Tissue-resident macrophages provide a pro-tumorigenic niche to early NSCLC cells.

Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment1,2. However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis3-5, whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions1. How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial-mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8+ T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions.

Structure and Function of Negri Bodies.

Replication and assembly of many viruses occur in viral factories which are specialized intracellular compartments formed during viral infection. For rabies virus, those viral factories are called Negri bodies (NBs). NBs are cytoplasmic inclusion bodies in which viral RNAs (mRNAs as well as genomic and antigenomic RNAs) are synthesized. NBs are spherical, they can fuse together, and can reversibly deform when encountering a physical barrier. All these characteristics are similar to those of eukaryotic membrane-less liquid organelles which contribute to the compartmentalization of the cell interior. Indeed, the liquid nature of NBs has been confirmed by FRAP experiments. The co-expression of rabies virus nucleoprotein N and phosphoprotein P is sufficient to induce the formation of cytoplasmic inclusions recapitulating NBs properties. Remarkably, P and N have features similar to those of cellular proteins involved in liquid organelles formation: N is an RNA-binding protein and P contains intrinsically disordered domains. An overview of the literature indicates that formation of liquid viral factories by phase separation is probably common among Mononegavirales. This allows specific recruitment and concentration of viral proteins. Finally, as virus-associated molecular patterns recognized by cellular sensors of RNA virus replication are probably essentially present in the viral factory, there should be a subtle interplay (which remains to be characterized) between those liquid structures and the cellular proteins which trigger the innate immune response.

Rabies Virus Infection Induces the Formation of Stress Granules Closely Connected to the Viral Factories.

Stress granules (SGs) are membrane-less dynamic structures consisting of mRNA and protein aggregates that form rapidly in response to a wide range of environmental cellular stresses and viral infections. They act as storage sites for translationally silenced mRNAs under stress conditions. During viral infection, SG formation results in the modulation of innate antiviral immune responses, and several viruses have the ability to either promote or prevent SG assembly. Here, we show that rabies virus (RABV) induces SG formation in infected cells, as revealed by the detection of SG-marker proteins Ras GTPase-activating protein-binding protein 1 (G3BP1), T-cell intracellular antigen 1 (TIA-1) and poly(A)-binding protein (PABP) in the RNA granules formed during viral infection. As shown by live cell imaging, RABV-induced SGs are highly dynamic structures that increase in number, grow in size by fusion events, and undergo assembly/disassembly cycles. Some SGs localize in close proximity to cytoplasmic viral factories, known as Negri bodies (NBs). Three dimensional reconstructions reveal that both structures remain distinct even when they are in close contact. In addition, viral mRNAs synthesized in NBs accumulate in the SGs during viral infection, revealing material exchange between both compartments. Although RABV-induced SG formation is not affected in MEFs lacking TIA-1, TIA-1 depletion promotes viral translation which results in an increase of viral replication indicating that TIA-1 has an antiviral effect. Inhibition of PKR expression significantly prevents RABV-SG formation and favors viral replication by increasing viral translation. This is correlated with a drastic inhibition of IFN-B gene expression indicating that SGs likely mediate an antiviral response which is however not sufficient to fully counteract RABV infection.

Focal adhesion kinase is involved in rabies virus infection through its interaction with viral phosphoprotein P.

UNLABELLED: The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential role in viral transcription and replication, and in addition, RABV P has been identified as an interferon antagonist. Here, a yeast two-hybrid screen revealed that RABV P interacts with the focal adhesion kinase (FAK). The binding involved the 106-to-131 domain, corresponding to the dimerization domain of P and the C-terminal domain of FAK containing the proline-rich domains PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by coimmunoprecipitation and colocalization of FAK with P in Negri bodies. By alanine scanning, we identified a single mutation in the P protein that abolishes this interaction. The mutant virus containing a substitution of Ala for Arg in position 109 in P (P.R109A), which did not interact with FAK, is affected at a posttranscriptional step involving protein synthesis and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in infected cells. This provides the first evidence of an interaction of RABV with FAK that positively regulates infection. IMPORTANCE: Rabies virus exhibits a small genome that encodes a limited number of viral proteins. To maintain efficient virus replication, some of them are multifunctional, such as the phosphoprotein P. We and others have shown that P establishes complex networks of interactions with host cell components. These interactions have revealed much about the role of P and about host-pathogen interactions in infected cells. Here, we identified another cellular partner of P, the focal adhesion kinase (FAK). Our data shed light on the implication of FAK in RABV infection and provide evidence that P-FAK interaction has a proviral function.

Formalin-fixed paraffin-embedded tissue as a source for quantitation of carcinogen DNA adducts: aristolochic acid as a prototype carcinogen.

DNA adducts are a measure of internal exposure to genotoxicants. However, the measurement of DNA adducts in molecular epidemiology studies often is precluded by the lack of fresh tissue. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues frequently are accessible, although technical challenges remain in retrieval of high quality DNA suitable for biomonitoring of adducts. Aristolochic acids (AA) are human carcinogens found in Aristolochia plants, some of which have been used in the preparation of traditional Chinese herbal medicines. We previously established a method to measure DNA adducts of AA in FFPE tissue. In this study, we examine additional features of formalin fixation that could impact the quantity and quality of DNA and report on the recovery of AA-DNA adducts in mice exposed to AA. The yield of DNA isolated from tissues fixed with formalin decreased over 1 week; however, the levels of AA-DNA adducts were similar to those in fresh frozen tissue. Moreover, DNA from FFPE tissue served as a template for PCR amplification, yielding sequence data of comparable quality to DNA obtained from fresh frozen tissue. The estimates of AA-DNA adducts measured in freshly frozen tissue and matching FFPE tissue blocks of human kidney stored for 9 years showed good concordance. Thus, DNA isolated from FFPE tissues may be used to biomonitor DNA adducts and to amplify genes used for mutational analysis, providing clues regarding the origin of human cancers for which an environmental cause is suspected.

Consensus statement on screening, diagnosis, classification and treatment of endemic (Balkan) nephropathy.

Currently used diagnostic criteria in different endemic (Balkan) nephropathy (EN) centers involve different combinations of parameters, various cut-off values and many of them are not in agreement with proposed international guidelines. Leaders of EN centers began to address these problems at scientific meetings, and this paper is the outgrowth of those discussions. The main aim is to provide recommendations for clinical work on current knowledge and expertise. This document is developed for use by general physicians, nephrologists, urologist, public health experts and epidemiologist, and it is hoped that it will be adopted by responsible institutions in countries harboring EN. National medical providers should cover costs of screening and diagnostic procedures and treatment of EN patients with or without upper urothelial cancers.

Human formalin-fixed paraffin-embedded tissues: an untapped specimen for biomonitoring of carcinogen DNA adducts by mass spectrometry.

DNA adducts represent internal dosimeters to measure exposure to environmental and endogenous genotoxicants. Unfortunately, in molecular epidemiologic studies, measurements of DNA adducts often are precluded by the unavailability of fresh tissue. In contrast, formalin-fixed paraffin embedded (FFPE) tissues frequently are accessible for biomarker discovery. We report here that DNA adducts of aristolochic acids (AAs) can be measured in FFPE tissues at a level of sensitivity comparable to freshly frozen tissue. AAs are nephrotoxic and carcinogenic compounds found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. AAs are implicated in the etiology of aristolochic acid nephropathy and upper urinary tract carcinoma. 8-Methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxole-5-carboxylic acid (AA-I) is a component of Aristolochia herbs and a potent human urothelial carcinogen. AA-I reacts with DNA to form the aristolactam (AL-I)-DNA adduct 7-(deoxyadenosin-N(6)-yl) aristolactam I (dA-AL-I). We established a method to quantitatively retrieve dA-AL-I from FFPE tissue. Adducts were measured, using ultraperformance liquid chromatography/mass spectrometry, in liver and kidney tissues of mice exposed to AA-I, at doses ranging from 0.001 to 1 mg/kg body weight. dA-AL-I was then measured in 10-µm thick tissue-sections of FFPE kidney from patients with upper urinary tract cancers; the values were comparable to those observed in fresh frozen samples. The limit of quantification of dA-AL-I was 3 adducts per 10(9) DNA bases per 2.5 µg of DNA. The ability to retrospectively analyze FFPE tissues for DNA adducts may provide clues to the origin of human cancers for which an environmental cause is suspected.

RELA tunes innate-like interferon I/III responses in human T cells.

In innate immune cells, intracellular sensors such as cGAS-STING stimulate type I/III interferon (IFN) expression, which promotes antiviral defense and immune activation. However, how IFN-I/III expression is controlled in adaptive cells is poorly understood. Here, we identify a transcriptional rheostat orchestrated by RELA that confers human T cells with innate-like abilities to produce IFN-I/III. Despite intact cGAS-STING signaling, IFN-I/III responses are stunted in CD4+ T cells compared with dendritic cells or macrophages. We find that lysine residues in RELA tune the IFN-I/III response at baseline and in response to STING stimulation in CD4+ T cells. This response requires positive feedback driven by cGAS and IRF7 expression. By combining RELA with IRF3 and DNA demethylation, IFN-I/III production in CD4+ T cells reaches levels observed in dendritic cells. IFN-I/III production provides self-protection of CD4+ T cells against HIV infection and enhances the elimination of tumor cells by CAR T cells. Therefore, innate-like functions can be tuned and leveraged in human T cells.

Aristolactam-DNA adducts are a biomarker of environmental exposure to aristolochic acid.

Endemic (Balkan) nephropathy is a chronic tubulointerstitial disease frequently accompanied by urothelial cell carcinomas of the upper urinary tract. This disorder has recently been linked to exposure to aristolochic acid, a powerful nephrotoxin and human carcinogen. Following metabolic activation, aristolochic acid reacts with genomic DNA to form aristolactam-DNA adducts that generate a unique TP53 mutational spectrum in the urothelium. The aristolactam-DNA adducts are concentrated in the renal cortex, thus serving as biomarkers of internal exposure to aristolochic acid. Here, we present molecular epidemiologic evidence relating carcinomas of the upper urinary tract to dietary exposure to aristolochic acid. DNA was extracted from the renal cortex and urothelial tumor tissue of 67 patients that underwent nephroureterectomy for carcinomas of the upper urinary tract and resided in regions of known endemic nephropathy. Ten patients from nonendemic regions with carcinomas of the upper urinary tract served as controls. Aristolactam-DNA adducts were quantified by (32)P-postlabeling, the adduct was confirmed by mass spectrometry, and TP53 mutations in tumor tissues were identified by chip sequencing. Adducts were present in 70% of the endemic cohort and in 94% of patients with specific A:T to T:A mutations in TP53. In contrast, neither aristolactam-DNA adducts nor specific mutations were detected in tissues of patients residing in nonendemic regions. Thus, in genetically susceptible individuals, dietary exposure to aristolochic acid is causally related to endemic nephropathy and carcinomas of the upper urinary tract.

Cancer Immunotherapies Based on Genetically Engineered Macrophages.

Anticancer immunotherapies are therapeutics aimed at eliciting immune responses against tumor cells. Immunotherapies based on adoptive transfer of engineered immune cells have raised great hopes of cures because of the success of chimeric antigen receptor T-cell therapy in treating some hematologic malignancies. In parallel, advances in detailed analyses of the microenvironment of many solid tumors using high-dimensional approaches have established the origins and abundant presence of tumor-associated macrophages. These macrophages have an anti-inflammatory phenotype and promote tumor growth through a variety of mechanisms. Attempts have been made to engineer macrophages with chimeric receptors or transgenes to counteract their protumor activities and promote their antitumor functions such as phagocytosis of cancer cells, presentation of tumor antigens, and production of inflammatory cytokines. In this review, we cover current breakthroughs in engineering myeloid cells to combat cancer as well as potential prospects for myeloid-cell treatments.

Balkan Endemic Nephropathy and the Causative Role of Aristolochic Acid.

Balkan endemic nephropathy is a chronic tubulointerstitial disease with insidious onset, slowly progressing to end-stage renal disease and frequently associated with urothelial carcinoma of the upper urinary tract (UTUC). It was described in South-East Europe at the Balkan peninsula in rural areas around tributaries of the Danube River. After decades of intensive investigation, the causative factor was identified as the environmental phytotoxin aristolochic acid (AA) contained in Aristolochia clematitis, a common plant growing in wheat fields that was ingested through home-baked bread. AA initially was involved in the outbreak of cases of rapidly progressive renal fibrosis reported in Belgium after intake of root extracts of Aristolochia fangchi imported from China. A high prevalence of UTUC was found in these patients. The common molecular link between Balkan and Belgian nephropathy cases was the detection of aristolactam-DNA adducts in renal tissue and UTUC. These adducts are not only biomarkers of prior exposure to AA, but they also trigger urothelial malignancy by inducing specific mutations (A:T to T:A transversion) in critical genes of carcinogenesis, including the tumor-suppressor TP53. Such mutational signatures are found in other cases worldwide, particularly in Taiwan, highlighting the general public health issue of AA exposure by traditional phytotherapies.

Clonally Expanded T Cells Reveal Immunogenicity of Rhabdoid Tumors.

Rhabdoid tumors (RTs) are genomically simple pediatric cancers driven by the biallelic inactivation of SMARCB1, leading to SWI/SNF chromatin remodeler complex deficiency. Comprehensive evaluation of the immune infiltrates of human and mice RTs, including immunohistochemistry, bulk RNA sequencing and DNA methylation profiling studies showed a high rate of tumors infiltrated by T and myeloid cells. Single-cell RNA (scRNA) and T cell receptor sequencing highlighted the heterogeneity of these cells and revealed therapeutically targetable exhausted effector and clonally expanded tissue resident memory CD8+ T subpopulations, likely representing tumor-specific cells. Checkpoint blockade therapy in an experimental RT model induced the regression of established tumors and durable immune responses. Finally, we show that one mechanism mediating RTs immunogenicity involves SMARCB1-dependent re-expression of endogenous retroviruses and interferon-signaling activation.

Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein.

Vesicular stomatitis virus (VSV) is an oncolytic rhabdovirus and its glycoprotein G is widely used to pseudotype other viruses for gene therapy. Low-density lipoprotein receptor (LDL-R) serves as a major entry receptor for VSV. Here we report two crystal structures of VSV G in complex with two distinct cysteine-rich domains (CR2 and CR3) of LDL-R, showing that their binding sites on G are identical. We identify two basic residues on G, which are essential for its interaction with CR2 and CR3. Mutating these residues abolishes VSV infectivity even though VSV can use alternative receptors, indicating that all VSV receptors are members of the LDL-R family. Collectively, our data suggest that VSV G has specifically evolved to interact with receptor CR domains. These structural insights into the interaction between VSV G and host cell receptors provide a basis for the design of recombinant viruses with an altered tropism.

Negri bodies are viral factories with properties of liquid organelles.

Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell cytoplasm. For rabies virus, those inclusions are called Negri bodies (NBs). We report that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase is confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Coexpression of rabies virus N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for Negri bodies-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response.Negative strand RNA viruses, such as rabies virus, induce formation of cytoplasmic inclusions for genome replication. Here, Nikolic et al. show that these so-called Negri bodies (NBs) have characteristics of liquid organelles and they identify the minimal protein domains required for NB formation.

[Investigation of Balkan endemic nephropathy in Serbia: how to proceed?].

Balkan endemic nephropathy (BEN) presents an unsolved puzzle despite fifty years of its investigation. Academy of Medical Sciences of the Serbian Medical Society organized a round table discussion on current unsolved problems related to BEN. The present paper summarizes presentations, discussion and conclusions of this meeting. During the last fifty years, the course of BEN prolonged and it shifted towards the older age in all endemic foci. Data on the incidence of BEN have been controversial and frequently based on the data on the number of BEN patients starting haemodialysis treatment. In Serbia, BEN patients present 6.5% of haemodialysis population and this percentage differs among different centres ranging from 5% (Leskovac) to 46% (Lazarevac). Maintenance of high prevalence of BEN patients on regular haemodialysis indicates that BEN is not an expiring disease. In addition, recent data have shown more frequent microalbuminuria and low-molecular weight proteinuria in children from endemic than from nonendemic families. Aetiology of BEN is still unknown despite numerous investigations of environmental and genetic factors. Today, there is a very current hypothesis on the aetiological role of aristolochic acid but the role of viruses, geochemical factors and genetic factors must not be neglected. Morphological features of BEN are nonspecific and characterized by acellular interstitial fibrosis, tubular atrophy and changes on pre- and postglomerular vessels. New immunohistochemical and molecular biology methods offer a new approach to BEN investigation. Association of BEN with high incidence of upper-urothelial tumours is well-known. Recent studies have shown significant changes of demographic characteristics of patients suffering upper-urothelial tumours, their prevalence in different endemic foci and characteristics of tumours. Further studies of BEN should be directed to determination of incidence and prevalence of disease in different endemic foci, investigations of different insufficiently examined aetiological factors as well as pathomorphological features of the disease by the use of modern methods.

Diagnostic criteria for Balkan endemic nephropathy: proposal by an international panel.

Balkan endemic nephropathy (BEN) is a familial chronic tubulointerstitial disease with insidious onset and slow progression to terminal renal failure. Diagnostic criteria for BEN have been described more than 40 years ago. Research groups on BEN use one of at least three described lists of criteria. Comparison of studies using such criteria is difficult, and a recent meeting of investigators (Zagreb, October 2006) has suggested that unified criteria have to be elaborated. In this paper, an International Panel of BEN Investigators agreed on criteria appropriate to epidemiologic studies and clinical investigations of BEN. A screening procedure of BEN in endemic settlements is proposed.

Upper urothelial tumors in emigrants from Balkan endemic nephropathy areas in Serbia.

AIM: The aim of the study was to investigate upper urothelial tumors (UUT) in emigrants from Balkan endemic nephropathy (BEN) areas in Serbia and compare them with UUT from both endemic and nonendemic areas. MATERIALS AND METHODS: A total of 1,121 patients from the state cancer database, between 1960 and 1998, were investigated. Sixty of them were emigrants from BEN areas. RESULTS: UUT in emigrants from BEN areas occurred after 21.7+/-9.9 years (median 20) spent in a rural environment. The time spent outside of the BEN regions was 33.2+/-12.8 years (median 31, range 10-72). Age at surgery was 55 years (range 31-89). In emigrants from BEN areas, there was a significant association of other diseases with UUT: renal failure in 63%, bladder tumors in 23.3% and bilateralism in 6.7% of the patients. Bilateralism was statistically more frequent in emigrants from BEN areas (p=0.04), as were low-grade tumors (p=0.03). There was no statistical difference in tumor stage between patients from BEN areas and from outside of them. Relatives of the emigrants from BEN areas were also affected by BEN, UUT and both of them (33%). CONCLUSIONS: It is concluded that hereditary as well as environmental factors are important for the expression and evolution of the disease. An early period of life spent in the endemic region seems important for the later development of UUT in emigrants from BEN regions. Different time spans spent in the endemic region have no effect on the age of appearance of UUT.

[Bilateral tumors of the upper urothelium].

INTRODUCTION: The incidence of tumors of the upper urothelium is high in our country, apart from their relation to specific regions (BEN and PBEN) and their frequent bilateralism. Bilateral forms are present in significant percentage and are followed, in most cases, by renal failure, which speaks in favor of conservative surgery, if possible. OBJECTIVE: The aim of the study was to present epidemiological, pathoanatomical and clinical characteristics of bilateral tumors of the upper urothelium and evaluate the results of their treatment. METHOD: Our retrospective study analyzed 12 patients with bilateral tumors of the upper urothelium who were treated in the period from 1992 to 1996, according to their epidemiological, clinical, pathoanatomical and pathohistological characteristics, type of surgical treatment and relevant success. RESULTS: In the observed period, bilateral tumors of the upper urothelium were found in 8.2% of our patients. In the group of 12 patients, 5 females and 7 males, 11 cases were from the region of Balkan Endemic Nephropathy (BEN). Renal failure was recorded in high percentage (66%). Radical surgical treatment--total nephroureterectomy was performed in 9 kidney units, and conservative operation in 15 units. Relapse significantly depended on tumor stage and grade, not on type of surgical treatment in the majority of cases. Five-year survival was 58.33%; major cause of death was associated with further evolution of tumor, recurrence and tumor dissemination, respectively, while renal failure complications were the cause of death in one case. CONCLUSION: The success of treatment mainly depends on tumor stage and grade and not on type of surgical method in conservative treatment, but renal failure and its complications are an important risk factor in these patients.