7-Dehydrocholesterol is an endogenous suppressor of ferroptosis.

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

Carfilzomib suppressed LDHA-mediated metabolic reprogramming by targeting ATF3 in esophageal squamous cell carcinoma.

Carfilzomib, a second-generation proteasome inhibitor, has been approved as a treatment for relapsed and/or refractory multiple myeloma. Nevertheless, the molecular mechanism by which Carfilzomib inhibits esophageal squamous cell carcinoma (ESCC) progression largely remains to be determined. In the present study, we found that Carfilzomib demonstrated potent anti-tumor activity against esophageal squamous cell carcinoma both in vitro and in vivo. Mechanistically, carfilzomib triggers mitochondrial apoptosis and reprograms cellular metabolism in ESCC cells. Moreover, it has been identified that activating transcription factor 3 (ATF3) plays a crucial cellular target role in ESCC cells treated with Carfilzomib. Overexpression of ATF3 effectively antagonized the effects of carfilzomib on ESCC cell proliferation, apoptosis, and metabolic reprogramming. Furthermore, the ATF3 protein is specifically bound to lactate dehydrogenase A (LDHA) to effectively suppress LDHA-mediated metabolic reprogramming in response to carfilzomib treatment. Research conducted in xenograft models demonstrates that ATF3 mediates the anti-tumor activity of Carfilzomib. The examination of human esophageal squamous cell carcinoma indicated that ATF3 and LDHA have the potential to function as innovative targets for therapeutic intervention in the treatment of ESCC. Our findings demonstrate the novel function of Carfilzomib in modulating ESCC metabolism and progression, highlighting the potential of Carfilzomib as a promising therapeutic agent for the treatment of ESCC.

Protein phosphatase 2A-B55ß mediated mitochondrial p-GPX4 dephosphorylation promoted sorafenib-induced ferroptosis in hepatocellular carcinoma via regulating p53 retrograde signaling.

Rationale: As a key endogenous negative regulator of ferroptosis, glutathione peroxidase 4 (GPX4) can regulate its antioxidant function through multiple post-translational modification pathways. However, the effects of the phosphorylation/dephosphorylation status of GPX4 on the regulation of inducible ferroptosis in hepatocellular carcinoma (HCC) remain unclear. Methods: To investigate the effects and molecular mechanism of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells. Sorafenib (Sora) was used to establish the ferroptosis model in HCC cells in vitro. Using the site-directed mutagenesis method, we generated the mimic GPX4 phosphorylation or dephosphorylation HCC cell lines at specific serine sites of GPX4. The effects of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells were examined. The interrelationships among GPX4, p53, and protein phosphatase 2A-B55ß subunit (PP2A-B55ß) were also explored. To explore the synergistic anti-tumor effects of PP2A activation on Sora-administered HCC, we established PP2A-B55ß overexpression xenograft tumors in a nude mice model in vivo. Results: In the Sora-induced ferroptosis model of HCC in vitro, decreased levels of cytoplasmic and mitochondrial GPX4, mitochondrial dysfunction, and enhanced p53 retrograde signaling occurred under Sora treatment. Further, we found that mitochondrial p53 retrograded remarkably into the nucleus and aggravated Sora-induced ferroptosis. The phosphorylation status of GPX4 at the serine 2 site (GPX4Ser2) revealed that mitochondrial p-GPX4Ser2 dephosphorylation was positively associated with ferroptosis, and the mechanism might be related to mitochondrial p53 retrograding into the nucleus. In HCC cells overexpressing PP2A-B55ß, it was found that PP2A-B55ß directly interacted with mitochondrial GPX4 and promoted Sora-induced ferroptosis in HCC. Further, PP2A-B55ß reduced the interaction between mitochondrial GPX4 and p53, leading to mitochondrial p53 retrograding into the nucleus. Moreover, it was confirmed that PP2A-B55ß enhanced the ferroptosis-mediated tumor growth inhibition and mitochondrial p53 retrograde signaling in the Sora-treated HCC xenograft tumors. Conclusion: Our data uncovered that the PP2A-B55ß/p-GPX4Ser2/p53 axis was a novel regulatory pathway of Sora-induced ferroptosis. Mitochondrial p-GPX4Ser2 dephosphorylation triggered ferroptosis via inducing mitochondrial p53 retrograding into the nucleus, and PP2A-B55ß was an upstream signal modulator responsible for mitochondrial p-GPX4Ser2 dephosphorylation. Our findings might serve as a potential theranostic strategy to enhance the efficacy of Sora in HCC treatment through the targeted intervention of p-GPX4 dephosphorylation via PP2A-B55ß activation.

Effects and mechanisms of trifluridine alone or in combination with cryptotanshinone in inhibiting malignant biological behavior of gastric cancer.

BACKGROUND: The incidence of gastric cancer (GC) ranks fourth among all malignant tumors worldwide, and the fatality rate ranks second among all malignant tumors. Several Chinese traditional medicines have been used in the treatment of advanced gastric cancer. This study aims to investigate the effect of combinational use of natural product cryptotanshinone (CTS) with anti-cancer drug trifluorothymidine (FTD) in GC. METHODS: Cell Counting Kit-8 assay was used to detect the inhibitory effect of the combinational or separate use of FTD and CTS on the growth of HGC-27 and AGS GC cells. The combined index of FTD and CTS was calculated using CompuSyn software. To understand the mechanism, we applied flow cytometry to study the cell cycle and cell apoptosis after treatment. We also investigated the amount of FTD incorporated into the DNA by immunofluorescence assay. The expression of relevant proteins was monitored using western blot. Furthermore, the effect of using TAS-102 in combination with CTS was studied in xenograft tumor nude mice model. RESULTS: FTD and CTS inhibited the growth of GC cells in a dose-dependent manner, respectively. They both exhibited low to sub-micromolar potency in HGC-27 and AGS cells. The combination of FTD and CTS showed synergistic anticancer effect in HGC-27 cells and AGS cells. Our mechanism studies indicate that FTD could block HGC-27 cells at G2/M phase, while CTS could block HGC-27 cells at G1/G0 phase, while FTD combined with CTS could mainly block HGC-27 cells at G2 phase. FTD in combination with CTS significantly increased the apoptosis of HGC-27 cells. We observed that CTS treatment increased the incorporation of FTD into the DNA HGC-27 cell. FTD treatment activated STAT3 phosphorylation in HGC-27 cells, while CTS treatment down-regulated the concentration of p-STAT3. Interestingly, the combination of CTS and FTD reduced STAT3 phosphorylation induced by FTD. In the in vivo experiments, we observed that the combination of TAS-102 with CTS was significantly more potent than TAS-102 on tumor growth inhibition. CONCLUSIONS: FTD combined with CTS has a synergistic anti-gastric cancer effect as shown by in vitro and in vivo experiments, and the combined treatment of FTD and CTS will be a promising treatment option for advanced gastric cancer.

Glioblastoma Stem Cell Targeting Peptide Isolated Through Phage Display Binds Cadherin 2.

Glioblastoma stem cells (GSCs) have unique properties of self-renewal and tumor initiation that make them potential therapeutic targets. Development of effective therapeutic strategies against GSCs requires both specificity of targeting and intracranial penetration through the blood-brain barrier. We have previously demonstrated the use of in vitro and in vivo phage display biopanning strategies to isolate glioblastoma targeting peptides. Here we selected a 7-amino acid peptide, AWEFYFP, which was independently isolated in both the in vitro and in vivo screens and demonstrated that it was able to target GSCs over differentiated glioma cells and non-neoplastic brain cells. When conjugated to Cyanine 5.5 and intravenously injected into mice with intracranially xenografted glioblastoma, the peptide localized to the site of the tumor, demonstrating intracranial tumor targeting specificity. Immunoprecipitation of the peptide with GSC proteins revealed Cadherin 2 as the glioblastoma cell surface receptor targeted by the peptides. Peptide targeting of Cadherin 2 on GSCs was confirmed through ELISA and in vitro binding analysis. Interrogation of glioblastoma databases demonstrated that Cadherin 2 expression correlated with tumor grade and survival. These results confirm that phage display can be used to isolate unique tumor-targeting peptides specific for glioblastoma. Furthermore, analysis of these cell specific peptides can lead to the discovery of cell specific receptor targets that may serve as the focus of future theragnostic tumor-homing modalities for the development of precision strategies for the treatment and diagnosis of glioblastomas.

A short peptide LINC00665_18aa encoded by lncRNA LINC00665 suppresses the proliferation and migration of osteosarcoma cells through the regulation of the CREB1/RPS6KA3 interaction.

Long noncoding RNAs (lncRNAs) encompass short open reading frames (sORFs) that can be translated into small peptides. Here, we investigated the encoding potential of lncRNA LINC00665 in osteosarcoma (OS) cells. Bioinformatic analyses were utilized to predict the lncRNAs with encoding potential in human U2OS cells. Protein expression was assessed by an immunoblotting or immunofluorescence method. Cell viability was assessed by cell counting Kit-8 (CCK-8). Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU) assay. Cell migration was gauged by transwell assay. The downstream effectors of the short peptide were verified using qualitative proteome analysis after immunoprecipitation (IP) experiments. The effect of the short peptide on protein interactions were confirmed by Co-Immunoprecipitation (CoIP) assays. We found that lncRNA LINC00665 encoded an 18-amino acid (aa)-long short peptide (named LINC00665_18aa). LINC00665_18aa suppressed the viability, proliferation, and migration of human MNNG-HOS and U2OS OS cells in vitro and diminished tumor growth in vivo. Mechanistically, LINC00665_18aa impaired the transcriptional activity, nuclear localization, and phosphorylation of cAMP response element-binding protein 1 (CREB1). Moreover, LINC00665_18aa weakened the interaction between CREB1 and ribosomal protein S6 kinase A3 (RPS6KA3, RSK2). Additionally, increased expression of CREB1 reversed the inhibitory effects of LINC00665_18aa on OS cell proliferation and migration. Our findings show that the short peptide LINC00665_18aa exerts a tumor-inhibitory function in OS, providing a new basis for cancer therapeutics through the functions of the short peptides encoded by lncRNAs.

On Target: An Intrapulmonary Transplantation Method for Modelling Lung Tumor Development in its Native Microenvironment.

The development of in vivo lung cancer models that faithfully mimic the human disease is a crucial research tool for understanding the molecular mechanisms driving tumorigenesis. Subcutaneous transplantation assays are commonly employed, likely due to their amenability to easily monitor tumor growth and the simplistic nature of the technique to deliver tumor cells. Importantly however, subcutaneous tumors grow in a microenvironment that differs from that resident within the lung. To circumvent this limitation, here we describe the development of an intrapulmonary (iPUL) orthotopic transplantation method that enables the delivery of lung cancer cells, with precision, to the left lung lobe of recipient mice. Critically, this allows for the growth of lung cancer cells within their native microenvironment. The coupling of iPUL transplantation with position emission tomography (PET) imaging permits the serial detection of tumors in vivo and serves as a powerful tool to trace lung tumor growth and dissemination over time in mouse disease models.

Loss of NLRP3 reduces oxidative stress and polarizes intratumor macrophages to attenuate immune attack on endometrial cancer.

Introduction: The interaction between endometrial cancer (EMC) cells and intratumoral macrophages plays a significant role in the development of the disease. PYD domains-containing protein 3 (NLRP3) inflammasome formation triggers caspase-1/IL-1ß signaling pathways and produces reactive oxygen species (ROS) in macrophages. However, the role of NLRP3-regulated ROS production in macrophage polarization and the subsequent growth and metastasis of EMC remains unknown. Methods: We conducted bioinformatic analysis to compare NLRP3 levels in intratumoral macrophages from EMC and normal endometrium. In vitro experiments involved knocking out NLRP3 in macrophages to shift the polarization from an anti-inflammatory M1-like phenotype to a proinflammatory M2-like phenotype and reduce ROS production. The impact of NLRP3 depletion on the growth, invasion, and metastasis of co-cultured EMC cells was assessed. We also evaluated the effect of NLRP3 depletion in macrophages on the growth and metastasis of implanted EMC cells in mice. Results: Our bioinformatic analysis showed significantly lower NLRP3 levels in intratumoral macrophages from EMC than those from normal endometrium. Knocking out NLRP3 in macrophages shifted their polarization to a proinflammatory M2-like phenotype and significantly reduced ROS production. NLRP3 depletion in M2-polarized macrophages increased the growth, invasion, and metastasis of co-cultured EMC cells. NLRP3 depletion in M1-polarized macrophages reduced phagocytic potential, which resulted in weakened immune defense against EMC. Additionally, NLRP3 depletion in macrophages significantly increased the growth and metastasis of implanted EMC cells in mice, likely due to compromised phagocytosis by macrophages and a reduction in cytotoxic CD8+ T cells. Discussion: Our results suggest that NLRP3 plays a significant role in regulating macrophage polarization, oxidative stress, and immune response against EMC. NLRP3 depletion alters the polarization of intratumoral macrophages, leading to weakened immune defense against EMC cells. The reduction in ROS production by the loss of NLRP3 may have implications for the development of novel treatment strategies for EMC.

Establishment of a rabbit liver metastasis model by percutaneous puncture of the spleen and implantation of the VX2 tumor strain under CT guidance.

This study aimed to compare the feasibility, success rate, and safety of establishing a rabbit VX2 liver metastasis model by percutaneous splenic implantation under CT guidance and open splenic implantation of the VX2 tumor strain. Fifty-two New Zealand white rabbits were randomly divided into group A (the percutaneous puncture group) (n = 26) and group B (the laparotomy group) (n = 26). In group A, 26 New Zealand white rabbits were implanted with tumor strains by percutaneous splenic puncture under CT guidance. In group B, 26 New Zealand white rabbits were implanted with tumor strains in the spleen by laparotomy. After 2-3 weeks of implantation, both group A and group B underwent MRI to confirm tumor growth in the spleen and metastasis to the liver. Two experimental rabbits randomly selected from groups A and B were killed for pathological examination. The success rate, complication rate, and operation time in groups A and B were compared and analyzed. A total of 23 rabbits in group A were successfully induced, and the success rate was 88.5% (23/26). The average time of operation was 14.42 ± 3.26 min. A total of 22 rabbits in group B were successfully induced, and the success rate was 84.6% (22/26). The average time of operation in group B was 23.69 ± 5.27 min. There was no significant difference in the success rate of induction between the two groups (P > 0.05). The MRI manifestations of liver metastases were multiple nodular and punctate abnormal signal shadows in the liver. Hematoxylin-eosin (HE) staining showed a large number of tumor cells in the tumor area. CT-guided percutaneous splenic implantation of the VX2 tumor strain to establish a rabbit liver metastasis model is a minimally invasive and feasible inducing method. The success rate of this technique is not lower than that of open splenic implantation, with low incidence of complications, and short operation time.

Repeated activation of Trpv1-positive sensory neurons facilitates tumor growth associated with changes in tumor-infiltrating immune cells.

It is considered that sensory neurons extend into the tumor microenvironment (TME), which could be associated with tumor growth. However, little is known about how sensory signaling could promote tumor progression. In this study, chemogenetic activation of transient receptor potential vanilloid 1 (Trpv1)-positive sensory neurons (C-fibers) by the microinjection of AAV-hSyn-FLEX-hM3Dq-mCherry into the sciatic nerve dramatically increased tumor volume in tumor-bearing Trpv1-Cre mice. This activation in Trpv1::hM3Dq mice that had undergone tumor transplantation significantly reduced the population of tumor-infiltrating CD4+ T cells and increased the mRNA level of the M2-macrophage marker, CX3C motif chemokine receptor 1 (Cx3cr1) in immunosuppressive cells, such as tumor-associated macrophages (TAMs) and tumor-infiltrating monocytic myeloid-derived suppressor cells (M-MDSCs). Under these conditions, we found a significant correlation between the decreased expression of the M1-macrophage marker Tnf and tumor volume. These findings suggest that repeated activation of Trpv1-positive sensory neurons may facilitate tumor growth along with changes in tumor-infiltrating immune cells.

An intrasplenic injection model of pancreatic cancer metastasis to the liver in mice.

Here, we provide a protocol for an intrasplenic injection model to establish pancreatic tumors in the mouse liver. We describe the steps to inject tumor cells into mouse spleen and to perform a splenectomy, followed by animal recovery and end point analysis of tumors in the liver. This model allows rapid and reproducible tumor growth in a clinically relevant metastatic site, providing a platform to evaluate the efficacy of anti-cancer drugs. This technique can be expanded to other cancer cell lines. For complete details on the use and execution of this protocol, please refer to Poh et al. (2022).1.

Detecting and monitoring tumors in orthotopic colorectal liver metastatic animal models with high-resolution ultrasound.

The ability to noninvasively detect and monitor the growth of orthotopic liver transplantation tumors is critical for replicating advanced colorectal cancer liver metastases (CRLMs) in animal models. We assessed the use of high-resolution ultrasound (HRU) to monitor CRLMs transplanted using various cell concentrations. Sixty BALB/c female mice were randomly divided into 3 groups, and murine colonic CT26 cells were injected into the left liver lobe at concentrations of 1 × 102 (group 1), 1 × 103 (group 2), or 1 × 104 (group 3). Tumor presentation, location, number, size, shape, and echogenicity were assessed daily with 24-MHz center frequency HRU starting 6 days after injection. Animals were sacrificed when the largest tumor was ≥ 1 cm in diameter. Sensitivity, specificity, and area under curve (AUC) of CRLMs diagnosed with HRU were calculated using receiver operating characteristic curve analysis. In group 1, 94% of mice formed < 5 tumors, and 41% formed a single tumor. Tumors were first detected with HRU on day 12 in group 1, day 10 in group 2, and day 7 in group 3; tumor volume doubling times were 14-15 days, 11-12 days, and 7-8 days, respectively. With a long diameter threshold of 2.4 mm, diagnostic sensitivity and specificity of HRU were 94.1% and 88.7%, respectively, and the AUC was 0.962. These findings suggest that HRU can be used to accurately detect and monitor the growth of CRLMs in an orthotopic transplantation mouse model, especially when a lower concentration of cells is used.

Establishment and evaluation of ectopic and orthotopic prostate cancer models using cell sheet technology.

BACKGROUND: The traditional prostate cancer (PCa) model is established by injecting cell suspension and is associated with a low tumor formation rate. Cell sheet technology is one of the advancements in tissue engineering for 3D cell-based therapy. In this study, we established ectopic and orthotopic PCa models by cell sheet technology, and then compared the efficiency of tumor formation with cell suspension injection. METHODS: DU145 cells were seeded on 35 mm temperature-sensitive dishes to form PCa cell sheets, while the cell suspension with the same cell density was prepared. After transplanting into the nude mice, the tumor volumes were measured every 3 days and the tumor growth curves were conducted. At the time points of 2 weeks and 4 weeks after the transplantation, magnetic resonance imaging (MRI) was used to evaluate the transplanting site and distant metastasis. Finally, the mice were sacrificed, and the related tissues were harvested for the further histological evaluation. RESULTS: The orthotopic tumor formation rate of the cell sheet injection group was obviously better than that in cell suspension injection group (100% vs 67%). Compared with cell suspension injection, the tumors of DU145 cell sheet fragments injection had the higher density of micro-vessels, more collagen deposition, and lower apoptosis rate. There was no evidence of metastasis in forelimb, lung and liver was found by MRI and histological tests. CONCLUSION: We successfully cultured the DU145 cell sheet and can be used to establish ectopic and orthotopic PCa tumor-bearing models, which provide an application potential for preclinical drug development, drug-resistance mechanisms and patient individualized therapy.

Effects of Whole-Body Vibration on Breast Cancer Bone Metastasis and Vascularization in Mice.

We evaluated whether whole-body vibration (WBV) prevented bone loss induced by breast cancer (BC) metastasis and the involvement of bone marrow vasculature. One day after orthotopic transplantation of mammary 4T1 tumor cells, 8-week-old BALB/c mice were subjected to 0.3 g/90 Hz vertical vibration for 20 min/day for 5 days/week (BC-WBV) or sham-handled (BC-Sham) over 3 weeks. Age-matched intact mice (Intact) were also sham-handled. Both tibiae were harvested from BC-WBV (n = 7), BC-Sham (n = 9), and Intact (n = 5) mice for bone structure imaging by synchrotron radiation-based computed tomography (SRCT) and hematoxylin and eosin staining, whereas right tibiae were harvested from other BC-WBV and BC-Sham (n = 6 each) mice for vascular imaging by SRCT. Tumor cells were similarly widespread in the marrow in BC-WBV and BC-Sham mice. In BC-Sham mice, cortical bone volume, trabecular volume fraction, trabecular thickness, trabecular number density, and bone mineral density were smaller, and marrow volume and trabecular separation were larger than in Intact mice. However, although trabecular thickness was smaller in BC-WBV than Intact mice, the others did not differ between the two groups. Serum osteocalcin tended to be higher in BC-WBV than BC-Sham mice. Compared with BC-Sham mice, BC-WBV mice had a smaller vessel diameter, a trend of a larger vessel number density, and smaller vessel diameter heterogeneity. In conclusion, WBV mitigates bone loss in BC bone metastasis, which may be partly due to increased bone anabolism. The alteration of marrow vasculature appears to be favorable for anti-tumor drug delivery. Further studies are needed to clarify the multiple actions of WBV on bone, tumor, and marrow vasculature and how they contribute to bone protection in BC metastasis.

In vivo accurate detection of the liver tumor with pharmacokinetic parametric images from dynamic fluorescence molecular tomography.

SIGNIFICANCE: Pharmacokinetic parametric images in dynamic fluorescence molecular tomography (FMT) can describe three-dimensional (3D) physiological and pathological information inside biological tissues, potentially providing quantitative assessment tools for biological research and drug development. AIM: In vivo imaging of the liver tumor with pharmacokinetic parametric images from dynamic FMT based on the differences in metabolic properties of indocyanine green (ICG) between normal liver cells and tumor liver cells inside biological tissues. APPROACH: First, an orthotopic liver tumor mouse model was constructed. Then, with the help of the FMT/computer tomography (CT) dual-modality imaging system and the direct reconstruction algorithm, 3D imaging of liver metabolic parameters in nude mice was achieved to distinguish liver tumors from normal tissues. Finally, pharmacokinetic parametric imaging results were validated against in vitro anatomical results. RESULTS: This letter demonstrates the ability of dynamic FMT to monitor the pharmacokinetic delivery of the fluorescent dye ICG in vivo, thus, enabling the distinction between normal and tumor tissues based on the pharmacokinetic parametric images derived from dynamic FMT. CONCLUSIONS: Compared with CT structural imaging technology, dynamic FMT combined with compartmental modeling as an analytical method can obtain quantitative images of pharmacokinetic parameters, thus providing a more powerful research tool for organ function assessment, disease diagnosis and new drug development.

Topical therapy for regression and melanoma prevention of congenital giant nevi.

Giant congenital melanocytic nevi are NRAS-driven proliferations that may cover up to 80% of the body surface. Their most dangerous consequence is progression to melanoma. This risk often triggers preemptive extensive surgical excisions in childhood, producing severe lifelong challenges. We have presented preclinical models, including multiple genetically engineered mice and xenografted human lesions, which enabled testing locally applied pharmacologic agents to avoid surgery. The murine models permitted the identification of proliferative versus senescent nevus phases and treatments targeting both. These nevi recapitulated the histologic and molecular features of human giant congenital nevi, including the risk of melanoma transformation. Cutaneously delivered MEK, PI3K, and c-KIT inhibitors or proinflammatory squaric acid dibutylester (SADBE) achieved major regressions. SADBE triggered innate immunity that ablated detectable nevocytes, fully prevented melanoma, and regressed human giant nevus xenografts. These findings reveal nevus mechanistic vulnerabilities and suggest opportunities for topical interventions that may alter the therapeutic options for children with congenital giant nevi.

[Establishment of An Animal Model of Acute B Lymphoblastic Leukemia with Minimal Residual Disease].

OBJECTIVE: To establish an animal model of acute B lymphoblastic leukemia (B-ALL) with minimal residual disease. METHODS: The transplanted tumor was formed by subcutaneous injection of 2×107 Nalm-6 cells, and the body weight, activity status and tumor formation status of nude mice were observed. Peripheral blood, bone marrow, liver and spleen and other tissues of nude mice were taken for pathological examination to understand whether the success of subcutaneous modeling was accompanied by systemic metastasis. RESULTS: There were 2×107 Nalm-6 cells injected subcutaneously in nude mice, (11.0±2.5) days later, the tumors of (3-4) × (3-4) mm were observed, the body weight of the nude mice was reduced and activity showed no limited. Infiltration of tumor cells in liver, spleen and bone marrow were observed in pathological sections. CONCLUSION: The animal model of subcutaneous tumor of B-ALL was successfully established in nude mice.

Establishment of Embryonic Zebrafish Xenograft Assays to Investigate TGF-ß Family Signaling in Human Breast Cancer Progression.

Transforming growth factor-ß (TGF-ß) family members have pivotal functions in controlling breast cancer progression, acting not only on cancer cells but also on other cells within the tumor microenvironment. Here we describe embryonic zebrafish xenograft assays to investigate how TGF-ß family signaling controls breast cancer cell intravasation, extravasation and regulates tumor angiogenesis. Fluorescently mCherry-labeled breast cancer cells are injected in the perivitelline space or Duct of Cuvier of Tg (fli:EGFP) transgenic Casper zebrafish embryos, in which the zebrafish express enhanced green fluorescent protein in the entire vasculature. The dynamic responses of migratory and invasive human cancer cells, and the induction of new blood vessel formation by the cancer cells in zebrafish host, are visualized using a fluorescent microscope. These assays provide efficient, reliable, low-cost models to investigate the effect of (epi)genetic modulators and pharmacological compounds that perturb the activity of TGF-ß family signaling components on breast cancer cell metastasis and angiogenesis.