Jeannine Alexander

Characterization of tumor-infiltrating lymphocyte subsets from human renal cell carcinoma: Specific reactivity defined by cytotoxicity, interferon-γ secretion, and proliferation

The detection of T cells with specificity for human renal cell carcinoma (RCC) has been difficult to document. In an attempt to improve our identification of RCC-reactive T cells, tumor-infiltrating lymphocytes (TIL) were expanded in interleukin-2/interleukin-4 (IL-2/IL-4) and then separated into CD4+ and CD8+ subsets using antibody-coated biomagnetic beads. TIL grown in IL-2/IL-4 expanded to greater numbers than TIL grown in IL-2 alone. From 16 patients in whom subset separation was performed, three CD4+ and three CD8+ TIL consistently had specificity for RCC that was detected by cytotoxicity, proliferation, or interferon-gamma (IFN-gamma) production. Four of the six lines were derived from the IL-2/IL-4 cultures. Two CD8+ TIL lines displayed specific lytic activity, lysing the autologous tumor but not allogeneic RCC or nonrenal tumors. Moreover, the lytic activity of these lines was blocked by anti-CD3 antibody, suggesting that tumor recognition was through the TCR/CD3 complex. Two additional TIL lines showed preferential lysis of RCC because they were cytotoxic for autologous tumor and one or more allogeneic RCC but not other tumor types. Two nonlytic CD4+ lines as well as the two CD8+ lines that were specifically lytic also produced IFN-gamma in response to the autologous tumor but not allogeneic RCC. Although these TIL lines produce IFN-gamma when stimulated with tumor alone, the addition of 5 U/ml of IL-2 significantly enhanced IFN-gamma secretion. The four TIL lines that showed specificity for RCC in terms of IFN-gamma production also had enhanced proliferation to the autologous RCC plus IL-2 but not to multiple allogeneic RCC plus IL-2. These studies demonstrate that TIL from RCC patients contain both CD4+ and CD8+ T cells that have specificity for RCC. In addition to cytotoxicity, specificity to RCC can be defined by IFN-gamma production and proliferation.

Intercellular Adhesion Molecule-1 Expression by Bladder Cancer Cells: Functional Effects

The role of intercellular adhesion molecule-1 (ICAM-1) and its ligand, leukocyte function-associated antigen-1 (LFA-1), in the interaction between bladder cancer cells and lymphokine activated killer (LAK) cells was investigated. Expression and modulation of ICAM-1 by cytokine treatment was assessed by immunocytometry and Northern blot analysis. Four of five human bladder cancer cell lines expressed ICAM-1 constitutively and responded to cytokine stimulation. Expression of ICAM-1 was upregulated most consistently by treatment with interferon-gamma (IFN gamma) and tumor necrosis factor-alpha (TNF alpha), cytokines that are released into the urine after intravesical BCG treatment. In contrast, interleukin-1 and phorbol myristate acetate exhibited variable effects on ICAM-1 expression, and interferon-alpha had no effect. The adherence of LAK cells to bladder cancer cell monolayers and LAK cell-mediated cytolysis were then studied. Monoclonal antibodies to ICAM-1 and LFA-1 significantly decreased the binding of LAK cells to the cell lines that express ICAM-1 (37 to 75% reduction, p < 0.05), and cytokine treatment (IFN gamma, TNF alpha) of these cells enhanced ICAM-1 dependent adherence (18 to 39% increase, p < 0.05). In contrast, these manipulations had no effect on the binding of LAK cells to the UMUC3 cell line, which does not express ICAM-1. Monoclonal antibodies to LFA-1 decreased LAK cell mediated cytolysis of the bladder cancer cells from 27 to 65% (p < 0.05), but anti-ICAM-1 antibodies were much less effective (0 to 25% decrease in cytolysis). Cytokine treatment (IFN gamma, TNF alpha) of the tumor cells did not significantly increase LAK cell-mediated cytolysis, despite upregulation of ICAM-1. These data demonstrate that ICAM-1 plays a role in the binding of LAK cells to bladder cancer cells but is only marginally involved in the process of LAK cell-mediated cytolysis. These findings suggest that adhesion molecules may be important mediators of the immune response to bladder cancer after intravesical BCG therapy.

The Influence of Cytokines on the Adhesion of Renal Cancer Cells to Endothelium

PURPOSE The development of tumor metastasis requires direct adhesive interactions between tumor cells and vascular endothelium. We examined the adherence of renal cell carcinoma (RCC) lines to endothelium after stimulation with different cytokines that induce expression of the vascular adhesion molecules endothelial leukocyte adhesion molecule (ELAM)-1, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1, MATERIALS AND METHODS Human umbilical vein endothelial cells (HUVEC) were used to determine the adhesion of the RCC lines CCF-RC1, 2 and 7 to endothelium. Expression of cell adhesion molecules (CAM) on HUVEC and RCC lines was measured with immunoflowcytometry. RESULTS Stimulation of HUVEC with rIl-1 beta, rTNF-alpha, or PMA resulted in a time-dependent 1.4- to 2.9-fold increase of RCC adhesion to HUVEC. Significant increased tumor cell binding was observed after 4 hours and paralleled the time-dependent induction of ELAM-1 and VCAM-1. Immunocytometry demonstrated the presence of the ligands sialyl Lewis X and VLA-4 on RCC, and blocking studies with monoclonal antibodies directed against tumor cell-endothelial interactions mediated by VCAM-1/VLA-4 and ELAM-1/sialyl Lewis X demonstrated marked inhibition of tumor cell adherence to cytokine-stimulated HUVEC. CONCLUSIONS This study demonstrates that cytokine-induced increases in RCC adherence to HUVEC are mediated in part by VCAM-1/VLA-4 and ELAM-1/sialyl Lewis X interactions and suggest that these molecules may play an important role in the ability of RCC to metastasize.

Molecular regulation of intercellular adhesion molecule 1 (ICAM-1) expression in renal cell carcinoma

Intercellular adhesion molecule-1 (ICAM-1) mediates two important functional. aspects of tumor biology, namely enhancement of tumor metastasis and mediation of host defense mechanisms such as lymphocyte-mediated tumor cytotoxicity. Since ICAM-1 is expressed by most renal cell carcinomas (RCC), the regulation of ICAM-1 expression is important in understanding the biological behavior of RCC. We report an investigation on ICAM-1 expression and molecular regulation by cytokines and protein kinase C activator on RCC cell lines. Of the various cytokines, tumor necrosis factor αa (TNFα), interferon-γ (IFNγ), and phorbol myristate acetate (PMA) strongly upregulated ICAM-1 protein expression on RCC. The kinetics of ICAM-1 message induction was studied by Northern analysis of total RNA extracted from RCC and normal kidney proximal tubular (NKPT) cells. Time course studies showed that ICAM-1 mRNA was upregulated by INFγ, TNFα, and PMA, plateaued after 2 h, and remained increased for up to 24 h. Although ICAM-1 mRNA in NKPT cells was upregulated by these cytokines, their messages returned to basal levels after 24 h. ICAM-1 mRNA stability assays showed that both unstimulated and stimulated RCC cells had very stable ICAM-1 mRNA up to 24 h. In order to investigate whether increased gene transcription contributes to ICAM-1 upregulation, RCC cells were treated with TNFα, IFNγ, or PMA with or without simultaneous addition of actinomycin D. ICAM-1 message induction-blocking studies suggested that primary upregulation of ICAM-1 mRNA may be caused by transcriptional upregulation. These results suggest that long-lasting ICAM-1 message upregulation in response to cytokines or PMA may be due to transcriptional upregulation in the early phase and stabilization of ICAM-1 message in the later phase (after 4 h). These observations suggest that RCC may lack the normal downregulatory mechanisms which control ICAM-1 expression and may explain the high frequency of ICAM-1 expression observed on primary human RCC.

Expression of cell adhesion molecules in an established and characterized new human renal cell cancer line, CCF-RC7

In order to investigate the importance of cell adhesion molecules (CAMs) in renal cell carcinoma (RCC), a cell line, designated as CCF-RC7, was established from a human RCC of the clear cell type. CCF-RC7 was passaged over 50 times in vitro for 31/2 years. The cell line has an epithelial morphology and a doubling time of 30 h, forming colonies in soft agar with an average efficiency of 10.4% and producing clear cell tumors in athymic nude mice. CCF-RC7 cells have an aneuploid-hypotetraploid karyotype with a modal chromosome number of 82 and rearrangements in chromosomes 9, 12 and 14. Immunohistochemical and flow immunocytometric analyses revealed high expression of ICAM-1 (CD54), and Hermes antigen (CD44), which was significantly upregulated by cytokine and PMA treatment. VLA-4 was expressed on approximately 20% of tumor cells and could not be altered by cytokine or PMA stimulation. High expression of sialyl Lewis X was also demonstrated by immunohistological examination. This newly characterized cell line will serve as a useful model for the study of CAMs during hematogenous metastasis and host defense mechanisms in human RCC.

Retroviral transduction of intercellular adhesion molecule-1 enhances endothelial attachment of bladder cancer

An intercellular adhesion molecule-1 (ICAM-1)-negative RT4 transitional cell carcinoma (TCC) cell line was transducted with full-length ICAM-1 cDNA via a retroviral vector. Flow cytometry showed that a sense-oriented clone (S20) highly expressed ICAM-1 while an anti-sense clone (AS6) did not. Both S20 and AS6 bound with equal frequency (30 ± 8.7% vs 30 ± 9.4%) to unstimulated human umbilical vein endothelial cells (HUVECs) in cell attachment assays. However, when phorbol myristate acetate (PMA)-activated T lymphocytes, which express lymphocyte function-associated antigen-1 (LFA-1), were cocultured with tumor cells, attachment of S20 increased twofold (60 ± 11.9%) but AS6 showed no change (32 ± 11%). Blocking studies with anti-LFA-1 and anti-ICAM-1 monoclonal antibodies caused an inhibition of the attachment to baseline levels, demonstrating that the enhancement of S20 attachment was dependent upon the LFA-1/ICAM-1 interaction. Enhanced attachment of S20 was not inhibited by the addition of isotypic immunoglobulin G. These results suggest that LFA-1-expressing leukocytes may act as a bridge between the endothelium and tumor cells which express ICAM-1 and, thereby, enhance the potential for hematogenous metastasis.

Adhesion Molecules in Renal Cell Carcinoma

Cell adhesion molecules (CAM) are cell surface molecules which define the temporal, spatial, and cellular selectivity patterns of cell-cell interactions. During ontogenesis, cell adhesion molecules provide tissue- and organ specific information and serve as contact molecules in the arrangement of complex tissues. In mature tissue CAM are important for the antigen-specific and non-specific immune response, mediating the extravasation of lymphocytes into perivascular tissue and the interaction between lymphocytes and other cells such as antigen presenting cells. (1)

IL-2 Based Combination Therapy of Malignant Disease: Summary of the Phase I Experience at the Cleveland Clinic

We have performed Phase I trials of rIL-2 in combination with IFNα or doxorubicin (DOX), based upon the independent activity of these agents and preclinical evidence of therapeutic synergy. In a Phase I trial of the combination of doxorubicin (DOX) and rIL-2, myelosuppression prevented dose escalation beyond the MTD of DOX 40 mg/m2 IV bolus day 1 and rIL-2 3.0 MU/m2/24 hr IV on days 2–5, 9–12, and 16–19. Significant increases in peripheral blood NK and LAK precursor activities were observed, but no clinical responses were produced in this patient population with largely gastrointestinal malignancies. In other Phase I studies, four week cycles of rIL-2 given by intravenous (IV) bolus injection three times weekly (TIW) have been administered in combination with rHuIFNα2a given intramuscularly (IM) TIW. With aggressive supportive care, the Maximum Tolerated Dose (MTD) of rIL-2 that could be given with rHuIFNα2a 10 MU/m2 IM TIW was 22.0 x 106 BRMP Units (MU)/m2 IV TIW. Dose limiting toxicities were CNS, pulmonary and cardiovascular. When given with rHuIFNα2a 10.0 MU/m2 TIW, the MTD of infusion rIL-2 was 3.0 MU/m2/24 hr x 5 days, weekly x 4. We have also treated pts with RCC and MM in Phase II trials of rIL-2 3.0 MU/m2/24 hrs D1–4 and rHuIFNα2a 5.0 MU/m2 SQ D1–4. Overall, 3/7 pts with RCC treated with infusion schedule rIL-2 and rHuIFNα2a have responded in our Phase I study, as opposed to 3/33 pts with RCC treated with bolus schedule rIL-2 and rHuIFNα2a. Of pts with MM, 6/23 pts have responded to bolus schedule rIL-2 and rHuIFNα2a while 0/3 have responded to infusion schedule rIL-2 with rHuIFNα2a in our Phase I studies. We have also entered patients on multi-center Phase II studies utilizing a somewhat less dose-intense schedule of infusion schedule rIL-2 and rHuIFNα2a. Among patients entered on these trials from the Cleveland Clinic, 3/11 pts with RCC have responded, while 2/10 pts with MM have responded. Further studies of the mechanisms by which responses are mediated are needed if IL-2 based therapies are to be improved.