Combining immunotherapy with high-dose radiation therapy (HDRT) significantly inhibits tumor growth in a syngeneic mouse model of high-risk neuroblastoma

Abstract

Purpose: The mortality in patients with MYCN-amplified high-risk neuroblastoma remains greater than 50% despite advances in multimodal therapy. Novel therapies are urgently needed that requires preclinical evaluation in appropriate mice models. Combinatorial treatment with high-dose radiotherapy (HDRT) and immunotherapy has emerged as an effective treatment option in a variety of cancers. Current models of neuroblastoma do not recapitulate the anatomic and immune environment in which multimodal therapies can be effectively tested, and there is a need for an appropriate syngeneic neuroblastoma mice model to study interaction of immunotherapy with host immune cells. Here, we develop a novel syngeneic mouse model of MYCN-amplified neuroblastoma and report the relevance and opportunities of this model to study radiotherapy and immunotherapy.

Materials and methods: A syngeneic allograft tumor model was developed using the murine neuroblastoma cell line 9464D derived a tumor from TH-MYCN transgenic mouse. Tumors were generated by transplanting 1 mm³ portions of 9464D flank tumors into the left kidney of C57Bl/6 mice. We investigated the effect of combining HDRT with anti-PD1 antibody on tumor growth and tumor microenvironment. HDRT (8 Gy x 3) was delivered by the small animal radiation research platform (SARRP). Tumor growth was monitored by ultrasound. To assess the effect on immune cells tumors sections were co-imuunostained for six biomarkers using the Vectra multispectral imaging platform.

Results: Tumor growth was uniform and confined to the kidney in 100% of transplanted tumors. HDRT was largely restricted to the tumor region with minimal scattered out-of-field dose. Combinatorial treatment with HDRT and PD-1 blockade significantly inhibited tumor growth and prolonged mice survival. We observed augmented T-lymphocyte infiltration, especially CD3⁺CD8⁺ lymphocytes, in tumors of mice which received combination treatment.

Conclusion: We have developed a novel syngeneic mouse model of MYCN amplified high-risk neuroblastoma. We have utilized this model to show that combining immunotherapy with HDRT inhibits tumor growth and prolongs mice survival.

Keywords: Cancer therapeutics; Combination therapy; High dose radiation therapy; Immunotherapy; MYCN-amplified neuroblastoma; Neuroblastoma; Novel syngeneic mouse model.

Fig. 1

Intrarenal syngeneic transplantation mouse model of neuroblastoma. (A) A flow diagram shows the generation of mouse model. 9464D cells were initially injected into the flank of C57BL/6 mice. Once the flank tumor size reached 1 cm³ , ^, as measured by ultrasound, it was explanted, divided into 1 mm³portions and placed in DMEM culture medium. White arrows designate the portions of explanted tumor. Simultaneous to the tumor explant, recipient mice were prepared for tumor implant. One piece of the explanted tumor was surgically implanted within the renal parenchyma of a recipient C57BL/6 mouse (n = 15). White arrow points to the implantation site within the kidney. (C) Representative ultrasound image of a tumor. (D) Tumor volumes measured by ultrasound on day 13 post implantation (n = 15).

Fig. 2

3D computed tomography (CT) scan based radiation treatment planning. (A) Two 170°arcs were designed to deliver 8Gy radiation, using a 1 cm × 1 cm collimator. Images were taken from the coronal plane, the sagittal plane, and the axial plane. (B) Isodose lines from an axial plan. Computed tomographic simulation was performed, and a representative mouse CT imaging scan was used to contour the gross tumor volume (GTV). Two 170° arcs were created targeting the GTV. (C) Phantom mouse construct from the coronal plane view. (D) Radiochromic film after coronal radiation delivery. Isodose lines were created based on the inverse intensity projected on the phantom based radiochromic films. (E) Measured dosage on the coronal radiochromic film. (F) Phantom mouse construct from the sagittal plane view. (G) Radiochromic film after sagittal radiation delivery. (H) Measured dosage on the sagittal radiochromic film.

Fig. 3

HDRT inhibits tumor growth at early time point. (A) Schema of anti-PD1 antibody and radiation treatments in 9464D syngeneic intrarenal tumors. Mice were enrolled on day 0. Mice were treated with anti-PD1 antibody on day 0, 3, 6 and 8 Gy fractions on day 3, 5 and 7 post-enrollment. Mice were subjected to early sacrifice (early time point) on day 9 and late sacrifice at 1.5 cm³ tumor size. (B) Representative gross images of tumors for each treatment group at early sacrifice time point. Mice were sacrificed on Day 9 as described in A. (C) Tumor volume at early time point (Day 9). Tumor volume was measured by ultrasound. (D) Tumor weight at early time point (Day 9).

Fig. 4

Combination of anti-PD-1 blockade and HDRT inhibits 9464D tumor growth. (A) Tumor growth with time for 4 treatment groups. Tumor volume was measured by ultrasound. Mice were enrolled on day 0. Mice were treated with anti-PD1 antibody on day 0, 3, 6 and 8 Gy fractions on day 3, 5 and 7 post-enrollment. (B) Kaplan–Meier analysis of mice, bearing 9464D tumors, from 4 treatment groups. Survival was defined as the day when tumor volume reached 1.5 cm³ and mouse was sacrificed.

Fig. 5

Combining HDRT with PD-1 inhibition increased cytotoxic CD3⁺CD8⁺ T-cell infiltration at early time point. (A) Representative images of quantitative multiplex immunofluorescence staining for DAPI (blue), CD3(cyan), CD4 (orange), CD8 (magneta), FOXP3 (yellow) and endomucin (red) at early time point. Mice were sacrificed at day 9 of the treatment regimen, tumors were formalin fixed and stained using Opal multiplex 6-plex kits with antibodies for CD3 (cyan), CD4 (orange), CD8 (Magenta), FOXP3 (yellow) and endomucin (red). DAPI is used as counterstain. Quantification of cell number per high-powered field for (B) CD3⁺ T cells, (C) CD3⁺CD8⁺ T cells, (D) CD3⁺CD4⁺ T cells and (E) CD3⁺CD4+FOXP3+ Treg cell. Mean ± SD. *, p < 0.05; **, p < 0.01; ****, p < 0.0001. For details of staining and quantification processes please see Material and Methods. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Elevated numbers of cytotoxic T-cells and T-helper cells in the combination group at late time point. A) Quantitative multiplex immunofluorescence images of T cell population in 9464D tumors. Tumors were coimuunostained for 6 markers: DAPI, blue; CD3, cyan; CD4, orange; CD8, magenta; FOXP3, yellow; Endomucin, red. Mice were sacrificed at 1.5cm3 tumor size and tumors were subjected to multiplex immunofluorescence staining (details of staining in Material and Methods). Quantification of cell number per high-powered field for (B) CD3⁺ T cells, (C) CD3⁺CD8⁺ T cells, (D) CD3⁺CD4⁺ T cells and (E) CD3⁺CD4+FOXP3+ Treg cells. Mean ± SD. *, p < 0.05; **, p < 0.01. Mice were subjected to early sacrifice (early time point) on day 9 and late sacrifice at 1.5 cm³ tumor size. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)