Distant Tumor Cells Research Articles

The dynamic duo: A narrative review on the synergy between stereotactic body radiotherapy and immunotherapy in lung cancer treatment (Review).

Immunotherapy, particularly immune checkpoint inhibitors (ICIs), is undoubtedly one of the major breakthroughs in lung cancer research. Patient survival and prognosis have all been improved as a result, although numerous patients do not respond to immunotherapy due to various immune escape mechanisms of the tumor cells. Recent preclinical and clinical evidence has shown that stereotactic body radiotherapy (SBRT), also known as stereotactic ablative radiotherapy, has a prominent immune priming effect that could elicit antitumor immunity against specific tumor antigens and destroy distant tumor cells, thereby achieving the elusive abscopal effect, with the resulting immuno‑active tumor environment also being more conducive to ICIs. Some landmark trials have already demonstrated the survival benefit of the dynamic duo of SBRT plus immunotherapy in metastatic non‑small‑cell lung cancer, while others such as PEMBRO‑RT further suggest that the addition of SBRT to immunotherapy could expand the current indication to those who have historically responded poorly to ICIs. In the present review, the biological mechanisms that drive the synergistic effect of SBRT and immunotherapy were first briefly outlined; then, the current understanding from clinical trials was summarized and new insight into the evolving role of immunotherapy and SBRT synergy in lung cancer treatment was provided. Finally, novel avenues for discovery were highlighted. The innovation of the present review lies in the inclusion of non‑ICI immunotherapy in the discussion, which provides a more comprehensive view on the current development and future trend of SBRT + immunotherapy synergy.

Ultrasmall AuCe nanozyme adjuvant boosted ROS for three-pronged tumor immunotherapy

Catalytic tumor immunotherapy presents a new combination of nanozymes and immunotherapy agents, but the final therapeutic outcomes are usually diminished by insufficient nanozyme activity, immunosuppressive tumor microenvironment (TME), and poor antigen delivery efficiency. Herein, a bimetallic nanozyme-based tumor immunotherapy nanovaccine is first proposed to address these limitations by attenuating the immunosuppression of TME and effectively delivering tumor-specific antigens and catalytic tumor nanomedicine. The tumor-specific antigen OVA-templated bimetallic AuCe nanozyme (denoted as ACO) is a nanodot composed of Au cluster and CeOx. The high Ce3+/Ce4+ ratio and charge transfer from Au to Ce synergistically enhance the peroxidase-like activity of ACO. The boosted reactive oxygen species level inhibits the proliferation of B16-OVA cells and simultaneously promotes the maturation of dendritic cells. The functionalization of PD-L1 inhibitory D-peptide on ACO (named ACOD) increases tumor targeting specificity and prolongs tumor site retention, facilitating apoptosis of primary and distant tumor cells and conquering immune evasion. Moreover, ACOD catalytic nanovaccine induces long-term immune memory against tumor recurrence and lung metastasis. Therefore, the bimetallic catalytic nanovaccine is a promising nanoformulation to eradicate cancer and safeguard against tumor relapse.

Quantifying the Abscopal Effect in Breast Cancer Using Granzyme B PET Imaging

<h3>Purpose/Objective(s)</h3> Radiation can alter the tumor microenvironment and produce a corresponding immune response in tumors that have not been irradiated by generating a local inflammatory response and enhancing recognition of distant tumor cells, which is termed the "abscopal effect". We previously designed a positron emission tomography (PET) tracer, [⁶⁸Ga]-NOTA-GZP, which non-invasively detects granzyme B, a cytotoxic protease released by cytotoxic CD8⁺ lymphocytes and NK cells that initiates apoptosis of target tumor cells. [⁶⁸Ga]-NOTA-GZP PET imaging has been previously demonstrated to be predictive of response to checkpoint blockade in murine models, and is currently in clinical trials. We hypothesized that the abscopal effect could be induced by a single fraction of 12Gy irradiation and quantified by [⁶⁸Ga]-NOTA-GZP PET imaging. <h3>Materials/Methods</h3> To test this hypothesis, 4T1 breast cancer cells were implanted bilaterally in the mammary fat pad and one of the tumors received a single fraction of 12Gy irradiation followed by combination of PD-1 and CTLA-4 blockade concurrently. Tumor growth of the irradiated and contralateral tumors was measured and compared to irradiation only, immunotherapy only and control groups. Changes in granzyme B were assessed with [⁶⁸Ga]-NOTA-GZP PET imaging every three days until day 9. <h3>Results</h3> From days 0 to 9, there was no obvious change of PET uptake in the control and immunotherapy only groups. However, for the irradiation group and concurrent combination treatment group, PET uptake of the irradiated side increased slightly but not significantly. A significant elevation in the non-irradiated tumor of concurrent combination treatment group (p<0.05) was observed on day 9. Accordingly, a single fraction of 12Gy irradiation caused growth delay of the primary tumors (57.94±20.42 mm³) but had no effect on the contralateral tumors outside the radiation field (782.61±16.37 mm³) by day 30. Additionally, no tumor control was observed in the immunotherapy only group (p=0.7641). For the concurrent combination treatment group, both the irradiated tumor (17.02±5.71 mm³, p <0.0001) and the non-irradiated tumor volume decreased significantly (23.059±1.70 mm³, p <0.0001), consistent with the abscopal effect. The mean number of lung metastasis was lower in concurrent combination treatment group than other groups (p=0.037). <h3>Conclusion</h3> Concurrent combination treatment of a single fraction of 12Gy irradiation and PD-1 and CTLA-4 blockade activated the abscopal effect on non-irradiated tumors. [⁶⁸Ga]-NOTA-GZP PET imaging was able to non-invasively quantify the abscopal effect prior to subsequent tumor volume changes. This clinically relevant non-invasive approach is a promising potential tool to guiding combination radiation and immunotherapy to better understand and activate the abscopal effect in patients.

The Biogenesis, Biology, and Clinical Significance of Exosomal PD-L1 in Cancer.

The exosome serves as a trafficking vehicle for transport of programmed death-ligand 1 (PD-L1) into receptor cells. In tumor microenvironment, distant tumor cells can remotely attack activated T cells by exosomal PD-L1. Here, we summerize the biogenesis and transport process of exosomal PD-L1. Then, we focus on the cancer biology of exosomal PD-L1 in immunosuppression and the mechanism by which it inhibits T cells. Finally, we highlight the prospects of exosomal PD-L1 as a tumor biomarker and its significance in immunotherapy. In addition, we discuss the new challenges faced in researching and utilizing exosomal PD-L1. This review may shed light on the exosomal PD-L1 from the bench to the clinic. Exosomes serve as trafficking vehicles for transport of programmed death-ligand 1 (PD-L1) into receptor cells. In tumor microenvironment, distant tumor cells can remotely attack activated T cells through exosomal PD-L1. Here, we have summarized the biogenesis and transport of exosomal PD-L1. Next, we focused on the cancer biology of exosomal PD-L1 in immunosuppression and the mechanism by which it inhibits T cells. Finally, we highlighted the prospects of exosomal PD-L1 as a tumor biomarker and its significance in immunotherapy. In addition, we have discussed the new challenges faced in studying and utilizing exosomal PD-L1. This review may shed light on the translation of exosomal PD-L1 from bench to clinic.

Sustained release of anti-PD-1 peptide for perdurable immunotherapy together with photothermal ablation against primary and distant tumors

Immune checkpoint PD-1/PD-L1 blockade has emerged as a successful immunotherapy strategy for treating several types of malignant tumors. A constant and proper drug concentration during the treatment is important because the long-term activation of the immune system is urgently needed to perdurably recognize and attack cancer cells for a better therapeutic effect with minimum side effects. However, practically few related studies have been reported to date. In this study, we constructed a therapeutic strategy combining PD-1 blocking with photothermal ablation for malignant tumors by co-encapsulating anti-PD-1 peptide (APP) and hollow gold nanoshell (HAuNS) into biodegradable Poly (d, l-lactic-co-glycolide) nanoparticles (APP- and HAuNS-loaded PLGA nanoparticles, AA@PN). Slow and continuous release of APP from AA@PN could be obtained from 0 to 40 days, and this release was easily accelerated by illumination with a near-infrared (NIR) laser. A clear killing effect on distant tumor cells was observed after treatment of the co-culture system of PMBCs and tumor cells with AA@PN plus an NIR laser, reflecting the activated immune response. AA@PN followed by multiple irradiations with an NIR laser showed the strongest antitumor effect, with the elimination of most primary tumors compared with other treatments, and significantly inhibited the growth of the distant uninjected primary tumors, similarly to free APP with frequent injections, which induced the longest survival time for the mice in this group.

Abstract 4936: Modelling clinical issues by using patient-derived xenografts: Evaluation of partial hepatectomy on hepatoblastoma intrahepatic and distant growth

Abstract Hepatoblastoma (HB) is a rare disease that represents the most frequent liver malignancy during childhood. HB incidence is approximately 1.5 cases per million children less than 15-years old in Western countries. HB is histologically classified as epithelial or mixed depending on the presence of mesenchymal component. The most common epithelial components are the embryonal and fetal phenotype, reminiscent of at early or late stages of liver development. The improvement of the clinical management in the last 30 years has allowed 5-year survival rate to pass from 35 to 80%,due to the introduction of cisplatin-based chemotherapy. More than 50% of tumors are either inoperable or are metastatic at diagnosis. Neoadjuvant chemotherapy allows tumor size reduction and sometimes leads to complete regression of pulmonary metastasis. In Europe, the SIOPEL (International Childhood Liver Tumors Strategy Group) protocols recommend the use of cisplatinum-based neoadjuvant chemotherapy followed by surgery and adjuvant chemotherapy. All international studies recommend resecting all the metastases remaining after neoadjuvant chemotherapy, however, to date no indication is provided that defines whether metatases removal must take place before or after primary tumor resection by partial or total hepatectomy. As liver regeneration is a complex process that involves the expression and release of numerous factors that induce cell hypertrophy and proliferation, the risk that hepatectomy-induced release of regenerating factors might help the survival and proliferation of microscopic intrahepatic or distant tumor cells leads several clinicians to perform hepatectomy only after the removal of all detectable metastases. To challenge this paradigm, we used a panel of HB patient-derived xenograft and evaluated the impact of partial hepatectomy on intrahepatic and extrahepatic tumor growth. Partial hepatectomy performed either concomitantly with interscapular implantation of HB-217 xenograft, a model established from a patient with intrahepatic recurrence, or at the time of tumor appearance in mice failed to increase tumor growth. When hepatectomy was realized on several HB models concomitantly with either intrahepatic injection of bioluminescent HB cells derived from HB PDXs or by tumor fragments’ direct implantation, one of these models, HB-243, also a model established from a patient with intrahepatic recurrence, showed increased tumor growth in mice concomitantly subjected to partial hepatectomy. These results suggest that increased tumor growth induced by hepatectomy can occur in HB, albeit heterogeneously. We are now evaluating the molecular features of this hepatectomy-sensitive model and the impact of different cytokines on HB-243 PDX-derived tumor cells. The aim of this study will be to identify molecular markers and drivers of tumor sensitivity to hepatectomy. Citation Format: Marianna Cornet, Anais Delaitre Delattre, Laura Brulle Soumare, Victorine Boissay, Thierry Tordjmann, Antoinette Lemoine, Olivier Deas, Jean-Gabriel Judde, Sophie Branchereau, Stefano Cairo. Modelling clinical issues by using patient-derived xenografts: Evaluation of partial hepatectomy on hepatoblastoma intrahepatic and distant growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4936. doi:10.1158/1538-7445.AM2017-4936

High Precision Imaging of Microscopic Spread of Glioblastoma with a Targeted Ultrasensitive SERRS Molecular Imaging Probe

The dismal prognosis of patients with malignant brain tumors such as glioblastoma multiforme (GBM) is attributed mostly to their diffuse growth pattern and early microscopic tumor spread to distant regions of the brain. Because the microscopic tumor foci cannot be visualized with current imaging modalities, it remains impossible to direct treatments optimally. Here we explored the ability of integrin-targeted surface-enhanced resonance Raman spectroscopy (SERRS) nanoparticles to depict the true tumor extent in a GBM mouse model that closely mimics the pathology in humans. The recently developed SERRS-nanoparticles have a sensitivity of detection in the femtomolar range. An RGD-peptide-conjugated version for integrin-targeting (RGD-SERRS) was compared directly to its non-targeted RAD-SERRS control in the same mice via Raman multiplexing. Pre-blocking with RGD peptide before injection of RGD-SERRS nanoparticles was used to verify the specificity of integrin-targeting. In contrast to the current belief that the enhanced permeability and retention (EPR) effect results in a baseline uptake of nanoparticles regardless of their surface chemistry, integrin-targeting was shown to be highly specific, with markedly lower accumulation after pre-blocking. While the non-targeted SERRS particles enabled delineation of the main tumor, the RGD-SERRS nanoparticles afforded a major improvement in visualization of the true extent and the diffuse margins of the main tumor. This included the detection of unexpected tumor areas distant to the main tumor, tracks of migrating cells of 2-3 cells in diameter, and even isolated distant tumor cell clusters of less than 5 cells. This Raman spectroscopy-based nanoparticle-imaging technology holds promise to allow high precision visualization of the true extent of malignant brain tumors.

Abstract P1-07-05: Triple negative breast cancer metastases: Protein signal transduction networks within the tumor-stromal microenvironment complement genomic analysis and stratify local versus distant metastasis

Abstract Background: Genomic profiling of primary TNBC may not reflect the lethal metastatic (mTNBC) lesions that constitute a selected subclone of the primary tumor. Moreover, while the stroma microenvironment of primary tumors has been the subject of study, almost nothing is known about the stromal microenvironment of metastasis. We conducted proteomic signal pathway network analysis of microdissected tumor cells and stroma from mTNBC samples for which whole genome sequencing was known. We addressed the following questions: 1) Can potentially actionable driver mutations in the metastasis be inferred from the activated/ suppressed protein signaling pathways in the tumor cells? 2) Are the tumor cell signaling pathways different between local and distant metastasis? 3) Does the proteomic signaling profile of the stroma provide strategies for stromal therapy of mTNBC? Methods: Enriched populations of mTNBC cells and adjacent stromal cells were collected by Laser Capture Microdissection from 13 fresh frozen mTNBC samples. Signal pathway profiling of 130 protein and/or phosphoprotein endpoints were quantified by reverse phase protein arrays (RPPA). The samples represented local (regional lymph node and chest wall) (n=7) and distant (n=6) metastasis. Spearman rho correlation analysis identified pair-wise protein linakges (rho=0.8, p&amp;lt;0.01). Whole genome sequencing was also performed on the mTNBC samples (Craig DW. Mol Cancer Ther, 2012). Results: We compared the protein signaling network of the tumor and matched stroma to investigate tumor-stroma crosstalk, and also to the genomic alterations (GAs) in the same tumor. GAs in mTNBC fell into two major categories: DNA repair or cell cycle/growth factor signaling. Proteomic analysis confirmed the down-regulation of endocrine (ER, AR, PR) and HER2 pathways. PhosphoJAK1/2 and phosphoHer3 were markedly down-regulated in mTNBCs, while EGFR and Her4 signaling were not. Spearman rho pairwise correlations revealed differences between local and distant metastases as well as between local and distant stroma. Cyclin D1 in locally metastatic tumor cells was linked with Autotaxin, PLCgamma and RUNX1, whereas in distant metastatic tumor cells, Cyclin D1 was linked with HIF-1alpha, phosphoJAK1, and HSP70. Growth factor signaling via phosphoEFGR was more prevalent in distant mTNBC tumor samples (322 linkages) compared to local mTNBC tumor samples (146 linkages). Distant stromal cell linkages were dominated by growth factor (phospho mTOR) and immune cell crosstalk, e.g. 21 linkages between CD63 in distant stromal samples compared to 1 CD63 linkage in local stromal cells. Discussion: Proteomic signal pathway data can stratify metastatic lesions into functionally important groups based on a) clinical phenotype (distant versus local metastasis) and b) GA subtype (DNA repair vs growth factor signaling), thereby providing new actionable strategies for novel therapies, e.g. anti-JAK1 and anti-HSP70, and providing a means to prioritize potential driver mutations. Profiling the signal pathway network of mTNBC stroma, for the first time, provides novel strategies for targeting immune cell and mTOR pathways, for new classes of stromal therapy. Citation Format: Virginia Espina, Maren K Levin, John D Carpten, David W Craig, Daniel Von Hoff, Lance A Liotta, Joyce O'Shaughnessy. Triple negative breast cancer metastases: Protein signal transduction networks within the tumor-stromal microenvironment complement genomic analysis and stratify local versus distant metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-05.

Cell type- and tumor zone-specific expression of pVEGFR-1 and its ligands influence colon cancer metastasis

BackgroundDetailed knowledge of the essential pro-angiogenic biomolecules, the vascular endothelial growth factor (VEGF) family and its receptors, in the characteristically heterogeneous tumor tissue is a pre-requisite for an effective personalized target therapy. The effects of VEGF receptors after ligand binding are mediated through receptor tyrosine autophosphorylation. We determined the relevance of the VEGFR-1 activating pathway for colon cancer (CC) metastasis.MethodsThe expression profiles of VEGFR-1, phosphorylated (activated) VEGFR-1 (pVEGFR-1Tyr1048, pVEGFR-1Tyr1213 and pVEGFR-1Tyr1333) and the VEGFR-1 ligands (VEGF, PlGF and VEGF-B) were investigated using immunohistochemistry in different tumor compartments (intratumoral - invasive front - extratumoral), cell types (tumor cells – macro- (large and small vessels) and the microvasculature (capillaries) - inflammatory cells) in human sporadic non-metastatic, lymphogenous metastatic and haematogenous metastatic CC.ResultsVEGF and PlGF produced by tumor cells have an autocrine affinity for their receptor VEGFR-1. Subsequent PlGF-mediated receptor activation by autophosphorylation at Tyr1048 and Tyr1213 is a potential signaling pathway, which in turn seems to protect against distant metastasis and, in regions of tumor budding, additionally against lymph node metastasis. This autocrine link could be supported by possible formation of PlGF-VEGF heterodimers and PlGF-PlGF homodimers, which are known to have anti-metastatic properties. In contrast, in order to enhance their potential for distant metastasis tumor cells produce paracrine-acting VEGF-B. VEGFR-1 activation in tumor-associated macrovasculature but not capillaries appears to affect metastatic ability. Paracrine-mediated receptor autophosphorylation at Tyr1048 and Tyr1213 in small vessels located intratumorally and along the invasive front appears to be inversely correlated with metastasis, especially distant metastasis. Additionally, macrovessels are able to produce VEGFR-1 ligands, which influence the metastatic potential. Paracrine-acting VEGF-B production by intratumorally located small vessels and autocrine-acting PlGF production by extratumorally located small vessels seem to be associated with the non-metastatic phenotype. In contrast, VEGF-B-expressing extratumoral large and small vessels correlate with distant metastasis. Lymphocyte-associated VEGFR-1 expression in the invasive front without accompanying autophosphorylation could prevent against distant metastasis possibly by acting as a decoy and scavenger receptor.ConclusionVEGFR-1 and its ligands participate in vascular, tumor cell-mediated and immuno-inflammatory processes in a complex biomolecule-dependent and tumor zone-specific manner and hence could influence metastatic behavior in CC.

Immune response after photodynamic therapy increases anti-cancer and anti-bacterial effects.

Photodynamic therapy (PDT) is a clinically approved procedure for treatment of cancer and infections. PDT involves systemic or topical administration of a photosensitizer (PS), followed by irradiation of the diseased area with light of a wavelength corresponding to an absorbance band of the PS. In the presence of oxygen, a photochemical reaction is initiated, leading to the generation of reactive oxygen species and cell death. Besides causing direct cytotoxic effects on illuminated tumor cells, PDT is known to cause damage to the tumor vasculature and induce the release of pro-inflammatory molecules. Pre-clinical and clinical studies have demonstrated that PDT is capable of affecting both the innate and adaptive arms of the immune system. Immune stimulatory properties of PDT may increase its beneficial effects giving the therapy wider potential to become more extensively used in clinical practice. Be sides stimulating tumor-specific cytotoxic T-cells capable to destroy distant untreated tumor cells, PDT leads to development of anti-tumor memory immunity that can potentially prevent the recurrence of cancer. The immunological effects of PDT make the therapy more effective also when used for treatment of bacterial infections, due to an augmented infiltration of neutrophils into the infected regions that seems to potentiate the outcome of the treatment.

Abstract 4979: Multidimensional crosstalk regulates myeloid cell hyperplasia, immune escape, and tumor progression.

Abstract Myeloid-derived suppressor cells (MDSCs) encompass a heterogeneous class of cells in different maturation states that proliferate and accumulate in tumors, secondary lymphoid organs and some epithelial organs during tumor growth. Tumor secretion of growth factors and chemokines regulate myeloid cell proliferation, apoptosis, differentiation, circulation and biodistribution both locally and systemically. There is a reciprocal interaction that occurs between tumor and myeloid cells, which can facilitate tumor growth and metastasis and myeloid cell proliferation and survival via unique and tumor specific arrays of growth factors and chemokines. The tumor microenvironment, a complex, highly dynamic environment composed of host-derived MDSCs, endothelial cells, and fibroblasts; contributes to the microenvironmental crosstalk regulating host immunity and tumor progression. However, the macroenvironment is also a critical to myeloid cell expansion, tumor invasion and immune escape and tumor progression. The primary site of MDSC expansion and immune suppression is splenic extramedullary hematopoiesis, which is regulated by growth factors secreted by the distant primary tumor cells. Similarly, regulation of tumor infiltration by myeloid cells is by tumor secretion of CCL2; however, this occurs at the macroenvironmental level as CCL2/CCR2 blockade inhibits medullary myeloid cell mobilization and recruitment from secondary lymphoid organs and not at the tumor. Further, regulatory crosstalk is bidirectional with tumor-secreted factors differentially upregulating myeloid and lymphoid cell growth factors and enzymes (G-CSF, CXCL1, CCL2, arginase) by medullary myeloid cells and splenic lymphocytes. In tumor bearing, mice the tumor-secreted growth factors support hematopoietic stem/progenitor cell proliferation, limit their apoptosis and inhibit T-cell function. Conversely, leukocyte-secreted cytokines limit immune and hematopoietic growth factor transcript levels by tumor cells (G-CSF, IL-23, CXCL1 and NOS-2) with associated effects on tumor malignancy and leukocyte proliferation and differentiation. In summary, tumors form both local and systemic, bidirectional, regulatory loop(s) between metastatic tumor cells and MDSCs that regulates their proliferation and differentiation supporting tumor metastasis and systemic and regional T-cell suppression. Citation Format: Ibrahim H. Younos, Alicia J. Dafferner, Holly Britton, James E. Talmadge. Multidimensional crosstalk regulates myeloid cell hyperplasia, immune escape, and tumor progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4979. doi:10.1158/1538-7445.AM2013-4979

Single cell molecular recognition of migrating and invading tumor cells using a targeted fluorescent probe to receptor PTPmu

Detection of an extracellular cleaved fragment of a cell-cell adhesion molecule represents a new paradigm in molecular recognition and imaging of tumors. We previously demonstrated that probes that recognize the cleaved extracellular domain of receptor protein tyrosine phosphatase mu (PTPmu) label human glioblastoma brain tumor sections and the main tumor mass of intracranial xenograft gliomas. In this article, we examine whether one of these probes, SBK2, can label dispersed glioma cells that are no longer connected to the main tumor mass. Live mice with highly dispersive glioma tumors were injected intravenously with the fluorescent PTPmu probe to test the ability of the probe to label the dispersive glioma cells in vivo. Analysis was performed using a unique three-dimensional (3D) cryo-imaging technique to reveal highly migratory and invasive glioma cell dispersal within the brain and the extent of colabeling by the PTPmu probe. The PTPmu probe labeled the main tumor site and dispersed cells up to 3.5 mm away. The cryo-images of tumors labeled with the PTPmu probe provide a novel, high-resolution view of molecular tumor recognition, with excellent 3D detail regarding the pathways of tumor cell migration. Our data demonstrate that the PTPmu probe recognizes distant tumor cells even in parts of the brain where the blood-brain barrier is likely intact. The PTPmu probe has potential translational significance for recognizing tumor cells to facilitate molecular imaging, a more complete tumor resection and to serve as a molecular targeting agent to deliver chemotherapeutics to the main tumor mass and distant dispersive tumor cells.

Intercellular adhering junctions with an asymmetric molecular composition: desmosomes connecting Merkel cells and keratinocytes

Merkel cells (MCs) are special neuroendocrine epithelial cells that occur as individual cells or as cell groups within the confinements of a major epithelium formed and dominated by other epithelial cells. In the epidermis and some of its appendages MCs are mostly located in the basal cell layer, occasionally also in suprabasal layers and generally occur in linear arrays in outer root sheath cell layers of hair follicles. As MCs are connected to the adjacent keratinocytes by a series of adhering junctions (AJs), of which the desmosomes are the most prominent, these junctions represent heterotypic cell-cell connections, i.e. a kind of structure not yet elucidated in molecular terms. Therefore, we have studied these AJs in order to examine the molecular composition of the desmosomal halves. Using light- and electron-microscopic immunolocalization and keratin 20 as the MC-specific cell type marker we show that the plaques of the MC half of the desmosomes specifically and constitutively contain plakophilin Pkp2. This protein, however, is absent in the keratinocyte half of such heterotypic desmosomes which instead contains Pkp1 and/or Pkp3. We discuss the developmental, tissue-architectonic and functional importance of such asymmetric junctions in normal physiology as well as in diseases, in particular in the formation of distant tumor cell metastasis.

Expression and significance of Cyr61 in distant metastasis cells of human primary salivary adenoid cystic carcinoma

The objective of this study was to examine the expression of cysteine-rich protein 61 (Cyr61) in the distant metastatic tumor cells of human primary salivary adenoid cystic carcinoma (SACC) and its relationship with tumor angiogensis and metastasis. The experimental group comprised 35 paraffin-embedded tumor specimens of distant metastasis from primary SACC, with their corresponding primary tumor tissues and matched normal salivary gland tissues used as the control groups. Immunohistochemical staining was used to detect the expression of Cyr61 and vascular endothelial growth factor in the experimental and control groups. Vascular endothelial cells were highlighted by the anti-CD34 antibody, and the Weidner method was used to quantify microvessel density (MVD). Cyr61 was overexpressed in distant metastatic tumor cells of primary SACC. Positive expression of Cyr61 and vascular endothelial growth factor (VEGF) progressively increased in normal salivary gland tissues, primary tumor tissues, and tumor tissues of distant metastasis (P < .05). Compared with primary tumor tissues, Cyr61 expression and VEGF expression showed significant increase in tumor tissues of distant metastasis (P < .05). Cyr61 expression significantly correlated with VEGF expression and MVD (P < .05). Cyr61 appeared to have a significant association with tumor angiogenesis and metastasis in SACC and may be an important target in tumor antiangiogenesis therapy.

Stimulation of anti-tumor immunity by photodynamic therapy

Photodynamic therapy (PDT) is a rapidly developing cancer treatment that utilizes the combination of nontoxic dyes and harmless visible light to destroy tumors by generating reactive oxygen species. PDT produces tumor-cell destruction in the context of acute inflammation that acts as a ‘danger signal’ to the innate immune system. Activation of the innate immune system increases the priming of tumor-specific T lymphocytes that have the ability to recognize and destroy distant tumor cells and, in addition, lead to the development of an immune memory that can combat recurrence of the cancer at a later point in time. PDT may be also successfully combined with immunomodulating strategies that are capable of overcoming or bypassing the escape mechanisms employed by the progressing tumor to evade immune attack. This article will cover the role of the immune response in PDT anti-tumor effectiveness. It will highlight the milestones in the development of PDT-mediated anti-tumor immunity and emphasize the combination strategies that may improve this therapy.

ZEB1 in Pancreatic Cancer

Pancreatic cancer is one of the most malignant human neoplasias. On the molecular level, epithelial-mesenchymal transition (EMT) has been demonstrated to contribute to the malignant phenotype of pancreatic cancer cells. ZEB1 is a transcriptional repressor that has been identified as an inducer of EMT. A negative feedback loop between ZEB1 and microRNA-200c has been shown to regulate this EMT induction in various models. With respect to pancreatic cancer, primary effects of EMT comprise increased local and distant tumor cell dissemination. Another recently described feature of the EMT is the acquisition of cancer stem cell traits. For pancreatic cancer cells, antagonism between ZEB1 and stemness-inhibiting micro-RNAs has been demonstrated to contribute to this process, providing experimental support for the migrating cancer stem cell (MCSC) hypothesis. ZEB1 has also been shown to be associated with drug resistance of pancreatic cancer cells. This article reviews the biological functions of ZEB1 with a focus on pancreatic cancer.

Implications for clinical treatment from the micrometer site dosimetric calculations in boron neutron capture therapy

Boron neutron capture therapy has now been used for several malignancies. Most clinical trials have addressed its use for the treatment of glioblastoma multiforme. A few trials have focused on the treatment of malignant melanoma with brain metastases. Trial results for the treatment of glioblastoma multiforme have been encouraging, but have not achieved the success anticipated. Results of trials for the treatment of malignant melanoma have been very promising, though with too few patients for conclusions to be drawn. Subsequent to these trials, regimens for undifferentiated thyroid carcinoma, hepatic metastases from adenocarcinoma of the colon, and head and neck malignancies have been developed. These tumors have also responded well to boron neutron capture therapy. Glioblastoma is an infiltrative tumor with distant individual tumor cells that might create a mechanism for therapeutic failure though recurrences are often local. The microdosimetry of boron neutron capture therapy can provide an explanation for this observation. Codes written to examine the micrometer scale energy deposition in boron neutron capture therapy have been used to explore the effects of near neighbor cells. Near neighbor cells can contribute a significantly increased dose depending on the geometric relationships. Different geometries demonstrate that tumors which grow by direct extension have a greater near neighbor effect, whereas infiltrative tumors lose this near neighbor dose which can be a significant decrease in dose to the cells that do not achieve optimal boron loading. This understanding helps to explain prior trial results and implies that tumors with small, closely packed cells that grow by direct extension will be the most amenable to boron neutron capture therapy.

Activation of CCR9/CCL25 in Cutaneous Melanoma Mediates Preferential Metastasis to the Small Intestine

Specific chemokines and their respective receptors have been implicated in distant tumor cell metastasis. Cutaneous melanoma has a distinct pattern of metastasis, preferentially targeting the submucosa of the small intestine. However, the underlying pathogenic mechanism remains unknown. Migration of CCR9(+) lymphocytes to the small intestine is known to occur in response to the chemoattractant effects of CCL25 (thymus-expressed chemokine). The integrin heterodimers alphabeta are also known to be important mediators of cellular adhesion. We hypothesize that the mechanism of small intestinal metastasis by melanoma is via the CCR9-CCL25 axis and specific integrins. Quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry were used to assess melanoma tumors for CCR9 and CCL25. Integrin expression was assessed using flow cytometry. CCR9 expression by quantitative reverse transcription-PCR was assessed in primary (n = 23) and metastatic (n = 198) melanomas, and melanoma lines derived from small intestinal metastases (n = 23). We showed CCR9 expression in 88 of 102 paraffin-embedded metastatic melanomas from the small intestine, 8 of 8 melanoma lines derived from metastases in the small intestine, and 0 of 96 metastatic melanomas from other sites. In vitro migration and invasion studies done on CCR9(+) melanoma lines showed migration in response to CCL25 that was inhibited by anti-CCR9 antibody or by short interfering RNA CCR9. Flow cytometric analysis confirmed CCR9 expression by melanomas to the small intestine and showed concomitant alpha(4)beta(1) integrin expression. Our findings show that functionally active CCR9 on melanoma cells facilitates metastasis to the small intestine. The CCR9-CCL25 axis may explain the high incidence of melanoma metastasis to this specific location.

TGF beta secreted by B16 melanoma antagonizes cancer gene immunotherapy bystander effect.

Tumor-targeted delivery of immune stimulatory genes, such as pro-inflammatory cytokines and suicide genes, has shown to cure mouse models of cancer. Total tumor eradication was also found to occur despite subtotal tumor engineering; a phenomenon coined the "bystander effect". The bystander effect in immune competent animals arises mostly from recruitment of a cancer lytic cell-mediated immune response to local and distant tumor cells which escaped gene modification. We have previously described a Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin 2 (IL2) fusokine (aka GIFT2) which serves as a potent anticancer cytokine and it here served as a means to understand the mechanistic underpinnings to the immune bystander effect in an immune competent model of B16 melanoma. As expected, we observed that GIFT2 secreted by genetically engineered B16 tumor cells induces a bystander effect on non modified B16 cells, when admixed in a 1:1 ratio. However, despite keeping the 1:1 ratio constant, the immune bystander effect was completely lost as the total B16 cell number was increased from 10(4) to 10(6) which correlated with a sharp reduction in the number of tumor-infiltrating NK cells. We found that B16 secrete biologically active TGFbeta which in turn inhibited GIFT2 dependent immune cell proliferation in vitro and downregulated IL-2R beta expression and IFN gamma secretion by NK cells. In vivo blockade of B16 originating TGFbeta significantly improved the immune bystander effect arising from GIFT2. We propose that cancer gene immunotherapy of pre-established tumors will be enhanced by blockade of tumor-derived TGFbeta.

Expression of chemokine receptor CXCR4 correlates with progression of pancreatic cancer

14018 Background: Despite many pathophysiological analyses, the process of tumor dissemination of pancreatic cancer remains vague. In diverse other tumor entities, expression of the chemokine receptor CXCR4 has been linked to tumor dissemination and poor prognosis. Therefore, we evaluated, if the expression of this chemokine receptor exerts similar effects in human pancreatic cancer. Methods: Expression of CXCR4 was evaluated in 120 patients with histologically confirmed pancreatic cancer and eight different pancreatic cancer cell lines. Expression intensities of tumor samples were correlated with both, tumor and patients characteristics. Results: Human pancreatic cancer samples and cell lines displayed variable intensities of CXCR4. CXCR4 revealed predominantly a cytoplasmatic, and in few specimens an additional weak membranous location. Strong CXCR4 expression was significantly associated with progressed disease as indicated by UICC status (P=0.031) and revealed a strong, yet not significant trend with distant tumor cell dissemination and locally progressed tumors (P=0.08; P=0.11, respectively). Conclusion: Strong expression of CXCR4 by pancreatic cancer is significantly associated with progressed tumor stages. No significant financial relationships to disclose.