Depletion of peripheral macrophages and brain microglia increases brain tumor titers of oncolytic viruses

Abstract

Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma.

Figure 1

MR imaging and quantification of tumor-associated phagocytic cells. A, tumors of animals treated with mock solution (PBS/PBS, top), intratumoral OV (PBS/OV, middle), or CL (CL/OV, bottom) before intratumoral OV were imaged for peripheral macrophages infiltrating the tumor. Imaging was done 72 h after OV injection. MR signals were color coded. Shift toward red, decreased T₂-weighted signal; shift toward blue, increased signal. The darker the T₂-weighted signal, the more superparamagnetic iron oxide particles are incorporated in the tumor by infiltrating peripheral phagocytic cells. B, columns, mean T₂ value (expressed in milliseconds) from 16 images of three rats per treatment group; bars, SD. Higher T₂ values correspond to decreased intratumoral infiltration of peripheral macrophages. Significance of pair means comparison was determined by ANOVA followed by post hoc Tukey’s test (n = 4). Asterisks, significant differences.

Figure 2

CL-mediated depletion of tumor-associated phagocytic cells. A, FACS analysis of the brain hemispheres harboring gliomas from rats treated with mock solution (PBS/PBS), OV alone (PBS/OV), or CL (CL/OV) before OV delivery. The graph shows the average percentage of CD68⁺ and CD163⁺ cells, detected by FACS, within the whole brain hemisphere of each treatment group (n = 3). For each cell type, a 3-fold increase is observed following OV treatment, and this increase is inhibited by CL treatment only for CD163⁺ cells. Significance of pair means comparison was determined by ANOVA followed by post hoc Tukey’s test for each cell type (CD68: P = 0.0085; CD163: P = 0.011). Asterisks, significant differences. No significant difference was observed for CD68⁺ cells in animals treated with PBS/OV and CL/OV. B, microphotographs of brains with established D74/HveC syngeneic gliomas for rats treated with mock vehicle (PBS/PBS, top row), OV alone (PBS/OV, middle row), and CL (CL/OV, bottom row) before OV injection. CL-mediated macrophage depletion is observed only for the CD163 antigen (right column), whose intratumoral distribution varies somewhat from that of the CD68⁺ cells (left column). Black arrows, accumulation of CD68⁺ cells around the tumor edge. C, microphotographs of OV-infected cancer cells in a rat tumor treated with OV for 72 h. The brains were stained with antibodies recognizing HSV1 antigens (green) and either anti-CD68 or anti-CD163 antibodies (red). Clones of OV-infected cancer cells are surrounded by CD68⁺ and CD163⁺ cells. Some of these are localized in the proximity of HSV and remain red (red arrows), but others have clearly incorporated HSV-infected cells, and colocalization of the two markers gives an orange-yellow staining (yellow arrows). The incorporation of HSV1 into either CD68⁺ or CD163⁺ cells was confirmed by scanning the laser through the slide with 1-μm intervals.

Figure 3

Effects of macrophage depletion on intratumoral spread of OV and tumor size. A, intratumoral OV-mediated LacZ transgene expression in animals treated with PBS (controls; PBS/OV), CL (CL/OV), and CPA (CPA/OV ) before OV delivery. The microphotographs represent the tumor area within the brain hemisphere (purple hematoxylin counterstaining) and LacZ transgene expression within the tumor (blue staining for β-galactosidase activity). B, a, columns, mean percentage of tumor area infected by OV from the treatment groups described in (A); bars, SD. This experiment was repeated thrice, and one representative result is shown. Significance of pair means comparison was determined by ANOVA followed by post hoc Tukey’s test (n = 4). Asterisks, significant differences. b, quantitative RT-PCR for the transcript encoding LacZ gene. Columns, mean fold induction (n = 4) for the transcript encoding LacZ in tumors of rats treated with OV alone (controls; PBS/OV), both CL and OV (CL/OV), and CPA and OV (CPA/OV); bars, SD. The baseline value of one was attributed to the expression of each gene in control gliomas, and the increase in LacZ expression was evaluated (Materials and Methods). The experiment was repeated thrice; one representative experiment is shown. Asterisks, means comparisons done with Tukey’s test showed significant difference among the three treatment groups. c, viral titers from tumors treated with PBS and OV (controls; PBS/OV), CL and OV (CL/OV), and CPA and OV (CPA/OV) were quantified on VERO cells (n =8). Columns, average of OV titers obtained; bars, SD. Viral titers of animals pretreated with CL increased 5-fold compared with those of animals treated with OV alone, whereas those treated with CPA increased 100-fold compared with control tumors. Asterisks, means comparisons done with ANOVA followed by a post hoc Tukey’s test showed significant difference between the three treatment groups. C, brain tumor volume (mm³) was measured by MR imaging in animals treated with mock solution (PBS/PBS ), CL alone (CL/PBS), OV alone (PBS/OV), and CL and OV (CL/OV); n = 3. Asterisks, comparisons of the means with Tukey’s test showed significant changes, with α = 0.05 among the three pair comparisons. No significant difference was present between PBS/PBS and CL/PBS treatment groups.

Figure 4

CL-mediated microglia depletion on brain slices cultured in vitro. CD68 (top) and CD163 (bottom) immunofluorescence in the tumor area of rat brain slices cultured ex vivo and treated with mock solution (PBS/PBS), mock and OV (PBS/OV), and CL and OV (CL/OV). The images clearly show OV-mediated intratumoral infiltration of CD68⁺ cells and their depletion by CL treatment. No CD163⁺ cells were detected in the whole brain slice, confirming their peripheral origin.

Figure 5

CL-mediated increase in OV titers explanted from ex vivo brain slices with established gliomas. A, the graph shows the viral titers obtained from brain slices with established D74/HveC gliomas infected with hrR3 OV strain after PBS or CL treatment at 3, 5, and 8 d after virus infection. Columns, average of three slices per treatment group; bars, SD. The experiment was done twice. Comparison of the means with ANOVA test showed significant changes in virus titers with α = 0.05. Post hoc Tukey’s test indicated significant changes at each time point between PBS- and CL-treated brain slices. B, representative photomicrographs of athymic nude mice brain slices implanted with human X12 glioblastoma cells treated with CL or control. CL-treated slices were thus depleted of resident phagocytes (microglia). rQNestin34.5 was then added to each slice, and its presence was detected by expression of GFP (the transgene is encoded by the virus). Positive signal is indicated by the green fluorescence.

Figure 6

Macrophage infiltration in human brain. A, CD68 (top row) and CD163 (bottom row) immunopositivity in tumors excised from a patient with glioblastoma multiforme before treatment with ONYX-015 (left column) and 1 mo after treatment (right column). CD68- and CD163-positive cells are indicated by the brown staining over the hematoxylin purple nuclear counterstaining. The figure shows OV-mediated induction of both CD68⁺ and CD163⁺ cells in this patient. B, the brain from a patient with active HSV1 encephalitis was stained by H&E using antibodies against HSV1 and against the CD68 and CD163 antigens. The figure shows HSV-induced infiltration of CD68⁺ and CD163⁺ cells in the brain parenchyma.