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Hypofractionated radiotherapy (hRT) can induce a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade (ICB). However, clinically, this effect is still rare, and ICB-mediated adverse events are common. Lenalidomide (lena) is an anti-angiogenic and immunomodulatory drug used in the treatment of hematologic malignancies. We here investigated in solid tumor models whether lena can enhance the abscopal effect in double combination with hRT. In two syngeneic bilateral tumor models (B16-CD133 melanoma and MC38 colon carcinoma), the primary tumor was treated with hRT. Lena was given daily for 3 weeks. Besides tumor size and survival, the dependence of the antitumor effects on CD8 cells, type-I IFN signaling, and T cell costimulation was determined with depleting or blocking antibodies. Tumor-specific CD8 T cells were quantified, and their differentiation and effector status were characterized by multicolor flow cytometry using MHC-I tetramers and various antibodies. In addition, dendritic cell (DC)-mediated tumor antigen cross-presentation and directly and the composition of tumor-associated vascular endothelial cells were investigated. In both tumor models, the hRT/lena double combination induced a significant abscopal effect. Control of the non-irradiated secondary tumor and survival were considerably better than with the respective monotherapies. The abscopal effect was strongly dependent on CD8 cells and associated with an increase in tumor-specific CD8 T cells in the non-irradiated tumor and its draining lymph nodes. Additionally, we found more tumor-specific T cells with a stem-like (TCF1 TIM3 PD1 ) and a transitory (TCF1 TIM3 CD101 PD1 ) exhausted phenotype and more expressing effector molecules such as GzmB, IFNγ, and TNFα. Moreover, in the non-irradiated tumor, hRT/lena treatment also increased DCs cross-presenting a tumor model antigen. Blocking type-I IFN signaling, which is essential for cross-presentation, completely abrogated the abscopal effect. A gene expression analysis of bone marrow-derived DCs revealed that lena augmented the expression of IFN response genes and genes associated with differentiation, maturation (including CD70, CD83, and CD86), migration to lymph nodes, and T cell activation. Flow cytometry confirmed an increase in CD70 CD83 CD86 DCs in both irradiated and abscopal tumors. Moreover, the hRT/lena-induced abscopal effect was diminished when these costimulatory molecules were blocked simultaneously using antibodies. In line with the enhanced infiltration by DCs and tumor-specific CD8 T cells, including more stem-like cells, hRT/lena also increased tumor-associated high endothelial cells (TA-HECs) in the non-irradiated tumor. We demonstrate that lena can augment the hRT-induced abscopal effect in mouse solid tumor models in a CD8 T cell- and IFN-I-dependent manner, correlating with enhanced anti-tumor CD8 T cell immunity, DC cross-presentation, and TA-HEC numbers. Our findings may be helpful for the planning of clinical trials in (oligo)metastatic patients.

Immune checkpoint inhibitors (ICIs) have emerged as a highly effective treatment option for patients with metastatic melanoma. As not all patients respond to ICI immunotherapy, imaging biomarkers are required to accurately monitor early response to therapy. Therefore, the aim of this study was to evaluate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring the effects of combined anti-PD-L1/anti-CTLA-4 immunotherapy in a murine melanoma model. Murine melanoma allografts (B16-F10) were implanted subcutaneously in n = 10 therapy and n = 10 control female C57BL/6 mice. CEUS with VEGFR2-targeted microbubbles was performed on day 7 and 12. The therapy group received 3 intraperitoneal injections on days 7, 9, 11 of combined anti-PD-L1/anti-CTLA-4 immunotherapy, the control group received a placebo. CEUS assessed tumour perfusion during an early vascular phase (wash-in area under the curve = WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity at 8 minutes (SI8min) and 10 minutes (SI10min)). For pathophysiological validation immunohistochemistry was performed. At follow-up, the CEUS perfusion parameter WiAUC demonstrated a significantly higher decrease in the therapy than in the control group (p = 0.021). At follow-up, the signal enhancement in the late phase was significantly lower in the therapy than in the control group (SI8min p = 0.003; SI10min p = 0.002). Immunohistochemistry revealed significantly more apoptotic tumour cells (p = 0.001), more tumour infiltrating lymphocytes (p = 0.049), lower tumour cell proliferation (p = 0.001), lower microvascular density (p = 0.003) and lower VEGFR2 expression (p = 0.003) in the therapy than in the control group. CEUS with VEGFR2-targeted microbubbles allowed for monitoring early treatment effects of a combined anti-PD-L1/anti-CTLA-4 immunotherapy on melanoma allografts with significantly lower tumour perfusion and significantly lower binding of VEGFR2-targeted microbubbles in the therapy than in the control group.

Combination radiotherapy (RT) and αPD-L1 therapy has potential to enhance local and distant (abscopal) tumor control, however, clinical results in humans have been variable. Using murine melanoma models, we found RT + αPD-L1 increases intra-tumor progenitor CD8+ PD-1+ TCF-1+ T cells. This increase depends on trafficking of the PD-1+ TCF-1+ cells from the tumor-draining lymph node (TdLN) to the tumor. RT alone promotes the expansion and differentiation of the TdLN derived PD-1+ TCF-1+ cells into TIM-3+ GZMB+ TCF-1- effector-like cells in the tumor with further enhancement after the addition of αPD-L1. In the TdLN, combination therapy enriches for a novel PD-1+ TCF-1+ TOX- LY6A+ subset with expression of a type I interferon and migratory signature. This subset is able to traffic to the tumor and differentiate into TIM-3+ TCF-1- cells. Finally, we found that ablation of the PD-1+ TCF-1+ T cell population attenuates the enhanced tumor control observed with combination RT + αPD-L1. These results suggest that abscopal response failures may be secondary to impaired stimulation of TdLN CD8+ PD-1 + TCF-1+ T cells or an inability of PD-1+ TCF-1+ cells in the TdLN to traffic to the tumor. Combination radiotherapy (RT) + αPD-L1 enhances tumor control via a tumor-draining lymph node (TdLN)-derived CD8+ PD-1+ TCF-1+ T cells. RT + αPD-L1 induces a novel LY6A+ subset in the TdLN that migrates to the tumor and differentiates into effectors.

Immunogenic cell death (ICD) is a programmed pathway leading to cell death and promotion of immunological responses. Melanoma is resistant to chemotherapy and radiotherapy (RT). Disulfiram (DSF), which forms complexes with copper (Cu), has been shown to induce ICD of many tumor types. Here, we aim to investigate whether DSF/Cu combined with irradiation (IR) can induce ICD and exert anti-cancer effects in melanoma. In vitro experiments, treatment of MV3 and B16F10 melanoma cells with DSF/Cu + IR significantly increased the cellular apoptosis and increased ICD markers: damage-associated molecular pattern molecule (DAMP) exposure and release, including calreticulin cell surface expression, high-mobility group box 1 (HMGB1) release, and decreased intracellular ATP levels. In addition, DSF/Cu combined with IR treatment inhibited tumor growth and enhanced tumor-infiltrating immune cells in the B16F10-bearing C57BL/6 model. Our findings reveal that combining IR with DSF/Cu induces ICD and inhibits tumor growth in melanoma, providing a promising strategy to overcome the inherent resistance of RT in melanoma.

Surgical resection is a primary treatment for solid tumors, but high rates of tumor recurrence and metastasis post-surgery present significant challenges. Manganese (Mn ), known to enhance dendritic cell-mediated cancer immunotherapy by activating the cGAS-STING pathway, has potential in post-operative cancer management. However, achieving prolonged and localized delivery of Mn to stimulate immune responses without systemic toxicity remains a challenge. We developed a post-operative microenvironment-responsive dendrobium polysaccharide hydrogel embedded with Mn -pectin microspheres (MnP@DOP-Gel). This hydrogel system releases Mn -pectin microspheres (MnP) in response to ROS, and MnP shows a dual effect : promoting immunogenic cell death and activating immune cells (dendritic cells and macrophages). The efficacy of MnP@DOP-Gel as a post-surgical treatment and its potential for immune activation were assessed in both subcutaneous and metastatic melanoma models in mice, exploring its synergistic effect with anti-PD1 antibody. MnP@DOP-Gel exhibited ROS-responsive release of MnP, which could exert dual effects by inducing immunogenic cell death of tumor cells and activating dendritic cells and macrophages to initiate a cascade of anti-tumor immune responses. experiments showed that the implanted MnP@DOP-Gel significantly inhibited residual tumor growth and metastasis. Moreover, the combination of MnP@DOP-Gel and anti-PD1 antibody displayed superior therapeutic potency in preventing either metastasis or abscopal brain tumor growth. MnP@DOP-Gel represents a promising drug-free strategy for cancer post-operative management. Utilizing this Mn -embedding and ROS-responsive delivery system, it regulates surgery-induced immune responses and promotes sustained anti-tumor responses, potentially increasing the effectiveness of surgical cancer treatments.

Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and MDA5; this results in growth inhibition and tumour inflammation, respectively. Loss of ADAR1 overcomes resistance to PD-1 checkpoint blockade caused by inactivation of antigen presentation by tumour cells. Thus, effective anti-tumour immunity is constrained by inhibitory checkpoints such as ADAR1 that limit the sensing of innate ligands. The induction of sufficient inflammation in tumours that are sensitized to interferon can bypass the therapeutic requirement for CD8 + T cell recognition of cancer cells and may provide a general strategy to overcome immunotherapy resistance. Deletion of the A-to-I double-stranded RNA-editing enzyme ADAR1 sensitizes tumour cells to immunotherapy.

Lipid nanoparticles (LNPs) can be designed to potentiate cancer immunotherapy by promoting their uptake by antigen-presenting cells, stimulating the maturation of these cells and modulating the activity of adjuvants. Here we report an LNP-screening method for the optimization of the type of helper lipid and of lipid-component ratios to enhance the delivery of tumour-antigen-encoding mRNA to dendritic cells and their immune-activation profile towards enhanced antitumour activity. The method involves screening for LNPs that enhance the maturation of bone-marrow-derived dendritic cells and antigen presentation in vitro, followed by assessing immune activation and tumour-growth suppression in a mouse model of melanoma after subcutaneous or intramuscular delivery of the LNPs. We found that the most potent antitumour activity, especially when combined with immune checkpoint inhibitors, resulted from a coordinated attack by T cells and NK cells, triggered by LNPs that elicited strong immune activity in both type-1 and type-2 T helper cells. Our findings highlight the importance of optimizing the LNP composition of mRNA-based cancer vaccines to tailor antigen-specific immune-activation profiles. The composition of lipid nanoparticles for the delivery of tumour-antigen-encoding mRNA can be optimized via a screening method to enhance antitumour activity via the modulation of the immune activity of helper T cells.

Immunotherapy is one of the most promising anti-cancer treatment. It involves activating the host's own immune system to eliminate cancer cells. Activation of cGAS-STING pathway is promising therapeutic approach for cancer immunotherapy. However, in human clinical trials, targeting cGAS-STING pathway results in insufficient or unsustainable anti-tumor response. To enhance its effectiveness, combination with other anti-cancer therapies seems essential to achieve synergistic systemic anti-tumor response.The aim of this study was to evaluate whether the combination of STING agonist-cGAMP with anti-vascular RGD-(KLAKLAK)2 peptide results in a better anti-tumor response in poorly immunogenic tumors with various STING protein and αvβ3 integrin status.Combination therapy inhibited growth of murine breast carcinoma more effectively than melanoma. In melanoma, the administration of STING agonist alone was sufficient to obtain a satisfactory therapeutic effect. In both tumor models we have noted stimulation of innate immune response following cGAMP administration alone or in combination. The largest population of immune cells infiltrating the TME after therapy were activated NK cells. Increased infiltration of cytotoxic CD8+ T lymphocytes within the TME was only observed in melanoma tumors. However, they also expressed the “exhaustion” PD-1 receptor. In contrast, in breast carcinoma tumors each therapy caused the drop in the number of infiltrating CD8+ T cells.The obtained results indicate an additional therapeutic benefit from combining STING agonist with an anti-vascular agent. However, this effect depends on the type of tumor, the status of its microenvironment and the expression of specific proteins such as STING and αvβ3 family integrin.

Melanoma is one of the most aggressive skin cancers worldwide. Although there has been much effort toward improving treatment options over the past few years, there remains an urgent need for effective therapy. Immunotherapy combined with chemotherapy has shown great promise in clinical trials. Here, we studied the cooperative effects of the small molecule drug pimozide, which has a therapeutic effect in melanoma, and RNA interference (RNAi) targeting PD-1, an important immune checkpoint molecule involved in tumor immune escape. PD-1 siRNA was delivered by attenuated Salmonella to melanoma-bearing mice in combination with pimozide. Our results demonstrated that the combination therapy had the optimal therapeutic effect on melanoma. The mechanisms underlying the efficacy involved the induction of apoptosis and an enhanced immune response. This study suggests that immunotherapy based on PD-1 inhibition combined with anticancer drugs could be a promising clinical strategy for the treatment of melanoma.

The phenomenon known as bipolar cancellation is observed when biphasic nanosecond electric field pulses are used, which results in reduced electroporation efficiency when compared to unipolar pulses of the same parameters. Basically, the negative phase of the bipolar pulse diminishes the effect of the positive phase. Our study aimed to investigate how bipolar cancellation affects Ca2+ electrochemotherapy and cellular response under varying electric field intensities and pulse durations (3–7 kV/cm, 100, 300, and 500 ns bipolar 1 MHz repetition frequency pulse bursts, n = 100). As a reference, standard microsecond range parametric protocols were used (100 µs × 8 pulses). We have shown that the cancellation effect is extremely strong when the pulses are closely spaced (1 MHz frequency), which results in a lack of cell membrane permeabilization and consequent failure of electrochemotherapy in vitro. To validate the observations, we have performed a pilot in vivo study where we compared the efficacy of monophasic (5 kV/cm × ↑500 ns × 100) and biphasic sequences (5 kV/cm × ↑500 ns + ↓500 ns × 100) delivered at 1 MHz frequency in the context of Ca2+ electrochemotherapy (B16-F10 cell line, C57BL/6 mice, n = 24). Mice treated with bipolar pulses did not exhibit prolonged survival when compared to the untreated control (tumor-bearing mice); therefore, the bipolar cancellation phenomenon was also occurrent in vivo, significantly impairing electrochemotherapy. At the same time, the efficacy of monophasic nanosecond pulses was comparable to 1.4 kV/cm × 100 µs × 8 pulses sequence, resulting in tumor reduction following the treatment and prolonged survival of the animals.