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Much of the bacterial anticancer therapy being developed relies on the ability of bacteria to specifically colonise tumours. Initial attempts to translate promising Salmonella enterica Typhimurium ( S . Typhimurium) preclinical results to the clinical setting failed, primarily due to lack of tumour colonisation and the significant toxicities from systemically administered Gram-negative bacteria. To address the difference in results between preclinical experiments performed in mice with transplant tumours and clinical trials in human volunteers with autochthonous tumours, a genetically engineered mouse model of breast cancer (BALB-neuT) was utilised to develop a strain of virulence-attenuated S . Typhimurium capable of robust colonisation of autochthonous tumours. Several genes that code for bacterial surface molecules, responsible for signalling a toxic immune response against the bacteria, were mutated. The resulting S. Typhimurium strain, BCT2, allowed non-toxic intravenous administration of 3 × 10 6 colony forming units of bacteria in tumour-burdened mice when combined with a vascular disruption agent to induce intratumoral necrotic space and facilitate bacterial colonisation.

Attenuated bacteria-mediated tumor targeting diagnosis will provide a novel strategy for further cancer treatments owing to the intrinsic facultative anaerobic characteristic of bacteria and rapid proliferation in the tumor sites. In this work, we firstly investigate the in vivo behaviour of the attenuated Salmonella typhimurium (S. typhimuriumΔppGpp/lux) after intravenous injection. S. typhimurium exhibits rapid proliferation in tumor sites after three days of injection through bioluminescence imaging, the Luria-Bertani plate and the Gram-staining assay. In contrast, S. typhimurium does not proliferate in the normal tissues and could be excreted from the body of mice. Afterwards, a targeting peptide ubiquicidin (UBI) labeled with fluorescent dye Cy5.5 or radionuclide 125 I was intravenously injected into the mice with or without S. typhimurium treatments for in vivo fluorescence imaging and single-photon emission computed tomography (SPECT/CT) imaging, respectively. The results show that the peptide UBI could specifically target the two independent bacteria-infected tumor models, the 4T1 murine breast cancer model and the CT26 mouse colon cancer model, realizing the sensitive multimodal imaging of tumors. Such a strategy (bacteria-mediated tumor targeting) may further improve the sensitivity to early diagnosis of tumors. We hope that our developed strategy could further be extended to cancer theranostics in the future, bringing good news for cancer patients.

The hypoxic region microenvironment reduces the susceptibility of the cancer cells to radiotherapy and anticancer drugs of the solid tumors. However, the reduced oxygen surroundings provide an appreciable habitat for anaerobic bacteria to colonize and proliferate. Herein, we present a biocompatible bacteriabased system that can deliver poly(lactic-co-glycolic acid)(PLGA) nanoparticles(PLGA NPs) specifically targeting into solid tumor to achieve precision imaging and treatment. In our strategy, anaerobic bacterium Bifidobacterium longum (B. longum) that colonizes selectively in hypoxic regions of animal body was successfully used as a vehicle to conjugate with PLGA NPs and transported into solid tumors. To improve the efficacy and specificity of tumor therapy, low-boiling point perfluorohexane (PFH) liquid was wrapped in the core of PLGA NPs (PFH/PLGA NPs), which could increase the deposition of energy by affecting the acoustic environment of the tumor and destroy cells after liquid-gas phase transition during High Intensity Focused Ultrasound (HIFU) irradiation. This strategy shows an effective diagnosis and treatment integration for giving stronger imaging, longer retention period and more effective tumor therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Salmonella is a Gram-negative, facultative anaerobe that is a common cause of host intestinal infections. Salmonella grows under aerobic and anaerobic conditions, and it has been proven capable of inhibiting tumor growth. However, the molecular mechanism by which Salmonella inhibits tumor growth is still unclear. Angiogenesis plays an important role in the development and progression of tumors. We investigated the antitumor effect of Salmonella in a syngeneic murine tumor model. Hypoxia-inducible factor-1 (HIF-1)α plays a significant role in tumor angiogenesis. We examined the molecular mechanism by which Salmonella regulated vascular endothelial growth factor (VEGF), which is an important angiogenic factor. The expression of VEGF in tumor cells was decreased by treatment with Salmonella. The conditioned medium from Salmonella-treated cells inhibited the proliferation of endothelial cells. Salmonella inhibited the expression of HIF-1α as well as downregulated its upstream signal mediator protein kinase B (AKT). Salmonella significantly inhibited tumor growth in vivo, and immunohistochemical studies of the tumors revealed decreased intratumoral microvessel density. These results suggest that Salmonella therapy, which exerts anti-angiogenic activities, represents a promising strategy for the treatment of tumors., Competing Interests: The authors declare no conflict of interest.

Therapeutic attenuated strains of Salmonella Typhimurium target and eradicate tumors in mouse models. However, the mechanism of S. Typhimurium for tumor targeting is still poorly understood. We performed a high-throughput screening of single-gene deletion mutants of S. Typhimurium in an orthotopic, syngeneic murine mammary model of breast cancer. The mutants under selection in this system were classified into functional categories to identify bacterial processes involved in Salmonella accumulation within tumors. Niche-specific genes involved in preferential tumor colonization were identified and exemplars were confirmed by competitive infection assays. Our results show that the chemotaxis gene cheY and the motility genes motAB confer an advantage for colonization of Salmonella within orthotopic syngeneic breast tumors. In addition, eutC, a gene belonging to the ethanolamine metabolic pathway, also confers an advantage for Salmonella within tumors, perhaps by exploiting either ethanolamine or an alternative nutrient in the inflamed tumor environment., Competing Interests: The authors declare no conflicts of interest.

Background/aim: Cancer treatment with attenuated Salmonella enterica Typhimurium (S. Typhimurium) has gained momentum in recent years. However, the effectiveness of this treatment has not been explored in autochthonous models. We report the efficacy of S. Typhimurium in mice with autochthonous mammary tumors., Materials and Methods: S. Typhimurium attenuated by deletion of cyclic adenosine monophosphate signaling, SalpNG.1, was injected into female BALB-neuT tumor-bearing mice. Mice were monitored for efficacy and sacrificed for mechanistic studies., Results: In treated mice, seven-week post-treatment tumor burden was reduced by 85% and median survival was increased by 88%. Efficacy was correlated with increased tumor-infiltrating CD8 and natural killer cells. In addition, SalpNG.1 treatment caused a systemic increase of monocytic myeloid-derived suppressor cells that accumulated to high numbers within tumor tissue. Bacteria were not detected in tumor tissue, suggesting that the observed efficacy was due to a systemic rather than a tumor-specific effect of the bacteria., Conclusion: S. Typhimurium treatment reduces tumor burden and increases survival in an autochthonous breast cancer model., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Unlabelled: Clostridium sporogenes ATCC 3584 is an obligate anaerobe that has been reported to possess excellent tumour-targeting capacity. Here, we use Cl. sporogenes as a vector to deliver IL-12, a potent antitumour cytokine that bears numerous antitumour properties but that has limited clinical applications due to its strong toxicity when delivered systemically. In this study, Cl. sporogenes was genetically engineered to secrete murine IL-12, and its antitumour efficacy and toxicity were investigated in a murine EMT6 mammary carcinoma model. After intravenous injection, Cl. sporogenes was able to selectively settle and reproduce in the tumours without encroaching on normal tissues, resulting in a clear delay of tumour growth and a 14·3% cure rate. Importantly, the mice showed no obvious toxicity-associated side effects, such as diarrhoea and weight loss, during the treatment process. The significant antitumour efficacy and low toxicity of this treatment may be explained by the selective tumour-targeting properties of Cl. sporogenes and by the sustained release of IL-12 accompanying bacterial proliferation. This moderate local IL-12 concentration would not induce the severe response in the entire body, that is inevitable when IL-12 is administered directly., Significance and Impact of the Study: Interleukin-12 (IL-12) is a potent antitumour cytokine, but it is toxic when administrated systemically. This study demonstrates that murine IL-12 can be systemically delivered to hypoxic sites in solid tumours by Clostridium sporogenes, producing a clear delay in tumour growth and a 14·3% cure rate in a mouse tumour model. Importantly, there is no obvious toxicity associated with IL-12 during the treatment process. This result may be accounted for by the excellent tumour-targeting capacity of Cl. sporogenes, targeting IL-12 directly to the tumour site instead of to the entire body., (© 2014 The Society for Applied Microbiology.)

The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2',2'-difluoro-2'-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2'-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors.

Background: Myeloid-derived suppressor cells (MDSCs) are present in large numbers in blood of mice and humans with cancer, and they strongly inhibit T-cell and natural killer (NK) cell responses, at young and old age. We found that a highly attenuated bacterium Listeria monocytogenes (Listeria(at))-infected MDSC and altered the immune-suppressing function of MDSC., Methods: Young (3 months) and old (18 months) BALB/cByJ mice with metastatic breast cancer (4T1 model) were immunised with Listeria(at) semi-therapeutically (once before and twice after tumour development), and analysed for growth of metastases and primary tumour, in relation to MDSC-, CD8 T-cell and NK cell responses., Results: We found that Listeria(at)-infected MDSC, which delivered Listeria(at) predominantly to the microenvironment of metastases and primary tumours, where they spread from MDSC into tumour cells (infected tumour cells will ultimately become a target for Listeria-activated immune cells). Immunotherapy with Listeria(at) significantly reduced the population of MDSC in blood and primary tumours, and converted a remaining subpopulation of MDSC into an immune-stimulating phenotype producing IL-12, in correlation with significantly improved T-cell and NK cell responses to Listeria(at) at both ages. This was accompanied with a dramatic reduction in the number of metastases and tumour growth at young and old age., Conclusions: Although preclinical studies show that immunotherapy is less effective at old than at young age, our study demonstrates that Listeria(at)-based immunotherapy can be equally effective against metastatic breast cancer at both young and old age by targeting MDSC.

Breast cancer patients are susceptible to infections such as candidiasis. Due to the importance and the role of matrix metalloproteinases (MMP) in breast cancer progression and its correlation with tumor metastasis, we analyzed the serum level of MMPs -2, -3, -9 and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in breast cancer bearing mice in the presence of systemic Candida albicans infection. Female BALB/c mice were divided into 4 groups: group I had tumor + candidiasis; group II, tumor only; group III, candidiasis only and group IV as negative control. Tumor tissue was separated from stock breast cancer bearing mice and transplanted subcutaneously into the groups I and II mice. Two weeks after tumor transplantation, groups I and III were infected with Candida albicans by intravenous injection. One week after systemic infection, the sera of the experimental groups were prepared and analyzed with ELISA for MMP-2, -3, -9 and TIMP-1 levels. The results showed that the levels of MMP-3, MMP-9 and TIMP-1 were increased in groups I, II and III, as compared to the control group. However, the level of MMP-2 was decreased in mice infected with Candida albicans and in infected mice bearing tumor. These data suggest that candidiasis may have a positive effect on tumor progression and metastasis.

Many studies have demonstrated that intravenously administered bacteria can target and proliferate in solid tumors and then quickly be released from other organs. Here, we employed the tumor-targeting property of Escherichia coli Nissle 1917 to inhibit mouse B16 melanoma and 4T1 breast tumors through the expression of azurin protein. For this purpose, recombinant azurin-expressing E. coli Nissle 1917 was developed. The levels of in vitro and in vivo azurin secretion in the engineered bacterium were determined by immunochemistry. Our results demonstrated that B16 melanoma and orthotopic 4T1 breast tumor growth were remarkably restrained and pulmonary metastasis was prevented in immunocompetent mice. It is worth noting that this therapeutic effect partially resulted from the antitumor activity of neutrophils and lymphocytes due to inflammatory responses caused by bacterial infections. No toxicity was observed in the animal during the experiments. This study indicates that E. coli Nissle 1917 could be a potential carrier to deliver antitumor drugs effectively for cancer therapy.

Background: An effective cancer therapeutic must selectively target tumours with minimal systemic toxicity. Expression of a cytotoxic protein using Salmonella typhimurium would enable spatial and temporal control of delivery because these bacteria preferentially target tumours over normal tissue., Methods: We engineered non-pathogenic S. typhimurium to secrete murine TNF-related apoptosis-inducing ligand (TRAIL) under the control of the prokaryotic radiation-inducible RecA promoter. The response of the RecA promoter to radiation was measured using fluorometry and immunoblotting. TRAIL toxicity was determined using flow cytometry and by measuring caspase-3 activation. A syngeneic murine tumour model was used to determine bacterial accumulation and the response to expressed TRAIL., Results: After irradiation, engineered S. typhimurium secreted TRAIL, which caused caspase-3-mediated apoptosis and death in 4T1 mammary carcinoma cells in culture. Systemic injection of Salmonella and induction of TRAIL expression using 2 Gy gamma-irradiation caused a significant delay in mammary tumour growth and reduced the risk of death by 76% when compared with irradiated controls. Repeated dosing with TRAIL-bearing Salmonella in conjunction with radiation improved the 30-day survival from 0 to 100%., Conclusion: These results show the pre-clinical utility of S. typhimurium as a TRAIL expression vector that effectively reduces tumour growth and extends host survival.

The successful application of live bacteria in cancer therapy requires a more detailed understanding of bacterial interaction with the tumour microenvironment. Here, we analysed the effect of Escherichia coli K-12 colonization on the tumour microenvironment by immunohistochemistry and fluorescence microscopy in the murine 4T1 breast carcinoma model. We described the colonization of tumour-bearing mice, as well as the spatiotemporal distribution of E. coli K-12 in the 4T1 tumour tissue over a period of 14 days. The colonization resulted within 3 days in large avascular necrotic tissue, redistribution of hypoxic areas and an enhanced collagen IV deposition within the colonized tumour tissue, which changed the tumoral perfusion of systemically injected immunoglobulins. In addition, E. coli K-12 colonization led to the redistribution of tumour-associated macrophages, forming a granulation tissue around bacterial colonies, and also to an increase in TNFalpha and matrix metalloproteinase 9 expression. Colonization of 4T1 tumours by E. coli K-12 resulted in strong reduction of pulmonary metastatic events. These new insights will contribute to the general understanding of the tumour-microbe cross-talk and to the design of bacterial strains with enhanced anticancer efficiency.

Systemic administration of microorganisms into tumor-bearing mice revealed preferential accumulation in tumors in comparison to clearance in organs such as spleen and liver. Here we compared the efficiency of tumor-specific colonization of pathogenic Salmonella typhimurium strains 14028 and SL1344 to the enteroinvasive Escherichia coli 4608-58 strain and to the attenuated Salmonella flexneri 2a SC602 strain, as well as to the uropathogenic E. coli CFT073, the non-pathogenic E. coli Top10, and the probiotic E. coli Nissle 1917 strain. All strains colonized and replicated in tumors efficiently each resulting in more than 1 x 10(8) colony-forming units per gram tumor tissue. Colonization of spleen and liver were significantly lower when E. coli strains were used in comparison to S. typhimurium and the non-pathogenic strains did not colonize those organs at all. Further investigation of E. coli Nissle 1917 showed that no drastic differences in colonization and amplification were seen when immunocompetent and immunocompromised animals were used, and we were able to show that E. coli Nissle 1917 replicates at the border of live and necrotic tumor tissue. We also demonstrated exogenously applied L-arabinose-dependent gene activation in colonized tumors in live mice. These findings will prepare the way for bacterium-mediated controlled protein delivery to solid tumors.

Inflammation associated with bacterial infections is a risk factor for cancers in humans, yet its role in breast cancer remains poorly understood. We have previously shown that innate immune inflammatory response against intestinal bacteria is sufficient to induce colon cancer. Here we report that infecting Rag2-deficient C57BL/6 Apc(Min/+) mice with an intestinal bacterial pathogen, Helicobacter hepaticus, significantly promotes mammary carcinoma in females and enhances intestinal adenoma multiplicity by a tumor necrosis factor alpha (TNFalpha)-dependent mechanism. The mammary and intestinal tumor development as well as the increase in proinflammatory mediators is suppressed by adoptive transfer of interleukin 10-competent CD4+CD45RB(lo)CD25+ regulatory (T(R)) cells. Furthermore, prior exposure of donor mice to H. hepaticus significantly enhances antitumor potency of their T(R) cells. Interestingly, these microbially experienced T(R) cells suppress tumorigenesis more effectively in recipient mice irrespective of their tumor etiology. These data suggest that infections with enteric pathogens enhance T(R)-cell potency and protect against epithelial cancers later in life, potentially explaining paradoxical increases in cancer risk in developed countries having more stringent hygiene practices. The possibility that dysregulated gut microbial infections in humans may lead to cancer in anatomically distant organs, such as breast, highlights the need for novel immune-based strategies in cancer prevention and treatment.

Background: Attenuated Salmonella typhimurium has been demonstrated as a potential gene delivery vector. Previous findings induce the necessity to optimize tumor selectivity and bacterial dosing in relation to tumor volume and intratumoral therapeutic gene expression., Materials and Methods: Attenuated Salmonella VNP20009 and VNP20047 (expressing cytosine deaminase) were systemically administered to tumor-bearing rats. The bacteria were quantified in tumor and normal organs. Conversion of 5-fluorocytosine to 5-fluorouracil was evaluated using thin layer chromatography., Results: Tumor colonization efficiency was dependent on Salmonella density, administration route and tumor volume. Colonization of normal tissues gradually decreased with time, while intratumoral proliferation of bacteria remained high during the follow-up period. The Optimal Therapeutic Dose (OTD) was found to be 5.10(7) cfu/rat. Intratumoral VNP20047-expressed CDase leading to the conversion of 5-FC to 5-FU was detected in vivo., Conclusion: Our results indicate the need to define an OTD, probably for each species, when using genetically engineered Salmonella as a tumor- and species-selective vector in cancer therapy.

Our experiments were concerned with formation of antitumor cross-resistance by Trichinella spiralis infection in rats. Prophylactic infection was followed by an insignificant decrease in the number of 3,4-benzpyrene-induced malignant fibrous histiocytoma. It also inhibited mammary gland cancer induced by 7,12-dimethylbenzo(a)anthracene. No significant influence on the formation of fibroadenomas as a side effect was reported.

Mouse mammary tumor virus is a replication-competent B-type murine retrovirus responsible for mammary gland tumorigenesis in some strains of laboratory mice. Mouse mammary tumor virus is transmitted horizontally through the milk (exogenous or milk-borne virus) to susceptible offspring or vertically through the germ line (endogenous provirus). Exogenously acquired and some endogenous mouse mammary tumor viruses are expressed at high levels in lactating mammary glands. We show here that there is packaging of the endogenous Mtv-1 virus, which is expressed at high levels in the lactating mammary glands of C3H/HeN mice, by the virions of exogenous C3H mouse mammary tumor virus [MMTV(C3H)]. The mammary tumors induced in C3H/HeN mice infected with exogenous MMTV (C3H) virus contained integrated copies of recombinant virus containing a region of the env gene from an endogenous virus. This finding indicates that there was copackaging of the Mtv-1 and MMTV(C3H) RNAs in the same virions. Moreover, because Mtv-1 encodes a superantigen protein with a V beta specificity different from that encoded by the exogenous virus, the packaging of Mtv-1 results in an infectious virus with a broader host range than MMTV(C3H).

The effects of caloric restriction on the incidence of mammary tumor development, the levels of the expression of mouse mammary tumor virus (MMTV)- and prolactin-RNA, as well as the levels of serum prolactin, were investigated in virgin RIII/Sa mice, a strain known to display a high incidence of spontaneous mammary tumor development. Of the 54 mice fed a low-calorie (LC; 10 kcal/day) diet containing low fat (LF; 5% corn oil) for a period of 72 weeks, only seven mice were found to develop mammary tumors, an incidence of 13%. By contrast, the cumulative tumor incidence in a similar sized group of mice, fed a high-calorie (HC; 16 kcal/day) low fat-containing diet was 73%. Estimation of the relative levels of MMTV-RNA, as determined by Northern and slot blot hybridizations, in the mammary glands of mice fed LCLF and HCLF diets for 8, 10, 16, 28, and 36 weeks revealed that the LCLF diet-fed mice expressed 4-15-fold less RNA than the HCLF diet-fed mice. Interestingly, however, the LCLF diet did not appear to exert any effect on the expression of prolactin RNA even though it reduced the levels of serum prolactin. We suggest that in RII/Sa mice the modulation of MMTV-induced mammary tumors by dietary calorie is linked to the secretion of serum prolactin which, in turn, affects the replication of MMTV required for mammary cell transformation.

Lymphocytic infiltrates of BALB/c C4 hyperplastic alveolar nodule (HAN) have elevated natural killer (NK) activity, which correlates positively with the progression of C4 HAN to tumor: C4 HAN produces an infectious mouse mammary tumor virus, MMTV(C4), which encodes a superantigen that activates and deletes T-cells with the V beta 2 segment in the T-cell receptor. In this report, NK activation by both MMTV(C4) and MMTV(C4) superantigen was tested. NK activity was measured in naive BALB/c mice, BALB/c mice depleted of V beta 2+ T-cell, or V beta 2-transgenic mice after they received injections of either purified MMTV(C4) or MMTV(C4)-infected splenocytes. Elevated NK activity was observed in BALB/c mice receiving MMTV(C4) or MMTV(C4)-infected splenocytes. Depletion of V beta 2+, but not V beta 8+, T-cells by specific anti-V beta hybridoma before injection of MMTV(C4)-infected cells reduced but did not eliminate NK activation. NK activation in V beta 2-transgenic mice occurred before massive CD4 T-cell deletion took place and was more pronounced than that in the nontransgenic littermates. These results indicate that MMTV activates NK cells through superantigen-dependent and -independent pathways and supports the role of MMTV(C4) in the augmented NK activity observed in C4 HAN infiltrates. The progression of C4 HAN to tumor represents a model system for the analysis of how tumorigenesis may be affected by lesion-associated viruses.

The transgenic mouse has emerged as an important model system to assess the transforming potential of oncogenes in the mammary epithelium. Mammary gland-specific expression of oncogenes in transgenic mice has resulted in the induction of a variety of phenotypes ranging from benign epithelial hyperplasias to metastatic mammary tumors. The induction of tumors in most of these transgenic models is a multi-step process where transgene expression, although required, is not sufficient for conversion of the primary mammary epithelial cell to the transformed phenotype. While the identity of many of these collaborating genetic events is obscure, several approaches have been applied with might shed light on their nature. In a few exceptional transgenic strains, tumor progression can occur very rapidly suggesting that, if additional genetic events are required, they occur very frequently. Recent genetic and biochemical characterization of these strains offers insight into the molecular mechanisms that may underlie the complex phenotypic features exhibited by these transgenic strains.

Mammary tumours developed in 110 (95.7%) of 115 DDD/1-Mtv-2/Mtv-2 (DDD/1-Mtv-2) and 24 (47.1%) of 51 DDD/1fDDD/1-Mtv-2 (DDD/1fMtv-2) force-bred female mice during a one-year period. The mean tumour age +/- SE was 220 +/- 7 and 269 +/- 7 days, respectively. These tumours were examined for responsiveness to pregnancies by comparing their growth after transplantation between virgin and breeding recipients. Of 73 tumours from DDD/1-Mtv-2 mice, 9 (12%) were completely pregnancy-dependent (CPD), 3 (4%) pregnancy-dependent (PD), 9 (12%) pregnancy-responsive (PR), and 52 (71%) pregnancy-independent (PI), and of 25 tumours from DDD/1fMtv-2 mice, one (4%) was CPD, one (4%) PR, and 23 (92%) PI. Although most tumours were heterogeneous in morphology and there was no clear relation between morphology and PD properties, most CPD tumours were type P and considered to be connected with mammary plaques. When 9 CPD tumours from DDD/1-Mtv-2 mice were serially transplanted in breeders, 6, 2 and one progressed to lose pregnancy dependence within 3, 8 and 18 generations, respectively. DDD/1-Mtv-2 mice will provide a model for studies on progression of mammary tumours from hormone-dependent to autonomous states.

We have previously characterized an infectious mouse mammary tumor virus [(MMTV(SW)] which induces a strong superantigen response in vivo. Here we describe the isolation and characterization of MMTV(C4) which was derived from milk of mice implanted with hyperplastic alveolar nodules. MMTV(C4) stimulates V beta 2 expressing T cells after local injection in vivo. Comparison with known open reading frame (orf) sequences revealed high homology to Mtv-6, an endogenous virus interacting with V beta 3-expressing T cells. The carboxyl-terminal amino acids were, however, altered. High homology including the carboxyl-terminal orf amino acids were found with MMTV(C3H-K). We show here that MMTV(C3H-K) has lost its superantigen function. Sequence comparisons permitted the characterization of few key amino acids which could be important for T cell receptor interaction and superantigen processing.

Linoleic acid, cholesterol, dexamethasone and progesterone were tested by immunocytochemistry and immunoprecipitation for their single and combined effects in vitro on mouse mammary tumor virus (MMTV) gp52 distribution among three compartments: cell-associated antigen, extracellular virus particles and extracellular shed antigen unassociated with virus particles. Results indicated that all additives significantly increased total MMTV gp52 levels and altered the distribution. Linoleic acid and dexamethasone induced the greatest relative proportion of extracellular gp52, whereas cholesterol and progesterone induced the greatest proportion of cell-associated gp52. The implications of these findings for the immune response to mammary tumors is discussed.

To study a possible role(s) played by the trisomy and pentasomy of chromosome 13 in murine mammary tumors, we examined, in eight cloned established cell lines derived from a single BALB/c mammary tumor induced by MTV, a correlation between the presence of trisomy or pentasomy 13 and transformation parameters and in vivo tumorigenicity in syngeneic mice. We found that cell lines with a higher incidence of trisomy or pentasomy 13 in cells of diploid and tetraploid chromosome numbers, respectively, grew to a much higher cell density in flasks than did those with low incidence, and they formed tumors in syngeneic BALB/c mice, whereas those with a low incidence of trisomy or pentasomy 13 were poorly tumorigenic. The presence in the tumorigenic cells of trisomy or pentasomy 13 was not correlated with their growth in soft agar. Furthermore, other chromosomes manifested a wide range of copy numbers in the presence of trisomy or pentasomy 13, indicating that no chromosomes counteracted chromosome 13 to prevent the tumorigenicity. In light of the tumorigenic growth of the cells that maintain gene dosage of chromosome 13 at different ploidy levels, the possibility of the yeast G1 cyclin-like roles played by the gene(s) residing on chromosome 13 in murine mammary tumorigenesis is discussed.

Tumorigenesis by mouse mammary-tumor virus (MMTV) involves proviral disruption and transcriptional activation of a number of cellular oncogenes, generically termed Int. The frequencies with which different Int genes are activated in different mouse strains can be quite variable, and previous surveys have suggested that insertions at Int-2/Fgf-3 occur primarily in strains that develop pregnancy-dependent mammary tumors. To address this issue, we have determined the relative contributions of 5 known Int genes (Wnt-1, Wnt-3, Fgf-3, Fgf-4 and Int-3) in mammary tumors from virgin BR6 and multiparous BR6, BALB/cfBR6 and RIII mice. Whereas Fgf-3 was implicated in 66%, 80% and 92% of the tumors from the respective parous animals, only 20% of the tumors from virgin mice expressed Fgf-3. This reduced involvement of Fgf-3 was compensated by proviral insertions in Fgf-4, Int-3 and Wnt-3, but the frequency of Wnt-1 activation was relatively constant. These data strengthen the link between Fgf-3 and a pregnancy-dependent phenotype and suggest that, in the strains analyzed, the frequency of Int-gene activation was influenced more by the hormonal status than by the genetic background.

Endogenous and infectious mouse mammary tumor viruses (MMTVs) encode in their 3' long terminal repeat a protein that exerts superantigen activity; that is, it is able to interact with T cells via the variable domain of the T cell receptor (TCR) beta chain. We show here that transmission of an infectious MMTV is prevented when superantigen-reactive cells are absent through either clonal deletion due to the expression of an endogenous MTV with identical superantigen specificity or exclusion due to expression of a transgenic TCR beta chain that does not interact with the viral superantigen. A strict requirement for superantigen-reactive T cells is also seen for a local immune response following MMTV infection. This immune response locally amplifies the number of MMTV-infected B cells, most likely owing to their clonal expansion. Collectively, our data indicate that a superantigen-induced immune response is critical for the MMTV life cycle.

Transgenic mice carrying the Wnt-1 protooncogene modified for expression in mammary epithelial cells exhibit hyperplastic mammary glands and stochastically develop mammary carcinomas, suggesting that additional events are necessary for tumorigenesis. To induce such events and to identify the genes involved, we have infected Wnt-1 transgenic mice with mouse mammary tumor virus (MMTV), intending to insertionally activate, and thereby molecularly tag, cooperating protooncogenes. Infection of breeding female Wnt-1 transgenics decreased the average age at which tumors appeared from approximately 4 months to approximately 2.5 months and increased the average number of primary tumors per mouse from 1-2 to > 5. A smaller effect was observed in virgin females, and infection of transgenic males showed no significant effect on tumor latency. More than half of the tumors from the infected breeding group contained one or more newly acquired MMTV proviruses in a pattern suggesting that most cells in tumors arose from a single infected cell. Analyses of provirus-containing tumors for induced or altered expression of int-2/Fgf-3, hst/Fgf-4, int-3, and Wnt-3 showed activation of int-2 in 39% of tumors, hst in 3%, and both int-2 and hst in 3%. DNA analyses with probes for protooncogenes and MMTV confirmed that the activations resulted from proviral insertions. There was no evidence for proviral insertions at the int-3, Wnt-3, or Wnt-1 loci. These findings provide further evidence that fibroblast growth factors Int-2 and Hst can cooperate with Wnt-1, another secreted factor, in mammary tumorigenesis, and they illustrate the capacity of this system to identify cooperating oncogenes.

Alkylphosphocholines, and especially their main representative hexadecylphosphocholine (HPC), show high anticancer activity in methylnitrosourea (MNU)-induced autochthonous rat mammary carcinoma. The regression of MNU-induced rat mammary carcinoma during HPC treatment can be evaluated by computed tomography and sonography. This allows a noninvasive monitoring of therapy in vivo (tumor size, morphology, and blood supply). Both diagnostic modalities can show a rapid concentric decrease in tumor volume as well as the appearance of cystic, scarry, and necrotic areas in the tumor tissue as a result of HPC treatment. In addition, prior to, during and after therapy tumor perfusion can be assessed by color Doppler sonography in vivo. A more than 4-fold difference in HPC efficacy was observed when the colony growth of explanted MNU-induced mammary carcinoma cells was measured in the methylcellulose colony assay (IC50 = 180 mumol HPC/l) and the Hamburger Salmon colony assay (IC50 = 740 mumol HPC/l). In the latter assay, growth of concomitantly seeded untransformed cells, especially of fibroblasts, is much lower than in the methyl-cellulose colony assay. We therefore assume that the antitumor efficacy of HPC against MNU-induced mammary carcinoma is enhanced by neighboring cells such as fibroblasts. Cell culture experiments with the three MNU-induced rat mammary carcinoma cell clones 1-C-2, 1-C-30, and 1-C-32 revealed IC50 values in the range of 50-70 mumol HPC/l. The volume of 1-C-2 cells increased up to 4-fold after 72 h of permanent exposure to 100 mumol HPC/l, a concentration that completely inhibited proliferation of tumor cell numbers without being cytotoxic. Nucleotide triphosphate levels dropped significantly after 24 h and were slowly restored in spite of continued exposure. After 72 h, they nearly reached those levels observed in plateau-phase cells. This suggests that HPC-induced growth inhibition has similarities with physiologically occurring growth arrest. Finally, replication of RNA viruses and DNA viruses was suppressed 30-fold and 7-fold, respectively, at low concentrations of HPC (12 mumol/l), which caused no or negligible growth inhibition in the virus-harboring cells, thus demonstrating specific antiviral activity of HPC. From these observations we conclude that HPC differs in many important aspects from conventional cytostatic agents and is certainly worth following-up in further investigations.

DDD/1 mice free from exogenous mouse mammary tumor virus (MMTV) do not develop any neoplastic mammary lesions. In GR mice, the expression of Mtv-2, an endogenous proviral MMTV, leads to 100% incidence of mammary ductal hyperplasias and tumors. An Mtv-2 congenic line, DDD/1-Mtv-2/Mtv-2, was established by introducing Mtv-2 from GR into DDD/1 to elucidate its function. Development of mammary plaques (MPQ) characterized by ductal hyperplasias was investigated in 152 congenic females on day 17 to 19 of the first pregnancy. The incidence of MPQ was 48.0% and most MPQ-positive mice (75.3%) had only one MPQ. Generally, MPQ were small in size: the diameter was as small as < or = 3 mm in 77.6% of them. Of 84 MPQ implanted into intact fat pads, 43 (51.2%), 38 (45.2%) and 3 (3.6%) showed undetectable, pregnancy-dependent and autonomous growths; respectively when the hosts underwent pregnancy. Almost all MPQ produced normal-appearing ductal-alveolar outgrowths in mammary epithelium-divested or cleared fat pads of virgins. MPQ implanted into cleared fat pads were very similar to normal mammary glands in the responses to progesterone (P) and estradiol (E) alone or in combination except for association of ductal hyperplasias in 4 of 12 MPQ under E+P treatment. These findings revealed the preneoplastic nature of MPQ. Exogenous MMTV proviruses were demonstrated in all MPQ. The int-2 DNA rearrangement was found in 2 of 10 MPQ but in none of 9 mammary carcinomas and the int-1 DNA rearrangement in none of 10 MPQ but in 5 of 10 carcinomas. It is thus likely that the Mtv-2 gene participates in a very early stage of mammary tumorigenesis not directly but indirectly through insertion mutation of host genes, while the cellular oncogenes, int-2 and int-1, may contribute to preneoplastic transformation of mammary epithelium and progression from preneoplastic to more malignant states, respectively.

Oncogenic viruses demonstrating a strict tropism for the mammary gland provide special opportunities to study the susceptibility of this tissue to neoplasia. In rats, human adenovirus type 9 (Ad9) elicits mammary fibroadenomas that are similar to common breast tumors in women, as well as phyllodes-like tumors and mammary sarcomas. By constructing recombinant adenoviruses between Ad9 and Ad26 (a related nontumorigenic virus), it was shown that the Ad9 E4 region was absolutely required to produce these mammary tumors. This indicates that an adenovirus gene located outside the classic transforming region (E1) can significantly influence the in vivo oncogenicity of an adenovirus. Consistent with a direct role in mammary gland oncogenesis, the Ad9 E4 region also exhibited transforming properties in vitro. Therefore, the Ad9 E4 region is a viral oncogene specifically involved in mammary gland tumorigenesis.

Low levels of anti-viral activity, mainly interferon alpha/beta (IFN-alpha/beta), are regularly found in lymphoid tissues of BALB/c mice infected with the C3H strain of mammary tumor virus. At the time of tumor development, significant amounts of anti-viral activity were detected in homogenates of spleen and mammary tumors, but not in blood and normal mammary glands. This activity is pH2-resistant and neutralized by antibody to IFN/alpha-beta. The pathogenetic role of IFN in mammary carcinogenesis was investigated in 2 ways: (a) by treating virus-infected newborn mice with antibody to IFN-alpha/beta, and (b) by giving either the latter antibody or IFN-alpha/beta to virus-free animals transplanted with pre-neoplastic lesions. Mice were treated only for 2 months, starting either 1 week after birth or immediately after tumor transplant. In case (a), treatment with antibody to IFN-alpha/beta shortened the incubation period of mammary carcinomas and decreased the mean survival time. In case (b), anti-IFN antibody did not significantly affect the development of mammary tumors. However, exogenous IFN-alpha/beta markedly reduced both tumor incidence and mortality rate. These results indicate that endogenous IFN-alpha/beta plays a crucial role in the in vivo restriction of the early infectious phase of spontaneous carcinogenesis and that relatively high doses of IFN-alpha/beta may inhibit the progression of pre-neoplastic lesions.

We have characterized mammary oncogenesis induced after polyomavirus infection of adult female nude mice regarding histopathogenesis, viral replication and viral and cellular oncogene expression. A unique transient generalized epithelial hyperplasia was observed (starting at 2 weeks post infection), preceding the development of dysplasias (onset 6 weeks post infection) and multiple neoplasias (onset 6 weeks post infection) in all glands. The ductal epithelium was the target for neoplastic transformation, and the occurrence of numerous ductal dysplasias coincided with the appearance of frank tumors. Stromal abnormalities were also seen. Tumor growth was not dependent upon ovarian hormones, and new tumors continued to develop in ovariectomized mice. Viral replication, high although variable, preceded but did not correlate with oncogenesis. Most but not all tumors contained high levels of unintegrated viral DNA. Tumors produced very low levels of live virus. Viral gene expression was markedly increased in the tumors compared with the infected but morphologically normal glands. The expression of c-myc was moderately increased (fourfold); changes in c-int-2 and c-Ha-ras expression were slight and inconsistent, while expression of c-neu and c-int-1 was unchanged.

Retroviruses have been implicated as causative agents for a range of diseases including neoplasia, autoimmunity and immunosuppression. No two retroviruses carry the same complement of genes and for this reason it is not surprising that they induce a variety of different disease states. One common element in retroviral evolution has been the need to avoid immune recognition in order to persist within the host. A comparative approach, looking at various persistent retroviruses, has been used to pin-point the types of genetic adaptations adopted by retroviruses to remain hidden, often within the T cell compartment. Most of these retroviruses are T-cell-tropic and the diseases which they induce usually reflect the effect of the retrovirus on normal lymphocyte function.

Murine mammary tumors induced by the Murine Mammary Tumor Virus (MMTV) were chosen to study the expression of the NM23 gene during the metastatic process because of their viral etiology, different from that of the previously reported experimental tumor systems. NM23 mRNA levels are higher in non metastatic tumors than in metastatic ones. Moreover, the NM23 expression is higher in tumors induced by the C3H variant of the MMTV than in tumors induced by the RIII variant. These data are a further support to the hypothesis of a basic role of the NM23 gene in the down-regulation of tumor progression.

Female C3H/He mice carrying the mammary tumor virus (MTV) were monitored for mammary tumor incidence and latent periods while submitted to a daily subcutaneous injection with amphetamine (0,4 mg/kg/day). Results show that amphetamine caused an increase in incidence and a decrease in latency of tumors compared with placebo. There was also appreciated a correlation with the lethality of mice.

We found that the number of peripheral blood polymorphonuclear leukocytes (PMN) dramatically increased in both sarcoma 180 (S-180) and MM-46 mammary carcinoma (MM-46) ascites tumor-bearing mice, and mice required a remarkable resistance to Candida albicans infection via intravenous route. When the resistance was determined by number of cells of C. albicans in the kidney, a significant decrease in the number of fungal cells was observed in the kidneys of infected ascites tumor-bearing mice. An increase of active oxygen levels of PMN from ascites tumor-bearing mice was observed, suggesting that this factor is important in developing of resistance in ascites tumor-bearing mice. Additionally, a culture supernatant of tumor cells co-cultivated with bone-marrow cells in vitro increased the number of granulocytes and macrophages differentiated from the bone-marrow cells.

The uniformly lethal development of mammary tumors in polyomavirus-infected adult female nude mice was prevented by adoptive cell transfer of polyomavirus-immune splenocytes or peritoneal cells. Transferred immune cells also lowered the growth rate of emerging tumors. The induction of other relatively less frequent tumors of the skin and bone was decreased as well. Using in situ hybridization of whole-body sections as well as hybridization of nucleic acids from the mammary glands, we show for the first time that transferred immune cells, but not normal cells, virtually eliminated virus signal in the whole mouse and in the mammary glands. Since infected and tumorous mammary glands produce very little infectious virus, it appears that a major mechanism mediating the prevention of polyomavirus oncogenesis involves the immunological elimination of nonproductively and persistently infected cells.

The distribution of mouse mammary tumor virus (MMTV) antigens was studied by the immunoperoxidase method in the II-TES and I-TES mouse strains as well as their progenitors, CS and DBA/2 strains. In the II-TES, I-TES and CS strains, and BALB/c mice foster-nursed with these strains, MMTV antigens were found not only in epithelial cells of the mammary glands but also in those of other tissues including the seminal vesicle, vas deferens, epididymis, prostate, parotid, submandibular, lacrimal, sebaceous, and urethral glands. In DBA/2 and BALB/cfDBA/2 mice, however, the MMTV antigens were found only in the mammary glands. Electron microscopic examination showed MMTV particles in these organs. When we examined the presence of Mtv-1 and 2 proviruses, which are known to be responsible for MMTV expression, in the genomes of the II-TES, I-TES, CS and DBA/2 strains by Southern blotting, Mtv-2 was not found in any of the mice and Mtv-1 was found in the II-TES and DBA/2 mice but not in the I-TES and CS mice. Instead, four new endogenous MMTV loci, which have never previously been reported in laboratory mouse strains, were detected in the genomes of the II-TES, I-TES and CS strains. One (designated Mtv-42) was common in the three strains and the other three (designated Mtv-43, 44 and 45) were common in the II-TEX and I-TES strains or the II-TES and CS strains. These results thus suggest that new endogenous MMTV loci may be responsible for MMTV expression in a variety of tissues of these three strains.

RNA expressions of common integration site (int) genes and several oncogenes were investigated in mammary carcinomas spontaneously developed in different three strains of mice; DD/Tbr, NIH Swiss and BALB/c which harbor DD-MMTV derived from DD/Tbr mouse. Latter two strains of mice were designated NIH/Mtv+ and BALB/Mtv+, respectively. An increased expression of int-1 (wnt-1) and int-2 genes was observed in 56% (9/16) and 50% (8/16) of mammary carcinomas of DD/Tbr mice, respectively. Either int-1 or int-2 RNAs were expressed in 81% (13/16) of the carcinomas of DD/Tbr mice. IN NIH/Mtv+ mice, activation of int-1 and int-2 was observed in 41% (7/17) and 24% (4/17) of mammary carcinomas, respectively. Either int-1 or int-2 RNAs were expressed in 47% (8/17) of the carcinomas examined in this strain. In BALB/Mtv+ mice, on the other hand, either int-1 or int-2 gene were transcribed into RNAs at low frequency (33%: 3/9). These results suggest that the frequency of activation of int genes in mammary carcinomas induced by the same DD-MMTV in three strains of mice is genetically defined characteristics of these strains, and that the involvement of int-1 and int-2 genes in virus-induced mammary carcinogenesis may be influenced by genetic properties of animals. The activation of int-1 and int-2 genes did not clearly correlate with an increase in the expression of oncogenes examined; H-ras, K-ras, N-ras, myc, raf, fgr, fms, erB, mos, and src genes.

The frequency with which int-1 and int-2 are rearranged in mouse mammary tumors by mouse mammary tumor virus (MMTV)-induced insertional mutagenesis is a consequence of the host genetic background. In 75% of C3H mammary tumors, int-1 is rearranged by MMTV insertion, whereas only 30% of BALB/cfC3H tumors contain a virus-induced rearrangement of int-1. This difference is significant (P less than 0.005) and could not be accounted for by the potentially additive effect of the genetically transmitted Mtv-1-encoded virus in C3H mice. Similarly, MMTV-induced rearrangement of the int-2 gene in mammary tumors of the R111 mouse strain (59%) occurred at a significantly (P less than 0.025) higher frequency than in BALB/cfR111 (25%) mammary tumors. Moreover, in BALB/cfR111 mammary tumors, there is evidence that rearrangement of int-1 and int-2 does not occur independently (P less than 0.025). These results suggest that the long history of inbreeding for high tumor incidence of C3H and R111 mouse strains has selected for the fixation of host mutations which either complement the action of the particular int gene or affect the sensitivity of specific subpopulations of mammary epithelium to infection by particular strains of MMTV.

Transcripts related to the human carcinoembryonic antigen were found in mRNA isolated from both dimethylbenzanthracene-induced and mouse mammary tumor virus-induced mammary tumors. A cDNA library was prepared from a dimethylbenzanthracene-induced tumor, and a clone was isolated by hybridization with a human carcinoembryonic antigen cDNA probe. Its sequence, when compared to those of members of the human carcinoembryonic antigen gene family, was most homologous to the sequence of the human biliary glycoprotein (BGP) gene. Thus, this clone appears to encode a portion of the mouse biliary glycoprotein gene. Southern blot analysis of EcoRI-digested mouse cellular DNA with this probe detected four restriction fragments, all of which appear to be located on mouse chromosome 7, Northern blot analysis using the mouse probe demonstrated that related mRNA species were expressed in some normal adult mouse tissues.

During the culturing of a mouse mammary tumor cell line, MuMT73, maintained in vitro for more than a decade, we found morphological heterogeneity in its cells; some showed contact inhibition in their growth, some formed domes and some grew criss cross and piled up. In trying to clone the cell line to isolate cells showing contact inhibition or dome formation, we were able to establish six clonal cell lines. These six cell lines were categorically divided into three groups according to their phenotypical behavior, Groups A, B and C. Group A (clones 1, 5 and 7) cells had a property of contact inhibition. They induced no tumor when injected into the subcutaneous tissue of the back, nor even when injected into the mammary fat pads or under the kidney capsule of syngenic or nude mice, and therefore were thought to be non-malignant in nature. They were positively stained by anti-keratin antiserum and had mouse mammary tumor viruses (MMTVs). Group B (clone 6) cells grew in a crisscross pattern and piled up, and they induced tumors when injected into the subcutaneous tissue of the back of mice. Group C (clones 3 and 4) cells formed domes in their growth and induced some tumors in the mammary fat pads and under the kidney capsule of KSN nude mice. In Southern blots with MMTV-env probe, numerous exogenous MMTV proviruses were detected in these cell lines. The insertion patterns of these proviruses in cells of non-malignant clonal lines (Group A) resembled those of malignant lines (Group B), except one band (about 26 Kb), but were considerably different from those of intermediate lines (Group C). On the other hand, no difference was detected in Southern blots with int-1 or int-2 probes among the non-malignant, intermediate and malignant clonal cell lines.

Three chemically-induced precancerous mammary hyperplasias, independently isolated in BALB/c mice, all contained mouse mammary tumor virus (MMTV) proviral DNA integrated into a common region in chromosomal DNA, designated Int-5 (Formerly Int-H, Gray et al., 1986). This site was cloned from a hyperplastic outgrowth (D2) into lambda phage. A 1.7 Kb Hind III DNA fragment, which flanks the 5' end of the MMTV insert, was generated from the cloned Int-5 region. This fragment was used as probe (IH-2) to localize Int-5 to mouse chromosome 9. The IH-2 sequence was highly conserved in DNA of several mammalian species including man, in three other widely divergent vertebrate phyla, and in C. elegans. The Int-5 region, containing 5.6 Kb 5' and 12.8 Kb 3' to the MMTV integration site in D2, was cloned in EMBL-3 from a BALB/c genomic DNA library. cDNA complementary to poly A+ lactating mammary gland RNA, annealed with Sst-1 fragments spanning most of the BALB/c Int-5 clone. The highest level of Int-5 specific poly A+ mRNA was detected in D2 tumor. Lactating mammary gland and D2 hyperplastic alveolar nodule contained 5-fold less Int-5 RNA while liver contained 8-fold less Int-5 RNA. Int-5 cDNA (IH-3) annealed with two RNA species of approximately 3.3 and 4.0 Kb. These data are consistent with the hypothesis that Int-5 contains an oncogene, different from any other previously described, involved in early events in some models of chemical carcinogenesis.