These studies demonstrate the feasibility and safety of using retroviral gene transduction for human gene therapy and have implications for the design of TIL with improved antitumor potency, as well as for the possible use of lymphocytes for the gene therapy of other diseases.

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We report that human breast cancer cells secrete a growth factor that is biologically and immunologically similar to platelet-derived growth factor (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or "competence" activity that is capable of inducing incorporation of [3H]thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. In addition, the conditioned medium contains an activity that competes with 1251-labeled PDGF for binding to PDGF receptors on normal human fibroblasts. The secretion of PDGF-like activity by the hormone-responsive cell line MCF-7 is stimulated by 17.3-estradiol. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (950C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of "30 kDa on NaDodSO4/polyacrylamide gels. Immunoprecipitation with PDGF antiserum of proteins from metabolically labeled cell lysates and conditioned medium followed by analysis on nonreducing NaDod-S04/polyacrylamide gels identified proteins of 30 and 34 kDa.Upon reduction, the 30-and 34-kDa bands were converted to 15-and 16-kDa bands suggesting that the immunoprecipitated proteins were made up of two disulfide-linked polypeptides similar to PDGF. Hybridization studies with cDNA probes for the A chain of PDGF and the B chain of PDGF/S1S identified transcripts for both PDGF chains in the MCF-7 and MDA-MB-231 cells. The data summarized above provide conclusive evidence for the synthesis and hormonally regulated secretion of a PDGF-like mitogen by breast carcinoma cells. Production of a PDGF-like growth factor by breast cancer cell lines may be important in mediating paracrine stimulation of tumor growth.

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The natural history of estrogen-responsive breast cancers often involves a phenotypic change to an estrogen-unresponsive, more aggressive tumor. The human breast cancer cell line, MCF-7, which requires estradiol for tumor formation in vivo and shows growth stimulation in response to estradiol in vitro, is a model for hormone-responsive tumors. The v-rasH onc gene was transfected into MCF-7 cells. The cloned MCF-7ras transfectants, which expressed the v-rasH messenger RNA and v-rasH p21 protein (21,000 daltons), were characterized. In contrast to the parental cell line, MCF-7ras cells no longer responded to exogenous estrogen in culture and their growth was minimally inhibited by exogenous antiestrogens. When tested in the nude mouse, the MCF-7ras cells were fully tumorigenic in the absence of estrogen supplementation. Thus, cells acquiring an activated onc gene can bypass the hormonal regulatory signals that trigger the neoplastic growth of a human breast cancer cell line.

The MCF-7 human breast cancer cell line responds to estrogen stimulation in vitro by increased secretion of growth factors and proliferation and in vivo by tumor formation in the nude mouse. To test a possible role of growth factor secretion in expression of the tumorigenic phenotype, we stably transfected MCF-7 cells with the v-Ha-ras oncogene to produce the MCF-7ras cell line. The MCF-7ras cell line was tumorigenic in the absence of estrogens and secreted 3-to 5-fold elevated levels of a high molecular weight form of a type a transforming growth factor-like growth factor, type ,B transforming growth factor, and insulin-like growth factor I. MCF-7ras cells, in contrast to MCF-7, were less sensitive to further growth stimulation by estrogen, type a transforming growth factor, and insulin-like growth factor I and showed little change in receptor levels for these hormones. Conditioned medium from MCF-7ras cells as well as two of its component growth factors (insulin-like growth factor I and type a transforming growth factor) replaced estrogen in stimulating MCF-7 colony formation in vitro. A coordinate increase in growth factor secretion by human breast cancer may contribute to its escape from estrogen dependence.

The application of bone marrow gene therapy has been stalled by the inability to achieve stable high-level gene transfer and expression in the totipotent stem cells. We show that retroviral vectors can stably introduce genes into antigenspecific murine and human T lymphocytes in culture. Murine helper T cells were transduced with the retroviral vector SAX to express both neomycin-resistance and human adenosine deaminase genes. These cells were expanded in culture and selected for expression of neomycin resistance with G418.

Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible, we transduced human TILs with the bacterial gene for neomycin-resistance (NeoR) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact NeoR gene integration and expressed high levels of neomycin phosphotransferase activity. The NeoR gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for 13-and y-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the (3 and y chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors (a and (3) and interleukin 2 receptor P55 but not for interleukin 1/3, granulocyte/macrophage colony-stimulating factor, interleukin 6, and interferon 'y when grown with high-dose interleukin 2 without subsequent activation with mitogen or specific antigen. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The NeoR gene-transduced TILs could thus be used to follow the trafficking and survival of TILs in vivo, and clinical protocols using these transduced TILs in cancer patients have begun. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs.Tumor-infiltrating lymphocytes (TILs) are lymphoid populations found associated with solid tumors and can be selectively grown in the presence of interleukin 2 (IL-2) (1, 2). Recent clinical trials have shown that the adoptive transfer of TILs in conjunction with IL-2 can result in cancer regression in some patients with advanced, otherwise refractory, cancers (1). Studies of the administration of human TILs labeled with "'In have shown that TILs recirculate and preferentially localize at tumor sites by several days after infusion (3). There has been no method available, however, to study long-term distribution and survival of autologous human TILs in vivo. We have proposed to use an inserted neomycinresistance (NeoR) gene (4, 5) into human TILs as a cell label to answer these questions. It has recently been shown that the NeoR gene can be inserted into murine lymphocytes and human TILs (K. Culver, K. Cornetta, S.M., P.A., S.F., A.K., R. Morgan, M.T.L., S.A.R., W.F.A., and R.M.B., unpublished results). This report represents results of studies to transduce and characterize the NeoR-transduced TIL populatio...