A therapeutic microparticle-based tumor lysate vaccine reduces spontaneous metastases in murine breast cancer

Abstract

Metastatic breast cancer is currently incurable, and available therapies are associated with severe toxicities. Induction of protective anti-tumor immunity is a promising therapeutic approach for disseminated breast cancer, as immune responses are (i) systemic; (ii) antigen-specific; and (iii) capable of generating long-lived "memory" populations that protect against future tumor recurrences. Pursuant with this approach, we have developed a novel heterologous prime/boost vaccination regimen that reduces spontaneous lung metastases in mice with established murine 4T1 adenocarcinoma breast tumors. In our studies, mice were orthotopically challenged with luciferase-expressing 4T1 tumor cells; luciferase expression was retained in vivo, enabling us to quantitatively track metastatic tumor growth via bioluminescent imaging. On day 6 post-challenge, mice received a therapeutic "prime" consisting of bulk tumor lysates encapsulated in poly(lactic-co-glycolic) acid (PLGA) microparticles (MPs). On day 11, mice received a "boost" composed of free tumor lysates plus a cocktail of Toll-like receptor (TLR)-stimulating adjuvants. Tumor progression was monitored in vaccinated and untreated mice for 25 days, a time at which 100% of untreated mice had detectable lung tumors. PLGA MPs injected subcutaneously trafficked to draining lymph nodes and were efficiently phagocytosed by dendritic cells (DCs) within 48 h. Our combination therapy reduced metastatic lung tumor burdens by 42% and did not induce autoimmunity. These findings illustrate that vaccines based upon MP delivery of tumor lysates can form the basis of an effective treatment for metastatic breast cancer and suggest that similar approaches may be both efficacious and well-tolerated in the clinic.

Fig. 1

Characterization of spontaneous lung metastases in the 4T1-fLUC model. a Excised lungs from ten tumor-free mice were IVIS-imaged to assess background luminescence. The metastatic incidence threshold (dashed red line) was defined as 3 S.D. above this mean. In the absence of therapy, 100% (n = 52) of mice have lung metastases by day 25 after s.c. 4T1-fLUC challenge. Mean ± S.E.M. b Excised lungs of 4T1-fLUC-challenged mice were IVIS-imaged to determine metastatic kinetics. Black down-pointing triangle: incidence-free mice; red up-pointing triangle: incidence-positive mice. Data are pooled from two to ten experiments using three to five mice per time point. c Excised lungs of 4T1-fLUC-challenged mice were IVIS-imaged on day 25 then homogenized and cultured for 6-thioguanine colony-forming assays. Linear regression analysis indicates a positive correlation between these two independent measures of lung tumor burden. Data from one experiment, representative of two, are shown. d H&E-stained lung sections from 4T1-fLUC-bearing mice indicate the presence of multiple metastatic tumors (arrows). a–c Each data point represents bioluminescent tumor burdens from excised lungs

Fig. 2

PLGA MPs are efficiently phagocytosed by DCs and MΦs in vivo. a Scanning electron photomicrograph of 4T1-loaded PLGA MP. b 4T1 lysate release profile from PLGA MPs cultured in PBS at 37°C. Data from three batches of 4T1 MPs. Mean ± S.E.M. c In vivo trafficking of Rhodamine B-loaded PLGA MP. Left: Representative flow cytometry plots indicating the gating strategy for DC and MΦ populations in the vaccine-dLN (inguinal). Center: Rhodamine B gating defined using FMO controls. Right: Frequency of Rhodamine B-positive phagocytes in the dLN 24 and 48 h after MP injection. Data shown (mean ± S.E.M.) are pooled from two experiments using a total of eight mice/time point

Fig. 3

Overview of heterologous prime/boost vaccination regimen. a Schematic outline of vaccine protocol. b, c Established 4T1-fLUC tumors at day 6 in the same mouse. b IVIS image of the primary tumor showing standardized region of interest. c Orthotopic placement of 4T1-fLUC tumor (black outline) within mammary gland #9 (dashed red outline). Asterisk denotes inguinal lymph node

Fig. 4

Combinatorial vaccine therapy reduces spontaneous lung metastasis. a Primary tumor kinetics measured by calipers. Data from two to nine experiments using the total number of mice are indicated in parentheses. b Standardized representation of IVIS-imaged excised lungs from vaccinated and untreated 4T1-fLUC-bearing mice at day 25. c Experimentally matched lung tumor burdens from 4T1-fLUC-challenged mice that either did or did not receive therapy, as indicated. Dashed red line indicates the metastatic incidence threshold, as defined in Fig. 1a. Data points indicate tumor burdens of individual excised lungs, pooled from two to six experiments with significance evaluated by two-tailed unpaired Student’s t tests. d Lung metastases normalized for inter-experimental variation. Overlaid numbers indicate the mean lung tumor burdens for each treatment group relative to matched untreated controls. Groups compared using Mann–Whitney U tests. a–c Mean ± S.E.M. NS = not significant; *p < 0.05; **p < 0.01

Fig. 5

Vaccine therapy does not induce autoimmunity. Tumor-free mice were fully vaccinated as summarized in Fig. 3a. Lungs and mammary pads were collected 100 days later. H&E-stained tissues appear normal at ×2 magnification, with no evidence of autoimmunity. Representative images selected from three vaccinated and two control mice, with six distinct sections evaluated per organ