Generation of bone marrow chimeras using X-ray irradiation: comparison to cesium irradiation and use in immunotherapy

Abstract

Bone marrow chimeras represent a key tool employed to understand biological contributions stemming from the hematopoietic versus the stromal compartment. In most institutions, cesium irradiators are used to lethally irradiate recipient animals prior to the injection of donor bone marrow. Cesium irradiators, however, have significant liabilities-including concerns around domestic security. Recently, X-ray irradiators have been implemented as a potential alternative to cesium sources. Only a small number of publications in the literature have attempted to compare these two modalities and, in most cases, the emphasis was on irradiation of human blood productions. We were able to find only a single study that directly compared X-ray and cesium technologies in the generation of murine bone marrow chimeras, a standard laboratory practice. This study focused on chimerism in the blood of recipient animals. In the present study, we begin by comparing cesium and X-ray based sources for irradiation, then transition to using X-ray-based systems for immunology models with an emphasis on immunotherapy of cancer in immunocompetent mouse models-specifically evaluating chimerism in the blood, spleen, and tumor microenvironment. While our data demonstrate that the two platforms are functionally comparable and suggest that X-ray based technology is a suitable alternative to cesium sources. We also highlight a difference in chimerism between the peripheral (blood, spleen) and tumor compartments that is observed using both technologies. While the overall degree of chimerism in the peripheral tissues is very high, the degree of chimerism in the tumor is cell type specific with T and NK cells showing lower chimerism than other cell types.

Keywords: X-ray irradiation; bone marrow chimera; cesium irradiation; immunotherapy; syngeneic; tumor model.

Figure 1.

Chimerism achieved with various doses of X-ray irradiation. Animals were irradiated with varying doses of X-ray irradiation and analyzed 8 weeks post reconstitution to evaluate chimerism. The reconstitution of the donor hematopoietic cells (CD45.2⁺; closed circles) or residual recipient hematopoietic cells (CD45.1⁺; open squares) is shown as a percent of total cells in the blood (A-C) or in the spleen (D-F) of animals. In general, the range of irradiation used demonstrated comparable reconstitution. This figure is representative of two independent experiments. Data represents mean +/- standard deviation with 5 mice per group.

Figure 2.

Irradiation and reconstitution results in a delay in B16F10 tumor growth dependent of initial radiation dose. 6 week old (A), age matched (B), or 7-11 Gy irradiated and reconstituted (C-E) C57Bl6 animals were implanted with 2 × 10⁵ B16F10 syngeneic melanoma tumor cells. In animals irradiated with 11 Gy, 5/9 tumors failed to engraft (E). In groups irradiated with 7 Gy and 9 Gy, all tumors (10/10) grew out. The growth rate of the tumors, however, was slower with increasing radiation doses (F). This figure is representative of at least two independent experiments.

Figure 3.

Chimerism at 8 weeks post reconstitution in the blood of X-ray and cesium irradiated animals. Donor marrow reconstitution was evaluated in animals that were irradiated with X-ray (A) or Cesium¹³⁷ (B). Donor hematopoietic cells (CD45.2⁺; closed circles) were differentiated from recipient hematopoietic cells (CD45.1⁺; open squares) by flow cytometry using congenic markers. Hematopoietic cells were identified based on commonly expressed leukocyte antigens. The figure is representative of at least two independent experiments. Data represents mean +/- standard deviation with 10 mice per group.

Figure 4.

Starting cell implantation has a minimal effect on tumor growth rate post irradiation and reconstitution. Unirradiated, age matched animals were used as a control comparison (A). Animals were irradiated with 7 grays of X-ray irradiation and reconstituted with congenic labeled bone marrow. 8 weeks post reconstitution, animals were implanted with varying doses of B16F10 syngeneic melanoma tumor cells ranging from 2 × 10⁵ to 3.2 × 10⁶ cells per mouse (B-F).

Figure 5.

The tumor microenvironment in reconstituted animals is comparable. Age matched unirradiated animals (control; open circles), Cesium¹³⁷ irradiated and reconstituted (Cs 9 Gy; open squares), and X-ray irradiated and reconstituted (X-ray 7 Gy; closed circles) animals were implanted with 4 × 10⁶ B16F10 syngeneic tumor cells subcutaneously. 13 d post implantation, blood (A), spleen (B), and tumors (C) were analyzed for the degree of donor cell reconstitution. Absolute cell number is shown for CD11b⁺ dendritic cells, B220⁺ B cells, NKp46⁺ NK cells, CD4⁺ T cells, CD8⁺ T cells, Ly6G⁺ neutrophils, and CD11b⁺Ly6C⁺Ly6G^- myeloid cells. Data represents mean +/- standard deviation with 10 mice per group.

Figure 6.

Chimerism at 8 weeks post reconstitution in the tumor microenvironment of X-ray and cesium irradiated animals. Donor cell reconstitution was evaluated in animals that were irradiated with X-ray (A) or Cesium¹³⁷ (B) and implanted with 4 × 10⁵ B16F10 melanoma tumor cells. Tumors were implanted and allowed to establish before dissociation and analysis on day 13 post implantation (approximately 140–220 mm³. Donor hematopoietic cells (CD45.2⁺; closed circles) were differentiated from recipient hematopoietic cells (CD45.1⁺; open squares) by flow cytometry using congenic markers. Hematopoietic cells were identified based on commonly expressed leukocyte antigens. The figure is representative of at least two independent experiments.

Figure 7.

Response to checkpoint therapy in B16F10 is increased in reconstituted animals, consistent with increases in baseline inflammation. Unirradiated, age matched controls (A and B) and animals that were irradiated with 7 gray of X-Ray irradiation and reconstituted with donor bone marrow (C and D) were implanted with 4 × 10⁵ B16F10 syngeneic melanoma cells subcutaneously. On day 11 post-implantation, animals were randomized into treatment groups that received either isotype antibody (A and C) or anti-CTLA-4 antibody (B and D). Tumor volumes were measured twice a week until the tumors exceeded 800 mm³.