The central idea behind the work is that cancer cells possess cytoprotective machinery that helps them survive stress and evade the immune system. One of the most important of these mechanisms is the unfolded protein response, or UPR, a signaling network that restores protein homeostasis when the endoplasmic reticulum becomes overloaded with misfolded proteins. The researchers hypothesized that if they could intensify and prolong proteostatic stress inside tumor cells without killing them outright, they could disrupt that survival machinery and simultaneously make the tumor more visible to the immune system. LOFU offered an ideal tool for the job. Unlike high-intensity focused ultrasound, or HIFU, which ablates tissue through coagulative necrosis, LOFU induces thermal and mechanical stress in cells while leaving them intact—enough stress to trigger immunogenic changes, but not enough to destroy the tissue.

To test this, the team used their in-house ultrasound platform, called APTx, which combines two coaxially mounted transducers operating at 1.1 megahertz and 250 kilohertz, focused simultaneously on the tumor center for a continuous three-second exposure. They studied three escalating intensities—80, 160, and 240 watts per square centimeter—in mice bearing orthotopic TSA tumors on a BALB/c background and E0771 tumors on a C57BL/6 background. A fine-gauge thermocouple implanted in the tumor core revealed a dose-dependent thermal response: untreated tumors sat at roughly 25 degrees Celsius, and each increase in intensity raised the maximum internal tumor temperature by about 5 degrees, reaching approximately 38 degrees Celsius at the highest setting. That temperature is warm enough to stress cells but far below the threshold for ablation, which is precisely the balance the researchers were seeking.

The molecular readouts confirmed that the acoustic stress was registering inside the tumor cells. Five hours after a single LOFU treatment, the team harvested tumor tissue and used quantitative reverse-transcription PCR to measure the expression of 62 genes associated with the unfolded protein response and 62 genes linked to heat shock proteins. Both gene sets were upregulated in a direct dose-dependent fashion with increasing intensity, and the most pronounced increase—without any tissue ablation—occurred at 240 watts per square centimeter, which became the standard dose for all subsequent experiments. Heatmaps of individual genes showed statistically significant upregulation across key branches of the UPR, including the PERK-ATF4 signaling pathway. Notably, the gene Eif2ak, which encodes PERK, was among those upregulated; PERK phosphorylates eIF2α, attenuating canonical translation and shifting cells toward non-canonical mRNA translation, a process previously linked to the generation of peptides presented on major histocompatibility complex class I molecules.

The stress response also translated into immunogenic cell death signals. Within 24 hours of a three-day LOFU regimen, the researchers observed cytosolic heat shock proteins—specifically BiP, HSP90, and HSP40—translocating to the surface of tumor cells, where they can act as damage-associated molecular patterns that activate dendritic cells and promote cross-presentation of tumor antigens to CD8-positive T cells. Using TSA cells engineered to carry a red fluorescent protein tag on the chromatin protein HMGB1, the team showed that LOFU treatment progressively increased the release of HMGB1 into the culture medium over 72 hours, another hallmark of immunogenic cell death. Interestingly, LOFU alone did not induce surface expression of calreticulin, an eat-me signal that radiation therapy does trigger, which is one reason the researchers chose to combine the two modalities rather than rely on ultrasound alone.

When the team added radiation to the protocol—delivering sub-ablative doses of 8 or 10 gray per fraction via a small-animal radiation platform two to four hours after each LOFU session—the combination produced changes that neither therapy achieved on its own. Radiation increased surface expression of the co-stimulatory molecules CD40 and CD80 on tumor cells, and the LOFU-plus-radiation combination additionally reduced expression of CD47, the do-not-eat-me signal that normally shields tumor cells from phagocytosis. In survival studies, mice receiving the combination therapy lived significantly longer than those receiving radiation alone, and tumor volumes in the combination group were substantially lower than in the radiation monotherapy group at later time points in both the TSA and E0771 models. LOFU monotherapy, by contrast, was less effective at controlling tumors than no treatment at all, underscoring that the ultrasound works best as a priming agent rather than a standalone therapy.

Two experiments pointed decisively to T cells as the mediators of the combination’s benefit. First, the survival advantage seen in wild-type mice disappeared entirely in athymic nude mice, which lack a functional T cell population. Second, in a dual-tumor model in which only the primary tumor was treated, the combination therapy reduced not only the treated tumor but also the untreated secondary tumor on the opposite flank—a systemic abscopal effect that radiation monotherapy failed to produce. Immunophenotyping of tumors ten days after treatment revealed why: while radiation increased the overall percentage of CD8-positive T cells infiltrating both tumor types, it also raised the proportion of regulatory T cells, or Tregs, in TSA tumors, an immunosuppressive population that can blunt the anti-tumor response. Adding LOFU prevented that radiation-induced Treg expansion, resulting in a significantly elevated ratio of CD8-positive T cells to Tregs in the combination group compared with untreated controls in both models. That ratio is clinically meaningful in breast cancer, where a higher CD8-to-FOXP3-positive Treg ratio in the tumor stroma correlates with improved survival.

The team also probed the quality of the T cell response, not just its quantity. Within the effector memory T cell subset—antigen-primed cells marked by CD44-positive, CD62L-negative expression that can position themselves in both lymphoid and peripheral tissues—the combination therapy reduced the percentage of cells expressing FAS in both CD8-positive and CD4-positive populations and increased the percentage of CD8-positive effector memory cells expressing the transcription factor Tbet, a marker of a Th1-biased cytotoxic program. Radiation alone produced some of these changes, but the combination was more effective. Cytokine analysis told a similar story: radiation increased interferon-gamma secretion in both CD8-positive and CD4-positive T cells, and the combination showed a trend toward even greater interferon-gamma production. Curiously, the radiation-induced upregulation of tumor necrosis factor alpha seen in monotherapy was not observed in the combination groups, hinting at a nuanced interplay between the two modalities in shaping the cytokine milieu of the tumor microenvironment.

Perhaps most striking was the evidence that the combination therapy generates a genuinely tumor-specific adaptive immune response. Using tetramer staining to identify T cells recognizing defined tumor-associated antigens, the researchers found that the combination significantly increased the infiltration of anti-gp70 CD8-positive T cells in TSA tumors—gp70 being an overexpressed epitope derived from an endogenous murine leukemia retrovirus—and significantly raised the anti-gp70 CD8-to-Treg ratio above both untreated and radiation-only groups. In E0771 tumors, the combination group showed the highest percentage of anti-p15E CD8-positive T cells, though that trend did not reach statistical significance, possibly because p15E is not an established tumor-associated antigen in that model. The quality of the anti-gp70 cells also improved under the combination, with fewer cells expressing FAS and more expressing Tbet compared with radiation alone.

The authors are careful to note that the combination is not curative: tumors that regressed to non-palpable sizes frequently recurred, echoing the clinical reality that roughly 15 percent of early-stage breast cancer patients experience local recurrence within 15 years. Still, the findings carry significant translational weight. The team showed that LOFU allows a 20 percent reduction in radiation dose while maintaining tumor control comparable to the higher dose alone—a meaningful margin, given that a 20 percent dose reduction could virtually eliminate serious side effects in radiation-sensitive patients. The researchers plan to explore whether adding anti-PD-1 immunotherapy or surgical resection after treatment can convert transient regression into durable control, and to dissect the role of tumor-associated macrophage polarization in the ultrasound-primed microenvironment. For now, the study positions LOFU as a promising non-ablative priming tool that, paired with radiation, turns the tumor itself into something resembling an in-situ vaccine—one that teaches the immune system to recognize and attack cancer both at the treated site and, potentially, far beyond it.

Subject of Research: Low-intensity focused ultrasound combined with radiation therapy enhances antitumor immunity in murine breast cancer models

Article Title: Enhancing tumoricidal effects of radiation therapy with low-intensity focused ultrasound in murine breast cancers

Article References: Schumacher, M. M., Gutierrez Chavez, C., Malachowska, B., Pandey, S., Tchaikovskaya, T., Singh, S., Barry, S., Macedo, R., & Guha, C. (2025). Enhancing tumoricidal effects of radiation therapy with low-intensity focused ultrasound in murine breast cancers. Frontiers in Acoustics, 3, Article 1616297. https://doi.org/10.3389/facou.2025.1616297

Image Credits: AI Generated

DOI: 10.3389/facou.2025.1616297

Keywords: low-intensity focused ultrasound, LOFU, radiation therapy, breast cancer, tumor microenvironment, immunotherapy, CD8-positive T cells, regulatory T cells, unfolded protein response, heat shock proteins, immunogenic cell death, abscopal effect