Table 2.

Studies of FUS-immunomodulation outside the CNS

	Ref	Year	Model	Ultrasound Parameters	Key Observations
Pre-Clinical and Clinical FUS Thermal Ablation.	53	2005	MC-38 mouse colon adenocarcinoma cell line	Frequency: 1.1 MHz Focal length: 63 mm Acoustic exposure conditions: Thermal HIFU: P- = 6.7 MPa, 30% duty cycle, 5 s Mechanical HIFU: P- = 10.7 MPa, 3% duty cycle, 30 s	HIFU treatment in vitro caused increased expression of ATP and Hsp60 APCs exposed to supernatant isolated from HIFU-treated tumor cells elevated CD80 and CD86 expression DCs and macrophages increased IL-12 and TNF-α secretion, respectively, in response to supernatant exposure Mechanical HIFU exposure condition surpassed its thermal counterpart in terms of ability to activate APCs
	54	2008	Reporter FVB mice transgenic for Hsp70-luc2A-eGFP	Frequency: 1.5 MHz Focal length: 5.1 cm Acoustic intensity: 53-352 W/cm2 Exposure time: 1s	HIFU can induce Hsp70 expression up to 96 hours post-heating Peak expression levels are observed between 6-48 hours following exposure
	55	1998	LNCaP cells, prostatic stromal cells (in vitro studies) 5 patients with clinically localized prostate cancer (clinical studies)	In vitro studies: Sublethal heating at 43°C, 46°C, or 49°C for 60 min in a water bath Clinical studies: Frequency: 4.0 MHz Available focal lengths: 2.5, 3.0, 3.5, and 4.0 cm Acoustic intensity: 1,260-2,200 W/cm2 Exposure time: 4s on followed by 12s off for re-positioning	Sublethal heat shock caused elevated Hsp27 expression by 3-4-fold in LNCaP cells Hsp27 expression was consistently observed at the borders of thermonecrosis in vivo, with strongest levels occurring at 2-3 hours following transrectal HIFU
	56	2006	23 patients with biopsy-proven breast cancer	Frequency: 1.6 MHz Focal length: 90 mm, Acoustic intensity: 5,000-15,000 W/cm2 Exposure time: 45-150 mins (median: 1.3 h)	All tumors treated with HIFU stained positive for epithelial membrane antigen and Hsp70 No tumors treated with HIFU stained positive for CD44v6, MMP9, or PCNA
	57	2004	6 patients with clinically localized prostate cancer	Frequency: 4 MHz Focal lengths: 3.0, 3.5 or 4.0 cm Acoustic intensity: 1260-2000 W/cm2 Exposure time: 4s on followed by 12s off for re-positioning	Hsp72, Hsp73, GRP-75, and GRP78 were overexpressed at the margins of HIFU treated regions
	58	2015	Subcutaneous B16F10 melanoma in female C57BL/6J mice	Frequency: 9.3 MHz Acoustic intensity: 4.5 W Focal length: Not provided Exposure time; 10s per location (120s total per tumor nodule)	HIFU treatment resulted in increased circulating TNF-α and IFN-γ, decreased circulating tumor cells, reduced pulmonary metastatic burden, and cumulative survival benefit. In vitro studies revealed a role for CD86 in driving anti-tumor immune effects in response to lifting of inhibition by miR-134.
	59	2012	Subcutaneous H22 hepatocellular carcinoma in female C57BL/6J mice	Frequency: 9.5 MHz Focal length: 8 mm Acoustic intensity: 5W Exposure time: 180-240s (median: 220s)	HIFU treatment elevated CTLs, TNF-α and IFN-γ secretion, and MHC class I/CD8+ cells versus sham and control
	60	2010	Subcutaneous H22 hepatocellular carcinoma in male and female C57BL/6J mice	Frequency: 9.5 MHz Focal length: 8 mm Acoustic intensity: 5 W Exposure time: 180-240s (median: 220s)	Mice immunized with DCs loaded with HIFU-ablated tumor lysate demonstrated increased magnitude of mature DCs and greater IL-12 and IFN-γ secretion compared to those immunized with mouse serum-loaded DCs. CTL cytotoxicity and TNF-α and IFN-γ secretion were significantly higher in mice immunized with HIFU tumor debris-loaded DCs.
	61	2010	Subcutaneous H22 hepatocellular carcinoma in male and female C57BL/6J mice	Frequency: 9.5 MHz Focal length: 8 mm Acoustic intensity: 5 W Exposure time: 180-240s (median: 220s)	Vaccination with HIFU-ablated tumor lysate resulted in elevated tumor-specific cytolytic activity compared to untreated tumor lysate vaccination, HIFU treatment alone, and control. HIFU-generated vaccine significantly reduced tumor growth and conferred 100% survival. Elevated expression of MHCII, CD80, CD86 and cytokine secretion (IL-12, IFN-γ) resulted from exposure of bone marrow DCs to HIFU-ablated or untreated tumor lysates in vitro.
	64	1992	Subcutaneous Cl300 neuroblastoma in male Ajax inbred mice	Frequency: 4 MHz Focal length: 8 cm Acoustic intensity: 550 W/cm2 Exposure time: 5s on followed by 5s off	Tumors ablated with thermal HIFU underwent significant growth inhibition and extended survival compared to untreated controls. Mice challenged with contralateral tumors displayed secondary (untreated) tumor growth reduction in response to treatment of primary tumor with HIFU.
	65	2010	Subcutaneous MC38 colon adenocarcinoma and B16 melanoma in female C57BL/6 mice	Frequency: 3.3 MHz Focal length: 63 mm Acoustic intensity: P+ / P- = 19.5/7.2 MPa Exposure time: 4s	Application of thermal HIFU to tumors mediated greater recruitment of DCs to lesion periphery (<55 oC) than center (up to 80 oC), with spare-scan technique yielding stronger anti-tumor immune response compared to dense-scan technique
	66	2017	Orthotopic neu exon deletion line model of mammary adenocarcinoma in FVB/n mice	Frequency: 3 MHz Focal length: Not provided Acoustic intensity: 5W (3.1 MPa) Scan speed: 1 revolution/s	Priming with immunotherapy 7 days prior to HIFU treatment resulted in decreased macrophages and MDSCs, increased CD8+ T cells secreting IFN-γ and PDL1+CD45+ cells, and elevated proportion of M1 macrophages Abscopal effect in the presence of increased tumor burden was more robust when immunotherapy priming preceded HIFU and lost when immunotherapy and HIFU were administered concomitantly.
	71	2009	48 female patients with biopsy-proven breast cancer	Frequency: 1.6 MHz Focal length: Not provided Acoustic intensity: 5,000- 15,000 W/cm2 Exposure time: 45-150 mins (mean: 1.3 h)	Neoplasms treated with HIFU expressed elevated NK cells as well as CD3+, CD4+, CD8+, and B lymphocytes in the ablated periphery. TILs positive for granzyme, FasL, and perforin were also greater in response to HIFU as compared with untreated control tumors.
	72	2004	16 patients with solid malignancies (osteosarcoma, hepatocellular carcinoma, renal cell carcinoma)	Frequency: 0.8 MHz Focal length: 135 mm Acoustic intensity: 5000-20000 W/cm2 Exposure time: Variable Therapeutic time: 2.5-8 h (median: 5.2 h)	Circulating CD4+ lymphocytes as well as the CD4+/CD8+ ratio increased in patients receiving HIFU
	73	2009	48 female patients with biopsy-proven breast cancer	Frequency: 1.6 MHz Focal length: Not provided Acoustic intensity: 5,000- 15,000 W/cm2 Exposure time: 45-150 mins (mean: 1.3 h)	HIFU-treated tumors were observed to have APCs infiltrating along the margins of ablation, with an overall increase in DCs, macrophages, and B cells as compared with control. CD80, CD86, and HLA-DR were more highly expressed on DCs and macrophages infiltrating HIFU-treated tumors.
	74	2008	15 patients with solid malignancies	Frequency: 0.8 MHz Focal length: Not provided Acoustic intensity: 5000-20,000 W/cm2 Exposure time: 0.78-3.62 h (mean: 2.74 h)	Patients exposed to complete or partial HIFU ablation experienced a reduction in serum immunosuppressive cytokine expression levels, with nonmetastatic patients experiencing lower expression levels as compared with metastatic patients VEGF, TGF-β1, and TGF-β2 were significantly reduced following HIFU treatment
Pre-Clinical FUS Mechanical Ablation.	63	2012	Subcutaneous RM-9 prostate cancer in C57BL/6J mice	Frequency: 3.3 MHz Focal length: Not provided Acoustic intensity: P+ / P- = 32/10 MPa (60 W) Exposure time: 20s (2% duty cycle)	Mechanical HIFU treatment (<45oC) and subsequent primary tumor resection attenuated intratumoral STAT3 activity, resulting in increased CTLs in spleens and TDLNs, and tumor growth inhibition upon rechallenge Number and activity of DCs was increased as a function of HIFU+surgery compared to surgery alone while immunosuppressive burden was alleviated
	67	2007	Subcutaneous H22 hepatocellular carcinoma in male and female C57BL/6J mice	Frequency: 3.3 MHz Focal length: 63 mm Acoustic exposure conditions: Thermal HIFU P+ / P- = 19.9/7.7 MPa, 3s Mechanical HIFU P+ / P- = 34.1/12.5 MPa, 2% duty cycle, 30s	Ablation with thermal and mechanical HIFU resulted in 3.1- and 4.1-fold increases in CD11c+ DCs, respectively, and 5- and 10-fold increases in TDLN CFSE+ DC accumulation, respectively. Both ablative protocols controlled tumor growth and conferred protection against tumor rechallenge Tumors ablated under mechanical HIFU protocol had stronger elevation tumor-specific CTL activity and IFN-γ secreting cells
Pre-Clinical Low-Intensity FUS.	17	2012	Subcutaneous CT-26 colon carcinoma in BALB/cByJNarl mice	Frequency: 0.5 MHz Focal length: Not provided Acoustic intensity: P- = 0.6 MPa (5 We) or 1.4 MPa (30 We) Exposure time:20s (total sonication time between 180-240s) Microbubble type: Sonovue	Tumors exposed to low-intensity FUS and microbubbles experienced a transient increase in non-regulatory T cell infiltration as well as sustained elevation of CTLs, which further translated to restriction of tumor growth.
	68	2015	Subcutaneous K1735 melanoma in C3H/HeN mice	Frequency: 3 MHz (unfocused) Acoustic intensity: 2.3 W/cm2 (0.22 MPa) Exposure time: 1 or 3 mins Microbubble type: Definity	Low-intensity antivascular US treatment significantly reduced tumor perfusion at both exposure times, while increasing HIF1A+ cells and CD45+CD3+ T cell infiltration in tumors
	69	2016	B16 melanoma in C57BL/6 and BALB/c nude mice	Frequency: 1 MHz Non-ablative low-intensity FUS: Focal length: 80* or 85** mm Exposure time: 1.5 s (5 min total per tumor) Acoustic intensity: 550 W/cm2 *P- = 2.93 MPa (3W) **P- = 3.81 MPa (3W) High-intensity ablation Focal length: 80 mm Exposure time: 4s (75% duty cycle) Acoustic intensity: P- = 5.42 MPa (12.5W)	Non-ablative, low-intensity FUS conferred increased tumor antigen presentation and Hsp70 presence on tumor cell membranes, and led to reversal of T cell tolerance within tumors. Combination of this regimen with fractionated radiation therapy led to control of pulmonary metastatic burden and extended recurrence-free survival.
	70	2015	Orthotopic neu exon deletion line model of mammary adenocarcinoma in FVB/n mice	Frequency: 1.54 MHz Focal length: Not provided Acoustic intensity: P- = 1.1 MPa Exposure time: 5 mins	In mice with multiple tumor sites, the combination of ultrasound with copper-doxorubicin liposomes and CpG controlled tumor growth and extended survival in the context of systemic disease. CD4+ and CD8+ T cell magnitudes increased and MDSCs decreased as a function of treatment in both primary (treated) and contralateral tumors.
	16	2015	Subcutaneous xenograft model of CEA-expressing LS-174T human colorectal adenocarcinoma in female NSG mice	Frequency: 510 kHz Focal length: Not provided Acoustic intensity: 0.25 and 0.5 MPa Exposure time: 10 ms every second for 1 min; Microbubble type: Optison	Low-intensity focused ultrasound with microbubbles conferred significant accumulation of adoptively transferred iron-oxide labeled human NK cells at 0.5 MPa. Accumulation in the tumors lasted up to 24 hours.