Microbubble-Protected Oncolytic Virotherapy Targeted by Sonoporation Induces Tumor Necrosis and T-Lymphocyte Infiltration in Humanized Mice Bearing Triple-Negative Breast Cancer

Abstract

Oncolytic virotherapy has shown great promise in mediating targeted tumor destruction through tumor-selective replication and induction of anti-tumor immunity; however, obstacles remain for virus candidates to reach the clinic. These include avoiding neutralizing antibodies, preventing stimulation of the adaptive immune response during intravenous administration, and inducing sufficient apoptosis and immune activation so that the body's defense can work to eradicate systemic disease. We have developed a co-formulation of oncolytic viruses (OVs) with Imagent^® lipid-encapsulated, perfluorocarbon microbubbles (MBs) to protect the OVs from the innate and adaptive immune system. Once inside the MB, the viral particles become acoustically active such that external ultrasound can target the delivery of the virus locally within the tumor. Humanized NSG female mice (Hu-CD34⁺ NSG-SGM3) engrafted in their flanks with MDA-MB-231-Luc triple-negative breast cancer (TNBC) cells were transduced with MB/OVs, with or without adjuvant Pembrolizumab treatment, and tumor sizes and tumor necrosis were assessed. The presence of CD8⁺ (cytotoxic T-cells), CD4⁺ (helper T-cells), and CD25⁺ (Tregs) tumor-infiltrating lymphocytes (TILs) was quantified in the tumor samples by immunohistochemistry. In an in vivo model of humanized mice engrafted with a human immune system, we observed significantly greater tumor necrosis and smaller tumor mass in human TNBC xenografts systemically treated with MB/OV complexes in the presence or absence of pembrolizumab adjuvant treatment, compared to controls. Additionally, we observed a low ratio of CD4⁺/CD8⁺ TILs and a high ratio of CD8⁺/CD25⁺ TILs in the MDA-MB-231 xenografts treated with MB/OVs complexes with or without pembrolizumab adjuvant treatment, compared to controls. Our study demonstrated the feasibility of using MBs to target OVs to TNBC through diagnostic ultrasound, which decreased tumor mass by increasing tumor necrosis and stimulated a local and systemic antitumoral immune response by increasing intratumoral CD8⁺ T-cytotoxic lymphocyte infiltration and decreasing CD25⁺ Treg cells.

Keywords: cavitation; gene therapy; immunotherapy; microbubbles; microspheres; oncolytic virus; ultrasound; ultrasound contrast agent.

Figure 1

Study treatment schedule. Treatments started when tumors were 150 mm³. Created with Biorender.com.

Figure 2

Detection of Adenovirus-5 immunohistochemistry. Adenovirus-5 Hexon protein expression was negative in the controls (MBs and Pembrolizumab groups). Adenovirus-5 Hexon expression significantly increased in the OV group, with a 50–75% rate in infected cells in the IV groups. The magnification of the upper panels is 100×, and the lower panels is 200×.

Figure 3

Tumor measurements by caliper and by IVIS.

Figure 4

Hematoxylin and eosin staining of tumor xenografts (right flank). A montage of the treated tumor is shown on the upper panels, where the areas of necrosis are evident; lower panels show a low magnification view of the tumors in which the different sizes of the necrotic areas can be observed (original magnification 100×).

Figure 5

Detection of tumor-infiltrating CD4⁺ cells by immunohistochemistry. CD4-positive immune cells are few in the control (saline) group and sporadic in the Pembrolizumab-treated group, but their numbers significantly increase in the OV groups and dramatically increase in the MB groups. The upper panel magnification is 100×, and the lower panel magnification is 200×.

Figure 6

Immunohistochemical detection of CD8⁺ T-cells in tumors. CD8⁺ T cell tumor infiltrates (TILs) are few in the control (saline) group, and sporadic in the Pembrolizumab treated group; however, their expression significantly increases in both OV groups, and their number is dramatically increased in both MB groups. The magnification of the upper panels is 100×, and the magnification of the lower panels is 200×.

Figure 7

Immunohistochemistry for IL-R2 alpha (CD25) in tumors. CD25⁺ cells are regularly distributed in the control MB group and are abundant in the Pembrolizumab group, mainly located within or surrounding areas of necrosis; however, their number plummeted in both MB groups, coinciding with these tumors having significantly smaller areas of necrosis. The magnification of the upper panels is 100×, and the magnification of the lower panels is 200×.