Systemic Antitumor Effects Research Articles

Synergistic anti-tumor effects of oncolytic virus and anti-programmed cell death protein 1 antibody combination therapy: For suppression of lymph node and distant metastasis in a murine melanoma model

It is believed that oncolytic viruses (OVs) exert both direct anti-tumor effects by intratumoral injection as well as indirect anti-tumor effects by activating systemic immunity. In phase III clinical trials, OV and anti-programmed cell death-1 (aPD-1) antibody combination therapy showed no significant differences in overall survival and progression-free survival in patients with unresectable advanced melanoma. In the study, OVs can exert only indirect anti-tumor effects in non-injected, systemic lesions. If the tumor is at a stage where both direct and indirect anti-tumor effects of OVs can be expected, OVs may further enhance the therapeutic effect, in addition to the clinically expected therapeutic effect. Therefore, we investigated whether canerpaturev (C-REV) and aPD-1 antibody combination therapy suppresses tumor progression in a murine melanoma model. Our findings showed that the C-REV and aPD-1 antibody combination therapy suppressed tumor progression in a murine melanoma model. The combination therapy stimulated systemic immunity in lymphoid tissues by activating helper T cells and B cells to enhance adaptive and humoral immunity, as well as by increasing effector/memory T cell fractions. Synergistically enhanced systemic anti-tumor effects suppressed lymph node and lung metastases. These findings suggest that direct anti-tumor effects by infecting and destroying cancer cells from within and indirect anti-tumor effects enhanced by the combination therapy worked simultaneously to suppress tumor progression. Our results may provide evidence to support the usefulness of OV and aPD-1 antibody combination therapy as a neoadjuvant therapy in the surgical treatment of melanoma.

Combined Radiotherapy and Hyperthermia: A Systematic Review of Immunological Synergies for Amplifying Radiation-Induced Abscopal Effects.

The abscopal effect is a systemic immune response characterized by metastases regression at sites distant from the irradiated lesion. This systematic review aims to explore the immunological mechanisms of action underlying the abscopal effect and to investigate how hyperthermia (HT) can increase the chances of radiotherapy (RT) triggering systemic anti-tumor immune responses. This review is created in accordance with the PRISMA guidelines. HT and RT have both complementary and synergistic immunological effects. Both methods trigger danger signal release, promoting cytokine and chemokine secretion, which increases T-cell infiltration and facilitates cell death. Both treatments upregulate extracellular tumor HSP70, which could amplify DAMP recognition by macrophages and DCs, leading to stronger tumor antigen presentation and CTL-mediated immune responses. Additionally, the combined increase in cell adhesion molecules (VCAM-1, ICAM-1, E-selectin, L-selectin) could enhance leukocyte adhesion to tumors, improving lymphocyte trafficking and boosting systemic anti-tumor effects. Lastly, HT causes vasodilation and improves blood flow, which might exacerbate those distant effects. We suggest the combination of local radiotherapy with fever-range whole-body hyperthermia to optimally enhance the chances of triggering the abscopal effect mediated by the immune system.

Prodrugs of paclitaxel improve insitu photo-vaccination.

Photodynamic therapy (PDT) effectively kills cancer cells and initiates immune responses that promote anticancer effects locally and systemically. Primarily developed for local and regional cancers, the potential of PDT for systemic antitumor effects [in situ photo-vaccination (ISPV)] remains underexplored. This study investigates: (1) the comparative effectiveness of paclitaxel (PTX) prodrug [Pc-(L-PTX)2] for PDT and site-specific PTX effects versus its pseudo-prodrug [Pc-(NCL-PTX)2] for PDT combined with checkpoint inhibitors; (2) mechanisms driving systemic antitumor effects; and (3) the prophylactic impact on preventing cancer recurrence. A bilateral tumor model was established in BALB/c mice through subcutaneous injection of CT26 cells. Mice received the PTX prodrug (0.5 μmole kg-1, i.v.), and tumors were treated with a 690-nm laser (75 mW cm-2 for 30 min, drug-light interval 0.5 h, light does 135 J cm-1), followed by anti-CTLA-4 (100 μg dose-1, i.p.) on days 1, 4, and 7. Notable enhancement in both local and systemic antitumor effectiveness was observed with [Pc-(L-PTX)2] compared to [Pc-(NCL-PTX)2] with checkpoint inhibitor. Immune cell depletion and immunohistochemistry confirmed neutrophils and CD8+ T cells are effectors for systemic antitumor effects. Treatment-induced immune memory resisted newly rechallenged CT26, showcasing prophylactic benefits. ISPV with a PTX prodrug and anti-CTLA-4 is a promising approach for treating metastatic cancers and preventing recurrence.

Near-infrared photoimmunotherapy as a complementary modality to in situ vaccine in a preclinical pancreatic cancer model

Pancreatic cancer is one of the most refractory malignancies. In situ vaccines (ISV), in which intratumorally injected immunostimulatory adjuvants activate innate immunity at the tumor site, utilize tumor-derived patient-specific antigens, thereby allowing for the development of vaccines in patients themselves. Near-infrared photoimmunotherapy (NIR-PIT) is a novel therapy that selectively kills cancer cells exclusively in the NIR-irradiated region. Extending our previous research showing that ISV using the unique nanoparticulate Toll-like receptor 9 (TLR9) ligand K3-SPG induced effective antitumor immunity, here we incorporated NIR-PIT into K3-SPG-ISV so that local tumor destruction by NIR-PIT augments the antitumor effect of ISV. In the mouse model of pancreatic cancer, the combination of K3-SPG-ISV and CD44-targeting NIR-PIT showed synergistic systemic antitumor effects and enhanced anti-programmed cell death-1 (PD-1) blockade. Mechanistically, strong intratumoral upregulation of interferon-related genes and dependency on CD8+ T cells were observed, suggesting the possible role of interferon and cytotoxic T cell responses in the induction of antitumor immunity. Importantly, this combination induced immunological memory in therapeutic and neoadjuvant settings. This study represents the first attempt to integrate NIR-PIT with ISV, offering a promising new direction for cancer immunotherapy, particularly for pancreatic cancer.

Proton radiation boosts the efficacy of mesothelin-targeting chimeric antigen receptor T cell therapy in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) represents a challenge in oncology, with limited treatment options for advanced-stage patients. Chimeric antigen receptor T cell (CAR T) therapy targeting mesothelin (MSLN) shows promise, but challenges such as the hostile immunosuppressive tumor microenvironment (TME) hinder its efficacy. This study explores the synergistic potential of combining proton radiation therapy (RT) with MSLN-targeting CAR T therapy in a syngeneic PDAC model. Proton RT significantly increased MSLN expression in tumor cells and caused a significant increase in CAR T cell infiltration into tumors. The combination therapy reshaped the immunosuppressive TME, promoting antitumorigenic M1 polarized macrophages and reducing myeloid-derived suppressor cells (MDSC). In a flank PDAC model, the combination therapy demonstrated superior attenuation of tumor growth and improved survival compared to individual treatments alone. In an orthotopic PDAC model treated with image-guided proton RT, tumor growth was significantly reduced in the combination group compared to the RT treatment alone. Further, the combination therapy induced an abscopal effect in a dual-flank tumor model, with increased serum interferon-γ levels and enhanced proliferation of extratumoral CAR T cells. In conclusion, combining proton RT with MSLN-targeting CAR T therapy proves effective in modulating the TME, enhancing CAR T cell trafficking, and exerting systemic antitumor effects. Thus, this combinatorial approach could present a promising strategy for improving outcomes in unresectable PDAC.

Abscopal Effect Demonstrated on [18F]FDG PET/CT in a Case of Renal Cell Carcinoma Postnephrectomy

Renal cell carcinoma accounts for 3% of all malignancies with many of them presenting with metastasis at the time of presentation. The abscopal effect, a phenomenon characterized by systemic bystander effects on nontargeted lesions due to local therapy, has been extensively studied in the context of radiotherapy and immunotherapy. However, documentation of the abscopal effect following surgery remains limited. We present a case of a 67-year-old Indian male diagnosed with clear cell renal cell carcinoma (RCC), who underwent left radical nephrectomy. Baseline staging and postnephrectomy follow-up scans with fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET-CT) revealed an FDG-avid primary lesion in the left kidney with metastasis to the left lung. Surprisingly, the follow-up FDG PET-CT scan demonstrated the resolution of the left lung metastasis, indicating the occurrence of the abscopal effect resulting from the local nephrectomy. This rare clinical observation highlights the potential of surgery to induce immunogenic tumor neoantigens release and inflammatory factors, leading to systemic antitumor effects. While the abscopal effect has been extensively studied in the context of radiotherapy and immunotherapy, there is a scarcity of studies reporting nephrectomy promoting such systemic effects. The combination of radiotherapy and immunotherapy has shown promising results in enhancing tumor immunosuppression and facilitating the abscopal effect. We report a case of RCC with lung metastasis showing abscopal effect with resolution of lung nodule postnephrectomy on 18-F-FDG PET-CT.

Biomimetic Hydrogel-Mediated Mechano-Immunometabolic Therapy for Inhibition of ccRCC Recurrence After Surgery.

The unique physical tumor microenvironment (TME) and aberrant immune metabolic status are two obstacles that must be overcome in cancer immunotherapy to improve clinical outcomes. Here, an in situ mechano-immunometabolic therapy involving the injection of a biomimetic hydrogel is presented with sequential release of the anti-fibrotic agent pirfenidone, which softens the stiff extracellular matrix, and small interfering RNA IDO1, which disrupts kynurenine-mediated immunosuppressive metabolic pathways, together with the multi-kinase inhibitor sorafenib, which induces immunogenic cell death. This combination synergistically augmented tumor immunogenicity and induced anti-tumor immunity. In mouse models of clear cell renal cell carcinoma, a single-dose peritumoral injection of a biomimetic hydrogel facilitated the perioperative TME toward a more immunostimulatory landscape, which prevented tumor relapse post-surgery and prolonged mouse survival. Additionally, the systemic anti-tumor surveillance effect induced by local treatment decreased lung metastasis by inhibiting epithelial-mesenchymal transition conversion. The versatile localized mechano-immunometabolic therapy can serve as a universal strategy for conferring efficient tumoricidal immunity in "cold" tumor postoperative interventions.

An exhaustive pre-clinical study of a circRNA drug candidate (cmRNA1210) encoding IL-12sc for anti-tumor therapeutics.

e14569 Background: Circular RNA as emerging technology hasn't been approved of acceptance of any IND application. What’s the pharmacokinetics, pharmacodynamics and toxicity study of circular RNA drug candidate would be like is still unpublished. Here we provide an exhaustive pre-clinical study of a novel circular RNA drug candidate encoding IL-12sc, the immune stimulatory effector. Methods: Here, we performed an exhaustive pre-clinical study of a circular RNA drug candidate(cmRNA1210) encoding IL-12sc, including pharmacokinetics, pharmacodynamics, pharmacology and toxicology study. Meanwhile, we explored the bioactivity and cross reactivity of cmRNA1210 in vitro and the pharmacokinetics in vivo. Results: cmRNA1210 and its replace molecule exhibits robust therapeutic effect and good safety in syngeneic mouse tumor model and humanized human tumor mouse model. In situ administration of cmRNA1210 achieves abscopal effect and inhibits lung metastasis progression. Treatment with cmRNA1210 leads to cytokines change in plasma and tumor, cytotoxic immune cells accumulation in tumor and tumor tissue necrosis. Dynamic transcriptome profiling of tumor tissue post cmRNA1210 treatment. Combination with checkpoint inhibitor exhibits synergy effect and significantly decreases tumor relapse rate. Kaplan-Meier survival curve in TCGA dataset shows strong correlation of high IL-12A/B expression and long survival. Conclusions: The bioactivity and cross reactivity in rodents and primates of cmRNA1210 were validated in vitro. The advantage of pharmacokinetics of circular RNA was confirmed in vivo. cmRNA1210 exhibits robust anti-tumor effect in various tumor models, indicating its wild application situation in clinical. In situ treatment achieves abscopal effect and significantly inhibits lung metastasis progression. Systemic anti-tumor effect has been successfully initiated. The pharmacodynamics study reveals there are cytokine changes, immune cells activation or accumulation at tumor site and tumor tissue necrosis after cmRNA1210 treatment. cmRNA1210 as immune stimulatory effector works synergistically with checkpoint inhibitor. Kaplan-Meier survival analysis of IL-12 expression based on TCGA dataset shows there is a strong correlation of high expression and long survival. Overall, this pre-clinical study provides elaborate results of cmRNA1210 as anti-tumor therapeutics and demonstrates the great potential to enter clinical trial in the future.

Combination of MHI148 Targeted Photodynamic Therapy and STING Activation Inhibits Tumor Metastasis and Recurrence.

Metastasis and recurrence are notable contributors to mortality associated with breast cancer. Although immunotherapy has shown promise in mitigating these risks after conventional treatments, its effectiveness remains constrained by significant challenges, such as impaired antigen presentation by dendritic cells (DCs) and inadequate T cell infiltration into tumor tissues. To address these limitations, we developed a multifunctional nanoparticle platform, termed GM@P, which consisted of a hydrophobic shell encapsulating the photosensitizer MHI148 and a hydrophilic core containing the STING agonist 2'3'-cGAMP. This design elicited robust type I interferon responses to activate antitumor immunity. The GM@P nanoparticles loaded with MHI148 specifically targeted breast cancer cells. Upon exposure to 808 nm laser irradiation, the MHI148-loaded nanoparticles produced toxic reactive oxygen species (ROS) to eradicate tumor cells through photodynamic therapy (PDT). Notably, PDT stimulated immunogenic cell death (ICD) to foster the potency of antitumor immune responses. Furthermore, the superior photoacoustic imaging (PAI) capabilities of MHI148 enabled the simultaneous visualization of diagnostic and therapeutic procedures. Collectively, our findings uncovered that the combination of PDT and STING activation facilitated a more conducive immune microenvironment, characterized by enhanced DC maturation, infiltration of CD8+ T cells, and proinflammatory cytokine release. This strategy stimulated local immune responses to augment systemic antitumor effects, offering a promising approach to suppress tumor growth, inhibit metastasis, and prevent recurrence.

Sustained Secretion of CCL21 via an Implantable Cell Reservoir Hydrogel Enhances the Systemic Antitumor Effect of Radiotherapy.

The combination of radiotherapy (RT) and immunotherapy shows promise in improving the clinical treatment of solid tumors; however, it faces challenges of low response rates and systemic toxicity. Herein, an implantable alginate/collagen hydrogel encapsulating C-C motif ligand 21 (CCL21)-expressing dendritic cells (CCL21-DCs@gel) was developed to potentiate the systemic antitumor effects of RT. The hydrogel functioned as a suitable reservoir for in vivo culture and proliferation of CCL21-DCs, thereby enabling sustained CCL21 release. The local CCL21 gradient induced by CCL21-DCs@gel significantly enhanced the efficacy of RT in suppressing primary tumor growth and inhibiting distant metastasis across several mouse models. Furthermore, the combination of RT with CCL21-DCs@gel provided complete prophylactic protection to mice. Mechanistic investigations revealed that CCL21-DCs@gel potentiated RT by promoting tumor lymphangiogenesis and attracting immune cell infiltration into the tumor. Collectively, these results suggest that CCL21-DCs@gel is a promising adjunct to RT for effectively eradicating tumors and preventing tumor recurrence.

Abstract 6679: Deciphering the mechanism of action of a live biotherapeutic product for the treatment of cancer

Abstract Intestinal microbiota is essential in shaping the immune system and its functions. In our effort to harness the potential of beneficial gut microbiota, we identified specific bacterial species that are less abundant in colorectal cancer patients compared to healthy individuals. Oral supplementation of these bacteria causes an effective anti-tumor activity in various cancer models through specific activation of cytotoxic T-cells. Based on these results, we aimed to identify the mechanism of action of this bacterial treatment. In vivo studies were performed in C57BL/6 and BALB/c mice. MC-38, CT-26, B16, LLC1.1 and 4T1 cells were used for the heterotopic tumor injections. Immunophenotyping was performed using flow cytometry and immunohistochemistry. Metagenomics analysis and whole genome sequence were performed to identify the bacterial strains. Metabolomics analyses were conducted to analyze bacterial supernatant and serum compositions of mice and patients. Since we found that the bacterial treatment led to a systemic anti-tumor effect, we analyzed serum of the mice that received the bacteria and found that a specific metabolite was highly upregulated compared to untreated mice. Analysis of the medium supernatant from cultures of the bacterial strains led to the discovery that metabolite identified in the serum is being produced by our bacteria. Applying this bacterial metabolite alone in the mouse cancer models led to a similar immune activation and anti-tumor effect as the supplementation with our bacterial strains. Furthermore, we identified the receptor that is targeted by this molecule through experiments with known agonists and antagonists for the target. The anti-tumor effect of the bacteria as well as the bacterial metabolite was blocked through the application of a receptor antagonist, proving that activation of this receptor is the mechanism that drives the enhanced anti-tumor immune response. Activation of the receptor, which is mainly expressed on macrophages led to enhanced IL-12 production and recruitment of cytotoxic T-cells into the tumor. Together, we have fully characterized the mechanism of action of our bacterial treatment, including the secreted active metabolite, the target receptor, and the activated immune cells. We are developing a live biotherapeutic product for the treatment of cancer based on oral administration of lyophilized bacteria. The known mechanism of action of our bacteria helps us to optimize the efficacy of our product, define the right dosage and understand possible side effects. Additionally, it enables us to improve patient selection and prediction of treatment response. Since our product is based on commensal bacteria that are commonly present in healthy individuals, we expect a very good safety and tolerability profile. Therefore, our therapy aims to significantly improve the efficacy of cancer treatment as well as the quality of life of cancer patients. Citation Format: Egle Katkeviciute, Francesca Ferraro, Martin Schwill, Philipp Busenhart, Ana Montalban-Arques. Deciphering the mechanism of action of a live biotherapeutic product for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6679.

Annotation of CD8+ T-cell function via ICAM-1 imaging identifies FAK inhibition as an adjuvant to augment the antitumor immunity of radiotherapy.

Background: Radiotherapy (RT) may trigger systemic antitumor immunity, manifesting as regression of non-irradiated lesions (abscopal effect). Intracellular adhesion molecule-1 (ICAM-1) is a key molecule involved in the abscopal effect of RT. However, the specific function of ICAM-1 in CD8+ T cells during antitumor immune responses remains unclear. Herein, we investigated whether noninvasive imaging of ICAM-1 can be used to annotate CD8+ T-cell function, thereby better selecting combinational therapy to enhance the antitumor immunity induced by RT. Methods: Using knockout mouse models, we investigated the role of ICAM-1 expressed on CD8+ T cells in the antitumor immunity of RT and conducted drug screening guided by ICAM-1-targeted noninvasive imaging. Results: The systemic antitumor effect of RT relies on the expression of ICAM-1 on CD8+ T cells. ICAM-1 expression is essential for CD8+ T-cell activation, proliferation, and effector function. Noninvasive annotation of the proliferation and effector function of CD8+ T cells by ICAM-1-targeted imaging identified VS-6063, a focal adhesion kinase inhibitor, as a new adjuvant to augment systemic antitumor immunity of RT in an immunologically "cold" tumor model. Mechanistically, VS-6063 overcomes the physical barriers in tumors and promotes the migration and infiltration of CD8+ T cells primed by RT into distant tumors. Conclusion: Our findings highlight that molecular imaging of ICAM-1 levels provides a dynamic readout of the proliferation and effector function of tumor-infiltrating CD8+ T cells, which facilitates the high-throughput exploitation of new combinational drugs to maximize the systemic antitumor effect of RT.

Preclinical Mouse Metastatic Model Established Through Induced Lung Metastases.

Metastatic disease is the major cause of cancer death, and the lung is one of the most common sites of cancer metastases. To investigate systemic antitumor effects or protective potential of local therapies, mouse models with induced metastases are indispensable in preclinical cancer research. Here, we describe the protocol for the metastatic mouse model established through induced 4T1 mammary carcinoma metastases. With minor prior optimization, it can be applied to other tumor cell lines of interest.

Low-dose radiation therapy mobilizes antitumor immunity: New findings and future perspectives.

Radiotherapy has unique immunostimulatory and immunosuppressive effects. Although high-dose radiotherapy has been found to have systemic antitumor effects, clinically significant abscopal effects were uncommon on the basis of irradiating single lesion. Low-dose radiation therapy (LDRT) emerges as a novel approach to enhance the antitumor immune response due to its role as a leverage to reshape the tumor immune microenvironment (TIME). In this article, from bench to bedside, we reviewed the possible immunomodulatory role of LDRT on TIME and systemic tumor immune environment, and outlined preclinical evidence and clinical application. We also discussed the current challenges when LDRT is used as a combination therapy, including the optimal dose, fraction, frequency, and combination of drugs. The advantage of low toxicity makes LDRT potential to be applied in multiple lesions to amplify antitumor immune response in polymetastatic disease, and its intersection with other disciplines might also make it a direction for radiotherapy-combined modalities.

Olaparib enhances radiation-induced systemic anti-tumor effects via activating STING-chemokine signaling in hepatocellular carcinoma

Although Poly (ADP-ribose) polymerase (PARP) inhibitors have been clinically approved for cancers with BRCA mutations and are known to augment radiotherapy responses, their roles in promoting the abscopal effect and mediating immunotherapy in BRCA-proficient hepatocellular carcinoma (HCC) remain underexplored. Our study elucidates that olaparib enhances the radio-sensitivity of HCC cells. Coadministration of olaparib and irradiation induces significant DNA damage by generating double-strand breaks (DSBs), as revealed both in vitro and in immune-deficient mice. These DSBs activate the cGAS-STING pathway, initiating immunogenic cell death in abscopal tumors. STING activation reprograms the immune microenvironment in the abscopal tumors, triggering the release of type I interferon and chemokines, including CXCL9, CXCL10, CXCL11, and CCL5. This in turn amplifies T cell priming against tumor neoantigens, leading to an influx of activated, neoantigen-specific CD8+ T-cells within the abscopal tumors. Furthermore, olaparib attenuated the immune exhaustion induced by radiation and enhances the responsiveness of HCC to immune checkpoint inhibitors. Collectively, our data advocate that a synergistic regimen of PARP inhibitors and radiotherapy can strategically reinforce both local (primary) and systemic (abscopal) tumor control, bolstering HCC susceptibility to immunotherapy.

The worthy role of hepatic arterial infusion chemotherapy in combination with anti-programmed cell death protein 1 monoclonal antibody immunotherapy in advanced hepatocellular carcinoma.

Systemic therapy remains the primary therapeutic approach for advanced hepatocellular carcinoma (HCC). Nonetheless, its efficacy in achieving control of intrahepatic lesions is constrained. Hepatic arterial infusion chemotherapy (HAIC) is a therapeutic approach that combines localized treatment with systemic antitumor effects, which aim is to effectively manage the progression of cancerous lesions within the liver, particularly in patients with portal vein tumor thrombosis (PVTT). Combining HAIC with anti-programmed cell death protein 1 (anti-PD-1) monoclonal antibody (mAb) immunotherapy is anticipated to emerge as a novel therapeutic approach aimed at augmenting the response inside the localized tumor site and achieving prolonged survival advantages. In order to assess the effectiveness, safety, and applicability of various therapeutic modalities and to address potential molecular mechanisms underlying the efficacy of HAIC-sensitizing immunotherapy, we reviewed the literature about the combination of HAIC with anti-PD-1 mAb therapies.

Electrical stimulation induces anti-tumor immunomodulation via a flexible microneedle-array-integrated interdigital electrode

Immunotherapy has revolutionized cancer therapy, using chemical or biological agents to reinvigorate the immune system. However, most of these agents have poor tumor penetration and inevitable side effects that complicate therapeutic outcomes. Electrical stimulation (ES) is a promising alternative therapy against cancers that does not involve chemical or biological agents but is limited in the fabrication and operation of complex micrometer-scale ES devices. Here, we present an optically microprinted flexible interdigital electrode with a gold-plated polymer microneedle array to generate alternating electric fields for cancer treatment. A flexible microneedle-array-integrated interdigital electrode (FMIE) was fabricated by combining optical 3D microprinting and electroless plating processes. FMIE-mediated ES of cancer cells induced necrotic cell death through mitochondrial Ca2+ overload and increased intracellular reactive oxygen species (ROS) production. This led to the release of damage-associated molecular patterns that activated the immune response and potentiated immunogenic cell death (ICD). FMIE-based ES has an excellent safety profile and systemic anti-tumor effects, inhibiting the growth of primary and distant tumors as well as melanoma lung metastasis. FMIE-based ES-driven cancer immunomodulation provides a new pathway for drug-free cancer therapy.

Comparison of Two Triple Therapy Regimens for Enhancing the Abscopal Effect in Mice

Localized radiotherapy (RT) can cause a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade (ICB). However, this effect is still clinically rare and improvements are highly desirable. We here compared two triple therapy regimens (RT+⍺PD1+⍺CTLA4 vs. RT+⍺PD1+IL2/⍺IL2 complexes (IL2c)) for their efficacy to improve systemic (abscopal) anti-tumor effects. In mice bearing bilateral subcutaneous C51 colon carcinoma tumors, the primary tumor was irradiated with two fractions of 8 Gy. ⍺PD1 and ⍺CTLA4 were given concomitantly and weekly thereafter; IL2c was given i.p. for 3 consecutive days. Besides tumor size and survival, tumor-specific CD8+ T cells were determined flow cytometrically using MHC-I tetramers and various antibodies. In addition, isolated TILs were cultured with PMA, ionomycin, and Brefeldin A in vitro to assess polyfunctionality (based on cytokine production) of CD8+ and CD4+ TILs. The abscopal effect was significantly stronger in mice treated with the ⍺CTLA4-containing triple combination (n = 13) than in mice treated with the IL2c-containing triple combination (n = 9) (p<0,05), and compared to mice treated with RT+⍺PD1 (n = 8) (p<0,05) or RT+⍺CTLA4 (n = 10) (p<0,05), respectively. The IL2c triple combination induced the abscopal effect better than RT+⍺PD1 (p<0,05). The ⍺CTLA4 triple therapy improved survival and resulted in complete cures of 8/13 mice. In mice treated with ⍺CTLA4-containing triple therapy, control of the irradiated tumor was partially dependent and that of the non-irradiated tumor was strongly dependent on T cells, primarily CD8 T cells but also CD4 T cells. With ⍺CTLA4 triple treatment (n = 9), the frequency and absolute numbers of polyfunctional TNF-⍺+IFN-γ+ AH-1 tumor-specific CD8+ T cells, IFN-γ+IL2+ AH-1 tumor-specific CD8+ T cells, TNF-⍺+IFN-γ+ CD4+ and IFN-γ+IL2+ CD4+ effector T cells were higher than with the IL2c triple combination (n = 9) (p<0,05), particularly in the non-irradiated tumors. RT+⍺PD1+⍺CTLA4 triple treatment induced more cytotoxic effector TILs than RT+⍺PD1+IL2c triple treatment, enhancing the abscopal effect and inducing a high abscopal cure rate. More experiments underway to reveal mechanisms.

Targeting XPO1 Combined with Radiotherapy to Enhance Systemic Anti-tumor Effects in Non-Small Cell Lung Cancer

The combination of radiation and radiosensitizing chemotherapeutic agents have shown promising anti-tumor effects in NSCLC. Acting as an oncogenic driver, XPO1 is frequently overexpressed and/or mutated in lung cancer. Thus, suppression of XPO1-mediated nuclear export presents a unique therapeutic strategy. We hypothesize that XPO1 inhibition combined with radiotherapy (XRT) may remodel the tumor immune microenvironment (TIME) and reduce radioresistance, thus enhance systemic anti-tumor effects. Herein, we optimized a small molecule inhibitor, WJ01024, which can bind to XPO1 and antagonize its activity to inhibit nuclear export. In the C57BL/6 mouse subcutaneous tumor model, we evaluated the ability of different treatment regimens containing oral WJ01014 single or combined with XRT (one fractions of 15 Gy) in tumor control and tumor recurrence inhibition. The effects of each treatment regimen on the alterations of immunophenotypes, including the quantification, activation, proliferative capacity, exhaustion marker expression, and memory status, were evaluated by flow cytometry. In our study, we found that the overexpression of XPO1 was associated with poor prognosis and survival in radioresistant patients with NSCLC. The combination therapy of WJ01024 and XRT resulted in an increase of apoptosis and a decrease of proliferation compared to monotherapy, which was closely correlated with tumor regression and improved survival in the C57BL/6 mouse subcutaneous tumor model. Notably, we found that WJ01024 were shown to enhance the therapeutic effect of XRT by remodeling TIME. Compared with XRT, the addition of WJ01024 increased the infiltration and proliferation of radiation-stimulated CD8+ T cells, which especially promoted the production of interferon-γ and granzyme B. Moreover, the combination therapy also reversed the immunosuppressive effect of radiation on the percentage of Tregs and exhausted T cells in mouse xenografts. Thus, the TIME was significantly improved in combination therapy. Strikingly, mechanistic studies suggested that the activation of cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) signaling pathway is required to reshape TIME and produce synergistic anti-tumor effect with the combination of WJ01024 and XRT. Our findings suggest that WJ01024 might be a potential synergistic treatment for radiotherapy to control the proliferation of NSCLC cells, promote tumor regression and prolong survival in mouse model of NSCLC by activating cGAS/STING signaling, and this in turn potentiate the immune microenvironment.

Irradiated Tumor Cells-Derived Exosomes Modulate Macrophage Polarization by Targeting SHP-2 Mediated Metabolic Reprogramming

Tumor-associated macrophages (TAMs) constitute a plastic and heterogeneous cell population of the tumor microenvironment (TME) that can regulate tumor proliferation and support resistance to therapy, constituting promising targets for the development of novel anticancer agents. The efficacy of radiotherapy, a mainstay of cancer treatment, can strongly influence TAMs recruitment and phenotype. Our previous results demonstrated that SHP-2 and PD-L1 inhibition combined with radiotherapy enhances systemic antitumor effects in non-small cell lung cancer (NSCLC). Especially, SHP-2 has an important effect on the polarization of TAM in the context of radiotherapy. However, the immune mechanisms of SHP-2 in TAM remain largely unknown, and this leads us to implement this project. Transmission electron microscopy and differential ultracentrifugation were used to verify the existence of exosomes. The bone marrow-derived macrophages (BMDM) and peritoneal macrophages (PM) were derived from C57BL/6 mice for vitro tests. In vivo and in vitro assays were used to identify roles of exosomal miRNA targeting SHP-2. To investigate the regulating function of SHP-2 in TAMs, co-culture experiments, qPCR, Western Blot, Flow Cytometry and Oxygraph-2k were employed. And we also explore tumor growth and tumor environment changes in SHP-2 flox/floxLyz-Cre+/- (CKO) mice. We found that irradiated tumor cells-derived exosomes reprogramed their energy metabolism and polarized primary macrophages to an anti-inflammatory phenotype. Furthermore, SHP-2 in macrophages was a direct target of exosomal miR-138-5p from irradiated tumor cells. In vitro study also demonstrates that miR-138-5p can down-regulate SHP-2 in the BMDMs and PMs. Further research has shown that SHP-2 negatively regulated glycolysis through dephosphorylating Pyruvate kinase M2 (PKM2) at the Tyr105 site. In addition, SHP-2 can inhabit PKM2 translocation to the nucleus by dephosphorylating PKM2 at the Ser37 site. Thus, the SHP099 (a SHP-2 inhibitor) can uptake and utilization of glucose by SHP-2/PKM2(Tyr105) (Ser37)/β-catenin/LDHA/Glut-1 axis, suggesting that SHP099 plays positive roles on glycolysis and M1-polarized. In vivo study showed that SHP-2 flox/floxLyz-Cre+/- (CKO) mice display enhanced control of solid tumor growth, accompanied by increased the proportion of M1-like macrophages. Our study demonstrates that exosomal miR-138-5p from irradiated tumor cells can modulate macrophage polarization by targeting SHP-2. And SHP-2 negatively regulates glycolysis and polarize macrophage to an M2 phenotype by SHP-2/PKM2(Tyr105) (Ser37)/β-catenin/LDHA/Glut-1 axis.