Sensitivity profile to chemotherapeutic drugs of each PC cell line
We conducted cytotoxicity assay by treating ten common PC cell lines, namely AsPC-1, BxPC-3, Capan-1, CFPAC, HPAF-II, MIA PaCa-2, PANC-1, Patu-8988, SW1990 and T3M4, with five first-line chemotherapeutic drugs of PC, namely gemcitabine, 5-FU, paclitaxel, irinotecan and cisplatin. Inhibition curves based on cell inhibition ratio were shown classified by cell lines (Figure 1). As for a single PC cell line, the IC50 values for each chemotherapeutic drug varied much from several nanomoles to thousands of micromoles. Nine of the ten PC cells were most resistant to cisplatin, except for AsPC-1, whose IC50 for 5-FU was much greater than that for the other four drugs. The two most toxic drugs for PC cell lines were paclitaxel and gemcitabine. CFPAC, MIA PaCa-2 and Patu-8988 were most sensitive to gemcitabine and the other seven cell lines were most sensitive to paclitaxel.
Sensitivity profile of PC cell lines to each chemotherapeutic drug
We then drew the inhibition curves classified by the five first-line chemotherapeutic drugs in order to compare sensitivity of the ten PC cell lines to a single drug visually (Figure 2). For some drugs, such as 5-FU and irinotecan, the IC50 values of the ten PC cell lines had little difference and the maximum was less than 100 times of the minimum. While for the drugs including gemcitabine, paclitaxel and cisplatin, the IC50 values of cell lines varied much. PC cell lines were generally sensitive to gemcitabine and paclitaxel, as the maximal IC50 value was less than 20μM and several of the cell lines were nearly totally sensitive to the two drugs in vitro, correlating with the fact that the two drugs could be combined into an effective and widely used regimen. Eight of the ten PC cell lines were extremely resistant to cisplatin, yet MIA PaCa-2 and AsPC-1 were sensitive to it. In this regard, genetic variants such as BRCA1/2 mutation have confirmed that PC is a conglomerate of multiple subtypes. Indeed, the alternations of BRCA1/2 have been noted to increase sensitivity to platinum-based chemotherapy in breast, ovarian cancer and PC [7-11].
IC50 values were summarized in the form of heatmap (Figure 3A). SW1990 was the most resistant PC cell line as its IC50 values for three drugs, gemcitabine, 5-FU and paclitaxel ranked top three. MIA PaCa-2 and BxPC-3 were the most sensitive PC cell lines. Regimen based on gemcitabine and regimen based on 5-FU are the two main chemotherapeutic regimens for PC. Out of this importance, we found that resistant PC cell lines to gemcitabine were SW1990, AsPC-1 and T3M4, while the sensitive cell lines were CFPAC, BxPC-3, HPAFII and Patu-8988 (Figure 3B). As for 5-FU, Patu-8988, SW1990 and PANC-1 were resistance to it and CFPAC and MIA PaCa-2 were sensitive.
Identification and validation of DEGs in drug sensitive and resistant PC cell lines
In order to explore genes associated with drug resistance in PC, we compared the transcriptome data acquired from the CCLE database of the resistant and sensitive cell lines of each chemotherapeutic drug (Figure 4A, Supplementary Figure 1). DEGs associated with cisplatin sensitivity had the largest amount of 642 genes, in which 181 genes were up-regulated in the cisplatin resistant cell lines and 461 genes were down-regulated. The least DEGs were associated with gemcitabine sensitivity, in which 37 genes expressed higher in gemcitabine resistant PC cell lines and 25 genes expressed lower.
Treatment strategy of PC tends towards combined regimen rather than a single drug. Gemcitabine and paclitaxel combined regimen is a widely applied first-line chemotherapeutic regimen recommended by the National Comprehensive Cancer Network (NCCN) guideline. Current clinical chemotherapy for PC is mostly based on 5-FU and gemcitabine[12, 13]. Hence, to identify genes that cause chemoresistance in multidrug combination regimens, DEGs for the five drugs were intersected using a Venn diagram (Figure 4B, Supplementary Table 1). The results show that several genes that have been shown to cause chemoresistance in tumors may also play an important role in multidrug combination chemotherapy for PC.
Several multidrug resistance associated genes were confirmed by qRT-PCR. We stimulated MIA PaCa-2 and BxPC-3 PC cell lines with 5-FU and gemcitabine, respectively, and observed a time-dependent increase in the expression of drug resistance associated genes, including TMEM178B, ANPEP, DNALI1, TFPI2, UCP2, GATA5, VSTM1, FAM196B, DZIP1, DNER and RGS5 (Fig 5A-B). Furthermore, some of the DEGs were also verified in PC cell lines (Fig 5C-D). Some of the validated DEGs have been studied in tumor drug resistance. The transcription factors in GATA family have been reported to regulate tumor development. TCR signaling activates a signaling axis that includes ITK, NF-κB, and GATA-3 and promotes chemotherapy resistance in non-Hodgkin lymphomas [14]. Uncoupling protein 2 (UCP2) promotes proliferation and chemoresistance via NF-κB/β-catenin axis in gallbladder cancer[15].
Enrichment analysis reveals extracellular matrix and cell-cell junction matters in PC chemoresistance
GO and KEGG pathway enrichment analyses were applied to discover the functions of the DEGs between drug sensitive and resistance PC cell lines (Fig 6A, Supplementary Table 2). The DEGs were significantly enriched in biological processes associated with extracellular matrix and cell adhesion. Previous studies have proposed multiple mechanisms of drug resistance in PC, including abnormal gene expression, mutations, dysregulation of key signaling pathways (such as NF-kB, Akt and apoptosis-related pathways), EMT and the role of extracellular stromal cells and cancer stem cells[16]. This finding is consistent with the accepted mechanism of drug resistance in PC. KEGG pathway analysis revealed that DEGs are mainly involved in extracellular matrix, external encapsulating structure, collagen-containing extracellular matrix, cell junction organization, cell-cell adhesion via plasma-membrane and cell junction assembly extracellular matrix structural constituent. In addition, pathways related to synaptic structure and calcium binding are also enriched (Fig 6B).