Priming versus propagating: distinct immune effects of alpha- versus beta-particle emitting radiopharmaceuticals when combined with immune checkpoint inhibition in mice

Domain: neuroscience
Published: 2026-01-26
Editorial outcome: minor_revision

Abstract
Radiopharmaceutical therapy (RPT) synergises with immune checkpoint inhibitors (ICI), but comparison of immunomodulation by different radioisotopes is lacking. Here, we evaluate mechanisms of response and timing of ICI administration relative to α-(225Ac) and β-emitting (90Y, 177Lu) radioisotope therapy, coupled with alkylphosphocholine NM600, when combined with dual (anti-PD-L1 and anti-CTLA4) ICI, using syngeneic poorly immunogenic (B78 and Myc-CaP) and immunogenic (MC38) murine models. Regardless of the isotope, RPT delivering 2 Gy mean tumor dose promotes tumor regression and improves survival in B78 or MC38 tumor-bearing mice when combined with early ICI administration. Greatest anti-tumor responses are seen in MC38 to 90Y-NM600+ICI and in B78 and Myc-CaP to 225Ac-NM600+ICI. Flow cytometry and single-cell RNA and T cell receptor sequencing reveal that, combined with ICI, β-emitting radioisotopes expand existing adaptive immunity, whereas α-emitting radiopharmaceuticals initiate immune priming. Thus, appropriate application of α- or β-emitting RPT in combination with ICI achieves distinct antitumor immune responses.

Introduction
Combinations of radiation therapy (RT) and immune checkpoint inhibitors (ICI) have shown efficacy preclinically and in certain clinical scenarios 1–5. Preclinical mechanisms by which RT enhances response to immunotherapy include inducing immunogenic cell death, releasing tumor-specific antigens and damage-associated molecular pattern molecules, and causing phenotypic changes on surviving tumor cells (upregulation of MHC-I and FAS, type I interferon secretion) 6–11. However, adding RT to a single tumor site in patients with metastatic disease has not improved the rate or duration of systemic response to standard-of-care ICIs 12. Several mechanisms have been proposed to account for this translational discrepancy, including patient selection (e.g., patients with immune exhausted tumor microenvironments have worse outcomes 2), radiation of tumor draining lymph nodes (necessary for propagation of an adaptive immune response 13), and non-optimal dosing or timing of radiation 14,15.

With a growing understanding of how RT enhances responses to immunotherapy preclinically and clinically in locally advanced disease settings, it is important to consider how these therapies may be combined to improve outcomes in patients with metastatic disease.

The role of external beam radiation therapy (EBRT) is limited in treatment of metastatic disease due to potentially prohibitive toxicities associated with irradiating multiple tumor sites or the whole body to cover radiographically occult micrometastatic disease. Additionally, unirradiated distant tumor sites have been shown to abrogate the in situ vaccine effect and resulting adaptive immune response following RT at one tumor site in certain preclinical studies 16,17.

Radiopharmaceutical therapy (RPT), which delivers a radioactive isotope conjugated to a tumor-targeting vector, is administered systemically to deposit radiation absorbed dose at all sites of uptake. RPT agents have the potential to overcome some of the limitations of EBRT in metastatic disease settings. Physical properties of radiopharmaceuticals vary widely, with beta (β)—(e.g., 90Y, 177Lu) and alpha (α)-particle (e.g., 223Ra, 225Ac) emitting radioisotopes being the focus of much clinical interest presently. β particles are characterized by low linear energy transfer (LET) (0.2 keV/µm) and travel millimeters in tissue with sparse ionization events, whereas α particles are high LET particles (50–230 keV/µm) that travel micrometers in tissue with densely clustered ionization events along their track 18. RPT agents have been studied both preclinically and in early clinical studies in combination with immunotherapy 19. To capitalize on the therapeutic potential of combinations of RPT+ICIs, it is critical to understand the effects of RPT properties (e.g., radionuclide, vector), disease characteristics (e.g., cancer type, immune environment), and RPT dosing and timing so that combinations with ICI may be optimized prior to advanced phase clinical investigation.

An unmet question is to determine the radiation absorbed dose of RPT and sequencing of administration of combination RPT and ICI therapies resulting in effective anti-tumor immune responses through comparative studies of 90Y-, 177Lu-, and 225Ac-NM600. NM600 is an alkylphosphocholine analog with selective uptake and retention in a wide array of tumors 20. The capacity of 90Y-, 177Lu-, and 225Ac-NM600 to generate type I interferon (IFN1) responses and immunomodulate the tumor microenvironment has been reported 15,21–23. 90Y-, 177Lu-, and 225Ac-NM600 can be utilized to examine RPT+ICI combinations in both poorly immunogenic (B78 melanoma and Myc-CaP prostate cancer) and immunogenic (MC38 colorectal carcinoma) syngeneic murine tumor models. B78, derived from the spontaneous melanocytic tumor B16F10, is characterized as poorly immunogenic, given its low levels of MHC-I expression, low numbers of baseline tumor-infiltrating lymphocytes, and limited response to immune checkpoint inhibition alone 15,24,25. MC38, a carcinogen-induced model, exhibits mismatch repair deficiency 26 and is characterized as immunogenic, having the highest mutational load amongst ten syngeneic tumor models and a propensity to respond to anti-CTLA4 27 and anti-PD-L1 alone 28.

In the current study, using these models and NM600, we seek to evaluate the development, efficacy, and key mechanisms of anti-tumor immunity in response to differing radionuclides and varied timing of ICI delivery. Systematically delineating differing tumor responses to 225Ac- 90Y- or 177Lu-NM600 in combination with dual ICI therapy (anti-PD-L1 and anti-CTLA4) in syngeneic murine tumor models is critical for efficacious translation to patients. Mechanistic insights from the immunogenicity of the tumor model may impact future study design and isotope selection and influence treatment regimens and patient selection as these therapies progress through clinical translation. We observe that 90Y-NM600+ICI promotes the greatest tumor regression in an immunogenic tumor model by propagating existing antitumor immune responses, while 225Ac-NM600+ICI leads to superior anti-tumor responses in poorly immunogenic tumor models by priming CD8+ T cells. These results underscore the relevance of understanding tumor mechanisms of resistance to ICI in order to employ effective RPT+ICI combination therapy.

Results
Murine tumor models exhibit tumor growth delay and survival improvement following low absorbed dose 225Ac-NM600+ICI

To perform absorbed dose-matched comparative immunologic studies of 90Y-, 177Lu-, and 225Ac-NM600, image-based radiation dosimetry studies were performed (NM600 chemical structure: Fig. 1A). Tumor uptake and dosimetry of 86Y/90Y-NM600 15 and 177Lu/225Ac-NM600 in mice bearing B78 melanoma tumors were reported previously 23. Maximum intensity projections of PET or SPECT scans showing MC38 selective uptake of 86Y-NM600 (imaging surrogate for 90Y-NM600) and 177Lu-NM600, respectively, are shown in Fig. 1B, C. Absorbed doses (mean±standard deviation) of 2.03±0.05, 0.50±0.01, 1.24±0.08, and 2.11±0.21 Gy/MBq 90Y-NM600 and 0.47±0.04, 0.08±0.01, 0.46±0.05, and 1.40±0.24 Gy/MBq 177Lu-NM600 were determined for blood, bone, spleen, and MC38 tumors, respectively (Fig. 1D, E). These absorbed dose estimates were obtained using a Monte Carlo voxel-based dosimetry method 29 and serial PET/CT or SPECT/CT data as well as ex vivo biodistribution data at one timepoint (Supplementary Fig. 1A, B). Estimates of absorbed dose to MC38 and normal tissues for 225Ac-NM600 were obtained by S-value calculations based on the Medical Internal Radiation Dose (MIRD) formalism using serial ex vivo biodistribution data (Supplementary Fig. 1C) 30. Absorbed doses of 0.163, 0.248, and 0.437 Gy/kBq 225Ac-NM600 were estimated for blood, bone, and MC38 tumor, respectively (Fig. 1F). Tumor absorbed dose estimates for all studies in this work are reported in Table 1

To determine the effects of different radionuclides on the heterogeneity of the spatial distribution of ionization radiation events in MC38 and B78 tumors, ionizing-radiation quantum imaging detector (iQID) scans of ionizing radiation event distribution were obtained on RPT treated tumors (Fig. 1G–K). Microdosimetric heterogeneity that may be present given the short tissue range of 225Ac compared to 90Y/177Lu (90Y: 11.3 mm, 177Lu: 1.8 mm, 225Ac: <0.1 mm) and not resolved by in vivo imaging can be assessed by digital autoradiography with the iQID system. These studies showed greater heterogeneity in ionizing radiation event distribution following 225Ac-NM600 than 177Lu-NM600 and 86Y-/90Y-NM600, resulting in “cold,” unirradiated regions within the B78 tumor adjacent to “hot” regions receiving high linear energy transfer (LET) radiation from 225Ac-NM600. No large differences in 90Y- or 177Lu-NM600 ionizing radiation event distribution between MC38 and B78 tumor types were observed.

Blood and additional normal tissue toxicity assessments for 90Y-NM600 and 177Lu-NM600 have previously been reported 22,31. The acute toxicity profile for the maximum tolerated activity (MTA) of 225Ac-NM600 in naïve C57BL/6 mice (18.5 kBq) is shown in Supplementary Fig. 2. 225Ac-NM600 could be delivered to naïve mice without physical signs of toxicity during the therapy studies but induced transient cytopenias (Supplementary Fig. 2A). Importantly, no mice treated in any of the studies reported in this manuscript were identified by veterinary staff as exhibiting signs or symptoms of illness/distress and no mice lost >20% of their body weight.

Although acute hepatotoxicity was not observed following 225Ac-NM600 (Supplementary Fig. 2B), we investigated late toxicities of 225Ac-NM600 (Supplementary Fig. 3A–D), and noted elevated liver transaminases and serum creatinine, suggesting hepatic and renal dysfunction. One parameter of radiopharmaceuticals that can be adjusted to improve toxicities and lesion: non-lesion residence times is molar activity (Am). Frequently, high Am is desired to reduce toxicities associated with certain RPT vectors or with target saturation at low Am 32. However, in the case of 111I-J591, lowering molar activity of the RPT agent by adding unlabeled antibody increased the lesion residence time and the ratio of lesion: liver residence times 33. For this reason, we investigated the effect of lowering the Am of 225Ac-NM600 (Supplementary Fig. 3). The liver uptake of 225Ac-NM600 was significantly decreased in the low Am group (0.011 MBq/nmol 225Ac-NM600) vs. high Am (0.336 MBq/nmol 225Ac-NM600), and the MC38 uptake was not significantly different. However, late (6 month post administration) changes in mouse weight and hepatic and renal toxicity were similar in low and high Am treatment groups. Thus, this approach of lowering the Am did not markedly improve the biodistribution of 225Ac-NM600. A possible explanation for the minor impact on biodistribution of altering NM600 Am is that NM600 has a lipid raft cellular uptake mechanism distinct from the receptor-ligand interaction employed by 111I-J591 and many other RPTs, which may not be saturable.

We also examined the cytotoxicity of 225Ac relative to EBRT in both MC38 and B78, observing an expected high relative biological effectiveness (RBE) of 225Ac (Supplementary Fig. 4A, B). Additionally, we confirmed that anti-PD-L1 antibody uptake and distribution was similar between MC38 and B78 tumors (Supplementary Fig. 5). We determined that MC38 and B78 tumor mRNA expression of Pdl1 and Ctla4 was not significantly changed following treatment with 90Y-, 177Lu-, or 225Ac-NM600+ICI (Supplementary Fig. 6A–D). Given that 225Ac-NM600 was tolerable with evidence of hematologic recovery and stable weight, we began testing 225Ac-NM600 in combination with ICI (Fig. 1L–S).

In MC38 (Fig. 1L–O and Supplementary Tables 1–3) and B78 (Fig. 1P–S and Supplementary Tables 4–6), dose finding studies were conducted to determine an absorbed dose of 225Ac-NM600 that could be administered in each tumor model with a therapeutic benefit. From our prior observation that a 2 Gy mean tumor absorbed dose was optimal for response to 90Y-NM600+anti-CTLA-4 15, we investigated 2 and 0.2 Gy 225Ac-NM600 in MC38 and B78. B78 has lower uptake of 225Ac-NM600 (0.10 Gy/kBq) 23 than MC38 (0.437 Gy/MBq), and an estimated tumor absorbed dose greater than 2 Gy could not be achieved without exceeding toxicity limits. In the MC38 model, 2 Gy alone provided only nominal survival benefit, and while addition of ICI to 2 Gy increased survival compared to both 2 Gy alone and the control, it was not significantly different from ICI alone (Fig. 1O). In the B78 model, however, while 2 Gy provide a moderate increase in survival relative to control, addition of ICI to the 2 Gy treatment increased survival compared to control, 2 Gy, and ICI alone. Because 2 Gy 225Ac-NM600 was tolerable and demonstrated a cooperative therapeutic interaction with ICI in the B78 model, we selected this dose for further study (225Ac-NM600+ICI vs. 225Ac-NM600 alone, overall survival) (Fig. 1M–O, Q–S). The differences in the patterns of response among MC38 and B78 tumors led us to hypothesize that the effects of RPT+ICI may be different based on the immunogenicity of the tumor model.

90Y-NM600+early/intermediate-timed ICI enhances therapeutic response in the immunogenic MC38 colorectal carcinoma model

Three different timings of ICI (dual anti-CTLA4 and anti-PD-L1) were investigated relative to treatment day 1 injection of activities of 90Y-NM600, 177Lu-NM600, or 225Ac-NM600 that were estimated from dosimetry to deliver 2 Gy mean tumor absorbed dose. ICI delivery was defined as early (days −3/0/3), intermediate (days 4/7/10), or delayed (days 11/14/17). The intermediate-timed ICI has been shown to be safe and effective in combination with all three RPT compounds and EBRT in murine tumor models 15,21,23,34. Considering the open clinical question of optimal sequencing of RT+ICI and effects of radioisotope half-life on tumor immunomodulation 23, we compared the effect of sequencing of RPT+ICI on tumor growth and survival (Fig. 2A–I); 16 treatment groups, repeated two separate times). Comparisons between varied ICI timings are shown in Fig. 2F, H and Supplementary Tables 7–13, and comparisons between varied radioisotopes are shown in Fig. 2G, I and Supplementary Tables 14–20.

90Y- and 177Lu-NM600+early ICI reduced MC38 growth rate and prolonged overall survival relative to all other groups. For 225Ac-NM600, overall survival prolongation was observed following only 225Ac-NM600+early ICI (Fig. 2F, H). For early ICI, 90Y-NM600+ICI slowed tumor growth rate and prolonged overall survival compared to 225Ac-NM600+ICI (Fig. 2G, I). For intermediate-timed ICI, 90Y-NM600 combination therapy showed the greatest therapeutic benefit. For delayed ICI, there were a few differences between any RPT+ICI combination therapy and the control groups (Fig. 2G, I). Rates of complete response (CR) for all mice included in Fig. 2 therapy studies are shown in Supplementary Fig. 7A–D, E demonstrates the rates at which mice rendered disease-free subsequently rejected re-engraftment on the contralateral flank with the same tumor cell line they had been cured from (as a measure of immunologic memory), relative to age-matched naïve control mice. This re-engraftment was performed on day 100 after initial RPT treatment, and rates of rejection were: 90Y-NM600+early ICI: 8/9; 90Y-NM600+intermediate ICI: 4/4; 177Lu-NM600+early ICI: 5/5; 177Lu-NM600+intermediate ICI: 1/1; early ICI: 8/10; naïve C57BL/6 mice: 0/15.

225Ac-NM600+early ICI prolongs overall survival in the poorly immunogenic B78 melanoma model

We next turned to a poorly immunogenic tumor model, B78 melanoma 24,25, to test whether the immunogenicity of a tumor model may impact patterns of response to RPT+ICI (Fig. 3A–I; 14 treatment groups). Comparisons between varied ICI timings are shown in Fig. 3F, H and Supplementary Tables 21–27, and comparisons between varied radioisotopes are shown in Fig. 3G, I and Supplementary Tables 28–34. For each radioisotope delivering 2 Gy mean tumor absorbed dose, early ICI administration led to a statistically significant reduction in tumor growth rate and prolongation of overall survival compared to RPT monotherapy and an untreated control group. 225Ac-NM600+ICI groups demonstrated the slowest tumor growth rates for each ICI timing. 225Ac-NM600+delayed ICI resulted in a significant extension of overall survival compared to 90Y-NM600+delayed ICI. Supplementary Fig. 8A–D shows the complete response and rechallenge rejection rates for B78 tumor cells injected on the contralateral flank of the complete responders or naïve mice on day 120: 90Y-NM600+early ICI: 2/2; 90Y-NM600+intermediate ICI: 2/2; 177Lu-NM600+early ICI: 1/1; 225Ac-NM600+intermediate ICI: 1/1; naïve C57BL/6 mice: 0/5.

CD8+ T cells are key immune mediators of effector and memory immune response to 90Y-, 177Lu-, and 225Ac-NM600+ICI

In prior work with B78 melanoma, we have shown the necessity of T cells for the therapeutic response to 90Y-NM600+anti-CTLA4 15. Separately, in response to immunotherapy, including ICIs, CD8+ T cells have been shown to be major mediators of the antitumor and memory response in mice bearing MC38 tumors 35–37. Radiation therapy directed against MC38 tumors has also been shown to enhance the antitumor effect of adoptively transferred CD8+ T cells 38. In MC38 colorectal carcinoma, the therapeutic benefit of 90Y-, 177Lu-, and 225Ac-NM600+ICI as compared to RPT monotherapy was abrogated when CD8+ T cells were depleted (Fig. 4A–K and Supplementary Tables 35–41). This suggests the necessity of functional CD8+ T cells for mediation of antitumor responses to these combination systemic therapies in mice bearing MC38 tumors.

MC38 tumors were harvested and dissociated for flow cytometry at various timepoints over the course of RPT+early ICI to evaluate longitudinal changes in the tumor immune infiltrate, including pre-treatment (day −3), and one harvest timepoint at the half-life of each radioisotope: day 4 (90Y half-life: 2.7 days), day 8 (177Lu: 6.7 days), and day 11 (225Ac: 10 days), following 2 Gy RPT administration on day 1 (Fig. 4L–N). Overall, the frequency of CD45+ tumor infiltrating immune cells was similar with or without RPT, with a slight reduction in CD45+ cell frequency in the 177Lu- and 225Ac-NM600+ICI tumors compared to ICI and 90Y-NM600+ICI on day 11. The 90Y-NM600+ICI treated tumors additionally demonstrated an increased CD8+/Treg ratio compared to ICI alone, and increased activation marker expression on CD8+ T cells and interferon-γ (IFNγ) production by CD8+ and CD4+ T cells compared to other groups. These results suggest that 90Y-NM600+ICI treatment leads to an inflamed tumor immune microenvironment in MC38 colorectal carcinoma. Notably, all groups had a reduction in Tregs following ICI on days −3/0/3, as expected following treatment with a Treg depleting anti-CTLA4 antibody (IgG2c, clone 9D9).

We next aimed to determine whether a tumor-specific memory response was elicited following RPT+ICI. To do this, we investigated the functionality of peripheral CD8+ effector memory (EM) cells 23 days after 2 Gy mean tumor absorbed dose from 90Y-, 177Lu-, or 225Ac-NM600+ICI or ICI alone compared to CD8+ EM cells isolated from naïve C57BL/6 mice (Fig. 4O–Q). In this assay, peripheral blood cells were co-cultured with or without MC38 tumor cells for 24 h, and flow cytometry was performed to measure TNF and IFNγ effector cytokine production by CD8+ EM cells (CD8+CD44+CD62L−). Overall, 90Y-NM600+ICI resulted in the greatest induction of TNF and IFNγ compared to other treatment groups, and treatment with 177Lu-NM600 did not induce increased TNF and IFNγ production by CD8+ EM cells. There were few or no differences in the level of TNF and IFNγ production by peripheral CD8+ EM cells between the groups in the absence of MC38 co-culture, consistent with tumor-specific activation.

Long-term immune memory response following 225Ac-NM600 dose escalation in immunogenic MC38 tumors

In prior studies, we had observed no improvement in tumor response to 90Y-NM600 RPT+ICI combo with dose escalation beyond 2 Gy of 90Y-NM600—a low LET, longer range RPT 15. Given that dose escalation of 225Ac-NM600 to a mean tumor absorbed dose of 8 Gy was tolerable with evidence of hematologic recovery and stable weight in mice bearing MC38 tumors, we began testing 225Ac-NM600 in combination with ICI in MC38 (Fig. 5A, B). A dose-dependent increase in TNF and IFNγ production by peripheral CD8+ EM cells following 0.2, 2, or 8 Gy mean tumor dose from 225Ac-NM600+ICI was observed (Fig. 5C).

We sought to investigate whether a memory response could be observed following 8 Gy mean tumor dose from 225Ac-NM600+ICI. Mice bearing MC38 colorectal carcinoma were treated with 225Ac-NM600 on day 1±ICI on days −3/0 (Fig. 5D–L and Supplementary Tables 42–44). Combination therapy significantly prolonged overall survival compared to RPT monotherapy and control (Fig. 5F). 225Ac-NM600+ICI enhanced the CR rate of primary MC38 tumors (5/5 CR) relative to RPT (0/5 CR) and ICI (3/5 CR) monotherapies (Fig. 5G). CRs were rechallenged with injection of MC38 cells in the flank on day 100. However, only 3/5 225Ac-NM600+ICI CR mice rejected tumor rechallenge as compared to 3/3 ICI CR mice (Fig. 5H).

From mice showing rejection of this rechallenge, blood and splenocyte cell populations were evaluated by flow cytometry 24 h following an intravenous injection of MC38 tumor cells on day 136 after RPT. Flow cytometry revealed a significant increase in activation of circulating CD8+ memory T cells by IFNγ and TNF expression compared to naïve mice following MC38 re-exposure for all 225Ac-NM600+ICI mice regardless of rechallenge response (Fig. 5I). No CD4+ memory cell activation differences were observed relative to age-matched naïve mice (Fig. 5J), suggesting the memory response maybe mediated by CD8+ cells. Increased CD8+ CM/EM cell ratios have been reported to be associated with long-term immunologic memory 39. Circulating lymphocyte percentages were maintained in 225Ac-NM600+ICI treated mice (Fig. 5K). Additionally, we noted increased CD4+ and CD8+ central memory (CM)/EM ratios in spleens of all 225Ac-NM600+ICI CR mice as compared to ICI monotherapy CR mice (Fig. 5L), regardless of whether the CR mice rejected rechallenge (Ac+ICI no tumor) or not (Ac+ICI tumor). Thus, long-term memory immune memory responses were observed following 225Ac-NM600 dose escalation in the immune sensitive MC38 tumor model.

225Ac-NM600+ICI showed therapeutic benefit over 90Y-NM600+ICI and did not decrease immune cell populations in immune-resistant tumors

To test our hypothesis that the immunogenicity of the tumor model may impact the optimal radioisotope for antitumor responses, we evaluated the therapeutic efficacy of low absorbed dose 225Ac-NM600 vs. 90Y-NM600+dual anti-CTLA4 and anti-PD-L1 in a second poorly immunogenic model. The Myc-CaP prostate cancer model was selected, as it has an immunosuppressive tumor microenvironment and limited responses to RPT or ICI alone 40. Tumor uptake and dosimetry of 86Y/90Y-NM600 20 and 225Ac-NM600 41 in mice bearing Myc-CaP prostate cancer tumors were reported previously. As was observed in B78 melanoma, Mice bearing Myc-CaP tumors showed a statistically significant reduction in tumor growth rate and prolonged overall survival when treated with 2 Gy 225Ac-NM600+ICI as compared to 2 Gy 90Y-NM600+ICI or monotherapies (Fig. 6A–C and Supplementary Tables 45–47). This finding supported the hypothesis that tumor immunogenicity may impact therapeutic response to 225Ac alpha-particle emitting vs. 90Y or 177Lu beta-particle emitting RPT when combined with ICI therapy.

In the immune resistant B78 melanoma, we sought to identify tumor immune microenvironment changes following 90Y-, 177Lu-, or 225Ac-NM600+ICI combination therapy that would explicate the relative therapeutic benefit of alpha-emitting low absorbed dose 225Ac-NM600 compared to low absorbed dose 90Y-NM600 observed in Fig. 3. We performed paired single cell (sc)RNA-sequencing (seq) and T cell receptor (TCR) seq among tumor-infiltrating immune cells from mice bearing B78 melanoma tumors receiving 2 Gy mean tumor absorbed dose from either 90Y-, 177Lu-, 225Ac-NM600 or EBRT+early ICI or ICI alone. Tumors were harvested, and 3 tumors/treatment group were pooled for CD45+ cell isolation and scRNA-seq and TCR seq on treatment day 15 (Fig. 6D–F). Supplementary Fig. 9A, B shows the tumor growth curves of the B78 melanoma tumors harvested and pooled for scRNA-seq analysis. Following filtering and quality control, data were analyzed for 40,672 cells with 11,312 mean reads per cell. The number of cells (independent biological replicates) used for scRNA-seq analysis per treatment group is indicated in Supplementary Table 48. Dimensionality reduction and graph-based clustering of all cells were performed to distinguish cells with unique transcriptional profiles. UMAPs showing identical cell populations (overall cell population analyzed) with each treatment group highlighted are shown in Fig. 6F.

Lymphoid cell type fractions are shown in Fig. 6D. Of note, the frequency of follicular B cells, which participate in T-cell-dependent antibody responses, was increased in the 225Ac-NM600+ICI and ICI alone treatment groups. The 225Ac-NM600 follicular B cells had a 2.57-fold increase compared to ICI alone in expression of the MHC-I gene, H2-K1. Frequencies of both CD8+ effector T cells and Tregs were highest in the 90Y-NM600+ICI treatment group. CD8+/Treg ratios for each treatment group were similar between groups in this analysis: 225Ac-NM600+ICI: 0.44, 177Lu-NM600+ICI: 0.37, 90Y-NM600+ICI: 0.49, EBRT+ICI: 0.44, ICI: 0.58. With portions of the same tumor samples that were used for single cell RNA sequencing, CD4+, CD8+, Tregs (FOXP3+ cells), and NK1.1+ cells were quantified by IHC (Supplementary Fig. 9). Consistent with the single cell RNA sequencing results, there were no statistically significant differences in these lymphoid cell populations between treatment groups (Fig. 6D and Supplementary Fig. 10A–D).

External beam radiation to tumor-draining lymph nodes (TDLN) decreases the effectiveness of RT+anti-CD25 immunotherapy 13. In metastatic settings, TDLNs may not be readily identifiable or avoidable. Furthermore, it is unknown what effect EBRT may have on other immune structures, including tertiary lymphoid structures (TLS), which have been shown to be critical to antitumor immunity with ICIs 42. We hypothesize that RPT, by molecularly rather than spatially targeting tumors, could avoid such structures. EBRT+ICI disrupted existing CD20+-rich lymphoid clusters in B78 melanoma (Fig. 6G, H). These CD20+ structures were present in a significantly greater number in the 225Ac-NM600+ICI tumor, which is the radioisotope with the shortest emission path length (<100 µm) 23. The short tissue path length signifies that irradiation is minimized to non-targeted cells in the tumor, including stroma and islands of immune cells, as well as neighboring structures, including lymph nodes.

225Ac-NM600+ICI leads to diversified effector T cell responses with key shared clonotypes

With the same cells used for scRNA-seq analysis, we performed TCR seq analysis to gain insight into the functional status of CD8+ T cells following 90Y-, 177Lu-, 225Ac-NM600, or EBRT+ICI. After quality control, TCR sequencing analysis provided data for 3,517 cells with productive V-J spanning pairs. The number of cells (independent biological replicates) used for TCR sequencing analysis per treatment group is shown in Supplementary Table 49. UMAPs of all T cells stratified by sample are shown in Fig. 7A. Clonotype expansion ranges for Fig. 7 are represented in Fig. 7B. UMAPs stratified by T cell subtype are shown in Fig. 7C, verifying that most medium and large T cell clones pertain to CD8+ effector and memory subtypes. Clonal expansion by T cell subtype and CDR3 sequence diversity measurement (D50) are shown in Fig. 7D, E. 177Lu- and 90Y-NM600+ICI have more clonal expansion of CD8+ effector and memory T cells, with 225Ac-NM600 and EBRT+ICI showing more diversity and a higher D50 score for CD8+ effector T cells. Figure 7F presents the clonal expansion data in a different way, demonstrating the % of cells with clonotypes per expansion. These results indicate that the majority of effector CD8+ clones in the 177Lu- and 90Y-NM600+ICI groups are medium or large clones. These findings are consistent with previously reported CD8+ clonal expansion following low absorbed dose 90Y-NM600+ICI 15. Finally, shared clonotypes between various subsets of T cells are shown in Fig. 7G, H, with 225Ac-NM600+ICI treated tumors having the greatest number of shared clonotypes between effector and memory CD8+ T cells (11), as compared to seven shared clonotypes for the same T cell subsets in each other treatment group.

While 225Ac-NM600+ICI treated tumors had greater T cell diversity overall, they exhibited shared clonotypes between effector and memory CD8+ T cells. This is consistent with an in situ vaccine effect and greater priming of an adaptive T cell response with high LET α-particle RPT, as compared to the greater clonal expansion or propagation of existing immune response in the absence of apparent T cell priming with low absorbed dose, low LET β-particle RPT.

225Ac-NM600+ICI enhances CD8+ cell expression of interferon-α and γ response genes compared to 90Y-/177Lu-NM600/EBRT+ICI

Pathway analysis and differential gene expression analysis were performed to evaluate the relative expression of genes in each combination RT+ICI treatment vs. ICI alone. These results for CD8+ effector T cells and M1 macrophages for genes involved in IFNγ and IFNα responses are shown in Fig. 8A–D and Supplementary Fig. 11A–C. In the 225Ac-NM600+ICI treatment group, both the hallmark interferon alpha response and hallmark interferon gamma response pathways were noted to be altered compared to the other treatment groups (CD8+ cells: Fig. 8A, M1 macrophages: Supplementary Fig. 11A). These pathways were selected for further differential gene expression analysis (Fig. 8B, C). Following 225Ac-NM600+ICI, several CD8+ effector T cell genes were upregulated to a greater degree than other RT+ICI groups: Rnf213, Txnip, Isg15, Bst2, Rtp4, Epsti1, Zbp1, Stat1, Irf7, and Ly6e. Of these, Rnf213, Isg15, Rpt4, and Stat1 are interferon-induced genes; IRF7 regulates several interferon α genes; and ZBP1 activates both IFN1 responses and NF-κB signaling 43. This IFN1 activation reflects the reported IFN1 signaling following radiation due to the accumulation of cytosolic micronuclei 44. TXNIP has been reported to be associated with increased tumor-infiltrating immune cells and a carcinostatic role overall 45. Epithelial-stromal interaction 1 (EPST1) is a protein that promotes monocyte adhesion to endothelial cells 46. BST2 and LY6E are pro-tumor proteins. B2m, which encodes β2 microglobulin, a component of MHC-I molecules, was upregulated for all groups relative to ICI alone. This increase in expression for all RT+ICI groups reflects the well-documented ability of RT to enhance MHC-I expression 47.

For M1 macrophages (Supplementary Fig. 11B, C), there were several genes upregulated following 225Ac-NM600+ICI but downregulated for other groups, including Ly6e, Irf7, Isg15, Bst2, and unique from CD8+ effector T cells, Ccl7 and Ifitm3. Ccl7 has been shown to facilitate antitumor responses to anti-PD-1 through recruitment of classical dendritic cells 48, and Ifitm3 encodes an interferon-inducible transmembrane protein. Conversely, both the MHC-II gene (H2-Aa) and Cd74, involved in the formation and transport of MHC-II 49, and were downregulated following 225Ac-NM600+ICI in M1 macrophages but upregulated in all other RT+ICI groups (Supplementary Fig. 11B, C). Taken together, genes induced by 225Ac-NM600+ICI have mixed roles in tumor progression and immune response, with a clear prevalence of MHC-I expression among all RT groups, and IFN1-inducible genes following 225Ac-NM600+ICI, consistent with the greater amount of DNA damage induced by the α-emitting radioisotope.

Finally, we performed differential gene expression analysis on CD8+ cells with shared clonotypes within effector and memory cells (the population of cells highlighted in Fig. 7G, H). The number of cells (independent biological replicates) with clonotypes shared within effector and memory CD8+ T cells, stratified by treatment group, is shown in Supplementary Table 50. Interestingly, one gene that was upregulated in this cell population in the 225Ac-NM600+ICI treatment group over each other treatment group was an interferon inducible gene, Ifi27l2a (red arrow, Fig. 8D). This observation further suggested that an active adaptive T cell response was present in the 225Ac-NM600+ICI-treated tumor.

Methods
Cell lines and culture
The murine melanoma B78-D14 (B78) cell line, derived from B16 melanoma, was obtained from Dr. Ralph Reisfeld (Scripps Research Institute) in 2002 58. The murine colorectal carcinoma MC38 cell line, isolated from a female C57BL/6 mouse, was obtained from Millipore Sigma (Cat# SCC172), and the murine prostate cancer Myc-CaP cell line, isolated from a male FVB mouse, was obtained from American Type Culture Collection (ATCC) (Cat # CRL-3255). B78 cells were grown in a humidified incubator at 37 °C with 5% CO2, in RPMI-1640 (Corning, Cat# 10-040-CV) supplemented with 10% FBS (Avantor, Cat # 97068-085), and 100 U/mL penicillin+100 µg/mL streptomycin (Corning, Cat# 30-002-CI); MC38 and Myc-CaP cells were grown in DMEM (Corning, Cat# 10-013-CV) supplemented with 10% FBS, 100 U/mL penicillin+100 µg/mL streptomycin. Cell line authentication was performed per ATCC guidelines using morphology, growth curves, and Mycoplasma testing within 6 months of use.

Murine tumor models
Mice were housed in a specific pathogen-free animal facility and treated under a protocol approved by the Institutional Animal Care and Use Committee at the University of Wisconsin-Madison (protocol: M005670). Control and experimental mice were co-housed. All mice were monitored daily by independent veterinary staff and weighed weekly. All mice were maintained under a tightly controlled temperature (22 °C), humidity (40–50%), light/dark (12/12 h) cycle conditions, with water and food ad libitum. C57BL/6NTac (C57BL/6 genetic background, stock number F151) and FVB/NTac (FVB genetic background, stock number F59) mice were purchased at age 6–8 weeks from Taconic. Both male and female C57BL/6 mice were included in MC38 and B78 therapy studies. Female mice were used for correlate studies. Male FVB/N mice only were included in Myc-CaP prostate cancer therapy studies as the prostate gland is present in male mammals only.

Mouse therapy studies
MC38 and Myc-CaP tumors were engrafted by subcutaneous flank injection of 1×106 tumor cells. B78 tumors were engrafted by subcutaneous flank injection of 2×106 tumor cells. Tumor size was determined using Vernier calipers, and volume approximated as (width2×length)/2. Mice were randomized immediately before treatment by permuted block randomization. Only mice with palpable flank tumors the day before treatment began were included in the study. Treatment began when tumors were well-established (~50–100 mm3), approximately 12 days after tumor implantation for MC38, 17 days for Myc-CaP, and 4 weeks for B78. The day of EBRT or RPT was defined as “day 1” of treatment. Anti-murine CTLA-4 (IgG2c, clone 9D9, produced by NeoClone) and anti-murine PD-L1 (IgG2b, clone 10F.9G2, BioXCell, Cat# BE0101) were administered by 100 µg intraperitoneal injection on days indicated in figures and figure legends. Tumors were measured twice weekly for at least 60 days after starting treatment unless mice died or were euthanized. Mice were euthanized via CO2 asphyxiation when tumor size exceeded 20 mm in the longest dimension or recommended by an independent animal health monitor for morbidity or moribund behavior. Due to institutional radiation safety protocols and the mice being treated with radioactive isotopes, all investigators were aware of mouse treatment groups. The number of mice in the therapy groups was based on anticipated effect size of outcomes to power experiments, determined in collaboration with biostatisticians, and therefore may vary between treatment groups and tumor models. Mouse therapy experiments were repeated in duplicate (two independent experiments). Final replicates are presented for tumor response and aggregate data for survival; number of animals (independent biological replicates) per group is indicated in figure legends.

Mouse CD8+ depletion studies
CD8+ T cell depletion was performed by injection of anti-murine CD8α (Rat IgG2b, κ; Clone 2.43; BioXCell, Cat# BE0061) or IgG2b control (Rat IgG2b, κ; Clone LTF-2; BioXCell, Cat# BE0090) by 300 µg intraperitoneal injection on days −5, 0, and 5. Depletion was confirmed on treatment day −3 by flow cytometry.

Mouse immune memory studies
Mice without palpable tumors on day 100 were considered complete responders (CR). CR mice were rechallenged with 1×106 MC38 cells or 2×106 B78 cells (whichever cell line matched the original tumor), contralateral flank injection on day 100. For each therapy study, n=5 age-matched naïve female C57BL/6 mice (independent biological replicates) were injected with either 1×106 MC38 cells or 2×106 B78 cells as a control. On day 136, CR mice and age-matched naïve controls (n=4 independent biological replicates) received 2×105 MC38 cells IV. An additional n=4 naïve controls (independent biological replicates) received no cells IV. Twenty-four hours post antigen re-exposure, blood and splenocytes were harvested for flow cytometry.

Radionuclides and radiochemistry
90Y was purchased as 90YCl3 from Eckert and Ziegler. 177Lu and 225Ac were purchased as 177LuCl3 and solid 225Ac(NO3)3 from Oak Ridge National Laboratory. The radiolabeling of 2-[[hydroxy[[18-[4-[[2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]phenyl]octadecyl]oxy]phosphinyl]oxy]-N,N,N-trimethyl ethanaminium (NM600) with 90Y, 177Lu, or 225Ac proceeded by mixing the radiometal with NM600 (0.336–3.36 MBq/nmol) in 0.1 M NaOAc buffer (pH 5.5) for 30 min at 90 °C for 90Y-NM600 22. The labeled compounds were purified using a reverse-phase Waters Oasis HLB Light cartridge (Milford, MA), eluted using 100% ethanol, dried with a stream of N2, and reconstituted in 0.9% NaCl with 0.1% v/v Tween 20. Yield and purity were determined with instant thin-layer chromatography using 50 mM EDTA and iTLC-SG glass microfiber chromatography paper impregnated with silica gel (Agilent Technologies). A cyclone phosphor image reader was used to analyze the chromatograms; the labeled compound remained at the spotting point while free radiometals moved with the solvent front.

PET/CT imaging
Mice bearing MC38 (n=3 male, n=3 female independent biological replicates) flank tumors (100–150 mm3) were injected via tail vein with 9.25 MBq of 86Y-NM600 and imaged with an Inveon microPET/microCT scanner (Siemens Medical Solutions, Knoxville, TN) at 3, 24, 48, and 72 h post injection of the radiotracer. For each scan, mice were anesthetized with isoflurane and placed in the prone position on the scanner bed. Sequential CT (80 kVp; 1000 mAs; 220 angles) and static PET scans (80 million coincidence events; time window: 3.432 ns; energy window: 350–650 keV) were collected. A three-dimensional ordered subset expectation maximization algorithm was used to reconstruct the PET images. These were then fused with corresponding CT images for attenuation correction and anatomical referencing. Tumor and organs of interest were contoured for region-of-interest analysis of the PET images to determine the magnitude and kinetics of 86Y-NM600 uptake, which is reported as percent injected activity per gram of tissue (%IA/g; mean±SEM). An ex vivo biodistribution study was carried out after the last scan timepoint.

SPECT/CT imaging
Animals with subcutaneous MC38 (C57BL/6) tumors were administered 18.5 MBq of 177Lu-NM600 in the lateral tail vein. Individual mice (n=4 independent biological replicates) were placed prone into a MILabs U-SPECT6/CT Uhr system (Houten, The Netherlands) under 2% isoflurane for longitudinal scans at 3, 24, 96, and 180 h post-injection. CT scans (5 min) were acquired for anatomical reference and attenuation correction and fused with the SPECT scans (45 min). Image reconstruction used a similarity-regulated ordered-subset expectation maximization (SROSEM) algorithm. Images were quantitatively analyzed by drawing volumes of interest (VOI) over the tumor and organs of interest to determine the percent injected activity (IA) per gram (%IA/g) for each tissue. An ex vivo biodistribution study was carried out after the last scan timepoint.

Ex vivo biodistribution
Mice bearing MC38 subcutaneous tumors were injected with 9.25 MBq 86Y-NM600, 18.5 MBq 177Lu-NM600, or 9.25 kBq 225Ac-NM600, then euthanized via CO2 asphyxiation at 72 h (86Y-NM600), 180 h (177Lu-NM600), or 2, 48, and 168 h (n=3 mice (independent biological replicates)/timepoint) post-injection, and organs of interest were collected for ex vivo biodistribution for dosimetry. For comparison between low Am and high Am 225Ac-NM600 biodistributions, MC38 tumor bearing mice were injected with 9.25 kBq of low Am (81.1 µg NM600/MBq 225Ac; n=3 independent biological replicates) or high Am (2.70 µg NM600/MBq 225Ac; n=2 independent biological replicates) 225Ac-NM600, and organs of interest were collected 12 days after injection. Organs from animals that received 225Ac-NM600 were allowed to decay at 4 °C overnight to reach secular equilibrium with 213Bi, which could be quantified. Organs were wet weighed, analyzed with a Perkin Elmer Wizard2 Gamma Counter (Westham, MA), and decay corrected to calculate the %IA/g for each tissue.

In vivo dosimetry estimation
To estimate the dosimetry of 225Ac-NM600, ex vivo biodistribution results were used. Allometric scaling was first performed to estimate the organ mass with respect to body mass, assuming 20 g for C57BL/6. Then, the residence time in each organ, MBq-sec/MBq(inj), was determined using trapezoidal integration, assuming only physical decay after the last timepoint. Lastly, the residence time for each organ was multiplied by the dose factor, mGy/MBq-sec, for each organ, which was assumed to be a sphere of equal mass that only receives self-dose. The total dose for each organ was computed by summing all contributions from the complete decay chain of 225Ac, considering negligible redistribution of daughter isotopes. Total absorbed dose estimates were not multiplied by an RBE/weighting factor. SPECT/CT-based 177Lu-NM600 dosimetry was estimated according to a previously described method 29,31. Total injected activity within each organ was calculated from the extrapolation of %IA/g at a given timepoint. The standard mouse model was used to convert organ-specific cumulative activity into absorbed dose per injected activity (Gy/MBq). Dose contributions from surrounding organs were also included in calculations.

Ionizing-radiation quantum imaging detector scanning
Mice bearing subcutaneous MC38 tumors were injected with 3.70 MBq 86Y-NM600 and 18.5 MBq 90Y-NM600 or 13.0 MBq 177Lu-NM600, then euthanized via CO2 asphyxiation at 48 h (n=2 mice (independent biological replicates)/treatment group). Tumors were harvested and frozen over dry ice in Tissue Tek Optimal Cutting Temperature (OCT) compound (Cat# 4583). Each tumor was sectioned in 10 µm sections on a Leica CM1950 cryostat and placed on charged glass slides (Fisher Scientific, Cat# NC1693867) for imaging on Ionizing-radiation Quantum Imaging Detector (iQID). The 86Y/90Y-NM600 tumor was sectioned at a depth of 1520 µm from the edge of the tissue. The 177Lu-NM600 tumor was sectioned at a depth of 1500 µm from the edge of the tissue. Two serial sections were placed on an individual slide. For 177Lu and 90Y, an image intensifying screen to detect low-and-medium-energy beta particles was used (Carestream BioMax TranScreen LE). For this study, an in-house slide holder was used for the scans in order to fix scan bed position of the glass slides for subsequent image registration purposes between other imaging modalities. One scan was performed per slide; scan durations: 12 h for 86Y/90Y-NM600 and 24 h for 177Lu-NM600. Histological images of each tumor section were captured and stitched together in the Infinity Analyze 7 software using an Infinity2-2M monochrome USB camera coupled to an Olympus CKX41 Inverted Brightfield Microscope using a 2× objective. Once histological and iQID autoradiography images were acquired, shadowgraph images used to overlay iQID images and histological images were obtained. Using a phosphorescent disk attached to the lid of the iQID enclosure, shadowgraphs were obtained. Shadowgraphs and histology images were imported into ImageJ. Once imported, the use of the SIFT feature extraction identified shared image features between the shadowgraph and the corresponding histological image. Rigid transform was selected between the two images, with the shadowgraph being used as the source image and the histological image being used as the target image. Once sufficient features were identified, bunWarpJ, a deformable image registration application, was used to register histology images to their corresponding shadowgraphs. Adobe Photoshop was used to produce a comprehensive iQID/histology overlay.

Mice bearing subcutaneous B78 tumors were injected with 0.74 MBq 90Y-NM600, 1.70 MBq 177Lu-NM600, or 18.5 kBq 225Ac-NM600, then euthanized via CO2 asphyxiation at 72 h (n=2 mice (independent biological replicates)/treatment group). Tumors were harvested and frozen over dry ice in Tissue Tek Optimal Cutting Temperature (OCT) compound (Cat# 4583). Each tumor was sectioned in 20 µm sections on a Leica CM1950 cryostat and placed on charged glass slides (Fisher Scientific, Cat# NC1693867) for imaging on iQID. Four serial sections from each tumor were placed on a single slide. Slides were placed against the iQID detector window separated from the input window by a thin sheet of mylar (Ludlum Measurements 01–5859) and the scintillator of choice depending on the radioisotope being scanned. The same scintillator/mylar setup as for the MC38 study was utilized for 90Y- and 177Lu-NM600 tumors. For 225Ac, a ZnS:Ag detector was used (Eljen Technology EJ-440). One scan was performed per slide; scan durations: 1 h for 90Y-NM600, 2 h for 177Lu-NM600, 12 h for 225Ac-NM600.

Toxicity assessments
To evaluate 225Ac-NM600 toxicity, comprehensive metabolic panel (CMP) and complete blood count (CBC) studies were performed in control and therapy mice. Groups of naïve C57BL/6 mice (n=21/group) were administered 18.5 kBq (MTA) high Am (2.70 µg NM600/MBq 225Ac) 225Ac-NM600 or 20 ng cold NM600 intravenously. Weights were recorded for n=6 mice (independent biological replicates) on days 0, 7, 14, 22, 29, 36, 43, and 6 months post-injection. Three mice (independent biological replicates) were culled from each cohort on days 0 (pre-injection), 7, 22, 28, 43, and 6 months post-injection, and 500 µL of blood was collected via axillary bleed. CBC analysis was completed using whole blood and an Abaxis VetScan HM5 hematology analyzer (Union City, CA). The remaining blood was centrifuged at 1968×g for 10 min to separate the serum from red blood cells; the serum was then run on an Abaxis VetScan VS2 analyzer (Union City, CA). Additionally, n=3 mice (independent biological replicates) were administered 18.5 kBq low As (81.1 µg NM600/MBq 225Ac) 225Ac-NM600 intravenously. Weights were recorded for n=3 mice (independent biological replicates) on days 0, 14, 41, and 6 months post-injection. Six months post injection, CBC and CMP analyses were performed by the same method as the other treatment groups.

In vitro radiation therapy
Delivery of EBRT (195 kV) in vitro was performed using a RS225 Cell Irradiator (Xstrahl). Delivery of in vitro radionuclide therapy was performed as follows. Briefly, a stock solution of 225Ac (unconjugated radionuclide)-containing cell culture media was prepared based on previously described dosimetry calculations 23 to deliver either 0.25 or 1 Gy absorbed dose in 24 h to a cell monolayer in a six-well plate. For 0.25 Gy, 6.882 kBq 225Ac was added to each well; for 1 Gy, 27.75 kBq 225Ac was added to each well.

In vivo radiation therapy
90Y-, 177Lu-, or 225Ac-NM600 therapy was administered via intravenous tail vein injection on treatment day 1 for MC38, B78, or Myc-CaP tumor-bearing mice. Table 1 shows injected activity for each RPT absorbed dose studied.

Delivery of EBRT (300 kV) in vivo was performed using an X-ray biological cabinet irradiator X-RAD CIX3 (Xstrahl). The dose rate for EBRT delivery in all experiments was approximately 2 Gy/min. Dosimetric calibration and monthly quality assurance checks were performed on these irradiators by the University of Wisconsin Medical Physics staff. Tumor-targeted EBRT was delivered at a dose of 2 Gy on day 1 with the right flank tumor exposed and the rest of the animal shielded using custom lead blocks.

Clonogenic assays
In vitro clonogenic assay of B78 and MC38 cells was performed 25. Briefly, cells were plated at a density of 100–4000 cells/well in a six-well plate (MIDSCI, Cat# TP92006), and irradiated with either 0, 2, 4, 6, or 8 Gy EBRT or 0.25 or 1 Gy 225Ac (n=6 wells/treatment group; n=12 wells/0 Gy control group). For EBRT-treated cells, cells were washed with phosphate-buffered saline (PBS) (Corning, Cat# 21-040-CV), and cell culture media was exchanged immediately following radiation. For 225Ac-treated cells, cells were washed with PBS and radioactive cell culture media was exchanged with non-radioactive cell culture media 24 h following start of radiation. Surviving colonies were stained with crystal violet to aid colony counting. Colonies containing >50 cells were scored to determine plating efficiency and the fractions of the cells surviving after each radiation dose. The log surviving fraction of control and irradiated colonies were calculated and plotted.

In vitro co-culture
MC38 cells or no cells were plated in 24 well plates (20,000 cells per well) containing DMEM media. Fresh media was exchanged after 24 h of culture, and peripheral blood mononuclear cells (PBMCs) harvested from treated or naïve mice were added. Blood (200 µL) was collected by submandibular vein collection. Fifty microliters of whole blood was used for CBC analysis. The remaining ~150 µL whole blood treated with RBC lysis buffer (BioLegend, Cat# 420301) and washed with PBS prior to co-culture. 200,000 PBMCs were added per well, for a 10:1 effector to target ratio. After 48 h of co-culture, PBMCs were harvested for analysis of activation markers using flow cytometry. Number of mice (independent biological replicates)/group is indicated in figure legend.

Flow cytometry
Flow cytometry was performed 59 using fluorescent beads (UltraComp eBeads, Invitrogen Cat# 01-2222-42) to determine compensation and fluorescence minus one (FMO) methodology to determine gating. Rainbow beads (Spherotech, Cat# NC9025287) were used to set voltages for each flow cytometry timepoint. For in vivo analysis, tumors were harvested and gently dissociated. Spleens and tumor draining lymph nodes were harvested and manually dissociated. Blood was collected by submandibular vein collection. Spleens and blood were treated with RBC lysis buffer and washed with PBS prior to staining. Cells were treated with CD16/32 antibody (BioLegend, Cat# 156603) to prevent non-specific binding. Live cell staining was performed using Ghost Red Dye 780 (Tonbo Biosciences, Cat# 50-105-2988) according to manufacturer’s instructions. After live-dead staining, a single cell suspension was labeled with the surface antibodies at 4 °C for 30 min and washed three times using flow buffer (2% FBS + 2 mM EDTA (Thermo Fisher, Cat# 15575020) in PBS). For intracellular staining, the cells were fixed and stained for internal markers with permeabilization solution according to manufacturer’s instructions (BD Cytofix/Cytoperm, BD Biosciences, Cat# 554714). Flow cytometry was performed using an Attune NxT Flow Cytometer (Thermo Fisher). Data were analyzed using FlowJo Software. A complete list of antibody targets, clones, and fluorophores is provided in Supplementary Table 51. Gating strategies for each flow cytometry experiment are shown in Supplementary Figs. 12–16. The number of mice (independent biological replicates) per group is indicated in figure legends.

Immunohistochemistry
Standard immunohistochemistry (IHC) methods were performed for Fig. 6 and Supplementary Fig. 10 25,60. IHC antibody targets and clones are listed in Supplementary Table 52. All labeling was performed with no primary antibody negative controls. CD20+ cell clusters were counted for an entire tumor cross-section, with groupings of ≥5 cells being considered a cell cluster. N=3 (90Y-NM600+ICI, 225Ac-NM600+ICI, EBRT+ICI, ICI) or n=4 (177Lu-NM600+ICI) tumors (independent biological replicates) were quantified for positive labeling by a blinded observer. For Supplementary Fig. 5 PD-L1 IHC, mice bearing 6–10 mm MC38 or B78 tumors were administered 100 µg anti-murine PD-L1 (rat IgG2b, clone 10F.9G2, BioXCell, Cat# BE0101) or PBS by intraperitoneal injection. Mice were euthanized via CO2 asphyxiation, and tumors were collected 24 h post-injection of anti-PD-L1 or PBS. Tumors were embedded (unfixed) in OCT and sectioned at a thickness of 8 µm. The anti-rat detection ImmPRESS kit (Vector Laboratories, Cat# MP-7444-15) was used for anti-PD-L1 detection according to manufacturer instructions.

Quantitative messenger RNA analysis
Mouse tumors were dissected and placed in tubes containing 2.8 mm ceramic beads (Fisher, Cat# 15-340-154) with TRIzol reagent (Invitrogen, Cat# 15596018). The tumors were homogenized for 60 s using a Bead Ruptor Elite (OMNI). Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Cat# 74106) according to manufacturer instructions. Isolated RNA was subjected to complementary DNA synthesis using the QuantiTect Reverse Transcription Kit (QIAGEN, Cat# 205313) according to manufacturer instructions. Quantitative PCR (qPCR) was performed using the CFX96 real-time system (Bio-Rad) with PowerUp SYBR Green master mix (Applied Biosystems, Cat# A25779). Relative messenger RNA (mRNA) expression of target genes was determined according to the 2−ΔΔCT method using Hprt as a reference gene 61. Forward and reverse primer sequences provided in Supplementary Table 53 were used to detect mRNA expression 15,21. The number of mice (independent biological replicates) per group is indicated in the figure legend.

Single-cell RNA sequencing
Tumors were dissected from three mice/treatment group, pooled by treatment, minced, and digested using the Miltenyi Biotech tumor dissociation kit (mouse, tough tumor dissociation protocol, Cat# 130-096-730) for 40 min at 37 °C. Cells were then strained through a 70 µm filter (Fisher, Cat# 08-771-2) and washed with RPMI-1640, and resuspended in 6 mL of RPMI-1640. Tumor infiltrating lymphocytes were isolated by Ficoll-Paque density gradient centrifugation 62. Briefly, 4 mL of Ficoll-Paque (Cytiva, Cat# 17544602) was added to a 15 mL tube, and the entire cell suspension was carefully layered on top. The samples were centrifuged at 600×g for 20 min with no brake or accelerator. Cells were collected from the layer interface, rinsed with RPMI-1640 at 375 ×g, strained through a 40 µm filter, and counted on a Countess 3 automated cell counter (Invitrogen). All samples had viability >90% and were resuspended at a concentration of 1.2×106 cells/mL in RPMI-1640 for single-cell RNA sequencing.

scRNA-seq was then performed 63. Briefly, single-cell suspensions from isolated CD45+ cells were loaded on a Chromium iX single cell instrument (10x Genomics) to generate single-cell beads in emulsion and scRNA-seq libraries were prepared. For Gel bead in EMulsion (GEM) generation and library preparation, the following materials from 10x Genomics were used: Chromium Next GEM Single Cell 5′ Kit v2 (PN-1000263); VDJ Amplification, Mouse TCR Kit (PN-1000254); 5′ Feature Barcode Kit (PN-1000256); Library Construction Kit (PN-1000190); Chromium Next GEM Chip K Single Cell Kit (PN-1000286); Dual Index Kit TT Set A (PN-1000215); Dual Index Kit TN Set A (PN-1000254). The Chromium Next GEM Single Cell 5′ v2 User Guide was followed. Single-cell barcoded cDNA libraries were quantified using a Qubit Fluorometer with Qubit dsDNA HS reagent (Invitrogen, Cat# Q32851), assayed using a Tapestation D1000 screen tape (Agilent) and sequenced on a NovaSeq 6000 S2 flow cell (Illumina) according to 10X Genomics recommendations (26 cycles read 1, 10 cycles i7 index read, 10 cycles i5 index read, and 90 cycles read 2). Cells were sequenced to greater than 50,000 reads per cell for the 5′ gene expression library and greater than 5000 reads per cell for the V(D)J library as recommended by manufacturer.

Cell Ranger (version 7.2.0) was used for demultiplexing, barcode processing, and gene counting. Reads were aligned to the mouse genome (version mm10). Doublets were removed by scDblFinder (version 1.14.0). We removed low-quality cells that had total read counts fewer than 1000, had log(“total read counts”+1), log(“number of genes with read count”+1), or fraction of read counts in the top 20% of genes beyond five times of median absolute deviation (MAD), or had fraction of read counts from mitochondria beyond three times of MAD. Read counts were normalized across all samples by the logNormCounts function in scuttle (version 1.10.1). Dimensionality reduction was performed by Scanpy (version 1.9.1). Cell types were predicted by SingleR (version 2.2.0) using the Immunological Genome Project reference data from celldex (version 1.10.1). Gene differential expression was analyzed by MAST (version 1.26.0) 64. Gene set enrichment analysis was performed by fgsea (version 1.26.0) using the mouse hallmark gene sets from MSigDB (version 2022.1). Following filtering and quality control, the number of cells (independent biological replicates) used for scRNA-seq analysis per treatment group is indicated in Supplementary Table 48. For the TCR data, for each cell the cellranger multi pipeline assembles the V(D)J transcripts into contigs, aligns the contigs to the TCR reference sequence, and determines whether the contigs correspond to a CDR3 sequence by annotating both ends of the CDR3 sequences with V and J genes.

We analyzed each sample independently and considered high confidence, productive and full length CDR3 sequences. Following quality control, the number of cells (independent biological replicates) used for TCR analysis per treatment group is indicated in Supplementary Table 49. We defined a clonotype as the unique combination of TRA and TRB CDR3 sequences annotated by different V and J genes. Then, we stratified the T cells according to their clonal expansion (Fig. 7B). To summarize the diversity and clonal expansion at the T cell type level, we counted the number of T cells annotated with a clonotype in each cell type and then calculated the Shannon diversity, clonal expansion, and the D50 index 65,66. We examined the T cell combinations sharing the same clonotypes per sample by using the ComplexUpset R package (v1.3.3) 67,68. The number of cells (independent biological replicates) with clonotypes shared within effector and memory CD8+ T cells is shown in Supplementary Table 50.

Statistical analysis
Prism 10 (GraphPad Software) and R 4.1.2 (R Foundation) were used for all statistical analyses. Student’s two-sided t-test was used for two-group comparisons. One-way or two-way ANOVA with Tukey’s honestly significant difference (HSD) test was used to assess the statistical significance of mean differences in CBC, CMP, flow cytometry, immunohistochemistry, complete response rate, rechallenge rejection rate, and mouse body weight data. For tumor growth analysis, all available data was used. To compare treatment groups, linear mixed models after log base 10 transformation of tumor volume were fitted on cell line, time in days, and their interaction. Tumor volumes of 0 were present in all experiments except those represented by Figs. 1Q and 3G. In such cases, a small constant (1×10−4) was added prior to log transformation. Mouse ID was also included as a random intercept, accounting for correlation between measurements taken from the same mouse (Supplementary Tables 1, 4, 7, 9, 11, 14, 16, 18, 21, 23, 25, 28, 30, 32, 35, 37, 39, 42 and 45). Pairwise contrasts were adjusted using Tukey’s method (Supplementary Tables 2, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26, 29, 31, 33, 36, 38, 40, 43 and 46). Kaplan–Meier method was used to estimate the survival distribution for the overall survival. Then, pairwise comparison of the overall survival was made using a log-rank test with Benjamini–Hochberg adjustment of p-values between levels of factors (Supplementary Tables 3, 6, 13, 20, 27, 34, 41, 44 and 47). All data are reported as mean ± standard error of the mean (SEM) unless otherwise noted. For all graphs, *, P<0.05; **, P<0.01; ***, P<0.001; and ****, P<0.0001. Number of data points and independent experiments are stated in all figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "General Comments\nIn this rigorous examination of the interaction between checkpoint inhibitors and radiopharmaceutical therapy (RPT), the\nauthors examine the role of high versus low LET Radiopharmaceutical therapy, the absorbed dose and the timing of ICI\ndelivery. The studies performed have anticipated most of the concerns of this reviewer.\nThe concept of an average absorbed dose for an alpha particle emitter in an intact macroscopic tumor is problematic\nhowever because of the short range and high potency of alphas. Probably better to present or discuss/reference studies that\nexamine absorbed dose as a function of radial distance. The authors overcome this to some extent by showing an\nequivalent biological effect at the selected mean absorbed dose in the absence of ICI (Fig 2E).\nThey interpret the difference between figures 2E and 3E as an RBE effect. An alternative explanation could be differential\npenetration of the ICE in the different tumor types. If they have controls that address this issue it would be good to include\nthem in the discussion.\nLikewise, the results of early versus intermediate versus delayed ICE could be interpreted as a result of differential\npenetration of ICI. Again, it would be good to highlight controls that address this and include this issue in the discussion.\nNM600 is a fundamentally different type of targeting agent that is independent of a receptor ligand interaction employed by\nother RPTs. The discussion of the extent to which the observed results from a lipid raft binding mechanism apply to receptor\nligand based RPTs should be expanded. The former is probably not saturable, while the latter, depending on the As is.\nIn general, this paper would be strengthened substantially if the authors took a greater account of penetration and the\nresulting absorbed dose (or ICI distribution) within the different tumor types for the different agents. This could be done in the\ndiscussion, by providing references where penetration/dose distribution is addressed for these agents/tumor types or by\nincluding studies that address this question in the manuscript.\nThe issue of penetration also speaks to the mouse to human translation since human tumors are generally larger than\nmurine tumors and should be alluded to in the discussion.\nSpecific Comments\n“Radiopharmaceutical therapy (RPT) is a modality of RT … “\nMany would disagree with this characterization – RPT is a multi-disciplinary modality, that draws on both RT and NM\nexpertise, the latter for imaging and pharmacokinetics, the former for radiobiology and treatment planning\nAre the reported absorbed doses multiplied by an RBE? If so what value?\n“However, in the case of 111In-J591…”\nIn manuscript text, reference to Fig S7 should be S6.", "reviewer_2": "The paper examines in detail the anti-tumor effects of combined targeted radionuclide therapies (RPT) and immune\ncheckpoint inhibition (ICI) with anti-CTLA-4 or anti-CTLA-4 and targeting of the PD-1/PD-L1 axis for cancer therapy.\nParticularly focus is set on alpha- versus beta-particle emitting radiopharmaceuticals.\nThe tested radioisotopes were 90Y-, 177Lu-, and 225Ac which were coupled with an alkylphosphocholine (NM600). As\npreclinical tumor model system, ectopic and syngeneic tumors were chosen, B78 melanoma as example for a poorly\nimmunogenic and MC38 colorectal cancer as example for an immunogenic tumor.\nOn what is the statement based on that B78 is poorly immunogenic as melanoma usually has high TMB and is considered to\nbe immunogenic and responsive to immune checkpoint inhibitors (ICIs)?\nTo perform dose-matched comparative immunologic studies, first convincing image-based radiation dosimetry studies were\nperformed.\nRegarding the data displayed in Figure 1 the authors state that 2Gy 225Ac-NM600 demonstrated an immune contribution to\ntumor response. However, no immune analyses are displayed, just tumor growth delay curves and survival curves. In\naddition, why was a 2-flank model used for B78 tumors and 1-flank model for MC38? Please clarify these issues.\nThe analyses further revealed that combination of radiation exposure of the tumor and ICI was most effective when ICI was\napplied early after irradiation in both tumor models. Again, tumor growth and survival data are shown.\nWhat was the rationale to choose in some cases only anti-CTLA-4 as ICI and in some cases combination of anti-CTLA-4\nand PD-L1?\nThe authors should include data about expression of distinct immune checkpoint molecules on the chosen tumor cell lines\nfollowing RPT.\nAs new and most intriguing finding the authors revealed that alpha particle radiation with high linear energy transfer was\nmost effective against B78 tumors. Mechanistically this was connected promoting immune priming of 225Ac-NM600 when\ncombined with ICI. Further, diversified effector T cell responses are suggested based on single cell RNA sequencing\nanalyses.\nAs these are key findings for B78 as “non-immunogenic” tumour, they should be validated\n- with another non-immunogenic tumor model\n- selected RNA-seq based findings should be validated by IHC stainings\nMinor: Please mention the ICIs which were used already in the abstract.", "reviewer_3": "Review Kerr et al. ‘Priming versus propagating: distinct immune effects of an alpha- versus beta-particle emitting\nradiopharmaceutical when combined with immune checkpoint inhibition’\nThis is the next in a series of reports on immune effects and combination with ICI of NM600. Here, in a highly extensive\nreport, the authors compare the effects of 90Y, 177Lu, and 225Ac labelled versions of the compound, in two allograft tumor\nmodels. They show distinct mechanisms of interplay between TRT and ICI, depending on the quality of emission.\nMA the same for all compounds? Or equitoxic dose, or same radiation absorbed dose?\n225Ac dosimetry -> how modelled? Did this include potential re-distribution of daughter isotopes?\nBioD is not the same between 90Y, 177Lu, and 225Ac? How is tumor vs normal tissue dose balanced when determining\nsame dose effects?\nWould 161Tb result in a different outcome?\nThe use of dose and radiation absorbed dose may be confusing.\nWhy should 2 Gy be the optimal for 177Lu and 225Ac, given their different RBEs?\nHow did ICI combination and scheduling influence toxicity?\nThe very MMR deficient nature of the tumor affecting immunogenicity also affects radiation response. It is therefore a large\ncaveat on interpreting the results.\nInvolvement of CD8+ T-cells is not new information. Was involvement of any other immune cell type disproved?\nMemory effects: what were the differences in immunotyping between tumor rejected and non-rejected animals? Control\nversus naïve animals is useful, but not the interesting comparison.\nWhy was the immune microenvironment studied in B78, but not MC38 tumors?\nWere there any clone-specific immune effects between radionuclides observed?\nWhat was the differential gene expression of RPT+ICI vs RPT alone, or naïve vs RPT alone? Now, only ICI vs RPT+ICI is\nconsidered. Was pathway analysis not considered?\nTo what degree are tumor infiltrating immune cells killed by TAT? Expression as percentages of infiltrates obscures the\neffect on absolute numbers. There may be a large survival bias, or an effect of newly infiltrated cells measured here.\nWould effects be different in immune excluded or immune desert models?\nPlease use ‘molar activity’, in compliance with nomenclature guidelines.\nDifference in combo effect is partially explained by the very different response to 225Ac-compound alone.\nFig 1: The different scheduling between MC38 and B78 models makes for a complicated comparison.\nAddition of individual mouse tumor growth curves can be moved to SI. It clutters the figures.\nFig 4M-N: normalisation to ICI treatment only may obscure significant effects, and non-linearities in the combination of RPT\nand ICI. Consider normalising to treatment-naïve animals.\nFig 5B: is too small.\nFig 6: comparison vs what? Naïve only, or vs RPT only? N= 3 (as suggested by FigS6)? Errors on analysis?\nFig 6: 2 tumors in the same animal assume no crosstalk between tumors. This is not necessarily the case.\nWas EBRT administered at the same dose? Would this result in the same response at all?\nFig S1: use the same scale throughout for ease of comparison\nFigS2: safety profile of RPT + ICI?\nFigS5: no control for innate immune response to RPT\nFigS6: why is there no tumor size measurement at the time of harvesting?"}, "author_response": "RESPONSE TO REVIEWERS’ COMMENTS\nReviewer #1 (Remarks to the Author):\nGeneral Comments\nIn this rigorous examination of the interaction between checkpoint inhibitors and\nradiopharmaceutical therapy (RPT), the authors examine the role of high versus low LET\nRadiopharmaceutical therapy, the absorbed dose and the timing of ICI delivery. The studies\nperformed have anticipated most of the concerns of this reviewer.\nThank you for your careful review and your time spent reviewing this manuscript.\nThe concept of an average absorbed dose for an alpha particle emitter in an intact macroscopic\ntumor is problematic however because of the short range and high potency of alphas. Probably\nbetter to present or discuss/reference studies that examine absorbed dose as a function of radial\ndistance. The authors overcome this to some extent by showing an equivalent biological effect at\nthe selected mean absorbed dose in the absence of ICI (Fig 2E).\nDosimetry for alpha-emitters, i.e., Ac-225, was performed using the S-value-based MIRD\nmethod calculations. The cumulated activity (total number of disintegrations) per injected\nactivity (MBq-s/MBq_injected) was determined via trapezoidal integration of the measured\n%ID/g data over time. S-values (dose factor, mGy/MBq-s) were then derived using OLINDA’s\nsphere model (liquid water material), interpolating self-dose S-values based on tumor mass–\nderived from spherical radii of each tumor (see Fig. R1 and Table R1). The corresponding S-\nvalues were applied to calculate the final absorbed dose.\nThe reviewer is correct in noting the fundamental differences between the microscopic behavior\nof beta particles (e.g., Lu-177 and Y-90) and alpha particles (e.g., Ac-225). These differences\nresult in distinct biological responses, even when the same absorbed dose is delivered.\nTraditionally, such variations are accounted for using a relative biological effectiveness (RBE)\nfactor. Reported RBE values for alpha particles vary widely in the literature and depend on\nseveral factors, including absorbed dose, dose rate, biological endpoint, reference radiation, and\ncell type (R1,R2). Although the clinically accepted RBE for alpha particles is currently 5 (R3),\napplying this—or any fixed RBE—in our study would be inappropriate. Our research focuses on\nnon-classical cell-kill mechanisms driven by immunogenic effects, which are not adequately\ncaptured by conventional RBE frameworks. As a result, we chose to report only the physical\nabsorbed dose, allowing readers to interpret the biological implications of beta- versus alpha-\nemitting radionuclides within the context of their own understanding or specific applications.\nFigure R1. Interpolated self-dose S-value for Ac-225 including full decay chain.\nTable R1. Self-dose S-value for Ac-225, including full decay chain, from OLINDA Sphere.\nSelf-dose converting factor\nSphere mass (g) Radius (mm)\nmGy/MBq-s rad/μCi-h\n0.01 1.34 4.48E+02 5.97E+03\n0.1 2.88 4.50E+01 6.00E+02\n0.5 4.92 9.02E+00 1.20E+02\n1 6.20 4.51E+00 6.01E+01\n2 7.82 2.26E+00 3.01E+01\n4 9.85 1.13E+00 1.50E+01\n6 11.27 7.53E-01 1.00E+01\n8 12.41 5.64E-01 7.52E+00\n10 13.37 4.51E-01 6.02E+00\n20 16.84 2.26E-01 3.01E+00\n40 21.22 1.13E-01 1.50E+00\n60 24.29 7.54E-02 1.00E+00\n80 26.73 5.64E-02 7.52E-01\n100 28.79 4.51E-02 6.02E-01\n300 41.53 1.51E-02 2.01E-01\n400 45.71 1.13E-02 1.51E-01\n500 49.24 9.04E-03 1.20E-01\n600 52.32 7.54E-03 1.00E-01\n1000 62.04 4.52E-03 6.02E-02\n2000 78.16 2.27E-03 3.01E-02\n3000 89.47 1.51E-03 2.01E-02\n4000 98.47 1.13E-03 1.51E-02\n5000 106.08 9.05E-04 1.21E-02\n6000 112.73 7.55E-04 1.01E-02\nR1) Sgouros G, Roeske JC, McDevitt MR, Palm S, Allen BJ, Fisher DR, Brill AB, Song H,\nHowell RW, Akabani G. MIRD pamphlet no. 22 (abridged): radiobiology and dosimetry of α-\nparticle emitters for targeted radionuclide therapy. J Nucl Med. 2010;51(2):311–28.\nR2) Hobbs RF, Howell RW, Song H, Baechler S, Sgouros G. Redefining relative biological\neffectiveness in the context of the EQDX formalism: implications for alpha-particle emitter\ntherapy. Radiat Res. 2014;181(1):90–8.\nR3) Kratochwil C, Bruchertseifer F, Rathke H, Bronzel M, Apostolidis C, Weichert W,\nHaberkorn U, Giesel FL, Morgenstern A. Targeted α-therapy of metastatic castration-resistant\nprostate cancer with 225Ac-PSMA-617: dosimetry estimate and empiric dose finding. J Nucl\nMed.\nThey interpret the difference between figures 2E and 3E as an RBE effect. An alternative\nexplanation could be differential penetration of the ICE in the different tumor types. If they have\ncontrols that address this issue it would be good to include them in the discussion.\nThis is an important alternative explanation to consider. Please see our response below regarding\nICI uptake and penetration, and the addition of Figure S4, where IHC shows no major\ndifferences between MC38 and B78 tumor types regarding anti-PD-L1 antibody distribution. We\nalso added an analysis of tumor mRNA expression of Ctla4 and Pdl1 following treatment with\n90Y/177Lu/225Ac-NM600 in Figure S5, wherein we found no significant differences in the\nexpression level of these checkpoint molecules due to RPT treatment in either MC38 or B78\ntumors. As a clarification, no ICI was used in the treatment groups shown in Figure 2E and 3E,\nwhich is why the RBE effect was considered.\nLikewise, the results of early versus intermediate versus delayed ICE could be interpreted as a\nresult of differential penetration of ICI. Again, it would be good to highlight controls that address\nthis and include this issue in the discussion.\nThanks for bringing forward this consideration. Please see our discussion on ICI uptake and\npenetration below. Additionally, we added this discussion regarding ICI timing controls to the\ndiscussion: “Timing of administration of RPT and ICI agents warrants further investigation as\nfactors including tumor size and host immune fitness change quickly in the short life cycle of a\nmouse. However, efforts were made to control the influence of tumor size in the therapeutic\nstudies including permuted block randomization by tumor size and separate ICI control treatment\ngroups for each administration timing (Figure 2G-I).” (lines 466-470)\nNM600 is a fundamentally different type of targeting agent that is independent of a receptor\nligand interaction employed by other RPTs. The discussion of the extent to which the observed\nresults from a lipid raft binding mechanism apply to receptor ligand based RPTs should be\nexpanded. The former is probably not saturable, while the latter, depending on the As is.\nYou raise an interesting and complex point. We added a statement to address this difference of\nNM600 from many other receptor-targeting RPTs to the discussion: “A possible explanation for\nthe minor impact on biodistribution of altering NM600 A is that NM600 has a lipid raft cellular\nm\nuptake mechanism distinct from the receptor-ligand interaction employed by 111I-J591 and many\nother RPTs, which may not be saturable.” (lines 512-515)\nIn general, this paper would be strengthened substantially if the authors took a greater account of\npenetration and the resulting absorbed dose (or ICI distribution) within the different tumor types\nfor the different agents. This could be done in the discussion, by providing references where\npenetration/dose distribution is addressed for these agents/tumor types or by including studies\nthat address this question in the manuscript.\nThanks for this helpful suggestion to clarify our work. We added studies addressing the question\nof penetration/dose distribution in Figures 1G-K, S4, and S5, and the following text to the\ndiscussion: “Additionally, we acknowledge that many features of syngeneic tumor lines aside\nfrom immunogenicity, including differential penetration, uptake, and resulting absorbed doses of\ntreatments, impact responses to any given treatment. These potential confounding factors were\ninvestigated in the following ways: 1) imaging (90Y, 177Lu) or biodistribution (225Ac)-based\ndosimetry methods were used to estimate injected activities to achieve the same average tumor\nradiation absorbed doses across tumor models and RPTs (Table 1); 2) iQID scans of NM600\ncompounds in MC38 and B78 tumors confirmed no major differences in NM600 uptake across\ntumor types (Figure 1G-K); 3) immunohistochemistry of anti-PD-L1 antibody penetration in\nMC38 and B78 tumors showed a similar distribution of anti-PD-L1 antibody uptake across the\ntwo tumor types (Figure S4); 4) mRNA expression of checkpoint molecules on B78 and MC38\nwas confirmed by qPCR to not significantly change following treatment with 2 Gy 90Y-, 177Lu-,\nor 225Ac-NM600 (Figure S5). Following these analyses, immunologic differences between MC38\nand B78 and Myc-CaP remain a key tumor feature that may relate to the therapeutic divergence\nobserved in response to 2 Gy 225Ac alpha-particle emitting vs 90Y or 177Lu beta-particle emitting\nRPT when combined with ICI therapy.” (lines 481-495)\nThe issue of penetration also speaks to the mouse to human translation since human tumors are\ngenerally larger than murine tumors and should be alluded to in the discussion.\nTumor size difference between species is an important consideration for RPT translation. To this\npoint, we added this sentence to the discussion: “Based on prior work with companion canines\nand human cell lines, we anticipate that the image-based dosimetry methods employed in this\nstudy will enable safe translation of RPT dosing studied here to larger tumors, including human\ntumors15,20,54, however the effects of specific isotopes may vary as a function of tumor size given\nthe fixed physical range of emitted radiation from these.” (lines 462-466)\nSpecific Comments\n“Radiopharmaceutical therapy (RPT) is a modality of RT … “\nMany would disagree with this characterization – RPT is a multi-disciplinary modality, that\ndraws on both RT and NM expertise, the latter for imaging and pharmacokinetics, the former for\nradiobiology and treatment planning\nWe have omitted this characterization, which was not pertinent to the focus of the manuscript.\nAre the reported absorbed doses multiplied by an RBE? If so what value?\nThe reported absorbed doses were not multiplied by an RBE. We kept the absorbed doses for\nresult interpretation. The following sentence has been added to the methods: “Total absorbed\ndose estimates were not multiplied by an RBE/weighting factor.” (lines 639-640)\n“However, in the case of 111In-J591…”\nIn manuscript text, reference to Fig S7 should be S6.\nThanks for catching this typo. We have now re-numbered the supplement and have correctly\nreferenced Figure S11 in the manuscript text. (line 507)\nReviewer #2 (Remarks to the Author):\nThe paper examines in detail the anti-tumor effects of combined targeted radionuclide therapies\n(RPT) and immune checkpoint inhibition (ICI) with anti-CTLA-4 or anti-CTLA-4 and targeting\nof the PD-1/PD-L1 axis for cancer therapy.\nParticularly focus is set on alpha- versus beta-particle emitting radiopharmaceuticals.\nThe tested radioisotopes were 90Y-, 177Lu-, and 225Ac which were coupled with an\nalkylphosphocholine (NM600). As preclinical tumor model system, ectopic and syngeneic\ntumors were chosen, B78 melanoma as example for a poorly immunogenic and MC38 colorectal\ncancer as example for an immunogenic tumor.\nThank you for your insightful comments and time spent reviewing this manuscript.\nOn what is the statement based on that B78 is poorly immunogenic as melanoma usually has\nhigh TMB and is considered to be immunogenic and responsive to immune checkpoint inhibitors\n(ICIs)?\nWe agree that melanoma, as a disease, is relatively immunogenic and ICI responsive, but there is\nclinically much variability that underlies this, as demonstrated by ~ 40-60% response rates and\nmuch lower complete response rates among patients with melanoma to ICIs. In our published\nworks we have observed that the B78 variant of B16 is relatively poorly immunogenic. We have\nnow expanded the discussion of the basis for our description of B78’s immunogenicity in the\nintroduction: “B78, derived from the spontaneous melanocytic tumor B16F10, is characterized as\npoorly immunogenic given its low levels of MHC-I expression, low numbers of baseline tumor\ninfiltrating lymphocytes, and limited response to immune checkpoint inhibition alone15,24,25.”\n(lines 91-93)\nTo perform dose-matched comparative immunologic studies, first convincing image-based\nradiation dosimetry studies were performed. Regarding the data displayed in Figure 1 the\nauthors state that 2Gy 225Ac-NM600 demonstrated an immune contribution to tumor\nresponse. However, no immune analyses are displayed, just tumor growth delay curves and\nsurvival curves. In addition, why was a 2-flank model used for B78 tumors and 1-flank\nmodel for MC38? Please clarify these issues.\nThanks for raising these points. Our description of these findings were not presented as we\nintended nor were they clearly stated. We feel based on your comment that the data require\na more detailed description in the results section. We have made the following revisions to\ncorrect this:\nWe revised the sentence to read: “2 Gy 225Ac-NM600 was tolerable and demonstrated a\ncooperative therapeutic interaction with ICI in the B78 model” (lines 156-157) in place of\n“immune contribution” to accurately represent the data being described. We broke down\nthe results more carefully in the presentation, so that the final passage reads:\nIn the MC38 model, 2 Gy alone provided only nominal survival benefit, and while addition\nof ICI to 2 Gy increased survival compared to both 2 Gy alone and the control, it was not\nsignificantly different from ICI alone (Figure 1O). In the B78 model, however, while 2 Gy\nprovide a moderate increase in survival relative to control, addition of ICI to the 2 Gy\ntreatment increased survival compared to control, 2 Gy, and ICI alone. Because 2 Gy 225Ac-\nNM600 was tolerable and demonstrated a cooperative therapeutic interaction with ICI in\nthe B78 model, we selected this dose for further study (225Ac-NM600+ICI vs. 225Ac-NM600\nalone, overall survival) (Figure 1M-O, Q-S). The differences in the patterns of response\namong MC38 and B78 tumors led us to hypothesize that the effects of RPT+ICI may be\ndifferent based on the immunogenicity of the tumor model.\nIn general, we would prefer to study multiple tumors per mouse for all RPT studies as\nperhaps the most impactful clinical application of this approach will be in the metastatic\ndisease setting. However, the presence of a second (or third, etc.) tumor can affect\nengraftment rates. For very large studies with multiple variables such as the ones in\nFigures 2 and 3, to meet the numbers of animals needed to power the study, we started with\na more reliable single tumor model to study therapeutic effect interactions in a more\ncontrolled manner. The pace of murine tumor growth in some models, such as MC38, can\nprevent the opportunity to see treatment effect from a long half-life RPT agent such as\n225Ac-NM600 when tumor burden is high as in multiple tumor models as these mice can\nrapidly become too ill to benefit from a therapeutic intervention. This is a limitation of such\nmodels. B78 is slower growing and does not have this limitation. For these reasons, in\nFigure 1 a 2-flank model was used for B78 tumors, whereas a 1-flank model was used for\nMC38 and most studies in the manuscript.\nThe analyses further revealed that combination of radiation exposure of the tumor and ICI was\nmost effective when ICI was applied early after irradiation in both tumor models.\nWhat was the rationale to choose in some cases only anti-CTLA-4 as ICI and in some cases\ncombination of anti-CTLA-4 and PD-L1?\nDual anti-CTLA-4 and anti-PD-L1 were used in all experiments in the manuscript based on\nrationale from Twyman-Saint Victor et al. (Nature, 2015) regarding utility of RT and both anti-\nPD-L1 and anti-CTLA-4 and from Potluri et al. (JITC, 2022) regarding the necessity of both\nanti-PD-L1 and anti-CTLA-4 in combination with 90Y-NM600 to achieve antitumor responses in\nimmune resistant prostate cancer models. We revised the manuscript to make it clear that prior\nwork, not the current work, used only anti-CTLA-4: “In prior work with B78 melanoma, we\nhave shown the necessity of T cells for the therapeutic response to 90Y-NM600+anti-CTLA415.”\n(lines 204-205)\nThe authors should include data about expression of distinct immune checkpoint molecules on\nthe chosen tumor cell lines following RPT.\nThank you for this suggestion. We added these data in Figure S5. Tumor mRNA expression of\nPdl1 and Ctla4 was not significantly changed following treatment with 90Y-/177Lu-/225Ac-\nNM600+ICI. (lines 144-146)\nAs new and most intriguing finding the authors revealed that alpha particle radiation with high\nlinear energy transfer was most effective against B78 tumors. Mechanistically this was connected\npromoting immune priming of 225Ac-NM600 when combined with ICI. Further, diversified\neffector T cell responses are suggested based on single cell RNA sequencing analyses.\nAs these are key findings for B78 as “non-immunogenic” tumour, they should be validated\n- with another non-immunogenic tumor model\nThank you for this important comment. We have added data to validate the therapeutic response\nobserved in B78 (225Ac-NM600+ICI > 90Y-NM600+ICI) in the poorly immunogenic Myc-CaP\nprostate cancer model (Figure 6A-C): “To test our hypothesis that the immunogenicity of the\ntumor model may impact the optimal radioisotope for antitumor responses, we evaluated the\ntherapeutic efficacy of low absorbed dose 225Ac-NM600 vs. 90Y-NM600 + dual anti-CTLA4 and\nanti-PD-L1 in a second poorly immunogenic model. The Myc-CaP prostate cancer model was\nselected, as it has an immunosuppressive tumor microenvironment and limited responses to RPT\nor ICI alone38. Tumor uptake and dosimetry of 86Y/90Y-NM60020 and 225Ac-NM60039 in mice\nbearing Myc-CaP prostate cancer tumors were reported previously. As was observed in B78\nmelanoma, Mice bearing Myc-CaP tumors showed a statistically significant reduction in tumor\ngrowth rate and prolonged overall survival when treated with 2 Gy 225Ac-NM600+ICI as\ncompared to 2 Gy 90Y-NM600+ICI or monotherapies (Figure 6A-C, Tables S45-S47). This\nfinding supported the hypothesis that tumor immunogenicity may impact therapeutic response to\n225Ac alpha-particle emitting vs. 90Y or 177Lu beta-particle emitting RPT when combined with\nICI therapy.” (lines 274-285)\n- selected RNA-seq based findings should be validated by IHC stainings\nWe performed IHC staining of portions of the tumor samples that were used for single cell RNA\nsequencing to validate the findings reported in Figure 6D (IHC results in Figure S9): “With\nportions of the same tumor samples that were used for single cell RNA sequencing, CD4+,\nCD8+, Tregs (FOXP3+ cells), and NK1.1+ cells were quantified by IHC (Figure S9). Consistent with the\nsingle cell RNA sequencing results, there were no statistically significant differences in these\nlymphoid cell populations between treatment groups (Figures 6D, S9).” (lines 309-312)\nMinor: Please mention the ICIs which were used already in the abstract.\nWe have added the following text to the abstract: “Effects of 90Y-, 177Lu-, and 225Ac-NM600 +\ndual anti-PD-L1 and anti-CTLA4 ICIs were compared…” (lines 33-34)\nReviewer #3 (Remarks to the Author):\nReview Kerr et al. ‘Priming versus propagating: distinct immune effects of an alpha- versus beta-\nparticle emitting radiopharmaceutical when combined with immune checkpoint inhibition’\nThis is the next in a series of reports on immune effects and combination with ICI of NM600.\nHere, in a highly extensive report, the authors compare the effects of 90Y, 177Lu, and 225Ac\nlabelled versions of the compound, in two allograft tumor models. They show distinct\nmechanisms of interplay between TRT and ICI, depending on the quality of emission.\nThank you for your thoughtful comments and time spent reviewing this manuscript.\nMA the same for all compounds? Or equitoxic dose, or same radiation absorbed dose?\nIn all the comparative therapy studies between radioisotopes (90Y- vs. 177Lu- vs. 225Ac-NM600),\nthe same radiation absorbed dose to the tumor (2 Gy) is used. The molar activity is different\nbetween compounds (225Ac-NM600: 0.336 MBq/nmol, 90Y-/177Lu-NM600: 3.36 MBq/nmol).\nInjected activities for each tumor radiation absorbed dose studied for each agent in each tumor\nmodel are listed in Table 1. (lines 713-714)\n225Ac dosimetry -> how modelled? Did this include potential re-distribution of daughter\nisotopes?\n225Ac dosimetry modeling is described in the methods section. Potential redistribution of\ndaughter isotopes was not included. The model assumed daughters in the same location as initial\nactivity: “To estimate dosimetry of 225Ac-NM600, ex vivo biodistribution results were used.\nAllometric scaling was first performed to estimate the organ mass with respect to body mass,\nassuming 20 g for C57BL/6. Then, the residence time in each organ, MBq-sec/MBq(inj), was\ndetermined using trapezoidal integration, assuming only physical decay after the last timepoint.\nLastly, the residence time for each organ was multiplied by the dose factor, mGy/MBq-sec, for\neach organ, which was assumed to be a sphere of equal mass that only receives self-dose. The\ntotal dose for each organ was computed by summing all contributions from the complete decay\nchain of 225Ac, considering negligible redistribution of daughter isotopes. Total absorbed dose\nestimates were not multiplied by an RBE/weighting factor.” (lines 632-640)\nBioD is not the same between 90Y, 177Lu, and 225Ac? How is tumor vs normal tissue dose\nbalanced when determining same dose effects?\nCorrect, BioD is not the same between 90Y-, 177Lu-, and 225Ac-NM600, as the size of the\nradioisotope alters the pharmacokinetics of the compound. In this manuscript, we elected to\nfocus on the immune response at the tumor while understanding and noting in the discussion that\nthe differing normal tissue doses may confound the immune response. We acknowledge that with\nother agents, tumor immune responses may occur at different times depending on normal\ntissue:tumor ratios. For example, the initial drop in white blood cells reaches a nadir on day 14\nfollowing 90Y-NM600 and 177Lu-NM600 and begins to recover by day 21 after treatment. With\n225Ac-NM600, WBC counts remained low through day 43 (Figure S2A) but recovered by 6\nmonths after treatment. All mice were monitored daily by independent veterinary staff and\nweighed weekly, and no mice treated in the studies presented here were requested to be\neuthanized by veterinary staff for signs of illness/distress or lost >20% of their body weight.\nWould 161Tb result in a different outcome?\nThis is an interesting question and would require an additional experiment outside the scope of\nthe current project. However, we speculate that 161Tb would have a similar beta-particle dose\ndistribution to 177Lu, but that conversion electrons would provide more local dose. We suspect\nthe co-emission of conversion electrons by 161Tb may provide 161Tb an advantage over 177Lu in a\npoorly immunogenic tumor by enhancing the local inflammation and IFN1 pathway activation,\nwhich may result in better responses to combination therapy with ICI in a tumor like B78 or\nMyc-CaP. This is an intriguing future direction that will be investigated in follow-up studies.\nThe use of dose and radiation absorbed dose may be confusing.\nWe revised all sentences throughout the manuscript to read “absorbed dose” instead of “dose” for\nconsistency and clarity.\nWhy should 2 Gy be the optimal for 177Lu and 225Ac, given their different RBEs?\nOne possibility why 2 Gy may be the optimal dose for the antitumor immune response to both\n177Lu-NM600+dual anti-CTLA-4 and anti-PD-L1 and 225Ac-NM600+dual ICI that we have\nconsidered is that the RBE is ~1 for lymphocytes for x-rays, beta particles, and alpha particles.\nAs a result, the RBE difference at the tumor may have less of an impact on the antitumor\nimmune response than it would if we were studying the tumor response to RPT alone (no\nimmunotherapy) or in an immunodeficient mouse model.\nHow did ICI combination and scheduling influence toxicity?\nAll mice were monitored daily by independent veterinary staff and weighed weekly. No mice\ntreated in any of the studies presented here were identified by these veterinary staff as exhibiting\nsigns or symptoms of illness/distress and no mice lost >20% of their body weight. This is now\ndocumented in the manuscript on lines 138-140 and 539-540.\nThe very MMR deficient nature of the tumor affecting immunogenicity also affects radiation\nresponse. It is therefore a large caveat on interpreting the results.\nThank you for raising this important point. We have added a sentence acknowledging this\nlimitation to the discussion: “Certain aspects of tumor immunogenicity, including mismatch\nrepair status, directly impact radiation response and the capacity of cells to repair DNA damage,\nmaking the mechanism of therapeutic response to combination RPT+ICI therapy difficult to\ndisentangle.” (line 495-498)\nInvolvement of CD8+ T-cells is not new information. Was involvement of any other immune\ncell type disproved?\nWe agree with the reviewer in their interest in other immune cell types. In this study, we\ntested only CD8+ T cell involvement with a CD8+ T cell depletion antibody. Our aim was\nnot to dismiss effects of other immune cells, and we would not seek to disprove their\ninvolvement as they are most certainly involved and are vital to the CD8 T cell response.\nFigure 6 (Lines 324-328) highlights an example of this important influence of CD20+ cells,\nwhich differed between radioligands. Future studies to delineate the mechanisms of action\nand interactions between T cells, RPT, and other immune cells will be of critical\nimportance. We therefore added this to the Discussion to highlight this point, which now\nincludes this sentence: “Future studies delineating interactions of other immune cells with\nRPTs will provide better mechanistic insight to further optimize its combination with\nICIs.” (Line 462-464) However, we focused on the CD8 T cell effector cell population in this\nstudy because our previously published studies identified this as a key mediator of response\nto the combination of RPT + ICIs. In much of tumor immunology, CD8+ T cells are the\ncritical effector of anti-tumor immunity given their unique capacity to eradicate cells on the\nbasis of tumor mutation-associated neoantigens. What is new and reported for the first\ntime in this manuscript is the differential mechanistic effect of 225Ac RPT in promoting the\nprocess of CD8+ T cell priming and the absence of this with 90Y and 177Lu RPT, which\nappear to propagate the response of pre-existing CD8+ T cell clones.\nMemory effects: what were the differences in immunotyping between tumor rejected and non-\nrejected animals? Control versus naïve animals is useful, but not the interesting comparison.\nThis is an interesting question. Interestingly, there were no differences in the immunotyping\nmarkers we studied between tumor rejected and non-rejected animals when evaluating immune\ncells in spleen or blood. We separated these categories in Figure 5K, showing similar spleen CD4\nCM/EM and CD8 CM/EM ratios, and include all complete responder mice (tumor rejected and\nnon-rejected) in the “MC38, Ac+ICI” category in black in Figure 5I-J, L, with no appreciable\nimmune-phenotypic differences in the blood or spleen 24 hours after re-exposure to tumor. It is\npossible that at the time of this assessment the memory cell populations were a minority of total\nimmune cells and that if we had measured circulating immune cell response at some later time\npoint then perhaps we would detect differences in non-rejecting/engrafted mouse populations\nsuch as lower levels of circulating memory T cells and/or lower levels of IFNγ- and TNFα-\nexpressing activated T cells. However, we do not have specimens available to test this possibility.\nWhy was the immune microenvironment studied in B78, but not MC38 tumors?\nThe immune microenvironment of MC38 tumors was studied in Figures 4L-Q and Figure 5B-L\nby analyzing: the MC38 tumor immune infiltrate by flow cytometry (Figure 4L-N), the response\nof circulating immune cells harvested from MC38-tumor bearing mice to co-culture with or\nwithout MC38 cells (Figure 4O-Q, 5C), circulating blood cell counts (Figure 5B), and by\nstudying the immune response of circulating blood cells and splenocytes to IV-injected MC38\ntumor cells (Figure 5D-L).\nWere there any clone-specific immune effects between radionuclides observed?\nWe performed expanded differential gene expression analysis within the population of CD8+\ncells with shared clonotypes within effector and memory cells (# of cells analyzed/group in Table\nS50) and observed the following: “Interestingly, one gene that was upregulated in this cell\npopulation in the 225Ac-NM600+ICI treatment group over each other treatment group was an\ninterferon inducible gene, Ifi27l2a (red arrow, Figure 8D). This observation further suggested\nthat an active adaptive T cell response was present in the 225Ac-NM600+ICI-treated tumor.”\n(lines 386-389)\nWhat was the differential gene expression of RPT+ICI vs RPT alone, or naïve vs RPT alone?\nNow, only ICI vs RPT+ICI is considered.\nSingle cell RNA sequencing was performed on RPT+ICI and ICI alone groups only due largely\nto cost constraints on the design of this experiment.\nWas pathway analysis not considered?\nPathway analysis was performed in addition to differential gene expression analysis. These\nresults have been added as Figure 8A and Figure S10A.\nTo what degree are tumor infiltrating immune cells killed by TAT? Expression as percentages of\ninfiltrates obscures the effect on absolute numbers. There may be a large survival bias, or an\neffect of newly infiltrated cells measured here.\nWe acknowledge that TAT kills cells and that immune cells are continually circulating and\nproliferating. Even in the monotherapy setting this is difficult to disentangle, as 225Ac-NM600\nmay introduce an additional component of the immune-mediated effect of priming. To at least\npartially address this, we have now listed absolute cell counts of CD8+ cells and Tregs for Figure\n4M-N tumor infiltrating immune cell flow cytometry experiments in the Source Data file, now\nuploaded with the manuscript.\nWould effects be different in immune excluded or immune desert models?\nWe would view B78 melanoma as largely consistent with an immune excluded or immune desert\nmodel. We have added reference to prior publications supporting this characterization (see\nresponse to point # 1, reviewer 2). We do however acknowledge the broader point, that poor\nresponsiveness to ICIs can derive from varied mechanisms and it is possible that distinct forms\nof RPT may be better than others for overcoming different mechanism of inherent or acquired\nresistance to ICIs. This is an important area for further investigation both in the context of\ndifferent tumor models and diseases and in the context of different forms of immunotherapy.\nPlease use ‘molar activity’, in compliance with nomenclature guidelines.\nMolar activity (A ) is now used throughout the manuscript and figures.\nm\nDifference in combo effect is partially explained by the very different response to 225Ac-\ncompound alone.\nWe have included additional consideration of this important point in the discussion section: “The\nMC38 response to RPT alone (Figure 2E) was not affected by difference in RBE, suggesting that\nthe therapeutic response in MC38 may be predominantly mediated by tumor-resident immune\ncells.” (lines 394-396) and “225Ac-NM600 is a high LET radioisotope with a greater RBE in the\nB78 tumor model as a monotherapy (Figure 3E).” (lines 415-416)\nFig 1: The different scheduling between MC38 and B78 models makes for a complicated\ncomparison.\nIn this figure, our intention was to show why 2 Gy was selected over 0.2 Gy in each MC38 and\nB78 for the remainder of the 225Ac-NM600 experiments, not to compare between MC38 and\nB78.\nAddition of individual mouse tumor growth curves can be moved to SI. It clutters the figures.\nThank you for this suggestion. Other reviewers have previously requested raw data be presented,\nwhich is why we have included individual tumor growth curves in most instances. If the editors\nwish, we are happy to adjust the presentation of this data.\nFig 4M-N: normalisation to ICI treatment only may obscure significant effects, and non-\nlinearities in the combination of RPT and ICI. Consider normalising to treatment-naïve animals.\nIn this study, we did not include treatment-naïve animals because we were strictly focused on the\neffect of RPT in the setting of ICI therapy and therefore set out to compare between ICI with\nRPT versus ICI without RPT.\nFig 5B: is too small.\nThis figure has been made larger.\nFig 6: comparison vs what? Naïve only, or vs RPT only? N= 3 (as suggested by FigS6)? Errors\non analysis?\nThe comparisons in the differential gene expression analyses in Figure 8 and S10 (formerly\nFigure 6) are between RPT+ICI groups and ICI only groups. Naïve and RPT only groups were\nnot included in the single cell RNA sequencing experiment due to the significant resource and\ncost burden of this experiment. N=3 tumors from three mice/group were harvested for single cell\nRNA sequencing (the same tumors that are shown in Figure (now) S8). The numbers of cells\nindividually analyzed and used to produce the plots in Figures 6-8, S10 number in the thousands\n(cell numbers in Table S48).\nFig 6: 2 tumors in the same animal assume no crosstalk between tumors. This is not necessarily\nthe case.\nThank you for raising this point, we fully agree. We discuss our rational for 1 vs. 2 tumor\nselection above in response to a question from Reviewer 2. In general, we would prefer to study\nmultiple tumors per mouse for all RPT studies as clinical translation of these approaches will be\nfirst tested in the metastatic disease setting. However, the presence of a second (or third, etc.)\ntumor can affect tumor engraftment rates and may have additional confounding effects on\nimmune response and overall host fitness/organ function - as reviewer 3 notes. For this reason, in\nall models we present data with single flank tumors and to explore therapeutic effects and\nmechanisms. We test our findings in the setting of multiple tumors using the B78 model, which\nis slower growing than MC38 and allows sufficient time for a therapeutic effect to be observed in\nthe absence of confounding changes in host fitness (not the case with MC38 two tumor models).\nWas EBRT administered at the same dose? Would this result in the same response at all?\nEBRT was administered at 2 Gy, which we have seen to result in similar immune responses (e.g.,\nIFN1 response activation) with differing time courses and magnitudes to RPT+ICI in other\nstudies (Patel et al, STM, 2021; Jagodinsky et al, Theranostics, 2021; Kerr et al, CIR, 2025).\nFig S1: use the same scale throughout for ease of comparison\nThanks for this suggestion, we have adjusted the figure so the scales are now the same.\nFigS2: safety profile of RPT + ICI?\nThe safety profile of RPT+ICI that was studied and reported in Figure 5A-B. All mice were\nmonitored daily by independent veterinary staff and weighed weekly. No mice treated in any of\nthe studies presented here were identified by these veterinary staff as exhibiting signs or\nsymptoms of illness/distress and no mice lost >20% of their body weight. This is now\ndocumented in the manuscript on lines 138-140 and 539-540.\nFigS5: no control for innate immune response to RPT\nIn future studies of immunologic memory and rechallenge rejection rates, it would be interesting\nto include RPT-treated tumor-naïve mice as well as untreated tumor-naïve mice as controls. RPT\ndoes not render mice disease-free so we are not able to investigate memory response or persistent\ninnate immune response following this therapy alone.\nFigS6: why is there no tumor size measurement at the time of harvesting?\nWe regret that tumor size was not measured on the day of harvesting for this study."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have addressed the majority of the reviewers’ comments and have revised the manuscript with care. Most of the\npreviously raised concerns have been satisfactorily resolved, and the manuscript now demonstrates improved clarity,\ncoherence, and completeness.", "reviewer_4": "The paper by Kerr et al reports on a detailed study to compare the effects of alpha vs beta particle emitting isotopes\ncombined with immunotherapy in preclinical models.\nThe authors have very thoughtfully responded to the reviewers comments in particular to comments about RPh and ICI\ntumor penetration."}, "author_response": ""}]

Decision letter