Tumor-Infiltrating Lymphocytes in Melanoma

Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) is arguably the most effective treatment for patients with metastatic melanoma. With higher response rates than ipilimumab or IL-2, and longer durations of response than vemurafenib, TIL therapy carries the potential to transform current outcomes in melanoma, while also defining the way cell-based immunotherapy gets incorporated into mainstream cancer treatment. This paper will review the current state of TIL therapy in melanoma, the strategies to improve its efficacy, the current obstacles, and future directions to expand the availability of TIL to the general patient population.     

Splenic Lymphoma with Villous Lymphocytes

Splenic lymphoma with villous lymphocytes (SLVL) is a recently identified B cell lymphoproliferative disorder characterised by splenomegaly and the presence in the peripheral blood of lymphocytes with 'villous' projections. These villous lymphocytes are slightly larger than those found in CLL, have a round nucleus, a visible nucleolus in 50% of cases and have a variable amount of basophilic cytoplasm and characteristically, an irregular cell surface with thin, short membrane villi that are unevenly distributed and most often seen at the poles of the cell. Small numbers of plasmacytoid cells are present in most cases. There may be a variable degree of anaemia or thrombocytopenia. Immunologically the cells display a B cell phenotype with surface immunoglobulin of moderate to strong intensity, CD19, CD20, CD22, class II MHC antigens and FMC-7 expression, but they are often negative with CD5 and CD23, commonly positive in CLL, and they also are negative with CD25, HC2 and B-ly-7, typical markers in hairy cell leukaemia (HCL). The expression of CD11c, another HCL marker is variable. The bone marrow is easily aspirated and splenic infiltration is predominantly in the white pulp, both features distinct from HCL. A monoclonal gammopathy (M-band) is identified in the serum in two-thirds of patients, occasionally with free urinary light chains, but is generally less than 20 g/l. The disease usually follows a benign clinical course but may undergo transformation to large cell lymphoma in a minority of cases. When treatment is required, splenectomy is the treatment of choice and should be considered before splenic irradiation or chemotherapy which are less effective modalities.     

肝素对小鼠肝癌细胞Hca-F 和Hca-P淋巴道转移的抑制作用

 目的 观察肝素对小鼠肝癌高转移株Hca-F和低转移株Hca-P细胞与冰冻淋巴结切片体外粘附和体内淋巴道转移的影响,探讨其作用的分子机制。方法 采用细胞黏附检测（Stamper-WoodruffMethod）、组织化学、流式细胞分析等方法,观察和检测肿瘤细胞粘附和转移的情况。结果 肝素体外对小鼠肝癌细胞Hca-F和Hca-P与冰冻淋巴结切片粘附有明显的抑制作用,100U、150U肝素组与对照组比较有高度统计学意义（P〈0.01）;肝素体内对小鼠肝癌细胞Hca-F淋巴道转移也有明显的抑制作用,100U肝素组与对照组比较有统计学意义（P〈0.05）。结论 小鼠肝癌细胞Hca-F和Hca-P均有L-选择蛋白的表达,仅程度不同,L-选择蛋白的表达水平高低与Hca-F和Hca-P细胞的淋巴道转移潜能正相关,肝素竞争性抑制L-选择蛋白与其配体的结合过程,从而达到抑制肿瘤细胞转移的目的,这可能是肝素抑制肿瘤及其转移作用的分子机制之一,由此也说明L-选择蛋白介导和协助了Hca-F和Hca-P细胞的淋巴道转移。     

The Biology of Melanoma Brain Metastasis

Brain metastases are clinically diagnosed in the majority of patients with metastatic melanoma. The prognosis for patients with melanoma brain metastasis is poor with a median survival time of 6 months after diagnosis. Development of better therapies requires a better understanding of the biology of melanoma brain metastasis. The development of a relevant in vivo model offers this possibility. The intracarotid injection of different murine or human melanoma cells into syngeneic or nude mice produces metastases in different regions of the brain. This site-specific metastasis is not due to patterns of initial cell arrest, motility, or invasiveness, but rather to the ability of melanoma cells to proliferate in the brain parenchyma or the meninges. The blood–brain barrier is intact in metastases that are smaller than 0.25mm in diameter. Although in larger metastases the blood–brain barrier is leaky, the lesions are resistant to many chemotherapeutic drugs. We have also analyzed the malignant behavior of several melanoma cell lines isolated from brain or visceral metastases of patients. The cells from brain metastases showed a slower growth rate and exhibited lower metastatic potential than cells from visceral metastases, indicating that brain metastases do not necessarily represent the end stage in the metastatic cascade. Rather, brain metastases are likely to originate from a unique subpopulation of cells within the primary neoplasm.     

LKB1 Loss Promotes Melanoma Formation and Metastasis

Lkb1 deletion in a Kras-mutant background causes highly metastatic melanoma in 100% of mice.     

Expression of EphA2 and E-cadherin in Gastric Cancer: Correlated with Tumor Progression and Lymphogenous Metastasis

In this study, gastric cancer progression was correlated with the over-expression of erythropoietin-producing hepatocellular (Eph)A2 receptor and down-expression of epithelial cadherin (E-cadherin). Immunohistochemistry of EphA2 and E-cadherin were performed on these tumor samples from 165 primary lesions of gastric cancer. The results showed that expression of EphA2 was obviously increased in gastric cancer tissues (P < 0.01), which was positively correlated with the depth of cancer invasion, tumor-node-metastasis (TNM) stage and lymph node metastasis (P < 0.05). Meanwhile, the expression of E-cadherin was significantly reduced (P < 0.01), which was negatively correlated with the depth of cancer invasion, grade of tumor differentiation, TNM stage and lymph node metastasis (P < 0.05). The correlation between EphA2 and E-cadherin expression was negative (r = −0.198, P = 0.011). In conclusion, either the over-expression of EphA2 or the down-expression of E-cadherin is correlated with cancer progression and lymphogenous metastasis in gastric cancer, suggesting that both of them may play an important role in tumor progression and metastasis.     

Inhibitory Effect of Chitin Heparinoids on the Lung Metastasis of B16-BL6 Melanoma

Structure-function studies for the antimetastatic activity of chemically modified chitin heparinoids composed of N-acetyl glucosamine units were performed in an experimental lung metastasis model. 6-O-Sulfatcd chitin (S-chitin) significantly inhibited the lung tumor colonization in proportion to the degree of sulfation. However, 6-O- and N-sulfated but partially N-deacetylated chitin (S-chitosan), and 6-O-carboxymethylated chitin (CM-chitin) had no effect. 6-O-Sulfated CM-chitin (SCM-chitin), which exhibited fairly low levels of anticoagulant activity, was also more effective than intact heparin. Furthermore, SCM-chitin with a high degree of sulfation (SCM-chitin III) caused a marked decrease of the number of lung tumor colonies in the spontaneous lung metastasis model. These results strongly suggest that 6-O-sulfate and N-acetyl groups in the glucosamine unit were required for the antimetastatic effect of chitin heparinoids as well as heparin, and SCM-chitin III may be of therapeutic benefit for the prevention of tumor metastasis.     

Differential Induction of Apoptosis in B16 Melanoma and EL-4 Lymphoma Cells by Cytostatin and Bactobolin

Most solid tumor cells are less sensitive to apoptosis induced by anticancer drugs than hematopoietic cancer cells. However, the mechanisms of the different responses to apoptosis in these cell types remain unknown. To explore this question, we used B16 melanoma and EL-4 lymphoma cells as solid tumor- and hematopoietic cancer-derived cell lines, and examined the effects of two apoptosis inducers, cytostatin and bactobolin, on both cell lines. Apoptosis in B16 cells was induced strongly by bactobolin, but weakly by cytostatin. In contrast, apoptosis in EL-4 cells was induced strongly by cytostatin, but weakly by bactobolin. While caspase-3 was activated upon induction of apoptosis in both cell lines, Ac-DEVD-CHO, a specific inhibitor of caspase-3, suppressed only the apoptosis in B16 cells. In B16 cells, cyclins E, A, and B1 were decreased by strongly apoptosis-inducing bactobolin prior to apoptosis commitment, but cyclin E was not decreased by weakly apoptosis-inducing cytostatin. On the other hand, in EL-4 cells cyclins D1, E, A, and B1 were decreased by strongly apoptosis-inducing cytostatin prior to apoptosis commitment, but neither cyclin A nor B1 was decreased by weakly apoptosis-inducing bactobolin. These results indicate that the dependency of apoptosis induction on caspase activity is different between the two cell lines. Furthermore, there may be an inverse correlation between specific cyclins and apoptosis induction in the two cell lines.     

Barbigerone Inhibits Tumor Angiogenesis,Growth and Metastasis in Melanoma

Tumor angiogenesis, growth and metastasis are three closely related processes. We therefore investigated theeffects of barbigerone on all three in the B16F10 tumor model established in both zebrafish and mouse models,and explored underlying molecular mechanisms. In vitro, barbigerone inhibited B16F10 cell proliferation,survival, migration and invasion and suppressed human umbilical vascular endothelial cell migration, invasionand tube formation in concentration-dependent manners. In the transgenic zebrafish model, treatment with10μM barbigerone remarkably inhibited angiogenesis and tumor-associated angiogenesis by reducing blood vesseldevelopment more than 90%. In vivo, barbigerone significantly suppressed angiogenesis as measured by H andE staining of matrigel plugs and CD31 staining of B16F10 melanoma tumors in C57BL/6 mice. Furthermore,it exhibited highly potent activity at inhibiting tumor growth and metastasis to the lung of B16F10 melanomacells injected into C57BL/6 mice. Western blotting revealed that barbigerone inhibited phosphorylation of AKT,FAK and MAPK family members, including ERK, JNK, and p38 MAPKs, in B16F10 cells mainly through theMEK3/6/p38 MAPK signaling pathway. These findings suggested for the first time that barbigerone could inhibittumor-angiogenesis, tumor growth and lung metastasis via downregulation of the MEK3/6/p38 MAPK signalingpathway. The findings support further investigation of barbigerone as a potential anti-cancer drug.     

Intracellular Localization and Biochemical Function of Variant β-Actin, which Inhibits Metastasis of B16 Melanoma

We analyzed the biochemical nature of βm-actin protein found in mouse B16 melanoma. When we carried out immunostaining with the antibody specific to βm-actin, filamentous immunofluorescence was observed in B16-F1, a low-metastatic cell line expressing βm-actin, but not in highly metastatic B16-F10 that did not express βm-actin. When a purified actin fraction containing βm-actin was polymerized and immunoprecipitated with anti-βm-actin antibody, the immunoprecipitate contained βm-, β- and γ-actin. This indicated that the βm-actin was incorporated into an actin filament together with β- and γ-actin in vitro , and this phenomenon was consistently suggested by cellular double immunostaining with anti-βm-actin and common anti-actin antibody. When the actin fraction containing βm-actin under a regular depolymerizing condition was subjected to immuno-adsorption assay using anti-βm antibody and protein-A Sepharose, the immunoadsorbed aggregates contained βm-, β-and γ-actin. This indicates that the actin fraction was not completely depolymerized and contained βm-actin-containing oligomers, which were too small to be precipitated with anti-βm-actin antibody alone. The incomplete depolymerization of the βm-actin-containing fraction was also suggested by the much lower DNase 1 inhibition activity of the βm-actin-containing fraction than that of β- and γ-actin fraction. Furthermore, a DNase 1 binding assay showed that cytoplasmic supernatant prepared from B16-F1 under a low-ionic condition contained less monomeric actin than the cytoplasmic preparation from B16-F10. These results suggested that βm-actin protein in B16 melanoma probably inhibits the dynamic conversion between the monomeric and polymerized forms of actin, leading to a decrease in cell motility and consequently the suppression of invasiveness and metastasis.     

肿瘤相关巨噬细胞促进宫颈癌侵袭转移的机制研究

 目的 研究肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs）对宫颈癌侵袭转移的影响及分子机制。方法 免疫组织化学法检测宫颈病变组织TAMs的浸润情况。对人单核巨噬细胞系THP1进行体外诱导分化，使其转化为M2型TAMs。取TAMs上清液刺激宫颈癌细胞SiHa和C33a。划痕法、Transwell法分别检测TAMs对宫颈癌细胞系迁移和侵袭能力的影响。Western blot检测刺激后宫颈癌细胞上皮间质转化进程及MMP-9的表达。结果 TAMs浸润数目与宫颈病变进展呈正相关。TAMs上清液刺激后，SiHa和C33a细胞出现间质样改变，而且迁移和侵袭能力显著增强（均P<0.5）。TAMs可下调E-Cadherin的表达，上调N-Cadherin、Vimentin及MMP-9的表达。结论 TAMs浸润与宫颈上皮恶性转化和进展密切相关，TAMs可能通过上调MMP-9的表达促进宫颈癌细胞侵袭转移。     

高、低淋巴道转移能力小鼠肝癌细胞株抑制性消减杂交文库的构建

应用抑制性消减杂交（suppression subtracted hybridization,SSH）技术,分别构建2个不同淋巴道转移能力小鼠肝癌细胞株的SSH文库,高转移文库以高转移能力细胞株Hca-F为检测子（tester）,同源的低转移能力细胞株Hca-P为驱赶子（driver）;低转移文库则相反.随机筛选2个文库阳性克隆进行测序,并在GenBank数据库中进行同源性比较.成功构建高、低淋巴道转移能力小鼠肝癌细胞株SSH文库,高、低转移文库中分别包含995个及967个阳性克隆;其中,95%的阳性克隆含有300bp～1 000bp不等的插入片段;随机测序显示,文库中含有已知的基因、ESTs及与任何序列无同源性的新基因片段.     

Relationship between Malignant Melanoma and Chromosome Damage in Human Peripheral Blood Lymphocytes

The incidence of malignant melanoma increases with age. One significiant effect of aging processes is anaccumulation of oxidative damage in the genetical material. In this study, the relationship between malignantmelanoma and damage in chromosomes and proliferative effectiveness of human peripheral lymphocytes wereinvestigated by the micronucleus (MN) technique. A total of 15 malignant melanoma patients and appropriatelymatching 15 healthy controls were involved in the study. MN frequencies and proliferative indexes (PI) afternon toxic levels of hydrogen peroxide treatment were also measured to determine damaging effect of oxidativestress in genome in addition to measuring the spontenous levels of micronuclei and PI. The patient group hada significantly higher rate of spontaneous MN than the control group (p<0.01). After treatment with H2O2, MNfrequencies in the patient group was significantly decreased (p<0.01) although there was no difference betweenthe treated and untreated results of control group (p=0.29). There was also difference (p<0.01) between the MNfrequencies of the patient and the control group either in the spontaneous levels or in the H2O2 treated groups.The same significant difference persisted when the PI values were compared between patient and control groups.Increase in the MN frequency in patients could mean the alterations in the chromosomal structure which maylead to the chromosome instability and therefore genetic susceptibility to cancer. This increased number ofmicronuclei can also be used for cytological marker in identifying high risk cases for malignant melanoma.     

SOX4表达与恶性黑素瘤临床病理特征及其转移的关系

目的探讨SOX4表达与恶性黑素瘤临床病理特征及其转移的相关性。方法选择恶性黑素瘤患者70例,同期收集正常皮肤组织30例作为对照组。免疫组织化学染色检测两组标本中SOX4阳性表达。比较两组的SOX4阳性积分。分析SOX4阳性积分与恶性黑素瘤临床病理特征的相关性。分析SOX4表达与恶性黑素瘤转移的关系。结果恶性黑素瘤患者的SOX4阳性表达积分明显高于对照组(P<0.01)。恶性黑素瘤组织SOX4阳性表达与年龄、性别无明显相关(P>0.05),与Breslow分级、Clark分级和溃疡形成显著相关(P<0.01)。3组恶性黑素瘤患者SOX4阳性表达率分别为69.23%、95.45%、100.00%,转移组明显高于原位组(P<0.01)。结论 SOX4在恶性黑素瘤组织中高表达,且与肿瘤的分期、进展及转移呈正相关。     

Murine Liver Metastasis Model Using L5178Y-ML Lymphoma and the Effect of Antitumor Agents on the Metastasis

A reproducible tumor model for liver metastasis has been developed from murine L5178Y lymphoma line by sequential cycles of subcutaneous inoculation of liver tumor cells, that were originally generated in livers of female (BALB/c × DBA/2)F₁ mice by injecting the parental cells into the tail vein. This variant (L5178Y-ML) metastasized predominantly to the liver after intravenous or subcutaneous injection. The livers of the animals killed 9 days after intravenous implantation of 5 × 10⁵ tumor cells were about 3 times the weight of control livers. All tumor-bearing mice died 10 to 12 days after inoculation. Subcutaneous implantation of L5178Y-ML in the side flank of mice induced metastatic nodules spontaneously in the livers. The tumor cells proliferated more in livers than in the implanted sites, compared with the parental L5178Y cells. The effects of 5-fluorouracil, mitomycin C, cis -platinum and doxorubicin on the liver metastasis of L5178Y-ML were examined at subtoxic doses; 5-fluorouracil was the most effective in both inhibiting the tumor growth in livers and prolonging the survival period of mice. This model provides a useful tool for the experimental therapy of hepatic tumors in mice.     

上腭恶性黑色素瘤广泛转移1例报道

资料患者,男,53岁.于2003年6月自觉进食时上腭不适,咽痛而就诊于秦皇岛市第一医院.查体:左上腭可见大小约6.0cm×6.0cm暗红色肿物,活检病理示:上腭恶性黑色素瘤.于7月2日行放疗:放疗量5000cGy,200cGy/次,共25天,放疗期间给予罗扰素300万UIH qod.放疗结束后即于8月1日复查CT示:(1)左侧硬腭软组织肿较前缩小,现局部仍可见软组织肿,约1.6cm×2.0cm(横截面),并有强化.肿物缩小.(2)硬腭骨质未见破坏.(3)左侧上颌窦炎症较前好转.     

抑制整合素α9基因的表达对黑色素瘤细胞B16F1的生长和肺转移的影响

目的 观察shRNA干扰整合素α9（ITGA9）的表达对黑色素瘤细胞B16F1的生长和肺转移的影响。方法 用RNA干扰技术下调B16F1中ITGA9的表达,建立小鼠皮下成瘤和肺转移模型,观察肿瘤生长情况,计数肺转移灶数量。结果 ITGA9-shRNA转染组的肿瘤生长速度减慢（P＜0.05）,实验终点,该组肿瘤平均体积与scramble-shRNA组相比下降36%;肺转移灶数量显著减少（P＜0.05）。结论 下调ITGA9的表达可抑制黑色素瘤细胞B16F1在小鼠体内的生长和肺转移。ITGA9可能成为黑色素瘤的治疗靶点。