Ultra-high dose-rate proton FLASH improves tumor control

Abstract

Background and purpose: Proton radiotherapy (PRT) offers potential benefits over other radiation modalities, including photon and electron radiotherapy. Increasing the rate at which proton radiation is delivered may provide a therapeutic advantage. Here, we compared the efficacy of conventional proton therapy (CONV^pr) to ultrahigh dose-rate proton therapy, FLASH^pr, in a mouse model of non-small cell lung cancers (NSCLC).

Materials and methods: Mice bearing orthotopic lung tumors received thoracic radiation therapy using CONV^pr (<0.05 Gy/s) and FLASH^pr (>60 Gy/s) dose rates.

Results: Compared to CONV^pr, FLASH^pr was more effective in reducing tumor burden and decreasing tumor cell proliferation. Furthermore, FLASH^pr was more efficient in increasing the infiltration of cytotoxic CD8⁺ T-lymphocytes inside the tumor while simultaneously reducing the percentage of immunosuppressive regulatory T-cells (Tregs) among T-lymphocytes. Also, compared to CONV^pr, FLASH^pr was more effective in decreasing pro-tumorigenic M2-like macrophages in lung tumors, while increasing infiltration of anti-tumor M1-like macrophages. Finally, FLASH^pr treatment reduced expression of checkpoint inhibitors in lung tumors, indicating reduced immune tolerance.

Conclusions: Our results suggest that FLASH dose-rate proton delivery modulates the immune system to improve tumor control and might thus be a promising new alternative to conventional dose rates for NSCLC treatment.

Keywords: FLASH; Lung cancer; Mouse model; Proton radiotherapy; Ultrahigh dose-rate proton.

Figure 1.. FLASH^pr has better efficacy in reducing lung tumor burden compared to CONV^pr.

(A) Experimental schematic for LLC orthotopic model of lung cancer, and timeline of irradiation and tissue harvest. (B) Mice treated with FLASH^pr and CONV^pr radiation have decreased lung tumor diameters compared to control tumor bearing mice. Tumors were measured using calipers at day 5 (3 mice per group) and day 8 (8 mice per group). (C) Representative images of tumor-bearing left lung lobe from each group at day 5 and day 8 post radiation. (D) Representative images after H&E staining of lung tumors at day 5 and day 8. (Scale bar 100 μm). (E-F) Immunofluorescence staining for mCherry-labelled tumor cells shows significant reduction of tumor burden at day 5 and day 8 in FLASH^pr group compared to control and CONV^pr. (Scale bar 500 μm). *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 2.. FLASH decreases the number of proliferating tumor cells compared to conventional dose-rate proton treatment.

(A-B) Number of Ki67 positive tumor cells is decreased in lung tumors treated with FLASH^pr compared to CONV^pr. Number of mCherry and Ki67 double positive cells per field was counted for each tumor and presented as average ± SEM (Scale bar 100 μm). Each dot represents one biological replicate. (C-D) FLASH^pr increases the percentage of mCherry/γH2A.X-double positive tumor cells compared to CONV^pr. Nuclei are counterstained with DAPI. Percentage of double positive cells were calculated for each tumor and presented as mean percentage ± SEM (Scale bar 100 μm). Each dot represents one biological replicate. (E-F) FLASH^pr increases the CD45⁺ immune cells to mCherry⁺ tumor cell ratio in lung tumors. Number of mCherry⁺ tumor cells and CD45⁺ immune cells were counted for each tumor and presented as the mean ratio of immune cells to tumor cells ± SEM (Scale bar 100 μm). Each dot represents one biological replicate. N = 3-8 mice per group; *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 3.. FLASH proton irradiation increase infiltration of CD3⁺ T-cells into tumors.

(A-B) Immunostaining with anti-CD3 antibody shows increased ratio between the number of T-lymphocytes and tumor cells in FLASH^pr-treated mice compared to CONV^pr at day 5 post radiation. The lower panel shows corresponding mCherry-positive tumor. Each dot represents the number of CD3⁺ T-cells/field for one biological replicate and data is presented as group mean ± SEM. N=3 mice per group (Scale bar 500 μm). (C-D) FLASH^pr increases the ratio of T-cells to tumor cells at day 8 after irradiation. Each dot represents the ratio of CD3⁺ T-cells to tumor cells for one biological replicate and data is presented as group mean ± SEM. (Scale bar 500 μm). N=8 mice per group. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 4.. FLASH^pr increases infiltration of CD8⁺ cytotoxic T-cell into the tumor and reduces the number of Tregs.

(A-C) Co-localization studies show the distribution of CD3⁺ T-cells (white), CD4⁺ helper T-cells (red) and CD8⁺ cytotoxic T-cells (green) inside lung tumors of different treatment groups at day 5 post radiation. Average numbers of CD4⁺, CD8⁺ and CD3⁺ T-lymphocytes in the lung tumors of different treatment groups were calculated for each biological replicate and presented as mean ratio ± SEM (Scale bar 100 μm). N=3 per group. (D-F) Co-localization studies show the distribution of CD4⁺ helper T-cells (red) and CD8⁺ cytotoxic T-cells (green) inside lung tumors of different treatment groups at day 8 post radiation. Average numbers of CD4⁺, CD8⁺ and CD3⁺ T-lymphocytes in the lung tumors of different treatment groups were calculated for each biological replicate and presented as mean ratio ± SEM (Scale bar 100 μm). N=8 per group. (G-I) Co-localization studies show the distribution of FOXP3⁺ Tregs (red) among CD3⁺ T-lymphocytes (green) inside lung tumors of different proton-irradiated groups at day 5 and day 8 (Scale bar 50 μm). Graph shows the percentage of FOXP3⁺ Tregs among total tumor associated CD3⁺ T-lymphocytes. Percentage of FOXP3⁺ Tregs for each biological replicate was counted and presented as mean ± SEM. N=3-8 per group. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 5.. FLASH^pr reduces the number of macrophages and increases the infiltration of T cells in tumors.

(A-B) Representative images show distribution of F4/80⁺ macrophages (white) in proton-treated and untreated lung tumors at day 8. LLC tumor cells are shown in red and nuclei are counterstained with DAPI (Scale bar 200 μm). Number of mCherry⁺ tumor cells and F4/80⁺ macrophages were counted for each biological replicate and presented as the ratio of macrophages to tumor cells per field ± SEM. (C-D) Co-localization studies show the distribution of CD8⁺ cytotoxic T-cells (green) and F4/80⁺ tumor-associated macrophages (white) among the mCherry-labelled tumor cells (red) in lung tumors of different treatment groups at day 8 (Scale bar 200 μm). Inserts are 20X images showing CD8+ cytotoxic T cells (green) and F4/80⁺ tumor-associated macrophages (white). Graphs show the ratio of CD8⁺ T-lymphocytes to F4/80⁺ macrophages per tumor. Data is representative of complete tumor fields and presented as mean ratio ± SEM. N= 8 per group. *, P<0.05; **, P<0.01; ***, P<0.001. (E-F) Co-localization studies demonstrate the decrease in the number of CD163⁺ cells (red) and the increase in the number of CD3⁺ T-lymphocytes (green) in the irradiated lung tumors compared to untreated tumors. Representative images of CD3⁺ T-cells and CD163⁺ cells at day 8 (Scale bar 100 μm). Graph shows correlation between the numbers of CD3⁺ T-cells and CD163⁺ M2-like macrophages in lung tumors. (G-H) Co-localization studies demonstrate the increase in the number of iNOS⁺ cells (red) and the increase in the number of CD3⁺ T-lymphocytes (green) in the radiation-treated lung tumors compared to untreated tumors. Representative images of CD3⁺ T-cells and iNOS⁺ cells distribution at day 8 (Scale bar 100 μm). Graph shows correlation between the number of CD3⁺ T-cells and iNOS⁺ M1-like macrophages in the lung tumors.

Figure 6.. FLASH^pr inhibits expression of checkpoint inhibitors PD-1 and PD-L1.

(A-B) Co-localization studies show the expression of PD-1 (red) among CD3⁺ T-lymphocytes (green) in lung tumors of different treatment groups at day 8 (Scale bar 50 μm). Nuclei were counterstained with DAPI. At least five tumors per group were used to calculate percentage of PD-1⁺ T-lymphocytes and presented as mean ratio ± SEM. N=5-7 per group. *, P<0.05; **, P<0.01; ***, P<0.001. (C-D) Co-localization studies show the expression of PD-L1 (green) among lung tumor cells (red) in different treatment groups at day 8 (Scale bar 50 μm). Nuclei were counterstained with DAPI. At least five tumors per group were used to calculate percentage of PD-1⁺ T-lymphocytes and presented as mean ratio ± SEM. *, P<0.05; **, P<0.01; ***, P<0.001.