Antitumor Effects of PD-1 Blockade Combined with Mild Hyperthermia in a Murine Osteosarcoma Model

TL;DR

Combining PD-1 blockade with mild hyperthermia improves tumor suppression and immune response in a mouse model of osteosarcoma.

Contribution

Demonstrates that mild hyperthermia enhances the antitumor effects of PD-1 blockade in osteosarcoma through immune modulation.

Findings

Combination therapy significantly suppressed tumor growth and reduced lung metastasis in mice.

Survival rates improved significantly in mice receiving combination therapy.

Immune profiling showed increased CD8+ T cells and pro-inflammatory macrophage profiles.

Abstract

Background: Osteosarcoma remains largely refractory to immune checkpoint inhibitor (ICI) monotherapy, and strategies to modulate the tumor immune microenvironment are being actively explored. Mild hyperthermia has been reported to influence antitumor immune responses; however, its impact in combination with PD-1 blockade in osteosarcoma has not been well characterized. Methods: Murine LM8 osteosarcoma cells were subjected to mild thermal stimulation, and changes in PD-L1 expression were evaluated. LM8-bearing mice were treated with mild hyperthermia, anti-PD-1 antibody, or their combination. Tumor growth, lung metastasis, and survival were assessed. Tumor-infiltrating immune cells were profiled using single-cell RNA sequencing to descriptively characterize immune-associated transcriptional features under each treatment condition. Results: Mild thermal stimulation (42 °C, 30 min) increased PD-L1 expression in LM8 cells in vitro. In vivo, combination therapy significantly suppressed primary tumor growth compared with control (χ2 = 29.75, p = 1.6 × 10−6) and reduced lung metastasis burden, with a significant decrease in metastatic nodules (p < 0.01). Kaplan–Meier analysis demonstrated a significant survival benefit in the combination group (log-rank p < 0.001). Single-cell RNA sequencing revealed an increased proportion of CD8+ T cells with reduced exhaustion-associated gene expression and a shift toward pro-inflammatory (M1-like) macrophage transcriptional profiles. Conclusions: PD-1 blockade combined with mild hyperthermia was associated with enhanced antitumor efficacy and immune-associated transcriptional remodeling in a murine osteosarcoma model, supporting further preclinical evaluation of this combination strategy.

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1. Introduction

Osteosarcoma (OS) represents the most frequent primary malignant bone tumor among pediatric and adolescent patients [1]. Globally, OS accounts for approximately 3–5 cases per million individuals per year, with a bimodal age distribution affecting adolescents and older adults. Current standard treatment for OS consists of wide surgical resection combined with multi-agent chemotherapy, typically including methotrexate, doxorubicin, and cisplatin. Advances in systemic chemotherapy have enhanced the long-term prognosis of high-grade OS patients. A previous study demonstrated that the combination of surgery with modern, dose-intensive, multi-agent chemotherapy achieved approximately 70% 5-year recurrence-free survival in patients with strictly localized, non-metastatic disease [2]. Nevertheless, subsequent progress in treatment modalities has been limited since 2000. OS treatment remains challenging due to the elevated recurrence rate and poor prognosis, with overall survival limited to approximately 20% in patients who present with metastasis at diagnosis [3]. Despite aggressive multimodal treatment, survival outcomes for metastatic or recurrent disease remain poor worldwide [4].

Recently, immune checkpoint inhibitors (ICIs) have demonstrated clinical benefit across multiple cancer types, including malignant melanoma, non-small-cell lung cancer, and renal cell carcinoma [5,6,7]. Multiple clinical trials have assessed the therapeutic potential of ICIs in OS. For instance, Davis et al. reported the clinical outcomes of nivolumab treatment in 13 patients with OS and showed that the drug was well-tolerated by children; however, no therapeutic effect was observed [8]. Further, Le Cesne et al. reported outcomes in 17 patients with advanced OS who received pembrolizumab, only 1 patient exhibited partial response to treatment [9]. The therapeutic effectiveness of ICI-based cancer immunotherapy is strongly influenced by PD-L1 expression within tumor cells and the degree of tumor-infiltrating lymphocyte (TIL) recruitment [10]. Accordingly, previous research reported that PD-L1 expression was detected in over 80% of OS patient samples, with high levels in 24% of samples, and that PD-L1 expression correlates with the presence of TILs [11]. However, clinical studies have demonstrated that PD-1 blockade as monotherapy shows limited antitumor activity in advanced OS [12]. Collectively, these results suggest that an immunosuppressive microenvironment exists in OS and that ICI monotherapy might be inadequate to treat high-grade advanced OS. In addition to surgery and multi-agent chemotherapy, emerging strategies such as targeted therapy, nanoparticle-based drug delivery, and immunomodulatory approaches have been explored for OS. Recent nanotechnology-based approaches, including fungal-derived chitosan nanoparticles, have demonstrated multi-target molecular interactions in OS cells, highlighting ongoing efforts to overcome therapeutic resistance [13].

Thermal-based cancer treatments encompass a broad range of modalities, including ablative approaches such as radiofrequency ablation (RFA) and microwave ablation (MWA), which induce rapid tumor necrosis through high-temperature exposure. These techniques are widely used in clinical oncology for local tumor control; however, their application is generally limited to focal ablation and is not primarily intended to modulate systemic antitumor immunity [14]. In contrast, mild hyperthermia directly damages tumor cells by damaging cell structures, inhibiting DNA repair, and inducing apoptosis [15,16,17]. This process also modifies the tumor microenvironment by activating the immune system [18], increasing blood flow, and improving drug delivery [19]. Consequently, hyperthermia monotherapy has been shown to have clinical tumor-shrinking effects. Furthermore, several recent studies have reported that the therapeutic efficacy of hyperthermia is enhanced when it is combined with chemotherapy or radiotherapy [20,21]. Thus, hyperthermia combined with chemotherapy or radiotherapy provides a clinically feasible therapeutic approach for solid tumors, including cervical [22], bladder [23], rectal [24], breast [25], and head and neck cancers [26]. Moreover, the integration of regional mild hyperthermia into neoadjuvant chemotherapy regimens in patients with localized high-risk soft tissue sarcomas has been reported to prolong survival and local progression-free survival [27]. In this respect, Nakano et al. reported that hyperthermic isolated regional perfusion in the lower extremities could help control OS growth locally [28]. Beyond its direct cytotoxic and vascular effects, mild hyperthermia has increasingly been recognized for its potential to modulate antitumor immunity, providing a rationale for combination with immunotherapeutic approaches.

Although ICI monotherapy is not expected to provide sufficient therapeutic effects against OS, we hypothesized that combinatorial therapy with ICIs and mild hyperthermia could modulate the tumor microenvironment and augment treatment effectiveness. Combinatorial treatment with ICIs and hyperthermia has been documented in other malignancies [29]; however, the same strategy has not been used to manage OS. Accordingly, we examined the therapeutic efficacy of combination therapy with anti-PD-1 and mild hyperthermia in OS and investigated the underlying mechanisms.

2. Materials and Methods

2.1. Cell Lines

In vitro experiments were performed using LM8 cells to assess tumor cell-intrinsic responses to mild thermal stimulation, such as changes in proliferation and PD-L1 expression. While additional OS cell lines could provide broader validation, the present study focused on LM8 cells to maintain consistency with the in vivo model. The mouse OS cell line LM8 was obtained from the RIKEN BioResource Research Center (Tsukuba, Japan). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; FUJIFILIM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and in a humidified atmosphere at 37 °C with 5% CO_2_.

2.2. Animals (Mice) and Ethics Statement

The LM8 murine OS model was selected because it is syngeneic to immunocompetent C3H mice and reliably recapitulates aggressive tumor growth and spontaneous lung metastasis, making it suitable for evaluating tumor–immune interactions under immune checkpoint blockade. Although other in vitro and in vivo OS models exist, including human xenograft and genetically engineered mouse models, the LM8 system allows assessment of immune-mediated effects in an intact immune environment.

Four- to five-week-old female C3H/HeNSlc (C3H) mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). All animals were housed in a temperature- and humidity-controlled specific pathogen–free (SPF) facility (22 ± 2 °C, 40–60% humidity) under a 12 h light/12 h dark cycle with ad libitum access to food and water. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, the ARRIVE guidelines 2.0 [30] and the AVMA Guidelines for the Euthanasia of Animals (2020) [31]. All procedures were approved by the Animal Experiment Committee of the University of Fukui (approval number R07029).

Mice were monitored at least once daily for signs of pain or distress, including reduced spontaneous activity, abnormal posture, decreased food and water intake, poor grooming or ruffled fur, impaired limb use related to tumor growth, and other abnormal behaviors. Body weight was not recorded as a welfare indicator because tumor burden substantially influences total body weight and may misrepresent systemic condition in subcutaneous tumor-bearing models. Instead, clinical condition, food intake, fur appearance, limb use, and overall activity were used as the primary welfare parameters.

Humane endpoints were predefined as follows: (1) a maximum tumor diameter of 20 mm, (2) evidence of tumor ulceration, self-destruction, or active bleeding, or (3) marked reduction in spontaneous activity indicating distress. None of the mice developed tumor ulceration during the study. If any humane endpoint was reached, mice were immediately euthanized.

During experimental procedures, anesthesia was administered as required; however, analgesics were not administered because analgesic agents can alter antitumor immune responses and interfere with the interpretation of tumor–immune interactions in this immunotherapy model. The absence of pain-related behaviors, such as persistent guarding or severely impaired limb use, was confirmed during daily monitoring.

Mice were humanely euthanized under deep isoflurane anesthesia (3–5% via open-drop induction), followed by cervical dislocation to ensure death, in accordance with AVMA guidelines for the Euthanasia of Animals.

2.3. Thermal Stimulation for Cultured Cells

LM8 cells were seeded into culture flasks and incubated at 37 °C in a humidified atmosphere containing 5% CO_2_. When cultures reached half confluence, the medium was exchanged for fresh medium preheated to 42 °C to induce thermal stimulation. The flasks were subsequently immersed in a thermostatically controlled circulating water bath adjusted to 42 °C for continued thermal stimulation for 30 min. Subsequently, the medium was exchanged for fresh medium at 37 °C, and the cultures were maintained at 37 °C in a 5% CO_2_ atmosphere.

2.4. Cell Proliferation Analysis

LM8 cells were plated in 6-well culture plates (2 × 10^4^ cells per well). The cells were then thermally stimulated at 42 °C for 30 min and cultured in an incubator. Cells were counted at intervals of 1, 2, 5, and 6 d after cell seeding using the Countess II automatic cell counter (Thermo Fisher Scientific, Waltham, MA, USA) to assess the effect of thermal stimulation on cellular proliferation.

2.5. Western Blotting

Western blotting was carried out following established protocols [32] with minor modifications. PD-L1 expression was analyzed by preparing whole-cell lysates in RIPA buffer (#08714; Nacalai Tesque, Inc., Kyoto, Japan) with added protease and phosphatase inhibitors. Lysates were maintained on ice for 30 min and then centrifuged at 15,000× g for 10 min at 4 °C to remove insoluble material. Protein levels in the supernatants were quantified by the Bradford assay (Bio-Rad Laboratories GmbH, Munich, Germany). Protein samples (20 μg) were resolved by SDS-PAGE on 10% or 10–20% polyacrylamide gradient gels (ATTO, Tokyo, Japan) at a constant current of 20 mA for 80 min, and transferred onto PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA or Immobilon-E, Millipore, MA, USA). Blocking was performed using EzBlock Chemi (ATTO, Tokyo, Japan) supplemented with 0.5% goat serum for 1 h at room temperature (RT). Subsequently, cells were maintained at 4 °C overnight with the anti-PD-L1 antibody (1:1000; #BE0101; Bio X Cell, Lebanon, NH, USA). β-actin (β-Actin (8H10D10) mAb; 1:1000 dilution; #3700; Cell Signaling Technology, Danvers, MA, USA,) was used as the loading control. Following washes, the membranes were exposed to an HRP-conjugated secondary antibody (1:10,000; anti-rat IgG, #112-035-167; Jackson ImmunoResearch Inc., West Grove, PA, USA) for 1 h at RT. Protein bands were revealed by the ECL Pro chemiluminescent substrate (PerkinElmer, Waltham, MA, USA) and captured with a Fusion FX imaging system (Vilber, FUSION model No. 2). With respect to the tumor tissues, they were harvested from mice, minced with scissors, and homogenized using a tissue homogenizer. The homogenates were lysed in RIPA buffer (Nacalai Tesque) on ice for 1 h; this was followed by protein extraction and Western blotting, as described above. For in vitro Western blotting, protein lysates were prepared from pooled cell cultures (three wells per condition). For in vivo Western blotting, tumors from three mice per group were pooled prior to lysis to obtain a representative sample while conserving tissue for additional analyses. Therefore, Western blot data are presented as qualitative, representative results rather than quantitatively analyzed biological replicates.

2.6. Hyperthermic Treatment for Mice

Egg-shaped plastic capsules were prepared to restrain mice during mild hyperthermia treatment (OS implanted around the knee joint). The capsules measured 9 cm × 5 cm and had an air hole at the top (Figure 1A,B). Two holes (1.5 cm in diameter) were created at the lower portion of the capsule to allow both hind limbs distal to the inguinal region to exit. The tumor-bearing hind limb was immobilized on a plastic rod connected to the capsule. After the mouse was placed inside the capsule, the lower openings were sealed with oil clay to prevent the ingression of warm water. Mild hyperthermia was induced by immersing the capsule in a thermostatically controlled water bath maintained at 42.0 °C. Hyperthermia was applied for 30 min per session, once every other day, for a total of four treatments. Water bath temperature was continuously monitored and maintained throughout each session. Intratumoral temperature was not directly measured in this study. Analgesic agents were not administered because of their potential immunomodulatory effects.

2.7. Treatment Protocol and Assessment of Tumor Volume and Lung Metastases in the Mice OS Model

Briefly, subcutaneous implantation of 1.0 × 10^7^ LM8 cells was performed in the dorsal region of C3H mice (Figure 1E). Two weeks after the initial injection, the tumor on the back was excised, cut into 3 mm-sized pieces, and transplanted into the lower limb of another mouse. Twelve days after tumor transplantation, mice with established tumors were distributed randomly to four groups, and treatment was initiated. Mice assigned to the control group (Ctrl) received an intraperitoneal injection of 100 μL of PBS (Thermo Fisher Scientific, Waltham, MA, USA). Mice in the PD-1 antibody group (PD1) were administered anti-PD-1 antibody (clone RMP1-14, rat IgG2a, κ; Bio X Cell, West Lebanon, NH, USA; BE0146; RRID: AB_10949053) at a dose of 200 µg per mouse, administered intraperitoneally four times per week. Mice in the hyperthermia group (HT) were anesthetized by isoflurane inhalation 12 days after tumor transplantation and placed in plastic capsules.

Hyperthermia was performed by immersing the tumor-bearing limb in a thermostatically controlled circulating water bath maintained at 42 °C for 30 min. The water temperature was continuously monitored to ensure stability, and pilot testing confirmed that it remained constant throughout each session. Treatments were administered on days 12, 14, 16, and 18 after tumor implantation. Mice were anesthetized with isoflurane during placement into the HT device. To minimize factors related to handling or restraint in between-group differences, control (Ctrl) mice and anti-PD-1 mice underwent sham hyperthermia treatment by placing them in similar capsules within a water bath maintained at room temperature. Mice in the combination therapy group (Comb) received treatment with both mild hyperthermia and anti-PD-1 antibody. Anti-PD-1 antibody was delivered via intraperitoneal injection four times weekly, immediately after hyperthermia. Tumor diameters were measured using Vernier calipers. Tumor volume was assessed according to the formula: (length × width^2^)/2. Tumor volumes were compared statistically as described in the Statistical Analyses section. Euthanasia was performed 30 days following tumor transplantation, and both lungs were harvested to assess lung metastases. Harvested lung tissues were fixed in paraformaldehyde, paraffin-embedded, sectioned, deparaffinized, and subjected to hematoxylin and eosin (H&E) staining, followed by microscopic evaluation. The number and size of the lung metastases were measured using the largest cross-sectional cut surfaces of the lungs (n = 5).

2.8. Immunohistochemistry

Histopathological examination was performed on resected tissue specimens fixed in 4% paraformaldehyde at room temperature for 24 h. Paraffin-embedded tissues were sectioned at 4 µm, deparaffinized, and stained with H&E or processed for immunohistochemistry. For PD-L1 immunohistochemistry, sections were incubated with an anti-PD-L1 antibody (#BE0101; Bio X Cell, Lebanon, NH, USA) at a dilution of 1:50, according to the manufacturer’s protocol. The same antibody was also used for Western blot analysis.

2.9. Statistical Analyses

Descriptive statistics are presented as mean ± SD. An unpaired two-tailed Welch’s t-test was used for comparisons between two groups, and one-way ANOVA with Tukey’s post hoc test was used for multiple group comparisons. Tumor growth data were analyzed using a linear mixed-effects model (LMM) to account for repeated measurements within animals, with Treatment as a between-subject factor, Time as a within-subject factor, and Mouse ID as a random effect. Fixed effects were evaluated using likelihood-ratio tests comparing full and reduced models, with post hoc multiple comparisons adjusted using the Holm–Šidák method when appropriate. Survival was evaluated using Kaplan–Meier survival curves, and intergroup differences were assessed using the log-rank test. ANOVA, t-tests, Kaplan–Meier analysis, and log-rank tests were performed using BellCurve for Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan), while the LMM was conducted using Python (version 3.x; statsmodels package). Significance levels are indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001. Western blot analyses were performed using pooled samples and were interpreted descriptively without statistical testing.

2.10. Single-Cell RNA Sequencing and Data Bioinformatic Analysis

Tumor-bearing mice received treatment beginning on day 12 after tumor implantation. Tumor samples for scRNA-seq were harvested on day 21 from mice in the vehicle control, anti-PD-1, and combination (hyperthermia + anti-PD-1) groups. All tumors were collected on the same day. Tumor-infiltrating immune cells were isolated from three independent tumors (n = 3 mice) in each experimental group. All tumor tissues were mechanically minced into small fragments on ice. The tissue fragments were enzymatically digested using Liberase™ TH Research Grade (0.2 Wünsch units/mL; Sigma-Aldrich, St. Louis, MO, USA; #5401135001)—diluted in RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA; Cat# 11875093)—at 37 °C for 1 h, with gentle agitation.

Subsequently, the suspension was filtered with a 100-µm cell strainer to remove the debris. To remove extracellular DNA, the filtered cells were treated with DNase I (1 mg/mL; Sigma-Aldrich) on ice for 5 min. The suspension was subsequently processed through a 40-µm cell strainer and rinsed twice with PBS supplemented with 0.04% BSA. The resulting single-cell suspension was subjected to density gradient centrifugation using a 20%/65% Percoll (Cytiva, Marlborough, MA, USA) discontinuous gradient to remove dead cells and debris. Centrifugation was performed using a swinging-bucket rotor at 1000× g for 20 min at RT with the deceleration setting set to “SLOW” to minimize disturbance of the gradient layers.

Enriched live cells were collected from the interface, washed, and resuspended in PBS supplemented 0.04% BSA. Cell viability was evaluated using the trypan blue exclusion assay. After mechanical and enzymatic dissociation, cells from the three tumors were pooled prior to library preparation and sequencing.

Subsequently, approximately 5000 cells with a viability of 71–76% were loaded onto a Chromium Controller (10x Genomics, Pleasanton, CA, USA) for Gel Bead-in-Emulsion (GEM) generation, targeting the capture of ~3000 cells per droplet together with reverse transcription reagents and uniquely barcoded gel beads. Reverse transcription within each emulsion enabled cDNA labeling with cell-specific barcodes. Amplification of cDNA and library construction for single-cell gene expression profiling were performed using the Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (10x Genomics). Libraries were quantified with a Qubit dsDNA assay (Thermo Fisher Scientific) and TapeStation D5000 ScreenTape (Agilent Technologies, Santa Clara, CA, USA) and subsequently sequenced on a DNBSEQ platform (MGI Tech Co., Shenzhen, China) with a configuration of Read1: 28 bp and Read2: 91 bp, yielding ~350 million paired-end reads per sample. Raw sequencing data were processed using the Cell Ranger pipeline (v6.1.2; 10x Genomics) for read alignment, gene expression quantification, cell calling, and clustering analysis. Cells from the three samples were jointly embedded in a common UMAP space, and treatment group labels were used to visualize the distribution of cells from each condition. Quality control, normalization, and subsequent analyses were conducted with the Seurat R package (v5.1.0). Cells with high mitochondrial gene content (>10%) or fewer than 200 detected genes were excluded. Data integration across samples was performed using the Harmony R package (v1.2.0) to correct for batch effects while preserving biological variation. For dimensionality reduction and visualization, Uniform Manifold Approximation and Projection (UMAP) was employed. Annotation of cell types was guided by the expression signatures of canonical marker genes. T cells and macrophages were grouped into subsets and re-clustered for further analysis. T-cell subpopulations were defined based on Cd4 and Cd8a expression. Macrophage polarization states were characterized using established M1 (Il1b, Nos2, and Tnf) and M2 markers (Mrc1, Arg1, and Mgl2). Module scores for the M1 and M2 phenotypes were calculated using the Add-Module-Score Function in Seurat. For comparisons between treatment groups, non-parametric tests were employed, with p < 0.05 regarded as statistically significant.

3. Results

3.1. Mild Hyperthermia Suppresses LM8 Cell Proliferation and Induces PD-L1 Expression In Vitro

To evaluate the direct effects of mild hyperthermia on OS cells, we first examined LM8 cell proliferation and PD-L1 expression in vitro.

First, we examined the effect of thermal stimulation—at 42 °C for 30 min—on LM8 murine OS cell proliferation (Figure 2A). Thermally stimulated cells exhibited a statistically significant but modest reduction in cell proliferation compared with cells without thermal stimulation; however, cell proliferation continued under the conditions tested. Under these experimental conditions, no marked increase in detached or floating cells was observed, and cells largely retained adherent morphology; however, cell death was not quantitatively assessed using dedicated apoptosis or necrosis assays.

Next, in vitro evaluation was conducted to determine how thermal stimulation affects PD-L1 expression. When thermal stimulation was not employed, PD-L1 expression was identified in LM8 cells and remained constant during culture. However, when thermal stimulation was employed, PD-L1 expression increased over time (Figure 2B). The data imply that in vitro thermal stimulation enhances PD-L1 expression, potentially improving the therapeutic impact of immune checkpoint inhibition in vivo.

3.2. Combination Therapy Is Associated with Reduced Tumor Progression and Reduced Lung Metastasis In Vivo

As PD-L1 expression was upregulated in LM8 cells following thermal stimulation, we assessed the impact of hyperthermia, anti-PD-1 antibody, and combinatorial therapy with hyperthermia and anti-PD1 antibody on LM8 cells on LM8-transplanted mice (Figure 3A). Tumor growth was analyzed using a linear mixed-effects model (LMM) with Treatment as a between-subject factor, Time as a within-subject factor, and MouseID as a random effect (Figure 3A). The analysis revealed significant main effects of Treatment (χ^2^ = 29.75, p = 1.6 × 10^−6^) and Time (χ^2^ = 122.65, p = 2.1 × 10^−26^), as well as a significant Treatment × Time interaction (χ^2^ = 19.08, p = 0.0245), indicating that tumor growth trajectories differed among treatment groups over time. Post hoc simple-effects analyses using Tukey’s HSD test demonstrated that tumor volumes in the combination therapy group were significantly smaller than those in the control group at days 19, 26, and 33 (adjusted p < 0.05), whereas no significant differences were detected at day 12. Detailed pairwise comparisons are provided in Supplementary Table S1.

PD-L1 expression following hyperthermia, anti-PD-1 antibody treatment, or combination therapy was examined by Western blotting of tumor samples from LM8-bearing mice (Figure 3B). PD-L1 protein expression following hyperthermia, anti-PD-1 antibody treatment, or combination therapy was examined by western blotting using pooled tumor lysates from LM8-bearing mice (Figure 3B). Western blot analysis provided supportive, qualitative confirmation of PD-L1 protein presence across treatment groups. Western blotting showed increased PD-L1 expression in the Comb and HT groups compared with that in the Ctrl group. Notably, PD-L1 expression was higher in the Comb group than that in the HT and PD-1 groups. Given the pooled nature of the samples, these data were not intended to assess biological variability or statistical significance and are therefore presented for descriptive purposes only. The induction of PD-L1 expression after combinatorial therapy was further confirmed by immunohistochemistry using an anti-PD-L1 antibody (Figure 3C).

Next, we examine the effect of each therapy on lung metastasis and mice survival. Lung metastasis was assessed in mice treated with hyperthermia, anti-PD-1 antibody treatment, or combination therapy. Figure 4A shows the microscopy findings of lung metastases in each group 31 days following tumor transplantation. The size of lung metastases in the HT, PD1, and Comb groups was smaller than that in the Ctrl group. The number of lung metastases was reduced in all groups (PD1, HT, and Comb) and this was significant in the Comb group (p < 0.01) (Figure 4B).

The influence of hyperthermia, anti-PD-1 antibody treatment, and combination therapy on the survival of LM8-transplanted mice were examined (Figure 4C). Kaplan–Meier survival curves indicated a significant survival benefit in the Comb group relative to the monotherapy and control groups (p < 0.001, n = 13).

No overt treatment-related toxicities were observed during the experimental period. Mice receiving combination therapy did not exhibit significant body weight loss, abnormal behavior, or signs of distress compared with control or monotherapy groups. No treatment-related mortality was observed.

3.3. Single-Cell RNA Sequencing Reveals Treatment-Associated Immune Landscape Changes

Single-cell RNA sequencing was performed to assess the effect of combinatorial therapy on the tumor immune microenvironment. Integration of data from all treatment conditions revealed distinct cell populations, including T cells, macrophages, and tumor cells (Figure 5A).

A focused analysis of T-cell populations revealed an altered CD4/CD8 T cell ratio following combinatorial therapy (Figure 5B). In the Comb group, an increase was observed in the proportion of CD8^+^ T cells, accompanied by a relative reduction in CD4^+^ T cells, compared with the Ctrl and PD1 groups (Figure 5C). The untreated control group shown in Figure 5C is presented to provide a descriptive overview of immune cell composition in the tumor microenvironment and was not intended to serve as a quantitative baseline for T-cell subset analysis. Additionally, the analysis of T-cell exhaustion markers (Tox and Lag3) showed that combinatorial therapy reduced the expression of Tox and Lag3 in CD8^+^ T cells (compared to Ctrl and PD1 groups) (Figure 5D), suggesting the alleviation of T-cell exhaustion in the tumor microenvironment. Comparisons of exhaustion-associated gene expression in Figure 5D are restricted to relative differences among treated groups and should not be interpreted as definitive deviations from an untreated baseline state.

Across the treatment conditions, the most notable changes were observed in macrophage cluster distribution, particularly in the Comb group. Macrophage polarization analysis revealed three distinct subpopulations, M1, M2-like, and M2, across all treatment conditions (Figure 6A, upper panels). Notably, the Comb group showed a marked shift in the distribution of these populations compared to the Ctrl and PD1 groups (Figure 6A, lower panels). Quantitative assessment of the polarization scores confirmed that the Comb group exhibited significantly higher M1 scores and lower M2 scores than the other treatment groups (Figure 6B), indicating the reprogramming of macrophages toward a pro-inflammatory phenotype. Protein-level assessment was limited to PD-L1 expression, which was evaluated by immunohistochemistry and Western blotting. Other immune-associated features identified by single-cell RNA sequencing were analyzed at the transcriptomic level and were not independently validated at the protein or functional level.

4. Discussion

In this study, we evaluated the effects of combining PD-1 blockade with mild hyperthermia in a murine OS model, with a focus on tumor outcomes and associated immune-related transcriptional features. Using a combination of in vivo tumor growth and metastasis analyses together with single-cell RNA sequencing, we provide a descriptive characterization of tumor and immune responses under different treatment conditions. We found that the combination of mild hyperthermia and PD-1 blockade was associated with increased CD8^+^ T-cell representation, reduced exhaustion-associated transcriptional features, and a shift toward pro-inflammatory macrophage phenotypes. Rather than proposing a specific mechanistic pathway, this work aims to place OS within the broader context of hyperthermia-based immunomodulation by documenting treatment-associated changes in immune cell composition and gene expression profiles.

The OS cell line LM8 showed only a limited decrease in cell proliferation, even after 30 min of thermal stimulation at 42 °C, without evidence of overt cytotoxicity under the conditions tested (Figure 2A). This may reflect a relative resistance of LM8 cells to mild thermal stimulation, given that the sensitivity to mild thermal stimulation varies among cells [33,34]. Notably, PD-L1 levels were elevated in LM8 cells exposed to thermal stimulation compared with unstimulated cells (Figure 2B). This finding is consistent with previous reports indicating that thermal stimulation induces PD-L1 expression in various tumor cells [35,36]. For example, in a mouse mammary tumor model (MDA-MB-231, MCF-7), raising the temperature from 39 to 44 °C increased PD-L1 expression on tumor cell surfaces. The molecular mechanisms that enhance PD-L1 expression under thermal stimulation are thought to involve heat shock proteins and heat shock factor-mediated pathways [37], as well as NF-κB [38], MAPK, and STAT3 pathways [18]. In the present study, we did not directly investigate the mechanism underlying increased PD-L1 expression in LM8 cells. Pathway-level mechanistic studies, including inhibition or phospho-protein analyses, will be important future directions to elucidate the upstream regulation of PD-L1.

In vivo, combination therapy with anti-PD-1 antibody and mild hyperthermia significantly inhibited tumor growth in LM8-transplanted mice (Figure 3A). Western blotting with pooled samples demonstrated higher PD-L1 expression in the Comb group than in the PD1, HT, and control groups (Figure 3B). The increased induction of PD-L1 protein expression after combination therapy was further confirmed by immunohistochemical analysis using an anti-PD-L1 antibody (Figure 3C). Previous studies have reported modulation of PD-L1 expression after hyperthermia in gastric cancer [35], breast cancer [36], head and neck squamous cell carcinoma [39], hepatocellular carcinoma [40], and non-small cell lung cancer [41]; however, no such study in OS has been available. Therefore, this is, to our knowledge, the first report to describe the antitumor efficacy of combination therapy with anti-PD-1 antibody and mild hyperthermia in OS.

To ensure clinically safe hyperthermia treatment for OS, we employed a relatively mild hyperthermic protocol involving immersion in a 42 °C water bath for 30 min. In the present study, intratumoral temperature was not directly measured, but previous hyperthermia experiments using the same heating protocol as in the present study have been conducted in various tumor models [42,43]. In these studies, intratumoral temperature was measured using a thermocouple (Model 6500; Mallinckrodt Medical, St. Louis, MO, USA). Although it took approximately 3 min for the temperature to reach the target level, the average intratumoral temperature was confirmed to be 42 °C [43]. In contrast, O’Hara et al. [44] reported that, in C3H mammary tumors implanted in the mouse hindlimb and heated via a 42–43 °C water bath, intratumoral temperatures measured with multiple microthermocouples were approximately 0.1 to 0.8 °C lower than the bath temperature. Therefore, we are currently planning to perform intratumoral temperature monitoring using microthermocouples in future experiments.

Tumor-suppressive effects of the combination therapy were observed not only in locally implanted tumors but also in lung metastases. In the Comb group, both the size and number of lung metastases were significantly decreased relative to the other groups (Figure 4A,B). In radiation therapy, abscopal effect is a phenomenon wherein a reduction in the size of lesions outside the radiation field is observed and is thought to be due to the activation of anticancer immunity [45,46]. Recent studies have shown that the combination of radiation therapy and ICIs can enhance the incidence and efficacy of the abscopal effect [47]. Similarly, in our study, mice treated with combination therapy showed improved survival compared with other groups (Figure 4C). This is thought to be due to the suppression of primary tumor growth and the suppression of lung metastases.

Although hyperthermia combined with ICIs has been evaluated in several other tumor models, OS exhibits distinct immunobiological features and is generally considered an immunologically cold tumor. To our knowledge, no prior studies have examined whether hyperthermia can enhance the response to PD-1 blockade in OS or characterized hyperthermia-induced immune remodeling at single-cell resolution. Our single-cell RNA sequencing analysis provides important insights into CD8^+^ T-cell activation, exhaustion-associated gene expression, and macrophage phenotype shifts following hyperthermia treatment. Single-cell gene expression analysis further demonstrated that combination therapy with anti-PD-1 antibody and mild hyperthermia modulated the tumor immune microenvironment to promote antitumor immunity. (Figure 5). The observed increase in CD8^+^ T-cell proportion, together with reduced expression of exhaustion-associated genes, reflects transcriptional features that are consistent with a less exhausted T-cell phenotype.

The control group, which received no treatment, served as the untreated baseline for assessing T-cell exhaustion states. Consistent with the highly immunosuppressive microenvironment characteristic of untreated LM8 tumors, exhaustion-associated markers such as Tox and Lag3 showed the highest expression in this group. These results align with those of previous studies showing that thermal stress can enhance T cell activation and function through multiple mechanisms, including increased antigen presentation and cytokine signaling [35,48].

Additionally, the observed shift in macrophage polarization, from an immunosuppressive M2 phenotype to a pro-inflammatory M1 phenotype, represents another important mechanism by which combination therapy may enhance antitumor immunity. M1 macrophages are generally characterized by the release of pro-inflammatory cytokines and reactive oxygen species responsible for antitumor cytotoxicity, while M2 macrophages support tumor progression and immune suppression [49,50,51]. Thus, macrophage polarization patterns observed in this study were associated with a more pro-inflammatory immune profile within the tumor microenvironment. Major immune-related biomarkers, including PD-L1 expression, T-cell exhaustion-associated genes, and macrophage polarization markers, were evaluated at the transcriptional or protein level. Furthermore, these findings suggest that mild hyperthermia may improve the antitumor response to PD-1 blockade. Particularly, modulation of both adaptive (T cells) and innate (macrophage) immune components may contribute to the improved therapeutic effect observed with the combination therapy. Because this scRNA-seq analysis was performed on pooled samples, it should be noted that while this analysis reflects changes at the cellular level, it may not reflect variation between individual mice.

Immune remodeling induced by hyperthermia and immune checkpoint blockade has been described in other tumor models using bulk transcriptomic analyses, immunohistochemistry, or flow cytometry, including increased CD8^+^ T-cell infiltration and macrophage polarization toward a pro-inflammatory phenotype [39,52]. Therefore, the immune phenotypes observed in the present study are not entirely unique to OS or to the use of single-cell RNA sequencing per se. However, the present study differs in that it applies single-cell RNA sequencing to an OS-specific context to comprehensively characterize immune remodeling induced by the combination of mild hyperthermia and PD-1 blockade at single-cell resolution. The immune-associated transcriptional features observed in the present OS model are consistent with these previous observations, extending them to an OS-specific context.

No overt treatment-related toxicities, abnormal behavior, or increased mortality were observed during combination therapy, suggesting that the regimen was well tolerated under the present experimental conditions.

This study has several limitations. First, the study uses only one OS model (LM8) and a single hyperthermia regimen (42 °C, 30 min). The findings are promising, but the biological scope is narrow. Broader validation across different OS models, temperatures, and exposure times will be needed in future studies. Second, to control for handling- and restraint-related effects, sham hyperthermia procedures were performed in the control and anti-PD-1 antibody treated groups using identical capsules at room temperature under general anesthesia; however, because postoperative analgesia was not administered because of potential immunomodulatory effects, subtle physiological effects related to heating itself cannot be completely excluded. The systemic stress responses during hyperthermia treatment may influence immune parameters. Stress-related biomarkers such as corticosterone, catecholamines, and proinflammatory cytokines were not directly measured, and their contribution cannot be completely excluded. Third, due to practical limitations related to cell recovery and sequencing depth, the scRNA-seq data set of HT group is absent. For the same reason, we were unable to include a baseline control group in the analysis of T cell exhaustion markers. These limitations restrict direct assessment of immune transcriptional changes induced by HT alone and limit precise contextualization of treatment-induced modulation of T-cell exhaustion relative to the natural tumor immune microenvironment. Accordingly, immune transcriptional changes observed in the combination group should be interpreted as associations under combination treatment conditions rather than as definitive evidence of synergy or combination-specific effects. Fourth, the scRNA-seq data showed phenotypic changes in T cells and macrophages, but we didn’t perform the protein-level validation, such as flow cytometry or immunohistochemistry, which would further strengthen the conclusions regarding T-cell exhaustion and macrophage polarization. Additionally, the functional contribution of these immune changes to tumor suppression was not explored. To clarify the functional contribution of these immune changes, the mechanistic validation experiments including CD8^+^ T-cell depletion experiments and assessing tumor infiltrating T-cell cytotoxicity or cytokine production, such as IFN-γ/TNF/IL2 following ex vivo re-stimulation with PMA/Ionomycin are necessary. Moreover, protein-level validation and functional assays were not performed, and therefore causal relationships between immune phenotypic changes and tumor suppression could not be established.

Despite these limitations, this study provides the first OS-specific, single-cell-level characterization of immune remodeling induced by the combination of mild hyperthermia and PD-1 blockade. Our findings support the concept that hyperthermia can function as an immunomodulatory adjunct to immune checkpoint inhibition and warrant further investigation of this combination strategy in OS and other immunologically cold tumors.

5. Conclusions

In conclusion, PD-1 blockade combined with mild hyperthermia was associated with suppressed tumor growth, reduced lung metastasis, and immune-associated transcriptional remodeling in a murine OS model. Key challenges include validation across additional OS models and mechanistic confirmation of immune-mediated antitumor effects. Future studies integrating functional immune assays and optimized hyperthermia platforms will be essential to advance this combination strategy toward clinical translation.