Invasion of spheroid aggregate of mouse lung adenocarcinoma (LA 795) cells into embryonic chick heart fragments]

An in vitro invasion model of tumor cell spheroid aggregate was established by using mouse lung adenocarcinoma cell line (LA795) and precultured embryonic chick heart fragments (PHF). The spheroid aggregates of LA795 cells were prepared by incubating a suspension of trypsinized LA795 cells on a gyratory shaker. Spheroid aggregates of LA795 cells in diameter of 0.2 mm were selected and confronted with PHF (diameter of 0.4 mm) on semisolid medium for 3-4 hours, then, individual confronting pairs were transferred into fluid medium for further co-culture on gyratory shaker. After 1, 3, 5 and 7 days, multiplicated confronting pairs were processed for histological and ultrastructural study. The invasive capacity and the invasion process of LA795 cells were examined. The results demonstrated that LA795 cell line has a high capacity of invasion and malignancy in vitro. This spheroid invasion model is very useful for studying invasiveness of tumor cells in vitro.     

小鼠肺腺癌（LA795）细胞球体移植于同基因鼠耳廓皮下后侵袭特性的研究

To evaluate invasion quantitatively and qualitatively in vivo, tumor cell aggregate invasion model was established. Mouse lung adenocarcinoma cell line (LA795) cells were allowed to form tumor cell aggregates after 3 days in a gyratory shaker culture system. LA795 cell aggregates, 0.3 mm in diameter, were transplanted subcutaneously to the auricles of syngenic T739 mice, which were then sacrificed at different intervals. Tumorigenicity was 100%. The process of invasion was divided into 5 stages: latent, proliferation, early invasion, mid and late invasion stages. In the mid and late invasion stages, cartilage and its capsule were invaded and progressively destroyed by tumor cells. Cartilage could be used as a criterion of judging invasion. Metastasis into the submaxillary lymph nodes (2/5) was found in the late invasion stage. The tumor cell aggregate invasion model is very useful in studying the invasion in vivo.     

Establishment, characteristics, and utilization of a new in vivo-in vitro system

Radiotherapy and chemotherapy are two important methods for malignant tumor treatment. To research radiobiological response in therapy, we have established a better experimental method in contrast to the traditional ones such as TCD50, regrowth delay, cell survival curve, etc, all with their limitations. A new mouse tumor in vivo-in vitro system LA795 Vv-Vt has been developed for studies on radiobiology. Such a system could be used to study the in vivo response of a solid tumor by the in vitro cloning assay. For the purpose of increasing the PE in vitro, LA795 Vv-Vt tumor line was purified through culturing the cells as a clonogenic spheroid. The spheroids were then injected into the flank of mouse subcutaneously for tumor growth. The in vivo-in vitro system LA795 Vv-Vt is an excellent model dissecting and analyzing the various factors which affect tumor development and determine the response of tumor to specific agent and regimens.     

In vivo experiments, in vitro assays in experimental oncology: establishment of LA 795 Vv-Vt

In vivo experiment, in vitro assays (in vivo-in vitro system) is a kind of experimental technique which is different from both in vitro cell line and tumor line. The new model can be grown and studied either as an animal tumor or a cell culture. The specimen could be assayed for cell survival in vitro. This model can be used to study the response of malignant cells to the treatment in a precision and depth that is impossible in tumors grown in vivo. LA 795 Vv-Vt is the first in vivo-in vitro system developed in China. It was established with lung adenocarcinoma of T-739 mouse. The tumor cells could grow freely both in vivo and in vitro with a plating efficiency of 20%. Characteristics of LA 795 Vv-Vt tumor and cell in culture were described and compared. In order to estimate the radiation response of different doses, cell survival curves of tumors irradiated under oxic and hypoxic conditions were drawn and compared. The oxygen enhance ratio was 2.98. The experiments indicate that in vivo-in vitro system has a good dependent relation in dose and effect and is worth extensive application.     

Tumor cell invasion and gap junctional communication. II. Normal and malignant cells confronted in multicell spheroids

The invasive behavior of five tumor cell lines was investigated with an in vitro invasion assay developed by Mareel et al. Spheroids of all 5 cell lines readily attached to precultured heart fragments (PHF), resulting in confronting pairs. Tumor cells (BICR/M1Rk, C6, and EMT6/Ro) which communicated with the host tissue via gap junctions, rapidly invaded the PHF within 3 days. Population doubling time or migratory activities had no influence on the invasion process. The noncoupled HeLa and L cells formed a cellular capsule around the PHF and showed no invasive activities. HeLa cells, however, started to destroy the PHF after 4 days. We suggest a mechanism different from that of coupled tumor cells. Epithelioid HeLa cells are linked by numerous tight junctions and may, therefore, cut off the nutrition supply for the inner-laying PHF, resulting in a disintegration of the heart tissue.     

恶性肿瘤体内外生长和发展过程与细胞外基质相关性的研究

目的探讨细胞外基质在肿瘤侵袭转移中的作用。方法利用小鼠肺腺癌细胞母系ＬＡ７９５移植于Ｔ７３９小鼠皮下和肾包膜下,以及人鼻咽癌细胞系ＣＮＥ－２Ｚ移植于裸小鼠皮下等体内移植模型,通过间接免疫酶标和间接免疫荧光技术,测定纤维粘连蛋白（ＦＮ）、层粘连蛋白（ＬＮ）与Ⅳ型胶原（ⅣＣ）在肿瘤移植后不同时间的表达,并利用多种体外实验方法（琼脂糖滴瘤细胞移动实验、软琼脂上瘤细胞集落生长速度测定、斑点杂交等）,分析瘤细胞运动能力、瘤细胞集落生长速度等与ＬＮ、ＦＮ和ⅣＣ之间的关系。结果随着肿瘤的生长,ＦＮ、ＬＮ与ⅣＣ的表达均增强,且呈不同的分布;外源性ＦＮ、ＬＮ及ⅣＣ能提高瘤细胞的体外运动能力和促进瘤细胞集落的体外生长。结论细胞外基质的分布及其合成和降解的变化,与恶性肿瘤的侵袭和转移有关,对预测肿瘤生物学行为有参考价值。观察细胞外基质与瘤细胞侵袭的关系,肾包膜下移植模型较为理想     

ANTITUMOR EFFECTS OF HUMAN IL-15 GENE MODIFIED LUNG CANCER CELL LINE

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Invasion of rat neurogenic cell lines in embryonic chick heart fragments in vitro

Invasive properties of 15 continuous neurogenic rat cell lines were investigated in vitro and were compared to their tumorigenicity in inbred BD IX rats. The cell lines were obtained by treating animals with a single transplacental dose of the carcinogen N-ethyl-N-nitrosourea (ENU) and 1) subsequently transferring brain cells into cell culture shortly after treatment or 2) explanting the resultant tumors from the offspring to monolayer cultures. In addition, one continuous nonneoplastic rat fibroblast line and three samples of untreated fetal rat brain cells were investigated. Cells from monolayer cultures were suspended and allowed to form aggregates for 24 hours on a gyratory shaker. The cell aggregates were then brought into contact with and allowed to attach to fragments from 9-days embryonic chick heart and were cultured on a gyratory shaker. All tumorigenic cell lines invaded the heart fragments, as characterized by progressive replacement of heart cells by invading cells. The heart tissue degenerated irreversibly. Nontumorigenic cell lines did not show invasiveness in vitro. Confrontation of cell aggregates and heart fragments in organotypic culture appeared to be a useful method to study directly the invasive properties of malignant transformants of neurogenic cells. The method might also permit prediction of tumorigenicity in the animal.     

小鼠肺癌B7疫苗细胞FLB2C诱导的抗肿瘤免疫应答

目的：研究小鼠肺癌细胞与活化B淋巴细胞融合的疫苗细胞FLB2c诱导细胞免疫反应情况。方法：用母本细胞LA795作对照，采用体外混合淋巴细胞培养，观察FLB2c细胞刺激T细胞增殖情况，通过CTLL细胞MTT法测定FLB2c刺激T细胞产生分泌IL－2的作用，用FLB2c免疫小鼠，观察疫苗体内诱导小鼠CTL活性的能力。结果：FLB2c细胞在体外刺激T细胞增殖的作用明显比LA795强；FLB2c能刺激T细胞分泌IL－2，而LA795则不能；FLB2c细胞在体内能诱导CTL活性，其诱导CTL在体外对FLB2c细胞和LA795的杀伤率分别为34％－25．3％，而LA795诱导的CTL对FLB2c和LA795杀伤率分别为10．5％和12．25％，两者的杀伤率有显著性差异。结论：FLB2c细胞在体内外均能刺激T细胞免疫应答，其以能力明显强于未融合的LA795小鼠肺癌细胞，将其作为疫苗细胞将有可能通过提高机体免疫应答而达到治疗肺癌的效果。本研究为进一步探讨该疫苗的应用价值奠定了免疫学基础。     

Inhibition of the invasion capacity of carcinoma cells by WX-UK1, a novel synthetic inhibitor of the urokinase-type plasminogen activator system

The overall survival rate of patients suffering from carcinomas has remained poor and nearly unchanged over the last decades. This is mainly due to the so-called minimal residual disease, i.e., remaining tumor cells that overcome surgery and/or radiotherapy and are the cause of locoregional and distant metastases. To metastasize, tumor cells take advantage of proteases to invade and remodel surrounding tissues. Here, we analyzed the efficiency of WX-UK1, a novel 3-amidinophenylalanine-based inhibitor of the uPA system, at inhibiting the invasive capacity of carcinoma cells. First, uPAR expression was characterized in different carcinoma cell lines, including SCCHN, breast and cervical carcinoma. Thereafter, the invasive potential of these cell lines was determined using Matrigel invasion chambers and a spheroid cocultivation model with human fibroblasts. uPAR expression levels correlated positively with invasion capacity, which could be significantly inhibited by WX-UK1. A decrease of tumor cell invasion by up to 50% was achieved in both models with the SCCHN line FaDu and the cervical carcinoma line HeLa after treatment with WX-UK1. Thus, our results demonstrate the potential of WX-UK1 in vitro as a promising adjuvant antimetastatic therapy of carcinomas.     

An in vitro model for cancer invasion: 1. Grading and quantitation of aggressiveness

Invasion, destruction and replacement of normal tissues by cancer cells is the first critical step in the metastatic cascade and is the result of complex interactions between tumor and host factors. In an attempt to understand the complex mechanism of local invasion, we have developed a simple, reliable and generally applicable in vitro system using the confrontation of precultured heart fragments (PHF) with a constant known number of potentially invading (10(5)) cells. This grading system is based essentially on quantitative data standardized both for the portion of the explant invaded by the neoplastic cells and the type of invasion demonstrated. Using this system we could measure the ability of an aggressor cell to (a) adhere or attach to PHF (Grade 1), (b) invade the outer fibroblastic layers (Grade II) and/or the cardiac muscle cells (Grade III) and (c) destroy and completely replace the PHF (Grade IV). It was at once apparent from these experiments that, irrespective of their invasive capacity and/or their neoplastic state, both malignant (SKBR-2 III, BT-20, MCF-7, ZR-75-30 LoVo and YAC-1) and non-malignant (HBL-100) cells attach to the PHF. Only malignant cells, however, showed substantial local invasion of both the outer fibroblastic layers and/or the cardiac muscle cells. Most important for the actual problems of invasion in vivo, is the fact that malignant cells from different cell lines demonstrate a wide range in their invasion capacity: three different patterns of invasion were thus established; a highly invasive, a slowly invasive and a poorly invasive pattern. We also show that invasion and proliferation, as defined for the purposes of this study, are two different and independent properties of a given cell line. SKBR-2 III and YAC-1 are here shown to possess the most aggressive potential; they both invade the PHF very early and completely. The rapid proliferation of these aggressive cells and the destruction of the host tissue lead to the rapid disappearance of the myoblasts and their complete replacement by the invading cells. Non-malignant epithelial cells attached to but could not invade even the outer fibroblastic layers of the PHF.     

A novel immunocompetent murine tumor model for the evaluation of RCAd-enhanced RDAd transduction efficacy

Low gene transfer rate in tumors, high dose-induced acute inflammatory response, and lack of an immunocompetent preclinical animal model to accurately reflect the therapeutic efficacy are prominent reasons for the lack of clinical success of adenoviral (Ad) vectors. In this study, we tested whether human replication-competent adenovirus (RCAd) can replicate in T739 mouse bladder transitional tumor cells (BTT) and lung adenocarcinoma cells (LA795), and whether RCAd can enhance the transduction rate and transgene expression of human replication defective adenoviruses (RDAd) in these tumor cells in vitro and in vivo. We demonstrated that human RCAd exhibited good infectability and cytopathologic effects in mouse BTT and LA795 cells, which was comparable to that in A549 and NCIH460 human tumor cells. In contrast, no infectability and cytopathologic effects were observed in other three mouse tumor cells such as 4T1, B16, and Lewis cells. The combined use of RCAd with RDAd significantly enhanced RDAd transduction efficiency in BTT and LA795 tumor cells in vitro and in vivo. When BTT and LA795 cells were co-infected with RDAd Ad-EGFP and RCAd, a large amount of E1a expression and 2-3 orders of increases in Ad-EGFP genomic DNA were observed. In contrast, the expression of the late gene Hexon is very low, which may explain ineffective packaging of viral particles. In conclusion, our study provided a novel immunocompetent animal model which is useful for evaluating RCAd infectability, cytopathy, and replication. The combined use of RCAd and RDAd provided a new solution for cancer gene therapy.     

Inhibition of embryonic cell aggregation by neoplastic cells.

The effects of normal and malignant cells on the aggregation of embryonic cells in gyratory shaker cultures were compared. The addition of 1 times 10-5 simian virus 40 (SV40)-transformed BALB/3T3 (SV40-3T3) cells to 6 times 10-6 embryonic neural retina cells caused a highly significant greater reduct on (22.7 percent) in aggregate diameter than the addition of untransformed BALB/3T3 (3T3) cells. The ratio of the number of single cells to the number of aggregates was significantly higher for cultures containing SV40-3T3 cells than for the cultures containing 3T3 cells. This effect was concentration dependent in the presence of cultured Ehrlich-Lettre hyperdiploid (ELD) ascites cells; however, media from ELD cell cultures or ELD cell sonicates resulted in aggregates of greater diameter and lower ratios of single cells to aggregates. This approach may provide a sensitive assay system for the interactions of tumor and other cells in vitro.     

Hypoxia-inducible factor-1α enhances the malignant phenotype of multicellular spheroid HeLa cells in vitro

The purpose of this study was to clarify the direct effect of hypoxia-inducible factor-1α (HIF-1α) on tumor growth, apoptosis and migration in vitro. To achieve this aim, a comparison was made of the differences in growth rates, apoptotic indices and cell invasive ability in the human cervical cancer cell line HeLa and the HIF-1α-blocked counterpart in a three-dimensional spheroid culture. A significant decrease in cell proliferation and invasion, and an increase in cell apoptosis were observed in HIF-1α-blocked cells in the three-dimensional culture. The data indicated that a multicellular spheroid culture is an ideal model of hypoxia in vitro and that HIF-1α is a significant regulator of adaptive processes that promote tumor cell malignant phenotypes, such as proliferation, anti-apoptosis and invasive ability.     

两种人类鼻咽癌细胞系CNE—1 CNE—2Z体外侵袭过程的定量分析

Computer-image processing system was used in the quantitative study on invasion in organ culture of two human nasopharyngeal carcinoma cell lines of different degree of differentiation, CNE-1 and CNE-2Z. For invasion of tumor cells into precultured heart fragments (PHF), observations were done on days 1,3,5 and 7 after organ culture. The results showed that the invasion of both CNE-1 and CNE-2Z cells was progressive. The invasion of CNE-1 tumor cells covered 82.22% of PHF on day 7, but on days 1 to 3 the invasion of CNE-2Z tumor cells covered 80-92% of PHF. This indicates that CNE-2Z cells are more invasive then CNE-1 cells.     

Spheroid Preparation from Hanging Drops: Characterization of a Model of Brain Tumor Invasion

BACKGROUND: The use of three-dimensional in vitro models of brain tumor invasion has provided a system for reconstructing some of the cellular microenvironments present in the tumor mass. While spheroids of murine and human astrocytoma cells can be prepared using spinning cultures, spheroid preparation using many cell lines is not amenable to this method. We have developed a reproducible system of creating implantable spheroids that is applicable to different cell lines, and is independent of cell line characteristics. METHODS: For murine and human brain tumor cell lines, 20 microl drops containing predetermined cell concentrations were suspended from the lids of culture dishes and the resulting aggregates were transferred to culture dishes base-coated with agar. The two-dimensional aggregates formed three-dimensional spheroids on the non-permissive agar substrate, and were then implanted into three-dimensional collagen I gels and the invasive activity assessed. The invasive activity of C6 and U251 spheroids prepared by hanging drops was compared to spheroids of similar size prepared by spinner culture. RESULTS: The hanging drop method produced implantable spheroids capable of sustained invasion using all cell lines tested. Most cell lines required initial hanging drop cell concentrations of 45,000 cells/drop, suspension times of 48, and 72 h on agar. C6 spheroids had the same invasive capacity regardless of the model utilized, however U251 spheroids produced by hanging drops had significantly increased invasion compared to those prepared by spinner culture. Only spheroids prepared by spinner culture showed histological evidence of central necrosis. CONCLUSIONS: This model represents a reproducible approach to the preparation of implantable spheroids with invasive potential that compares with those produced using spinner culture. The use of hanging drops broadens the applicability of three-dimensional in vitro assays examining brain tumor invasiveness.     

高转移肺癌细胞株的建立及其肿瘤干细胞生物学特性

 目的 研究高转移肺腺癌细胞株的肿瘤干细胞生物学特征。方法 肺腺癌细胞系A549接种裸鼠皮下成瘤后,取肺的转移灶,通过机械分离法获取肺转移细胞,体外扩大培养后,再次接种裸鼠。如此反复接种裸鼠,获得稳定肺转移的细胞株A549-V13。采用无血清悬浮培养及PKH26染色确定A549-V13存在肿瘤干细胞。流式细胞术（FACS）检测和分选A549和A549-V13中肿瘤干细胞标志物CD133的表达并进行体内外生物学特征实验。结果 建立稳定高转移A549-V13细胞株。无血清悬浮培养A549-V13,7天后形成的球形细胞团中存在单个PKH26阳性细胞。FACS检测显示,与A549中CD133⁺细胞相比,高转移A549-V13细胞株中CD133⁺细胞的表达比例显著提高4.85倍。A549-V13中CD133⁺细胞具有更强的自我更新能力,侵袭能力提高1.42倍,耐药能力也显著增强,其IC₅₀提高1.26倍,A549-V13的CD133⁺细胞在裸鼠皮下接种2×102个细胞3月致瘤4/6,而接种2×10²个A549的CD133⁺细胞3月才致瘤2/6。结论 建立高转移肺癌细胞株A549-V13,伴随CD133⁺细胞表达比例的增加,其体内外生物学功能显著增强。     

Vaccination with allogeneic GM-CSF gene-modified lung cancer cells: antitumor activity comparing with that induced by autologous vaccine

OBJECTIVE: The aim of this study was to investigate the whole allogeneic (differing tissue-type) tumor cells as vaccine in the mouse lung cancer model. The immunogenic and antitumor activity of allogeneic vaccine was compared with that of autologous cancer cell vaccine. METHODS: C57/BL mice inoculated with Lewis lung cancer (LLC) cells were used as the animal model to test the effects of allogeneic vaccination. LA795 and LLC lung cancer cell lines, which were transfected with the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, were administered as allogeneic and autologous tumor vaccine, respectively. The irradiated tumor cells were administered as subcutaneous vaccines before the tumor challenge. The immunity of cancer vaccine was tested by mouse interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) lactate dehydrogenase (LDH) assays. The serum level of IFN-gamma and interleukin (IL)-4 was tested using the enzyme-linked immunosorbent assay method. RESULTS: Prophylactic vaccination with allogeneic LA795 cells protected against the LLC tumor challenge in C57/BL. The tumor growth was inhibited and the survival was accordingly prolonged. The cytotoxicity of the spleen cells or the purified CD(8)(+) T-cells against LLC cells in the mice immunized with either the autologous or allogeneic cancer cell vaccine was significantly increased, relative to that of the control, untreated group (p<0.05). ELISPOT IFN-gamma assays showed that spleen cells from mice immunized with LA795 cells could be activated after coculture with irradiated LLC cells. In addition, the serum level of Th1-king cytokine IFN-gamma significantly increased after vaccination; however, no statistically difference was found in Th2-kind cytokine IL-4. CONCLUSIONS: The allogeneic cancer vaccine could induce immune responses and protection against lung cancer, which had no significant difference with that of autologous vaccine.     

Adenovirus-mediated expression of antisense MMP-9 in glioma cells inhibits tumor growth and invasion

Matrix metalloproteinase 9 (MMP-9) is known to play a major role in cell migration and invasion in both physiological and pathological processes. Our previous work has shown that increased MMP-9 levels are associated with human glioma tumor progression. In this study, we evaluated the ability of an adenovirus containing a 528 bp cDNA sequence in antisense orientation to the 5' end of the human MMP-9 gene (Ad-MMP-9AS) to inhibit the invasiveness and migratory capacity of the human glioblastoma cell line SBN19 in in vitro and in vivo models. Infection of glioma cells with Ad-MMP-9AS reduced MMP-9 enzyme activity by approximately 90% compared with mock- or Ad-CMV-infected cells. Migration and invasion of glioblastoma cells infected with Ad-MMP-9AS were significantly inhibited relative to Ad-CMV-infected controls in spheroid and Matrigel assays. Intracranial injections of SNB19 cells infected with Ad-MMP-9AS did not produce tumors in nude mice. However, injecting the Ad-MMP-9AS construct into subcutaneous U87MG tumors in nude mice caused regression of tumor growth. These results support the theory that adenoviral-mediated delivery of the MMP-9 gene in the antisense orientation has therapeutic potential for treating gliomas.     

An integrated computational/experimental model of tumor invasion

The intracellular and extracellular dynamics that govern tumor growth and invasiveness in vivo remain poorly understood. Cell genotype and phenotype, and nutrient, oxygen, and growth factor concentrations are key variables. In previous work, using a reaction-diffusion mathematical model based on variables that directly describe tumor cell cycle and biology, we formulated the hypothesis that tumor morphology is determined by the competition between heterogeneous cell proliferation caused by spatial diffusion gradients, e.g., of cell nutrients, driving shape instability and invasive tumor morphologies, and stabilizing mechanical forces, e.g., cell-to-cell and cell-to-matrix adhesion. To test this hypothesis, we here obtain variable-based statistics for input to the mathematical model from in vitro human and rat glioblastoma cultures. A linear stability analysis of the model predicts that glioma spheroid morphology is marginally stable. In agreement with this prediction, for a range of variable values, unbounded growth of the tumor mass and invasion of the environment are observed in vitro. The mechanism of invasion is recursive subspheroid component development at the tumor viable rim and separation from the parent spheroid. Results of computer simulations of the mathematical model closely resemble the morphologies and spatial arrangement of tumor cells from the in vitro model. We propose that tumor morphogenesis in vivo may be a function of marginally stable environmental conditions caused by spatial variations in cell nutrients, oxygen, and growth factors, and that controlling these conditions by decreasing spatial gradients could benefit treatment outcomes, whereas current treatment, and especially antiangiogenic therapy, may trigger spatial heterogeneity (e.g., local hypoxia), thus causing invasive instability.