Figure 4. STAT3 signaling in the human tumor cells can mediate human T-cell trafficking into the tumor in vivo to increase the tumor growth rate in an allogeneic reconstitution system.

a. Lymphocytes from HNSCC patients undergoing surgery were harvested and assayed for migration towards conditioned supernatants from Cal27 tumor cell lines in this chemotaxis assay (5 experiments performed in triplicates) [19]. Negative controls consisted of media with no exposure to tumor cells (“no treatment”), and 100% serum as positive controls. Cal27 cells were transduced with scrambled lentivirus (“tumor cells”) or STAT3 siRNA lentivirus (“Stat3 knockout”). b. HLA-A2+ Cal27 HNSCC cell lines were transduced with either scrambled siRNA or STAT3 siRNA, and these tumor cells were injected into humanized mice (7-10 mice/group). Growth rates were followed after xenotransplantation (left panel, *P<0.01). These tumor cells were also injected into non-humanized NOG-A2 mice (right panel). These experiments were independently repeated 3 times. c. Harvested tumors were stained with H&E and analyzed. The bar scale is 100µ. d. Tumor transduced with STAT3 siRNA showed greater number of tumor infiltrating human CD45+ cells (left panel). Harvested tumor from the two groups of tumor from humanized mice (scrambled siRNA control and siRNA treated Cal27) were stained with human CD45-Alexa Fluor 568 conjugate and CD45+cells were counted over 10 fields and averaged. We also ensured that transduced tumor cells had stable suppression of STAT3 at the time of tumor harvest (right panel) by performing pSTAT3 expression analysis in the two groups of tumors from Fig. 4b at the time of tumor harvest on day 36. The bar scale is 100µ.