Subsequent Metastasis Research Articles

Tumor growth following genetic modifications of mouse prostate tumor cells

Abstract Objectives 1) To determine the growth features of MyC-CaP mouse prostate tumors induced in immunodeficient nude mice and in immunocompetent FVB mice; and 2) To assess growth characteristics of MyC-CaP-p53 null tumor cells in vitro. Methods MyC-CaP cells were transfected with enhanced green fluorescent protein (eGFP), and the MyC-CaP-eGFP cells were injected intraperitoneally into naive immunodeficient nude recipients and into naive immunocompetetent FVB recipients. The transplanted tumor cells were subsequently tracked by fluorescent stereo microscopy. MyC-CaP-p53 null tumor cells were generated by inducible CRISPR/Caspase-9 gene deletion targeted to the native mutated p53 tumor suppressor gene. Results 1) Increased growth of MyC-CaP-eGFP tumors occurred in immunodeficient nude mice compared to growth in immunocompetent FVB mice; 2) decreased metastasis of MyC-CaP-eGFP tumors compared to MyC-CaP tumors occurred following injection into immunocompetent FVB mice; and 3) MyC-CaP-p53 null cells showed reduced proliferation and growth in vitro compared to non-modified MyC-CaP tumor cells. Conclusions Three conclusions can be drawn from these studies: 1) Injection of MyC-CaP-eGFP tumor cells into naive immunodeficient nude recipients allowed for adequate examination of initial tumor viability and subsequent tumor growth and metastasis; 2) Decreased MyC-CaP-eGFP tumor growth in immunocompetent FVB mice compared to immunodeficient nude mice indicates that an immune response to the eGFP protein inhibited growth of the MyC-CaP-eGFP tumor; and 3) The growth inhibition following deletion of the mutated p53 tumor suppressor gene in MyC-CaP tumor cells indicates that the mutated allele helps drive the malignancy.

Cancer

Cancer is a malignant neoplasm based on the uncontrolled proliferation of cells that are capable of invasion into surrounding tissues and subsequent metastasis. These cells are characterized by a low degree of differentiation – the lower it is, the more malignant the process is. Cancer has become a pandemic worldwide, yielding only to the diseases of cardiovascular system in terms of mortality. Etiology of this pathological process remains unclear. Development of medications and effective methods for the treatment of malignant neoplasms is extremely important today and it is still an unsolved scientific problem.

Remodeling of tight junction protein zonula occludens (ZO)‐1 by glutamine under glucose deprivation in colorectal cancer cells

Malignant cells exhibit significant metabolic alteration in nutrients to meet the energetic and biosynthetic demands. Dual routes of nutrients uptake (blood supply and luminal source) are unique characteristics of the cancerous colonic cells. Recently, several lines of evidence have suggested that glutamine can support primary cancer cell growth and serve as an alternate energy fuel during distant metastasis when available glucose concentrations are limited. Tight junctions are specialized epithelial structures for the cell‐cell adhesion and interaction. Tumor dissociation and subsequent metastasis in the tumor microenvironment may occur due to the tight junction remodeling. The current study aims to investigate the role of glutamine in colorectal cancer cells under glucose deprivation. Human colorectal cancer Caco‐2 cells were grown in the transwells to confluency and cultured in DMEM with various glutamine concentration (0 – 50 mM) in the absence of glucose. Apical administration of glutamine in Caco‐2 cells displayed a dose‐dependent drop of TER at 24 hours with no changes of the apical‐to‐basolateral FITC‐labeled dextran 4000 flux. Immunofluorescence staining of tight junction protein zonula occludens (ZO)‐1 and claudin‐1 showed lateral undulations and punctate of the structure accompanied with enlargement of cellular and nuclear size in Caco‐2 cells treated with glutamine (50 mM). Quantification of undulations and cell size revealed significant changes of tight junctional morphological after glutamine treatment in Caco‐2 cells. Decreased protein levels of ZO‐1, but not claudin‐1, were found in detergent‐insoluble fractions of cell preparation of Western blotting. The decline of TER and alteration of the tight junctional structure was not influencing the cell viability. However, the addition of glutamate, which is the amino acid produced during the first step in glutamine catabolism, has no impact on TER. In conclusion, these results suggested that the enteral administration of glutamine may play a role in the modulation of tight junction dynamics in colorectal cancer cells.

IMMUNOHISTOCHEMICAL EVALUATION OF LAMININ-5 AND MYOFIBROBLASTS IN EPITHELIAL DYSPLASIA AND ORAL SQUAMOUS CELL CARCINOMA

Background: Oral squamous cell carcinoma is one of the most prevalent cancers worldwide. The transformation of the normal epithelial cell into a tumor cell bestows upon them certain features at the cellular and molecular level which aids in its survival and proliferation. Invasion of the altered tumor cells through the basement membrane into the connective tissue stroma and their subsequent spread and metastasis is an important prognostic indicator. Laminin-5 is a protein associated with a migratory phenotype in epithelial neoplastic cells. Along with laminin, the stromal myofibroblasts play a significant role in tumor invasion, due to its ability to modify the extracellular matrix.
 Aim: To evaluate the role of laminin 5 and stromal myofibroblasts in oral epithelial dysplasia and oral squamous cell carcinoma.
 Methods: Paraffin-embedded archival samples of 25 normal, 30 oral epithelial dysplasia and 30 oral squamous cell carcinoma (OSCC) were evaluated for laminin-5 and α- smooth muscle actin (SMA) using standard immunohistochemistry. Semi-quantitative assessment of the expression of laminin and alpha SMA was done in all the study samples. The area of staining and the staining intensity was evaluated in order to determine the staining index which were then statistically analyzed between the three groups.
 Results: All the cases of laminin showed cytoplasmic staining in the basal cell layer and basement membrane. Expression of laminin was observed in the basal cell layer of normal and epithelial dysplasia study group and mainly around the tumor islands in OSCC group. α- SMA was seen with increasing intensity with increasing grade of the disease. Comparison of laminin expression between the three groups showed a statistically significant decrease in the staining index from normal to epithelial dysplasia to OSCC (p < 0.01). Statistical comparison of α-SMA in between the three groups using Kruskal- Wallis test showed a significant increase in the expression of α-SMA from normal to epithelial dysplasia to OSCC (p < 0.01)
 Conclusion: Decreased laminin expression in the basement membrane and increased expression of α-SMA favors tumor invasion, establishment of an invasive phenotype of neoplastic cells and a permissive environment for tumor invasion.
 Key words: Epithelial dysplasia, Oral cancer, Laminin, Alpha- Smooth muscle actin, Immunohistochemistry, Myofibroblasts

XIAOPI formula inhibits the pre-metastatic niche formation in breast cancer via suppressing TAMs/CXCL1 signaling

BackgroundRecent findings suggested that premetastatic niche (PMN) is a prerequisite in mediating cancer metastasis. Previously we demonstrated that XIAOPI formula could inhibit breast cancer lung metastasis via inhibiting tumor associated macrophages (TAMs)-secreted CXCL1. Herein, we aimed to explore the effects of XIAOPI formula on preventing breast cancer PMN formation and its underlying molecular mechanisms.MethodsCXCL1 expression of TAMs was detected by qPCR and Western blotting assay. The influences of XIAOPI formula on the proliferation of TAMs and 4 T1 in the co-culture system were tested by CCK8 or EdU staining. Transwell experiment was applied to determine the effects of XIAOPI formula on the invasion ability of HSPCs and 4 T1. Breast cancer xenografts were built by inoculating 4 T1 cells into the mammary pads of Balb/c mice and lung metastasis was monitored by luciferase imaging. Immune fluorescence assay was used to test the epithelial-mesenchymal transition process and PMN formation in the lung tissues. The effects of XIAOPI formula on TAMs phenotype, hematopoietic stem/progenitor cells (HSPCs) and myeloid-derived suppressor cells (MDSCs) were determined by flow cytometry.ResultsIt was found that XIAOPI formula could inhibit the proliferation and polarization of M2 phenotype macrophages, and reduce CXCL1 expression in a dose-dependent manner. However, M1 phenotype macrophages were not significantly affected by XIAOPI formula. TAMs/CXCL1 signaling was subsequently found to stimulate the recruitment of c-Kit+/Sca-1+ HSPCs and their differentiation into CD11b+/Gr-1+ MDSCs, which were symbolic events accounting for PMN formation. Moreover, XIAOPI formula was effective in inhibiting HSPCs activation and suppressing the proliferation and metastasis of breast cancer cells 4 T1 induced by HSPCs and TAMs co-culture system, implying that XIAOPI was effective in preventing PMN formation in vitro. Breast cancer xenograft experiments further demonstrated that XIAOPI formula could inhibit breast cancer PMN formation and subsequent lung metastasis in vivo. The populations of HSPCs in the bone marrow and MDSCs in the lung tissues were all remarkably declined by XIAOPI formula treatment. However, the inhibitory effects of XIAOPI formula could be relieved by CXCL1 overexpression in the TAMs.ConclusionsTaken together, our study provided preclinical evidence supporting the application of XIAOPI formula in preventing breast cancer PMN formation, and highlighted TAMs/CXCL1 as a potential therapeutic strategy for PMN targeting therapy.4rAD-F1p2pRhmA-5xKKeeVVideo Graphical abstract

Central Nervous System–Invading Eccrine Gland Carcinoma: A Clinicopathologic Case Series and Literature Review

Eccrine carcinoma involvement of the central nervous system (CNS) is exceedingly rare. The prognosis and response to treatment of this pathology remain poorly characterized. A retrospective case series and literature review were conducted. CNS-invading eccrine carcinoma was diagnosed in 3 patients (2 male and 1 female; age range, 60-79 years), including 2 cases of brain metastases and 1 case of brain-invading skull metastasis with subsequent spinal metastasis. The interval from primary tumor to CNS invasion was 18-51 months. All patients received multimodal therapy following diagnosis of CNS involvement. One patient who harbored a NOTCH1 mutation demonstrated a durable oncologic response after treatment with the immune checkpoint inhibitor pembrolizumab and lived 39 months after CNS invasion. The other 2 patients were discharged to hospice care within 1 month after the diagnosis of eccrine carcinoma brain metastasis. Including this case series, 23 cases of eccrine carcinoma invasion or metastasis to the CNS have been reported, with survival after diagnosis of CNS involvement ranging from a few weeks to 4 years. We present 3 cases of eccrine carcinoma metastatic to the CNS, including the first reported case to our knowledge of eccrine carcinoma treated with immunotherapy. This case, harboring a NOTCH1 mutation, demonstrated the longest durable oncologic response reported in this rare disease. Genomic and molecular testing may play increasingly important roles in the evaluation of these metastases.

Abstract P2-21-02: Implementation of a breast cancer post-mortem tissue donation program

Abstract BACKGROUND: Histologic and molecular differences between primary breast cancer and their subsequent metastases are well-established. Characterization of this heterogeneity may provide important information for both diagnosis and prognosis. Tissue donation via autopsy can aid in characterizing the microenvironment and understanding organ tropism. We describe the implementation, results, and challenges of a cohesive program of post-mortem tissue procurement in patients with breast cancer. METHODS: Prior to formalization of the program, three autopsies were performed without specific operating procedures. In 2016, formal input from patients, their families, clinicians, and pathology technicians helped to operationalize a formal tissue donation program. Ten autopsies have been performed to date. Demographic and clinical data were collected for these patients through retrospective chart reviews. When available, primary tumor tissue and any prior metastasis which were biopsied or surgically resected were acquired from clinical archives at our institution, or requested from outside facilities for future analyses. RESULTS: Between October 2011 and April 2019, autopsies were performed on 1 male and 9 female patients. Time from primary to metastatic disease was an average of 4 years. The median age at the time of death was 56 years (range 37-73). The average time from diagnosis of metastatic disease to consent to autopsy was 30 months. Although the majority of patients died outside the hospital (70%), the mean time between death and the start of autopsy was 4.7 hours. Samples of metastatic tissue were collected in all patients postmortem. There was an average of 16 samples collected from various sites that included both normal and metastatic tissue. The most common sites were the liver (80%), lung (70%), and lymph nodes (70%). Immunohistochemical analysis of metastatic tumors revealed intrapatient heterogeneity in ER, HER2 and Ki67 staining. TABLE 1: CLINICAL COURSE OF PATIENTSN=10 patientsPatients (%)Age at Diagnosis, years (median)46.7 (24.9-57.8)GenderFemale9 (90)Male1 (10)AJCC stage at initial presentationStage 13 (30)Stage 23 (30)Stage 33 (30)Stage 41 (10)Molecular SubtypesHR+/HER 2-6 (60)HR+/HER 2+2 (20)HR-/HER 2-2 (20)HR-/HER 2+0 (0)Time from initial diagnosis to metastatic disease, years (median)4.3 (0-12.8)Tissue of Metastases Obtained While AliveYes4 (40)No6 (60)Median age at time of death, years56.5 (37-73)Total Survival (dx to death), years (median)7.7Metastatic Survival (mets to death), years (median)3.3 TABLE 2: POST MORTEM TISSUE DONATION PROGRAMTime from Metastatic Diagnosis to Consent for Autopsy, months (median)30.3Time from Consent for Autopsy to Death, months (median)10.25Location of DeathHome5 (50)Hospital3 (30)Inpatient Hospice Facility2 (20)Time from death to starting autopsy, hours (median)4.68Time to complete autopsy, hours (median) N=72.82Number of Samples Collected During Autopsy (median)16.2 (3-27)Sites for Metastatic Tissue Procurement Postmortem8 (80)Liver7 (70)Lung7 (70)Lymph Nodes4 (40)Brain4 (40)Ascitic Fluid3 (30)Bone2 (20)Adrenal2 (20)Pericardium2 (20)Pleural Effusion1 (10)Chest Wall1 (10)Gall Bladder1 (10) CONCLUSION: Post-mortem tissue donation programs allow procurement of tissue that would otherwise be inaccessible in the living patient. Histological examination and immunohistochemical assessment of key biomarkers will be used to further characterize the tissue collected ante-mortem and post-mortem. This data combined with the clinical history and longitudinal specimens will help us better understand how intra- and inter-tumor heterogeneity play a role in the clinical course of breast cancer. Also, as we establish the program, we will identify barriers that need to be addressed in order to optimize the process. Citation Format: Tara Hyder, Lori Miller, Jennifer M Atkinson, Zoe Wecht, Ashuvinee Elangovan, Kai Ding, Nolan Priedigkeit, Zheqi Li, Tiantong Liu, Laura Savariau, Fangyuan Chen, Oscar Lopez, Dayne Ashman, Daniel Geisler, Jacob A Smith, Rachel Jankowitz, Priscilla F McAuliffe, Shannon Puhalla, Humberto E Trejo Bittar, Jeffrey S Nine, Peter C Lucas, Adam Brufsky, Steffi Oesterreich, Adrian V Lee, Margaret Rosenzweig. Implementation of a breast cancer post-mortem tissue donation program [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-21-02.

Effect of Varying Mechanical Environment in 2D Culture on Subsequent Metastasis Process of Ovarian Cancer

The epithelial ovarian cancer metastasizes through a unique transcoelomic routes, wherein cancer cells crumble out to body cavity, survive in an anchorage-independent manner as spheroids, and then finally stick to the outermost layer of another organ to develop a secondary tumor. The later stage mechanisms have already been elucidated in vitro and in vivo recently. Comparatively, only few clues exist to explain what happens during the early stage of metastasis. Recent studies reported marked heterogeneities in junctional proteins and cytoskeleton composition even within a single spheroid. Also, some of these intra-tumor heterogeneities are known to be sustained throughout the whole metastasis steps. Since the intra-tumor heterogeneity arises at that stage, herein, we focused on varying the mechanical environment of early-stage to induce cancer cells to have different cytoskeletal composition and so on. We cultured a non-serous high-grade ovarian cancer cell line, SK-OV-3, on polyacrylamide gels of different stiffness to establish two groups of distinct phenotypes. The difference between these two population of cells was characterized by immunostaining and migratory phenotype. To mimic the floating situation in the body cavity, we adopted the hanging drop method to generate the cellular spheroids. Afterward, the cells cultured with different stiffness were made into cellular spheroids, together or separately. The resultant spheroids were also tested with the adhesiveness and spreading ability on a physiologically relevant environment. Through these experimental setting, we examined whether different mechanical microenvironmental conditions in 2D culture, mimicking the heterogeneous conditions of the primary tumor, affect the subsequent spheroidal compaction and dissemination.

The role of opioids in cancer progression.

The role of opioids in cancer progression.

A Case of Colonic Endocrine Cell Carcinoma with Subsequent Axillary Lymph Node Metastasis of which Resection Led to a Long-term Survival

A Case of Colonic Endocrine Cell Carcinoma with Subsequent Axillary Lymph Node Metastasis of which Resection Led to a Long-term Survival

Role of Matrix Metalloproteinases in Angiogenesis and Cancer.

During angiogenesis, new vessels emerge from existing endothelial lined vessels to promote the degradation of the vascular basement membrane and remodel the extracellular matrix (ECM), followed by endothelial cell migration, and proliferation and the new generation of matrix components. Matrix metalloproteinases (MMPs) participate in the disruption, tumor neovascularization, and subsequent metastasis while tissue inhibitors of metalloproteinases (TIMPs) downregulate the activity of these MMPs. Then, the angiogenic response can be directly or indirectly mediated by MMPs through the modulation of the balance between pro- and anti-angiogenic factors. This review analyzes recent knowledge on MMPs and their participation in angiogenesis.

Extracellular vesicles of carcinoma-associated fibroblasts creates a pre-metastatic niche in the lung through activating fibroblasts

ObjectivesCarcinoma-associated fibroblasts (CAFs) have been known to promote cancer progression by modifying the primary tumor microenvironment. We aimed to elucidate the intercellular communication between CAFs and secondary organs in salivary adenoid cystic carcinoma (SACC) metastasis.MethodsPre-metastatic and metastatic animal models of SACC were established using extracellular vesicles (EVs) from CAFs and SACC cells. Lung fibroblasts (LFs) were treated with EVs and their transcriptomic alterations were identified by RNA sequencing. ITRAQ were performed to analyze EV cargos. TC I-15 was used to inhibit EV uptake by LFs and SACC lung metastasis in vivo.ResultsHere, we show that CAF EVs induced lung pre-metastatic niche formation in mice and consequently increased SACC lung metastasis. The pre-metastatic niche induced by CAF EVs was different from that induced by SACC EVs. CAF EVs presented a great ability for matrix remodeling and periostin is a potential biomarker characterizing the CAF EV-induced pre-metastatic niche. We found that lung fibroblast activation promoted by CAF EVs was a critical event at the pre-metastatic niche. Integrin α2β1 mediated CAF EV uptake by lung fibroblasts, and its blockage by TC I-15 prevented lung pre-metastatic niche formation and subsequent metastasis. Plasma EV integrin β1 was considerably upregulated in the mice bearing xenografts with high risk of lung metastasis.ConclusionsWe demonstrated that CAF EVs participated in the pre-metastatic niche formation in the lung. Plasma EV integrin β1 might be a promising biomarker to predict SACC metastasis at an early stage. An integrated strategy targeting both tumor and stromal cells is necessary to prevent SACC metastasis.

The impact of systemic treatment on brain metastasis in patients with non-small-cell lung cancer: A retrospective nationwide population-based cohort study

To compare the incidence of brain metastases of advanced non-small cell lung cancer (NSCLC) treated with systemic cytotoxic chemotherapy (CC) and targeted therapy (TT), we performed a large-scale, retrospective, nationwide, cohort study. The population data were extracted from the Health Insurance Review and Assessment Service of Korea database from January 1, 2011, to November 30, 2016. Of the 29,174 patients newly diagnosed with stage IIIB or IV NSCLC who received systemic treatment, we investigated the initial and subsequent incidence of brain metastases. Besides, among 22,458 patients without initial brain metastasis, the overall cumulative incidence of subsequent brain metastases was compared according to systemic treatment administered. In total, 1,126 (5.0%) patients subsequently developed brain metastasis. The overall cumulative incidence of brain metastasis was significantly higher in the TT group than in the CC group (1-year cumulative incidence: 8.7% vs. 3.8%; 3-year: 17.2% vs. 5.0%; P < 0.001). Younger age, female sex, and first-line TT were significant risk factors for subsequent brain metastasis. In conclusion, the overall cumulative incidence of brain metastasis was significantly higher in patients received TT as the first-line treatment than in those received CC.

The dynamics of the inflammatory response during BBN-induced bladder carcinogenesis in mice

BackgroundBladder cancer (BC) is the most common malignant disease of the urinary tract. Recurrent high grade non muscle invasive BC carries a serious risk for progression and subsequent metastases. The most common preclinical mouse model for bladder cancer relies on administration of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) to mice. BBN-induced tumors in mice recapitulate the histology of human BC and were characterized with an overexpression of markers typical for basal-like cancer subtype in addition to a high mutational burden with frequent mutations in Trp53, similar to human muscle invasive BC.MethodsBladder cancer was induced in C57BL/6J male mice by administering the BBN in the drinking water. A thorough histopathological analysis of bladder specimen during and post BBN treatment was performed at 2, 4, 16, 20 and 25 weeks. RNA sequencing and qPCR was performed to assess the levels of expression of immunologically relevant genes at 2 weeks and 20 weeks during and post BBN treatment.ResultsWe characterized the dynamics of the inflammatory response in the BBN-induced BC in mice. The treatment with BBN had gradually induced a robust inflammation in the first 2 weeks of administration, however, the inflammatory response was progressively silenced in the following weeks of the treatment, until the progression of the primary carcinoma. Tumors at 20 weeks were characterized with a marked upregulation of IL18 when compared to premalignant inflammatory response at 2 weeks. In accordance with this, we observed an increase in expression of IFNγ-responsive genes coupled to a pronounced lymphocytic infiltrate during the early stages of malignant transformation in bladder. Similar to human basal-like BC, BBN-induced murine tumors displayed an upregulated expression of immunoinhibitory molecules such as CTLA-4, PD-L1, and IDO1 which can lead to cytotoxic resistance and tumor escape.ConclusionsDespite the recent advances in bladder cancer therapy which include the use of checkpoint inhibitors, the treatment options for patients with locally advanced and metastatic BC remain limited. BBN-induced BC in mice displays an immunological profile which shares similarities with human MIBC thus representing an optimal model for preclinical studies on immunomodulation in management of BC.

Risk stratification system for groups with a low, intermediate, and high risk of subsequent distant metastasis and death following isolated locoregional recurrence of breast cancer.

This study aimed to identify prognostic factors for long-term outcomes among patients with isolated locoregional recurrence (ILRR) of breast cancer as their first failure event. Many prognostic tools have been developed to inform systemic treatment choices in the adjuvant setting, but tools for predicting post-ILRR prognosis are scarce. A total of 495 patients who experienced ILRR after primary surgery at the Asan Medical Center between 1989 and 2008 were included. All patient information and tumor characteristics at the initial surgery were retrieved from our retrospectively collected database, and ILRRs were categorized as local recurrence or regional recurrence (RR). Distant metastasis-free survival (DMFS), breast cancer-specific survival (BCSS), and overall survival post-ILRR were calculated. The median follow-up from the ILRR was 65months (range 1-249months), and the 5-year post-ILRR DMFS rate was 58.9%. We found three factors-lymph node metastasis, a disease-free interval < 30months, and RR as the ILRR type-that were independent prognostic factors for both DMFS [hazard ratio (HR) = 2.08, 1.60, and 1.59; P < 0.001, 0.002, and 0.003, respectively] and BCSS (HR = 2.28, 1.99, and 1.48; P < 0.001, < 0.001, and 0.016, respectively) post-ILRR. Patients were classified into four groups according to the presence these three prognostic indicators: the low-, intermediate-, high-, and extremely high-risk groups. The 5-year post-ILRR DMFS rates were 79.4%, 68.1%, 47.6%, and 36.0%, respectively. This risk stratification system for subsequent distant metastases after ILRR can be used to make more informed decisions regarding prognosis-based local or systemic management strategies.

EP1.15-13 Prognosis of Colorectal Cancer Cases That Underwent Lung and Liver Metastasectomy and Therapeutic Outcomes of Brain Metastasis

The resectability is often debated in cases of lung and liver metastases. Further, we had reported previously that the incidence of brain metastasis is significantly higher in colorectal cancer with lung metastasis than cases with liver metastasis (lung: 7.7%, liver: 1.6%). We compared and investigated the therapeutic outcomes of cases of lung, liver and brain metastasis. Between 2002 and 2013, we retrospectively studied the prognosis of 90 cases of colorectal cancer cases that underwent lung metastasectomy, and 148 cases that underwent liver metastasectomy. The course of treatment in 8 cases of subsequent brain metastasis was also evaluated. The 5-year survival rate (5-SR)for 90 cases of lung metastasectomy overall was 66.7%. 63 cases were initial metastasis and the 5-SR was 66.7%. Out of these, there were 7 cases of synchronous metastasis with a 5-SR of 57.1%, and 56 cases of metachronous metastasis with a 5-SR of 67.9%, indicating no significant difference. The regions of secondary recurrence after lung resection were as follows: lung: 28 (cases), liver: 7, brain: 3, mediastinal lymph nodes: 5, other: 5. The 5-SR for 148 cases of liver metastasectomy overall was 54.7%. 141 cases were initial metastasis and the 5-SR was 55.3%. Cases who underwent lung metastasectomy had a significantly higher survival rate (p=0.0068). Out of these, 70 cases were synchronous metastasis with a 5-SR of 35.7%, and 71 cases were metachronous metastasis with a significantly higher 5-SR of 67.9% (p=0.011). The regions of secondary recurrence after liver resection were as follows: lung: 47 (cases), liver: 66,brain: 2, abdominal lymph nodes: 9, dissemination: 6, other: 7. While there were 5 cases of subsequent brain metastasis after lung resection, no subsequent brain metastasis was found after liver resection. All but one of the 8 cases of brain metastasis after lung resection were treated with surgery or CyberKnife. The total survival period was 1141 - 3848 days (median: 1913.5 days), and the survival period after detection of brain metastasis was 15 - 852 days (median: 401 days). In addition, all cases were experiencing associated symptoms when brain metastasis was detected, and only one patient was undergoing regular examinations to detect brain metastasis. While appropriate surgical intervention is recommended in cases of lung metastasis and metachronous liver metastasis,it is debatable in cases of synchronous liver metastasis. Cases of lung metastasis should give attention to brain metastasis and recieve early detection and intervention.

Focal Adhesion Kinase (FAK) Overexpression and Phosphorylation in Oral Squamous Cell Carcinoma and their Clinicopathological Significance.

Focal adhesion kinase (FAK) is involved in progression of various cancers, and FAK overexpression has been associated with cancer invasion and metastasis. However, the involvement of FAK expression in the clinicopathological malignancy of oral squamous cell carcinoma (OSCC) remains unknown. In addition, there is no consensus regarding the role of p16 expression in OSCC. In this study, the immunohistochemically measured expression of FAK, phosphorylated FAK (FAKpY397) and p16 expressions and their associations with clinicopathological features and 5-year survival rates were examined in surgical samples from 70 patients with primary OSCC. FAK and FAKpY397 were expressed at high levels in 42 cases (60.0%) and 34 cases (48.6%), respectively, and 9 cases (12.9%) were positive for p16. FAK expression was significantly correlated with local recurrence, subsequent metastasis, and the mode of invasion. FAKpY397 expression was significantly correlated with both N classification and the mode of invasion. p16 expression was significantly correlated with clinical stage only. Patients having high expression of FAK, FAKpY397, or both showed significantly worse prognosis, but p16 expression showed no significant relation to prognosis. The results suggested that overexpression and phosphorylation of FAK in OSCC may affect cancer progression, such as local invasion and lymph node metastasis, and thereby contribute to life prognosis.

Values of alkaline phosphatase at the diagnosis of castration resistance and response to primary androgen deprivation therapy as predictors of subsequent metastasis in non-metastatic castration-resistant prostate cancer.

Among various therapeutic options available for metastatic castration-resistant prostate cancer (mCRPC), only apalutamide and enzalutamide have shown evidences of improved metastasis-free survival (MFS) for non-metastatic castration-resistant prostate cancer (nmCRPC). However, there is a paucity of evidence to indicate who may be targeted for aggressive therapy among patients with nmCRPC. The objectives of this retrospective study were to explore predictors of metastasis in patients with nmCRPC and to identify a subpopulation of patients with nmCRPC who may benefit from aggressive therapy. A total of 115 patients with CRPC who had no metastasis detected at the time of diagnosis of CRPC were included in this retrospective study. All patients were treated at Jikei University and its affiliated hospitals. The primary outcome measure was MFS from the time of diagnosis of CRPC. Predictors of MFS were also explored with a multivariate Cox hazard model. The median observation period after diagnosis of CRPC was 30months (range 2-143months). Kaplan-Meier analysis revealed a median MFS of 76. Multivariate analysis demonstrated that low alkaline phosphatase (ALP) values at diagnosis of CRPC and favorable response to primary androgen deprivation therapy (ADT) were significant predictors of longer MFS (P = 0.011, and 0.031, respectively). Results of this study suggest that high ALP values at diagnosis of CRPC and poor response to primary ADT may predict the propensity of metastasis in patients with nmCRPC. Further prospective studies will be required enrolling more patients to confirm our findings.

Metastatic Tumor Cells Exploit Their Adhesion Repertoire to Counteract Shear Forces during Intravascular Arrest

Cancer metastasis is a process whereby a primary tumor spreads to distant organs. We have demonstrated previously that blood flow controls the intravascular arrest of circulating tumor cells (CTCs) through stable adhesion to endothelial cells. We now aim to define the contribution of cell adhesion potential and identify adhesion receptors at play. Early arrest is mediated by the formation of weak adhesion, depending on CD44 and integrin αvβ3. Stabilization of this arrest uses integrin α5β1-dependent adhesions with higher adhesion strength, which allows CTCs to stop in vascular regions with lower shear forces. Moreover, blood flow favors luminal deposition of fibronectin on endothelial cells, an integrin α5β1 ligand. Finally, we show that only receptors involved in stable adhesion are required for subsequent extravasation and metastasis. In conclusion, we identified the molecular partners that are sequentially exploited by CTCs to arrest and extravasate in vascular regions with permissive flow regimes.

Novel ADAM-17 inhibitor ZLDI-8 inhibits the proliferation and metastasis of chemo-resistant non-small-cell lung cancer by reversing Notch and epithelial mesenchymal transition in vitro and in vivo

Acquired drug-resistant non-small cell lung cancer (NSCLC) has strong proliferation ability and is prone to epithelial-mesenchymal transition (EMT) and subsequent metastasis. Notch pathway mediates cell survival and EMT and is involved in the induction of multidrug resistance (MDR). ZLDI-8 is an inhibitor of Notch activating/cleaving enzyme ADAM-17 we found before. However, the effects of ZLDI-8 on resistant NSCLC was unclear. Here, we demonstrated for the first time that ZLDI-8 could induce apoptosis in lung cancer, especially in chemotherapy-resistant non-small cell lung cancer cells, and also inhibit migration, invasion and EMT phenotype of drug-resistant lung cancer. ZLDI-8 inhibits the Notch signaling pathway, thereby regulating the expression of survival/apoptosis and EMT-related proteins. Moreover, ZLDI-8 suppresses multidrug-resistant lung cancer xenograft growth in vivo and blocks metastasis in a tail vein injection mice model. Therefore, ZLDI-8 is expected to be an effective agent in the treatment of drug-resistant lung cancer.