New tools for the quantitative assessment of prodrug delivery and neurotoxicity.

Systemic off-target toxicities, including neurotoxicity, are prevalent side effects in cancer patients treated with a number of otherwise highly efficacious anticancer drugs. In the current study, we have: (1) developed a new analytical metric for the in vivo preclinical assessment of systemic toxicities/neurotoxicity of new drugs and delivery systems; and (2) evaluated, in mice, the in vivo efficacy and toxicity of a versatile and modular NanoDendron (ND) drug delivery and imaging platform that we recently developed. Our paclitaxel-carrying ND prodrug, ND(PXL), is activated following proteolytic cleavage by MMP9, resulting in localized cytotoxic chemotherapy. Using click chemistry, we combined ND(PXL) with a traceable beacon, ND(PB), yielding ND(PXL)-ND(PB) that functions as a theranostic compound. In vivo fluorescence FRET imaging of this theranostic platform was used to confirm localized delivery to tumors and to assess the efficiency of drug delivery to tumors, achieving 25-30% activation in the tumors of an immunocompetent mouse model of breast cancer. In this model, ND-drug exhibited anti-tumor efficacy comparable to nab-paclitaxel, a clinical formulation. In addition, we combined neurobehavioral metrics of nociception and sensorimotor performance of individual mice to develop a novel composite toxicity score that reveals and quantifies peripheral neurotoxicity, a debilitating long-term systemic toxicity of paclitaxel therapy. Importantly, mice treated with nab-paclitaxel developed changes in behavioral metrics with significantly higher toxicity scores indicative of peripheral neuropathy, while mice treated with ND(PXL) showed no significant changes in behavioral responses or toxicity score. Our ND formulation was designed to be readily adaptable to incorporate different drugs, imaging modalities and/or targeting motifs. This formulation has significant potential for preclinical and clinical tools across multiple disease states. The studies presented here report a novel toxicity score for assessing peripheral neuropathy and demonstrate that our targeted, theranostic NDs are safe and effective, providing localized tumor delivery of a chemotherapeutic and with reduced common neurotoxic side-effects.

Stromal matrix metalloproteinase 2 regulates collagen expression and promotes the outgrowth of experimental metastases.

Breast cancer survival rates decrease from 99% for patients with local disease to 25% for those with distant metastases. Matrix metalloproteinases (MMPs), including MMP2, are associated with metastatic progression. We found that loss of host MMP2 reduces the proliferation of experimental metastases in the lungs and identified fibroblasts in tumour-bearing lungs as the major source of MMP2. In vitro, spheroidal mammary tumour growth was increased by co-culture with control fibroblasts isolated from tumour-bearing lungs, but not when fibroblasts with stable Mmp2 knockdown were used. This result prompted us to assess whether MMP2 was responsible for a tumour-proliferative, activated fibroblast phenotype. To test this, we evaluated: (a) fibroblasts from wild-type tumour-bearing lungs, with or without shRNA-mediated MMP2 knockdown; and (b) normal, quiescent fibroblasts isolated from either WT or Mmp2(-/-) mice. Quantitative PCR revealed that Mmp2 knockdown attenuated expression of two markers of activation (α-smooth muscle actin and vimentin), but there was minimal expression in quiescent WT or Mmp2(-/-) fibroblasts, as expected. Placing quiescent fibroblasts under activating conditions led to increases in activation-associated transcripts in WT but not Mmp2(-/-) fibroblasts. Additionally, Mmp2 knockdown fibroblasts showed significantly decreased expression of the matrix transcripts collagen I, collagen IV and fibronectin. Addition of active TGFß was sufficient to rescue the MMP2-dependent collagen I and IV expression, while MMP2-induced collagen expression was blocked by the addition of TGFß1-neutralizing antibody. Gene expression data in stromal cells of human breast cancers reveal that MMP2 expression is also positively correlated with activation and matrix transcripts. Thus, we present a model whereby MMP2 production in tumour fibroblasts is important for TGFß1 activity and subsequent activation of fibroblasts to a matrix-producing, proliferation-supportive phenotype. Overall, our results reveal a previously undefined role for MMP2 in metastatic outgrowth mediated by fibroblasts, and extend the mechanisms by which MMPs contribute to tumour progression.

IL4 receptor ILR4α regulates metastatic colonization by mammary tumors through multiple signaling pathways.

IL4, a cytokine produced mainly by immune cells, may promote the growth of epithelial tumors by mediating increased proliferation and survival. Here, we show that the type II IL4 receptor (IL4R) is expressed and activated in human breast cancer and mouse models of breast cancer. In metastatic mouse breast cancer cells, RNAi-mediated silencing of IL4Rα, a component of the IL4R, was sufficient to attenuate growth at metastatic sites. Similar results were obtained with control tumor cells in IL4-deficient mice. Decreased metastatic capacity of IL4Rα "knockdown" cells was attributed, in part, to reductions in proliferation and survival of breast cancer cells. In addition, we observed an overall increase in immune infiltrates within IL4Rα knockdown tumors, indicating that enhanced clearance of knockdown tumor cells could also contribute to the reduction in knockdown tumor size. Pharmacologic investigations suggested that IL4-induced cancer cell colonization was mediated, in part, by activation of Erk1/2, Akt, and mTOR. Reduced levels of pAkt and pErk1/2 in IL4Rα knockdown tumor metastases were associated with limited outgrowth, supporting roles for Akt and Erk activation in mediating the tumor-promoting effects of IL4Rα. Collectively, our results offer a preclinical proof-of-concept for targeting IL4/IL4Rα signaling as a therapeutic strategy to limit breast cancer metastasis.

Lack of MMP10 exacerbates experimental colitis and promotes development of inflammation-associated colonic dysplasia.

Inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) represent serious health burdens because of both the tissue-damaging disease itself and an elevated risk of colon cancer. The increased expression of many members of the matrix metalloproteinase (MMP) family of enzymes that occurs in colitis has long been associated with the destructive nature of the disease. Recent findings in cancer and other MMP-associated diseases, however, led us to question whether MMPs are indeed detrimental in the setting of colitis. Here, we focus on a single MMP family member, MMP10, and assess its role in a murine model of colonic tissue damage induced by dextran sulfate sodium (DSS) treatment. Using mice genetically deficient for MMP10, we find that absence of this enzyme leads to significantly worse disease scores and failure to resolve inflammation even after extended recovery periods. We show that MMP10 is produced predominantly by infiltrating myeloid cells in both murine and human colitis. Through bone marrow transplant experiments, we confirm that bone marrow-derived MMP10 contributes to colitis severity. Mice lacking MMP10 have a significantly higher propensity for development of dysplastic lesions in the colon after two rounds of DSS exposure. Thus, we conclude that MMP10 is required for resolution of DSS-induced colonic damage, and in its absence, chronic inflammation and ultimately dysplasia occurs.

Residents Learn to Improve Care Using the ACGME Core Competencies and Institute of Medicine Aims for Improvement: the Health Care Matrix.

OBJECTIVES: This article describes how internal medicine residents at Vanderbilt University Medical Center learn to assess and improve care using the Institute of Medicine aims for improvement and the Accreditation Council for Graduate Medical Education core competencies combined in a tool called the health care matrix. The most important and popular use of the health care matrix has been with suboptimal care, in which care is not safe, timely, effective, efficient, equitable, or patient centered. BACKGROUND: The core competencies provide a means of defining why care was not safe, timely, effective, efficient, equitable, or patient centered. The Institute of Medicine aims for improvement are also important because they are used to frame most publicly reported measures of quality. Few residents have an understanding of these public measures and how their futures will be affected by the growing trend toward quality report cards. INTERVENTION: To help the residents understand the significance of public measures of quality, they learn to assess their patients as a "panel," looking at the care they provide for patients with coronary artery disease and diabetes mellitus. Residents use the health care matrix to analyze 1 of their patients, and then as a group they select a health care matrix for their improvement project. The way the health care matrix is formatted and the sequencing of the core competencies allow for the analysis of the cells to lead to the final question "What was learned and what needs to be improved?" The residents are then taught the tools and methods of quality improvement and complete their project. Some of these projects have had a significant influence on external measures of quality for this organization. The article describes the 8-week course that residents complete, the use of the health care matrix, the analysis of the patient panel, and finally an example of a completed project in which they improve the timeliness of antibiotics administration to patients with pneumonia (a public measure of quality).