Biological characteristics of gene expression features in pancreatic cancer cells induced by proton and X-ray irradiation

Background: Radiation therapy is an important alternative treatment for advanced cancer. The aim of the current study was to disclose distinct alterations of the biological characteristics of gene expression features in pancreatic cancer cells, MIAPaCa-2, following proton and X-ray irradiation. Materials and methods: Using cDNA microarray, we examined the gene expression alterations of MIAPaCa-2 cells following proton or X-ray irradiation. We also isolated the surviving MIAPaCa-2 cells after irradiation and analyzed their gene expression profiles. Results: Although the cytocidal effects of both types of irradiation were similar at sufficient doses in vitro and in vivo, the affected gene expression profile alterations of MIAPaCa-2 cells irradiated with protons were distinct from those irradiated with X-ray. Interestingly, clustering analysis of gene expression of the surviving MIAPaCa-2 cells was also completely discernible between the two types of irradiation. However, a similar cytocidal effect was still observed in the proton- and X-ray-irradiated surviving cells after re-irradiation, commonly showing biological effects related to apoptosis and cell cycle processes. Conclusions: Proton irradiation treatment for pancreatic cancer provides the distinct biological effect of steady gene expression alterations compared to X-ray irradiation; however, surviving cells from both types of irradiation were still susceptible to the cytocidal effects induced by proton re-irradiation treatment.

Keywords: Proton; X-ray; irradiation; gene expression profile; pancreatic cancer

Cancer is the leading cause of death in the developed countries (Soerjomataram et al. [20]). The incidence of pancreatic cancer (PC) is increasing, and the prognosis of patients with PC is extremely poor, with a 5-year survival rate of less than 10% (Vincent et al. [25]). Surgical treatment is the most effective and only radical treatment when no local invasive or remote lesion is found. Diagnosis of PC in the early stage is extremely rare; ∼80% of PC are diagnosed at stage IV (Paulson et al. [14]), leading to no indication for surgical treatment. As such, chemotherapy is currently only used as an alternative; however, it cannot achieve a radical cure to all viable PC cells, and the clinical outcome is mainly a partial response or stable disease at best (Reni et al. [16]; Suker et al. [21]), leaving a fraction of viable cancer cells, that rapidly recur and grow, leading to a lethal prognosis.

Chemotherapy agents are based on DNA damage, caused by miscellaneous functions that vary among drug (Swift and Golsteyn [22]): including alkylating agents, platinum drugs, antimetabolites, and topoisomerase inhibitors. Alternatively, radiation therapy (RT) is unique (Withers [26]); the mechanism of its cytocidal effect differs from those of anti-cancer drugs (Goldstein and Kastan [3]), and involves DNA damage, which occurs directly by breaking double strands and indirectly by producing reactive oxygen species (Swift and Golsteyn [22]). Several types of irradiation are available, such as X-ray, gamma-ray, and proton (Aoki-Nakano et al. [1]). Each irradiation type differs in terms of its radiological and physical characteristics (Marx [10]). X-ray treatment has long been used as an alternative for cancer treatment (Rosenman et al. [17]). X-ray therapy is based on the electromagnetic interaction through the passage. In contrast, proton therapy is based on a dose concentration at a range of protons and is an alternative to X-ray treatment. The unique feature of proton cancer irradiation therapy is the Bragg peak (Cuaron et al. [2]), which generates the most energy loss when the proton particle stops at the site of target tissues, avoiding damage to the normal tissues. Thus, localized accumulated cytocidal effects induced by proton radiation with efficient delivery of a relatively high radiation-effective dose is considered more beneficial than X-ray. While the attractive biological features of proton RT are intriguing, the different biological responses between proton and X-ray radiation have not been fully elucidated.

In this study, we aimed to elucidate the biological characteristics of gene expression features provided by proton and X-ray irradiation on the PC cell line, MIAPaCa-2. Although proton and X-ray irradiation delivered to MIAPaCa-2 cells showed similar cytocidal and tumoricidal effects, the altered gene expression profile (GEP) of irradiated MIAPaCa-2 cells was apparently different. We also found that the GEP of surviving cells after proton irradiation was distinct from that of surviving cells after X-ray irradiation. Despite the distinct difference in GEPs of surviving cells between proton and X-ray irradiation, a similar cytocidal effect was observed following proton re-irradiation, revealing alterations in apoptosis and cell cycle-related processes in GEPs.

Human PC cell line, MIAPaCa-2 cells (ATCC, Manassas, VA, USA), were cultured in Dulbecco's modified Eagle's medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco).

MIAPaCa-2 cells were irradiated by proton or X-ray at various doses. To generate proton irradiation, 200 MeV proton was applied, delivered from a synchrotron accelerator at the Wakasa Wan Energy Research Center (WERC). The accelerator complex has been described elsewhere (Aoki-Nakano et al. [1]; Tomita et al. [24]). X-ray irradiation was performed using a device for use with small animals (MBR-1520R-3, HITACHI, Tokyo, Japan). The culture plates with cells were located on the place where the designated physical dose was irradiated in the equipment. We re-irradiated the proton- or X-ray irradiated surviving MIAPaCa-2 cells with 8 Gy (physical dose) proton: since these cells were surviving cells after 8 Gy irradiation.

For irradiation, 100, 200, and 400 cells were plated onto 6 well culture dishes. After 7 to 17 days, the number of colonies was counted, and the ratio of the number of colonies to the number of initially disseminated cells was calculated. The relative biological effectiveness (RBE) of proton versus X-ray was calculated using equation (1), where D(X) stands for the X-ray dose that reduced the number of surviving cells by 10% compared to non-irradiated cells, and D(p) stand for the proton dose with the same cytocidal effect.

(1)

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MIAPaCa-2 cells were irradiated with 8 Gy of X-ray or protons. One hour later, 2 × 103 harvested cells were seeded onto a 96-well plate (Falcon, Franklin Lakes, NJ, USA), and cell proliferation was assessed after 24 h, 48 h, 96 h, and 144 h using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), followed by color absorbance on a microplate reader.

RNA was isolated from cells using RNA isolation kit (RNeasy Mini Kit, Qiagen, Heidelberg, Germany). Quantity and quality of the obtained RNA were assessed using NanoDrop 2000c spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Gene expression analysis was performed by cDNA microarray (one-color) as previously described (Komura et al. [6]). One sample per the designated experimental sample was analyzed for cDNA microarray. Briefly, isolated RNA was amplified and labeled with Cy3 using the Quick Amp Labeling Kit (Agilent Technologies, Palo Alto, CA, USA). The prepared target pacomplementary RNA was hybridized to the cDNA microarray plate using the Whole Human Genome Microarray Kit, 4x44K (Agilent Technologies). Analysis of the obtained gene expression data was performed using the BRB array tool (NCI, http://linus.nci.nih.gov/BRB-ArrayTools.html) for unsupervised clustering and class comparison. The normalization methods, either quantile normalization or the relative to reference array which was median array in the experiment, were used in accordance with the BRB-Array tools version 4.5.1. The intensity filter threshold was set at a minimum value of 10. Replicate spots within an array were averaged. Genes were excluded if missing values exceeded 50% between arrays and if a minimum fold change was not met (<20% of expression data values having an at least a 1.5-fold change in either direction from the gene's median value). Multiple probe sets were reduced to one per gene symbol by using the most variable probe set across arrays, measured by the interquartile range. Significant genes whose expression was altered by class comparison were analyzed for their relevant biological processes by MetaCore software suite (GeneGo, Carlsbad, CA, USA) as previously described (Komura et al. [6]). The obtained gene expression data were deposited into GEO (http://www.ncbi.nlm.nih.gov/geo/) as GSE107444 (comprised of GSE107440, GSE107441, GSE10742, and GSE 10743).

Total RNA was isolated from cells using the microRNA Isolation kit (Stratagene, San Diego, CA, USA) and reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. Real-time detection PCR (RTD-PCR) was performed using proprietary TaqMan gene expression probes (Applied Biosystems). Data were collected and analyzed with Rotor-gene Q (QIAGEN). Relative gene expression levels were calculated with the 2−△△Ct method using GAPDH as the control gene.

BALB/c athymic mice (Charles River Laboratories Japan, Inc., Yokohama, Japan) were implanted on the footpad with 5 × 106 MIAPaCa-2 cells. After 10 days, when tumors with a 3–4 mm diameter were visible, proton or X-ray was irradiated to the tumor at doses of 5 Gy or 10 Gy with the use of a lead plate shield to avoid the other organs. The longest and shortest diameters of the tumors were measured, and the tumor volume was calculated by equation (2) (Sakai, Kaneko, Nakamoto et al. [18], Sakai, Kaneko, Sato et al. [19]), where Vt stands for tumor volume (mm3), Ds is the shortest diameter (mm), and Dl is the longest diameter (mm).

(2)

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Animal experiments were approved by our institutional animal experiment review board (code # AP-081183).

MIaPaCa-2 cells were irradiated with protons or X-ray at a dose of 8 Gy. After 7 to 17 days, colonies of surviving cells were picked and expanded for use in subsequent experiments.

Student's t-test was performed using SPSS statistics 23 (IBM, Armonk, NY, USA). Statistical significance was determined as p < .05.

MIAPaCa-2 cells were irradiated by proton or X-ray up to 8 Gy in vitro, and the number of surviving colonies was counted for 7 to 17 days. Proton and X-ray irradiation showed almost equivalent cytocidal effects (Figure 1). The RBE of proton irradiation was 1.3 compared to X-ray, which was similar to the generally recognized value 1.1 (Paganetti et al. [13]).

Graph: Figure 1. Colony assay of the irradiated MIAPaCa-2 cells. MIAPaCa-2 cells were irradiated with doses of 1, 2, 4, 6, and 8 Gy. Irradiated cells (100, 200, and 400 cells) were plated onto a 6 well culture dish. After 7 to 17 days, the number of colonies formed was counted, and the ratio of colonies formed to the number of cells without irradiation was calculated. Open circle: X-ray irradiation, Open quadrant: proton irradiation. Bars: standard error.

Cell proliferation capability was assessed by the MTS assay. The suppression of MIAPaCa cells irradiated with either over 4 Gy X-ray or proton was observed after 96 h, indicating that rapid and sufficient suppression of cell proliferation could be achieved by over 4 Gy radiation, of either X-ray or proton, after 3 days (Figure 2).

Graph: Figure 2. MTS assay of the irradiated MIAPaCa-2 cells. MIAPaCa-2 cells were irradiated with various doses of X-rays (a) or protons (b). One hour after irradiation, 2 × 103 cells were seeded onto a 96-well plate, subjected to the MTS assay after 24 h, 48 h, 96 h, and 144 h. Physical irradiation doses are expressed and the proton RBE was 1.3. Bars: standard error.

We also examined the therapeutic effect of proton and X-ray irradiation on the implanted MIAPaCa-2 tumor on murine foot pads. The suppressive effect of the implanted MIAPaCa-2 cells on tumor formation and growth in vivo was also similar when they were irradiated by either protons or X-ray (Figure 3).

Graph: Figure 3. Anti-tumor effect of irradiation on the implanted MIAPaCa-2 tumor in vivo. MIAPaCa-2 cells (5 × 106) were implanted on the footpad of BALB/c athymic mice. Ten days later, proton (a) or X-ray (b) irradiation was delivered to the irradiated to the implanted tumors, and the resulting tumor size was measured. Rhombus: no irradiation, quadrant: 5 Gy, triangle: 10 Gy. Values are expressed as physical doses, and the proton RBE was 1.3.

We next examined how the gene expression of MIAPaCa-2 cells was affected by proton and X-ray irradiation using cDNA microarray analysis (GEO accession no. GSE107440). We obtained RNA from the cells 3 h or 12 h after irradiation. By unsupervised clustering analysis using 3623 genes that passed expression quality, we found two distinct, clear clusters representing proton and X-ray irradiation that formed, irrespective of the time elapsed after irradiation and the dose (Figure 4). By class comparison analysis of the proton- and X-ray- irradiated cells, we found that 1472 genes were differentially expressed at p < .01 (Supplementary Table 1). Regarding these 1472 genes, 676 were up-regulated by proton irradiation, and 796 were up-regulated by X-ray irradiation. Biological process analysis of the up-regulated genes in MIAPaCa-2 cells irradiated by protons or X-ray was distinct; the biological processes related to the 676 genes up-regulated by proton irradiation compared to X-ray irradiation included cell cycle regulation, epithelial-mesenchymal transition, chromatin modification and blood vessel morphogenesis, as well as platelet aggregation, implying the biological potential to affect surrounding tissue (Supplementary Table 2; Supplementary Figure 1). Significant alteration in the expression of randomly selected genes (CCR3 (C-C chemokine receptor type 3), ID2 (Inhibitor of DNA binding 2), PLXNA2 (Plexin A2), and CXCR4 (C-X-C chemokine receptor type 4)) were confirmed by RTD-PCR (Figure 5). The biological process of the 796 genes that were up-regulated by X-ray irradiation compared to proton irradiation involved extra-and intracellular components, cadherins, actin filaments, and developmental-related processes, such as WNT and NOTCH signaling (Supplementary Table 3; Supplementary Figure 2). Significant alterations in the expression of randomly selected genes (HEYL (Hes related family BHLH transcription factor with YHRW motif-like), CDH12 (Cadherin 12), PLAUR (Plasminogen activator, urokinase receptor), and STAT5A (Signal transducer and activator of transcription 5A)) were confirmed by RTD-PCR (Figure 6).

Graph: Figure 4. Unsupervised clustering analysis of the gene expression of MIAPaCa-2 cells irradiated with protons or X-ray. MIAPaCa-2 cells were irradiated with protons or X-ray at 2 Gy or 8 Gy. After 3 h or 12 h of irradiation, RNA was isolated and the GEP was examined by the cDNA microarray, followed by unsupervised clustering analysis. Physical irradiation doses are expressed and the proton RBE was 1.3.

Graph: Figure 5. RTD-PCR analysis of genes whose expression was up-regulated in MIAPaCa-2 cells irradiated with protons compared to X-rays. Cells were irradiated with protons or X-rays at doses of 2 or 8 Gy. After 3 or 12 h, the cells were harvested and the RNA was extracted, followed by RTD-PCR analysis for the indicated genes. Four samples of each type were studied. The y-axis, in logarithmic scale, shows relative gene expression levels normalized to that of GAPDH. p values were calculated using Student's t-test. Bars: standard deviations.

Graph: Figure 6. RTD-PCR analysis of genes whose expression was up-regulated in MIAPaCa-2 cells irradiated with X-rays compared to protons. Cells were irradiated with protons or X-rays at doses of 2 or 8 Gy. After 3 or 12 h, the cells were harvested and the RNA was extracted, followed by RTD-PCR analysis for the indicated genes. Four samples of each type were studied. The y-axis, in logarithmic scale, shows relative gene expression levels normalized to that of GAPDH. p values were calculated using Student's t-test. Bars: standard deviations.

These gene expression analysis data suggest that the irradiation of MIAPaCa-2 PC cells exerts different biological effects depending on the type of irradiation, despite similar cytocidal effects being obtained following both types of irradiation.

The cytocidal effect of irradiation is not sufficiently lethal to cancer cells, leaving residual viable cancer cells. In this context, we assessed the consequent gene expression features of MIAPaCa-2 cells that survived proton or X-ray irradiation. We isolated the surviving colonies of MIAPaCa-2 cells irradiated with protons (n = 5) or X-ray (n = 4), and expanded them for GEP analysis by cDNA microarray (GEO accession no. GSE107441). By clustering analysis of gene expression of the surviving cells with all 6991 genes whose expression passed the quality threshold, two clusters formed that discerned the proton-irradiated surviving cells from the X-ray-irradiated surviving cells (Figure 7). Expression of the 901 genes was significantly different (p < .01) between proton- and X-ray-irradiated surviving cells (Supplementary Table 4). Regarding the 490 up-regulated genes in proton-irradiated surviving cells, the relevant biological processes were development, blood vessel and angiogenesis processes, NOTCH signaling, and IL-2 inflammatory processes (Supplementary Table 5; Supplementary Figure 3). Significant alterations in the expression of randomly selected genes (FZD4 (Fizzled class receptor 4), STAT5A, FGF1 (Fibroblast growth factor 1), MMP19 (Matrix metalloproteinase 19), and TGM2 (Transglutaminase 2)) were confirmed by RTD-PCR (Figure 8). The 411 up-regulated genes in X-ray irradiated surviving cells were also similarly related to blood coagulation, and developmental-related processes, including WNT signaling. Significant alterations in the expression of randomly selected genes (MMP1 (Matrix metalloproteinase 1), CD86 (CTLA-4 counter-receptor B7.2), NANOG, PLCB1 (Phospholipase C beta 1), and AKAP12 (A-kinase anchoring protein 12)) were confirmed by RTD-PCR (Figure 9). In addition to these similar biological features, the characteristic features of biological processes of the up-regulated genes in X-ray irradiated surviving cells were antigen presentation, involving MHC class I, II and the costimulatory molecule CD86, and leukocyte chemotaxis, involving CX3CR1 (C-X3-C Motif Chemokine Receptor 1) and LFA-3 (CD58) (Supplementary Table 6; Supplementary Figure 4), indicating that X-ray-irradiated surviving cells are immunogenic.

Graph: Figure 7. Unsupervised clustering analysis of the gene expression of proton- and X-ray-irradiated surviving MIAPaCa-2 cells. MIAPaCa-2 cells were irradiated with protons or X-ray at a dose of 8 Gy in vitro. After 7 to 17 days, the colonies with surviving cells were picked and expanded. RNA was isolated from each expanded surviving cell, followed by gene expression analysis. Unsupervised clustering analysis of gene expression was performed. Physical irradiation doses are expressed and the proton RBE was 1.3. P-surviving cell: proton-irradiated surviving cell, X-surviving cell: X-ray-irradiated surviving cell.

Graph: Figure 8. RTD-PCR analysis of genes whose expression was up-regulated in MIAPaCa-2 cells that survived proton irradiation compared to cells that survived X-ray irradiation. Proton-irradiated surviving cell; n = 5, X-ray-irradiated surviving cell; n = 4. The y-axis, in logarithmic scale, shows relative gene expression levels normalized to that of GAPDH. p values were calculated using Student's t-test. Bars: standard deviations.

Graph: Figure 9. RTD-PCR analysis of genes whose expression was up-regulated in MIAPaCa-2 cells that survived X-ray irradiation compared to cells that survived proton irradiation. Proton-irradiated surviving cells; n = 5, X-ray-irradiated surviving cells; n = 4. The y-axis, in logarithmic scale, shows relative gene expression levels normalized to that of GAPDH. p values were calculated using Student's t-test. Bars: standard deviations.

We next assessed how proton re-irradiation affected proton- and X-ray irradiated surviving cells. Since cells survived after 8 Gy X-ray irradiation or equivalent proton irradiation (8 Gy, RBE = 1.3), we re-irradiated cells with the same dose. Unsupervised clustering analysis of the GEP of proton irradiated surviving cells by proton re-irradiation, using filtered 9510 genes resulted in two complete clusters that discerned re-irradiated from non-re-irradiated cells (GEO accession no. GSE107442) (Figure 10(a)). In contrast, clustering analysis of the GEP using 13,672 filtered genes of X-ray-irradiated surviving MIAPaCa-2 cells that were re-irradiated by proton did not result in the formation of obvious clusters (GEO accession no. GSE107443) (Figure 10(b)), suggesting that the GEP of X-ray-irradiated surviving cells is relatively consistent, even after proton re-irradiation.

Graph: Figure 10. Unsupervised clustering analysis of the gene expression of proton- and X-ray-irradiated surviving cells re-irradiated with protons. Surviving MIAPaCa-2 cells after proton or X-ray were expanded. The expanded cells were re-irradiated by proton at a dose of 8 Gy. After 4 h, RNA was extracted, followed by gene expression analysis. Unsupervised clustering analysis of gene expression was performed. (a) Proton irradiated surviving cells that were re-irradiated with protons. (b) X-ray irradiated surviving cells that were re-irradiated with protons. Physical irradiation doses are expressed and the proton RBE was 1.3. P-surviving cell: proton-irradiated surviving cell, X-surviving cell: X-ray-irradiated surviving cell.

We next assessed how proton re-irradiation affects proton- or X-ray-irradiated surviving cells. The expression of 653 genes was significantly altered (p < .01) in proton-irradiated surviving cells that were re-irradiated by protons compared to those not re-irradiated (Supplementary Table 7). Significant alterations in the expression of randomly selected genes (FOS (Fos proto-oncogene, AP-1 transcription factor subunit), TNF (Tumor necrosis factor), EGR1 (Early growth response 1), FGFR1 (Fibroblast growth factor receptor 1), and POU5F1 (POU class 5 homeobox 1) were confirmed by RTD-PCR (Figure 11). The biological processes of these 653 genes were indicative of apoptosis-related processes, as well as cell cycle regulation, inflammation, and development (Supplementary Table 8; Supplementary Figure 5). We also found that the expression of 323 genes was significantly altered (p < .05) in X-ray irradiated surviving cells that were re-irradiated by protons compared to those not re-irradiated (Supplementary Table 9). Significant alterations in the expression of randomly selected genes (FOS, TNF, EGR1, JUN (Jun proto-oncogene, AP-1 transcription factor subunit), SOCS1 (Suppressor of cytokine signaling 1), and CXCL8 (C-X-C motif chemokine ligand 8)) were confirmed by RTD-PCR (Figure 12). The biological processes related to these genes were similarly indicative of apoptosis, cell cycle regulation, inflammation, and development (Supplementary Table 10; Supplementary Figure 6).

Graph: Figure 11. RTD-PCR analysis of genes whose expression was significantly affected in proton-irradiated surviving MIAPaCa-2 cells which were subjected to proton re-irradiation. Physical re-irradiation dose: 8 Gy, the proton RBE was 1.3. Biological replicates; n = 5. The y-axis, in logarithmic scale, shows relative gene expression levels normalized to that of GAPDH. p values were calculated using Student's t-test. Bars: standard deviations.

Graph: Figure 12. RTD-PCR analysis of genes whose expression was significantly affected in X-ray-irradiated surviving MIAPaCa-2 cells which were subjected to re-irradiated with protons. Physical re-irradiation dose: 8 Gy, the proton RBE was 1.3. Biological replicates; n = 4. The y-axis, in logarithmic scale, shows relative gene expression levels normalized to that of GAPDH. p values were calculated using Student's t-test. Bars: standard deviations.

To confirm the cytocidal effect of proton re-irradiation of proton- or X-ray-irradiated surviving cells, we performed a colony assay in vitro to assess surviving cells for 14 days after proton re-irradiation. Three out of four X-ray irradiated surviving cells were completely susceptible to the cytocidal effect by proton (8 Gy) re-irradiation. A cytocidal effect was also observed in all surviving cells following proton re-irradiation (Figure 13). Thus, after the initial irradiation, surviving cells were still susceptible to the cytocidal effect of proton re-irradiation, regardless of the initial irradiation type (proton or X-ray).

Graph: Figure 13. Colony assay of proton and x-ray irradiated surviving MIAPaCa-2 cells that were re-irradiated with protons. Proton- and X-ray irradiated surviving MIAPaCa-2 cells (n = 4, n = 5, respectively) were re-irradiated with doses of 2, 4, 6, and 8 Gy of protons. Irradiated cells (100, 200, and 400 cells) were plated onto a 6-well culture dish. After 7 to 17 days, the number of colonies formed was counted, and the ratio of colonies formed to the number of cells without irradiation was calculated. Open symbol: X-ray-irradiated surviving cells, Filled symbol: proton-irradiated surviving cells. Physical irradiation doses are expressed and the proton RBE was 1.3.

In this study, we assessed the biological effects of proton and X-ray therapy on MIAPaCa-2 PC cells. Although the cytocidal effects of proton and X-ray irradiation were similar, the gene expression alterations were distinct. We also isolated and expanded the surviving MIAPaCa-2 cells after irradiation. Expression feature analysis showed that the GEP was distinct between proton- and X-ray-irradiated surviving cells. However, the cytocidal effect was similar in both types of surviving cell following proton re-irradiation, implicating apoptosis and cell cycle processes in the altered GEPs.

The response to the initial irradiation (proton or X-ray) was distinct: the response by protons of cancer cells mainly involved cell cycle regulation, epithelial-mesenchymal transition, chromatin modification, blood vessel morphogenesis, and platelet aggregation, implying its potential to affect the cancer microenvironment. The biological response induced by X-ray involved extracellular and intracellular components, cadherin, actin filaments, and developmental-related processes, such as WNT and NOTCH signaling. The reasons for this difference are not clear, and the consequences of these effects should be further investigated, especially in the context of clinical cancer treatment benefits (Levin et al. [8]).

One intriguing finding from this study was that GEPs of proton- and X-ray-irradiated surviving cells showed both similarities and differences. The similarities included blood vessel, coagulation, NOTCH and WNT-related developmental processes. These processes are suggestive of the survival of cancer cells escaping from irradiation damage. While these similar processes were observed, a difference was found in the context of immunological processes: the proton-irradiated surviving cells indicated IL-2-related processes, which are indicative of a T cell immune response, whereas the X-ray-irradiated surviving cells indicated an antigen presentation immune response involving MHC classes I and II, as well as costimulatory molecules, suggesting an antigenic immune response. Since MIAPaCa-2 is a human PC cell, this unique response should be assessed in other syngeneic cancer models. The detailed molecular mechanism by which these stable biological features of surviving cells occur after single high-dose irradiation by protons and X-rays has yet to be investigated.

In the animal experiment, we used footpad tumor models and single high-dose radiation was applied. In clinical practice for RT, the standard protocol is 25 to 28 times with 2 Gy-equivalent (GyE) doses or 33 times with 1.8 GyE for proton radiation, and 25 to 28 times with 1.8 to 2 Gy for X-ray irradiation (Nichols et al. [12]; Nichols et al. [11]; Terashima et al. [23]). Considering the Bragg peak feature of proton radiation, an increased dose may be necessary, although adverse effects in adjacent organs (such as the duodenum and stomach in cases of PC) should be considered. In these aspects, the dose and frequency should be further investigated to achieve the optimal therapeutic effect and sufficient safety of proton RT for PC, since DNA double-strand breakage induced by ionizing radiation not only leads to cell death, but may also cause a carcinogenic effect (Little et al. [9]) in addition to these clinical anatomical issues.

Cancer treatment mainly relies on anti-cancer drugs (Reni et al. [16]; Suker et al. [21]), when surgical treatment is not curatively or palliatively conducted, leaving a residual viable cancer lesion. Therefore, alternative treatments should be explored to distinguish remaining cancer. In this respect, RT would be a unique alternative approach not only because of its cytocidal effects, achieved by inducing apoptosis, but also because of its abscopal effect, as reported in some cancers (Postow et al. [15]). We examined the biological effects of proton and X-ray irradiation on MIAPaCa-2 cells, a human PC cell line. PC is the most lethal malignancy (Kamisawa et al. [5]); the natural overall survival is extremely poor. Currently, successful surgical eradicative treatment is the only way to provide a curative effect in PC (Hartwig et al. [4]): Otherwise, remaining therapies including chemotherapy (e.g., the gemcitabine-based anti-cancer drug and the emerging chemotherapeutic regime, FOLFIRINOX (Lee and Park [7])) are still limited in the context of therapeutic efficacy, not achieving complete remission of PC. Thus, a novel treatment including irradiation should be explored as an alternative therapy to improve the prognosis of PC patients.

The biological response induced by proton irradiation in MIAPaCa-2 PC cells was distinct from that induced by X-ray irradiation, and proton-irradiated surviving cells were different from X-ray-irradiated surviving cells in the context of their resultant steady GEPs after the initial irradiation. However, the altered GEPs in proton- or X-ray-irradiated surviving cells are not directly related to the susceptibility to re-irradiation treatment, implying a complex mechanism involving biological alterations, including of the GEP, in cancer cells. Further analysis is needed to elucidate the biological effects of irradiation on cancer cells, which will provide important details regarding the effect of irradiation on cancer and increase the opportunities for cancer treatment to improve prognosis.

We would like to thank Ms. Sachie Yamazaki for her excellent technical assistance.

No potential conflict of interest was reported by the authors.

Haruo Fujinaga , MD, is awaiting conferral of a PhD in Medical Sciences from Kanazawa University. His research was developed within the department of Disease control and homeostasis, Kanazawa University, Graduate school of medical sciences, and focusses on radio- and proton-therapies of tumors of the gastrointestinal tract.

Associate Professor Yoshio Sakai , MD, PhD., is a gastroenterologist working in the Department of Gastrroenterology, Kanazawa University.

Associate Professor Tatsuya Yamashita , MD, PhD, is working in the Department of Gastroenterology, Kanazawa University Hospital, focussing his research in hepatology research. He worked at Global Hepatitis Programme in WHO Headquarters as first secondee from Kanazawa University. He contributed to parts of algorithm and monitoring for drug toxicity in WHO HBV guidelines.

Associate Professor Kuniaki Arai , MD, PhD, is working in the Department of Gastroenterology, Kanazawa University Hospital, focussing his research on the treatments of hepatocellular carcinoma with radiofrequency ablation (RFA) and chemotherapy.

Associate Professor Takeshi Terashima , MD, PhD, is working in the Department of Gastroenterology, Kanazawa University Hospital, focussing his research in hepatology research.

Takuya Komura , MD, PhD, is working in the department of System Biology, Kanazawa University. His expertise is in gastroenterology, infectious diseases and surgery.

Akihiro Seki , MD, PhD, is a Post-Doctoral Fellow studying the role of the microbiome in affecting dendritic cell function. He started his clinical practice as gastroenterologist, and his basic science career at the Graduate School of Medical Science, Kanazawa University, Japan. He obtained his PhD in Medical Science at the Kanazawa University.

Assistant Professor Kazunori Kawaguchi , MD, PhD, is working as physician and researcher in the Department of Gastroenterology, Kanazawa University Hospital, focussing on viral hepatitis and liver regeneration therapies. He worked as Research Fellow in the National Institute of Allergy and Infectious Diseases (NIAID).

Alessandro Nasti , MPharm, PhD, is working as Postdoctoral Researcher in the in the department of System Biology, Kanazawa University. His basic research is focussed on immunotherapies for the diseases of the gastrointestinal tract.

Keiko Yoshida , MSc, is enrolled as PhD student in the department of Disease control and homeostasis, Kanazawa University. Her basic research is focussed on the development of new chemoimmunotherapies for pancreatic ductal adenocarcinomas.

Professor Takashi Wada , MD, PhD, is Head of Department of Nephrology and Laboratory Medicine, Kanazawa University Hospital. He specializes in Nephrology, Laboratory Medicine and Internal Medicine with a focus in Nephrology Clinical immunology, therapeutic application of novel anti-inflammatory drugs and development of novel laboratory medicine and biomarkers.

Kazutaka Yamamoto , MD, PhD, is a radiation oncologist as well as a basic radiologist. He worked for The Wakasa Wan Energy Research Center, Tsuruga, Japan, as a leading researcher of particle therapy in addition to his former carrier in PET diagnosis of nuclear medicine.

Kyo Kume , PhD, is a chief researcher as well as a medical physicist at the Proton Medical Researcher Division, The Wakasa Wan Energy Research Center, Tsuruga, Japan, focusing on radiation and medical physics, especially in the field of particle therapy and beam application engineering.

Takashi Hasegawa is a research member of The Wakasa Wan Energy Research Canter, Tsuruga, Japan, focusing on accelerator engineering.

Takushi Takata , PhD, was a research member of The Wakasa Wan Energy Research Center, Fukui, Japan, focusing on particle therapy in medical physics.

Professor Masao Honda , MD, PhD is affiliated to the Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University. He specializes in Liver Disease Gastroenterology, Collagenous pathology/Allergology, Virology with a focus on viral hepatitis and the molecular biology of hepatocellular carcinoma.

Professor Shuichi Kaneko , MD, PhD, is affiliated to the Faculty of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University. He specializes in Experimental Pathology, Gastroenterology, Virology, and Cancer, with a focus on Cancer Research, Liver metabolism and systemic illness, and Viral Hepatitis.