Brachytherapy for targeting the immune system in cervical cancer patients.

BACKGROUND: New combinations based on standard therapeutic modalities and immunotherapy require understanding the immunomodulatory properties of traditional treatments. The objective was to evaluate the impact of brachytherapy (BT) on the immune system of cervical cancer and to identify the best modality, High-dose-rate brachytherapy (HDR-BT) vs. Pulsed-dose-rate (PDR-BT), to target it. METHODS: Nineteen patients enrolled in a prospective study received chemoradiation (CRT) and subsequently HDR-BT or PDR-BT. Peripheral blood samples were obtained for immunophenotyping analysis by flow-cytometry before CRT, BT, and two and four weeks after BT. The Friedman one-way ANOVA, Conover post hoc test, and the Wilcoxon signed-rank test were used to compare changes in cell populations at different periods, perform multiple pairwise comparisons and assess differences between treatment groups (PDR and HDR). RESULTS: Natural killer cells (NKs) were the best target for BT. Patients receiving HDR-BT achieved significantly higher values ​​and longer time of the CD56dimCD16 + NK cells with greater cytotoxic capacity than the PDR-BT group, which presented their highest elevation of CD56-CD16 + NK cells. Furthermore, both BT modalities were associated with an increase in myeloid-derived suppressor cells (MDSCs), related to a worse clinical prognosis. However, there was a decrease in the percentage of CD4 + CD25 + Foxp3 + CD45RA + regulatory T cells (Tregs) in patients receiving HDR-BT, although there were no significant differences between BT. CONCLUSIONS: Immune biomarkers are important predictive determinants in cervical cancer. Higher cytotoxic NK cells and a trend toward lower values of Tregs might support the use of HDR-BT to the detriment of PDR-BT and help develop effective combinations with immunotherapy.

Clinical and prognostic significance of CD14 (+) HLA-DR (-/low) myeloid-derived suppressor cells in patients with hepatocellular carcinoma received transarterial radioembolization with Yttrium-90.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. For unresectable HCC, transarterial radioembolization (TARE) with Yttrium-90 is a widely used treatment. The aim of this study was to investigate whether monocytic myeloid-derived suppressor cells (M-MDSC) and CD39+ T cells can be non-invasive predictive biomarkers of radiological response and prognosis in patients with HCC treated with TARE. This study was conducted on 39 patients with HCC who were treated with TARE between August 2018 and December 2019 and the control group consisted of 23 healthy volunteers. CD4+, CD8+, CD39+ T cells, Natural killer (NK) cells, myeloid cells (MC) and M-MDSC parameters are examined in the course of TARE treatment with student t test and Kaplan-Meier method. There were statistically significant differences in M-MDSC, CD39+ T cells and MC values between healthy controls and HCC patients. A statistically significant difference was found in M-MDSC and CD4+ T cells values in the HCC patient group who responded to the treatment compared to those who did not. Survival analysis found that patients with lower frequencies (under 3.81%) of M-MDSC showed more prominent differences of overall survival (OS) compared to patients with all high groups. We found that M-MDSC in the peripheral blood might be a useful non-invasive biomarker to predict OS. We have shown for the first time that M-MDSC is correlated with treatment response in HCC patients treated with TARE. Additionally, we have found that the percentage of CD39+ T cells is high in HCC patients and these cells are positively correlated with M-MDSC.

Polymorphonuclear-MDSCs Facilitate Tumor Regrowth After Radiation by Suppressing CD8+ T Cells.

PURPOSE: Radiation therapy (RT) is widely used in the treatment of cancer. Unfortunately, RT alone is insufficient to control the disease in most cases, as regrowth after irradiation still occur. Thus, it would be meaningful to explore the underlying mechanism of tumor regrowth after irradiation. Myeloid-derived suppressor cells (MDSCs) contribute to the immunosuppressive tumor microenvironment and hinder the therapeutic efficacy of RT. However, it is unclear whether MDSCs-mediated immune suppression contributes to local relapse after irradiation. In this article, we tried to figure out how MDSCs sabotage the therapeutic effect of RT, and tried to determine the potential synergistic effect of combination between targeting MDSCs and RT. METHODS AND MATERIALS: A syngeneic murine model of Lewis lung cancer was used. The abundance of tumor infiltrating MDSCs and tumor growth after irradiation was assessed. The percentage and functional state of CD8+ T cells were measured by flow cytometry, with or without polymorphonuclear (PMN)-MDSCs depletion. Arginase 1 (ARG1) expression and activity of MDSCs were examined by hematoxylin and eosin staining and flow cytometry. ARG1 inhibitor and phosphodiesterase 5 inhibitor sildenafil were administered after RT to figure out the underlying mechanism of MDSCs-mediated immunosuppression. RESULTS: We demonstrated that irradiation recruited MDSCs, especially the polymorphonuclear subset, into the tumor microenvironment. PMN-MDSCs inhibited the CD8+ T cell response by elevating ARG1 expression. Selective depletion of PMN-MDSCs or inhibition on ARG1 promoted the infiltration and activation of intratumoral CD8+ T cells, and delayed tumor regrowth after irradiation. We showed that sildenafil reduced the accumulation and ARG1 expression of PMN-MDSCs after irradiation, thus abrogating the MDSCs-mediated immunosuppression. CONCLUSIONS: Our results have suggested that PMN-MDSCs participate in the irradiation-induced immune suppression through ARG1 activation. We have also found that sildenafil has the potential to facilitate antitumor immunity, which provides a new alternative to delay tumor recurrence after RT.

Role of Myeloid-Derived Suppressor Cells in High-Dose-Irradiated TRAMP-C1 Tumors: A Therapeutic Target and an Index for Assessing Tumor Microenvironment.

PURPOSE: To investigate the temporal and spatial infiltration of TRAMP-C1 tumors by myeloid-derived suppressor cells (MDSCs) after high-dose radiation therapy (RT), and to explore their effect on tumor growth. METHODS AND MATERIALS: TRAMP-C1 intramuscularly tumors were irradiated with a single dose of 8 Gy or 25 Gy. The dynamics of infiltrated MDSCs and their intratumoral spatial distribution were assessed by immunohistochemistry and flow cytometry. Cytokine levels in the blood and tumor were analyzed by multiplex immunoassay. Mice were injected with anti-Gr-1 antibody to determine whether MDSCs affect tumor growth after RT. RESULTS: CD11b+Gr-1+ MDSCs infiltrated TRAMP-C1 tumors irradiated with 25 Gy, but not 8 Gy, within 4 hours and recruitment persisted for at least 2 weeks. Both CD11b+Ly6G+Ly6C+ polymorphonuclear-MDSCs (PMN-MDSCs) and CD11b+Ly6G-Ly6Chi monocytic-MDSCs (M-MDSCs) were involved. Tumor RT also increased the representation of both MDSC subpopulations in the spleen and peripheral blood. Levels of multiple cytokines were increased in the tumors at 2 weeks, including GM-CSF, G-CSF, CCL-3, CCL-5, CXCL-5, IL-6, IL-17α, and VEGF-a; while G-CSF, IL-6, and TNF-α levels increased in the blood. PMN-MDSCs aggregated in the central necrotic region of the irradiated tumors over time, where they were associated with avascular hypoxia (CD31-PIMO+). MDSCs expressed the proangiogenic factor, matrix metalloproteinase-9, and, within the necrotic area, high levels of arginase-1 and indoleamine 2,3-dioxygenase. Depletion of PMN-MDSCs by Gr-1 antibody increased the efficacy of high-dose RT. CONCLUSIONS: PMN-MDSCs infiltrate TRAMP-C1 tumors after high-dose RT. Their spatial distribution suggests they are involved in the evolution of an intratumoral state of necrosis associated with avascular hypoxia, and their phenotype is consistent with them being immunosuppressive. They appear to promote tumor growth after RT, making them a prime therapeutic target for therapeutic intervention. Assessment of MDSCs and cytokine levels in blood could be an index of the need for such an intervention.

STAT3 Modulation of Regulatory T Cells in Response to Radiation Therapy in Head and Neck Cancer.

BACKGROUND: Radioresistance represents a major problem in the treatment of head and neck cancer (HNC) patients. To improve response, understanding tumor microenvironmental factors that contribute to radiation resistance is important. Regulatory T cells (Tregs) are enriched in numerous cancers and can dampen the response to radiation by creating an immune-inhibitory microenvironment. The purpose of this study was to investigate mechanisms of Treg modulation by radiation in HNC. METHODS: We utilized an orthotopic mouse model of HNC. Anti-CD25 was used for Treg depletion. Image-guided radiation was delivered to a dose of 10 Gy. Flow cytometry was used to analyze abundance and function of intratumoral immune cells. Enzyme-linked immunosorbent assay was performed to assess secreted factors. For immune-modulating therapies, anti-PD-L1, anti-CTLA-4, and STAT3 antisense oligonucleotide (ASO) were used. All statistical tests were two-sided. RESULTS: Treatment with anti-CD25 and radiation led to tumor eradication (57.1%, n = 4 of 7 mice), enhanced T-cell cytotoxicity compared with RT alone (CD4 effector T cells [Teff]: RT group mean = 5.37 [ 0.58] vs RT + αCD25 group mean =10.71 [0.67], P = .005; CD8 Teff: RT group mean = 9.98 [0.81] vs RT + αCD25 group mean =16.88 [2.49], P = .01) and induced tumor antigen-specific memory response (100.0%, n = 4 mice). In contrast, radiation alone or when combined with anti-CTLA4 did not lead to durable tumor control (0.0%, n = 7 mice). STAT3 inhibition in combination with radiation, but not as a single agent, improved tumor growth delay, decreased Tregs, myeloid-derived suppressor cells, and M2 macrophages and enhanced effector T cells and M1 macrophages. Experiments in nude mice inhibited the benefit of STAT3 ASO and radiation. CONCLUSION: We propose that STAT3 inhibition is a viable and potent therapeutic target against Tregs. Our data support the design of clinical trials integrating STAT3 ASO in the standard of care for cancer patients receiving radiation.

Bendamustine with Total Body Irradiation Limits Murine Graft-versus-Host Disease in Part Through Effects on Myeloid-Derived Suppressor Cells.

Graft-versus-host disease (GVHD) remains a significant challenge in allogeneic hematopoietic cell transplantation (HCT). An underinvestigated strategy to reduce GVHD is the modification of the preparative conditioning regimen. In the present study, we aimed to evaluate GVHD associated with bendamustine (BEN) conditioning in conjunction with total body irradiation (TBI) as an alternative to the standard myeloablative regimen of cyclophosphamide (CY) and TBI. We demonstrate that BEN-TBI conditioning, although facilitating complete donor chimerism, results in significantly less GVHD compared with CY-TBI. In BEN-TBI-conditioned mice, suppressive CD11b+Gr-1high myeloid cells are increased in the blood, bone marrow, spleen, and intestines. When Gr-1high cells are depleted before transplantation, the beneficial effects of BEN-TBI are partially lost. Alternatively, administration of granulocyte colony-stimulating factor, which promotes CD11b+Gr-1+ myeloid cell expansion, is associated with a trend toward increased survival in BEN-TBI-conditioned mice. These findings indicate a potential role of myeloid-derived suppressor cells in the mechanism by which BEN allows engraftment with reduced GVHD. BEN-TBI conditioning may present a safer alternative to CY-TBI conditioning for allogeneic HCT.

Targeting Myeloid-derived Suppressor Cells and Programmed Death Ligand 1 Confers Therapeutic Advantage of Ablative Hypofractionated Radiation Therapy Compared With Conventional Fractionated Radiation Therapy.

PURPOSE: Ablative hypofractionated radiation therapy (AHFRT) presents a therapeutic advantage compared with conventional fractionated radiation therapy (CFRT) for primary and oligometastatic cancers. However, the underlying mechanisms remain largely unknown. In the present study, we compared the immune alterations in response to AHFRT versus CFRT and examined the significance of immune regulations contributing to the efficacy of AHFRT. METHODS AND MATERIALS: We established subcutaneous tumors using syngeneic lung cancer and melanoma cells in both immunocompetent and immunocompromised mice and treated them with AHFRT and CFRT under the same biologically equivalent dose. RESULTS: Compared with CFRT, AHFRT significantly inhibited tumor growth in immunocompetent, but not immunocompromised, mice. On the cellular level, AHFRT reduced the recruitment of myeloid-derived suppressor cells (MDSCs) into tumors and decreased the expression of programmed death-ligand 1 (PD-L1) on those cells, which unlashed the cytotoxicity of CD8+ T cells. Through the downregulation of vascular endothelial growth factor (VEGF), AHFRT inhibited VEGF/VEGF receptor signaling, which was essential for MDSC recruitment. When combined with anti-PD-L1 antibody, AHFRT presented with greater efficacy in controlling tumor growth and improving mouse survival. By altering immune regulation, AHFRT, but not CFRT, significantly delayed the growth of secondary tumors implanted outside the irradiation field. CONCLUSIONS: Targeting MDSC recruitment and enhancing antitumor immunity are crucial for the therapeutic efficacy of AHFRT. When combined with anti-PD-L1 immunotherapy, AHFRT was more potent for cancer treatment.

[Effect of concurrent chemoradiotherapy and radiotherapy alone on peripheral myeloid-derived suppressor and T regulatory cells in patients with nasopharyngeal cancer].

Objective: To investigate the percentage of myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg) in peripheral blood of nasopharyngeal cancer (NPC) patients undergoing concurrent chemoradiotherapy or radiotherapy alone. Methods: Sixty NPC patients who received radiotherapy or concurrent chemoradiotherapy from September 2012 to November 2015 and 20 healthy individuals were included in this study. For the patients, the blood samples were collected at four time points: pre-radiation (Pre-RT), reaching a dose of 40 Gy (RT-40 Gy), finishing radiation (RT-finish) and three months after finishing radiation (3m-post-RT). Flow cytometry was used to evaluate the percentage of Treg (CD4(+) CD25(+) CD127(low/-)) and MDSC (HLA-DR(-)CD11b(+) CD33(+) ) cells in peripheral blood. Results: Treg and MDSC cells were present in peripheral blood lymphocytes of healthy individuals as a percentage of (7.50±1.62)% and (1.08±0.48)%, respectively. The proportions of peripheral Treg cells in patients at Pre-RT, RT-40 Gy, RT-finish and 3m-post-RT time points were (8.42± 1.52)%, (9.10±1.57)%, (8.87±1.56)% and (7.31±1.43)%, respectively, showing a statistically significant difference between Pre-RT and the other groups (P<0.05). At Pre-RT point, the percentage of Treg cells in Stage Ⅲ-Ⅳ patients [(8.63±1.39)%] was higher than that in Stage Ⅰ-Ⅱ [(7.65±1.94)%, P=0.042]. Moreover, the proportions of peripheral MDSC cells in patients at Pre-RT, RT-40 Gy, RT-finish and 3m-post-RT time points were (2.14±1.21)%, (4.08±1.90)%, (3.76±1.31)% and (1.52±0.88)%, respectively. The percentages of MDSC cells at RT-40 Gy and RT-finish points were significantly higher than those at Pre-RT, while the percentage of MDSC cells at 3m-post-RT was significantly lower than those at Pre-RT (P<0.05). At Pre-RT point, the percentage of MDSC cells in Stage Ⅲ-Ⅳ patients [(2.25±1.26)%] was higher than that in Stage Ⅰ-Ⅱ [(1.35±0.66)%, P=0.007]. At RT-finish point, the proportions of MDSC and Treg cells in patients with Ⅲ-Ⅳ grade of radiation induced oral mucositis [(4.41±1.27)% and (9.91±1.23)%] were significantly higher than those in Ⅰ-Ⅱ grade patients [(3.15±1.04)% and (8.41±1.52)%, both of P<0.05]. Conclusions: The proportions of MDSC and Treg cells in initial treated NPC patients are higher than healthy individuals, and they are also associated with the tumor stages. During the concurrent chemoradiotherapy and radiation, the percentage of MDSC and Treg cells is elevated, suggesting a decreased immune activity. The increase of MDSC and Treg cells is related to radiation induced oral mucositis.

Altered Biological Potential and Radioresponse of Murine Tumors in Different Microenvironments.

PURPOSE: This study was conducted to evaluate the biological features of murine hepatocarcinoma according to different tumor microenvironmental models and to determine the change in molecular and immunologic responses after radiation. MATERIALS AND METHODS: Tumor models were established in the liver (orthotopic) and thigh (heterotopic) of male C3H/HeN mice. Tumor growth and lung metastasis were assessed in these models. To evaluate the radiation effect, the tumors were irradiated with 10 Gy. Factors associated with tumor microenvironment including vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), transforming growth factor beta1 (TGF-ß1), CD31, and serum interleukin-6 (IL-6) were evaluated. Tumor-infiltrating regulatory immune cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs) were also analyzed. RESULTS: A higher number of lung metastases were observed in the orthotopic tumor model than in the heterotopic tumor model. VEGF, CD31, COX-2, and TGF-ß1 expression was more prominent in the orthotopic tumor model than in the heterotopic tumor model. Expression of the angiogenic factor VEGF and key regulatory molecules (TGF-ß1 and COX-2) decreased following radiation in the orthotopic tumor model, while the serum IL-6 level increased after radiation. In the orthotopic tumor model, the number of both Tregs and MDSCs in the tumor burden decreased after radiation. CONCLUSION: The orthotopic tumor model showed higher metastatic potential and more aggressive molecular features than the heterotopic tumor model. These findings suggest that the orthotopic tumor mouse model may be more reflective of the tumor microenvironment and suitable for use in the translational research of radiation treatment.