Inhibition of Macrophage-Specific CHIT1 as an Approach to Treat Airway Remodeling in Severe Asthma.

Chitotriosidase (CHIT1) is an enzyme produced by macrophages that regulates their differentiation and polarization. Lung macrophages have been implicated in asthma development; therefore, we asked whether pharmacological inhibition of macrophage-specific CHIT1 would have beneficial effects in asthma, as it has been shown previously in other lung disorders. CHIT1 expression was evaluated in the lung tissues of deceased individuals with severe, uncontrolled, steroid-naïve asthma. OATD-01, a chitinase inhibitor, was tested in a 7-week-long house dust mite (HDM) murine model of chronic asthma characterized by accumulation of CHIT1-expressing macrophages. CHIT1 is a dominant chitinase activated in fibrotic areas of the lungs of individuals with fatal asthma. OATD-01 given in a therapeutic treatment regimen inhibited both inflammatory and airway remodeling features of asthma in the HDM model. These changes were accompanied by a significant and dose-dependent decrease in chitinolytic activity in BAL fluid and plasma, confirming in vivo target engagement. Both IL-13 expression and TGFß1 levels in BAL fluid were decreased and a significant reduction in subepithelial airway fibrosis and airway wall thickness was observed. These results suggest that pharmacological chitinase inhibition offers protection against the development of fibrotic airway remodeling in severe asthma.

Arginase 1/2 Inhibitor OATD-02: From Discovery to First-in-man Setup in Cancer Immunotherapy.

Pharmacologic inhibition of the controlling immunity pathway enzymes arginases 1 and 2 (ARG1 and ARG2) is a promising strategy for cancer immunotherapy. Here, we report the discovery and development of OATD-02, an orally bioavailable, potent arginases inhibitor. The unique pharmacologic properties of OATD-02 are evidenced by targeting intracellular ARG1 and ARG2, as well as long drug-target residence time, moderate to high volume of distribution, and low clearance, which may jointly provide a weapon against arginase-related tumor immunosuppression and ARG2-dependent tumor cell growth. OATD-02 monotherapy had an antitumor effect in multiple tumor models and enhanced an efficacy of the other immunomodulators. Completed nonclinical studies and human pharmacokinetic predictions indicate a feasible therapeutic window and allow for proposing a dose range for the first-in-human clinical study in patients with cancer. SIGNIFICANCE: We have developed an orally available, small-molecule intracellular arginase 1 and 2 inhibitor as a potential enhancer in cancer immunotherapy. Because of its favorable pharmacologic properties shown in nonclinical studies, OATD-02 abolishes tumor immunosuppression induced by both arginases, making it a promising drug candidate entering clinical trials.

The Role of Chitinases in Chronic Airway Inflammation Associated with Tobacco Smoke Exposure.

Chitinases and chitinase-like proteins are thought to play a role in innate inflammatory responses. Our study aimed to assess whether chitinase concentration and activity in induced sputum (IS) of patients exposed to tobacco smoke are related to the level of airway inflammation including the level and activity of chitinases and chitinase-like proteins. The study included 22 patients with chronic obstructive pulmonary disease (COPD), 12 non-COPD smokers, and nine nonsmoking subjects. Sputum CHIT1 and YKL-40 levels and chitinolytic activity were compared with sputum IL-6, IL-8, IL-18, and MMP-9 levels. A hierarchical cluster analysis was also performed. Sputum YKL-40 was higher in COPD patients than in the control groups. Sputum CHIT1 and YKL-40 levels correlated with IS inflammatory cell count as well as with MMP-9 and IL-8 levels. Two main clusters were revealed: Cluster 1 had lower chitinase levels and activity, lower IS macrophage and neutrophil count, and lower IS IL-8, IL-18, and MMP-9 than Cluster 2. Comparison of COPD patients from both clusters revealed significant differences in the IS inflammatory profile despite comparable clinical and functional data. Our findings seem to confirm the involvement of chitinases in smoking-associated chronic airway inflammation and show that airway chitinases may be a potential novel marker in COPD phenotyping.

Pharmacological Inhibition of Chitotriosidase (CHIT1) as a Novel Therapeutic Approach for Sarcoidosis.

Introduction: Sarcoidosis is a systemic disease of unknown etiology characterized by granuloma formation in the affected tissues. The pathologically activated macrophages are causatively implicated in disease pathogenesis and play important role in granuloma formation. Chitotriosidase (CHIT1), macrophage-derived protein, is upregulated in sarcoidosis and its levels correlate with disease severity implicating CHIT1 in pathology. Methods: CHIT1 was evaluated in serum and bronchial mucosa and mediastinal lymph nodes specimens from sarcoidosis patients. The therapeutic efficacy of OATD-01 was assessed ex vivo on human bronchoalveolar lavage fluid (BALF) macrophages and in vivo in the murine models of granulomatous inflammation. Results: CHIT1 activity was significantly upregulated in serum from sarcoidosis patients. CHIT1 expression was restricted to granulomas and localized in macrophages. Ex vivo OATD-01 inhibited pro-inflammatory mediators' production (CCL4, IL-15) by lung macrophages. In the acute model of granulomatous inflammation in mice, OATD-01 showed anti-inflammatory effects reducing the percentage of neutrophils and CCL4 concentration in BALF. In the chronic model, inhibition of CHIT1 led to a decrease in the number of organized lung granulomas and the expression of sarcoidosis-associated genes. Conclusion: In summary, CHIT1 activity was increased in sarcoidosis patients and OATD-01, a first-in-class CHIT1 inhibitor, demonstrated efficacy in murine models of granulomatous inflammation providing a proof-of-concept for its clinical evaluation in sarcoidosis.

Inhibition of CHIT1 as a novel therapeutic approach in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and eventually fatal lung disease with a complex etiology. Approved drugs, nintedanib and pirfenidone, modify disease progression, but IPF remains incurable and there is an urgent need for new therapies. We identified chitotriosidase (CHIT1) as new driver of fibrosis in IPF and a novel therapeutic target. We demonstrate that CHIT1 activity and expression are significantly increased in serum (3-fold) and induced sputum (4-fold) from IPF patients. In the lungs CHIT1 is expressed in a distinct subpopulation of profibrotic, disease-specific macrophages, which are only present in patients with ILDs and CHIT1 is one of the defining markers of this fibrosis-associated gene cluster. To define CHIT1 role in fibrosis, we used the therapeutic protocol of the bleomycin-induced pulmonary fibrosis mouse model. We demonstrate that in the context of chitinase induction and the macrophage-specific expression of CHIT1, this model recapitulates lung fibrosis in ILDs. Genetic inactivation of Chit1 attenuated bleomycin-induced fibrosis (decreasing the Ashcroft scoring by 28%) and decreased expression of profibrotic factors in lung tissues. Pharmacological inhibition of chitinases by OATD-01 reduced fibrosis and soluble collagen concentration. OATD-01 exhibited anti-fibrotic activity comparable to pirfenidone resulting in the reduction of the Ashcroft score by 32% and 31%, respectively. These studies provide a preclinical proof-of-concept for the antifibrotic effects of OATD-01 and establish CHIT1 as a potential new therapeutic target for IPF.

Discovery of OATD-01, a First-in-Class Chitinase Inhibitor as Potential New Therapeutics for Idiopathic Pulmonary Fibrosis.

Chitotriosidase (CHIT1) and acidic mammalian chitinase (AMCase) are the enzymatically active chitinases that have been implicated in the pathology of chronic lung diseases such as asthma and interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF) and sarcoidosis. The clinical and preclinical data suggest that pharmacological inhibition of CHIT1 might represent a novel therapeutic approach in IPF. Structural modification of an advanced lead molecule 3 led to the identification of compound 9 (OATD-01), a highly active CHIT1 inhibitor with both an excellent PK profile in multiple species and selectivity against a panel of other off-targets. OATD-01 given orally once daily in a range of doses between 30 and 100 mg/kg showed significant antifibrotic efficacy in an animal model of bleomycin-induced pulmonary fibrosis. OATD-01 is the first-in-class CHIT1 inhibitor, currently completed phase 1b of clinical trials, to be a potential treatment for IPF.

Benzoxazepine-Derived Selective, Orally Bioavailable Inhibitor of Human Acidic Mammalian Chitinase.

Human acidic mammalian chitinase (hAMCase) is one of two true chitinases in humans, the function of which remains elusive. In addition to the defense against highly antigenic chitin and chitin-containing pathogens in the gastric and intestinal contents, AMCase has been implicated in asthma, allergic inflammation, and ocular pathologies. Potent and selective small-molecule inhibitors of this enzyme have not been identified to date. Here we describe structural modifications of compound OAT-177, a previously developed inhibitor of mouse AMCase, leading to OAT-1441, which displays high activity and selectivity toward hAMCase. Significantly reduced off-target activity toward the human ether-à-go-go-related gene (hERG) and a good pharmacokinetic profile make OAT-1441 a potential candidate for further preclinical development as well as a useful tool compound to study the physiological role of hAMCase.

Discovery and Pharmacokinetics of Sulfamides and Guanidines as Potent Human Arginase 1 Inhibitors.

We designed and synthesized a series of arginase inhibitors as derivatives of the well-known 2-(S)-amino-6-boronohexanoic acid (ABH) with basic and neutral side chains in the α-position relative to the amino acid group. In an effort to improve the pharmacokinetic profile of literature examples and retain potent enzymatic activity, sulfamido moieties were introduced to generate hydrogen bond interaction with the aspartic acid residue in the arginase active site. The compounds with basic guanidine-containing side chains were even more potent arginase inhibitors. Both groups of compounds, as designed, demonstrated low clearance in their pharmacokinetic profile. The most active inhibitor 15aa showed high nanomolar potency with IC50 = 32 nM toward human arginase 1 and demonstrated low clearance (4.2 mL/min/kg), long t 1/2, and moderate volume of distribution in rat pharmacokinetic studies.

Development of Dual Chitinase Inhibitors as Potential New Treatment for Respiratory System Diseases.

Acidic mammalian chitinase (AMCase) and chitotriosidase-1 (CHIT1) are two enzymatically active proteins produced by mammals capable of cleaving the glycosidic bond in chitin. Based on the clinical findings and animal model studies, involvement of chitinases has been suggested in several respiratory system diseases including asthma, COPD, and idiopathic pulmonary fibrosis. Exploration of structure-activity relationships within the series of 1-(3-amino-1H-1,2,4-triazol-5-yl)-piperidin-4-amines, which was earlier identified as a scaffold of potent AMCase inhibitors, led us to discover highly active dual (i.e., AMCase and CHIT1) inhibitors with very good pharmacokinetic properties. Among them, compound 30 was shown to reduce the total number of cells in bronchoalveolar lavage fluid of mice challenged with house dust mite extract after oral administration (50 mg/kg, qd). In addition, affinity toward the hERG potassium channel of compound 30 was significantly reduced when compared to the earlier reported chitinase inhibitors.

Targeting peroxiredoxin 1 impairs growth of breast cancer cells and potently sensitises these cells to prooxidant agents.

BACKGROUND: Our previous work has shown peroxiredoxin-1 (PRDX1), one of major antioxidant enzymes, to be a biomarker in human breast cancer. Hereby, we further investigate the role of PRDX1, compared to its close homolog PRDX2, in mammary malignant cells. METHODS: CRISPR/Cas9- or RNAi-based methods were used for genetic targeting PRDX1/2. Cell growth was assessed by crystal violet, EdU incorporation or colony formation assays. In vivo growth was assessed by a xenotransplantation model. Adenanthin was used to inhibit the thioredoxin-dependent antioxidant defense system. The prooxidant agents used were hydrogen peroxide, glucose oxidase and sodium L-ascorbate. A PY1 probe or HyPer-3 biosensor were used to detect hydrogen peroxide content in samples. RESULTS: PRDX1 downregulation significantly impaired the growth rate of MCF-7 and ZR-75-1 breast cancer cells. Likewise, xenotransplanted PRDX1-deficient MCF-7 cells presented a retarded tumour growth. Furthermore, genetic targeting of PRDX1 or adenanthin, but not PRDX2, potently sensitised all six cancer cell lines studied, but not the non-cancerous cells, to glucose oxidase and ascorbate. CONCLUSIONS: Our study pinpoints the dominant role for PRDX1 in management of exogeneous oxidative stress by breast cancer cells and substantiates further exploration of PRDX1 as a target in this disease, especially when combined with prooxidant agents.

FOXO1 promotes resistance of non-Hodgkin lymphomas to anti-CD20-based therapy.

Diminished overall survival rate of non-Hodgkin lymphoma (NHL) patients treated with a combination regimen of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) has been recently linked to recurrent somatic mutations activating FOXO1. Despite of the clinical relevance of this finding, the molecular mechanism driving resistance to R-CHOP therapy remains largely unknown. Herein, we investigated the potential role of FOXO1 in the therapeutic efficacy of rituximab, the only targeted therapy included in the R-CHOP regimen. We found CD20 transcription is negatively regulated by FOXO1 in NHL cell lines and in human lymphoma specimens carrying activating mutations of FOXO1. Furthermore, both the expression of exogenous mutants of FOXO1 and the inhibition of AKT led to FOXO1 activation in lymphoma cells, increased binding to MS4A1 promoter and diminished CD20 expression levels. In contrast, a disruption of FOXO1 with CRISPR/Cas9 genome-editing (sgFOXO1) resulted in CD20 upregulation, improved the cytotoxicity induced by rituximab and the survival of mice with sgFOXO1 tumors. Accordingly, pharmacological inhibition of FOXO1 activity in primary samples upregulated surface CD20 levels. Importantly, FOXO1 was required for the downregulation of CD20 levels by the clinically tested inhibitors of BTK, SYK, PI3K and AKT. Taken together, these results indicate for the first time that the AKT-unresponsive mutants of FOXO1 are important determinant of cell response to rituximab-induced cytotoxicity, and suggest that the genetic status of FOXO1 together with its transcriptional activity need further attention while designing anti-CD20 antibodies based regimens for the therapy of pre-selected lymphomas.

Kinase-independent function of E-type cyclins in liver cancer.

E-type cyclins (cyclins E1 and E2) are components of the core cell cycle machinery and are overexpressed in many human tumor types. E cyclins are thought to drive tumor cell proliferation by activating the cyclin-dependent kinase 2 (CDK2). The cyclin E1 gene represents the site of recurrent integration of the hepatitis B virus in the pathogenesis of hepatocellular carcinoma, and this event is associated with strong up-regulation of cyclin E1 expression. Regardless of the underlying mechanism of tumorigenesis, the majority of liver cancers overexpress E-type cyclins. Here we used conditional cyclin E knockout mice and a liver cancer model to test the requirement for the function of E cyclins in liver tumorigenesis. We show that a ubiquitous, global shutdown of E cyclins did not visibly affect postnatal development or physiology of adult mice. However, an acute ablation of E cyclins halted liver cancer progression. We demonstrated that also human liver cancer cells critically depend on E cyclins for proliferation. In contrast, we found that the function of the cyclin E catalytic partner, CDK2, is dispensable in liver cancer cells. We observed that E cyclins drive proliferation of tumor cells in a CDK2- and kinase-independent mechanism. Our study suggests that compounds which degrade or inhibit cyclin E might represent a highly selective therapeutic strategy for patients with liver cancer, as these compounds would selectively cripple proliferation of tumor cells, while sparing normal tissues.

Discovery of selective, orally bioavailable inhibitor of mouse chitotriosidase.

This article describes our work towards the identification of a potent and selective inhibitor of mouse chitotriosidase (mCHIT1). A series of small molecule inhibitors of mCHIT1 and mAMCase have been developed from early lead compound 1. Examination of synthetized analogues led to discovery of several novel highly potent compounds. Among them compound 9 (OAT-2068) displays a remarkable 143-fold mCHIT1 vs. mAMCase selectivity. To explain the observed SAR molecular docking experiments were performed, which were in line with the experimental data from the enzymatic assays. Inhibitor 9 (OAT-2068) was found to have an excellent pharmacokinetic profile. This, together with high activity and selectivity, makes the compound an ideal and unique tool for studying the role of CHIT1 in biological models.

Targeting Acidic Mammalian chitinase Is Effective in Animal Model of Asthma.

This article highlights our work toward the identification of a potent, selective, and efficacious acidic mammalian chitinase (AMCase) inhibitor. Rational design, guided by X-ray analysis of several inhibitors bound to human chitotriosidase (hCHIT1), led to the identification of compound 7f as a highly potent AMCase inhibitor (IC50 values of 14 and 19 nM against human and mouse enzyme, respectively) and selective (>150× against mCHIT1) with very good PK properties. This compound dosed once daily at 30 mg/kg po showed significant anti-inflammatory efficacy in HDM-induced allergic airway inflammation in mice, reducing inflammatory cell influx in the BALF and total IgE concentration in plasma, which correlated with decrease of chitinolytic activity. Therapeutic efficacy of compound 7f in the clinically relevant aeroallergen-induced acute asthma model in mice provides a rationale for developing AMCase inhibitor for the treatment of asthma.

HDAC6 inhibition upregulates CD20 levels and increases the efficacy of anti-CD20 monoclonal antibodies.

Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.

Selection of an optimal promoter for gene transfer in normal B cells.

Gene transfer into normal quiescent human B cells is a challenging procedure. The present study aimed to investigate whether it is possible to increase the levels of transgene expression by using various types of promoters to drive the expression of selected genes­of­interest. To produce lentiviral particles, the present study used the 2nd generation psPAX2 packaging vector and the vesicular stomatitis virus ­expressing envelope vector pMD2.G. Subsequently, lentiviral vectors were generated containing various promoters, including cytomegalovirus (CMV), elongation factor­1 alpha (EF1α) and spleen focus­forming virus (SFFV). The present study was unable to induce satisfactory transduction efficiency in quiescent normal B cells; however, infection of normal B cells with Epstein­Barr virus resulted in increased susceptibility to lentiviral transduction. In addition, the SFFV promoter resulted in a higher level of transgene expression compared with CMV or EF1α promoters. As a proof­of concept that this approach allows for stable gene expression in normal B cells, the present study used bicistronic lentiviral vectors with genes encoding fluorescent reporter proteins, as well as X­box binding protein­1 and binding immunoglobulin protein.

Inhibitors of SRC kinases impair antitumor activity of anti-CD20 monoclonal antibodies.

Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.

Cyclin C is a haploinsufficient tumour suppressor.

Cyclin C was cloned as a growth-promoting G1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumour suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an 'orphan' CDK19 kinase, as well as CDK8 and CDK3. These cyclin-C-CDK complexes phosphorylate the Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin-C-knockout mice. Cyclin C ablation or heterozygosity collaborates with other oncogenic lesions and accelerates development of T-cell acute lymphoblastic leukaemia (T-ALL). Furthermore, the cyclin C encoding gene CCNC is heterozygously deleted in a significant fraction of human T-ALLs, and these tumours express reduced cyclin C levels. We also describe point mutations in human T-ALL that render cyclin-C-CDK unable to phosphorylate ICN1. Hence, tumour cells may develop different strategies to evade inhibition by cyclin C.

Peroxiredoxin-1 protects estrogen receptor α from oxidative stress-induced suppression and is a protein biomarker of favorable prognosis in breast cancer.

INTRODUCTION: Peroxiredoxin-1 (PRDX1) is a multifunctional protein, acting as a hydrogen peroxide (H2O2) scavenger, molecular chaperone and immune modulator. Although differential PRDX1 expression has been described in many tumors, the potential role of PRDX1 in breast cancer remains highly ambiguous. Using a comprehensive antibody-based proteomics approach, we interrogated PRDX1 protein as a putative biomarker in estrogen receptor (ER)-positive breast cancer. METHODS: An anti-PRDX1 antibody was validated in breast cancer cell lines using immunoblotting, immunohistochemistry and reverse phase protein array (RPPA) technology. PRDX1 protein expression was evaluated in two independent breast cancer cohorts, represented on a screening RPPA (n = 712) and a validation tissue microarray (n = 498). In vitro assays were performed exploring the functional contribution of PRDX1, with oxidative stress conditions mimicked via treatment with H2O2, peroxynitrite, or adenanthin, a PRDX1/2 inhibitor. RESULTS: In ER-positive cases, high PRDX1 protein expression is a biomarker of improved prognosis across both cohorts. In the validation cohort, high PRDX1 expression was an independent predictor of improved relapse-free survival (hazard ratio (HR) = 0.62, 95% confidence interval (CI) = 0.40 to 0.96, P = 0.032), breast cancer-specific survival (HR = 0.44, 95% CI = 0.24 to 0.79, P = 0.006) and overall survival (HR = 0.61, 95% CI = 0.44 to 0.85, P = 0.004). RPPA screening of cancer signaling proteins showed that ERα protein was upregulated in PRDX1 high tumors. Exogenous H2O2 treatment decreased ERα protein levels in ER-positive cells. PRDX1 knockdown further sensitized cells to H2O2- and peroxynitrite-mediated effects, whilst PRDX1 overexpression protected against this response. Inhibition of PRDX1/2 antioxidant activity with adenanthin dramatically reduced ERα levels in breast cancer cells. CONCLUSIONS: PRDX1 is shown to be an independent predictor of improved outcomes in ER-positive breast cancer. Through its antioxidant function, PRDX1 may prevent oxidative stress-mediated ERα loss, thereby potentially contributing to maintenance of an ER-positive phenotype in mammary tumors. These results for the first time imply a close connection between biological activity of PRDX1 and regulation of estrogen-mediated signaling in breast cancer.

Generation of a new bioluminescent model for visualisation of mammary tumour development in transgenic mice.

BACKGROUND: Numerous transgenic models have been generated to study breast cancer. However, despite many advantages, traditional transgenic models for breast cancer are also burdened with difficulties in early detection and longitudinal observation of transgene-induced tumours, which in most cases are randomly located and occur at various time points. Methods such as palpation followed by mechanical measurement of the tumours are of limited value in transgenic models. There is a crucial need for making these previously generated models suitable for modern methods of tumour visualisation and monitoring, e.g. by bioluminescence-based techniques. This approach was successfully used in the current study. RESULTS: A new mouse strain (MMTV-Luc2 mice) expressing Luc2 luciferase primarily in mammary tissue in females, with low-level background expression in internal organs, was generated and bred to homozygosity. After these mice were intercrossed with MMTV-PyVT mice, all double transgenic females developed mammary tumours by the age of 10 weeks, the localisation and progression of which could be effectively monitored using the luminescence-based in vivo imaging. Luminescence-based readout allowed for early visualisation of the locally overgrown mammary tissue and for longitudinal evaluation of local progression of the tumours. When sampled ex vivo at the age of 10 weeks, all tumours derived from MMTV-Luc2PyVT females displayed robust bioluminescent signal. CONCLUSIONS: We have created a novel transgenic strain for visualisation and longitudinal monitoring of mammary tumour development in transgenic mice as an addition and/or a new and more advanced alternative to manual methods. Generation of this mouse strain is vital for making many of the existing mammary tumour transgenic models applicable for in vivo imaging techniques.