Pulsed-Focused Ultrasound Slows B16 Melanoma and 4T1 Breast Tumor Growth through Differential Tumor Microenvironmental Changes

Abstract

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.

Keywords: 4T1 breast cancer; B16 melanoma; focused ultrasound; immune response; tumor microenvironment.

Figure 1

(A) Schematic diagram of the experimental set-ups. Murine melanoma B16 or breast 4T1 cells were inoculated into C57BL/6 or BALB/c mice (n = 6 mice/tumor type/time point), respectively. Mice were treated with 6 MPa pFUS once tumor sizes reached ~5 mm in diameter. Tumors were harvested at different time points post-pFUS and were subjected to proteomic (top), transcriptomic (middle), immunohistochemistry, and flow cytometry (bottom) analyses. Tumor size volumes of (B) B16 melanoma (C) or 4T1 breast flank tumor model acquired from the third cohort study. Black lines indicate tumor size (mean ± SD) following pFUS treatment on day 0. Dashed black lines indicated an untreated control group. Asterisks indicate statistical significance (p < 0.05; t-test).

Figure 2

Time-course analysis of proteomic changes in TIME of mouse B16 melanoma and 4T1 breast tumor models (n = 6 mice/tumor type/time point) following pFUS. Heat maps depicting fold changes in CCTFs over time of mouse (A) B16 melanoma or (C) 4T1 breast tumor models 1, 8, or 24 h post-pFUS treatment. For each protein, time points were normalized to values detected on un-sonicated tumors at day 0. See also Figures S1 and S3 for raw data. Temporal proteomic profile of (B) B16 and (D) 4T1 flank tumor models harvested at 1, 3, or 5 days post-pFUS. Data presented as fold changes between the mean value detected from the pFUS-treated group to their time-matched untreated controls. Increased fold changes are indicated in red, while decreased fold changes are shown in blue. Fold change > 3.1 is displayed as dark red. Time points with p-values < 0.05 were considered statistically significant (ANOVA) and marked by asterisks IL: Interleukin; CCL: Chemokine (C-C motif) ligand; CXCL: Chemokine (C-X-C motif) ligand; ICAM: Intercellular adhesion molecule; IFNγ: Interferon-gamma; G-CSF: Granulocyte-colony stimulating factor; GM-CSF: Granulocyte macrophage-CSF; M-CSF: macrophage-CSF; LIF: Leukemic inducible factor; TGFβ: Transforming growth factor-beta; TNFα: Tumor necrosis factor-alpha; VCAM: vascular cell adhesion molecule; and VEGF: Vascular endothelial growth factor. See also Figures S2 and S4 for raw data.

Figure 3

Immune cell profiling following pFUS in B16 tumors. (A) Heat map of flow cytometry analysis (n = 6 mice/time point) for B16 tumor (B16), spleen (Sp) or lymph node (LN) compared to time-matched control tissues. Tissues were harvested 1 (left), 3 (middle), or 5 (right) days following pFUS; Increased fold changes are indicated in red while decreased fold changes are shown in blue. Fold change > 3.1 is displayed as dark red; (B) Quantitative values of immune cell profiles (mean ± SD) of mouse B16 tumor, Sp, or LN, dissected 1, 3, or 5 days following pFUS. y-axis represents the relationship between each cell population to the total cells detected; x-axis represents days post-pFUS. Asterisks indicate statistical significance (p < 0.05; ANOVA) between total cells detected in pFUS-treated mice (black squares) samples to time-matched controls (black circles). Helper T cells (T_h1), cytotoxic T lymphocytes (CTL), regulatory T cells (T_reg), natural killer (NK) cells, dendritic cells (DC), F_4/80 macrophages (M₁ and M₂), myeloid-derived suppressor cells (MDSC), cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1).

Figure 4

Immune cell profiling following pFUS in 4T1 tumors. (A) Heat map of flow cytometry analysis (n = 6 mice/time point) results for 4T1 tumor (4T1), spleen (Sp) or lymph node (LN) compared to time-matched control tissues. Tissues were harvested 1 (left), 3 (middle) or 5 (right) days following pFUS; Increased fold changes are indicated in red while decreased fold changes are shown in blue. Fold change > 3.1 is displayed as dark red; (B) Quantitative values of immune cell profiles (mean ± SD) of mouse 4T1, Sp, or LN, right, dissected 1, 3 or 5 days following pFUS. y-axis represents the relationship between each cell population to the total cells detected; x-axis represents days post-pFUS. Asterisks indicate statistical significance (p < 0.05; ANOVA) between total cells detected in pFUS-treated mice (black squares) samples to time-matched controls (black circles). Helper T cells (T_h1), cytotoxic T lymphocytes (CTL), regulatory T cells (T_reg), natural killer (NK) cells, dendritic cells (DC), F_4/80 macrophages (M₁ and M₂), myeloid-derived suppressor cells (MDSC), cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1).

Figure 5

(A) Gene ontology (GO) categories of top significantly enrichment obtained from B16 (red bars) and 4T1 (black bars) flank tumors 1 (left) or 12 (right) h following pFUS treatment compared to un-sonicated controls. Log₂ fold change for each gene is shown in its respective bar. (B) Hierarchical clustering of gene expression signature associated with hypoxia, KRAS signaling, epithelial to mesenchymal transition (EMT), and Ca²⁺ signaling. GO of biological processes gene sets for B16 (C) or 4T1 (D) tumors 1 (left) or 12 (right) h post-pFUS compared to un-sonicated controls. (n = 5 mice/tumor type/time point).

Figure 6

Representative images of Ki-67 localization within sectioned (A) B16 melanoma or (B) 4T1 breast flank tumor sections 1 (top), 3 (middle), or 5 (bottom) days following pFUS treatment (right) or untreated control tumors (left) (n = 6 sections/tumor type/time point). Quantitative analysis of Ki-67 localization within (C) B16 tumors or (D) 4T1 tumor area. The upper and lower bounds of the boxplots denote the 25th and 75th percentiles, while the midlines indicating the mean values. Whiskers indicate values outside the upper/lower quartile and within standard deviations. Asterisks indicate statistical significance (p < 0.05; unpaired t-test) between tumor-bearing mice treated with pFUS to time-matched untreated controls. (scale 100 µm).