Modelling the tumor immune microenvironment for precision immunotherapy.

The complexity of the cellular and acellular players within the tumor microenvironment (TME) allows for significant variation in TME constitution and role in anticancer treatment response. Spatial alterations in populations of tumor cells and adjacent non-malignant cells, including endothelial cells, fibroblasts and tissue-infiltrating immune cells, often have a major role in determining disease progression and treatment response in cancer. Many current standard systemic antineoplastic treatments target the cancer cells and could be further refined to directly target commonly dysregulated cell populations of the TME. Recent developments in immuno-oncology and bioengineering have created an attractive potential to model these complexities at the level of the individual patient. These developments, along with the increasing momentum in precision medicine research and application, have catalysed exciting new discoveries in understanding drug-TME interactions, target identification, and improved efficacy of therapies. While rapid progress has been made, there are still many challenges to overcome in the development of accurate in vitro, in vivo and ex vivo models incorporating the cellular interactions that take place in the TME. In this review, we describe how advances in immuno-oncology and patient-derived models, such as patient-derived organoids and explant cultures, have enhanced the landscape of personalised immunotherapy prediction and treatment of solid organ malignancies. We describe and compare different immunological targets and perspectives on two-dimensional and three-dimensional modelling approaches that may be used to better rationalise immunotherapy use, ultimately providing a knowledge base for the integration of the autologous TME into these predictive models.

Patient-Derived Explants as a Precision Medicine Patient-Proximal Testing Platform Informing Cancer Management.

Precision medicine approaches that inform clinical management of individuals with cancer are progressively advancing. Patient-derived explants (PDEs) provide a patient-proximal ex vivo platform that can be used to assess sensitivity to standard of care (SOC) therapies and novel agents. PDEs have several advantages as a patient-proximal model compared to current preclinical models, as they maintain the phenotype and microenvironment of the individual tumor. However, the longevity of PDEs is not compatible with the timeframe required to incorporate candidate therapeutic options identified by whole exome sequencing (WES) of the patient's tumor. This review investigates how PDE longevity varies across tumor streams and how this is influenced by tissue preparation. Improving longevity of PDEs will enable individualized therapeutics testing, and thus contribute to improving outcomes for people with cancer.

Culture of Bladder Cancer Organoids as Precision Medicine Tools.

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as two-dimensional (2D) cultures on tissue culture plastic. There is a critical need for more representative models of tumor complexity that can accurately predict therapeutic response and sensitivity. The development of three-dimensional (3D) ex vivo culture of patient-derived organoids (PDOs), derived from fresh tumor tissues, aims to address these shortcomings. Organoid cultures can be used as tumor surrogates in parallel to routine clinical management to inform therapeutic decisions by identifying potential effective interventions and indicating therapies that may be futile. Here, this procedure aims to describe strategies and a detailed step-by-step protocol to establish bladder cancer PDOs from fresh, viable clinical tissue. Our well-established, optimized protocols are practical to set up 3D cultures for experiments using limited and diverse starting material directly from patients or patient-derived xenograft (PDX) tumor material. This procedure can also be employed by most laboratories equipped with standard tissue culture equipment. The organoids generated using this protocol can be used as ex vivo surrogates to understand both the molecular mechanisms underpinning urological cancer pathology and to evaluate treatments to inform clinical management.

The mitochondrial genome of the black-tailed dusky antechinus (Antechinus arktos).

In this study, we report the mitochondrial genome of the black-tailed antechinus (Antechinus arktos), a recently-discovered, endangered carnivorous marsupial inhabiting a caldera that straddles the border of Australia's mid-east coast. The circular A. arktos genome is 17,334 bp in length and has an AT content of 63.3%. Its gene content and arrangement are consistent with reported marsupial mitogenome assemblies.

The long non-coding RNA GHSROS facilitates breast cancer cell migration and orthotopic xenograft tumour growth.

Recent evidence suggests that numerous long non­coding RNAs (lncRNAs) are dysregulated in cancer, and have critical roles in tumour development and progression. The present study investigated the ghrelin receptor antisense lncRNA growth hormone secretagogue receptor opposite strand (GHSROS) in breast cancer. Reverse transcription­quantitative polymerase chain reaction revealed that GHSROS expression was significantly upregulated in breast tumour tissues compared with normal breast tissue. Induced overexpression of GHSROS in the MDA­MB­231 breast cancer cell line significantly increased cell migration in vitro, without affecting cell proliferation, a finding similar to our previous study on lung cancer cell lines. Microarray analysis revealed a significant repression of a small cluster of major histocompatibility class II genes and enrichment of immune response pathways; this phenomenon may allow tumour cells to better evade the immune system. Ectopic overexpression of GHSROS in the MDA­MB­231 cell line significantly increased orthotopic xenograft growth in mice, suggesting that in vitro culture does not fully capture the function of this lncRNA. This study demonstrated that GHSROS may serve a relevant role in breast cancer. Further studies are warranted to explore the function and therapeutic potential of this lncRNA in breast cancer progression.

The mitochondrial genome of the black-tailed dasyure (Murexia melanurus).

In this study, we report the mitochondrial genome of the black-tailed dasyure (Murexia melanurus) of New Guinea. The circular genome is 17,736 bp in length and has an AT content of 60.5%. Its gene content - 13 protein-coding genes (PCGs), 2 ribosomal (rRNA) genes, 21 transfer RNA (tRNA) genes, a tRNA pseudogene (tRNALys ), and a non-coding control region (CR) - and gene arrangement are consistent with previous marsupial mitogenome assemblies.

Limited short-term effects on human prostate cancer xenograft growth and epidermal growth factor receptor gene expression by the ghrelin receptor antagonist [D-Lys3]-GHRP-6.

PURPOSE: The ghrelin axis regulates many physiological functions (including appetite, metabolism, and energy balance) and plays a role in disease processes. As ghrelin stimulates prostate cancer proliferation, the ghrelin receptor antagonist [D-Lys3]-GHRP-6 is a potential treatment for castrate-resistant prostate cancer and for preventing the metabolic consequences of androgen-targeted therapies. We therefore explored the effect of [D-Lys3]-GHRP-6 on PC3 prostate cancer xenograft growth. METHODS: NOD/SCID mice with PC3 prostate cancer xenografts were administered 20 nmoles/mouse [D-Lys3]-GHRP-6 daily by intraperitoneal injection for 14 days and tumour volume and weight were measured. RNA sequencing of tumours was conducted to investigate expression changes following [D-Lys3]-GHRP-6 treatment. A second experiment, extending treatment time to 18 days and including a higher dose of [D-Lys3]-GHRP-6 (200 nmoles/mouse/day), was undertaken to ensure repeatability. RESULTS: We demonstrate here that daily intraperitoneal injection of 20 nmoles/mouse [D-Lys3]-GHRP-6 reduces PC3 prostate cancer xenograft tumour volume and weight in NOD/SCID mice at two weeks post treatment initiation. RNA-sequencing revealed reduced expression of epidermal growth factor receptor (EGFR) in these tumours. Further experiments demonstrated that the effects of [D-Lys3]-GHRP-6 are transitory and lost after 18 days of treatment. CONCLUSIONS: We show that [D-Lys3]-GHRP-6 has transitory effects on prostate xenograft tumours in mice, which rapidly develop an apparent resistance to the antagonist. Although further studies on [D-Lys3]-GHRP-6 are warranted, we suggest that daily treatment with the antagonist is not a suitable treatment for advanced prostate cancer.

No effect of unacylated ghrelin administration on subcutaneous PC3 xenograft growth or metabolic parameters in a Rag1-/- mouse model of metabolic dysfunction.

Ghrelin is a peptide hormone which, when acylated, regulates appetite, energy balance and a range of other biological processes. Ghrelin predominately circulates in its unacylated form (unacylated ghrelin; UAG). UAG has a number of functions independent of acylated ghrelin, including modulation of metabolic parameters and cancer progression. UAG has also been postulated to antagonise some of the metabolic effects of acyl-ghrelin, including its effects on glucose and insulin regulation. In this study, Rag1-/- mice with high-fat diet-induced obesity and hyperinsulinaemia were subcutaneously implanted with PC3 prostate cancer xenografts to investigate the effect of UAG treatment on metabolic parameters and xenograft growth. Daily intraperitoneal injection of 100 µg/kg UAG had no effect on xenograft tumour growth in mice fed normal rodent chow or 23% high-fat diet. UAG significantly improved glucose tolerance in host Rag1-/- mice on a high-fat diet, but did not significantly improve other metabolic parameters. We propose that UAG is not likely to be an effective treatment for prostate cancer, with or without associated metabolic syndrome.

Whole-Genome Sequence of the Metastatic PC3 and LNCaP Human Prostate Cancer Cell Lines.

The bone metastasis-derived PC3 and the lymph node metastasis-derived LNCaP prostate cancer cell lines are widely studied, having been described in thousands of publications over the last four decades. Here, we report short-read whole-genome sequencing (WGS) and de novo assembly of PC3 (ATCC CRL-1435) and LNCaP (clone FGC; ATCC CRL-1740) at ∼70 × coverage. A known homozygous mutation in TP53 and homozygous loss of PTEN were robustly identified in the PC3 cell line, whereas the LNCaP cell line exhibited a larger number of putative inactivating somatic point and indel mutations (and in particular a loss of stop codon events). This study also provides preliminary evidence that loss of one or both copies of the tumor suppressor Capicua (CIC) contributes to primary tumor relapse and metastatic progression, potentially offering a treatment target for castration-resistant prostate cancer (CRPC). Our work provides a resource for genetic, genomic, and biological studies employing two commonly-used prostate cancer cell lines.

Insights from engraftable immunodeficient mouse models of hyperinsulinaemia.

Hyperinsulinaemia, obesity and dyslipidaemia are independent and collective risk factors for many cancers. Here, the long-term effects of a 23% Western high-fat diet (HFD) in two immunodeficient mouse strains (NOD/SCID and Rag1 -/-) suitable for engraftment with human-derived tissue xenografts, and the effect of diet-induced hyperinsulinaemia on human prostate cancer cell line xenograft growth, were investigated. Rag1 -/-and NOD/SCID HFD-fed mice demonstrated diet-induced impairments in glucose tolerance at 16 and 23 weeks post weaning. Rag1 -/- mice developed significantly higher fasting insulin levels (2.16 ± 1.01 ng/ml, P = 0.01) and increased insulin resistance (6.70 ± 1.68 HOMA-IR, P = 0.01) compared to low-fat chow-fed mice (0.71 ± 0.12 ng/ml and 2.91 ± 0.42 HOMA-IR). This was not observed in the NOD/SCID strain. Hepatic steatosis was more extensive in Rag1 -/- HFD-fed mice compared to NOD/SCID mice. Intramyocellular lipid storage was increased in Rag1 -/- HFD-fed mice, but not in NOD/SCID mice. In Rag1 -/- HFD-fed mice, LNCaP xenograft tumours grew more rapidly compared to low-fat chow-fed mice. This is the first characterisation of the metabolic effects of long-term Western HFD in two mouse strains suitable for xenograft studies. We conclude that Rag1 -/- mice are an appropriate and novel xenograft model for studying the relationship between cancer and hyperinsulinaemia.

MUC13 overexpression in renal cell carcinoma plays a central role in tumor progression and drug resistance.

Metastatic renal cell carcinoma is a largely incurable disease, and existing treatments targeting angiogenesis and tyrosine kinase receptors are only partially effective. Here we reveal that MUC13, a cell surface mucin glycoprotein, is aberrantly expressed by most renal cell carcinomas, with increasing expression positively correlating with tumor grade. Importantly, we demonstrated that high MUC13 expression was a statistically significant independent predictor of poor survival in two independent cohorts, particularly in stage 1 cancers. In cultured renal cell carcinoma cells MUC13 promoted proliferation and induced the cell cycle regulator, cyclin D1, and inhibited apoptosis by inducing the anti-apoptotic proteins, BCL-xL and survivin. Silencing of MUC13 expression inhibited migration and invasion, and sensitized renal cancer cells to killing by the multi-kinase inhibitors used clinically, sorafenib and sunitinib, and reversed acquired resistance to these drugs. Furthermore, we demonstrated that MUC13 promotion of renal cancer cell growth and survival is mediated by activation of nuclear factor κB, a transcription factor known to regulate the expression of genes that play key roles in the development and progression of cancer. These results show that MUC13 has potential as a prognostic marker for aggressive early stage renal cell cancer and is a plausible target to sensitize these tumors to therapy.

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide.

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

Rapid method for growth hormone receptor exon 3 delete (GHRd3) SNP genotyping from archival human placental samples.

Analysis of archival samples from cohorts of pregnant women may be key to discovering prognosticators of stillbirth and pregnancy/perinatal complications. Growth hormone (GH) and its receptor (GHR) are pivotal in feto-placental development and pregnancy maintenance. We report a rapid, optimized method for genotyping the GHR full-length versus exon 3-deleted isoform (GHRd3). TaqMan single nucleotide polymorphism (SNP) genotyping proved superior to standard multiplex polymerase chain reaction (PCR) in allele detection and GHR genotyping from archived samples, including those with poor genomic deoxyribonucleic acid quality/quantity such as formalin fixed, paraffin embedded, blood, and serum. Furthermore, this assay is suitable for high through put 96 or 384-well plate quantitative PCR machines with automated genotype calling software. The TaqMan genotyping assay can increase the data obtained from precious archival human samples.

Comparative analysis reveals loss of the appetite-regulating peptide hormone ghrelin in falcons.

Ghrelin and leptin are key peripherally secreted appetite-regulating hormones in vertebrates. Here we consider the ghrelin gene (GHRL) of birds (class Aves), where it has been reported that ghrelin inhibits rather than augments feeding. Thirty-one bird species were compared, revealing that most species harbour a functional copy of GHRL and the coding region for its derived peptides ghrelin and obestatin. We provide evidence for loss of GHRL in saker and peregrine falcons, and this is likely to result from the insertion of an ERVK retrotransposon in intron 0. We hypothesise that the loss of anorexigenic ghrelin is a predatory adaptation that results in increased food-seeking behaviour and feeding in falcons.

Ghrelin O-acyltransferase (GOAT) is expressed in prostate cancer tissues and cell lines and expression is differentially regulated in vitro by ghrelin.

BACKGROUND: Ghrelin is a 28 amino acid peptide hormone that is expressed in the stomach and a range of peripheral tissues, where it frequently acts as an autocrine/paracrine growth factor. Ghrelin is modified by a unique acylation required for it to activate its cognate receptor, the growth hormone secretagogue receptor (GHSR), which mediates many of the actions of ghrelin. Recently, the enzyme responsible for adding the fatty acid residue (octanoyl/acyl group) to the third amino acid of ghrelin, GOAT (ghrelin O-acyltransferase), was identified. METHODS: We used cell culture, quantitative real-time reverse transcription (RT)-PCR and immunohistochemistry to demonstrate the expression of GOAT in prostate cancer cell lines and tissues from patients. Real-time RT-PCR was used to demonstrate the expression of prohormone convertase (PC)1/3, PC2 and furin in prostate cancer cell lines. Prostate-derived cell lines were treated with ghrelin and desacyl ghrelin and the effect on GOAT expression was measured using quantitative RT-PCR. RESULTS: We have demonstrated that GOAT mRNA and protein are expressed in the normal prostate and human prostate cancer tissue samples. The RWPE-1 and RWPE-2 normal prostate-derived cell lines and the LNCaP, DU145, and PC3 prostate cancer cell lines express GOAT and at least one other enzyme that is necessary to produce mature, acylated ghrelin from proghrelin (PC1/3, PC2 or furin). Finally, ghrelin, but not desacyl ghrelin (unacylated ghrelin), can directly regulate the expression of GOAT in the RWPE-1 normal prostate derived cell line and the PC3 prostate cancer cell line. Ghrelin treatment (100nM) for 6 hours significantly decreased GOAT mRNA expression two-fold (P < 0.05) in the PC3 prostate cancer cell line, however, ghrelin did not regulate GOAT expression in the DU145 and LNCaP prostate cancer cell lines. CONCLUSIONS: This study demonstrates that GOAT is expressed in prostate cancer specimens and cell lines. Ghrelin regulates GOAT expression, however, this is likely to be cell-type specific. The expression of GOAT in prostate cancer supports the hypothesis that the ghrelin axis has autocrine/paracrine roles. We propose that the RWPE-1 prostate cell line and the PC3 prostate cancer cell line may be useful for investigating GOAT regulation and function.

Silencing of ghrelin receptor expression inhibits endometrial cancer cell growth in vitro and in vivo.

Ghrelin is a 28-amino acid peptide hormone produced predominantly in the stomach but also in a range of normal cell types and tumors, where it has endocrine, paracrine, and autocrine roles. Previously, we have demonstrated that ghrelin has proliferative and antiapoptotic effects in endometrial cancer cell lines, suggesting a potential role in promoting tumor growth. In the present study, we investigated the effect of ghrelin receptor, GHSR, and gene silencing in vitro and in vivo and characterized ghrelin and GHSR1a protein expression in human endometrial tumors. GHSR gene silencing was achieved in the Ishikawa and KLE endometrial cancer cell lines, using a lentiviral short-hairpin RNA targeting GHSR. The effects of GHSR1a knockdown were further analyzed in vivo using the Ishikawa cell line in a NOD/SCID xenograft model. Cell proliferation was reduced in cultured GHSR1a knockdown Ishikawa and KLE cells compared with scrambled controls in the absence of exogenously applied ghrelin and in response to exogenous ghrelin (1,000 nM). The tumor volumes were reduced significantly in GHSR1a knockdown Ishikawa mouse xenograft tumors compared with scrambled control tumours. Using immunohistochemistry, we demonstrated that ghrelin and GHSR1a are expressed in benign and cancerous glands in human endometrial tissue specimens, although there was no correlation between the intensity of staining and cancer grade. These data indicate that downregulation of GHSR expression significantly inhibits endometrial cancer cell line and mouse xenograft tumour growth. This is the first preclinical evidence that downregulation of GHSR may be therapeutic in endometrial cancer.

IL-10 promotes production of intestinal mucus by suppressing protein misfolding and endoplasmic reticulum stress in goblet cells.

BACKGROUND & AIMS: Protein misfolding and endoplasmic reticulum (ER) stress have been observed in intestinal secretory cells from patients with inflammatory bowel diseases and induce intestinal inflammation in mice. However, it is not clear how immune factors affect ER stress and therefore disease symptoms. METHODS: We analyzed the effects of interleukin (IL)-10 on ER stress in intestinal tissues in wild-type C57BL/6, Winnie, IL-10(-/-), and Winnie × IL-10(+/-) mice. In Winnie mice, misfolding of the intestinal mucin Muc2 initiates ER stress and inflammation. We also analyzed the effects of different inhibitors of IL-10 signaling and the N-glycosylation inhibitor tunicamycin in cultured human LS174T goblet cells. RESULTS: Administration of neutralizing antibodies against IL-10 or its receptor (IL-10R1) to Winnie mice rapidly exacerbated ER stress and intestinal inflammation compared with mice given vehicle (controls). Antibodies against IL-10 also increased accumulation of misfolded Muc2 in the ER of goblet cells of Winnie mice and increased T-cell production of inflammatory cytokines. Winnie × IL-10(+/-) mice and IL-10(-/-) mice with a single Winnie allele each developed more severe inflammation than Winnie mice or IL-10(-/-) mice. Administration of tunicamycin to wild-type mice caused intestinal ER stress, which increased when IL-10R1 was blocked. In LS174T cells, induction of ER stress with tunicamycin and misfolding of MUC2 were reduced by administration of IL-10; this reduction required STAT1 and STAT3. In LS174T cells incubated with tunicamycin, IL-10 up-regulated genes involved in MUC2 folding and in ER-associated degradation and maintained correct folding of MUC2, its transport from the ER, and its O-glycosylation and secretion. CONCLUSIONS: IL-10 prevents protein misfolding and ER stress by maintaining mucin production in goblet cells and helps the intestine preserve the mucus barrier.

The MUC13 cell-surface mucin protects against intestinal inflammation by inhibiting epithelial cell apoptosis.

BACKGROUND AND AIMS: The MUC13 transmembrane mucin is highly and constitutively expressed in the small and large intestine. Although MUC13 polymorphisms have been associated with human inflammatory bowel diseases and susceptibility to Escherichia coli infection in pigs, the biological functions of MUC13 are unknown. This study aimed to explore whether MUC13 modulates intestinal inflammation. METHODS: Muc13(-/-) mice were generated, phenotyped and challenged with the colitis-inducing agent, dextran sodium sulphate (DSS). Colitis was assessed by clinical symptoms and intestinal histopathology. Intestinal epithelial cell apoptosis and proliferation, macrophage infiltration and cytokine production were also quantified. Apoptosis of human LS513 intestinal epithelial cells in response to apoptotic agents, including DSS, was also measured, following knockdown of MUC13 with siRNA. RESULTS: Muc13(-/-) mice were viable, fertile and developed normally, with no spontaneous intestinal pathology except mild focal neutrophilic inflammation in the small and large intestines of old mice. In response to DSS challenge, Muc13(-/-) mice developed more severe acute colitis, as reflected by increased weight loss, rectal bleeding, diarrhoea and histological colitis scores compared with wild-type mice. Increased numbers of F4/80(+) macrophages in inflamed mucosa of Muc13(-/-) mice were accompanied by increased expression of intestinal IL-1ß and TNFα mRNA. Muc13(-/-) mice had significantly increased intestinal epithelial cell apoptosis within 3 days of DSS exposure. LS513 cells were more susceptible to DSS, actinomycin-D, ultraviolet irradiation and TRAIL-induced apoptosis when MUC13 was knocked down by siRNA. CONCLUSIONS: These novel findings indicate a protective role for Muc13 in the colonic epithelium by inhibiting toxin-induced apoptosis and have important implications for intestinal infections, inflammatory diseases and the development of intestinal cancer.

Endocrine impact of Helicobacter pylori: focus on ghrelin and ghrelin o-acyltransferase.

Ghrelin is predominantly produced by the gastric enteroendocrine cell compartment and is octanoylated by the recently discovered ghrelin o-acyltransferase (GOAT) before secretion into the bloodstream. This octanoylation is essential for many of the biological properties of ghrelin including appetite stimulation and anti-inflammatory properties as only the acylated form of ghrelin binds to the ghrelin receptor, the growth hormone secretagogue receptor (GHS-R). Given the gastric location of ghrelin production, it is perhaps not surprising that insult to the gastric mucosa affects circulating ghrelin levels in humans. Helicobacter pylori (H. pylori) infects more than fifty percent of the world's population and once established within the gastric mucosa, can persist for life. Infection is associated with chronic gastritis, gastric atrophy and ulceration, reduced appetite and a lower body mass index (BMI). The large majority of studies investigating levels of circulating ghrelin and ghrelin expression in the stomach in patients with H. pylori infection indicate that the bacterium has a negative impact on ghrelin production and/or secretion. Eradication of infection restores ghrelin, improves appetite and increases BMI in some studies, however, a causative relationship between H. pylori-associated serum ghrelin decline and food intake and obesity has not been established. Most studies measure total ghrelin in the circulation although the measurement of the ratio of acyl/total ghrelin gives a clearer indication that the ghrelin acylation process is altered during infection and atrophy. GOAT is essential for the production of biologically-active, acyl ghrelin and the impact of H. pylori on GOAT expression and activity will be highly informative in the future.