Cancer Cell Implantation Research Articles

Comparison of Fixation Methods for Preservation of Morphology, RNAs, and Proteins From Paraffin-Embedded Human Cancer Cell-Implanted Mouse Models

Xenograft transplantation of human tumor cells into immunodeficient mice is an important method to clarify the roles of specific molecules or chemicals in vivo. Recently, this method has been reported as a definitive examination to identify tumor stem cells. In this study, the authors compared the morphology and the quality and quantity of ribonucleic acid (RNA) and protein in paraffin-embedded tissues of nude mice implanted with human uterine cervical cancer cells, followed by fixation with commonly used fixatives, including 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF), 20% NBF, and 99% ethanol (EtOH). The quality of the isolated RNA from PFA- and NBF-fixed paraffin-embedded tissues was high, while EtOH-fixed tissues showed degradation of RNA. NBF-fixed tissues showed excellent quality of morphology, but EtOH-fixed tissues showed contraction of cells. Immunohistochemical results showed differences depending on fixations. The 99% EtOH-fixed samples showed decreases of Ki-67 and VEGF-A immunoreactivities, but improved cytokeratin immunoreactivity. This study indicated that formalin fixation is better than alcohol fixation for RNA preservation in paraffin-embedded cancer cell implantation models. Immunohistochemical results differed markedly depending on fixation materials and antibodies; therefore, suitable fixations are needed to quantify and compare the results of immunohistochemical staining on cancer cell implanted nude mice tissues.

The sensitization of peripheral C-fibers to lysophosphatidic acid in bone cancer pain

Aims Lysophosphatidic acid (LPA) is released from injured tissue and cancer cells and is involved in the induction of neuropathic pain. The present study explores whether LPA plays a role in the development of osteocarcinoma-induced pain. Main methods The bone cancer model was established using the Walker 256 mammary gland carcinoma cell line, and cancer-related behavioral and physiological changes were observed using von Frey, X-ray and immunohistochemical methods. The role of LPA in the bone cancer model and related mechanisms were examined by using in vitro single fiber recording and western blot. Key findings Rats exhibited severe hyperalgesia 2 weeks after the cancer cell implantation. Several changes were observed at this time point including: ipsilateral dorsal root ganglion (DRG) neurons were labeled by injured neurons marker ATF3; LPA 1 receptor expression in DRG neurons was increased; sural C-fibers were more sensitive to LPA stimuli, and this response could be blocked by LPA receptor and substance P receptor antagonists. Significance These data indicate that LPA is involved in the induction of bone cancer pain through mechanisms of peripheral C-fibers sensitization. LPA and its downstream molecules possibly are promising therapeutic targets for treatment of cancer pain.

胃瘻部に転移した下咽頭癌例

Percutaneous endoscopic gastrostomy (PEG) tube emplacement is commonly used to feed those with head, neck, or esophageal cancer. An 80-year-old man with hypopharyngeal cancer underwent PEG before chemoradiotherapy. After 4 months, tissue granulation extended to the gastrostomy site. The pathological lesion findings showed squamous cell carcinoma, suspected to have metastasized from hypopharyngeal cancer. To avoid cancer cell implantation, it is important to select the type of gastrostomy carefully.

Intravesical Ultrasonography for Tumor Staging in an Orthotopically Implanted Rat Model of Bladder Cancer

Noninvasive and serial evaluation of tumor development and growth is important in an orthotopic animal bladder tumor model. However, to our knowledge a reliable method has not been established. We determined the usefulness of intravesical ultrasonography for tumor staging and volumetric assessment in an animal model. Tumors with various stages were formed in 20 female Fischer F344 rats by implanting AY-27 rat bladder carcinoma cells. Cells were implanted by instilling a suspension (4x10 cells) into the bladder cavity after urothelial denudation using hydrochloric acid or by directly injecting the cell suspension into the bladder wall. Tumor volume and invasion depth were measured by intravesical ultrasonography using an ultrathin 2.5Fr intraluminal ultrasound catheter via the urethra 7 to 10 days after cancer cell implantation. The rats were then sacrificed for histopathological examination. All rats showed bladder tumors 7 to 10 days after cancer cell implantation, of which stages varied from superficial to advanced disease. Intravesical ultrasonography showed a clear cross-sectional view of all layers of the bladder wall and enabled visualization of bladder tumors (minimal 0.5 mm in diameter). This approach also provided an accurate diagnosis of tumor invasion into muscle layers and perivesical tissues with precise invasion depth and tumor size. The positive predictive ratio regarding tumor staging reached 85%. Repeat examinations were feasible without noticeable adverse effects. Intravesical ultrasonography is a reliable and appropriate noninvasive method for evaluating tumor stage and size in an orthotopic rat bladder tumor model.

Liver Metastases in Rats: Chemoembolization Combined with Interstitial Laser Ablation for Treatment

To assess the effect of transcatheter arterial chemoembolization (TACE) combined with laser-induced thermotherapy (LITT) for treatment of liver metastases in an animal model. All experiments were approved by the German government and the institutional animal research review board. After subcapsular liver implantation of colorectal cancer cells in 30 WAG rats (on day 0), the animals were randomly assigned to three interventional treatment groups. In the 10 rats in group A, TACE was performed: Fourteen days after cancer cell implantation and within 20 minutes after laparotomy and retrograde placement of a microcatheter into the gastroduodenal artery, these rats were injected with mitomycin (0.1 mg), iodized oil (0.1 mL), and degradable starch microspheres (5.0 mg). In the 10 rats in group B, LITT was performed: Also on day 14, the tumors in these animals were exposed to Nd:YAG laser light of 1064 nm at 2 W for 5 minutes. In the 10 rats in group C, combined treatment was administered: TACE was performed on day 14, and LITT was performed on day 21. Tumor volumes were measured before (on day 13) and after (on day 28) treatment with magnetic resonance (MR) imaging, and the mean tumor growth ratio (day 13 tumor volume divided by day 28 tumor volume) was calculated. The mean tumor volumes measured before and after the treatments were, respectively, 0.11 and 0.60 cm(3) in group A, 0.11 and 0.68 cm(3) in group B, and 0.11 and 0.35 cm(3) in group C. The mean tumor growth ratio was 5.42 in group A, 6.14 in group B, and 3.15 in group C. According to Bonferroni test results, compared with the rats in groups A and B (controls), the group C rats had significantly inhibited tumor growth (P < .01 for both comparisons). Use of combined TACE-LITT treatment, compared with the use of TACE or LITT alone, significantly inhibits tumor growth.

Rationnels de la chirurgie oncologique au sein d’un traitement multimodal des cancers

The permanent dissemination of circulating cancer cells from infiltrative cancers renders the notion of localized cancer obsolete. This raises the question of the role of treatments such as surgery which are localized in time and space. Moreover, it is now proven that surgery increases the number of circulating cancers cells and produces transient immunodepression which promotes cancer cell implantation. In addition, the healing process stimulates growth factors, which also act on tumor growth. In spite of these deleterious effects, oncological surgery remains an important tool in the anti-cancer armamentarium. Optimal cytoreductive surgery facilitates the effect of adjuvant treatments by eliminating the vascular sanctuaries present in the center of any large tumor and by its effect as a striking-force very localized in time and space. Oncological surgery must evolve as an integrative part of our improving understanding of tumor biology, taking into account recent developments in functional imaging, genomics and proteomics. Modalities and timing must be reconsidered for oncological surgery to play its proper role as one of the many components contributing to multimodal cancer treatments. New modalities will undoubtedly be necessary with the development of future technologies.

MULTIVARIATE ANALYSIS OF CLINICAL PARAMETERS OF SYNCHRONOUS PRIMARY SUPERFICIAL BLADDER CANCER AND UPPER URINARY TRACT TUMOR

We determined the incidence and characteristic of synchronous upper urinary tract tumors (UUTTs) in patients with primary superficial bladder carcinoma and evaluated the characteristics of bladder tumors related to UUTTs. We performed a retrospective study of 1,529 patients with primary superficial bladder carcinoma who underwent initial examination of the upper urinary tract with excretory urography. Data were analyzed by multivariate analysis using logistic regression. Variables evaluated and related to the incidence of UUTT were multiplicity, carcinoma in situ, bladder tumor size, localization of tumor in the bladder, and tumor grade and stage. A total of 28 patients (1.8%) had simultaneous bladder tumor and UUTT. UUTTs showed no preferred location and 17.9% were multiple. Of UUTTs 46% were invasive and almost 87% were grade 2 or 3. The only significant variable related to UUTT was bladder tumor in the trigone (RR 5.8, 95% IC 2.18 to 15.9, p <0.0005). Of 147 tumors located in the trigone 11 (7.5%) were associated with UUTT, corresponding to 41% of the UUTTs first diagnosed. If multiplicity and tumors in the trigone (551 cases) had been considered, 66.7% of tumors would have been diagnosed. Synchronous UUTT and superficial bladder tumor are uncommon but 46% are invasive. Considering the possible examination of the upper urinary tract only in patients with tumor in the trigone or with multiple bladder tumors 41.4% or 69% of UUTTs, respectively, would have been diagnosed. Patients with tumor in the trigone are at almost 6-fold higher risk for a synchronous tumor in the upper urinary tract.

Early diagnosis of breast cancer by hair diffraction

A correlation between the incidence of breast cancer and an observed change in the X-ray diffraction pattern of hair from the afflicted individuals was first reported in 1999. Since that time, over 500 hair samples have been analyzed in double-blinded breast cancer studies with no false negatives being detected. To correlate this observed change with the presence of breast cancer, we examined whiskers removed from nude mice prior to and 8 weeks after subcutaneous implantation of a human breast cancer cell line. Here we show that the change observed in human hair was also evident in whiskers and that it appeared soon after cancer cell implantation.

Suppression of peritoneal implantation of gastric cancer cells by adenovirus vector-mediated NK4 expression.

Peritoneal dissemination is the most common mode of metastasis in gastric cancer. We previously reported the importance of milky spots (MS), peritoneal lymphoid tissues, as selective sites of cancer implantation in peritoneal dissemination. In the present study, we first demonstrated that intraperitoneal injection of adenovirus vector encoding the GFP gene into tumor-free nude mice resulted in GFP expression at omental and mesenteric MS; MS macrophages were target cells for adenovirus infection. We confirmed that intraperitoneal injection of adenovirus vector encoding the NK4 gene (AdNK4) resulted in NK4 production localized to the peritoneal cavity, especially the omentum. Adenovirus vector-mediated MS-selective transgene expression was markedly impaired in tumor-bearing mice whose MS had already been replaced by infiltrating cancer cells. However, prior injection of AdNK4 successfully inhibited MS-selective cancer cell implantation, resulting in suppression of peritoneal dissemination and prolongation of survival. Adenovirus vector-mediated MS-selective delivery of a therapeutic gene may prevent peritoneal dissemination of gastric cancer.

TIMP-1 Overexpression in Pancreatic Cancer Attenuates Tumor Growth, Decreases Implantation and Metastasis, and Inhibits Angiogenesis

Background. Matrix metalloproteinases (MMPs) are involved in pancreatic cancer progression. This study was undertaken to determine the effects of overexpression of a natural tissue inhibitor of MMP (TIMP-1) on pancreatic cancer cell growth, metastasis, and angiogenesis.Methods. A poorly differentiated human pancreatic cancer cell line (PANC-1) underwent gene transfection to overexpress TIMP-1 (CD-1 cells). One million PANC-1 or CD-1 cells were injected into 40 nude mice subcutaneously (N = 20) or orthotopically (N = 20). Mice were sacrificed at 120 days. TIMP-1 overexpression by CD-1 cells was confirmed prior to injection and after necropsy. Immunohistochemical staining was undertaken after necropsy to evaluate tumors for TIMP-1, MMP-2, apoptosis (TUNEL), and angiogenesis (CD-31).Results. Tumors of CD-1 cells were less likely to implant (35% vs 70%, P = 0.05) and grew to smaller size (0.5 g ± 0.03 vs 1.5 g ± 0.20, P < 0.001) than tumors of PANC-1 cells. As well, subcutaneous CD-1 tumors appeared later than PANC-1 tumors (45 days ± 2.0 vs 27 days ± 2.2, P < 0.001). Orthotopic CD-1 tumors metastasized less often than PANC-1 tumors (20% vs 60%, P < 0.05). MMP-2 expression was similar in PANC-1 and CD-1 tumors, while CD-1 tumors showed increased TIMP-1 expression, increased apoptosis, and decreased angiogenesis relative to PANC-1 tumors.Conclusions. TIMP-1 overexpression reduces pancreatic cancer cell implantation, growth, metastasis, and angiogenesis, while increasing tumor apoptosis, all without altering MMP-2 production. This study demonstrates the potential role of TIMP-1 as a target in gene therapy for pancreatic cancer.

Bladder cancer cell implantation in reconstructed bladder in vitro: a model of tumour recurrence

To evaluate the involvement of umbrella cells in tumour adhesion and growth, by examining whether human urinary bladder carcinoma cells (HUBCC) can grow on reconstructed urinary bladder mucosa in vitro, as the implantation of tumour cells after resection is thought to be a cause of bladder tumour recurrence. Normal transitional epithelial cells isolated from porcine bladder were cultured on reconstructed lamina propria using fibroblasts in type I collagen gel. The urothelium thus reconstructed was artificially injured either by a scalpel or by dilute acid, after which transitional epithelial cells began to grow in a stratified fashion within a few days of culture. A HUBCC line (HT-1197) was seeded onto this impaired mucosa to determine whether the cells could become implanted. Cultured cells on the reconstructed mucosa were evaluated by histological observation of vertical paraffin sections. The inoculated transitional epithelial cells grew in a stratified fashion and closely resembled urothelium in vivo. The superficial cells that were in contact with the medium solution differentiated into umbrella cells. HUBCC were unable to adhere to reconstructed mucosa which had not been injured, but these cells could adhere to and become implanted on the reconstructed mucosa after it had been injured either by a scalpel or by dilute acid. After acid injury, only the surface-covering cells were removed sporadically, while the lower epithelial cell layer remained intact. The bladder cancer cells adhered to and proliferated within these stripped regions. These results suggest that the urothelium, especially umbrella cells, seems to be important in preventing the adhesion and growth of urinary bladder tumour cells.

Bladder cancer cell implantation in reconstructed bladder in vitro: a model of tumour recurrence

Objective To evaluate the involvement of umbrella cells in tumour adhesion and growth, by examining whether human urinary bladder carcinoma cells (HUBCC) can grow on reconstructed urinary bladder mucosa in vitro, as the implantation of tumour cells after resection is thought to be a cause of bladder tumour recurrence. Materials and methods Normal transitional epithelial cells isolated from porcine bladder were cultured on reconstructed lamina propria using fibroblasts in type I collagen gel. The urothelium thus reconstructed was artificially injured either by a scalpel or by dilute acid, after which transitional epithelial cells began to grow in a stratified fashion within a few days of culture. A HUBCC line (HT‐1197) was seeded onto this impaired mucosa to determine whether the cells could become implanted. Cultured cells on the reconstructed mucosa were evaluated by histological observation of vertical paraffin sections. Results The inoculated transitional epithelial cells grew in a stratified fashion and closely resembled urothelium in vivo. The superficial cells that were in contact with the medium solution differentiated into umbrella cells. HUBCC were unable to adhere to reconstructed mucosa which had not been injured, but these cells could adhere to and become implanted on the reconstructed mucosa after it had been injured either by a scalpel or by dilute acid. After acid injury, only the surface‐covering cells were removed sporadically, while the lower epithelial cell layer remained intact. The bladder cancer cells adhered to and proliferated within these stripped regions. Conclusion These results suggest that the urothelium, especially umbrella cells, seems to be important in preventing the adhesion and growth of urinary bladder tumour cells.

β1-Integrins Partly Mediate Binding of Ovarian Cancer Cells to Peritoneal Mesothelium in Vitro

Ovarian cancer cells frequently metastasize by implanting onto the peritoneal mesothelial surface of the abdominal cavity. Although the CD44 molecule expressed by ovarian cancer cells is known to partly mediate this process, the role of other adhesion proteins such as β1-integrins has been previously difficult to demonstrate using the 4B4 anti-β1 neutralizing antibody. In view of the widespread expression of β1-integrins in ovarian cancer, however, we have further examined the potential role of this class of molecules in ovarian cancer cell implantation through the use of an alternative anti-β1 neutralizing antibody, MAB13. We now report that MAB13 is capable of inhibiting the binding of three separate human ovarian cancer cell lines (36M2, CAOV-3, SKOV-3) to mesothelium (mean 37 ± 4% inhibition, n = 21, P < 0.001). An additive inhibitory effect was observed when MAB13 was combined with anti-CD44 antibody (clone 515) (mean 63 ± 3% inhibition, n = 19, P < 0.001), suggesting that binding occurs through two independent pathways involving both β1-integrins and CD44. Because fibronectin is an extracellular matrix ligand recognized by many types of integrins and is abundantly expressed on mesothelial cells, the inhibitory effects of MAB13 and 4B4 on ovarian cancer cell binding to fibronectin were directly compared. MAB13 inhibited ovarian cancer cell binding to fibronectin to a significantly greater degree than did 4B4, suggesting that the discordant effects of these two antibodies on mesothelial adhesion may be partly related to their differential ability to neutralizing fibronectin-mediated binding. Studies using anti-α5 neutralizing antibody demonstrated that the α5β1 fibronectin receptor contributes to approximately 50% of integrin-mediated binding of 36M2 and CAOV-3 cells (which express the α5 chain in 54 and 58% of cells, respectively). Since the RGD sequence of fibronectin is a known recognition site for many types of integrins, including α5β1 and ανβ3, we performed binding in the continued presence of both anti-α5 and RGD peptide in order to exclude other types of fibronectin–integrin interactions. The addition of RGD peptides at concentrations known to be capable of blocking fibronectin binding resulted in no additional inhibitory effect over that observed with anti-α5 antibody alone, suggesting that α5β1 was the major receptor responsible for fibronectin-mediated ovarian cancer binding to mesothelium. These data demonstrate that ovarian cancer cell binding to peritoneal mesothelium is mediated by several adhesion pathways and that simultaneous inhibition of both β1-integrin and CD44 function may be necessary in order to significantly limit the intraabdominal spread of this tumor in vivo.

Sex prevalence of focal dystonias.

The sex prevalence of idiopathic focal dystonia is reported from a data base review of all patients seen at the National Hospital of Neurology, Queen Square and King's College, London up to 1993. There was a higher prevalence of females to males in all categories of focal dystonia involving the craniocervical region. The female to male ratio for cranial dystonia was 1.92:1 (P < 0.01) and 1.6:1 (P < 0.001) for spasmodic torticollis. On the other hand, twice as many men than women had writer's cramp (M:F = 2.0:1, P < 0.01). At present, there is no clear explanation to account for this differences in the sex prevalence of different types of focal dystonia.

Expression and Function of β1 and αvβ3 Integrins in Ovarian Cancer

Ovarian cancer cells disseminate by implanting onto the peritoneal mesothelial cell surface of the abdominal cavity. A common feature of these peritoneal implants is the presence of tumor cell invasion into the submesothelial extracellular matrix (ECM). In view of the important role of integrins in ECM recognition and cell migration, we were interested in defining the pattern of integrin expression and function in ovarian cancer cell lines and primary tissue samples. The β1 integrin chain was expressed by CAOV-3, SKOV-3, OVCAR-3, and SW626 ovarian cancer cell lines, associated with expression of α1, -2, -3, -5, and -6 chains. The α4 chain was also expressed by two of the four lines. In addition to β1 integrins, the αvβ3 integrin was also expressed, although there was no expression of β2, -4, and -7 chains. Immunoprecipitation of surface-labeled CAOV-3 cells with an anti-β2 antibody revealed a band at ≈110-130 kDa consistent with the known molecular mass of the β1 chain, as well as several associated bands consistent with noncovalently linked integrin α chains. A similar pattern of β1 and αvβ3 integrin expression was observed for primary ovarian cancer tissue samples. Ovarian cancer cell lines exhibited significant binding to collagen type I and laminin which was primarily mediated by β1 integrins. In contrast, ovarian cancer cell binding to fibronectin was mediated by both α5β1 and αvβ3 integrins. Even though mesothelial cells were observed to express fibronectin mRNA and protein, binding of ovarian cancer cells to peritoneal mesothelium was not blocked by neutralizing antibodies to β1 or αvβ3 integrins. These data suggest that functional integrins are commonly expressed by ovarian cancer cells, although they do not appear to mediate ovarian cancer cell implantation onto peritoneal mesothelium. The role that integrins play in the invasion of ovarian cancer cells into the submesothelial ECM deserves further investigation.