Although TRAMPC2/TR/CCL21-L2 (Line 2) cells had higher background expression relative to Line 1, GANT61 mw tetracycline induced much higher levels of CCL21 production. These data indicate that both these lines have the capacity to grow both in vitro and in vivo following orthotopic implantation. Fig. 2 Variable expression of CCL21 by TRAMPC2/TR/CCL21 cells following passage in vitro and tumor growth in vivo. a

TRAMPC2 cells were transfected with the repressor and CCL21 expression vectors using Fugene 6 transfection reagent and selected in antibiotic-containing media. Cloned antibiotic resistant cell lines (TRAMPC2/TR/CCL21, clones 4, 5 and 6) were tested for CCL21 expression with or without 2 ug/ml of tetracycline by ELISA. ELISA was performed after 3 and 8 passages to test whether these clones maintained inducible expression of the transgene. b Syngeneic mice were implanted orthotopically with TRAMPC2/TR/CCL21 tumor cells

(clone 4, 5 × 105). After several months following implantation, palpable tumors were excised, diced and explants cultured in vitro. Cloned lines were derived, expanded in selection media and tested for Bucladesine in vitro tet-induced secretion of CCL21. Clonal lines from six tumors isolated from individual mice (M1-6) were evaluated (see Table I). This panel illustrates the expression levels achieved by tetracycline in the 10 clones (10/103) that produced CCL21. None of the lines derived from two tumors (M5 and M6) displayed inducible expression of CCL21. c Eight clonal lines with weak induction derived from mouse tumors 1 and 4 were pooled to generate

L1. The remaining two lines (M3.2 and M4.2) were pooled to generate L2. These two pooled lines were then subjected to antibiotic selection using zeocin and blasticidin, expanded in vitro and then tested for inducible CCL21 production. Note different scales in panels B and C Table 1 Distribution of inducible expression of CCL21 in clonal lines derived from TRAMC2/TR/CCl21 tumors following orthotopic tumor growth Casein kinase 1 in vivo Mouse No. Clones No. Inducible Clones % Inducible Expression M1 22 6 27 M2 18 1 5.5 M3 19 1 5.3 M4 11 2 18 M5 15 0 0 M6 18 0 0 Total 103 10 9.7 Nine syngeneic mice were implanted orthotopically with TRAMPC2/TR/CCL21 tumor cells (5 × 105). One mouse died without any tumor and two mice never grew tumors. After several months following implantation, 6 palpable tumors were excised, diced and explants cultured in vitro. Cloned lines were derived, expanded in selection media and tested for tet-induced secretion of CCL21 by ELISA. Clonal lines from six tumors isolated from individual mice were evaluated (M1-6). Impact of Intratumoral Expression of CCL21 on Survival, Tumor Growth and Metastatic Disease TRAMPC2/TR/CCL21 clones (L1 and L2) displayed high levels of tet-inducible expression of CCL21 and grew in vivo. We next wanted to test whether CCL21 expression in the TME enhances survival of mice implanted orthotopically with prostate tumor cells.