Sandra J. Yokota

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.

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.

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.

Human CD4+ Effector Memory T Cells Persisting in the Microenvironment of Lung Cancer Xenografts Are Activated by Local Delivery of IL-12 to Proliferate, Produce IFN-γ, and Eradicate Tumor Cells

The implantation of small pieces of human primary lung tumor biopsy tissue into SCID mice results in a viable s.c. xenograft in which the tissue architecture, including tumor-associated leukocytes, tumor cells, and stromal cells, is preserved in a functional state. By monitoring changes in tumor volume, gene expression patterns, cell depletion analysis, and the use of function-blocking Abs, we previously established in this xenograft model that exogenous IL-12 mobilizes human tumor-associated leukocytes to kill tumor cells in situ by indirect mechanisms that are dependent upon IFN-γ. In this study immunohistochemistry and FACS characterize the early cellular events in the tumor microenvironment induced by IL-12. By 5 days post-IL-12 treatment, the constitutively present human CD45+ leukocytes have expanded and infiltrated into tumor-rich areas of the xenograft. Two weeks post-treatment, there is expansion of the human leukocytes and complete effacement of the tumor compared with tumor progression and gradual loss of most human leukocytes in control-treated xenografts. Immunohistochemical analyses reveal that the responding human leukocytes are primarily activated or memory T cells, with smaller populations of B cells, macrophages, plasma cells, and plasmacytoid dendritic cells capable of producing IFN-α. The predominant cell population was also characterized by FACS and was shown to have a phenotype consistent with a CD4+ effector memory T cell. We conclude that quiescent CD4+ effector memory T cells are present within the tumor microenvironment of human lung tumors and can be reactivated by the local and sustained release of IL-12 to proliferate and secrete IFN-γ, leading to tumor cell eradication.

T Cells and Stromal Fibroblasts in Human Tumor Microenvironments Represent Potential Therapeutic Targets

The immune system of cancer patients recognizes tumor-associated antigens expressed on solid tumors and these antigens are able to induce tumor-specific humoral and cellular immune responses. Diverse immunotherapeutic strategies have been used in an attempt to enhance both antibody and T cell responses to tumors. While several tumor vaccination strategies significantly increase the number of tumor-specific lymphocytes in the blood of cancer patients, most vaccinated patients ultimately experience tumor progression. CD4+ and CD8+ T cells with an effector memory phenotype infiltrate human tumor microenvironments, but most are hyporesponsive to stimulation via the T cell receptor (TCR) and CD28 under conditions that activate memory T cells derived from the peripheral blood of the cancer patients or normal donors. Attempts to identify cells and molecules responsible for the TCR signaling arrest of tumor-infiltrating T cells have focused largely upon the immunosuppressive effects of tumor cells, tolerogenic dendritic cells and regulatory T cells. Here we review potential mechanisms by which human T cell function is arrested in the tumor microenvironment with a focus on the immunomodulatory effects of stromal fibroblasts. Determining in vivo which cells and molecules are responsible for the TCR arrest in human tumor-infiltrating T cells will be necessary to formulate and test strategies to prevent or reverse the signaling arrest of the human T cells in situ for a more effective design of tumor vaccines. These questions are now addressable using novel human xenograft models of tumor microenvironments.

IL-12 Delivered Intratumorally by Multilamellar Liposomes Reactivates Memory T Cells in Human Tumor Microenvironments

Using a novel loading technique, IL-12 is reported here to be efficiently encapsulated within large multilamellar liposomes. The preclinical efficacy of the cytokine loaded liposomes to deliver IL-12 into human tumors and to reactive tumor-associated T cells in situ is tested using a human tumor xenograft model. IL-12 is released in vivo from these liposomes in a biologically active form when injected into tumor xenografts that are established by the subcutaneous implantation of non-disrupted pieces of human lung, breast or ovarian tumors into immunodeficient mice. The histological architecture of the original tumor tissue, including tumor-associated leukocytes, tumor cells and stromal cells is preserved anatomically and the cells remain functionally responsive to cytokines in these xenografts. The local and sustained release of IL-12 into the tumor microenvironment reactivates tumor-associated quiescent effector memory T cells to proliferate, produce and release IFN-gamma resulting in the killing of tumor cells in situ. Very little IL-12 is detected in the serum of mice for up to 5 days after an intratumoral injection of the IL-12 liposomes. We conclude that IL-12 loaded large multilamellar liposomes provide a safe method for the local and sustained delivery of IL-12 to tumors and a therapeutically effective way of reactivating existing tumor-associated T cells in human solid tumor microenvironments. The potential of this local in situ T cell re-stimulation to induce a systemic anti-tumor immunity is discussed.

The effect of free fatty acids on spectrin organization in lymphocytes.

Previous studies have shown thatcis unsaturated free fatty acids (uFFAs) are able to cause alterations in the normal distribution pattern of certain cytoskeletal proteins in lymphocytes, including tubulin, actin, α-actinin, and myosin. The cytoskeletal protein spectrin naturally possesses a marked heterogeneity of distribution among resting T and B lymphocytes isolated from all murine lymphoid organs. In some cells, spectrin is observed in a ring-like staining pattern at the periphery of the cell, reflecting a likely association with the cell membrane; in other cells, spectrin is found within the cytoplasm as a large single aggregate or in several smaller aggregates. Addition of uFFA to freshly isolated murine lymphocytes causes disruption in the latter pattern of spectrin organization. Following short-term incubation (15 min) of tissue-derived lymphocytes (from spleen, thymus, and lymph node) and 1 μg/mL uFFA (oleic [18:1cis], linoleic [18:2cis, cis], arachidonic [20:4], or elaidic [18:1trans] acid) there is a loss of cytoplasmic aggregates of spectrin and a concomitant increase in cells in which spectrin is diffusely distributed. This effect is not seen when two saturated FFAs (sFFAs) were used. When using DO11.10 cells, a T-cell hybridoma in which nearly all cells constitutively express a cytoplasmic aggregate of spectrin, a similar effect was observed, but greater concentrations (10–20 μg/mL) of FFA were needed to obtain the same effect. Addition of calcium to the incubation buffer substantially blocks spectrin reorganization. In several disease states, serum levels of FFA are observed to be excessively high; our data support the hypothesis that cytoskeletal reorganization in lymphocytes may be related to the altered immune function frequently observed in these conditions.

VH and VL Gene Complexes Encoding an Anti-Spectrin Antibody are Defined by Nucleotide Sequencing of cDNA from a Hybridoma Generated from Hu-PBL-SCID Mouse

Previously we reported that human imunocompetent cells engrafted into scid mice mount a sustained and vigorous humoral immune response to murine erythrocytes. One of the dominant and consistently observed reactivity pattern of these antibodies in immunoblot analysis is with the alpha and beta isoforms of spectrin. In order to define the human xenoreactive response more completely, a hybridoma was generated (from a hu-PBL-scid mouse) whose antibody reacted with two high molecular weight species 225 to 250 kDa. We report here that this conserved antibody species reacts with both the murine and human erythrocyte proteins and cDNA nucleotide sequence analysis of the light and heavy chain genes encoding this antibody reveals that the light chain variable region gene has been previously observed in association with an autoreactive antibody. In addition to characterizing a conserved human B cell clonotype this is the first report of a human monoclonal antibody being generated from the hu-PBL-scid model using the standard hybridoma technology.