Low Metastatic Potential Research Articles

Abstract 1045: Understanding breast cancer metastasis through circulating tumor cells

Abstract Due to the advance in technologies for rare cell isolation, circulating tumor cells (CTC) have recently received vast interest. During tumor progression tumor cells invade the primary tumor microenvironment and intravasate into blood vessels, where they are referred as CTCs. These CTCs disseminate to other organs and a subset of these cells will be able to form metastasis. The growing interest for CTC is confronted with the difficulty associated with their isolation and characterization. To address this challenge, our lab has recently optimized the ex vivo culture condition and was able to establish CTC lines from breast cancer patients. These CTC lines constitute a unique cell population in the metastatic process and gave us a rare opportunity to investigate the signal pathways involved in each step of the metastatic cascade. To assess the metastatic potential of breast cancer patient-derived CTC lines we utilized an experimental mouse model for metastasis by injecting CTCs directly into the left ventricle of the heart in female immuodeficient NSG mice. The ability of 4 CTC lines to arrest and colonize in organ was monitored by bioluminescence imaging once every 2 weeks for 5 months. CTC lines were capable of generating brain, lung, bone and ovary metastases. Most of those organs are common sites of metastases in breast cancer patients. Two patient-derived CTC lines have a high metastatic potential (over 80% of mice had metastases after 3 months) with generation of simultaneous metastases in multiple organs such as bone, lung and Ovary. These mice remained brain-metastases free for up to 8 months. However, two other patient-derived CTC lines demonstrated the brain as preferential site of metastasis despite their overall low metastatic potential. Interestingly, of 4 breast cancer patients where CTC lines were generated, one developed a metastatic brain tumor and her CTC line has the highest risk of brain metastases in our mouse model. We further investigated genetic and epigenetic determinants that regulate the organotropism of CTCs. We isolated metastatic variants corresponding to a subpopulation of CTCs with a preferential tropism for the brain, lung and bone. Gene expression analysis (RNA-seq) of these variants identified potential gene signatures of breast cancer metastasis. The pathways with the highest enrichment scores were glioblastoma multiform, ceramide biosynthesis and PCP pathway for the brain metastatic variants and were interferon and mTOR signaling for the lung metastatic variants. Next we used a method for assaying chromatin accessibility (ATAC-seq) and identified potential regulatory regions mediating the organ tropism in breast cancer. Together our data provide the evidence of a promising role of CTC as an early prognostic factor in metastasis. Additionally, we expect to develop novel organ tropism associated markers, which can be considered for potential therapeutic targets in breast cancers. Citation Format: Remi Klotz, Thomas Amal, Alan Wang, Matthew Mackay, Kathleen Heller, Lin Li, Maxwell Serowoky, Grace Lee, Jane Han, Andrew Smith, Min Yu. Understanding breast cancer metastasis through circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1045. doi:10.1158/1538-7445.AM2017-1045

CORR Insights®: Pathologically Benign Lymph Nodes Can Mimic Malignancy on Imaging in Patients With Angiomatoid Fibrous Histiocytoma.

CORR Insights®: Pathologically Benign Lymph Nodes Can Mimic Malignancy on Imaging in Patients With Angiomatoid Fibrous Histiocytoma.

Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma

BackgroundRetinoblastoma (Rb) is the most common primary intraocular tumor in children. Local treatment of the intraocular disease is usually effective if diagnosed early; however advanced Rb can metastasize through routes that involve invasion of the choroid, sclera and optic nerve or more broadly via the ocular vasculature. Metastatic Rb patients have very high mortality rates. While current therapy for Rb is directed toward blocking tumor cell division and tumor growth, there are no specific treatments targeted to block Rb metastasis. Two such targets are matrix metalloproteinases-2 and -9 (MMP-2, −9), which degrade extracellular matrix as a prerequisite for cellular invasion and have been shown to be involved in other types of cancer metastasis. Cancer Clinical Trials with an anti-MMP-9 therapeutic antibody were recently initiated, prompting us to investigate the role of MMP-2, −9 in Rb metastasis.MethodsWe compare MMP-2, −9 activity in two well-studied Rb cell lines: Y79, which exhibits high metastatic potential and Weri-1, which has low metastatic potential. The effects of inhibitors of MMP-2 (ARP100) and MMP-9 (AG-L-66085) on migration, angiogenesis, and production of immunomodulatory cytokines were determined in both cell lines using qPCR, and ELISA. Cellular migration and potential for invasion were evaluated by the classic wound-healing assay and a Boyden Chamber assay.ResultsOur results showed that both inhibitors had differential effects on the two cell lines, significantly reducing migration in the metastatic Y79 cell line and greatly affecting the viability of Weri-1 cells. The MMP-9 inhibitor (MMP9I) AG-L-66085, diminished the Y79 angiogenic response. In Weri-1 cells, VEGF was significantly reduced and cell viability was decreased by both MMP-2 and MMP-9 inhibitors. Furthermore, inhibition of MMP-2 significantly reduced secretion of TGF-β1 in both Rb models.ConclusionsCollectively, our data indicates MMP-2 and MMP-9 drive metastatic pathways, including migration, viability and secretion of angiogenic factors in Rb cells. These two subtypes of matrix metalloproteinases represent new potential candidates for targeted anti-metastatic therapy for Rb.

Influence of phosphoglycerate kinase 1 on metastasis and invasion of hepatoma cells and prognosis of liver cancer

Objective: To investigate the association between expression of phosphoglycerate kinase 1 (PGK1) in liver cancer tissue and prognosis, as well as its influence on metastasis and invasion of hepatocellular carcinoma (HCC) cells. Methods: Overexpression and downregulated expression of PGK1 in HCC cells were mediated by lentivirus to establish hepatoma cell lines with different expression levels of PGK1. The Transwell chamber invasion assay, wound healing assay, and colony-forming assay were used to investigate the influence of PGK1 on metastasis, invasion, and proliferation of HCC cells. Immunohistochemistry was used to measure the expression of PGK1 in liver cancer tissue samples from 116 patients with hepatocellular carcinoma who underwent radical surgery, and the Kaplan-Meier method and the log-rank test were used to determine the association between PGK1 expression and prognosis of patients with liver cancer. Results: HCCLM3 and MHCC97H HCC cells with high metastatic potential had significantly higher expression of PGK1 than Hep3B and Huh7 HCC cells with low metastatic potential. Downregulation of PGK1 expression significantly inhibited the migration (31.2% ± 2.4% vs 12.0% ± 1.3%, t = 21.57, P < 0.01), invasion (58 ± 11 vs 21 ± 8, t = 4.687, P < 0.05), and colony-forming ability (168.6 ± 15.1 vs 118.4 ± 8.1, t = 6.650, P < 0.05) of MHCC97H cells, while overexpression of PGK1 enhanced the migration (62.8% ± 4.4% vs 83.6% ± 6.1%, t = 20.56, P < 0.01), invasion (80 ± 12 vs 121 ± 15, t = 4.603, P < 0.05), and colony-forming ability (52.3 ± 8.6 vs 84.7 ± 9.0, t = 27.18, P < 0.01) of Hep3B cells. The high PGK1 expression group had significantly shorter median disease-free survival time and mean survival time than the low PGK1 expression group (22.00 ± 8.51 vs not reached, P < 0.05; 46.00 ± 16.87 vs not reached, P < 0.01). Conclusions: PGK1 is involved in the regulation of metastasis and invasion of HCC cells and can promote the migration and invasion of HCC cells. Therefore, PGK1 may be an important predictor of prognosis and postoperative recurrence in patients with liver cancer.

MiRNA expression profiles reveal the involvement of miR-26a, miR-548l and miR-34a in hepatocellular carcinoma progression through regulation of ST3GAL5

MicroRNAs (miRNAs) have key roles in comprehensive physiological and pathological processes by targeting specific genes through translational repression. Identification of miRNAs related to metastasis enables us to obtain better insight into cancer development. In the current study, we investigated the miRNA expressional profiles in the highly invasive human hepatocellular carcinoma cell line MHCC97-H and MHCC97-L with lower metastatic potential using miRNA microarrays. By quantitative real-time PCR, we confirmed the results of miRNA experiments. Thirteen differentially expressed miRNAs were identified between MHCC97-H and MHCC97-L cells; and the same results were found in clinical samples. Using bioinformatic analysis and luciferase reporter assay, we found that ST3GAL5, a sialyltransferase gene, was the direct target of miR-26a, miR-548l and miR-34a. Engineered expression of miR-26a, miR-548l or miR-34a in MHCC97-H or MHCC97-L cells could significantly change their malignant behaviors and oncogenicity in in vitro and in vivo assays. Manipulated expression of ST3GAL5 also led to the alteration of the metastatic potential of MHCC97-H and MHCC97-L cells, in agreement with the effects of above three miRNAs. Altogether, our data indicate that the levels of these miRNAs may be used as biological markers for evaluating hepatocellular carcinoma progression. miR-26a, miR-548l and miR-34a, acting as tumor suppressors, may exert their effects by regulating ST3GAL5.

SUMO-Specific Cysteine Protease 1 Promotes Epithelial Mesenchymal Transition of Prostate Cancer Cells via Regulating SMAD4 deSUMOylation.

In advanced prostate cancer, small ubiquitin-like modifier (SUMO)-specific cysteine protease 1 (SENP1) is up-regulated. However, the role of SENP1 in regulating deSUMOylation of TGF-β/SMADs signaling is unknown. In this study, we developed a lentiviral vector, PLKO.1-shSENP1, to silence SENP1 in prostate cancer cells with high metastatic characteristics (PC3M). Likewise, we also created an adenovirus vector, Ad5/F11p-SENP1 to over-express SENP1 in prostate cancer cells with low metastatic potential (LNCaP). We showed that silencing of SENP1 promoted cellular apoptosis, and inhibited proliferation and migration of PC3M cells. Moreover, SENP1 silencing increased the SMAD4 expression at protein level, up-regulated E-cadherin and down-regulated Vimentin expression, indicating the inhibition of epithelial mesenchymal transition (EMT). Furthermore, SMAD4 interference abolished SENP1-mediated up-regulation of E-cadherin, suggesting that SENP1 regulated E-cadherin expression via SMAD4. SENP1 over-expression in LNCaP cells reduced SMAD4 protein, and promoted EMT via decreasing E-cadherin and increasing Vimentin. Moreover, down-regulation of SMAD4 and E-cadherin were blocked, after transfection with two SUMOylation sites mutated SMAD4, suggesting that SENP1 might reduce SMAD4 levels to regulate E-cadherin expression via deSUMOylation of SMAD4. In conclusion, SENP1 deSUMOylated SMAD4 to promote EMT via up-regulating E-cadherin in prostate cancer cells. Therefore, SENP1 is a potential target for treatment of advanced prostate cancer.

C-Jun Contributes to Transcriptional Control of GNA12 Expression in Prostate Cancer Cells.

GNA12 is the α subunit of a heterotrimeric G protein that possesses oncogenic potential. Activated GNA12 also promotes prostate and breast cancer cell invasion in vitro and in vivo, and its expression is up-regulated in many tumors, particularly metastatic tissues. In this study, we explored the control of expression of GNA12 in prostate cancer cells. Initial studies on LnCAP (low metastatic potential, containing low levels of GNA12) and PC3 (high metastatic potential, containing high GNA12 levels) cells revealed that GNA12 mRNA levels correlated with protein levels, suggesting control at the transcriptional level. To identify potential factors controlling GNA12 transcription, we cloned the upstream 5′ regulatory region of the human GNA12 gene and examined its activity using reporter assays. Deletion analysis revealed the highest level of promoter activity in a 784 bp region, and subsequent in silico analysis indicated the presence of transcription factor binding sites for C/EBP (CCAAT/enhancer binding protein), CREB1 (cAMP-response-element-binding protein 1), and c-Jun in this minimal element for transcriptional control. A small interfering RNA (siRNA) knockdown approach revealed that silencing of c-Jun expression significantly reduced GNA12 5′ regulatory region reporter activity. In addition, chromatin immunoprecipitation assays confirmed that c-Jun binds to the GNA12 5′ regulatory region in PC3 cells. Silencing of c-Jun expression reduced mRNA and protein levels of GNA12, but not the closely-related GNA13, in prostate cancer cells. Understanding the mechanisms by which GNA12 expression is controlled may aid in the development of therapies that target key elements responsible for GNA12-mediated tumor progression.

MP22-12 POSTOPERATIVE OUTCOME OF CYSTIC RENAL CELL CARCINOMA DEFINED ON PREOPERATIVE IMAGING: A RETROSPECTIVE STUDY

You have accessJournal of UrologyKidney Cancer: Epidemiology & Evaluation/Staging I1 Apr 2017MP22-12 POSTOPERATIVE OUTCOME OF CYSTIC RENAL CELL CARCINOMA DEFINED ON PREOPERATIVE IMAGING: A RETROSPECTIVE STUDY Taesoo Choi, Jun Phil Na, CHUNG UN LEE, Hyunwoo Chung, SEUNGHEE YUM, Hyun Hwan Sung, Hwang Gyun Jeon, Seong Il Seo, Seong Soo Jeon, Hyun Moo Lee, Han Yong Choi, Chan Kyo Kim, and Byong Chang Jeong Taesoo ChoiTaesoo Choi More articles by this author , Jun Phil NaJun Phil Na More articles by this author , CHUNG UN LEECHUNG UN LEE More articles by this author , Hyunwoo ChungHyunwoo Chung More articles by this author , SEUNGHEE YUMSEUNGHEE YUM More articles by this author , Hyun Hwan SungHyun Hwan Sung More articles by this author , Hwang Gyun JeonHwang Gyun Jeon More articles by this author , Seong Il SeoSeong Il Seo More articles by this author , Seong Soo JeonSeong Soo Jeon More articles by this author , Hyun Moo LeeHyun Moo Lee More articles by this author , Han Yong ChoiHan Yong Choi More articles by this author , Chan Kyo KimChan Kyo Kim More articles by this author , and Byong Chang JeongByong Chang Jeong More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.666AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To evaluate postoperative outcome of cystic renal cell carcinoma (RCC) defined on preoperative computed tomography (CT), and to find the optimal cut-off of cystic cystic proportion in association with patients’ prognosis. METHODS In this institutional review board-approved study with waiver of informed consent, a total of 1315 patients who received surgery for single RCC with pre-operative CT were enrolled. The cystic proportion of RCC was determined on CT. The optimal cut-off of cystic proportion was explored regarding cancer-specific survival. The RCCs were categorized as cystic or non-cystic RCCs according to conventional (i.e. cystic proportion = 75%) and the optimal cut-off, and then post-operative outcomes were compared between the two groups. Multivariate Cox regression analysis was performed to determine the independent predictor for cancer-specific survival. RESULTS Of the 1315 lesions, 107 (8.1%) were identified as cystic RCCs according to a conventional cut-off. Postoperative outcome of cystic RCC was significantly better than that of non-cystic RCC (P<0.001). Neither metastasis nor recurrence in patients with cystic RCC was developed after surgery. In association with cancer-specific survival rate, the optimal cut-off of cystic proportion was 45%, and 197 (15.0%) were defined as cystic RCCs accordingly. On Cox regression analysis, cystic proportion = 45% in RCC was an independent predictor of favorable outcome regarding cancer-specific survival (hazard ratio 0.34, P = 0.03). CONCLUSIONS Cystic RCC defined on preoperative CT is associated with low metastatic potential and favorable outcomes after surgery. Particularly, cystic proportion = 45% is an independent prognostic factor for favorable survival. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e261 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Taesoo Choi More articles by this author Jun Phil Na More articles by this author CHUNG UN LEE More articles by this author Hyunwoo Chung More articles by this author SEUNGHEE YUM More articles by this author Hyun Hwan Sung More articles by this author Hwang Gyun Jeon More articles by this author Seong Il Seo More articles by this author Seong Soo Jeon More articles by this author Hyun Moo Lee More articles by this author Han Yong Choi More articles by this author Chan Kyo Kim More articles by this author Byong Chang Jeong More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

N-myc downstream-regulated gene 1 inhibits the proliferation and invasion of hepatocellular carcinoma cells via the regulation of integrin β3.

N-myc downstream-regulated gene 1 (NDRG1) is a multifunctional protein associated with carcinogenesis and tumor progression. The function of NDRG1 in hepatocellular carcinoma (HCC) cells remains controversial. The present study investigated the role of NDRG1 in HCC as well as its molecular mechanism using a range of techniques, including western blot analysis, cellular proliferation test, wound healing assay and Transwell assay. In HCC, the levels of NDRG1 expression were highest in the cytoplasm, followed by the membrane, and were lowest in the nucleus. NDRG1 was revealed to inhibit the proliferation and invasion of BEL7402 cells, which facilitated the hypothesis that NDRG1 expression levels may be lower in cell line with a high metastatic potential compared with those in cell lines with a low metastatic potential. However, the present study identified that NDRG1 expression was higher in detached BEL7402 cells and MHCC-97H cells compared with that in attached BEL7402 cells and MHCC-97L cells. Thus, this finding was contrary to what was expected, suggesting that NDRG1 overexpression in the HCC with a high metastatic potential may be the compensatory mechanism. The human HCC BEL7402 cell line demonstrated a significant increase in the capability of motility, invasion and cellular proliferation following NDRG1-short hairpin RNA transfection. Integrin β3 (ITGB3) protein expression was increased in NDRG1-downregulated BEL7402 cells and SMMC7721 cells compared with that in the control cells. The present study suggested that NDRG1 may be a potential anti-tumor target for the treatment of patients with HCC. A potential mechanism for these roles of NDRG1 is by regulating ITGB3 expression; however, this requires additional investigation.

A preliminary study of the relationship between autophagy and metastasis of nasopharyngeal carcinoma cells after X-ray irradiation

Objective To investigate the relationship between autophagy and metastasis of nasopharyngeal cancer (NPC) cell lines 5-8F and 6-10B after X-ray irradiation and the related mechanism. Methods Two substrains, 5-8F and 6-10B, of the NPC cell line SUNE1, with high and low metastatic potentials, respectively, were used in our study. After 4 Gy X-ray irradiation, 5-8F cells were treated with rapamycin (2.0 μmol/L) to induce autophagy and 6-10B cells were treated with LY294002(10 μmol/L) to inhibit autophagy. The autophagy and metastatic activity of NPC cells were determined using qRT-PCR, Western blot, Transwell assay, laser confocal microscopy, and transmission electron microscopy. Results 5-8F cells showed a lower level of autophagy than 6-10B cells after X-ray irradiation. Rapamycin increased the autophagy and inhibited the metastasis of 5-8F cells after irradiation, while LY294002 inhibited the autophagy and increased the metastasis of 6-10B cells. Conclusions NPC 5-8F cells, which have a high metastatic potential, have a lower level of autophagy than 6-10B cells, which have a low metastatic potential. Autophagic inhibition could increase the metastatic activity of NPC cells, while autophagic activation could reduce their metastatic activity. Mechanistic analysis indicates that the PI3K/AKT/mTOR pathway is involved in this process. Key words: X-ray irradiation; Cell line, nasopharyngeal; Autophagy; Metastasis

Sensitivity and specificity of self-reported cancer history compared to cancer registry.

234 Background: The Ontario Health Study (OHS) is a large prospective epidemiologic cohort study in which any Ontario resident eighteen years of older may enroll regardless of prior medical history. Baseline data are collected using web-based tools. As part of the consent process, participants are asked for consent to link study data with administrative and health care claims databases, including the Ontario Cancer Registry (OCR). There is an option to enter their Health Insurance Number (HIN) for this purpose. The purpose of this study was to link these data and evaluate the accuracy of self-reported cancer history compared to the cancer registry. Methods: Consenting participants that provided HINs were deterministically linked to the administrative data. Those that did not were probabilistically linked using name, sex and date of birth. Cancer registry records indicating a cancer diagnosed before the date of completion of the OHS baseline survey were considered the gold standard. Concordance, sensitivity, and specificity were assessed. Results: OHS records were successfully linked to administrative claims data and the Ontario Cancer Registry (OCR) with an 85.13% match rate. The final cohort consisted of 139,798 participants. A personal history of cancer was reported by 13,171 of these subjects, out of which 10,066 were found with a record in the OCR. The sensitivity of self-report was 77% and the specificity 95%. Excluding cancers diagnosed after the completion of the baseline survey increased sensitivity of self-report to 93%. The main area of discrepancy causing low sensitivity was the self-reporting of non-melanomatous skin cancers in the OHS questionnaire. Conclusions: While some self-over-reporting of cancer history may occur, cancers with lower metastatic potential tend to be under-captured in our provincial cancer registry. These findings have implications for cohort creation for research and quality improvement.

Proximity of Metastatic Cells Enhances Their Mechanobiological Invasiveness.

A critical step in metastases formation is cancer-cell invasion through tissue. During invasion, cells change morphology and apply forces to their surroundings. We have previously shown that single, metastatic breast-cancer cells will mechanically indent a synthetic, impenetrable polyacrylamide gel with physiological-stiffness in attempted invasion; benign breast cells do not indent the gels. In solid tumors, e.g., breast cancers, metastases occur predominantly by collective cell-invasion. Thus, here we evaluate the effects of cell proximity on mechanical invasiveness, specifically throughchanges in gel indention. Gel indentation is induced by 56, 33 and 2% (in >1000 cells), respectively, of adjacent high metastatic potential (MP), low MP and benign breast cells, being double the amounts observed in single, well-separated cells. Single cells exhibited a distribution of indentationdepths below 10µm, while adjacent cells also showed a second peak of deeper indentations. The second peak included 65% of indenting high MP cells as compared to 15% in the low MP cells, illustrating the difference in theirinvasiveness. Thus, proximity of the metastatic cells enhances their mechanical ability to invade, demonstrating why collective cancer-cell migration is likely more efficient. This could potentially provide a rapid, quantitative approach to identify metastatic cells, and to determine their metastatic potential.

Solid pseudopapillary neoplasm of the pancreas: Management and long-term outcome

BackgroundSolid pseudopapillary neoplasm (SPN) of pancreas is a rare pancreatic neoplasm with a low metastatic potential. Our aim was to study the clinical–pathological characteristics, and long-term outcome of this tumor. MaterialsRretrospective single center study of patients operated for SPN of pancreas. Clinical and pathological data were collected. ResultsFrom 1995 to 2016, 1320 patients underwent pancreatic resection. SPN was confirmed in 32 cases (2.46%), including 29 (90.6%) female and three (9.4%) male, with a mean age of 28.4 ± 12.2 years. SPN was the most common pathology among female patients under age of 40 (72.4%). Abdominal pain was the most frequent presenting symptom (48%), whereas none of the patients presented with jaundice. Mean tumor diameter was 5.9 cm (range, 0.9–14 cm). All patients underwent margin-negative surgical resection. Two patients demonstrated gross malignant features, including liver metastases at presentation (n = 1), and adjacent organ and vascular invasion (n = 1). Microscopic malignant features were present in thirteen patients (40.6%). Recurrence occurred in the retroperitoneal lymph nodes (n = 1, 7 years post resection) and in the liver (n = 2, 1 and 5 years post resection). Mean follow-up was 49.2 months (range, 1–228 months). Five and 10-year disease-free survival was 96.5% and 89.6% respectively. ConclusionsSPNs are low-grade tumors with a good prognosis. Margin-negative surgical resection is curative in most patients. However, almost 15% of patients demonstrate malignant features including invasion of adjacent organs or metastatic disease. Patients with malignant disease are still expected to have long survival, and aggressive surgical approach is advocated.

Mitochondrial Fluctuations as a Measure of Biomechanical Properties of Murine Cells

Introduction: The active mechanical properties of the cellular cytoskeleton are important in a number of significant cellular processes from migration to cell division, and are altered in cancer. A non-invasive single-cell assay of these properties would be of significant value for understanding these cellular processes, would give insights into mechanical heterogeneity between cells, and would be of therapeutic importance in cancer. A promising technique is derived from passive particle microrheology, wherein fluorescent beads are inserted into the cytoplasm, their mean square displacement (MSD) measured, and mechanical parameters calculated using viscoelastic theory. The non-invasive version of this method uses mitochondrial fluctuations towards the same goal which is potentially a very useful assay due to its relative simplicity. Methods: We carried out a detailed study of mitochondrial fluctuations in the C3H-10T1/2 cell line, a murine embryonic mesenchymal cell line, as well as two osteosarcoma cell lines with low and high metastatic potentials, DUNN and DLM8. Cells were cultured under standard growth condition for 24 hr, and then stained with MitoTracker Green FM (ThermoFisher Scientific). Random cells were selected for imaging using spinning disk confocal microscopy at a high temporal resolution (100 ms per frame) for 100 s. We optimized a multi-step image processing protocol to identify mitochondria with high fidelity. We selected the mitochondria with life-time spanning the entire imaging period and with no interaction with other mitochondria and calculated the MSD. Results: The MSD of isolated mitochondria resembles that of a particle in a viscoelastic medium, in agreement with previous results. However comparison of MSD between controls and cells treated with drugs that disrupt the actin and microtubule network showed surprisingly small effects, while treatment with ATP synthesis inhibitors significantly decreased the MSD. Our results imply that mitochondrial fluctuations are primarily driven by active fluctuations of the cytoskeleton, and are relatively unaffected by perturbing the actin or microtubule network, at least in the cell lines in our study. Comparison of MSDs between less invasive and more invasive murine osteosarcoma cells showed significant differences at long time scales. Hence active fluctuations differ significantly between different cell types. We also observed significant heterogeneity among cells of the same type and even more heterogeneity in the mitochondria from a single cell. Our analysis suggests that mitochondrial fluctuations reflect the active contractile properties of the cytoskeleton.

Astragaloside IV inhibits metastasis in hepatoma cells through the suppression of epithelial-mesenchymal transition via the Akt/GSK-3β/β-catenin pathway.

Our previous studies demonstrated that traditional Chinese herbal medicine 'Songyou Yin' inhibited the growth and invasion of hepatocellular carcinoma (HCC) cells, and altered epithelial‑mesenchymal transition (EMT) markers in oxaliplatin‑treated HCC tissues and cell lines. In the present study, we aimed to explore whether astragaloside IV (AS-IV), a component of 'Songyou Yin', can affect the growth and invasion of HCC cells and the underlying mechanism involved. Human HCC cell lines Huh7 and MHCC97-H, with low and high metastatic potential, respectively, were treated with increasing doses of AS-IV. The Cell Counting Kit-8 (CCK-8), plate clone formation, Transwell, wound healing and immunofluorescence assays were used to investigate the effects of AS-IV on HCC cell proliferation, migration and invasion. The protein expression levels were analyzed by western blotting and immunofluorescence assay. The CCK-8 and plate clone formation assays showed that AS-IV had little effect on the proliferation of HCC cells invitro. However, the Transwell and wound healing assays demonstrated that AS-IV inhibited the migration and invasion of HCC cells in a dose-dependent manner and the morphology of HCC cells was altered from spindle into oval shaped in the AS-IV pretreated groups. The upregulation of E-cadherin and downregulation of N-cadherin, vimentin, α-SMA and Slug were also observed in the AS-IV pretreated groups. Additionally, AS-IV treatment resulted in a profound decrease in the phosphorylated forms of Akt and GSK-3β, which in turn inhibited the expression of β-catenin. Thus, we conclude that AS-IV attenuates the invasive and migratory abilities of HCC cells through the inhibition of EMT by targeting the Akt/GSK-3β/β-catenin pathway.

Label-Free Raman Spectroscopy Detects Stromal Adaptations in Premetastatic Lungs Primed by Breast Cancer.

Recent advances in animal modeling, imaging technology, and functional genomics have permitted precise molecular observations of the metastatic process. However, a comprehensive understanding of the premetastatic niche remains elusive, owing to the limited tools that can map subtle differences in molecular mediators in organ-specific microenvironments. Here, we report the ability to detect premetastatic changes in the lung microenvironment, in response to primary breast tumors, using a combination of metastatic mouse models, Raman spectroscopy, and multivariate analysis of consistent patterns in molecular expression. We used tdTomato fluorescent protein expressing MDA-MB-231 and MCF-7 cells of high and low metastatic potential, respectively, to grow orthotopic xenografts in athymic nude mice and allow spontaneous dissemination from the primary mammary fat pad tumor. Label-free Raman spectroscopic mapping was used to record the molecular content of premetastatic lungs. These measurements show reliable distinctions in vibrational features, characteristic of the collageneous stroma and its cross-linkers as well as proteoglycans, which uniquely identify the metastatic potential of the primary tumor by recapitulating the compositional changes in the lungs. Consistent with histological assessment and gene expression analysis, our study suggests that remodeling of the extracellular matrix components may present promising markers for objective recognition of the premetastatic niche, independent of conventional clinical information. Cancer Res; 77(2); 247-56. ©2016 AACR.

Manganese-enhanced MRI for detection of metastatic potential in colorectal cancer

Objective To study manganese superoxide dismutase (MnSOD) expression in colorectal cancer (CRC) cells and observe its relation to metastatic potential. To investigate the diagnostic performance of manganese-enhanced magnetic resonance imaging (MEMRI) for detecting metastatic potential of CRC. Methods High and low metastatic potential CRC cell lines SW620, HCT116, LoVo and SW480, DLD-1, HCT15, Caco-2, as well as normal colon mucosal cell CCD841 CoN were cultured. MnSOD expression level in cells was detected by western blot and the measurement was repeated for three times. HCT15, DLD-1, LoVo and SW620 cells were selected to perform in vitro MEMRI and subcutaneous xenografts were developed for subsequent in vivo MEMRI. MnCl2·4H2O solution was utilized as the contrast agent and T1 shortening was calculated. The differences of MnSOD expression level in cells, average T1 value shortening of cells and xenografts were separately compared by one-way ANOVA. Results The difference of MnSOD expression level in CRC cells were significant (P 0.05). Conclusions MEMRI has the potential to noninvasively distinguish different metastatic potential CRC by revealing greater T1 value shortening in more aggressive one. However the MnSOD expression in CRC cells is not corresponding to malignant potential. Key words: Colorectal neoplasms; Magnetic resonance imaging; Metastatic potential

Dermatofibrosarcoma Protuberans.

Sir, A 42-year-old woman presented with history of multiple asymptomatic progressive nodular lesions over abdomen since 8 years. Patient had small nodular lesion at the same site, which was excised 9 years back. On cutaneous examination, multiple erythematous nodules of variable sizes were coalescing to form a large plaque over the abdomen [Figure 1]. A large solitary erythematous protuberant nodule measuring 10 × 8 cm was present over the plaque. Scaling and crusting were noticed over larger lesion. On palpation it was firm, non-tender, and not freely mobile over the underlying structures. There was no regional lymphadenopathy. On investigation, her complete blood count, liver function tests, and renal function tests were within normal limit. Serological tests for human immunodeficiency virus (HIV) and hepatitis B were negative. An incisional biopsy was performed under local anesthesia and tissue was sent for histopathology. Hematoxylin and eosin (H and E) stain showed spindle shaped tumor cells arranged in storiform pattern in the dermis [Figure 2]. Immunohistochemical (IHC) study showed positive for human progenitor cell Ag CD34 [Figure 3] and vimentin and negative for S100 protein, features suggestive of dermatofibrosarcoma protuberans (DFSP).Figure 1: Multinodular mass with protuberant lesion over abdomenFigure 2: Histopathology showing spindle shape tumor cells in dermis (H and E, ×10)Figure 3: Immunohistochemistry showing positivity to CD34 (IHC, ×40)DFSP is a locally aggressive, low grade, and relatively uncommon cutaneous neoplasm which has a high propensity for local relapse with low metastatic potential.[12] It accounts for <0.1% of all neoplasms, with an annual reported incidence of 0.8–4.5 cases per million.[34] It is most commonly seen in third or fourth decade with slight male preponderance.[5] Trunk is the most common site affected followed by proximal extremities and scalp. Head and neck, and genitals are rarely involved.[6] Clinically, it presents as pink or violet red plaque which may be surrounded by telangiectatic skin.[7] These lesions are fixed to skin, but move freely over underlying structures. They do not exhibit nodular growth pattern until late in their course. It becomes fixed to underlying structures in advanced and/or recurrent cases.[8] Clinically, it may be confused with other conditions such as sclerosing basal cell carcinoma, morphea, scar, and anetoderma.[89] On histopathology, it shows spindle-shaped neoplastic cells arranged in cartwheel or storiform pattern in the dermis.[7] Immunohistochemical stains are employed to differentiate DFSP from dermatofibroma, as sometimes it is difficult to differentiate the two on routine H and E stain. Studies have shown that CD-34 and vimentin are usually expressed in many cases of DFSP. Coagulation factor XIIIa is generally not expressed in DFSP, but is strongly expressed in dermatofibroma.[10] In our case it showed positive for both CD34 and vimentin. Surgical excision is the treatment of choice. Due to the high recurrence rate, radical excision is the preferred option. It involves complete surgical excision of the tumor with wide margins (>3 cm).[7] Mohs micrographic surgery is the most precise margin controlled approach for excision of DFSP.[11] Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Metastatic breast cancer cells adhere strongly on varying stiffness substrates, initially without adjusting their morphology

We show that metastatic breast cancer cells are quantitatively identifiable from benign cells during adherence onto soft, elastic gels. We identify differences in time-dependent morphology and strength of adherence of single breast cells that are likely related to their malignancy and metastatic potential (MP). Specifically, we compare high and low MP breast cancer cells with benign cells as a control on collagen-coated, polyacrylamide gels with Young's modulus in the physiological range of 2.4-10.6 kPa. We observe that the evaluated metastatic breast cancer cells remain rounded, with small contact area, up to 6.5 h following seeding. In contrast, the benign cells spread and become more elongated on stiffer gels. We identify measurable differences in the two-dimensional, lateral, traction forces exerted by the cells, where the rounded, metastatic cells apply significantly larger, traction forces, as compared to the benign cells, on gels stiffer than 2.4 kPa. The metastatic cell lines exhibited gel-stiffness-dependent differences in traction forces, strain energies, and morphologies during the initial stages of adhesion, which may relate to their MP or invasiveness.

LASS2 inhibits growth and invasion of bladder cancer by regulating ATPase activity.

Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2) is a novel suppressor of human cancer metastasis, and downregulation of LASS2 has been associated with a poor prognosis in patients with bladder cancer (BC). However, the molecular mechanism underlying LASS2-mediated inhibition of tumor invasion and metastasis in BC remains unclear. LASS2 has been reported to directly bind to subunit C of vacuolar H+-ATPase (V-ATPase) in various types of cancer, suggesting that LASS2 may inhibit cancer invasion and metastasis by regulating the function of V-ATPase. The present study investigated the effect of LASS2-specific small interfering (si)RNA on the invasion and metastasis of the RT4 human BC cell line, which has a low metastatic potential, and its functional interaction with V-ATPase. Silencing of LASS2 in RT4 cells was able to increase V-ATPase activity, the extracellular hydrogen ion concentration and, in turn, the activation of secreted matrix metalloproteinase (MMP)-2 and MMP-9, which occurred simultaneously with enhanced cell proliferation, cell survival and cell invasion in vitro, as well as acceleration of BC growth in vivo. In this process, it was found that siRNA-LASS2 treatment was able to suppress cell apoptosis induced by doxorubicin. These findings suggest that silencing of LASS2 may enhance the growth, invasion and metastasis of BC by regulating ATPase activity.