<figure><img src="image_64.png" /></figure>

For Stat3 ChIP, the protein G-bound immunocomplexes were washed four times with buffer containing 50mM HEPES pH 7.6, 500mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Sodium Deoxycholate and protease inhibitors, while for H3K27ac ChIP were washed with the following buffers: low salt wash buffer containing 20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl; high salt wash buffer containing 20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl; LiCl wash buffer containing 10 mM Tris-HCl pH 8.0, 1% Sodium Deoxycholate, 250 mM LiCl, 1 mM EDTA, 1% NP40 and protease inhibitors. Immunocomplexes were eluted from the beads and crosslinking was reversed by incubation for overnight at 65°C with TE buffer containing 1% SDS. DNA from immunoprecipitated samples as well as DNA from 10% input was purified by using QIAquick PCR Purification Kit (Qiagen), according to the manufacturer's instruction. The Stat3 ChIP and the H3K27ac ChIP were analyzed by ChIP-qPCR using SYBR GreenER kit (Invitrogen) on target genomic regions (Table S1). qPCR reactions were performed on a Rotor-Gene Q 2plex HRM Platform (Qiagen, 9001560). The data are expressed as a percentage of the DNA Input.

For *in vivo* chromatin immunoprecipitation, two entire frozen quadriceps per condition (CTR and C26 for pSTAT3, H3K27ac, IgG) were homogenized with a Bounce homogenizer in 1,200 µL nuclear extraction buffer composed by 10 mM Tris HCl, 10 mM NaCl, 3 mM MgCl₂, 10 mg/mL BSA, 0.1% IGEPAL, 0.5 mM DTT and protease inhibitors. Afterward, 37% formaldehyde was added to the lysates to reach a final concentration of 1%, and incubated at room temperature for 10 min, tapping occasionally to mix. The crosslinking reaction was interrupted by adding 75 µL of 1.25 M glycine on ice for 5 min. Samples were centrifuged at 1000g, 4°C for 10 min. Cell pellets were resuspended in SDS ChIP lysis buffer containing 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.3% SDS and protease inhibitors. Chromatin was sheared to an average DNA fragment length of 500 bp using Picoruptor sonicator (Diagenode) (15 cycles, 30 s on/off). After sonication, the samples were processed as *in vitro* ChIP-qPCR.

## Histology

Extracted gastrocnemii were immediately frozen in isopentane cooled in liquid nitrogen and stored at -80°C. Transversal sections of 10µm were cut at the midbelly with a cryostat. Cross sectional area (CSA) was determined by sections fixation for 10 min in PFA 4% PBS, then blocking with Triton X-100 0.1%, BSA 3% in PBS before incubating with primary antibodies against laminin (Santa Cruz Biotechnology sc-59854), followed by incubation with corresponding secondary antibodies (Alexa Fluor 488) and DAPI (Abcam 228549). Pictures were taken as mosaic of the full section with a confocal microscope (Leica SP5) and fiber areas were measured with MorphLibJ plugin for ImageJ.⁷⁹

## Neuromuscular junction analysis

Extensor digitorum longus (EDL) muscles were immediately fixed in PFA 4% PBS for 10 min and placed in cold PBS. The full muscles were stained with fluorescently tagged α-bungarotoxin - Alexa 488 (Thermo Fisher B13422) for 90 min 1:500 in PBS at room temperature. The whole muscles were mounted and pictured with fluorescence confocal microscopy (Leica SP5).

Each post-synaptic NMJ has been acquired through 1 µm thick z stack, and voxel were automatically quantified with ImageJ software.

For denervation quantification, fixed EDL muscles were permeabilized for 2 h at room temperature in 2% Triton X-100 in PBS, then blocked in 4% BSA - 1% Triton X-100 in PBS. The primary antibody anti-neurofilament-H (heavy) (Abcam #Ab4680) was incubated 1:1000 in blocking buffer at 4°C overnight. 3 x 20' PBS washes were followed by secondary antibody and fluorescently tagged α-bungarotoxin - Alexa 488 (Thermo Fisher B13422) incubation and whole mount. Pictures were acquired with fluorescence confocal microscopy (Leica SP8). Denervation was measured evaluating 60 to 90 NMJs per mouse (with a total of three mice per treatment group). Our scoring system, ranged from 0 to 2: 0 for complete denervation, 1 for partial denervation, and 2 for full innervation.

## RNA isolation and quantitative PCR (qPCR)

Total RNA was isolated from snap-frozen tissue samples using TRIzol reagent (Invitrogen) according to the manufacturer's guidelines. 0.5µg of total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcriptase kit (Applied Biosystems) following the manufacturer's instructions. cDNA was analyzed by Real Time Quantitative PCR (ABI PRISM 7900HT FAST or Quant Studio 6 Real-Time PCR system, Applied Biosystems) using the PowerUp SYBR Green master mix (Applied Biosystems #A25742) and primers were designed according to Harvard validated PrimerBank. Relative mRNA levels were calculated using the 2-ΔΔCT method and normalized to GAPDH mRNA. For human muscle biopsies, total RNA was extracted from approximately 20 mg of rectus abdominal muscle using TRIzol (Invitrogen). 1 µg of RNA was reverse transcribed using the SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). Gene expression was analyzed by qRT-PCR (Quant Studio 5 Real-Time PCR system, Applied Biosystems) using the PowerUp SYBR Green Master Mix (Applied Biosystems #A25742). Data were normalized to beta-actin gene expression. All RT-qPCR primers are listed in Table S1.

## Adeno-associated virus (AAV) shRNA cloning

For the *in vivo* adeno-associated virus (AAV9-shRNA) delivery, short hairpin sequences targeting ERFE (shERFE - TRCN0000178969), FKBP12 (shFKBP12 - TRCN0000012492) or scramble sequence (shCTR) were cloned in the AAV plasmid and assembled in viral particles as previously described.⁷⁸,⁸⁰
## FACS

Fresh primary C26 tumor specimens of 8–10 mm mean diameter were finely minced with blades and digested by incubation with 1 mg/mL collagenase IV (Sigma Aldrich) in RPMI-1640 at 37°C for 1 h in an orbital shaker. After washing in RPMI-1640 with 10% FBS, the cell suspension was passed through a 70 µm pore cell strainer, centrifuged at 1400 rpm for 10 min and incubated in erythrocyte lysing buffer (155 mM NH₄Cl, 15.8 mM Na₂CO₃, 1 mM EDTA, pH 7.3) for 10 min at room temperature. After washing in RPMI-1640 with 10% FBS, 10⁶ cells were collected, re-suspended in PBS, and treated with Fc-receptor blocker (anti-CD16/CD32 antibody, #101302, Biolegend). Splenocytes were collected by smashing spleens from vaccinated mice on a 40 µm pore cell strainer, centrifuging the resulting cells at 1,400 rpm for 10 min and incubating them in erythrocyte lysing buffer, followed by treatment with Fc-receptor blocker. 50 µL heparinized blood collected by intracardiac injection were incubated with erythrocyte lysing buffer, washed and incubated with Fc-receptor blocker. After Fc-receptor blocking, all samples were stained for 30 min at 4°C with the following antibodies, as reported previously⁸¹:

* anti-CD45-VioGreen (#130-123-900), anti-CD3-FITC (#130-119-135), anti-CD4-APC/Vio770 (#130-119-134), anti-CD8-VioBlue (#130-123-865), anti-CD49b-PE (#130-102-337), anti-PD1-APC (#130-102-263), anti-CD11b-FITC (#130-113-234), anti-F4/80-PE/Vio770 (#130-118-459), anti-Ly6C-APC/Vio770 (#130-111-917), anti-Ly6G-VioBlue (#130-119-986), anti-MHCII-APC (#130-112-388), anti-CD11b-FITC (#130-113-234), anti-CD11c APC (#130-110-839) (all from Miltenyi Biotec) anti-CD69-PE/Vio770 (#104512, Biolegend), anti-CD206-PE (#141706, Biolegend) and anti-PDL1-PE (# 558091, BD Bioscience). Samples were acquired on BD-FACSVerse and cell populations (NK: CD3⁻ CD49b⁺; NKT: CD3⁺ CD49b⁺; CD4⁺ T cells: CD3⁺ CD49b⁻ CD4⁺; CD8⁺ T cells: CD3⁺ CD49b⁻ CD8⁺; DC: CD11b⁻ CD11c⁺; macrophages: CD11b⁺ F4/80⁺; m-MDSC: CD11b⁺ F4/80⁻ Ly6G⁻ Ly6Chigh; gMDSC: CD11b⁺ F4/80⁻ Ly6G⁺ Ly6Cdim/neg) analyzed with FlowJO10.5.3 and reported as percentage on the CD45⁺ leukocyte population, following doublets and dead cells elimination.

## Vaccine preparation and mouse immunization

A plasmid coding for chimeric rat and human ErbB2 extracellular and transmembrane domains (RHuT)⁸² and the corresponding control empty plasmid pVAX1 (Invitrogen, Monza, Italy) were amplified and then purified using the Endofree Qiagen Plasmid-Giga (Qiagen Inc., Cjatsworth, CA) following manufacturer's instructions. Vaccination was performed by injecting 50 µg of DNA diluted in 20 µL of 0.9% NaCl into the quadriceps muscle of anesthetized mice. Immediately after the vaccine injection, the muscle was electroporated by using an array needle electrode connected to an electroporator (Cliniporator, IGEA, Carpi, Italy). Two 25-ms trans-cutaneous low voltage electric pulses with an amplitude of 150 V spaced by a 300-ms interval were applied.⁸³ Vaccination was repeated 14 days later. Two weeks after, mice were culled and sera and spleens were collected.

## Antibody response

Blood samples were collected 14 days after the last vaccination and serum obtained by centrifugation. The concentration of anti-rat Her2 antibodies was determined by flow cytometry as the ability of diluted sera (1:200) to bind 3T3/NKB cells (BALB/c 3T3 NKB, expressing the rat ErbB2, H-2Kd, and B7.1 molecules),⁸⁴ which were a generous gift from Dr. Wei-ZenWei (Karmanos Cancer Institute, Detroit, MI, USA). A FITC-conjugated rabbit anti-mouse IgG antibody (F313, Dako, Milano, Italy) was used to detect the bound primary antibodies. Flow cytometry was performed with a BD-FACSVerse and samples were analyzed with FlowJO10.5.3, and antibody titers reported as mean fluorescence intensity (MFI).

## IFN-γ enzyme-linked immunospot (ELISpot) assay

Splenocytes from vaccinated mice were plated at 1 × 10⁶/well into 96-well HTS IP plates (Millipore) precoated with 5 µg/mL of rat anti-mouse IFN-γ (clone R4-6A2, BD Biosciences). Cells were then stimulated with 15 µg/mL of rat ErbB2 immunodominant peptide (TYVPANASL) peptide for 16 h in the incubator, or with concanavalin A (2 µg/ml) or medium alone as positive and negative controls, respectively. Images of the wells were acquired, and IFN-γ spots enumerated, with a microplate reader, along with a computer-assisted image analysis system (Immunospot; CTL Europe, Bonn, Germany), as previously described.⁸²

## QUANTIFICATION AND STATISTICAL ANALYSIS

All graphs show mean ± SEM and were analyzed with GraphPad Prism (version 6.0, GraphPad Software). Detailed statistical tests are reported in figure legends, where n represents the total number of independent experiments. For in vivo data, n is referred to the number of mice per group.

Statistical significance was tested with unpaired t-test when two groups of data were compared, with one-way ANOVA followed by Sidak's multiple comparison test when more than two groups were analyzed. Two-way ANOVA followed by Sidak's multiple comparison test was performed when two variables were analyzed. When relevant, one sample t-test was performed. For human biopsies analysis non-parametric Kruskal-Wallis test followed by Benjamini, Krieger and Yekutieli multiple comparison test was performed. Significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001. The illustrations were created on Biorender.com and all figures were assembled using Adobe Illustrator (AI, 2022).
## FACS

Fresh primary C26 tumor specimens of 8–10 mm mean diameter were finely minced with blades and digested by incubation with 1 mg/mL collagenase IV (Sigma Aldrich) in RPMI-1640 at 37°C for 1 h in an orbital shaker. After washing in RPMI-1640 with 10% FBS, the cell suspension was passed through a 70 µm pore cell strainer, centrifuged at 1400 rpm for 10 min and incubated in erythrocyte lysing buffer (155 mM NH₄Cl, 15.8 mM Na₂CO₃, 1 mM EDTA, pH 7.3) for 10 min at room temperature. After washing in RPMI-1640 with 10% FBS, 10⁶ cells were collected, re-suspended in PBS, and treated with Fc-receptor blocker (anti-CD16/CD32 antibody, #101302, Biolegend). Splenocytes were collected by smashing spleens from vaccinated mice on a 40 µm pore cell strainer, centrifuging the resulting cells at 1,400 rpm for 10 min and incubating them in erythrocyte lysing buffer, followed by treatment with Fc-receptor blocker. 50 µL heparinized blood collected by intracardiac injection were incubated with erythrocyte lysing buffer, washed and incubated with Fc-receptor blocker. After Fc-receptor blocking, all samples were stained for 30 min at 4°C with the following antibodies, as reported previously⁸¹:

* anti-CD45-VioGreen (#130-123-900), anti-CD3-FITC (#130-119-135), anti-CD4-APC/Vio770 (#130-119-134), anti-CD8-VioBlue (#130-123-865), anti-CD49b-PE (#130-102-337), anti-PD1-APC (#130-102-263), anti-CD11b-FITC (#130-113-234), anti-F4/80-PE/Vio770 (#130-118-459), anti-Ly6C-APC/Vio770 (#130-111-917), anti-Ly6G-VioBlue (#130-119-986), anti-MHCII-APC (#130-112-388), anti-CD11b-FITC (#130-113-234), anti-CD11c APC (#130-110-839) (all from Miltenyi Biotec) anti-CD69-PE/Vio770 (#104512, Biolegend), anti-CD206-PE (#141706, Biolegend) and anti-PDL1-PE (# 558091, BD Bioscience). Samples were acquired on BD-FACSVerse and cell populations (NK: CD3⁻ CD49b⁺; NKT: CD3⁺ CD49b⁺; CD4⁺ T cells: CD3⁺ CD49b⁻ CD4⁺; CD8⁺ T cells: CD3⁺ CD49b⁻ CD8⁺; DC: CD11b⁻ CD11c⁺; macrophages: CD11b⁺ F4/80⁺; m-MDSC: CD11b⁺ F4/80⁻ Ly6G⁻ Ly6Chigh; gMDSC: CD11b⁺ F4/80⁻ Ly6G⁺ Ly6Cdim/neg) analyzed with FlowJO10.5.3 and reported as percentage on the CD45⁺ leukocyte population, following doublets and dead cells elimination.

## Vaccine preparation and mouse immunization

A plasmid coding for chimeric rat and human ErbB2 extracellular and transmembrane domains (RHuT)⁸² and the corresponding control empty plasmid pVAX1 (Invitrogen, Monza, Italy) were amplified and then purified using the Endofree Qiagen Plasmid-Giga (Qiagen Inc., Cjatsworth, CA) following manufacturer's instructions. Vaccination was performed by injecting 50 µg of DNA diluted in 20 µL of 0.9% NaCl into the quadriceps muscle of anesthetized mice. Immediately after the vaccine injection, the muscle was electroporated by using an array needle electrode connected to an electroporator (Cliniporator, IGEA, Carpi, Italy). Two 25-ms trans-cutaneous low voltage electric pulses with an amplitude of 150 V spaced by a 300-ms interval were applied.⁸³ Vaccination was repeated 14 days later. Two weeks after, mice were culled and sera and spleens were collected.

## Antibody response

Blood samples were collected 14 days after the last vaccination and serum obtained by centrifugation. The concentration of anti-rat Her2 antibodies was determined by flow cytometry as the ability of diluted sera (1:200) to bind 3T3/NKB cells (BALB/c 3T3 NKB, expressing the rat ErbB2, H-2Kd, and B7.1 molecules),⁸⁴ which were a generous gift from Dr. Wei-ZenWei (Karmanos Cancer Institute, Detroit, MI, USA). A FITC-conjugated rabbit anti-mouse IgG antibody (F313, Dako, Milano, Italy) was used to detect the bound primary antibodies. Flow cytometry was performed with a BD-FACSVerse and samples were analyzed with FlowJO10.5.3, and antibody titers reported as mean fluorescence intensity (MFI).

## IFN-γ enzyme-linked immunospot (ELISpot) assay

Splenocytes from vaccinated mice were plated at 1 × 10⁶/well into 96-well HTS IP plates (Millipore) precoated with 5 µg/mL of rat anti-mouse IFN-γ (clone R4-6A2, BD Biosciences). Cells were then stimulated with 15 µg/mL of rat ErbB2 immunodominant peptide (TYVPANASL) peptide for 16 h in the incubator, or with concanavalin A (2 µg/ml) or medium alone as positive and negative controls, respectively. Images of the wells were acquired, and IFN-γ spots enumerated, with a microplate reader, along with a computer-assisted image analysis system (Immunospot; CTL Europe, Bonn, Germany), as previously described.⁸²

## QUANTIFICATION AND STATISTICAL ANALYSIS

All graphs show mean ± SEM and were analyzed with GraphPad Prism (version 6.0, GraphPad Software). Detailed statistical tests are reported in figure legends, where n represents the total number of independent experiments. For in vivo data, n is referred to the number of mice per group.

Statistical significance was tested with unpaired t-test when two groups of data were compared, with one-way ANOVA followed by Sidak's multiple comparison test when more than two groups were analyzed. Two-way ANOVA followed by Sidak's multiple comparison test was performed when two variables were analyzed. When relevant, one sample t-test was performed. For human biopsies analysis non-parametric Kruskal-Wallis test followed by Benjamini, Krieger and Yekutieli multiple comparison test was performed. Significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001. The illustrations were created on Biorender.com and all figures were assembled using Adobe Illustrator (AI, 2022).