Colorimetric histology using plasmonically active microscope slides.

The human eye can distinguish as many as 10,000 different colours but is far less sensitive to variations in intensity1, meaning that colour is highly desirable when interpreting images. However, most biological samples are essentially transparent, and nearly invisible when viewed using a standard optical microscope2. It is therefore highly desirable to be able to produce coloured images without needing to add any stains or dyes, which can alter the sample properties. Here we demonstrate that colorimetric histology images can be generated using full-sized plasmonically active microscope slides. These slides translate subtle changes in the dielectric constant into striking colour contrast when samples are placed upon them. We demonstrate the biomedical potential of this technique, which we term histoplasmonics, by distinguishing neoplastic cells from normal breast epithelium during the earliest stages of tumorigenesis in the mouse MMTV-PyMT mammary tumour model. We then apply this method to human diagnostic tissue and validate its utility in distinguishing normal epithelium, usual ductal hyperplasia, and early-stage breast cancer (ductal carcinoma in situ). The colorimetric output of the image pixels is compared to conventional histopathology. The results we report here support the hypothesis that histoplasmonics can be used as a novel alternative or adjunct to general staining. The widespread availability of this technique and its incorporation into standard laboratory workflows may prove transformative for applications extending well beyond tissue diagnostics. This work also highlights opportunities for improvements to digital pathology that have yet to be explored.

Prostate cancer cell-intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone.

The latency associated with bone metastasis emergence in castrate-resistant prostate cancer is attributed to dormancy, a state in which cancer cells persist prior to overt lesion formation. Using single-cell transcriptomics and ex vivo profiling, we have uncovered the critical role of tumor-intrinsic immune signaling in the retention of cancer cell dormancy. We demonstrate that loss of tumor-intrinsic type I IFN occurs in proliferating prostate cancer cells in bone. This loss suppresses tumor immunogenicity and therapeutic response and promotes bone cell activation to drive cancer progression. Restoration of tumor-intrinsic IFN signaling by HDAC inhibition increased tumor cell visibility, promoted long-term antitumor immunity, and blocked cancer growth in bone. Key findings were validated in patients, including loss of tumor-intrinsic IFN signaling and immunogenicity in bone metastases compared to primary tumors. Data herein provide a rationale as to why current immunotherapeutics fail in bone-metastatic prostate cancer, and provide a new therapeutic strategy to overcome the inefficacy of immune-based therapies in solid cancers.

Loss of type I IFN responsiveness impairs natural killer cell antitumor activity in breast cancer.

Competent type I IFN signaling is the lynchpin of most immune surveillance mechanisms and has recently proven critical to the efficacy of several anticancer agents. Expression of the type I IFN receptor, IFNAR, underpins type I IFN responsiveness in all cells and facilitates the activation and cytotoxic potential of lymphocytes, while loss of IFNAR on lymphocytes has previously been associated with tumor progression and poor patient survival. This study underscores the importance of intact type I IFN signaling to NK cells in the regulation of tumorigenesis and metastasis, whereby ablation of NK cell IFNAR1 impairs antitumor activity and tumor clearance. Using a preclinical model of triple negative breast cancer, we identified that intact IFNAR on NK cells is required for an effective response to type I IFN-inducing immunotherapeutics that may be mediated by pathways associated with NK cell degranulation. Taken together, these data provide a rationale for considering the IFNAR status on NK cells when devising therapeutic strategies aimed at inducing systemic type I IFN signaling in breast cancer.

Smac mimetics LCL161 and GDC-0152 inhibit osteosarcoma growth and metastasis in mice.

BACKGROUND: Current therapies fail to cure over a third of osteosarcoma patients and around three quarters of those with metastatic disease. "Smac mimetics" (also known as "IAP antagonists") are a new class of anti-cancer agents. Previous work revealed that cells from murine osteosarcomas were efficiently sensitized by physiologically achievable concentrations of some Smac mimetics (including GDC-0152 and LCL161) to killing by the inflammatory cytokine TNFα in vitro, but survived exposure to Smac mimetics as sole agents. METHODS: Nude mice were subcutaneously or intramuscularly implanted with luciferase-expressing murine 1029H or human KRIB osteosarcoma cells. The impacts of treatment with GDC-0152, LCL161 and/or doxorubicin were assessed by caliper measurements, bioluminescence, 18FDG-PET and MRI imaging, and by weighing resected tumors at the experimental endpoint. Metastatic burden was examined by quantitative PCR, through amplification of a region of the luciferase gene from lung DNA. ATP levels in treated and untreated osteosarcoma cells were compared to assess in vitro sensitivity. Immunophenotyping of cells within treated and untreated tumors was performed by flow cytometry, and TNFα levels in blood and tumors were measured using cytokine bead arrays. RESULTS: Treatment with GDC-0152 or LCL161 suppressed the growth of subcutaneously or intramuscularly implanted osteosarcomas. In both models, co-treatment with doxorubicin and Smac mimetics impeded average osteosarcoma growth to a greater extent than either drug alone, although these differences were not statistically significant. Co-treatments were also more toxic. Co-treatment with LCL161 and doxorubicin was particularly effective in the KRIB intramuscular model, impeding primary tumor growth and delaying or preventing metastasis. Although the Smac mimetics were effective in vivo, in vitro they only efficiently killed osteosarcoma cells when TNFα was supplied. Implanted tumors contained high levels of TNFα, produced by infiltrating immune cells. Spontaneous osteosarcomas that arose in genetically-engineered immunocompetent mice also contained abundant TNFα. CONCLUSIONS: These data imply that Smac mimetics can cooperate with TNFα secreted by tumor-associated immune cells to kill osteosarcoma cells in vivo. Smac mimetics may therefore benefit osteosarcoma patients whose tumors contain Smac mimetic-responsive cancer cells and TNFα-producing infiltrating cells.

Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion.

Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Trim33 Binds and Silences a Class of Young Endogenous Retroviruses in the Mouse Testis; a Novel Component of the Arms Race between Retrotransposons and the Host Genome.

Transposable elements (TEs) have been active in the mammalian genome for millions of years and the silencing of these elements in the germline is important for the survival of the host. Mice carrying reporter transgenes can be used to model transcriptional silencing. A mutagenesis screen for modifiers of epigenetic gene silencing produced a line with a mutation in Trim33; the mutants displayed increased expression of the reporter transgene. ChIP-seq of Trim33 in testis revealed 9,109 peaks, mostly at promoters. This is the first report of ChIP-seq for Trim33 in any tissue. Comparison with ENCODE datasets showed that regions of high read density for Trim33 had high read density for histone marks associated with transcriptional activity and mapping to TE consensus sequences revealed Trim33 enrichment at RLTR10B, the LTR of one of the youngest retrotransposons in the mouse genome, MMERVK10C. We identified consensus sequences from the 266 regions at which Trim33 ChIP-seq peaks overlapped RLTR10B elements and found a match to the A-Myb DNA-binding site. We found that TRIM33 has E3 ubiquitin ligase activity for A-MYB and regulates its abundance. RNA-seq revealed that mice haploinsufficient for Trim33 had altered expression of a small group of genes in the testis and the gene with the most significant increase was found to be transcribed from an upstream RLTR10B. These studies provide the first evidence that A-Myb has a role in the actions of Trim33 and suggest a role for both A-Myb and Trim33 in the arms race between the transposon and the host. This the first report of any factor specifically regulating RLTR10B and adds to the current literature on the silencing of MMERVK10C retrotransposons. This is also the first report that A-Myb has a role in the transcription of any retrotransposon.

A switch in mechanism of action prevents doxorubicin-mediated cardiac damage.

Cancer patients treated with doxorubicin are at risk of congestive heart failure due to doxorubicin-mediated cardiotoxicity via topoisomerase IIß poisoning. Acute cardiac muscle damage occurs in response to the very first dose of doxorubicin, however, cardioprotection has been reported after co-treatment of doxorubicin with acyloxyalkyl ester prodrugs. The aim of this study was to examine the role played by various forms of acute cardiac damage mediated by doxorubicin and determine a mechanism for the cardioprotective effect of formaldehyde-releasing prodrug AN-9 (pivaloyloxymethyl butyrate). Doxorubicin-induced cardiac damage in BALB/c mice bearing mammary tumours was established with a single dose of doxorubicin (4 or 16 mg/kg) administered alone or in combination with AN-9 (100 mg/kg). AN-9 protected the heart from doxorubicin-induced myocardial apoptosis and also significantly reduced dsDNA breaks, independent from the level of doxorubicin biodistribution to the heart. Covalent incorporation of [14C]doxorubicin into DNA showed that the combination treatment yielded significantly higher levels of formaldehyde-mediated doxorubicin-DNA adducts compared to doxorubicin alone, yet this form of damage was associated with cardioprotection from apoptosis. The cardiac transcriptomic analysis indicates that the combination treatment initiates inflammatory response signalling pathways. Doxorubicin and AN-9 combination treatments were cardioprotective, yet preserved doxorubicin-mediated anti-tumour proliferation and apoptosis in mammary tumours. This was associated with a switch in doxorubicin action from cardiac topoisomerase IIß poisoning to covalent-DNA adduct formation. Co-administration of doxorubicin and formaldehyde-releasing prodrugs, such as AN-9, may be a promising cardioprotective therapy while maintaining doxorubicin activity in primary mammary tumours.

Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis.

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.

Intratumoral administration of the Toll-like receptor 7/8 agonist 3M-052 enhances interferon-driven tumor immunogenicity and suppresses metastatic spread in preclinical triple-negative breast cancer.

OBJECTIVES: Loss of tumor-inherent type I interferon (IFN) signalling has been closely linked to accelerated metastatic progression via decreased immunogenicity and antitumor immunity. Previous studies in murine models of triple-negative breast cancer (TNBC) demonstrate that systemic IFN inducers are effective antimetastatic agents, via sustained antitumor CD8+ T-cell responses. Repeated systemic dosing with recombinant IFNs or IFN inducers is associated with significant toxicities; hence, the use of alternate intratumoral agents is an active area of investigation. It is critical to investigate the impact of intratumoral agents on subsequent metastatic spread to predict clinical impact. METHODS: In this study, the local and systemic impact of the intratumoral Toll-like receptor (TLR) 7/8 agonist 3M-052 alone or in combination with anti-PD1 was evaluated in metastatic TNBC models. The IFN-α receptor (IFNAR1) blocking antibody, MAR1-5A3, along with immune-deficient mice and ex vivo assays are utilised to examine the key targets of this agent that are critical for an antimetastatic response. RESULTS: Single intratumoral administration of 3M-052 reduced mammary tumor growth, induced a T-cell-inflamed tumor microenvironment (TME) and reduced metastatic spread to lung. Metastasis suppression was reliant on IFN signalling and an antitumor immune response, in contrast to primary tumor growth inhibition, which was retained in NSG and CD8+ T-cell-depleted mice. 3M-052 action was demonstrated via dendritic cell activation and production of type I IFN and other pro-inflammatory cytokines to initiate a T-cell-inflamed TME and promote tumor cell antigen presentation. CONCLUSION: This work supports neoadjuvant TLR agonist-based immunotherapeutics as realistic options for immune activation in the TME and long-term metastatic protection in TNBC.

Tumor inherent interferon regulators as biomarkers of long-term chemotherapeutic response in TNBC.

Patients diagnosed with triple negative breast cancer (TNBC) have an increased risk of rapid metastasis compared to other subtypes. Predicting long-term survival post-chemotherapy in patients with TNBC is difficult, yet enhanced infiltration of tumor infiltrating lymphocytes (TILs) has been associated with therapeutic response and reduced risk of metastatic relapse. Immune biomarkers that predict the immune state of a tumor and risk of metastatic relapse pre- or mid-neoadjuvant chemotherapy are urgently needed to allow earlier implementation of alternate therapies that may reduce TNBC patient mortality. Utilizing a neoadjuvant chemotherapy trial where TNBC patients had sequential biopsies taken, we demonstrate that measurement of T-cell subsets and effector function, specifically CD45RO expression, throughout chemotherapy predicts risk of metastatic relapse. Furthermore, we identified the tumor inherent interferon regulatory factor IRF9 as a marker of active intratumoral type I and II interferon (IFN) signaling and reduced risk of distant relapse. Functional implications of tumor intrinsic IFN signaling were demonstrated using an immunocompetent mouse model of TNBC, where enhanced type I IFN signaling increased anti-tumor immunity and metastasis-free survival post-chemotherapy. Using two independent adjuvant cohorts we were able to validate loss of IRF9 as a poor prognostic biomarker pre-chemotherapy. Thus, IRF9 expression may offer early insight into TNBC patient prognosis and tumor heat, allowing for identification of patients that are unlikely to respond to chemotherapy alone and could benefit from further immune-based therapeutic intervention.

Discriminating the earliest stages of mammary carcinoma using myoepithelial and proliferative markers.

Mammographic screening has led to increased detection of breast cancer at a pre-invasive state, hence modelling the earliest stages of breast cancer invasion is important in defining candidate biomarkers to predict risk of relapse. Discrimination of pre-invasive from invasive lesions is critically important for such studies. Myoepithelial cells are the barrier between epithelial cells and the surrounding stroma in the breast ductal system. A number of myoepithelial immunohistochemistry markers have been identified and validated in human tissue for use by pathologists as diagnostic tools to distinguish in situ carcinoma from invasive breast cancer. However, robust myoepithelial markers for mouse mammary tissue have been largely under-utilised. Here, we investigated the utility of the myoepithelial markers smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC), cytokeratin-14 (CK14) and p63 to discriminate mammary intraepithelial neoplasia (MIN) from invasive disease in the C57BL/6J MMTV-PyMT transgenic model of mammary carcinoma. We identified that SMMHC and CK14 are retained in early in situ neoplasia and are appropriate markers for distinguishing MIN from invasive disease in this model. Additionally, the proliferation marker Ki67 is a superior marker for differentiating between normal and hyperplastic ducts, prior to the development of MIN. Based on this, we developed a scoring matrix for discriminating normal, hyperplasia, MIN and invasive lesions in this spontaneous mammary tumorigenesis model. This study demonstrates heterogeneous expression of myoepithelial proteins throughout tumour development, and highlights the need to characterise the most appropriate markers in other models of early breast cancer to allow accurate classification of disease state.

Encapsulation of Mitoxantrone within Cucurbit[8]uril Decreases Toxicity and Enhances Survival in a Mouse Model of Cancer.

Mitoxantrone was efficiently encapsulated within cucurbit[8]uril in a 2:1 complex where the two mitoxantrone molecules were symmetrically located through both portals of a cucurbit[8]uril cage. The novel complex facilitates increased mitoxantrone uptake in mouse breast cancer cells and decreases the toxicity of the drug in healthy mice. In an orthotopic mouse model of metastatic breast cancer the complex still maintains in vivo anticancer activity compared to the free drug, yet provides a statistically significant increase in the survival of these mice compared to conventional mitoxantrone treatment. This new low toxicity formulation offers the possibility to increase mitoxantrone dose and thus maximize efficacy while managing the dose limiting side effects.

Neoadjuvant Interferons: Critical for Effective PD-1-Based Immunotherapy in TNBC.

The lack of targeted therapies available for triple-negative breast cancer (TNBC) patients who fail to respond to first-line chemotherapy has sparked interest in immunotherapeutic approaches. However, trials utilizing checkpoint inhibitors targeting the PD-1/PD-L1 axis in TNBC have had underwhelming responses. Here, we investigated the interplay between type I IFN signaling and the PD-1/PD-L1 axis and tested the impact of combining IFN inducers, as immune activators, with anti-PD-1, to induce an antimetastatic immune response. Using models of TNBC, we demonstrated an interplay between type I IFN signaling and tumor cell PD-L1 expression that affected therapeutic response. The data revealed that the type I IFN-inducer poly(I:C) was an effective immune activator and antimetastatic agent, functioning better than anti-PD-1, which was ineffective as a single agent. Poly(I:C) treatment induced PD-L1 expression on TNBC cells, and combined poly(I:C) and anti-PD-1 treatment prolonged metastasis-free survival in a neoadjuvant setting via the induction of a tumor-specific T-cell response. Use of this combination in a late treatment setting did not impact metastasis-free survival, indicating that timing was critical for immunotherapeutic benefit. Together, these data demonstrated anti-PD-1 as an ineffective single agent in preclinical models of TNBC. However, type I IFN inducers were effective immune activators, and neoadjuvant trials combining them with anti-PD-1 to induce a sustained antitumor immune response are warranted. Cancer Immunol Res; 5(10); 871-84. ©2017 AACR.

Wiz binds active promoters and CTCF-binding sites and is required for normal behaviour in the mouse.

We previously identified Wiz in a mouse screen for epigenetic modifiers. Due to its known association with G9a/GLP, Wiz is generally considered a transcriptional repressor. Here, we provide evidence that it may also function as a transcriptional activator. Wiz levels are high in the brain, but its function and direct targets are unknown. ChIP-seq was performed in adult cerebellum and Wiz peaks were found at promoters and transcription factor CTCF binding sites. RNA-seq in Wiz mutant mice identified genes differentially regulated in adult cerebellum and embryonic brain. In embryonic brain most decreased in expression and included clustered protocadherin genes. These also decreased in adult cerebellum and showed strong Wiz ChIP-seq enrichment. Because a precise pattern of protocadherin gene expression is required for neuronal development, behavioural tests were carried out on mutant mice, revealing an anxiety-like phenotype. This is the first evidence of a role for Wiz in neural function.

Hypomethylation of ERVs in the sperm of mice haploinsufficient for the histone methyltransferase Setdb1 correlates with a paternal effect on phenotype.

The number of reports of paternal epigenetic influences on the phenotype of offspring in rodents is increasing but the molecular events involved remain unclear. Here, we show that haploinsufficiency for the histone 3 lysine 9 methyltransferase Setdb1 in the sire can influence the coat colour phenotype of wild type offspring. This effect occurs when the allele that directly drives coat colour is inherited from the dam, inferring that the effect involves an "in trans" step. The implication of this finding is that epigenetic state of the sperm can alter the expression of genes inherited on the maternally derived chromosomes. Whole genome bisulphite sequencing revealed that Setdb1 mutant mice show DNA hypomethylation at specific classes of transposable elements in the sperm. Our results identify Setdb1 as a paternal effect gene in the mouse and suggest that epigenetic inheritance may be more likely in individuals with altered levels of epigenetic modifiers.