Introduction

Pancreatic cancer remains one of the most life-threatening malignancies in the world. Statistics by the American Cancer Society shows that 53,670 new cases of pancreatic cancer were diagnosed in 2017 and 43,090 will die from the disease [1]. Most pancreatic cancers do not present early specific sign and symptoms and are usually diagnosed at the very advanced stage with distant metastasis to other organs. Consequently, the majority of patients diagnosed with pancreatic cancers are unresectable [2]. Gemcitabine-based chemotherapy has been accepted as the first line of treatment for the patients with advanced pancreatic cancers [3]. However, the current work revealed that gemcitabine therapy caused different efficacy [4, 5], so it is highly desirable to develop a reliable technique to evaluate the early response to chemotherapy, which is essential for optimizing the regimen.

Nowadays, the development of advanced imaging technique has been emerging as the most widely used non-invasive technique for evaluating the response of tumors to therapies [6,7,8,9]. Diffusion-weighted (DW) imaging modality is sensitive to the random Brownian motion of free water molecules and is quantified by the calculation of apparent diffusion coefficients (ADCs). It has been increasingly used for assessing the therapeutic effect of treatments in tumors [10, 11]. However, ADC values are influenced by both tissue diffusivity and pseudorandom motion caused by microcapillary perfusion, also known as pseudodiffusion [12].

The intravoxel incoherent motion (IVIM) model, separate measurement of the perfusion-related parameters at low b values (pseudodiffusion coefficient Dfast and the pseudodiffusion factor fp) and the pure molecular-based diffusion coefficient Dslow at b values higher than 100 s/mm2 can also be obtained with biexponential fitting of the signal intensity versus b curve using multi-b DWI [13,14,15]. This advanced imaging technique has been shown to be useful for the evaluation of treatment response to nasopharyngeal carcinoma, hepatocellular carcinoma, breast cancer liver metastases, and others [16,17,18]

The main mechanism of gemcitabine is to inhibit cell proliferation and promote apoptosis in pancreatic cancer cells [19]. Based on this mechanism, the number of tumor cells can be reduced and the diffusion of water molecules can be accelerated so there would be anticipated change in the diffusion-related coefficient compared to perfusion-related coefficient when using gemcitabine. In this study, we used diffusion-weighted imaging (DWI) and intravoxel incoherent motion DWI (IVIM-DWI) to assess the chemotherapy response of pancreatic cancer in an orthotopic mouse model to prove our hypotheses.

Materials and methods

Animals and animal model with orthotopic pancreatic cancer

All the experimental protocols were approved by the Ethics Committee of Shanghai Jiao Tong University (Approval no. 201801013) and were conducted in strict accordance with the Guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals. Twenty-four male BALB/C nu/nu mice (5 weeks old, 15–17 g body weight) were used for creating orthotopic pancreatic cancer model.

The human pancreatic adenocarcinoma cell line SW1990 was purchased from American Type Culture Collection (ATCC). The mice were anesthetized with 100 µl of 2% pentobarbital sodium injected into the abdominal cavity. Then the abdominal cavity was opened by a 5–10-mm transverse incision on the left flank. The tail of the pancreas was exposed through this incision. Tumor cells (2 × 106) were inoculated in the joint portion between the body and tail of the pancreas.

DW MRI and IVIM DWI of the tumors

Twenty-four mice with pancreatic cancers were randomly allocated to two groups after 1 week of inoculation. Twelve mice were treated with gemcitabine and twelve mice were treated with saline as a control. Gemcitabine was intravenously administrated to the mice at a dose of 50 mg/kg once a week for 3 weeks and then 1 week interval without chemotherapy. MRI was performed 1 day prior to the administration of the first dose of gemcitabine (Day − 1), 1 day after the first dose of gemcitabine (Day 1) and 10 days after the initiation of the therapy (Day 10). MR imaging was performed on a 1.5-Tesla MR scanner (MAGNETOM Aera, SIEMENS Healthcare, Erlangen, Germany) with a 16-channel Hand/Wrist coil (SIEMENS Healthcare, Erlangen, Germany). Mice were anesthetized with intraperitoneal injection with 100 µl of 2% pentobarbital sodium, and to minimize the distortion artifact in echo planar images, mice were kept in 37.0 ± 0.2 °C water in a plastic box with only head outside of the water for breathing during the MR scanning.

First, transverse and coronal MR images of the upper abdomen were acquired, including T2-weighted fast spin echo images, and free-breathing routine DW echo planar images with three b values (b = 50, 400 and 800 s/mm2) for ADC measurement. Second, free-breathing multi-b DW MRI (b = 0, 25, 50, 80, 100, 300, 500, 800 s/mm2) for IVIM measurement were acquired. The routine 3-b DWI sequence has its own advantage of shorter scanning time and less motion sensitivity, while IVIM sequence acquires more b values, needs longer scanning time and might be more motion-sensitive. Using ADC generated from the subset b values of the IVIM sequence might underestimate the performance of the conventional ADC. Thus, we use two separated sequences to acquired ADC and IVIM. Details regarding all sequence parameters are summarized in Table 1.

Table 1 Parameters of the MR sequences
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MR imaging analysis

Tumor volume was calculated using the formula for ellipsoid tumors, V = L × W × D × (π/6), where L and W are the largest length and width measured on the coronal image, D represents the largest depth measured on the axial image [20].

ADC maps were generated from DW images at b = 50,400 and 800 s/mm2 automatically using the Syngo software (SIEMENS Healthcare, Erlangen, Germany), and IVIM-derived maps were generated using a prototype post-processing program integrated in Syngo software (SIEMENS Healthcare, Erlangen, Germany).

The ADC values were calculated by fitting the signal intensity to a mono-exponential model as: S(b) = S0 exp(− bADC), where S(b) is the signal intensity with a given b value and S0 is the signal intensity without diffusion weighting [21]. For IVIM-derived parameter maps, a biexponential model was used for the calculation of the molecular diffusion-related coefficient Dslow, pseudodiffusion coefficient Dfast, and the pseudodiffusion fraction fp as follows [22] :

$$S \, \left( b \right) \, = \, S_{0} \left( {f_{\text{p}} { \exp }\left( { - b \, \left( {D_{\text{fast}} + D_{\text{slow}} } \right)} \right) \, + \, \left( { 1- f_{\text{p}} } \right){ \exp }\left( { - bD_{\text{slow}} } \right)} \right)$$

To generate three parametric images (Dslow, Dfast and fp), the above biexponential model was fit using the following approach: initial estimation of Dslow using a reduced set of b values larger than a predetermined value (200 s/mm2) and then using the resulting Dslow as a fix parameter to fit the missing parameters similar to what was described in [22].

ROIs were manually drawn to encompass the entire tumor area on DW images with b = 800 s/mm2 by two image reviewers (an MRI physicist and an abdominal radiologist, with 7 and 5 years of experience in abdomen MR imaging, respectively). Then ROIs were copied to ADC maps and three IVIM-derived parametric maps. For each ROI, mean values were calculated by Syngo software on each parametric map.

Histology

The mice were euthanized for harvesting the tumor at day 28. Tumors were fixed in 10% buffered formalin and were cut in half before embedding in paraffin. The halves were processed separately, with the cut edge ultimately facing the microtome surface. Serial 4 μm-thick sections were cut on a rotary microtome. Sections were deparaffinized in xylene and washed in gradient alcohol.

For Ki67 and VEGF staining, the sections were incubated in 3% hydrogen peroxide (H2O2) for 15 min for endogenous peroxidase activity blockade and heated in an autoclave in citrate buffer (pH 6.0) for 10 min for antigen retrieval, followed by blocking non-specific antigen with 10% normal goat serum (NGS) for 1 h, then the sections were incubated with the primary antibody against CD31 and Ki67 (abcam, Abcam Hong Kong Ltd, China) overnight at 4 °C. HRP-linked anti-rabbit/mouse antibodies (EnViSion Detection Kit, Gene Tech company, China) were used as secondary antibodies,

For TUNEL staining, incubate tissue sections for 15 min at 37 °C with proteinase K working solution, followed by which add 50 μl TUNEL reaction mixture for 60 min at 37 °C in a humidified atmosphere in the dark, then the sections were incubated in a humidified chamber for 60 min at 37 °C with 50 μl Converter-POD.

All tissue sections were developed using diaminobenzidine (DAB) as the chromogen and counterstained with hematoxylin, dehydrated, and cover-slipped. Obvious vascular endothelial cell coloring marked with CD31 and nucleus coloring marked with Ki67 were determined positive. The counting method described by Mehta et al. [23] was adapted for evaluation of MVD and Ki67 expression. Apoptotic cell number was also calculated.

Statistical analysis

Quantitative variables were presented as mean ± SEM. We performed statistical analysis using the statistical software SPSS (SPSS version 16.0, IBM Corporation, New York, USA). the Kolmogorov–Smirnov test was used to determine if the data have a normal distribution. Normally distributed variables were analyzed using independent Student’s t test. For the data which were not normally distributed, the nonparametric Mann–Whitney sum rank test was used to compare the difference. For all statistical analyses, a P value less than 0.05 was considered to indicate a significant difference.

Results

MRI findings

The T2WI and representative parameter maps of orthotopic pancreatic tumor were successfully obtained with minimal motion artifact (Fig. 1). To study the size of tumors, high-resolution T2WI in the coronal and transverse planes were analyzed. Tumors could be reliably identified in all tumor-bearing animals. The growth pattern of the tumors resembled a spherical growth pattern, and the tumors showed a substantially uniform high-intensity signal. The results of the ADC and IVIM-related parameters for gemcitabine-treated group and saline-untreated group at day − 1, 1, 10 were shown in Table 2. As shown in Table 2 the values of ADC and Dslow in the entire tumor region were significantly increased in gemcitabine-treated group in contrast to saline-untreated group at day 1 (1.88 ± 0.34 × 10−3 s/mm2 vs 1.45 ± 0.16 × 10−3 s/mm2, P = 0.028, and 1.74 ± 0.29 × 10−3 s/mm2 vs 1.34 ± 0.26 × 10−3 s/mm2, P =0.030). No significant difference of pseudodiffusion coefficient Dfast and pseudodiffusion fraction fp was found between the gemcitabine-treated group and saline-untreated group (Fig. 2).

Fig. 1
figure 1

T2WI and parameter maps of one mouse. a Transversal T2WI, b coronal T2WI, tumor was indicated with black arrow. c ADC map, dDslow map, an ROI was drawn around entire tumor area at the mid-level

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Table 2 Mean (SD) of the diffusion parameters at − 1, 1, 10 day and P values between the two groups
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Fig. 2
figure 2

The effect of treatment on ADC (a), Dfast (b), Dslow (c), and fp (d) measured with DWI and IVIM. Statistical differences between groups  were indicated by asterisks above bars (*P< 0.05). G gemcitabine group, U saline-untreated group

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Effects of gemcitabine on orthotopic pancreatic tumor growth

Gemcitabine (Met-Gem) can effectively inhibit the growth of pancreatic tumor compared to the saline-untreated group (Fig. 3). When tumor was excised from sacrificed mice after 4 weeks, the tumor volumes in gemcitabine-treated group were significantly lower than those in saline-untreated group at day 28 (P = 0.0015). Tumor in control group showed liquefaction necrosis at day 10 after treatment, while being more significant in gemcitabine group (Fig. 4).

Fig. 3
figure 3

Effects of gemcitabine on orthotopic pancreatic tumor growth. a Tumor volume size throughout the treatment schedule. b Gross morphorology of tumor at endpoint after gemcitabine (G) or saline (U) dosed. Statistical differences between groups were indicated by asterisks above bars (*P<0.05). G gemcitabine group, U saline-untreated group

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Fig. 4
figure 4

T2WI of tumor in mice at day 10 (a, c) and day 28 (b, d) after therapy with gemcitabine. Axial T2WI (a, b), Coronal T2WI (c, d), high intensity in the center of tumor (arrow) indicated necrosis and liquefaction

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Histological and immunohistochemistry assessment

The histology of orthotopic tumor tissues was examined using hematoxylin and eosin (H&E) staining. The tissues in the gemcitabine-treated group and saline-untreated group showed a typical histological appearance of pancreatic cancer (Fig. 5a). Boundaries between tumor tissue and surrounding normal tissue were unclear. Cancer cells were larger showing polygonal, spindle or irregular shape with cytoplasm lightly stained, the nucleus larger, the karyoplasmic ratio  increased, and nucleoli were obvious. Tumor cells were arranged in a tubular or solid nest, no adenoid structure, consistent with the characteristics of poorly differentiated adenocarcinoma of the pancreas. Residual normal tissue remained around tumor. In addition, Fig. 5a showed representative microphotographs of Ki-67, TUNEL and CD31 staining of tumor tissues of two groups collected at day 28, with the proliferating cells, apoptosis cells and microvessel areas indicated with black arrows in each subfigure. Quantifications of proliferating cell (Ki-67 positive), apoptosis cell (TUNEL positive) and MVD of two groups presented in Fig. 5b–d, respectively. The apoptosis cell of saline-untreated group was significantly lower than that of gemcitabine-treated group (P < 0.01) (Fig. 5b), whereas the proliferating cell was significantly higher than that of gemcitabine-treated group (P < 0.01) (Fig. 5c). No significant differences in MVD were observed between the two groups (Fig. 5d).

Fig. 5
figure 5

Gemcitabine induces tumor responses through reducing proliferation and increasing apoptosis. a Representative microphotographs of HE (original magnification, × 200), Ki-67, TUNEL and CD31 staining (× 400) in tumors dosed by Gemcitabine and saline. The proliferating cells, apoptosis cells and microvessel areas were indicated with black arrows in each row. b–d Proliferating (Ki-67 expressed) cell, apoptosis (TUNEL expressed) cell and microvessel (CD31 expressed) densities of gemcitabine and saline groups were presented; statistical differences between groups were indicated by asterisks above bars (**P < 0.01). G gemcitabine group, U saline-untreated group

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Discussion and conclusion

As the first-line drugs approved for pancreatic cancer by FDA, gemcitabine can significantly improve the quality of life of patients with pancreatic cancer and prolong life span, and is the gold standard for the treatment of pancreatic cancer [3, 24]. However, in the clinical treatment, many patients with pancreatic cancer acquire resistance to chemotherapy gemcitabine [25], which reduces the effects in the clinical application of gemcitabine, so it is important to evaluate the treatment of gemcitabine earlier.

As functional magnetic resonance imaging, diffusion-weighted imaging (DWI) can timely, accurately and reliably reflect the microscopic alterations of tissue composition and functional status of water exchange among the tissue components pathophysiologically by studying the microscopic movement of water molecules. The diffusion coefficient of DWI is called the apparent diffusion coefficient, reflecting the diffusion capacity of the tissue structure. Due to its large cell density, ADC value of solid tumor is smaller than that of normal tissue [26]. Recent studies have found signal attenuation is affected not only by the diffusion of water molecules but also by local capillary microcirculation when b value is lower [27, 28].

In this study, orthotropic model of pancreatic cancer in nude mice was established successfully. Compared with the subcutaneous model, orthotropic xenograft models are an ideal biological system for studying pancreatic cancer, as they can establish a human pancreatic cancer in its native site with the ability to expand relevance to drug evaluations. Referring to gemcitabine regimen in the clinical treatment, the value in the early assessment of efficacy was compared between IVIM-DWI and DWI. In this study, we used two different sequences instead of one sequence for IVIM and ADC acquisition. The reason is that we attempted to compare the parameters derived from the multiple-b IVIM sequence with the ADC derived from 3-b DWI sequence. The 3-b DWI sequence has its own advantage of shorter scanning time and less motion sensitivity. While IVIM sequence acquires more b values it needs longer scanning time and might be more motion-sensitive. Using ADC generated from the subset b values of the IVIM sequence might underestimate the performance of the conventional ADC. Thus, we use a separated sequence to acquired ADC. As can be seen from Table 2, the Dslow values and the ADC values were both statistically significant compared to the control group after 1 day of gemcitabine therapy. With rapid growth, tumor angiogenesis cannot keep up with the rate of tumor cell proliferation. There was liquefaction necrosis within the tumor formation and diffusion of water molecules enhanced in the corresponding region (Fig. 4). The increased diffusion of water molecules caused by the tumor necrosis is counteracted by the one caused by the intervention of gemcitabine. This may explain why the Dslow values and ADC values were not statistically significant at day 10 between two groups. Our results also showed that Dfast values and fp values of gemcitabine-treated group were not statistically significant compared with the saline-untreated group at day 1 and day 10. Studies have found Dfast values and fp values were positively correlated with MVD [29]. Gemcitabine is not an angiogenesis inhibitor and its main mechanism is to inhibit cell proliferation and induce cell apoptosis by interfering with DNA replication to cause DNA damage and block cell cycle progression, which was proved by tumor tissue immunohistochemistry indicators after the treatment period. Compared with saline-untreated group, cells in gemcitabine-treated group proliferated slower, apoptosis occured faster, and MVD changed less.

It is well known that when DWI is used on small experimental animals, the poor inhomogeneity of the magnetic field at the air–tissue interface produces severe susceptibility artefacts, with higher magnetic field intensity resulting in more severe artefacts [30, 31]. Some researchers have attempted examining animals using alginate moulding and ultrasonic coupling medium to improving signal intensity [32]. In this study, we choose a relatively simple and convenient method using 1.5 T MR, immersing experimental animals in liquid to isolate such air–tissue interface effects and achieving relatively satisfied results. Two points should be mentioned during the scanning process. First, scanning starts when the water is calm, the second is to keep the water temperature at 37.0 ± 0.2° C. The short scanning time in our plan, the maximum scanning time of the sequence is not more than 5 min, makes the two points feasible.

In conclusion, changes in ADC and Dslow value can be detected before gemcitabine treatment-induced reduction in tumor size. The earlier imaging technologies were used, the less ADC and Dslow value were affected by tumor growth. Monitoring ADC and Dslow value could be important methods of assessing the early therapy efficacy of pancreatic cancer.