Aberrant activation of Rho/ROCK signaling in impaired polarity switching of colorectal micropapillary carcinoma.

Micropapillary carcinoma (MPC) is a morphologically distinctive form of carcinoma, composed of small nests of cancer cells surrounded by lacunar spaces. Invasive MPC is associated with poor prognosis. The nests of tumor cells in MPC reportedly exhibit reverse polarity, although the molecular mechanisms underlying MPC patterns are poorly understood. Using the cancer tissue-originated spheroid (CTOS) method, we previously reported polarity switching in colorectal cancer (CRC). When cultured in suspension, the apical membrane promptly switches from the outside surface of the CTOSs to the surface of the lumen inside the CTOSs under extracellular matrix (ECM)-embedded conditions, and vice versa. Here, we investigated two CTOS lines from CRC patient tumors with MPC lesions. Xenograft tumors from the CTOSs exhibited the MPC phenotype. The MPC-CTOSs did not switch polarity in vitro. Time-course analysis of polarity switching using real-time imaging of the apical membrane revealed that local switching was continually propagated in non-MPC-CTOSs, while MPC-CTOSs were unable to complete the process. Integrin ß4 translocated to the outer membrane when embedded in ECM in both MPC and non-MPC-CTOSs. Protein levels, as well as the active form of RhoA, were higher in MPC-CTOSs. The suppression of RhoA activity by GAP overexpression enabled MPC-CTOSs to complete polarity switching both in vitro and in vivo, while overexpression of active RhoA did not affect polarity switching in non-MPC-CTOSs. Pretreatment with a ROCK inhibitor enabled MPC-CTOSs to complete polarity switching both in vitro and in vivo, although delayed treatment after becoming embedded in ECM failed to do so. Thus, the inability to switch polarity might be a cause of MPC, in which the aberrant activation of RhoA plays a critical role. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

High-throughput screening in colorectal cancer tissue-originated spheroids.

Patient-derived cancer organoid culture is an important live material that reflects clinical heterogeneity. However, the limited amount of organoids available for each case as well as the considerable amount of time and cost to expand in vitro makes it impractical to perform high-throughput drug screening using organoid cultures from multiple patients. Here, we report an advanced system for the high-throughput screening of 2427 drugs using the cancer tissue-originated spheroid (CTOS) method. In this system, we apply the CTOS method in an ex vivo platform from xenograft tumors, using machines to handle CTOS and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. CTOS passages in xenograft tumors resulted in minimal changes of morphological and genomic status, and xenograft tumor generation efficiently expanded the number of CTOS to evaluate multiple drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease.

Dynamic Change of Polarity in Primary Cultured Spheroids of Human Colorectal Adenocarcinoma and Its Role in Metastasis.

Intestinal epithelial cells possess apical-basal polarity, which governs the exchange of nutrients and waste. Perturbation of cell polarity appears to be a general feature of cancers, although most colorectal cancers are differentiated adenocarcinomas, in which polarity is maintained to some extent. Little is known about the role of dysregulated polarity in cancer. The cancer tissue-originated spheroid method was applied to the preparation and culture of spheroids. Spheroids were cultured in suspension or in type I collagen gel. Polarity was assessed by IHC of apical markers and electron microscopy. Two types of polarity status in spheroids were observed: apical-in, with apical membrane located at cavities inside the spheroids in type I collagen gel; and apical-out, with apical membrane located at the outermost layer of spheroids in suspension. These polarities were highly interchangeable. Inhibitors of Src and dynamin attenuated the polarity switch. In patients, clusters of cancer cells that invaded vessels had both apical-in and apical-out morphologic features, whereas primary and metastatic tumors had apical-in features. In a mouse liver metastasis model, apical-out spheroids injected into the portal vein became apical-in spheroids in the liver within a few days. Inhibitors of Src and dynamin significantly decreased liver metastasis. Polarity switching was observed in spheroids and human cancer. The polarity switch was critical in an experimental liver metastasis model.

Drug screening and grouping by sensitivity with a panel of primary cultured cancer spheroids derived from endometrial cancer.

Several molecular targeting drugs are being evaluated for endometrial cancer; selecting patients whose cancers are sensitive to these agents is of paramount importance. Previously, we developed the cancer tissue-originated spheroid method for primary cancer cells taken from patients' tumors as well as patient-derived xenografts. In this study, we successfully prepared and cultured cancer tissue-originated spheroids from endometrial cancers. Characteristics of the original tumors were well retained in cancer tissue-originated spheroids including morphology and expression of p53 or neuroendocrine markers. We screened 79 molecular targeting drugs using two cancer tissue-originated spheroid lines derived from endometrioid adenocarcinoma grade 3 and serous adenocarcinoma. Among several hits, we focused on everolimus, a mammalian target of rapamycin complex 1 inhibitor, and YM155, a survivin inhibitor. When sensitivity to everolimus or YM155 was assessed in 12 or 11 cancer tissue-originated spheroids, respectively, from different endometrial cancer patients, the sensitivity varied substantially. The cancer tissue-originated spheroids sensitive to everolimus showed remarkable suppression of proliferation. The phosphorylation status of the mammalian target of rapamycin complex 1 downstream molecules before and after everolimus treatment did not predict the effect of the drug. In contrast, the cancer tissue-originated spheroids sensitive to YM155 showed remarkable cell death. The effect of YM155 was also confirmed in vivo. The histological type correlated with YM155 sensitivity; non-endometrioid adenocarcinomas were sensitive and endometrioid adenocarcinomas were resistant. Non-canonical autophagic cell death was the most likely cause of cell death in a sensitive cancer tissue-originated spheroid. Thus, sensitivity assays using cancer tissue-originated spheroids from endometrial cancers may be useful for screening drugs and finding biomarkers.

Radiation sensitivity assay with a panel of patient-derived spheroids of small cell carcinoma of the cervix.

Small cell carcinoma of the uterine cervix (SCCC) is a rare cancer with a poor prognosis for which no standard treatment exists. Here, we successfully established panels of patient-derived spheroid cultures from six SCCC patient samples by cancer tissue-originated spheroids (CTOS) method. To assess the intrinsic radiosensitivity and mechanism of radioresistance in individual SCCC patients, we further developed an in vitro sensitivity assay for radiation. Radiation sensitivity in the CTOS assay varied among individual cases and was consistent with in vivo radiation sensitivity using CTOS-derived xenograft tumors in the examined cases. Furthermore, by comparing gene expression in CTOSs with different radiosensitivity, we found that expression of hypoxia-inducible factor-1α (HIF-1α) target genes was upregulated in resistant CTOSs. HIF-1α protein levels increased several hours after irradiation. In a radioresistant CTOS, an inhibitor of heat shock protein 90 (HSP90) suppressed radiation-induced HIF-1α expression. Suppression of HIF-1α by small hairpin RNA significantly enhanced the effect of radiation, at least in part by promoting radiation-induced apoptosis. HSP90 inhibitor also increased radiation sensitivity. Our results indicate that radiation-induced HIF-1α upregulation was one mechanism of radioresistance in a radioresistant SCCC CTOS. Accumulating CTOS lines may provide a good platform to study characters of rare cancers like SCCC.

High-dose chemotherapeutics of intravesical chemotherapy rapidly induce mitochondrial dysfunction in bladder cancer-derived spheroids.

Non-muscle invasive bladder cancer is treated with intravesical chemotherapy (IVC) after transurethral resection (TUR) to reduce the probability of recurrence. Despite improvement, the recurrence rate remains high. Intravesical chemotherapeutics at high doses are expected to ablate unresected tumors and floating cancer cells after TUR, although the fate of bladder cancer cells exposed to high-dose chemotherapeutics remains unclear. In this study, we utilized cancer tissue-originated spheroids (CTOS) prepared from bladder cancers or patient-derived xenografts, which may recapitulate human tumors better than 2-D cultures of established cell lines. We exposed CTOS to 1 mg/mL of epirubicin (EPI) or mitomycin C (MMC) for 2 h. EPI was promptly and homogeneously distributed into cancer cells in the CTOS. Two hours after exposure to MMC, the mitochondrial membrane potential decreased and the mitochondria were fragmented, while plasma membrane integrity was maintained. ATP levels rapidly decreased in CTOS after exposure to EPI or MMC. Although activation of the apoptotic pathway was confirmed by the advent of cleaved poly (ADP-ribose) polymerase, fragmentation of DNA (a hallmark of apoptosis) was not observed in CTOS after exposure to EPI and MMC. In the CTOS prepared directly from 19 surgical specimens exposed to EPI and MMC, the decrease of ATP levels varied among patients. Further establishment of the test might help the drug selection and the prediction of recurrence for individual patients.

Retaining cell-cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer.

Primary culture of the cancer cells from patients' tumors can provide crucial information of individual tumors, yet the technology has not been optimized until now. We developed an innovative culture method for primary colorectal cancer cells, based on the principle that cell-cell contact of cancer cells was maintained throughout the process. When tumor tissue was dissociated into cell clusters, in which cell-cell contact was retained, they rapidly formed spheroids that we termed cancer tissue-originated spheroids (CTOSs). CTOSs of colorectal cancer consisted of highly purified and viable cancer cells, and they were prepared with high efficiency. In immunodeficient mice, CTOSs formed xenograft tumors that retained the features of the parental tumors. Moreover, CTOSs were able to be cultured and expanded in vitro using a 3D culture system and stem cell culture medium. This method allowed evaluation of chemosensitivity and signal pathway activation in cancer cells from individual patients. Easy preparation and culture of pure primary cancer cells provides an innovative platform for studying cancer biology and developing personalized medicine.

Involvement of heregulin/HER3 in the primary culture of human urothelial cancer.

PURPOSE: We previously established a novel method of human colorectal cancer primary culture. This method, termed the cancer tissue originated spheroid method, involves the preparation of multicellular spheroids of primary cancer cells that are cultured so that cell-cell contact is maintained. We applied this method to human urothelial cancer. MATERIALS AND METHODS: Cancer tissue originated spheroids were prepared from xenografts or primary human bladder urothelial cancer tumors following the same protocol used for human colorectal cancer. Cancer tissue originated spheroids were characterized using immunohistochemistry, Western blot and polymerase chain reaction. RESULTS: We established a xenograft from a primary bladder urothelial cancer, and isolated and cultured cancer tissue originated spheroids from the xenograft tumor. Cancer tissue originated spheroids retained the characteristics of the original tumor and those of the xenograft. Heregulin promoted cancer tissue originated spheroid growth, and inhibitors of PI3K and mTOR inhibited heregulin induced growth, as did lapatinib but not erlotinib. We also prepared cancer tissue originated spheroids from primary bladder urothelial cancer. The success rate of establishing primary cancer tissue originated spheroids from nonmuscle invasive urothelial cancer was 90.7% and that from muscle invasive cancer was 68.2%. The overall success rate was 84.2%. Heregulin promoted the growth of primary cancer tissue originated spheroids from 4 of 7 patients. CONCLUSIONS: We report a method of establishing primary cultures of human urothelial cancer cells. Growth stimulation by heregulin in cancer tissue originated spheroids from xenografts and primary tumors suggests the possibility of molecular targeting therapy against HER3 signaling for human urothelial cancer. The cancer tissue originated spheroid method might be useful for selecting patients for molecular targeting drugs such as lapatinib.

Cellular hypoxia of pancreatic beta-cells due to high levels of oxygen consumption for insulin secretion in vitro.

Cellular oxygen consumption is a determinant of intracellular oxygen levels. Because of the high demand of mitochondrial respiration during insulin secretion, pancreatic ß-cells consume large amounts of oxygen in a short time period. We examined the effect of insulin secretion on cellular oxygen tension in vitro. We confirmed that Western blotting of pimonidazole adduct was more sensitive than immunostaining for detection of cellular hypoxia in vitro and in vivo. The islets of the diabetic mice but not those of normal mice were hypoxic, especially when a high dose of glucose was loaded. In MIN6 cells, a pancreatic ß-cell line, pimonidazole adduct formation and stabilization of hypoxia-inducible factor-1α (HIF-1α) were detected under mildly hypoxic conditions. Inhibition of respiration rescued the cells from becoming hypoxic. Glucose stimulation decreased cellular oxygen levels in parallel with increased insulin secretion and mitochondrial respiration. The cellular hypoxia by glucose stimulation was also observed in the isolated islets from mice. The MIN6 cells overexpressing HIF-1α were resistant to becoming hypoxic after glucose stimulation. Thus, glucose-stimulated ß-cells can become hypoxic by oxygen consumption, especially when the oxygen supply is impaired.

[Hypoxic microenvironment and cancer dormancy].

The microenvironment of solid tumors is heterogeneous, and hypoxia is associated with the malignancy of cancers. In this review, we introduce the characteristics of cancer cells in hypoxic areas. The hypoxic response of cancer cells in vivo is different from that in vitro. The expression of MYC is downregulated in the cancer cells in hypoxic regions. Cancer cell proliferation and its metabolism are dramatically suppressed in the hypoxic area, and are in a stage of cellular dormancy. Dual deficiency of oxygen and glucose in vitro induces dormancy of cancer cells in hypoxic regions. Researchers have focused on the proliferation of cancer cells so far, but aside from proliferation, it is important for the survival of cancer cells that they become inactive/ dormant in the hypoxic regions. Dormant cancer cells become resistant to chemotherapy and irradiation, leading to recurrence and metastasis. Therefore, we need to characterize and target dormant cancer cells in the hypoxic regions.

Dose response of a radiophotoluminescent glass dosimeter for TomoTherapy, CyberKnife, and flattening-filter-free linear accelerator output measurements in dosimetry audit.

PURPOSE: We experimentally determined the radiophotoluminescent glass dosimeter (RPLD) dose responses for TomoTherapy, CyberKnife, and flattening-filter-free (FFF) linear accelerator (linac) outputs for dosimetry audits in Japan. METHODS: A custom-made solid phantom with a narrow central-axis spacing of three RPLD elements was used for output measurement to minimise the dose-gradient effect of the non-flattening filter beams. For RPLD dose estimation, we used the ISO 22127 formalism. Additional unit-specific correction factors were introduced and determined via the measured data. For TomoTherapy (7 units) and CyberKnife (4 units), the doses were measured under machine-specific reference fields. For FFF linac (5 units), in addition to the reference condition, we obtained the field-size effects for the range from 5×5 cm to 25×25 cm. RESULTS: The correction factors were estimated as 1.008 and 0.999 for TomoTherapy and CyberKnife, respectively. For FFF linac, they ranged from 1.011 to 0.988 for 6 MV and from 1.011 to 0.997 for 10 MV as a function of the side length of the square field from 5 to 25 cm. The estimated uncertainties of the absorbed dose to water measured by RPLD for the units were 1.32%, 1.35%, and 1.30% for TomoTherapy, CyberKnife, and FFF linac, respectively. A summary of the dosimetry audits of these treatment units using the obtained correction factors is also presented. The average percentage differences between the measured and hospital-stated doses were <1% under all conditions. CONCLUSION: RPLD can be successfully used as a dosimetry audit tool for modern treatment units.

Early reduction of glucose uptake after cisplatin treatment is a marker of cisplatin sensitivity in ovarian cancer.

Cisplatin is an effective chemotherapeutic agent for ovarian cancer, but the sensitivity of cancers differs in individual cases. Because cisplatin is reported to suppress glucose uptake, we investigated the correlation between glucose uptake and sensitivity to the drug. A fluorescent derivative of D-glucose, 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl) amino]-2-deoxyglucose), was used to evaluate glucose uptake. Two ovarian cancer cell lines, SKOV-3 as a relatively resistant line and OVCAR-3 as a relatively sensitive line, were analyzed. Both cell lines had a decreased number of cells accompanied by cell death 24 h after cisplatin treatment, but not at 3 h. In contrast, glucose uptake was decreased 3 h after high-dose cisplatin treatment, which correlated with the sensitivity to the drug at 24 h. The protein levels of glucose transporter 1 (GLUT1) did not change with cisplatin treatment. In contrast, the membrane localization of GLUT1 disappeared after cisplatin treatment. Other cisplatin-resistant cell lines did not show an early decrease in glucose uptake after cisplatin treatment. The early decrease in glucose uptake and later cell death also correlated in cultured cancer cells from ovarian cancer patients. Thus, the decrease in glucose uptake at an early time point after high dose cisplatin treatment reflected cisplatin chemosensitivity in ovarian cancer cells. Measuring glucose uptake might be useful as a rapid evaluation of cisplatin chemosensitivity in ovarian cancer patients.

Thioredoxin binding protein-2 mediates metabolic adaptation in response to lipopolysaccharide in vivo.

OBJECTIVES: Endotoxin triggers a reorganization of the energy metabolic pathway, including the promotion of fatty acid utilization to adapt to a high energy demand during endotoxemia. However, the factors responsible for the metabolic adaptation and characteristic pathologies resulting from defective utilization fatty acids during endotoxin response have not been fully clarified. The thioredoxin binding protein-2 (TBP-2) knockout (TBP-2) mouse is an animal model of fatty acid oxidation disorder. The aim of this study was to determine whether and how TBP-2 is involved in metabolic regulation in a lipopolysaccharide (LPS)-induced endotoxemia model in mice. DESIGN: Prospective animal trial. SETTING: Research laboratory. SUBJECTS: TBP-2 and wild control mice. INTERVENTION: TBP-2 and wild control mice were intraperitoneally injected with LPS. Mortality, serum levels of markers of hepatorenal injuries, cytokines, insulin, glucose and lipid derivatives, and the hepatic signaling pathway regulating gluconeogenesis were investigated. MEASUREMENTS AND MAIN RESULTS: Following the administration of LPS, TBP-2 mice showed a predisposition for death without any significant elevation of inflammatory cytokines, compared to the wild mice. LPS-challenged TBP-2 mice showed fat deposition in the liver and kidney, organ injuries, glycogen depletion, and elevation of serum lipid derivatives such as free fatty acids, triglyceride and cholesterol. Hyperinsulinemia and hypoglycemia were observed in TBP-2 mice after LPS injection. Death due to the LPS administration was prevented by supplementation of glucose. Phosphorylation of Akt and FoxO1, an inhibitory pathway of gluconeogenesis in the liver of LPS-challenged TBP-2 mice was demonstrated, suggesting the enhancement of insulin signaling. CONCLUSIONS: TBP-2 is involved in metabolic control during LPS-induced endotoxemia. After the LPS challenge, TBP-2 mice showed several characteristic aspects, such as hepatorenal injuries, and dysregulation of the lipid and glucose metabolisms. Furthermore, hypoglycemia promoted by hyperinsulinemia may be a critical risk factor for mortality in circumstances in which fatty acid utilization is impaired during endotoxemia.

Effects of aging and hypoxia-inducible factor-1 activity on angiogenic cell mobilization and recovery of perfusion after limb ischemia.

Ischemia is a stimulus for production of angiogenic cytokines that activate local vascular cells and mobilize angiogenic cells to the circulation. These responses are impaired in elderly patients with peripheral arterial disease. Hypoxia-inducible factor (HIF)-1 mediates adaptive responses to ischemia, including production of angiogenic cytokines. In this study, we demonstrate that aging and HIF-1 loss-of-function impair the expression of multiple angiogenic cytokines, mobilization of angiogenic cells, maintenance of tissue viability, and recovery of limb perfusion following femoral artery ligation. We show that HIF-1 directly activates transcription of the gene encoding stem cell factor and that mice lacking the cognate receptor C-KIT have impaired recovery from ischemia. Administration of AdCA5, an adenovirus encoding a constitutively active form of HIF-1alpha, improved the recovery of perfusion in older mice to levels similar to those in young mice. Injection of AdCA5 into nonischemic limb was sufficient to increase the number of circulating angiogenic cells. These results indicate that HIF-1 activity is necessary and sufficient for the mobilization of angiogenic cells and that HIF-1alpha gene therapy can counteract the pathological effects of aging in a mouse model of limb ischemia.

Thioredoxin and thioredoxin binding protein 2 in the liver.

Thioredoxin (TRX) is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys- and constitutes a major thiol reducing system. TRX protects cells from stress-induced damage through antioxidative, antiapoptotic, and anti-inflammatory effect. In animal models, thioacetamide (TAA)-induced acute hepatitis and TAA-induced liver fibrosis was attenuated in TRX transgenic (TRXTG) mice. Plasma level of TRX is a good marker for hepatitis and nonalcoholic steatohepatitis (NASH) in human patients. Recently, we identified TRX binding protein 2 (TBP2) in a yeast two-hybrid screening. TBP2 regulates both the expression and reducing activity of TRX as well as cell growth. TBP2 knockout (TBP2KO) mice showed disorder in lipid metabolism. TBP2 plays a multiple role on cell growth and lipid and glucose metabolism. Thus, TRX and TBP2 play important roles in the pathophysiology of liver diseases, including NASH, indicating that ratio of TRX and TBP2 expression could be a novel marker of liver diseases like NASH.

Hypoxia-inducible factor-1-dependent repression of E-cadherin in von Hippel-Lindau tumor suppressor-null renal cell carcinoma mediated by TCF3, ZFHX1A, and ZFHX1B.

A critical event in the pathogenesis of invasive and metastatic cancer is E-cadherin loss of function. Renal clear cell carcinoma (RCC) is characterized by loss of function of the von Hippel-Lindau tumor suppressor (VHL), which negatively regulates hypoxia-inducible factor-1 (HIF-1). Loss of E-cadherin expression and decreased cell-cell adhesion in VHL-null RCC4 cells were corrected by enforced expression of VHL, a dominant-negative HIF-1alpha mutant, or a short hairpin RNA directed against HIF-1alpha. In human RCC biopsies, expression of E-cadherin and HIF-1alpha was mutually exclusive. The expression of mRNAs encoding TCF3, ZFHX1A, and ZFHX1B, which repress E-cadherin gene transcription, was increased in VHL-null RCC4 cells in a HIF-1-dependent manner. Thus, HIF-1 contributes to the epithelial-mesenchymal transition in VHL-null RCC by indirect repression of E-cadherin.

Spontaneous transformation of cultured mouse bone marrow-derived stromal cells.

Bone marrow-derived stromal cells have engendered interest because of their therapeutic potential for promoting tissue vascularization and repair. When mononuclear cells isolated from mouse bone marrow were cultured in DMEM supplemented with 10% fetal bovine serum, cell populations arose that showed rapid proliferation and loss of contact inhibition. These cells formed invasive soft tissue sarcomas after i.m. injection into nude or scid mice. I.v. injection resulted in the formation of tumor foci in the lungs. The tumors were transplantable into syngeneic immunocompetent mice. Direct injection of cultured cells into immunocompetent mice also resulted in tumor formation. Karyotype analysis showed that increased chromosome number and multiple Robertsonian translocations occurred at passage 3 coincident with the loss of contact inhibition. The remarkably rapid malignant transformation of cultured mouse bone marrow cells may have important implications for ongoing clinical trials of cell therapy and for models of oncogenesis.

Spheroid Cultures of Primary Urothelial Cancer Cells: Cancer Tissue-Originated Spheroid (CTOS) Method.

Increasingly, it has been recognized that studying cancer samples from individual patients is important for the development of effective therapeutic strategies and in endeavors to overcome therapy resistance. Primary cultures of cancer cells acutely dissected from individual patients can provide a platform that enables the study and characterization of individual tumors. To that end, we have developed a method for preparing cancer cells in the form of multi-cellular spheroids. The cells can be derived from patient tumors (primary cells), from patient-derived xenografts, or from genetically- or chemically induced animal tumors. This method of culturing spheroids composed of cells derived from cancer tissues can be applied to various types of cancer, including urothelial cancer. The method is based on the principle of retaining cell-cell contact throughout cancer cell preparation and culturing. The first step is a partial digestion of the tumor specimen into small fragments; these fragments spontaneously form spheroidal shapes within several hours. The spheroid is referred to as a cancer tissue-originated spheroid (CTOS). The advantage of the CTOS method is that it allows one to prepare pure cancer cells at high yield. CTOSs can be stably cultured in serum-free conditions. The CTOS method can be applied to drug sensitivity assays, drug screening, and analyses of intracellular signaling. Moreover, the CTOS method provides a platform for studying the nature of cancer cell clusters.

Promotion of malignant phenotype after disruption of the three-dimensional structure of cultured spheroids from colorectal cancer.

Individual and small clusters of cancer cells may detach from the edges of a main tumor and invade vessels, which can act as the origin of metastasis; however, the mechanism for this phenomenon is not well understood. Using cancer tissue-originated spheroids, we studied whether disturbing the 3D architecture of cancer spheroids can provoke the reformation process and progression of malignancy. We developed a mechanical disruption method to achieve homogenous disruption of the spheroids while maintaining cell-cell contact. After the disruption, 9 spheroid lines from 9 patient samples reformed within a few hours, and 3 of the 9 lines exhibited accelerated spheroid growth. Marker expression, spheroid forming capacity, and tumorigenesis indicated that stemness increased after spheroid disruption. In addition, the spheroid forming capacity increased in 6 of 11 spheroid lines. The disruption signature determined by gene expression profiling supported the incidence of remodeling and predicted the prognosis of patients with colorectal cancer. Furthermore, WNT and HER3 signaling were increased in the reformed spheroids, and suppression of these signaling pathways attenuated the increased proliferation and stemness after the disruption. Overall, the disruption and subsequent reformation of cancer spheroids promoted malignancy-related phenotypes through the activation of the WNT and ERBB pathways.

Cell-surface thioredoxin-1: possible involvement in thiol-mediated leukocyte-endothelial cell interaction through lipid rafts.

Human thioredoxin-1 (hTrx) exhibits a disulfide reducing activity and was originally identified as a soluble cytokine-like factor secreted from cells of a human T-cell leukemia virus type I (HTLV-I)-transformed cell line. Recent studies have revealed that endogenous Trx plays an important role in cytoprotection against various oxidative stress-associated disorders. However, the function of exogenous Trx is still not fully understood. We report here that a cysteine-modified mutant of recombinant human Trx (rhTrx-C35S) binds to human umbilical vein endothelial cells (HUVECs) as well as stimulated T cells and rapidly enters these cells via lipid rafts. In addition, we found that endogenous Trx is expressed on the surface of HUVECs, including lipid rafts. These events suggest cell-surface Trx as a possible target of rhTrx-C35S. Furthermore, we found that anti-human Trx mouse monoclonal antibody inhibits adherence of LPS-stimulated human peripheral blood polymorphonuclear cells (PMNs) to HUVECs. This adherence was also suppressed by a recombinant human Trx (rhTrx), but not by a mutant rhTrx (rhTrx-C32S/C35S) with no reducing activity. Cell-surface Trx may be involved in the process of interaction between PMNs and HUVECs and a possible target of cysteine-modified exogenous Trx as well as wild-type exogenous Trx through redox regulation.