Liposomal oncolytic adenovirus as a neoadjuvant therapy for triple-negative breast cancer

Abstract

Breast cancer remains one of the leading causes of cancer-related death, with triple-negative breast cancer (TNBC) accounting for 15-20% of cases. TNBC, characterized by the absence of ER, PR, and HER2 protein, is an aggressive form of breast cancer that is unresponsive to hormonal therapies and HER2-targeted treatments, with fewer treatment options and poorer prognosis. Oncolytic adenoviruses (Ad) are a potential treatment option for TNBC but require coxsackievirus and adenovirus receptors (CAR) to effectively enter and transduce cancer cells. This study investigates a novel neoadjuvant therapy to improve the efficacy of an oncolytic Ad with human telomerase reverse transcriptase (Ad-hTERT) in CAR-low TNBC tumors using folate surface-modified liposomes to enhance delivery. This therapy helps deescalate treatment by reducing or eliminating the need for checkpoint inhibitors or toxic chemotherapy combinations. In vitro studies using CAR-low TNBC murine 4T1-eGFP cells, CAR-high TNBC human MDA-MB-231-GFP cells and several other TNBC human cancer cell lines with varying CAR expression demonstrated significantly higher cytotoxicity with encapsulated Ad-hTERT compared to Ad-hTERT. Similar results were observed in patient-derived primary TNBC cells. In vivo studies in immunocompetent mice with CAR-low 4T1-eGFP tumors revealed that encapsulated Ad-hTERT, administered as neoadjuvant therapy, resulted in stable or reduced tumor sizes, improved survival rates, higher apoptosis of cancer cells, lower cancer cell proliferation, and increased T-cell infiltration in resected tumors. Furthermore, encapsulated Ad-hTERT prevented lung metastasis and tumor recurrence at the primary site, resulting in higher survival rates in mice. Thus, liposomal encapsulation of Ad may be a viable strategy for treating TNBC.

Keywords: Coxsackievirus and adenovirus receptor; Liposomes; Metastasis; Neoadjuvant therapy; Oncolytic adenovirus; Triple-negative breast cancer.

Fig. 1

Mechanism of Ad and liposome-encapsulated Ad internalization in cancer cells. (A) Ad relies heavily on the coxsackievirus and adenovirus receptor (CAR) for cell entry. (B) Ad transduce inefficiently in cancer cells with low or absent CAR expression. Low CAR expression is notable in tumors such as TNBC, which causes resistance to Ad-based gene therapy. (C) The process of encapsulating Ad in liposomes: The virus becomes encapsulated within liposomes due to favorable charge interactions. (D) Cationic liposome containing negatively charged Ad. (E) Encapsulated Ad can enter tumor cells via folate receptor-mediated endocytosis and membrane fusion, even in the absence of CAR expression.

Fig. 2

In vitro transduction of Ad-hTERT and encapsulated Ad-hTERT in TNBC cells. (A) Schematic of Ad-hTERT, a telomerase-specific, replication-competent adenovirus with E1A and E1B genes driven by the hTERT promoter and linked via an IRES. (B) Cell viability of 4T1-eGFP cells treated with Ad-hTERT (red) and encapsulated (blue) Ad-hTERT at various MOIs for 72 hours (n = 3). Encapsulated Ad-hTERT showed significantly higher cytotoxicity at MOI 10, 50, 100, and 200 (****p ≤ 0.0001). (C) Cell viability of MDA-MB-231-GFP cells under identical conditions (n = 3). Encapsulated Ad-hTERT demonstrated significantly higher cytotoxicity at MOIs of 10, 50, and 100 (****p ≤ 0.0001), and at an MOI of 200 (**p ≤ 0.01). In (C), the blue bar is not visible due to near-complete cell death.

Fig. 3

In vitro transduction of Ad-hTERT and encapsulated Ad-hTERT in additional TNBC cell lines. (A–C) Cell viability of MDA-MB-436, HCC1937, and SUM159PT cells, respectively, following treatment with Ad-hTERT (red) and encapsulated (blue) Ad-hTERT at various MOIs for 72 hours (n = 3). Encapsulated Ad-hTERT exhibited significantly higher cytotoxicity at MOI 10 and 100 in all cell lines: MDA-MB-436 (****p ≤ 0.0001), HCC1937 (***p ≤ 0.001), and SUM159PT (***p ≤ 0.001). .

Fig. 4

In vitro transduction of Ad-hTERT and encapsulated Ad-hTERT in patient-derived primary TNBC cells. (A) DAPI-stained fluorescence microscopy images of cells treated with Ad-hTERT and encapsulated Ad-hTERT at MOI 100 for 72 hours. Scale bar = 100 μm. (B) Cell viability of patient-derived primary TNBC cells treated with Ad-hTERT (red) and encapsulated (blue) Ad-hTERT under the same conditions (n = 3). Encapsulated Ad-hTERT induced significantly higher cytotoxicity (**p ≤ 0.01).

Fig. 5

Comparative in vivo biodistribution of Ad-hTERT and encapsulated Ad-hTERT on CAR-low 4T1-eGFP TNBC tumors. (A) Treatment model: CAR-low 1 × 10⁶ 4T1-eGFP TNBC tumors were inoculated into the mammary fat pad of BALB/c mice. The tumors were treated with intratumoral injection of PBS, empty liposomes, Ad-hTERT, or encapsulated Ad-hTERT as neoadjuvant therapy. 2 days after the treatment, mice were sacrificed and liver and tumor tissues were isolated for E1A gene copy numbers vis RT-qPCR (B) E1A gene copy numbers in tumors treated with PBS (n = 3), control empty liposomes (n = 3), Ad-hTERT (n = 5), and encapsulated Ad-hTERT (n = 5). Encapsulated Ad-hTERT demonstrated significantly higher E1A copy numbers (p-value: **** ≤ 0.0001) compared to the Ad-hTERT. (C) E1A gene copy numbers in liver tissues demonstrated significantly lower E1A copy numbers (p-value: *** ≤ 0.001) in mice treated with encapsulated AD-hTERT compared to the Ad-hTERT.

Fig. 6

Comparative in vivo therapeutic efficacy of Ad-hTERT and encapsulated Ad-hTERT on CAR-low 4T1-eGFP TNBC tumors. (A) Treatment model: CAR-low 1 × 10⁶ 4T1-eGFP TNBC tumors were inoculated into the mammary fat pad of BALB/c mice. The tumors were treated with intratumoral injections of PBS, empty liposomes, Ad-hTERT, or encapsulated Ad-hTERT as neoadjuvant therapy. Upon completion of treatment, the tumors were resected, and the mice were monitored for survival probability and metastasis evaluation. (B) Average tumor growth curves: Tumor growth curves for mice treated with control PBS (n = 4), control empty liposomes (n = 4), Ad-hTERT (n = 5), and encapsulated Ad-hTERT (n = 5). Encapsulated Ad-hTERT demonstrated significantly higher therapeutic efficacy (p-value: ** ≤ 0.01) compared to the Ad-hTERT. (C) Individual tumor growth curves of each group. (D) Images of surgically removed tumors. (E) Tumor weights: Weights of surgically removed tumors upon completion of intratumoral injection treatment, shown for each treatment group. Encapsulated Ad-hTERT demonstrated significantly lower tumor weights (p-value: *** ≤ 0.001) compared to the Ad-hTERT.

Fig. 7

Comparative in vivo therapeutic efficacy of Ad-hTERT and encapsulated Ad-hTERT on CAR-low 4T1-eGFP TNBC tumors recurrence and lung metastases in immunocompetent mice. (A) Representative bright-field images, Dino-Lite GFP digital microscope images, and H&E-stained images of isolated lungs. White arrows in the GFP images and black arrows in the H&E-stained images indicate metastases present only in the lungs of the control and Ad-hTERT groups. Scale bar = 60 μm. (B) Lung metastasis was observed in all groups except the mice treated with encapsulated Ad-hTERT. (C) After 1 week of tumor resection and up to 30 days post-surgery, primary murine tumor recurrence was observed in all groups except mice treated with encapsulated Ad-hTERT. (D) Survival curves for mice treated with control PBS (n = 4), control empty liposomes (n = 4), Ad-hTERT (n = 5), and encapsulated Ad-hTERT (n = 5). Encapsulated Ad-hTERT demonstrated significantly higher survival probability compared to Ad-hTERT (p-value: * ≤ 0.05) and control PBS (p-value: ** ≤ 0.01).

Fig. 8

Caspase-3 IHC, Ki-67 IF, CD3 IF, and CD8 IF staining of primary murine tumors: (A) Caspase-3 IHC, Ki-67 IF, CD3 IF, and CD8 IF staining of primary murine tumors. IF staining: Immunofluorescent microscopy images show DAPI-stained cells are blue, Ki-67-stained cells are yellow, CD3-stained cells are red, and CD8-stained cells are magenta. Black scale bar = 60 μm, and white scale bar = 100 μm. (B) Comparative quantitative analysis of caspase-3, Ki-67, CD3, and CD8 positive cells in control PBS (n = 4), control empty liposomes (n = 4), Ad-hTERT (n = 5), and encapsulated Ad-hTERT (n = 5). The entire tumor sections were used for calculation. The percentage of caspase-3-positive cells was calculated relative to hematoxylin-positive cells. The Ki-67, CD3, and CD8 positive cell percentages were calculated relative to DAPI-positive cells.

Fig. 9

Application of encapsulated Ad-hTERT as neoadjuvant therapy for TNBC.