Post-operative unadjuvanted therapeutic xenovaccination with chicken whole embryo vaccine suppresses distant micrometastases and prolongs survival in a murine Lewis lung carcinoma model

Abstract

Immunotherapy in the form of anticancer vaccination relies on the mobilization of the patient's immune system against specific cancer antigens. Instead of focusing on an autologous cell lysate, which is not always available in clinical practice, the present study investigates vaccines utilizing xenogeneic foetal tissue that are rich in oncofoetal antigens. Lewis lung carcinoma (LLC)-challenged C57BL/6 mice were treated with either a xenogeneic vaccine made from chicken whole embryo, or a xenogeneic vaccine made from rat embryonic brain tissue, supplemented with a Bacillus subtilis protein fraction as an adjuvant. Median and overall survival, size of metastatic foci in lung tissue and levels of circulating CD8a⁺ T cells were evaluated and compared with untreated control mice. Following primary tumour removal, a course of three subcutaneous vaccinations with xenogeneic chicken embryo vaccine led to significant increase in overall survival rate (100% after 70 days of follow-up vs. 40% in untreated control mice), significant increase in circulating CD8a⁺ T cells (18.18 vs. 12.6% in untreated control mice), and a significant decrease in the area and incidence of metastasis foci. The xenogeneic rat brain tissue-based vaccine did not improve any of the investigated parameters, despite promising reports in other models. We hypothesize that the proper selection of antigen source (tissue) can constitute an effective immunotherapeutic product.

Keywords: Lewis lung carcinoma; cytotoxic lymphocytes; metastasis; mice; vaccination; xenogeneic.

Figure 1.

Experimental anticancer vaccination and sampling scheme for C57BL/6 mice challenged with LLC cells. Experiment design depicted from the time of tumor challenge (day 0) to the end of the survival observation (day 70). Figure presented previously (39).

Figure 2.

Kaplan-Meier survival curves for mice receiving adjuvant treatment with different xenogeneic vaccines following the removal of primary LLC-derived tumors. The mice that were treated with the xeno chicken vaccine survived significantly longer (mOS, 70 days) compared with the mice that were treated with the xeno rat vaccine (mOS, 60 days; P=0.003) and control group animals (mOS, 55 days; P=0.003). There was no significant difference in survival between the xeno rat and control groups. For each treatment arm, n=10. LLC, Lewis lung carcinoma; mOS, mean overall survival.

Figure 3.

Percentage of CD8a⁺ T cells in circulating peripheral blood monocytes prior to therapeutic xenovaccination (day 16, white column), 3 days after the completion of xenovaccination (day 26, black columns) and at the end of the study (day 70, grey columns). There were no significant changes in the levels of circulating CD8a⁺ T cells 3 days after the completion of xenovaccination, compared with the levels prior to the start of vaccination (within and between the experimental arms). However, on day 70 (at the end of the observation period), the levels of circulating CD8a⁺ T cells were significantly higher in the surviving mice vaccinated with the xeno chicken vaccine (n=10) compared with surviving mice (n=4) in the control group (P=0.002) or in the Xeno Rat group (P=0.049). In xeno chicken vaccine-treated mice, the level of CD8a⁺ T cells on day 70 was significantly higher compared with the level on day 16 (following tumor removal, prior to xenovaccination) (P=0.036) and on day 26 (3 days after the completion of xenovaccination) (P=0.003). There were no significant differences in the levels of circulating CD8a⁺ T cells in the mice that were treated with xeno rat vaccines evaluated at different time points or comparing T-cell levels with those in control group animals. Additionally, there was a significantly lower CD8a⁺ T-cell population in the control mice at the end of the study (day 70) compared with the level following the completion of vaccination (day 16) (P=0.028). CD8a, cluster of differentiation 8a. *P≤0.05.

Figure 4.

CD8a⁺ flow cytometric analysis. Histograms for the (A) control, (B) xeno rat and (C) xeno chicken treatment groups. CD8a, cluster of differentiation 8a.

Figure 5.

Histological analysis of lung samples from mice surviving the experiment (day 70). Histological slides from (A) control mice, (B) mice in the xeno rat experimental arm and (C) mice in the xeno chicken experimental arm. Metastatic foci (A and B) fill the whole field of view or are (C) indicated by an arrow. Magnification, ×100.