# Towards melanoma in situ vaccination with multiple ultra-narrow X-ray beams

Verdiana Trappetti, Cristian Fernández-Palomo, Prateek Arora, Marine

Potez, Paolo Pellicioli, Jennifer Fazzari, Nahoko Shintani, Ismael

Sanchez-Gonzalez, Cheuk Ting Wu, Bettina de Breuyn Dietler, et al.

► To cite this version:

Verdiana Trappetti, Cristian Fernández-Palomo, Prateek Arora, Marine Potez, Paolo Pellicioli, et al. "Towards melanoma in situ vaccination with multiple ultra-narrow X-ray beams." Cancer Letters, 2025, 608, pp.217326. 10.1016/j.canlet.2024.217326. hal-05122156

HAL Id: hal-05122156

https://hal.science/hal-05122156v1

Submitted on 8 Jul 2025

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Original Articles

# Towards melanoma *in situ* vaccination with multiple ultra-narrow X-ray beams

<figure><img src="image_3.png" /></figure>

Verdiana Trappetti<sup>a</sup>, Cristian Fernández-Palomo<sup>a</sup>, Prateek Arora<sup>a,b</sup>, Marine Potez<sup>c</sup>, Paolo Pellicioli<sup>a,d</sup>, Jennifer Fazzari<sup>a</sup>, Nahoko Shintani<sup>a</sup>, Ismael Sanchez-Gonzalez<sup>a</sup>, Cheuk Ting Wu<sup>a</sup>, Bettina de Breuyn Dietler<sup>a</sup>, Nadia Mercader-Huber<sup>a,b</sup>, Olga A. Martin<sup>a,e</sup>, Stephan von Gunten<sup>f</sup>, Vladislav Volarevic<sup>g</sup>, Valentin Djonov<sup>a,*</sup>

ARTICLE INFO

Keywords:

Melanoma

In situ vaccination

Microbeam radiotherapy

Cytotoxic T lymphocytes

Abscopal effects

ABSTRACT

Despite the recent progress, current treatment modalities are not able to eradicate cancer. We show that Microbeam Radiotherapy (MRT), an innovative type of Spatially Fractionated Radiotherapy, can control murine melanoma by activating the host's own immune system. The beneficial effects are very pronounced in comparison to uniform radiotherapy traditionally employed in the clinic. Our results show that MRT increased antigen presentation, activating Cytotoxic T Lymphocytes (CTLs) which are essential to MRT's treatment efficacy in melanoma. Depletion of CTLs abrogated treatment response. Multiplex nucleic acid hybridization technology revealed key features of lymphocyte populations such as proliferation, differentiation, and ligand-receptor interactions. In addition, CTLs were shown to be essential for locoregional metastatic control and systemic abscopal effects confirmed by activation of antigen presenting cells and CTL trafficking in the tumour-draining lymph nodes. MRT also showed a synergistic effect with immunotherapy. Overall, MRT induces a robust antitumour immune response, acting like an *in situ* vaccination, which could be exploited to treat a variety of treatment-resistant malignancies.

# 1. Introduction

Cancer remains a leading cause of death worldwide, underlying the urgent need for therapeutic innovations. Radiation Therapy (RT) is a key anti-cancer treatment option, with an estimated 50 % of all cancer patients receiving RT as part of their therapeutic regimen [1]. However, RT, typically involving X-ray beams with a uniform dose profile, has limitations in the total dose that can be delivered in order to mitigate

normal tissue toxicity. Its administration in multiple low dose fractions over several weeks helps reduce collateral normal tissue toxicity but can lead to tumour radioresistance.

Innovative technological approaches are currently under development to overcome the limitations of traditional RT. One of the most promising strategies is Spatially Fractionated RT (SFRT), a method which, unlike conventional RT, simultaneously administers high and low doses creating a non-uniform radiation field. This unique approach

<footnote><sup>a</sup> Institute of Anatomy, University of Bern, 3012, Bern, Switzerland</footnote>

<footnote><sup>b</sup> Department of Biomedical Research, University of Bern, 3008, Bern, Switzerland</footnote>

<footnote><sup>c</sup> H. Lee Moffitt Cancer Center and Research Institute, 33612, Tampa, FL, USA</footnote>

<footnote><sup>d</sup> Biomedical Beamline ID17, ESRF, The European Synchrotron, 38000, Grenoble, France</footnote>

<footnote><sup>e</sup> Centre for Medical Radiation Physics (CMRP), University of Wollongong, 2522, NSW, Australia</footnote>

<footnote><sup>f</sup> Institute of Pharmacology, University of Bern, 3010, Bern, Switzerland</footnote>

<footnote><sup>g</sup> Departments of Genetics, Microbiology and Immunology, Center for Research on Harmful Effects of Biological and Chemical Hazards, Faculty of Medical Sciences University of Kragujevac, 34000, Kragujevac, Serbia</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail addresses: verdiana.trappetti@unibe.ch (V. Trappetti), cristian.fernandez@unibe.ch (C. Fernández-Palomo), prateek.arora@unibe.ch (P. Arora), marine.potez@gmail.com (M. Potez), paolo.pellicioli@unibe.ch (P. Pellicioli), Fazzari.Jennifer@mayo.edu (J. Fazzari), nahoko.shintani@unibe.ch (N. Shintani), ismael.sanchez@unibe.ch (I. Sanchez-Gonzalez), cheukting.wu@unibe.ch (C.T. Wu), bettina.debreuyn@unibe.ch (B. de Breuyn Dietler), nadia.mercader@unibe.ch (N. Mercader-Huber), omartin@uow.edu.au (O.A. Martin), stephan.vongunten@unibe.ch (S. von Gunten), drvolarevic@yahoo.com (V. Volarevic), valentin.djonov@unibe.ch (V. Djonov).</footnote>
improves tumour control while minimizing radiation-induced toxicity to normal tissues. Microbeam radiation therapy (MRT) became one of the most effective preclinical SFRT modalities [2]. MRT delivers a parallel array of ultra-narrow beams in the micrometric range (25–100 µm), depositing high radiation doses (peaks) in their path and low radiation doses (valleys) in the regions between them [3]. This specific attribute is key to the normal tissue sparing effect and exceptional tumour control observed in a variety of preclinical models [4–11]. We have demonstrated that MRT efficiently delays growth of B16-F10 murine melanoma and even induces complete tumour remission [12–14]. After a single MRT irradiation melanomas undergo a significant volume reduction that starts 5–6 days post-treatment [12]. Some of the biological mechanisms governing MRT efficacy identified so far include its selective disruption of tumour vasculature, reduction of tumour cell proliferation, and induction of cellular senescence [12,15]. However, evidence from our recent work suggests the immune system plays a role in this treatment response. In particular, overexpression of some immunostimulatory cytokines and immune markers were induced following MRT [12]. Additionally, we have recently shown that treatment of B16-F10 melanoma with MRT administered in a double fraction regimen is more effective than a single fraction for disease control, leading to 25 % complete remission and reduction of locoregional metastasis [14]. It was also seen that double fraction MRT further boosted the immune response relative to a single fraction [14]. In summary, MRT is emerging as a paradigm-shifting technique in the field, as it aims to not only directly kill tumour cells, but also to induce a strong anti-tumour immune responses [16].

The idea of using RT to induce such immune responses has been around for over 20 years and is also described as radiation-induced cancer *in situ* vaccination [17,18]. Cancer *in situ* vaccination is known as the induction of an adaptive immune response against the tumour in response to tumour-antigens (TAs). The steps that characterize this process are: (i) immunogenic tumour cell death followed by (ii) the release and presentation of the TAs by antigen-presenting cells (APCs), which will promote (iii) *in situ* anti-tumour immune responses mediated predominantly by Cytotoxic T cells (CTLs) leading to (iv) systemic effects inducing metastatic control [19]. In the field of radiation oncology, the induction of systemic effects on distant cancer lesions after localized RT to the primary tumour is known as the abscopal effect [20]. Ideally, RT would systematically trigger abscopal effects in patients, but unfortunately, this phenomenon occurs in only a fraction of patients and usually requires the use of adjuvant immunotherapy, which can have notable toxic side effects [21–23].

Given the potential of MRT to modulate immune responses and the known benefits of therapies that trigger abscopal effects in certain cancers such as melanoma, we hypothesize that MRT may have the capacity to elicit a more potent response than traditional (uniform) RT.

We propose that MRT could induce *in situ* vaccination, thereby triggering an abscopal effect in murine melanoma. To test our hypothesis, we investigated whether key immune populations play a prominent role in the MRT-mediated treatment effects. Furthermore, we studied the radioimmunological mechanisms induced after one MRT fraction up to a week post-treatment in comparison to uniform RT. Once we underlined the main immune cell types contributing to MRT treatment efficacy and characterized their status, we investigated their role in survival, tumour growth and development of locoregional metastasis. We investigated the involvement of proximal tumour draining lymph nodes (LNs) to validate the potential of MRT-induced anti-tumour abscopal effects.

# 2. Material and methods

## 2.1. Tumour model

Animal experiments were conducted under the Swiss permits BE61/15, BE61/18 and BE131/19 and the French permits 14_ethax22, 16_ethax9 and 20_ethax4. C57BL/6 J mice (females, 8 weeks old) were purchased from Charles River Laboratories. Mice were allowed 2 weeks of acclimatisation period upon arrival to the conventional animal facility and were socially housed. All cages were equipped with wood chip bedding material, soft tissue nesting material and red plastic housing. Facility conditions included 12 hours day/night cycle, a temperature of 23 °C and 50 % humidity. Animals were given access to mice maintenance food and water *ad libitum*. Mice were labelled and identified via tail-labelling with permanent colour markers. The first labelling occurred when the mice were under general anaesthesia, required for the tumour implantations. Melanomas from the B16F10 cell line were established as previously published [24]. For survival and metastasis studies, a single melanoma was implanted in the left ear of each mouse, while for the *in-situ* characterization studies 2 melanomas were implanted, one in each ear of the mouse. Mice were randomized before the beginning of any treatment to ensure equal tumour size in all groups.

## 2.2. Irradiations and dosimetry

All irradiations were performed at the ID17 Biomedical beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The uniform RT irradiation field was 5.5 mm wide and 14 mm high, while the MRT irradiations consisted of 26 vertical beamlets, 14 mm high, 50 µm wide and spaced 200 µm centre to centre. The multislit collimator (MSC) used to spatially fractionate the irradiation field at the ID17 beamline defines a pattern of 50 µm wide apertures spaced by 400 µm [25], therefore a double passage irradiation approach was used. This approach consists of a first irradiation characterized by the collimator geometry pattern, followed by a horizontal translation of the MSC by 200 µm, and a second irradiation to achieve the desired microbeam spacing.

In the uniform RT configuration, a dose of 6.4 Gy was delivered to the tumour centre, while MRT delivered a peak dose of 400 Gy and a valley dose of 6.4 Gy. Due to the different irradiation geometries and doses delivered, two different irradiation spectrum configurations were defined. For the uniform RT, a polychromatic spectrum with a mean photon energy of 86 keV and a dose rate to target of 4045 Gy/s was used. For MRT, a spectrum with a mean photon energy of 102 keV and a dose rate to target of 14,250 Gy/s was employed [26]. Thus, both MRT and uniform RT were performed using an ultra-high dose rate [27] with total delivery times under 60 ms. Melanomas from the control (CTR) group received no radiation treatment (0 Gy). For the longitudinal tumour study, each treatment group consisted of 20 mice, 10 mice underwent CD8⁺ T cell depletion while the other 10 did not.

## 2.3. Flow cytometry analysis

Mice were humanely euthanised with an intraperitoneal injection of sodium pentobarbital (200 mg/kg body weight), and 6-8 melanomas per time point were harvested at day 2, 5 and 7 post-irradiations (PI). The tumours were placed in a 0.1 % Collagenase IV solution, minced with fine scissors, and incubated for 45 min at 37 °C. The digested tissue was then filtered with 100 µm and, subsequently, 40 µm cell strainers (Falcon by Corning, Glendale, AZ, USA). The solution was centrifuged at 450×g for 5 min at 4 °C. To eliminate red blood cells, the pellet was resuspended in Red Blood Cells Lysis Buffer Solution (eBioscience, Hatfield, United Kingdom), then washed and centrifuged. To exclude dead cells from the analysis, a LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit for 405 nm excitation (Thermo Fisher Scientific, Fremont, CA, USA) was used, according to product datasheet guidelines. Fc receptors were blocked by incubating the cell suspension with Mouse FC Block (BD, Franklin Lakes, NJ, USA cat. 553142, clone 2.4G2) 1/100 in flow cytometry (FC) buffer (FC Buffer: 5 % FBS and 2 mM EDTA in Phosphate-buffered saline (PBS) 1X solution) for 30 min at 4 °C. The samples were washed with FC buffer, centrifuged, and stained for membrane proteins with a mix of antibodies for 30 min at 4 °C. Finally,
the cells were washed with the FC Buffer. Fixation and permeabilization of the single cell suspension were performed with the Fixation/Permeabilization Solution Kit Cytofix/Cytoperm™ Plus (BD). Cells were then stained for the nuclear protein FOXP3 in FC buffer for 30 min at 4 °C. Flow cytometry data were acquired on a BD LSR II (Special Order System (SORP)) and analysed with FlowJo V10 Software. A preliminary gating strategy was applied to all samples: physical gating for leucocytes, gating to select single cells and gating for negative cells, identified by LIVE/DEAD™ Fixable Violet Dead Cell Stain, to select only live cells. Leucocytes of the myeloid lineage were identified as CD45⁺ (Biolegend cat. 103147, clone 30-F11), CD11b⁺ (Biolegend cat. 101245, clone M1/70) cells. Tumour-associated macrophages (TAMs) were identified as CD45⁺, CD11b⁺ and F4/80high (Biolegend cat. 123149, clone BM8) cells. Granulocytes and monocytes together were identified as CD45⁺, CD11b⁺ and F4/80low cells. Subpopulations of M1-like and M2-like TAMs were differentiated evaluating the expression of the markers CD40 (Biolegend cat. 124612, clone 3/23) and CD206 (Biolegend cat. 141723, clone C068C2) in the TAM population respectively. The standardized Mean Fluorescent Intensity (MFI) was calculated with the following formula:

$$Standardized MFI = (Mean_{positive} - Mean_{negative}) / 2*SD_{negative}$$

Where Mean<sub>positive</sub> is the mean fluorescent intensity of the antibody of interest within the selected population, Mean<sub>negative</sub> is the mean fluorescent intensity of the negative control and SD<sub>negative</sub> is the standard deviation of the intensity of the negative control [28]. To identify T cells, CD3⁺ (Biolegend cat. 100349, clone 145-2C11) cells were gated over the total live cells and further set apart into two main T cell subtypes: CD3+/CD4+ (Biolegend cat. 100555, clone RM4-5) (T-holders) and CD3+/CD8+ (Biolegend cat. 100744, clone 53-6.7) CTLs. T regulatory cells (Tregs) were differentiated evaluating the expression of FOXP3 (Biolegend cat. 126408, clone MF-14) in T-helper cells.

## 2.4. Nanostring nCounter analysis

The NanoString nCounter gene expression assay was performed with a 785-gene pre-designed “Immune Exhaustion” panel to which we added an additional 9 genes: Bag 6, Calr, Cgas, Mlkl, Ripk1, Ripk3, Sigele, Sting 1 and Trex1. This panel aims at the characterization of the immune status of the tumours and the complex interplay between immune cells and its tumour microenvironment. We analysed 24 melanomas from 5 different groups: unirradiated controls collected 12 days post-tumour implantation (n = 4), 2 and 7 days post-MRT (n = 5/group), 2 and 7 days post-uniform RT (n = 5/group). Melanomas were snap frozen in isopentane and kept at -80 °C degrees immediately after being harvested. RNA was extracted from frozen mouse melanoma tissue using the NucleoSpin RNA Mini kit (Takara Bio Inc., Shiga, Japan) employing a modified version of the manufacturer's protocol for Isolation of RNA from Fibrous Tissue. Tissue was homogenized using the Bead Ruptor Elite (Omni International Inc., Kennesaw, GA, USA). Proteinase K was added to the homogenate and the samples underwent an incubation at room temperature followed by heating at 55 °C. Tissue debris was removed, and RNA was then bound, washed, DNase treated, and eluted off the Nucleospin RNA Column. For gene expression analysis on the NanoString nCounter system, 100-160 ng of RNA was hybridized to a multiplexed nucleotide probe pool for 16 hours at 65 °C. Enriched targets were purified and quantified using the nCounter MAX Analysis System.

## 2.5. Bioinformatic analysis

The raw data was analysed by ROSALIND® (https://rosalind.bio/). The normalization, fold changes and p values for the data were calculated using the nCounter® Advanced Analysis protocol. In brief, the counts within a lane were divided by the geometric mean of the

normalizer probes from the same lane. For normalization genes, housekeeping probes were selected based on the geNorm algorithm as implemented in the NormqPCR R library [29]. Fold changes and p values were calculated using the fast method and Generalized Linear Model (GLM) as described in the nCounter® Advanced Analysis 2.0 User Manual. P value adjustment was performed using the Benjamini-Hochberg method of estimating false discovery rates (FDR).

Downstream analysis was performed using R v4.3.2 with the data wrangling performed using the tidyverse [30,31]. The differentially expressed genes obtained from ROSALIND were subject to pathway analysis using overrepresentation analyses with the Gene Ontology (GO) and Reactome terms using the ClusterProfiler R package [32-34]. Top 10 GO terms were chosen for the categories Biological Processes and Cellular Components. Ten relevant pathways were chosen within the top 20 Reactome terms for the figure. The unbiased pathway enrichment can be found in the supplementary table (Supplementary Table S2). To identify the ligand-receptor interactions, OmniPath database was used, as this database has experimentally identified and curated ligands and receptors [35]. The ligand-receptor pairs in which both the partners were differentially expressed were chosen for the analyses. The pairs were plotted as a chord diagram [36]. The functional annotation of transcriptional factors, kinases, or genes related to cell death were derived using text mining of the GO annotations derived from Ensembl database using biomaRt [37-39]. To obtain the protein-protein interaction network, the STRING database was used. The network was then imported into igraph, shaped according to the molecule classes (kinases, transcription factor, and others), and coloured according to the log 2 fold change [40,41]. For the gene selection we used the terms coming from the overrepresentation analysis with GO Biological Processes performed on DEGs between the uniform RT and the MRT groups at 7 days post-treatment. Out of the 19 selected genes for the network all belonged to the term “lymphocyte proliferation”, 10 genes also belonged to the GO biological process term “lymphocyte differentiation”. Heatmaps were plotted using Complex heatmap package in R [42,43]. The viridis package of R was used for colourblind friendly colour palettes [44].

For cellular deconvolution, we used the web version of CIBERSORTx [45]. As input for deconvolution, the normalized counts were obtained from the Rosalind software. For the cell signature genes, “LM22 signature matrix” (from CIBERSORTX) for immune cells was used. The algorithm was run in batch correction B-mode, quantile normalization disabled, and with 100 permutations. Post-run, the data were imported in R where macrophages were considered as a sum of the different subpopulations, and the cell populations renormalized from 0 to 1. The data were then plotted using ggplot 2 [31]. The genes related to abscopal effect, were obtained from the literature [46,47]. MHC-II and T cell exhaustion genes were chosen as annotated in the panel and cell profiling groups as assigned by NanoString. For cell death genes, we pulled the GO names, and description from Ensemble using biomaRt and searched for the terms containing apoptosis, entosis, methuosis, paraptoxis, mitoptosis, ferroptosis, pyroptosis, NETosis, necroptosis or cell death [38,39]. The code used for the analysis can be found in GitHub at https://github.com/MercaderLabAnatomy/PUB-Trappetti et al_2024. The Nanostring nCounter raw data have been deposited in Gene Expression Omnibus (GEO) genomics data repository under accession number GSE268311.

## 2.6. CD8⁺ T cell depletion

We depleted CD8⁺ T cells from mice using an InvivoMab CD8α compound (clone 2.43, BioXcell), to specifically deplete CD8⁺ T cells in vivo. For the pilot trial, the compound (each dose was 300 µg in 100 µl Ringer's solution for 20 g mouse body weight) was administered via intraperitoneal injection at day 7, 10, and 13 post-implantation. CD8⁺ T cell depletion was combined with MRT, uniform RT or no radiation treatment and compared to undepleted counterparts. For the longitudinal study, the InvivoMab CD8α compound was administered 3 days
before irradiation, on the day of irradiation and every third day after that, until euthanasia.

## 2.7. Immunotherapy administration

We administered immunotherapy to the mice as a combination of two immune checkpoint inhibitors (ICIs): *InvivoMab* anti-mouse PD-1 (clone RMP1-14, BioXcell) and *InvivoMab* anti-mouse CTLA-4 (clone 9D9, BioXcell). Each dose for each ICI was 200 µg in 100 µl Sodium Chloride 0.9 % solution for 20 g of mouse body weight. Three doses of ICIs were administered via intraperitoneal injection at day 0 (day of MRT treatment), at day 3 and at day 6 after MRT.

## 2.8. Survival analysis

Mice in the longitudinal survival study were culled upon reaching humane endpoints, which were defined by ulceration of melanomas and/or tumour volume exceeding 100 mm³. Mice were monitored daily for appearance, consumption of food and water, passive behaviour, behaviour after stimulation and tumour size (starting at 7 days post-implantation). Kaplan-Meier survival curves were generated and analysed using Prism V10 (GraphPad Software, San Diego, CA, USA). The percentage increase in lifespan (ILS) was calculated with the following formula: $ILS = ((MS - CTR) / CTR) * 100$ where MS is the mean survival (in days) of the specific treatment group and CTR is the mean survival (in days) of the unirradiated, undepleted control.

## 2.9. Tumour growth curve

The tumour growth curves were obtained by digital calliper measurements. Measurements were taken daily starting at day 8 post-implantations (day 2 prior to irradiation). The maximum dimensions of the tumour in the sagittal, coronal, and transverse planes were recorded. The tumour volume (in mm³) was calculated with the formula: $V = \frac{4\pi}{3} * \frac{a}{2} * \frac{b}{2} * \frac{c}{2}$ where a, b, and c are the maximum recorded lengths of the tumour in the sagittal, coronal, and transverse planes, respectively.

## 2.10. Analysis of metastatic volume and score in LNs

Both the right and left largest cervical LNs from melanoma-bearing mice were harvested and examined. LNs were fixed in a 4 % paraformaldehyde (PFA) solution and stored in 70 % EtOH solution and examined macroscopically. The volume of the LNs was measured as described above for the volume of the melanomas and the volume occupied by metastases relative to the total LN volume was estimated according to our scoring system adopted from our previous study [14].

## 2.11. Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from 4 % PFA-fixed lymph nodes using the RNeasy FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. cDNA was synthesized from 2 µg for each sample of RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA United States). RT-qPCR was performed on 100 ng of cDNA using the QuantStudio 6 system (Applied Biosystems) with PowerUp SYBR Green Master Mix (Applied Biosystems). Relative expression levels were calculated using the 2⁻ΔCt method, where ΔCt represents the difference between the Ct values of the target gene and the reference gene. Primer sequences used were: for the housekeeping gene *B2m* forward: TGTATGCTATCCAGAAAACCCC, reverse: GCAGTTCAGTATGTTCGGCTTC; for the housekeeping gene *Actb* forward: CGCAGCCACTGTCGAGTC, reverse: GTCATCCATGGCGAACTGGT; and for the targeted gene *Mlana* forward: CCTGGGGATTGCTCTGCTTA, reverse: TGACGCCTTTTGTCATCAAG.

## 2.12. Immunostaining

Frozen sections of 100 µm thickness, from melanomas harvested at 2 hours post-MRT, were cut and processed for whole-mount fluorescent immunostaining against γH2AX to visually confirm the irradiation with the microbeams. Moreover, thin frozen sections were cut at 12 µm thickness for the ears bearing melanoma and at 5 µm for the cervical LNs (both were harvested at 2, 5, and 7 days post-treatment). The sections were placed in two different blocking solutions: (i) a 2 % bovine serum albumin (BSA) (Sigma-Aldrich, cat. A4503-50G) and 0,3 % Triton X-100 in PBS 1X solution and (ii) a 10 % donkey serum (Sigma-Aldrich, cat. D9663), 3 % milk powder and 0,3 % Triton X-100 in PBS 1X solution. The sections were first incubated with blocking solution number one for 15 min and subsequently incubated with blocking solution number two for 1 hour at room temperature under soft rocking. The sections were then incubated overnight at 4 °C with the primary antibodies: rabbit polyclonal antibodies against γH2AX (Abcam, Cambridge, UK, cat. ab11174) or CD86 (Bioss Antibodies, Woburn, MA, cat. bs-1035R), rabbit monoclonal antibody against CD8a (Abcam, cat. ab217344), or rat monoclonal antibodies against MHC class I (Abcam, cat. ab281903), or MHC class II (Thermo Fisher Scientific, cat. 14-5321-82), or CD62L (Thermo Fisher Scientific, cat. MA1-10262) or Ki67 (Thermo Fisher Scientific, cat. 14-5698-82) in blocking solution number two. For double staining, antibodies against CD8 and MHC class I (for ears bearing melanoma), CD8a and CD62L, and CD86 and MHC class II (for cervical LNs) were applied together. On the next day, sections were washed 3 times for 10 min in a 0,1 % Triton X-100 in PBS 1X solution and then incubated for 6 hours at 4 °C with the secondary antibodies: donkey anti-rabbit IgG (H + L) Alexa Fluor 488 (Thermo Fisher Scientific) and/or anti-rat IgG (H + L) Alexa Fluor 594 (Thermo Fisher Scientific) with 4',6-diamidino-2-phenylindole (DAPI) in blocking solution number two. After a second round of washings (3 × 10 min), the sections were mounted with ProLong™ Glass Antifade Mountant (Thermo Fisher Scientific, cat. P36984). For the TdT-mediated dUTP Nick End Labeling (TUNEL) staining we used In Situ Apoptosis Detection Kit from Takara (cat. MK505) according to manufacturer's instructions. The sections were imaged with a Zeiss LSM 880 (Carl Zeiss Microscopy GmbH, Jena, Germany) point scanning confocal microscope. To image the entire tissue sections of melanomas and LNs, tilescans with multichannel Z-stacks (4 slices, 3 µm step size) were acquired. For the MHC-II staining in melanomas a 10X objective with Numerical Aperture (NA) of 0.45 was employed and image pixel size was 0.69 µm × 0.69 µm. For the all the remaining stainings in melanomas and LNs a 20X objective with NA of 0.8 and image pixel size was 0.35 µm × 0.35 µm. Tilescans were subsequently stitched with the ZEN Blue software.

## 2.13. Quantification of CD8, MHC I, MHC II, CD62L and CD86 immunoreactivities

To assess the amount of CD8, MHC-I, MHC-II, CD62L, CD86, Ki67 and TUNEL positive cells, quantification of positive areas for each antibody was carried out using Fiji software [48]. First, the maximum intensity projection of z-stack images was generated, and the region of interest (ROI) (whole area of a tumour or a LN) was manually drawn with the polygon tool for each sample individually using DAPI and transmission light (TL) images. The positive area for each marker was segmented after setting a user-defined threshold. The same threshold setting was used for all samples in an experiment. Subsequently, the segmented area was measured.

To assess the amount of double positive cells, a composite image of 2 colours from the two selected markers (green and red colours to make yellow) was converted to a RGB colour image after defining the ROI. The double positive yellow area was selected after setting a user-defined colour threshold (same setting for each sample in the experiment) and then measured. Quantification results are expressed as a percentage of positive areas over the total area of the ROI.
## 2.14. Statistical analysis

Sample sizes, statistical test employed, and p values are indicated in the figure legends per each individual panel. Statistically significant outliers were underlined in a few cases by the ROUT test (Q = 1 %) and excluded from statistical analysis. Data normality was determined before using parametric or non-parametric statistical test. All statistical tests were performed using Prism V10 (GraphPad Software, San Diego, CA, USA) except for the gene expression data coming from the Nano-string nCounter assay (see above under "Bioinformatic analysis").

## 3. Results

### 3.1. MRT increases infiltration of CD8⁺ T cells in melanomas

Previous work from our group has shown MRT is highly effective in controlling murine melanoma [12]. To investigate the role of the immune response behind this efficacy, tumour-infiltrating immune populations were characterized at 2, 5 and 7 days post-treatment (unirradiated CTR, uniform RT and MRT) by flow cytometry-based immunophenotyping analysis. The preliminary gating strategy applied to all flow cytometry data of the presented study is reported in Fig. S1A. The data revealed that at 7 days there was a higher CD3⁺ T cell

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** *Immune-phenotype of melanomas reveals a higher infiltration of Cytotoxic CD8⁺ T cells after MRT.* Quantification of (A) CD3⁺ T cells; (B) CD3⁺CD4⁺ T-helper cells; (C) Tregs as FOXP3 MFI in T-helper cells; (D) CD3⁺CD8⁺ CTLs for the unirradiated CTR, uniform RT and MRT groups at day 2, 5 and 7 post-treatment. (E and F) Adopted gating strategy for CD3⁺ T cells and CD3⁺ T cell subpopulations: CD8⁺ CTLs and CD4⁺ helper T cells and comparison between unirradiated control, uniform RT and MRT (one representative melanoma for each group is reported). (G) Quantification of CTLs for the unirradiated CTR and uniform RT and MRT groups at day 2, 5 and 7 post-treatment by immunofluorescent staining. The CD8⁺ positive area within the whole tumour was evaluated. (H) Immunostained sections of one representative melanoma belonging to the unirradiated CTR, uniform RT and MRT groups at day 7 post-treatment showing CD8⁺ cells. Quantification of (I) CD45+/CD11b<sup>high</sup>/F4/80<sup>low</sup> granulocytes/monocytes (gran/mono); (J) Tumour-associated macrophages (TAMs) and relative abundance of (K) M1-like TAMs as CD40 MFI within the population and (L) M2-like TAMs as CD206 MFI within the population, for the unirradiated CTR, uniform RT and MRT groups at day 2, 5 and 7 post-treatment. Analysis from A to J were performed by flow cytometry assay. Data are presented as individual values and bars represent the mean ± SD. N = 6 to 8 per each group for the flow cytometry assays while n = 5 to 7 per each group for the immunofluorescence assays. One-way ANOVA with Tukey's multiple comparison test within the groups was performed for the significance analysis. \* = *p* < 0.05; \*\* = *p* < 0.01; \*\*\* = *p* < 0.001; \*\*\*\* = *p* < 0.0001. MFI = mean fluorescent intensity; CTR = unirradiated control; RT = radiotherapy; MRT = microbeam radiation therapy.</figcaption></figure>
infiltration in the MRT group compared to the uniform RT group (Fig. 1A). When we further analysed T cell subpopulations, such as CD4⁺ T helper cells, CD4⁺ FOXP3-expressing regulatory T cells (Tregs) and CD8⁺ CTLs (Fig. 1B, C and D respectivelyF), we found that the main contributors to this increase were CD3⁺CD8⁺ CTLs. Significantly higher infiltration of CTLs was observed in the MRT group compared to the uniform RT group at 7 days post-treatment (Fig. 1D). One representative plot of a tumour for each treatment group at 7 days post-irradiation is reported for CD3⁺ T cells (Fig. 1E) and CD3⁺ T cell subpopulations: CD8⁺ CTLs and CD4⁺ helper T cells (Fig. 1F). The specific increase of CD8⁺ cells at 7 days post-MRT was also confirmed by immunofluorescent staining (Fig. 1G and H). Furthermore, CD3⁺CD4⁺ T helper cells were observed to be lower in both uniform RT and MRT treatment compared to unirradiated CTRs at 7 days post-treatment, with a tendency to be higher following MRT relative to uniform RT (Fig. 1B). Tregs were found to be higher in the MRT group at all time points compared to the uniform RT group, but lower compared to the unirradiated group at 7 days (Fig. 1C).

In terms of myeloid cells (adopted gating strategies reported in Fig. S1B), we noted that both RT treatments elicited a significant decrease of F4/80<sup>low</sup>/CD11b<sup>+</sup> granulocytes/monocytes in the tumours at 5 days post-treatment compared to unirradiated CTR (Fig. 1I). We also

found that there was a more significant infiltration of F4/80<sup>high</sup>/CD11b<sup>+</sup> TAMs in the MRT compared to the CTR group (p = ****) than for the uniform RT group compared to the CTR group (p = ***) at 5 days post-treatment (Fig. 1J). At the earliest time point (2 days post-irradiation), TAMs showed a higher M1-like pro-inflammatory profile after MRT compared to the other two groups, according to the levels of CD40 expression within this subpopulation (Fig. 1K). In contrast, at 5 days post-treatment, the uniform RT group showed a more prominent M1-like phenotype amongst the infiltrating TAMs (Fig. 1K). At the same time, after both treatments (MRT and uniform RT compared to unirradiated CTR) there was a decrease of M2-like CD206<sup>+</sup> TAMs already present at 2 days post-treatment and persisting up to 7 days (Fig. 1L).

Our most important finding coming from the melanoma immunophenotyping analysis is the distinct increased infiltration of T cells specifically CTLs at 7 days post-MRT.

## 3.2. MRT enhances antigen presentation by MHC-I and MHC-II complexes

In order to determine the mechanism behind this CTL infiltration we investigated early DNA damage and antigen presenting activity within the tumours. Two hours post-MRT, the DNA damage marker, γH2AX,

<figure><img src="image_5.png" /></figure>

**Fig. 2. Increased antigen presentation after MRT.** (A) Quantification of MHC-I positive area and (B) CD8 and MHC-I positive area within the melanomas assessed by immunofluorescent staining. (C) A high magnification representative image of a melanoma at day 7 post-MRT is reported: MHC-I Alexa Fluor 594 (magenta) CD8 Alexa Fluor 488 (green) and DAPI (blue). (D) Quantification of MHC-II positive area within the tumours assessed by immunofluorescent staining. (E) A high magnification representative image of a melanoma at day 7 post-MRT is reported: MHC-II Alexa Fluor 594 (red) and DAPI (blue). Data are presented as individual values (n = 5 to 7 per each group) and bars represent the mean ± SD. One-way ANOVA with Tukey's multiple comparison test was performed for the significance analysis. (F) Expression level heatmap of all genes encoding for proteins involved in antigen processing and presentation and receptor activity within the MHC class I (in pink), class Ib (in yellow) and class II (in light blue) complexes, coming from the Nanostring nCounter assay. # symbol indicates genes encoding for associated proteins to the antigen presenting complexes. Heatmap shows the comparison among uniform RT and MRT at 7 days post-treatment and unirradiated CTR. P values for DEGs were calculated as described in materials and methods. \* = *p* < 0.05; \*\* = *p* < 0.01; \*\*\* = *p* < 0.001; \*\*\*\* = *p* < 0.0001. (G) CIBERSORTx immune cell profiling plot showing the estimated relative abundance of 14 main tumour infiltrating immune subpopulations per each of the treatment group: unirradiated CTR, uniform RT and MRT at 2 days post-treatment and uniform RT and MRT at 7 days post-treatment. NKs = natural killer cells; MCs = mast cells; MFs = macrophages; Tregs = T regulatory cells; Mono = monocytes; DC = dendritic cells; Eos = eosinophils; MHC = major histocompatibility complex; CTR = unirradiated control; DAPI = 4',6-diamidino-2-phenylindole; RT = radiotherapy; MRT = microbeam radiation therapy.
confirms the heterogenous MRT dose distribution indicating the high-dose peak microbeam regions, as has been previously documented in several preclinical models [49–51] (Fig. S1C). The nuclear saturation of the γH2AX foci revealed the magnitude of the MRT-induced DNA damage within these regions. With the signal specifically localized to the peak regions, it is also possible to clearly appreciate the MRT geometry employed with a centre-to-centre distance between microbeams of 200 µm (Fig. S1C). Immunofluorescent staining and quantification of immunoreactivity for the MHC-I complex showed that there was a tendency to have more MHC-I in the MRT-treated melanomas at 7 days post-treatment (Fig. 2A), and this difference was statistically significant when we measured the double positive MHC-I/CD8 staining (Fig. 2B and C). Immunofluorescent staining and quantification of immunoreactivity for the MHC-II complex also showed the same tendency to increase at 7 days post-irradiation in the MRT group (Fig. 2D and E).

Furthermore, to gain a deeper understanding of the immune status of the irradiated melanomas, we performed a NanoString nCounter gene expression assay. Of the genes in the panel, we selected and plotted in a heatmap all the genes encoding proteins of the MHC-I, MHC-Ib and MHC-II complexes or known to be directly associated with their activity. We observed that there is tendency for all the selected genes to be overexpressed in MRT-treated tumours (specifically at 7 days post-treatment) and most of the transcripts are significantly higher in the MRT group when compared to uniform RT (Fig. 2F). To further confirm increased antigen presentation post-MRT, we performed a gene set enrichment analysis (GSEA) on differentially expressed genes (DEGs) between uniform RT and MRT-treated tumours at 7 days post-treatment. GSEA with the Gene Ontology (GO) biological process gene set showed significant enrichment for antigen presenting-related terms (Fig. S2).

To estimate the immune cell populations in the tissue, we employed the cell deconvolution method of CIBERSORTx on the NanoString nCounter data. This method takes the key gene signatures of various cell types and estimates their presence in the given tissue. This analysis revealed an increase in the proportion of B cell and dendritic cell (DC) populations 7 days after RT, with the latter being the main immune population at this time point, with no apparent differences between uniform RT- and MRT-treated groups (Fig. 2G). However, when the MRT-treated melanomas were compared to the uniform RT-treated ones, they showed significantly higher MHC-I and MHC-II expression by immunofluorescence and increased Cd80 and Cd86 gene expression by NanoString nCounter assay (Supplementary Table S1).

These results suggest that MRT treated melanomas display enhanced antigen-presenting and T cell priming activity.

### 3.3. T cells are active and proliferating

To broaden our knowledge on the status and functions of lymphocytes that might underly the higher treatment efficacy of MRT in the melanoma model, we investigated the transcripts measured in the NanoString nCounter gene expression assay. Upon plotting the gene expression levels of the entire nCounter panel, we could observe a unique gene profile belonging to the MRT group at 7 days post-treatment in comparison to all the other groups, characterized by an increase in almost all genes (Fig. S3A). At 2 days post-treatment, there were significant DEGs between the unirradiated CTR and the uniform RT groups as well as between the unirradiated CTR and the MRT groups but no significant DEGs were found in the direct comparison between uniform RT and MRT (Supplementary Table S1). To better understand the changes happening in the tumour microenvironment at 7 days post-treatment we performed a Gene Overrepresentation analysis with GO Biological Processes gene set on DEGs between the uniform RT and the MRT groups at 7 days post-treatment. The terms “lymphocyte proliferation” and “lymphocyte differentiation” were enriched within the top 10 significantly enriched terms (Fig. S3B and complete DEGs list in Supplementary Table S1). We then obtained the protein-protein interaction network from the STRING database and subset it for 19 genes from the

GO biological process term “lymphocyte proliferation”. Within the network (Fig. 3A) there are 4 genes encoding transcription factors (Cebpb, Foxp3, Lef 1 and Nfatc2) and 9 for protein kinases (Btk, Fyn, Jak2, Jak 3, Lyn, Ripk3, Syk, Tyk2, Zap 70) while the remaining genes encode for receptors (Cd28, Cd40, Cd86, Il2ra, Tnfrsf14 and Tnfrsf9). We plotted the expression levels of these genes in the network for the unirradiated CTR group and for the uniform RT and MRT group at 7 days post-treatment. For the whole gene set, we observed a sequential increase in the expression levels from unirradiated, to RT-treated group with the largest increase observed 7 days post-MRT. Specifically, we saw a pronounced expression in the kinases such as Syk, Fyn and Jak2 suggesting a posttranscriptional kinase-mediated signalling change. Experimental data validated augmented proliferation of CD8⁺ T cells at 7 days post-MRT compared to uniform RT (Figs. S3C and C'). The GSEA on the GO cellular component gene set on DEGs between uniform RT and MRT at 7 days post-treatment revealed that within the top 10 significantly enriched terms, most are related to membrane components such as rafts or microdomains (Fig. S3D), suggesting a possible role of these structures in immune cell activation [52]. Despite the previous indications of T cell activation and proliferation, we also observed that 4 markers of CD8⁺ T cell exhaustion were overexpressed at 7 days post-MRT: Lag 3, Eomes, Cd244a and Ptger4, specified in the immune cell profiling feature by Nanostring (Fig. S3D). All these results suggest that lymphocytes in the melanomas at 7 days post-MRT were activated and had the capacity to proliferate.

### 3.4. CD8⁺ T cells induce melanoma cell death

Once we assessed the presence of CD8⁺ T cells and their activation and proliferation status in MRT-treated melanomas, we wanted to check if they had the capability of inducing tumour cell death contributing to MRT-induced tumour shrinkage and prolonged survival. Higher expression of cell-death-related genes was observed following MRT, specifically 7 days post-treatment (Fig. 3B left panel) in comparison to the uniform RT-treated mice and unirradiated CTR. Specifically, the following four molecules known to participate in CTL-dependent elimination of tumour cells were found to be significantly overexpressed in the MRT group (Fig. 3B right panel): Lamp1 a marker of CD8⁺ T cell degranulation following stimulation by tumour antigens [53], Casp3 a key player for CD8⁺ T cell-induced immunogenic cell death [54], Fas-associated death domain (Fadd) and Fas, responsible for activation-induced and lymphocyte-mediated cell death [55]. Experimental data validated augmented cell death in melanomas at 7 days post-MRT compared to uniform RT (Fig. 3C).

### 3.5. T cells interact with other tumour immune populations

To understand the intercellular interaction among the different cell populations within the melanomas, as well as the signalling pathways governing the observed treatment efficacy of MRT treatment, we performed a ligand-receptor analysis. The differentially expressed genes (DEGs) between MRT and uniform RT at 7 days post-treatment were subsetsed for ligands and receptors (LRs). For this analysis, we used the OmniPath database which contains a compilation of experimentally identified LRs from various data sources [35]. These data are presented as a chord diagram (Fig. 3D), where each connection represents a LR interaction. Expression levels for these receptors (28 receptors) and ligands (29 ligands) are reported in Fig. 3E and F respectively. Remarkably, we found evidence for enhanced CD28⁻CD80 receptor-ligand interactions upon MRT, which is fundamental to T cell activation and eventual establishment of CD8⁺ T cell memory responses [56]. In this regard it has to be mentioned that transcripts for markers of CD8⁺ effector memory T cells (Tem), such as the transcription factors (Blimp1) [57] and Id2 [58], as well as markers for CD8⁺ central memory T cells (Tcm) such as Ccr 7 [59] and transcription factor Bcl6 [60] were overexpressed 7 days after MRT as well. Also, CD4 receptor transcripts
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** *Study of lymphocytes functions and interactions with other immune cell populations.* (A) Core regulatory network of lymphocytes proliferation composed by 19 genes (each node represents a gene). The status of the network in terms of gene expression levels for each node is reported for the CTR, the uniform RT and MRT at 7 days post-treatment (going from left to right within the panel). Genes encoding transcription factors are represented in round boxes and kinases in squared boxes, the rest of the genes encodes for receptors. (B) Heatmap of cell death related genes for the groups: CTR, and uniform RT and MRT at 7 days post-treatment. Out of the total, the normalized gene expression profile of 4 selected genes are represented with violin plots. The selected genes (Lamp 1, Casp3, Fadd and Fas) are compatible to CD8⁺ Cytotoxic T cell-induced cell death. Data are represented as individual values (n = 4 to 5) and significance between uniform RT and MRT 7 days post-treatment is indicated. P values for DEGs were calculated as described in materials and methods. *p* < 0.0001. (C) Quantification of TUNEL+ cells for the unirradiated CTR, uniform RT and MRT groups at day 7 post-treatment by immunofluorescent staining. Representative images depict TUNEL+ immunostaining in the unirradiated CTR, uniform RT and MRT groups at Day 7 post-treatment. Data are presented as individual values (n = 5 to 7 per each group) and bars represent the mean ± SD. One-way ANOVA with Tukey's multiple comparison test was performed for the significance analysis. * = *p* < 0.05; ** = *p* < 0.001. (D) Ligand receptor chord plot shows all known interactions between significant DEGs annotated either as ligand or receptor in the comparison between uniform RT and MRT at 7 days post-treatment. (E) Gene expression heatmap for the genes encoding receptors and (F) ligands. (G) Reactome overrepresentation analysis of DEGs between the groups uniform RT and MRT at 7 days post-treatment. CTR = unirradiated control; Unif = uniform; RT = radiotherapy; MRT = microbeam radiation therapy; DEGs = differentially expressed genes; TUNEL: TdT-mediated dUTP Nick End Labeling.</figcaption></figure>

(interacting with its ligand Il16) were overexpressed in the MRT group. Furthermore, our analysis indicates enhanced cytokine (e.g. *Il*-1*a*, *Il*-18) and chemokine (e.g. *Ccl*3, *Ccl*4) signalling pathways following MRT, which may enhance recruitment and effector functions of both myeloid and lymphoid cells. Presence of *Ccl*3 cytokine expression could also relate to the portion of B cells underlined in Fig. 2G [61]. Other pathways upregulated in MRT are associated with immune cell adhesion and extravasation (*Icam*1) [62] or apoptosis (tumour-necrosis factor; *Tnf*). At 2 days post-treatment, no ligands nor receptors were found in the DEGs

from the direct comparison between uniform RT and MRT, however, when both groups were compared to the unirradiated CTR at 2 days post-treatment, there was a higher number of interactions in the case of the MRT-treated group (Fig. S3E). LRs specific to the MRT group at 2 days post-treatment found in the analysis were *Ccl*3 and *Icam*1 as well as *Ccl*2 and *Il*1*a* as ligands and *Cd*80 but also *Ccr*2 and *Ccr*5 among the receptors upregulated following MRT, compared to the unirradiated CTR group (Fig. S3F).

In line with these findings, results from reactome overrepresentation
analyses reported in Fig. 3G (DEGs between uniform RT and MRT at 7 days post-treatment) indicated enhanced cytokine/interleukin signaling and signatures related to immune cell activation, including T and B cell-specific pathways, involving T cell and B cell Receptors (TCR and BCR) and CD28 stimulation. All these findings point to higher lymphocyte activity and functionality in MRT-treated melanomas and predict interactions between several immune cell populations in the tumour microenvironment, such as CTLs with APCs (Cd28/Cd80 and Cxcr3/Ccl20) and CTLs with T helpers (Cd4/Il16).

### 3.6. CTL depletion abrogates MRT efficacy

After underlining the increased presence of CTLs post-MRT we wanted to investigate whether they were essential for treatment outcomes by depleting them from melanoma-bearing mice. CD8⁺ CTL depletion (CD8(-)) with the InvivoMab CD8α compound was confirmed by flow cytometry (Figs. S4A and B) in several animals (Fig. S4C). We performed a longitudinal survival study following uniform RT, MRT or no treatment in CD8(-) and undepleted animals. The experimental design for the longitudinal survival study is outlined in Fig. 4A. MRT-

treated mice that underwent CD8(-) showed a significant decrease in the survival rate (Fig. 4B) and median survival time (MST) (Fig. 4C) when compared to the MRT-treated undepleted counterparts. CTR CD8(-) mice had decreased lifespan compared to the CTR group while the rest of the groups had an increased lifespan, with the MRT-treated, undepleted mice having the longest lifespan (Fig. 4D). The MRT-treated group with CD8⁺ CTLs showed significantly longer survival with respect to all the other experimental groups (Fig. 4B). CD8(-) had no significant effect on survival in the unirradiated CTR or in the uniform RT-irradiated animals (Fig. 4B).

CD8(-) abrogated the MRT-induced tumour growth delay observed in the undepleted irradiated animals (Fig. 4E). MRT-treated mice that underwent CD8(-) showed a similar tumour growth delay to the mice in the uniform RT groups, with or without CD8⁺ cells (Fig. 4E). Comparison of MRT-treated groups with and without CD8⁺ CTLs revealed significant divergence in tumour growth delay starting as early as 5 days post-MRT (Fig. 4F). This difference in response to MRT is even more evident when analysing the response of each individual animal (n = 10) in the MRT with CD8⁺ T cells group vs. the MRT CD8(-) group (Fig. 4G–G'). Only 2 out of 10 tumours in the MRT-treated group (with

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** CD8⁺ Cytotoxic T cells (CTLs) depletion abrogates MRT efficacy. (A) Experimental design and timeline of the survival study: melanomas were implanted 10 days prior to radiation treatment; the InvivoMab CD8α compound for CD8⁺ CTL depletion was administered to half of the mice belonging to each treatment group (n = 10) 3 days prior to treatment, on the day of the treatment and every third day after that until mice euthanasia. Panel created with BioRender.com. (B) Kaplan-Meier survival curves for each treatment group (n = 10). The log-rank (Mantel-Cox) test showed that the differences in survival between MRT CD8(-) versus MRT (**p**<0.01) were statistically significant. Significant differences in terms of survival were also underlined between the MRT group and all the other groups (***p*<0.001). (C) Median survival times for each treatment group (n = 10). (D) Percentage increase in average lifespan relative to the undepleted CTR; (n = 10). (E) Tumour growth curves for each treatment group (n = 10). (F) Comparison between the MRT and the MRT CD8(-) tumour growth curves. Bars represent the Standard Error of the Mean (SEM). Two-way ANOVA multiple comparison test was performed for the significance analysis (**p** = 0.0063 at day 5, *p* = 0.0058 at day 6 and *p* = 0.0288 at day 7 post-treatment). (G) The growth of each single tumour belonging to the MRT group is reported. (G') The growth of each single melanoma in the MRT CD8(-) group is shown. Tumours underlined with # show a different response to the treatment from the rest of the group. CTR = unirradiated control; RT = radiotherapy; CD8(-) = CD8⁺ CTL depleted; MRT = microbeam radiation therapy.</figcaption></figure>
CD8⁺ CTLs) showed no response to the irradiation. The remaining 8 tumours showed the typical reduction in tumour volume following MRT starting from approximately 5 days post-treatment (Fig. 4G). On the contrary, in the MRT-treated CD8⁻ group only 2 out of 10 tumours showed a partial response to MRT irradiation while the remaining 8 did not show any response (Fig. 4G').

### 3.7. CTL depletion abrogates MRT-induced metastatic reduction

The largest locoregional sentinel cervical LNs, draining LNs for the ears [63], per mouse were harvested and evaluated for the presence of metastatic melanoma.

We quantified by RT-qPCR the expression of the melanoma-restricted antigen *Mlana* (also known as *Melan-A*), which we have previously shown to be a specific marker for metastatic B16F10 melanoma cells [14], in LNs collected at 5 and 7 days post-treatment. It was found that at day 5, there were no significant differences in *Mlana* expression levels among the treatment groups (Fig. S5A). However, by day 7 LN metastasis, as indicated by *Mlana* gene expression, was significantly reduced in mice treated with MRT compared to those receiving uniform RT (Fig. S5A'). Notably, in contrast to the uniform RT group, the MRT-treated group showed a significant decrease in locoregional metastasis over time (Figs. S5B and S5B').

For the longitudinal survival study, metastatic burden was scored as

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Evaluation of systemic effects post-MRT. (A) Illustration showing the location of the analysed locoregional LNs and images of two representative metastatic LN samples of score 3 and score 5. The illustration was created with **BioRender.com**. (B) Multiple variables plot of the metastatic score (score from 0 to 5) for each one of the treatment groups (n = 10/group) over time. The metastatic score observed in the locoregional LNs for each of the melanoma bearing mouse is reported at the day of mouse euthanasia. (C) Bar plot of the LN volumes per each treatment group. CD8⁺ CTLs depletion worsen metastatic formation post-MRT, while there are no significant differences between the groups CTR and CTR CD8⁻, uniform RT and uniform RT CD8⁻. Data are presented as individual values (n = 10) and bars represent the mean ± SD. \*p = 0.0121. Kruskal-Wallis with Dunn's multiple comparison test among the treatment groups was performed for the significance analysis. (D) Bar plot of ratio between the average LN volume and the MST per each treatment group showing that the best ratio for the MRT treated group and the worst ratio for the MRT CD8⁻ group. (E) Bar plot of the mean value of the ratio between the LN score and the day of euthanasia for each mouse per each treatment group showing the lowest value for the MRT treated group and the highest value for the MRT CD8⁻ group. (F) Heatmap of expression levels of abscopal related genes within the melanomas for the unirradiated CTR and the uniform RT and MRT at 7 days post-treatment. P values for DEGs were calculated as described in materials and methods. \* = p < 0.05; \*\* = p < 0.01; \*\*\* = p < 0.0001. For the *Cgas* gene \# = p of 0.0538. (G) Quantification of CD8 and CD62L double positive area within the LNs. \*p = 0.0296. (H) A high magnification representative image of a LN at day 5 post-MRT is reported: CD8 Alexa Fluor 488 (green), CD62L Alexa Fluor 594 (magenta) and DAPI (blue). (I) Quantification of MHC-II and CD86 double positive area within the LNs. \*\*\*\*p < 0.0001. For (G) and (I) Data are represented as individual values (n = 10 to 13 per each group) and bars represent the mean ± SD. One-way ANOVA with Dunnett's multiple comparison test (all group compared to CTR) was performed for the significance analysis. (J) A high magnification representative image of a LN at day 5 post-MRT is reported: CD86 Alexa Fluor 488 (green), MHC-II Alexa Fluor 594 (magenta) and DAPI (blue). LNs = lymph nodes; DAPI = 4',6-diamidino-2-phenylindole; CTR = unirradiated control; RT = radiotherapy; CD8⁻ = CD8⁺ CTLs depleted; MRT = microbeam radiation therapy; MST = median survival time; DEGs = differentially expressed genes.</figcaption></figure>
previously published [14]. Briefly, scores range from 0 to 5, where 0 represents LNs with no visible melanoma cells and a score of 5 represents enlarged LNs completely invaded by melanoma cells. A schematic representation of anatomical metastatic location and examples of metastatic LNs assigned a score of 3 and 5 are reported in Fig. 5A. In a preliminary analysis, the volume of all the LNs included in the study (considering both left and right) positively correlated to the score of the LNs with a Pearson r value of 0.5634. Details about the correlation study can be found in Supplementary Table S3. Comparing all the tumour-draining LNs (corresponding to tumour-bearing ear on the left side) with the contralateral LNs (corresponding to non-tumour-bearing ear), regardless of the radiation treatment or CD8⁺ T cell depletion, revealed that the volume of the left LNs is significantly greater than that of the right LNs (Fig. S5C). In addition, when we analysed the score of left LNs, regardless of the radiation treatment and CD8⁺ T cell depletion, we observed that most of them had a metastatic score of 2 or greater than 2, while the right LNs had a maximum score of 2 (Fig. S5D). These analyses indicated that the presence of the tumour on the left ear directly influenced the formation of the metastases in the corresponding, locoregional cervical LNs.

Furthermore, all the groups (CTR, uniform RT, and MRT) that underwent CD8(-), exhibited a general increase in metastatic formation in the left cervical LNs compared to those groups that did not receive depletion treatment (Fig. S5E). The right LNs were not affected by the CD8(-) (Fig. S5F). Interestingly, grouping the LNs according to their anatomical side and whether they received CD8(-) or not, independently of the radiation treatment received, revealed that all the LNs with a maximum metastatic score (score 5) belonged to the left LNs with CD8(-) (Fig. S5G). These data indicate that the CD8(-) alone has an impact on metastasis formation in the mice tumour-draining LNs, regardless of the type of radiation treatment received.

Given the results of this preliminary analysis, only the left cervical LNs were used for comparison among individual treatment groups: CTR, CTR CD8(-), uniform RT, uniform RT CD8(-), MRT and MRT CD8(-). The metastatic scores recorded per animal on the day of euthanasia revealed that LNs from the MRT group had higher scores (mostly score 3 and 4) relative to those in the other treatment groups that were euthanized at earlier timepoints. This excludes the groups with CD8(-), where most LNs with a score of 5 were found (Fig. 5B). However, it should be considered that the mice in the MRT group had significantly longer time to develop metastases (MST is increased at least 3 times compared to all the other groups). MRT CD8(-) mice had the highest number of LNs with a score 5 (Fig. 5B). MRT CD8(-) mice showed a significant increase in locoregional metastatic burden in terms of LN volume, compared to the animals that did not receive the depletion treatment (Fig. 5C). The CD8(-) had no significant effect on metastasis formation in unirradiated CTR and uniform RT groups (Fig. 5C).

Finally, when comparing the median LN volume:MST ratio (Fig. 5D) and the mean of the LN score:day of euthanasia ratio (Fig. 5E) for each of the six experimental groups, it was found that the MRT-treated mice with CD8⁺ T cells had the lowest values in both cases.

These data, together with the *Mlana* RT-qPCR quantification at early time points, indicate that MRT has the capacity to control formation of locoregional metastasis in addition to controlling the primary tumour and that CTLs are essential to this control.

### 3.8. MRT triggers systemic anti-tumour responses that involve tumour draining lymph nodes

Given that MRT can control locoregional metastasis formation, we investigated the expression levels of genes already known to be involved in the regulation of abscopal effects [46,47] in the unirradiated CTR, uniform RT and MRT groups at 7 days post-treatment (Fig. 5F). *Hmgb1*, *Trex1*, *Nfkb 1*, *Stng* and *Il1rl1* were found to be significantly overexpressed when comparing MRT to the uniform RT group. *Cgas* also showed a similar trend (p value of 0.0538) as did most of the remaining

genes. Furthermore, we analysed the immune activation status within the tumour draining LNs. Most importantly, we observed an increase in double positive immunoreactivities for the markers CD8 and CD62L (also known as SELL) (Fig. 5G and H) suggesting the presence of activated CD8⁺ T cells able to migrate, specifically at 5 days post-MRT. To corroborate our finding about the probable migration of CTLs to the primary tumour, we found that the transcript for the gene *Cd62l* was significantly increased within the tumours in the MRT group relative to uniform RT at 7 days post-treatment (Supplementary Table S1). CD62L is also a marker for memory T lymphocytes [64].

At 5 days post-treatment in the MRT group, there was also an increase in MHC-II/CD86 double positive cells within the tumour draining LNs (Fig. 5I and J) suggesting increased activity of DCs.

In the end, we found indications, both in the primary tumour and in the locoregional LNs, of a systemic immune response underlying MRT-induced abscopal effects.

### 3.9. MRT shows a synergistic effect in combination with immunotherapy

To predict the potential benefits of combining MRT with immunotherapy, we looked at the gene expression data for immune checkpoints (ICs) such as *Ctla-4* and *Pd-1*, which were found overexpressed in the MRT group compared to the uniform RT group at 7 days post-treatment (Fig. 6A). To test our hypothesis that ICs would enhance MRT treatment efficacy, we designed an experiment in which we compared tumour growth in mice treated with MRT only to mice treated with MRT plus combination of three doses of anti-CTLA-4 (αCTLA-4) and anti-PD-1 (αPD-1) together. The experimental design is reported in Fig. 6B. Tumour growth curves show a improved tumour growth delay for the MRT+αCTLA-4/αPD-1 group (Fig. 6C) in comparison to the MRT only group. Analysis of survival revealed a 40 % long-term survival, corresponding to complete melanoma remission in the MRT+ αCTLA-4/αPD-1 group (Fig. 6D). These findings suggest that MRT and immunotherapy exhibit a synergistic effect.

## 4. Discussion

To the best of our knowledge this study is the most comprehensive to date investigating the immune mechanisms behind the known efficacy of MRT in a preclinical cancer model. While previous studies partially described the involvement of anti-tumour immune responses post-MRT, here we have shown that CTLs are the indispensable mediators of MRT efficacy in radioresistant murine melanoma and described the most important lymphocyte features, and immune population interactions. In addition, the presence of activated CTLs is also required to control locoregional metastasis formation. This control was observed after MRT and indicates the presence of systemic abscopal effects confirmed by the activation of APCs and trafficking of CTLs to the locoregional LNs themselves. Overall, it was demonstrated that MRT acts as a tool for inducing melanoma *in situ* vaccination by (i) inducing immunogenic cell death that leads to (ii) increased antigen presentation by APCs, which activates (iii) local anti-tumour immune responses mediated by CTLs, which finally triggers (iv) systemic effects on metastatic lesions.

### 4.1. CD8⁺ T cells as main mediators of the MRT-effect

CD8⁺ T cells or CTLs represent the main cellular mediators of the adaptive immune defence against malignant cells, having a crucially important role in the therapy of melanoma [65]. CTLs kill antigen-presenting tumour cells and, therefore, they are a fundamental component in anti-tumour immune responses [66]. We showed increased MHC-I positive cells, and specifically a significant increase in double positive MHC-I and CD8 areas in melanoma 7 days post-irradiation with MRT. These double positive areas can be attributed to interactions between antigen presenting tumour cells and CTLs themselves. This increase of CTLs and their activation status could
<figure><img src="image_9.png" /><figcaption>**Fig. 6. Synergistic effect between MRT and immunotherapy.** (A) Normalized gene expression profile of *Ctla-4* and *Pd-1* genes are represented with violin plots. Data are represented as individual values (n = 4 to 5) and significance between uniform RT and MRT 7 days post-treatment is indicated. P values for DEGs were calculated as described in materials and methods. \*\* = *p* < 0.01; and for the *Ctla-4* gene \# = *p* of 0.0663. (B) Experimental design and timeline of the experiment: melanomas were implanted 10 days prior to radiation treatment; the immunotherapy (αCTLA-4 and αPD-1 together) was administered on the day of MRT treatment, three days and six days post-MRT. Panel created with BioRender.com. (C) Tumour growth curves for the unirradiated CTR, MRT and MRT+αCTLA-4/αPD-1 groups (n = 5 to 10). (D) Plot representing survival for the MRT and MRT+αCTLA-4/αPD-1 groups (n = 5 to 10). “Dead” refers to mice euthanised due to meeting the humane endpoint criteria. “Survived” indicates mice who had complete tumour remission and therefore long-term survival. CTR = unirradiated control; MRT = microbeam radiation therapy; α = anti.</figcaption></figure>

explain the remarkable reduction in tumour volume starting from day 5/6 post-MRT [12]. CTLs can efficiently eliminate melanoma cells in a juxtracrine (cell-to-cell-dependent) and paracrine manner (through the delivery of anti-tumourigenic and cytotoxic cytokines). Binding of FasL, expressed on tumour-infiltrating CTLs, with the Fas receptor of tumour cells (its transcript was found increased at 7 days post-MRT in our model) initiates formation of the death-inducing signalling complex and leads to the caspase-8-dependent apoptosis of malignant cells [67]. CTL-derived perforins generate transmembrane pores, enabling entry of pro-apoptotic granzymes in the cytosol and initiate an apoptotic series of events in the tumour cells [67].

Effector functions and anti-tumour properties of CTLs correlate with their density in the tumour microenvironment [68]. High numbers of tumour-infiltrating CTLs increases CTL-tumour cell interactions. In addition, release of cytotoxic cytokines (IFN-γ and TNF-α) is positively associated with high-density CTL infiltration in the tumour microenvironment, resulting in enhanced malignant cell death [68]. Our results are in line with these findings, showing that an MRT-dependent increase in total number of tumour-infiltrating CTLs correlated with melanoma regression. In addition, cell death related genes, including CTL-induced cell death genes, were found to increase and the TUNEL assay confirmed experimentally the increased cell death rates in MRT-treated melanomas.

CTLs are not only present and functional within the melanomas, but we also demonstrated that they are indispensable to the remarkable MRT-induced tumour regression exhibited in previous preclinical melanoma studies. We hypothesize that this potent anti-tumour immune response mediated by CTLs is the result of the unique features related to the radiation dose heterogenicity of MRT comprising supraclinical peak dose regions (400 Gy), in which there is prominent immunogenic cell death following extensive DNA damage. Immunogenic cell death leads to production of damage-associated molecular patterns (DAMPs) in the tumours. DAMPs are known to promote T cell activation and

recruitment of myeloid cells [69–71]. In our case, MRT specifically boosts the cytotoxic activity of CD8⁺ T cells and this results in tumour shrinkage. Expression of CCL3 and CCL2 (also known as MIP1-α and MCP1 respectively) within the tumour microenvironment shows that signals involved in monocyte recruitment (possible to be differentiated into APCs in tumour tissues) are triggered as early as 2 days post-MRT. Higher protein levels of CCL2 in mouse melanomas at 5 days post-MRT were also underlined by a previously published bioplex analysis [12]. High plasma levels of CCL2, creating a systemic inflammatory state, are already known to have a crucial role in inducing abscopal DNA damage in tissues distant from those bearing melanoma even without the application of RT [72]. CCL3 chemokine transcript, found to be increased at 7 days post-MRT, is known to be associated with the recruitment of macrophages and DCs (APCs) but also to mediate interactions between CD4⁺ T helper cells, DCs and naïve CD8⁺ T cells [73]. The CCL4 chemokine has long been known as a lymphocyte chemoattractant, specifically inducing the transendothelial migration of T lymphocytes [74]. CCL3 and CCL4 (also known as MIP1-B) were previously investigated at the protein level within the melanoma microenvironment and found to be elevated as early as 5 days post-MRT [12]. With our ligand-receptor analysis it was predicted that both CCL3 and CCL4 would preferentially bind to the CCR1 receptor, whose gene expression was also significantly increased in tumours 7 days post-MRT. Additional experiments depleting other types of immune subpopulations (such as CD4⁺ T cells, DCs, etc.) will provide further insight into the immune response mechanisms following MRT. Our data from the GO enrichment analysis suggest an involvement of membrane rafts in the melanoma response to MRT. Membrane rafts are lipid clusters in the cell membrane controlled by interactions with cytoskeletal proteins [75] and are also known to play an essential role in TCR signalling, and therefore, in several aspects of T cell activation [52]. Membrane raft clustering is also involved in the activity of CD28, the T cell coreceptor to TCR, that binds CD80 and CD86<sup>76</sup>, molecules whose
transcripts where found specifically overexpressed after MRT in our analysis (Supplementary Table S1).

Unfortunately, after a single fraction of MRT this potent CTL response may not last longer than 7 days. In fact, levels of Tregs are higher in the MRT treated tumours compared to the uniform RT-treated ones (although still lower than what was found in the unirradiated CTR group) and CTL exhaustion markers are high. The increase of Tregs overtime along with tumour progression (peak at day 7) found in the unirradiated CTR group is in line with previously published data in a preclinical B16-F10 melanoma model [76], where this increase was shown to correlate with dysfunctional CD8⁺ T cell infiltration displaying poor effector activity. In the case of a strong anti-tumour response Tregs are the brake system that comes into play to avoid an “exaggerated” immune response [77], while CTLs are inevitably getting exhausted after prolonged exposure to antigenic stimulation [78]. Although these are both immune suppressive mechanisms and eventually promote tumour progression, they are also well-known targets for immunotherapies, opening possibilities for combinatorial MRT treatment.

## 4.2. Markers predicting success of MRT treatment

This study clearly underlined that the increase of CD8⁺ T cells in

melanomas can be exploited as a marker to assess MRT efficacy. Indeed, their presence in tumour tissues is already considered a good prognostic factor for certain cancer types, including melanoma [79]. At the same time, an increase in tumour infiltrating lymphocytes is a good prognostic marker for response to immune checkpoint inhibitors (ICI) [80], which could be a suitable option to combine with MRT to further enhance treatment response, as underlined in our promising preclinical data. In our case, a combination of αCTLA-4 (the human equivalent of Ipilimumab) and αPD-1 (the human equivalent of Nivolumab) is what showed a synergistic effect with MRT. We are confident that the prominent enhancement of anti-tumour immune cell recruitment would also benefit “cold tumours”, those with few infiltrating T cells, by shifting their phenotype towards a “hot”, immunogenic one, with a higher content of T cells. Further preclinical studies should be carried out in a variety of tumour models to confirm the reproducibility of these MRT-induced immune effects. Promising results about the MRT-induced anti-tumour responses in murine mammary carcinoma (historically considered a “cold” tumour) came from Yang and colleagues [81] as they found evidence of increased T cells after MRT in comparison to a uniform RT. Similarities between our results with other preclinical studies employing different types of SFRT and immune responses are also encouraging for clinical translation. In these studies wider beams

<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Graphical abstract-MRT as a potential tool for in situ vaccination of melanoma. Following MRT treatment of melanoma, a cascade of events resembling in situ vaccination unfolds. Post-MRT, there is a significant increase in the production of cytokines, such as CCL3, CCL4 and CCL5, within the tumour microenvironment. Simultaneously, an increase in double positive MHC-II+/CD8+ activated antigen-presenting cells (APCs) is observed in the locoregional cervical lymph nodes (LNs), possibly relating to the activation and migration of CD8+ T cells. By day 7 post-MRT, there is an increase in MHC-II-expressing APCs within the melanomas. Additionally, CD8+ T cells infiltrate the tumour site and are activated via MHC-I antigen presentation, along with co-stimulation through CD28-CD80 ligand receptor interaction, which was predicted by the ligand-receptor analysis of the transcriptomic data. Upregulation of genes involved in T cell activation and proliferation was demonstrated by the transcriptomic analysis, and this could further lead to cytokine production, cytotoxic function, and memory formation. Ultimately, this process leads to tumour cell death induction, underlined by the upregulation of genes associated with CD8+ cytotoxic T cell-induced cell death and subsequent tumour shrinkage post-MRT. At the conclusion of the longitudinal survival experiment, the metastatic score observed in the tumour draining LNs for each mouse shows a lower grade associated with a longer survival time after MRT. This is in contrast with the rest of the experimental groups where the metastatic score is higher in relation to a survival time that is more than 3 times shorter compared to the one of the MRT group. As a result, MRT treatment in this melanoma model mirrors the efficacy of in situ vaccination, not only inducing tumour cell death at the primary site but also reducing metastasis in the locoregional LNs, via a series of anti-tumour immune responses involving CTLs as the main mediators. The illustration was created with BioRender.com.</figcaption></figure>
and consequently lower radiation peak doses were employed [82,83].

Other markers predicting a positive response to treatment after MRT emerging from this study are the chemokines Ccl3, Ccl4 and the intercellular adhesion molecule 1. ICAM1 is also associated with inflammation post vascular damage specifically induced by the vascular endothelium [84], and this might connect also to the MRT-induced selective tumour vascular disruption mechanisms [15].

Further emerging markers encoding for surface receptors to be exploited as indicators of MRT efficacy are Cd28 and Cd80.

### 4.3. MRT as potential new tool for melanoma in situ vaccination

All things considered, MRT proves to be an effective tool for inducing a "sterile" *in situ* vaccination, as summarized in the graphical abstract presented in Fig. 7. The abundant DNA damage justifies the presence of subsequent immunogenic cell death and the activation of a downstream anti-tumour immune response. We hypothesize that supraclinical radiation doses within the peak microbeam regions are also able to trigger an enhanced release and presentation of TAs. This was observed as an augmentation of MHC-I and MHC-II complexes post-MRT. MHC-I is present mostly on tumour cells and MHC class II on B cells, macrophages and DCs and our analyses suggested an abundance of all these immune cell populations within the tumours post-MRT.

We also found evidence of activation of the cGAS/STING pathway classically related to late abscopal effects. However, we also saw the overexpression of transcript for the DNA exonuclease TREX1, whose activation limits the STING-induced abscopal effects [47]. In our case, given the potent CTL response observed, we hypothesize that the action of TREX1 is probably insufficient to limit the anti-tumour effects.

*In situ* vaccination requires antigen-loaded DCs to be transferred to secondary lymphoid tissue and presentation or cross-presentation to antigen-specific T cells [19]. Indeed, in our case we saw in the untreated out-of-field cervical LNs an increase in MHC-II/CD86 double positive cells at 5 days post-MRT. These DCs would activate T cell priming. In this regard, we showed the presence of CD8+/CD62L+ cells in tumour draining LNs 2 days before we observed the peak of CTL infiltration in the MRT-treated primary melanomas. We suggest that these cells in the LNs might be involved in mechanisms preventing the formation of metastasis following MRT treatment. Activation of the CD28/CD80 axis, overexpression of Tem and Tcm markers within the tumour microenvironment, as well as the presence of B cells, suggest the development of T cell memory.

To use MRT as tool for *in situ* vaccination could also have economic advantages for two reasons: (i) delivering MRT has relatively low costs and we do not expect the need for patients to receive daily fractions but ideally to undergo MRT weekly [14]; (ii) *in situ* vaccinations exploit the available TAs avoiding additional costs for identification, isolation, and *in vitro* processing of one specific tumour antigen that is commonly needed to generate a cancer vaccine.

### 4.4. Future perspective towards clinical use

Keeping in mind that we obtained anti-tumour mechanisms resembling tumour *in situ* vaccination employing only a single fraction of MRT and the synergistic effect with a single fraction MRT combined with ICIs, suggests this technique has a great potential to be used as metronomic treatment over a few weeks or with the right combinatorial treatment approach. Combinatory treatments, such as ICIs, could enhance immunogenic cell death and/or evasion of the tumour cells from the tumour immune surveillance mechanisms. We already know that repeating a second MRT at 10 days after the first irradiation resulted in 25 % complete melanoma remission and there are indications that the second fraction further boosts the immune response explaining the efficacy of the fractionated treatment [14].

Although 3rd generation synchrotrons are the most favourable radiation sources capable of providing the optimal beam geometry,

energy, and dose rate characteristics for MRT studies and implementation, the need for more compact and less expensive sources has driven research towards alternative solutions that can be considered a reasonable compromise in terms of performance. High-power rotating-anode X-ray tubes, designed to deliver FLASH radiation (dose rate of about 100 Gy/s) and potentially suitable for the installation of a colli-mator to obtain SFRT, are commercially available (Xstrahl, Xstrahl_FLASH_MKGLSBFLASHP V1.0.pdf). Thanks to the compact dimensions of this type of system, it has been installed in various laboratories worldwide. In addition, line focus X-ray tube technology is being developed at the Technical University of Munich [85] with promising radiation characteristics that could meet the requirements of MRT for clinical applications.

Of extreme importance for future clinical translation would be to underline the minimum peak and valley doses necessary to elicit this increase in CTLs and related systemic effects. We might be able to achieve tumour *in situ* vaccination with a peak dose lower than 400 Gy, building an easier path for the employment of compact radiation sources, to benefit as many cancer patients as possible, all over the world.

### CRediT authorship contribution statement

**Verdiana Trappetti:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Cristian Fernández-Palomomo:** Writing – review & editing, Validation, Methodology, Investigation, Funding acquisition, Conceptualization. **Prateek Arora:** Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Resources, Methodology, Investigation, Formal analysis, Data curation. **Marine Potez:** Writing – review & editing, Methodology, Investigation. **Paolo Pelli-cioli:** Writing – review & editing, Writing – original draft, Software, Resources, Methodology, Investigation, Formal analysis. **Jennifer Faz-zari:** Writing – review & editing, Writing – original draft, Methodology, Investigation. **Nahoko Shintani:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis. **Ismael Sanchez-Gonzalez:** Investigation, Methodology, Writing – review & editing. **Cheuk Ting Wu:** Writing – review & editing, Visualization. **Bettina de Breuyn Dietler:** Methodology, Investigation. **Nadia Mercader-Huber:** Writing – review & editing, Supervision. **Olga A. Martin:** Writing – review & editing, Supervision, Investigation, Formal analysis. **Stephan von Gunten:** Writing – review & editing, Conceptualization. **Vladislav Volarevic:** Writing – review & editing, Supervision, Conceptualization. **Valentin Djonov:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Investigation, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgement

This research was funded by the Swiss National Foundation (Grant nr. 310030-220064/1), Bernese Cancer Liga (Grant number 190), European Union Grant BG-RRP-2.004-0004-C01 and Scientific Association of Swiss Radiation Oncology (SASRO) research grant 2023 awarded to Verdiana Trappetti. We thank Canopy Biosciences (a Bruker company) for provision of the Nanostring nCounter assay and related services.

We thank Dr David Repaz Pernaut and Ms Nivia Barontini for their assistance during the revision of the manuscript.

We acknowledge the European Synchrotron Radiation Facility (ESRF) for provision of beam time, the staff of the Biomedical Beamline ID17 and the staff of the animal facility at the ESRF. We wish dedicate
this work to the memory of Dr Elke Bräuer-Krisch, enthusiastic scientist and great MRT-promoter [86].

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.canlet.2024.217326.

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