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3.2. Single dosing with activatable drug delivery and immunotherapy or immunotherapy alone enhanced tumor infiltrating CD8+ T-cells and myeloid recruitment in treated and distant lesions
In order to elucidate a protocol for coupling chemotherapy with immunotherapy (CpG+αPD-1, abbreviated here as IT), we first sur-veyed the immune environment one week after a single administration of ADD, CpG, and αPD-1 (ADD-IT) in a murine model of primary and distant site mammary adenocarcinoma. NDL tumors were orthotopi-cally transplanted into the fourth and ninth mammary fat pads of FVB mice, where a single tumor was treated 21 days after transplantation. Dox was delivered to the tumor via ultrasound-mediated release from CuDox-TSL and CpG was injected intratumorally; αPD-1 was then ad-ministered intraperitoneally 3 days after treatment of a single tumor (Fig. 3A). One week after the start of treatment, ADD-IT significantly reduced the total fraction of live Streptozocin in the treated tumor (pooled Journal of Controlled Release 303 (2019) 42–54
Fig. 2. Tumor-specific antigen presentation increased in tumor and blood after treatment. A) Schematic diagram illustrating the treatment protocol of CuDox +US (ADD) in C57BL/6 mice (n = 13) bearing one B16-OVA tumor (locally treated tumor) and one control B16-F10 tumor on the opposite side (distant tumor). B-D) Frequency of SIINFEKL-labeled immune cells, and specifically DCs, and macrophages as a percentage of leukocytes in tumors directly treated with ADD or in the distant tumor of the treated mice (n = 5) compared to B16-OVA and B16-F10 tumors of untreated control mice (n = 5), respectively. E) Frequency of SIINFEKL-labeled blood immune cells as a percentage of leuko-cytes in blood of mice treated with ADD compared to no-treatment (NT) control mice (n = 3). *p < 0.05, **p < 0.01.
tumor and draining lymph node) compared to the no-treatment (NT) control (Fig. 3B). While IT and ADD-IT enhanced the fraction of leu-kocytes (CD45+ cells) in the treated tumor (Fig. 3C), ADD-IT increased the frequency of CD8+ T-cells in both the treated and distant tumor compared to the NT control (Fig. 3D). Despite the presence of tumor infiltrating CD8+ T-cells observed with ADD-IT treatment, the fraction of activated CD8+ T-cells secreting IFN-ɣ showed only an insignificant upward trend but was significantly enhanced in tumors directly treated with IT compared to NT control tumors (Fig. 3E). Although the fre-quency of antigen-presenting dendritic cells (DCs) was significantly increased by αPD-1 alone and not with other treatments (Fig. 3F), the number of infiltrating macrophages increased in the treated and distant tumors of the ADD-IT cohort on flow cytometry (Fig. 3G). IHC verified the infiltration of CD8+ T-cells in mice treated with the ADD-IT pro-tocol (Fig. 3H). This was accompanied by a massive recruitment of myeloid cells (F4/80+ cells) into the tumor periphery and pockets of infiltration into non-viable tumor tissue. Foxp3 expression as a marker of regulatory T-cells was not increased by the ADD-IT treatment.
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Fig. 3. Combining ADD with CpG and αPD-1 increased cytotoxic T lymphocytes in local and distant NDL tumors. A) Representative treatment protocol and timeline for treatment of NDL tumor-bearing mice. One tumor of bilateral tumors of NDL orthotopically transplanted into FVB/n mice was treated with one complete administration of each component of CuDox+US+CpG (ADD-CpG) followed by αPD-1 post three days. B-G) On day 28 after one complete treatment of ADD-IT (n = 4) or IT (n = 4) or a single administration of αPD-1 (n = 4), tumors/inguinal lymph nodes of treated mice were stained with the antibody cocktail as listed below and analyzed via flow cytometry and compared with untreated control tumors. Fraction of live cells given as a percentage of total isolated cells (B), frequency of leukocytes (CD45+ cells) as a percentage of live cells (C) and frequency of cytotoxic T lymphocytes (CD3+CD8+ cells) as a percentage of total leukocytes (D), populations of IFN-γ producing CD8+ (CD3+CD8+IFN-γ+ cells) T-cells as a percentage of total leukocytes (E), fraction of DCs (CD11c+MHCII+ F4/80− cells) as a percentage of leukocytes (F), fraction of macrophages (CD11b+F4/80+Gr-1− cells) as a percentage of leukocytes (G) across various treatments. H) Histological sections of the no-treatment control, the tumor locally treated with CuDox+US+CpG+αPD-1 (ADD-IT) and the distant tumor on day 28 were stained with H&E (the left first column), CD8 (second column), Foxp3 (third column), F4/80 (fourth column) and the magnified images (fifth column), respectively. CD8, F4/80, and Foxp3 staining (brown) identify CD8+ T-cells, macrophages, and T regs, respectively. Red arrowheads denote the inguinal lymph nodes. Scale bars are 3 mm for whole tumor image and 100 μm for the magnified image. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)