DSTYK depletion sensitizes lung cancer cells to TNF- α CD8 + killing. (A) Syngeneic mouse models for sh#DSTYK LUAD (left) and squamous carcinoma cell lines (right) respectively. sh#DSTYK was activated when tumors reached 50–100 mm3. Analysis by ANOVA. LLC parental vs. sh#3 and parental vs. sh#4, P < 0.001; UN-SCC680 parental vs. sh#3, P = 0.04; and parental vs. sh#4, P < 0.001. Number of C57BL/6J or A/J mice per group = 7. Scale bar: 1 cm. (B) LLC tumor–bearing mice were treated with doxycycline to induce sh#DSTYK in the presence or absence of depleting antibodies (anti-CD8). Statistical analysis was performed by ANOVA comparing tumor volumes of experimental groups at the end point day. LLC parental vs. sh#3, P < 0.001, while no differences were found in other comparations. Number of C57BL/6J mice per group = 7. (C) LLC tumor–bearing mice were treated with doxycycline to induce sh#DSTYK in the presence or absence of depleting antibodies (anti-CD4, anti-NK). Statistical analysis was performed by ANOVA comparing tumor volumes of experimental groups at the end point day. LLC parental vs. sh#3, sh#3+anti-CD4, and sh#3+anti-NKs, P < 0.001. Number of C57BL/6J mice per group = 7. (D) UN-SCC679 tumor–bearing mice were treated with doxycycline to induce sh#DSTYK in the presence or absence of depleting antibodies (anti-CD8). Statistical analysis was performed by ANOVA. Parental vs. sh#4, P < 0.001, while no differences were found in other comparations. Number of A/J mice per group = 7. (E) Control or DSTYK KO LLC cells were pulsed with Ova peptide and incubated with OT-1 T cells at the indicated E:T ratios for 96 h. The dot plot graph shows the relative area under the curve (n = 3) compared with cells incubated in the absence of T cells. t test was used to compared experimental groups and rendered P < 0.001 in every treated condition. (F) LLC T cell killing was performed in the presence of the TNF-α–blocking molecule etanercept. Analysis by t test. KO untreated vs. KO 1:3, P < 0.001. (G) T cell killing of LLC or H2009 parental and KO cell lines was performed in the presence or absence of TNF-α. Analysis by t test. LLC parental vs. KO cells treated with TNF-α, P = 0.02; H2009 parental vs. KO cells treated with TNF-α, P < 0.001. (H) Parental and DSTYK inhibited LLC tumor–bearing mice were treated with etanercept. Statistical analysis was performed by ANOVA. Parental vs. KO, P < 0.0001, while no differences were found in other comparations. Number of C57BL/6J mice per group = 7. (I) Intravital microscopy of T cell responses in LLC DSTYK wildtype and deficient tumors. GFP-LLC#sh tumor cells were injected in the dorsal part of the ear of hCD2dsRED mice in which T cells are red fluorescent. 7 d later, animals were given or not given doxycycline in the drinking water for 24 h and then imaged in a confocal microscope. Images of a representative experiment and corresponding to Video 2. Scale bar: 50 µm. (J) Dot plot quantification of percentage of apoptotic tumor cells per field and number of intratumor T cells in LLC DSTYK wildtype and deficient tumors from F. n = 4 mice per group; each dot represents a single quantified HPF. Statistical analysis of apoptotic tumor cells per field was performed by t test; P < 0.001 and no differences were found in the number of intratumor T cells. *, P < 0.05; ***, P < 0.001.
