Selman Ali

Chemokines induce migrational responses in human breast carcinoma cell lines

Chemokines have been shown to chemoattract and activate different leukocyte populations. Here we report the in vitro effect of macrophage inflammatory protein (MIP)‐1α, MIP‐1β, regulated on activation, normal T‐cells, expressed and secreted (RANTES), monocyte chemotactic protein‐1 (MCP‐1), interleukin‐8 (IL‐8), interferon inducible protein‐10 (IP‐10), neutrophil‐activating peptide‐2 (NAP‐2), growth‐related protein (GRO)‐α and GRO‐γ, on the migration of 3 human breast carcinoma cell lines, MCF‐7, T47D and ZR‐75‐1, using a microchemotaxis chamber to assess migration across fibronectin‐coated polycarbonate membranes. MCF‐7 cells responded chemotactically to all chemokines tested in a pattern which was dose and time dependent, although responses to the different chemokines were variable. ZR‐75‐1 responded to MIP‐1β and GRO‐α, giving maximum migration indices of 3.7 and 5.3, respectively, and exhibited a migratory response to MIP‐Iα, IL‐8 and MCP‐1 although to a lower degree. T47D was unresponsive to the chemokines tested, but both MCF‐7 and T47D cells bound radiolabelled ligands with binding constants (Kd) ranging from 0.6 to 2.2 nM and 0.6 to 2.1 nM, respectively. The specificity of the chemotactic response of MCF‐7 to MIP‐1α and MIP‐1β was confirmed using chemokine‐specific neutralising antibodies and heat denaturation, and was demonstrated to involve G protein and cyclic AMP signalling pathways. MIP‐1β and MIP‐1α were shown to induce changes in the organisation of the actin cytoskeleton and the level of F‐actin in MCF‐7 cells, as determined using flow cytometric analysis and confocal microscopy. Our results show that breast carcinoma cells can respond to chemokines, and suggests a potential role for these molecules in the process of tumour cell migration, invasion and metastasis. Int. J. Cancer 71:257–266, 1997.

Escape from immunotherapy: possible mechanisms that influence tumor regression/progression

Tumor escape is one major obstacle that has to be addressed prior to designing and delivering successful immunotherapy. There is compelling evidence to support the notion that immunogenic tumors, in murine models and cancer patients, can be rejected by the immune system under optimum conditions for activating adaptive and nonadaptive antitumor immune responses. Despite this capability, a large number of tumors continue to grow and evade recognition and/or destruction by the immune system. The limited success in current immunotherapeutic strategies may be due to a variety of reasons: failure of effector cells to compete with the growing tumor burden, production of humoral factors by tumors that locally block cytotoxicity, antigen/MHC loss, T-cell dysfunction, production of suppressor T cells—to name but a few causes for therapeutic ineffectiveness for the particular malignancy being treated. To optimize immunotherapy strategies, correction of immune-activating signals, eradication of inhibitory factors, and the evasion from newly developed immunoresistant tumor phenotypes need to be simultaneously considered.

Disabled Infectious Single-Cycle Herpes Simplex Virus as an Oncolytic Vector for Immunotherapy of Colorectal Cancer

New modalities of treatment for colorectal cancer are required to support and improve those currently available. One such approach is immunotherapy by transfer of immunostimulatory genes to tumor cells. Here, we report the use of a herpes simplex virus (HSV) vector that is capable of a single round of infection (disabled infectious single-cycle [DISC]-HSV) as a gene transfer vehicle for colorectal cancer. This vector has potential advantages over other vectors for cancer immunotherapy in that it lyses infected tumor cells. Infection with DISC-HSV inhibited tumor cell growth both in vitro and in vivo. In addition, DISC-HSV-mediated cell killing occurs by both apoptotic and necrotic mechanisms. A range of colorectal tumor cell lines could be rapidly transduced with DISC-HSV/lacZ (14-90% in 4 hr). Both tumor prevention and tumor therapy protocols showed clear antitumor effects with DISC-HSV/mGM-CSF. In the prophylactic approach, an infected/irradiated whole cell vaccine protected up to 80% of mice from rechallenge. In addition, intratumoral injection of established tumors with DISC-HSV/GM-CSF caused rejection in 40% of mice and generated some protection from subsequent rechallenge. In both cases, however, it is clear that a dominant therapeutic effect of the DISC-HSV vector derives from its oncolytic properties, irrespective of the transduced gene. As a prelude to taking these studies forward to human clinical trials, we demonstrate that tumor cells could be successfully grown from freshly obtained human colorectal cancer resections (within 1 week of surgery), were transduced with DISC-HSV/hGM-CSF, and secreted the cytokine. This study provides the preclinical basis for trials of immunotherapy of colorectal cancer using DISC-HSV.

Tumor Regression Induced by Intratumor Therapy with a Disabled Infectious Single Cycle (DISC) Herpes Simplex Virus (HSV) Vector, DISC/HSV/Murine Granulocyte-Macrophage Colony-Stimulating Factor, Correlates with Antigen-Specific Adaptive Immunity

Direct intratumor injection of a disabled infectious single cycle HSV-2 virus encoding the murine GM-CSF gene (DISC/mGM-CSF) into established murine colon carcinoma CT26 tumors induced a significant delay in tumor growth and complete tumor regression in up to 70% of animals. Pre-existing immunity to HSV did not reduce the therapeutic efficacy of DISC/mGM-CSF, and, when administered in combination with syngeneic dendritic cells, further decreased tumor growth and increased the incidence of complete tumor regression. Direct intratumor injection of DISC/mGM-CSF also inhibited the growth of CT26 tumor cells implanted on the contralateral flank or seeded into the lungs following i.v. injection of tumor cells (experimental lung metastasis). Proliferation of splenocytes in response to Con A was impaired in progressor and tumor-bearer, but not regressor, mice. A potent tumor-specific CTL response was generated from splenocytes of all mice with regressing, but not progressing tumors following in vitro peptide stimulation; this response was specific for the gp70 AH-1 peptide SPSYVYHQF and correlated with IFN-γ, but not IL-4 cytokine production. Depletion of CD8+ T cells from regressor splenocytes before in vitro stimulation with the relevant peptide abolished their cytolytic activity, while depletion of CD4+ T cells only partially inhibited CTL generation. Tumor regression induced by DISC/mGM-CSF virus immunotherapy provides a unique model for evaluating the immune mechanism(s) involved in tumor rejection, upon which tumor immunotherapy regimes may be based.

Peptide immunisation of HLA-DR–transgenic mice permits the identification of a novel HLA-DRβ1*0101– and HLA-DRβ1*0401–restricted epitope from p53

Because of the central role of CD4+ T cells in antitumour immunity, the identification of the MHC class II–restricted peptides to which CD4+ T cells respond has become a priority of tumour immunologists. Here, we describe a strategy permitting us to rapidly determine the immunogenicity of candidate HLA-DR–restricted peptides using peptide immunisation of HLA-DR–transgenic mice, followed by assessment of the response in vitro. This strategy was successfully applied to the reported haemaglutinin influenza peptide HA(307–319), and then extended to three candidate HLA-DR–restricted p53 peptides predicted by the evidence-based algorithm SYFPEITHI to bind to HLA-DRβ1*0101 (HLA-DR1) and HLA-DRβ1*0401 (HLA-DR4) molecules. One of these peptides, p53(108–122), consistently induced responses in HLA-DR1– and in HLA-DR4–transgenic mice. Moreover, this peptide was naturally processed by dendritic cells (DCs), and induced specific proliferation in the splenocytes of mice immunised with p53 cDNA, demonstrating that immune responses could be naturally mounted to the peptide. Furthermore, p53(108–122) peptide was also immunogenic in HLA-DR1 and HLA-DR4 healthy donors. Thus, the use of this transgenic model permitted the identification of a novel HLA-DR–restricted epitope from p53 and constitutes an attractive approach for the rapid identification of novel immunogenic MHC class II–restricted peptides from tumour antigens, which can ultimately be incorporated in immunotherapeutic protocols.

Immunotherapeutic potential of DISC-HSV and OX40L in cancer

Several vectors, viral and bacterial, have been developed over the past few years for means of generating an effective antitumor immune response. We have developed and studied a “model for immunotherapy” using a viral vector disabled infectious single cycle-herpes simplex virus (DISC-HSV), which efficiently transduces various tumor cell lines and offers a useful vehicle for the further development of cell-based vaccines. The immunotherapeutic potential of DISC-HSV encoding granulocyte macrophage colony stimulating factor (GM-CSF) was demonstrated in a number of murine carcinoma models, leading to complete regression of well-established tumors in up to 70% of the mice. Moreover, the therapeutic potential of DISC-HSV-GM-CSF was significantly enhanced when used in combination therapy with either OX40L or dendritic cells (DC), even in a poorly immunogenic tumor model. The ability of this vector to accept large gene inserts, its good safety profile, its ability to undergo only a single round of infection, the inherent viral immunostimulatory properties and its ability to infect various tumor cell lines efficiently, make DISC-HSV an ideal candidate vector for immunotherapy. The DISC- CT-26 tumor model was used to investigate the mechanisms associated with immunotherapy induced tumor rejection. Although CTL induction, was positively correlated with regression, MHC class I down regulation and accumulation of immature Gr1+ myeloid cells were shown to be the main immuno-suppressor mechanisms operating against regression and associated with progressive tumor growth. The CTL response was associated with the immuno-dominant AH-1 peptide of the retroviral glycoprotein gp70. This model of immunotherapy has provided an opportunity to dissect further the immunological events associated with tumor-rejection and escape. Since other antigens may be important in initiating tumor rejection, we have investigated the expression of MTA-1, an antigen that appears to be expressed widely in human and murine tumors. The immunogenicity of MTA-1 was studied and its potential as a tumor rejection antigen is under investigation.

Trafficking of tumor peptide-specific cytotoxic T lymphocytes into the tumor microcirculation

The major histocompatibility complex class I‐restricted CD8+ cytotoxic T‐lymphocyte (CTL) effector arm of the adaptive immune response can specifically recognize and destroy tumor cells expressing peptide antigens. Although adoptive T‐cell therapy has been successfully used for the treatment of viral and malignant diseases, little is known of the trafficking and fate of adoptively transferred antigen‐specific T cells. In the present study, splenocytes derived from mice that rejected their tumors (CT26 or CT26‐clone 25 tumors) in response to direct intratumor injection of disabled infectious single‐cycle herpes simplex virus (DISC‐HSV) encoding murine GM‐CSF were restimulated with peptide in vitro. CTLs specific for the AH‐1 and β‐gal peptides expressed by CT26 and CT26‐clone 25 tumor cells, respectively, were generated and used for adoptive cellular therapy and trafficking studies. Intravenous administration of AH‐1‐specific CTLs 3 days following i.v. injection of CT26 cells resulted in significant tumor growth inhibition, whereas administration of control CTLs generated against a bacterial β‐gal peptide did not inhibit the growth of tumors. Trafficking of AH‐1‐specific lymphocytes and their interaction with the CT26 tumor microcirculation was analyzed using real‐time in vivo microscopy (IVM). AH‐1‐specific but not β‐gal‐specific CTLs adhered and localized in the CT26 tumor microvasculature, but neither population adhered to the endothelium of the normal microcirculation. This study provides direct visual evidence suggesting that AH‐1‐specific CTLs that mediate a therapeutic response traffic to and localize within the tumor microenvironment.

Fibronectin is chemotactic for CT 26 colon carcinoma cells: sub-lines selected for increased chemotaxis to fibronectin display decreased tumorigenicity and lung colonization.

CT 26 murine colon carcinoma cells demonstrated directional migration (chemotaxis) in response to fibronectin (FN). Sub-lines were derived by positive and negative selection to FN across Transwell filters of 8 mm pore size. The FL6 sub-line (positively selected) demonstrated a significantly increased chemotactic response (P < 0.01) to FN compared with parental CT 26 cells, while the FU7 sub-line (negatively selected) showed a reduced chemotactic response to FN (P < 0.01). Comparable levels of a4, a5, av and b1 integrins, which mediate FN attachment, were expressed on positively and negatively selected sub-lines and parental CT 26 cells. Activation of integrins with Mn 2+ suggested that the integrins expressed on FL6 cells were in the fully activated state; in contrast FU7 cells displayed only partially activated integrins. Cell attachment and integrin activation status of the sub-lines correlated with their chemotactic response to FN. In vivo FL6 cells showed a significantly reduced tumour growth rate s.c. and a reduction in the number of lung colonies formed following i.v. injection compared with parental CT 26 and FU7 cells. In contrast FU7 cells displayed a sig-nificant increase in s.c. tumour growth and the number of lung colonies when compared with the parental line and FL6 sub-line. The results indicate that interaction between integrin receptors expressed on cancer cells and FN plays a central role in the chemotactic response of CT 26 colon carcinoma cells, and that in this model cells selected for chemotaxis to FN displayed a reduced malignant potential.© Kluwer Academic Publishers 1998

Disabled Infectious Single Cycle (DISC) Herpes Simplex Virus as a Vector for Immunotherapy of Cancer

Although the idea of immunotherapy for cancer is not new, recent advances in gene delivery systems and the discovery of new immunomodulatory genes has meant that the field is currently extremely active [1]. The delivery of genes which can modify the host’s response to its own tumour cells has been carried out both by using viral vectors and by non-viral delivery systems. In addition the genes have been delivered both directly to the tumours in vivo [2], or, more commonly, to tumour cells ex vivo, which are then re-injected into the tumour-bearing host [3]. There are two key features which may bear on the success of such approaches to the immunotherapy of cancer. Firstly the choice of immunomodulatory gene. Although there are a very large number of possible genes to test, and even larger number of potential combinations of genes, most reported work has concentrated on one or two key genes, principally cytokines such as IL-2 and GM-CSF. The second key element of the approach, and the subject of this article, is the choice of delivery system.

Virulence loss and amastigote transformation failure determine host cell responses to Leishmania mexicana

The effect of alterations in virulence and transformation by long‐term in vitro culture of Leishmania mexicana promastigotes on infectivity and immune responses was investigated. Fresh parasite cultures harvested from Balb/c mice were passaged 20 times in vitro. Infectivity was decreased and was completely avirulent after 20 passages. The qPCR results showed a down‐regulation of GP63, LPG2, CPC, CPB2, CPB2.8, CHT1, LACK and LDCEN3 genes after passage seven concomitant with a reduced and absence of infectivity by passages seven and 20, respectively. Parasites at passages one and 20 are referred to as virulent and avirulent, respectively. The growth of avirulent and virulent parasite was affected by conditioned media derived from macrophages or monocytes infected with parasites for 2 h. Giemsa staining showed the failure of avirulent but not virulent parasites to transform to the amastigote stage in infected host cells with both virulent and avirulent modulating the expression of CCL‐22, Tgad51, Cox2, IL‐1, IL‐10, TGF‐β, TNF‐α, Rab7, Rab9 and A2 genes; virulent but not avirulent L. mexicana significantly up‐regulated Th2‐associated cytokines, but down‐regulated Rab7 and Rab9 gene expression. In conclusion, a model for L. mexicana is reported, which is of potential value in studying host–parasite interaction.