The design of this experimental study was based on the survival of mice with induced MM comparing controls and BoxA-treated mice and the accuracy of ultrasound and OI in detecting peritoneal masses.
Murine MM AB1cells were obtained from Cell Bank Australia and cultured in RPMI 1640 (Life Technologies, New York, USA) supplemented with 5% volume/volume fetal bovine serum (Life Technologies), 2 mM l-glutamine and 100 U/mL penicillin/streptomycin and subsequently intraperitoneally injected in “Bag ALBino” (BALB/c) mice. Masses grown in BALB/c mice were explanted and the procedure to obtain AB1 cells expressing the gene for the luciferase enzyme (AB1-B/c-LUC) cells was previously described .
HMGB1 contains two deoxyribonucleic acid-binding domains, named BoxA (BoxA alone behaves as a HMGB1 competitor  while it contains an epitope that promotes inflammation) and an acidic tail. BoxA corresponds to amino acids 2–89 of HMGB1, where amino acid 1 is methionine and is removed both in mammalian HMGB1 and in BoxA; the sequence of this segment of HMGB1 is identical in all mammals .
Animal experiments have been reviewed and approved by the Institutional Animal Care and Use Committees of Ospedale S. Raffaele and Istituto di Ricerche Farmacologiche “Mario Negri,” which include ad hoc members for ethical issues. Animals were housed in the Institutes Animal Care Facilities, which meet international standards. Certified veterinarians who are responsible for health monitoring, animal welfare supervision, experimental protocols, and procedure revision regularly checked them, in both institutions.
The following two experiments were performed.
First preliminary experiment. Fourteen mice were intraperitoneally injected with 7 × 104 AB1-B/c-LUC cells (in 500 μL), and on the same day, the 1st OI was performed (intraperitoneal 200 μL luciferin) to confirm the success of injection. The mice were randomized into two groups (n = 7 + 7) and treated intraperitoneally, every other day for 28 days, as follows: 7 mice with BoxA wild type (400 μL, 2 mg/mL = 800 μg, corresponding to 32 mg/kg); 7 mice with 400 μL of phosphate-buffered saline (controls).
Second experiment. Forty mice were injected with MM cells and divided into two groups (n = 20 + 20), which were treated intraperitoneally with BoxA or phosphate-buffered saline as above every other days for 33 days. Some mice that survived to the second experiment were rechallenged after 2 months with murine MM cells to confirm the tumor rejection. The second experiment was developed to increase the robustness of the preliminary data, increasing the number of the mice.
Tumor response and survival were assessed in both experiments using experimental ultrasound- and OI-based parameters.
Ultrasound examination was performed using a 40-MHz linear probe (Vevo 2100, Fujifilm Visualsonics, Toronto, Canada), both using freehand and fixed support. The ultrasound protocol was based on the study of the abdomen of the mouse divided into 9 quadrants which were likely to correspond to those of human and each quadrant was given a number (from 1 to 9): right hypochondrium, epigastric region, left hypochondrium, right flank, mesogastric region, left flank, right iliac fossa, hypogastric region, and left iliac fossa.
Tumor response was evaluated with ultrasound through the presence or absence of masses; number, volume, distribution, growth and shrinkage of mass, and presence or absence of ascites were also considered. All the following mass parameters were evaluated using brightness-mode ultrasound and color Doppler: echogenicity, shape, margins, vascularization, and diameter. In the first experiment, ultrasound was performed on day 5, 22, and 32 and in the second experiment, instead, on day 30.
Optical imaging protocol
The system was equipped with a low noise, back-thinned, back-illuminated charge-coupled-device camera cooled to − 90 °C (quantum efficiency in the visible range above 85%). Before the OI procedure, each mouse received an intraperitoneal injection of 6 g of luciferin (d-luciferin potassium salt, Perkin Elmer, Milan, Italy) per kilogram of body weight. Luciferase oxidizes luciferin to generate light, which is produced because the reaction forms oxyluciferin in an electronically excited state. The reaction releases a photon of light as oxyluciferin goes back to the ground state  and can be quantified by OI.
During image acquisition, the animals were kept at 37 °C and under gaseous anesthesia (2–3% isoflurane and 1 L/min O2). After luciferin injection, OI was performed from 0 to 30 min by acquiring images every 2 min in order to detect the highest OI signal, with the following technical setting: exposure time “auto”; binning8, focal ratio 1; and a field of view of 13 cm (field C).
Optical imaging was performed using the Living Image 4.4 software (PerkinElmer’s IVIS Spectrum Pre-clinical in Vivo Imaging System, Milan, Italy) by measuring the total light flux (photons/s) in a region of interest which included the abdomen and excluded limbs and testicles of mice. OI was also performed to evaluate the presence or absence of masses according to their luminescence intensity. The timing of OI execution was day 1, 6, 14, 20, 27, 31, 41, and 48 for the first experiment and day 1, 6, 13, 30, 34, 61, 68, 76, and 84 for the second experiment.
Comparison between ultrasound and OI
Detection (presence/absence) of masses was recorded for both techniques and the findings were compared using pathology as a reference standard. Suspicious masses and samples from abdominal organs were histopathologically and immunohistochemically studied after the mice were sacrificed . For histopathological analysis, samples of explanted tumor masses were fixed in 10% neutral buffered formalin for 24–48 h, processed with a Tissue Processor (Leica, Buccinasco-Milan, Italy) and paraffin embedded. Sections of 4 μm were cut, stained with hematoxylin-eosin, and evaluated under a light microscope.
Contingency tables and Fisher exact test were used to assess difference both in the first and in the second experiment between the two mouse populations both for mortality and for the presence of masses at ultrasound evaluation. Concordance between mass detection at imaging (ultrasound and OI) and histology was investigated using simple contingency tables. A two-tailed p value lower than 0.05 was considered significant. Statistical analyses were performed using the SPSS software, version 25.0 (IBM, Armonk, New York, USA).