Case report: Diverse immune responses in advanced pancreatic ductal adenocarcinoma treated with immune checkpoint inhibitor-based conversion therapies.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at an advanced stage, presenting limited therapeutic options and a grim prognosis due to its aggressive nature. Despite ongoing exploration of various combination therapies, a standardized treatment approach after the first-line treatment progress remains elusive. This report details the cases of two patients with unresectable advanced PDAC who underwent distinct conversion treatment regimens involving immune checkpoint inhibitors (ICIs). Remarkably, both patients became eligible for surgery following different anti-PD-1 antibody-based conversion therapies, ultimately achieving R0 resection. In essence, our findings highlight the efficacy of the anti-PD-1 antibody combined with a tyrosine kinase inhibitor (TKI) regimen and chemotherapy alongside anti-PD-1 antibody as viable conversion therapies for preoperative advanced PDAC. Tumor immune microenvironment (TIME) analysis underscores the intratumoral and intertumoral heterogeneity observed in the postoperative immune landscape of surgical specimens. This insight contributes to a deeper understanding of the potential benefits of these conversion therapies in addressing the challenging landscape of advanced PDAC.

Zebrafish functional xenograft vasculature platform identifies PF-502 as a durable vasculature normalization drug.

Tumor vasculature often exhibits disorder and inefficiency. Vascular normalization offers potential for alleviating hypoxia and optimizing drug delivery in tumors. However, identifying effective agents is hindered by a lack of robust screening. We aimed to establish a comprehensive method using the zebrafish functional xenograft vasculature platform (zFXVP) to visualize and quantify tumor vasculature changes. Employing zFXVP, we systematically screened compounds, identifying PF-502 as a robust vascular normalization agent. Mechanistic studies showed PF-502 induces endothelial cell-cycle arrest, streamlines vasculature, and activates Notch1 signaling, enhancing stability and hemodynamics. In murine models, PF-502 exhibited pronounced vascular normalization and improved drug delivery at a sub-maximum tolerated dose. These findings highlight zFXVP's utility and suggest PF-502 as a promising adjunctive for vascular normalization in clinical settings.

Targeting Microglial Metabolic Rewiring Synergizes with Immune-Checkpoint Blockade Therapy for Glioblastoma.

Glioblastoma (GBM) constitutes the most lethal primary brain tumor for which immunotherapy has provided limited benefit. The unique brain immune landscape is reflected in a complex tumor immune microenvironment (TIME) in GBM. Here, single-cell sequencing of the GBM TIME revealed that microglia were under severe oxidative stress, which induced nuclear receptor subfamily 4 group A member 2 (NR4A2)-dependent transcriptional activity in microglia. Heterozygous Nr4a2 (Nr4a2+/-) or CX3CR1+ myeloid cell-specific Nr4a2 (Nr4a2fl/flCx3cr1Cre) genetic targeting reshaped microglia plasticity in vivo by reducing alternatively activated microglia and enhancing antigen presentation capacity for CD8+ T cells in GBM. In microglia, NR4A2 activated squalene monooxygenase (SQLE) to dysregulate cholesterol homeostasis. Pharmacologic NR4A2 inhibition attenuated the protumorigenic TIME, and targeting the NR4A2 or SQLE enhanced the therapeutic efficacy of immune-checkpoint blockade in vivo. Collectively, oxidative stress promotes tumor growth through NR4A2-SQLE activity in microglia, informing novel immune therapy paradigms in brain cancer. SIGNIFICANCE: Metabolic reprogramming of microglia in GBM informs synergistic vulnerabilities for immune-checkpoint blockade therapy in this immunologically cold brain tumor. This article is highlighted in the In This Issue feature, p. 799.

Regenerative failure of intrahepatic biliary cells in Alagille syndrome rescued by elevated Jagged/Notch/Sox9 signaling.

Despite the robust healing capacity of the liver, regenerative failure underlies numerous hepatic diseases, including the JAG1 haploinsufficient disorder, Alagille syndrome (ALGS). Cholestasis due to intrahepatic duct (IHD) paucity resolves in certain ALGS cases but fails in most with no clear mechanisms or therapeutic interventions. We find that modulating jag1b and jag2b allele dosage is sufficient to stratify these distinct outcomes, which can be either exacerbated or rescued with genetic manipulation of Notch signaling, demonstrating that perturbations of Jag/Notch signaling may be causal for the spectrum of ALGS liver severities. Although regenerating IHD cells proliferate, they remain clustered in mutants that fail to recover due to a blunted elevation of Notch signaling in the distal-most IHD cells. Increased Notch signaling is required for regenerating IHD cells to branch and segregate into the peripheral region of the growing liver, where biliary paucity is commonly observed in ALGS. Mosaic loss- and-gain-of-function analysis reveals Sox9b to be a key Notch transcriptional effector required cell autonomously to regulate these cellular dynamics during IHD regeneration. Treatment with a small-molecule putative Notch agonist stimulates Sox9 expression in ALGS patient fibroblasts and enhances hepatic sox9b expression, rescues IHD paucity and cholestasis, and increases survival in zebrafish mutants, thereby providing a proof-of-concept therapeutic avenue for this disorder.

The inhibition of protein translation promotes tumor angiogenic switch.

The 'angiogenic switch' is critical for tumor progression. However, the pathological details and molecular mechanisms remain incompletely characterized. In this study, we established mammal xenografts in zebrafish to visually investigate the first vessel growth (angiogenic switch) in real-time, by inoculating tumor cells into the perivitelline space of live optically transparent Transgenic (flk1:EGFP) zebrafish larvae. Using this model, we found that hypoxia and hypoxia-inducible factor (HIF) signaling were unnecessary for the angiogenic switch, whereas vascular endothelial growth factor A gene (Vegfa) played a crucial role. Mechanistically, transcriptome analysis showed that the angiogenic switch was characterized by inhibition of translation, but not hypoxia. Phosphorylation of eukaryotic translation initiation factor 2 alpha (Eif2α) and the expression of Vegfa were increased in the angiogenic switch microtumors, and 3D tumor spheroids, and puromycin-treated tumor cells. Vegfa overexpression promoted early onset of the angiogenic switch, whereas Vegfa knockout prevented the first tumor vessel from sprouting. Pretreatment of tumor cells with puromycin promoted the angiogenic switch in vivo similarly to Vegfa overexpression, whereas Vegfa knockdown suppressed the increase. This study provides direc and dynamic in vivo evidences that inhibition of translation, but not hypoxia or HIF signaling promotes the angiogenic switch in tumor by increasing Vegfa transcription.

Clinically relevant orthotopic xenograft models of patient-derived glioblastoma in zebrafish.

An accurate prediction of the intracranial infiltration tendency and drug response of individual glioblastoma (GBM) cells is essential for personalized prognosis and treatment for this disease. However, the clinical utility of mouse patient-derived orthotopic xenograft (PDOX) models remains limited given current technical constraints, including difficulty in generating sufficient sample numbers from small tissue samples and a long latency period for results. To overcome these issues, we established zebrafish GBM xenografts of diverse origin, which can tolerate intracranial engraftment and maintain their unique histological features. Subsequent single-cell RNA-sequencing (scRNA-seq) analysis confirmed significant transcriptional identity to that of invading GBM microtumors observed in the proportionally larger brains of model animals and humans. Endothelial scRNA-seq confirmed that the zebrafish blood-brain barrier is homologous to the mammalian blood-brain barrier. Finally, we established a rapid and efficient zebrafish PDOX (zPDOX) model, which can predict long-term outcomes of GBM patients within 20 days. The zPDOX model provides a novel avenue for precision medicine of GBM, especially for the evaluation of intracranial infiltration tendency and prediction of individual drug sensitivity.