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Resistance to immune-checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold-, myeloid cell-, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell-subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC.

Supplementary figures, tables, material and methods, and references

Current pharmacological treatments for endometriosis are limited to hormonal agents that can relieve pain but cannot cure the disease. Therefore, the development of a disease-modifying drug for endometriosis is an unmet medical need. By studying human endometriotic samples, we found that the progression of endometriosis was associated with the development of inflammation and fibrosis. In addition, IL-8 expression was highly up-regulated in endometriotic tissues and closely correlated with disease progression. We created a long-acting recycling antibody against IL-8 (AMY109) and evaluated its clinical potency. Because rodents do not produce IL-8 and do not experience menstruation, we analyzed the lesions in cynomolgus monkeys that spontaneously developed endometriosis and in a surgically induced endometriosis monkey model. Both spontaneously developed and surgically induced endometriotic lesions demonstrated pathophysiology that was highly similar to that of human endometriosis. Once-a-month subcutaneous injection of AMY109 to monkeys with surgically induced endometriosis reduced the volume of nodular lesions, lowered the Revised American Society for Reproductive Medicine score as modified for monkeys, and ameliorated fibrosis and adhesions. In addition, experiments using cells derived from human endometriosis revealed that AMY109 inhibited the recruitment of neutrophils to endometriotic lesions and the production of monocyte chemoattractant protein-1 from neutrophils. Thus, AMY109 may represent a disease-modifying therapy for patients with endometriosis.

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy.Significance:Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1

New cancer characteristics can be discovered by focusing on the process of tumor formation. Cancer stem cells (CSCs) are a key subpopulation, as they are theorized to be at the apex of the tumor hierarchy. We can better understand their function in the tumor hierarchy by using sectioned samples to observe the growth of tumors from their origins as CSCs. In this study, we evaluated the growth of moderate differentiated colorectal cancer from LGR5-positive cells, which is a CSC marker of colorectal cancer, using xenograft and three-dimensional culture models spatiotemporally. These cells express LGR5 at high levels and show CSC phenotypes. To detect them, we used a previously generated antibody that specifically targets LGR5, and were therefore able to observe LGR5-positive cells aggregating into small clusters (sCLs) over the course of tumor growth. Because these LGR5-expressing sCLs formed continuously during growth mainly in the invasive front, we concluded that the structure must contribute significantly to the expansion of CSCs and to tumor growth overall. We confirmed the formation of sCLs from gland structures using a three-dimensional culture model. In addition, sCLs exhibited upregulated genes related to stress response and partial/hybrid epithelial-mesenchymal transition (EMT), as well as genes reported to be prognosis factors. Finally, sCLs with high LGR5 expression were identified in clinical samples. Based on these results, we elucidate how sCLs are an important contributors to tumor growth and the expansion of CSCs.

Tumor research has largely relied on xenograft models created by the engraftment of cultured cell lines derived from tumor tissues into immunodeficient mice for in vivo studies. Like in vitro models, such models retain the ability of tumor cells to continuously proliferate, so they have been used to predict the clinical relevance of studies on proliferating cells. However, these models are composed of a limited population of tumor cells, which include only those tumor cells that are able to adapt to culture conditions, and thus they do not reflect the diversity and heterogeneity of tumors. This, at least in part, explains the poor predictivity of non-clinical data in the research and development of molecularly targeted drugs. Recently, research focus has been directed towards patient-derived xenograft (PDX) models created by directly engrafting tumor tissues, which have not been cultured in vitro, into immunodeficient mice. PDX models reflect the diversity and heterogeneity of tumors, and the evidence they provide can be verified in the patient tissues from which they were derived originally. PDX models are anticipated to efficiently bridge non-clinical and clinical data in translational research. Based on the evidence obtained from our research experience, this review describes the characteristics of PDX models for acting as tumor models, and elucidates the points to consider when attempting to establish these models.

Lupus nephritis (LN) is a renal disease presented in systemic lupus erythematosus (SLE) and is divided into 6 classes based on histopathological criteria set by the International Society of Nephrology/Renal Pathology Society. Major mouse models of SLE usually develop class III/IV LN, which are characterized by subendothelial deposits and endocapillary hypercellularity. We examined the pathological features of kidneys in a mouse model of SLE induced by a toll-like receptor 7 agonist, imiquimod (IMQ). In experiment 1, eleven-female FVB/NJcl wild type mice were treated with IMQ on their ear skin 3 times per week. Plasma anti-dsDNA increased from 2 weeks after first IMQ treatment and 2 mice exhibited nephrotic syndrome from 6 weeks. Histopathology revealed eosinophilic mesangial deposits in all mice from 4 weeks. Subsequently, podocytes showed enlargement with vacuolation and their numbers decreased in 6 mice. There was no infiltration of inflammatory cells, subendothelial deposits in glomeruli, or subepithelial deposits in glomeruli. In experiment 2 using 10 mice at 8 weeks after IMQ treatment, the mesangial deposits were observed in all mice and confirmed to be IgG, IgA, IgM, C1q and C3 by immunofluorescence, which corresponds to class II LN. Foot process effacement was detected by transmission electron microscopy and was considered to lead to proteinuria. These mice exhibited pathological characteristics corresponding to class II LN and podocyte injury, which make it distinct from other mouse models of SLE.

Vestibular organs consist of the maculae staticae, which are located in both the utricle and saccule, as well as the semicircular ducts and their ampullas. There have been no reports on specimen preparation methods for vestibular organs, including maculae staticae or semicircular ducts. In this study, we investigated highly reproducible methods of preparing vestibular organ specimens for histopathological examinations. We established a method that allows researchers to observe the utricle and saccule, including otoliths, the ampulla of a semicircular duct, and parts of semicircular ducts. This highly reproducible method is useful for histopathological analysis of mice with symptoms of abnormal equilibrium caused by medical toxicity and genetic modification.

Recurrence after chemotherapy is one of the biggest obstacles in cancer therapy, along with metastasis. Although histopathological evaluation in preclinical models like the xenograft model help us to understand the pathophysiological process of tumor growth, there are not enough detailed histopathological analyses of such models. In this study, to learn how a tumor recovers the typical tumor structure after structural corruption during chemo-treatment, xenografted tumors originating from a patient-derived xenograft of colorectal cancer (CRC) were analyzed histopathologically over time. There were many Duct (Flattened) at Day 1 (one day after the final administration of Irinotecan), but the ratio of Duct (Columnar) and Cribriform-structures typically found in colorectal adenocarcinoma-increased over time. Finally, at Day 15 (15 days after the final administration of Irinotecan), tumor structure and size were once again the same as in the control group. LGR5, a known cancer stem cell (CSC) marker for CRC, was highly expressed on protruding structures observed from Duct (Flattened) during their transformation into Duct (Columnar) and Cribriform. In addition, these LGR5-expressing protruding structures were either Ki67 negative or positive. These results suggest that the formation of protruding structures on Duct (Flattened) is a pivotal first step in the regrowth of tumors.

Background: Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway inhibition may overcome chemoresistance of metastatic pancreatic cancer (MPC) refractory to gemcitabine + nab-paclitaxel (GN) due to suppression of cancer associated fibroblast (CAF) and increase of drug infiltration in tumor. We sought to determine the safety, recommended dose of tocilizumab (TCZ), an IL-6 receptor monoclonal antibody, and biological correlates of tumor shrinkage in patients with GN-refractory MPC. Methods: This phase 1, dose finding trial was conducted following preclinical study of IL-6 signaling blockade in genetically engineered mouse model of pancreatic cancer (pGEMM) and patients-derived CAF, enrolled 10 patients with MPC that had progressed after GN. During the dose-finding part to determine dose-limiting toxicity (DLT), patients initially received TCZ 8 mg/kg on Day 1 and nab-paclitaxel 100 mg/m2 + gemcitabine 750 mg/m2 on Days 2, 9, and 16. The subsequent dose-expansion part used the TCZ dose identified in the dose-finding study. Before Cycle 1 and at the end of Cycle 1, biopsy of liver metastasis was performed 3 to 5 hours after levofloxacin (LVXF) administration to measure LVFX infiltration in tumor using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). Results: In pGEMM tumor, mouse IL-6 receptor antibody plus GN led pathological response. The growth of CAFs from patients with pancreatic cancer was inhibited by TCZ. In this phase I study, no DLTs occurred and the recommended dosage of TCZ was determined to be 8 mg/kg. Treatment-emergent adverse events (TEAEs) occurred in 80% of patients (Grade ≥3 in 60%) with decreased neutrophil count (n=5, 7 events) being the most common TEAE. Disease control rate was 80·0% (95%CI: 44·4-97·5) and tumor reduction during cycle 1 was observed in 4 patients who were defined as responder. In paired-biopsied samples, decrease of phosphorylated (p) STAT3 expression in tumor was observed in 7 of 8 patients (88%). Responder-related biological activities were increase of cleaved PARP expression of tumor nuclei (P = 0.01), decrease of proliferative CAF (P = 0.08), and increase of LVFX infiltration in tumor (P = 0.04). Decrease of pSTAT3 expression (P = 0.02) was favor to increase of LVFX infiltration against increase of gamma-H2AX, an index of gemcitabine exposure (P = 0.20). Conclusion: TCZ+GN-rechallenge had a manageable safety profile and showed preliminary activity via inhibition of CAF and gain of intratumoral drug infiltration in MPC. Citation Format: Shuichi Mitsunaga, Masafumi Ikeda, Kazunori Aoki, Kyoko Yamaguchi, Noriaki Sawada, Etsuko Fujii, Masanobu Nishidate, Takashi Fujitomo, Hideaki Mizuno, Yoko Kayukawa, Atsuhiko Kato, Mayu Makikawa, Hiroshi Imaoka, Mitsuhito Sasaki, Kazuo Watanabe, Hiroyuki Tsunoda, Kimio Terao, Atsushi Ochiai. Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT565.

Assessing how gene expression analysis by RNA sequencing (RNA-Seq) correlates to a unique morphology is increasingly necessary, and laser capture microdissection (LCM) is a critical research tool for discovering the genes responsible in a region of interest (ROI). Because RNA-Seq requires high-quality RNA, a sample preparation procedure that can preserve morphology and give the required quality of RNA is essential. A PAXgene®-fixed paraffin-embedded (XFPE) block can satisfy the need for high-quality RNA, but there are few reports on adapting the method for LCM, such as how small an ROI is analyzable by RNA-Seq. In this study, we confirmed the morphology and preservation of RNA in XFPE and then assessed the relationship between the size of pieces cut by LCM and their RNA quality. In XFPE, the morphology was similar to that in alcohol-based fixed samples, the quality of the RNA extracted from a whole sample was excellent, that is equivalent to that of a fresh frozen sample, and the quality was maintained over one year later. Three sizes of pieces-large (25,000 µm2), medium (5,000 µm2), and small (1,000 µm2)-were cut by LCM so that the total areas of the sections cut per size were the same. RNA quality was found to be best preserved when tissue was cut into pieces of over 5,000 µm2. In summary, XFPE exhibits good morphology and excellent preservation of RNA quality. Furthermore, it can be a good tool when used with LCM and RNA-Seq, giving well-balanced RNA quality and tissue morphology in the ROI.

In vitro-cultured 3D structures called organoids have become important tools for biological research, but there is little information concerning simple and efficient methods to evaluate organoid morphology. To address this issue, we attempted to establish a simple method by applying conventional histopathology that enables observation of multiple organoids on a single cross section, maintains good morphology, and is applicable to various histopathological stains. By centrifugation in unsolidified agarose solution, we were able to accumulate the organoids onto a single plane. The morphology was well preserved, and the various morphological types and sizes of organoid structures were identified. This method was also applicable for special staining, immunohistochemistry, and immunofluorescence staining. This method makes it possible to utilize the advantages of conventional pathological methods when studying organoids.

Desmoglein-3 (DSG3) is a potential target of cytotoxic antibody therapy for squamous cell carcinomas but is also expressed in various normal squamous epithelia. We obtained information about DSG3 distribution in mouse tissues by immunohistochemistry and conducted an intravenous multiple-dose study in mouse to estimate the toxic potential of anti-DSG3 therapy. DSG3 was expressed in the squamous epithelium of several organs including the skin, esophagus, tongue, forestomach, eye, and vagina. It was expressed at all estrous cycles of the vagina with changes in distribution patterns along with the structural changes in each cycle, and expression was reduced in ovariectomized (OVX) mice. On the administration of the antibody, there was disarrangement of the vaginal mucosal epithelium with formation of miroabscess, increased granulocyte infiltration, and single cell necrosis. Despite similar expression levels of DSG3 in other tissues, histopathological changes were limited to the vagina. The severity of the changes was reduced by ovariectomy. From these findings, the lesions were thought to be related to the drastic change in the histological structure of the vaginal mucosa accompanying the estrous cycle. Thus, we have shown that the changing expression of target antigen distribution and its relationship with physiological changes in tissue structure are important features for estimating the toxic potential of cytotoxic antibody therapy.

Pathological evaluation of juvenile toxicity studies requires the understanding of normal tissue development at different ages. Here, we report the morphological features of the neonatal mouse intestine, focusing on crypt fission. Postnatal day (PND) 7 and 14 mice showed fewer crypts and less mature epithelial morphology compared to PND 21 and 28. Crypt fission occurred in three stages: 1) flattening of the crypt base into a skirt shape, 2) penetration of myofibroblasts into the crypt base center, and 3) complete separation of a single crypt into two daughter crypts. The ratio of crypt fission to total number of crypts was the highest at PND 14 and 7 in the jejunum and colon, respectively. Crypt fission, a key phenomenon for balance or imbalance in epithelial cell hierarchy, including stem and differentiated cells, is related to tissue injury repair and tumorigenesis. Therefore, examining crypt fission can provide valuable insights into current conditions of intestine.

The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.

Correction to: Scientific Reports 7: Article number: 45839; published online: 03 April 2017; updated: 19 March 2018 In this Article, the Authors neglected to include the Data and Material Availability section. This section should read: “Data and material availability The novel mouse strain generatedin this study is available on request from Chugai Pharmaceutical Co.

Leucine-rich repeat-containing G-protein coupled receptor 5, or LGR5, is a molecule that recognizes stem cells in multiple organs and also in colon cancer. Previously, we have developed monoclonal antibodies specific to LGR5 protein that can be used for immunofluorescence staining, but because a very low level of LGR5 protein is expressed, the visualization technique needed to be enhanced. To develop procedures to detect LGR5 protein in various specimens by immunofluorescence staining, we evaluated the Alexa-labeled streptavidin biotin (LSAB), the Qdot, and the tyramide methods. The detection sensitivity was highest in the tyramide method followed by the Qdot method, whereas subcellular localization of the protein was most clear in the Qdot method, because the Qdot method gave a high S/N ratio that could show a low background. Thus, the tyramide method is superior to the Q-dot method for intensifying the signal of a low expression protein, and the Qdot method is superior to the tyramide method for identifying the subcellular localization of the target protein. The results of the present study will be helpful in providing more insight into the pathophysiological roles of LGR5-positive cancer stem cells and in developing therapeutic approaches for targeting cancer stem cells.

Antibodies can swiftly provide therapeutics to target disease-related molecules discovered in genomic research. Antibody engineering techniques have been actively developed and these technological innovations have intensified the development of therapeutic antibodies. From the mid-1990’s, a series of therapeutic antibodies were launched that are now being used in clinic. The disease areas that therapeutic antibodies can target have subsequently expanded, and antibodies are currently utilized as pharmaceuticals for cancer, inflammatory disease, organ transplantation, cardiovascular disease, infection, respiratory disease, ophthalmologic disease, and so on. This paper briefly describes the modes of action of therapeutic antibodies. Several non-clinical study results of the pathological changes induced by therapeutic antibodies are also presented to aid the future assessment of the toxic potential of an antibody developed as a therapeutic.

Recently, large-scale gene expression profiling is often performed using RNA extracted from unfixed frozen or formalin-fixed paraffin embedded (FFPE) samples. However, both types of samples have drawbacks in terms of the morphological preservation and RNA quality. In the present study, we investigated 30 human prostate tissues using the PFA-AMeX method (fixation using paraformaldehyde (PFA) followed by embedding in paraffin by AMeX) with a DNA microarray combined with laser-capture microdissection. Morphologically, in contrast to the case of atypical adenomatous hyperplasia, loss of basal cells in prostate adenocarcinomas was as obvious in PFA-AMeX samples as in FFPE samples. As for quality, the loss of rRNA peaks 18S and 28S on the capillary electropherograms from both FFPE and PFA-AMeX samples showed that the RNA was degraded equally during processing. However, qRT-PCR with 3’ and 5’ primer sets designed against human beta-actin revealed that, although RNA degradation occurred in both methods, it occurred more mildly in the PFA-AMeX samples. In conclusion, the PFA-AMeX method is good with respect to morphology and RNA quality, which makes it a promising tool for DNA microarrays combined with laser-capture microdissection, and if the appropriate RNA quality criteria are used, the capture of credible GeneChip data is well over 80% efficient, at least in human prostate specimens.

Tissue cross-reactivity (TCR) studies are conducted when developing therapeutic antibodies, but their value is sometimes questioned because the positive organs often do not match the target organs of toxicity. We conducted TCR studies in human and cynomolgus monkey tissues for the development of an anti-human tissue factor antibody (TFAb) and also for a commercially available antibody, to clarify the true distribution of the target antigen. Tissue factor (TF) was found to be distributed in a wide variety of organs and tissues, including the heart and urinary bladder, in human and monkey. Administration of the TFAb to cynomolgus monkey caused hemorrhagic lesions mainly in the heart and urinary bladder in an incidental manner. This was thought to show the physiological role of TF in regulating hemostasis in these organs. Because the distribution of antigen in human and monkey was similar, the possibility that the TFAb would have similar effects in human was judged to be high, and because of the incidental nature of the effects, that they would be difficult to avoid. Thus it was possible to prospectively characterize the hazardous potential of a therapeutic antibody by accurately evaluating the tissue distribution of the target antigen and understanding its biological nature.

T cell–mediated immunotherapy is an attractive strategy for treatment in various disease areas. In this therapeutic approach, the CD3 complex is one of the key molecules to modulate T cell functions; however, in many cases, we cannot evaluate the drug candidates in animal experiments because the therapeutics, usually monoclonal antibodies specific to human CD3, cannot react to mouse endogenous Cd3. Although immunodeficient mice transfused with human hematopoietic stem or precursor cells, known as humanized mice, are available for these studies, mice humanized in this manner are not completely immune competent. In this study we have succeeded in establishing a novel mouse strain in which all the three components of the Cd3 complex — Cd3ε, Cd3δ, and Cd3γ — are replaced by their human counterparts, CD3E, CD3D, and CD3G. Basic immunological assessments have confirmed that this strain of human CD3 EDG–replaced mice are entirely immune competent, and we have also demonstrated that a bispecific antibody that simultaneously binds to human CD3 and a tumor-associated antigen (e.g. ERBB2 or GPC3) can be evaluated in human CD3 EDG–replaced mice engrafted with tumors. Our mouse model provides a novel means to evaluate the in vivo efficacy of human CD3–mediated therapy.

Summary Hepatocellular carcinoma (HCC) is a common cancer worldwide and represents the outcome of the natural history of chronic liver disease. The growing rates of HCC may be partially attributable to increased numbers of people with non‐alcoholic fatty liver disease (NAFLD) and non‐alcoholic steatohepatitis (NASH). However, details of the liver‐specific molecular mechanisms responsible for the NAFLD–NASH–HCC progression remain unclear, and mouse models that can be used to explore the exact factors that influence the progression of NAFLD/NASH to the more chronic stages of liver disease and subsequent HCC are not yet fully established. We have previously reported a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) as a dietary NASH model with rapidly progressive liver fibrosis in mice. The current study in C57BL/6J mice fed CDAHFD provided evidence for the chronic persistence of advanced hepatic fibrosis in NASH and disease progression towards HCC in a period of 36 weeks. When mice fed CDAHFD were switched back to a standard diet, hepatic steatosis was normalized and NAFLD activity score improved, but HCC incidence increased and the phenotype of fibrosis‐associated HCC development was observed. Moreover, when mice continued to be fed CDAHFD for 60 weeks, HCC further developed without severe body weight loss or carcinogenesis in other organs. The autochthonous tumours showed a variety of histological features and architectural patterns including trabecular, pseudoglandular and solid growth. The CDAHFD mouse model might be a useful tool for studying the development of HCC from NAFLD/NASH, and potentially useful for better understanding pathological changes during hepatocarcinogenesis.

Background To understand the clinical segments of IL-31 signaling blockade therapy in pruritus of atopic dermatitis (AD), direct detection of the target proteins in the diseased tissues will provide crucial information. There is a lack of direct evidence concerning the cellular origin of IL-31 in AD skins, and data on the expression of IL-31RA in AD are inconsistent. Also, there is no available information regarding IL-31RA protein expression in human dorsal root ganglia (DRG), which mediates the sensation of itch and is the long-suspected source of the protein. Objective We sought to obtain direct evidence concerning the distribution of IL-31- and IL-31RA-protein expressing cells and their characteristics in AD skin samples and in human DRG. Methods IL-31 was detected immunohistochemically in AD skins, and representative sections were double stained with IL-31 and several immune-markers. IL-31RA was stained immunohistochemically in AD skins and normal human DRG, and representative AD skins were double stained with IL-31RA and PGP9.5 (a nerve marker). Results IL-31-positive cells were observed as mononuclear infiltrating cells and as CD11b co-expressing cells in severe AD samples. As for IL-31RA, positive reactions were detected in keratinocytes and nerve fibers in the dermis of AD and in the neurons of normal DRG. Conclusion The detection of IL-31 in infiltrating cells of severe AD skin and of IL-31RA in nerve fibers of AD dermis and normal DRG indicates IL-31 signaling may be a contributing factor in the persistence and exacerbation of AD skin lesions.

Some therapeutic antibodies as anticancer agents exert their effects through the host immune system, but the factors that predict their cytotoxicity, including complement-dependent cytotoxicity (CDC), are unclear. In the present study, we attempted to elucidate some of these factors in a preclinical model. CDC-related mesangiolysis caused by administration of the anti-Thy-1.1 antibody can be studied in the rat anti-Thy-1 glomerulonephritis model, so the model was used in this study. Three animals each were sacrificed at 0.5, 1, 8, 24 and 48 hours after i.v. administration of the anti-Thy-1.1 antibody at 1mg/kg. The distribution of the Thy-1.1 antigen and 2 membrane complement regulatory proteins (mCRPs), Crry and CD55, in three non-treated animals and the distribution of the injected antibody and C3 in the model was studied by immunohistochemistry. In the mesangial cells of the kidney, both expression of the antigen and distribution of the antibody with C3 deposition were observed with weak expression of mCRPs. There was also antigen and antibody distribution in the medullary cells of the adrenal gland and in the lymphocytes of the thymus but no C3 deposition, which was thought to be related to high expression of mCRPs. The antigen was observed in several other organs and tissues without distribution of the antibody. Cell death was only observed in the mesangial cells. These results clearly demonstrate that activation of CDC is regulated by several factors, such as distribution of the target molecule, antibody distribution and the balance among the molecules of the CDC cascade and mCRPs.

Increased incidence of adrenal pheochromocytoma is frequently encountered in rat carcinogenicity studies. In some of the studies, the finding is judged to be due to a rat-specific mechanism of carcinogenesis caused by a disturbance of calcium homeostasis. However, direct evidence that the proliferation of chromaffin cells in the adrenal medulla is induced solely by hypercalcemia is not available. In this study, calcium gluconate was intravenously infused for 7 days to rat chromaffin cells by a tail cuff method, and cumulative labeling with bromodeoxyuridine (BrdU) was carried out to evaluate the proliferative activity. The serum calcium concentration was dose-dependently increased, and a high calcium concentration was stably sustained from day 2 to 7. In the adrenal medulla, BrdU-positive chromaffin cells increased in the calcium gluconate-treated animals, and the BrdU-labeling index increased in a dose-dependent manner. In addition, an increased BrdU-labeling index of chromaffin cells was shown to correlate with the serum calcium concentration. Our results demonstrate that hypercalcemia directly enhances the proliferative activity of chromaffin cells and that the proliferative activity is correlated with the serum calcium concentration.

Spontaneous progressive nephropathy dominated by glomerular lesions in common marmosets has been reported. However, the histopathologic characteristics, including the relationship between glomerular and tubulointerstitial lesions, have not been described in detail. In the present study, the authors examined the histopathologic characteristics of the background renal lesions in common marmosets (3 males and 9 females, 3 to 8 years old). The severity of glomerular lesions was graded into 3 classes: grade I, no alteration; grade II, hilar/focal increase of mesangial matrix; grade III, global/diffuse increase of mesangial matrix. Tubulointerstitial lesions (tubular regeneration and hyperplasia and interstitial inflammation and fibrosis) were scored according to the area of each lesion. The renal lesions were characterized by enlargement of glomeruli, expanded mesangial area with increase of periodic acid–Schiff reaction-positive matrix, tubular regeneration and hyperplasia, and interstitial inflammation and fibrosis. Glomerular lesions progressed with increasing mesangial matrix and aging. Additionally, the tubulointerstitial lesions became exacerbated with progressing glomerular lesions. Tubular hyperplasia was divided into 4 types according to the structure of the cell layer (simple or stratified-like), the area of increased lining cells (partial or entire), cytoplasmic staining (eosinophilic or basophilic), brush border and thickness of basement membrane, and the activity of cell proliferation. In conclusion, the background renal lesions in common marmosets were characterized by glomerular lesions with increase of mesangial matrix, which progressed with aging, and secondary tubulointerstitial lesions, including tubular hyperplasia. Those lesions were thus diagnosed as progressive glomerulonephropathy in common marmosets.

Fluorescein isothiocyanate (FITC)-labeled antibodies are widely used as primary antibodies in the tissue cross-reactivity (TCR) studies for the development of therapeutic antibodies. However, the effects of FITC-labeling on the characteristics of an antibody are poorly understood. The present study was performed to examine the effect of FITC-labeling on the binding affinity and immunohistochemical staining profile of an antibody, using several antibodies with different FITC-labeling indices. The results showed that the FITC-labeling index in antibody was negatively correlated with the binding affinity for its target antigen. Immunohistochemically, an antibody with a higher labeling index had a tendency to be more sensitive, but was also more likely to yield non-specific staining. Based on these findings, we recommend that a FITC-labeled antibody used as a primary antibody in a TCR study should be carefully selected from several differently labeled antibodies to minimize the decrease in the binding affinity and achieve the appropriate sensitivity and interpretation of the immunohistochemistry.

Background: GC33 is a recently developed monoclonal antibody against human glypican-3 (GPC3), which is significantly upregulated in hepatocellular carcinoma (HCC). GC33 recognizes a GPC3 ectodomain and shows significant antitumour activity in vivo. Thus, humanized GC33 antibody may be a promising tool for treating HCC having cell surface GPC3 expression. Aims: This study aims to determine the specificity, subcellular localization and prognostic impact of GPC3 immunoreactivity detected by GC33 in HCC clinical specimens. Methods: Immunohistochemical analysis was performed for 194 cases of resected HCC and prognostic analysis was performed for 185 eligible cases. Two antigen retrieval methods (autoclave and protease pretreatments) were used for immunohistochemistry and compared. The immunoscore system reflecting circumferential membranous GPC3 immunoreactivity was developed using either the autoclave or protease methods. The GPC3 mRNA level was analysed by quantitative real-time reverse transcription-polymerase chain reaction. Results: GC33 immunostaining after autoclave is a sensitive method and revealed the GPC3 expression (≥20% of tumour cells) in the majority (77%) of HCC samples tested. Alternatively, protease pretreatment showed lower sensitivity, but was superior for evaluating the intensity and subcellular localization of GPC3. Correlation between immunoscores and the GPC3 mRNA level was also confirmed. Subsequent clinicopathological analysis revealed worse prognoses in HCC patients with circumferential membranous GPC3 immunoreactivity. For HCC patients with hepatitis C virus (HCV) infection in particular, the high membranous GPC3 immunoreactivity was an independent prognostic factor for disease-free survival. Conclusions: Circumferential membranous GPC3 immunoreactivity in HCC indicates poorer prognosis particularly in patients with HCV infection.

Background: ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody (TRAB) currently in a Phase 1 clinical trial (NCT02748837) in patients with solid tumors that are glypican-3 (GPC3)-positive. ERY974 consists of a common light chain but has two different heavy chains that each recognize a different protein, GPC3 or CD3. ERY974 simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface to induce cellular cytotoxicity mediated by the potent effector function of T cells. The Fc portion of ERY974 is modified to eliminate FcγR binding and prevent GPC3-independent Fc-mediated effector function. However, binding to FcRn, an important factor in the PK profile of IgG, is maintained. ERY974 shows strong antitumor activity against gastric, lung, ovarian, head & neck, hepatic, and esophageal cancer-derived tumors in a non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mouse model injected with human T cells. In a cohort expansion of the Phase 1 trial, the activity of ERY974 will be examined in gastric cancer; however, individual clinical samples do not show uniform expression levels of GPC3 in tumor lesions, reflecting the heterogenous and complex structure of patients' tumors. To predict the potential efficacy of ERY974 in gastric cancer patients, we examined whether ERY974 alone or in combination with chemotherapy, could show substantial activity against heterogenous tumors with different GPC3 expression profiles using patient-derived xenografts (PDX) of gastric cancer. Method & results: We evaluated the antitumor effect of ERY974 in PDX gastric cancer tumors that have high, moderate, or low expression levels of GPC3 in a NOD-SCID mouse model injected with human T cells that were expanded in vitro using CD3/CD28 beads. The expression of GPC3 was evaluated by immunohistochemistry and quantitative RT-PCR. The efficacy of ERY974 monotherapy seemed to correlate with the GPC3 expression levels in each PDX tumor. The combination of ERY974 and chemotherapy, such as paclitaxcel, cisplatin or capecitabine, showed increased antitumor activity compared with ERY974 or chemotherapy alone. Conclusion: These preclinical data support the possibility that ERY974 alone or in combination with chemotherapy will demonstrate activity in patients with gastric cancer, and the GPC3 expression profile might be a useful predictive biomarker of ERY974 efficacy for patient selection. Citation Format: Azuma Yumiko, Yuji Sano, Toshiaki Tsunenari, Yasuko Kinoshita, Yoko Miyazaki, Junko Shinozuka, Etsuko Fujii, Atsuhiko Kato, Takahiro Ishiguro, Shohei Kishishita, Junichi Nezu, Yoshiki Kawabe, Mika Endo. ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, shows antitumor activity in gastric cancer patient-derived xenograft models with varying glypican-3 expression [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 5609.

Introduction: Codrituzumab/GC33/RO5137382 (GC33) is a humanized monoclonal antibody that targets glypican-3 (GPC3), an oncofetal protein expressed on the cell surface of hepatocellular carcinoma (HCC). GC33 interacts with CD16/FcγR3 and triggers antibody-dependent cellular cytotoxicity. Because anti-PD-L1/PD-1 agents have shown marked antitumor effect in various cancer types including HCC, we investigated if GC33 plus anti-PD-L1 mAb combination can augment antitumor efficacy in a mouse hepatoma syngeneic model transfected with human GPC3, named Hepa1-6/hGPC3. Methods: The Hepa1-6/hGPC3 cells were subcutaneously inoculated into C57BL/6J mice. After tumor mass was established, anti-mouse GPC3 mAb (mGC33; once-weekly), anti-mouse PD-L1 mAb (anti-mPD-L1 mAb; once-weekly), or a combination was administered on the first day of treatment (Day 0). Tumor tissues were collected on Day 21 for immunohistochemical (IHC) of F4/80 and PD-L1. To analyze tumor infiltrating lymphocytes (TILs), mGC33, anti-mPD-L1 mAb, or combination was administered to the Hepa1-6/hGPC3 mice. After 3 and 8 days from the 2nd dosing, TILs were analyzed to quantify the CD45-, CD3ε-, CD4-, or CD8α-positive TILs and CD11b+F4/80+ macrophages by flow cytometry. Results: In the Hepa1-6/hGPC3 model, combination therapy demonstrated a marked antitumor effect compared to the corresponding dose of mGC33 or anti-mPD-L1 mAb alone. Pathological complete responses were observed only in combination groups. The necrosis was more marked with combination therapy than with mGC33 or anti-mPD-L1 mAb alone. Though F4/80-positive cells existed mainly in the stroma in the vehicle group, these cells infiltrated the tumor periphery after mGC33 treatment. Most tumor-infiltrating immune cells, including macrophages and multinucleated giant cells, were PD-L1-positive. The combination increased CD45-, CD3ε-, and CD8α-positive T lymphocytes, but not CD4-positive T lymphocytes on Days 3 and 8 after the 2nd dosing. TILs were not increased in mice treated with either mGC33 or anti-mPD-L1 mAb. Conclusions: In this mouse model, mGC33 plus anti-mPD-L1 mAb combination therapy showed more potent antitumor efficacy than either monotherapy. mGC33 treatment enhanced tumor infiltration of PD-L1-positive immune cells, such as macrophages and multinucleated giant cells. Because anti-mPD-L1 mAb can block the binding between PD-L1 on macrophages and PD-1 on T cells, the CD8-positive T lymphocytes may be increased by combination therapy. These results suggest that the combination therapy of GC33 and anti-PD-L1 mAb may be clinically useful as a treatment for HCC. Citation Format: Mika Endo, Yasuko Kinoshita, Kenji Adachi, Yoshinori Narita, Jun Amano, Atsuhiko Kato, Takeshi Watanabe, Yoko Kayukawa, Yoko Miyazaki, Toshihiko Ohtomo. Anti-glypican-3 monoclonal antibody (codrituzumab/GC33/RO5137382) treatment enhances tumor infiltration of PD-L1-positive macrophages, and combination therapy with anti-PD-L1 monoclonal antibody promotes antitumor effects [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 2747.

Emphysematous cystitis is a rare disorder caused by bacterial infection and characterized by gas accumulation within the bladder wall with cyst formation. This report describes the histopathological characteristics of emphysematous cystitis found in a diabetic female beagle induced by streptozotocin and alloxan. Macroscopically, multiple cyst-like structures were observed on the cut surface of the urinary mucosa. During fixation, small specimens cut from the mucosa floated on the surface of the fixative solution. Histopathologically, multiple cysts were lined with a single layer of flattened cells found to be immunohistochemically positive for vimentin, partially positive for α-smooth muscle actin or macrophage scavenger receptor, class A, and thought to be myofibroblasts, fibroblasts or macrophages. Multinucleated giant cells were observed around the cysts, and gram-negative short bacilli were observed in the lumen of the urinary bladder. From these findings, this case was diagnosed as emphysematous cystitis.

Background: Glypican-3 (GPC3) is frequently upregulated in hepatocellular carcinoma (HCC). Analysis of GPC3-deficient mice implies GPC3 involvement in macrophage-lineage cells. Aim: In this study, we first assessed the association of GPC3 expression with the macrophage population in liver tissues from 30 HCC patients using immunohistochemistry. Methods: The GPC3 expression was categorized into three patterns – one with GPC3-negative staining and two with GPC3-positive staining (one with unclear membrane staining and one with clear membrane staining, designated GPC3+/C). The number of macrophages that were stained with resident macrophage (rMϕ) or pan-macrophage (pMϕ) markers was counted for each GPC3 expression pattern. Results: GPC3 immunoreactivity was observed in 76.7% of the HCC specimens. No significant differences were observed in the number of rMϕ marker-positive cells among the three expression patterns. In contrast, the GPC3+/C pattern showed a significantly higher number of pMϕ-positive cells compared with the other two patterns, most of which tended to take on the morphology of migrating macrophages. A second experiment conducted to compare macrophage infiltration between the xenograft tissues of a GPC3-transfected HCC cell line and its parent GPC3-nonexpressing cell line revealed that the increase in macrophages was stimulated by membrane expression of GPC3. Conclusion: The observations suggest that the increased macrophages in the GPC3+/C pattern are likely to be recruited macrophages, not resident macrophages, and that the expression of GPC3 in the membrane is involved in macrophage recruitment.

Previously, we demonstrated the antitumor efficacy of the anti-glypican-3 (GPC3) antibody GC33 in several human liver cancer xenograft models and the important role of antibody-dependent cellular cytotoxicity (ADCC) in the antitumor mechanism of GC33. Involvement of other mechanisms such as modulation of the functions of GPC3 in antitumor activity remains to be elucidated. In this study, we investigated histopathologically time-course changes in xenografts in mice following a single administration of GC33 to clarify the morphological changes contributing to the tumor growth inhibition of GC33, including the changes in GPC3-related factors/components [proliferation, extracellular matrices (ECMs) and macrophage]. Histopathological changes peaked 3-5 d after GC33 administration and included increased tumor cell death, tumor cells with round morphology, multinucleated tumor cells and small spindle/round-like cells (mostly F4/80-positive macrophages). No direct effects of GC33 on proliferation activity of tumor cells were observed. Meanwhile, alteration of ECM structures and a remarkable increase in macrophages was noted in the GC33-treated group. Increase in macrophages was observed mainly in the outer layer of tumor nodules; the area of the increase approximately included the area where the change in tumor cells and ECMs were observed. Interestingly, depletion of macrophages in the xenograft models resulted in a marked reduction of the antitumor activity of GC33. In the in vitro ADCC assay, ADCC was only slightly induced by mouse peritoneal macrophages. These data suggest that macrophages play an important role in the antitumor activity of GC33, which is not likely to be direct ADCC by macrophages themselves.

Interleukin-6 (IL-6) is a pleiotropic cytokine and a contributing factor in many diseases such as rheumatoid arthritis, Castleman's disease, Crohn's disease, and multiple myeloma. Since the blockade of the signaling pathway of the IL-6/interleukin-6 receptor (IL-6R)/gp130 complex is considered to have therapeutic value in such diseases, we developed an IL-6R humanized antibody (tocilizumab). In the current report, distribution of IL-6R in both normal human and cynomolgus monkey tissues was assessed as fundamental data to support preclinical and clinical studies of tocilizumab. Human and cynomolgus monkey tissue panels were stained with commercially available anti-human IL-6R and a species- and isotype-matched negative antibody, as well as assay control slides. The detection system applied used an Envision immunoperoxidase staining procedure with DAB reaction. Positive reactions were observed in the tissue elements of lymphatic, hematopoietic, digestive, reproductive, exocrine, endocrine, neural, muscular, epidermal, respiratory, and urinary systems of the human and cynomolgus monkey tissue panels. The current report is inclusive of a wide variety of tissues and shows the distribution of IL-6R to be similar for both human and monkey tissues. We consider this information fundamental for the support and interpretation of preclinical and clinical studies of anti-IL-6R antibody therapy.

Mitemcinal is an erythromycin derivative with motilin agonistic action, developed as a gastrointestinal motor-activating agent. The characteristics of mitemcinal-induced multinuclear hepatocytes (MNHs, hepatocytes with three or more nuclei per cell) from detailed morphological observations together with the results of a study on the mechanisms of MNH formation by combining cytocentrifuge preparations with 5-bromo-2’-deoxyuridine cumulative labeling are reported. MNHs were observed only in rats in the high-dose groups of the subchronic study, with a higher incidence in females and reversibility after twenty-eight days of drug withdrawal, but not observed in dogs. In the chronic study, the incidence increased relative to the dose. Histopathologically, MNHs were preferentially observed in the centrilobular zone, without nuclear atypia or mitotic figures. In the cell kinetic study, the labeling pattern of MNHs included all-positive, all-negative, and mixed labeling patterns of nuclei. The all-negative pattern indicated that the cells were formed by fusion of nondividing cells. The current results indicate that the cell kinetic approach effectively demonstrated the mechanism of mitemcinal-induced MNHs as fusion of hepatocytes and that drug-induced disturbance of mitosis is not involved in the multinucleation of MNHs by mitemcinal.

To understand the contribution of IL-6/IL-6R to subchondral bone and bone marrow abnormality in RA patients and the effects of tocilizumab on those abnormalities, we evaluated early change in a collagen-induced arthritis (CIA) monkey model with or without a single administration of tocilizumab. Six CIA cynomolgus monkeys received tocilizumab and 3 CIA monkeys received vehicle only. Their interphalangeal joints were analyzed using HE, silver impregnation (SI), or immunohistochemistry (RANKL) staining. The number of osteoclasts increased in the arthritis control but was suppressed in the tocilizumab-treated animals. Osteoblast/stromal cells of the arthritis control monkeys were of monolayer, while in the tocilizumab-treated monkeys, the cells were multi-layer or differentiated osteoblasts, and the meshwork of the reticulum fibers showed recovery in the SI. Hematopoietic marrow was replaced by interstitial fluid and reticulum fibers were eliminated in the arthritic model but showed recovery in the tocilizumab-treated animals. RANKL showed overproduction with arthritis and suppressed with tocilizumab treatment. The evidence indicates that IL-6/IL-6R is involved in subchondral bone and bone marrow change in RA patients. Tocilizumab treatment recovered changes in the CIA monkeys as a result of the co-differentiation between the osteoclasts and the osteoblast/stramal cells, at least partially through the suppression of RANKL overproduction.

Mitemcinal is an erythromycin derivative, which acts as an agonist of the motilin receptor. For assessment of the carcinogenicity of mitemcinal, we conducted a short-term carcinogenicity study in p53 (+/-) C57BL/6 mice and a 104-week carcinogenicity study in CD(SD)IGS rats. There was no evidence of a carcinogenic potential in mouse when administered for 26 consecutive weeks at levels up to 250 mg/kg/day. In the rat study, an increased incidence of lymphoma was noted in 5/60 males and 8/60 females of the high dose group (60 mg/kg/day) compared to 1/60 and 0/60 in control males and females, respectively, with statistical significance in females. Rat lymphomas include different immunomorphologic types (T- or B-cell lineage). Immunohistochemical analysis revealed that lymphomas from mitemcinal-treated rats and spontaneous cases were of T-cell lineage. The overall weight of evidence suggests that the incidence of spontaneous lymphoma was enhanced in the rat study. They also indicate that the increased incidence of lymphomas was based on a non-genotoxic effect with a threshold dose-response and that the tumorigenesis was based on the strain or species specificity of background factors. The high dose in the rat study is approximately 1600-fold higher (AUC) than that of the clinical dose, a sufficient margin of safety for the clinical dose. We conclude that the risk of carcinogenesis due to mitemcinal in humans can be considered to be minimal and is to represent an acceptable risk for the continued administration of mitemcinal to humans.

Histiocytic sarcoma is characterized by a malignant invasive proliferation of atypical cells showing morphologic and immunophenotypic features similar to those of mature tissue histiocytes. We report a case of histiocytic sarcoma in 11-year-old male cynomolgus macaque fed with a high-fat diet for 6 years. Invasive proliferation of neoplastic cells was localized in systemic organs such as liver, spleen, aorta, kidney, lung, peritonea, and mesenterium. Diffuse cellular foci of invasive proliferation were mainly observed in the peri/intra vascular area, sinus and serous membrane frequently accompanying arteriosclerosis. The tumor cells showed a round to spindle or polygonal shape with abundant foamy to vacuolated cytoplasm. Hemophagocytosis was noted infrequently. In immunohistochemical examinations, some medium to large-sized histiocytic atypical cells were positive for histiocytic markers such as CD68 and Lysozyme. Our case raises the possibility that histiocytic proliferation might be related to hypercholesterolemia and atherosclerosis. This is the first case report of histiocytic sarcoma in non-human primates.