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 longterm 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.

Material

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. ' 2007 Wiley-Liss, Inc.Key words: DNA aneuploidy; DNA diploidy; flow-sorted colorectal tumors; CGH; chromosomal aberrations; FISH; 20q13 gain Colorectal tumorigenesis is characterized by the sequential accumulation of multiple and extensive genomic changes in normal colonic mucosal cells, 1 which ultimately lead to high-level aneuploidy in the majority of tumors. 2 The order of appearance of different genetic aberrations and the molecular mechanisms underlying the shift from diploidy to aneuploidy are not yet clear. The origin of high-level aneuploidy has been suggested to occur via tetraploidization of near-diploid aberrant colonic mucosal cells followed by chromosome loss. 3 Genes responsible for DNA double-strand break repair, chromosomal stability, chromosomal segregation and chromosome disjunction have also been suggested as likely candidates in the development of aneuploidy. 4,5 TP53 gene alterations may also be permissive for aneuploidy, 6 since the resulting loss of wild-type TP53 function may facilitate the formation and survival of cells with abnormal DNA content. Aneuploid colorectal tumors exhibit considerable chromosomal instability as evidenced by frequent gains of chromosomes 20 and 13 and frequent losses of chromosomes 18 and 4. 7,8 Late-stage colorectal adenomas harbor some of the same chromosomal aberrations as seen in colorectal carcinomas, although their incidence is less frequent. 7,9 The putative oncogenes and tumor suppressor genes localized to these frequently-amplified or -deleted chromosomal regions are still mostly unidentified.The resolution of flow cytometric DNA content measurements is limited to the detection of a variation in DNA content of about 5%. Thus cell populations with gain or loss of only 1 or 2 chromosomes may be measured by flow cytometry as having diploid DNA content, even if such near-diploid populations predominate in a cell sample. Flow cytometric DNA content histograms of aneuploid tumors typically show a cell population with ''diploid'' DNA content (Figure 1a). This DNA diploid cell population consists of infiltrating leucocytes and colonic mucosal cells. Some of these colonic mucosal cells may possibly be late-stage adenoma cells or abnormal cells (with some few chromosomal aberrations) that are at an early stage of tumor progression, and which may stil...

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.