-α/IFN-γ-stimulated HaCaT keratinocytes. FC alleviated AD apparent symptoms, such as dermatitis score, transepidermal water loss, epidermal thickness, and mast cell infiltration upon declining pro-inflammatory cytokines and mediators, IL-6, IL-5, IL-13, TSLP, and TNF-α. The skin barrier protein, filaggrin, was also recovered by FC administration in vivo and in vitro. Immune response and skin barrier dysfunction are both mitigated by three routes of FC administration: oral, topical, and both routes via the regulation of IκB, MAPKs, and STATs pathways. In summary, FC could be a potential therapeutic agent for AD by regulating immune balance and skin barrier function.

Preventive Effects of Exosome-Rich Conditioned Medium From Amniotic Membrane-Derived Mesenchymal Stem Cells for Diabetic Retinopathy in Rats.

Purpose: Diabetic retinopathy (DR) is an important disease that causes vision loss in many diabetic patients. Stem cell therapy has been attempted for treatment of this disease; however, it has some limitations. This study aimed to evaluate the preventive efficacy of exosome-rich conditioned medium (ERCM) derived from amniotic membrane stem cells for DR in rats. Methods: Twenty-eight 8-week-old male Sprague-Dawley rats were divided into three groups: group 1, normal control (Con) group; group 2, diabetes mellitus (DM) group; and group 3, DM with ERCM-treated (DM-ERCM) group. DM was induced by intraperitoneal injection of streptozotocin. The DM-ERCM group received ERCM containing 1.2 × 109 exosomes into subconjunctival a total of four times every 2 weeks. Results: On electroretinogram, the DM-ERCM group had significantly higher b-wave and flicker amplitudes than those in the DM group. In fundoscopy, retinal vascular attenuation was found in both the DM and DM-ERCM groups; however, was more severe in the DM group. On histology, the ganglion cell and nerve fiber layer rates of the total retinal layer significantly increased in the DM group compared with the Con group, whereas the DM-ERCM group showed no significant difference compared with the Con group. Cataracts progressed significantly more in the DM group than that in the DM-ERCM group and there was no uveitis in the DM-ERCM group. Conclusions: Subconjunctival ERCM delayed the progression of DR and cataracts and significantly reduced the incidence of uveitis. Translational Relevance: Our study shows the clinical potential of minimally invasive exosome-rich conditioned medium treatment to prevent diabetic retinopathy.

Modified Technique of Chemoport Placement in Severely Obese Patient for Administration of Chemotherapy.

A common site of chemoport implantation is the anterior chest wall. However, it is difficult to needle chemoport and to maintain needles in severely obese patients. Because the skin is thick, it is difficult to find the port and the needle easily comes off. We describe a different safe and easy to replicate method of chemoport placement in a severely obese patient. We placed the chemopot directly above the sternum. It is particularly more useful for very obese patients. This technique is safe and an easy to replicate method of chemoport placement.

Anti-cancer effects of metformin in a 3D co-culture model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) presents with condensed stroma that contributes to its high invasive capability. Although metformin adjuvant treatment has been suggested to improve the survival times of patients with PDAC, the mechanism responsible for that benefit has been investigated only in two-dimensional cell lines. We assessed the anti-cancer effect of metformin in a three-dimensional (3D) co-culture model to quantify the migration behavior of patient-derived PDAC organoids and primary pancreatic stellate cells (PSCs). At a concentration of 10 µM, metformin reduced the migratory ability of the PSCs by downregulating the expression of matrix metalloproteinase-2 (MMP2). In the 3D direct co-cultivation of PDAC organoids and PSCs, metformin attenuated the transcription of cancer stemness-related genes. The reduced stromal migratory ability of PSCs was associated with the downregulation of MMP2, and MMP2 knockdown in PSCs reproduced their attenuated migratory ability. The anti-migration effect of a clinically relevant concentration of metformin was demonstrable in a 3D indirect co-culture model of PDAC consisting of patient-derived PDAC organoids and primary human PSCs. The metformin suppressed PSC migration via MMP2 downregulation and attenuated cancer stemness factors. Furthermore, oral administration of metformin (30 mg/kg) strikingly suppressed the growth of PDAC organoids xenograft in immunosuppressed mice. These results indicate metformin could offer the potential approach as an effective therapeutic drug for PDAC.

Antibodies against endogenous retroviruses promote lung cancer immunotherapy.

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS)1,2. Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive1,2. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma3. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response.

Intracellular Adhesion Molecule-1 Improves Responsiveness to Immune Checkpoint Inhibitor by Activating CD8+ T Cells.

Immune checkpoint inhibitor (ICI) clinically benefits cancer treatment. However, the ICI responses are only achieved in a subset of patients, and the underlying mechanisms of the limited response remain unclear. 160 patients with non-small cell lung cancer treated with anti-programmed cell death protein-1 (anti-PD-1) or anti-programmed death ligand-1 (anti-PD-L1) are analyzed to understand the early determinants of response to ICI. It is observed that high levels of intracellular adhesion molecule-1 (ICAM-1) in tumors and plasma of patients are associated with prolonged survival. Further reverse translational studies using murine syngeneic tumor models reveal that soluble ICAM-1 (sICAM-1) is a key molecule that increases the efficacy of anti-PD-1 via activation of cytotoxic T cells. Moreover, chemokine (CXC motif) ligand 13 (CXCL13) in tumors and plasma is correlated with the level of ICAM-1 and ICI efficacy, suggesting that CXCL13 might be involved in the ICAM-1-mediated anti-tumor pathway. Using sICAM-1 alone and in combination with anti-PD-1 enhances anti-tumor efficacy in anti-PD-1-responsive tumors in murine models. Notably, combinatorial therapy with sICAM-1 and anti-PD-1 converts anti-PD-1-resistant tumors to responsive ones in a preclinical study. These findings provide a new immunotherapeutic strategy for treating cancers using ICAM-1.

Bimetallic Hyaluronate-Modified Au@Pt Nanoparticles for Noninvasive Photoacoustic Imaging and Photothermal Therapy of Skin Cancer.

Although spherical gold (Au) nanoparticles have remarkable photothermal conversion efficiency and photostability, their weak absorption in the near-infrared (NIR) region and poor penetration into deep tissues have limited further applications to NIR light-mediated photoacoustic (PA) imaging and noninvasive photothermal cancer therapy. Here, we developed bimetallic hyaluronate-modified Au-platinum (HA-Au@Pt) nanoparticles for noninvasive cancer theranostics by NIR light-mediated PA imaging and photothermal therapy (PTT). The growth of Pt nanodots on the surface of spherical Au nanoparticles enhanced the absorbance in the NIR region and broadened the absorption bandwidth of HA-Au@Pt nanoparticles by the surface plasmon resonance (SPR) coupling effect. In addition, HA facilitated the transdermal delivery of HA-Au@Pt nanoparticles through the skin barrier and enabled clear tumor-targeted PA imaging. Compared to conventional PTT via injection, HA-Au@Pt nanoparticles were noninvasively delivered into deep tumor tissues and completely ablated the targeted tumor tissues by NIR light irradiation. Taken together, we could confirm the feasibility of HA-Au@Pt nanoparticles as a NIR light-mediated biophotonic agent for noninvasive skin cancer theranostics.

Generation of multilineage liver organoids with luminal vasculature and bile ducts from human pluripotent stem cells via modulation of Notch signaling.

BACKGROUND: The generation of liver organoids recapitulating parenchymal and non-parenchymal cell interplay is essential for the precise in vitro modeling of liver diseases. Although different types of multilineage liver organoids (mLOs) have been generated from human pluripotent stem cells (hPSCs), the assembly and concurrent differentiation of multiple cell types in individual mLOs remain a major challenge. Particularly, most studies focused on the vascularization of mLOs in host tissue after transplantation in vivo. However, relatively little information is available on the in vitro formation of luminal vasculature in mLOs themselves. METHODS: The mLOs with luminal blood vessels and bile ducts were generated by assembling hepatic endoderm, hepatic stellate cell-like cells (HscLCs), and endothelial cells derived entirely from hPSCs using 96-well ultra-low attachment plates. We analyzed the effect of HscLC incorporation and Notch signaling modulation on the formation of both bile ducts and vasculature in mLOs using immunofluorescence staining, qRT-PCR, ELISA, and live-perfusion imaging. The potential use of the mLOs in fibrosis modeling was evaluated by histological and gene expression analyses after treatment with pro-fibrotic cytokines. RESULTS: We found that hPSC-derived HscLCs are crucial for generating functional microvasculature in mLOs. HscLC incorporation and subsequent vascularization substantially reduced apoptotic cell death and promoted the survival and growth of mLOs with microvessels. In particular, precise modulation of Notch signaling during a specific time window in organoid differentiation was critical for generating both bile ducts and vasculature. Live-cell imaging, a series of confocal scans, and electron microscopy demonstrated that blood vessels were well distributed inside mLOs and had perfusable lumens in vitro. In addition, exposure of mLOs to pro-fibrotic cytokines induced early fibrosis-associated events, including upregulation of genes associated with fibrotic induction and endothelial cell activation (i.e., collagen I, α-SMA, and ICAM) together with destruction of tissue architecture and organoid shrinkage. CONCLUSION: Our results demonstrate that mLOs can reproduce parenchymal and non-parenchymal cell interactions and suggest that their application can advance the precise modeling of liver diseases in vitro.