Table 1.
Real time RT-PCR and Protein level in whole tumors
| PCR | Control | α-CCL2 | AdE7 | AdE7 + α-CCL2 | p-value (Ade7 Vs. combination) |
|---|---|---|---|---|---|
| TNF-α | 1 | 0.8 | 1.8 | 2.8 | < 0.01 |
| TGF-β | 1 | 0.6 | 0.6 | 0.9 | < 0.01 |
| IFN-γ | 1 | 0.8 | 4.1 | 7 | < 0.01 |
| IL10 | 1 | 1 | 0.95 | 1.8 | 0.01 |
| IL-12 | 1 | 1.3 | 0.8 | 1.5 | < 0.01 |
| CXCL-10 (IP-10) | 1 | 2.3 | 1.7 | 3 | < 0.01 |
| CCL2 (MCP-1) | 1 | 1.2 | 2.2 | 2.2 | NS |
| CCL12 | 1 | 0.6 | 1.1 | 1 | NS |
| CCL5 (RANTES) | 1 | 0.9 | 1 | 1 | NS |
| ICAM-1 | 1 | 0.7 | 0.8 | 1.6 | < 0.01 |
| CD8 | 1 | 0.7 | 2.4 | 4.7 | < 0.01 |
| Protein | |||||
| TNF (pg/ml/g) | 331 | 180 | 373 | 999 | 0.02 |
Mice (n = 4–5 for each group) bearing large (average size of 200–250 mm3) TC1 tumors, were treated in one of four ways: 1) control no treatment (Control); 2) I.P. α-CCL2 mAb twice per week starting at day 13 and throughout the experiment (α-CCL2); 3) S.Q. vaccine with Ad.E7, and a booster vaccine after a week (Ad.E7); and 4) combination of Ad.E7 and α-CCL2 mAb (Combo). Two days after the second (booster) Ad.E7 vaccine, tumors were harvested, digested, and RNA was extracted. Equal amounts of RNA from each tumor in each group were pooled, cDNA generated, and subjected to real time RT-PCR analysis. RNA was normalized using β-actin levels. Each assay was run in at least quadruplicate. Fold change with each treatment compared to control is shown. Major changes between immunotherapy alone and the combination with α-CCL2 mAb are highlighted.
For the evaluation of TNF-α protein levels (bottom line), explants from individual tumors of each treatment (n=8 of each subgroup) were plated in medium. After 24 hours, the level of TNF-α was evaluated using an ELISA kit. Mean levels are shown for each treatment, adjusted to tumor weight and medium volume.