Stromal stiffening accompanies malignancy, compromises treatment, and promotes tumor aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumors should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase- and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbor the highest number of tumor-associated macrophages (TAM), whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumors, whereas stromal cell targeting did. Stromal cells in microdissected human tumors expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of a cohort of breast cancer patient biopsies revealed that stromal expression of lysyl hydroxylase two, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumor aggression and identify lysyl hydroxylase two as a stromal biomarker.

Hormographiella is a rare fungal pathogen in humans; however, case reports have described disseminated infection in immunocompromised hosts. This pathogen has been described to yield poor prognosis in patients who harbor it. Herein, we present a case report of autopsy-proven disseminated Hormographiella aspergillata infection, confirmed by DNA sequencing, in a patient experiencing a relapse of leukemia. This 54-year-old Caucasian man with chronic myelogenous leukemia (CML) that had been diagnosed in 1989, after having received a hematopoietic cell allotransplant from a compatible sibling donor, had B-cell lymphoid-blast phase of CML in April of 2013, with multiple relapses. His most recent relapse was in September of 2016, when bone marrow biopsy showed 90% blasts. The results of bronchoalveolar lavage (BAL) cultures were positive for filamentous fungus infection. The patient developed encephalopathy and worsening respiratory statusand tachycardia with flutter and hypotension, which resulted in his death. At autopsy, bilateral pleural effusions, multiple right pleural nodules, and subarachnoid hemorrhage were noted. Angioinvasive hyphal fungi were found in the right frontal lobe of the brain and the right upper lobe of the lung. Morphologically, the fungi had multiseptate, branching hyphae. The bronchoalveolar lavage specimen grew a fungus for which the colony morphologic characteristics and microscopic features were compatible with a Hormographiella species. H. aspergillata from the bronchoalveolar lavage was further identified by sequencing the D2 hypervariable region of the large-subunit (LSU) ribosomal DNA gene and the full internal transcribed spacer (ITS) regions.

1Collagen deposition and stromal stiffening accompany malignancy, compromise treatment, and 2 promote tumor aggression. Clarifying the molecular nature of and the factors that regulate 3 extracellular matrix stiffening in tumors should identify biomarkers to stratify patients for therapy 4 and therapeutic interventions to improve outcome. We profiled lysyl hydroxylase-and lysyl 5 oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex 6 collagen crosslinks in more aggressive human breast cancer subtypes with the stiffest stroma. 7These tissues also harbored the highest number of tumor-associated macrophages (TAM), whose 8 therapeutic ablation not only reduced metastasis, but also concomitantly decreased accumulation 9 of collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking 10 enzyme lysyl oxidase had no impact on collagen crosslinking in PyMT mammary tumors, whereas 11 stromal cell targeting did. Consistently, stromal cells in microdissected human tumors expressed 12 the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of a cohort of 13 breast cancer patient biopsies revealed that stromal expression of lysyl hydroxylase two, an 14 enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening 15 correlated significantly disease specific mortality. The findings link tissue inflammation, stromal 16 cell-mediated collagen crosslinking and stiffening to tumor aggression and identify lysyl 17 hydroxylase two as a novel stromal biomarker. 18 19Significance 20We show infiltrating macrophages induce stromal fibroblast, and not epithelial, expression of 21 collagen crosslinking enzymes that drive tumor stiffening. Stromal enzyme LH2 is significantly 22 upregulated in breast cancer patients with the stiffest stroma, the most trivalent HLCCs and the 23 worst prognosis, underscoring its potential as a biomarker and therapeutic target. 24 25 Her2/Neu mice or genetic reduction of PLOD2 in subcutaneously-injected lung tumor epithelial 52 cells reduce tissue fibrosis, stromal stiffening and collagen crosslinking and concomitantly 53 decrease tumor incidence and aggression(12,23). Moreover, elevating LOX or LH2-mediated 54 collagen crosslinking enhances fibrosis and stromal stiffness and promotes malignant 55 transformation and tumor aggression in lung and mammary xenografted tumors(12,23). These 56 observations suggest that the direct targeting of specific collagen crosslinking enzymes has 57 clinical merit for the treatment of cancer. However, given caveats with recent clinical trials 58 targeting ECM modifiers including suboptimal activity of inhibitory treatments and the risk of off-59 target effects, strategies designed to interfere with the induction and activation of these 60 crosslinking enzymes offer an attractive alternative(26). Towards this goal, the identification and 61 causal implication of additional factors that regulate the levels and/or activity of collagen 62 crosslinking...

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