ADVANCED DIAGNOSTIC IMAGING AND PATHOLOGIC FINDINGS OF THYROID LESIONS IN TWO SHORT-BEAKED ECHIDNAS (TACHYGLOSSUS ACULEATUS).

Two zoo-maintained short-beaked echidnas (Tachyglossus aculeatus) had long histories of intermittent anorexia and lethargy. Case 1 presented with a recurrence of these signs after transfer to another facility and died shortly after arrival. A focal area of hyperattenuation within the paratracheal tissue of the cranial mediastinum was noted antemortem on CT. Postmortem, this corresponded with severe thyroid follicular hyperplasia with lymphoplasmacytic thyroiditis. Additional findings included a systemic fungal infection without an inflammatory response, suggesting underlying factors such as torpor or immunosuppression. In Case 2, an intrathoracic mass was identified during a preshipment examination. CT confirmed a contrast-enhanced mass compressing the cranial vena cava and right atrium, and the animal was euthanized. The mass was diagnosed histologically as thyroid adenocarcinoma. These cases report thyroiditis and thyroid adenocarcinoma in echidna and describe the use of IV contrast and CT as a diagnostic aid in this species.

Differentiation signals induce APOBEC3A expression via GRHL3 in squamous epithelia and squamous cell carcinoma.

Two APOBEC (apolipoprotein-B mRNA editing enzyme catalytic polypeptide-like) DNA cytosine deaminase enzymes (APOBEC3A and APOBEC3B) generate somatic mutations in cancer, driving tumour development and drug resistance. Here we used single cell RNA sequencing to study APOBEC3A and APOBEC3B expression in healthy and malignant mucosal epithelia, validating key observations with immunohistochemistry, spatial transcriptomics and functional experiments. Whereas APOBEC3B is expressed in keratinocytes entering mitosis, we show that APOBEC3A expression is confined largely to terminally differentiating cells and requires Grainyhead-like transcription factor 3 (GRHL3). Thus, in normal tissue, neither deaminase appears to be expressed at high levels during DNA replication, the cell cycle stage associated with APOBEC-mediated mutagenesis. In contrast, we show that in squamous cell carcinoma tissues, there is expansion of GRHL3 expression and activity to a subset of cells undergoing DNA replication and concomitant extension of APOBEC3A expression to proliferating cells. These findings indicate a mechanism for acquisition of APOBEC3A mutagenic activity in tumours.

Single-cell analysis reveals prognostic fibroblast subpopulations linked to molecular and immunological subtypes of lung cancer.

Fibroblasts are poorly characterised cells that variably impact tumour progression. Here, we use single cell RNA-sequencing, multiplexed immunohistochemistry and digital cytometry (CIBERSORTx) to identify and characterise three major fibroblast subpopulations in human non-small cell lung cancer: adventitial, alveolar and myofibroblasts. Alveolar and adventitial fibroblasts (enriched in control tissue samples) localise to discrete spatial niches in histologically normal lung tissue and indicate improved overall survival rates when present in lung adenocarcinomas (LUAD). Trajectory inference identifies three phases of control tissue fibroblast activation, leading to myofibroblast enrichment in tumour samples: initial upregulation of inflammatory cytokines, followed by stress-response signalling and ultimately increased expression of fibrillar collagens. Myofibroblasts correlate with poor overall survival rates in LUAD, associated with loss of epithelial differentiation, TP53 mutations, proximal molecular subtypes and myeloid cell recruitment. In squamous carcinomas myofibroblasts were not prognostic despite being transcriptomically equivalent. These findings have important implications for developing fibroblast-targeting strategies for cancer therapy.

ATM Regulates Differentiation of Myofibroblastic Cancer-Associated Fibroblasts and Can Be Targeted to Overcome Immunotherapy Resistance.

Myofibroblastic cancer-associated fibroblast (myoCAF)-rich tumors generally contain few T cells and respond poorly to immune-checkpoint blockade. Although myoCAFs are associated with poor outcome in most solid tumors, the molecular mechanisms regulating myoCAF accumulation remain unclear, limiting the potential for therapeutic intervention. Here, we identify ataxia-telangiectasia mutated (ATM) as a central regulator of the myoCAF phenotype. Differentiating myofibroblasts in vitro and myoCAFs cultured ex vivo display activated ATM signaling, and targeting ATM genetically or pharmacologically could suppress and reverse differentiation. ATM activation was regulated by the reactive oxygen species-producing enzyme NOX4, both through DNA damage and increased oxidative stress. Targeting fibroblast ATM in vivo suppressed myoCAF-rich tumor growth, promoted intratumoral CD8 T-cell infiltration, and potentiated the response to anti-PD-1 blockade and antitumor vaccination. This work identifies a novel pathway regulating myoCAF differentiation and provides a rationale for using ATM inhibitors to overcome CAF-mediated immunotherapy resistance. SIGNIFICANCE: ATM signaling supports the differentiation of myoCAFs to suppress T-cell infiltration and antitumor immunity, supporting the potential clinical use of ATM inhibitors in combination with checkpoint inhibition in myoCAF-rich, immune-cold tumors.

Targeting cancer-associated fibroblasts: Challenges, opportunities and future directions.

Cancer-associated fibroblasts (CAFs) are a common cell in the tumour microenvironment with diverse tumour-promoting functions. Their presence in tumours is commonly associated with poor prognosis making them attractive therapeutic targets, particularly in the context of immunotherapy where CAFs have been shown to promote resistance to checkpoint blockade. Previous attempts to inhibit CAFs clinically have not been successful, however, in part due to a lack of understanding of CAF heterogeneity and function, with some fibroblast populations potentially being tumour suppressive. Recent single-cell transcriptomic studies have advanced our understanding of fibroblast phenotypes in normal tissues and cancers, allowing for a more precise characterisation of CAF subsets and providing opportunities to develop new therapies. Here we review recent advances in the field, focusing on the evolving area of therapeutic CAF targeting.

Targeting cancer associated fibroblasts to enhance immunotherapy: emerging strategies and future perspectives.

Cancer associated fibroblasts are a prominent component of the tumour microenvironment in most solid cancers. This heterogeneous population of cells are known to play an important role in tumour progression and recent studies have demonstrated that CAFs may confer resistance to checkpoint immunotherapy, suggesting that targeting these cells could improve response rates. However, effective clinical strategies for CAF targeting have yet to be identified. In this editorial, we highlight current limitations in our understanding of CAF heterogeneity, and discuss the potential and possible approaches for CAF-directed therapy.

POPULATION PHARMACOKINETICS OF CEFTAZIDIME AFTER A SINGLE SUBCUTANEOUS INJECTION AND NORMAL ORAL AND CLOACAL BACTERIAL FLORA SURVEY IN EASTERN HELLBENDERS (CRYPTOBRANCHUS ALLEGANIENSIS ALLEGANIENSIS).

Population pharmacokinetics utilizing sparse sampling were used to determine pharmacokinetics of ceftazidime in eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) due to their slow growth rate and the limited number of appropriately sized individuals in the zoo-housed population. Twenty-five eastern hellbenders received a single subcutaneous injection of ceftazidime at 20 mg/kg. Each animal had blood samples collected up to four times between 0 and 192 hr postinjection. Plasma samples were analyzed by high-pressure liquid chromatography. A nonlinear mixed-effects model was fitted to the data to determine typical values for population parameters, an ideal method due to the sampling limitation of each hellbender. Results indicate an elimination half-life of 36.63 hr and volume of distribution of 0.31 L/kg. Antibiotic concentrations were above a minimum inhibitory concentration (MIC) value of 8 µg/ml for 120 hr. Prior to antibiotic administration, six hellbenders had oral and six other individuals had cloacal swabs taken for aerobic culture. Fifty-five bacterial isolates were obtained (24 cloacal, 31 oral) with 10/12 (83%) individuals growing three or more different isolates and 11/12 (92%) growing Shewanella putrefaciens. Twelve isolates had susceptibility testing performed and all were susceptible to ceftazidime. These results indicate that ceftazidime is an appropriate choice of antibiotic in hellbenders and when given at a dosage of 20 mg/kg subcutaneously, maintains concentrations above the MIC of susceptible bacteria for up to 5 days.

Cancer-Associated Fibroblasts in Oral Cancer: A Current Perspective on Function and Potential for Therapeutic Targeting.

The role of the tumour microenvironement (TME) in cancer progression and resistance to therapies is now widely recognized. The most prominent non-immune cell type in the microenvironment of oral cancer (OSCC) is cancer-associated fibroblasts (CAF). Although CAF are a poorly characterised and heterogenous cell population, those with an "activated" myofibroblastic phenotype have been shown to support OSCC progression, promoting growth, invasion and numerous other "hallmarks of malignancy." CAF also confer broad resistance to different types of therapy, including chemo/radiotherapy and EGFR inhibitors; consistent with this, CAF-rich OSCC are associated with poor prognosis. In recent years, much CAF research has focused on their immunological role in the tumour microenvironment, showing that CAF shield tumours from immune attack through multiple mechanisms, and particularly on their role in promoting resistance to anti-PD-1/PD-L1 checkpoint inhibitors, an exciting development for the treatment of recurrent/metastatic oral cancer, but which fails in most patients. This review summarises our current understanding of CAF subtypes and function in OSCC and discusses the potential for targeting these cells therapeutically.

CTEN Induces Tumour Cell Invasion and Survival and Is Prognostic in Radiotherapy-Treated Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is a heterogenous disease treated with surgery and/or (chemo) radiotherapy, but up to 50% of patients with late-stage disease develop locoregional recurrence. Determining the mechanisms underpinning treatment resistance could identify new therapeutic targets and aid treatment selection. C-terminal tensin-like (CTEN) is a member of the tensin family, upregulated in several cancers, although its expression and function in HNSCC are unknown. We found that CTEN is commonly upregulated in HNSCC, particularly HPV-ve tumours. In vitro CTEN was upregulated in HPV-ve (n = 5) and HPV+ve (n = 2) HNSCC cell lines. Stable shRNA knockdown of CTEN in vivo significantly reduced tumour growth (SCC-25), and functional analyses in vitro showed that CTEN promoted tumour cell invasion, colony formation and growth in 3D-culture (SCC-25, Detroit 562). RNA sequencing of SCC-25 cells following CTEN siRNA knockdown identified 349 differentially expressed genes (logFC > 1, p < 0.05). Gene ontology analysis highlighted terms relating to cell locomotion and apoptosis, consistent with in vitro findings. A membrane-based antibody array confirmed that CTEN regulated multiple apoptosis-associated proteins, including HSP60 and cleaved caspase-3. Notably, in a mixed cohort of HPV+ve and HPV-ve HNSCC patients (n = 259), we found a significant, independent negative association of CTEN with prognosis, limited to those patients treated with (chemo)radiotherapy, not surgery, irrespective of human papillomavirus (HPV) status. These data show that CTEN is commonly upregulated in HNSCC and exerts several functional effects. Its potential role in modulating apoptotic response to therapy suggests utility as a predictive biomarker or radio-sensitising target.

Tumor-Resident Stromal Cells Promote Breast Cancer Invasion through Regulation of the Basal Phenotype.

Collective invasion can be led by breast cancer cells expressing basal epithelial markers, typified by keratin-14 (KRT14). We analyzed gene expression data from The Cancer Genome Atlas and demonstrated a significant correlation between a KRT14+ invasion signature and a stromal-mediated extracellular matrix (ECM) organization module. We then developed a novel coculture model of tumor organoids with autologous stromal cells. Coculture significantly increased KRT14 expression and invasion of organoids from both luminal and basal murine breast cancer models. However, stromal cell conditioned medium induced invasion but not KRT14 expression. Cancer cells released TGFß and that signaling pathway was required for stromal cell-induced invasion and KRT14 expression. Mechanistically, TGFß induced NOX4 expression in stromal cells and NOX4 inhibition reduced invasion and KRT14 expression. In summary, we developed a novel coculture model and revealed dynamic molecular interactions between stromal cells and cancer cells that regulate both basal gene expression and invasive behavior. IMPLICATIONS: Fibroblasts within mammary tumors can regulate the molecular phenotype and invasive behavior of breast cancer cells. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/11/1615/F1.large.jpg.

T-cell tumour exclusion and immunotherapy resistance: a role for CAF targeting.

Recent studies have highlighted a major role for cancer-associated fibroblasts (CAFs) in promoting immunotherapy resistance by excluding T cells from tumours. Recently, we showed that CAFs can be effectively targeted by inhibiting the enzyme NOX4; this 'normalises' CAFs and overcomes immunotherapy resistance. Here we discuss our study and other strategies for CAF targeting.

Hyperglycemic hyperosmolar state in a chimpanzee (Pan troglodytes).

A 19-year-old female chimpanzee (Pan troglodytes) presented for cachexia, acute weakness, hyporexia, icterus, and polyuria. The animal was diagnosed with a hyperglycemic hyperosmolar state, which is a well-recognized syndrome in diabetic humans that is rarely diagnosed in animals. This case documents an important and likely under-reported syndrome in non-human primates.

NOX4 Inhibition Potentiates Immunotherapy by Overcoming Cancer-Associated Fibroblast-Mediated CD8 T-cell Exclusion from Tumors.

Determining mechanisms of resistance to αPD-1/PD-L1 immune-checkpoint immunotherapy is key to developing new treatment strategies. Cancer-associated fibroblasts (CAF) have many tumor-promoting functions and promote immune evasion through multiple mechanisms, but as yet, no CAF-specific inhibitors are clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, and 4T1) to investigate how CAFs influence the immune microenvironment and affect response to different immunotherapy modalities [anticancer vaccination, TC1 (HPV E7 DNA vaccine), αPD-1, and MC38] and found that CAFs broadly suppressed response by specifically excluding CD8+ T cells from tumors (not CD4+ T cells or macrophages); CD8+ T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8+ T cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a nondepleting antibody overcame the CD8+ T-cell exclusion effect without affecting Tregs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human cancers, and compared this with TGFß1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacologic inhibition [GKT137831 (Setanaxib)] of NOX4 "normalized" CAF to a quiescent phenotype and promoted intratumoral CD8+ T-cell infiltration, overcoming the exclusion effect; TGFß1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition and could improve outcome in a broad range of cancers. SIGNIFICANCE: NOX4 is critical for maintaining the immune-suppressive CAF phenotype in tumors. Pharmacologic inhibition of NOX4 potentiates immunotherapy by overcoming CAF-mediated CD8+ T-cell exclusion. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/9/1846/F1.large.jpg.See related commentary by Hayward, p. 1799.

An optimised tissue disaggregation and data processing pipeline for characterising fibroblast phenotypes using single-cell RNA sequencing.

Single-cell RNA sequencing (scRNA-Seq) provides a valuable platform for characterising multicellular ecosystems. Fibroblasts are a heterogeneous cell type involved in many physiological and pathological processes, but remain poorly-characterised. Analysis of fibroblasts is challenging: these cells are difficult to isolate from tissues, and are therefore commonly under-represented in scRNA-seq datasets. Here, we describe an optimised approach for fibroblast isolation from human lung tissues. We demonstrate the potential for this procedure in characterising stromal cell phenotypes using scRNA-Seq, analyse the effect of tissue disaggregation on gene expression, and optimise data processing to improve clustering quality. We also assess the impact of in vitro culture conditions on stromal cell gene expression and proliferation, showing that altering these conditions can skew phenotypes.

Single-cell transcriptomic analysis of tissue-resident memory T cells in human lung cancer.

High numbers of tissue-resident memory T (TRM) cells are associated with better clinical outcomes in cancer patients. However, the molecular characteristics that drive their efficient immune response to tumors are poorly understood. Here, single-cell and bulk transcriptomic analysis of TRM and non-TRM cells present in tumor and normal lung tissue from patients with lung cancer revealed that PD-1-expressing TRM cells in tumors were clonally expanded and enriched for transcripts linked to cell proliferation and cytotoxicity when compared with PD-1-expressing non-TRM cells. This feature was more prominent in the TRM cell subset coexpressing PD-1 and TIM-3, and it was validated by functional assays ex vivo and also reflected in their chromatin accessibility profile. This PD-1+TIM-3+ TRM cell subset was enriched in responders to PD-1 inhibitors and in tumors with a greater magnitude of CTL responses. These data highlight that not all CTLs expressing PD-1 are dysfunctional; on the contrary, TRM cells with PD-1 expression were enriched for features suggestive of superior functionality.

Bladder teratoma in a maned wolf (Chrysocyon brachyurus).

Background: Teratomas are germ cell tumors, comprised of a mixture of tissue types and with tissue foreign to their site of origin. Case Description: A 5.5-year-old intact female maned wolf (Chrysocyon brachyurus) was treated for recurrent stranguria and suspected cystitis. Due to lack of resolution, the wolf was anesthetized for further evaluation. The urinary bladder was firm on palpation, with a markedly thickened wall and no observable lumen on ultrasound. Neoplastic infiltration was suspected on double contrast cystogram and confirmed via surgical exploration. The lesion was inoperable and the wolf was euthanized. Gross necropsy revealed two poorly distinguished masses infiltrating the urinary bladder dorsally and caudoventrally, with minimal remaining lumen. Histopathologic examination of the bladder and associated masses revealed a neoplasm comprised of multiple tissue types. Vascular invasion was noted. Conclusion: The neoplasm was diagnosed as an extragonadal teratoma. Few extragonadal teratomas have been described and this is the first report of a teratoma originating in the urinary bladder of a non-human species.

An Optimized Method to Isolate Human Fibroblasts from Tissue for ex vivo Analysis.

Despite their involvement in many physiological and pathological processes, fibroblasts remain a poorly-characterized cell type. Analysis of primary fibroblasts while maintaining their in vivo phenotype is challenging: standard methods for fibroblast isolation require cell culture in vitro, which is known to alter phenotypes. Previously-described protocols for the dissociation of primary tissues fail to extract sufficient numbers of fibroblasts, instead largely yielding immune cells. Here, we describe an optimized method for generating a fibroblast-enriched single-cell suspension from human tissues using combined mechanical and enzymatic dissociation. This allows analysis of ex vivo fibroblasts without the need for culture in vitro.

COMPARISON OF PROPOFOL CONSTANT RATE INFUSION AND ISOFLURANE FOR MAINTENANCE OF ANESTHESIA IN SPEKE'S GAZELLE, GAZELLA SPEKEI.

The aims of this study were to determine if a propofol constant rate infusion (CRI) in Speke's gazelle, Gazella spekei, would serve as an effective alternative maintenance anesthetic, result in shorter recovery times, and improve anesthetic recovery quality when compared with isoflurane. Eight adult gazelle were enrolled in this complete crossover study with a minimum 3-wk washout period. All gazelle were induced with 10 mg/kg intravenous propofol and maintained with either propofol CRI (0.4 mg/kg/min) or isoflurane (1-3%) for 45 min. Animals were monitored for anesthetic depth and physiologic variables including heart and respiratory rates, oxygen saturation, end-tidal carbon dioxide, indirect blood pressure, and temperature every 5 min. Blood gas samples were analyzed within the first 10 min following anesthetic induction and within the last 10 min of anesthesia. Recovery times were recorded. Recovery quality was classified by a residual ataxia grading scale. Seven gazelle completed the study by undergoing both anesthetic treatments; one female (12 yr old) developed complications 2 days after isoflurane anesthesia, consisting of seizures, azotemia, leukocytosis, hypocalcemia, and hypomagnesemia but was treated successfully. Propofol anesthesia resulted in lower respiratory rates compared with isoflurane and a decrease in respiratory rate over time. Propofol CRI maintained blood pressure values closer to physiologically normal ranges compared with isoflurane for 45 min of anesthesia. Recovery times were comparable between propofol and isoflurane treatments. While individuals receiving propofol had higher residual ataxia scores compared with individuals receiving isoflurane, differences were not clinically important. This study demonstrated that propofol CRI (0.4 mg/kg/min) is an effective maintenance anesthetic agent in healthy adult Speke's gazelle for noninvasive procedures with endotracheal intubation and intermittent positive pressure ventilation.