Non-classical Monocytes Enhance the Efficacy of Immune Checkpoint Inhibitors on Colon Cancer in a Syngeneic Mouse Model.

BACKGROUND/AIM: The response rate to immune checkpoint inhibitors (ICIs) is approximately 10%-30% and only in a few cancer types. In the present study, we determined whether non-classical monocytes (NCMs) could enhance ICI efficacy in colon cancer using a syngeneic mouse model. MATERIALS AND METHODS: The MC38 C57BL/6 mouse colon cancer model was used. Cells collected from the bone marrow of C57BL/6 mice were cultured, and NCMs were fractionated by cell sorting and administered via the tail veins to the mice implanted with MC38 cells. The anti-mouse PD-L1 antibody was administered three times, and tumor volume and overall survival were observed. RESULTS: More tumors were eradicated and more complete response occurred, after cotreatment with ICIs and NCMs than after treatment with ICIs alone. Moreover, no efficacy was observed when NCMs were administered alone. CONCLUSION: NCMs enhance ICI efficacy. The underlying mechanisms and clinical applications will be studied in the future.

Novel quantitative immunohistochemical analysis for evaluating PD-L1 expression with phosphor-integrated dots for predicting the efficacy of patients with cancer treated with immune checkpoint inhibitors.

Introduction: Programmed cell death ligand 1 (PD-L1) expression in tumor tissues is measured as a predictor of the therapeutic efficacy of immune checkpoint inhibitors (ICIs) in many cancer types. PD-L1 expression is evaluated by immunohistochemical staining using 3,3´-diaminobenzidine (DAB) chronogenesis (IHC-DAB); however, quantitative and reproducibility issues remain. We focused on a highly sensitive quantitative immunohistochemical method using phosphor-integrated dots (PIDs), which are fluorescent nanoparticles, and evaluated PD-L1 expression between the PID method and conventional DAB method. Methods: In total, 155 patients with metastatic or recurrent cancer treated with ICIs were enrolled from four university hospitals. Tumor tissue specimens collected before treatment were subjected to immunohistochemical staining with both the PID and conventional DAB methods to evaluate PD-L1 protein expression. Results: PD-L1 expression assessed using the PID and DAB methods was positively correlated. We quantified PD-L1 expression using the PID method and calculated PD-L1 PID scores. The PID score was significantly higher in the responder group than in the non-responder group. Survival analysis demonstrated that PD-L1 expression evaluated using the IHC-DAB method was not associated with progression-free survival (PFS) or overall survival (OS). Yet, PFS and OS were strikingly prolonged in the high PD-L1 PID score group. Conclusion: Quantification of PD-L1 expression as a PID score was more effective in predicting the treatment efficacy and prognosis of patients with cancer treated with ICIs. The quantitative evaluation of PD-L1 expression using the PID method is a novel strategy for protein detection. It is highly significant that the PID method was able to identify a group of patients with a favorable prognosis who could not be identified by the conventional DAB method.

Monocyte subsets associated with the efficacy of anti­PD­1 antibody monotherapy.

Immune checkpoint inhibitors (ICIs) are among the most notable advances in cancer immunotherapy; however, reliable biomarkers for the efficacy of ICIs are yet to be reported. Programmed death (PD)-ligand 1 (L1)-expressing CD14+ monocytes are associated with shorter overall survival (OS) time in patients with cancer treated with anti-PD-1 antibodies. The present study focused on the classification of monocytes into three subsets: Classical, intermediate and non-classical. A total of 44 patients with different types of cancer treated with anti-PD-1 monotherapy (pembrolizumab or nivolumab) were enrolled in the present study. The percentage of each monocyte subset was investigated, and the percentage of cells expressing PD-L1 or PD-1 within each of the three subsets was further analyzed. Higher pretreatment classical monocyte percentages were correlated with shorter OS (r=-0.32; P=0.032), whereas higher non-classical monocyte percentages were correlated with a favorable OS (r=0.39; P=0.0083). PD-L1-expressing classical monocytes accounted for a higher percentage of the total monocytes than non-classical monocytes with PD-L1 expression. In patients with non-small cell lung cancer (NSCLC), a higher percentage of PD-L1-expressing classical monocytes was correlated with shorter OS (r=-0.60; P=0.012), which is similar to the observation for the whole patient cohort. Comparatively, higher percentages of non-classical monocytes expressing PD-L1 were significantly associated with better OS, especially in patients with NSCLC (r=0.60; P=0.010). Moreover, a higher percentage of non-classical monocytes contributed to prolonged progression-free survival in patients with NSCLC (r=0.50; P=0.042), with similar results for PD-L1-expressing non-classical monocytes. The results suggested that the percentage of monocyte subsets in patients with cancer before anti-PD-1 monotherapy may predict the treatment efficacy and prognosis. Furthermore, more classical monocytes and fewer non-classical monocytes, especially those expressing PD-L1, are involved in shortening OS time, which may indicate the poor efficiency of anti-PD-1 treatment approaches.

The Sympathetic Nervous System Contributes to the Establishment of Pre-Metastatic Pulmonary Microenvironments.

Emerging evidence suggests that neural activity contributes to tumor initiation and its acquisition of metastatic properties. More specifically, it has been reported that the sympathetic nervous system regulates tumor angiogenesis, tumor growth, and metastasis. The function of the sympathetic nervous system in primary tumors has been gradually elucidated. However, its functions in pre-metastatic environments and/or the preparation of metastatic environments far from the primary sites are still unknown. To investigate the role of the sympathetic nervous system in pre-metastatic environments, we performed chemical sympathectomy using 6-OHDA in mice and observed a decrease in lung metastasis by attenuating the recruitment of myeloid-derived suppressor cells. Furthermore, we note that neuro-immune cell interactions could be observed in tumor-bearing mouse lungs in conjunction with the decreased expression of Sema3A. These data indicate that the sympathetic nervous system contributes to the preparation of pre-metastatic microenvironments in the lungs, which are mediated by neuro-immune cell interactions.

Increased Plasma Soluble PD-1 Concentration Correlates with Disease Progression in Patients with Cancer Treated with Anti-PD-1 Antibodies.

Immune checkpoint inhibitors (ICIs) confer remarkable therapeutic benefits to patients with various cancers. However, many patients are non-responders or develop resistance following an initial response to ICIs. There are no reliable biomarkers to predict the therapeutic effect of ICIs. Therefore, this study investigated the clinical implications of plasma levels of soluble anti-programmed death-1 (sPD-1) in patients with cancer treated with ICIs. In total, 22 patients (13 with non-small-cell lung carcinoma, 8 with gastric cancer, and 1 with bladder cancer) were evaluated for sPD-1 concentration using enzyme-linked immunosorbent assays for diagnostic and anti-PD-1 antibody analyses. sPD-1 levels were low before the administration of anti-PD-1 antibodies. After two and four cycles of anti-PD-1 antibody therapy, sPD-1 levels significantly increased compared with pretreatment levels (p = 0.0348 vs. 0.0232). We observed an increased rate of change in plasma sPD-1 concentrations after two and four cycles of anti-PD-1 antibody therapy that significantly correlated with tumor size progression (p = 0.024). sPD-1 may be involved in resistance to anti-PD-1 antibody therapy, suggesting that changes in sPD-1 levels can identify primary ICI non-responders early in treatment. Detailed analysis of each cancer type revealed the potential of sPD-1 as a predictive biomarker of response to ICI treatment in patients with cancer.

Analysis of ADAM12-Mediated Ephrin-A1 Cleavage and Its Biological Functions.

Accumulating evidence indicates that an elevated ephrin-A1 expression is positively correlated with a worse prognosis in some cancers such as colon and liver cancer. The detailed mechanism of an elevated ephrin-A1 expression in a worse prognosis still remains to be fully elucidated. We previously reported that ADAM12-cleaved ephrin-A1 enhanced lung vascular permeability and thereby induced lung metastasis. However, it is still unclear whether or not cleaved forms of ephrin-A1 are derived from primary tumors and have biological activities. We identified the ADAM12-mediated cleavage site of ephrin-A1 by a Matrix-assisted laser desorption ionization mass spectrometry and checked levels of ephrin-A1 in the serum and the urine derived from the primary tumors by using a mouse model. We found elevated levels of tumor-derived ephrin-A1 in the serum and the urine in the tumor-bearing mice. Moreover, inhibition of ADAM-mediated cleavage of ephrin-A1 or antagonization of the EphA receptors resulted in a significant reduction of lung metastasis. The results suggest that tumor-derived ephrin-A1 is not only a potential biomarker to predict lung metastasis from the primary tumor highly expressing ephrin-A1 but also a therapeutic target of lung metastasis.

Eph/Ephrin Signaling in the Tumor Microenvironment.

The Eph/ephrin system plays a vital role in diverse physiological events such as neurogenesis, vasculogenesis, and cell adhesion. Expression analysis of mRNA and protein in clinical samples revealed the involvement of the Eph/ephrin system in tumorigenesis, Alzheimer's disease, and atherosclerosis. Therefore, the Eph/ephrin system is considered a promising therapeutic target. However, no molecularly targeted drug against Ephs and ephrins is being used in the clinic thus far.Tumors are composed of various types of cells, including fibroblasts, immune cells, and endothelial cells. Recent studies showed the contribution of these cells to tumor growth, tumor progression, drug resistance, and metastasis. In this chapter, we discuss the role of Eph/ephrin system in the tumor microenvironment and describe its functions in tumor initiation, angiogenesis, cancer stem cell, tumor immunity, and also the metastatic environment.

Lung Tumor Cell Recruitment Assay.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

Roles of EphA1/A2 and ephrin-A1 in cancer.

The biological functions of the Eph/ephrin system have been intensively investigated and well documented so far since its discovery in 1987. Although the Eph/ephrin system has been implicated in pathological settings such as Alzheimer's disease and cancer, the molecular mechanism of the Eph/ephrin system in those diseases is not well understood. Especially in cancer, recent studies have demonstrated that most of Eph and ephrin are up- or down-regulated in various types of cancer, and have been implicated in tumor progression, tumor malignancy, and prognosis. However, they lack consistency and are in controversy. The localization patterns of EphA1 and EphA2 in mouse lungs are very similar, and both knockout mice showed similar phenotypes in the lungs. Ephrin-A1 that is a membrane-anchored ligand for EphAs was co-localized with EphA1 and EphA2 in lung vascular endothelial cells. We recently uncovered the molecular mechanism of ephrin-A1-induced lung metastasis by understanding the physiological function of ephrin-A1 in lungs. This review focuses on the function of EphA1, EphA2, and ephrin-A1 in tumors and an establishment of pre-metastatic microenvironment in the lungs.

Paradoxical counteraction by imatinib against cell death in myeloid progenitor 32D cells expressing p210BCR-ABL.

Chronic myeloid leukemia (CML) is believed to be caused by the tyrosine kinase p210BCR-ABL, which exhibits growth-promoting and anti-apoptotic activities. However, mechanisms that allow cell differentiation in CML still remain elusive. Here we established tetracycline (Tet)-regulatable p210BCR-ABL-expressing murine 32D myeloid progenitor (32D/TetOff-p210) cells to explore p210BCR-ABL-induced cell death and differentiation. Tet-regulatable overexpression of p210BCR-ABL induced cell death due to the activation of both caspase-1 and caspase-3, coincident with the differentiation from myeloid progenitors into CD11b+Ly6C+Ly6G+ cells with segmented nuclei, exemplified as granulocytic myeloid-derived suppressor cells (G-MDSC), and the ability to secrete IL-1ß, TNF-α, and S100A8/A9 into the culture supernatant. Treatment with imatinib almost completely abrogated all these phenotypes. Moreover, overexpression of a sensor of activated caspase-1 based on fluorescence resonance energy transfer (FRET) probe enabled us to detect activation of caspase-1 in a human CML cell line, K562. Furthermore, increased numbers of splenic G-MDSC associated with enhancement of S100A8/A9 production were observed in transgenic mice expressing p210BCR-ABL compared with that in wild-type mice. We also propose the novel mode of cell death in this 32D/TetOff-p210 system termed as myeloptosis.

C1D is not directly involved in the repair of UV-damaged DNA but protects cells from oxidative stress by regulating gene expressions in human cell lines.

A small nuclear protein, C1D, has roles in various cellular processes, transcription regulation, genome stability surveillance, DNA repair and RNA processing, all of which are required to maintain the host life cycles. In the previous report, C1D directly interacts with XPB, a component of the nucleotide excision repair complex, and C1D knockdown reduced cell survival of 27-1 cells, CHO derivative cells, after UV irradiation. To find out the role of C1D in UV-damaged cells, we used human cell lines with siRNA or shRNA to knockdown C1D. C1D knockdown reduced cell survival rates of LU99 and 786-O after UV irradiation, although C1D knockdown did not affect the efficiency of the nucleotide excision repair. Immunostaining data support that C1D is not directly involved in the DNA repair process in UV-damaged cells. However, H2O2 treatment reduced cell viability in LU99 and 786-O cells. We also found that C1D knockdown upregulated DDIT3 expression in LU99 cells and downregulated APEX1 in 786-O cells, suggesting that C1D functions as a co-repressor/activator. The data accounts for the reduction of cell survival rates upon UV irradiation.

Intracellular LINGO-1 negatively regulates Trk neurotrophin receptor signaling.

Neurotrophins, essential regulators of many aspects of neuronal differentiation and function, signal via four receptors, p75, TrkA, TrkB and TrkC. The three Trk paralogs are members of the LIG superfamily of membrane proteins, which share extracellular domains consisting of leucine-rich repeat and C2 Ig domains. Another LIG protein, LINGO-1 has been reported to bind and influence signaling of p75 as well as TrkA, TrkB and TrkC. Here we examine the manner in which LINGO-1 influences the function of TrkA, TrkB and TrkC. We report that Trk activation promotes Trk association with LINGO-1, and that this association promotes Trk degradation by a lysosomal mechanism. This mechanism resembles the mechanism by which another LIG protein, LRIG1, promotes lysosomal degradation of receptor tyrosine kinases such as the EGF receptor. We present evidence indicating that the Trk/LINGO-1 interaction occurs, in part, within recycling endosomes. We show that a mutant form of LINGO-1, with much of the extracellular domain deleted, has the capacity to enhance TrkA signaling in PC12 cells, possibly by acting as an inhibitor of Trk down-regulation by full length LINGO-1. We propose that LINGO-1 functions as a negative feedback regulator of signaling by cognate receptor tyrosine kinases including TrkA, TrkB and TrkC.

Drug Targeting Based on a New Concept-Targeting Against TLR4 as an Example.

TLRs are very important players to regulate innate immune responses. TLR4 controls the host defense by sensing an exotic pathogen, such as lipopolysaccharides. At the same time, some endogenous proteins, including HMGB1 and S100A8, could also function to be a ligand to elicit inflammatory reactions. These facts make TLR4 signaling system very complicated. For instance, the application of TLR4 ligands in cancer therapies is desirable for enhancement of anti-tumor immunity in terms of its reparative nature, but undesirable for enhancement of metastatic growth of cancer cells. In this manuscript, in order to make a novel molecular design to disrupt an interaction between TLR4/MD-2 and endogenous ligands, we provide a potential binding style of the TLR4/MD-2 complex with HMGB1 by using their 3D structural data and docking simulations, and also discuss S100A8 binding to TLR4/MD-2.

Human serum amyloid A3 (SAA3) protein, expressed as a fusion protein with SAA2, binds the oxidized low density lipoprotein receptor.

Serum amyloid A3 (SAA3) possesses characteristics distinct from the other serum amyloid A isoforms, SAA1, SAA2, and SAA4. High density lipoprotein contains the latter three isoforms, but not SAA3. The expression of mouse SAA3 (mSAA3) is known to be up-regulated extrahepatically in inflammatory responses, and acts as an endogenous ligand for the toll-like receptor 4/MD-2 complex. We previously reported that mSAA3 plays an important role in facilitating tumor metastasis by attracting circulating tumor cells and enhancing hyperpermeability in the lungs. On the other hand, human SAA3 (hSAA3) has long been regarded as a pseudogene, which is in contrast to the abundant expression levels of the other isoforms. Although the nucleotide sequence of hSAA3 is very similar to that of the other SAAs, a single oligonucleotide insertion in exon 2 causes a frame-shift to generate a unique amino acid sequence. In the present study, we identified that hSAA3 was transcribed in the hSAA2-SAA3 fusion transcripts of several human cell lines. In the fusion transcript, hSAA2 exon 3 was connected to hSAA3 exon 1 or hSAA3 exon 2, located approximately 130kb downstream from hSAA2 exon 3 in the genome, which suggested that it is produced by alternative splicing. Furthermore, we succeeded in detecting and isolating hSAA3 protein for the first time by an immunoprecipitation-enzyme linked immune assay system using monoclonal and polyclonal antibodies that recognize the hSAA3 unique amino acid sequence. We also demonstrated that hSAA3 bound oxidized low density lipoprotein receptor (oxLDL receptor, LOX-1) and elevated the phosphorylation of ERK, the intracellular MAP-kinase signaling protein.

Eph as a target in inflammation.

Evidence to show that the Eph/ephrin system is involved in inflammation induced by infection, injury, inflammatory diseases, and atherosclerosis has been increased. Although the roles of the Eph/ephrin system in both neural and vascular development as well as cell motility are well documented, its involvement in inflammatory processes has not yet been elucidated in detail. Moreover, the soluble form of artificially oligomerized or dimerized Fc-fused ephrin-A1 has been widely used in in vitro and/or in vivo studies to activate the EphA receptors, whereas its physiological functions as a membrane-anchored protein remain largely unknown. Recent studies using clinical samples reported that the overexpression of Ephs and ephrins in some tumors such as hepatocellular carcinoma positively correlated with both malignancy of tumors and the poor prognosis of cancer patients. However, the molecular mechanisms underlying malignancy of tumors are not fully understood. The author herein summarizes the molecular mechanisms of the Eph/ephrin system involved in the immune system and inflammatory processes. Especially, the author focuses on inflammation-induced physiological changes in vascular endothelial cells leading to vascular hyper-permeability and described them in this review. The author also introduces those that contribute to ephrin-A1-mediated lung metastasis.

LINGO-1 protein interacts with the p75 neurotrophin receptor in intracellular membrane compartments.

Axon outgrowth inhibition in response to trauma is thought to be mediated via the binding of myelin-associated inhibitory factors (e.g. Nogo-66, myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and myelin basic protein) to a putative tripartite LINGO-1·p75(NTR)·Nogo-66 receptor (NgR) complex at the cell surface. We found that endogenous LINGO-1 expression in neurons in the cortex and cerebellum is intracellular. Mutation or truncation of the highly conserved LINGO-1 C terminus altered this intracellular localization, causing poor intracellular retention and increased plasma membrane expression. p75(NTR) associated predominantly with natively expressed LINGO-1 containing immature N-glycans, characteristic of protein that has not completed trans-Golgi-mediated processing, whereas mutant forms of LINGO-1 with enhanced plasma membrane expression did not associate with p75(NTR). Co-immunoprecipitation experiments demonstrated that LINGO-1 and NgR competed for binding to p75(NTR) in a manner that is difficult to reconcile with the existence of a LINGO-1·p75(NTR)·NgR ternary complex. These findings contradict models postulating functional LINGO-1·p75(NTR)·NgR complexes in the plasma membrane.

ZFC3H1, a zinc finger protein, modulates IL-8 transcription by binding with celastramycin A, a potential immune suppressor.

Celastramycin A, a small molecule that inhibits the production of antibacterial peptides in an ex vivo culture system of Drosophila, suppresses the TNFα-mediated induction of IL-8 in mammalian cells. To understand its molecular mechanism, we examined Celastramycin A binding proteins and investigated their biological functions. Our screening and subsequent pull-down assay revealed ZFC3H1 (also known as CCDC131 or CSRC2), an uncharacterized zinc finger protein, as a Celastramycin A binding protein. The knockdown of ZFC3H1 reduced IL-8 expression levels in the TNFα-stimulated lung carcinoma cell line, LU99, and UV-irradiated HeLa cells. Based on reporter assay results, we concluded that ZFC3H1 participates in the transcriptional activation of IL-8. The findings of our UV-irradiation experiments implied that ZFC3H1 may indirectly interact with ERCC1 in an activated DNA repair complex. Thus, we designated ZFC3H1 as a mammalian target of Celastramycin A (mTOC).

Ephrin-A1 expression induced by S100A8 is mediated by the toll-like receptor 4.

The deregulation of Eph/ephrin protein expression has been shown to lead to tumor development and progression. Both mRNA and protein expression analyses using clinical samples have demonstrated that ephrin-A1 is over-expressed in various cancers and positively correlates with a poor prognosis for cancer patients. The prognosis of cancer patients depends on metastasis to distant organs. We previously demonstrated that ADAM12 metalloproteinase cleaved ephrin-A1 and ADAM12-cleaved ephrin-A1 enhanced vascular permeability by degrading VE-cadherin and the EphA2 receptor at the plasma membrane. An increase of soluble ephrin-A1 levels in the serum facilitated tumor cell recruitment to the lungs, which resulted in lung metastasis. We also found that ephrin-A1 was overexpressed in 3LL tumors, a highly metastatic tumor, in mice and TNFα, an authentic positive regulator of ephrin-A1, was not elevated in the tumors, whereas S100A8 was. Moreover, S100A8 induced ephrin-A1 expression mediated by the toll-like receptor 4 (TLR4). S100A8 is known to be an endogenous ligand for TLR4 and its expression was shown to be increased in the lungs at the premetastatic phase. Thus, S100A8 and ephrin-A1 contribute to lung metastasis. Therefore, elucidating the regulation mechanism of ephrin-A1 overexpression is of importance and may lead to the development of therapeutic drugs against tumor growth and metastasis.