This study utilized a newly optimized mouse model of chronic lung disease using long-term exposure to HDE, which was adapted from prior acute and repetitive murine dust exposure models. We used a total of 20 A/J mice for the preliminary study with 10 of them being HDE-treated and the other 10 saline-treated. Each mouse was lightly anesthetized by isoflurane inhalation prior to receiving a single intranasal chal-lenge of either 50 uL of 12.5% HDE or 1x PBS, thrice weekly for 24 consecutive weeks. To study the tumorigenic potential of the dust in a chronic exposure setting, we used the tobacco-specific carcinogen NNK to induce lung tumorigenesis in the mice. At the start of week 4, we administered a one-time intraperitoneal injection (i.p.) of 100 mg/kg NNK, which has been well-established to generate lung adenomas in A/J mice 21 weeks post-injection. Of the 20 exposed mice, 5 HDE-exposed and 5 saline-exposed were administered an i.p injection of NNK while the other 5 from each respective group received 1x PBS via i.p injection as the control.
Growth protocol
Male and female A/J mice 8-12 weeks of age were received from The Jackson Laboratory (Bar Harbor, ME, USA) and housed in micro-isolator cages (five per cage) at the University of California, Riverside animal barrier facilities. Mice were allowed unrestricted access to food and water, monitored for any behavioral or physiological changes, and weighed weekly. All experiments and procedures were approved by the University of California River-side Institutional Animal Care and Use Committee.
Extracted molecule
total RNA
Extraction protocol
Tumors were counted and excised from the left lungs following collection and half of the left lung was then stored in RNA later (Thermo Fisher, Waltham, MA, USA) for gene expression analyses. Left lung tissues were homogenized and RNA was extracted using the PureLink RNA Mini Kit (Invitrogen, Carlsbad, California, USA) and sample quality was quantified using the NanoDrop ND-100 (NanoDrop Technologies, Inc, Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (UC Riverside Core Facilities, Agilent Technologies, Santa Clara, CA, USA).
Label
n.a.
Label protocol
We utilized the mouse PanCancer Immune Profiling Panel (NanoString Technologies, Seattle, WA, USA) codeset which is a pre-designed panel containing 770 target genes related to immune response and carcinogenesis. Sample preparation was performed by mixing 50 ng of total RNA with the codeset and reporter probes and hybridizing the mixture for 16 hrs to form the desired target-probe complex. The hybridized complex was then plated and run on a nCounter Sprint profiler to image and quantify the data.
Hybridization protocol
n.a.
Scan protocol
Chip was imaged and quantified by a nCounter Sprint profiler.
Data processing
Gene expression data analysis was performed using the nCounter Analysis System, nSolver 4.0 software. Manual normalization of the expression data was performed using the geometric mean of the housekeeping genes EEF1G, OAZ1, RPL19, and SDHA. Proceeding sample nor-malization, 21 of the 24 samples passed the normalization parameters and were deemed viable for subsequent analyses. The samples breakdown are as follows: 6 saline-only, 4 HDE-only, 6 saline+NNK, and 5 HDE+NNK.
