Image-guided focused ultrasound (FUS) has been successfully employed as an ablative treatment for solid malignancies by exposing immune cells to tumor debris/antigens, consequently inducing an immune response within the tumor microenvironment (TME). To date, immunomodulation effects of non-ablative pulsed-FUS (pFUS) on the TME are poorly understood. In this study, the temporal differences of cytokines, chemokines, and trophic factors (CCTFs) and immune cell populations induced by pFUS were interrogated in murine B16 melanoma or 4T1 breast cancer cells subcutaneously inoculated into C57BL/6 or BALB/c mice. Natural history growth characteristics during the course of 11 days showed a progressive increase in size for both tumors, and proteomic analysis revealed a shift toward an immunosuppressive TME. With respect to tumor natural growth, pFUS applied to tumors on days 1, 5, or 9 demonstrated a decrease in the growth rate 24 h post-sonication. Flow cytometry analysis of tumors, LNs, and Sp, as well as CCTF profiles, relative DNA damage, and adaptive T-cell localization within tumors, demonstrated dynamic innate and adaptive immune-modulation following pFUS in early time points of B16 tumors and in advanced 4T1 tumors. These results provide insight into the temporal dynamics in the treatment-associated TME, which could be used to evaluate an immunomodulatory approach in different tumor types.

Focused ultrasound (FUS) has shown promise as a non-invasive treatment modality for solid malignancies. FUS targeting to tumors has been shown to initiate pro-inflammatory immune responses within the tumor microenvironment. Pulsed FUS (pFUS) can alter the expression of cytokines, chemokines, trophic factors, cell adhesion molecules, and immune cell phenotypes within tissues. Here, we investigated the molecular and immune cell effects of pFUS on murine B16 melanoma and 4T1 breast cancer flank tumors. Temporal changes following sonication were evaluated by proteomics, RNA-seq, flow-cytometry, and histological analyses. Proteomic profiling revealed molecular changes occurring over 24 h post-pFUS that were consistent with a shift toward inflamed tumor microenvironment. Over 5 days post-pFUS, tumor growth rates were significantly decreased while flow cytometric analysis revealed differences in the temporal migration of immune cells. Transcriptomic analyses following sonication identified differences in gene expression patterns between the two tumor types. Histological analyses further demonstrated reduction of proliferation marker, Ki-67 in 4T1, but not in B16 tumors, and activated cleaved-caspase 3 for apoptosis remained elevated up to 3 days post-pFUS in both tumor types. This study revealed diverse biological mechanisms following pFUS treatment and supports its use as a possible adjuvant to ablative tumor treatment to elicit enhanced anti-tumor responses and slow tumor growth.

Focused ultrasound (FUS) ablation has been successfully used for thermal ablation of solid malignancies. However, non-ablative pulsed-FUS (pFUS) has received less attention but could be less destructive anti-tumor therapy by stimulating a pro-inflammatory immune response within the tumor microenvironment (TME). Clinically, such an approach could be useful to treat peripheral disease (e.g. micro-metastases) in otherwise normal-appearing tissues. Here, we explored the alterations of cytokines, chemokines and trophic factors (CCTF) and cell adhesion molecules (CAM) in the TME of mouse flank tumor models following pFUS treatment at different time points of tumor progression. Mice were bilaterally inoculated with subcutaneous B16 melanoma or 4T1 breast tumors into the hind limbs and allowed to reach diameters of ~5 mm (n=6/group/timepoint) before performing experiments (Day 1). Separate groups of mice were subjected to pFUS (1 MHz; Peak negative pressure = 6 MPa; Duty cycle = 10%, 100 pulses per site) at Days 1, 5, or 10 and euthanized 24 hr post-pFUS for proteomic analyses. Separate groups of time-matched tumor-bearing mice that did not receive pFUS were also euthanized at the same time points both to assess relative changes due to pFUS at each time point and provide information regarding the natural history of TME progression in each tumor type. Proteomic analyses in untreated control tumors revealed an overall shift toward immunosuppressive (pro-tumor) TME over the 10 days. When comparing proteomic effects of pFUS treatment in compared to untreated time-matched controls, tumors treated on Day 1 exhibited greater upregulation of pro-inflammatory (anti-tumor) CCTF and CAM and pFUS had less pronounced effects on the TME when administered in groups of mice treated at Days 5 or 10. To further examine the acute effects of pFUS at on CCTF and CAM over 72hr. pFUS treatment in elicited unique CCTF and CAM profiles for the two tumor types, but both could be considered shifted towards anti-tumoral TME, especially within the first 24 hrs. These data suggest that pFUS can potentially modulate TME in a therapeutically beneficial way. However, they reveal two important caveats that require substantial investigation: 1) Different tumor types have unique molecular responses and imply pFUS may be more suitable for certain tumor types; 2) Natural histories encompass dynamic TME changes as part of disease progression and appropriate timing of pFUS in each tumor type will be necessary to maximizing the potential therapeutic benefits of pFUS. Citation Format: Gadi Cohen, Parwathay Chandran, Lauren E. Tomlinson, Maggie E. Sundby, Rebecca M. Lorsung, Scott R. Burks, Joseph A. Frank. Characterization of temporal proteomic dynamics of murine breast and melanoma tumor microenvironments without and with pulsed focused ultrasound [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5606.

Various cellular and biological immunotherapies have revolutionized the treatment of human malignancies by augmenting endogenous immune responses within the tumor microenvironment (TME). Although non-ablative pulsed-FUS (pFUS) has received less attention, it has been successfully used as a noninvasive treatment of solid malignancies by stimulating a pro-inflammatory immune response within the TME. Such an approach could be useful to treat peripheral micro-metastases in normal-appearing tissues. In the present study, we interrogated the immune-modulatory changes that occur within the TME following pFUS exposure. Murine B16 melanoma and 4T1 mammary carcinoma cells were subcutaneously implanted into both mice flanks (n=10 tumors/group/time point). The natural histories cytokine, chemokine, trophic factor (CCTF), and cell adhesion molecule (CAM) expression was evaluated within the TME of untreated flank tumors over 11 days. Ultrasound-guided pFUS at 1 MHz and peak negative pressures of 6 MPa was administered to tumors after reaching ~5 mm in diameter. Temporal immune-related changes in tumors were assessed following sonication using using proteomic, transcriptomic and histological analyses. Natural history growth characteristics showed a progressive increase in size for both tumors, and proteomic analysis revealed a shift toward an immunosuppressive TME. An anti-tumor TME was detected following pFUS treatment with increased expression of various pro-inflammatory CCTF and CAM as well as reduced tumor growth rates. Transcriptomic analyses following sonication identified inverted gene expression patterns in the two tumor types. Functional analysis of B16 tumors revealed increased intracellular signaling pathways associated with immune response regulations, while 4T1 tumors demonstrated reduced expression of proliferation genes. This study provides a macroscopic overview on the temporal dynamics of the TME and underline the profound magnitude of tumoral heterogeneity between tumor types as well as through progressive stages of similar primary tumors. Nevertheless, the induced immune-modulation changes suggest that pFUS may alter the expression of pro-inflammatory CCTF, CAM, towards an anti-tumoral TME, and supports the potential use of pFUS as a neoadjuvant treatment approach. Citation Format: Gadi Cohen, Parwathy Chandran, Rebecca M. Lorsung, Lauren E. Tomlinson, Robert B. Rosenblatt, Scott R. Burks, Joseph A. Frank. Temporal immune changes of murine breast and melanoma tumor microenvironments following pulsed focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2790.