ASMase is Essential for the Immune Response to Partial-Tumor Radiation Exposure.

BACKGROUND/AIMS: Tumor response to radiation is thought to depend on the direct killing of tumor cells. Our laboratory has called this into question. Firstly, we showed that the biology of the host, specifically the endothelial expression of acid sphingomyelinase (ASMase), was critical in determining tumor radiocurability. Secondly, we have shown that the immune system can enhance radiation response by allowing a complete tumor control in hemi-irradiated tumors. In this paper, we focus on the integration of these two findings. METHODS: We used Lewis Lung Carcinoma (LLC) cells, injected in the flank of either: (i) ASMase knockout or (ii) WT of matched background (sv129xBl/6) or (iii) C57Bl/6 mice. Radiation therapy (RT) was delivered to 50% or 100% of the LLC tumor volume. Tumor response, immune infiltration (CD8+ T cells), ICAM-1, and STING activation were measured. Radiotherapy was also combined with methyl-cyclodextrin, to inhibit the ASMase-mediated formation of ceramide-enriched lipid rafts. RESULTS: We recapitulated our previous finding, namely that tumor hemi-irradiation was sufficient for tumor control in the LLC/C57Bl/6 model. However, in ASMase KO mice hemi-irradiation was ineffective. Likewise, pharmacological inhibition of ASMase significantly reduced the tumor response to hemi-irradiation. Further, we demonstrated elevated ICAM-1 expression, increased levels of CD8+ T cells, ICAM-1, and STING activation in tumors growing in C57Bl/6 mice, as well as the ASMase WT strain. However, no such changes were seen in tumors growing in ASMase KO mice. CONCLUSION: ASMase and ceramide generation are necessary to mediate a radiation-induced anti-tumor immune response via STING activation.

Single-cell RNA sequencing reveals recruitment of the M2-like CCL8high macrophages in Lewis lung carcinoma-bearing mice following hypofractionated radiotherapy.

BACKGROUND: Tumor-associated macrophages (TAMs) play a pivotal role in reshaping the tumor microenvironment following radiotherapy. The mechanisms underlying this reprogramming process remain to be elucidated. METHODS: Subcutaneous Lewis lung carcinoma (LLC) murine model was treated with hypofrationated radiotherapy (8 Gy × 3F). Single-cell RNA sequencing was utilized to identify subclusters and functions of TAMs. Multiplex assay and enzyme-linked immunosorbent assay (ELISA) were employed to measure serum chemokine levels. Bindarit was used to inhibit CCL8, CCL7, and CCL2. The infiltration of TAMs after combination treatment with hypofractionated radiotherapy and Bindarit was quantified with flow cytometry, while the influx of CD206 and CCL8 was assessed by immunostaining. RESULTS: Transcriptome analysis identified a distinct subset of M2-like macrophages characterized by elevated Ccl8 expression level following hypofractionated radiotherapy in LLC-bearing mice. Remarkbly, hypofractionated radiotherapy not only promoted CCL8high macrophages infiltration but also reprogrammed them by upregulating immunosuppressive genes, thereby fostering an immunosuppressive tumor microenvironment. Additioinally, hypofractionated radiotherapy enhanced the CCL signaling pathway, augmenting the pro-tumorigenic functions of CCL8high macrophages and boosting TAMs recruitment. The adjunctive treatment combining hypofractionated radiotherapy with Bindarit effectively reduced M2 macrophages infiltration and prolonged the duration of local tumor control. CONCLUSIONS: Hypofractionated radiotherapy enhances the infiltration of CCL8high macrophages and amplifies their roles in macrophage recruitment through the CCL signaling pathway, leading to an immunosuppressive tumor microenvironment. These findings highlight the potential of targeting TAMs and introduces a novel combination to improve the efficacy of hypofractionated radiotherapy.

[Effects of high-fat and low-carbohydrate diet combined with radiotherapy on tumor microenvironment of Lewis lung cancer bearing mice].

Objective: To investigate the effect of high-fat and low-carbohydrate diet combined with radiotherapy on the tumor microenvironment of mice with lung xenografts. Methods: C57BL/6J mice were selected to establish the Lewis lung cancer model, and they were divided into the normal diet group, the high-fat and low-carbohydrate diet group, the normal diet + radiotherapy group, and the high-fat and low-carbohydrate diet + radiotherapy group, with 18 mice in each group. The mice in the normal diet group and the normal diet + radiotherapy group were fed with the normal diet with 12.11% fat for energy supply, and the mice in the high-fat and low-carbohydrate diet group and the high-fat and low-carbohydrate diet + radiotherapy group were fed with high-fat and low-carbohydratediet with 45.00% fat for energy. On the 12th to 14th days, the tumor sites of the mice in the normal diet + radiotherapy group and the high-fat and low-carbohydrate diet + radiotherapy group were treated with radiotherapy, and the irradiation dose was 24 Gy/3f. The body weight, tumor volume, blood glucose and blood ketone level, liver and kidney function, and survival status of the mice were observed and monitored. Immunohistochemical staining was used to detect the tumor-associated microangiogenesis molecule (CD34) and lymphatic endothelial hyaluronan receptor 1 (LYVE-1), Sirius staining was used to detect collagen fibers, and multiplex immunofluorescence was used to detect CD8 and programmed death-1 (PD-1). Expression of immune cell phenotypes (CD3, CD4, CD8, and Treg) was detected by flow cytometry. Results: On the 27th day after inoculation, the body weigh of the common diet group was(24.78±2.22)g, which was significantly higher than that of the common diet + radiotherapy group [(22.15±0.48)g, P=0.030] and high-fat low-carbohydrate diet + radiotherapy group [(22.02±0.77)g, P=0.031)]. On the 15th day after inoculation, the tumor volume of the high-fat and low-carbohydrate diet + radiotherapy group was (220.88±130.05) mm3, which was significantly smaller than that of the normal diet group [(504.37±328.48) mm3, P=0.042)] and the high-fat, low-carbohydrate diet group [(534.26±230.42) mm3, P=0.016], but there was no statistically significant difference compared with the normal diet + radiotherapy group [(274.64±160.97) mm3]. In the 4th week, the blood glucose values of the mice in the high-fat and low-carbohydrate diet group were lower than those in the normal diet group, with the value being (8.00±0.36) mmol/L and (9.57±0.40) mmol/L, respectively, and the difference was statistically significant (P＜0.05). The blood ketone values of the mice in the high-fat and low-carbohydrate diet group were higher than those in the normal diet group, with the value being (1.00±0.20) mmol/L and (0.63±0.06) mmol/L, respectively, in the second week. In the third week, the blood ketone values of the two groups of mice were (0.90±0.17) mmol/L and (0.70±0.10) mmol/L, respectively, and the difference was statistically significant (P＜0.05). On the 30th day after inoculation, there were no significant differences in aspartate aminotransferase, alanine aminotransferase, creatinine, and urea between the normal diet group and the high-fat, low-carbohydrate diet group (all P＞0.05). The hearts, livers, spleens, lungs, and kidneys of the mice in each group had no obvious toxic changes and tumor metastasis. In the high-fat and low-carbohydrate diet + radiotherapy group, the expression of CD8 was up-regulated in the tumor tissues of mice, and the expressions of PD-1, CD34, LYVE-1, and collagen fibers were down-regulated. The proportion of CD8+ T cells in the paratumoral lymph nodes of the high-fat and low-carbohydrate diet + radiotherapy group was (25.13±0.97)%, higher than that of the normal diet group [(20.60±2.23)%, P＜0.050] and the normal diet + radiotherapy group [(19.26±3.07)%, P＜0.05], but there was no statistically significant difference with the high-fat and low-carbohydrate diet group [(22.03±1.75)%, P＞0.05]. The proportion, of CD4+ T cells in the lymph nodes adjacent to the tumor in the normal diet + radiotherapy group (31.33±5.16)% and the high-fat and low-carbohydrate diet + radiotherapy group (30.63±1.70)% were higher than that in the normal diet group [(20.27±2.15)%, P＜0.05] and the high-fat and low-carbohydrate diet group (23.70±2.62, P＜0.05). Treg cells accounted for the highest (16.58±5.10)% of T cells in the para-tumor lymph nodes of the normal diet + radiotherapy group, but compared with the normal diet group, the high-fat and low-carbohydrate diet group, and the high-fat and low-carbohydrate diet + radiotherapy group, there was no statistically significant difference (all P＞0.05). Conclusion: High-fat and low-carbohydrate diet plus radiotherapy can enhance the recruitment and function of immune effector cells in the tumor microenvironment, inhibit tumor microangiogenesis, and thus inhibit tumor growth.

Differential therapeutic effects of PARP and ATR inhibition combined with radiotherapy in the treatment of subcutaneous versus orthotopic lung tumour models.

BACKGROUND: Subcutaneous mouse tumour models are widely used for the screening of novel antitumour treatments, although these models are poor surrogate models of human cancers. METHODS: We compared the antitumour efficacy of the combination of ionising radiation (IR) with two DNA damage response inhibitors, the PARP inhibitor olaparib and the ATR inhibitor AZD6738 (ceralasertib), in subcutaneous versus orthotopic cancer models. RESULTS: Olaparib delayed the growth of irradiated Lewis lung carcinoma (LL2) subcutaneous tumours, in agreement with previous reports in human cell lines. However, the olaparib plus IR combination showed a very narrow therapeutic window against LL2 lung orthotopic tumours, with nearly no additional antitumour effect compared with that of IR alone, and tolerability issues emerged at high doses. The addition of AZD6738 greatly enhanced the efficacy of the olaparib plus IR combination treatment against subcutaneous but not orthotopic LL2 tumours. Moreover, olaparib plus AZD6738 administration concomitant with IR even worsened the response to radiation of head and neck orthotopic tumours and induced mucositis. CONCLUSIONS: These major differences in the responses to treatments between subcutaneous and orthotopic models highlight the importance of using more pathologically relevant models, such as syngeneic orthotopic models, to determine the most appropriate therapeutic approaches for translation to the clinic.

Synergistic effect and reduced toxicity by intratumoral injection of cytarabine-loaded hyaluronic acid hydrogel conjugates combined with radiotherapy on lung cancer.

The aim of this study was to explore the synergistic anti-tumor effects of cytarabine hyaluronic acid-tyramine (Ara-HA-Tyr) hydrogel conjugates and radiotherapy (RT) in the Lewis lung cancer (LLC) xenograft model, and the mechanisms involved. The radiotherapy sensitization ratio (SER) of 0.5 µg cytarabine (Ara-C) was 1.619 in the LLC cells. Ara-HA-Tyr was prepared by encapsulating Ara-C into hyaluronic acid-tyramine (HA-Tyr) conjugates. The hydrogels were formed through the oxidative coupling of tyramines by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). Mice engrafted with the LLC cells were given intra-tumoral injections of saline, Ara-C or Ara-HA-Tyr, with or without RT. The combination of Ara-HA-Tyr and RT increased survival compared to free Ara-C and RT (p < 0.05), and prolonged tumor growth delay (TGD). Furthermore, the RT + Ara-HA-Tyr combination therapy significantly reduced 18F-FDG uptake, induced cell cycle arrest at G2/M-phase, increased apoptosis and histone H2AX phosphorylation (γ-H2AX), and decreased the proliferation index (Ki67) in tumor cells compared to either monotherapy. Taken together, Ara-C encapsulated with HA-Tyr effectively sensitized tumor xenografts to RT and showed significantly less systemic toxicity. Graphical abstract In this work, Ara-C encapsulated with hyaluronic acid-tyramine conjugates (HA-Tyr) was prepared and used to investigate its synergistic anti-tumor efficacy by combination with radiotherapy in the Lewis lung cancer xenograft model. The synergistic mechanism may be related to tumor cell cycle redistribution, apoptosis and expression of histone H2AX phosphorylation.

Toll-like receptor 3 signal augments radiation-induced tumor growth retardation in a murine model.

Radiotherapy induces anti-tumor immunity by induction of tumor antigens and damage-associated molecular patterns (DAMP). DNA, a representative DAMP in radiotherapy, activates the stimulator of interferon genes (STING) pathway which enhances the immune response. However, the immune response does not always parallel the inflammation associated with radiotherapy. This lack of correspondence may, in part, explain the radiation-resistance of tumors. Additive immunotherapy is expected to revive tumor-specific CTL facilitating radiation-resistant tumor shrinkage. Herein pre-administration of the double-stranded RNA, polyinosinic-polycytidylic acid (polyI:C), in conjunction with radiotherapy, was shown to foster tumor suppression in mice bearing radioresistant, ovalbumin-expressing Lewis lung carcinoma (LLC). Extrinsic injection of tumor antigen was not required for tumor suppression. No STING- and CTL-response was induced by radiation in the implant tumor. PolyI:C was more effective for induction of tumor growth retardation at 1 day before radiation than at post-treatment. PolyI:C targeted Toll-like receptor 3 with minimal effect on the mitochondrial antiviral-signaling protein pathway. Likewise, the STING pathway barely contributed to LLC tumor suppression. PolyI:C primed antigen-presenting dendritic cells in draining lymph nodes to induce proliferation of antigen-specific CTL. By combination therapy, CTL efficiently infiltrated into tumors with upregulation of relevant chemokine transcripts. Batf3-positive DC and CD8+ T cells were essential for therapeutic efficacy. Furthermore, polyI:C was shown to stimulate tumor-associated macrophages and release tumor necrosis factor alpha, which acted on tumor cells and increased sensitivity to radiation. Hence, polyI:C treatment prior to radiotherapy potentially induces tumor suppression by boosting CTL-dependent and macrophage-mediated anti-tumor responses. Eventually, polyI:C and radiotherapy in combination would be a promising therapeutic strategy for radiation-resistant tumors.

Combination radiotherapy and cantharidin inhibits lung cancer growth through altering tumor infiltrating lymphocytes.

This study aimed to detect the effect of combination radiotherapy and cantharidin on lung cancer growth. We found that combination therapy with radiotherapy and cantharidin was more effective in inhibiting the tumor growth than radiotherapy or cantharidin alone. It decreased the percentage of CD4+ Tregs and enhanced the percentage of CD8+ T cells, CD4+ Teff cells when comparing to that of single treatment. Combination therapy promoted a great increase in double producing CD8+ T cells and CD4+ Teff cells in tumor infiltrating lymphocytes. Overexpression of CTLA4 reversed the inhibitory action of combination treatment on cancer growth. Our data suggest that combining radiotherapy and cantharidin may have synergistic effects in driving tumor rejection by increasing T-cell infiltration, proliferation and cytokine production.

[Effects of simvastatin on the proliferation, invasion and radiosensitivity in Lewis lung cancer cell line].

Objective: To investigate the effects of simvastatin on proliferation, invasion and radiosensitivity of mouse Lewis lung cancer cell line in vitro. Methods: The inhibitory effects of simvastatin on proliferation of Lewis lung cancer cells were detected by MTT assay. Matrigel invasion and migration assay was used to determine the invasion and motility ability of the Lewis cells. P38 activity was measured by p38 activity detection kit, and the expressions of p-p38, MKP-1, RhoA and MMP-2 were analyzed by Western blot. Lung cancer xenograft model was established in C57BL/6 mice. The mice were randomly divided into control group, simvastatin group, radiotherapy alone group and combined treatment group. The mice were killed 27 days after inoculation. The tumor mass, volume and lung metastatic nodules in the mice were compared. Results: The cell proliferation rates of 0 µmol/L, 10 µmol/L, 20 µmol/L and 30 µmol/L simvastatin groups were 100%, (87.0±9.0)%, (76.5±8.1)% and (67.0±7.3)%, respectively (P<0.05). Invasive cell numbers of the above groups were 298±30, 251±26, 207±20 and 132±19 per field, respectively (P<0.05). The intracellular p38 activities were 100%, (83.1±8.8)%, (70.2±8.2)% and (59.0±6.4)%, respectively. The relative expressions of p-p38 were 100%, (76.2±6.7)%, (56.4±5.4)% and (36.5±3.2)%, respectively. The expressions of RhoA were 100%, (80.1±5.3)%, (55.3±6.2)% and (38.6±4.8)%, respectively. The expressions of MMP-2 were 100%, (89.6±8.6)%, (51.9±4.7)% and (42.7±3.1)%, respectively, while the expressions of MKP-1 were 100%, (136.5±12.2)%, (168.8±15.3)% and (187.7±13.4)%, respectively (all P<0.05). Lung metastatic nodules and mass in the control, simvastatin, radiotherapy group and combined treatment groups were 6.24±1.09, 3.07±0.71 g, 5.09±1.16, 2.43±0.53 g, 3.12±0.68, 1.96±0.62 g and 2.65±0.38, 1.12±0.43 g, respectively (all P<0.05). The tumor inhibition rates were 39.0%, 48.1% and 26.5%, respectively, in the radiotherapy alone, combined treatment and simvastatin groups (all P<0.05). Conclusions: Simvastatin inhibits the proliferation of Lewis cell line by inhibiting the activity of p38 and expression of p-p38. Meanwhile, simvastatin reduces the invasion and motility of Lewis cell line through down-regulating the expression of RhoA and MMP-2. When combined with radiotherapy, simvastatin can inhibit tumor growth and metastasis, and improve the treatment efficacy of radiotherapy synergistically.

Pirfenidone enhances the efficacy of combined radiation and sunitinib therapy.

Radiotherapy is a widely used treatment for many tumors. Combination therapy using anti-angiogenic agents and radiation has shown promise; however, these combined therapies are reported to have many limitations in clinical trials. Here, we show that radiation transformed tumor endothelial cells (ECs) to fibroblasts, resulting in reduced vascular endothelial growth factor (VEGF) response and increased Snail1, Twist1, Type I collagen, and transforming growth factor (TGF)-ß release. Irradiation of radioresistant Lewis lung carcinoma (LLC) tumors greater than 250 mm³ increased collagen levels, particularly in large tumor vessels. Furthermore, concomitant sunitinib therapy did not show a significant difference in tumor inhibition versus radiation alone. Thus, we evaluated multimodal therapy that combined pirfenidone, an inhibitor of TGF-induced collagen production, with radiation and sunitinib treatment. This trimodal therapy significantly reduced tumor growth, as compared to radiation alone. Immunohistochemical analysis revealed that radiation-induced collagen deposition and tumor microvessel density were significantly reduced with trimodal therapy, as compared to radiation alone. These data suggest that combined therapy using pirfenidone may modulate the radiation-altered tumor microenvironment, thereby enhancing the efficacy of radiation therapy and concurrent chemotherapy.

Radiotherapy promotes tumor-specific effector CD8+ T cells via dendritic cell activation.

Radiotherapy is an important treatment for cancer. The main mode of action is thought to be the irreversible damage to tumor cell DNA, but there is evidence that irradiation mobilizes tumor-specific immunity, and recent studies showed that the efficacy of high-dose radiotherapy depends on the presence of CD8(+) T cells. We show in this study that the efficacy of radiotherapy given as a single, high dose (10 Gy) crucially depends on dendritic cells and CD8(+) T cells, whereas CD4(+) T cells or macrophages are dispensable. We show that local high-dose irradiation results in activation of tumor-associated dendritic cells that in turn support tumor-specific effector CD8(+) T cells, thus identifying the mechanism that underlies radiotherapy-induced mobilization of tumor-specific immunity. We propose that in the absence of irradiation, the activation status of dendritic cells rather than the amount of tumor-derived Ag is the bottleneck, which precludes efficient anti-tumor immunity.

[The bio-effects of high single-dose radiation on xenografts of Lewis lung carcinoma].

OBJECTIVE: To investigate the bio-effects of high single-dose radiation on xenografts of Lewis lung carcinoma. METHODS: Female 8-week-old C57 mice bearing 4-6 mm diameter Lewis lung carcinoma tumors in the hind legs were divided into 3 groups, control group (0 Gy), high single-dose group (12 Gy/one fraction/day) and routine radiation group (22 Gy/11 fraction/15 d). The mean biological effective dose (BED) of both radiation groups was 26.4 Gy. Changes in hypoxia, DNA damage and cell cycle of the tumor cells at 1, 3, 8, 15 and 21 d after first irradiation was assessed by immunofluorescence and flow-cytometry and the tumor growth curve was also made. RESULTS: Compared to the fractionated treatment, the tumor growth was delayed after single dose irradiation. The percent of hypoxic cells after single dose radiation was lower than fractioned irradiation at 3, 8, 15 d after first radiation. The foci of gamma-H2AX showed that the single dose caused heavier DNA damages than fractioned irradiation at 1, 3 d after first radiation. The decline of G0/G1 percentage and increase of G2/M percentage of cells was found in both radiation schedules, but the G2/M percentage after single dose radiation was higher. CONCLUSION: In the C57 mice bearing Lewis lung carcinoma, the high single-dose regimen inhibits the tumor growth more than fractioned irradiation. We hypothesized that conversion of high single-dose to BED using the LQ formalism under estimated the in vivo effect of hypofractionated radiation.

MHY1485 potentiates immunogenic cell death induction and anti-cancer immunity following irradiation.

Recent in vitro experiments showed that combined treatment with MHY1485, a low-molecular-weight compound, and X-ray irradiation significantly increased apoptosis and senescence in tumor cells, which was associated with oxidative stress, endoplasmic reticulum (ER) stress and p21 stabilization, compared to radiation treatment alone. However, evidence for MHY1485 treatment-mediated suppression of tumor growth in animals is still lacking. Furthermore, it has been shown that ER stress enhances immunogenic cell death (ICD) in tumor cells, as it can exert a favorable influence on the anti-cancer immune system. In the present study, we examined whether co-treatment of MHY1485 and X-ray irradiation induces ICD and in vivo tumor growth suppression using the CT26 and Lewis lung carcinoma murine tumor cell lines. We found that MHY1485 + X-ray treatment promotes ICD more effectively than X-ray treatment alone. MHY1485 suppresses tumor growth in vivo under co-treatment with X-rays and increases INF-γ, tumor necrosis factor, interleukin-2 and interleukin-12 levels in the spleen as well as the presence of CD8+ cells in the tumor. The results suggest that MHY1485 treatment leads to the conversion of irradiated tumors into effective vaccines. Thus, MHY1485 is a promising lead compound for use in combination with radiotherapy.

C-C chemokine receptor 4 (CCR4)-positive regulatory T cells interact with tumor-associated macrophages to facilitate metastatic potential after radiation.

PURPOSE: Our previous study revealed that elevated C-C motif chemokine ligand 2 (CCL2) secretion by irradiated cancer cells recruited C-C motif chemokine receptor 2 (CCR2)-positive myeloid cells and polarized M2-type tumor-associated macrophages (TAMs), promoting lung metastasis in an established mouse model. This study investigated the impact of CCL2 and TAMs on adaptive immunity. METHODS: We assessed the influence of CCL2 and TAMs on adaptive immunity through two ectopic allograft mouse models constructed with MB49 bladder cancer cells and Lewis lung carcinoma cells. Both models exhibited delayed primary tumor growth following radiation therapy (RT), but RT promoted the development of pulmonary metastases in C57BL/6 mice. Additionally, we employed a direct coculture system to investigate the interaction between macrophages and target cells in the context of adaptive immunity. RESULTS: C-C motif chemokine receptor 4 (CCR4)-positive regulatory T cells (Tregs) were recruited to the postirradiated tumor microenvironment (TME). Utilizing a CCR4 antagonist to inhibit CCL2-CCR4 activation reversed the infiltration of CCR4 + Tregs and reduced the incidence of pulmonary metastases. In addition, a positive feedback loop between M2-type TAMs and Tregs was observed. The combined blockade of the CCL2-CCR4 and CCL2-CCR2 signaling pathways further decreased the risk of RT-promoted lung metastasis. CONCLUSION: The recruitment of CCR4 + Tregs to the postirradiated TME increases the metastatic potential of tumor cells through increased interactions with M2-type TAMs. A significant reduction in post-RT lung metastases in ectopic mouse models was achieved by disrupting the recruitment of both CCR4 + Tregs and CCR2 + myeloid cells, which are TAM precursors.

Imaging the efficacy of UVC irradiation on superficial brain tumors and metastasis in live mice at the subcellular level.

The effect of UVC irradiation was investigated on a model of brain cancer and a model of experimental brain metastasis. For the brain cancer model, brain cancer cells were injected stereotactically into the brain. For the brain metastasis model, lung cancer cells were injected intra-carotidally or stereotactically. The U87 human glioma cell line was used for the brain cancer model, and the Lewis lung carcinoma (LLC) was used for the experimental brain metastasis model. Both cancer cell types were labeled with GFP in the nucleus and RFP in the cytoplasm. A craniotomy open window was used to image single cancer cells in the brain. This double labeling of the cancer cells with GFP and RFP enabled apoptosis of single cells to be imaged at the subcellular level through the craniotomy open window. UVC irradiation, beamed through the craniotomy open window, induced apoptosis in the cancer cells. UVC irradiation was effective on LLC and significantly extended survival of the mice with experimental brain metastasis. In contrast, the U87 glioma was relatively resistant to UVC irradiation. The results of this study suggest the use of UVC for treatment of superficial brain cancer or metastasis.

Cisplatin and Albumin-Based Gold-Cisplatin Nanoparticles Enhance Ablative Radiation Therapy-Induced Antitumor Immunity in Local and Distant Tumor Microenvironment.

PURPOSE: Ablative radiation therapy (RT) is an important strategy to eliminate primary tumor and can potentially induce the abscopal effect. Human serum albumin nanoparticle (NP) was used for controlled release of cisplatin to decrease cisplatin's systemic toxicity, and gold (Au) was added to increase RT-induced immunogenic cell death and potentiate the abscopal antitumor immunity. METHODS AND MATERIALS: The designed albumin-based cisplatin-conjugated AuNPs were administered concurrently with ablative RT. C57BL/6 mice implanted with syngeneic murine Lewis lung carcinoma or murine MB49 tumor models were treated with ablative RT (12 Gy per fraction for 2 fractions, total 24 Gy), cisplatin, or Au-cisplatin NPs. RESULTS: Combining ablative RT with cisplatin or Au-cisplatin NPs both destroyed the primary tumor effectively and elicited immunogenic cell death accompanied by release of danger-associated molecular patterns. This enhanced recruitment of effector tumor-infiltrating immune cells, including natural killer T cells and CD8+ T cells, and elicited an increased percentage of professional antigen-presenting CD11c+ dendritic cells. Transient weight loss, accompanying hepatotoxicity, nephrotoxicity, and hematopoietic suppression, was observed as a systemic adverse event in the cisplatin but not the Au-cisplatin NPs group. Cisplatin and Au-cisplatin NPs both showed equivalent ability to reduce metastatic potential when combined with ablative RT, confirmed by suppressed unirradiated flank tumor growth and decreased metastatic lung tumor burden, which translated to improved survival. Mobilization and abundance of effector tumor-infiltrating immune cells including CD8+ T cells and dendritic cells were observed in the distant lung tumor microenvironment after ablative RT with cisplatin or Au-cisplatin NPs, demonstrating increased antitumor immunotherapeutic activity as an abscopal effect. CONCLUSIONS: Compared with cisplatin, the albumin-based Au-cisplatin NPs exhibited equivalent but no superior antitumor immunotherapeutic activity while reducing systemic adverse events and can be safely administered concurrently with ablative RT. Alternative NP formulations may be designed to further improve anticancer outcomes.

Low- dose Apatinib promotes vascular normalization and hypoxia reduction and sensitizes radiotherapy in lung cancer.

BACKGROUND AND PURPOSE: Abnormal vascular network of tumor can create a hypoxic microenvironment, and reduce radiotherapy sensitivity. Normalization of tumor vasculature can be a new therapeutic strategy for sensitizing radiotherapy. This study aimed to explore the effect of apatinib on vascular normalization, as well as the syngeneic effect with radiotherapy on lung cancer. MATERIALS AND METHODS: Lewis lung carcinoma (LLC) xenograft-bearing female C57BL/6 mice were treated with different doses of apatinib (30, 60, and 120 mg/kg per day) and/or radiation therapy (8 Gy/1F) and then sacrificed to harvest tumor tissue for immunohistochemical test. Further 18 F-FMISO micro- PET in vivo explored the degree of hypoxia. RESULTS: Immunohistochemistry of CD31 and alpha-smooth muscle actin (α-SMA) proved that low-dose apatinib can normalize vasculature in tumor, especially on Day 10. Tissue staining of hypoxyprobe-1 and 18 F-FMISO micro- PET in vivo showed that 60 mg/kg/day of apatinib significantly alleviates hypoxia. Moreover, this study further proved that low-dose apatinib (60 mg/kg/day) can enhance the radio-response of LLC xenograft mice. CONCLUSION: Our data suggested that low- dose apatinib can successfully induce a vascular normalization window and function as a radio- sensitizer in the lung cancer xenografts model.

Anlotinib Enhances the Antitumor Activity of High-Dose Irradiation Combined with Anti-PD-L1 by Potentiating the Tumor Immune Microenvironment in Murine Lung Cancer.

BACKGROUND: Radioimmunotherapy has become one of the most promising strategies for cancer treatment. Preclinical and clinical studies have demonstrated that antiangiogenic therapy can improve the efficacy of immunotherapy and sensitize radiotherapy through a variety of mechanisms. However, it is undefined whether angiogenesis inhibitors can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of anlotinib (AL3818) on the combination of radiotherapy and immune checkpoint inhibitors in Lewis lung carcinoma mouse. METHODS: C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control. Immune response and immunophenotyping including the quantification and activation were determined by flow cytometry, multiplex immunofluorescence, and multiplex immunoassay. RESULTS: Triple therapy (radiotherapy combined with anti-PD-L1 and anlotinib) increased tumor-infiltrating lymphocytes and reversed the immunosuppressive effect of radiation on the tumor microenvironment in mouse model. Compared with radioimmunotherapy, the addition of anlotinib also boosted the infiltration of CD8+ T cells and M1 cells and caused a decrease in the number of MDSCs and M2 cells in mice. The levels of IFN-gamma and IL-18 were the highest in the triple therapy group, while the levels of IL-23, IL-13, IL-1 beta, IL-2, IL-6, IL-10, and Arg-1 were significantly reduced. NF-κB, MAPK, and AKT pathways were downregulated in triple therapy compared with radioimmunotherapy. Thus, the tumor immune microenvironment was significantly improved. As a consequence, triple therapy displayed greater benefit in antitumor efficacy. CONCLUSION: Our findings indicate that anlotinib might be a potential synergistic treatment for radioimmunotherapy to achieve better antitumor efficacy in NSCLC patients by potentiating the tumor immune microenvironment.

Transcutaneous Vagal Nerve Stimulation Alone or in Combination With Radiotherapy Stimulates Lung Tumor Infiltrating Lymphocytes But Fails to Suppress Tumor Growth.

The combination of radiotherapy (RT) with immunotherapy represents a promising treatment modality for non-small cell lung cancer (NSCLC) patients. As only a minority of patients shows a persistent response today, a spacious optimization window remains to be explored. Previously we showed that fractionated RT can induce a local immunosuppressive profile. Based on the evolving concept of an immunomodulatory role for vagal nerve stimulation (VNS), we tested its therapeutic and immunological effects alone and in combination with fractionated RT in a preclinical-translational study. Lewis lung carcinoma-bearing C57Bl/6 mice were treated with VNS, fractionated RT or the combination while a patient cohort with locally advanced NSCLC receiving concurrent radiochemotherapy (ccRTCT) was enrolled in a clinical trial to receive either sham or effective VNS daily during their 6 weeks of ccRTCT treatment. Preclinically, VNS alone or with RT showed no therapeutic effect yet VNS alone significantly enhanced the activation profile of intratumoral CD8+ T cells by upregulating their IFN-γ and CD137 expression. In the periphery, VNS reduced the RT-mediated rise of splenic, but not blood-derived, regulatory T cells (Treg) and monocytes. In accordance, the serological levels of protumoral CXCL5 next to two Treg-attracting chemokines CCL1 and CCL22 were reduced upon VNS monotherapy. In line with our preclinical findings on the lack of immunological changes in blood circulating immune cells upon VNS, immune monitoring of the peripheral blood of VNS treated NSCLC patients (n=7) did not show any significant changes compared to ccRTCT alone. As our preclinical data do suggest that VNS intensifies the stimulatory profile of the tumor infiltrated CD8+ T cells, this favors further research into non-invasive VNS to optimize current response rates to RT-immunotherapy in lung cancer patients.

Pre-treatment with Bifidobacterium infantis and its specific antibodies enhance targeted radiosensitization in a murine model for lung cancer.

PURPOSE: The hypoxic microenvironments of solid tumours are complex and reduce the susceptibility of cancer cells to chemo- and radiotherapy. Conventional radiosensitisers have poor specificity, unsatisfactory therapeutic effects, and significant side effects. Anaerobic bacteria colonise and destroy hypoxic areas of the tumour and consequently enhance the effects of radiation. METHODS: In this study, we treated a Lewis lung carcinoma transplant mouse model with Bifidobacterium infantis (Bi) combined with its specific monoclonal antibody (mAb) and radiotherapy (RT) to investigate its ability to radiosensitise the tumour. The tumour metabolism and hypoxia in the tumour tissue were monitored by micro-18F-FDG and 18F-FMISO PET/CT imaging. Immunohistochemistry was used to detect phosphorylated histone (γ-H2AX), proliferation (Ki-67), platelet endothelial cell adhesion molecules (CD31), tumour necrosis factor-α (TNF-α), hypoxia-inducible factor-1α (HIF-1α), and glucose transporter 1 (Glut-1) levels. RESULTS: Tumour growth was slowed and survival time was markedly prolonged in mice subjected to the combination of B. infantis, specific antibody, and radiotherapy. Levels of HIF-1α, Glut-1, Ki-67, and CD31 expression, as well as uptake of FDG and FMISO, were the lowest in the combination-treated mice. In contrast, γ-H2AX and TNF-α expression levels were elevated and hypoxia in tumour tissue was reduced compared with controls. CONCLUSION: In conclusion, our data indicated that the curative effect of radiotherapy for lung cancer was enhanced by pre-treating mice with a combination of B. infantis and its specific monoclonal antibody.

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.