Combination of ultrasound-based mechanical disruption of tumor with immune checkpoint blockade modifies tumor microenvironment and augments systemic antitumor immunity

Abstract

BackgroundDespite multimodal adjuvant management with radiotherapy, chemotherapy and hormonal therapies, most surgically resected primary breast cancers relapse or metastasize. A potential solution to late and distant recurrence is to augment systemic antitumor immunity, in part by appropriately presenting tumor antigens, but also by modulating the immunosuppressive tumor microenvironment (TME). We previously validated this concept in models of murine carcinoma treated with a novel predominately microcavitating version of high-intensity focused ultrasound (HIFU), mechanical high-intensity focused ultrasound (M-HIFU). Here we elucidated the mechanisms of enhanced antitumor immunity by M-HIFU over conventional thermal high-intensity focused ultrasound (T-HIFU) and investigated the potential of the combinatorial strategy with an immune checkpoint inhibitor, anti-PD-L1 antibody.MethodsThe antitumor efficacy of treatments was investigated in syngeneic murine breast cancer models using triple-negative (E0771) or human ErbB-2 (HER2) expressing (MM3MG-HER2) tumors in C57BL/6 or BALB/c mice, respectively. Induction of systemic antitumor immunity by the treatments was tested using bilateral tumor implantation models. Flow cytometry, immunohistochemistry, and single-cell RNA sequencing were performed to elucidate detailed effects of HIFU treatments or combination treatment on TME, including the activation status of CD8 T cells and polarization of tumor-associated macrophages (TAMs).ResultsMore potent systemic antitumor immunity and tumor growth suppression were induced by M-HIFU compared with T-HIFU. Molecular characterization of the TME after M-HIFU by single-cell RNA sequencing demonstrated repolarization of TAM to the immunostimulatory M1 subtype compared with TME post-T-HIFU. Concurrent anti-PD-L1 antibody administration or depletion of CD4+ T cells containing a population of regulatory T cells markedly increased T cell-mediated antitumor immunity and tumor growth suppression at distant, untreated tumor sites in M-HIFU treated mice compared with M-HIFU monotherapy. CD8 T and natural killer cells played major roles as effector cells in the combination treatment.ConclusionsPhysical disruption of the TME by M-HIFU repolarizes TAM, enhances T-cell infiltration, and, when combined with anti-PD-L1 antibody, mediates superior systemic antitumor immune responses and distant tumor growth suppression. These findings suggest M-HIFU combined with anti-PD-L1 may be useful in reducing late recurrence or metastasis when applied to primary tumors.

Rational Nanomedicine Design Enhances Clinically Physical Treatment‐Inspired or Combined Immunotherapy

“…As they were used in mouse neuroblastoma, immune escape blockade using aPD-L1 and mAb was implemented after HIFU ablation, and they cooperatively caused the potent anti-tumor immune responses of the whole body and reduced the recurrence and metastasis of advanced tumors. [120,125] Similar results were obtained in the mouse pancreatic cancer model. [126] PEI-PLGA-NaHCO 3 -NPs developed by the Wang et al showed an effective tumor-targeting effect in the 4T1 breast cancer model, which was much preferable for enhancing HIFU treatment.…”

Combination of a novel heat shock protein 90-targeted photodynamic therapy with PD-1/PD-L1 blockade induces potent systemic antitumor efficacy and abscopal effect against breast cancers

“…Cells were washed with PBS, stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, Rockford, Illinois, USA), and followed by intracellular staining and/or cell surface staining as previously described. 22 23 26 Detailed staining methods are shown in online supplemental methods . Cells were acquired by LSRII flow cytometer (BD Biosciences, San Jose, California, USA) and analyzed by FlowJo software IX (BD Biosciences).…”

“…Mouse IFN-γ ELISPOT assays (Mabtech, Cincinnati, Ohio, USA) were done according to the manufacturer’s instructions as previously described. 22 27 Overlapping HER2 or HER3 mixed peptides (JPT Peptide Technologies, Berlin, Germany) or OVA257-264 (SIINFEKL) peptide were used to detect anti-HER2, anti-HER3 or anti-OVA cellular responses. HIV peptide mix (BD Bioscience) was used as a negative control of the assay.…”

“…The amount of serum antibodies specific to the target antigen were assessed by either ELISA (OVA) or cell-based ELISA (HER2 and HER3) as previously described. 22 27 Briefly, ELISA for anti-OVA antibodies in serum was performed using OVA protein (2.5 µg/50 µL/well) coated plates (Immulon 4HBX, Thermo Scientific, Massachusetts, USA). Cell-based ELISA was performed using 96-well plates seeded with BC cells (4T1-HER2, 4T1-HER3, or parental 4T1, 10,000 cells/well).…”

“…[14][15][16][17][18][19][20][21] For example, we recently showed that mechanical disruption of tumor tissue using HIFU modified the TME, by repolarizing macrophages to an M1-like phenotype and inducing intratumoral infiltration of CD8+T cells, and elicited strong systemic antitumor immunity. 22 In common, these approaches seek to either activate more potent effector T cells or modify the TME to allow intratumoral T cell migration; however, few effectively enhance the signals for T cell infiltration. Another tumor ablative approach is photodynamic therapy (PDT) which consists of systemic administration of a photosensitizer compound followed by application of laser light resulting in the generation of reactive oxygen species that damages the target cells.…”