Tumor Model Research Articles

Heat-Inducible CAR-T Overcomes Adverse Mechanical Tumor Microenvironment in a 3D Bioprinted Glioblastoma Model

Glioblastoma (GBM) presents a significant therapeutic challenge due to the limited efficacy of existing treatments. Chimeric antigen receptor (CAR) T-cell therapy offers promise, but its potential in solid tumors like GBM is undermined by the physical barrier posed by the extracellular matrix (ECM). To address the inadequacies of traditional 2D cell culture, animal models, and Matrigel-based 3D culture in mimicking the mechanical characteristics of tumor tissues, we employed biomaterials and digital light processing-based 3D bioprinting to fabricate biomimetic tumor models with finely tunable ECM stiffness independent of ECM composition. Our results demonstrated that increased material stiffness markedly impeded CAR-T cell penetration and tumor cell cytotoxicity in GBM models. The 3D bioprinted models enabled us to examine the influence of ECM stiffness on CAR-T cell therapy effectiveness, providing a clinically pertinent evaluation tool for CAR-T cell development in stiff solid tumors. Furthermore, we developed an innovative heat-inducible CAR-T cell therapy, effectively overcoming the challenges posed by the stiff tumor microenvironment.

Discovery of novel EGFR-PROTACs capable of degradation of multiple EGFR-mutated proteins

Although three generations of Epidermal growth factor receptor (EGFR) - TK inhibitors have been approved for the treatment of Non-small-cell lung cancers (NSCLC), their clinical application is still largely hindered by acquired drug resistance mediated new EGFR mutations and side effects. The Proteolysis targeting chimera (PROTAC) technology has the potential to overcome acquired resistance from mutant EGFR through a novel mechanism of action. In this study, we developed the candidate degrader IV-3 by structural modifications of the lead compound 13, which exhibited limited antiproliferative activity against HCC-827 cells. Compared to compound 13, IV-3 exhibited remarkable anti-proliferative activity against HCC-827 cells, NCI–H1975 cells, and NCI–H1975-TM cells (IC50 = 0.009 μM, 0.49 μM and 3.24 μM, respectively), as well as significantly inducing degradation of EGFR protein in these cell lines (DC50 = 17.93 nM, 0.25 μM and 0.63 μM, respectively). Further investigations confirmed that IV-3 exhibited superior anti-tumor activity in all xenograft tumor models through the degradation of mutant EGFR protein. Moreover, IV-3 showed no inhibitory activity against A431 and A549 cells expressing wild-type EGFR, thereby eliminating potential toxic side effects emerging from wild-type EGFR inhibition. Overall, our study provides promising insights into EGFR-PROTACs as a potential therapeutic strategy against EGFR-acquired mutation.

Polymer Micropatches as B-Cell Engagers.

B cells, despite their several unique functionalities, remain largely untapped for use as an adoptive cell therapy and are limited to in vitro use for antibody production. B cells can be easily sourced, they possess excellent lymphoid-homing capabilities, and they can act as antigen-presenting cells (APCs), offering an alternative to dendritic cells (DCs), which have shown limited efficacy in the clinical setting. Soluble factors such as IL-4 and anti-CD40 antibody can enhance the activation, survival, and antigen-presenting capabilities of B cells; however, it is difficult to attain sufficiently high concentrations of these biologics to stimulate B cells in vivo. Micropatches as Cell Engagers (MACE) are polymeric microparticles, surface functionalized with anti-CD40 and anti-IgM, which can attach to B cells and simultaneously engage multiple B-cell receptors (BCR) and CD40 receptors. Stimulation of these receptors through MACE, unlike free antibodies, enhanced the display of costimulatory molecules on the B-cell surface, increased B-cell viability, and improved antigen presentation by B cells to T cells in vitro. B-cell activation by MACE further synergized with soluble IL-4 and anti-CD40. MACE also elicited T-cell chemokine secretion by B cells. Upon intravenous adoptive transfer, MACE-bound B cells homed to the spleen and lymph nodes, key sites for antigen presentation to T cells. Adoptive transfer of MACE-B cells pulsed with the CD4+ and CD8+ epitopes of ovalbumin significantly delayed tumor progression in a murine subcutaneous EG7-OVA tumor model, demonstrating the functional benefit conferred to B cells by MACE.

Novel lactylation-related signature to predict prognosis for pancreatic adenocarcinoma

BACKGROUND Lactate, previously considered a metabolic byproduct, is pivotal in cancer progression and maintaining the immunosuppressive tumor microenvironment. Further investigations confirmed that lactate is a primary regulator, introducing recently described post-translational modifications of histone and non-histone proteins, termed lysine lactylation. Pancreatic adenocarcinomas are characterized by increased glycolysis and lactate accumulation. However, our understanding of lactylation-related genes in pancreatic adenocarcinomas remains limited. AIM To construct a novel lactylation-related gene signature to predict the survival of patients with pancreatic cancer. METHODS RNA-seq and clinical data of pancreatic adenocarcinoma (PDAC) were obtained from the GTEx (Genotype-Tissue Expression) and TCGA (The Cancer Genome Atlas) databases via Xena Explorer, and GSE62452 datasets from GEO. Data on lactylation-related genes were obtained from publicly available sources. Differential expressed genes (DEGs) were acquired by using R package “DESeq2” in R. Univariate COX regression analysis, LASSO Cox and multivariate Cox regressions were produced to construct the lactylation-related prognostic model. Further analyses, including functional enrichment, ESTIMATE, and CIBERSORT, were performed to analyze immune status and treatment responses in patients with pancreatic cancer. PDAC and normal human cell lines were subjected to western blot analysis under lactic acid intervention; two PDAC cell lines with the most pronounced lactylation were selected. Subsequently, RT-PCR was employed to assess the expression of LRGs genes; SLC16A1, which showed the highest expression, was selected for further investigation. SLC16A1-mediated lactylation was analyzed by immunofluorescence, lactate production analysis, colony formation, transwell, and wound healing assays to investigate its role in promoting the proliferation and migration of PDAC cells. In vivo validation was performed using an established tumor model. RESULTS In this study, we successfully identified 10 differentially expressed lactylation-related genes (LRGs) with prognostic value. Subsequently, a lactylation-related signature was developed based on five OS-related lactylation-related genes (SLC16A1, HLA-DRB1, KCNN4, KIF23, and HPDL ) using Lasso Cox hazard regression analysis. Subsequently, we evaluated the clinical significance of the lactylation-related genes in pancreatic adenocarcinoma. A comprehensive examination of infiltrating immune cells and tumor mutation burden was conducted across different subgroups. Furthermore, we demonstrated that SLC16A1 modulates lactylation in pancreatic cancer cells through lactate transport. Both in vivo and in vitro experiments showed that decreasing SLC16A1 Level and its lactylation significantly inhibited tumor progression, indicating the potential of targeting the SLC16A1/ Lactylation-associated signaling pathway as a therapeutic strategy against pancreatic adenocarcinoma. CONCLUSION We constructed a novel lactylation-related prognostic signature to predict OS, immune status, and treatment response of patients with pancreatic adenocarcinoma, providing new strategic directions and antitumor immunotherapies.

Alkenyl oxindole is a novel PROTAC moiety that recruits the CRL4DCAF11 E3 ubiquitin ligase complex for targeted protein degradation.

Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of heterobifunctional compounds by conjugating different alkenyl oxindoles with bromodomain-containing protein 4 (BRD4) inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent heterobifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in a mouse xenograft tumor model. Our research provides new employable proteolysis targeting chimera (PROTAC) moieties for targeted protein degradation, providing new possibilities for drug discovery.

An NGS-based assay for accurate detection and quantification of immune gene expression in mouse tumor models.

Tumor microenvironment (TME) is a complex dynamic system with many tumor-interacting components including tumor-infiltrating leukocytes (TILs), cancer associated fibroblasts, blood vessels, and other stromal constituents. It intrinsically affects tumor development and pharmacology of oncology therapeutics, particularly immune-oncology (IO) treatments. Accurate measurement of TME is therefore of great importance for understanding the tumor immunity, identifying IO treatment mechanisms, developing predictive biomarkers, and ultimately, improving the treatment of cancer. Here, we introduce a mouse-IO NGS-based (NGSmIO) assay for accurately detecting and quantifying the mRNA expression of 1080 TME related genes in mouse tumor models. The NGSmIO panel was shown to be superior to the commonly used microarray approach by hosting 300 more relevant genes to better characterize various lineage of immune cells, exhibits improved mRNA and protein expression correlation to flow cytometry, shows stronger correlation with mRNA expression than RNAseq with 10x higher sequencing depth, and demonstrates higher sensitivity in measuring low-expressed genes. We describe two studies; firstly, detecting the pharmacodynamic change of interferon-γ expression levels upon anti-PD-1: anti-CD4 combination treatment in MC38 and Hepa 1-6 tumors; and secondly, benchmarking baseline TILs in 14 syngeneic tumors using transcript level expression of lineage specific genes, which demonstrate effective and robust applications of the NGSmIO panel.

3D Bioprinted Tumor-Stroma Models of Triple-Negative Breast Cancer Stem Cells for Preclinical Targeted Therapy Evaluation.

Breast cancer stem cells (CSCs) play a pivotal role in therapy resistance and tumor relapse, emphasizing the need for reliable in vitro models that recapitulate the complexity of the CSC tumor microenvironment to accelerate drug discovery. We present a bioprinted breast CSC tumor-stroma model incorporating triple-negative breast CSCs (TNB-CSCs) and stromal cells (human breast fibroblasts), within a breast-derived decellularized extracellular matrix bioink. Comparison of molecular signatures in this model with different clinical subtypes of bioprinted tumor-stroma models unveils a unique molecular profile for artificial CSC tumor models. We additionally demonstrate that the model can recapitulate the invasive potential of TNB-CSC. Surface-enhanced Raman scattering imaging allowed us to monitor the invasive potential of tumor cells in deep z-axis planes, thereby overcoming the depth-imaging limitations of confocal fluorescence microscopy. As a proof-of-concept application, we conducted high-throughput drug testing analysis to assess the efficacy of CSC-targeted therapy in combination with conventional chemotherapeutic compounds. The results highlight the usefulness of tumor-stroma models as a promising drug-screening platform, providing insights into therapeutic efficacy against CSC populations resistant to conventional therapies.

Pluronic-Based Nanoparticles for Delivery of Doxorubicin to the Tumor Microenvironment by Binding to Macrophages.

The active targeting drug delivery system based on special types of endogenous cells such as macrophages has emerged as a promising strategy for tumor therapy, owing to its tumor homing property and biocompatibility. In this work, the active tumor-targeting drug delivery system carrying doxorubicin-loaded nanoparticles (DOX@MPF127-MCP-1, DMPM) on macrophage (RAW264.7) surfaces via the mediation of interaction with the CCR2/MCP-1 axis was exploited. Initially, the amphiphilic block copolymer Pluronic F127 (PF127) was carboxylated to MPF127 at the hydroxyl terminus. Subsequently, MPF127 was modified with MCP-1 peptide to prepare MPF127-MCP-1 (MPM). The DOX was wrapped in MPM to form DMPM nanomicelles (approximately 100 nm) during the self-assembly process of MPM. The DMPM spontaneously bound to macrophages (RAW264.7), which resulted in the construction of an actively targeting delivery system (macrophage-DMPM, MA-DMPM) in vitro and in vivo. The DOX in MA-DMPM was released in the acidic tumor microenvironment (TME) in a pH-responsive manner to increase DOX accumulation and enhance the tumor treatment effect. The ratio of MA-DMPM homing reached 220% in vitro compared with the control group, indicating that the MA-DMPM was excellently capable of tumor-targeting delivery. In in vivo experiments, nonsmall cell lung cancer cell (NCI-H1299) tumor models were established. The results of the fluorescence imaging system (IVIS) showed that MA-DMPM demonstrated tremendous tumor-targeting ability in vivo. The antitumor effects of MA-DMPM in vivo indicated that the proportion of tumor cell apoptosis in the DMPM-treated group was 63.33%. The findings of the tumor-bearing mouse experiment proved that MA-DMPM significantly suppressed tumor cell growth, which confirmed its immense potential and promising applications in tumor therapy.

Abstract A021: The role of MGAT5 in sex-dependent regulation of CD8+ T cells in muscle-invasive bladder cancer

Abstract Biological sex significantly affects bladder cancer incidence and outcome. Although men have much greater incidence of bladder cancer than women, females tend to be diagnosed with more advanced tumors and have worse outcomes. The mechanisms driving these differences are likely multifactorial, with biological factors playing an important role, however, much of the biology underlying these differences is unknown. The immune system is a critical regulator of cancer biology, and immune features have been associated with bladder cancer grade, stage, progression, and response to therapies. Sex dramatically affects the immune system including anti-tumor immunity. However, how sex and immunity intersect to affect clinical outcomes in bladder cancer patients remains unclear. CD8+ T cells are a key part of anti-tumor immune responses and the importance of inhibitory receptors such as programmed cell death-1 (PD-1), is now well established as a mechanism restricting T cell responses in MIBC. However, there are a variety of other regulatory pathways that restrict their anti-tumor function, which have yet to be investigated. One such pathway involves alterations in protein glycosylation, which can affect T cell function at many levels. The activity of the glycosyltransferase N-acetylglucosaminyltransferase V (MGAT5), responsible for complex N-glycan branching, has been shown to regulate T cell function in autoimmunity and chronic viral infections, however, its role in regulating anti-tumor CD8+ T cell responses has yet to be explored. We assessed the activity of MGAT5 in circulating and tumor-infiltrating CD8+ T cells in mice using a panel of cell lines in a syngeneic orthotopic model of muscle-invasive bladder cancer (MIBC). We found that in all tumor models, MGAT5 activity was upregulated in tumor infiltrating CD8+ T cells compared with circulating cells. Furthermore, using high parameter flow cytometry, we assessed the activity of MGAT5 in CD8+ T cells from mice with bladder cancer induced by the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), and in patients with MIBC. In both mice and humans, we found that sex affects MGAT5 activity in memory CD8+ T cells, with females exhibiting decreased MGAT5 activity in these cells compared to males. The functional significance of altering MGAT5 activity in T cells was also assessed in vivo. To do this, chimeric mice were generated by mixed bone-marrow transplant resulting in mice with loss of Mgat5 in T cells. Bladder cancer was then induced using BBN, and morbidity (defined as either 10% weight loss in one week, 10% weight loss post-BBN, or humane endpoint), as a proxy for mortality, was assessed over time. In this model, male mice were protected from BBN-induced morbidity compared to female mice, and this protection was abrogated by homozygous or heterozygous loss of Mgat5 in T cells. Overall, our data suggest that sex-dependent regulation of MGAT5 activity in CD8+ T cells may be an important underlying factor affecting outcomes in bladder cancer. Citation Format: Morgan E. Roberts, Igor Moskalev, Peter C. Black. The role of MGAT5 in sex-dependent regulation of CD8+ T cells in muscle-invasive bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A021.

Abstract B026: APOBEC3 promotes squamous differentiation via IL1A/AP-1 signaling

Abstract The APOBEC family of cytidine deaminase enzymes catalyze the conversion of cytosine to uracil in ssDNA and RNA substrates. The resulting C to U transition has been noted in the innate immune response to viral infection, and somatic hypermutation. However, APOBEC family members APOBEC3A (hA3A) and APOBEC3B (hA3B) have been implicated as drivers of the APOBEC3 mutational signatures in numerous cancer types. Notably, approximately 70% of single nucleotide variant mutations in bladder cancer can be attributed to APOBEC3 induced mutagenesis. Studies have implicated various APOBEC3 family members to be drivers of tumorigenesis, disease progression, and therapeutic resistance. To investigate APOBEC3’s role in bladder cancer, we have generated a novel, cre-inducible, pten and p53 knockout, to overexpress mouse Apobec3(mA3) in Upk3a+ cells (UPPA), compared to a model that does not overexpress mA3 (UPP). Apobec3 expression was shown to accelerate bladder tumor formation, indicated by a shorter median time of tumor latency in the UPPA model (43.3 weeks) compared to the UPP model (53.6 weeks). While both tumor models had presence of squamous differentiation, the extensiveness was more profound in the UPPA tumors. Transcriptional analysis of UPP and UPPA tumors, via scRNA sequencing, revealed an enrichment of squamous gene expression in the UPPA tumors. Utilizing mouse syngeneic bladder cancer cell lines and normal bladder urothelial cells, we overexpressed mA3 in a doxycycline inducible manner. Overexpression of mA3 was shown to prevent luminal differentiation. Additionally, mA3 overexpression resulted in the increase of IL-1a production. IL-1a production was sufficient to drive squamous differentiation in BBN963 and normal bladder urothelial cells. Blocking cFOS and IL-1R, individually, prevented expression of squamous genes. These results suggest that DNA damage induced by mA3 is sufficient to drive squamous differentiation via signaling through IL-1a resulting in AP-1 transcription factor activation. We then analyzed bladder cancer samples from The Cancer Genome Atlas and found that hA3A was the only APOBEC family member to be significantly associated with squamous gene expression. Interestingly, hA3A expression was enriched in the basal/squamous consensus subtype. To further interrogate the potential of hA3A being a driver of squamous differentiation in bladder cancer, we have also generated doxycycline inducible expression of hA3A and hA3B in syngeneic bladder cancer cell lines. Targeting IL-1a in various autoimmune diseases has resulted in FDA approval of antibodies targeting IL-1a and IL-1R. Given the clinical utility of targeting IL-1a, it is plausible to target this signaling axis for therapeutic benefit in bladder cancers with ongoing hA3A activity. In aggregate, our work demonstrates the promotion of an aggressive tumor state, driven by APOBEC mutagenesis and implicates hA3A as a possible driver of squamous differentiation in urothelial carcinoma. Citation Format: Andrew S. Truong, Michael S. Sturdivant, Mi Zhou, Wolfgang A. Beckabir, John Raupp, Ujjawal Manocha, Elliot D. Toomer, Ibardo A. Zambrano, Hung-Jui Tan, Marc A. Bjurlin, Angela B. Smith, Tracy L. Rose, Matthew I. Milowsky, Sara E. Wobker, Kathryn H. Gessner, Jeffrey S. Damrauer, William Y. Kim. APOBEC3 promotes squamous differentiation via IL1A/AP-1 signaling [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B026.

Abstract A016: NRF2 activation promotes a fitness disadvantage in normal urothelium and drives a basal-like phenotype

Abstract Introduction: Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays a key role in protecting cells from oxidative stress. NRF2 is negatively controlled by Kelch-like ECH-associated protein 1 (KEAP1), which facilitates NRF2 ubiquitination and proteosomal degradation under homeostatic conditions. NRF2 activating mutations occur at a frequency of 5.9% in muscle-invasive bladder cancer (MIBC). However, the role of NRF2 in MIBC has not been fully understood. The aim of this study is to clarify how NRF2 affect to the normal epithelial cells and the tumor cells in bladder. Methods: We used genetically engineered mice (GEM) with a Cre inducible Nrf2 activating mutation (LSL-Nrf2-E79Q). To evaluate how Nrf2 affects the tumorigenesis process in vivo, the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) was administered to Upk3a-CreERT2; LSL-Nrf2-E79Q/+ (UN) mice. We also established normal urothelial organoids and BBN tumor derived organoids from wild-type (WT) and Krt5-CreERT2; LSL-Nrf2-E79Q/+ (K5N) mice for ex vivo study. To induce overexpression of NRF2, we utilized KI696 (KEAP1-NRF2 inhibitor) as a pharmacological agent and performed NAD/NADH assay to quantify the reductive stress. Additionally, we performed single cell RNA sequencing (scRNA-seq) with WT, UN and K5N mice bladder to identify the effect of Nrf2E79Q on the normal epithelial cells. Results: In BBN induced tumor model in vivo, UN mice did not differ in time to tumor development from the control WT mice. Furthermore, UN tumors had lost the post-recombination allele of LSL-Nrf2-E79Q in genomic PCR, meaning cells expressing Nrf2E79Q are selected against during the process. In ex vivo study, KI696 induced reductive stress in both WT normal urothelial organoids and BBN tumor derived organoids, but inhibited cell proliferation only in normal urothelial organoids. In K5N organoids, Nrf2E79Q activation enhanced relative mRNA expression of a number of basal related genes. In addition to that, vehicle organoids in differentiation media showed phenotypic differentiation, whereas those treated with tamoxifen did not demonstrate phenotypic signs of differentiation suggesting Nrf2 activation promotes a basal-like phenotype. Furthermore, we annotated three clusters in mice bladder epithelial cells as luminal, intermediate and basal using scRNA-seq and looked at their proportion among WT, UN and K5N mice. We found that the percentage of luminal cells was decreased and intermediate and basal cells increased in both UN and K5N mice compared to WT mice. This indicated that Nrf2E79Q promotes disadvantage in luminal cells in UN mice, whereas it contributes to the increase in intermediate and basal cells in K5N mice. Conclusion: These results suggested that Nrf2E79Q promotes a fitness disadvantage in normal mice urothelium. Furthermore, Nrf2E79Q inhibits differentiation of K5N organoids and drives a basal-like phenotype in GEM models. Further investigation is needed to clarify the mechanism of NRF2 activating mutation contributing the tumorigenesis in bladder. Citation Format: Akihiro Hamada, Yuki Kita, Di Wu, Mi Zhou, Sean Clark-Garvey, Andrew Gdowski, Michael Sturdivant, Wolfgang Beckabir, Elliott Drew Toomer, Emily Kounlavong, Lucia Lim, Kathryn Gessner, Jeffrey Damrauer, William Kim. NRF2 activation promotes a fitness disadvantage in normal urothelium and drives a basal-like phenotype [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A016.

GZ17-6.02 kills PDX isolates of uveal melanoma.

GZ17-6.02 has undergone phase I evaluation in patients with solid tumors (NCT03775525). The RP2D is 375 mg PO BID, with an uveal melanoma patient exhibiting a 15% reduction in tumor mass for 5 months at this dose. Studies in this manuscript have defined the biology of GZ17-6.02 in PDX isolates of uveal melanoma cells. GZ17-6.02 killed uveal melanoma cells through multiple convergent signals including enhanced ATM-AMPK-mTORC1 activity, inactivation of YAP/TAZ and inactivation of eIF2α. GZ17-6.02 significantly enhanced the expression of BAP1, predictive to reduce metastasis, and reduced the levels of ERBB family RTKs, predicted to reduce growth. GZ17-6.02 interacted with doxorubicin or ERBB family inhibitors to significantly enhance tumor cell killing which was associated with greater levels of autophagosome formation and autophagic flux. Knock down of Beclin1, ATG5 or eIF2α were more protective than knock down of ATM, AMPKα, CD95 or FADD, however, over-expression of FLIP-s provided greater protection compared to knock down of CD95 or FADD. Expression of activated forms of mTOR and STAT3 significantly reduced tumor cell killing. GZ17-6.02 reduced the expression of PD-L1 in uveal melanoma cells to a similar extent as observed in cutaneous melanoma cells whereas it was less effective at enhancing the levels of MHCA. The components of GZ17-6.02 were detected in tumors using a syngeneic tumor model. Our data support future testing GZ17-6.02 in uveal melanoma as a single agent, in combination with ERBB family inhibitors, in combination with cytotoxic drugs, or with an anti-PD1 immunotherapy.

IFNγ at the early stage induced after cryo-thermal therapy maintains CD4+ Th1-prone differentiation, leading to long-term antitumor immunity

IntroductionRecently, more and more research illustrated the importance of inducing CD4+ T helper type (Th)-1 dominant immunity for the success of tumor immunotherapy. Our prior studies revealed the crucial role of CD4+ Th1 cells in orchestrating systemic and durable antitumor immunity, which contributes to the satisfactory outcomes of the novel cryo-thermal therapy in the B16F10 tumor model. However, the mechanism for maintaining the cryo-thermal therapy-mediated durable CD4+ Th1-dominant response remains uncovered. Additionally, cryo-thermal-induced early-stage CD4+ Th1-dominant T cell response showed a correlation with the favorable prognosis in patients with colorectal cancer liver metastasis (CRCLM). We hypothesized that CD4+ Th1-dominant differentiation induced during the early stage post cryo-thermal therapy would affect the balance of CD4+ subsets at the late phase.MethodsTo understand the role of interferon (IFN)-γ, the major effector of Th1 subsets, in maintaining long-term CD4+ Th1-prone polarization, B16F10 melanoma model was established in this study and a monoclonal antibody was used at the early stage post cryo-thermal therapy for interferon (IFN)-γ signaling blockade, and the influence on the phenotypic and functional change of immune cells was evaluated.ResultsIFNγ at the early stage after cryo-thermal therapy maintained long-lasting CD4+ Th1-prone immunity by directly controlling Th17, Tfh, and Tregs polarization, leading to the hyperactivation of Myeloid-derived suppressor cells (MDSCs) represented by abundant interleukin (IL)-1β generation, and thereby further amplifying Th1 response.DiscussionOur finding emphasized the key role of early-phase IFNγ abundance post cryo-thermal therapy, which could be a biomarker for better prognosis after cryo-thermal therapy.

E3 ubiquitin ligase UBR5 promotes gemcitabine resistance in pancreatic cancer by inducing O-GlcNAcylation-mediated EMT via destabilization of OGA.

Pancreatic cancer (PC) is among the deadliest malignancies, with an extremely poor diagnosis and prognosis. Gemcitabine (GEM) remains the first-line drug for treating PC; however, only a small percentage of patients benefit from current immunotherapies or targeted therapies. Resistance to GEM is prevalent and affects long-term survival. We found that ubiquitin-protein ligase E3 module N-recognition 5 (UBR5) is a therapeutic target against GEM resistance. UBR5 was markedly upregulated in clinical GEM-resistant PC samples and GEM-resistant PC cells. UBR5 knockdown markedly increased GEM sensitivity in GEM-resistant PC cell lines. UBR5-mediated GEM resistance was accompanied by activation of epithelial-mesenchymal transition (EMT) and could be mitigated by inhibiting EMT. Further analysis revealed that UBR5 promoted GEM resistance in PC cells by enhancing O-GlcNAcylation-mediated EMT. In addition, UBR5 knockdown resulted in increased O-GlcNAase (OGA) levels, an essential negatively regulated enzyme in the O-GlcNAcylation process. We identified a negative association between OGA and UBR5 levels, which further supported the hypothesis that O-GlcNAcylation-mediated GEM resistance induced by UBR5 is OGA-dependent in PC cells. Mechanistic studies revealed that UBR5 acts as an E3 ubiquitin ligase of OGA and regulates O-GlcNAcylation by binding and modulating OGA, facilitating its degradation and ubiquitination. Additionally, high-throughput compound library screening using three-dimensional protein structure analysis and drug screening identified a Food and Drug Administration drug, Y-39983 dihydrochloride, as a potent GEM sensitiser and UBR5 inhibitor. The combination of Y-39983 dihydrochloride and GEM attenuated tumour growth in a mouse xenograft tumour model. Collectively, these data demonstrated that UBR5 plays a pivotal role in the sensitisation of PC to GEM and provides a potential therapeutic strategy to overcome GEM resistance.

Novel locally nebulized indocyanine green for simultaneous identification of tumor margin and intersegmental plane.

Segmentectomy, recommended for early-stage lung cancer or compromised lung function, demands precise tumor detection and intersegmental plane identification. While Indocyanine green (ICG) commonly aids in these aspects using near-infrared (NIR) imaging, its separate administrations through different routes and times can lead to complications and patient anxiety. This study aims to develop a lung-specific delivery method by nebulizing low-dose ICG to targeted lung segments, allowing simultaneous detection of lung tumors and intersegmental planes across diverse animal models. To optimizing the dose of ICG for lung tumor and interlobar fissure detection, different doses of ICG (0.25, 0.1 and 0.05mg/kg) were nebulized to rabbit lung tumor models. The distribution of locally nebulized ICG in targeted segments was studied to evaluate the feasibility of detecting lung tumor and intersegmental planes in canine lung pseudotumor models. NIR fluorescence imaging demonstrated clear visualization of lung tumor margin and interlobar fissure using local nebulization of 0.1mg/kg ICG for only 4 min during surgery in the rabbit models. In the canine model, the local nebulization of 0.05mg/kg of ICG into the target segment enabled clear visualization of pseudotumor and intersegmental planes for 30 min. This innovative approach achieves a reduction in ICG dose and prolonged the visualization time of the intersegmental plane and effectively eliminates the need for the hurried marking of tumors and intersegmental planes. We anticipate that lung specific delivery of ICG will prove valuable for image-guided limited resection of lung tumors in clinical practice.

Cascade loop of ferroptosis induction and immunotherapy based on metal‐phenolic networks for combined therapy of colorectal cancer

AbstractCancer immunotherapy is the most promising method for tumor therapy, while ferroptosis could activate the immunogenicity of cancer and strengthen the cellular immune response. However, limited by the complex tumor microenvironment, the abundant glutathione (GSH) and low reactive oxygen species (ROS) seriously weaken ferroptosis and the immune response. Herein, the authors report photothermal metal‐phenolic networks (MPNs) supplied with buthionine sulfoximine (BSO) by reducing levels of GSH and then trapping the tumor cells in the ferroptosis and immunotherapy cascade loop to eliminate colorectal cancer (CRC). The MPNs coated with the model antigen ovalbumin can accumulate at the tumor site, mediate immunogenic cell death (ICD) under NIR irradiation, and initiate tumoricidal immunity. Then the activated CD8+ T cells would release IFN‐γ to inhibit GPX4 and promote the immunogenic ferroptosis induced by Fe3+ and BSO. Finally, the tumor cells at intertumoral and intratumoral levels would be involved in the ferroptosis‐dominated cancer‐immunity circle for CRC eradication, resulting in outstanding therapeutic outcomes in both primary and distant tumor models. Overall, this strategy employs a photothermal nanoplatform to rapidly stimulate ICD and restrain the oxidation defense system, which provides a promising approach to significantly amplify the “cascade loop” of ferroptosis induction and immunotherapy for treatment of CRC.

METTL5 promotes gastric cancer progression via sphingomyelin metabolism.

The treatment of gastric cancer (GC) has caused an enormous social burden worldwide. Accumulating studies have reported that N6-methyladenosine (m6A) is closely related to tumor progression. METTL5 is a m6A methyltransferase that plays a pivotal role in maintaining the metabolic stability of cells. However, its aberrant regulation in GC has not been fully elucidated. To excavate the role of METTL5 in the development of GC. METTL5 expression and clinicopathological characteristics were analyzed via The Cancer Genome Atlas dataset and further verified via immunohistochemistry, western blotting and real-time quantitative polymerase chain reaction in tissue microarrays and clinical samples. The tumor-promoting effect of METTL5 on HGC-27 and AGS cells was explored in vitro by Cell Counting Kit-8 assays, colony formation assays, scratch healing assays, transwell assays and flow cytometry. The tumor-promoting role of METTL5 in vivo was evaluated in a xenograft tumor model. The EpiQuik m6A RNA Methylation Quantification Kit was used for m6A quantification. Next, liquid chromatography-mass spectrometry was used to evaluate the association between METTL5 and sphingomyelin metabolism, which was confirmed by Enzyme-linked immunosorbent assay and rescue tests. In addition, we investigated whether METTL5 affects the sensitivity of GC cells to cisplatin via colony formation and transwell experiments. Our research revealed substantial upregulation of METTL5, which suggested a poor prognosis of GC patients. Increased METTL5 expression indicated distant lymph node metastasis, advanced cancer stage and pathological grade. An increased level of METTL5 correlated with a high degree of m6A methylation. METTL5 markedly promotes the proliferation, migration, and invasion of GC cells in vitro. METTL5 also promotes the growth of GC in animal models. METTL5 knockdown resulted in significant changes in sphingomyelin metabolism, which implies that METTL5 may impact the development of GC via sphingomyelin metabolism. In addition, high METTL5 expression led to cisplatin resistance. METTL5 was found to be an oncogenic driver of GC and may be a new target for therapy since it facilitates GC carcinogenesis through sphingomyelin metabolism and cisplatin resistance.

Discovery of Small and Bifunctional Molecules Targeting PD-L1/CD73 for Cancer Dual Immunotherapy.

In this work, a series of bifunctional PD-L1/CD73 (cluster of differentiation 73) small-molecule inhibitors were designed and synthesized. Among them, CC-5 showed the strongest PD-L1 inhibitory effects with an IC50 of 6 nM and potent anti-CD73 activity with an IC50 of 0.773 μM. The high PD-L1/CD73 inhibitory activity of CC-5 was further confirmed by SPR assays with KD of 182 nM for human PD-L1 and 101 nM for CD73, respectively. Importantly, CC-5 significantly suppressed tumor growth in a CT26 and B16-F10 tumor model with TGI of 64.3% and 39.6%, respectively. Immunohistochemical (IHC) and flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) indicated that CC-5 exerted anticancer effects via activating the tumor immune microenvironment. Collectively, CC-5 represents the first dual PD-L1/CD73 inhibitor worthy of further research as a bifunctional immunotherapeutic agent.

Tumor microenvironment-modulating oncolytic adenovirus combined with GSK-3β inhibitor enhances antitumor immune response against bladder cancer

Bladder cancer is a common type of cancer around the world, and the majority of patients are diagnosed with non-muscle-invasive bladder cancer (NMIBC). Although low-risk NMIBC has a good prognosis, the disease recurrence rate and development of treatment-refractory disease remain high in intermediate- to high-risk NMIBC patients. To address these challenges for the treatment of NMIBC, a novel combination therapy composed of an oncolytic adenovirus (oAd) co-expressing interleukin (IL)-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), and relaxin (RLX; HY-oAd) and a clinical-stage glycogen synthase kinase (GSK)-3β inhibitor (9-ING-41; elraglusib) was investigated in the present report. Our findings demonstrate that HY-oAd and 9-ING-41 combination therapy (HY-oAd+9-ING-41) exerted superior inhibition of tumor growth compared with respective monotherapy in a syngeneic NMIBC tumor model. HY-oAd+9-ING-41 induced high-level tumor extracellular matrix (ECM) degradation and a more potent antitumor immune response than the respective monotherapy. In detail, HY-oAd+9-ING-41 induced superior accumulation of intratumoral T cells, prevention of immune cell exhaustion, and induction of tumor-specific adaptive immune response compared to either monotherapy. Collectively, these results demonstrate that the combination of HY-oAd and 9-ING-41 may be a promising approach to elicit a potent antitumor immune response against bladder cancer.

Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKβ independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.