Cryo-thermal therapy reshaped the tumor immune microenvironment to enhance the efficacy of adoptive T cell therapy

Abstract

Background

Adoptive cell therapies (ACT) exhibit excellent efficacy in hematological malignancy. However, its application in solid tumors still has many challenges partly due to the tumor immune microenvironment. Cryo-thermal therapy (CTT) can induce an acute inflammatory response and remold the immune environment, providing an appropriate environment for the activation of adaptive immunity. However, it remains unclear whether CTT can enhance the efficacy of ACT.

Methods

A bilateral B16F10 tumor-bearing mouse model was used to assess whether CTT could enhance the efficacy of ACT. The right large tumor was subjected to CTT, and the left small tumor was collected for flow cytometry, RNA-seq, immunohistochemistry and TCR Vβ sequencing. Finally, bilateral B16F10 tumor-bearing mice and 4T1 tumor-bearing mice were used to assess the efficacy after CTT combined with ACT.

Results

CTT dramatically reshaped the immune microenvironment in distal tumors to an acute inflammatory state by promoting innate cell infiltration, increasing cytokine production by macrophages and DCs. The remodeling of the tumor immune microenvironment further enhanced the antitumor efficiency of ACT by increasing the proliferation of T cells, promoting activation of the effector functions of T cells and boosting the expansion of TCR clones.

Conclusions

Our results suggest that CTT can significantly reshape the tumor immunosuppressive microenvironment and convert “cold tumors” into “hot tumors,” thereby enhancing ACT-induced immune responses and maximizing the therapeutic effect of ACT.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00262-024-03884-2.

Background

Over the past 30 years, the rapid development of adoptive cell therapies (ACT), including chimeric antigen receptor therapy (CAR-T), TCR-engineered T cell (TCR-T) therapy and tumor-infiltrating lymphocyte (TIL) therapy, has produced a wide range of prospects for curing cancer [1]. While ACT has been used successfully in the treatment of hematological malignancies, its use in solid tumors is still in its infancy, partly due to the unique microenvironment of solid tumors [2, 3].

The tumor microenvironment inhibits the antitumor capacity of ACT in a variety of ways. First, T cells have difficulty infiltrating the tumor to perform their killing function due to physical and metabolic barriers and the lack of chemotactic signals [3, 4]. Even when infiltrating tumors, T cells are easily exhausted by soluble molecules such as TGF-β and PEG₂ and coinhibitory molecules expressed by tumor cells and immunosuppressive cells [3, 5]. Most importantly, in addition to local immunosuppression, patients with tumors exhibit systemic immunosuppression [6], which can directly inhibit the effector function of T cells after ACT. Therefore, overcoming tumor-induced immunosuppression is key to improving the therapeutic effects of ACT in solid tumors.

Cryo-thermal therapy (CTT), which is a novel tumor ablation therapy approved by the China National Medicine Products Administration (No. 20233010773), can disrupt tumor cells in situ through alternating cooling and heating [7]. Our previous study showed that CTT prompted a Th1-dominant antitumor immune response and improve the long-term survival of tumor-bearing mice [8, 9]. Moreover, CTT induced an acute proinflammatory response and remodeled the immune environment [10–12]. Thus, we hypothesized that CTT could potentially enhance the efficacy of ACT by modifying the immune environment.

Previously, we found that ACT using expanded T cell from CTT-treated mice inhibited experimental lung metastases in mice [13]. In this study, using the same ACT strategy, we investigated whether CTT could enhance the efficacy of ACT using bilateral B16F10 tumor-bearing mice. We found that CTT reshaped the immune microenvironment in distal tumors and prompted innate immune cell infiltration and activation. In addition, CTT combined with ACT (combination therapy) promoted the effector function and the intratumor proliferation of T cells and induced the clonal expansion of T cells. Moreover, combination therapy inhibited tumor growth in bilateral B16F10 tumor-bearing mice and improved long-term survival in 4T1 tumor-bearing mice susceptible to spontaneous metastasis. The insights from this study provide evidence that CTT could be developed as a platform technology to enhance the efficacy of ACT.

Methods

Animal model

Female C57BL/6 mice and BALB/c mice (Shanghai Slaccas Experimental Animal Co., Ltd, China) were housed and fed sterile food with standard mice nutritional formula and sterile watering the isolated cages of 12-h light/dark cycle environment. B16F10 cells and 4T1 cells were cultured in DMEM medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products) and penicillin–streptomycin (Hyclone). To prepare the B16F10 bilateral tumor-bearing mice, 5 × 10⁵ and 5 × 10⁴ B16F10 cells were injected subcutaneously (s.c.) into right flank and left flank of mice respectively when the mice were 6–8 weeks old and weighed approximately 20 g. To prepare the 4T1 tumor-bearing mice, cells (4 × 10⁵) were injected s.c. into right flank of mice when the mice were 6–8 weeks old and weighed approximately 20 g. Tumor-bearing mice were then randomly assigned to housing cages, and subjected to different treatments. Mice were euthanized when the humanitarian endpoint was reached. Tumor sizes were monitored every 2 days and its volume was estimated using the following formula: V (cm³) = π × L (major axis) ×  W (minor axis) × H (vertical axis)/6.

The cryo-thermal therapy procedures

Cryo-thermal therapy (CTT) was performed as described previously when the average tumor size reached about 0.2 cm³ [12]. Briefly, the subcutaneous tumor of mice was frozen with liquid nitrogen at − 20 ℃ for 5 min and then heated with radiofrequency at − 50 ℃ for 10 min.

The expansion of T cell and adoptive T cells transfer therapy

At 14 d after CTT, mice were killed and the spleen were collected for T cells expansion [13]. Briefly, the plates were precoated with 10 µg/mL anti-CD3 mAb (Biolegend, clone 2c11) at 4 ℃ overnight. Single splenocytes were resuspended at 2 × 10⁶ cells per mL in RPMI 1640 medium (Hyclone) supplemented with 15% FBS, 100 units/mL penicillin and 100 µg/mL streptomycin and cultured in anti-CD3-coated plates for 2 days. Then, cells were harvested and expanded in RPMI 1640 medium supplemented with 15% FBS, 100 units/mL penicillin, 100 µg/mL streptomycin, 60 IU/mL recombinant human interleukin 2 (rhIL-2, novoprotein) and 50 µM beta-mercaptoethanol and cultured for three additional days.

For adoptive T cells transfer therapy, 5 × 10⁶ expanded T cells were adopted to tumor-bearing mice or mice 1 day after CTT via tail vein injection. After T cell adoption, 90 000 IU IL-2 was given to all mice per day for three continuous days.

Flow cytometry

The spleen and tumors were collected after therapy. A single-cell suspension of splenocytes was prepared using GentleMACS dissociator (Miltenyi Biotec). The tumors were digested with collagenase I (Yeasen, China), hyaluronidase (Yeasen, China) and DNase I (Yeasen, China), and the tissues were mashed through a 70-mm cell strainer (Falcon, USA). Red blood cells were removed by erythrocyte-lysing reagent containing 0.15 M NH₄Cl, 1.0 M KHCO₃, and 0.1 mM Na₂EDTA. For cell surface staining, cells were stained with antibodies for 30 min at 4 °C. For intracellular staining, cells were stimulated for 4 h with Cell Activation Cocktail (Biolegend) according to the manufacturer’s protocol. The cells were surface stained with antibody binding cell-specific surface marker and fixed and permeabilized using Fixative Buffer (Biolegend) and Intracellular Staining Perm Wash Buffer (Biolegend), respectively. True-Nuclear™ Transcription Factor Buffer Set (Biolegend) was used to analyze the expression of transcription factors. The following antibodies were purchased from Biolegend: PE/Dazzle 594-CD45 (30-F11), PE/Cy7-Ly6G (clone 1A8), FITC-Ly6C (clone HK1.4), Pacific blue-CD11b (clone M1/70), BV711-F4/80 (clone BM8), PE-CD11c (clone N418), Percp-Cy5.5-I-A/I-E (clone M5/114.15.2), Percp-Cy5.5-CD3 (clone 145-2411), APC/Cy7-CD4 (clone RM4-5), Pacific blue-CD8 (clone 53-6.7), PE/Dazzle 594-IFN-γ (clone XMG1.2), BV421-IL4 (clone 11B11), PE-IL-17 (clone TC11-18H10.1), AF647-granzyme (clone GB11), PE-Perforin (clone S16009A), PE-FoxP3 (clone MF-14), AF700-CD8 (clone 53-6.7), BV605-IFN-γ (clone XMG1.2) and FITC-granzyme B (clone GB11). BUV395-CD45 (clone 30-F11), BUV395-Ki67 (clone 11F6) and APC-CD4 (clone RM4-5) were purchased from BD bioscience. Cell fluorescence was assessed with a LSRFortessa (BD Biosciences) and analyzed with FlowJo software (version 10.6.2).

Hematoxylin & Eosin (H&E) staining and immunohistochemical staining

For H&E staining, tissues were fixed for 24 h in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. Tissue sections (3–5 µm) were stained with H&E. Images were acquired with KFBIO KF-PRO-120 digital pathology slide scanner. Areas exhibiting pyknosis, karyorrhexis and karyolysis were identified as necrotic regions. The percentage of necrotic area was calculated for 10 randomly obtained fields using the ImageJ software.

For immunohistochemical staining, heat-induced antigen retrieval was performed using sodium citrate buffer. Then endogenous peroxidase was removed using Endogenous Peroxidase Blocking Buffer (Beyotime Biotechnology). Slides were blocked with 2% bovine serum albumin. Purified anti-CD11b (clone M1/70, Biolegend), anti-mouse CD3 (clone SP162, Abcam) and HRP anti-rat IgG (Beyotime Biotechnology) were used for immunohistochemical staining. Then the slides were stained with diaminobenzidine (DAB) kit (Beyotime Biotechnology) at room temperature for 10 min in the dark, followed by counterstaining with hematoxylin for cell nuclei. Images were acquired with a Leica microscope (Leica DM6 B).

RNA sequencing and analysis

Total RNA was extracted using the TRIzol reagent (Invitrogen), and the purity and quantification of RNA were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific). RNA integrity was assessed by using the Agilent 2100 Bioanalyzer (Agilent Technologies). Then the libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. PCA analysis was performed using R (v 3.2.0) to evaluate the biological duplication of samples. Gene Set Enrichment Analysis (GSEA) was performed using GSEA software [14]. Bioinformatic analysis was performed using the OECloud tools at https://cloud.oebiotech.com.

TCR Vβ sequencing and analysis

RNA samples were analyzed by high-throughput sequencing of TRB using the ImmuHub® TCR profiling system at a deep level (ImmuQuad Biotech). Briefly, a 5’ RACE unbiased amplification protocol was used. Sequencing was performed on an Illumina NovaSeq® system with PE150 mode (Illumina). Map V, D, J and C segments with NCBI and then extract CDR3 regions and assemble clonotype for all clones. We further defined amounts of each TRB clonotype by adding numbers of TRB clones sharing the same nucleotide sequence of CDR3. Further analysis of the TCR repertoires was performed in R using the Immunarch package.

Statistical analysis

The results were expressed as the mean ± standard deviation (SD). Statistical analyses were conducted with Student’s t test (for two groups comparisons) or one-way ANOVA test (for multiple groups comparisons). A P-value of < 0.05 was regarded as statistically significant. Exact n values were provided in the figure legends.

Results

CTT reshapes the remote tumor immune microenvironment

Our previous study showed that CTT induced an acute inflammatory environment to promote antitumor immunity [10, 11]. To investigate whether CTT could reshape the immune environment in distal tumors, we established a bilateral tumor model by inoculating mice with 5 × 10⁵ and 5 × 10⁴ B16F10 cells in their right and left flanks, respectively. At 12 days post-inoculation, the right (large) tumor was subjected to CTT and the left (small) tumor was collected for RNA sequencing (RNA-seq) (Fig. 1A). A dramatic change in the gene expression profile of the untreated left tumor after CTT was found compared to untreated controls, in which 336 genes were upregulated and 142 genes were downregulated (Fig. 1B–C). Gene set enrichment analysis (GSEA) based on hallmark gene sets from the Mouse Molecular Signatures Database (MSigDB) revealed that allograft rejection, interferon alpha and gamma response, inflammatory response, and IL-6/JAK/STAT3 signaling pathways were significantly enriched in the untreated left tumor from the CTT group compared to that of the control group (Fig. 1D). Meanwhile, GSEA based on Gene Ontology (GO) gene sets indicated that the neutrophil chemotaxis, migration, extravasation and monocyte migration pathways as well as the activation and M1 polarization of macrophages and cytokine production in macrophages and dendritic cells (DCs) pathways were unregulated in the untreated left tumors of CTT group compared with those of control group (Fig. 1E and Figure S1). Therefore, the infiltration of innate immune cells within distal tumors of mice after receiving different treatments was examined using flow cytometry. We found that innate immune cells, including neutrophils, macrophages, and DCs, were significantly increased within the tumor as early as 24 h after CTT (Fig. 1F and see Figure S2; gating strategy). Next, immunohistochemical staining of the untreated left tumors from the CTT group and control group was performed. Compared to the control group, the infiltration of CD11b⁺ myeloid cells was increased after CTT (Fig. 1G). These results suggest that CTT induced an acute inflammatory response and promoted the infiltration of innate immune cells, leading to reshaping of the immune environment in remote tumors.

Fig. 1.

CTT reshaped the tumor immune environment. A Scheme of experiment design. The bilateral tumor model was established by inoculating 5*10⁵ and 5*10⁴ B16F10 cells on the right and left flanks of C57BL/6 mice, respectively. On day 12 after inoculation, the right tumor was received CTT, then the left tumor was collected for the RNA-seq at 7 days after CTT. B Heatmap of differentially expressed genes, encompassing gene clustering. C Volcano plot of gene expression change of the left tumor from CTT group over untreated control mice, encompassing p-value and fold change. D Enriched gene sets (FDR < 0.05) of tumor from the CTT group compared with the control group were analyzed by GSEA based on HALLMARK terms in MSigDB. E The neutrophil chemotaxis (GO: 0030593), monocyte chemotaxis (GO:0002548), macrophage cytokine production (GO:0010934) and dendritic cell cytokine production (GO:0002371) pathways in MSigDB of tumor from the CTT group compared with the control group were analyzed by GSEA. F The proportions of neutrophils, monocytes, macrophages and DCs in tumor at 24 h after CTT were measured by flow cytometry. G Representative slides showing CD11b immunohistochemical staining. *p < 0.05, **p < 0.01, ***p < 0.001. n = 3 for RNA-seq and n  = 5 for flow cytomery

Combination therapy leads to distal tumor necrosis

Previously, we demonstrated that CTT induced the production of specific T cells and that expanded splenic T cell from mice receiving CTT protected against experimental lung metastases [13]. However, the phenotype and inhibitory effect of these expanded T cells on established solid tumors are still unknown. Thus, the function of expanded splenic T cell after CTT was further evaluated by using flow cytometry. Compared to pre-expansion T cells, the expression of CD69 on CD8⁺ T cells and the proportions of IFN-γ⁺ CD4⁺ T cells and IFN-γ⁺ CD8⁺ T cells were significantly increased after in vitro expansion, indicating that T cells were highly activated after in vitro expansion (Fig. 2A). Meanwhile, the expression of perforin in CD8⁺ T cells and granzyme B (GZMB) in CD4⁺ T cells and CD8⁺ T cells was dramatically unregulated after in vitro expansion (Fig. 2B). The proportions of Th2 cells, Th17 cells and Tregs were very low and unchanged before and after amplification (Figure S3). Thus, given the protective effect against lung metastases [13] and the high expression levels of activating and cytotoxic molecules, we hypothesized that the expanded splenic T cells from mice receiving CTT would be able to serve as a cell source for ACT.

Fig. 2.

CTT combined with ACT promoted distal tumor necrosis. At 14 days after CTT, mouse spleens were collected for T cell expansion. The splenocytes were stimulated for 2 days in plates coated with anti-CD3 antibody and subsequently expanded for 3 days in complete medium containing recombinant human IL-2 (60 IU/mL). The cells were then collected for subsequent flow cytometry assays or adoptive transfers. A–B Expression of CD69, IFN-γ, perforin and granzyme B in CD4⁺ and CD8.⁺ T cells before and after expansion was measured by flow cytometry. C Scheme of experiment design. Splenic T cells from CTT-treated mice were expanded and were adopted to tumor-bearing mice or to mice 1 d after CTT treatment. At 7 days after CTT, the left tumors were collected for H&E staining. D Representative slides showing the H&E staining of the left tumor and quantification of necrotic areas by using ImageJ. *p < 0.05, ***p < 0.001, ****p < 0.0001. n = 4

To verify the antitumor efficacy of ACT in vivo, expanded splenic T cells were adoptively transferred to a bilateral mouse tumor model, and necrosis in left untreated tumor was evaluated by H&E staining 7 days after CTT (Fig. 2C). Unexpectedly, there was no necrosis in the tumors from mice received ACT alone (Fig. 2D). The main obstacle for ACT in solid tumors is the immunosuppressive tumor microenvironment [2]. Our above results suggested that CTT could reshape the immune environment of distal tumors, and we hypothesized that CTT could improve the antitumor effects of ACT. Although CTT alone failed to promote necrosis of the left tumor, the combination therapy caused extensive necrosis of the left tumor, as evidenced by the substantial pyknosis, karyorrhexis and karyolysis observed following the combination therapy (Fig. 2D). These results suggest that CTT could effectively enhance the antitumor capacity of ACT.

Combination therapy promoted activation of antigen presentation cells in tumor

To further investigate the immunological mechanisms by which CTT enhanced the antitumor capacity of ACT, RNA-seq was conducted on the left tumors from the control, CTT, ACT, and combination therapy groups on day 7 after CTT. Consistent with the above results described, GSEA based on hallmark gene sets in MsigDB revealed that combination therapy significantly upregulated the interferon gamma and alpha response, allograft rejection, inflammatory response, and IL-6/JAK/STAT3 signaling pathways in the untreated left tumor compared to ACT alone (Fig. 3A). Meanwhile, neutrophil, monocyte and macrophage chemotaxis pathways (GO:0030593, GO:0002548 and GO:0048246) as well as cytokine production pathways in DCs (GO:0002371) were enriched in the combination therapy group compared with the ACT group (Fig. 3B). These results reaffirmed that CTT reshaped the immune environment in remote tumors. In addition, genes related to the antigen processing and presentation pathway (GO:0019882) were significantly enriched in the combination therapy group compared to ACT alone. (Fig. 3C). Thus, the expression of MHC-II on antigen-presenting cells within the tumor was measured by using flow cytometry. Only those in the combination therapy group demonstrated a statistically significant upregulation of MHC-II expression on DCs, monocytes, and macrophages in comparison with the control group (Fig. 3D–F). Consequently, following the CTT-mediated remodeling of the tumor immune environment, the combination therapy further enhanced the antigen-presenting function of innate immune cells.

Fig. 3.

CTT combined with ACT prompted the activation of antigen presentation cells in tumors. A Enriched gene sets (FDR < 0.05) of tumor from the CTT combined with ACT group compared with the ACT group were analyzed by GSEA based on hallmark gene sets in MSigDB. B–C The neutrophil chemotaxis (GO:0030593), monocyte chemotaxis (GO:0002548), macrophage chemotaxis (GO:0048246), dendritic cell cytokine production (GO:0002371) pathways (B) and antigen processing and presentation (GO:0019882) pathway (C) of tumor from the CTT combined with ACT group compared with the ACT group were analyzed by GSEA. D–F The expression of MHC-II on DCs (D), monocytes (E) and macrophages (F) in tumor was measured by flow cytometry. *p < 0.05, **p < 0.01. n = 3 for RNA-seq and n = 5 for flow cytometry

Combination therapy promoted the proliferation and effector function of T cells in tumor

As shown in Fig. 3A, TNF signaling via the NF-κB and IL-2/STAT5 signaling pathways was significantly enriched in the combination therapy group as compared to the ACT group. TNF and IL-2 are Th1 cytokines associated with T cell cytotoxicity, activation and proliferation [15, 16], suggesting that CTT could promote the function of T cells after ACT. Thus, T cell-associated immune pathways in GO biological process gene sets were analyzed using GSEA. T cell-mediated immunity and cytotoxicity pathways were extensively unregulated in the combination therapy group compared to ACT alone (Fig. 4A). In addition, the proportion of CD4⁺ T cells and CD8⁺ T cells in tumor was significantly increased after combination therapy compared to ACT alone (Fig. 4B and see Figure S4; gating strategy). The elevated proportion of T cells might be attributed to their active proliferation, as the expression of Ki67 within CD4⁺ and CD8⁺ T cells was significantly increased (Fig. 4C). Immunohistochemical staining revealed a notable increase in the number of CD3⁺ T cells within the tumor following combination therapy, when compared to the other group (Fig. 4D). Effector T cells within the tumor are the mainstay of tumor cell killing. Therefore, the effector function of intratumoral T cells was examined using flow cytometry. A significant increase in the proportion of intratumoral CD62⁻CD44⁺ effector memory T cells (T_EM) was observed in the combination therapy group in comparison to those in the control and CTT groups (Fig. 4E). Meanwhile, combination therapy demonstrated a notable increase in the expression of IFN-γ, granzyme B, and perforin in CD8⁺ T cells in comparison to the other groups (Fig. 4F). Moreover, RNA-seq results showed that the expression of genes that encoding granzyme and perforin such as GZMA, GZMB, GZMG and PRF1 was increased after combination therapy compared with that in other groups (Fig. 4G). These results suggested that combination therapy could further facilitate the proliferation and effector function of T cells leading to the maximization of T cell-mediated antitumor immunity.

Fig. 4.

CTT combined with ACT enhanced proliferation and effector function of T cells in tumor. A T cell-associated immune pathways in GO biological process gene sets (GO:0002250, GO:0002449, GO:0031341, GO:0001906, GO:0002821, GO:0002706, GO:0002819, GO:0001913, GO:0001914, GO:0002711, GO:0002456 and GO:0002709) were analyzed using GSEA. The color of the bubbles reflects the p-value, while the size reflects the number of genes. B The proportions of CD4⁺ T cells and CD8⁺ T cells in tumor were measured by flow cytometry. C The expression of Ki67 in CD4⁺ T cells and CD8⁺ T cells in tumor. D Representative slides showing CD3 immunohistochemical staining. n = 3. E The proportion of CD62⁻CD44⁺ T_EM in tumors. F The expression of IFN-γ, GZMB and perforin in CD8⁺ T cells was measured by flow cytometry. G Heatmap of mean expression of cytotoxicity-related genes. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 3 for RNA-seq and n = 5 for flow cytomery

Combination therapy promoted clonal expansion of T cells

The T cell receptor (TCR) determines the specificities of T cells; thus, the TCR repertoire has an important impact on the patient disease progression and therapeutic response [17]. To investigate the influence of different treatments on the TCR Vβ repertoire of tumor-infiltrating T cells, TCR Vβ sequencing was performed on the left tumors of the control, ACT, CTT and combination therapy groups on day 7 after CTT. As shown in Fig. 5A, ACT and CTT significantly increased the frequency of the top5 TCR clones from 9.78 to 17.35% and 15.49%, respectively, while combination therapy further increased the frequency of the top5 TCR clones (22.09%). Meanwhile, the top5 TCR clones in the combination therapy could be detected within tumors from other groups, suggesting that these amplified TCR clones have potential tumor specificity (Fig. 5B). These results suggest that combination therapy promoted infiltration and expansion of T cells within the tumor.

Fig. 5.

CTT combined with ACT increased the proportion of top5 TCR clones. A At 7 days after CTT, the left tumors from different group were collected for TCR Vβ sequencing. The pie chart showed the proportion of top5 TCR clones in each group and the bubble diagram showed the frequency of each clonal species. B Clonotype tracking showed that the top5 TCR clones in CTT combined with ACT group were also present in other groups of tumors

Combination therapy promoted suppression of distal tumors in B16F10 models and spontaneously metastatic tumors in 4T1 models

The above results suggested that CTT reshaped the tumor immunosuppressive microenvironment and could be combined with ACT to promote antitumor immunity. To further demonstrate the therapeutic efficacy of the combination therapy, the right tumors of B16F10 bilaterally tumor-bearing mice were treated with CTT as the target lesion, and the left tumors were used as the observation lesion to monitor tumor size. Splenic T cells from CTT-treated mice were simulated for 2 days with and anti-CD3 antibody, expanded for 3 days in the presence of IL-2 and administered to mice 1 day after CTT treatment (Fig. 6A). ACT alone failed to inhibit the growth of the observation lesion (Fig. 6B). CTT-treated mice showed a slight reduction in observed lesion growth, but no significant difference (Fig. 6B). Importantly, the combination therapy significantly inhibited the growth of the left observed lesion (Fig. 6B). To verify the efficacy of combination therapy on malignancies, a 4T1 mouse breast cancer model was used, which is a murine model of stage IV breast cancer and an spontaneously form micrometastases after subcutaneous inoculation [18, 19]. CTT significantly improved survival rates in mice compared to untreated control mice (Fig. 6C). Importantly, combination therapy increased the survival rate of mice from approximately 50% to approximately 90% compared to CTT alone for a long-term observation period (Fig. 6C). These data indicate that combination therapy can restrict distal tumors growth and eliminate micrometastasis.

Fig. 6.

CTT combined with ACT promoted the suppression of distal tumors in B16F10 tumor and 4T1 tumor models. A Scheme of experiment design. Splenic T cells from CTT-treated mice were expanded and were adopted to mice 1 day after CTT treatment. B Growth curve of the left tumor of the B16F10 bilateral tumor model. n = 5. C Survival of 4T1 tumor-bearing mice following CTT or CTT combined with ACT. Ctrl, n = 6; CTT, n = 15; Comb, n = 19. **p < 0.01

Discussion

To date, improving the efficacy of ACT is a major challenge in solid tumor treatment. In this study, we found that CTT, a localized thermal tumor physiotherapy, had remarkable success in enhancing the efficacy of ACT. Mechanistically, CTT remodeled the systemic immune microenvironment to an acute inflammatory state, thereby enhancing the antitumor efficacy of ACT by increasing the proliferation, effector functions and clonal expansion of T cells.

Previously, we found that CTT remodeled the systemic immune environment and induced an “acute” phenotypic environment that resulted in the induction of Th1-dominant antitumor immunity [9, 10, 12]. However, whether CTT-induced systemic antitumor immunity can modulate the immune microenvironment of tumors is not clear. In this study, a bilateral B16F10 tumor-bearing mouse model was established to evaluate whether the therapeutic effect of CTT on primary tumors could modulate the immune microenvironment in distal tumors. We found that CTT dramatically altered the immune microenvironment to an acute inflammatory state in the distal tumor, characterized by massive infiltration of myeloid cells and an enrichment with inflammatory-related pathways. These findings support that CTT not only improves the systemic immune environment, but also reshapes the immune microenvironment of tumors.

CTT has been shown to reduce pulmonary micrometastases in B16F10 tumor models [12]. However, in a bilateral B16F10 tumor-bearing mouse model with visible tumor, CTT alone could not promote the infiltration of T cells and was unable to effectively inhibit tumor growth in this study. Although the infiltration of myeloid cells in distal tumors was increased and highly activated after CTT, few effector T cells were recruited in response to myeloid cells at this time point, possibly because T cells were adequately activated at 14 days after CTT [8, 9]. Therefore, ACT performed after CTT can rapidly replenish large numbers of effector T cells, thereby enabling a prompt response to the acute inflammatory response induced by CTT.

In recent years, ACT has shown promising efficacy in B-cell leukemia and lymphoma, demonstrating its promising anticancer activity and potential therapeutic prospects [20]. However, the use of ACT in the treatment of solid tumors is still in its early stages [2]. The first obstacle to ACT in the treatment of solid tumors is the immunosuppressive microenvironment. Tumor-induced immunosuppression significantly limits the effector function of ACT [21–23]. In this study, ACT alone could not effectively inhibit the growth of established tumors. Thus, we hypothesized that CTT could facilitate the efficacy of ACT by remodeling the tumor immune microenvironment. Compared to ACT alone, combination therapy significantly promoted the tumor infiltration and activation of innate immune cells. We therefore suggest that CTT-induced acute myeloid cell infiltration and activation of innate cells are critical for improving the efficacy of ACT. The acute activation of innate immunity could set the stage for the activation of more complex, antigen-committed, adaptive immune responses [24]. Meanwhile, activated innate immune cells can produce chemokines such as CXCL9, CXCL10 to recruit T cells and induce the priming and expansion of tumor-specific T cells [25, 26]. Indeed, in this study, CTT significantly promoted the intratumor proliferation, effector function and the clonal expansion of T cells after ACT.These findings suggested that CTT could be developed as a platform technology to enhance the efficacy of ACT.

Conclusions

Collectively, our results suggested that CTT could significantly reshape the tumor immunosuppressive microenvironment and convert “cold tumors” into “hot tumors,” which promoted the proliferation and effector function of T cells and thereby maximizing the therapeutic effect of ACT. The insights from this study provide evidence for the clinical application of CTT in combination with ACT including CAR-T, TCR-T and TIL therapies.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

ACT

Adoptive cell therapies

CTT

Cryo-thermal therapy

DCs

Dendritic cells

GSEA

Gene set enrichment analysis

MFI

Median fluorescence intensity

TCR-T

TCR-engineered T cell

TIL

Tumor-infiltrating lymphocyte

Author contributions

SW, PP, JW and ZZ performed the experiments. PL coordinated the project. PL, SW and PP designed the experiments. The manuscript was written by SW and revised by PL, and LXX. All authors reviewed the manuscript.

Funding

This study was supported by the National Key Research and Development Program of China (Grant No. 2023YFC2411403), the Shanghai Science and Technology Commission of Shanghai Municipality (Grant No. ZJ2021-ZD-007) and the National Natural Science Foundation of China (Grant No. 82072085).

Data availability

No datasets were generated or analyzed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethics approval

All animal experiments were approved by the Animal Welfare Committee of Shanghai Jiao Tong University, and experimental methods were performed in accordance with the guidelines of Shanghai Jiao Tong University Animal Care (approved by Shanghai Jiao Tong University Scientific Ethics Committee, Registration No. 2020017).