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. 2024 Jun;35(6):900-908.e2.
doi: 10.1016/j.jvir.2024.03.012. Epub 2024 Mar 18.

Histoplasty Modification of the Tumor Microenvironment in a Murine Preclinical Model of Breast Cancer

Alexander A Pieper  1 , Nicholas A Stowe  2 , Sarvesh Periyasamy  3 , Brian M Burkel  4 , Noah W Tsarovsky  5 , Ajay P Singh  6 , Alexander L Rakhmilevich  5 , Paul M Sondel  7 , Suzanne M Ponik  4 , Paul F Laeseke  8 , John-Paul J Yu  9
Affiliations

Histoplasty Modification of the Tumor Microenvironment in a Murine Preclinical Model of Breast Cancer

Alexander A Pieper et al. J Vasc Interv Radiol. 2024 Jun.

Abstract

Purpose: To develop a noninvasive therapeutic approach able to alter the biophysical organization and physiology of the extracellular matrix (ECM) in breast cancer.

Materials and methods: In a 4T1 murine model of breast cancer, histoplasty treatment with a proprietary 700-kHz multielement therapy transducer using a coaxially aligned ultrasound (US) imaging probe was used to target the center of an ex vivo tumor and deliver subablative acoustic energy. Tumor collagen morphology was qualitatively evaluated before and after histoplasty with second harmonic generation. Separately, mice bearing bilateral 4T1 tumors (n = 4; total tumors = 8) were intravenously injected with liposomal doxorubicin. The right flank tumor was histoplasty-treated, and tumors were fluorescently imaged to detect doxorubicin uptake after histoplasty treatment. Next, 4T1 tumor-bearing mice were randomized into 2 treatment groups (sham vs histoplasty, n = 3 per group). Forty-eight hours after sham/histoplasty treatment, tumors were harvested and analyzed using flow cytometry.

Results: Histoplasty significantly increased (P = .002) liposomal doxorubicin diffusion into 4T1 tumors compared with untreated tumors (2.12- vs 1.66-fold increase over control). Flow cytometry on histoplasty-treated tumors (n = 3) demonstrated a significant increase in tumor macrophage frequency (42% of CD45 vs 33%; P = .022) and a significant decrease in myeloid-derived suppressive cell frequency (7.1% of CD45 vs 10.3%; P = .044). Histoplasty-treated tumors demonstrated increased CD8+ (5.1% of CD45 vs 3.1%; P = .117) and CD4+ (14.1% of CD45 vs 11.8%; P = .075) T-cell frequency.

Conclusions: Histoplasty is a nonablative focused US approach to noninvasively modify the tumor ECM, increase chemotherapeutic uptake, and alter the tumor immune microenvironment.

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Figures

Figure 1.
Figure 1.
Histoplasty qualitatively altered the tumor extracellular matrix and decreased collagen fiber density. (a) Overview of the hypothesized effect of histoplasty on tumoral extracellular matrix destruction. After histoplasty treatment, decreased collagen within the tumor stroma was hypothesized to result in improved immune cell infiltration and chemotherapeutic tumor uptake. (b) Second harmonic generation (SHG) imaging of murine breast cancer collagen matrix before and after ex vivo tumor treatment with histoplasty. CT-FIRE quantification of collagen fiber morphology before and after histoplasty treatment with SHG. Of note, there was a nearly 30% decrease in the number of fibers present in the imaged field after histoplasty treatment. (Figure 1a was created with BioRender.)
Figure 2.
Figure 2.
Histoplasty improved chemotherapeutic uptake. (a) Experimental overview of histoplasty treatment and measurement of chemotherapeutic tumor uptake and retention. (b) Relative fold-change of murine breast cancer tumor fluorescence (±SEM) over the course of 2 days in tumors not treated with histoplasty or exposed to doxorubicin (Doxil; blue, control), tumors not treated with histoplasty and exposed to doxorubicin (red), and tumors treated with histoplasty and exposed to doxorubicin (green). P values were calculated using 2-way analysis of variance with Tukey multiple comparison correction: *P < .05; **P < .01; ***P < .001; ****P < .0001. Group comparisons not specifically outlined within the figure were not significantly different. (Figure 2a was created with BioRender.)
Figure 3.
Figure 3.
Histoplasty increased the ratio of antitumor immune cells in the tumor microenvironment while decreasing the ratio of immune suppressive myeloid-derived suppressive cells (MDSCs). (a) Experimental schema and representative flow cytometry dot plots of untreated (black) and histoplasty-treated (blue) tumor-associated immune cells. Flow cytometry analysis showed the ratio of (b) CD4+ and CD8+ T cells to CD45+ cells, macrophages to CD45+ cells, dendritic cells to CD45+ cells, and MDSCs to CD45+ cells in murine breast cancer tumors 2 days after treatment with histoplasty. P values were calculated using unpaired t test: *P < .05. Group comparisons not specifically outlined within the figure were not significantly different. (Figure 3a was created with BioRender.)
Figure 4.
Figure 4.
Antitumor effect of histoplasty in the 4T1 murine breast cancer model. (a) Mean tumor volume plots (±SEM) and (b) overall survival in the 4T1 breast cancer model from a single experiment, with 4–5 mice per group, showed group responses to no treatment (black) and histoplasty (blue). P value was calculated using log-rank testing. Group comparisons not specifically outlined within the figure were not significantly different.
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