Potentiality of multiple modalities for single-cell analyses to evaluate the tumor microenvironment in clinical specimens.

Single-cell level analysis is powerful tool to assess the heterogeneity of cellular components in tumor microenvironments (TME). In this study, we investigated immune-profiles using the single-cell analyses of endoscopically- or surgically-resected tumors, and peripheral blood mononuclear cells from gastric cancer patients. Furthermore, we technically characterized two distinct platforms of the single-cell analysis; RNA-seq-based analysis (scRNA-seq), and mass cytometry-based analysis (CyTOF), both of which are broadly embraced technologies. Our study revealed that the scRNA-seq analysis could cover a broader range of immune cells of TME in the biopsy-resected small samples of tumors, detecting even small subgroups of B cells or Treg cells in the tumors, although CyTOF could distinguish the specific populations in more depth. These findings demonstrate that scRNA-seq analysis is a highly-feasible platform for elucidating the complexity of TME in small biopsy tumors, which would provide a novel strategies to overcome a therapeutic difficulties against cancer heterogeneity in TME.

Cotton rat (Sigmodon hispidus) develops metabolic disorders associated with visceral adipose inflammation and fatty pancreas without obesity.

Obesity induces metabolic disorders such as type 2 diabetes, hypertension, and cardiovascular diseases and has become a global health concern. Recent studies imply that fat accumulation in nonadipose tissue correlates with metabolic disorders. However, there are no suitable animal models to evaluate this phenomenon. This study investigated the characteristics of metabolic disorders found in cotton rat (Sigmodon hispidus). Blood biochemical examinations revealed that cotton rats, predominantly males, developed hyperinsulinemia, hyperglycemia, and dyslipidemia when fed a normal diet. The islets increased in size through ß-cell hyperplasia, which was associated with serum insulin level in both sexes, strongly indicating insulin resistance. In male cotton rats, oxidative stress was observed in ß cells, and macrophage infiltration into the visceral white adipose tissue was reported, both of which were associated with serum insulin level without visceral obesity. In contrast, female cotton rats developed hyperinsulinemia without histopathological changes that were reported in males. Adipocytes were found to be accumulated in the pancreas but not in the liver of both sexes during aging. Pancreatic fat accumulation was associated with the serum insulin level only in females. Taken together, cotton rats developed metabolic disorders associated with visceral fat inflammation in the absence of obesity. In addition, pancreatic ectopic fat may also be related to the early stages of these conditions. Thus, the cotton rat may serve as a novel and useful model for metabolic disorders characterized by visceral adipose inflammation and ectopic fat accumulation in the pancreas without obesity.

Ovarian mast cells migrate toward ovary-fimbria connection in neonatal MRL/MpJ mice.

MRL/MpJ mice have abundant ovarian mast cells (MCs) as compared with other strains at postnatal day 0 (P0); however, they sharply decrease after birth. These ovarian MCs, particularly beneath the ovarian surface epithelium (SE), which express mucosal MC (MMC) marker, might participate in early follicular development. This study investigated the changes in spatiotemporal distribution of MCs in the perinatal MRL/MpJ mouse ovaries. At P0 to P7, the MCs were densely localized to the ovary, especially their caudomedial region around the ovary-fimbria connection. The neonatal ovarian MCs showed intermediate characteristics of MMC and connective tissue MC (CTMC), and the latter phenotype became evident with aging. However, the expression ratio of the MMC to CTMC marker increased from P0 to P4 in the MRL/MpJ mouse ovary. Similarly, the ratio of MCs facing SE to total MC number increased with aging, although the number of ovarian MCs decreased, indicating the relative increase in MMC phenotypes in the early neonatal ovary. Neither proliferating nor apoptotic MCs were found in the MRL/MpJ mouse ovaries. The parenchymal cells surrounding MCs at ovary-fimbria connection showed similar molecular expression patterns (E-cadherin+/Foxl2-/Gata4+) as that of the ovarian surface epithelial cells. At P2, around the ovary-fimbria connection, c-kit- immature oocytes formed clusters called nests, and some MCs localized adjacent to c-kit- oocytes within the nests. These results indicated that in postnatal MRL/MpJ mice, ovarian MCs changed their distribution by migrating toward the parenchymal cells composing ovary-fimbria connection, which possessed similar characteristics to the ovarian surface epithelium. Thus, we elucidated the spatiotemporal alterations of the ovarian MCs in MRL/MpJ mice, and suggested their importance during the early follicular development by migrating toward the ovary-fimbria connection. MRL/MpJ mice would be useful to elucidate the relationship between neonatal immunity and reproductive systems.

Hydronephrosis with ureteritis developed in C57BL/6N mice carrying the congenic region derived from MRL/MpJ-type chromosome 11.

Inbred MRL/MpJ mice show several unique phenotypes in tissue regeneration processes and the urogenital and immune systems. Clarifying the genetic and molecular bases of these phenotypes requires the analysis of their genetic susceptibility locus. Herein, hydronephrosis development was incidentally observed in MRL/MpJ-derived chromosome 11 (D11Mit21-212)-carrying C57BL/6N-based congenic mice, which developed bilateral or unilateral hydronephrosis in both males and females with 23.5% and 12.5% prevalence, respectively. Histopathologically, papillary malformations of the transitional epithelium in the pelvic-ureteric junction seemed to constrict the ureter luminal entrance. Characteristically, eosinophilic crystals were observed in the lumen of diseased ureters. These ureters were surrounded by infiltrating cells mainly composed of numerous CD3+ T-cells and B220+ B-cells. Furthermore, several Iba-1+ macrophages, Gr-1+ granulocytes, mast cells and chitinase 3-like 3/Ym1 (an important inflammatory lectin)-positive cells were detected. Eosinophils also accumulated to these lesions in diseased ureters. Some B6.MRL-(D11Mit21-D11Mit212) mice had duplicated ureters. We determined >100 single nucleotide variants between C57BL/6N- and MRL/MpJ-type chromosome 11 congenic regions, which were associated with nonsynonymous substitution, frameshift or stopgain of coding proteins. In conclusion, B6.MRL-(D11Mit21-D11Mit212) mice spontaneously developed hydronephrosis due to obstructive uropathy with inflammation. Thus, this mouse line would be useful for molecular pathological analysis of obstructive uropathy in experimental medicine.

MRL/MpJ-Fas(lpr) mice show abnormalities in ovarian function and morphology with the progression of autoimmune disease.

The immune system is known to affect reproductive function, and maternal-fetal immune tolerance is essential for a successful pregnancy. To investigate the relationship between autoimmune disease and female reproductive function, we performed a comparative analysis of the ovarian phenotypes for C57BL/6 mice, autoimmune disease-prone MRL/MpJ (MRL/+) mice and congenic MRL/MpJ-Fas(lpr) (MRL/lpr) mice harboring a mutation in the Fas gene that speeds disease onset. Both MRL-background strains showed earlier vaginal opening than C57BL/6 mice. The estrous cycle became irregular by 6 and 12 months of age in MRL/lpr mice and mice of the other two strains, respectively. Histological analysis at 3 months revealed that the number of primordial follicles was smaller in MRL-background mice than in C57BL/6 mice after 3 months. In addition, MRL/lpr and MRL/+ mice displayed lower numbers of ovarian follicles and corpora lutea at 3 and 6 months, and 6 and 12 months, respectively, than that in age-matched C57BL/6 mice. MRL/lpr and MRL/+ mice developed ovarian interstitial glands after 3 and 6 months, respectively. In particular, MRL/lpr mice showed numerous infiltrating lymphocytes within the ovarian interstitia, and partially stratified ovarian surface epithelia with more developed microvilli than that observed in C57BL/6 mice at 6 months. No significant differences in serum hormone levels were observed between the strains. In conclusion, MRL/lpr mice display altered ovarian development, morphology and function consistent with the progression of severe autoimmune disease, as these findings are less severe in MRL/+ counterparts.

The onset of heat-induced testicular calcification in mice: involvement of the telomeric locus on chromosome 1.

Spermatocytes of MRL/MpJ mice are more heat resistant than those of C57BL/6 mice in experimental cryptorchidism. This phenotype depends in part on the locus at the 81-cM region of MRL/MpJ-type chromosome 1 (Chr 1). To evaluate the function of this locus, we examined pathological changes in mouse testes resulting from transient scrotal heat stress. Immediately after scrotal heat stress, meiosis progression and blood-testis barrier integrity were preserved in MRL/MpJ but not in C57BL/6 mice, nor in a C57BL/6-based congenic strain carrying the MRL/MpJ-derived Chr 1 locus (B6.MRLc1). Testicular damage was severe in the weeks after scrotal heat stress in all three strains; however, testicular calcification was observed only in C57BL/6 and MRL/MpJ mice (initially as nanocrystals in mitochondria of degenerating germ cells). In testes, expression of gremlin 2, a bone morphogenetic protein antagonist encoded on Chr 1, was markedly higher in B6.MRLc1 than in C57BL/6 or MRL/MpJ mice. Furthermore, gremlin-2 and bone morphogenetic protein 2 mRNA levels in heated testes correlated negatively and positively, respectively, with calcification. Thus, although the MRL/MpJ-derived locus on Chr 1 may play a pivotal role in recovery from heat-induced testicular damage, especially via inhibition of calcification, MRL/MpJ mice have a precipitating factor for testicular calcification and heat shock-resistant factors that reside outside the 81-cM region of Chr 1.

Genomic analysis of the appearance of ovarian mast cells in neonatal MRL/MpJ mice.

In MRL/MpJ mice, ovarian mast cells (OMCs) are more abundant than in other mouse strains, and tend to distribute beneath the ovarian surface epithelium at birth. This study investigated the factors regulating the appearance of neonatal OMCs in progeny of the cross between MRL/MpJ and C57BL/6N strains. F1 neonates had less than half the number of OMCs than MRL/MpJ. Interestingly, MRLB6F1 had more neonatal OMCs than B6MRLF1, although they were distributed over comparable areas. Furthermore, in MRL/MpJ fetuses for which parturition was delayed until embryonic day 21.5, the number of OMCs was significantly higher than in age-matched controls at postnatal day 2. These results suggest that the number of OMCs was influenced by the environmental factors during pregnancy. Quantitative trait locus analysis using N2 backcross progeny revealed two significant loci on chromosome 8: D8Mit343-D8Mit312 for the number of OMCs and D8Mit86-D8Mit89 for their distribution, designated as mast cell in the ovary of MRL/MpJ 1 (mcom1) and mcom2, respectively. Among MC migration-associated genes, ovarian expression of chemokine (C-C motif) ligand 17 at mcom1 locus was significantly higher in MRL/MpJ than in C57BL/6N, and positively correlated with the expression of OMC marker genes. These results indicate that the appearance of neonatal OMCs in MRL/MpJ is controlled by environmental factors and filial genetic factors, and that the abundance and distribution of OMCs are regulated by independent filial genetic elements.

Vitamin A deprivation affects the progression of the spermatogenic wave and initial formation of the blood-testis barrier, resulting in irreversible testicular degeneration in mice.

The blood testis-barrier (BTB) is essential for maintaining homeostasis in the seminiferous epithelium. Although many studies have reported that vitamin A (VA) is required for the maintenance of spermatogenesis, the relationships between the BTB, spermatogenesis and VA have not been elucidated. In this study, we analyzed BTB assembly and spermatogenesis in the testes of mice fed the VA-deficient (VAD) diet from the prepubertal period to adulthood. During the prepubertal period, no changes were observed in the initiation and progression of the first spermatogenic wave in mice fed the VAD diet. However, the numbers of preleptotene/leptotene spermatocytes derived from the second spermatogenic wave onwards were decreased, and initial BTB formation was also delayed, as evidenced by the decreased expression of mRNAs encoding BTB components and VA signaling molecules. From 60 days postpartum, mice fed the VAD diet exhibited apoptosis of germ cells, arrest of meiosis, disruption of the BTB, and dramatically decreased testis size. Furthermore, vacuolization and calcification were observed in the seminiferous epithelium of adult mice fed the VAD diet. Re-initiation of spermatogenesis by VA replenishment in adult mice fed the VAD diet rescued BTB assembly after when the second spermatogenic wave initiated from the arrested spermatogonia reached the preleptotene/leptotene spermatocytes. These results suggested that BTB integrity was regulated by VA metabolism with meiotic progression and that the impermeable BTB was required for persistent spermatogenesis rather than meiotic initiation. In conclusion, consumption of the VAD diet led to critical defects in spermatogenesis progression and altered the dynamics of BTB assembly.

Mice stage-specific claudin 3 expression regulates progression of meiosis in early stage spermatocytes.

Claudin 3 is a protein component of the tight junction strands. Tight junctions between adjacent Sertoli cells form the blood-testis barrier (BTB). During spermatogenesis, seminiferous stage-specific expression of claudin 3 is believed to regulate the migration of preleptotene/leptotene spermatocytes across the BTB. Here, we determined the cell types expressing claudin 3 in adult mouse testis and investigated spermatogenesis after testis-specific in vivo knockdown of claudin 3. The results of in situ hybridization revealed that claudin 3 mRNA was predominantly expressed in germ cells near the basal lamina of seminiferous tubules at stages VI-IX. Furthermore, claudin 3 protein was localized not only to the BTB but also to the cell membrane of STRA8-expressing preleptotene/leptotene spermatocytes in the testis of adult ICR.Cg-Tg(Stra8-EGFP)1Ysa/YsaRbrc mice. Although claudin 3 knockdown did not affect BTB integrity, it did cause a partial delay in spermatocyte migration across the BTB. Moreover, claudin 3 knockdown resulted in a prolonged preleptotene phase during spermatogenesis. These data indicate that the seminiferous stage-specific expression and localization of claudin 3 during spermatogenesis regulate the progression of meiosis by promoting germ cell migration across the BTB.

Distinct distribution of the tensin family in the mouse kidney and small intestine.

Tensin family members are cytoplasmic proteins that are localized to the integrin-mediated cell-basement membrane junctions and implicated in cytoskeletal organization, cell migration, and proliferation. The mammalian genome contains four paralogs, Tns1, Tns2, Tns3, and Tns4. Murine mutations in the Tns1 and Tns2 genes cause polycystic kidney disease and glomerular sclerosis, respectively, and Tns3-null mice exhibit an impaired intestinal epithelial development. However, the knowledge concerning the localization of each tensin is still fragmentary. In this study, the cellular and subcellular distributions of tensin members were defined and compared with each other. RT-PCR analysis indicated that Tns2 and Tns3 were more abundant in isolated glomeruli and that Tns1 was highly expressed in areas other than the glomeruli, but no Tns4 expression was observed in the kidney. All tensin members were detected in the small intestine. Immunohistochemical staining revealed that Tns1 was predominantly localized to the mesangium of glomeruli and renal tubules. In contrast, Tns2 and Tns3 were highly expressed in the podocytes and the partial collecting system. In the small intestine, Tns2 and Tns3 were highly expressed in crypt and villous epithelial cells. Furthermore, we found that Tns3 was colocalized with TJ protein ZO-1 in renal tubules. These results indicate distinct differences in the cellular expression of Tns1, Tns2, and Tns3, and suggest that they may be able to function independently of each other in the kidney and the small intestine.

Molecular dynamics of the blood-testis barrier components during murine spermatogenesis.

The blood-testis barrier (BTB) separates the seminiferous epithelium into the adluminal and basal compartments. During murine spermatogenesis, preleptotene/leptotene spermatocytes migrate from the basal to the adluminal compartment through the BTB during stages VIII-IX. In the present study, we focused on the tight junction (TJ) molecules and analyzed their spatiotemporal expression during the murine seminiferous epithelial cycle. Structural analysis revealed that the principal components of the BTB, for example, claudin-3, claudin-11, occludin, and zonula occludens-1 (ZO-1), were localized at the basal and luminal sides of the preleptotene/leptotene spermatocytes during the migration stages (VIII-IX). Although we detected claudin-11, occludin, and ZO-1 throughout spermatogenesis, claudin-3 was only detected during stages VI-IX. Quantitative PCR using dissected seminiferous tubules from three stages (Early: II-VI, Middle: VII-VIII, Late: IX-I) clarified that the mRNA levels of TJ molecules were not correlated with the histoplanimetrical protein levels during spermatogenesis. Additionally, tubulobulbar complexes, considered to be involved in the internalization of TJ, were observed at the BTB site. Furthermore, a significant reduction in the mRNA levels of genes for the degradation of occludin (Itch) and endocytic recycling (Rab13) were observed during the Late and Middle stages, respectively. Therefore, we hypothesized that the lag between mRNA and protein expression of TJ molecules may be due to posttranslational modulation, for example, tubulobulbar complexes and endocytic recycling processes. In conclusion, these findings indicate that the integrity of the BTB is maintained throughout spermatogenesis, and the stage-specific localization of claudin-3 protein plays an important role in regulating BTB permeability.