*2.1. Animals*

All animal experiments were performed in accordance with protocols approved by the University of Iowa Institutional Animal Care and Use Committee (IACUC) and adhere to the NIH Guide for the Care and Use of Laboratory Animals. C57BL/6 (stock #556) and BALB/c mice (stock #555) were purchased from Charles River Laboratories. 129X1 mice (stock #000691) were purchased from Jackson Laboratories. Wild-type 129Sv/Jae mice were bred and maintained at the University of Iowa.

#### *2.2. CRISPR*/*Cas9 Generated MPNSTs and Growth Analysis*

Adenovirus containing Cas9 and sgRNAs targeting *Nf1* and *p53* was purchased from ViraQuest (North Liberty, Iowa) [27]. Prior to injection, virus was mixed with DMEM and calcium phosphate as previously described [28–30]. Tumors were generated by injection of 25 uL of prepared virus into the left sciatic nerve of mice. When tumors reached a volume of 150 mm<sup>3</sup> (Day 1), they were measured by calipers 3 times weekly. Tumor volumes were calculated using the formula *V* = (π × *L* × *W* × *H*)/6, with *L, W*, and *H* representing the length, width, and height of the tumor in mm, respectively. Tumors were harvested when they reached a volume of 1500 mm<sup>3</sup> or earlier if animals showed signs of distress, in accordance with IACUC guidelines at the University of Iowa. Tissue was collected for histology, RNA, and generation of cell lines.

#### *2.3. Generation of Cell Lines from MPNSTs*

Cell lines were derived from terminally-harvested MPNSTs. Tumors were finely minced and digested in dissociation bu ffer Collagenase Type IV (700 units/mL, Thermo, 17104-019, Thermo Fisher Scientific, Waltham, MA, USA) and dispase (2.4 units/mL, Thermo, 17105-041, Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1–1.5 h at 37 ◦C on an orbital shaker. Dissociated tissue was passed through a sterile 70 μM cell strainer (Fisherbrand, 22363548, Thermo Fisher Scientific, Waltham, MA, USA), washed once with PBS, and resuspended in DMEM (Gibco, 11965-092, Thermo Fisher Scientific, Waltham, MA, USA). Cells were cultured in DMEM containing 10% FBS, 1% penicillin-streptomycin (Gibco, 15140-122, Thermo Fisher Scientific, Waltham, MA, USA) and 1% sodium pyruvate (Gibco, 11360-070, Thermo Fisher Scientific, Waltham, MA, USA). After 10 passages, cells were used for indel analysis and subsequent studies.

## *2.4. Indel Analysis*

Indel pattern analysis was previously described [31]. Genomic regions of *Nf1* and *p53* that spanned the gRNA target sites were amplified by PCR using Phusion high-fidelity DNA polymerase (NEB, M0530L). PCR primers for *Nf1* indels generate a 569 bp fragment in wild-type cells while those used to amplify *p53* indels result in a 520 bp fragment in wild-type cells. Primer sequences are listed in Supplementary Table S1. PCR amplicons were purified with the Monarch PCR and DNA Cleanup Kit (NEB T1030S). Sanger sequencing was performed by the Genomics Division of the Iowa Institute of Human Genetics at the University of Iowa. Indel frequencies were quantified from the chromatograms by sequence trace analysis using Synthego ICE [32]. Indels > 50 bp were determined by band size on a 2% agarose gel.

#### *2.5. Histology and Immunohistochemistry*

Upon harvest, a portion of tumor tissue was stored in 10% neutral bu ffered formalin for fixation and subsequent para ffin embedment. Formalin-fixed para ffin embedded tumors were sectioned and stained with hematoxylin (Vector Laboratories, H-3401, Burlingame, CA, USA) and eosin (Sigma-Aldrich, 586-X, St. Louis, MO, USA) to evaluate tissue morphology. All immunostaining was conducted with citrate-based antigen retrieval (Vector Laboratories, H-3300, Burlingame, CA, USA). The following antibodies were used: S100 (Abcam, ab4066, Cambridge, United Kingdom), Ki67 (BD Biosciences, 556003), CD4 (Abcam, ab183685), CD8a (Thermo Fischer Scientific, 14-0808-82, Waltham, MA, USA), Foxp3 (Thermo Fisher Scientific, 14-4777-82, Waltham, MA, USA), and F4/80 (Thermo Fisher Scientific 14-4801-82, Waltham, MA, USA). To visualize mast cells, slides were stained with toluidine blue solution (0.02% toluidine blue in 1% NaCl, pH 2.2) for 2 min, followed by two washes in distilled water and three washes in 100% ethanol. At least five tumors per group were analyzed, and quantification of cells staining positive was performed on 6 independent fields. The 20× fields were used for all analyses except for Ki67, which used 40× fields. Imaging was performed using an EVOS XL Core Imaging System (Thermo Fisher Scientific, AMEX1000, Waltham, MA, USA).

## *2.6. Quantitative RT-PCR*

Upon harvest, tumor tissue was stored in RNA Later (AM7020, Thermo Fisher Scientific) at −20 ◦C. Tumors (*n* = 5 per strain) were homogenized in liquid nitrogen and resuspended in Trizol (15596018, Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 1 ug of RNA using iScript (1708891, Bio-Rad). RT-qPCR was performed with Power-up Sybr Green 2x Master Mix (A25778, Thermo Fisher Scientific, Waltham, MA, USA) per the manufacturer's instructions on an Applied Biosystems 7900HT instrument using the ΔΔ Ct relative to B2M expression (Genomics Division of the Iowa Institute of Human Genetics, University of Iowa). Primer sequences are listed in Supplementary Table S1 [24].

## *2.7. Statistical Analysis*

Statistical analysis was performed using GraphPad Prism 8. Tumor growth kinetics, IHC quantification, and gene expression were analyzed using a one-way ANOVA with Tukey's multiple comparison test. Sample sizes for IHC and qRT-PCR analysis were 5 per group. Comparison of survival curves was performed using the log-rank (Mantel–Cox) test. For all studies, a *p* value of less than 0.05 was considered statistically significant.
