Drug delivery to tumors. A problem requiring microscopic resolution

One factor in the effectiveness of chemotherapy for both primary tumors and their metastases is the delivery of the drug to the cancer cells within the tumor. The uptake of Adriamycin, assayed in 8 cu mm samples from primary tumors, lung metastases and "normal" lung tissue (unaffected by the metastases) in mice, showed wide variations between different tumors, between primary tumors and their metastases and between different metastases. The heterogeneity of uptake was greater in tumors than in normal lung tissues, whose uptake was in the range of the metastases, i.e., higher than in the tumor itself. Studies of Adriamycin uptake within individual tumors showed a wide range of values; this variability was statistically significantly increased as compared with the variation in normal lung tissues. Since the lower levels found in some tumor areas were below the cytotoxic level, those areas may represent "drug-resistant" cells from which tumor recurrence could ensue. In a three-dimensional reconstruction, the areas of lowest uptake were found at both peripheral and internal regions, a finding not in accord with the concentric zonal concentration expected on the basis of considerations of tumor vascularity. It is suggested that the problem of drug delivery to tumors needs further study at the microscopic level and that automated microscopic image analysis and three-dimensional reconstructions of serial sections could greatly extend the macroscopic findings of this study.     

Release of Active Drug from Intracellular Liposomal Prodrug Depots in Macrophages Controlled by Liposome Composition

Abstract

Several drugs have limited potency due to their rapid elimination or inactivation. The anticancer drug 5-fluoro-deoxyuridine (FUdR), which is frequently used in therapeutic treatment of liver metastases from colon tumors, is an example of such drugs. It is rapidly eliminated from circulation and metabolized, mainly by the hepatocytes in the liver. Over the past few years we have investigated the possibility to keep the drug away from the hepatocytes and to save it from rapid inactivation by encapsulating it in liposomes. In this way the liposomal drug is expected to accumulate in the macrophages of the liver (Kupffer cells), which form a major target site for intravenously administered liposomes. There, as the liposomal structure is gradually degraded by lysosomal enzymes, the drug will be released, initially within the lysosomal compartment, while subsequently it will leak out of the lysosomes and eventually out of the cells so as to become available for uptake by intrahepatically situated tumor cells. In this contribution we describe this system for the prodrug dipalmitoyl-FUdR, incorporated in the liposomal bilayer, requiring an additional step for the drug to become available, i.e. the enzymatic deacylation of the prodrug. It is demonstrated that the rate of intralysosomal degradation of liposomes in Kupffer cells varies substantially with liposomal lipid composition and that the rate of release of active drug from the Kupffer cells parallels the rate of liposome degradation. In addition, it is demonstrated that in this way the antitumor activity of the FUdR can be enhanced by more than two orders of magnitude and that the degree of antitumor activity reflects, to a limited extent, the rate at which the liposomes are degraded.     

Claudin-2 promotes breast cancer liver metastasis by facilitating tumor cell interactions with hepatocytes

We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.     

Sensitivity and resistance in human metastatic melanoma to the new chloroethylnitrosourea anti-tumor drug Fotemustine

Fotemustine is a novel chloroethylnitrosourea derivative currently used in Phase III clinical trials for disseminated metastatic melanoma. This drug has been shown to inhibit enzymes in the ribonucleotide reduction pathway (i.e., thioredoxin reductase, glutathione reductase and ribonucleotide reductase). 14C chloroethyl-labelled Fotemustine covalently labels the thiolate active sites of thioredoxin reductase and glutathione reductase yielding 14C chloroethyl-thioether enzyme-inhibitor complexes. Enzyme activities can be restored by a reduced thioredoxin or reduced glutathione mediated beta-elimination of the chloroethyl group. 14C Fotemustine has been used to determine its reactivity and metabolism in drug sensitive and resistant melanoma metastases and in cultures of sensitive and resistant clones of human melanoma cells. Melanoma metastases from four different patients who were treated with Fotemustine could be labelled with radioactive drug only under reducing conditions with NADPH as electron donor and DTNB as substrate. FPLC analysis of these extracts revealed two radioactive proteins (I) glutathione reductase and (II) an unidentified protein with 95 and 50 kDa subunits. A similar labelling pattern was also found in extracts of Fotemustine sensitive melanoma cells (Cal 1). Fotemustine resistant tumors were melanotic and contained more glutathione reductase than thioredoxin reductase, whereas sensitive tumors were clinically amelanotic with more thioredoxin reductase than glutathione reductase. Fotemustine resistant melanoma cells (Cal 7) showed a slower uptake of 14C-label with 34% less isotope intracellularly in 1 h compared to sensitive melanoma cells (Cal 1). These results strongly indicate (I) the induction of alternate electron donors thioredoxin reductase or glutathione reductase for ribonucleotide reduction determines tumor and melanoma cell responses to the drug and (II) Fotemustine transport and the intracellular redox status seems to regulate resistance in melanoma cells and tissues.     

Influence of experimental hypothyroidism on the development of metastases in rats

The influence of experimental hypothyroidism induced by a synthetic antithyroid drug, methylthiouracil, upon the development of metastases was studied on male Wistar rats, inoculated intravenously with Walker 256 carcinosarcoma cells. The experimental results reveal obvious differences between the control group and the group previously treated with methylthiouracil, concerning the incidence, latent period, localization and tumor extension. Hypothyroidism promoted metastasis, increasing significantly the metastatic incidence (88% against 62% in controls), shortening the latent period and extending the localization of tumor metastases to unusual sites, such as liver and spinal column.     

Systemic Activation of Macrophages by Liposomes Containing Muramyltripeptide Phosphatidylethanolamine for therapy of Cancer Metastasis

Abstract

The heterogeneous response of metastases to conventional therapy is a major cause of failure in cancer treatment. Evidence that activated macrophages can recognize and destroy neoplastic cells in vitro without regard to their phenotypic diversity has stimulated efforts to develop effective approaches to the activation of macrophages in situ. Systemic administration of liposomes containing immunomodulators activates macrophages in situ and augments host destruction of spontaneous metastases.

Liposomes are a useful carrier system for the transport of agents to phagocytic cells in vivo. Once in the circulation, liposomes are cleared by phagocytic cells, and this passive localization provides an effective mechanism for targeting liposome-entrapped materials, such as muramyltripeptide phosphatidylethanolamine (MTP-PE), to macrophages.

Macrophage destruction of metastases in vivo is significant, provided that the total tumor burden at start of treatment is minimal. For this reason, we advocate using chemotherapy or radiotherapy first to reduce the tumor burden in patients with metastases. Tumoricidal macrophages that can differentiate neoplastic from bystander nonneoplastic cells are then used to destroy the few tumor cells that escape destruction by conventional therapeutic methods.     

Reversible regulation of metastasis by ROS-generating mtDNA mutations

It has been controversial whether mtDNA mutations are responsible for oncogenic transformation (normal cells to develop tumors), and for malignant progression (tumor cells to develop metastases). To clarify this issue, we created trans-mitochondrial cybrids with mtDNA exchanged between mouse tumor cells that express different metastatic phenotypes. The G13997A mutation in the ND6 gene of mtDNA from high metastatic tumor cells reversibly controlled development of metastases by overproduction of reactive oxygen species (ROS), but did not control development of tumors. The mtDNA-mediated reversible control of metastasis reveals a novel function of mtDNA, and suggests that ROS scavengers may be therapeutically effective in suppressing metastasis.     

The evolution of tumor metastases during clonal expansion

Cancer is a leading cause of morbidity and mortality in many countries. Solid tumors generally initiate at one particular site called the primary tumor, but eventually disseminate and form new colonies in other organs. The development of such metastases greatly diminishes the potential for a cure of patients and is thought to represent the final stage of the multi-stage progression of human cancer. The concept of early metastatic dissemination, however, postulates that cancer cell spread might arise early during the development of a tumor. It is important to know whether metastases are present at diagnosis since this determines treatment strategies and outcome. In this paper, we design a stochastic mathematical model of the evolution of tumor metastases in an expanding cancer cell population. We calculate the probability of metastasis at a given time during tumor evolution, the expected number of metastatic sites, and the total number of cancer cells as well as metastasized cells. Furthermore, we investigate the effect of drug administration and tumor resection on these quantities and predict the survival time of cancer patients. The model presented in this paper allows us to determine the probability and number of metastases at diagnosis and to identify the optimum treatment strategy to maximally prolong survival of cancer patients.     

The role of the thiol N-acetylcysteine in the prevention of tumor invasion and angiogenesis

We have extensively studied the effects of N-acetylcysteine (NAC), a cytoprotective drug that can prevent in vivo carcinogenesis. Here we review our findings NAC completely inhibits gelatinolytic activity of metalloproteases and chemotactic and invasive activities of tumor cells. In addition, NAC reduces the number of lung metastases when malignant murine melanoma cells are injected into nude mice. NAC treatment decreases the weight of primary tumors and produces a dose-related increase in tumor latency. Moreover, oral administration of NAC reduces the formation of spontaneous metastases. In experimental metastasis assays, we have found a synergistic reduction in the number of lung metastases after treatment with doxorubicin (DOX) and NAC in nude mice. In tumorigenicity and spontaneous metastasis assays, the combined administration of DOX and oral NAC again has shown synergistic effects on the frequency and weight of primary tumors and local recurrences and completely prevented the formation of lung metastases. The addition of NAC to endothelial cells strongly reduces their invasive activity in response to angiogenic stimuli. NAC inhibited the degradation and release of radiolabeled type IV collagen by activated endothelial cells, indicating that NAC blocks gelatinase activity. Oral administration of NAC reduces the angiogenic response induced by KS tumor cell products, confirming the ability of NAC to inhibit the invasive activity of endothelial cells in vivo and thereby blocking angiogenesis.     

Preferential Colonization of Metastases by Oncolytic Vaccinia Virus Strain GLV-1h68 in a Human PC-3 Prostate Cancer Model in Nude Mice

Recently, we showed that the oncolytic vaccinia virus GLV-1h68 has a significant therapeutic potential in treating lymph node metastases of human PC-3 prostate carcinoma in tumor xenografts. In this study, underlying mechanisms of the virus-mediated metastases reduction were analyzed. Immunohistochemistry demonstrated that virus-treatment resulted in a drastically decrease of blood and lymph vessels, representing essential routes for PC-3 cell migration, in both tumors and metastases. Thus, GLV-1h68 drastically reduced essential routes for the metastatic spread of PC-3 cells. Furthermore, analysis of viral distribution in GLV-1h68-injected tumor-bearing mice by plaque assays, revealed significantly higher virus titers in metastases compared to solid tumors. To elucidate conditions potentially mediating the preferential viral colonization and eradication of metastases, microenvironmental components of uninfected tumors and metastases were compared by microscopic studies. These analyses revealed that PC-3 lymph node metastases showed increased vascular permeability, higher proliferation status of tumor cells as determined by BrdU- and Ki-67 assays and lesser necrosis of PC-3 cells than solid tumors. Moreover, an increased number of immune cells (MHCII⁺/CD68⁺ macrophages, MHCII⁺/CD19⁺ B lymphocytes) combined with an up-regulated expression of pro-inflammatory cytokines was observed in metastases in comparison to primary PC-3 tumors. We propose that these microenvironmental components mediated the metastatic tropism of GLV-1h68. Therefore, vaccinia virus-based oncolytic virotherapy might offer a novel treatment of metastatic prostate carcinomas in humans.     

Identification of primary tumors of brain metastases by SIMCA classification of IR spectroscopic images

Brain metastases are secondary intracranial lesions which occur more frequently than primary brain tumors. The four most abundant types of brain metastasis originate from primary tumors of lung cancer, colorectal cancer, breast cancer and renal cell carcinoma. As metastatic cells contain the molecular information of the primary tissue cells and IR spectroscopy probes the molecular fingerprint of cells, IR spectroscopy based methods constitute a new approach to determine the origin of brain metastases. IR spectroscopic images of 4 by 4 mm2 tissue areas were recorded in transmission mode by a FTIR imaging spectrometer coupled to a focal plane array detector. Unsupervised cluster analysis revealed variances within each cryosection. Selected clusters of five IR images with known diagnoses trained a supervised classification model based on the algorithm soft independent modeling of class analogies (SIMCA). This model was applied to distinguish normal brain tissue from brain metastases and to identify the primary tumor of brain metastases in 15 independent IR images. All specimens were assigned to the correct tissue class. This proof-of-concept study demonstrates that IR spectroscopy can complement established methods such as histopathology or immunohistochemistry for diagnosis.     

Identification of primary tumors of brain metastases by SIMCA classification of IR spectroscopic images

Brain metastases are secondary intracranial lesions which occur more frequently than primary brain tumors. The four most abundant types of brain metastasis originate from primary tumors of lung cancer, colorectal cancer, breast cancer and renal cell carcinoma. As metastatic cells contain the molecular information of the primary tissue cells and IR spectroscopy probes the molecular fingerprint of cells, IR spectroscopy based methods constitute a new approach to determine the origin of brain metastases. IR spectroscopic images of 4 by 4 mm² tissue areas were recorded in transmission mode by a FTIR imaging spectrometer coupled to a focal plane array detector. Unsupervised cluster analysis revealed variances within each cryosection. Selected clusters of five IR images with known diagnoses trained a supervised classification model based on the algorithm soft independent modeling of class analogies (SIMCA). This model was applied to distinguish normal brain tissue from brain metastases and to identify the primary tumor of brain metastases in 15 independent IR images. All specimens were assigned to the correct tissue class. This proof-of-concept study demonstrates that IR spectroscopy can complement established methods such as histopathology or immunohistochemistry for diagnosis.     

Progression of mammary adenocarcinomas as reflected by nuclear DNA content

In 18 breast cancer patients the DNA histograms observed in the primary tumor at the date of diagnosis were compared with those found in the corresponding local and distant metastases at autopsy up to more than 12 yr later. All patients, except one, exhibited the same type of DNA histogram in both the primary tumor and its metastases. In one patient the DNA histogram changed from an euploid type in the primary breast carcinoma to an aneuploid type in the metastases. The results are interpreted as indicating that mammary adenocarcinoma in general exhibit a high degree of stability of the nuclear DNA content during the history of the disease. It is suggested that in breast cancer progression of the tumor disease is more likely due to a net increase and/or dissemination of tumor cells exhibiting similar genetic properties and malignancy potential than to a progressive dedifferentiation and increase of malignancy of the tumor cells.     

Purification of immunoprotective tumor antigens by preparative isotachophoresis

Following inoculation with 1 X 10(6) MOPC-315 tumor cells, a single injection of a very low dose of melphalan (L-PAM, L-phenylalanine mustard), 0.75 mg/kg, cured most of the mice bearing a day 11 large primary tumor (20 mm) and metastases, but failed to cure mice bearing a day 4 nonpalpable tumor. Treatment of mice bearing a nonpalpable tumor with the very low dose of drug compromised the ability of the mice to respond effectively to the same low dose of drug when the tumor became large (day 12). However, a nonpalpable tumor could be eradicated by treatment of tumor bearers with a low dose of L-PAM, if it was present concomitantly with a large tumor on the contralateral side. A high dose of L-PAM, 15 mg/kg, cured mice bearing either a nonpalpable or a large tumor. The eradication of the tumor induced by the high dose of L-PAM appeared to be due solely to the tumoricidal effect of the drug. On the other hand, the eradication of the tumor by the low dose of L-PAM also required the participation of antitumor immunity of the host, since subsequent injection of antithymocyte serum abrogated the curative effect of the drug in most mice. Mice cured by a high dose of L-PAM were not resistant to subsequent lethal tumor challenge. In contrast, mice cured by the low dose of L-PAM were able to reject a tumor challenge of 300 times the minimal lethal tumor dose. The results obtained with L-PAM therapy are similar to the results that we had previously reported with cyclophosphamide therapy. Thus, the timing of therapy with a low dose of drug for mice bearing a MOPC-315 tumor is critical for successful therapy. Moreover, the selection of a low dose rather than a high dose of drug to eradicate a large tumor offers the advantage that it results in long-lasting potent antitumor immunity as a consequence of the participation of host antitumor immunity in the eradication of the tumor.     

Src kinase activation by nitric oxide promotes resistance to anoikis in tumour cell lines

Tumour progression involves the establishment of tumour metastases at distant sites. Resistance to anoikis, a form of cell death that occurs when cells lose contact with the extracellular matrix and with neighbouring cells, is essential for metastases. NO has been associated with anoikis. NO treated HeLa cells and murine melanoma cells in suspension triggered a nitric oxide (NO)-Src kinase signalling circuitry that enabled resistance to anoikis. Two NO donors, sodium nitroprusside (SNP) (500 µM) and DETANO (125 µM), protected against cell death derived from detachment of a growth permissive surface (experimental anoikis). Under conditions of NO-mediated Src activation the following were observed: (a) down-regulation of the pro-apoptotic proteins Bim and cleaved caspase-3 and the cell surface protein, E-cadherin, (b) up-regulation of caveolin-1, and (c) the dissociation of cell aggregates formed when cells are detached from a growth permissive surface. Efficiency of reattachment of tumour cells in suspension and treated with different concentrations of an NO donor, was dependent on the NO concentration. These findings indicate that NO-activated Src kinase triggers a signalling circuitry that provides resistance to anoikis, and allows for metastases.     

Angioma fusiform cells stimulated by conditioned medium from melanoma cells secrete a neomatrix which plays a role in "in vitro metastasis".

The rebuilt tumor model is a three dimensional mass of tumoral cells and angioma fusiform cells in collagen. Rebuilt tumors can give rise to "in vitro metastases" and these metastases depend on the presence of a neomatrix secreted in vitro by rebuilt tumor cells. This study defines the origin of the neomatrix and its role in "in vitro metastasis". Fusiform cells of angioma origin (AF3cells) were stimulated ten-fold by growing them in conditioned medium from a human melanoma cell line (MM2). The stimulated AF3 cells produced a dense neomatrix that was firmly attached to the culture flask. The AF3 cells were removed and MM2 cells were grown on this neomatrix. They gave rise to tumorous nodules very like the "in vitro metastases" produced by rebuilt tumors. The MM2 conditioned medium contained basic fibroblast growth factor, which could account for the angiogenetic activity of the tumoral cells. The fusiform cells of angioma origin that are stimulated by cancerous conditioned medium, are responsible for secretion of the neomatrix which plays a role in "in vitro metastasis".     

Quantitative expression profiling of G-protein-coupled receptors (GPCRs) in metastatic melanoma: the constitutively active orphan GPCR GPR18 as novel drug target

G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma.     

Identification of Ovarian Cancer Metastatic miRNAs

Serous epithelial ovarian cancer (EOC) patients often succumb to aggressive metastatic disease, yet little is known about the behavior and genetics of ovarian cancer metastasis. Here, we aim to understand how omental metastases differ from primary tumors and how these differences may influence chemotherapy. We analyzed the miRNA expression profiles of primary EOC tumors and their respective omental metastases from 9 patients using miRNA Taqman qPCR arrays. We find 17 miRNAs with differential expression in omental lesions compared to primary tumors. miR-21, miR-150, and miR-146a have low expression in most primary tumors with significantly increased expression in omental lesions, with concomitant decreased expression of predicted mRNA targets based on mRNA expression. We find that miR-150 and miR-146a mediate spheroid size. Both miR-146a and miR-150 increase the number of residual surviving cells by 2–4 fold when challenged with lethal cisplatin concentrations. These observations suggest that at least two of the miRNAs, miR-146a and miR-150, up-regulated in omental lesions, stimulate survival and increase drug tolerance. Our observations suggest that cancer cells in omental tumors express key miRNAs differently than primary tumors, and that at least some of these microRNAs may be critical regulators of the emergence of drug resistant disease.     

Both cell-surface and secreted CSF-1 expressed by tumor cells metastatic to bone can contribute to osteoclast activation

Tumors metastatic to the bone produce factors that cause massive bone resorption mediated by osteoclasts in the bone microenvironment. Colony stimulating factor (CSF-1) is strictly required for the formation and survival of active osteoclasts, and is frequently produced by tumor cells. Here we hypothesize that the CSF-1 made by tumor cells contributes to bone destruction in osteolytic bone metastases. We show that high level CSF-1 protected osteoclasts from suppressive effects of transforming growth factor β (TGF-β). r3T cells, a mouse mammary tumor cell line that forms osteolytic bone metastases, express abundant CSF-1 in vitro as both a secreted and a membrane-spanning cell-surface glycoprotein, and we show that both the secreted and the cell-surface form of CSF-1 made by r3T cells can support osteoclast formation in co-culture experiments in the presence of RankL. Mice with r3T bone metastases have elevated levels of both circulating and bone-associated CSF-1, and the majority of CSF-1 found in bone metastases is associated with the tumor cells. These results support the idea that tumor-cell produced CSF-1 contributes to osteoclast development and survival in bone metastasis.