Richard B. Bankert

Characterization of Human Lung Tumor-Associated Fibroblasts and Their Ability to Modulate the Activation of Tumor-Associated T Cells

The tumor microenvironment of human non-small cell lung cancer (NSCLC) is composed largely of stromal cells, including fibroblasts, yet these cells have been the focus of few studies. In this study, we established stromal cell cultures from primary NSCLC through isolation of adherent cells. Characterization of these cells by flow cytometry demonstrated a population which expressed a human fibroblast-specific 112-kDa surface molecule, Thy1, α-smooth muscle actin, and fibroblast activation protein, but failed to express CD45 and CD11b, a phenotype consistent with that of an activated myofibroblast. A subset of the tumor-associated fibroblasts (TAF) was found to express B7H1 (PD-L1) and B7DC (PD-L2) constitutively, and this expression was up-regulated by IFN-γ. Production of cytokines and chemokines, including IFN-γ, monokine induced by IFN-γ, IFN-γ-inducible protein-10, RANTES, and TGF-β1 was also demonstrated in these cells. Together, these characteristics provide multiple opportunities for the TAF to influence cellular interactions within the tumor microenvironment. To evaluate the ability of TAF to modulate tumor-associated T cell (TAT) activation, we conducted coculture experiments between autologous TAF and TAT. In five of eight tumors, TAF elicited a contact-dependent enhancement of TAT activation, even in the presence of a TGF-β1-mediated suppressive effect. In the three other tumors, TAF had a net suppressive effect upon TAT activation, and, in one of these cases, blockade of B7H1 or B7DC was able to completely abrogate the TAF-mediated suppression. We conclude that TAF in human NSCLC are functionally and phenotypically heterogeneous and provide multiple complex regulatory signals that have the potential to enhance or suppress TAT function in the tumor microenvironment.

Human-SCID mouse chimeric models for the evaluation of anti-cancer therapies.

The ability to engraft human tumors and human immunocompetent cells successfully in severe combined immunodeficient (SCID) mice has spawned the development and use of human-mouse chimeric models to evaluate anti-cancer therapies. The lack of standardization and many other potential pitfalls have contributed to the current controversy surrounding the reliability of these different models. Five frequently used SCID mouse models and their specific applications are summarized with the specific aim of providing an objective discussion of the strengths and limitations of each model, together with suggestions for overcoming some of the variabilities and for improving the design and use of future models.

Follicular Lymphoma Intratumoral CD4+CD25+GITR+ Regulatory T Cells Potently Suppress CD3/CD28-Costimulated Autologous and Allogeneic CD8+CD25− and CD4+CD25− T Cells

Regulatory T cells (TR) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor-reactive effector T cells. In this study, we demonstrate that follicular lymphoma (FL)-infiltrating CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. We further identify a population of FL-infiltrating CD4+CD25+GITR+ TR that are significantly overrepresented within FL nodes (FLN) compared with that seen in normal (nonmalignant, nonlymphoid hyperplastic) or reactive (nonmalignant, lymphoid hyperplastic) nodes. These TR actively suppress both the proliferation of autologous nodal CD8+CD25− and CD4+CD25− T cells, as well as cytokine production (IFN-γ, TNF-α and IL-2), after CD3/CD28 costimulation. Removal of these cells in vitro by CD25+ magnetic bead depletion restores both the proliferation and cytokine production of the remaining T cells, demonstrating that FLN T cell hyporesponsiveness is reversible. In addition to suppressing autologous nodal T cells, these TR are also capable of suppressing the proliferation of allogeneic CD8+CD25− and CD4+CD25− T cells from normal lymph nodes as well as normal donor PBL, regardless of very robust stimulation of the target cells with plate-bound anti-CD3 and anti-CD28 Abs. The allogeneic suppression is not reciprocal, as equivalent numbers of CD25+FOXP3+ cells derived from either normal lymph nodes or PBL are not capable of suppressing allogeneic CD8+CD25− and CD4+CD25− T cells, suggesting that FLN TR are more suppressive than those derived from nonmalignant sources. Lastly, we demonstrate that inhibition of TGF-β signaling partially restores FLN T cell proliferation suggesting a mechanistic role for TGF-β in FLN TR-mediated suppression.

Human CD4+ T Cells Present Within the Microenvironment of Human Lung Tumors Are Mobilized by the Local and Sustained Release of IL-12 to Kill Tumors In Situ by Indirect Effects of IFN-γ

By implanting nondisrupted pieces of human lung tumor biopsy tissues into SCID mice, it has been possible to establish viable grafts of the tumor, as well as the tumor-associated microenvironment, including inflammatory cells, fibroblasts, tumor vasculature, and the extracellular matrix. Using this xenograft model, we have evaluated and characterized the effects of a local and sustained release of human rIL-12 (rhIL-12) from biodegradable microspheres. In response to rhIL-12, the human CD45+ inflammatory cells present within the xenograft mediate the suppression or the complete arrest of tumor growth in SCID mice. Analysis of the cellular events reveals that human CD4+ and CD8+ T cells are induced by rhIL-12 to produce and secrete IFN-γ. Serum levels of human IFN-γ in mice bearing rhIL-12-treated tumor xenografts correlate directly with the degree of tumor suppression, while neutralizing Abs to human IFN-γ abrogate the IL-12-mediated tumor suppression. Gene expression profiling of tumors responding to intratumoral rhIL-12 demonstrates an up-regulation of IFN-γ and IFN-γ-dependent genes not observed in control-treated tumors. Genes encoding a number of proinflammatory cytokines, chemokines (and their receptors), adhesion molecules, activation markers, and the inducible NO synthase are up-regulated following the introduction of rhIL-12, while genes associated with tumor growth, angiogenesis, and metastasis are decreased in expression. NO contributes to the tumor killing because an inhibitor of inducible NO synthase prevents IL-12-induced tumor suppression. Cell depletion studies reveal that the IL-12-induced tumor suppression, IFN-γ production, and the associated changes in gene expression are all dependent upon CD4+ T cells.

The next generation of liposome delivery systems: recent experience with tumor-targeted, sterically-stabilized immunoliposomes and active-loading gradients.

ABSTRACT Three topics are discussed. Enhanced anti-tumor efficacy of targeted doxorubicin-containing sterically-stabilized liposomes using an anti-β1 integrin Fab’ ligand. Use of tumor targeting with an internalizing ligand to improve the efficacy of a non-leaky cisplatin-containing sterically-stabilized liposome formulation. Formulation variables (remote-loading with dextran ammonium sulfate, rigid lipid bilayer) used to optimize in vivo performance of a liposomal camptothecin analog.

Long-Term Engraftment and Expansion of Tumor-Derived Memory T Cells Following the Implantation of Non-Disrupted Pieces of Human Lung Tumor into NOD-scid IL2Rγnull Mice

Non-disrupted pieces of primary human lung tumor implanted into NOD-scid IL2Rγnull mice consistently result in successful xenografts in which tissue architecture, including tumor-associated leukocytes, stromal fibroblasts, and tumor cells are preserved for prolonged periods with limited host-vs-graft interference. Human CD45+ tumor-associated leukocytes within the xenograft are predominantly CD3+ T cells with fewer CD138+ plasma cells. The effector memory T cells that had been shown to be quiescent in human lung tumor microenvironments can be activated in situ as determined by the production of human IFN-γ in response to exogenous IL-12. Plasma cells remain functional as evidenced by production of human Ig. Significant levels of human IFN-γ and Ig were detected in sera from xenograft-bearing mice for up to 9 wk postengraftment. Tumor-associated T cells were found to migrate from the microenvironment of the xenograft to the lung, liver, and primarily the spleen. At 8 wk postengraftment, a significant portion of cells isolated from the mouse spleens were found to be human CD45+ cells. The majority of CD45+ cells were CD3+ and expressed a phenotype consistent with an effector memory T cell, consisting of CD4+ or CD8+ T cells that were CD45RO+, CD44+, CD62L−, and CD25−. Following adoptive transfer into non-tumor bearing NOD-scid IL2Rγnull mice, these human T cells were found to expand in the spleen, produce IFN-γ, and maintain an effector memory phenotype. We conclude that the NOD-scid IL2Rγnull tumor xenograft model provides an opportunity to study tumor and tumor-stromal cell interactions in situ for prolonged periods.

Humanized Mouse Model of Ovarian Cancer Recapitulates Patient Solid Tumor Progression, Ascites Formation, and Metastasis

Ovarian cancer is the most common cause of death from gynecological cancer. Understanding the biology of this disease, particularly how tumor-associated lymphocytes and fibroblasts contribute to the progression and metastasis of the tumor, has been impeded by the lack of a suitable tumor xenograft model. We report a simple and reproducible system in which the tumor and tumor stroma are successfully engrafted into NOD-scid IL2Rγnull (NSG) mice. This is achieved by injecting tumor cell aggregates derived from fresh ovarian tumor biopsy tissues (including tumor cells, and tumor-associated lymphocytes and fibroblasts) i.p. into NSG mice. Tumor progression in these mice closely parallels many of the events that are observed in ovarian cancer patients. Tumors establish in the omentum, ovaries, liver, spleen, uterus, and pancreas. Tumor growth is initially very slow and progressive within the peritoneal cavity with an ultimate development of tumor ascites, spontaneous metastasis to the lung, increasing serum and ascites levels of CA125, and the retention of tumor-associated human fibroblasts and lymphocytes that remain functional and responsive to cytokines for prolonged periods. With this model one will be able to determine how fibroblasts and lymphocytes within the tumor microenvironment may contribute to tumor growth and metastasis, and will make it possible to evaluate the efficacy of therapies that are designed to target these cells in the tumor stroma.

Activation induces a rapid reorganization of spectrin in lymphocytes

Lymphocyte activation results in a rapid reorganization of the cytoskeletal protein spectrin. Immediately following an activation signal, there is fragmentation of a spectrin-rich cytoplasmic structure and subsequent translocation of the fragments to defined areas of the plasma membrane in both antigen-specific T cell hybridomas and lymph node T cells. These dramatic changes have been documented by light and electron microscopic immunolocalization and by immunoblot analysis of plasma membrane-enriched preparations. A T cell hybridoma variant lacking the spectrin-rich cytoplasmic structure of the parental line does not redistribute spectrin and produces little or no IL-2 in response to antigen-dependent activation. This suggests a functional link between spectrin distribution and activation potential. We propose that the cytoplasmic structure functions as an organizing center or reservoir for spectrin that is sensitive to signaling at the cell surface.

Cytokine immunotherapy of cancer with controlled release biodegradable microspheres in a human tumor xenograft/SCID mouse model.

Abstract A novel biodegradable poly(lactic acid) microsphere formulation was evaluated for in vivo cytokine immunotherapy of cancer in a human tumor xenograft/severe combined immunodeficiency (SCID) mouse model. Co-injection of interleukin-2 (IL-2)-loaded microspheres with tumor cells into a subcutaneous site resulted in the complete suppression of tumor engraftment in 80% of animals. In contrast, bovine-serum-albumin(BSA)-loaded particles or bolus injections of poly(ethylene glycol)/IL-2 were ineffective in preventing tumor growth. The antitumor effect of IL-2 released by the microspheres was shown to be mediated by the mouse natural killer cells. This is the first evidence that the rejection of human tumor xenografts can be provoked by the sustained in vivo delivery of IL-2 from biodegradable microspheres. The use of poly(lactic acid) microspheres to deliver cytokines to the tumor environment could provide a safer and simpler alternative to gene therapy protocols in the treatment of cancer.

Human lung tumors, patients' peripheral blood lymphocytes and tumor infiltrating lymphocytes propagated in scid mice.

Studies of the pathogenesis of human tumors and the evaluation of new therapeutic modalities have been limited by the lack of a suitable experimental animal model. Currently very little is known regarding patients’ immune response to their tumors and the consequences of this response to tumor growth. Further, the evaluation of novel tumor therapies has been largely restricted to animal tumor models which may not accurately reflect the efficacy of such therapies for human tumors (Bankert et al. 1989). While athymic homozygous nu/nu mice have been used extensively for the heterotransplantation of human tumors (Fogh and Trempe 1975), their use in evaluating therapies for human cancer is limited by their ability to respond to thymic independent antigens, by the presence of immunoglobulin in their circulation and tissues and by phenotypic and karyotypic changes in the tumor that have been observed during tumor growth in nude mice. We have previously reported (Reddy et al. 1987) that xenografts of human lung tumors can be successfully propagated in the B cell and T cell deficient mouse mutant strain, CB-17 scid (described by Bosma et al. 1983). Several characteristics of the scid mouse make it particularly attractive as a potential model to evaluate new therapeutic approaches to the treatment of human cancers. In this report we summarize our findings that scid mice support the growth of human tumors and we establish here that this mouse is a viable model with which to evaluate the antibody directed delivery of cytotoxic agents to human lung tumors.