Cell culture
Lewis lung carcinoma (LL/2) cells transduced with LV-Fluc-P2A-Puro (identifier: LL/2-Fluc-Puro) were purchased in 2018 directly from Imanis Life Sciences (Rochester, MN, USA), who authenticate the cell lines prior to shipment. The cell line was tested for mouse pathogens prior to use and were negative. LL/2 cells are mouse cells and do not require ethical approval. Cells were cultured at 37 °C in 5% CO2 using DMEM supplemented with 10% fetal bovine serum, 1x Penicillin/Streptomycin and 2 μg/mL puromycin.
Foam, talc foam (TF) and talc slurry (TS)
The foam delivery system is comprised of a triblock copolymer [18] hydrogel (Fig. 1a) in a saline solution that exhibits a reverse thermosensitive viscosity profile. A liquid at room temperature, the hydrogel undergoes a sol-gel transition at temperatures greater than ~ 25 °C (Fig. 1b). When foamed by agitating the cooled liquid hydrogel with air, the resultant foam is a thin liquid. At physiological temperatures, the foam rapidly collapses and forms a viscous, sticky gel.
The cooled liquid was loaded into a 10 cc syringe and aerated using a Disofix three-way stopcock (B. Braun Medical Inc., Bethlehem, PA) and a second 10 cc syringe filled with air at a 1:1 ratio of air and liquid. Foam was generated by mixing the air and gel through the three-way stopcock until the desired consistency of foam was obtained. The foam was freshly prepared shortly before injection and kept on ice until immediately before each injection.
For the talc foam, the above-mentioned cooled liquid was combined with talc (powder, 10 μm, Millipore-Sigma) within a 10 cc syringe, to achieve a concentration of 2 mg talc per gram of mouse body weight (2 mg/g). The mixture was aerated using a 1:1 ratio of air and the gel-talc mixture, to generate TF.
TS was prepared as previously described [19, 20] to a final concentration of 2 mg/g. In brief, talc was mixed with sterile saline using two 10 cc syringes connect via a three-way stopcock. TS was freshly prepared shortly before injection and kept on ice until right before each injection.
Rheology
Rheological studies of the triblock copolymer hydrogel (Fig. 1b) were performed on a DHR-3 rheometer, TA Instruments (Calumet, MI) using parallel plates, 40 mm in diameter, with a gap of 1 mm. A Peltier system was used to perform temperature ramps by heating the plates and all data were collected in the linear regime of the amplitude. A thin layer of silicone oil was added surrounding the plates to prevent evaporation during the experiment. A heating rate of 1.45 °C/min was used. The complex modulus, G*, storage modulus, G’, and loss modulus G”, were measured for each of the formulations. The complex modulus is related to the storage modulus and loss modulus by equation 1 (Eq. 1).
$$ {G}^{\ast }=\sqrt{G^{\hbox{'}2}+}\kern0.5em {G}^{"2} $$
(1)
Computer tomographic (CT) imaging and quantitative analysis
Mice were anesthetized with 2% isoflurane in O2 for induction and were then maintained in 0.5% isoflurane during the scan. Scanning was performed with a Sofie Biosciences CT/PET machine (G8 PET/CT, PerkinElmer/Sofie Biosciences, Culver City, CA) using the standard protocol specified by the manufacturer. To calculate airspace volume, VivoQuant software (inviCRO, Boston, MA) was used. To identify the total area of air-filled lung, areas with minimal contrast within a specific range, were automatically highlighted using the Connected Thresholding function. This setting was kept consistent for all analyzed mice. The airspace volume was calculated automatically in mm3. Trachea and other artifacts were excluded manually. Mice were scanned immediately after the injection of tumor cells and again at the end of the experiment. The pre-injection scan for each mouse served as the reference scan used to calculate the remaining lung volume for each treatment.
Murine model
All animal care and experimental procedures were prospectively approved by the Fox Chase Cancer Center Institutional Animal Care and Use Committee (IACUC). The Fox Chase Cancer Center staff tended to the mice daily and for the duration of the experiment. 6–8-week-old male and female C57BL/6 mice (obtained from The Jackson Laboratory, Bar Harbor, ME) were acclimatized for 1 week before use.
To first assess TF as a sclerosing agent, mice without tumors were randomly assigned into one of the following groups: control (foam or saline), TF, or TS. For the TS group or for TF (prepared as described above), the volume injected was adjusted based on body weight, to achieve a final injected dose of 2 mg talc per gram mouse body weight. The volume for foam or saline was calculated to match the volumes used for the talc treatment group. Talc mixtures (TF or TS) were then injected intrapleurally into the right pleural cavity. Mice were anesthetized with isoflurane and the right chest was cleaned with an alcohol solution. A 23-gauge needle attached to a 1 mL syringe was introduced into the right chest cavity at 1 cm lateral to the right parasternal line, as previously described [17]. The mice were euthanized 7 days post-treatment, and the chest cavity was evaluated for fibrosis as described below.
To generate MPE, intrapleural injection of LL/2 cells was performed. Mice were anesthetized with 1–3% isoflurane gas and the right chest was cleaned with an alcohol solution. A 23-gauge needle attached to a 1 mL syringe containing 1.5 × 105 of LL/2 cells in sterile PBS was introduced into the right chest cavity at 1 cm lateral to the right parasternal line. The needle was slowly advanced until it reached the pleural space. The cell suspension was then carefully injected. All mice were monitored until completely recovered from the procedure. Four days after the injection of LL/2 cells, all mice were randomly assigned into one of the following groups: control (foam or saline), TF, or TS, each of which were injected as described in the previous paragraph.
For effusion and survival analyses, mice were injected with LL/2 cells as described above, then underwent CT imaging 1 day and 14 days after the injection of cells, to assess lung volume status. Four days after the injection of cells, all mice were randomly assigned to receive intrapleural control foam/saline, TF, or TS, at the appropriate concentration and volume as noted above. The mice were euthanized (CO2 inhalation) according to the approved IACUC protocol upon showing signs of poor health, presentation with signs of pain or distress, or if they experienced a rapid loss of weight (> 20% of body weight over 7 days).
Histopathological evaluation
Immediately following euthanasia, the abdominal wall of the mice was opened, and the viscera were retracted to visualize the diaphragm, which was punctured with a 23-gauge needle to aspirate pleural fluid. The pleural fluid volume was measured. The thorax was dissected and removed en bloc. All lungs were imaged and collected for histology. Lungs were fixed in 10% phosphate-buffered formaldehyde for 24–48 h, dehydrated by incubation in ethanol followed by xylene (70% ethanol, 3 h; 95% ethanol, 2 h; 100% ethanol, 2 h; ethanol-xylene, 1 h; xylene, 3 h) then embedded in paraffin. 5 μm thick slices were cut, mounted on slides and stained with trichrome. Stained slides were scanned with Vectra 2.3 Automated Quantitative Pathology Imaging System (Perkin Elmer, Waltham, MA). Tissue segmentation (% fibrosis) was determined by automated quantitative analysis using InForm software (PerkinElmer Inc., Waltham, MA).
Statistics
For all experiments indicated, p values were calculated using one-way ANOVA (GraphPad Prism version 6.00 for Mac; GraphPad Software) or Student’s t test as specified. Survival curves were generated using the Kaplan and Meier method and tested for significance using log-rank tests.
In justification of animal number in experimental cohorts, we would set the Type I error rate to 5%/3 = 1.67% Type I error (2-sided). In this case with a relatively small number of multiple comparisons, the Bonferroni correction sets the p-value to a level that is more generous than using a 1% False Discovery Rate (FDR). With 10 animals per cohort, we would have 85% power to detect a standardized effect of 1.7 standard deviation units. We used generalized linear models (GLMs) for analysis, assuming normal distribution and identity link.
