Sophisticated genetically engineered macrophages, CAR-Macs, in hitting the bull's eye for solid cancer immunotherapy approaches.

Studies have begun to employ macrophages engineered with chimeric antigen receptor (CAR-Macs) against solid tumors since they can enter solid tumor tissue and interact with approximately all cellular components in the tumor microenvironment. The chimeric antigen receptor (CAR) has emerged as an appealing strategy for improving immune cells' ability to detect cancer. Tumor-associated macrophages (TAMs) generated with CAR designs exhibit appropriate potency based on their capacity to enter solid tumors and communicate through the inhibitory tumor microenvironment. CAR-Macs technology is a new therapeutic method for attacking cancer cells by switching pro-tumoral M2 macrophages to anti-tumoral M1 macrophages, enhancing macrophage phagocytosis, or increasing antigen presentation activity. CAR-Macs may have a prevailing impact on surrounding immune cells, indicating that they retain anti-tumor activity in the presence of human M2 macrophages, demonstrating their use in CAR technology. Understanding the biology of TAM and targeting novel domains for the advanced CAR-Macrophage platform, it will be feasible to add a new dimension to immunotherapy techniques used exclusively in solid malignancies. This review describes how CAR-Macs technologies modulate CAR-Macrophage production, potential target biomarkers on these platforms, their role in immunotherapeutic approaches, and tumor microenvironment.

Tailored modulation of stemness and drug resistance marker characteristics in K-Ras mutant lung cancer cells via PD-L1 gene suppression.

AIMS: We aimed to analyze the association of tumor cell-specific Programmed Cell Death Ligand 1 (PD-L1) expression with stemness markers and multi-drug resistance genes in non-small cell lung cancer. MAIN METHODS: ATP Binding Cassette Subfamily G Member 2 (ABCG2), Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), CD44, Epithelial cell adhesion molecule (EPCAM), Kruppel-like factor 4 (KLF4), MYC Proto-Oncogene (MYC), Nanog Homeobox (NANOG), SRY-Box Transcription Factor 2 (SOX2) as well as ATP Binding Cassette Subfamily C Member (ABCC) 1-6, ABCC10-12 with ATP Binding Cassette Subfamily B Member (ABCB) 1 and ABCB4-9 belongs to ABC transporters family were analyzed and their correlation with PD-L1 was evaluated using Cancer Cell Line Encyclopedia and The Cancer Genome Atlas data sets. We validated potential lung cancer stemness markers harboring ABCG2, CD44, ALDH1A1 and SOX-2, which are affected as a result of siRNA-mediated suppression of PD-L1 via flow cytometry. K-Ras downstream signaling proteins as well as multidrug resistance proteins were also investigated. KEY FINDINGS: PD-L1 was found to be positively correlated with CD44, whereas negatively correlated with ALDH1A1 (Pearson r = 0.44, r = -0.48; respectively) in 45 K-Ras mutated NSCLC cells based on CCLE database. While ABCC5 was dominantly decreased in K-Ras mutant lung cancer cells affected by PD-L1 gene suppression, expression changes were observed in the activation of distinct signaling pathways in a cell line-dependent manner. SIGNIFICANCE: The evaluation of markers in lung adenocarcinoma with different types of K-Ras mutations in terms of both stemness and drug resistance markers will contribute to the development of personalized immunotherapy regimens.

Mechanisms of Antitumor Immunity and Immunosurveillance.

The immune system has a well-defined role in all stages of carcinogenesis. The current chapter presents a discussion of various constituents of immunity involved in tumorigenesis along with their mechanisms, forming the basis for immunoprevention and immunotherapy.

Targets and Strategies for Cancer Immunoprevention.

The immune system plays a key role in cancer prevention, initiation, and progression. Antitumoral immune responses can be boosted by harnessing antitumorigenic immune activators and/or blocking tumorigenic proinflammatory factors. Here we define these targets as well as the strategies that could be developed for effective cancer immunoprevention.

SRGN-Triggered Aggressive and Immunosuppressive Phenotype in a Subset of TTF-1-Negative Lung Adenocarcinomas.

BACKGROUND: Approximately 20% of lung adenocarcinoma (LUAD) is negative for the lineage-specific oncogene Thyroid transcription factor 1 (TTF-1) and exhibits worse clinical outcome with a low frequency of actionable genomic alterations. To identify molecular features associated with TTF-1-negative LUAD, we compared the transcriptomic and proteomic profiles of LUAD cell lines. SRGN , a chondroitin sulfate proteoglycan Serglycin, was identified as a markedly overexpressed gene in TTF-1-negative LUAD. We therefore investigated the roles and regulation of SRGN in TTF-1-negative LUAD. METHODS: Proteomic and metabolomic analyses of 41 LUAD cell lines were done using mass spectrometry. The function of SRGN was investigated in 3 TTF-1-negative and 4 TTF-1-positive LUAD cell lines and in a syngeneic mouse model (n = 5 to 8 mice per group). Expression of SRGN was evaluated in 94 and 105 surgically resected LUAD tumor specimens using immunohistochemistry. All statistical tests were 2-sided. RESULTS: SRGN was markedly overexpressed at mRNA and protein levels in TTF-1-negative LUAD cell lines (P < .001 for both mRNA and protein levels). Expression of SRGN in LUAD tumor tissue was associated with poor outcome (hazard ratio = 4.22, 95% confidence interval = 1.12 to 15.86, likelihood ratio test, P = .03), and with higher expression of Programmed cell death 1 ligand 1 (PD-L1) in tumor cells and higher infiltration of Programmed cell death protein 1-positive lymphocytes. SRGN regulated expression of PD-L1 as well as proinflammatory cytokines, including Interleukin-6, Interleukin-8, and C-X-C motif chemokine 1 in LUAD cell lines; increased migratory and invasive properties of LUAD cells and fibroblasts; and enhanced angiogenesis. SRGN was induced by DNA demethylation resulting from Nicotinamide N-methyltransferase-mediated impairment of methionine metabolism. CONCLUSIONS: Our findings suggest that SRGN plays a pivotal role in tumor-stromal interaction and reprogramming into an aggressive and immunosuppressive tumor microenvironment in TTF-1-negative LUAD.

Revisiting CCL-type chemokines in breast cancer and its milieu: prominent targetable chemokines, CCL8 and CCL21.

The patterns of chemokine expression play a decisive role in both breast cancer prognosis and metastasis. In a recent article published in Bioscience Reports, 'Bioinformatics identification of CCL8/21 as potential prognostic biomarkers in breast cancer microenvironment', Chen et al. presented that expression of both CCL8 and CCL21 among CCL-type chemokines is prominent for prognosis of the breast cancer, metastasis and chemoresistance (Biosci Rep (2020) 40(11); DOI: 10.1042/BSR20202042). Identifying the sources of the CCL8 and CCL21 in the tumor microenvironment and developing targeting strategies for these chemokines to prevent tumor growth will improve both prognosis and therapeutic outcomes.

Identification of the dominant angiogenic CXCL class chemokines associated with non-small cell lung cancer via bioinformatics tools.

Chemokines play a critical role in lung cancer progression and metastasis. In non-small cell lung cancer, the determination of dominant angiogenic CXCL-type chemokines may increase the efficacy of targeted therapy and modulate the prognosis of lung cancer. Also, chemokine and chemokine receptors shape the immune response in the cross-talk between both cancer cells and immune cells in the tumor microenvironment. In this computational evaluation study based on databases containing mostly RNA-seq analyses, it is aimed to determine the dominant angiogenic CXCL-type chemokines with the highest expression in lung adenocarcinoma tissues and particularly in non-small cell lung cancer cells. CXCL1, CXCL5, CXCL7, and CXCL8, which can potentially be co-regulated and associated with poor survival, and phagocyte infiltration including neutrophils and macrophages are predominantly identified in non-small cell lung cancer. Moreover, the receptors of these chemokines, CXCR1 (binding CXCL8) and CXCR2 (binding CXCL1, 5, 7, 8), are positively correlated with the infiltration of neutrophils and macrophages. With the discovery of the common regulatory mechanisms of these angiogenic chemokines and validation studies in clinical examples, the chemokine panels specific to non-small cell lung cancer will become clear and have a decisive role in the prognosis of the disease.

Cancer Stemness as a Target for Immunotherapy is Shaped by Proinflammatory Stress.

Cancer stem cells represent a rare subpopulation of cancer cells carrying self-renewal and differentiation features in multi-step tumorigenesis, tumor recurrence and metastasis. Proinflammatory stress is highly associated with cancer stemness via induction of cytokines, tumorpromoting immune cells and cancer stemness-related signaling pathways. This review summarizes the major pro-inflammatory factors affecting cancer stem cell characteristics and the critical immunotherapeutic strategies to eliminate cancer stem cells.

Transcriptional splice variants of CD40 and its prognostic value in breast cancer.

CD40 is an important tumor necrosis factor receptor (TNFR) family protein for the development of antitumor response against cancer cells, apart from its role in the regulation of the immune system as a costimulatory molecule. It is broadly expressed on the surface of immune cells and in diverse cancer types, including breast cancer. Here, we analyzed both CD40/CD40 ligand expression in breast cancer cells and tissues using public data sets and overall survival analysis in ungrouped breast cancer patients, as well as in the triple-negative breast cancer subtype. We detected CD40 gene expression along with its 3 different splice variants (variants 1-3), predominantly in the triple-negative subgroup of breast cancer cell lines. The results of the overall survival analysis showed that high CD40 gene expression, particularly in the triple-negative subgroup of breast cancer patients, is associated with better survival. In addition to the transcriptional levels of CD40 splice variants, investigation of protein levels of these variants will allow the categorization of breast cancer cells and reveal their potential as an immunotherapeutic target.

Macrophage chemoattractants secreted by cancer cells: Sculptors of the tumor microenvironment and another crucial piece of the cancer secretome as a therapeutic target.

Beyond their essential role in leukocyte homing in the context of inflammation, chemokines orchestrate the host response to cancer progression. Chemokines are key accelerators in the amplification of inflammatory signals and metastasis in the distal zone of tumors, indicating possible immune editing of tumor cells in the microenvironment. This review summarizes the main macrophage-attracting chemokines secreted from cancer cells and how these mediators can be targeted to improve cancer immunotherapy in multiple cancer types.

IL22 Promotes Kras-Mutant Lung Cancer by Induction of a Protumor Immune Response and Protection of Stemness Properties.

Somatic KRAS mutations are the most common oncogenic variants in lung cancer and are associated with poor prognosis. Using a Kras-induced lung cancer mouse model, CC-LR, we previously showed a role for inflammation in lung tumorigenesis through activation of the NF-κB pathway, along with induction of interleukin 6 (IL6) and an IL17-producing CD4+ T-helper cell response. IL22 is an effector molecule secreted by CD4+ and γδ T cells that we previously found to be expressed in CC-LR mice. IL22 mostly signals through the STAT3 pathway and is thought to act exclusively on nonhematopoietic cells with basal IL22 receptor (IL22R) expression on epithelial cells. Here, we found that higher expression of IL22R1 in patients with KRAS-mutant lung adenocarcinoma was an independent indicator of poor recurrence-free survival. We then showed that genetic ablation of Il22 in CC-LR mice (CC-LR/IL22KO mice) caused a significant reduction in tumor number and size. This was accompanied by significantly lower tumor cell proliferation, angiogenesis, and STAT3 activation. Il22 ablation was also associated with significant reduction in lung-infiltrating inflammatory cells and expression of protumor inflammatory cytokines. Conversely, this was accompanied with increased antitumor Th1 and cytotoxic CD8+ T-cell responses, while suppressing the protumor immunosuppressive T regulatory cell response. In CC-LR/IL22KO mice, we found significantly reduced expression of core stemness genes and the number of prototypical SPC+CCSP+ stem cells. Thus, we conclude that IL22 promotes Kras-mutant lung tumorigenesis by driving a protumor inflammatory microenvironment with proliferative, angiogenic, and stemness contextual cues in epithelial/tumor cells. Cancer Immunol Res; 6(7); 788-97. ©2018 AACR.

IL-6 family cytokines: Key inflammatory mediators as biomarkers and potential therapeutic targets.

IL-6 is a critical cytokine in acute phase response and involved in the pathogenesis of several chronic inflammatory diseases including cancer. Studies have highlighted that levels of IL-6 and its family members can be useful for diagnosis, prognosis of relapse-free survival and recurrence. IL-6 family cytokines have been identified as cancer biomarkers through screening of inflammatory mediators in different fluids including saliva, serum, and bronchoalveolar lavage fluid (BALF). IL-6 can be modulated by chemopreventive drugs, small molecules, monoclonal antibodies and immune checkpoint inhibitors. Unveiling the different sources of IL-6, the interaction between IL-6 and its cellular targets, the IL-6-dependent tumor resistance mechanisms, and the identification of novel regulators of IL-6 are some of the highly complex topics included in this review and their understanding could aid cancer biomarkers and therapy development.

Reduced IL-6 levels and tumor-associated phospho-STAT3 are associated with reduced tumor development in a mouse model of lung cancer chemoprevention with myo-inositol.

Several promising chemopreventive agents have for lung cancer emerged in preclinical models and in retrospective trials. These agents have been shown to modulate pathways altered in carcinogenesis and reduce markers of carcinogenesis in animal and cell culture models. Cancer-prone transgenic mice with oncogenic Kras expressed in the airway epithelium (CcspCre/+ ; KrasLSL-G12D/+ ) were raised on diets compounded with myo-inositol. These animals form lung premalignant lesions in a stereotypical fashion over the ten weeks following weaning. Mice raised on myo-inositol containing diets showed potent reduction in the number, size, and stage of lesions as compared to those raised on control diets. myo-inositol has previously been reported to inhibit phosphoinositide 3-kinase (PI3K) signaling. However, in mice raised on myo-inositol, total PI3K signaling was largely unaffected. Proteomic and cytokine analyses revealed large reduction in IL-6 related pathways, including STAT3 phosphorylation. This effect was not due to direct inhibition of IL-6 production and autocrine signaling within the tumor cell, but rather through alteration in macrophage recruitment and in phenotype switching, with an increase in antitumoral M1 macrophages.

Investigation of hemophagocytic lymphohistiocytosis in severe sepsis patients.

PURPOSE: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition characterized by uncontrolled inflammation and has common clinical and laboratory features with sepsis. The aim of this study was to investigate patients treated with severe sepsis who had bicytopenia for the presence of HLH. MATERIALS AND METHODS: Patients with severe sepsis who were non-responsive to treatment and developed at least bicytopenia were included. Peripheral blood samples were collected and stored for later evaluation for natural killer (NK) activity and soluble interleukin-2 receptor levels. Diagnostic criteria of HLH were retrospectively analyzed. RESULTS: Seventy-five of 382 patients (20%) were followed as severe sepsis and septic shock. Among them, 40 patients had bicytopenia. Twenty-six of 40 patients were excluded due to the presence of active solid or hematological malignancies. Three patients died before fulfillment of HLH criteria and one patient denied to give consent. All of the remaining 10 patients had at least five of the eight criteria according to criteria of the Histiocyte Society. Only one of 10 patients was diagnosed as HLH and received treatment during intensive care unit stay. None of the 10 patients survived. CONCLUSIONS: This study emphasizes to consider the possibility of HLH and the need of rapid assessment of patients with severe sepsis who had bicytopenia and were resistant to treatment in intensive care.

IL6 Blockade Reprograms the Lung Tumor Microenvironment to Limit the Development and Progression of K-ras-Mutant Lung Cancer.

Activating mutations of K-ras are the most common oncogenic alterations found in lung cancer. Unfortunately, attempts to target K-ras-mutant lung tumors have thus far failed, clearly indicating the need for new approaches in patients with this molecular profile. We have previously shown NF-κB activation, release of IL6, and activation of its responsive transcription factor STAT3 in K-ras-mutant lung tumors, which was further amplified by the tumor-enhancing effect of chronic obstructive pulmonary disease (COPD)-type airway inflammation. These findings suggest an essential role for this inflammatory pathway in K-ras-mutant lung tumorigenesis and its enhancement by COPD. Therefore, here we blocked IL6 using a monoclonal anti-IL6 antibody in a K-ras-mutant mouse model of lung cancer in the absence or presence of COPD-type airway inflammation. IL6 blockade significantly inhibited lung cancer promotion, tumor cell-intrinsic STAT3 activation, tumor cell proliferation, and angiogenesis markers. Moreover, IL6 inhibition reduced expression of protumor type 2 molecules (arginase 1, Fizz 1, Mgl, and IDO), number of M2-type macrophages and granulocytic myeloid-derived suppressor cells, and protumor T-regulatory/Th17 cell responses. This was accompanied by increased expression of antitumor type 1 molecule (Nos2), and antitumor Th1/CD8 T-cell responses. Our study demonstrates that IL6 blockade not only has direct intrinsic inhibitory effect on tumor cells, but also reeducates the lung microenvironment toward an antitumor phenotype by altering the relative proportion between protumor and antitumor immune cells. This information introduces IL6 as a potential druggable target for prevention and treatment of K-ras-mutant lung tumors. Cancer Res; 76(11); 3189-99. ©2016 AACR.

CXCL7-induced macrophage infiltration in lung tumor is independent of CXCR2 expression: CXCL7-induced macrophage chemotaxis in LLC tumors.

Chemokines play diverse roles in modulating the immune response during tumor development. Levels of CXC chemokine ligand 7 (CXCL7) protein vary during tumorigenesis, and the evidence suggests that this chemokine serves as a novel biomarker of early-stage lung cancer. We investigated the effect of CXCL7 gene expression on the infiltration of myeloid cells into the tumor microenvironment in Lewis lung carcinoma (LLC). Tumors established from LLC cells overexpressing CXCL7 (CXCL7-LLC tumors) increased the infiltration of CD206(+) M2 macrophages at the early stages of tumorigenesis. This infiltration was independent of CXCR2 expression on either tumor cells or macrophages. CXCL7-LLC tumors developed faster than control-LLC tumors (IRES-LLC tumor) did. The extent of CD4(+) T cell, CD8(+) T cell, and natural killer T cell infiltration was similar between the two tumor groups. Our findings suggest that CXCL7 attracts macrophages especially at the tumor site and may accelerate lung tumor development in the early stages.

Molecular analysis of Chanarin-Dorfman syndrome (CDS) patients: Identification of novel mutations in the ABHD5 gene.

Chanarin-Dorfman syndrome (CDS) is an autosomal recessive metabolic disorder associated with congenital ichthyosis and a multisystemic accumulation of neutral lipids in various types of cells. Recently, mutations of the ABHD5 gene were identified as the cause of CDS. In this work, we carried out molecular analysis of the ABHD5 gene in 6 unrelated patients. We identified one previously reported mutation, N209X and two novel genetic alterations; a nonsense mutation (p.Y50X) and missense mutation (p.S73A).