A new in vitro model for investigation of tumor cell-platelet-endothelial cell interactions and concomitant eicosanoid biosynthesis

We have developed a new in vitro model system to examine tumor cell-platelet-endothelial cell interactions under dynamic conditions. Using the same model, we can determine endogenous eicosanoid metabolism and alterations in the prostacyclin-thromboxane A2 balance associated with interactions among tumor cells, platelets, and endothelial cells. The model consisted of cloned rat aortic endothelial cells grown on gelatin microcarrier beads under dynamic conditions (i.e., spinner culture). Interactions of these endothelial cells with platelets (heparinized rat platelet rich plasma) and/or tumor cells (rat Walker 256 carcinosarcoma) were assessed in an aggregometer. Gelatin beads alone or microcarrier grown endothelial cells did not elicit spontaneous aggregation of platelet rich plasma over a time period of 30 min. Microcarrier grown endothelial cells inhibited tumor cell induced platelet aggregation in a dose dependent fashion (i.e., depending on endothelial cell number). The ability of microcarrier grown endothelial cells to inhibit tumor cell induced platelet aggregation depended on endogenous production of prostacyclin. This conclusion is based on the following results: an increased number of microcarrier grown endothelial cells caused a prolongation of the aggregation lag time; an increased number of microcarrier grown endothelial cells caused a proportionate increase in 6-keto-prostaglandin F1 alpha concentration; an increased number of microcarrier grown endothelial cells was inversely correlated with thromboxane A2 production by platelets; indomethacin pretreatment of microcarrier grown endothelial cells caused a decrease in prostacyclin production and therefore overcame the associated inhibition of tumor cell induced platelet aggregation; and the inhibition of tumor cell induced platelet aggregation in the presence of endogenous prostacyclin produced by microcarrier grown endothelial cells was the same as that observed in the presence of exogenous prostacyclin. Scanning electron microscopy of aggregometry samples revealed: little or no platelet or tumor cell adhesion to gelatin beads alone, a low basal adhesion of tumor cells to microcarrier grown endothelial cells, and large aggregates of platelets and tumor cells located primarily at gaps in the monolayer of indomethacin treated microcarrier grown endothelial cells. This new in vitro model provides a method for examining the effects of eicosanoid metabolism by endothelial cells on tumor cell-platelet-endothelial cell interactions under dynamic conditions.     

Platelet enhancement of tumor cell adhesion to subendothelial matrix: role of platelet cytoskeleton and platelet membrane

Platelet involvement during tumor cell adhesion to subendothelial matrix was examined in vitro. Platelets were subjected to thrombin stimulation and mechanical lysis and examined for their effects on tumor cell adhesion. These treatments altered the platelet ultrastructure and cytoskeletal integrity. Untreated washed rat platelets (WRP) exhibited extensive adhesion to and spreading on substrates and substantially enhanced tumor cell adhesion to the same substrates (i.e., 250% greater than tumor cells without platelets). Thrombin prestimulation of platelets limited platelet adhesion and spreading and platelet facilitation of tumor cell adhesion. Complete mechanical lysis disrupted both the platelet membrane and the cytoskeleton and eliminated the ability of platelets to adhere or to enhance tumor cell adhesion. Partially lysed platelets resembled membrane ghosts and facilitated tumor cell adhesion by a mechanism independent of spreading and cytoskeletal rearrangement. Fractionation studies indicated that platelet cytoskeletal components played a role in the adhesion process. Pretreatment of WRP with cytochalasin A or B dose dependently inhibited microfilament-mediated platelet spreading and platelet-enhanced tumor cell adhesion. Colchicine and vinblastine induced microtubule depolymerization, but they had no observable effect on platelet spreading or platelet-enhanced tumor cell adhesion. It was concluded that platelet-enhanced tumor cell adhesion to subendothelial matrix depends on an intact platelet cytoskeleton and on a platelet membrane component(s) and is mediated by surface contact between platelets and tumor cells. Furthermore, platelet-mediated tumor cell adhesion to subendothelial matrix may involve two mechanisms: one dependent on, and one independent of, platelet spreading and cytoskeletal rearrangement.     

An in vivo functional genetic screen reveals a role for the TRK-T3 oncogene in tumor progression

Over the past decades, much has been learnt about the genes that contribute to oncogenic transformation of primary cells in vitro. However, much less is known about the genes that contribute to the later stages of tumor progression, in which cells of ever increasing malignancy arise through clonal selection in vivo. To search for genes that confer a tumor progression phenotype in vivo, we have used a functional genetic approach. We used adenovirus-transformed mouse embryo fibroblasts, which are tumorigenic in immunodeficient nude mice, but not in immunocompetent mice, due to strong cytotoxic T-cell-mediated immune rejection. We infected these cells in vitro with several high-complexity retroviral cDNA expression libraries and selected rare variants that formed tumors in immunocompetent mice. Using this approach, we identify here the TRK-T3 oncogene as a tumor progression gene. TRK-T3 does not inhibit T-cell reactivity towards the tumor cells. Instead, we find that cells expressing TRK-T3 enhances in vivo growth rate, most likely by stimulating anchorage-independent proliferation in growth factor-limiting conditions. Our data indicate that cDNA expression libraries can be used to identify tumor progression genes in vivo that cannot be readily identified using in vitro cell culture systems.     

The role of tumor markers in the treatment of germ cell tumor

Alfa fetoprotein (AFP), human chorionic gonadotropin (HCG), beta HCG and lactate dehydrogenase (LDH) are powerful markers of germ cell tumors. The role of tumor markers is very important in the diagnosis, treatment and follow-up of germ cell tumors, respectively. We can often deduce the histological typing of germ cell tumors by tumor marker elevation before surgery. Tumor markers also frequently provide clues as to outcome in individual cases before treatment. The half-life of tumor markers during chemotherapy indicate the effect of the treatment. The optimal regimen of chemotherapy should therefore be selected based on the half-life of tumor markers. Normalized tumor markers designate the phase of discussion on surgical indications. Determination of tumor markers is important in following patients after treatment of germ cell tumors. The elevation of serum tumor markers denotes recurrence and is often the first sign of treatment failure.     

An exponential-Gompertzian description of LoVo cell tumor growth from in vivo and in vitro data

The data of nine monolayer cultures, 48 multicellular spheroids, and 19 s.c. xenografts of LoVo cells were fitted, on an individual basis, by exponential and Gompertzian equations, respectively. The mean growth parameters alpha 0 (initial growth rate) and beta (retardation factor) of the three experimental systems presented a strong linear correlation alpha 0 = 6.88 beta + 0.56, r = 0.9843. This implies that, at the particular tumor size of 155 microns in diameter (mean +/- SD = 50-310 microns), the tumor growths described by Gompertzian curves (from spheroids as well as from s.c. xenografts) have the same growth rate as monolayer cultures. This occurrence strongly supports an exponential-Gompertzian growth model, where an exponential monolayer-like phase changes to a Gompertzian growth, controlled by environmental conditions, when the tumor size has reached a critical value.     

In vivo tumor cell adhesion in the pulmonary microvasculature is exclusively mediated by tumor cell - endothelial cell interaction

Background  

Metastasis formation is the leading cause of death among colon cancer patients. We established a new in-situ model of in vivo microscopy of the lung to analyse initiating events of metastatic tumor cell adhesion within this typical metastatic target of colon cancer.     

The role of the tumor suppressor RUNX3 in giant cell tumor of the bone

RUNX3 is a tumor suppressor gene localized in 1p36. In various human tumors, the region is frequently inactivated through hypermethylation, histone modulation and other processes. Recent studies have suggested that loss of RUNX3 expression is involved in stomach, colon and breast cancer. However, the relationship between RUNX3 expression and giant cell tumor of the bone (GCTB) remains elusive. The aim of our study was to elucidate the roles of RUNX3 expression in carcinogenesis and progression of giant cell tumor of the bone. The levels of RUNX3 mRNA and protein were evaluated in human GCTB specimens and cell lines. To assess RUNX3 methylation we employed methylation-specific polymerase chain reaction using GCTB specimens and cell lines. In addition, to examine the roles of RUNX3 in giant cell tumor of the bone, GCTB cells were transfected with pcDNA3.1-RUNX3 (RUNX3 was cloned into the pcDNA3.1 plasmid). Flow cytometry (FCM) was used to analyze the apoptosis and cell cycle. The mobility of cells was tested by transwell migration assay. The expression rates of RUNX3 in patients with GCTB were significanly lower than normal bone tissues. Thirty of 47 human cancer specimens exhibited suppression (P<0.05). Down-regulation of RUNX3 mRNA in the same GCTB cell lines was associated with RUNX3 DNA methylation. In in?vitro experiments, exogenous expression of RUNX3 strongly inhibited cell growth in GCTB by MTT (P<0.05), induced apoptosis as evidenced by Annexin?V-FITC and increased G1 phase ratio by PI (P<0.05). Transwell migration assay showed that less RUNX3 positive cells migrated to the lower side of the membrane than negative ones (P<0.05). These results show that RUNX3 is a tumor suppressor in GCTB. RUNX3 DNA methylation may be the molecular basis for its lower expression. These data may be applied in GCTB for diagnostics and therapeutics.     

An improved isoprenylcysteine carboxylmethyltransferase inhibitor induces cancer cell death and attenuates tumor growth in vivo

Inhibitors of isoprenylcysteine carboxylmethyltransferase (Icmt) are promising anti-cancer agents, as modification by Icmt is an essential component of the protein prenylation pathway for a group of proteins that includes Ras GTPases. Cysmethynil, a prototypical indole-based inhibitor of Icmt, effectively inhibits tumor cell growth. However, the physical properties of cysmethynil, such as its low aqueous solubility, make it a poor candidate for clinical development. A novel amino-derivative of cysmethynil with superior physical properties and marked improvement in efficacy, termed compound 8.12, has recently been reported. We report here that Icmt ^−/− mouse embryonic fibroblasts (MEFs) are much more resistant to compound 8.12-induced cell death than their wild-type counterparts, providing evidence that the anti-proliferative effects of this compound are mediated through an Icmt specific mechanism. Treatment of PC3 prostate and HepG2 liver cancer cells with compound 8.12 resulted in pre-lamin A accumulation and Ras delocalization from the plasma membrane, both expected outcomes from inhibition of the Icmt-catalyzed carboxylmethylation. Treatment with compound 8.12 induced cell cycle arrest, autophagy and cell death, and abolished anchorage-independent colony formation. Consistent with its greater in vitro efficacy, compound 8.12 inhibited tumor growth with greater potency than cysmethynil in a xenograft mouse model. Further, a drug combination study identified synergistic antitumor efficacy of compound 8.12 and the epithelial growth factor receptor (EGFR)-inhibitor gefitinib, possibly through enhancement of autophagy. This study establishes compound 8.12 as a pharmacological inhibitor of Icmt that is an attractive candidate for further preclinical and clinical development.     

肿瘤细胞在活体小鼠肝脏微循环中动态变化的可视性研究

目的:在小鼠活体内动态观察肿瘤细胞在肝脏微循环中的运动、分布及凋亡。方法:用Calcein AM荧光标记B16F1黑色素瘤细胞,将其注射入C57BL/6小鼠的肠系膜静脉,在荧光倒置显微镜下动态观察肿瘤细胞在肝脏微循环中的运动及分布,测量并计算肿瘤细胞在终末门静脉中的运动速度。注射肿瘤细胞8小时后,切取肝脏,用凋亡标记荧光素对肝组织切片进行原位DNA末端标记,在荧光显微镜下计算凋亡细胞百分比。结果:B16F1黑色素瘤细胞进入肝脏微循环中后,90%的细胞通过机械性嵌顿停留在肝窦中,10%的肿瘤细胞通过黏附停留在终末门静脉中,B16F1细胞在肝脏终末门静脉中的运动速度为648±53μm/s。在注射肿瘤细胞8小时后,在肝窦中约有30%的肿瘤细胞发生凋亡,在终末门静脉中约有10%的肿瘤细胞发生凋亡。结论:利用荧光标记的肿瘤细胞及荧光倒置显微镜为探讨肿瘤肝转移机制提供了一个良好的活体观察方法。     

肿瘤干细胞研究进展

干细胞是一类具有自我更新能力和多向分化潜能的原始细胞，干细胞的自我更新调节通路异常与肿瘤形成密切相关。恶性肿瘤的进行性生长、转移和复发与干细胞的基本特性相似，推测在肿瘤组织中存在少数能驱使肿瘤形成与发展的肿瘤干细胞。随着近年来正常干细胞研究的飞速发展，肿瘤干细胞的研究取得了令人鼓舞的成果，急性髓细胞白血病、乳腺癌及脑瘤中的肿瘤干细胞已相继得到分离和鉴定。现对近年来肿瘤干细胞的研究进展作简要综述。     

肿瘤细胞在活体小鼠肝脏微循环中动态变化的可视性研究

目的：在小鼠活体内动态观察肿瘤细胞在肝脏微循环中的运动、分布及凋亡。方法：用Calcein AM荧光标记B16F1黑色素瘤细胞,将其注射入C57BL/6小鼠的肠系膜静脉,在荧光倒置显微镜下动态观察肿瘤细胞在肝脏微循环中的运动及分布,测量并计算肿瘤细胞在终末门静脉中的运动速度。注射肿瘤细胞8小时后,切取肝脏,用凋亡标记荧光素对肝组织切片进行原位DNA末端标记,在荧光显微镜下计算凋亡细胞百分比。结果：B16F1黑色素瘤细胞进入肝脏微循环中后,90%的细胞通过机械性嵌顿停留在肝窦中,10%的肿瘤细胞通过黏附停留在终末门静脉中,B16F1细胞在肝脏终末门静脉中的运动速度为648±53μm/s。在注射肿瘤细胞8小时后,在肝窦中约有30%的肿瘤细胞发生凋亡,在终末门静脉中约有10%的肿瘤细胞发生凋亡。结论：利用荧光标记的肿瘤细胞及荧光倒置显微镜为探讨肿瘤肝转移机制提供了一个良好的活体观察方法。     

The role of pentoxifylline in the prevention of tumor cell implantation

Short-term administration of pentoxifylline concurrent with intraportal or intravenous instillation of W163 adenocarcinoma was ineffective in preventing tumor cell implantation in adult male rats.     

Role of tumor cytoskeleton and membrane glycoprotein IRGpIIb/IIIa in platelet adhesion to tumor cell membrane and tumor cell-induced platelet aggregation

Components of the tumor cell cytoskeleton (i.e., microtubules, microfilaments, and intermediate filaments) have been reported to affect metastatic ability, since disruption of these components leads to a decrease in metastasis. One mechanism of metastasis which has not been previously considered is the decreased interaction of tumor cells with platelets. We present evidence that disruption of the tumor cell cytoskeleton decreases the ability of tumor cells to aggregate homologous platelets. This effect is dependent upon the disruption of microfilaments/intermediate filaments but not disruption of microtubules. In addition, tumor cell platelet interactions require the lateral mobility of specific receptors (i.e., clustering) on the tumor cell plasma membrane. A membrane glycoprotein immunologically related to the platelet glycoprotein IIb/IIIa complex was identified on Walker 256 carcinosarcoma cells using specific polyclonal and monoclonal antibodies and Northern blot analysis using complementary DNA probes for IIb and IIIa. Mobility of this receptor is dependent upon tumor cell microfilaments/intermediate filaments, but not microtubules. Furthermore, treatment of tumor cells with specific antibodies to the platelet glycoprotein IIb/IIIa complex inhibits tumor cell-platelet interaction at the macroscopic level (i.e., aggregation) and at the ultrastructural level (i.e., platelet adhesion to the tumor cell surface). These results suggest that this immunologically related glycoprotein IIb/IIIa is a receptor for platelet binding to the tumor cell surface, an event which precedes overt platelet aggregation and is dependent upon an intact tumor cell microfilament and intermediate filament network. Therefore, the decreased metastasis observed by others following disruption of the tumor cell cytoskeleton may be due, in part, to a decreased tumor cell-platelet interaction.     

Kinetics of in vivo tumor cell destruction after specific immunization

The natural cytotoxic activity in vivo can be evaluated by measuring in vivo tumor cell destruction after injecting mice with 125IUdR-labeled tumor cells, measuring their total body radioactivity and calculating the % radioactivity lost. We have studied the in vivo destruction of 125IUdR-labeled L1210 leukemic cells by B10.D2 mice previously immunized 4 times with heavily irradiated L1210 leukemic cells. Mathematical analysis of our results indicates that the radiolabel loss on day 1 is similar in normal and immunized animals, but that it stays greater over the following days in immunized animals, indicating that the difference between the two groups is not the extent of the initial cell destruction but the durability of the response. There is a good correlation between the eventual survival, the % of 125IUdR lost and the number of tumor cells present in the peritoneal cavity three hours after their injection. Such a methodology provides a very early prediction of the survival of each mouse, thus identifying animals with a poor prognosis.     

An essential role of antigen-presenting cell/T-helper type 1 cell-cell interactions in draining lymph node during complete eradication of class II-negative tumor tissue by T-helper type 1 cell therapy

Prior studies have shown that transfer of ovalbumin (OVA)-specific T helper type 1 (Th1) cells into mice bearing MHC class II+ OVA-expressing tumor cells (A20-OVA) causes complete tumor rejection. Here we show that, although Th1 cell therapy alone was not effective against MHC class II- OVA-expressing tumor cells (EG-7), treatment of mice bearing established EG-7 tumors by i.v. transfer of Th1 cells combined with i.t. injection of the model tumor antigen OVA induced complete tumor rejection. Transferred Th1 cells enhanced the migration of tumor-infiltrating antigen-presenting cells (APC) that had processed OVA into the draining lymph node (DLN). Although transferred Th1 cells were randomly distributed in DLN, distal LN, spleen, and tumor tissue, active proliferation of Th1 cells always initiated in DLN, where Th1 cells efficiently interacted with APC that presented OVA. In parallel, OVA-tetramer+ CTLs, showing EG-7-specific cytotoxicity, were highly induced in DLN and the local tumor site. The OVA-tetramer+ CTL functioned systemically because two bilateral tumor masses were both completely rejected on treatment of one tumor. Furthermore, either active proliferation of transferred Th1 cells or generation of tetramer+ CTL was not induced in MHC class II-deficient mice and LN-deficient Aly/Aly mice. These results indicate that DLN is an indispensable organ for initiating active APC/Th1 cell interactions, which is critical for inducing complete eradication of tumor mass by tumor-specific CTL.     

Interaction between tumor and immune system: the role of tumor cell biology

In present work the role of tumor cell biology upon different conditions of their interaction with the cells of immune system is discussed. The presented data show that it tumor cell biology that in many cases determines tumor antigen recognition and realization of cytotoxic action of killer cells. Here also we discuss own data obtained with the use of experimental tumor models (transplantable MC-rhabdomyosarcoma, B16 melanoma) and human tumors (soft tissue sarcoma, melanoma, breast cancer, ovarian cancer, cervical cancer). It was demonstrated that various tumors have different sensitivity to antitumor action of activated (LAK) and non-activated lymphocytes. Recent studies on the role of tumor cells in expansion and activity of different suppressor cells (MDSC, Treg, Th17, M-2 macrophages, etc.) are overviewed. The role of organ-specificity and interaction of the components of microenvironment (cells of immune system and stroma, tumor cells, endothelial cells, extracellular matrix) are discussed in details. Along with the presentation of modern views on some patterns that characterize the development of drug resistance of different tumors and capabilities of immune system cells to participate in lysis of resistant tumor cells, the authors present their own data illustrating that resistant tumor cells reveal increased sensitivity to LAK. An analysis of the involvement of a number of molecules, lymphocytes, and tumor cells in the phenomenon of elevated sensitivity to LAK action allowed to conclude that tumor cells reveal high sensitivity at the background of decreased E-cadherin expression and increased CD40 expression.     

Snail2 promotes osteosarcoma cell motility through remodelling of the actin cytoskeleton and regulates tumor development

The function of Snail2 in mesenchymal tumors is, to date unknown. Using knockdown and overexpression studies, we show that Snail2 regulates migration and invasion of osteosarcoma cells. Knockdown resulted in significantly decreased motility, remodelling of the actin cytoskeleton, and loss of cellular protrusions. Over-expression increased motility, formation of actin-rich cellular protrusions, and altered expression of some non-canonical Wnt pathway components whilst decreasing expression of the adhesion molecule OB-cadherin. Unexpectedly, knockdown also resulted in significantly smaller tumors in an in vivo CAM assay. Therefore Snail2 may be a potential therapeutic target for clinical intervention of osteosarcoma.     

Relationship between in vivo drug exposure of the tumor interstitium and inhibition of tumor cell growth in vitro: a study in breast cancer patients

A novel approach is described to simulate effect site pharmacodynamics of anticancer drugs. This approach is based on (i) the in vivo measurement of unbound, interstitial drug pharmacokinetics (PK) in solid tumor lesions in patients and (ii) a subsequent pharmacodynamic (PD) simulation of the time versus drug concentration profile in an in vitro setting. For this purpose, breast cancer cells (MCF-7) were exposed in vitro to the time versus interstitial tumor concentration profiles of 5-fluorouracil (5-FU) and methotrexate (MTX) from primary breast cancer lesions in patients. This led to a maximal reduction in the viable cell count of 69 on day 4, and of 71 on day 7 for 5-FU and MTX, respectively. This effect was dependent on the initial cell count and was characterized by a high interindividual variability. For 5-FU there was a significant correlation between the maximum antitumor effect and the intratumoral AUC (r=0.82, p=0.0005), whereas no correlation could be shown for MTX (r=0.05, p=0.88). We conclude, that the in-vivo-PK / in-vitro-PD model presented in this study may provide a rational approach for describing and predicting pharmacodynamics of cytotoxic drugs at the target site. Data derived from this approach support the concept that tumor penetration of 5-FU may be a response-limiting event, while the response to MTX may be determined by events beyond interstitial fluid kinetics.