Nano-Pulse Treatment Overcomes the Immunosuppressive Tumor Microenvironment to Elicit In Situ Vaccination Protection against Breast Cancer

Abstract

We previously reported that nano-pulse treatment (NPT), a pulsed power technology, resulted in 4T1-luc mammary tumor elimination and a strong in situ vaccination, thereby completely protecting tumor-free animals against a second live tumor challenge. The mechanism whereby NPT mounts effective antitumor immune responses in the 4T1 breast cancer predominantly immunosuppressive tumor microenvironment (TME) remains unanswered. In this study, orthotopic 4T1 mouse breast tumors were treated with NPT (100 ns, 50 kV/cm, 1000 pulses, 3 Hz). Blood, spleen, draining lymph nodes, and tumors were harvested at 4-h, 8-h, 1-day, 3-day, 7-day, and 3-month post-treatment intervals for the analysis of frequencies, death, and functional markers of various immune cells in addition to the suppressor function of regulatory T cells (Tregs). NPT was verified to elicit strong in situ vaccination (ISV) against breast cancer and promote both acute and long-term T cell memory. NPT abolished immunosuppressive dominance systemically and in the TME by substantially reducing Tregs, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). NPT induced apoptosis in Tregs and TAMs. It also functionally diminished the Treg suppression capacity, explained by the downregulation of activation markers, particularly 4-1BB and TGFβ, and a phenotypic shift from predominantly activated (CD44⁺CD62L^-) to naïve (CD44^-CD62L⁺) Tregs. Importantly, NPT selectively induced apoptosis in activated Tregs and spared effector CD4⁺ and CD8⁺ T cells. These changes were followed by a concomitant rise in CD8⁺CD103⁺ tissue-resident memory T cells and TAM M1 polarization. These findings indicate that NPT effectively switches the TME and secondary lymphatic systems from an immunosuppressive to an immunostimulatory state, allowing cytotoxic T cell function and immune memory formation to eliminate cancer cells and account for the NPT in situ vaccination.

Keywords: apoptosis; breast cancer; immunosuppression; in situ vaccination; memory T cells; myeloid-derived suppressor cells; nano-pulse treatment (NPT); regulatory T cells; tumor microenvironment; tumor-associated macrophages.

Figure 1

NPT tumor ablation results in in situ vaccination protection. (A) NPT and tumor re-challenging scheme. Female BALB/c mice with orthotopic tumors (6–8 mm) are treated with NPT. Animals tumor-free over 7 weeks are challenged orthotopically (4T1-luc) with 0.5 × 10⁶ live tumor cells. Ctrl: age-matched naïve mice without prior NPT. NPT: mice tumor-free over 7 weeks after NPT (100 ns, 40–50 kV/cm, 3 Hz, and 1000 pulses) are rechallenged with live tumor cells. (B,C) 4T1 tumor growth and survival curves of animals following a second live tumor challenge: the number of tumor-free (red) vs. total mice is indicated. ***: p < 0.001.

Figure 2

NPT elicits antitumor memory response. (A) Cell counts of CD8⁺ Trms per million CD45⁺ cells in dLNs. D0, D3, and D7: Days 0, 3, and 7. (B,D) CD8⁺ (B) and CD4⁺ (D) memory T cells in the blood and spleens of mice. Tcm: CD44⁺CD62L⁺ T cells; Tem: CD44⁺CD62L⁻ T cells. (C,E) IFN-γ⁺ CD8⁺ (C) and CD4⁺ (E) T cells from splenocytes after 6 h incubations with plate-bound anti-CD3. (F) IFN-γ product of splenocytes after 24 h incubation with tumor lysate. Groups: Tumor: untreated tumor-bearing mice, and NPT: NP-treated mice. Note: in B-F, tumor mice were at the end point for euthanasia (age 15–16 weeks), while NPT mice were euthanized post-treatment at 3 months (age 21–22 weeks). N = 3–5. Error bars: SD. *: p < 0.05, **: p < 0.01, and ***: p < 0.001 (one-way ANOVA or t-test).

Figure 3

NPT reverses the Treg dominance locally and systemically. Breast tumors were established with an injection of 1 × 10⁶ 4T1-luc cells into the posterior part of the mammary fat pad. The control group (n = 4) received the tumor inoculation only. The remaining mice underwent NPT (100 ns pulses, 50 kV/cm, 3 Hz, 1000 pulses) on Day 11 following tumor inoculation. The treated mice were euthanized 4 h (4 h), 8 h (8 h), and on Day 1 (D1), Day 3 (D3), and Day 7 (D7) post-NPT. Their tumor tissues, tumor-draining lymph nodes (dLNs), blood, and spleens were harvested. Control tissues were obtained from mice with untreated tumors. (A) Summary flow plots represent Foxp3⁺ Tregs and Foxp3^{− _} Tconv among the total CD4⁺ T cell population in the blood. (B–D) Quantitative bar graphs depict the percentage of Tregs among the CD4⁺ T cell population in blood (B), spleens (C), or dLNs (D). (E) The Tconv/Treg ratios dLNs. (F) TIL Tregs are represented in quantitative bar graphs as the percentage of Tregs among CD4⁺ TILs. (G) A standardized CD4 Treg vs. CD4 Tconv cell count. N = 4 per group. Error bars, SD. **: p < 0.01 and *: p < 0.05 (one-way ANOVA).

Figure 4

Changes in apoptosis among T cell subsets following NPT. (A,B) Summary flow plots represent Annexin V expression among activated and naïve Treg subsets in the dLN at 4 h and 1-day post-NPT. (C) Quantitative graph shows Annexin V expression among CD8, CD4 Tconv, CD4 total Treg, activated Treg, and naïve Treg subsets in the dLN at 4 h and 1 day post-NPT. N = 4 per group. Error bars, SD. (D) Quantitative graph shows the percentage of Annexin V expression increase, among the above subsets, from the untreated control to 4 h post-treatment. Error bars, SE. *: p < 0.05 and **: p < 0.01 (one-way ANOVA).

Figure 5

NPT eradicates activated Tregs and impairs Treg function. (A) In vitro suppression assay showed a reduced functional suppression capacity of Tregs isolated from the NP-treated mice. Tregs isolated from dLNs of tumor-bearing (Ctrl) or NP-treated (NPT) mice were incubated with CFSE-labeled responder cells at the Treg/Tresponder ratios of 1:1, 1:2, and 1:4 for 60 h in the presence of CD3/CD28 activation beads. Responder cell proliferation was analyzed based on the dilution of the CFSE dye. The quantitative plots represent the percentage suppression at each Treg/Tresponder ratio in the control and treatment groups. (B,D) Changes in activated and naïve Treg distribution in the dLN are represented in the summary flow plots (B) and quantitative bar graphs (D). (C,E,F) Phenotypic changes in the 4-1BB activation marker expression among Foxp3⁺ Tregs are represented in the summary flow plots (C) and quantitative bar graphs (E,F) in the dLN (C,E) and spleen (F). (G) Changes in TGFβ expression among Tregs in the dLN are represented in the quantitative bar graph. N = 4 per group. Error bars, SD. *: p < 0.05 and **: p < 0.01 (one-way ANOVA).

Figure 6

NPT diminishes intratumoral TAMs and MDSCs with differential characteristics. (A) Changes in intratumoral TAMs and MDSCs distribution on Day 1 and Day 3 post-treatment are shown in a representative flow plot and quantitative graph. (B,C) Intratumoral TAM (B) and MDSC (C) apoptosis representative flow plot indicated with mean ± SD are shown at 4 h post-NPT. (D) Changes in CD86 and MHC-II costimulatory marker expression among TAMs were examined on Day 1 or Day 3 post-treatment. A representative flow plot with mean ± SD is shown. N = 3–5 per group. Error bars, SD. *: p < 0.05 and **: p < 0.01 (one-way ANOVA).

Figure 7

The impact of NPT on the TME in breast cancer. NPT shifts the immune cell dynamics from a pro-tumor to an antitumor environment. Suppressor cells, including Tregs, TAMs, and MDSCs, are diminished. Tregs undergo apoptosis, exhibit a shift from an activated 4-1BB⁺ to a naïve 4-1BB⁻ phenotype, and demonstrate reduced functional suppression capacity post-treatment. Antitumor CD4 Tconv and CD8⁺ CD103⁺ resident memory T cells increase in frequency.