Chika Iwamoto

CD34 + CD38 + CD19 + as well as CD34+CD38-CD19+ cells are leukemia-initiating cells with self-renewal capacity in human B-precursor ALL

The presence of rare malignant stem cells supplying a hierarchy of malignant cells has recently been reported. In human acute myelogenous leukemia (AML), the leukemia stem cells (LSCs) have been phenotypically restricted within the CD34+CD38− fraction. To understand the origin of malignant cells in primary human B-precursor acute lymphocytic leukemia (B-ALL), we established a novel in vivo xenotransplantation model. Purified CD34+CD38+CD19+, CD34+CD38−CD19+ and CD34+CD38−CD19− bone marrow (BM) or peripheral blood (PB) cells from three pediatric B-ALL patients were intravenously injected into sublethally irradiated newborn NOD/SCID/IL2rγnull mice. We found that both CD34+CD38+CD19+ and CD34+CD38−CD19+ cells initiate B-ALL in primary recipients, whereas the recipients of CD34+CD38−CD10−CD19− cells showed normal human hematopoietic repopulation. The extent of leukemic infiltration into the spleen, liver and kidney was similar between the recipients transplanted with CD34+CD38+CD19+ cells and those transplanted with CD34+CD38−CD19+ cells. In each of the three cases studied, transplantation of CD34+CD38+CD19+ cells resulted in the development of B-ALL in secondary recipients, demonstrating self-renewal capacity. The identification of CD34+CD38+CD19+ self-renewing B-ALL cells proposes a hierarchy of leukemia-initiating cells (LICs) distinct from that of AML. Recapitulation of patient B-ALL in NOD/SCID/IL2rγnull recipients provides a powerful tool for directly studying leukemogenesis and for developing therapeutic strategies.

Polymorphic Sirpa is the genetic determinant for NOD-based mouse lines to achieve efficient human cell engraftment

Current mouse lines efficient for human cell xenotransplantation are backcrossed into NOD mice to introduce its multiple immunodeficient phenotypes. Our positional genetic study has located the NOD-specific polymorphic Sirpa as a molecule responsible for its high xenograft efficiency: it recognizes human CD47 and the resultant signaling may cause NOD macrophages not to engulf human grafts. In the present study, we established C57BL/6.Rag2(nullIl2rgnull) mice harboring NOD-Sirpa (BRGS). BRGS mice engrafted human hematopoiesis with an efficiency that was equal to or even better than that of the NOD.Rag1(nullIl2rgnull) strain, one of the best xenograft models. Consequently, BRGS mice are free from other NOD-related abnormalities; for example, they have normalized C5 function that enables the evaluation of complement-dependent cytotoxicity of antibodies against human grafts in the humanized mouse model. Our data show that efficient human cell engraftment found in NOD-based models is mounted solely by their polymorphic Sirpa. The simplified BRGS line should be very useful in future studies of human stem cell biology.

JAK2 V617F uses distinct signalling pathways to induce cell proliferation and neutrophil activation

The acquired JAK2 V617F mutation is observed in the majority of patients with BCR‐ABL1 negative chronic myeloproliferative neoplasms (MPN). BCR‐ABL1 negative MPN displays myeloproliferation with an elevated leucocyte alkaline phosphatase (LAP) activity, a neutrophil activation marker. We tried to separate the downstream signalling of JAK2 V617F to stimulate myeloproliferation and LAP activity. NB4, a myeloid lineage cell line, was transduced with Jak2 V617F mutation or wild‐type Jak2. We found that Jak2 V617F mutation, but not wild‐type Jak2 enhanced LAP expression in NB4‐derived neutrophils and proliferation of NB4 cells. JAK2 V617F induces constitutive phosphorylation of STAT3 and STAT5, and uses signalling targets such as Ras/MEK/ERK and PI3K/Akt pathways. By using MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, and STAT3 or STAT5 siRNAs, JAK2 V617F was found to specifically use the STAT3 pathway to enhance LAP expression, while STAT5, Ras/MEK/ERK and PI3K/Akt, but not STAT3 pathways, were able to stimulate cell proliferation. These data strongly suggest that JAK2 V617F uses distinct signalling pathways to induce typical pathological features of MPN, such as high LAP activity and enhanced cell proliferation.

Extra-pancreatic invasion induces lipolytic and fibrotic changes in the adipose microenvironment, with released fatty acids enhancing the invasiveness of pancreatic cancer cells

Pancreatic cancer progression involves components of the tumor microenvironment, including stellate cells, immune cells, endothelial cells, and the extracellular matrix. Although peripancreatic fat is the main stromal component involved in extra-pancreatic invasion, its roles in local invasion and metastasis of pancreatic cancer remain unclear. This study investigated the role of adipose tissue in pancreatic cancer progression using genetically engineered mice (Pdx1-Cre; LSL-KrasG12D; Trp53R172H/+) and an in vitro model of organotypic fat invasion. Mice fed a high fat diet had significantly larger primary pancreatic tumors and a significantly higher rate of distant organ metastasis than mice fed a standard diet. In the organotypic fat invasion model, pancreatic cancer cell clusters were smaller and more elongated in shape and showed increased fibrosis. Adipose tissue-derived conditioned medium enhanced pancreatic cancer cell invasiveness and gemcitabine resistance, as well as inducing morphologic changes in cancer cells and increasing the numbers of lipid droplets in their cytoplasm. The concentrations of oleic, palmitoleic, and linoleic acids were higher in adipose tissue-derived conditioned medium than in normal medium, with these fatty acids significantly enhancing the migration of cancer cells. Mature adipocytes were smaller and the concentration of fatty acids in the medium higher when these cells were co-cultured with cancer cells. These findings indicate that lipolytic and fibrotic changes in peripancreatic adipose tissue enhance local invasiveness and metastasis via adipocyte-released fatty acids. Inhibition of fatty acid uptake by cancer cells may be a novel therapy targeting interactions between cancer and stromal cells.

Landmark-based reconstruction of 3D smooth structures from serial histological sections

Given microscope images, one can observe 2D cross-sections of 3D micro anatomical structures with high spatial resolutions. Each of the 2D microscope images alone is, though, not suitable for studying the 3D anatomical structures and hence many works have been done on a 3D image reconstruction from a given series of microscope images of histological sections obtained from a single target tissue. For the 3D image reconstruction, an image registration technique is necessary because there exists the independent translation, rotation, and non-rigid deformation of the histological sections. In this paper, a landmark-based method of fully non-rigid image registration for the 3D image reconstruction is proposed. The proposed method first detects landmarks corresponded between given images by using a template matching and then non-rigidly deforms the images so that the corresponding landmarks detected in different images are located along a single smooth curve in the reconstructed 3D image. Most of all conventional methods for the reconstruction of 3D microscope image registers two consecutive images at a time and many micro anatomical structures often have unnatural straight shape along the vertical (z) direction in the resultant 3D image because, roughly speaking, the conventional methods registers two given images so that pixels with the same coordinates in the two images have the same pixel value. The proposed method, on the other hand, determine the deformations of all given images by referring to the all images and deforms them simultaneously. In the experiments, a 3D microscope image of the pancreas of a KPC mouse was reconstructed from a series of microscope images of the histological sections.

Adipose tissue-derived stromal cells are sources of cancer-associated fibroblasts and enhance tumor progression by dense collagen matrix: Adipose tissue-derived stromal cells

Although recent studies revealed that adipose tissue accelerates pancreatic tumor progression with excessive extracellular matrix, key players for desmoplasia in the adipose microenvironment remains unknown. Here, we investigated the roles of adipose tissue‐derived stromal cells (ASCs) in desmoplastic lesions and tumor progression by in vitro and in vivo experiments. In a three‐dimensional (3‐D) organotypic fat invasion model using visceral fat from CAG‐EGFP mice, GFP‐positive fibroblastic cells infiltrated toward cancer cells. When tumor cells were inoculated into transplanted visceral fat pads in vivo, tumor weights and stromal components were enhanced compared to subcutaneous and orthotopic tumor cells inoculated without fat pads. Expression of αSMA in established human ASCs was lower compared to cancer associated fibroblasts, and the 3‐D collagen matrices produced by ASCs cultured in cancer cell‐conditioned medium changed from loose to dense structures that affected the motility of cancer cells. Microarray analyses revealed upregulation of S100A4 in ASCs, while S100A4‐positive stromal cells were observed at extrapancreatic invasion sites of human pancreatic cancer. The present findings indicate that ASCs are recruited to extrapancreatic invasion sites and produce dense collagen matrices that lead to enhanced tumor progression. Both inhibition of ASCs recruitment and activation could lead to a novel antistromal therapy.

Basement membrane destruction by pancreatic stellate cells leads to local invasion in pancreatic ductal adenocarcinoma

Stroma invasion is an important step in pancreatic cancer progression. However, how pancreatic ductal adenocarcinoma (PDAC) with ductal structure invades the surrounding stroma has not been clear. Here, we elucidated the mechanism of stromal invasion of PDAC, using organoids. From resected PDAC specimens, we established human PDAC organoids, which developed ductal and basement membrane (BM) structures. When the organoids were co-cultured with pancreatic stellate cells (PSCs) in a collagen matrix, organoids lost their BM and ductal structures, and invaded collagen matrix more frequently than did mono-cultured organoids. Interestingly, direct contact by PSCs to PDAC organoids was observed before BM destruction. Matrix metalloproteinase (MMP) 2 or membrane type-1 MMP (MT1MMP) knockdown in PSCs significantly attenuated BM destruction by PSCs, and retained the ductal structures in organoids. Our results imply that direct contact by PSCs induces BM destruction and stromal invasion of PDAC via MMP2 which binds to MT1MMP on PSCs.

Accurate 3D Reconstruction of a Whole Pancreatic Cancer Tumor from Pathology Images with Different Stains

When applied to 3D image reconstruction, conventional landmark-based registration methods tend to generate unnatural vertical structures due to inconsistencies between the employed model and the real tissue. This paper demonstrates a fully non-rigid image registration method for 3D image reconstruction which considers the spatial continuity and smoothness of each constituent part of the microstructures in the tissue. Corresponding landmarks are detected along the images, defining a set of trajectories, which are smoothed out in order to define a diffeomorphic mapping. The resulting reconstructed 3D image preserves the original tissue architecture, allowing the observation of fine details and structures.

Postmortem interval estimation using the animal model of postmortem gas volume changes

It is important to estimate the postmortem interval in forensic autopsy. Many methods to estimate the postmortem interval have been reported, and are typically associated with internal examination. However, there are issues such as rejection of autopsy by the family and a lack of forensic doctor in internal examination. Therefore, it is necessary to develop new methods, such as autopsy imaging, that can substitute for internal examination. Here, we first evaluated whether gas volume in the body increased with postmortem interval. Time-dependent X-ray CT imaging of euthanized Crl:CD (SD) rats (n = 3) was performed immediately after euthanasia and at seven subsequent time points up to 168 h (7 days) at 24-hour intervals. The data revealed that gas volume in the body increased in a time-dependent manner. Next, we reconstructed 3D images of isolated gas and calculated the gas volume using Amira software. In all cases, the volume of both portal venous gas and intestinal gas increased in a time-dependent manner. The volume of portal venous gas increased exponentially, while the volume of intestinal gas increased in a linearly with time. These data might be suggested that the postmortem gas volume change is one of indicators for estimating the postmortem interval. In addition, it would be possible to estimate more accurate postmortem interval by combining not only gas volume changes at the above two sites but also gas volume changes of the other sites such as heart cavities, kidney parenchyma, or abdominal aorta.