Molecular subtypes of lung adenocarcinoma present distinct immune tumor microenvironments.

Overcoming resistance to immune checkpoint inhibitors is an important issue in patients with non-small-cell lung cancer (NSCLC). Transcriptome analysis shows that adenocarcinoma can be divided into three molecular subtypes: terminal respiratory unit (TRU), proximal proliferative (PP), and proximal inflammatory (PI), and squamous cell carcinoma (LUSQ) into four. However, the immunological characteristics of these subtypes are not fully understood. In this study, we investigated the immune landscape of NSCLC tissues in molecular subtypes using a multi-omics dataset, including tumor-infiltrating leukocytes (TILs) analyzed using flow cytometry, RNA sequences, whole exome sequences, metabolomic analysis, and clinicopathologic findings. In the PI subtype, the number of TILs increased and the immune response in the tumor microenvironment (TME) was activated, as indicated by high levels of tertiary lymphoid structures, and high cytotoxic marker levels. Patient prognosis was worse in the PP subtype than in other adenocarcinoma subtypes. Glucose transporter 1 (GLUT1) expression levels were upregulated and lactate accumulated in the TME of the PP subtype. This could lead to the formation of an immunosuppressive TME, including the inactivation of antigen-presenting cells. The TRU subtype had low biological malignancy and "cold" tumor-immune phenotypes. Squamous cell carcinoma (LUSQ) did not show distinct immunological characteristics in its respective subtypes. Elucidation of the immune characteristics of molecular subtypes could lead to the development of personalized immune therapy for lung cancer. Immune checkpoint inhibitors could be an effective treatment for the PI subtype. Glycolysis is a potential target for converting an immunosuppressive TME into an antitumorigenic TME in the PP subtype.

Effects of two weeks of food restriction on toxicological parameters in cynomolgus monkeys.

Animals frequently eat less after a test-article treatment in nonclinical toxicological studies, and it can be difficult to distinguish test article-derived toxicities from secondary changes related to this reduced food intake. Therefore, in this study, we restricted the food intake of cynomolgus monkeys (Cambodian, male, n=2 or 3, 48 ± 3 months old) to 25% of the control for two weeks and evaluated the effects on toxicological parameters (general conditions, body weight, electrocardiography, urinalysis, hematology, blood chemistry, bone marrow analysis, pathological examination). After 2 weeks, the monkeys exhibited decreases in bone marrow erythropoiesis (e.g., decreases in reticulocytes and bone marrow erythrocytes), as well as glycogenesis induction (e.g., increase in aspartate aminotransferase (AST)) and malnutrition (e.g., decrease in triglyceride and systemic adipocytes atrophy). Additionally, histopathological analysis revealed granuloma and inflammatory cell infiltration in coronary fat, which had never been found in previous food restriction studies using other animal species. These findings will enable researchers to more accurately evaluate the toxicological risks of test articles that simultaneously induce food intake reduction.

Fibroblast inhibition by tocilizumab enabled gemcitabine/nab-paclitaxel rechallenge for pancreatic cancer.

Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway inhibition may overcome chemoresistance of metastatic pancreatic cancer (MPC). We sought to determine the safety and recommended dose of tocilizumab (TCZ), an IL-6 receptor monoclonal antibody, and biological correlates of tumor shrinkage in patients with gemcitabine (GEM)/nanoparticle albumin-bound paclitaxel (nab-PTX)-refractory MPC. This phase 1 study enrolled 10 patients with MPC who had progressed after GEM/nab-PTX. Patients initially received TCZ 8 mg/kg on Day 1 and nab-PTX 100 mg/m2 + GEM 750 mg/m2 on Days 2, 9, and 16. Before and at the end of Cycle 1, biopsy of liver metastases was performed 3-5 h after levofloxacin (LVFX) administration to measure LVFX infiltration into tumor tissue. No dose-limited toxicities occurred, and the recommended dosage of TCZ was determined to be 8 mg/kg. Treatment-emergent adverse events occurred in 80% of patients, of which decreased neutrophil count was the most common. Tumor reduction during Cycle 1 was observed in four patients, who were defined as responders. In paired-biopsy samples, responder-related biological activities were an increase of cleaved PARP expression of tumor nuclei (p = 0.01), a decrease of proliferative cancer-associated fibroblasts (CAFs) (p = 0.08), and an increase of LVFX infiltration in the tumor (p = 0.04). A decrease of phosphorylated STAT3 expression (p = 0.02) favored an increase in LVFX infiltration. In conclusion, TCZ + GEM/nab-PTX-rechallenge had a manageable safety profile and showed preliminary activity via inhibition of CAF and improved intratumoral drug infiltration in MPC.

Tumor-infiltrating Leukocyte Profiling Defines Three Immune Subtypes of NSCLC with Distinct Signaling Pathways and Genetic Alterations.

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-dominant, 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. Significance: The precise TIL profiling classified NSCLC into novel three immune subtypes that correlates with patient outcome, identifying subtype-specific molecular pathways and genomic alterations that should play important roles in constructing subtype-specific immune tumor microenvironments. These classifications of NSCLC based on TIL status are useful for developing personalized immune therapies for NSCLC.

A long-acting anti-IL-8 antibody improves inflammation and fibrosis in endometriosis.

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.

Digital workflows for pathological assessment of rat estrous cycle stage using images of uterine horn and vaginal tissue.

Assessment of the estrous cycle of mature female mammals is an important component of verifying the efficacy and safety of drug candidates. The common pathological approach of relying on expert observation has several drawbacks, including laborious work and inter-viewer variability. The recent advent of image recognition technologies using deep learning is expected to bring substantial benefits to such pathological assessments. We herein propose 2 distinct deep learning-based workflows to classify the estrous cycle stage from tissue images of the uterine horn and vagina, respectively. These constructed models were able to classify the estrous cycle stages with accuracy comparable with that of expert pathologists. Our digital workflows allow efficient pathological assessments of the estrous cycle stage in rats and are thus expected to accelerate drug research and development.

A simple specimen preparation method for histopathological evaluation of vestibular organs.

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.

EOS789, pan-phosphate transporter inhibitor, ameliorates the progression of kidney injury in anti-GBM-induced glomerulonephritis rats.

Hyperphosphatemia associated with chronic kidney disease (CKD) not only dysregulates mineral metabolism and bone diseases, but also strongly contributes to the progression of kidney disease itself. We have identified a novel drug for hyperphosphatemia, EOS789, that interacts with several sodium-dependent phosphate transporters (NaPi-IIb, PiT-1, and PiT-2) known to contribute to intestinal phosphate absorption. In this study, we investigated whether EOS789 could ameliorate kidney disease progression in glomerulonephritis rats. Anti-glomerular basement membrane (GBM) nephritis was induced in rats by intravenously administering two types of anti-rat GBM antibodies. We evaluated the effect of EOS789 administered in food admixture on hyperphosphatemia and kidney disease progression. In an anti-GBM nephritis rats, which exhibit a significant increase in serum phosphate and a decline in renal function, EOS789 dose-dependently improved hyperphosphatemia and EOS789 at 0.3% food admixture significantly ameliorated kidney dysfunction as shown in the decline of serum creatinine and BUN. Renal histopathology analysis showed that EOS789 significantly decreased crescent formation in glomeruli. To elucidate the mechanism underlying glomerular disease progression, human mesangial cells were used. High phosphate concentration in media significantly increased the expression of Collagen 1A1, 3A1, and αSMA mRNA in human mesangial cells and EOS789 dose-dependently suppressed these fibrotic markers. These results indicate that EOS789 prevented glomerular crescent formation caused by mesangial fibrosis by ameliorating hyperphosphatemia. In conclusion, EOS789 would not only be useful against hyperphosphatemia but may also have the potential to relieve mesangial proliferative glomerulonephritis with crescent formation.

Class II lupus nephritis with podocyte injury in imiquimod-induced lupus-prone mice.

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.

A case of spontaneous autoimmune skin disease in a cynomolgus monkey (Macaca fascicularis).

Pemphigus is an autoimmune blistering disease characterized by lesions on the skin and mucous membranes. To date, no spontaneous cases of this disease have been reported in cynomolgus monkeys. This report describes the histopathological characteristics of spontaneous pemphigus in a cynomolgus monkey. Macroscopically, redness and scaling with pruritus were observed on the skin of the entire body. Histopathologically, the epidermis showed intercellular edema, and eosinophils and mononuclear cells infiltrated the epidermis. There was no obvious acantholysis in the epidermis. The perivascular area showed edema, and eosinophils and mononuclear cells infiltrated the vessels in the dermis. Immunohistochemically, the intercellular area in the epidermis was positive for Immunoglobulin G and Complement component 3. Serologically, anti-desmoglein 1 and desmoglein 3 antibodies in the serum were negative. From these findings, this case was diagnosed as an autoimmune skin disease, suspected to be pemphigus, and concluded as lesions being similar to those in human "pemphigus herpetiformis".

Lipomatosis of axillary lymph nodes in a cynomolgus monkey (Macaca fascicularis).

Lipomatosis of lymph nodes is defined as the replacement of the lymphatic parenchyma by adipose tissue which grows in the node from the hilus toward the cortical zone. In humans, it is considered as part of the normal aging process and is common in obese patients, but there are no reports in non-human primates. In this report, we describe the first case of lymph node lipomatosis in the bilateral axillary lymph nodes of a young adult cynomolgus monkey. Macroscopically, there were no apparent abnormalities in the axillary lymph nodes on either side, and their volumes were unchanged. At the cut surface, pale yellow fat-like tissue was observed in the medullary area. Histopathologically, well differentiated adipocytes replaced a large part of the lymphatic parenchyma in the area from the hilus to the medulla without any malignant findings. Based on these findings, the patient was diagnosed with lipomatosis of the lymph nodes.

Protruding structures with high expression of LGR5 are formed during regrowth phase after chemo-treatment in xenograft model of colorectal adenocarcinoma.

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.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Minipig.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project (www.toxpath.org/inhand.asp) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in most tissues and organs from the minipig used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

Antibody to CD137 Activated by Extracellular Adenosine Triphosphate Is Tumor Selective and Broadly Effective In Vivo without Systemic Immune Activation.

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.

Continuous formation of small clusters with LGR5-positive cells contributes to tumor growth in a colorectal cancer xenograft model.

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.

Exploitation of Elevated Extracellular ATP to Specifically Direct Antibody to Tumor Microenvironment.

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.

Patient-derived xenograft (PDX) models: characteristics and points to consider for the process of establishment.

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.

Tissue-specific effects of an anti-desmoglein-3 ADCC antibody due to expression of the target antigen and physiological characteristics of the mouse vagina.

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.

Histopathological evaluation of crypt fission during intestinal development in neonatal mice.

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.

Difference in morphology and interactome profiles between orthotopic and subcutaneous gastric cancer xenograft models.

In xenograft models, orthotopic (ORT) engraftment is thought to provide a different tumor microenvironment compared with subcutaneous (SC) engraftment. We attempted to characterize the biological difference between OE19 (adenocarcinoma of the gastroesophageal junction) SC and ORT models by pathological analysis and CASTIN (CAncer-STromal INteractome) analysis, which is a novel method developed to analyze the tumor-stroma interactome framework. In SC models, SCID mice were inoculated subcutaneously with OE19 cells, and tumor tissues were sampled at 3 weeks. In ORT models, SCID mice were inoculated under the serosal membrane of the stomach wall, and tumor tissues were sampled at 3 and 6 weeks after engraftment. Results from the two models were then compared. Histopathologically, the SC tumors were well circumscribed from the adjacent tissue, with scant stroma and the formation of large ductal structures. In contrast, the ORT tumors were less circumscribed, with small ductal structures invading into abundant stroma. Then we compared the transcriptome profiles of human tumor cells with the mouse stromal cells of each model by species-specific RNA sequencing. With CASTIN analysis, we successfully identified several interactions that are known to affect the tumor microenvironment as being selectively enhanced in the ORT model. In conclusion, pathological analysis and CASTIN analysis revealed that ORT models of OE19 cells have a more invasive character and enhanced interaction with stromal cells compared with SC models.