3.1. Compare the differential proteins between baseline and best response in anlotinib responders
Plasma samples from 28 patients were used for proteomics analysis. In this cohort, 14 patients without any anlotinib response (Median PFS: 35.5 days; Median OS: 158.5 days), and 14 patients with good anlotinib response (Median PFS: 189 days; Median OS: 373 days) (Fig. 1, Supplementary Table 2). The biomarkers using plasma proteomics to screen responders and non-responders remain unclear. In order to screen the effective plasma biomarkers, we firstly compared the plasma protein levels those from responders between the two time points including baseline and best response. Total 528 proteins were detected via quantitative proteomics. Of 528 proteins, 28 protein levels were relative higher and 30 protein levels were relative lower at the time point of best response, as compared with those from baseline (Fig. 2a). Heat map analysis of those 58 protein differential levels indicated there are some sample differences between the duplicates, but still found lots of proteins showed remarkably differences (Fig. 2b). Here, we performed biological process analysis and cell component analysis on those 58 differential proteins. Large parts of those proteins played roles in single-multicellular organism process and multi-cellular organismal process after biological process analysis, which also played roles in extracellular region part and extracellular region after cell component analysis (Fig. 2c). Furthermore, biological process analysis suggested the up-regulated proteins enriched in the items of single-multicellular organism process, multi-cellular organismal process, biological regulation, and so on, and the down-regulated proteins enriched in the items of protein metabolic process, organonitrogen compound metabolic process, nitrogen compound metabolic process, and so on (Fig. 2d). These results suggested that anlotinib performed its anti-tumor effect may associated with the 58 differential proteins, and potentially be used for anlotinib biomarker screening.
3.2. Differential proteomics analysis throughout anlotinib administration in anlotinib responders
To analysis the changes of plasma protein levels after anlotinib administration, we compared the plasma protein levels those from responders at three time points including baseline, best response and progression disease. Total 18 proteins were screened out, and showed significantly alteration (Fig. 3a). Heat map analysis indicated 7 protein levels increased at the time point of best response, then decreased at time point of progression disease. 7 protein levels increased continually and 1 protein levels decreased continually after anlotinib administration. 3 protein levels decreased at the time point of best response, and then increased at time point of progression disease (Fig. 3b). Biological process analysis suggested those 18 proteins enriched in the items of platelet activation, cell activation, blood coagulation, and so on. Cell component analysis suggested those proteins enriched in membrane-bounded vesicle, extracellular space, vesicle, and so on (Fig. 3c). KEGG pathway analysis indicated those proteins enriched in the signaling pathways including shigellosis, complement and coagulation cascades, salmonella infection, and salivary secretion (Fig. 3d). These results suggested that anlotinib-induced plasma protein level alterations may affect the different biological processes, cell components, and signaling pathways which were potentially involved in acquired resistance.
3.3. Analysis of resistance to anlotinib via proteomics characterization in non-responders
Next, we compared the plasma protein levels those from non-responders at two time points including baseline and best response. Totally 41 plasma protein levels changed including 20 protein levels significantly enhanced and 21 protein levels remarkably dropped, after administration of anlotinib (Fig. 4a). Biological process analysis suggested those proteins enriched in the items including cell migration, localization of cell, cell motility, and so on (Fig. 4b). Further analysis indicated that the up-regulated proteins enriched in the items of metabolic process, organic substance metabolic process, cellular process, and so on. The down-regulated proteins enriched in the items of endocytosis, receptor-mediated endocytosis, chemical homeostasis, and so on (Fig. 4c). Cell component analysis suggested those proteins enriched in the items including extracellular region, extracellular region part, vesicle, and so on (Fig. 4b). These results provided hypothesis that those proteins may play an important role in compensatory effect of anlotinib-induced tumor cell process inhibition.
3.4. Biomarker screening between anlotinib responders and non-responders via proteomics analysis
Understand the baseline plasma protein levels between responders and non-responders will contribute to provide potential biomarker screening for anlotinib responsive stratification. Here, we found that 514 common proteins both existed in the plasma samples from responders and non-responders. Of 514 proteins, 23 proteins with higher level and 21 proteins with lower levels from responders, compared with those from non-responders (Fig. 5a). Heat map analysis suggested that majority of those differential proteins stably existed in the duplicated samples (Fig. 5b). Those differential proteins enriched in the biological process items including peptide cross-linking, regulation of peptidase activity, cornification, and so on (Fig. 5c). Those higher level proteins from responders at baseline enriched in the items of cellular process, metabolic process, single-organism process, and so on. And those lower level proteins enriched in the items of response to stimulus, biological regulation, regulation of biological process, and so on (Fig. 5d). Cell component analysis suggested those differential proteins enriched in extracellular space, extracellular region part, extracellular region, and so on (Fig. 5c). Furthermore, we compared the plasma protein levels between responders and non-responders at the time point of progression disease. Results suggested that there are 4 proteins with higher levels in non-responders than those in responders, and 15 proteins with lower levels in non-responders than those in responders (Supplementary Fig. 2). These results suggested potential biomarkers could be screened out for anlotinib stratification.
3.5. Integrative analysis reveals ARHGDIB levels potentially be used for anlotinib responder screening
To further screen out the potential plasma biomarker, integrative analysis was performed on those protein levels at three time points of baseline, best response, and progression disease from responders, and at the time point of baseline from non-responders. After filtered, we found 43 proteins showed important potential value (Fig. 6a). For the samples from non-responders at the time point of baseline, of 43 proteins, 5 proteins with lower level and 38 proteins with higher level (Fig. 6b). Biological process analysis suggested these proteins enriched in the items of receptor-mediated endocytosis, platelet degranulation, innate immune response, and so on. Cellular component analysis suggested these proteins enriched in the items of protein binding, Poly (A) RNA binding, calcium ion binding, and so on. Molecular function analysis suggested these proteins enriched in the items of extracellular exosome, extracellular region, extracellular space, and so on (Fig. 6c). Among these proteins, we selected the ARHGDIB maybe have some predictive value due to anlotinib can down-regulate ARHGDIB expression in NCI-H1975 cells, and those patients with lower ARHGDIB expression have longer OS outcome in TCGA cohort (Fig. 6d, 6e). Furthermore, we detected the plasma ARHGDIB levels at baseline on those responders and non-responders, and found that high levels of plasma ARHGDIB in NSCLC patients have a better response to anlotinib than those patients with a low level of plasma ARHGDIB [High (n = 14), Median PFS = 189 days versus High (n = 14), Median PFS = 40.5 days, P < 0.001; High (n = 14), Median OS = 375 days versus High (n = 14), Median PFS = 158.5 days, P = 0.034] (Fig. 6f, 6g). These results suggested proteomics analysis potential used for anlotinib responsive stratification.