Experimental pancreatic cancer develops in soft pancreas: novel leads for an individualized diagnosis by ultrafast elasticity imaging

Rapid, easy and early pancreatic cancer diagnosis and therapeutic follow up continue to necessitate an increasing attention towards the development of effective treatment strategies for this lethal disease. The non invasive quantitative assessment of pancreatic heterogeneity is limited. Here, we report the development of a preclinical imaging protocol using ultrasonography and shear wave technology in an experimental in situ pancreatic cancer model to measure the evolution of pancreatic rigidity. Methods: Intrapancreatic tumors were genetically induced by mutated Kras and p53 in KPC mice. We evaluated the feasiblity of a live imaging protocol by assessing pancreas evolution with Aixplorer technology accross 36 weeks. Lethality induced by in situ pancreatic cancer was heterogeneous in time. Results: The developed method successfully detected tumor mass from 26 weeks onwards at minimal 0.029 cm3 size. Elastography measurements using shear wave methodology had a wide detection range from 4.7kPa to 166.1kPa. Protumorigenic mutations induced a significant decrease of the rigidity of pancreatic tissue before tumors developed in correlation with the detection of senescent marker p16-positive cells. An intratumoral increased rigidity was quantified and found surprisingly heterogeneous. Tumors also presented a huge inter-individual heterogeneity in their rigidity parameters; tumors with low and high rigidity at detection evolve very heterogeneously in their rigidity parameters, as well as in their volume. Increase in rigidity in tumors detected by ultrafast elasticity imaging coincided with detection of tumors by echography and with the detection of the inflammatory protumoral systemic condition by non invasive follow-up and of collagen fibers by post-processing tumoral IHC analysis. Conclusion: Our promising results indicate the potential of the shear wave elastography to support individualization of diagnosis in this most aggressive disease.


Supplementary Legends
Supplemental  Table 1: Parameters of optimized settings adapted to internal abdominal organs.
Supplemental Table 2: Raw data and analysis of Ctrl and KPC mice in time.

Materials
Specific Reagents : • Experimental murine models of PDAC. The use of animals for these experiments requires the approval of Ethics Committee or an equivalent regulatory organization.
• Isofluorane (Virbac, Vetflurane 1 000MG/G, cat. No. BE-V166896) ! CAUTION volatile anesthetic agent which is administered to the mask with anesthetic spray. The pancreas is an organ whose position and limits are difficult to observe. It is just below the stomach, stretched between the two kidneys, slightly above the left curve of the spleen. To validate the location, dissection is recommended. Train your eyes to detect pancreas on wild-type animals. Perform a cervical dislocation and a dissection. Add gel and observe the pancreas without skin interference to better locate the pancreas and its specific imaging. II.
To train on animals without dissection, 1 to 2 mL of saline solution can be injected i.p., which increases the rate of optimal imaging; this procedure requires a specific ethical authorization. b. New settings III.
The search for adapted parameters was performed and described in supplementary Table 2. To vary the parameters, use the touchscreen (including "Other Settings") and the control panel.

IV.
Record the settings as a "New Preset". To configure the Aixplorer with the new settings, press the "Probe" button on the Control Panel, select the probe used, go to "New Preset", enter the new one with the Touch Screen, and confirm.

2-ANALYSIS MOUSE OF INTEREST
a. Mouse preparation • TIMING 5 min I.
Open the air and oxygen inlets that feed the MiniTag station. II.
Turn on the Minitag and the heating plate at 36 ° C, with the isoflurane flux between 2 and 3%. III.
Anesthetize the mouse of interest in the induction box, before placing it in the mask. IV.
Shave the abdomen from the middle of the sternum to the bladder down well on the sides of the mouse. Wipe the cream with a water-soaked dressing before starting the observation.  CRITICAL STEP To properly observe the spleen and better locate the pancreas, which are both internal organs, shave properly the left side of the animal. V.
Apply a sufficient amount of ultrasound gel on the abdomen and on the probe.  CRITICAL STEP Avoid air bubbles that would deteriorate the quality of the image. (Suppl Figure 3D,E) b. Setting • TIMING 1 min I.
Briefly press the ON / OFF button located on the Control Panel. (Suppl Figure 3F, blue circle) Press the "Probe" button on the Control Panel. (Suppl Figure 3F, yellow circle). Select the SL22-7lab probe and the default setting or the new setting as described in ► 1b-IV. II.
Create a new "Patient" file to record all images of the same mouse in a single folder. Press the "Patient" key on the left of the Control Panel. (Suppl Figure 3F, red circle). Enter the name of the desired "Patient". III.
Increase the gain percentage of B-mode to 34% (brighter image, Suppl Figure, 3F, B button). IV.

d. Pancreas imaging • TIMING 5-10 min
I. B-Mode -Press the "B" button and search for the pancreas and kidney. Save an image in Bmode. ? TROUBLESHOOTING.

II.
Doppler color mode -start an analysis in COL mode to avoid the localizations which contain the blood vessels. To enlarge the observation screen, use the "Select" key and then roll the TrackBall to vary the length. Decrease the Gain in COL mode, to focus on the main blood vessels. (Suppl Figure 3F, C button). III.
SWE-Mode -Press the SWE mode. Place the visualizing panel (Area) on the part of the pancreas to be analyzed and on the left kidney of the mouse. Wait for the elastography measurements to cover the entire measurement screen and then save the image. (Suppl Figure 3F, SW button).  CRITICAL STEP Avoid pressure on the probe. ? TROUBLESHOOTING.

IV.
Then reduce the Area Size, to "Medium". Save another image.

V.
Once the series of the images are carried out, check the mouse until awake. VI.
Select the desired image on the Monitor. V.
Several measurements are possible. 1. Q-Box plot, allows freehand drawing of the measuring surface. 2. Q-Box, allows a unique measure of elastography in a predefined circle area. 3. Q-Box ratio, measures the ratio of two or more quantitative boxes. VI.
Press the "Save Image" is necessary. VII.
In B mode, measure tumoral volume. VIII.
Press the "End Exam." IX.
Select the desired patients, and export the raw image ("Export to DICOM"), images ("Export to JPEG / AVI") or measures ("Clinical data"). X.
switch off the Aixplorer.

B-mode analysis
I. It sometimes happens that the pancreas is difficult to observe. The first and most recurrent reason is a swollen and filled stomach that forms a black shadow on the pancreas or even on the kidney controls. It is then necessary to find the best moment of the day to go to observe the mice in order to avoid this problem. (Suppl Figure 2D).

SWE analysis
I.
The first difficulty lies in the fact that if the target organ / tumor is not measured, this is assessed by an absence of color coded SWE image superimposed to B mode image; move the probe to reach a satisfying image (Suppl Figure 2A,B). II.
If a SWE color gradient is visible outside the mouse, verify that there is a sufficient amount of gel, change the pressure exerted on the probe or its position / inclination (Suppl Figure 2C).   Figure 6 Therville N, et al