Background/Objectives: Clear cell renal cell carcinoma (ccRCC), the predominant malignant subtype of kidney cancer, is the leading cause of death among renal cell carcinoma patients. Although a subset of ccRCC patients benefit from select immune checkpoint inhibitors (ICIs), prognosis remains poor. While PD-1 and PD-L1 have been extensively studied, the prevalence and distribution of other immune checkpoints (ICs) and their relationship with epithelial-to-mesenchymal transition (EMT) remain poorly characterised. Here, we investigated the interplay between twenty ICs and EMT markers and assessed their combined prognostic relevance in ccRCC patients. Methods: Transcriptomic profiling and integrated bioinformatic analyses were performed, including differential expression, correlation analyses, survival analyses, forest plot analyses, ROC curve evaluation, and OncoPrint visualisation, complemented by analysis of single-cell RNA sequencing data, immunohistochemistry, and multiplex secretory IC (LegendPlex) assays. Results: Transcriptomic profiling of over 500 ccRCC tumours versus normal kidney tissue revealed dysregulation of ICs, particularly LAG3 and NT5E. Notably, expression of ICs, including LAG3 and NT5E, was associated with poor overall survival in 415 ccRCC patients. ICs that synergised with the EMT phenotype provided improved prognostic discrimination compared to individual ICs. Correlation analyses, single-cell RNA sequencing, and immunohistochemistry demonstrated an association between EMT-associated tumours and expression of LAG3 and NT5E. ROC analysis indicated modest prognostic performance of LAG3 and NT5E. Conclusions: Collectively, this study identifies an EMT-IC axis in ccRCC and demonstrates its relevance to tumour biology and patient outcomes, highlighting LAG3 and NT5E as potential prognostic markers and therapeutic targets that warrant further investigation.

Glioblastoma is the most aggressive form of brain tumor resulting in low overall patient survival rates of 12–15 months post diagnosis. Several factors contribute to the complexity of the tumor, including tumor heterogeneity, blood–brain barrier complications, genetic defects, cancer stem cell generation, and immune evasion. These factors can result in the progression of glioblastoma and are controlled by signaling pathways. Some of the signaling pathways involved in glioblastoma progression include ERK, NF-κB, Wnt, and PI3K/AKT/mTOR. Our and others’ previous studies have found that TIM-3 and TGF-β signaling is altered in glioblastoma patients and may contribute to cancer progression. Immune promoting pathways such as STING have also been studied in glioblastoma to enhance anti-tumor immunity; however the interconnecting roles of these pathways are not well described. This review highlights the role of these three key cancer-related pathways in glioblastoma and their mechanistic link. Better understanding these links may result in improved treatment targets or disease progression biomarkers.

Abstract Background ADAMTS1, a disintegrin and metalloproteinase with thrombospondin motif 1, plays a role in inflammation, organogenesis, and ovulation. Up or downregulation of ADAMTS1 has been implicated in tissue remodelling leading to cancer. We analysed the expression of ADAMTS1 in different stages/grades of primary and metastatic serous ovarian tumours and ascites-derived tumour cells from patients and assessed the functional role of ADAMTS1 in ovarian cancer (OC) cell lines. Methods The expression and localisation of ADAMTS1 was assessed by immunohistochemistry (IHC) and Opal Multiplex IHC staining of OC tissues. Functional roles of ADAMTS1 in OC cell lines were assessed by using siRNA-mediated knockdown (KD), MTT assay, cell migration by xCELLigence, cell adhesion, ELISA, qRT-PCR, Western blot, immunofluorescence (IF) and activity of Cdc42 GTPase. Results The expression of ADAMTS1 was significantly enhanced in higher stages/grades of ovarian tumours compared to benign tumours. In high-grade tumours, ADAMTS1 was localised more in the nucleus of epithelial cells while localisation in stromal cells was mostly in the cytoplasm. Significantly higher mRNA expression of ADAMTS1 was noted in epithelial compared to mesenchymal ascites-derived tumour cells. The expression of ADAMTS1 was significantly higher in metastatic high-grade tumours compared to primary tumours. KD of ADAMTS1 expression by siRNA in OC cell lines had no effect on cell proliferation but resulted in decreased cell adhesion, increased cell migration accompanied by increased expression of markers (CDH1 and EPCAM) associated with epithelial plasticity. Remodelling of ECM accompanied by increased intra- and extracellular production of VCAN and enhanced Cdc42 GTPase activity was also noted in cell lines with ADAMTS1 KD. Cdc42 GTPase specific inhibitor, ML141, reversed ADAMTS1 KD-mediated enhanced migration. On the other hand, VCAN KD inhibited ADAMTS1 KD-mediated migration and reversed the effect on cell adhesion. Conclusions These results suggest that the expression of ADAMTS1 progressively enriches ovarian tumours and promotes OC progression. Its knock down in in vitro cell culture impacts ECM remodelling through enhanced Cdc42GTPase activity and VCAN production resulting in epithelial cell plasticity, accelerated migration, and reduced cell adhesion.

Glioblastoma is the most aggressive primary brain malignancy, characterised by extensive intra-tumoural heterogeneity, therapy resistance, and a profoundly immunosuppressive tumour microenvironment. The galectin family, a group of β-galactoside-binding lectins, has emerged as a key regulator of tumour biology, influencing oncogenesis, immune modulation, and therapy resistance. In this study, we performed an integrative bioinformatics analysis to systematically evaluate the expression patterns, prognostic significance, genetic alterations, and functional roles of galectin family members in glioblastoma. We utilised publicly available genomic datasets and computational tools to perform our analysis, including UALCAN, GEPIA, cBioPortal, STRING, GeneMANIA, DAVID, and TIMER. We identified LGALS1, LGALS3, and LGALS9 as significantly upregulated in glioblastoma, with their overexpression correlating with adverse patient survival. Functional enrichment analysis highlighted galectin-mediated pathways involved in extracellular matrix remodelling, immune dysregulation, tumour-promoting pathways, and protein processing, suggesting their pivotal role in glioblastoma pathogenesis. We also show that transcriptional and immunological signatures suggest that galectins may regulate glioblastoma immunosuppression, extracellular matrix remodelling, and protein homeostasis. Our findings provide novel insights into the oncogenic and immunoregulatory roles of galectins in glioblastoma, establishing their potential as prognostic biomarkers and therapeutic targets.

Several immunoregulatory or immune checkpoint receptors including T cell immunoglobulin and mucin domain 3 (TIM-3) have been implicated in glioblastoma progression. Rigorous investigation over the last decade has elucidated TIM-3 as a key player in inhibiting immune cell activation and several key associated molecules have been identified both upstream and downstream that mediate immune cell dysfunction mechanistically. However, despite several reviews being published on other immune checkpoint molecules such as PD-1 and CTLA-4 in the glioblastoma setting, no such extensive review exists that specifically focuses on the role of TIM-3 in glioblastoma progression and immunosuppression. Here, we critically summarize the current literature regarding TIM-3 expression as a prognostic marker for glioblastoma, its expression profile on immune cells in glioblastoma patients and the exploration of anti-TIM-3 agents in glioblastoma pre-clinical models for potential clinical application.

183 Background: MNK inhibition has been shown to downregulate phosphorylation at serine 209 of eIF4E, a potent regulatory point of CAP-mediated translation of 5’ polyadenylated mRNA associated with growth factor and pro-oncogenic signals in cells. Elevated levels of eIF4E phosphorylation are observed in a broad range of tumors. MNK1/2 knock out mice are healthy and viable, as cellular house-keeping mRNA translation occurs readily using the IRES mechanism, whereas mRNA with more complex and longer 5’ untranslated ends such as those involved in growth factor and pro-oncogenic signaling, are MNK activated. Cells from MNK knockout animals become relatively resistant to subsequent oncogenic transformation. In a program of SAD and MAD Phase I studies conducted in normal healthy volunteers, Tinodasertib was found to be safe and well tolerated with no dose-limiting toxicity (DLT). The objectives of this ongoing Phase 2 study are to evaluate safety, and preliminary efficacy of Tinodasertib as monotherapy and in combination with either pembrolizumab or irinotecan in patients with advanced colorectal cancer (CRC). Methods: Patients had to have advanced CRC and previously received ≥2 lines of therapy. The dose escalation phase of the study was open to all patients with CRC. As of 23 July 2024, 22 patients were dosed in a modified 3X3 dose escalation design with Tinodasertib from 20 to 80 mg on alternate days. Out of 22 patients, 12 received monotherapy, 4 were treated with Tinodasertib and Irinotecan and 6 were treated with Tinodasertib and pembrolizumab. Majority (19) had MSS CRC and 3 had unknown status. Nine patients had KRAS-mut CRC. Results: No DLTs were observed and MTD was not reached. Grade 3 treatment-related adverse events (TRAEs) were observed in 2 (9%) patients and were related to irinotecan while no Grade 3 AEs were attributed to Tinodasertib by either the investigator or the sponsor. Most common TRAEs were related to gastrointestinal system organ class. There were no Grade 4-5 TRAEs. Of the 22 patients, 18 were evaluable for RECIST 1.1 response. No patient had an objective response to treatment, 12 had stable disease with disease control rate of 67% and a progression free survival of 2.99 months. Patients remained on therapy for up to 28 weeks. Overall survival (OS) at 52 weeks was 52% (CI 29 to 93). Conclusions: Tinodasertib either as monotherapy or combined with irinotecan or pembrolizumab, was well tolerated with no DLTs at the dose levels evaluated. Prolonged median time to progression and OS compared to historical controls were observed even during the dose escalation phase for the study population as a whole and for each of the monotherapy and combination arms. Enrolment in the dose escalation phase continues. Clinical trial information: NCT05462236 .

Unconventional T cells are a subset of T cells that recognize a range of non-peptide antigens, such as lipids, vitamin B metabolites, and phosphoantigens. These antigens are presented by non-classical antigen-presenting molecules, including CD1, MHC-related 1 (MR1), and butyrophilin molecules. Unconventional T cells include CD1-restricted T cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells. Unconventional T cells can rapidly produce large amounts of cytokines upon activation and are suggested as being crucial in the interface of innate and adaptive immunity. These cells are implicated in various human diseases, including cancer and autoimmune disorders, with alterations in both their frequency and function noted. Therefore, understanding their developmental pathways is critical for uncovering potential mechanisms behind these diseases. This knowledge could inform the development of targeted therapies to modulate unconventional T cells and their responses for clinical benefit. Using a combination of high-dimensional flow cytometry, transcriptomic and functional analysis, and precursor-product experiments, we have mapped the postnatal thymic development pathways of MAIT, Vγ9Vδ2, and NKT cells. This revealed shared and unique features of their development, providing insights into their unique biology. This new understanding could pave the way for novel therapeutic strategies that leverage the unique properties of unconventional T cells in disease contexts. NHMRC ideas grant Hematopoiesis and Immune System Development (HEM)

Mitochondrial-associated granulocyte macrophage colony-stimulating factor (Magmas) is a unique protein located in the inner membrane of mitochondria, with an active role in scavenging reactive oxygen species (ROS) in cellular systems. Ovarian cancer (OC), one of the deadliest gynaecological cancers, is characterised by genomic instability, affected by ROS production in the tumour microenvironment. This manuscript discusses the role of Magmas and efficacy of its novel small molecule inhibitor BT#9 in OC progression, metastasis, and chemoresistance. Magmas expression levels were significantly elevated in high-grade human OC compared to benign tumours by immunohistochemistry. The inhibition of Magmas by BT#9 enhanced ROS production and reduced mitochondrial membrane permeability, basal respiration, mitochondrial ATP production, and cellular functions, such as the proliferation and migration of OC cell lines in vitro. Oral administration of BT#9 in vivo significantly reduced tumour growth and spread and enhanced the survival of mice without having any effect on the peritoneal organs. These data suggest that Magmas is functionally important for OC growth and spread by affecting ROS levels and that the inhibition of Magmas activity by BT#9 may provide novel clinical benefits for patients with this malignancy.

Cell and gene therapy are innovative biomedical strategies aimed at addressing diseases at their genetic origins. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems have become a groundbreaking tool in cell and gene therapy, offering unprecedented precision and versatility in genome editing. This chapter explores the role of CRISPR in gene editing, tracing its historical development and discussing biomolecular formats such as plasmid, RNA, and protein-based approaches. Next, we discuss CRISPR delivery methods, including viral and non-viral vectors, followed by examining the various engineered CRISPR variants for their potential in gene therapy. Finally, we outline emerging clinical applications, highlighting the advancements in CRISPR for breakthrough medical treatments.

Glioblastoma is highly proliferative and invasive. However, the regulatory cytokine networks that promote glioblastoma cell proliferation and invasion into other areas of the brain are not fully defined. In the present study, we define a critical role for the IL-11/IL-11Rα signalling axis in glioblastoma proliferation, epithelial to mesenchymal transition, and invasion. We identified enhanced IL-11/IL-11Rα expression correlated with reduced overall survival in glioblastoma patients using TCGA datasets. Proteomic analysis of glioblastoma cell lines overexpressing IL-11Rα displayed a proteome that favoured enhanced proliferation and invasion. These cells also displayed greater proliferation and migration, while the knockdown of IL-11Rα reversed these tumourigenic characteristics. In addition, these IL-11Rα overexpressing cells displayed enhanced invasion in transwell invasion assays and in 3D spheroid invasion assays, while knockdown of IL-11Rα resulted in reduced invasion. Furthermore, IL-11Rα-overexpressing cells displayed a more mesenchymal-like phenotype compared to parental cells and expressed greater levels of the mesenchymal marker Vimentin. Overall, our study identified that the IL-11/IL-11Rα pathway promotes glioblastoma cell proliferation, EMT, and invasion.

Pregnancy associated plasma protein-A (PAPP-A) plays an integral role in breast cancer (BC), especially triple negative breast cancer (TNBC). This subtype accounts for the most aggressive BC, possesses high tumor heterogeneity, is least responsive to standard treatments and has the poorest clinical outcomes. There is a critical need to address the lack of effective targeted therapeutic options available. PAPP-A is a protein that is highly elevated during pregnancy. Frequently, higher PAPP-A expression is detected in tumors than in healthy tissues. The increase in expression coincides with increased rates of aggressive cancers. In BC, PAPP-A has been demonstrated to play a role in tumor initiation, progression, metastasis including epithelial-mesenchymal transition (EMT), as well as acting as a biomarker for predicting patient outcomes. In this review, we present the role of PAPP-A, with specific focus on TNBC. The structure and function of PAPP-A, belonging to the pappalysin subfamily, and its proteolytic activity are assessed. We highlight the link of BC and PAPP-A with respect to the IGFBP/IGF axis, EMT, the window of susceptibility and the impact of pregnancy. Importantly, the relevance of PAPP-A as a TNBC clinical marker is reviewed and its influence on immune-related pathways are explored. The relationship and mechanisms involving PAPP-A reveal the potential for more treatment options that can lead to successful immunotherapeutic targets and the ability to assist with better predicting clinical outcomes in TNBC.

Epithelial ovarian cancer is aggressive and causes high mortality among women worldwide. Members of the plakin family are essential to maintain cytoskeletal integrity and key cellular processes. In this study we characterised the expression of plakins, particularly plectin (PLEC), periplakin (PPL), envoplakin (EVPL), and EMT-related proteins by immunohistochemistry in n = 48 patients’ samples to evaluate a potential correlation of plakin expression with EMT as EOC progresses. These tissue plakin and EMT expression analyses were further evaluated by in vitro cell line expression and correlated with the expression of these molecules using publicly available datasets such as Cancer Genome Atlas (TCGA) and Clinical Proteome Tumour Analysis Consortium (CPTAC) datasets. We demonstrate that the expression of PPL and PLEC plakins is decreased in high-grade compared to low-grade EOCs with mixed EMT marker protein expression. This is supported by the correlation of high PPL and PLEC expression with an epithelial rather than mesenchymal phenotype. Our data suggest a partial loss of plakin expression as EOC tumours progress. This may impact the connections of plakins with membrane-bound receptors, which impede the downstream signalling required for the initiation of EMT as the tumours progress.

Inhibitory receptors are critical for regulating immune cell function. In cancer, these receptors are often over-expressed on the cell surface of T and NK cells, leading to reduced anti-tumor activity. Here, through the analysis of 11 commonly studied checkpoint and inhibitory receptors, we discern that only HAVCR2 (TIM3) and ENTPD1 (CD39) display significantly greater gene expression in glioblastoma compared to normal brain and lower grade glioma. Cell surface TIM-3, but not ENTPD1, was also elevated on activated CD4+ and CD8+ T cells, as well as on NK cells from glioblastoma patients compared to healthy donor T and NK cells. A subsequent analysis of molecules known to co-ordinate TIM-3 function and regulation was performed, which revealed that BAT3 expression was significantly reduced in CD4+ and CD8+ T cells, as well as NK cells from glioblastoma patients compared to counterparts from healthy donors. These pro-inhibitory changes are also correlated with reduced levels of the activation marker CD69 and the pro-inflammatory cytokine IFNγ in CD4+ and CD8+ T cells, as well as NK cells from glioblastoma patients. Collectively, these data reveal that glioblastoma-mediated CD4+ and CD8+ T cell and NK cell suppression is due, at least in part, to dysregulated TIM-3 and BAT3 expression and the associated downstream immunoregulatory and dysfunctional effects.

The zeolitic imidazolate framework (ZIF) is one of the most explored metal-organic-framework-based systems for nucleic acid delivery to cancer cells. Current nucleic acid delivery tools exhibit several drawbacks, such as high manufacturing costs, endosomal entrapment, toxicity, and immunogenicity. However, the biomimetic mineralization of Zn-based ZIFs offers a low-cost and facile encapsulation of nucleic acids at room temperature in aqueous conditions. The efficiency of nucleic acid delivery and its subsequent impact on inflammation in cells are influenced by the physicochemical properties of the material. The imidazole content determines the formation and crystallinity of ZIF, and an optimal ratio ensures the formation of well-defined and highly crystalline structures. In this study, a series of siRNA-encapsulated ZIFs (siRNA@ZIF) were systematically prepared by varying ligand-to-metal (L/M) molar ratios. Our study demonstrates that variations in ligand concentrations influence the crystalline structures, particle size, and shape of siRNA@ZIF particles. At low L/M, two-dimensional siRNA@ZIF particles form with a size of 1 μm. As the L/M ratio increases gradually, the particle size decreases, resulting in three-dimensional particles ∼200 nm in size. We also observed better stability of siRNA@ZIF in water prepared using high L/M values and time-dependent cellular uptake by the cells. Additionally, no significant impact of the biocomposites on inflammation was found, indicating the lack of an unwanted immune response and nonimmunotoxic nature over longer periods (96 h). These findings highlight the necessity of fine-tuning ligand concentrations and synthesis chemistry in designing efficient and optimal ZIF-based systems as versatile delivery platforms for nucleic acids.

The process of epithelial-mesenchymal transition (EMT) involves the phenotypic transformation of cells from epithelial to mesenchymal status. The cells exhibiting EMT contain features of cancer stem cells (CSC), and the dual processes are responsible for progressive cancers. Activation of hypoxia-inducible factors (HIF) is fundamental to the pathogenesis of clear cell renal cell carcinoma (ccRCC), and their role in promoting EMT and CSCs is crucial for ccRCC tumour cell survival, disease progression, and metastatic spread. In this study, we explored the status of HIF genes and their downstream targets, EMT and CSC markers, by immunohistochemistry on in-house accrued ccRCC biopsies and adjacent non-tumorous tissues from patients undergoing partial or radical nephrectomy. In combination, we comprehensively analysed the expression of HIF genes and its downstream EMT and CSC-associated targets relevant to ccRCC by using publicly available datasets, the cancer genome atlas (TCGA) and the clinical proteome tumour analysis consortium (CPTAC). The aim was to search for novel biological prognostic markers that can stratify high-risk patients likely to experience metastatic disease. Using the above two approaches, we report the development of novel gene signatures that may help to identify patients at a high risk of developing metastatic and progressive disease.