Fig 2: Gas6 blockade increases NK cell numbers in metastatic lungs and in tumor draining lymph nodes. (A) Immunohistochemical staining of NK cells in metastatic lungs from pancreatic tumor bearing mice treated with control IgG or anti-Gas6 antibody. Lesions indicated by dashed line and NK cells by red asterisk. Scale bar, 50 µm. (B) Quantification of NK cells in metastatic lung tissues from control IgG and anti-Gas6 treated mice. Values shown are the mean and SEM (n = 6 mice in IgG treatment group, n = 7 mice in anti-Gas6 treatment group). **p = 0.01, using unpaired student T-test. (C) Immunofluorescent staining of NK cells in mesenteric lymph nodes from pancreatic tumor bearing mice treated with control IgG or anti-Gas6 antibody. NK marker NKp46 is shown in red, Ki67 is shown in green and nuclei were stained with DAPI (in blue). Scale bar, 50 µm. (D) Quantification of NK cells in tumor draining lymph nodes from control IgG and anti-Gas6 treated mice. Values shown are the mean and SEM (n = 6 mice IgG treatment group and n = 7 mice anti-Gas6 treatment group, 3–6 fields/ mouse tissue were quantified). *p = 0.05, using unpaired student T-test.

Fig 3: Serum deprivation induces RSK-driven mTOR activation and correlates with autocrine GAS6 level. (A) Western blot analysis of 2D cultivated MDA-MB231, Caliper, H292, and Hs578T cells that are shown being either cultivated for 24 h with FBS (?1+) or without FBS for 24 (?1-) and 48 h (?2-). The lack of growth factors activates ERK T202/Y204 and SRC Y416 and specifically in H292 EGFR Y1173 and AKT S473 phosphorylation. TAM receptor analyses were performed to substantiate a direct link to the PDK-RSK-dependent mTOR activation. ACTIN served as loading control for all western blots. A representative sample of multiple biological replicates is displayed in the bottom line. (B and C) High autocrine GAS6, and not PROS1, matches with pRSK S380 activation in tumor cells. Supernatants of 2D cultivated tumor cells were analyzed by GAS6 ELISA (B) and PROS1 ELISA (C). The results represent the average of fourfold determination.

Fig 4: Axl augments ZIKV infection of human SC. (A) ZIKV progeny titers measured in the supernatant of SC treated with anti-Gas6 neutralizing antibody (50 µg/ml) or anti-Axl blocking antibody (10 µg/ml) or goat IgG control (50 µg/ml) by plaque assay at 24 h postinfection. (B) ZIKV progeny titers were also determined 24 h postinfection in SC grown in the presence of serum (FBS) or absence of serum (-FBS) and compared to serum-deprived SC treated with rhGas6 (5 nM) upon infection (-FBS +rhGas6). (C) ZIKV RNA copies measured in infected SC in the presence and absence of Axl kinase inhibitor R428 (1 µM) by qRT-PCR and expressed as ZIKV PFU equivalents per microgram of RNA. (D) ZIKV progeny titers measured by plaque assay at 24 h and 72 h postinfection of SC treated with R428. (E) ZIKV infection of SC treated with R428 (1 µM) was also determined by IFA via staining of ZIKV (anti-ZIKV-E [green]) at 48 h postinfection. (F) Cell viability was evaluated following R428 treatment (1 µM) in mock-infected and infected SC by CellTiter 96 AQueous One Solution at 48 h postinfection, and the percent cell viability was calculated by comparison to the value for mock-infected cells at the corresponding time point. DMSO vehicle was used as untreated control for R428 experiments. Values are averages ± standard deviations (SD) (error bars) from 3 to 5 independent experiments with cells infected at an MOI of 1. Values that are significantly different are indicated by a bar and asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 5: Schematics depicting the multifunctional role of stroma-derived Gas6 in pancreatic cancer. In vivo blockade of Gas6 signaling with a neutralizing anti-Gas6 antibody or warfarin, partially reverses tumor cells EMT and activates NK cells, leading to a decrease in pancreatic cancer metastasis.