The tumour microenvironment, treatment resistance and recurrence in glioblastoma.

The adaptability of glioblastoma (GBM) cells, encouraged by complex interactions with the tumour microenvironment (TME), currently renders GBM an incurable cancer. Despite intensive research, with many clinical trials, GBM patients rely on standard treatments including surgery followed by radiation and chemotherapy, which have been observed to induce a more aggressive phenotype in recurrent tumours. This failure to improve treatments is undoubtedly a result of insufficient models which fail to incorporate components of the human brain TME. Research has increasingly uncovered mechanisms of tumour-TME interactions that correlate to worsened patient prognoses, including tumour-associated astrocyte mitochondrial transfer, neuronal circuit remodelling and immunosuppression. This tumour hijacked TME is highly implicated in driving therapy resistance, with further alterations within the TME and tumour resulting from therapy exposure inducing increased tumour growth and invasion. Recent developments improving organoid models, including aspects of the TME, are paving an exciting future for the research and drug development for GBM, with the hopes of improving patient survival growing closer. This review focuses on GBMs interactions with the TME and their effect on tumour pathology and treatment efficiency, with a look at challenges GBM models face in sufficiently recapitulating this complex and highly adaptive cancer.

Recent Advances in Understanding the Molecular Basis of Infantile Haemangioma Development.

Infantile haemangioma (IH), the most common vascular tumour of infancy, is comprised of diverse cell types including endothelial cells, pericytes, fibroblasts and immune cells. IH is characterized by rapid proliferation followed by slow involution over 1 - 10 years. Most lesions regress spontaneously, but up to 10% can be disfiguring with complications that require further medical treatment. Recent research has revealed the biological characteristics of IH, highlighting the involvement of angiogenesis and vasculogenesis during tumour formation. Gene expression profiling has provided vital insights into these underlying biological processes, with some of the key IH-related pathways identified, including VEGF, RAAS, HIF-1α, Notch, PDGF, PI3K/Akt/mTOR, JAK/STAT, FGF, PPARγ, IGF. Further evidence suggests extracellular matrix factors and hormone receptors regulate IH progression. In this review, we explore the molecular mechanisms involved in the proliferating, plateau and involuting phases of IH. This involves identifying differentially expressed genes, targeted proteins, and key signalling pathways. This knowledge will increase the broader understanding of vascular development, tissue remodelling and angiogenesis.

Unlocking hidden potential: advancements, approaches, and obstacles in repurposing drugs for cancer therapy.

High rates of failure, exorbitant costs, and the sluggish pace of new drug discovery and development have led to a growing interest in repurposing "old" drugs to treat both common and rare diseases, particularly cancer. Cancer, a complex and heterogeneous disease, often necessitates a combination of different treatment modalities to achieve optimal outcomes. The intrinsic polygenicity of cancer, intricate biological signalling networks, and feedback loops make the inhibition of a single target frequently insufficient for achieving the desired therapeutic impact. As a result, addressing these complex or "smart" malignancies demands equally sophisticated treatment strategies. Combinatory treatments that target the multifaceted oncogenic signalling network hold immense promise. Repurposed drugs offer a potential solution to this challenge, harnessing known compounds for new indications. By avoiding the prohibitive costs and long development timelines associated with novel cancer drugs, this approach holds the potential to usher in more effective, efficient, and cost-effective cancer treatments. The pursuit of combinatory therapies through drug repurposing may hold the key to achieving superior outcomes for cancer patients. However, drug repurposing faces significant commercial, technological and regulatory challenges that need to be addressed. This review explores the diverse approaches employed in drug repurposing, delves into the challenges faced by the drug repurposing community, and presents innovative solutions to overcome these obstacles. By emphasising the significance of combinatory treatments within the context of drug repurposing, we aim to unlock the full potential of this approach for enhancing cancer therapy. The positive aspects of drug repurposing in oncology are underscored here; encompassing personalized treatment, accelerated development, market opportunities for shelved drugs, cancer prevention, expanded patient reach, improved patient access, multi-partner collaborations, increased likelihood of approval, reduced costs, and enhanced combination therapy.

The helminth TGF-ß mimic TGM4 is a modular ligand that binds CD44, CD49d and TGF-ß receptors to preferentially target myeloid cells.

The murine helminth parasite Heligmosomoides polygyrus expresses a family of modular proteins which, replicating the functional activity of the immunomodulatory cytokine TGF-ß, have been named TGM (TGF-ß Μimic). Multiple domains bind to different receptors, including TGF-ß receptors TßRI (ALK5) and TßRII through domains 1-3, and prototypic family member TGM1 binds the cell surface co-receptor CD44 through domains 4-5. This allows TGM1 to induce T lymphocyte Foxp3 expression, characteristic of regulatory (Treg) cells, and to activate a range of TGF-ß-responsive cell types. In contrast, a related protein, TGM4, targets a much more restricted cell repertoire, primarily acting on myeloid cells, with less potent effects on T cells and lacking activity on other TGF-ß-responsive cell types. TGM4 binds avidly to myeloid cells by flow cytometry, and can outcompete TGM1 for cell binding. Analysis of receptor binding in comparison to TGM1 reveals a 10-fold higher affinity than TGM1 for TGFßR-I (TßRI), but a 100-fold lower affinity for TßRII through Domain 3. Consequently, TGM4 is more dependent on co-receptor binding; in addition to CD44, TGM4 also engages CD49d (Itga4) through Domains 1-3, as well as CD206 and Neuropilin-1 through Domains 4 and 5. TGM4 was found to effectively modulate macrophage populations, inhibiting lipopolysaccharide-driven inflammatory cytokine production and boosting interleukin (IL)-4-stimulated responses such as Arginase-1 in vitro and in vivo. These results reveal that the modular nature of TGMs has allowed the fine tuning of the binding affinities of the TßR- and co-receptor binding domains to establish cell specificity for TGF-ß signalling in a manner that cannot be attained by the mammalian cytokine.

CD44 acts as a coreceptor for cell-specific enhancement of signaling and regulatory T cell induction by TGM1, a parasite TGF-ß mimic.

Long-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, Heligmosomoides polygyrus, down-modulates the host immune system through release of an immunosuppressive TGF-ß mimic, TGM1, which is a divergent member of the CCP (Sushi) protein family. TGM1 comprises 5 domains, of which domains 1-3 (D1/2/3) bind mammalian TGF-ß receptors, acting on T cells to induce Foxp3+ regulatory T cells; however, the roles of domains 4 and 5 (D4/5) remain unknown. We noted that truncated TGM1, lacking D4/5, showed reduced potency. Combination of D1/2/3 and D4/5 as separate proteins did not alter potency, suggesting that a physical linkage is required and that these domains do not deliver an independent signal. Coprecipitation from cells treated with biotinylated D4/5, followed by mass spectrometry, identified the cell surface protein CD44 as a coreceptor for TGM1. Both full-length and D4/5 bound strongly to a range of primary cells and cell lines, to a greater degree than D1/2/3 alone, although some cell lines did not respond to TGM1. Ectopic expression of CD44 in nonresponding cells conferred responsiveness, while genetic depletion of CD44 abolished enhancement by D4/5 and ablated the ability of full-length TGM1 to bind to cell surfaces. Moreover, CD44-deficient T cells showed attenuated induction of Foxp3 by full-length TGM1, to levels similar to those induced by D1/2/3. Hence, a parasite protein known to bind two host cytokine receptor subunits has evolved a third receptor specificity, which serves to raise the avidity and cell type-specific potency of TGF-ß signaling in mammalian cells.

Protection from T cell-dependent colitis by the helminth-derived immunomodulatory mimic of transforming growth factor-ß, Hp-TGM.

In animal models of inflammatory colitis, pathology can be ameliorated by several intestinal helminth parasites, including the mouse nematode Heligmosomoides polygyrus. To identify parasite products that may exert anti-inflammatory effects in vivo, we tested H. polygyrus excretory-secretory (HES) products, as well as a recombinantly expressed parasite protein, transforming growth factor mimic (TGM), that functionally mimics the mammalian immunomodulatory cytokine TGF-ß. HES and TGM showed a degree of protection in dextran sodium sulphate-induced colitis, with a reduction in inflammatory cytokines, but did not fully block the development of pathology. HES also showed little benefit in a similar acute trinitrobenzene sulphonic acid-induced model. However, in a T cell transfer-mediated model with recombination activation gene (RAG)-deficient mice, HES-reduced disease scores if administered throughout the first 2 or 4 weeks following transfer but was less effective if treatment was delayed until 14 days after T cell transfer. Recombinant TGM similarly dampened colitis in RAG-deficient recipients of effector T cells, and was effective even if introduced only once symptoms had begun to be manifest. These results are a promising indication that TGM may replicate, and even surpass, the modulatory properties of native parasite HES.

TGM6, a helminth secretory product, mimics TGF-ß binding to TßRII to antagonize TGF-ß signaling in fibroblasts.

The murine helminth parasite Heligmosomoides polygyrus expresses a family of proteins structurally related to TGF-ß Mimic 1 (TGM1), a secreted five domain protein that activates the TGF-ß pathway and converts naïve T lymphocytes to immunosuppressive Tregs. TGM1 signals through the TGF-ß type I and type II receptors, TßRI and TßRII, with domains 1-2 and 3 binding TßRI and TßRII, respectively, and domains 4-5 binding CD44, a co-receptor abundant on T cells. TGM6 is a homologue of TGM1 that is co-expressed with TGM1, but lacks domains 1 and 2. Herein, we show that TGM6 binds TßRII through domain 3, but does not bind TßRI, or other type I or type II receptors of the TGF-ß family. In TGF-ß reporter assays in fibroblasts, TGM6, but not truncated TGM6 lacking domains 4 and 5, potently inhibits TGF-ß- and TGM1-induced signaling, consistent with its ability to bind TßRII but not TßRI or other receptors of the TGF-ß family. However, TGM6 does not bind CD44 and is unable to inhibit TGF-ß and TGM1 signaling in T cells. To understand how TGM6 binds TßRII, the X-ray crystal structure of the TGM6 domain 3 bound to TßRII was determined at 1.4 Å. This showed that TGM6 domain 3 binds TßRII through an interface remarkably similar to the TGF-ß:TßRII interface. These results suggest that TGM6 has adapted its domain structure and sequence to mimic TGF-ß binding to TßRII and function as a potent TGF-ß and TGM1 antagonist in fibroblasts. The coexpression of TGM6, along with the immunosuppressive TGMs that activate the TGF-ß pathway, may prevent tissue damage caused by the parasite as it progresses through its life cycle from the intestinal lumen to submucosal tissues and back again.

The IL-25-dependent tuft cell circuit driven by intestinal helminths requires macrophage migration inhibitory factor (MIF).

Macrophage migration inhibitory factor (MIF) is a key innate immune mediator with chemokine- and cytokine-like properties in the inflammatory pathway. While its actions on macrophages are well-studied, its effects on other cell types are less understood. Here we report that MIF is required for expansion of intestinal tuft cells during infection with the helminth Nippostrongylus brasiliensis. MIF-deficient mice show defective innate responses following infection, lacking intestinal epithelial tuft cell hyperplasia or upregulation of goblet cell RELMß, and fail to expand eosinophil, type 2 innate lymphoid cell (ILC2) and macrophage (M2) populations. Similar effects were observed in MIF-sufficient wild-type mice given the MIF inhibitor 4-IPP. MIF had no direct effect on epithelial cells in organoid cultures, and MIF-deficient intestinal stem cells could generate tuft cells in vitro in the presence of type 2 cytokines. In vivo the lack of MIF could be fully compensated by administration of IL-25, restoring tuft cell differentiation and goblet cell expression of RELM-ß, demonstrating its requirement upstream of the ILC2-tuft cell circuit. Both ILC2s and macrophages expressed the MIF receptor CXCR4, indicating that MIF may act as an essential co-factor on both cell types to activate responses to IL-25 in helminth infection.

Oral delivery of a functional algal-expressed TGF-ß mimic halts colitis in a murine DSS model.

Inflammatory bowel disease (IBD) is a set of immunological disorders which can generate chronic pain and fatigue associated with the inflammatory symptoms. The treatment of IBD remains a significant hurdle with current therapies being only partially effective or having significant side effects, suggesting that new therapies that elicit different modes of action and delivery strategies are required. TGM1 is a TGF-ß mimic that was discovered from the intestinal helminth parasite Heligmosomoides polygyrus and is thought to be produced by the parasite to suppress the intestinal inflammation response to help evade host immunity, making it an ideal candidate to be developed as a novel anti-inflammatory bio-therapeutic. Here we utilized the expression system of the edible green algae Chlamydomonas reinhardtii in order to recombinantly produce active TGM1 in a form that could be ingested. C. reinhardtii robustly expressed TGM1, and the resultant recombinant protein is biologically active as measured by regulatory T cell induction. When delivered orally to mice, the algal expressed TGM1 is able to ameliorate weight loss, lymphadenopathy, and disease symptoms in a mouse model of DSS-induced colitis, demonstrating the potential of this biologic as a novel treatment of IBD.

The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function.

T cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired. In the absence of CCT-controlled protein folding, Th2 polarization diverges from normal differentiation with paradoxical continued IFN-γ expression. As a result, CCT-deficient T cells fail to generate an efficient immune protection against helminths as they are unable to sustain a coordinated recruitment of the innate and adaptive immune systems. These findings thus demonstrate that normal T cell biology is critically dependent on CCT-controlled proteostasis and that its absence is incompatible with protective immunity.

The parasite cytokine mimic Hp-TGM potently replicates the regulatory effects of TGF-ß on murine CD4+ T cells.

Transforming growth factor-beta (TGF-ß) family proteins mediate many vital biological functions in growth, development and regulation of the immune system. TGF-ß itself controls immune homeostasis and inflammation, including conversion of naïve CD4+ T cells into Foxp3+ regulatory T cells (Tregs) in the presence of interleukin-2 and T-cell receptor ligands. The helminth parasite Heligmosomoides polygyrus exploits this pathway through a structurally novel TGF-ß mimic (Hp-TGM), which binds to mammalian TGF-ß receptors and induces Tregs. Here, we performed detailed comparisons of Hp-TGM with mammalian TGF-ß. Compared with TGF-ß, Hp-TGM induced greater numbers of Foxp3+ Tregs (iTregs), with more intense Foxp3 expression. Both ligands upregulated Treg functional markers CD73, CD103 and programmed death-ligand 1, but Hp-TGM induced significantly higher CD39 expression than did TGF-ß. Interestingly, in contrast to canonical TGF-ß signaling through Smad2/3, Hp-TGM stimulation was slower and more sustained. Gene expression profiles induced by TGF-ß and Hp-TGM were remarkably similar, and both types of iTregs suppressed T-cell responses in vitro and experimental autoimmune encephalomyelitis-driven inflammation in vivo. In vitro, both types of iTregs were equally stable under inflammatory conditions, but Hp-TGM-induced iTregs were more stable in vivo during dextran sodium sulfate-induced colitis, with greater retention of Foxp3 expression and lower conversion to a ROR-γt+ phenotype. Altogether, results from this study suggest that the parasite cytokine mimic, Hp-TGM, may deliver a qualitatively different signal to CD4+ T cells with downstream consequences for the long-term stability of iTregs. These data highlight the potential of Hp-TGM as a new modulator of T-cell responses in vitro and in vivo.

The Helminth Parasite Heligmosomoides polygyrus Attenuates EAE in an IL-4Rα-Dependent Manner.

Helminth parasites are effective in biasing Th2 immunity and inducing regulatory pathways that minimize excessive inflammation within their hosts, thus allowing chronic infection to occur whilst also suppressing bystander atopic or autoimmune diseases. Multiple sclerosis (MS) is a severe autoimmune disease characterized by inflammatory lesions within the central nervous system; there are very limited therapeutic options for the progressive forms of the disease and none are curative. Here, we used the experimental autoimmune encephalomyelitis (EAE) model to examine if the intestinal helminth Heligmosomoides polygyrus and its excretory/secretory products (HES) are able to suppress inflammatory disease. Mice infected with H. polygyrus at the time of immunization with the peptide used to induce EAE (myelin-oligodendrocyte glycoprotein, pMOG), showed a delay in the onset and peak severity of EAE disease, however, treatment with HES only showed a marginal delay in disease onset. Mice that received H. polygyrus 4 weeks prior to EAE induction were also not significantly protected. H. polygyrus secretes a known TGF-ß mimic (Hp-TGM) and simultaneous H. polygyrus infection with pMOG immunization led to a significant expansion of Tregs; however, administering the recombinant Hp-TGM to EAE mice failed to replicate the EAE protection seen during infection, indicating that this may not be central to the disease protecting mechanism. Mice infected with H. polygyrus also showed a systemic Th2 biasing, and restimulating splenocytes with pMOG showed release of pMOG-specific IL-4 as well as suppression of inflammatory IL-17A. Notably, a Th2-skewed response was found only in mice infected with H. polygyrus at the time of EAE induction and not those with a chronic infection. Furthermore, H. polygyrus failed to protect against disease in IL-4Rα-/- mice. Together these results indicate that the EAE disease protective mechanism of H. polygyrus is likely to be predominantly Th2 deviation, and further highlights Th2-biasing as a future therapeutic strategy for MS.

Regulatory T-cells in helminth infection: induction, function and therapeutic potential.

Helminth parasites infect an alarmingly large proportion of the world's population, primarily within tropical regions, and their ability to down-modulate host immunity is key to their persistence. Helminths have developed multiple mechanisms that induce a state of hyporesponsiveness or immune suppression within the host; of particular interest are mechanisms that drive the induction of regulatory T-cells (Tregs). Helminths actively induce Tregs either directly by secreting factors, such as the TGF-ß mimic Hp-TGM, or indirectly by interacting with bystander cell types such as dendritic cells and macrophages that then induce Tregs. Expansion of Tregs not only enhances parasite survival but, in cases such as filarial infection, Tregs also play a role in preventing parasite-associated pathologies. Furthermore, Tregs generated during helminth infection have been associated with suppression of bystander immunopathologies in a range of inflammatory conditions such as allergy and autoimmune disease. In this review, we discuss evidence from natural and experimental infections that point to the pathways and molecules involved in helminth Treg induction, and postulate how parasite-derived molecules and/or Tregs might be applied as anti-inflammatory therapies in the future.

TGF-ß mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus.

We recently reported the discovery of a new parasite-derived protein that functionally mimics the immunosuppressive cytokine transforming growth factor (TGF)-ß. The Heligmosomoides polygyrus TGF-ß Mimic (Hp-TGM) shares no homology to any TGF-ß family member, however it binds the mammalian TGF-ß receptor and induces expression of Foxp3, the canonical transcription factor of both mouse and human regulatory T cells. Hp-TGM consists of five atypical Complement Control Protein (CCP, Pfam 00084) domains, each lacking certain conserved residues and 12-15 amino acids longer than the 60-70 amino acids consensus domain, but with a recognizable 3-cysteine, tryptophan, cysteine motif. We now report on the identification of a family of nine related Hp-TGM homologues represented in the secreted proteome and transcriptome of H. polygyrus. Recombinant proteins from five of the nine new TGM members were tested for TGF-ß activity, but only two were functionally active in an MFB-F11 reporter assay, and by the induction of T cell Foxp3 expression. Sequence comparisons reveal that proteins with functional activity are similar or identical to Hp-TGM across the first three CCP domains, but more variable in domains 4 and 5. Inactive proteins diverged in all domains, or lacked some domains entirely. Testing truncated versions of Hp-TGM confirmed that domains 1-3 are essential for full activity in vitro, while domains 4 and 5 are not required. Further studies will elucidate whether these latter domains fulfill other functions in promoting host immune regulation during infection and if the more divergent family members play other roles in immunomodulation.

Innate IFN-γ ameliorates experimental autoimmune encephalomyelitis and promotes myeloid expansion and PDL-1 expression.

The innate immune system plays a central role in the immune-mediated pathology of multiple sclerosis, and is a therapeutic target for progressive disease. Recently, it has been demonstrated that MIS416, a novel immunomodulatory microparticle that activates NOD-2 and TLR-9-signaling, has disease-modifying activity in multiple sclerosis models. This activity is dependent on innate IFN-γ; however, the precise immune regulatory mechanisms amplified by MIS416 have not previously been determined. Using the experimental autoimmune encephalomyelitis model, MIS416 treatment was associated with IFN-γ-dependant expansion of Treg number and increased suppressive function; however, these cells did not account for disease reduction. Additionally, MIS416 treatment stimulated increased nitric oxide production that was IFN-γ-dependant but dispensable for protection. Finally, MIS416-mediated protection was shown to correlate with IFN-γ-dependant expansion of PDL-1-expressing peripheral myeloid cells, a subset of which was found to be selectively recruited to the brain. This central nervous system trafficking was independent of neuro-inflammatory signals as it occurred in MIS416-treated healthy mice. Together, these findings provide insight into regulatory myeloid cell activities amplified by MIS416-mediated NOD-2 and TLR-9 signalling and highlight the potential importance of these cells in accessing the brain where they may act locally and contribute to the control of neuroinflammation.

A structurally distinct TGF-ß mimic from an intestinal helminth parasite potently induces regulatory T cells.

Helminth parasites defy immune exclusion through sophisticated evasion mechanisms, including activation of host immunosuppressive regulatory T (Treg) cells. The mouse parasite Heligmosomoides polygyrus can expand the host Treg population by secreting products that activate TGF-ß signalling, but the identity of the active molecule is unknown. Here we identify an H. polygyrus TGF-ß mimic (Hp-TGM) that replicates the biological and functional properties of TGF-ß, including binding to mammalian TGF-ß receptors and inducing mouse and human Foxp3+ Treg cells. Hp-TGM has no homology with mammalian TGF-ß or other members of the TGF-ß family, but is a member of the complement control protein superfamily. Thus, our data indicate that through convergent evolution, the parasite has acquired a protein with cytokine-like function that is able to exploit an endogenous pathway of immunoregulation in the host.