Search Results (83)

Glioblastoma (GBM) is the most aggressive primary brain tumor with limited therapeutic options and extremely poor prognosis. Aberrant activation of the receptor tyrosine kinase MET drives tumor progression, therapeutic resistance, and reduced survival, particularly in the mesenchymal GBM subtype. Given its crucial role in GBM recurrence and progression, we investigated the mechanisms of resistance to MET inhibition using patient-derived glioma-initiating cells (GICs) and orthotopic xenograft mouse models. GICs were treated with the MET inhibitor crizotinib to elucidate the mechanism of adaptive resistance. Prolonged MET inhibition induced a senescent-like phenotype in GICs, associated with downregulation of BNIP3, a mitochondrial protein regulating mitophagy. We showed that BNIP3 downregulation led to activation of mTOR signaling, promoting cellular survival and adaptive resistance. Combining crizotinib with the mTOR inhibitor everolimus effectively suppressed mTOR activity, reduced cell viability, and induced mitochondrial alterations, apoptosis, and necroptosis. In orthotopic GBM xenograft models, combined MET and mTOR inhibition significantly prolonged survival compared with single-agent treatments. Notably, sequential treatment—crizotinib followed by everolimus—further enhanced therapeutic efficacy. These effects were achieved without significant weight loss, supporting tolerability of the treatment regimen. Our findings identify the BNIP3-mTOR axis as a critical mediator of resistance to MET inhibition and demonstrate that combined inhibition of MET and mTOR exhibits significant synergy against GBM. Full article

Background/Objectives: Colorectal cancer (CRC) is the second most common cancer-causing death in the United States. The Mediterranean diet, rich in extra-virgin olive oil (EVOO), is associated with a lower risk of colorectal cancer (CRC). Earlier studies reported S-(−)-oleocanthal (OC), the major EVOO phenolic, to be effective in CRC progression and recurrence suppression in a subcutaneous xenograft model by targeting the SMYD2–EZH2/c-MET signaling axis and favorably modulating gut microbiota (GM). However, lead validation in an orthotopically xenografted model that mimics the tumor microenvironment is yet to be achieved. The GM contribution to OC anti-CRC activity remains unknown. Methods: We used an orthotopic intra-cecally xenografted nude mouse model to validate OC anti-KRAS-mutant CRC activity and assessed the contribution of OC GM modulation to this activity using an oral antibiotic depletion strategy. Results: Daily oral 10 mg/kg OC impressively suppressed KRAS^G13D-mutant CRC HCT-116-Luc progression and metastasis more effectively than intraperitoneal administration 3×/week. By contrast, GM depletion with a broad-spectrum antibiotics cocktail (ABC) partially attenuated this activity, suggesting GM contribution to OC anti-CRC activity. Thus, fecal microbiota transplantation (FMT) was conducted using fresh daily oral fecal GM treatments collected from 20 mg/kg OC-dosed nude mouse donors. A week before FMT dosing, orthotopic HCT-116-Luc tumor-bearing recipient mice were subjected to GM depletion using daily oral ABC dosing. Recipient mice treated with FMT from OC-treated donors exhibited dramatic >99% reductions in primary and near-complete suppression of multi-organ metastatic tumor burden versus FMT controls. Conclusions: Collectively, oral OC is validated as an effective anti-KRAS^G13D-mutant CRC lead. Future clinical trials can validate its anti-CRC potential in a human model. Full article

Preclinical bladder cancer models are often judged by tumor take, tumor growth or treatment inhibition, yet these endpoints do not reveal which bladder-specific constraints a given model preserves or bypasses. The bladder is shaped by a specialized urothelial barrier, urine exposure, cyclic filling and emptying, inflammatory injury, metabolic stress and intravesical treatment pressure. In this review, we use susceptibility engineering as an organizing framework for model selection and validation. We define susceptibility engineering as the deliberate definition, perturbation and reporting of model states that alter tumor initiation, adhesion, colonization, survival or therapeutic exposure. This framework groups cell lines, patient-derived organoids, cell-line-derived xenograft (CDX) and patient-derived xenograft (PDX) models, orthotopic transplantation, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced tumors, genetically engineered mouse models and large-animal platforms according to the biological constraints they test. We focus on three linked dimensions: urothelial barrier integrity and uroplakin-related tools; local colonization thresholds under bladder-specific selection; metabolic susceptibility involving peroxisome proliferator-activated receptor gamma (PPARG)-associated differentiation programs and candidate solute carrier family 25 (SLC25)-linked mitochondrial stress nodes. We further distinguish large-animal systems as platforms for local delivery, imaging, device testing and procedural scale rather than universal substitutes for mouse models. A susceptibility-based validation framework could improve model selection, explain divergent responses across systems and support tiered platforms that connect patient-derived biology, mechanistic mouse studies and clinically realistic intravesical evaluation. Full article

Background and Objectives: Sevoflurane, a widely used inhalational anesthetic, is frequently administered during hepatocellular carcinoma (HCC) surgery, including hepatic resection and orthotopic liver transplantation. Because such procedures often require prolonged anesthetic exposure, the potential influence of sevoflurane on HCC cell behavior is of clinical interest. We aimed to evaluate the effects of sevoflurane on the proliferation and migration of HepG2 cells in vitro and on tumor growth in a xenograft mouse model in vivo, and to explore whether hypoxia-inducible factor-1α (HIF-1α) might be involved in this process. Materials and Methods: For the in vitro experiments, HepG2 cells were exposed to room air (0%), 2%, or 4% sevoflurane. A scratch wound healing assay was used to assess cell migration, and the number of viable cells was quantified by hemocytometer counting on day 4 to estimate proliferation. For the in vivo experiments, BALB/c nude mice bearing HepG2 xenografts were exposed to room air, 2% sevoflurane, or 4% sevoflurane for 3 h, three times weekly for 5 weeks. Tumor size and tumor weight were measured at the end of the exposure period. HIF-1α protein levels in tumor tissue were measured by enzyme-linked immunosorbent assay (ELISA) in tumor lysates and normalized to total tumor protein as an exploratory mechanistic analysis. Given the small sample available for this endpoint, the analysis had limited sensitivity to detect modest differences. Results: When wound closure was quantified and pooled across the analyzable experiments, no statistically significant difference was detected among the room air, 2% sevoflurane, and 4% sevoflurane groups (day-2 closure 19.9 ± 32.1%, 22.1 ± 25.8%, and 22.3 ± 28.8%, respectively; repeated-measures ANOVA p = 0.82), with variability dominated by between-experiment rather than treatment differences. In the proliferation assay, the number of viable HepG2 cells on day 4 was significantly lower in the 2% sevoflurane group (62.6 ± 3.3 × 10⁵) than in the room air group (68.5 ± 4.2 × 10⁵; p < 0.05); the 4% sevoflurane group (66.0 ± 3.2 × 10⁵) showed an intermediate value that did not reach statistical significance. In the xenograft model, mean tumor size in the room air, 2% sevoflurane, and 4% sevoflurane groups was 7.1 ± 1.9, 2.7 ± 2.0, and 2.1 ± 0.9 cm³, respectively (p = 0.041 for room air vs. 2% sevoflurane; p = 0.034 for room air vs. 4% sevoflurane). Tumor weight was likewise lower in the sevoflurane groups (room air, 7.88 ± 2.2 g; 2% sevoflurane, 2.95 ± 2.1 g; 4% sevoflurane, 2.3 ± 1.6 g; p = 0.044 for room air vs. 2% sevoflurane; p = 0.067 for room air vs. 4% sevoflurane). No statistically significant differences in tumor HIF-1α protein levels were observed among the three groups. Conclusions: In this exploratory study, sevoflurane exposure was associated with reduced HepG2 xenograft tumor growth in vivo, whereas its in vitro effects were more limited: a reduction in viable cell number was observed only at 2% sevoflurane, and an effect on cell migration could not be confirmed when analyzed across experiments. Tumor HIF-1α levels did not differ significantly between groups, suggesting that other molecular pathways may be involved. Further mechanistic and clinical studies are warranted before any conclusions can be drawn about the relevance of these findings to the perioperative management of patients with HCC. Full article

Background: Tumor–stroma interactions play a critical role in renal cell carcinoma (RCC) progression. Cancer-associated fibroblasts (CAFs) are considered key components of the tumor microenvironment; however, their origin remains controversial. This study aimed to determine whether bone marrow-derived mesenchymal stem cells (MSCs) contribute to CAF-like stromal changes and RCC progression. Methods: An orthotopic xenograft mouse model was established using luciferase- and GFP-labeled Caki-1 cells. MSCs labeled with PKH26 were administered intravenously. Tumor growth was evaluated using an in vivo imaging system and tumor volume measurements. Immunohistochemical analyses were performed to assess MSC localization and α-smooth muscle actin (α-SMA) expression. In vitro proliferation and migration assays were conducted using direct and indirect co-culture systems. Results: The intravenous administration of MSCs significantly increased tumor growth and bioluminescence intensity in an orthotopic model. The tumor volumes were significantly larger in the MSC-treated versus control group. An immunofluorescence analysis demonstrated partial co-localization of PKH26-labeled MSCs with α-SMA-positive fibroblast-like cells, suggesting acquisition of CAF-like features. Direct co-culture with MSCs significantly enhanced RCC cell proliferation and migration in vitro, whereas culturing in conditioned medium alone did not produce similar effects. Conclusions: Exogenously administered bone marrow-derived MSCs may be recruited into RCC tissues and acquire CAF-like features through interactions with tumor cells. These findings suggest that stromal–tumor cell interactions within the tumor microenvironment may contribute to RCC progression and represent a potential therapeutic target. Full article

Background: Adipocytes play a critical role in the breast cancer tumorigenic microenvironment. However, their effects and underlying mechanisms remain unclear. This study aims to investigate the role of adipocytes in luminal A breast cancer invasiveness at the cellular and molecular levels. Methods: Various adipocyte types were co-cultured with MCF7 breast cancer cells in direct and indirect manners. Invasiveness was assessed via proliferation, migration, and invasion, with alterations examined at morphological, cellular, and molecular levels. The role of adipocytes on MCF7 was further explored using an orthotopic breast cancer xenograft mouse model. Results: MCF7 co-cultured with adipocytes, especially brown adipocytes (BAC), showed increased invasiveness and tumorigenic potential. Morphologically, co-cultivation with BAC increased the proliferation, EMT, and stemness of MCF7. Mechanistically, co-culture of MCF7 with BAC exhibited disturbed expression of genes related to adipogenesis and mitochondrial dynamics; notably, IRX3 was the most prominently elevated one. Knockdown of IRX3 restored balanced mitochondrial function and reduced both the invasiveness of breast cancer cells in vitro and tumor growth in vivo. Conclusions: Brown adipocytes promote breast cancer invasiveness by upregulating adipogenesis-related IRX3, which acts via the mitochondrial functional regulation. Full article

Background/Objectives: Advanced renal cell carcinoma (aRCC) remains incurable, with no established optimal sequence of targeted therapies due to interpatient heterogeneity and acquired resistance. We developed a luciferase-enabled patient-derived orthotopic xenograft (PDOX) avatar platform to evaluate sequential targeted therapies in individualized aRCC models that recapitulate tumor architecture, proliferation, angiogenesis, metastasis, and PD-L1 expression. Methods: Tumor specimens from two renal cell carcinoma (RCC) patients were expanded subcutaneously in NOD/SCID mice, transduced with luciferase/red fluorescent protein (Luc/RFP), and orthotopically implanted into mouse kidneys (KiCa-Pt58: sarcomatoid RCC, pT3aN1M1, Fuhrman grade 4; KiCa-Pt118: clear cell RCC with sarcomatoid component, pT3aNxM0, Fuhrman grade 4, respectively). Tumor growth and metastasis were monitored weekly by bioluminescence imaging (BLI). Mice were randomized into vehicle control or four sequential treatment groups (Everolimus→Sunitinib [E→S], Sunitinib→Everolimus [S→E], Pazopanib→Sunitinib [P→S], Pazopanib→Everolimus [P→E]). Drugs were administered orally three times weekly until resistance (>200% BLI increase), with one switch. At necropsy, tumor burden, ex vivo BLI metastasis, weights, H&E histology, and immunohistochemistry (Ki67, CD44, CD31, PD-L1) were assessed. Results: Two independent experiments were performed. In dosing optimization, PDOX tumors recapitulated parental histology and proliferative indices, mirroring patient trajectories. KiCa-Pt58 (metastatic sarcomatoid RCC; deceased 1-month post-nephrectomy) showed aggressive features: rapid engraftment at low doses, early growth (week 2), and lung metastases in 78% of mice (sacrifice day 34), reflecting a fulminant course. KiCa-Pt118 (non-metastatic; patient recurrence-free >8 years post nephrectomy) exhibited indolent behavior: delayed engraftment requiring higher doses plus lymph node stromal (HK) support, slower growth (week 4), no metastases, and later sacrifice (day 78), consistent with remission. In sequential therapy evaluation, for KiCa-Pt58, P→E yielded greatest reductions in tumor weight (p < 0.01), lung metastases (p < 0.01), Ki67+ proliferation, CD31+ angiogenesis, and PD-L1 expression versus control; E→S and S→E were also effective. For KiCa-Pt118, S→E and P→E reduced tumor burden (p < 0.01) and Ki67⁺ proliferation; S→E lowered CD31 and PD-L1. Conclusions: This RCC PDOX platform faithfully preserves patient-specific biology—including metastatic propensity, engraftment efficiency, growth kinetics, and stromal dependency—while enabling real-time evaluation of sequential targeted therapies. Given the limited number of models tested, these findings provide proof-of-concept for individualized treatment exploration in advanced RCC and support future investigation of rational combinations with immune checkpoint blockade in humanized or immunocompetent systems. Full article

The poor clinical outcomes of pediatric high-grade glioma (pHGG) highlight the urgent need for new therapies. Oligodendrocyte lineage transcription factor 2 (OLIG2) is a pro-mitotic transcription factor highly expressed in glioma stem cells and may represent a novel therapeutic target. To evaluate the therapeutic efficacy of an OLIG2 inhibitor CT-179 in pHGG, we determined the OLIG2 mRNA expression in 10 patient-derived orthotopic xenograft (PDOX) models. In vitro activities of CT-179 were analyzed in monolayer and neurosphere cells (0–10 µM) with and without radiation (XRT) (0–8 Gy), brain penetration was evaluated in tumor-bearing PDOX mice, and in vivo efficacy was determined at 15–240 mg/kg (oral) alone or combined with XRT (2 Gy/day × 5 days). Changes in animal survival times were analyzed using the Kaplan–Meier method, followed by pair-wise comparisons. Increased OLIG2 mRNA expression was detected in seven out of ten PDOX models. CT-179 inhibited cell viability in a time- and dose-dependent manner in all eight pGBM xenograft tumors (IC₅₀ 0.03–10 µM) and was potentiated by XRT (0.03–1 µM). Oral gavage (24 mg/kg) of CT-179 for 5 days led to effective penetration in mouse cerebrum (3232.7 ± 569.2 ng/g), cerebellum (1563.3 ± 269.6 ng/g), brain stem (1685.3 ± 309 ng/g), and PDOX tumors (1814 ± 110.3 ng/g) vs. 361.3 ± 1.5 ng/mL in serum. CT-179 alone was not active at 200 mg/kg in four models, although it was moderately effective at 240 mg/kg in one model. When combined with XRT, a significant extension of animal survival times was observed in two out of four models. Doses needed to eliminate OLIG2 expression in vitro varied from 0.3 to >1 µM in pGBM cells. In summary, our data showed that orally administered CT-179 penetrated the blood–brain barrier (BBB) and exhibited potential for inhibiting pGBM growth when combined with XRT. Full article

Background/Objectives: Glioblastoma (GBM) is an aggressive primary brain tumor with dismal prognosis and limited treatment options. Immunotherapy, including personalized approaches using tumor-infiltrating lymphocytes (TILs) and allogeneic natural (NK) or engineered killer cells (chimeric antigen receptor NK, NK-CAR), and oncolytic viruses (OV), has shown some potential in GBM. Combining different therapeutic strategies may enhance treatment efficacy. Here, we present a xenograft GBM mouse model with multiparametric detection for various immunotherapy research applications. Methods: In a xenograft GBM NOD-Prkdcs scid Il2rgem1/Smoc (NSG) mouse model based on orthotopic transplantation of patient-derived GBM cultures retaining tumor heterogeneity, intravenous and intratumor immunotherapeutic interventions by TIL and OV therapy were performed. Xenograft engraftment was evaluated using intravital MRI; delivery of OV and TILs to the tumor and changes in the tumor and peritumoral space were assessed using intravital confocal microscopy; and metabolic and structural changes in the tumor and peritumoral environment were assessed via fluorescence lifetime imaging microscopy (FLIM) and optical coherence tomography (OCT). The intravital imaging data were compared with the results of preliminary and final histological and immunocytochemical data. Results: Both OV and TILs demonstrated tumor-specific targeting and delivery across the blood–brain barrier. Further, we showed that in this model the xenograft response to both therapeutic treatments can be assessed using FLIM and OCT. Conclusions: Overall, this work presents an optimized mouse model suitable for assessing the effect of combined TIL immunotherapy and OV on GBM in translational studies. Full article

Background/Objectives: High-grade gliomas (HGGs), including glioblastomas, are among the most aggressive brain tumors due to their high intratumoral heterogeneity and extensive infiltration. Glioma stem-like cells (GSCs) frequently invade along white matter tracts such as the corpus callosum, but the molecular programs driving this region-specific invasion remain poorly defined. The aim of this study was to identify transcriptional signatures associated with GSC infiltration into the corpus callosum. Methods: We established an orthotopic xenograft model by implanting fluorescently labeled human GSCs into nude mouse brains. Tumor growth and invasion patterns were assessed using tissue clearing, light-sheet fluorescence microscopy, and histological analyses. To characterize region-specific molecular profiles, we performed microfluidic-based single-cell RNA expression analysis of 48 invasion- and stemness-related genes in cells isolated from the tumor bulk (TB) and corpus callosum (CC). Results: By six weeks post-implantation, GSCs displayed marked tropism for the corpus callosum, with distinct infiltration patterns captured by three-dimensional imaging. Single-cell gene expression profiling revealed significant differences in 7 of the 48 genes (14.6%) between TB- and CC-derived GSCs. These genes—NES, CCND1, GUSB, NOTCH1, E2F1, EGFR, and TGFB1—collectively defined a “corpus callosum invasion signature” (CC-Iv). CC-derived cells showed a unimodal, high-expression profile of CC-Iv genes, whereas TB cells exhibited bimodal distributions, suggesting heterogeneous transcriptional states. Importantly, higher CC-Iv expression correlated with worse survival in patients with low-grade gliomas. Conclusions: This multimodal approach identified a corpus callosum-specific invasion signature in glioma stem-like cells, revealing how local microenvironmental cues shape transcriptional reprogramming during infiltration. These findings provide new insights into the spatial heterogeneity of gliomas and highlight potential molecular targets for therapies designed to limit tumor spread through white matter tracts. Full article

Background: Metastasis continues to be a leading cause of mortality in osteosarcoma (OS) among pediatric and young adult populations, with few effective therapeutic options available. Despite immunotherapy advancements, its efficacy in OS is hindered by an incomplete understanding of the immunosuppressive tumor microenvironment (TME). Methods: We utilized multiplex imaging mass cytometry and phenoplexing to characterize immune and stromal cell populations within the TME of a tissue microarray comprising 51 primary OS tumors. The prognostic significance of TME cell abundance and spatial cell–cell distance was evaluated using Kaplan–Meier and Cox regression analyses. To investigate macrophage functionality in vivo, we employed orthotopic xenograft mouse models by co-injecting THP-1-derived M0 or M2 macrophages with 143B OS cells to assess their impact on tumor growth and pulmonary metastasis. Mechanisms of macrophage-mediated metastasis were explored using Luminex, ELISA, and transwell migration assays. Results: Our results showed that macrophages dominated the TME, with M0 and M2 subtypes significantly outnumbering M1 macrophages (M1) and other myeloid cells. T cells and myeloid-derived suppressor cells (MDSC) were the second and third most abundant immune populations, respectively. Among stromal cells, endothelial cells predominated over fibroblasts. While individual immunosuppressive cell populations (M2, MDSC, and Treg) showed no direct correlation with clinical outcomes, the collective abundance of M2 and MDSC was significantly associated with reduced metastasis-free survival (MFS, p = 0.0244) and recurrence-free survival (RFS, p = 0.0040). Notably, closer spatial proximity between M2 macrophages and immunosuppressive cells (p = 0.0248) or Ki-67⁺ cells (p = 0.0321) correlated with decreased MFS, suggesting the formation of an M2-centric immunosuppressive and pro-tumor hub. In vivo, co-injection of M2 macrophages with 143B cells significantly enhanced pulmonary metastasis (p = 0.0140). Luminex analysis identified M2-derived MIP-1α (CCL3) as a candidate chemokine driving OS cell metastatic potential. Conclusions: This study provides a high-resolution map of the OS TME, highlighting the prognostic significance of M2 and immunosuppressive cell interactions in driving metastasis, potentially through MIP-1α signaling. These findings establish a foundation for developing targeted immunotherapies to improve outcomes in metastatic OS. Full article

Background: To overcome the limitations of conventional CRC (colorectal cancer) mouse models in replicating metastasis and enabling efficient therapeutic evaluation, we developed a novel implantation method using tissue adhesive to establish reproducible orthotopic and metastatic tumors. Conventional models using injection or suturing techniques often suffer from technical complexity, inconsistent tumor establishment, and limited metastatic reliability. Methods: We developed and validated a novel orthotopic and metastatic CRC model utilizing tissue adhesive for tumor transplantation. Uniform tumor fragments derived from bioluminescent HCT116/Luc xenografts were affixed to the cecum of nude mice. Tumor growth and metastasis were monitored through bioluminescence imaging and confirmed by the results of histological analysis of metastatic lesions. The model’s utility for therapeutic testing was evaluated using MK801, an NMDA receptor antagonist. Results: The biological-based model demonstrated rapid and reproducible tumor implantation (<5 min), consistent primary tumor growth, and robust metastasis to the liver and lungs. The biological-based approach achieved 80% tumor engraftment (4/5), with consistent metastasis to the liver and lungs in all mice, compared with lower and variable metastasis rates in injection (0%, 0/5) and suturing (20%, 1/5) methods. MK801 treatment significantly suppressed both primary tumor growth and metastasis, validating the model’s suitability for preclinical drug evaluation. Conclusions: By enabling rapid, reproducible, and spontaneous formation of metastatic lesions using a minimally invasive tissue adhesive technique, our model represents a significant methodological advancement that supports high-throughput therapeutic screening and bridges the gap between experimental modeling and clinical relevance in colorectal cancer research. Full article

Pancreatic cancer is the third leading cause of cancer-related death in the US. First-line chemotherapy regimens for pancreatic ductal adenocarcinoma (PDAC) include FOLFIRINOX or gemcitabine (Gem) with or without paclitaxel (Ptx); however, 5-year survival with these regimens remains poor. Previous work has demonstrated protein arginine methyltransferase 5 (PRMT5) to be a promising therapeutic target in combination with Gem for the treatment of PDAC; however, these findings have yet to be confirmed in relevant preclinical models of PDAC. To test the possibility of PRMT5 as a viable therapeutic target, clinically relevant orthotopic and metastatic patient-derived xenograft (PDX) mouse models of PDAC growth were utilized to evaluate the effect of PRMT5 knockout (KO) or pharmacologic inhibition on treatment with Gem alone or Gem with Ptx. Primary endpoints included tumor volume, tumor weight, or metastatic tumor burden as appropriate. The results showed that Gem-treated PRMT5 KO tumors exhibited decreased growth and were smaller in size compared to Gem-treated wild-type (WT) tumors. Similarly, the Gem-treated PRMT5 KO metastatic burden was lower than the Gem-treated WT metastatic burden. The addition of a PRMT5 pharmacologic inhibitor to Gem and Ptx therapy resulted in a lower final tumor weight and fewer metastatic tumors. The depletion of PRMT5 results in increased DNA damage in response to Gem and Ptx treatment. Thus, PRMT5 genetic depletion or inhibition in combination with Gem-based therapy improved the response in primary and metastatic PDAC in clinically relevant mouse models, suggesting that PRMT5 is a viable therapeutic target for combination therapy in PDAC. Full article

Background: Pancreatic cancer is the most lethal of all human cancers. The disease has no obvious symptoms in its early stages and in the majority of cases, the cancer goes undetected until it has advanced to the point that surgery is no longer a viable option or until it has metastasized to other organs. The absence of reliable and sensitive biomarkers for the early detection of pancreatic cancer contributes to the poor ability to detect the disease before it progresses to an untreatable stage. Objectives: Here, an orthotopic xenograft mouse model of pancreatic cancer was investigated to determine if urinary metabolic biomarkers could be identified and used to detect the early formation of pancreatic tumors. Methods: The orthotopic xenograft mouse model of pancreatic cancer was established by injecting human MiaPaCa-2 cells, derived from a male patient aged 65 years with pancreatic adenocarcinoma, into the pancreata of severe combined immunodeficient mice. Orthotopic pancreatic tumors, allowed to grow for eight weeks, were successfully established in the pancreata in 15 out of 20 mice. At the time of sacrifice, tumors were excised and histologically analyzed and the masses and volumes recorded. Urine samples were collected prior to injection, at one-week post injection, and every two weeks afterwards for eight weeks. Results: NMR-based metabolic profiling of the urine samples indicated that 31 metabolites changed significantly over the course of tumor initiation and growth. Longitudinal metabolic profiling analysis indicated an initial increase in activity of the metabolic pathways involved in energy production and/or cell synthesis by cancer cells as required to support tumor growth that was followed by a diminished difference between control and orthotopic mice associated with tumor senescence as the tumors reached 7–8 weeks post injection. Conclusions: The results indicate that NMR-based urinary metabolic profiling may be able to detect the earliest stages of pancreatic tumor initiation and growth, highlighting the potential for translation to human clinical studies. Full article

Background/Objective: Prostate cancer (PCa) remains a prevalent and deadly disease, particularly in its advanced stages. Despite various available treatments, resistance to drugs like enzalutamide continues to present significant challenges. This study aimed to investigate the role of Galectin-1 (Gal-1) in enzalutamide-resistant PCa and assess its potential as a therapeutic target to overcome resistance. Methods: The study utilized specific siRNA-mediated knockdown of Gal-1 in enzalutamide-resistant PCa cells to evaluate its effects on cell proliferation and response to enzalutamide treatment. An orthotopic mouse model was employed to examine the in vivo impact of Gal-1 knockdown. Pharmacological targeting of Gal-1 was conducted using LLS30, and its effects were assessed both in vitro and in vivo. RNA sequencing (RNA-seq) analysis was performed to explore the molecular mechanisms underlying the observed effects. Results: The findings demonstrated significant upregulation of Gal-1 in enzalutamide-resistant PCa cells. Gal-1 knockdown inhibited cell proliferation and resensitized resistant cells to enzalutamide treatment in the orthotopic mouse model. Elevated levels of androgen receptor full-length and AR-V7 are key mechanisms under-lying resistance to enzalutamide in PCa. Gal-1 knockdown suppressed AR and AR-V7 expression and their transcriptional activity. Treatment with LLS30 significantly suppressed the growth of enzalutamide-resistant PCa cells and exhibited synergistic effects when combined with enzalutamide. Notably, this combination therapy significantly inhibited the growth of enzalutamide-resistant xenografts in vivo. RNA-seq analysis revealed that LLS30 modulates AR and AR-V7 signaling through the inhibition of associated target genes. Conclusion: These findings highlight Gal-1 as a promising therapeutic target for overcoming enzalutamide resistance in PCa. Targeting the Gal-1/AR/AR-V7 axis with LLS30 presents a novel strategy to enhance enzalutamide efficacy and address drug resistance in advanced PCa. Full article