Inflammatory Response In Tumors Research Articles

Nicaraven protects against endotoxemia-induced inflammation and organ injury through modulation of AMPK/Sirt1 signaling in macrophages

Endotoxemia is a disease characterized by systemic inflammatory responses and organ injury caused by lipopolysaccharide (LPS) infection, with high mortality. Nicaraven (AVS), a potent hydroxyl radical scavenger, has been proven to regulate the inflammatory response in tumors. To investigate the protective effects and mechanisms of AVS in endotoxemia, mice were injected intraperitoneally with LPS to induce endotoxemia. AVS treatment significantly decreased the levels of pro-inflammatory cytokines in the serum, reduced neutrophil infiltration, attenuated multiple organ injury, and increased the survival rate in LPS-challenged mice. In the LPS-induced inflammatory model of macrophages, AVS inhibited macrophage activation, suppressed nitric oxide (NO) production, and inhibited the expression and secretion of pro-inflammatory cytokines. Mechanistically, AVS treatment up-regulated silence information regulator transcript-1 (Sirt1) expression in a time- and dose-dependent manner. AVS treatment activated the AMP-dependent protein kinase (AMPK)/Sirt1 signaling pathway and suppressed the activation of nuclear factor kappa B (NF-κB) in macrophages exposed to LPS. However, the anti-inflammatory effects of AVS could be reversed by the AMPK, the Sirt1 inhibitor, or the histone deacetylase inhibitor. We confirmed that the AMPK inhibitor inhibited AVS-mediated AMPK/Sirt1 activation and NF-κB p65 acetylation. These results suggested that AVS alleviated endotoxemia by activating the AMPK/Sirt1 signaling pathway in macrophages.

MAPK-pathway inhibition mediates inflammatory reprogramming and sensitizes tumors to targeted activation of innate immunity sensor RIG-I

Kinase inhibitors suppress the growth of oncogene driven cancer but also enforce the selection of treatment resistant cells that are thought to promote tumor relapse in patients. Here, we report transcriptomic and functional genomics analyses of cells and tumors within their microenvironment across different genotypes that persist during kinase inhibitor treatment. We uncover a conserved, MAPK/IRF1-mediated inflammatory response in tumors that undergo stemness- and senescence-associated reprogramming. In these tumor cells, activation of the innate immunity sensor RIG-I via its agonist IVT4, triggers an interferon and a pro-apoptotic response that synergize with concomitant kinase inhibition. In humanized lung cancer xenografts and a syngeneic Egfr-driven lung cancer model these effects translate into reduction of exhausted CD8+ T cells and robust tumor shrinkage. Overall, the mechanistic understanding of MAPK/IRF1-mediated intratumoral reprogramming may ultimately prolong the efficacy of targeted drugs in genetically defined cancer patients.

IFNγ-induced Chemokines Are Required for CXCR3-mediated T-Cell Recruitment and Antitumor Efficacy of Anti-HER2/CD3 Bispecific Antibody.

The response to cancer immune therapy is dependent on endogenous tumor-reactive T cells. To bypass this requirement, CD3-bispecific antibodies have been developed to induce a polyclonal T-cell response against the tumor. Anti-HER2/CD3 T-cell-dependent bispecific (TDB) antibody is highly efficacious in the treatment of HER2-overexpressing tumors in mice. Efficacy and immunologic effects of anti-HER2/CD3 TDB were investigated in mammary tumor model with very few T cells prior treatment. We further describe the mechanism for TDB-induced T-cell recruitment to tumors. The immunologic effects and the mechanism of CD3-bispecific antibody-induced T-cell recruitment into spontaneous HER2-overexpressing mammary tumors was studied using human HER2 transgenic, immunocompetent mouse models. Anti-HER2/CD3 TDB treatment induced an inflammatory response in tumors converting them from poorly infiltrated to an inflamed, T-cell abundant, phenotype. Multiple mechanisms accounted for the TDB-induced increase in T cells within tumors. TDB treatment induced CD8+ T-cell proliferation. T cells were also actively recruited post-TDB treatment by IFNγ-dependent T-cell chemokines mediated via CXCR3. This active T-cell recruitment by TDB-induced chemokine signaling was the dominant mechanism and necessary for the therapeutic activity of anti-HER2/CD3 TDB. In summary, we demonstrate that the activity of anti-HER2/CD3 TDB was not dependent on high-level baseline T-cell infiltration. Our results suggest that anti-HER2/CD3 TDB may be efficacious in patients and indications that respond poorly to checkpoint inhibitors. An active T-cell recruitment mediated by TDB-induced chemokine signaling was the major mechanism for T-cell recruitment.

Abstract 644: Validation of PD-L1 staining by IHC in solid tumors: A journey from xenograft model to ex vivo culture of clinical samples

Abstract Background: Blockade of tumor immune evasion by immune checkpoints inhibitors (ICI) which target PD-L1/2 and PD-1 signaling axis ensured durable clinical benefit in solid tumors including melanoma, Non-Small Cell Lung Cancer, and colorectal cancers which led to the approval of Atezolizumab, Nivolumab, and Pembrolizumab. Studies revealed that response to PD-1/PD-L1 blockade might correlate with PD-L1 expression in tumor cells (Herbst et al., Nature 2014; Robert et al., NEJM 2015a, 2015b; KEYNOTE-001; -012; -040 Trials). Currently, assays for the prognostic/predictive role of tumor PD-L1 expression are “not standardized with respect to either quantity or distribution of expression” (McLaughlin et al., JAMA Oncol. 2016). Objective: We aimed to standardize, validate and set up criteria for the evaluation of PD-L1 protein expression using conventional immunohistochemistry (IHC) staining to demonstrate PD-L1 protein distribution in cultured ex vivo tumors obtained from surgically resected tumor samples. Methods: We used cell lines, their xenografts tumors, and Formalin-Fixed Paraffin-Embedded tumor samples from patients with different solid tumors to compare results from two different PD-L1 antibodies (Rb Anti-Human PD-L1/CD274 Mono-Ab Clone SP142 from SPRING BIOSCIENCE CATALOG# M4420; Mono-Mouse Anti-Human PD-L1 [Concentrate] Clone 22C3 from DAKO Code: M3653).TNBC (MDA-MB231, MDA-MB468, SUM149) and NSCLC (H1975 and H2228) cell lines, human term placenta and tonsil whole tissue sections were used for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using diaminobenzidine chromogenic IHC in FFPE sections/ fixed cells. Tumor-infiltrating lymphocytes (TILs) in tissue and ex vivo samples were scored in hematoxylin-eosin slides using current consensus guidelines. Finally, we validated PD-L1 expression in ex vivo cultured live tumor samples from patients with lung cancers. Results: With both PD-L1 antibodies, we find that PD-L1 IHC expression was heterogeneous. When validated using human tonsil sections, a focal and sporadic predominantly membranous positivity was observed in xenograft tissues and tumor cells. Both antibodies showed limited concordance, significant intra-assay heterogeneity, and significant interassay discordance. We also observed a markedly high non-specific staining in both necrotic tumor areas and scars. Conclusion: We had standardized and validated IHC method for the PD-L1 protein expression in (1) xenografts of several breast cancer cell lines, (2) FFPE from different solid tumors and (3) finally in ex vivo cultured tumors from lung cancer patients. Expression of PD-L1 is currently being correlated with TILs / inflammatory response in tumors the result of which will be presented at the meeting. This evaluation of PD-L1 expression based on ex vivo model may help to stratify patients for the treatment with ICI. Citation Format: Xiaoqian lin, Jennifer Carlson, Megan Quast, Yuliang Sun, Tyler Jepperson, Casey Williams, Luis RojasEspaillat, Benjamin Solomon, David Starks, Amy Krie, Pradip De, Raed Sulaiman, Nandini Dey, Brian Leyland-Jones. Validation of PD-L1 staining by IHC in solid tumors: A journey from xenograft model to ex vivo culture of clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 644.

Nanoparticle-Based Magnetic Resonance Imaging on Tumor-Associated Macrophages and Inflammation.

The inflammatory response, mediated by tissue-resident or newly recruited macrophages, is an underlying pathophysiological condition for many diseases, including diabetes, obesity, neurodegeneration, atherosclerosis, and cancer. Paradoxically, inflammation is a double-edged sword in oncology. Macrophages are, generally speaking, the major drivers of inflammatory insult. For many solid tumors, high density of cells expressing macrophage-associated markers have generally been found in association with a poor clinical outcome, characterized by inflamed microenvironment, a high level of dissemination and resistance to conventional chemotherapies. On another hand, radiation treatment also triggers an inflammatory response in tumors (often referred to as pseudoprogression), which can be associated with a positive treatment response. As such, non-invasive imaging of cancer inflammation and tumor-associated macrophages (TAMs) provides a revolutionary diagnostic tool and monitoring strategy for anti-inflammatory, immuno- and radiotherapies. Recently, quantitative T2-weighted magnetic resonance imaging (qT2wMRI), using injection of superparamagnetic iron oxide nanoparticles (SPIONs), has been reported for the assessment of TAMs non-invasively in animal models and in human trials. The SPIONs are magnetic resonance imaging (MRI) contrast agents that significantly decrease T2 MR relaxation times in inflamed tissues due to the macrophage-specific uptake and retention. It has been shown that macrophage-populated tumors and metastases will accumulate iron oxide nanoparticles and decrease T2-relaxation time that will result in a negative (dark) contrast in qT2wMRI. Non-invasive imaging of TAMs using SPION holds a great promise for staging the inflammatory microenvironment of primary and metastatic tumors as well monitoring the treatment response of cancer patients treated with radiation and immunotherapy.

Abstract 3793: Effects of DCA and metformin on murine colon cancer growth

Abstract Background: Dichloroacetate (DCA) and metformin (M) have been reported to have many effects on cancer cells including alterations in pH homeostasis and glucose metabolism, cell cycle arrest, induction of autophagy and modulation of the tumor microenvironment. These effects have been shown to increase tumor apoptosis, inhibit neoplastic cell proliferation, improve the immune response against tumor cells and decrease the protective inflammatory response in tumors, which has led to the investigation of these drugs as anticancer agents. This study examines the effects of DCA and metformin in a murine colon cancer model both in vivo and in vitro. Methods: Murine colon cancer cell lines (ATCC CT26-CL25, CT26-WT) and cells isolated from subcutaneous metastatic colon cancer tumors (CT71911) were grown in the presence of DCA (serial concentrations from 12 mM to 192mM) and M (serial concentrations from 8 mM to 48 mM). Viable cells were counted and averaged for 3 separate assays. Balb/c mice in triplicate were injected IP with serial concentrations of DCA and M (8,24,48 mM) beginning one day prior to tail vein administration of 7.5 x 103 colon cancer cells (CT26-WT, CT71911). Daily IP injections of DCA and M were continued for 6 weeks or until the mice succumbed to metastatic cancer. All mice were autopsied and cause of death was recorded. Results: All three cell lines demonstrated a linear reduction in growth and increased apoptosis with increasing concentrations of DCA. All tumor cells were killed at concentrations above 96 mM. M was cytostatic but not cytotoxic for all three cell lines with minimal growth at 8 mM and no growth at higher concentrations. In vivo, administration of IP DCA prolonged survival by 80 - 89% (days after tumor challenge), while IP metformin prolonged survival by 35 - 38%. Increased survival with both DCA and M was not dose dependent with variation between concentrations less than 10%. All mice died of lung metastases. Conclusion: Both DCA and M demonstrated beneficial effects on murine colon cancer cells with reductions in cell growth and increased apoptosis in vitro and increased survival in vivo. M exhibited cytostatic activity and a lower improvement in survival while DCA exhibited cytotoxic activity and a longer survival benefit. Further investigation in the use of these agents as adjunct therapies for colon cancer is warranted. Citation Format: Robert P. Sticca, Tonya C. Murphy. Effects of DCA and metformin on murine colon cancer growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3793. doi:10.1158/1538-7445.AM2014-3793

Chronic inflammation in tumor stroma is an independent predictor of prolonged survival in epithelioid malignant pleural mesothelioma patients

This study aims to determine whether a semi-quantitative assessment of inflammatory response in tumor and stroma on routine hematoxylin and eosin-stained (H&E) slides can predict survival in patients with epithelioid malignant pleural mesothelioma (MPM). H&E sections of 175 epithelioid MPM specimens from a single institution (1989-2009) were reviewed. Patients who received neoadjuvant chemotherapy were excluded from analysis. Each tumor was histologically assessed for acute and chronic inflammatory response both within the tumor and the stromal component. Inflammatory response was graded: low (none to mild infiltrate) or high (moderate to severe infiltrate). Log-rank test and Cox proportional hazards regression were used to investigate the association between the degree of inflammation (acute/tumor, acute/stroma, chronic/tumor, and chronic/stroma) and overall survival (OS). Patients with high chronic inflammatory response in stroma (n = 59) had improved survival compared to low (n = 116) (median OS = 19.4 vs. 15.0 months, P = 0.01). This prognostic stratification remained significant in stage III patients (median OS = 16.0 vs. 9.3 months, P = 0.03). In multivariate analysis, chronic inflammation in stroma was an independent predictor of survival (HR = 0.659, 95% CI 0.464-0.937, P = 0.02). While high degree of chronic inflammatory cell infiltration in the stromal component was associated with improved overall survival, degree of other inflammatory responses did not show significant correlation with OS. Our study for the first time investigates inflammatory response in tumor and stroma and not only suggests the prognostic value of inflammatory response in epithelioid MPM but also provides rationale for investigation of immunotherapy to benefit epithelioid MPM patients.

Activated Monocytes in Peritumoral Stroma of Hepatocellular Carcinoma Promote Expansion of Memory T Helper 17 Cells

Although cancer patients exhibit a generalized immunosuppressive status, substantial evidence indicates that the inflammatory reaction at a tumor site can promote tumor growth and progression. Hepatocellular carcinoma (HCC) is usually derived from inflamed cirrhotic liver with extensive leukocyte infiltration. We recently found that proinflammatory T helper (Th)17 cells are accumulated in HCC tissue, where they promote disease progression by fostering angiogenesis. Here we show that interleukin (IL)-17-producing cells were enriched predominantly in peritumoral stroma of HCC tissues, and their levels were well correlated with monocyte/macrophage density in the same area. Most peritumoral CD68(+) cells exhibited an activated phenotype. Accordingly, tumor-activated monocytes were significantly superior to the suppressive tumor macrophages in inducing expansion of Th17 cells from circulating memory T cells in vitro with phenotypic features similar to those isolated from HCCs. Moreover, we found that tumor-activated monocytes secreted a set of key proinflammatory cytokines that triggered proliferation of functional Th17 cells. Inhibition of monocytes/macrophages inflammation in liver markedly reduced the level of tumor-infiltrating Th17 cells and tumor growth in vivo. The proinflammatory Th17 cells are generated and regulated by a fine-tuned collaborative action between different types of immune cells in distinct HCC microenvironments, and allows the inflammatory response of activated monocytes to be rerouted in a tumor-promoting direction. Selectively modulating the "context" of inflammatory response in tumors might provide a novel strategy for anticancer therapy.

Up-regulation of Interleukin-8 by Vascular Endothelial Growth Factor in Vasculatures in vivo

Background: Vascular endothelial growth factor (VEGF) plays an essential role in promoting angiogenesis during tumor development. In addition, VEGF can mediate the inflammatory response in tumors. VEGF increases the level of neutrophil migration by upregulating interleukin-8 (IL-8) in endothelial cells in vitro. However, it is unclear if VEGF can mediate IL-8 production in vivo. Methods: To address this issue, this study examined the effect of VEGF on IL-8 production in vivo using an adenovirus transduction and mouse ear assay. Results: Adenovirus-encoded VEGF (VEGF-Ad) increased the level of IL-8 production in endothelial cells in vitro compared to the control-adenovirus (CTL-Ad). The mouse ear assay showed that VEGF-Ad increased the level of IL-8 production in the endothelium. Immunohistochemistry showed that the IL-8 proteins were expressed in the vasculature within a human glioblastoma, which is known to strongly express VEGF. Conclusion: These results suggest that VEGF can mediate the inflammatory response in endothelial cells in vivo via the up-regulation of IL-8.

Implications for Tumor Control During Protection of Normal Tissues With Antioxidants

The use of antioxidants during antineoplastic radiation therapy has long been a controversial topic. Antioxidants are known for their ability to scavenge free radicals, potentially reducing the damage caused by ionizing radiation to treated tissues. Reduction of normal tissue injury from radiotherapy with antioxidant supplementation is intriguing; however, it raises significant concerns about the possible inadvertent simultaneous protection of tumor tissues. This issue is not limited to the clinical trial setting, given that increasing numbers of the American public take vitamin supplements and naturopathic remedies during therapy. DNA damage is the most lethal form of cellular damage caused by ionizing radiation. With the types of ionizing radiation used in clinical practice today, approximately two thirds of DNA damage is caused by short-lived (nanoseconds to microseconds) high-energy primary and secondary free radicals. To act as a radioprotector by scavenging these free radicals, an antioxidant needs to be present in sufficient concentrations and have efficient radical scavenging capabilities in proximity to the target (DNA) during the radiation exposure. -tocopherol and -carotene would not be expected to provide significant protection against radiationinduced primary and secondary reactive species compared with conventional radioprotectors. However, free-radical generation in tissues may continue after the radiation exposure as a consequence of an inflammatory response, with the generation of cytokines and sustained production of longer lived free radicals. In this setting, particularly in normal tissues, -tocopherol and -carotene may in fact be quite effective toward protecting against sustained freeradical damage. Interestingly, it is now becoming clear that free radicals can also serve as initiators (triggers) for complex signal transduction and gene expression pathways governing proand antisurvival responses. For example, -tocopherol can modulate transcriptional and posttranscriptional regulation of protein kinase C, cyclooxigenase, and lipoxygenase. Whether radiation induces a similar inflammatory response in tumors is less clear, given their markedly different redox status. To further complicate the issue of supplementation, the concentration of the agent or the surrounding environment can alter the antioxidant effects of many molecules. As for the microenvironment, -carotene is an antioxidant at low oxygen concentrations; however, it acts as a pro-oxidant at high oxygen concentrations. To illustrate this complex relationship of dose, agent, and microenvironment, consider the following two large randomized studies that have assessed the efficacy of -carotene as a preventative agent in patients at high risk of malignancy. The Alpha-Tocopherol, Beta-Carotene cancer prevention trial randomly assigned 29,133 male smokers to supplementation with -tocopherol, -carotene, both, or placebo, and unexpectedly found an increased risk of lung cancer in participants who received -carotene supplementation. Supplementation with -tocopherol alone in this study appeared to have no adverse effect on lung cancer incidence. In addition, the -Carotene and Retinol Efficacy trial randomly assigned 18,314 men and women at high risk of developing lung cancer to the combination of daily -carotene and retinyl palmitate (vitamin A) or placebo. This large study was stopped early because of evidence of a significant increase in the risk of lung cancer and lung cancer mortality in the treatment group compared with the placebo group. It is hypothesized that the increased incidence of lung cancers seen in both of these large, randomized studies is at least partially due to the pro-oxidant nature of -carotene when used at high doses or in oxygen-rich environments, such as the lung. How do the preclinical findings that antioxidants improve the efficacy of radiotherapy translate into clinical outcomes? Numerous clinical studies investigating the effects of antioxidants in combination with radiotherapy or JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 23 NUMBER 24 AUGUST 2