IFNγ at the early stage induced after cryo-thermal therapy maintains CD4+ Th1-prone differentiation, leading to long-term antitumor immunity.

Introduction: Recently, more and more research illustrated the importance of inducing CD4+ T helper type (Th)-1 dominant immunity for the success of tumor immunotherapy. Our prior studies revealed the crucial role of CD4+ Th1 cells in orchestrating systemic and durable antitumor immunity, which contributes to the satisfactory outcomes of the novel cryo-thermal therapy in the B16F10 tumor model. However, the mechanism for maintaining the cryo-thermal therapy-mediated durable CD4+ Th1-dominant response remains uncovered. Additionally, cryo-thermal-induced early-stage CD4+ Th1-dominant T cell response showed a correlation with the favorable prognosis in patients with colorectal cancer liver metastasis (CRCLM). We hypothesized that CD4+ Th1-dominant differentiation induced during the early stage post cryo-thermal therapy would affect the balance of CD4+ subsets at the late phase. Methods: To understand the role of interferon (IFN)-γ, the major effector of Th1 subsets, in maintaining long-term CD4+ Th1-prone polarization, B16F10 melanoma model was established in this study and a monoclonal antibody was used at the early stage post cryo-thermal therapy for interferon (IFN)-γ signaling blockade, and the influence on the phenotypic and functional change of immune cells was evaluated. Results: IFNγ at the early stage after cryo-thermal therapy maintained long-lasting CD4+ Th1-prone immunity by directly controlling Th17, Tfh, and Tregs polarization, leading to the hyperactivation of Myeloid-derived suppressor cells (MDSCs) represented by abundant interleukin (IL)-1ß generation, and thereby further amplifying Th1 response. Discussion: Our finding emphasized the key role of early-phase IFNγ abundance post cryo-thermal therapy, which could be a biomarker for better prognosis after cryo-thermal therapy.

Two-stage generative adversarial networks for metal artifact reduction and visualization in ablation therapy of liver tumors.

PURPOSE: The strong metal artifacts produced by the electrode needle cause poor image quality, thus preventing physicians from observing the surgical situation during the puncture process. To address this issue, we propose a metal artifact reduction and visualization framework for CT-guided ablation therapy of liver tumors. METHODS: Our framework contains a metal artifact reduction model and an ablation therapy visualization model. A two-stage generative adversarial network is proposed to reduce the metal artifacts of intraoperative CT images and avoid image blurring. To visualize the puncture process, the axis and tip of the needle are localized, and then the needle is rebuilt in 3D space intraoperatively. RESULTS: Experiments show that our proposed metal artifact reduction method achieves higher SSIM (0.891) and PSNR (26.920) values than the state-of-the-art methods. The accuracy of ablation needle reconstruction is 2.76 mm average in needle tip localization and 1.64° average in needle axis localization. CONCLUSION: We propose a novel metal artifact reduction and an ablation therapy visualization framework for CT-guided ablation therapy of liver cancer. The experiment results indicate that our approach can reduce metal artifacts and improve image quality. Furthermore, our proposed method demonstrates the potential for displaying the relative position of the tumor and the needle intraoperatively.

Exploration of the impact of multimode thermal therapy versus radiofrequency ablation on CD8+ T effector cells of liver malignancies based on single cell transcriptomics.

Introduction: Multimode thermal therapy (MTT) is an innovative interventional therapy developed for the treatment of liver malignancies. When compared to the conventional radiofrequency ablation (RFA), MTT typically offers improved prognosis for patients. However, the effect of MTT on the peripheral immune environment and the mechanisms underlying the enhanced prognosis have yet to be explored. The aim of this study was to further investigate the mechanisms responsible for the difference in prognosis between the two therapies. Methods: In this study, peripheral blood samples were collected from four patients treated with MTT and two patients treated with RFA for liver malignancies at different time points before and after the treatment. Single cell sequencing was performed on the blood samples to compare and analyze the activation pathways of peripheral immune cells following the MTT and RFA treatment. Results: There was no significant effect of either therapy on the composition of immune cells in peripheral blood. However, the differential gene expression and pathway enrichment analysis demonstrated enhanced activation of T cells in the MTT group compared to the RFA group. In particular, there was a remarkable increase in TNF-α signaling via NF-κB, as well as the expression of IFN-α and IFN-γ in the CD8+ effector T (CD8+ Teff) cells subpopulation, when compared to the RFA group. This may be related to the upregulation of PI3KR1 expression after MTT, which promotes the activation of PI3K-AKT-mTOR pathway. Conclusion: This study confirmed that MTT could more effectively activate peripheral CD8+ Teff cells in patients compared with RFA and promote the effector function, thus resulting in a better prognosis. These results provide a theoretical basis for the clinical application of MTT therapy.

Survival Analysis for Multimode Ablation Using Self-Adapted Deep Learning Network Based on Multisource Features.

Novel multimode thermal therapy by freezing before radio-frequency heating has achieved a desirable therapeutic effect in liver cancer. Compared with surgical resection, ablation treatment has a relatively high risk of tumor recurrence. To monitor tumor progression after ablation, we developed a novel survival analysis framework for survival prediction and efficacy assessment. We extracted preoperative and postoperative MRI radiomics features and vision transformer-based deep learning features. We also combined the immune features extracted from peripheral blood immune responses using flow cytometry and routine blood tests before and after treatment. We selected features using random survival forest and improved the deep Cox mixture (DCM) for survival analysis. To properly accommodate multitype input features, we proposed a self-adapted fully connected layer for locally and globally representing features. We evaluated the method using our clinical dataset. Of note, the immune features rank the highest feature importance and contribute significantly to the prediction accuracy. The results showed a promising C td-index of 0.885 ±0.040 and an integrated Brier score of 0.041 ±0.014, which outperformed state-of-the-art method combinations of survival prediction. For each patient, individual survival probability was accurately predicted over time, which provided clinicians with trustable prognosis suggestions.

Activated NK cells reprogram MDSCs via NKG2D-NKG2DL and IFN-γ to modulate antitumor T-cell response after cryo-thermal therapy.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) can potently inhibit T-cell activity, promote growth and metastasis of tumor and contribute to resistance to immunotherapy. Targeting MDSCs to alleviate their protumor functions and immunosuppressive activities is intimately associated with cancer immunotherapy. Natural killer (NK) cells can engage in crosstalk with multiple myeloid cells to alter adaptive immune responses, triggering T-cell immunity. However, whether the NK-cell-MDSC interaction can modulate the T-cell immune response requires further study. Cryo-thermal therapy could induce the maturation of MDSCs by creating an acute inflammatory environment to elicit a CD4+ Th1-dominant immune response, but the mechanism regulating this process remains unclear. METHODS: NK cells were depleted and NKG2D was blocked with monoclonal antibodies in vivo. MDSCs, NK cells and T cells were assessed by flow cytometry and isolated by magnetic-activated cell sorting (MACS). MDSCs and NK cells were cocultured with T cells to determine their immunological function. The transcriptional profiles of MDSCs were measured by qRT-PCR and RNA-sequencing. Isolated NK cells and MDSCs by MACS were cocultured to study the viability and maturation of MDSCs regulated by NK cells. TIMER was used to comprehensively examine the immunological, clinical, and genomic features of tumors. RESULTS: NK-cell activation after cryo-thermal therapy decreased MDSC accumulation and reprogrammed immunosuppressive MDSCs toward a mature phenotype to promote T cell antitumor immunity. Furthermore, we discovered that NK cells could kill MDSCs via the NKG2D-NKG2DL axis and promote MDSC maturation by interferon gamma (IFN-γ) in response to NKG2D. In addition, CD4+ Th1-dominant antitumor immune response was dependent on NKG2D, which promoted the major histocompatibility complex Ⅱ pathway of MDSCs. High activated NK-cell infiltration and NKG2D level in tumors were positively correlated with better clinical outcomes. CONCLUSIONS: Cryo-thermal therapy induces effective CD4+ Th1-dominant antitumor immunity by activating NK cells to reprogram MDSCs, providing a promising therapeutic strategy for cancer immunotherapy.

Combining all-trans retinoid acid treatment targeting myeloid-derived suppressive cells with cryo-thermal therapy enhances antitumor immunity in breast cancer.

Targeting myeloid-derived suppressive cells (MDSCs) has been considered a potential strategy in tumor therapy. However, a single drug targeting MDSCs remains a challenge in the clinic. An increasing number of studies have shown that combination agents targeting MDSCs and immunotherapy may provide exciting new insights and avenues to explore in tumor therapy. In our previous study, a novel cryo-thermal therapy was developed for metastatic tumors that systematically activate innate and adaptive immunity. Moreover, cryo-thermal therapy was shown to dramatically decrease the levels of MDSCs and induce their differentiation toward potent antigen-presenting cells. However, the therapeutic effects of cryo-thermal therapy on the 4T1 mouse breast cancer model were still not satisfactory because of the high level of MDSCs before and after treatment. Therefore, in this study, we combined cryo-thermal therapy with all-trans retinoid acid (ATRA), a small molecule drug that can induce the inflammatory differentiation of MDSCs. We found that combination therapy notably upregulated the long-term survival rate of mice. Mechanically, combination therapy promoted the phenotype and functional maturation of MDSCs, efficiently decreasing suppressive molecule expression and inhibiting glutamine and fatty acid metabolism. Moreover, MDSCs at an early stage after combination therapy significantly decreased the proportions of Th2 and Treg subsets, which eventually resulted in Th1-dominant CD4+ T-cell differentiation, as well as enhanced cytotoxicity of CD8+ T cells and natural killer cells at the late stage. This study suggests a potential therapeutic strategy for combination ATRA treatment targeting MDSCs with cryo-thermal therapy to overcome the resistance of MDSC-induced immunosuppression in the clinic.

Th1-Dominant CD4+ T Cells Orchestrate Endogenous Systematic Antitumor Immune Memory After Cryo-Thermal Therapy.

Recent studies suggest that highly activated, polyfunctional CD4+ T cells are incredibly effective in strengthening and sustaining overall host antitumor immunity, promoting tumor-specific CD4+ T-cell responses and effectively enhancing antitumor immunity by immunotherapy. Previously, we developed a novel cryo-thermal therapy for local tumor ablation and achieved long-term survival rates in several tumor models. It was discovered that cryo-thermal therapy remodeled the tumor microenvironment and induced an antigen-specific CD4+ T-cell response, which mediated stronger antitumor immunity in vivo. In this study, the phenotype of bulk T cells in spleen was analyzed by flow cytometry after cryo-thermal therapy and both CD4+ Th1 and CD8+ CTL were activated. In addition, by using T-cell depletion, isolation, and adoptive T-cell therapy, it was found that cryo-thermal therapy induced Th1-dominant CD4+ T cells that directly inhibited the growth of tumor cells, promoted the maturation of MDSCs via CD4+ T-cell-derived IFN-γ and enhanced the cytotoxic effector function of NK cells and CD8+ T cells, and promoted the maturation of APCs via cell-cell contact and CD4+ T-cell-derived IFN-γ. Considering the multiple roles of cryo-thermal-induced Th1-dominant CD4+ T cells in augmenting antitumor immune memory, we suggest that local cryo-thermal therapy is an attractive thermo-immunotherapy strategy to harness host antitumor immunity and has great potential for clinical application.

CCL5 Deficiency Enhanced Cryo-Thermal-Triggered Long-Term Anti-Tumor Immunity in 4T1 Murine Breast Cancer.

Breast cancer remains one of the most common solid tumors. Tumor immunosuppressive factors mainly hinder the control of tumors. We previously developed an innovative cryo-thermal therapy that was shown to significantly suppress distal metastasis and improve long-term survival in murine B16F10 melanoma and 4T1 mammary carcinoma models. However, the effect of cryo-thermal therapy on the 4T1 model was not excellent. CCL5 has been reported to help the progression of breast cancer, so in this study, CCL5-/- was used to explore the role of host-derived CCL5 after cryo-thermal therapy. CCL5-/- could not completely resist tumor development, but it significantly improved survival rates when combined with cryo-thermal therapy. Mechanically, CCL5-/- mildly decreases the percentage of MDSCs, increases DC maturation and macrophage's inflammatory function at an early stage after tumor inoculation, and later up-regulate the level of Th1 and down-regulate the level of Tregs. When combined with cryo-thermal therapy, CCL5-/- dramatically down-regulated the proportion of MDSCs and induced full M1 macrophage polarization, which further promoted Th1 differentiation and the cytotoxicity of CD8+ T cells. Our results indicated that CCL5-/- contributed to cryo-thermal-triggered, long-lasting anti-tumor memory immunity. The combination of cryo-thermal therapy and CCL5 blockades might extend the survival rates of patients with aggressive breast cancer.

A Novel Multi-Mode Thermal Therapy for Colorectal Cancer Liver Metastasis: A Pilot Study.

A novel multi-mode thermal therapy was developed for local tumor ablation and the systemic stimulation of anti-tumor immunity, consisting of a rapid liquid nitrogen freezing, and followed by the radiofrequency heating of target tumor tissue. This pilot study aimed to compare the therapeutic effects of the new therapy with conventional radiofrequency ablation (RFA) on patients with colorectal cancer liver metastasis (CRCLM). From August 2016 to September 2019, thirty-one patients with CRCLM received either multi-mode thermal therapy (n = 17) or RFA (n = 14). Triphasic contrast-enhanced magnetic resonance imaging (MRI), routine blood tests, and peripheral blood immune responses were evaluated before the treatment and in 1, 3, 6, and 12 months after. Local tumor response and progression-free survival (PFS) were assessed using the Kaplan-Meier method, and pre- and post-treatment immune cell counts were analyzed using Mann-Whitney U and Wilcoxon tests. A significantly longer PFS was observed in the multi-mode thermal therapy group in comparison to that of the conventional RFA group (median, 11.4 versus 3.4 months, p = 0.022). It was found that multi-mode therapy induced the functional maturation of dendritic cells, promoted CD4+ T cell-mediated antitumor responses, and decreased regulatory T cells, contributing to better therapeutic efficacy in CRCLM patients.

Numerical Study of Bubble Cloud and Thermal Lesion Evolution During Acoustic Droplet Vaporization Enhanced HIFU Treatment.

Acoustic droplet vaporization (ADV) has been proven to enhance high intensity focused ultrasound (HIFU) thermal ablation of tumor. It has also been demonstrated that triggering droplets before HIFU exposure could be a potential way to control both the size and the shape of the thermal lesion. In this paper, a numerical model is proposed to predict the thermal lesion created in ADV enhanced HIFU treatment. Bubble oscillation was coupled into a viscoelastic medium in the model to more closely represent real applications in tissues. Several physical processes caused by continuous wave ultrasound and elevated temperature during the HIFU exposure were considered, including rectified diffusion, gas solubility variation with temperature in the medium, and boiling. Four droplet concentrations spanning two orders of magnitude were calculated. The bubble cloud formed from triggering of the droplets by the pulse wave ultrasound, along with the evolution of the shape and location of the bubble cloud and thermal lesion during the following continuous wave exposure was obtained. The increase of bubble void fraction caused by continuous wave exposure was found to be consistent with the experimental observation. With the increase of droplet concentration, the predicted bubble cloud shapes vary from tadpole to triangular and double triangular, while the thermal lesions move toward the transducer. The results show that the assumptions used in this model increased the accuracy of the results. This model may be used for parametrical study of ADV enhanced HIFU treatment and be further used for treatment planning and optimization in the future.

Downregulated TNF-α Levels after Cryo-Thermal Therapy Drive Tregs Fragility to Promote Long-Term Antitumor Immunity.

Immunotherapy has emerged as a therapeutic pillar in tumor treatment, but only a minority of patients get benefit. Overcoming the limitations of immunosuppressive environment is effective for immunotherapy. Moreover, host T cell activation and longevity within tumor are required for the long-term efficacy. In our previous study, a novel cryo-thermal therapy was developed to improve long-term survival in B16F10 melanoma and s.q. 4T1 breast cancer mouse models. We determined that cryo-thermal therapy induced Th1-dominant CD4+ T cell differentiation and the downregulation of Tregs in B16F10 model, contributing to tumor-specific and long-lasting immune protection. However, whether cryo-thermal therapy can affect the differentiation and function of T cells in a s.q. 4T1 model remains unknown. In this study, we also found that cryo-thermal therapy induced Th1-dominant differentiation of CD4+ T cells and the downregulation of effector Tregs. In particular, cryo-thermal therapy drove the fragility of Tregs and impaired their function. Furthermore, we discovered the downregulated level of serum tumor necrosis factor-α at the late stage after cryo-thermal therapy which played an important role in driving Treg fragility. Our findings revealed that cryo-thermal therapy could reprogram the suppressive environment and induce strong and durable antitumor immunity, which facilitate the development of combination strategies in immunotherapy.

A New Model for Estimation of Individual Blood Flow Effect during Multimode Thermal Therapy of Tumor.

An accurate temperate control is the key during multimode thermal therapy of tumor. However, the tumor tissue temperature is greatly influenced by local blood flow changes of individuals. A simple but effective method is proposed for estimation of the local blood flow and its impact on the ablation boundary temperature. The proposed model is focused on the tumor domain, namely the targeted treatment region. In the natural thawing process post tumor freezing during the therapy, the main energy transferring to the tumor tissue comes from the blood flow of the surrounding normal tissue on the tumor boundary. By fitting the rewarming temperature measured in the tissue, the inversed problem is solved by the model to calculate the boundary convection condition and thus to predict the corresponding blood perfusion rate. The model is validated by the animal experimental data. The calculated blood perfusion rates are within the published range, but differ individually. The results prove that the new model and the estimated personalized convection coefficient can better predict the tissue temperature distribution during the therapy.Clinical Relevance-The model estimates the local blood flows around the tumor of individuals and the influence on heat transfer process. It can be used to better predict and control the temperature on the tumor boundary during the therapy that is critical to the therapeutic effect. The model also greatly cuts down the calculation time which facilitates the possibility of intraoperative real time monitoring.

Neoantigen-specific CD4+ T-cell response is critical for the therapeutic efficacy of cryo-thermal therapy.

BACKGROUND: Traditional tumor thermal ablations, such as radiofrequency ablation (RFA) and cryoablation, can result in good local control of tumor, but traditional tumor thermal ablations are limited by poor long-term survival due to the failure of control of distal metastasis. Our previous studies developed a novel cryo-thermal therapy to treat the B16F10 melanoma mouse model. Long-term survival and T-cell-mediated durable antitumor immunity were achieved after cryo-thermal therapy, but whether tumor antigen-specific T-cells were augmented by cryo-thermal therapy was not determined. METHODS: The long-term antitumor therapeutic efficacy of cryo-thermal therapy was performed in B16F10 murine melanoma models. Splenocytes derived from mice treated with RFA or cryo-thermal therapy were coincubated with tumor antigen peptides to detect the frequency of antigen specific CD4+ and CD8+ T-cells by flow cytometry. Splenocytes were then stimulated and expanded by αCD3 or peptides and adoptive T-cell therapy experiments were performed to identify the antitumor efficacy of T-cells induced by RFA and cryo-thermal therapy. Naïve mice and tumor-bearing mice were used as control groups. RESULTS: Local cryo-thermal therapy generated a stronger systematic antitumor immune response than RFA and a long-lasting antitumor immunity that protected against tumor rechallenge. In vitro studies showed that the antigen-specific CD8+ T-cell response was induced by both cryo-thermal therapy and RFA, but the strong neoantigen-specific CD4+ T-cell response was only induced by cryo-thermal therapy. Cryo-thermal therapy-induced strong antitumor immune response was mainly mediated by CD4+ T-cells, particularly neoantigen-specific CD4+ T-cells. CONCLUSION: Cryo-thermal therapy induced a stronger and broader antigen-specific memory T-cells. Specifically, cryo-thermal therapy, but not RFA, led to a strong neoantigen-specific CD4+ T-cell response that mediated the resistance to tumor challenge.

Iron promotes breast cancer cell migration via IL-6/JAK2/STAT3 signaling pathways in a paracrine or autocrine IL-6-rich inflammatory environment.

Iron overload can act as catalyst for the formation of free radicals, which may promote oxidant-mediated breast carcinogenesis. However, the association between iron and breast cancer has not been comprehensively elucidated. In this study, we found that iron overload upregulated the inflammatory cytokine interleukin-6 (IL-6) expression to activate Janus Kinases 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) signaling in triple negative breast cancer (TNBC) MDA-MB-231 cell lines, resulting in epithelial-mesenchymal transition (EMT) and cancer cell migration, but it had no effects on the estrogen receptor (ER)-positive breast cancer MCF-7 cells. However, in the presence of exogenous IL-6, iron overload could also dramatically induce an autocrine IL-6 loop in ER-positive MCF-7 cells to active IL-6/JAK2/STAT3 signaling, resulting in enhanced EMT and cell motility. In vivo animal studies also identified that iron overload promoted the progression of low metastatic breast cancer tumorigenicity and lung metastasis following the addition of exogenous IL-6. This study suggested that iron overload could result in inducible IL-6 expression leading to promote malignant transformation of breast cancer cells in an paracrine or autocrine IL-6-rich inflammatory environment. Anti-inflammation and iron depletion therapy would be an effective therapeutic/preventive strategy for suppressing breast cancer progression.

Tumor-related HSP70 released after cryo-thermal therapy targeted innate immune initiation in the antitumor immune response.

PURPOSE: In our previous study, a novel cryo-thermal therapy that could stimulate the maturation of innate immune cells to subsequently activate the CD4+Th1 cell-dominated antitumor response was developed. However, why cryo-thermal therapy can induce the maturation of innate immunity remains unknown. METHODS: In this study, western blot and ELISA were used to analyze the levels of damage-associated molecular patterns (DAMPs, including heat shock protein 70 (HSP70), calreticulin and high-mobility group box protein 1) in situ and in the peripheral blood at different times after cryo-thermal therapy or traditional radiofrequency ablation. The effects of these three DAMPs on myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs) and macrophages were investigated by antibody neutralization in vitro. The phenotypic and functional changes in MDSCs, DCs and macrophages were analyzed using FACS and qRT-PCR. An anti-HSP70 antibody was injected intravenously at 6 h after cryo-thermal therapy on days 1 and 2 and mouse survival was monitored. RESULTS: Cryo-thermal therapy could trigger the release of DAMPs in situ and in the peripheral circulation, which could downregulate the proportion and suppressive signature of MDSCs, and promote the M1 macrophages polarization and DCs maturation. Among three DAMPs, HSP70 played the most evident role in M1 macrophage polarization. In vivo neutralization of HSP70 in the early stage of treatment could significantly decrease the survival rate of cryo-thermal therapy treated mice. CONCLUSIONS: Local cryo-thermal therapy not only destroyed solid tumors thermally and mechanically but also induced the release of a large amount of DAMPs to effectively trigger a systemic antitumor response.

A new radiofrequency balloon angioplasty device for atherosclerosis treatment.

BACKGROUND: Restenosis remains a challenge in the treatment of atherosclerosis due to damage to the endothelial layer and induced proliferation of smooth muscle cells. A novel radiofrequency (RF) heating strategy was proposed to selectively ablate atherosclerosis plaque and to thermally inhibit the proliferation of smooth muscle cells while keeping the endothelial cells intact. METHODS: To realize the proposed strategy, a new radiofrequency balloon catheter, consisting of three ports, a three-channel tube, a balloon and an electrode patch, was designed. To evaluate the feasibility of this new design, a phantom experiment with thermocouples measuring temperatures with different voltages applied to the electrodes was conducted. A numerical model was established to obtain the 3D temperature distribution. The heating ability was also evaluated in ex vivo diseased artery samples. RESULTS: The experimental results showed that the highest temperature could be achieved in a distance from the surface of the balloon as designed. The temperature differences between the highest temperature at 0.78 mm and those of the surface reached 9.87 °C, 12.55 °C and 16.00 °C under applied 15 V, 17.5 V and 20 V heating, respectively. In the circumferential direction, the heating region (above 50 °C) spread from the middle of the two electrodes. The numerical results showed that the cooling effect counteracted the electrical energy deposition in the region close to the electrodes. The thermal lesion could be directed to cover the diseased media away from the catheter surface. The ex vivo heating experiment also confirmed the selective heating ability of the device. The temperature at the targeted site quickly reached the set value. The temperature of the external surface was higher than the inner wall surface temperature of the diseased artery lumen. CONCLUSION: Both the experimental and numerical results demonstrated the feasibility of the newly designed RF balloon catheter. The proposed RF microelectrodes heating together with the cooling water convection can realize the desired heating in the deeper site of the blood vessel wall while sparing the thin layer of the endothelium.

A role of eosinophils in mediating the anti-tumour effect of cryo-thermal treatment.

Previous, we established a novel therapeutic approach to tumour of cryo-thermal therapy, which can induce durable anti-tumour memory immunity mediated by CD4+ T cell, and contribute to prolonged survival in B16F10 murine melanoma model and 4T1 murine mammary carcinoma. It has become apparent that innate immune cells are involved in the regulation of adaptive T cell immunity. Our previous studies revealed that cryo-thermal therapy induced M1 macrophage polarization and DCs maturation were required for the shaping of systemic long-lived T cell mediated anti-tumour memory immunity. Eosinophils are multifunctional innate effector cells and there is lack of knowledge on the role of eosinophils in cryo-thermal-induced anti-tumour immunity. This study revealed that cryo-thermal therapy activated eosinophils in spleen at early stage following the treatment. Furthermore, cryo-thermal-activated eosinophils exerted versatile immunologic regulation from innate immunity to anti-tumour adaptive immunity, such as M1 macrophage polarization, DCs maturation, differentiation of CD4-CTL subtypes and enhanced cytotoxicity of CD8+ T cells. Our study indicated that the cryo-thermal-activated eosinophils was essential for the shaping of durable anti-tumour memory immunity. Thus, our results present a new concept for eosinophils mediated anti-tumour immunity after cryo-thermal therapy.

Deferoxamine-induced high expression of TfR1 and DMT1 enhanced iron uptake in triple-negative breast cancer cells by activating IL-6/PI3K/AKT pathway.

Background: Deferoxamine (DFO) is a commonly used iron chelator, which can reduce the iron levels in cells. DFO is normally used to treat iron-overload disease, including some types of cancer. However, our previous studies revealed that DFO treatment significantly increased the iron concentrations in triple-negative breast cancer cells (TNBCs) resulting in enhanced cell migration. But the mechanism of DFO-induced increasing iron uptake in aggressive TNBCs still remained unclear. Materials and methods: Iron metabolism-related proteins in aggressive breast cancer MDA-MB-231, HS578T and BT549 cells and nonaggressive breast cancer MCF-7 and T47D cells were examined by immunofluorescence and Western blotting. The possible regulatory mechanism was explored by Western blotting, co-incubation with neutralizing antibodies or inhibitors, and transwell assay. Results: In this study, we found that DFO treatment significantly increased the levels of iron uptake proteins, DMT1 and TfR1, in aggressive TNBCs. Moreover, both TfR1 and DMT1 expressed on cell membrane were involved in high iron uptake in TNBCs under DFO-induced iron deficient condition. For the possible regulatory mechanism, we found that DFO treatment could promote a high expression level of IL-6 in aggressive MDA-MB-231 cells. The activated IL-6/PI3K/AKT pathway upregulated the expression of iron-uptake related proteins, TfR1 and DMT1, leading to increased iron uptakes. Conclusion: We demonstrated that DFO could upregulate expression of TfR1 and DMT1 , which enhanced iron uptake via activating IL-6/PI3K/AKT signaling pathway in aggressive TNBCs.

Cryo-thermal therapy induces macrophage polarization for durable anti-tumor immunity.

Many cancer therapies are being developed for the induction of durable anti-tumor immunity, especially for malignant tumors. The activation of antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), can bridge innate and adaptive immune responses against tumors. However, APCs have an immunosuppressive phenotype and reversing it for effective tumor-specific antigen presenting is critical in developing new cancer treatment strategies. We previously developed a novel cryo-thermal therapy to treat malignant melanoma in a mouse model; long-term survival and durable anti-tumor immunity were achieved, but the mechanism involved was unclear. This study revealed cryo-thermal therapy-induced macrophage polarization to the M1 phenotype and modulated the phenotypic and functional maturation of DCs with high expression of co-stimulatory molecules, increased pro-inflammatory cytokine production, and downregulated immuno-inhibitory molecule expression. Further, we observed CD4+ T-cell differentiation into Th1 and cytotoxic T-cell sub-lineages and generation of cytotoxic CD8+ T cells, in which M1 macrophage polarization had a direct, important role. The results indicated that cryo-thermal-induced macrophage polarization to the M1 phenotype was essential to mediate durable anti-tumor immunity, leading to long-term survival. Thus, cryo-thermal therapy is a promising strategy to reshape host immunosuppression, trigger persistent memory immunity for tumor eradication, and inhibit metastasis in the long term.

Cryo-thermal therapy inducing MI macrophage polarization created CXCL10 and IL-6-rich pro-inflammatory environment for CD4+ T cell-mediated anti-tumor immunity.

PURPOSE: We previously developed a novel cryo-thermal therapy to treat malignant mammary carcinoma and melanoma in a mouse model; long-term survival and CD4+ T cell orchestrating anti-tumor immune memory response were achieved. Moreover, cryo-thermal-induced CD4+ T cell differentiation into Th1 and CD4+CTL sub-lineages, in which M1 macrophage polarization played a direct, important role. In particular, cryo-thermal therapy triggered M1 macrophage polarization with up-regulated expression of C-X-C motif ligand 10 (CXCL10) and Interleukin 6 (IL-6). But whether CXCL10 and IL-6 contribute to CD4+ T cell-mediated anti-tumor immunity remains unclear. In this study, the role of cryo-thermal-induced CXCL10 and IL-6 in anti-tumor immunity was determined. METHODS: The level of CXCL10 and IL-6 in spleen and serum was determined by RT-PCR and ELISA on day 14 after cryo-thermal therapy. Splenic dendritic cells (DCs) and macrophages were isolated from cryo-thermal-treated mice on day 5 and 14, and the level of CXCL10 and IL-6 in macrophages and DCs was determined by ELISA. The transwell migration assay was performed to study immune cell migration. In vivo neutralization of CXCL10 or IL-6 was performed to investigate the phenotypic changes of immune cells. RESULTS: Cryo-thermal therapy induced M1 macrophage polarization with up-regulation of CXCL10 and IL-6 expression in spleen. CXCL10 and IL-6 promoted DCs migration and maturation, and subsequently promoted CD4+ T cell migration and differentiation into Th1 and CD4+ CTL, moreover, reduced myeloid-derived suppressor cells (MDSCs) accumulation. CONCLUSIONS: Cryo-thermal-induced CXCL10 and IL-6 created acute inflammatory environment to initiate a systemically cascading innate and adaptive anti-tumor immunity, which was more permissive for tumor eradication.