ICAM-2 Expression Mediates a Membrane-Actin Link, Confers a Nonmetastatic Phenotype and Reflects Favorable Tumor Stage or Histology in Neuroblastoma

The actin cytoskeleton is a primary determinant of tumor cell motility and metastatic potential. Motility and metastasis are thought to be regulated, in large part, by the interaction of membrane proteins with cytoplasmic linker proteins and of these linker proteins, in turn, with actin. However, complete membrane-to-actin linkages have been difficult to identify. We used co-immunoprecipitation and competitive peptide assays to show that intercellular adhesion molecule-2 (ICAM-2)/α-actinin/actin may comprise such a linkage in neuroblastoma cells. ICAM-2 expression limited the motility of these cells and redistributed actin fibers in vitro, and suppressed development of disseminated tumors in an in vivo model of metastatic neuroblastoma. Consistent with these observations, immunohistochemical analysis demonstrated ICAM-2 expression in primary neuroblastoma tumors exhibiting features that are associated with limited metastatic disease and more favorable clinical outcome. In neuroblastoma cell lines, ICAM-2 expression did not affect AKT activation, tumorigenic potential or chemosensitivity, as has been reported for some types of transfected cells. The observed ICAM-2-mediated suppression of metastatic phenotype is a novel function for this protein, and the interaction of ICAM-2/α-actinin/actin represents the first complete membrane-linker protein-actin linkage to impact tumor cell motility in vitro and metastatic potential in an in vivo model. Current work focuses on identifying specific protein domains critical to the regulation of neuroblastoma cell motility and metastasis and on determining if these domains represent exploitable therapeutic targets

Development of a Tumor-Selective Approach to Treat Metastatic Cancer

BACKGROUND: Patients diagnosed with metastatic cancer have almost uniformly poor prognoses. The treatments available for patients with disseminated disease are usually not curative and have side effects that limit the therapy that can be given. A treatment that is selectively toxic to tumors would maximize the beneficial effects of therapy and minimize side effects, potentially enabling effective treatment to be administered. METHODS AND FINDINGS: We postulated that the tumor-tropic property of stem cells or progenitor cells could be exploited to selectively deliver a therapeutic gene to metastatic solid tumors, and that expression of an appropriate transgene at tumor loci might mediate cures of metastatic disease. To test this hypothesis, we injected HB1.F3.C1 cells transduced to express an enzyme that efficiently activates the anti-cancer prodrug CPT-11 intravenously into mice bearing disseminated neuroblastoma tumors. The HB1.F3.C1 cells migrated selectively to tumor sites regardless of the size or anatomical location of the tumors. Mice were then treated systemically with CPT-11, and the efficacy of treatment was monitored. Mice treated with the combination of HB1.F3.C1 cells expressing the CPT-11-activating enzyme and this prodrug produced tumor-free survival of 100% of the mice for >6 months (P<0.001 compared to control groups). CONCLUSIONS: The novel and significant finding of this study is that it may be possible to exploit the tumor-tropic property of stem or progenitor cells to mediate effective, tumor-selective therapy for metastatic tumors, for which no tolerated curative treatments are currently available

HB1.F3.C1 cells injected intravenously localized to microscopic bone marrow disease.

<div><p>Concordant detection of v-<i>myc</i> (HB1.F3.C1 cells) by PCR and TH expression (neuroblastoma cells) by RT-PCR in bone marrow specimens.</p> <p>Bone marrow samples isolated from animals injected with HB1.F3.C1 cells were analyzed for the presence of v-<i>myc</i> (HB1.F3.C1 cells) or the expression of TH (NB-1643 cells).</p> <p>HB1.F3.C1 cells were present in the bone marrow only when tumor cells were also present.</p> <p>HB1.F3.C1 cells were not detected in the bone marrow of non-tumor-bearing animals.</p> <p>The positive controls (+) were DNA extracted from HB1.F3.C1 cells for v-<i>myc</i>, and RNA extracted from NB-1691 cells for TH.</p> <p>The negative controls (−) contained no DNA or RNA template, respectively.</p></div

HB1.F3.C1 cells target macroscopic metastatic neuroblastoma in the bone marrow.

<div><p>X-ray image of hind limb of a mouse with advanced stage neuroblastoma (Day 82; left panel, scale bar: 1 cm).</p> <p>Confirmation of the tumor mass as human SK-N-AS neuroblastoma cells was performed by immunohistochemistry using anti-human mitochondria antibody (not shown).</p> <p>The CM-DiI-labeled HB1.F3.C1 cells (red cells, injected into the tail vein 3 days prior to sacrifice) localized to tumor in the marrow (right panel; scale bar: 200 µm).</p></div

HB1.F3.C1 cells transduced with an adenoviral multiplicity of infection of 5, 10, or 20 retain their tumor-tropism toward conditioned medium from SK-N-AS neuroblastoma cells.

<p>Bars represent the average number of migrated cells±SEM of triplicate wells in modified Boyden chamber assays.</p

Schematic diagram of the protocol for NDEPT.

<div><p>Human neuroblastoma tumor cells are injected intravenously to produce disseminated tumors.</p> <p>At an appropriate time after injection of neuroblastoma cells, neural stem cells or neural progenitor cells transduced with adenovirus to express a prodrug-activating enzyme (in this study, a secreted form of rabbit carboxylesterase [rCE]) are injected intravenously.</p> <p>Following migration of stem cells or progenitor cells to tumor foci and a delay of 3–4 days to allow relatively high level expression of the prodrug-activating enzyme into the extracellular milieu at the tumor sites, mice are treated with the prodrug (in this study, CPT-11).</p> <p>The prodrug is activated selectively at tumor foci, to increase the therapeutic index of the prodrug.</p></div

Treatment protocol of HB1.F3.C1/AdCMVrCE/CPT-11 NDEPT.

<div><p>One day prior to being used in treatments, cells were transduced with the AdCMVrCE construct (see text).</p> <p>The treatment schedule was based on the time-course of expression of the secreted form of rCE, following adenoviral transduction of HB1.F3.C1 cells (Danks, unpublished observation) and on a schedule of administration of CPT-11 that has been shown to be relatively effective for neuroblastoma patients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000023#pone.0000023-Furman1" target="_blank">[35]</a>.</p></div