Wataru Ikeda

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells (CSCs)¹, a lack of experimental platforms that enable prospective analysis of CSC dynamics with sufficient spatiotemporal resolution has hindered testing of this hypothesis. Here, we develop a live genetic lineage-tracing system that allows longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids and identify LGR5⁺ CSCs that display a dormant behavior in a chemo-naive state. Dormant LGR5⁺ cells are marked by p27 expression, and intravital imaging directly demonstrates the persistence of LGR5⁺p27⁺ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals an upregulation of COL17A1, a cell adhesion molecule that strengthens hemidesmosome, in dormant LGR5⁺p27⁺ cells. COL17A1-knockout organoids lose the dormant LGR5⁺p27⁺ subpopulation and become sensitive to chemotherapy, suggesting a role of cell-matrix interface in dormancy maintenance. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5⁺p27⁺ cells through FAK-YAP activation. Abrogation of YAP signaling restrains chemo-resistant cells from exiting dormancy and delays tumor regrowth, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome refractoriness of human colorectal cancer to conventional chemotherapy.

Objective: To elucidate the role of the fractalkine (FKN)/CX₃CR1 pathway in joint destruction in rheumatoid arthritis.Methods: We examined the effect of treatment with anti–mouse FKN (anti‐mFKN) monoclonal antibody (mAb) on joint destruction and the migration of osteoclast precursors (OCPs) into the joint, using the collagen‐induced arthritis (CIA) model.DBA/1 mice were immunized with bovine type II collagen to induce arthritis, and then treated with anti‐mFKN mAb. Disease severity was monitored by arthritis score, and joint destruction was evaluated by soft x‐ray and histologic analyses. Plasma levels of joint destruction markers were assessed by enzyme‐linked immunosorbent assay. FKN expression on endothelial cells was detected by immunohistochemistry. Bone marrow–derived OCPs were labeled with fluorescein and transferred to mice with CIA, and the migration of the OCPs to the joints was then analyzed.Results: Both prophylactic and therapeutic treatment with anti‐mFKN mAb significantly decreased the arthritis and soft x‐ray scores. Plasma levels of cartilage oligomeric matrix protein and matrix metalloproteinase 3 decreased after treatment with anti‐mFKN mAb. Histologic analysis revealed that anti‐mFKN mAb inhibited synovitis, pannus formation, and cartilage destruction, as well as suppressed bone damage, with a marked reduction in the number of tartrate‐resistant acid phosphatase–positive osteoclasts. Anti‐mFKN mAb strongly inhibited the migration of bone marrow–derived OCPs into the affected synovium.Conclusion: Anti‐mFKN mAb notably ameliorates arthritis and joint destruction in the CIA model, as well as inhibits migration of OCPs into the synovium. These results suggest that inhibition of the FKN/CX₃CR1 pathway could be a novel strategy for treatment of both synovitis and joint destruction in rheumatoid arthritis.

In vivo imaging of mouse retinas using two-photon or confocal laser scanning fluorescence microscopy is a powerful tool for time-lapse analyses of the dynamic movements of cell populations. However, acute and reversible opacification of the crystalline lenses of mouse eyes under anesthesia decreases the visibility of the ocular fundus. Therefore, we developed a customized contact lens for preventing cataract during continuous retinal imaging in anesthetized mice. This experimental approach will aid in the elucidation of cellular and molecular dynamics in the CNS under physiological and pathological conditions.

Most of preclinical cancer studies use xenograft models established from human cell lines or patient-derived cancer cells subcutaneously implanted into the flank of immunocompromised mice. These models are often assumed to represent the original diseases and are valuable tools, at least to some extent, for understanding both the basic biology of cancer and for proof-of-concept studies of molecularly targeted therapies. However, analyzing the cellular behavior of individual components within xenografts, including tumor cells, stromal cells, immune cells, and blood vessels, is challenging. In particular, it has been difficult and urgently required to trace the whole process of heterogeneous tumor microenvironment formation mediated by various components described above. Here we demonstrate a method for monitoring this process using a window device system that we have recently developed and a subcutaneous xenograft model that accurately recapitulates the histology of human lung adenocarcinoma. Use of our imaging window device and a multiphoton laser scanning microscope provides a powerful tool for investigating tumor heterogeneity and responses to drug treatments in an in vivo live imaging system.

In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRβ antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders.

Tight junctions (TJs) and adherens junctions (AJs) form an apical junctional complex at the apical side of the lateral membranes of epithelial cells, in which TJs are aligned at the apical side of AJs. Many cell adhesion molecules (CAMs) and cell polarity molecules (CPMs) cooperatively regulate the formation of the apical junctional complex, but the mechanism for the alignment of TJs at the apical side of AJs is not fully understood. We developed a cellular system with which epithelial‐like TJs and AJs were reconstituted in fibroblasts and analyzed the cooperative roles of CAMs and CPMs. We exogenously expressed various combinations of CAMs and CPMs in fibroblasts that express negligible amounts of these molecules endogenously. In these cells, the nectin‐based cell–cell adhesion was formed at the apical side of the junctional adhesion molecule (JAM)‐based cell–cell adhesion, and cadherin and claudin were recruited to the nectin‐3‐ and JAM‐based cell–cell adhesion sites to form AJ‐like and TJ‐like domains, respectively. This inversed alignment of the AJ‐like and TJ‐like domains was reversed by complementary expression of CPMs Par‐3, atypical protein kinase C, Par‐6, Crb3, Pals1 and Patj. We describe the cooperative roles of these CAMs and CPMs in the apico‐basal alignment of TJs and AJs in epithelial cells.