Targeting disseminated estrogen-receptor-positive breast cancer cells in bone marrow.

Estrogen receptor-positive (ER+) breast cancer can recur up to 20 years after initial diagnosis. Delayed recurrences arise from disseminated tumors cells (DTCs) in sites such as bone marrow that remain quiescent during endocrine therapy and subsequently proliferate to produce clinically detectable metastases. Identifying therapies that eliminate DTCs and/or effectively target cells transitioning to proliferation promises to reduce risk of recurrence. To tackle this problem, we utilized a 3D co-culture model incorporating ER+ breast cancer cells and bone marrow mesenchymal stem cells to represent DTCs in a bone marrow niche. 3D co-cultures maintained cancer cells in a quiescent, viable state as measured by both single-cell and population-scale imaging. Single-cell imaging methods for metabolism by fluorescence lifetime (FLIM) of NADH and signaling by kinases Akt and ERK revealed that breast cancer cells utilized oxidative phosphorylation and signaling by Akt to a greater extent both in 3D co-cultures and a mouse model of ER+ breast cancer cells in bone marrow. Using our 3D co-culture model, we discovered that combination therapies targeting oxidative phosphorylation via the thioredoxin reductase (TrxR) inhibitor, D9, and the Akt inhibitor, MK-2206, preferentially eliminated breast cancer cells without altering viability of bone marrow stromal cells. Treatment of mice with disseminated ER+ human breast cancer showed that D9 plus MK-2206 blocked formation of new metastases more effectively than tamoxifen. These data establish an integrated experimental system to investigate DTCs in bone marrow and identify combination therapy against metabolic and kinase targets as a promising approach to effectively target these cells and reduce risk of recurrence in breast cancer.

Acepromazine and Chlorpromazine as Pharmaceutical-grade Alternatives to Chlorprothixene for Pupillary Light Reflex Imaging in Mice.

Studies of visual responses in isoflurane-anesthetized mice often use the sedative chlorprothixene to decrease the amount of isoflurane used because excessive isoflurane could adversely affect light-evoked responses. However, data are not available to justify the use of this nonpharmaceutical-grade chemical. The current study tested whether pharmaceutical-grade sedatives would be appropriate alternatives for imaging pupillary light reflexes. Male 15-wk-old mice were injected intraperitoneally with 1 mg/kg chlorprothixene, 5 mg/kg acepromazine, 10 mg/kg chlorpromazine, or saline. After anesthetic induction, anesthesia maintenance used 0.5% and 1% isoflurane for sedative- and saline-injected mice, respectively. A photostimulus (16.0 log photons cm-2 s-1; 470 nm) was presented to the right eye for 20 min, during which the left eye was imaged for consensual pupillary constriction and involuntary pupil drift. Time to immobilization, loss of righting reflex, physiologic parameters, gain of righting reflex, and degree of recovery were assessed also. The sedative groups were statistically indistinguishable for all measures. By contrast, pupillary drift occurred far more often in saline-treated mice than in the sedative groups. Furthermore, saline-treated mice took longer to reach maximal pupil constriction than all sedative groups and had lower heart rates compared with chlorpromazine- and chlorprothixene-sedated mice. Full recovery (as defined by purposeful movement, response to tactile stimuli, and full alertness) was not regularly achieved in any sedative group. In conclusion, at the doses tested, acepromazine and chlorpromazine are suitable pharmaceutical-grade alternatives to chlorprothixene for pupil imaging and conceivably other in vivo photoresponse measurements; however, given the lack of full recovery, lower dosages should be investigated further for use in survival procedures.

Outbreak of Murine Infection with Clostridium difficile Associated with the Administration of a Pre- and Perinatal Methyl Donor Diet.

Between October 2016 and June 2017, a C57BL/6J mouse colony that was undergoing a pre- and perinatal methyl donor supplementation diet intervention to study the impact of parental nutrition on offspring susceptibility to disease was found to suffer from an epizootic of unexpected deaths. Necropsy revealed the presence of severe colitis, and further investigation linked these outbreak deaths to a Clostridium difficile strain of ribotype 027 that we term 16N203. C. difficile infection (CDI) is associated with antibiotic use in humans. Current murine models of CDI rely on antibiotic pretreatment to establish clinical phenotypes. In this report, the C. difficile outbreak occurs in F1 mice linked to alterations in the parental diet. The diagnosis of CDI in the affected mice was confirmed by cecal/colonic histopathology, the presence of C. difficile bacteria in fecal/colonic culture, and detection of C. difficile toxins. F1 mice from parents fed the methyl supplementation diet also had significantly reduced survival (P < 0.0001) compared with F1 mice from parents fed the control diet. When we tested the 16N203 outbreak strain in an established mouse model of antibiotic-induced CDI, we confirmed that this strain is pathogenic. Our serendipitous observations from this spontaneous outbreak of C. difficile in association with a pre- and perinatal methyl donor diet suggest the important role that diet may play in host defense and CDI risk factors.IMPORTANCEClostridium difficile infection (CDI) has become the leading cause of infectious diarrhea in hospitals worldwide, owing its preeminence to the emergence of hyperendemic strains, such as ribotype 027 (RT027). A major CDI risk factor is antibiotic exposure, which alters gut microbiota, resulting in the loss of colonization resistance. Current murine models of CDI also depend on pretreatment of animals with antibiotics to establish disease. The outbreak that we report here is unique in that the CDI occurred in mice with no antibiotic exposure and is associated with a pre- and perinatal methyl supplementation donor diet intervention study. Our investigation subsequently reveals that the outbreak strain that we term 16N203 is an RT027 strain, and this isolated strain is also pathogenic in an established murine model of CDI (with antibiotics). Our report of this spontaneous outbreak offers additional insight into the importance of environmental factors, such as diet, and CDI susceptibility.

Short-Term Environmental Conditioning Enhances Tumorigenic Potential of Triple-Negative Breast Cancer Cells.

Tumor microenvironments expose cancer cells to heterogeneous, dynamic environments by shifting availability of nutrients, growth factors, and metabolites. Cells integrate various inputs to generate cellular memory that determines trajectories of subsequent phenotypes. Here we report that short-term exposure of triple-negative breast cancer cells to growth factors or targeted inhibitors regulates subsequent tumor initiation. Using breast cancer cells with different driver mutations, we conditioned cells lines with various stimuli for 4 hours before implanting these cells as tumor xenografts and quantifying tumor progression by means of bioluminescence imaging. In the orthotopic model, conditioning a low number of cancer cells with fetal bovine serum led to enhancement of tumor-initiating potential, tumor volume, and liver metastases. Epidermal growth factor and the mTORC1 inhibitor ridaforolimus produced similar but relatively reduced effects on tumorigenic potential. These data show that a short-term stimulus increases tumorigenic phenotypes based on cellular memory. Conditioning regimens failed to alter proliferation or adhesion of cancer cells in vitro or kinase signaling through Akt and ERK measured by multiphoton microscopy in vivo, suggesting that other mechanisms enhanced tumorigenesis. Given the dynamic nature of the tumor environment and time-varying concentrations of small-molecule drugs, this work highlights how variable conditions in tumor environments shape tumor formation, metastasis, and response to therapy.