Previously, we showed that cortical mineralization is coordinately adjusted to mechanically offset external bone size differences between A/J (narrow) and C57BL/6J (wide) mouse femora to achieve whole bone strength equivalence at adulthood. The identity of the genes and their interactions that are responsible for establishing this homeostatic state (i.e., canalization) remain unknown. We hypothesize that these inbred strains, whose inter-individual differences in bone structure and material properties mimic that observed among humans, achieve functional homeostasis by differentially adjusting key molecular pathways regulating external bone size and mineralization throughout growth. The cortices of A/J and C57BL/6J male mouse femora were phenotyped and gene expression levels were assessed across growth (i.e. 2, 4, 6, 8, 12, 16-weeks of age). A difference in total cross-sectional area (p < 0.01) and cortical tissue mineral density were apparent between mouse strains by 2-weeks of age and maintained at adulthood (p < 0.01). These phenotypic dissimilarities corresponded to gene expression level differences among key regulatory pathways throughout growth. A/J mice had a 1.55 – 7.65 fold greater expression among genes inhibitory to Wnt pathway induction while genes involved in cortical mineralization were largely upregulated 1.50 – 3.77 fold to compensate for their narrow diaphysis. Additionally, both mouse strains showed an upregulation among Wnt pathway antagonists corresponding to the onset of adult ambulation (i.e., increased physiological loads). This contrasts with other studies showing an increase in Wnt pathway activation following functionally isolated, experimental in vivo loading regimens. A/J and C57BL/6J long bones provide a model to develop a systems-based approach to identify individual genes and the gene-gene interactions that contribute to trait differences between the strains while being involved in the process by which these traits are coordinately adjusted to establish similar levels of mechanical function; thus providing insight into the process of canalization.

We investigated the hypoxia-dependent cytotoxicity of AQ4N (banoxantrone) using a panel of 13 cancer cell lines and studied its relationship to the expression of the quinone reductase DTdiaphorase (NQO1), which is widely found in cancer cells. We also investigated pharmacologic treatments that increase tumor hypoxia in vivo and their impact on AQ4N chemosensitivity in a solid tumor xenograft model. AQ4N showed $8-fold higher cytotoxicity under hypoxia than normoxia in cultures of 9L rat gliosarcoma and H460 human non-small-cell lung carcinoma cells but not for 11 other human cancer cell lines. DT-diaphorase protein levels and AQ4N chemosensitivity were poorly correlated across the cancer cell line panel, and AQ4N chemosensitivity was not affected by DT-diaphorase inhibitors. The vasodilator hydralazine decreased tumor perfusion and increased tumor hypoxia in 9L tumor xenografts, and to a lesser extent in H460 tumor xenografts. However, hydralazine did not increase AQ4N-dependent antitumor activity. Combination of AQ4N with the angiogenesis inhibitor axitinib, which increases 9L tumor hypoxia, transiently increased antitumor activity but with an increase in host toxicity. These findings indicate that the capacity to bioactivate AQ4N is not dependent on DTdiaphorase and is not widespread in cultured cancer cell lines. Moreover, the activation of AQ4N cytotoxicity in vivo requires tumor hypoxia that is more extensive or prolonged than can readily be achieved by vasodilation or by antiangiogenic drug treatment.

Thrombospondin-2 (TSP2) is a matricellular protein component of the bone extracellular matrix. Long bones of adult TSP2-deficient mice have increased endosteal bone thickness due to expansion of the osteoblast progenitor cell pool, and these cells display deficits in osteoblastic potential. Here, we investigated the effects of TSP2 deficiency on whole bone geometric and mechanical properties in growing 6-wk-old male and female wild-type and TSP2-knockout (KO) mice. Microcomputed tomography and mechanical testing were conducted on femora and L2 vertebrae to assess morphology and whole bone mechanical properties. In a second series of experiments, femoral diaphyses were harvested from wild-type and TSP2-KO mice. Detergent-soluble type I collagen content was determined by Western blot of right femora. Total collagen content was determined by hydroxyproline analysis of left femora. In a third series of experiments, cortical bone was dissected from the anterior and posterior aspects of the femoral middiaphysis and imaged by transmission electron microscopy to visualize collagen fibrils. Microcomputed tomography revealed minimal structural effects of TSP2 deficiency. TSP2 deficiency imparted a brittle phenotype on cortical bone. Femoral tissue mineral density was not affected by TSP2 deficiency. Instead, transmission electron microscopy revealed less intensely stained collagen fibrils with altered morphology in the extracellular matrix assembled by osteoblasts on the anterior surface of TSP2-KO femora. Femoral diaphyseal bone displayed comparable amounts of total collagen, but the TSP2-KO bones had higher levels of detergent-extractable type I collagen. Together, our data suggest that TSP2 is required for optimal collagen fibrillogenesis in bone and thereby contributes to normal skeletal tissue quality.

Introduction To support the care of lung cancer patients, oncologists have needed to stay current on treatment advancements and build relationships with a new group of survivors in an era where lung cancer survivorship has been re-defined. The objectives of the study were to (1) understand the perspectives of advanced lung cancer patients whose tumors have oncogenic alterations about their care experiences with their oncologist(s) and (2) describe the perceptions of advanced lung cancer patients about seeking second opinions and navigating care decisions. Methods In this qualitative study, patients with advanced lung cancer ( n = 25) on targeted therapies were interviewed to discuss their ongoing experience with their oncologists. We used deductive and inductive qualitative approaches in the coding of the data. We organized the data using the self-determination framework. Results Patients described both positive and negative aspects of their care as related to autonomy, provider competency, and connectedness. Patients sought second opinions for three primary reasons: expertise, authoritative advice, and access to clinical trial opportunities. When there is disagreement in the treatment plan between the primary oncologist and the specialist, there can be confusion and tension, and patients have to make difficult choices about their path forward. Conclusions Patients value interactions that support their autonomy, demonstrate the competency of their providers, and foster connectedness. To ensure that patients receive quality and goal-concordant care, developing decision aids and education materials that help patients negotiate recommendations from two providers is an area that deserves further attention.

Cancer stem-like cells were isolated from several human tumor cell lines by limiting dilution assays and holoclone morphology, followed by assessment of self-renewal capacity, tumor growth, vascularity, and blood perfusion. H460 holoclone-derived tumors grew slower than parental H460 tumors, but displayed significantly increased microvessel density and tumor blood perfusion. Microarray analysis identified 177 differentially regulated genes in the holoclone-derived tumors, of which 47 were associated with angiogenesis. The dysregulated genes include several small leucine-rich proteoglycans that may modulate angiogenesis and serve as novel therapeutic targets for inhibiting cancer stem cell-driven angiogenesis.