Activated dendritic cells delivered in tissue compatible biomatrices induce in-situ anti-tumor CTL responses leading to tumor regression

Abstract

Dendritic cell (DC) based anti-cancer immunotherapy is well tolerated in patients with advanced cancers. However, the clinical responses seen after adoptive DC therapy have been suboptimal. Several factors including scarce DC numbers in tumors and immunosuppressive tumor microenvironments contribute to the inefficacy of DCs as cellular vaccines. Hence DC based vaccines can benefit from novel methods of cell delivery that would prevent the direct exposure of immune cells to suppressive tumor microenvironments. Here we evaluated the ability of DCs harbored in biocompatible scaffolds (referred to as biomatrix entrapped DCs; beDCs) in activating specific anti-tumor immune responses against primary and post-surgery secondary tumors. Using a preclinical cervical cancer and a melanoma model in mice, we show that single treatment of primary and post-surgery secondary tumors using beDCs resulted in significant tumor growth retardation while multiple inoculations were required to achieve a significant anti-tumor effect when DCs were given in free form. Additionally, we found that, compared to the tumor specific E6/E7 peptide vaccine, total tumor lysate induced higher expression of CD80 and CD40 on DCs that induced increased levels of IFNγ production upon interaction with host lymphocytes. Remarkably, a strong immunocyte infiltration into the host-implanted DC-scaffold was observed. Importantly, the host-implanted beDCs induced the anti-tumor immune responses in the absence of any stromal cell support, and the biomatrix structure was eventually absorbed into the surrounding host tissue. Collectively, these data indicate that the scaffold-based DC delivery may provide an efficient and safe way of delivering cell-based vaccines for treatment of primary and post-surgery secondary tumors.

Keywords: biomatrices; dendritic cells; immunotherapy; tumor.

Figure 1. Total tumor lysate induces DC activation

Bone marrow derived DCs were exposed to tumor associated antigen (TAA) or antigen specific E6/E7 peptides in presence or absence of proinflammatory cytokine cocktail. (A) Representative FACS analysis of costimulatory molecules on DCs after 18–24 hours of activation. (B) Statistical analysis of DC activation from A. (C) Levels of IFNγ induced after incubation of variously activated DCs with splenocytes for 3–4 days. Data are shown as + SEM. *p-value < 0.05, **p-value. < 0.01.

Figure 2. Treatment of TC1 primary (1°) tumors using DCs-in-scaffolds (beDC) or free DCs

Mice bearing TC1 or TC1-luciferase (luc) cell induced 1° tumors were treated with free DCs or DC-in-scaffolds (beDC). (A) TAA-activated DCs were harbored in the fibrin scaffold and placed near the tumor site at day 5–6 (black arrow) or were inoculated in free form subcutaneously at three occasions (red arrow). (B) DCs were activated with E6/E7 peptides and inoculated into tumor-bearing mice either in free form (red arrows) or after harboring in fibrin scaffolds. (C and D) Luciferase expression of TC1-luc tumors after treatment by TAA-activated DCs given in fibrin scaffolds (beDC). (E) Comparison of treatment efficacy of single inoculation of scaffold harbored DCs (beDC) or free DCs in TC1 induced 1^° tumors. Error bars represent ± SEM. *p-value < 0.05.

Figure 3. Induction and treatment of TC1 post-surgery secondary (2°) tumors using TAA activated DCs

TC1 primary tumors were resected at day 10 when the tumor became 500 mm³ in size. (A) Resected tumors were treated with three injections of TAA-activated DCs (red arrows) or single placement of beDCs. (B) Statistical analysis of tumor volume at day 26. (C) Mice with completely regressed tumors in be DC group in B were re-challenged with TC1 cells and tumor growth was compared with the control group at day 10. Luciferase expression (D) and quantification (E) in 2° tumor-bearing mice after the DC scaffold treatment. Data is represented as ± SEM. *p-value < 0.05, **p-value < 0.01.

Figure 4. DCs in fibrin scaffolds facilitate immune cell movement ex vivo and in vivo

(A) Migration profiles of DCs harbored in scaffolds (shaded bars) and lymphocytes (white bars) towards chemotactic signals in a transwell assay system. (B) Lymphocytic movement towards DCs in scaffolds under ex vivo conditions. Panel 1 shows the lymphocyte arrangement around the DC scaffold while 2 and 3 respectively shows the lymphocytic arrangement around biomatrix without DCs or in the absence of any stimulus. Dotted lines in 2 and 3 depicts the edge of biomatrix. Panel 4 shows the close association of infiltrated lymphocytes (white arrows) with biomatrix entrapped DCs (beDCs) (black arrows). Bar in 1 is equivalent to 100 μm and in 4 is equivalent to 25 μm. (C) Pie charts showing the absolute cell numbers (%) inside biomatrix harvested from various groups as shown in the figure. n = 3. (D) Representative FACS plots showing MFI and the statistical analysis of the plots (E) of the IFNγ induced upon activation of splenocytes harvested from various groups as shown in the figure.

Figure 5. Lymphocyte interaction inside the DC harboring scaffolds

DC scaffolds (beDC) or scaffolds only, placed in tumor bearing mice or DC-scaffold placed in normal mice were recovered two weeks post-implantation and processed for immunohistochemistry to demonstrate various cell types. (A) Relative size of the biomatrices harvested from various mice groups as depicted in the figure (far left panel). n = 3–5. The dotted line in ‘gross specimen’ differentiates the biomatrix from the reactive host-tissue. Representative images of biomatrix sections stained by the respective antibodies as indicated in the figure. The bar in the ‘gross structure’ is equivalent to 1 mm, whereas for other panels is equal to 100 μm. (B) Snapshots from Supplementary Movie S2 showing lymphocyte (green) movement and interaction with DCs (red) inside the biomatrix. The start point of the observation was set as the zero time point. (C) Lymphocyte division (white arrows) inside the biomatrix in the vicinity of DC (red). Representative snapshot from Supplementary Movie S3.