Fig 2: Silencing Src attenuated tumorigenic properties in H460R and A549R cells. a MTT assay showed that Src-silenced A549R and H460R cells became more sensitive towards CDDP treatment significantly as compared to their wildtype counterparts. b Src-silenced H460R and A549R cells showed a markedly reduced ability to form tumor spheres. c Aldefluor assay showed a significantly reduced ALDH1 activity in both A549R and H460R cells with Src-downregulated. The results are presented as mean ± SD. d Accompanied western blots showed that after Src was silenced in both cell lines, the expression of MIF, CD155 and ß-catenin was clearly suppressed. VC, vector control; shSrch, Src-silencing by shRNA. e Real-time PCR results showed that mRNA level of M2 markers, CCL13, TGF-ß was significantly down-regulated in the macrophage co-cultured with Src-silenced A549R and H460R cells. f ELISA assay showed that macrophages co-cultured with Src-silenced NSCLC cells secreted a significantly lower amount of M2 cytokines, IL-10 and CCL22, relative to their counterparts

Fig 3: High expression of Src, MIF and CD155 is associated with poor survival in patients with NSCLC. a A public database of NSCLC patients was queried for Src, MIF and CD155 expression. Kaplan-Meier overall survival (KM) plots were drawn for each marker. The clinical samples were stratified into high and low expression of Src, MIF and CD155 respectively. In all three KM plots showed that the high expression of Src, MIF or CD155 predicts for a shorter overall survival rate in the patients. b Immunohistochemical analyses of 20 pairs clinical samples from our own hospital. The staining intensity score was compared between cisplatin sensitive and resistant samples. A higher staining score of Src, MIF and CD155 was observed in the resistant samples as compared to their cisplatin-sensitive counterparts. The lower panels depict the representative micrograph of samples stained with respective markers. P-value is indicated. c An increased CD163high and CX3CR1high M2 TAMs (by flow cytometry) were identified within the CDDP-resistant tumor samples as compared to CDDP-sensitive counterparts. d A negative correlation was found between the percentage of CD163high and CX3CR1high M2 TAMs and CDDP-sensitivity. The green lines delineate the connection between high percentage of CD163high and CX3CR1high M2 TAMs and their corresponding CDDP-sensitivity

Fig 4: Prolonged cisplatin treatment enriched CDDP-resistant NSCLC cells with increased properties of tumorigenesis and cancer stemness. a Flow cytometry analysis showed that a marked increased CD133+ cell population in H460R and A549R cells as compared to their parental counterparts. The bar graph (right) is the quantitative representation of the flow cytometric experiments and showed the comparative level of CD133+ cell population. b, c Increased tumor sphere and colony forming abilities were also observed in H460R and A549R cells. d Real-time PCR profiling of H460R and A549R cells revealed that a significantly increased in oncogenic markers, Src, MIF, stemness markers, CD133, Notch1, ß-catenin (CTNNB1) and multiple drug resistant pumps, ABCG2 and ABCB1. *p < 0.05; **p < 0.01; ***p < 0.001

Fig 5: Dasatinib-treated NSCLC cells exhibited a lower ability to induce M2 polarization in macrophages. a Real-time PCR profiling of macrophages co-culture with NSCLC cells treated with dasatinib. qPCR results showed that the M2 markers, CCl13 and TGF-ß were significantly reduced when co-cultured with H460R and A549R treated with dasatinib (2 µM, 24 h prior to co-culture), while M1 markers, IL-1ß and TNF-a remained relatively unchanged. b Comparative ELISA assays of M1 M2 cytokines. Macrophages co-cultured with dasatinib-treated A549R and H460R cells showed a significantly lower secretion of M2 cytokines, IL-10 and CCL22 as compared to their counterparts which were co-cultured with A540R and H460R cells without prior dasatinib treatment. c ELISA assay showed that dasatinib treatment lowered the MIF and IL-6 secreted by both A549R and H460R cells