initiation-metastatic-breast-carcinoma-targeting-ductal-epithelium

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Wistar Institute

Environmental stress and endocrine control can affect pathogenesis of sexually transmitted diseases such as trichomoniasis. Acute Tritrichomonas foetus infection was compared in female BALB/c mice to infections in mice treated with high doses of estradiol or housed in constant bright illumination (stressed). In untreated mice, T. foetus readily colonized the reproductive tract, causing minimal epithelial damage and inflammation. Several fold increases of IFN-gamma, TNF-alpha, MCP-1, and IL-6 cytokines were detected after estradiol-treatment of mice, resulting in greatly enhanced inflammation and tissue damage throughout the reproductive tract. Interestingly, estradiol-treatment of mice resulted in reduced T. foetus colonization compared to untreated mice. Infection in stressed mice resulted in increased tissue damage, inflammation, and inflammatory cytokine expression, although parasite colonization within the reproductive tract was similar to that in untreated mice. These results indicate that either estradiol-treatment or stress result in pathogenesis often observed during severe disease. Alternatively, infection in non-treated mice results in chronic colonization, with little inflammation or pathology.

Tritrichomonas foetus: pathogenesis of acute infection in normal, estradiol-treated, and stressed mice.

Tritrichomonas foetus is the cause of trichomoniasis in cattle. Severe infection is often associated with heavy neutrophil and macrophage accumulation, although it is not known how this response protects during early parasite colonization. The goal of this study was to examine the effects of an early host response upon initial T. foetus colonization within the murine reproductive tract. Mice depleted of neutrophils before T. foetus infection had a significantly higher parasite burden within the reproductive tract compared with mock-depleted control mice. Additionally, gp91(phox-/-)/ iNOS(-/-), and iNOS(-/-) mice had substantially larger parasite burdens than C57BL/6 control mice, whereas gp91l(Phox-/-) mice had similar parasite burden to C57BL/6 control mice. Interestingly, phorbol 12-myristate 13-acetate-stimulated neutrophils and macrophages isolated from all groups of mice were unable to kill T. foetus in vitro. However, macrophages isolated from gp91l(phox-/-) and C57BL/6 mice stimulated with interferon-gamma and lipopolysaccharide were able to kill T. foetus in vitro, whereas macrophages isolated from gp91(phox(-/-)/ iNOS(-/-) and iNOS(-/-) mice were unable to kill T. foetus, suggesting the ability of macrophages to produce reactive nitrogen species but not reactive oxygen species (ROS) is critical for parasite killing during early infection in vivo and in vitro. Additionally, neutrophils seem to control early dissemination of T. foetus throughout the reproductive tract, although production of ROS is not critical for this process.

Neutrophils and inducible nitric-oxide synthase are critical for early resistance to the establishment of Tritrichomonas foetus infection.

Influenza virus infections increase susceptibility to secondary bacterial infections, such as pneumococcal pneumonia, resulting in increased morbidity and mortality. Influenza-induced tissue damage is hypothesized to increase susceptibility to Streptococcus pneumoniae infection by increasing adherence to the respiratory epithelium. Using a mouse model of influenza infection followed by S. pneumoniae infection, we found that an influenza infection does not increase the number of pneumococci initially present within the trachea, but does inhibit pneumococcal clearance by 2 hours after infection. To determine whether influenza damage increases pneumococcal adherence, we developed a novel murine tracheal explant system to determine influenza-induced tissue damage and subsequent pneumococcal adherence. Murine tracheas were kept viable ex vivo as shown by microscopic examination of ciliary beating and cellular morphology using continuous media flow for up to 8 days. Tracheas were infected with influenza virus for 0.5-5 days ex vivo, and influenza-induced tissue damage and the early stages of repair to the epithelium were assessed histologically. A prior influenza infection did not increase pneumococcal adherence, even when the basement membrane was maximally denuded or during the repopulation of the basement membrane with undifferentiated epithelial cells. We measured mucociliary clearance in vivo and found it was decreased in influenza-infected mice. Together, our results indicate that exposure of the tracheal basement membrane contributes minimally to pneumococcal adherence. Instead, an influenza infection results in decreased tracheal mucociliary velocity and initial clearance of pneumococci, leading to an increased pneumococcal burden as early as 2 hours after pneumococcal infection.

Influenza virus infection decreases tracheal mucociliary velocity and clearance of Streptococcus pneumoniae.

The biological significance of protective CD8 T-cell-mediated responses against non-traditional alternative reading frame epitopes remains relatively unknown. Cytolytic CD8 T cells (CTL) specific for a non-traditional cryptic MHC class I epitope, SYNTGRFPPL, are critically involved in the protection of mice during infection with the LP-BM5 murine retrovirus. The goal of this study was to determine the functional properties of the protective SYNTGRFPPL-specific CTL during LP-BM5 infection of susceptible BALB/c CD8(-/-) mice. Direct infection experiments and adoptive transfer of CD8 T cells derived from perforin (pfp)(-/-), IFN gamma(-/-), FasL(-/-) and, as a positive control, wild-type BALB/c mice, were utilized to assess the effector mechanisms responsible for protection. Our results indicate that SYNTGRFPPL-specific effector CTL preferentially utilize perforin-mediated cytolysis to provide protection against LP-BM5-induced pathogenesis, whereas CTL production of IFN gamma is not required. Our results also suggest a minimal contribution of FasL/Fas-mediated lysis during the effector response. Collectively, these results provide insight into effector mechanisms utilized by protective CTL directed against non-traditional cryptic epitopes during disease protection.

Defining the mechanism(s) of protection by cytolytic CD8 T cells against a cryptic epitope derived from a retroviral alternative reading frame.

T cell adoptive transfer strategies that have produced clinical remissions against specific tumors have so far produced disappointing results against ovarian cancer. Recent evidence suggests that adoptively transferred CD4(+) T cells can trigger endogenous immune responses in particular patients with ovarian cancer through unknown mechanisms. However, conflicting reports suggest that ovarian cancer-infiltrating CD4(+) T cells are associated with negative outcomes. In this study, we elucidate the phenotypic attributes that enable polyclonal CD4(+) T cells briefly primed against tumor Ags to induce therapeutically relevant endogenous antitumor immune responses. Our results unveil a therapeutic mechanism whereby tumor-primed CD4(+) T cells transferred into ovarian cancer-bearing mice secrete high levels of CCL5, which recruits endogenous CCR5(+) dendritic cells to tumor locations and activate them through CD40-CD40L interactions. These newly matured dendritic cells are then able to prime tumor-specific endogenous CD8(+) T cells, which mediate long-term protection. Correspondingly, administration of tumor-primed CD4(+) T cells significantly delayed progression of MHC class II(-) ovarian cancers, similarly to CD8(+) T cells only, and directly activated wild-type but not CD40-deficient dendritic cells recruited to the tumor microenvironment. Our results unveil a CCL5- and CD40L-dependent mechanism of transferring immunity from exogenously activated CD4(+) T cells to tumor-exposed host cells, resulting in sustained antitumor effects. Our data provide a mechanistic rationale for incorporating tumor-reactive CD4(+) T cells in adoptive cell transfer immunotherapies against ovarian cancer and underscore the importance of optimizing immunotherapeutic strategies for the specific microenvironment of individual tumors.

CD4+ T cells elicit host immune responses to MHC class II-negative ovarian cancer through CCL5 secretion and CD40-mediated licensing of dendritic cells.

CD277, a member of the butyrophilin subfamily 3 (BTN3), shares significant sequence similarities and predicted common structural features with inhibitory B7-H4 and other members of the B7 superfamily. Here we report that CD277 is consistently expressed in stromal, as well as tumor cells in the microenvironment of human advanced ovarian carcinoma specimens, both of primary and metastatic origin. MHC-II+ myeloid antigenpresenting leukocytes (dendritic cells and macrophages) express significantly higher levels of surface CD277, compared to other tumor-infiltrating leukocyte subsets, and this expression is significantly up-regulated by multiple common tumor microenvironmental signals, including VEGF and CCL3. Most importantly, engagement of CD277 on the surface of TCR-stimulated T cells inhibits their otherwise robust expansion and production of Th1 cytokines by preventing the up-regulation of cFLIP. Our results point to a role for CD277 up-regulated by microenvironmental signals in the acquisition of a regulatory phenotype by tumor-associated myeloid cells. Consequently, CD277, and likely other butyrophilins and butyrophilin-like molecules, emerge as regular players in the orchestration of immunosuppressive networks in ovarian cancer, and therefore new targets for interventions to overcome immune evasion and boost anti-tumor immunity in cancer patients.

CD277 is a negative co-stimulatory molecule universally expressed by ovarian cancer microenvironmental cells.

The immunodominant cryptic epitope SYNTGRFPPL, encoded within open reading frame 2 of the LP-BM5 retroviral gag gene, is critical for protection against retroviral-induced pathogenesis. The goal of this study was to dissect the memory response against this unique immunodominant cryptic epitope. Unlike the protective acute effector population of SYNTGRFPPL-specific CD8 T cells, long-lived SYNTGRFPPL-specific CD8 T cells lacked the ability to protect susceptible mice infected with LP-BM5 retrovirus. Compared to memory CD8 T cells against a conventional epitope with similar MHC-I specificity, primed and restimulated using similar conditions, long-lived SYNTGRFPPL-specific CD8 T cells were impaired in their ability to recall against antigen, with reduced cytolytic capabilities and cytokine production. Since similar priming and restimulation regimes were utilized to generate each effector CD8 T cell population, this study has potentially broad implications with regard to the selection criteria of potent, highly conserved cryptic epitopes for use in epitope-based vaccines.

Impaired memory CD8 T cell responses against an immunodominant retroviral cryptic epitope.

Modulating the activity of miRNAs provides opportunities for novel cancer interventions. However, low bioavailability and poor cellular uptake are major challenges for delivering miRNA mimetics specifically to tumor cells. Here, we took advantage of the spontaneous enhanced endocytic activity of ovarian cancer-associated dendritic cells (DC) to selectively supplement the immunostimulatory miRNA miR-155. In vivo processing of nanoparticles carrying oligonucleotide duplexes mimicking the bulged structure of endogenous pre-miRNA (but not siRNA-like oligonucleotides) dramatically augmented miR-155 activity without saturating the RNA-induced silencing complex. Endogenous processing of synthetic miR-155 favored Ago2 and, to a lesser extent, Ago4 loading, resulting in genome-wide transcriptional changes that included silencing of multiple immunosuppressive mediators. Correspondingly, tumor-infiltrating DCs were transformed from immunosuppressive to highly immunostimulatory cells capable of triggering potent antitumor responses that abrogated the progression of established ovarian cancers. Our results show both the feasibility and therapeutic potential of supplementing/replenishing miRNAs in vivo using nonviral approaches to boost protective immunity against lethal tumors. Thus, we provide a platform, an optimized design, and a mechanistic rationale for the clinical testing of nonviral miRNA mimetics.

Reprogramming tumor-associated dendritic cells in vivo using miRNA mimetics triggers protective immunity against ovarian cancer.

We characterized the initiation and evolution of the immune response against a new inducible p53-dependent model of aggressive ovarian carcinoma that recapitulates the leukocyte infiltrates and cytokine milieu of advanced human tumors. Unlike other models that initiate tumors before the development of a mature immune system, we detect measurable anti-tumor immunity from very early stages, which is driven by infiltrating dendritic cells (DCs) and prevents steady tumor growth for prolonged periods. Coinciding with a phenotypic switch in expanding DC infiltrates, tumors aggressively progress to terminal disease in a comparatively short time. Notably, tumor cells remain immunogenic at advanced stages, but anti-tumor T cells become less responsive, whereas their enduring activity is abrogated by different microenvironmental immunosuppressive DCs. Correspondingly, depleting DCs early in the disease course accelerates tumor expansion, but DC depletion at advanced stages significantly delays aggressive malignant progression. Our results indicate that phenotypically divergent DCs drive both immunosurveillance and accelerated malignant growth. We provide experimental support for the cancer immunoediting hypothesis, but we also show that aggressive cancer progression after a comparatively long latency period is primarily driven by the mobilization of immunosuppressive microenvironmental leukocytes, rather than loss of tumor immunogenicity.

Ovarian cancer progression is controlled by phenotypic changes in dendritic cells.

In the clinical setting, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene enhance the inflammatory response in the lung to Pseudomonas aeruginosa (P. aeruginosa) infection. However, studies on human airway epithelial cells in vitro have produced conflicting results regarding the effect of mutations in CFTR on the inflammatory response to P. aeruginosa, and there are no comprehensive studies evaluating the effect of P. aeruginosa on the inflammatory response in airway epithelial cells with the ΔF508/ΔF508 genotype and their matched CF cell line rescued with wild-type (wt)-CFTR. CFBE41o- cells (ΔF508/ΔF508) and CFBE41o- cells complemented with wt-CFTR (CFBE-wt-CFTR) have been used extensively as an experimental model to study CF. Thus the goal of this study was to examine the effect of P. aeruginosa on gene expression and cytokine/chemokine production in this pair of cells. P. aeruginosa elicited a more robust increase in cytokine and chemokine expression (e.g., IL-8, CXCL1, CXCL2 and TNF-α) in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells. These results demonstrate that CFBE41o- cells complemented with wt-CFTR mount a more robust inflammatory response to P. aeruginosa than CFBE41o-ΔF508/ΔF508-CFTR cells. Taken together with other published studies, our data demonstrate that there is no compelling evidence to support the view that mutations in CFTR induce a hyperinflammatory response in human airway epithelial cells in vivo. Although the lungs of patients with CF have abundant levels of proinflammatory cytokines and chemokines, because the lung is populated by immune cells and epithelial cells there is no way to know, a priori, whether airway epithelial cells in the CF lung in vivo are hyperinflammatory in response to P. aeruginosa compared with non-CF lung epithelial cells. Thus studies on human airway epithelial cell lines and primary cells in vitro that propose to examine the effect of mutations in CFTR on the inflammatory response to P. aeruginosa have uncertain clinical significance with regard to CF.

Does the F508-CFTR mutation induce a proinflammatory response in human airway epithelial cells?

The immune surveillance hypothesis proposed over 50 years ago that many precancerous lesions are eliminated without a histological trace due to immunological pressure. Since then, it has become apparent that both the tumor and the anti-cancer immune response evolve over a long period to allow the eventual escape of nascent precancerous lesions into full-blown tumors. Although primarily focusing on loss of antigenicity, the immunoediting hypothesis has gradually evolved to appreciate the role of active immunosuppression in tumor progression, where myeloid leukocytes are increasingly recognized as the major driving force. This review highlights recent studies implicating how myeloid cells with antigen-presenting capabilities are co-opted by tumors to promote malignant progression. Because at least some advanced tumors remain significantly immunogenic, these new studies add a tweak to the immunoediting hypothesis as well as a rationale to block immunosuppressive mechanisms as a first-line intervention in cancer patients.

Anti-tumor immunity: myeloid leukocytes control the immune landscape.

Reversing tumor-associated immunosuppression seems necessary to stimulate effective therapeutic immunity against lethal epithelial tumors. Here, we show this goal can be addressed using cps, an avirulent, nonreplicating uracil auxotroph strain of the parasite Toxoplasma gondii (T. gondii), which preferentially invades immunosuppressive CD11c(+) antigen-presenting cells in the ovarian carcinoma microenvironment. Tumor-associated CD11c(+) cells invaded by cps were converted to immunostimulatory phenotypes, which expressed increased levels of the T-cell receptor costimulatory molecules CD80 and CD86. In response to cps treatment of the immunosuppressive ovarian tumor environment, CD11c(+) cells regained the ability to efficiently cross-present antigen and prime CD8(+) T-cell responses. Correspondingly, cps treatment markedly increased tumor antigen-specific responses by CD8(+) T cells. Adoptive transfer experiments showed that these antitumor T-cell responses were effective in suppressing solid tumor development. Indeed, intraperitoneal cps treatment triggered rejection of established ID8-VegfA tumors, an aggressive xenograft model of ovarian carcinoma, also conferring a survival benefit in a related aggressive model (ID8-Defb29/Vegf-A). The therapeutic benefit of cps treatment relied on expression of IL-12, but it was unexpectedly independent of MyD88 signaling as well as immune experience with T. gondii. Taken together, our results establish that cps preferentially invades tumor-associated antigen-presenting cells and restores their ability to trigger potent antitumor CD8(+) T-cell responses. Immunochemotherapeutic applications of cps might be broadly useful to reawaken natural immunity in the highly immunosuppressive microenvironment of most solid tumors. Cancer Res; 73(13); 3842-51. Â©2013 AACR.

Avirulent Toxoplasma gondii Generates Therapeutic Antitumor Immunity by Reversing Immunosuppression in the Ovarian Cancer Microenvironment.

Inversion immunosuppression associ&eacute;es &agrave; la tumeur semble n&eacute;cessaire pour stimuler l&#39;immunit&eacute; th&eacute;rapeutique efficace contre les tumeurs &eacute;pith&eacute;liales l&eacute;tales. Nous montrons ici, que cet objectif peut &ecirc;tre adress&eacute; &agrave; l&#39;aide de la cps, une souche auxotrophe uracile avirulentes, il le parasite Toxoplasma gondii (T. gondii), qui envahit pr&eacute;f&eacute;rentiellement IMMUNOSUPPRESSEURS CD11c(+) des cellules pr&eacute;sentatrices d&#39;antig&egrave;ne dans le microenvironnement de cancer ovarien. Associ&eacute;es aux tumeurs CD11c(+) cellules envahies par la cps ont &eacute;t&eacute; converties aux ph&eacute;notypes immunostimulantes, qui a exprim&eacute; des niveaux accrus des mol&eacute;cules de costimulation de r&eacute;cepteur de T-cellule CD80 et CD86. R&eacute;ponse au traitement de la cps de l&#39;environnement de la tumeur ovarienne immunosuppresseur, CD11c(+) cellules retrouv&eacute; leur pouvoir efficacement Croix-aujourd &#39; hui l&#39;antig&egrave;ne et amorcer les r&eacute;ponses de cellules T CD8. Parall&egrave;lement, traitement de la cps a nettement r&eacute;ponses sp&eacute;cifiques de l&#39;antig&egrave;ne tumoral par les cellules T CD8. Des exp&eacute;riences de transfert adoptif a d&eacute;montr&eacute; que ces r&eacute;ponses de cellules T anti-tumorale efficaces pour supprimer le d&eacute;veloppement de tumeurs solides. En effet, traitement intrap&eacute;riton&eacute;al cps d&eacute;clench&eacute;e rejet des tumeurs &eacute;tablies de ID8-VegfA, un mod&egrave;le de x&eacute;nogreffe agressive de cancer de l&#39;ovaire, conf&eacute;rant un avantage de survie dans un mod&egrave;le agressif associ&eacute; (ID8-Defb29/Vegf-A), aussi. Les bienfaits th&eacute;rapeutiques du traitement de la cps s&#39;est appuy&eacute; sur l&#39;expression d&#39;IL-12, mais elle &eacute;tait inattendue ind&eacute;pendante de MyD88 signalisation ainsi que l&#39;exp&eacute;rience immunitaire avec T. gondii. Pris ensemble, nos r&eacute;sultats &eacute;tablissent que cps pr&eacute;f&eacute;rentiellement envahit les cellules pr&eacute;sentatrices d&#39;antig&egrave;ne associ&eacute; aux tumeurs et restaure leur capacit&eacute; &agrave; d&eacute;clencher des r&eacute;ponses de cellules T CD8 antitumorales puissantes. Immunochemotherapeutic applications des cps peuvent &ecirc;tre largement utiles pour r&eacute;veiller une immunit&eacute; naturelle dans le microenvironnement hautement immunosuppresseur des tumeurs plus solides. Cancer Res ; 73 ; 3842-51. &copy; 2013 AACR.

Avirulente Toxoplasma gondii g&eacute;n&egrave;re l&#39;immunit&eacute; antitumorale th&eacute;rapeutiques par inversion de l&#39;Immunosuppression dans le microenvironnement de Cancer ovarien.

Article	Year
Tritrichomonas foetus: pathogenesis of acute infection in normal, estradiol-treated, and stressed mice. Experimental parasitology\| PubMed ID: 17014850	2007
Neutrophils and inducible nitric-oxide synthase are critical for early resistance to the establishment of Tritrichomonas foetus infection. The Journal of parasitology\| PubMed ID: 17626348	2007
Influenza virus infection decreases tracheal mucociliary velocity and clearance of Streptococcus pneumoniae. American journal of respiratory cell and molecular biology\| PubMed ID: 19520922	2010
Defining the mechanism(s) of protection by cytolytic CD8 T cells against a cryptic epitope derived from a retroviral alternative reading frame. Virology\| PubMed ID: 19539970	2009
CD4+ T cells elicit host immune responses to MHC class II-negative ovarian cancer through CCL5 secretion and CD40-mediated licensing of dendritic cells. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 20400704	2010
CD277 is a negative co-stimulatory molecule universally expressed by ovarian cancer microenvironmental cells. Oncotarget\| PubMed ID: 21113407	2010
Impaired memory CD8 T cell responses against an immunodominant retroviral cryptic epitope. Virology\| PubMed ID: 21295815	2011
Reprogramming tumor-associated dendritic cells in vivo using miRNA mimetics triggers protective immunity against ovarian cancer. Cancer research\| PubMed ID: 22307839	2012
Ovarian cancer progression is controlled by phenotypic changes in dendritic cells. The Journal of experimental medicine\| PubMed ID: 22351930	2012
Does the F508-CFTR mutation induce a proinflammatory response in human airway epithelial cells? American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 22821996	2012
Anti-tumor immunity: myeloid leukocytes control the immune landscape. Cellular immunology\| PubMed ID: 23121972	2012
Avirulent Toxoplasma gondii Generates Therapeutic Antitumor Immunity by Reversing Immunosuppression in the Ovarian Cancer Microenvironment. Cancer research\| PubMed ID: 23704211	2013

Melanie R. Rutkowski

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