Radiotherapy (RT) combined with immunotherapy is promising; however, the immune response signature in the clinical setting after RT remains unclear. Here, by integrative spatial and single-cell analyses using multiplex immunostaining (CODEX), spatial transcriptome (VISIUM), and single-cell RNA sequencing, we substantiated the infiltration of immune cells into tumors with dynamic changes in immunostimulatory and immunosuppressive gene expression after RT. In addition, our comprehensive analysis uncovered time- and cell type–dependent alterations in the gene expression profile after RT. Furthermore, myeloid cells showed prominent up-regulation of immune response–associated genes after RT. Notably, a subset of infiltrating tumor-associated myeloid cells showing PD-L1 positivity exhibited significant up-regulation of immunostimulatory (HMGB1 and ISG15), immunosuppressive (SIRPA and IDO1), and protumor genes (CXCL8, CCL3, IL-6, and IL-1AB), which can be targets of immunotherapy in combination with PD-L1. These datasets will provide information on the RT-induced gene signature to seek an appropriate target for personalized immunotherapy combined with RT and guide the timing of combination therapy.

Background: The standard treatment for unresectable non-small cell lung cancer (NSCLC) is anti-PD-L1 therapy combined with chemoradiotherapy (anti-PD-L1-CRT). Although some patients achieve complete cancer eradication and cure, more than half of patients retain persistent cancer cells. Our research aimed to unravel the nuanced mechanisms involved in both immune attack and evasion induced by anti-PD-L1-CRT with single cell spatial transcriptome. Methods: Xenium is a cutting-edge single-cell spatial analysis tool that enables pathology-based and single-cell analyses while preserving spatial information. In our study, we used Xenium to identify the tumour microenvironment (TME), immune dynamics, and residual cancer cells at the single-cell level following treatment with anti-PD-L1-CRT. Results: Posttreatment alterations included a significant increase in CXCL9+ cells and CXCL13 + T cells, particularly around tumour cells. Additionally, we discovered that CXCL13 + T cells directly impact cancer cells in the posttreatment environment. Moreover, we identified clusters of immune-cold cancer cells posttreatment, revealing their activation of DNA repair pathways and high proliferative capacity. The novel spatial analysis tool Xenium enabled identification of the immune environment at the single-cell level following treatment with anti-PD-L1-CRT, elucidating its characteristics. Conclusions: These findings suggest potential advancements in developing new treatments to improve posttreatment immune responses and address resistance challenges.

Bone-modifying agents (BMAs) have been widely used to reduce skeletal-related events, including pathological fractures. Herein, we aimed to clarify the incidence of pathological fractures caused by high-risk femoral bone metastases after palliative radiotherapy (RT) in the BMA era and evaluate the necessity of prophylactic surgical stabilization. We assessed 90 patients with high-risk femoral bone metastases, indicated by Mirels’ scores ≥ 8, without pathological fractures and surgical fixations, who received palliative RT at our institution between January 2009 and December 2018. Pathological fracture incidence was analyzed using the Kaplan–Meier method and was 22.8% and 31.0% at 2 and 6 months, respectively. Pathological fractures were caused by 17 of 65 lesions (26.2%) and 9 of 25 lesions (36.0%) in patients who received BMAs and those who did not, respectively (p = 0.44). Additionally, 17 of 42 lesions (40.5%) and 9 of 48 lesions (18.8%) with axial cortical involvement ≥30 and <30 mm, respectively, caused pathological fractures (p = 0.02). The incidence of pathological fractures was high among patients with high-risk femoral bone metastases treated with palliative RT, particularly those with axial cortical involvement ≥30 mm. Therefore, aggressive indications for prophylactic surgical stabilization are warranted for high-risk femoral metastases despite BMA administration.