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Successful establishment of a tumour relies on a cascade of interactions between cancer cells and stromal cells within an evolving microenvironment. Both immune and nonimmune cellular components have key roles in this process. Here, we review the major anti‐ and pro‐tumour factors in the early tumour microenvironment and discuss how understanding this process may be exploited in the clinic., Successful establishment of a tumour relies on a cascade of interactions between cancer cells and stromal cells within an evolving microenvironment. Both immune and nonimmune cellular components are key factors in this process, and the individual players may change their role from tumour elimination to tumour promotion as the microenvironment develops. While the tumour–stroma crosstalk present in an established tumour is well‐studied, aspects in the early tumour or premalignant microenvironment have received less attention. This is in part due to the challenges in studying this process in the clinic or in mouse models. Here, we review the key anti‐ and pro‐tumour factors in the early microenvironment and discuss how understanding this process may be exploited in the clinic.

Components of the cellular and the humoral arm of the immune system are essential elements of the tumour microenvironment (TME). The TME includes tumour-associated macrophages which have served as a paradigm for the cancer-promoting inflammation. Cytokines, IL-1 in particular, and complement have emerged as important players in tumour promotion. On the other hand, myeloid cells, innate lymphoid cells and complement have the potential, if unleashed, to mediate anticancer resistance. Targeting checkpoints restraining innate immunity, macrophages and natural killer (NK) cells in particular holds promise as a therapeutic strategy.

Simple Summary Cytokines are a group of small proteins in the body that play an important part in boosting the immune system. Thymic stromal lymphopoietin (TSLP) is a cytokine that plays an important role in the maturation of T cells. Two variants of TSLP, long-form (lfTSLP) and short-form (sfTSLP), have been found, however their roles in cancers are not known. In this study, we discovered that sfTSLP, but not lfTSLP, is predominantly expressed in ovarian and endometrial cancers. The switch that turns the sfTSLP gene on or off is controlled by external modifications of DNA. Our results also found that sfTSLP promotes tumour growth through activating several signal pathways in cancer cells. Abstract Thymic stromal lymphopoietin (TSLP) is an epithelial cell derived cytokine belonging to the IL-7 family and a key initiator of allergic inflammation. Two main isoforms of TSLP, classified as long- (lfTSLP) and short-form (sfTSLP), have been reported in human, but their expression patterns and role(s) in cancers are not yet clear. mRNA expression was examined by isoform-specific RT-PCR and RNA in situ hybridisation. Epigenetic regulation was investigated by chromatin immunoprecipitation-PCR and bisulfite sequencing. Tumour progression was investigated by gene overexpression, cell viability assay, cancer organoid culture and transwell invasion. Signals were investigated by proteome profiler protein array and RNA-sequencing. With the use of isoform-specific primers and probes, we uncovered that only sfTSLP was expressed in the cell lines and tumour tissues of human ovarian and endometrial cancers. We also showed the epigenetic regulation of sfTSLP: sfTSLP transcription was regulated by histone acetylation at promoters in ovarian cancer cells, whereas silencing of the sfTSLP transcripts was regulated by promoter DNA methylation in endometrial cancer cells. In vitro study showed that ectopically overexpressing sfTSLP promoted tumour growth but not invasion. Human phosphokinase array application demonstrated that the sfTSLP overexpression activated phosphorylation of multiple intracellular kinases (including GSK3α/β, AMPKα1, p53, AKT1/2, ERK1/2 and Src) in ovarian cancer cells in a context-dependent manner. We further investigated the impact of sfTSLP overexpression on transcriptome by RNA-sequencing and found that EFNB2 and PBX1 were downregulated in ovarian and endometrial cancer cells, suggesting their role in sfTSLP-mediated tumour growth. In conclusion, sfTSLP is predominantly expressed in ovarian and endometrial cancers and promotes tumour growth.

Cancer prevention is a major issue in the field of public health. Ethanol extracts of the ginseng berry were found to inhibit the inflammation induced by 12- O -tetradecanoylphorbol-13-acetate (TPA), a tumour promoter. The extracts also inhibited tumour promotion by TPA in the mouse skin two-stage carcinogenesis model. The major components, ginsenosides Rd ( 3 ) and Re ( 4 ), inhibited inflammation induced by TPA in mice, while protopanaxadiol ( 1 ) and protopanaxatriol ( 2 ), the aglycones of these ginsenosides, markedly inhibited TPA-induced inflammatory ear oedema in mice. The inhibitory effects of these compounds were more potent than those of indomethacin.

The role of myeloid-derived suppressor cells (MDSCs) in cancer development has become clear over recent years, and MDSC targeting is an emerging opportunity for enhancing the effectiveness of current anticancer therapies. As MDSCs are not only able to limit anti-tumour T-cell responses, but also to promote tumour angiogenesis and invasion, their monitoring has prognostic and predictive value. Herein, we review the key features of MDSCs in cancer promotion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Autophagy is a tightly controlled process comprising lysosomal degradation and recycling of cellular proteins and organelles. In cancer, its paradoxical dual role of cytoprotection and cytotoxicity is context-dependent and controversial. Autophagy primarily acts as a mechanism of tumour suppression, by maintenance of genomic integrity and prevention of proliferation and inflammation. This, combined with immune-surveillance capabilities and autophagy's implicated role in cell death, acts to prevent tumour initiation. However, established tumours exploit autophagy to survive cellular stresses in the hostile tumour microenvironment. This can lead to therapy resistance, one of the biggest challenges facing current anti-cancer approaches. Autophagy modulation is an exciting area of clinical development, attempting to harness this fundamental process as an anti-cancer strategy. Autophagy induction could potentially prevent tumour formation and enhance anti-cancer immune responses. In addition, drug-induced autophagy could be used to kill cancer cells, particularly those in which the apoptotic machinery is defective. Conversely, autophagy inhibition may help to sensitise resistant cancer cells to conventional chemotherapies and specifically target autophagy-addicted tumours. Currently, hydroxychloroquine is in phase I and II clinical trials in combination with several standard chemotherapies, whereas direct, deliberate autophagy induction remains to be tested clinically. More comprehensive understanding of the roles of autophagy throughout different stages of carcinogenesis has potential to guide development of novel therapeutic strategies to eradicate cancer cells.

Cancer prevention is an important issue in various healthcare fields; thus, we continue searching for potential useful compounds. This study focuses on the utilization of Canadian goldenrod, which is commonly seen growing on vacant land. Methanol extracts of the flower of Canadian goldenrod inhibited inflammation by 12- O -tetradecanoylphorbol-13-acetate (TPA) and inhibited promotion in the mouse skin two-stage carcinogenesis model. Five inhibitory compounds were isolated from the active fraction, and these compounds were identified as flavonoids; kaempferol ( 1 ), quercetin ( 2 ), kaempferol-3- O -rutinoside ( 3 ), querectin-3- O -rutinoside ( 4 ), and isorhamnetin-3- O -rutinoside ( 5 ). These compounds inhibited the inflammation induced by TPA, and the inhibitory effects were similar to indomethacin.

This study assessed the toxicological and biological responses of aerosols from a novel hybrid tobacco product. Toxicological responses from the hybrid tobacco product were compared to those from a commercially available Tobacco Heating Product (c-THP), a prototype THP (p-THP) and a 3R4F reference cigarette, using in vitro test methods which were outlined as part of a framework to substantiate the risk reduction potential of novel tobacco and nicotine products. Exposure matrices used included total particulate matter (TPM), whole aerosol (WA), and aqueous aerosol extracts (AqE) obtained after machine-puffing the test products under the Health Canada Intense smoking regime. Levels of carbonyls and nicotine in these matrices were measured to understand the aerosol dosimetry of the products. The hybrid tobacco product tested negative across the in vitro assays including mutagenicity, genotoxicity, cytotoxicity, tumour promotion, oxidative stress and endothelial dysfunction. All the THPs tested demonstrated significantly reduced responses in these in vitro assays when compared to 3R4F. The findings suggest these products have the potential for reduced health risks. Further pre-clinical and clinical assessments are required to substantiate the risk reduction of these novel products at individual and population levels.

The role of myeloid‐derived suppressor cells (MDSCs) in cancer development has become clear over recent years, and MDSC targeting is an emerging opportunity for enhancing the effectiveness of current anticancer therapies. As MDSCs are not only able to limit anti‐tumour T‐cell responses, but also to promote tumour angiogenesis and invasion, their monitoring has prognostic and predictive value. Herein, we review the key features of MDSCs in cancer promotion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Since 1909, the cancer immunosurveillance concept has undergone four distinct eras. These include a general acceptance during 1957–1974, an abandonment during 1974–1996, resurrection during 1996–2001 in the form of an elegant theory of tumour immunoediting proposed by Robert Schreiber, and a retreat since 2006. Recently, in the Journal of Pathology, Ciampricotti et al reported an elegant experimental model designed by establishing RAG2−/−/MMTV-NeuT mice. Using this, they demonstrated that the development and metastasis of HER-2/neu-positive spontaneous mammary carcinoma were not altered by the presence or absence of the adaptive immune system. Their fascinating results are a call to revisit controversial reports as to an effective role of the adaptive immune system in tumour inhibition versus tumour promotion or tolerance in the development of spontaneous carcinomas. Ciampricotti and colleagues present a strong case for revising our ideas of cancer immunoediting and appreciating the complexity of the interaction between cancer and the immune system. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Invited commentary for Ciampricotti M et al. Development of metastatic HER2+ breast cancer is independent of the adaptive immune system. Journal of Pathology. 2011; 224: 56–66. http://dx.doi.org/10.1002/path.2837

In a case-control study of 148 cases of testicular cancer and 314 healthy controls information was collected on lifetime working histories and specific exposures. The main finding was a six-fold increase in the risk for seminoma, one type of testicular cancer, among plastic workers exposed to polyvinyl chloride (PVC). No overrisks were observed for other plastics than PVC. There may be an aetiological link to xenoestrogens, i.e. chemicals in the environment with oestrogenic properties. Phthalates are used in PVC as plasticizer and have oestrogenic properties that could promote the growth of endocrine sensitive tumour cells. However, this "endocrine disruptors" hypothesis must be further evaluated in experimental models with regard to the causative mechanisms of the oestrogenic tumour promotion.

Cellular senescence is a stable proliferation arrest that is associated with extensive cellular remodelling and an altered secretory pathway. Through its numerous inducers that lead to altered gene expression, senescence is able to influence many contrasting functions and pathologies, namely tumour suppression, tumour promotion, wound healing and ageing. As senescence is able to control such important tissue functions, it is now being pinpointed as a possible route for novel therapies. This article and accompanying poster aim to provide a summary of the initiators, pathways and roles of senescence, as well as present examples of senescence and a possible use for senescence in therapy.

Microcystin-LR (MCLR) is a peptide produced by freshwater cyanobacteria that induces severe hepatotoxicity in humans and animals. MCLR is also a potent tumour promoter and it has been proposed that this activity is mediated by the inhibition of protein phosphatases PP1/PP2A, possibly through the activation of proto-oncogenes c-jun, c-fos and c-myc. However, the mechanisms underlying MCLR-induced tumour promotion are still largely unknown, particularly in non-liver cells. In previous studies we have demonstrated that micromolar concentrations of MCLR induce cytotoxic effects in the kidney Vero-E6 cell line. The purpose of the present work was to evaluate whether the exposure to subcytotoxic concentrations of MCLR was sufficient to induce the proliferation of Vero-E6 cells. Through BrdU incorporation assay we show that at nanomolar concentrations MCLR stimulates cell cycle progression in Vero-E6 kidney cell line. Moreover, the analysis of mitogen-activated protein kinases p38, JNK and ERK1/2 activity revealed that the proliferative effect of MCLR is associated with the activation of the pro-proliferative ERK1/2 pathway. These results emphasise the importance to confirm in vivo the impact of MCLR on tumour promotion at kidney level.

In this study we investigated the modification of TCDD-induced enhancement of nitrosamine-initiated altered hepatic foci (AHF) development by dietary vitamin A supplementation and the type of diet. Vitamin A deficiency was shown to potentiate the effect of TCDD. Furthermore, severe vitamin A deficiency alone enhanced the development of AHF. A differential effect on AHF-development by the two types of diet used (purified vs cereal diet) was apparent. However, it was concluded that TCDD-induced depletion of hepatic stores of vitamin A is not a primary mechanism in liver tumour promotion induced by this compound. However, it may well be that TCDD-induced vitamin A deficiency acts as a tumour promoting stimulus in concert with an, as yet, unidentified mechanism of tumour promotion by TCDD.

KEYWORDS - CLASSIFICATION: Animals;Base Sequence;chemistry;Canada;Carcinogens;Cell Culture Techniques;Cell Survival;Cells,Cultured;Chromosomal Instability;Clone Cells;drug effects;Dna;DNA Fingerprinting;DNA Primers;Environmental Health;mechanisms of carcinogenesis;Mice;Mice,Inbred C3H;Mutagenesis;Oxidative Stress;pharmacology;Safety;Tetradecanoylphorbol Acetate. The ability of tumour promoters to alter DNA stability within regions that contain tandemly repeated sequences (TRSs), was studied in a cell culture model of multi-stage carcinogenesis. Non-cytotoxic concentrations of TPA (12-O-tetradecanoyl-phorbol-13-acetate) and xanthine oxidase with xanthine substrate, sufficient to promote morphological transformation in C3H/10T1/2 cultures, were tested for their effects on mutation frequencies in TRSs by a DNA fingerprinting approach. Specifically, restriction digests of genomic DNA samples from randomly selected, non-transformed clones, isolated from cultures after several days exposure to promoters, were visualized by Southern hybridizations with the multi-locus pentamer repeat sequence probe, Ms6-Hm (Pc-1). Basal and promoter-induced frequencies of sub-clone TRS fingerprint polymorphisms were estimated in five cell populations: an uncloned stock culture, three populations established from normal-appearing sub-clones, and one clonal population established from a 3-methylcholanthrene (MCA)-transformed focus. Basal variant fingerprint frequencies spanned a range from 0.0 to 0.43% mutants/band among cells from the four untransformed populations. Both TPA and xanthine oxidase treatments significantly increased recorded mutation frequencies, 2.3- and 2.7-fold, respectively, using combined data from the progenitor population and three untransformed clones. The untreated MCA-transformed clonal population appeared to contain a single, pre-existing mutant restriction fragment, but additional mutations were induced thereafter, in response to the promoting treatments. Taken together, the measured increases in mutations were highly significant and suggest that promoters of cell transformation in the C3H/10T1/2 cell line might induce a genome-wide instability targeted to regions containing Ms6-Hm sequence motifs.

Sixteen DNA microsatellites or simple sequence length polymorphisms (SSLPs), generated by polymerase chain reaction (PCR) were selected for use in the genetic quality control of the nine inbred SENCAR strains currently available. The SENCAR strains constitute a powerful tool for mechanistic studies of multi-stage skin carcinogenesis, as well as for studies to understand the underlying genetic basis of resistance to tumour promotion and progression. SSLP analysis is a fast and economical way for detecting genetic contamination (unexpected outcrosses) among these closely-related albino strains, where standard immunological and biochemical markers have been shown to be insufficient.

Growth factors in general and transforming growth factor-a in particular have been related to cell proliferation and cell differentiation. This study was designed to clarify the distribution pattem of TGF-a in chemically-induced hepatocarcinogenesis. Sprague- Dawley rats were subjected to different non-intensive or intensive carcinogenic treatments using diethylnitrosamine (DEN) as carcinogen and ethinyl estradiol (EE) as promoter. The livers were fixed in 2% paraformaldehyde, dehydrated in a series of ethanol solutions, embedded in paraffin and sectioned. In the preneoplastic lesions no TGF-a immunoreactive cells were identified, but in some hepatic tumours cell immunostained with TGF-a antibody were observed. These results suggest that the cells capable of expressing TGF-a constitutively may be involved in neoplastic development in vivo.

This study was conducted to determine the effects of nitrite (0.05% in drinking-water) and soy sauce (20% in a refined diet) on the initiation and promotion of benzo[a]pyrene-induced forestomach neoplasia in ICR mice. In two experiments nitrite and soy sauce together significantly reduced the number of neoplasms per animal. Soy sauce (without nitrite) produced a smaller apparent (non-significant) reduction whereas nitrite (without soy sauce) had no effect. Evidence suggested that soy sauce might contain factors that reduce calorie absoprtion or utilization, but this observation was independent of the inclusion of nitrite in the drinking-water and therefore could not by itself explain the significant reduction in neoplasms in mice given soy sauce plus nitrite. Protection appeared to primarily involve the tumour promotion stage.

The tumour-promoting activity of methyl ethyl ketone peroxide (MEKP) was tested on the skin of hairless mice using a two-stage initiation-promotion protocol. When ultraviolet radiation in the UVB region (280-320 nm) was used as tumour initiator, MEKP showed weak promoting activity. The promotional activity of MEKP was potentiated by diethyl maleate, which is known to deplete intracellular glutathione, suggesting that lipid peroxidation may be important in the tumour promotion.

TCDD-induced enhancement of altered hepatic foci was studied in nitrosamine initiated, female Sprague-Dawley rats. In rats maintained on a conventional, cereal-based diet, ten weeks exposure to TCDD by weekly S.C. injections (0.7 μg/kg) starting five weeks after initiation significantly enhanced foci development provided an initial loading dose (3.5 μg/kg) was applied. The same total dose administered for 14 weeks (0.7 μg/kg/w) starting one week after initiation was ineffective in this respect. However, if rats were shifted to a purified, casein-based diet one week after initiation, the ten weeks TCDD administration schedule with a initial loading dose did not significantly influence foci development.

Images Figs. 2-7 Figs. 8-11

Incomplete Carcinogens in Cigarette Smoke Condensate: Tumour-Promotion by a Phenolic Fraction

The organochlorine pesticide DDT is a liver tumour promoter and a potent inhibitor of intercellular communication. Present knowledge of the mechanism by which DDT inhibits intercellular communication is limited but it has been suggested that increased intracellular free calcium induced by DDT could be of importance. As the effects of calcium are closely associated with the multifunctional protein calmodulin (CaM) in most cells the potential binding of DDT to CaM and subsequent effects on CaM-stimulated Ca2+/Mg2+-ATPase activity were studied. DDT inhibited CaM-stimulated Ca2+/Mg2+-ATPase activity and bound to CaM in a manner similar to established CaM-inhibitors. Subsequently an in vitro assay for measuring inhibition of metabolic cooperation between 6-thioguanine (TG)-sensitive and TG-resistant Chinese hamster (V79) cells was used to investigate the possible involvement of CaM in the regulation of intercellular communication. Calmidazolium (CzM), a potent CaM inhibitor, was tested alone or in combination with the tumour promoters 12-O-tetradecanoyl phorbol-13-acetate (TPA) or DDT known inhibitors of intercellular communication. The results showed that CzM alone was without effect with regard to inhibition of metabolic cooperation but potentiated the response induced by TPA, an effect not noticed with DDT. These results suggest different mechanisms of action of TPA and DDT on metabolic cooperation and support the hypothesis that with calcium CaM may be of importance for drug-induced inhibition of intercellular communication and tumour promotion.

Mechanisms of Tumour Promotion and Co-carcinogenesis. (Vol. 2 of a series on Carcinogenesis—A comprehensive survey)