Isolation of lymph shows dysregulation of STAT3 and CREB pathways in the spleen and liver during leukemia development in a rat model.

BACKGROUND AND AIMS: Acute myeloid leukemia (AML) is a heterogeneous malignant condition characterized by massive infiltration of poorly differentiated white blood cells in the blood stream, bone marrow, and extramedullary sites. During leukemic development, hepatosplenomegaly is expected to occur because large blood volumes are continuously filtered through these organs. We asked whether infiltration of leukemic blasts initiated a response that could be detected in the interstitial fluid phase of the spleen and liver. MATERIAL AND METHODS: We used a rat model known to mimic human AML in growth characteristics and behavior. By cannulating efferent lymphatic vessels from the spleen and liver, we were able to monitor the response of the microenvironment during AML development. RESULTS AND DISCUSSION: Flow cytometric analysis of lymphocytes showed increased STAT3 and CREB signaling in spleen and depressed signaling in liver, and proteins related to these pathways were identified with a different profile in lymph and plasma in AML compared with control. Additionally, several proteins were differently regulated in the microenvironment of spleen and liver in AML when compared with control. CONCLUSION: Interstitial fluid, and its surrogate efferent lymph, can be used to provide unique information about responses in AML-infiltered organs and substances released to the general circulation during leukemia development.

Implantable microenvironments to attract hematopoietic stem/cancer cells.

The environments that harbor hematopoietic stem and progenitor cells are critical to explore for a better understanding of hematopoiesis during health and disease. These compartments often are inaccessible for controlled and rapid experimentation, thus limiting studies to the evaluation of conventional cell culture and transgenic animal models. Here we describe the manufacture and image-guided monitoring of an engineered microenvironment with user-defined properties that recruits hematopoietic progenitors into the implant. Using intravital imaging and fluorescence molecular tomography, we show in real time that the cell homing and retention process is efficient and durable for short- and long-term engraftment studies. Our results indicate that bone marrow stromal cells, precoated on the implant, accelerate the formation of new sinusoidal blood vessels with vascular integrity at the microcapillary level that enhances the recruitment hematopoietic progenitor cells to the site. This implantable construct can serve as a tool enabling the study of hematopoiesis.

Access to the spleen microenvironment through lymph shows local cytokine production, increased cell flux, and altered signaling of immune cells during lipopolysaccharide-induced acute inflammation.

The spleen is involved in fluid volume regulation, immune responses, and hematopoiesis. Yet, the composition of the fluid phase within the spleen microenviroment, the migratory routes of lymphocytes as well as the splenic response to bacterial endotoxin is incomplete. To address these issues, we isolated postnodal lymph in rats by cannulating an efferent lymphatic draining the spleen, and assessed the secretion of signaling substances during a septic response induced by LPS. Spleen lymph flow increased 8-fold after LPS exposure. The spleen exhibited a permeable microvasculature with low sieving of macromolecules that was absent after exposure to LPS. Furthermore, after LPS exposure the spleen contributed significantly to the production of pro- and anti-inflammatory cytokines, and experiments in splenectomized rats suggested it may induce a protracted inflammation because of a dominant role in IL-6 production. A significant amount of lymphocytes exited via lymphatics draining the spleen in control rats. LPS-induced inflammation resulted in increased T cell and reduced B cell subset fractions, and gave a significant increase in CD4(+) and CD8(+) subset T cell efflux and a reduced B cell efflux in spleen lymph. Exposure of leukocytes to the spleen microenvironment affected their signaling status, and by phosphorylation specific flow cytometry we could identify STAT3 and CREB as important mediators in the cellular signaling occurring during endotoxemia. We conclude that analysis of spleen lymph may unravel immune cell migration patterns and local signaling, and immune cells exit via lymph having acquired specific activation signatures after exposure to the spleen microenvironment.

Chromosomal 20q gain in the DNA diploid component of aneuploid colorectal carcinomas.

The order of appearance of different genetic aberrations during the shift from diploidy/near-diploidy to aneuploidy in colorectal cancers is not yet clear. We studied genetic alterations in flow cytometrically-sorted DNA diploid and corresponding aneuploid epithelial cell populations from each of 20 colorectal tumors using comparative genomic hybridization, FISH, and PCR. Analysis of the 19 cases in which aberrations were found in the flow-sorted diploid population indicated that large-scale aneuploidization in colorectal cancer was preceded by amplification of oncogene(s) localized to chromosome 20q13.2 and by KRAS mutations, but not by TP53 deletions or losses of large chromosomal regions such as 4q, 8p and 18q.