Targeting of adenovirus vectors to tumor cells does not enable efficient transduction of breast cancer metastases

Targeting of oncolytic adenoviruses to tumors can potentially increase their efficacy and safety profile after systemic application. We have developed recently a capsid-modified vector containing the adenovirus serotype 35 fiber shaft and knob inserted into an Ad5 capsid. This Ad5/35 vector infects cells via a coxsackievirus adenovirus receptor-independent pathway. Here we attempted to exploit this new tropism of Ad5/35 vectors for tumor-specific infection. In vitro, the Ad5/35 vector efficiently transduced human breast cancer cells that were refractory to infection with conventional Ad5-based vectors. Additionally, primary mouse hepatocytes were relatively refractory to Ad5/35 infection in vitro or after systemic vector application to mice. In an animal model of breast cancer metastasis, intraportal infusion of MDA-MB435 cells produced multiple hepatic metastases that were surrounded by extracellular matrix and developed blood vessels confined to the tumor stroma. Tail vein injection of a standard Ad5-based vector into tumor-bearing animals resulted in transduction of mouse hepatocytes but not metastases. However, the capsid-modified Ad5/35 vector transduced only approximately 8% of metastases. The metastases that were susceptible to Ad5/35 infection demonstrated blood vessels in close proximity to tumor nests without extracellular matrix separating endothelial and tumor cells. These findings indicate that transduction of liver metastases not only requires tumor-specific tropism but also new strategies to increase accessibility of tumor cells to systemically applied oncolytic adenoviruses.     

Volume of lymphatic metastases does not independently influence prognosis in colorectal cancer.

PURPOSE: To evaluate the prognostic relevance of the volume of nodal metastatic disease in colorectal cancer patients. PATIENTS AND METHODS: One hundred node-positive patients with T2 or T3 carcinoma of the colon or rectum after routine histologic examination of the regional nodes were studied. The metastatic tumor was measured with an ocular micrometer, and the tumor volume was determined. RESULTS: The mean lymph node metastatic tumor volume was 5.1 +/- 4.99 mm(3) (range, 0.05 to 83,434 mm(3)). There was only a weak positive correlation with number of nodes involved with metastatic disease and tumor volume in nodes (r =.45). Median follow-up was 39 months (range, 1 to 87 months). The number of nodes was highly predictive of outcome. Individuals with one to three positive nodes had a substantially better survival than individuals with four or more positive nodes (P <.001). The volume of nodal metastatic disease correlated with outcome (P =.019). Patients dying as a result of disease had substantially greater mean metastatic nodal volume than those who were alive (3,705 v 1,783 mm(3); P =.036). However, the total metastatic nodal volume did not, independent of positive nodes or number of positive nodes, predict outcome. Individuals with micrometastatic nodal volume did not have improved survival when compared with individuals with macrometastatic nodal volume (P =.79). CONCLUSION: The number of nodes involved with metastatic tumor, rather the volume of metastatic involvement of the regional lymph nodes, predicts outcome. These results suggest that micrometastatic disease may have a similar prognosis as macrometastatic disease when the same number of lymph nodes are involved with metastatic tumor.     

Irrigation does not dislodge or destroy tumor cells adherent to the tumor bed

Local recurrences in the surgical bed after tumor resection may be due to residual tumor cells “dropping” into the wound. Irrigation with water is often used to remove these cells. We designed experiments to determine whether irrigation would prevent tumor recurrence. Surgical wounds of uniform size in C57BL/6 mice were seeded with 5 × 10², 5 × 10³, 5 × 10⁴, 5 × 10⁵, or 5 × 10⁶ viable syngeneic B16-F10 melanoma cells to test the hypothesis that irrigation with water would decrease local tumor recurrence. The tumor-contaminated wounds were irrigated with distilled water or with saline (0.9% NaCl) immediately or 5, 30, 60, 120, or 240 min after seeding. Control wounds were seeded but not irrigated. The technique of irrigation was altered in a second group of experiments such that the amount of time the tumor cells were exposed to the water or saline was 5, 10, or 15 min. To determine the rapidity and durability of tumor cell attachment to host tissue, 1 × 10⁴ viable B16-F10 tumor cells were seeded in vitro onto freshly cut disks of syngeneic mouse dermis. The tissue was irrigated with saline or distilled water 0, 2, 5, 10, 15, 30, 60, 120, or 240 min later. Tumor growth was observed in all the mice and neither the mechanical action of irrigation nor the hypotonic effect of distilled water changed the rate of growth. Scanning electron microscopy (SEM) demonstrated stable and firm attachment to mouse tissue within seconds of seeding with no noticeable dislodgement or cytotoxicity by either saline or water irrigation. The data suggest that the commonly used technique of irrigating the bed of the resected tumor may not be of value in preventing local recurrences. © 1993 Wiley-Liss, Inc.     

Bladder opacification does not significantly influence dose distribution in conformal radiotherapy of prostate cancer.

PURPOSE: To compare the results of treatment planning with or without bladder contrast during simulation of three dimensional conformal radiotherapy (3D-CRT) for prostate cancer (18 MV X-rays, six field arrangement), and to assess the potential changes in dose distribution in the target and rectal volumes. METHODS AND MATERIALS: Based on CT-simulation using intravenous contrast media, 3-D conformal treatment planning was performed in five patients. To simulate a non-opacified bladder, the electron matrix density of the opacified bladder was virtually changed to water density. Two treatment plans were carried out, with and without bladder opacification. In each patient dose distributions were formally compared for both plans, and the increment in monitor units (MU) needed to compensate for the presence of contrast media was assessed. RESULTS: A mean dose variation of -0.03% (range, -0.03-0.14%) and -1.13% (range, -1.85-0%) was observed for the prostate and the rectum, respectively. The average mean MU increment without bladder contrast normalized to the case with bladder contrast was 0.31% +/- 0.52. CONCLUSIONS: Bladder opacification used during simulation does not significantly influence prostate or rectal dose distributions in prostate patients treated with 3D-CRT, 18 MV X-rays, and a six-beam arrangement.     

Recurrence dynamics does not depend on the recurrence site

Introduction  

The dynamics of breast cancer recurrence and death, indicating a bimodal hazard rate pattern, has been confirmed in various databases. A few explanations have been suggested to help interpret this finding, assuming that each peak is generated by clustering of similar recurrences and different peaks result from distinct categories of recurrence.     

Primary systemic therapy does not eradicate disseminated tumor cells in breast cancer patients

Introduction The presence of disseminated tumor cells in the bone marrow of breast cancer patients has proven to be an independent prognostic factor. The aim of this study was to investigate the status of tumor cell dissemination after primary systemic therapy in relation to therapy response. Methods Bone marrow aspirates were obtained from 120 patients after completion of primary systemic therapy. Disseminated tumor cells were detected by immunocytochemistry using the APAAP method. Bone marrow status was correlated with clinicopathological factors as well as tumor response to primary systemic therapy. Results Sixty out of 120 patients had disseminated tumor cells in their bone marrow aspirates (50%). Response rates were 18% for pathologic complete remission, 52% for partial remission, 28% for no change and 3% for progression. Despite complete remission, 36% of these patients were bone marrow positive. In the partial remission group, the positivity rate was 48%. About 61% of patients with stable disease had disseminated tumor cells in their bone marrow. A trend to higher positivity rates was observed in the poor responder group compared to responders (61% vs. 38%, P = 0.1). Conclusion Primary systemic therapy does not completely eradicate disseminated tumor cells in the bone marrow of breast cancer patients. The biological role of persistent disseminated tumor cells needs to be further investigated to optimize current and future therapeutic strategies.     

Hexadecylphosphocholine does not influence phospholipase D and sphingomyelinase activity in human leukemia cells

Hexadecylphosphocholine (HePC) is the first representative of the alkylphosphocholines (APC), a new group of biologically active compounds. HePC has pronounced antiproliferative effects on neoplastic cells in vitro and in vivo. The molecular mechanism by which HePC exerts its biological effects is still under investigation. Recently there has been growing evidence that HePC probably interferes with cellular signalling via phospholipases. It has been shown to inhibit both forms of phospholipase C (PLC), the phosphatidylinositol- and the phosphatidylcholine-specific PLC, and phospholipase A2. Here we present data showing that HePC inhibits the activity of phospholipase D in vitro, whereas the action of this enzyme in leukemic cell lines is not affected. Furthermore HePC does not seem to disturbed the activity of sphingomyelinase, another enzyme of phospholipid metabolism which has been shown to play an important role in cellular signalling as well.     

Surgery promotes implantation of disseminated tumor cells, but does not increase growth of tumor cell clusters

INTRODUCTION: Local recurrence and peritoneal dissemination is common after intentionally curative resection of colorectal carcinoma. It is not yet clear which mechanisms stimulate post-operative intra-abdominal tumor development. Enhanced adhesion or growth of tumor cells and/or post-operative immuno suppression may influence tumor recurrence. AIMS OF THE STUDY: In the present study, we evaluated effects of local and remote surgery on intra-abdominal tumor development. MATERIALS AND METHODS: A standardized intra-abdominal trauma was inflicted by rubbing both uterus horns in laparotomy groups, while a dorsolateral thoracotomy was performed in thoracotomy groups (on day -1, 0, or +3). To induce tumor development rats were injected intra-peritoneally with the coloncarcinoma cell line CC531s on day 0 and evaluated after 21 days. RESULTS: Rats undergoing laparotomy and injection on day 0 showed significantly higher tumorload than control rats (195 +/- 20 vs. 47 +/- 29, P < 0.001). When a laparotomy was performed, the day before tumor inoculation even higher tumorload was seen (245 +/- 37 vs. 195 +/- 20, P < 0.01). Strikingly, performing a thoracotomy on the day before or on the same day as tumor inoculation resulted in enhanced tumorload compared to controls as well (135 +/- 84 vs. 47 +/- 29; P < 0.001 and 88 +/- 38 vs. 47 +/- 29; P < 0.02, respectively). Either laparotomy or thoracotomy 3 days after tumor cell inoculation did not affect growth of pre-existing tumor cell clusters. CONCLUSIONS: The (post) surgical intra-peritoneal microenvironment enhances successful implantation of spilled tumor cells, whereas growth of adhered tumor cell clusters is not affected. The inflammatory response as a result of remote surgery promotes successful tumor development as well.     

Sentinel lymphadenectomy does not increase the incidence of in-transit metastases in primary melanoma.

PURPOSE: Recent reports by European investigators suggest that sentinel lymphadenectomy (SLND), a mainstay of melanoma diagnosis and treatment planning, increases the risk of in-transit metastasis (ITM) and should be abandoned. This study compared the incidence of ITM after wide local excision (WLE), WLE plus SLND (SLND), or WLE plus elective lymphadenectomy (ELND) for primary melanoma. PATIENTS AND METHODS: Review of our prospective database identified 4,412 patients who underwent WLE (n = 2,771), SLND (n = 1,016), or ELND (n = 625) for stage I/II melanoma (1971 through 2002). The incidence of ITM overall and as a first recurrence was examined before and after computerized prognostic matching of treatment groups. Intergroup statistical comparisons used chi(2) analysis and log-rank test. RESULTS: The incidence of ITM increased with Breslow depth, Clark level, and T stage. Although overall incidence of ITM was significantly higher (P = .0008) after ELND (6.56%) versus WLE (3.36%) or SLND (3.64%), the ELND group had higher risk primaries. Treatment groups matched by T stage (1,875 patients; 625 per group) or by age, sex, Breslow depth, and primary location (1,680 patients; 560 per group), showed no significant differences in ITM overall or as a first recurrence. CONCLUSION: There is no relationship between SLND and ITM. Recent reports to the contrary reflect analysis of significantly smaller cohorts not matched for confounding variables such as T stage. The phase III Multicenter Selective Lymphadenectomy Trial will definitively settle the issue; until then, use of SLND, the most accurate staging procedure for early-stage melanoma, should continue.     

The emergence of tumor metastases

The appearance of metastases is an ominous sign in the natural history of any malignant tumor. Their presence implies a high tumor burden and greatly decreases the probability of a cure. Metastasis development requires the evolution of tumor cells that can survive in an environment that is normally not supportive to their growth and such cells must leave the tumor to establish tumor niches elsewhere. The interactions between the appearance of cells with metastatic ability in the primary tumor and their exit from the tumor lead to complex dynamics that can be either beneficial or detrimental to the tumor. We develop a simple mathematical model to illustrate how the interplay between mutation rate and export probability affects the intratumoral dynamics of metastasis-enabled cells and the rate of metastases formation.     

Glucagon-like peptide-2 does not modify the growth or survival of murine or human intestinal tumor cells

Glucagon-like peptide-2 (GLP-2) secreted from enteroendocrine cells exerts proabsorptive, regenerative, and cytoprotective actions in the normal and injured gut epithelium. Hence, sustained GLP-2 receptor (GLP-2R) activation represents a strategy under investigation for the prevention and treatment of chemotherapy-induced mucositis. Nevertheless, the consequences of increased GLP-2R signaling for the growth and survival of intestinal tumor cells remain poorly understood. We studied the proliferative and cytoprotective actions of GLP-2 in human colon cancer cells stably transfected with the GLP-2R and in nude mice harboring GLP-2R(+) human colon cancer cells. The importance of the GLP-2R for tumor growth was also examined in Apc(Min/+) mice chronically treated with exogenous GLP-2 and in Apc(Min/+):Glp2r(-/-) mice. GLP-2 increased cyclic AMP accumulation and produced cell-specific activation of growth and survival pathways in DLD-1, SW480, and HT29 cells. However, GLP-2 did not stimulate cell growth or attenuate cycloheximide-, LY294002-, indomethacin-, or chemotherapy-induced cytotoxicity in vitro. Moreover, chronic GLP-2 administration had no effect on the growth of human colon cancer cell xenografts in nude mice in vivo. Daily GLP-2 treatment for 7 weeks increased growth of normal gut mucosa but did not increase the number or size of polyps in Apc(Min/+) mice, and genetic disruption of the Glp2r gene in Apc(Min/+) mice did not modify polyp size or number. Taken together, although GLP-2R activation engages signaling pathways promoting cell proliferation and cytoprotection in the normal gut epithelium, sustained direct or indirect modulation of GLP-2R signaling does not modify intestinal tumor cell growth or survival.     

Sentinel lymphonodectomy does not increase the risk of loco-regional cutaneous metastases of malignant melanomas

With regard to malignant melanoma, the impact of lymph node surgery on the development of loco-regional cutaneous metastases (LCM) has not yet been adequately addressed. However, this aspect is of interest, since sentinel lymphonodectomy (SLNE) has been suspected of causing LCM by inducing entrapment of melanoma cells. We analysed 244 patients with SLNE and compared the data with 199 patients treated with delayed lymph node dissection (DLND) for clinically palpable metastases. Analysis of both groups commenced at the time of excision of the primary tumour, using the Kaplan-Meier method. LCM that appeared as a first recurrence, as well as the overall probability of developing LCM, were recorded. For sentinel-negative patients with a primary melanoma >1mm thick, the 5-year probability of developing LCM as a first recurrence was 6.9 +/- 0.02% (+/-standard error of the mean (SEM)). The probability was 17.6 +/- 0.03% in the DLND group. Comparing the two node-positive subgroups, the probability of developing LCM as a first recurrence was significantly higher in patients with positive SLNE (27.3 +/- 0.05%, P = 0.03). However, the 5-year overall probability of developing LCM did not differ significantly in the node-positive groups (33.3% in the DLND group vs. 33.7% in patients with positive sentinel lymph nodes (SLNs)). Since early excision of lymphatic metastases by SLNE avoids nodal recurrences, thereby prolonging the recurrence-free interval, the chance of LCM to manifest as a first recurrence should inevitably increase. However, the overall in-transit probability is not increased after SLNE.     

The expression of P-glycoprotein does influence the distribution of novel fluorescent compounds in solid tumour models

Solid tumours display a complex drug resistance phenotype that involves inherent and acquired mechanisms. Multicellular resistance is an inherent feature of solid tumours and is known to present significant barriers to drug permeation in tumours. Given this barrier, do acquired resistance mechanisms such as P-glycoprotein (P-gp) contribute significantly to resistance? To address this question, the multicellular tumour spheroid (MCTS) model was used to examine the influence of P-gp on drug distribution in solid tissue. Tumour spheroids (TS) were generated from either drug-sensitive MCF7(WT) cells or a drug-resistant, P-gp-expressing derivative MCF7(Adr). Confocal microscopy was used to measure time courses and distribution patterns of three fluorescent compounds; calcein-AM, rhodamine123 and BODIPY-taxol. These compounds were chosen because they are all substrates for P-gp-mediated transport, exhibit high fluorescence and are chemically dissimilar. For example, BODIPY-taxol and rhodamine 123 showed high accumulation and distributed extensively throughout the TS(WT), whereas calcein-AM accumulation was restricted to the outermost layers. The presence of P-gp in TS(Adr) resulted in negligible accumulation, regardless of the compound. Moreover, the inhibition of P-gp by nicardipine restored intracellular accumulation and distribution patterns to levels observed in TS(WT). The results demonstrate the effectiveness of P-gp in modulating drug distribution in solid tumour models. However, the penetration of agents throughout the tissue is strongly determined by the physico-chemical properties of the individual compounds.     

Rapid induction of malignant tumor in Sprague-Dawley rats by injection of RK3E-ras cells

Several tumor animal models have been provided as a tool for developing cancer therapy. Here, we developed rapid, easy-to use, and cost-effective new rat animal model for invasion and metastasis of cancer using genetically k-ras-induced rat kidney cells (RK3E-ras). We observed tumor as early as 3 days after injection of RK3E-ras cells in subcutaneous of Sprague-Dawley rats. Tumor size and volume were increased exponentially for 2 weeks. The tail vein injected rats obtained the lethal infiltration in the lung within 2 weeks. This tumor animal model has great potential for studying cancer processes and short-term screening of variable cancer therapy strategy.     

Thyroxine administration during radiation therapy to the neck does not prevent subsequent thyroid dysfunction

In an attempt to reduce the incidence of hypothyroidism following irradiation of the neck, we administered oral L-thyroxine in doses sufficient to suppress serum TSH to 20 patients receiving radiation therapy for Hodgkin's disease or other lymphomas. L-thyroxine was discontinued when radiation therapy was completed. Twenty similar patients who did not receive L-thyroxine during radiation therapy served as a control group. After a mean follow-up period of 33 months, seven patients (35%) in the L-thyroxine group developed elevation of serum TSH and were started on chronic L-thyroxine therapy. In the control group, after mean follow-up of 19 months, five patients (25%) developed elevation of TSH and were started on chronic L-thyroxine. We conclude that suppression of serum TSH during neck irradiation does not prevent subsequent thyroid dysfunction.     

Transient Kupffer cell depletion and subsequent replacement by infiltrating monocyte-derived cells does not alter the induction or progression of hepatocellular carcinoma

Due to a combination of rapid disease progression and the lack of curative treatment options, hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Infiltrated, monocyte-derived, tumor-associated macrophages are known to play a role in HCC pathogenesis, but the involvement of Kupffer cells (KCs) remains elusive. Here, we used the Clec4F-diphteria toxin receptor transgenic mouse model to specifically investigate the effect of KC depletion on HCC initiation, progression and neoplastic growth following liver resection. For this purpose, several HCC mouse models with varying underlying etiologies were used and partial hepatectomy was performed. Our results show that in HCC, developed on a fibrotic or non-alcoholic steatohepatitis background, depletion of embryonic KCs at the onset of HCC induction and the subsequent replacement by monocyte-derived KCs does not affect the tumor burden, tumor microenvironment or the phenotype of isolated KCs at end-stage disease. In non-chronic liver disease-associated diethylnitrosamine-induced HCC, ablation of Clec4F⁺ KCs did not alter tumor progression or neoplastic growth following liver resection. Our results show that temporal ablation of resident KCs does not impact HCC pathogenesis, neither in the induction phase nor in advanced disease, and indicate that bone marrow-derived KCs are able to swiftly repopulate the available KC niche and adopt their phenotype.