The advent and success of immune checkpoint inhibitors (ICIs) in cancer treatment has broadened the spectrum of tumours that might be considered "immunogenic" and susceptible to immunotherapeutic (IT) intervention. Not all cancer types are sensitive, and not all patients with any given type respond. Combination treatment of ICIs with an established cytotoxic modality such as radiation therapy (RT) is a logical step towards improvement. For one, RT alone has been shown to be genuinely immunomodulatory and secondly pre-clinical data generally support combined ICI-RT approaches. This new integrated therapy for cancer treatment holds much promise, although there is still a lot to be learned about how best to schedule the treatments, manage the toxicities and determine what biomarkers might predict response, as well as many other issues. This review examines how RT alters the immune rheostat and how it might best be positioned to fully exploit IT.

Purpose

Glioblastoma (GBM) remains an incurable disease despite extensive treatment with surgical resection, irradiation, and temozolomide. In line with many other forms of aggressive cancers, GBM is currently under consideration as a target for immunotherapy. However, GBM tends to be nonimmunogenic and exhibits a microenvironment with few or no effector T cells, a relatively low nonsynonymous somatic mutational load, and a low predicted neoantigen burden. GBM also exploits a multitude of immunosuppressive strategies.

Methods and materials

A number of immunotherapeutic approaches have been tested with disappointing results. A rationale exists to combine immunotherapy and radiation therapy, which can induce an immunogenic form of cell death with T-cell activation and tumor infiltration.

Results

Various immunotherapy agents, including immune checkpoint modulators, transforming growth factor beta receptor inhibitors, and indoleamine-2,3-dioxygenase inhibitors, have been evaluated with irradiation in preclinical GBM models, with promising results, and are being further tested in clinical trials.

Conclusions

This review aims to present the basic rationale behind this emerging complementary therapeutic approach in GBM, appraise the current preclinical and clinical data, and discuss the future challenges in improving the antitumor immune response.

Background

The cyclooxygenase 2 (COX-2) pathway has been implicated in the molecular pathogenesis of many malignancies, including lung cancer. Apricoxib, a selective COX-2 inhibitor, has been described to inhibit epithelial-mesenchymal transition (EMT) in human malignancies. The mechanism by which apricoxib may alter the tumor microenvironment by affecting EMT through other important signaling pathways is poorly defined. IL-27 has been shown to have anti-tumor activity and our recent study showed that IL-27 inhibited EMT through a STAT1 dominant pathway.

Objective

The purpose of this study is to investigate the role of apricoxib combined with IL-27 in inhibiting lung carcinogenesis by modulation of EMT through STAT signaling.

Methods and results

Western blot analysis revealed that IL-27 stimulation of human non-small cell lung cancer (NSCLC) cell lines results in STAT1 and STAT3 activation, decreased Snail protein and mesenchymal markers (N-cadherin and vimentin) and a concomitant increase in expression of epithelial markers (E-cadherin, β-and γ-catenins), and inhibition of cell migration. The combination of apricoxib and IL-27 resulted in augmentation of STAT1 activation. However, IL-27 mediated STAT3 activation was decreased by the addition of apricoxib. STAT1 siRNA was used to determine the involvement of STAT1 pathway in the enhanced inhibition of EMT and cell migration by the combined IL-27 and apricoxib treatment. Pretreatment of cells with STAT1 siRNA inhibited the effect of combined IL-27 and apricoxib in the activation of STAT1 and STAT3. In addition, the augmented expression of epithelial markers, decreased expression mesenchymal markers, and inhibited cell migration by the combination treatment were also inhibited by STAT1 siRNA, suggesting that the STAT1 pathway is important in the enhanced effect from the combination treatment.

Conclusion

Combined apricoxib and IL-27 has an enhanced effect in inhibition of epithelial-mesenchymal transition and cell migration in human lung cancer cells through a STAT1 dominant pathway.

The purpose of this study was to determine the dynamic contributions of different immune cell subsets to primary and abscopal tumor regression after hypofractionated radiation therapy (hRT) and the impact of anti-PD-1 therapy. A bilateral syngeneic FSA1 fibrosarcoma model was used in immunocompetent C3H mice, with delayed inoculation to mimic primary and microscopic disease. The effect of tumor burden on intratumoral and splenic immune cell content was delineated as a prelude to hRT on macroscopic T1 tumors with 3 fractions of 8 Gy while microscopic T2 tumors were left untreated. This was performed with and without systemic anti-PD-1. Immune profiles within T1 and T2 tumors and in spleen changed drastically with tumor burden in untreated mice with infiltrating CD4+ content declining, while the proportion of CD4+ Tregs rose. Myeloid cell representation escalated in larger tumors, resulting in major decreases in the lymphoid:myeloid ratios. In general, activation of Tregs and myeloid-derived suppressor cells allow immunogenic tumors to grow, although their relative contributions change with time. The evidence suggests that primary T1 tumors self-regulate their immune content depending on their size and this can influence the lymphoid compartment of T2 tumors, especially with respect to Tregs. Tumor burden is a major confounding factor in immune analysis that has to be taken into consideration in experimental models and in the clinic. hRT caused complete local regression of primary tumors, which was accompanied by heavy infiltration of CD8+ T cells activated to express IFN-γ and PD-1; while certain myeloid populations diminished. In spite of this active infiltrate, primary hRT failed to generate the systemic conditions required to cause abscopal regression of unirradiated microscopic tumors unless PD-1 blockade, which on its own was ineffective, was added to the RT regimen. The combination further increased local and systemically activated CD8+ T cells, but few other changes. This study emphasizes the subtle interplay between the immune system and tumors as they grow and how difficult it is for local RT, which can generate a local immune response that may help with primary tumor regression, to overcome the systemic barriers that are generated so as to effect immune regression of even small abscopal lesions.

The purpose of this study was to determine the dynamic contributions of different immune cell subsets to primary and abscopal tumor regression after hypofractionated radiation therapy (hRT) and the impact of anti-PD-1 therapy. A bilateral syngeneic FSA1 fibrosarcoma model was used in immunocompetent C3H mice, with delayed inoculation to mimic primary and microscopic disease. The effect of tumor burden on intratumoral and splenic immune cell content was delineated as a prelude to hRT on macroscopic T1 tumors with 3 fractions of 8 Gy while microscopic T2 tumors were left untreated. This was performed with and without systemic anti-PD-1. Immune profiles within T1 and T2 tumors and in spleen changed drastically with tumor burden in untreated mice with infiltrating CD4+ content declining, while the proportion of CD4+ Tregs rose. Myeloid cell representation escalated in larger tumors, resulting in major decreases in the lymphoid:myeloid ratios. In general, activation of Tregs and myeloid-derived suppressor cells allow immunogenic tumors to grow, although their relative contributions change with time. The evidence suggests that primary T1 tumors self-regulate their immune content depending on their size and this can influence the lymphoid compartment of T2 tumors, especially with respect to Tregs. Tumor burden is a major confounding factor in immune analysis that has to be taken into consideration in experimental models and in the clinic. hRT caused complete local regression of primary tumors, which was accompanied by heavy infiltration of CD8+ T cells activated to express IFN-γ and PD-1; while certain myeloid populations diminished. In spite of this active infiltrate, primary hRT failed to generate the systemic conditions required to cause abscopal regression of unirradiated microscopic tumors unless PD-1 blockade, which on its own was ineffective, was added to the RT regimen. The combination further increased local and systemically activated CD8+ T cells, but few other changes. This study emphasizes the subtle interplay between the immune system and tumors as they grow and how difficult it is for local RT, which can generate a local immune response that may help with primary tumor regression, to overcome the systemic barriers that are generated so as to effect immune regression of even small abscopal lesions.

Abstract Background Interleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. Epithelial–mesenchymal transition (EMT) and angiogenesis are key processes in carcinogenesis. Although IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. In this study, we investigated the role of IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human non-small cell lung carcinoma (NSCLC) cells. Methods STAT activation following IL-27 exposure was measured in human NSCLC cell lines. Expression of epithelial (E-cadherin, γ-catenin) and mesenchymal (N-cadherin, vimentin) markers were assessed by Western blot analysis. Production of pro-angiogenic factors (VEGF, IL-8/CXCL8, CXCL5) were examined by ELISA. Cell motility was examined by an in vitro scratch and transwell migration assays. Selective inhibitors of STAT1 (STAT1 siRNAs) and STAT3 (Stattic) were used to determine whether both STAT1 and STAT3 are required for IL-27 mediated inhibition of EMT and secretion of angiogenic factors. Results Our results demonstrate that IL-27 stimulation in NSCLC resulted in 1) STAT1 and STAT3 activation in a JAK-dependent manner, 2) development of epithelial phenotypes, including a decrease in the expression of a transcriptional repressor for E-cadherin (SNAIL), and mesenchymal marker (vimentin) with a reciprocal increase in the expression of epithelial markers, 3) inhibition of cell migration, and 4) reduced production of pro-angiogenic factors. STAT1 inhibition in IL-27–treated cells reversed the IL-27 effect with resultant increased expression of Snail, vimentin and the pro-angiogenic factors. The inhibition of STAT3 activation had no effect on the development of the epithelial phenotype. Conclusion IL-27 induces mesenchymal to epithelial transition and inhibits the production of pro-angiogenic factors in a STAT1–dominant pathway. These findings highlight the importance of STAT1 in repressing lung carcinogenesis and describe a new anti-tumor mechanism of IL-27.

Oncogenic KRAS mutations are frequently found in non-small cell lung carcinoma (NSCLC) and cause constitutive activation of the MEK-ERK pathway. Many cancer types have been shown to overexpress PD-L1 to escape immune surveillance. FRA1 is a MEK/ERK-dependent oncogenic transcription factor and a member of the AP-1 transcriptional factor superfamily. This study assesses the hypothesis that KRAS mutation directly regulates PD-L1 expression through the MEK-ERK pathway mediated by FRA1. Premalignant human bronchial epithelial cell (HBEC) lines harboring the KRAS mutation^V12, EGFR mutation, p53 knock-down, or both KRAS mutation and p53 knock-down were tested for levels of PD-L1, FRA1, and ERK activation (pERK). Our results showed that KRAS mutation alone, but not other genetic alterations, induced significantly higher expression of PD-L1 compared to its vector counterparts. The increased PD-L1 expression in the KRAS mutated cells was dramatically reduced by inhibition of ERK activation. Furthermore, the MEK-ERK pathway-dependent PD-L1 expression was markedly reduced by FRA1 silencing. Interestingly, FRA1 silencing led to inhibition of ERK activation, indicating that FRA1 plays a role in PD-L1 regulation via positive feedback of ERK activation. Correlation of PD-L1 and FRA1 mRNA expression was validated using human lung cancer specimens from The Cancer Genome Atlas (TCGA) and established NSCLC cell lines from Cancer Cell Line Encyclopedia (CCLE). FRA1 expression was significantly associated with PD-L1 expression, and high FRA1 expression was correlated with poor overall survival. Our findings suggest that oncogenic KRAS-driven PD-L1 expression is dependent on MEK-ERK and FRA1 in high risk, premalignant HBEC.

Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated, including in non-small cell lung cancer (NSCLC). Here, we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo Snail-mediated transformation relied upon silencing of the tumor-suppressive RNA splicing regulatory protein ESRP1. In clinical specimens of NSCLC, ESRP1 loss was documented in Snail-expressing premalignant pulmonary lesions. Mechanistic investigations showed that Snail drives malignant progression in an ALDH⁺CD44⁺CD24^- pulmonary stem cell subset in which ESRP1 and stemness-repressing microRNAs are inhibited. Collectively, our results show how ESRP1 loss is a critical event in lung carcinogenesis, and they identify new candidate directions for targeted therapy of NSCLC.Significance: This study defines a Snail-ESRP1 cancer axis that is crucial for human lung carcinogenesis, with implications for new intervention strategies and translational opportunities. Cancer Res; 78(8); 1986-99. ©2018 AACR.

Ionizing radiation (IR) can reprogram proteasome structure and function in cells and tissues. In this article, we show that IR can promote immunoproteasome synthesis with important implications for Ag processing and presentation and tumor immunity. Irradiation of a murine fibrosarcoma (FSA) induced dose-dependent de novo biosynthesis of the immunoproteasome subunits LMP7, LMP2, and Mecl-1, in concert with other changes in the Ag-presentation machinery (APM) essential for CD8+ T cell-mediated immunity, including enhanced expression of MHC class I (MHC-I), β2-microglobulin, transporters associated with Ag processing molecules, and their key transcriptional activator NOD-like receptor family CARD domain containing 5. In contrast, in another less immunogenic, murine fibrosarcoma (NFSA), LMP7 transcripts and expression of components of the immunoproteasome and the APM were muted after IR, which affected MHC-I expression and CD8+ T lymphocyte infiltration into NFSA tumors in vivo. Introduction of LMP7 into NFSA largely corrected these deficiencies, enhancing MHC-I expression and in vivo tumor immunogenicity. The immune adaptation in response to IR mirrored many aspects of the response to IFN-γ in coordinating the transcriptional MHC-I program, albeit with notable differences. Further investigations showed divergent upstream pathways in that, unlike IFN-γ, IR failed to activate STAT-1 in either FSA or NFSA cells while heavily relying on NF-κB activation. The IR-induced shift toward immunoproteasome production within a tumor indicates that proteasomal reprogramming is part of an integrated and dynamic tumor-host response that is specific to the stressor and the tumor and therefore is of clinical relevance for radiation oncology.

Purpose: A phase I study was conducted to determine safety, clinical efficacy, and antitumor immune responses in patients with advanced non-small cell lung carcinoma (NSCLC) following intratumoral administration of autologous dendritic cells (DC) transduced with an adenoviral (Ad) vector expressing the CCL21 gene (Ad-CCL21-DC). We evaluated safety and tumor antigen-specific immune responses following in situ vaccination (ClinicalTrials.gov: NCT01574222).Experimental Design: Sixteen stage IIIB/IV NSCLC subjects received two vaccinations (1 × 10⁶, 5 × 10⁶, 1 × 10⁷, or 3 × 10⁷ DCs/injection) by CT- or bronchoscopic-guided intratumoral injections (days 0 and 7). Immune responses were assessed by tumor antigen-specific peripheral blood lymphocyte induction of IFNγ in ELISPOT assays. Tumor biopsies were evaluated for CD8⁺ T cells by IHC and for PD-L1 expression by IHC and real-time PCR (RT-PCR).Results: Twenty-five percent (4/16) of patients had stable disease at day 56. Median survival was 3.9 months. ELISPOT assays revealed 6 of 16 patients had systemic responses against tumor-associated antigens (TAA). Tumor CD8⁺ T-cell infiltration was induced in 54% of subjects (7/13; 3.4-fold average increase in the number of CD8⁺ T cells per mm²). Patients with increased CD8⁺ T cells following vaccination showed significantly increased PD-L1 mRNA expression.Conclusions: Intratumoral vaccination with Ad-CCL21-DC resulted in (i) induction of systemic tumor antigen-specific immune responses; (ii) enhanced tumor CD8⁺ T-cell infiltration; and (iii) increased tumor PD-L1 expression. Future studies will evaluate the role of combination therapies with PD-1/PD-L1 checkpoint inhibition combined with DC-CCL21 in situ vaccination. Clin Cancer Res; 23(16); 4556-68. ©2017 AACR.