Eradication of hepatoma and colon cancer in mice with Flt3L gene therapy in combination with 5-FU

Abstract

We developed a recombinant defective adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L (Ad-mFlt3L) under control of cytomegalovirus promoter to investigate the biological efficacy of Flt3L in combination with chemotherapeutical drug, 5-FU, in eliciting an effective anti-cancer immunity in mouse hepatoma and colon cancer model systems. The constructed Ad-mFlt3L efficiently infected hepatoma and colon cancer cells both in vitro and in vivo, leading to a high production of mFlt3L proteins in association with accumulation of DCs, NK cells and lymphocytes in local tumor tissues. Administration of Ad-mFlt3L can protect bone marrow injury caused by 5-Fu and stimulates proliferation and maturation of lymphocytes, APCs and NKs. Intratumoral injection of Ad-mFlt3L followed by an intraperitoneal administration of 5-Fu significantly inhibited tumor growth and cured established tumors. Adenovirus mediated Flt3L gene therapy synergies with chemotherapeutic drug, 5-Fu, in elicitation of long-lasting antitumor immunity. The tumor specific immunity can be adoptively transferred into naïve animals successfully by transfusion of CD3+CD8+ T cells from the treated mice. The data suggests that adenovirus mediated Flt3L gene therapy in combination with 5-Fu chemotherapy may open a new avenue for development of anti-cancer chemogenetherapy.

Fig. 1

Treatment of mouse hepatoma and colon carcinoma with Ad-mFL in combination with 5-Fu. 4 × 10⁶ SMCC-1 or Hepa1-6 cells in a volume of 100 μl of PBS were inoculated subcutaneously into the right lateral flank of syngeneic C57BL/6 mice, six groups, 20 per group. When the tumors reached 0.8–1 m in diameter (15 days after inoculation), 1 × 10⁹ fu of Ad-mFL or Ad-GFP, diluted in 0.1 l saline buffer (PBS) or PBS alone were intratumorally injected for two times (day 1 and 3). After last intratumoral injection, each group was followed by intraperitoneal administration of 5-Fu at a dose of 10mg/kg for 3 days. Tumor developments were monitored and animal survival was calculated. a Tumor size of SMCC-1 colon carcinoma; b tumor size of Hepa1-6 hepatoma; c survival of animals bearing SMCC-1 colon carcinoma; d survival of animals bearing Hepa1-6 hepatoma. These experiments were repeated from three times with comparable results. *P ≤ 0.05, **P ≤ 0.01. Student’s t test for a and b or Fisher’s exact test for c and d

Fig. 2

Infiltration of T lymphocytes, NK cells and Dendritic cells in tumor tissues treated with Ad-mFL in combination with 5-Fu. Animals were treated by different protocols. At the end of observation period, mice were killed and paraffin-embedded tumor sections were prepared. CD8⁺ T cells, CD56⁺ NK cells and CD11c⁺ DC were stained by a routine IHC protocol. A strong infiltration (+++) of CD8⁺ T cells, NK cells and DCs were observed

Fig. 3

Recovery of 5-Fu induced reduction of PWBC and PLTs by Ad-mFL. The peripheral WBC counts and PLT counts of the mice treated with different protocols (twenty mice per group) were measured every day. a PWBC counts of SMCC-1 xenografted mice; b PBWC counts of Hepa1-6 xenografted mice; c PPLTs counts of SMCC-1 xenografted mice; d PPLTs counts of Hepa1-6 xenografted mice. The results are the representative data from three comparable experiments. *P ≤ 0.05, **P ≤ 0.01 (Student’s t test)

Fig. 4

Enhancement of bone marrow damage induced by 5-Fu. Bone marrow cells from the mice treated with different protocols on day 7 and analyzed for the numbers of granulocyte-macrophage colony-forming unite (CFU-GM) and stem cell colony-forming unite (CFU-S). a CFU-GM and CFU-S of SMCC-1 xenografted mice; b CFU-GM and CFU-S of Hepa1-6 xenografted mice. These experiments are representative data from three comparable experiments, five mice per group. *P ≤ 0.05, **P ≤ 0.01 (Student’s t test)

Fig. 5

Induction of long-lasting anti-tumor specific immunity by Ad-mFL plus 5-Fu. Animals with complete regression of SMCC-1 or hepa1-6 xenografted tumors induced by administration of Ad-mFL in combination with 5-Fu therapy were maintained for 4 months. The animals were then divided into two groups, five per group, and re-challenged with 4 × 10⁶ parental Hepa1-6 or SMCC-1 cells, respectively. Tumor developments were monitored. These experiments were repeated for three times with similar results. *P ≤ 0.05, **P ≤ 0.01 (Student’s t test)

Fig. 6

Cytotoxicity assay of spleen cells from the treated animals. The purified splenic cells were co-cultured with γ-irradiated (5,000 rad) parental hepa1-6 or SMCC-1 cells for 9 days in complete RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% l-glutamine and 20 μm/ml recombinant IL-2. The cytotoxic activity of splenocytes from treated and untreated animals was determined using the ⁵¹Cr release assay. NK cell killing activity was measured by a standard procedure as reported previously. a Splenocytes from five mice with regression of SMCC-1 tumors; b splenocytes from five mice with regression of Hepa1-6 tumors; c Splenocytes from five naïve mice. The results are the representative data from three comparable experiments. *P ≤ 0.05, **P ≤ 0.01 (Student’s t test)