Immunotheranostic microbubbles (iMBs) - a modular platform for dendritic cell vaccine delivery applied to breast cancer immunotherapy

Abstract

Background: Therapeutic strategies engaging the immune system against malignant cells have revolutionized the field of oncology. Proficiency of dendritic cells (DCs) for antigen presentation and immune response has spurred interest on DC-based vaccines for anti-cancer therapy. However, despite favorable safety profiles in patients, current DC-vaccines have not yet presented significant outcome due to technical barriers in active DC delivery, tumor progression, and immune dysfunction. To maximize the therapeutic response, we present here a unique cell-free DC-based vaccine capable of lymphoid organ targeting and eliciting T-cell-mediated anti-tumor effect.

Methods: We developed this novel immunotheranostic platform using plasma membranes derived from activated DCs incorporated into ultrasound contrast microbubbles (MBs), thereby offering real-time visualization of MBs' trafficking and homing in vivo. Human PBMC-derived DCs were cultured ex vivo for controlled maturation and activation using cell membrane antigens from breast cancer cells. Following DC membrane isolation, immunotheranostic microbubbles, called DC-iMBs, were formed for triple negative breast cancer treatment in a mouse model harboring a human reconstituted immune system.

Results: Our results demonstrated that DC-iMBs can accumulate in lymphoid organs and induce anti-tumor immune response, which significantly reduced tumor growth via apoptosis while increasing survival length of the treated animals. The phenotypic changes in immune cell populations upon DC-iMBs delivery further confirmed the T-cell-mediated anti-tumor effect.

Conclusion: These early findings strongly support the potential of DC-iMBs as a novel immunotherapeutic cell-free vaccine for anti-cancer therapy.

Keywords: Breast Cancer; Dendritic cell vaccine; Immunotherapy; Microbubbles; Molecular imaging; Oncology; Ultrasound (US).

Scheme 1

Personalized DC-iMB strategy for TNBC immunotherapy. a Schematic illustration of DC-iMB showing the presence of synthetic phospholipids, DC membrane phospholipids and proteins, and gas core. b By nature, DC-iMB allows US imaging (e.g., tumor size and perfusion monitoring, spleen retention visualization), and targeted immunotherapy via TNBC specific antigen presentation and naïve T-cell activation in lymphoid organs (thymus, spleen, and lymph nodes). c DC-iMB vaccine preparation: Monocytes (CD14⁺) were isolated from patient’s peripheral blood for autologous DC generation (1a) while hPBMCs were used for human immune system reconstitution (i.e., T-cell engraftment) of immunodeficient NSG mice (1b). Immature monocyte-derived DCs (iMoDCs) were generated by culturing the isolated CD14⁺ cells with GM-CSF and IL-4 (2). Monocyte-derived DCs (MoDCs) were matured and pulsed with MDA-MB-231 cancer cell derived membrane antigens (3). The plasma membrane from MoDCs was then isolated (4) and used for DC-iMB formulation (5) before injection into a humanized mouse model of TNBC via several cycles of intravenous (i.v.) injections (6). CCM: cancer cell membrane; DCm: DC membrane; GM-CSF: granulocyte macrophage colony stimulating factor; LNs: lymph nodes; MHC: major histocompatibility complex; PLs: phospholipids; TLR: Toll-like receptor.

Fig. 1

CD14-positive cell enrichment from 3 healthy blood donors and their characterization for different surface markers. a Histogram plots showing CD14, CD11c, and CD45 marker specific fluorescence signals from cells pre- and post- cell sorting. b CD14⁺ sorted cell fraction purity and recovery quantification. c hPBMCs gating strategy in CD45 vs. SSC-H dot plot. d Percentage assessment of each major cell group. All data are shown as Mean ± SD. G = granulocyte; M = monocyte; L = lymphocyte; B = blast

Fig. 2

MoDC maturation and activation ex vivo. a FACS analysis of various cell surface markers (CD11c, CD14, CD33, MHC II, CD80, CD83, CD86 and CD206). Fluorescence intensity shift was compared to unstained control. b Cell morphology after complete maturation and activation (+) compared to control (−). Cells were photographed using a digital camera assembled on a bright field inverted microscope. Original magnification was 40×. Scale bar = 100 μm.*p < 0.05; **p < 0.005; ***p < 0.0001

Fig. 3

In vitro DC-iMB characterization. a Diameter size distribution and zeta potential of MB and DC-iMB_{donor 1,} DC-iMB_{donor 2,} and DC-iMB_{donor 3}; b Bright-field microscopic image of DC-iMBs. Scale bar is 50 μm; and c Particle concentration after formulation

Fig. 4

Therapeutic evaluation of DC-iMBs in TNBC bearing humanized immune system mice. a Schematic outline of the experimental design and timeline adopted for treatment, imaging, and blood collection. b (i-iv) Relative change in tumor volume over time. Black arrows indicate the starting date of therapeutic treatments. c Animals from different treatment groups measured for body weight over time (n = 4/group) to monitor the impact of treatments on animal health as well as xGVHD development. d Survival curves of animals from different treatment groups. All data are shown as Mean ± SD. *p < 0.05; **p < 0.005; ***p < 0.0001

Fig. 5

In vivo imaging evaluation of DC-iMB. a Bioluminescence images acquired at multiple time points during the treatments to assess therapeutic responses. b Representative B-mode US (top) and non-linear contrast images of a spleen pre-injection (middle) and post-injection (bottom) with DC-iMBs, conventional MBs, or PBS. Scale bar = 3 mm. c Quantification of tumor bioluminescence signal over time. d Quantification of US signal enhancement pre- and post- MBs, DC-iMBs, or PBS injection. CEUS: contrast enhanced ultrasound. ***p < 0.0001

Fig. 6

Terminal ex vivo size evaluation and apoptosis. a Tumor volume, and b spleen size measurements at study endpoint; c mean number of apoptotic cells per mm² of tumor tissue. *p < 0.05; **p < 0.005; ***p < 0.0001

Fig. 7

Ex vivo cell distribution in a spleen, b thymus, c tumor, d blood, e lymph nodes, and f lungs of mice treated or not with DC-iMBs. *p < 0.05; **p < 0.005; ***p < 0.0001

Fig. 8

In situ characterization of CD4⁺ and CD8⁺ T-cell migration by confocal microscopy. a Representative spleen images. Tissues were processed for histologic analysis and triple stained with hoechst33342 (blue), hCD4 (green), and hCD8 (red). Scale bar = 0.5 mm; b Magnification of spleen area treated by hPBMCs + DC-iMBs. Scale bar = 40 μm; c Splenic quantification of fluorescence intensity ratios (hCD4/Hoechst and hCD8/hoechst33342) for all animal groups; d Representative tumor sections with respective magnified areas. Scale bars = 0.5 mm and 40 μm, respectively. e Quantification of fluorescence intensity ratios (hCD4/Hoechst and hCD8/hoechst33342) in tumor sections for all animal groups. **p < 0.005