Fig 2: Methodological set-up and biological validation of non-homogenous tumor irradiation in immunocompetent mice bearing colorectal tumors.A Schematic representation of the different types of RT treatments, involving control tumors (Ctrl, 0 Gy), total tumor volume 2 Gy RT (TI2), total tumor volume 16 Gy RT (TI16), partial tumor volume 16/0 Gy RT (PI16/0), and partial tumor volume 16/2 Gy RT (PI16/2). The different portions of partially-irradiated tumors are also identified. Portions irradiated at 16 Gy and 0 Gy (unirradiated) from PI16/0 tumors (top right of the panel) are respectively called PI16/0 16 Gy and PI16/0 0 Gy. Reciprocally, portions irradiated at 16 Gy and 2 Gy from PI16/2 tumors (bottom right of the panel) are respectively called PI16/2 16 Gy and PI16/2 2 Gy. Black-dotted lines represent the limit between the two halves of partially-irradiated tumors. Created in BioRender. Mondini (2024) BioRender.com/j74g766. B Representative tumor segmentations generated with the help of CT imaging, representing all the irradiation regimens used in this article (TI2, TI16, PI16/0, PI16/2). The collimator (red squares in the figure) used for each isocenter (Iso, red points in the figure) is indicated below the associated panel. C Dose-volume histogram (DVH) showing dose distribution within the tumor volume, depending on the RT modality (dose, TI or PI) of the segmentations presented in (B). D Schematic representation of tumor staining and sampling method depending on the read-out. Both extremities of the tumors were identified and separated according to the procedure described in “Methods”. The intermediate portion (unstained) separating the extremities (green and yellow) was discarded. Created in BioRender. Mondini (2024) BioRender.com/n07j238. E Representative image of a γH2AX (dark blue) staining performed following partial tumor volume irradiation (PI16/0, 16 Gy and 0 Gy) on subcutaneous MC38 mouse tumors (n = 4 mice, two independent experiments). The red arrows show γH2AX foci. F H2AX phosphorylation on serine 139 was quantified by ELISA in Ctrl tumors (“Total IR”, 0 Gy), in TI16 tumors (“Total IR”, 16 Gy), and in both halves of PI16/0 tumors (“Partial IR”, 16 Gy and 0 Gy), with n = 4 mice in all groups. Data are shown as the mean ± SEM. Numbers on these graphs represent p values and were determined by two-way ANOVA. Source data are provided as a Source Data file.

Fig 3: Combining ERT and αPD-1 induces potent antitumor effects in mice with bulky tumors.a Dose distribution in a transverse section of LDRT, partial SBRT and ERT, along with the treatment scheme for mice. b, c Mouse positioning (b) and iERT dose distribution (c) of a bulky tumor. The SBRT (red dashed line) and LDRT (blue dashed line) zones were sampled along the sagittal plane, and the intermediate tissue at their interface (at least 2 mm in thickness, c, left, turquoise dashed line) was carefully removed. The intermediate tissue at the SBRT and LDRT interface along the transverse section (turquoise dashed line, c right and d left panels) was carefully removed. d Green marking ink on tumor (d left) and H&E staining (d, middle and right) confirming the SBRT-irradiated region. e, f γH2AX (green) immunofluorescence demonstrating DNA damage in iERT-treated tumors with distinct LDRT and SBRT zones, compared with untreated controls (f). Nuclei: DAPI (blue). The orange dashed line approximately delineates the SBRT zone. g Quantification of γH2AX foci in tumors 15 min post-ERT versus untreated tumors (n = 6 biologically independent samples per group, representative images shown in e and f). h, i CT26 tumors treated with ERT were dissected ex vivo into LDRT and SBRT zones. h Western blotting analysis of phospho-H2AX (Ser139) and GAPDH, with quantitative densitometry shown on the right (representative image from three independent experiments in which 6 different untreated and ERT-treated tumors were analyzed). i ELISA-based measurement of γH2AX levels presented as fold change relative to untreated tissue (n = 5 biologically independent samples per group). j–l Tumor growth curves for bulky CT26 (j, n = 11 mice per group), LLC1 (k, n = 5 mice per group) or B16-OVA (l, for untreated, n = 7 mice; for others, n = 6 mice per group) tumors in mice treated with partial SBRT/αPD-1, LDRT/αPD-1, or iERT. m–o Survival of mice bearing CT26 (m, n = 11 mice per group), LLC1 (n, n = 5 mice per group) or B16-OVA (o for untreated, n = 7 mice; for others, n = 6 mice per group) tumors. Data are presented as mean ± SEM. P- values (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001) were determined by one-way ANOVA (g–i), a two-tailed t test (j–l) and a log-rank test (m–o). Source data are provided as a Source Data file. a left Created in BioRender. https://biorender.com/7kwswlo.