Overcoming acquired resistance to PD-1 inhibitor with the addition of metformin in small cell lung cancer (SCLC).

Metformin has been widely used as the treatment of type II diabetes mellitus for its anti-hyperglycemic effect. In recent years, it has also been extensively studied for its anti-cancer effect as it diminishes immune exhaustion of CD8 + tumor-infiltrating lymphocytes (TILs). It decreases apoptosis of CD8 + TILs, thereby enhancing T cell-mediated immune response to tumor cells. Here, we present a unique case of a patient with small cell lung cancer (SCLC) who exhibited an overall partial response as per Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST 1.1) since starting metformin in combination with nivolumab therapy. Our patient had been treated with nivolumab monotherapy for 2 years until she had progression of disease. After she was started on metformin along with nivolumab therapy, she has shown a significant durable response for over 6 months. Many patients develop resistance to immunotherapy such as antibodies against cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death 1 (PD-1), and programmed cell death ligand 1 (PD-L1). Tumor hypoxia is one of the resistance factors. Signals activated by hypoxic environments in tumors are associated with decreased sensitivity to the PD-1 blockade. Metformin inhibits oxygen consumption in tumor cells in vitro and in vivo, reducing intratumoral hypoxia. These data suggest that metformin can improve susceptibility to anti-PD-1 treatment. To the best of our knowledge, our case is the first reported example demonstrating a proof-of-concept that metformin can contribute to overcoming acquired resistance to PD-1 inhibitors.

Osteopontin: correlation with phagocytosis by brain macrophages in a rat model of stroke.

Osteopontin (OPN) is an adhesive glycoprotein linked to a variety of pathophysiological processes. We investigated whether OPN might act as an opsonin in the diseased brain by studying the postischemic expression and localization of OPN mRNA and protein in a rat model of ischemic stroke. In addition, we characterized the subcellular localization of OPN protein in the ischemic brain core. Induction of OPN mRNA occurred in activated microglia/macrophages in the ischemic core on days 3-7 after reperfusion and this was sustained up to day 28, at least. OPN protein was synthesized and secreted by brain macrophages, which first surrounded damaged striatal white matter tracts and then infiltrated into them. Punctate OPN-immunoreactive profiles were scattered throughout the infarction core except in white matter bundles. Electron microscopy showed the localization of OPN protein along the membranes lining what appeared to be the debris of dead neurons. These were located in the extracellular space and within the cytoplasm of brain macrophages, indicating that the OPN protein accumulated selectively on the surface of dead cells, most of which were phagocytosed subsequently by brain macrophages. However, no significant induction of OPN occurred in degenerating striatal white matter tracts or in brain macrophage-engulfed axonic or myelin debris. These data suggest that OPN secreted by brain macrophages in this rat model of stroke might be involved in the phagocytosis of fragmented cell debris and possibly not in the phagocytosis of axonic or myelin debris.

Expression of vascular endothelial growth factor receptor-3 mRNA in the developing rat cerebellum.

Vascular endothelial growth factor receptor (VEGFR)-3, a receptor for VEGF-C and VEGF-D, has recently been suggested to play an important role during neuronal development. To characterize its potential role in CNS ontogenesis, we investigated the spatiotemporal and cellular expression of VEGFR-3 in developing and mature rat cerebellum using in situ hybridization. VEGFR-3 expression appeared as early as E15, and was restricted to the ventricular zone of the cerebellar primordium, the germinative neuroepithelium, but was absent by E20. Instead, the expression area of VEGFR-3 in the cerebellum grew in parallel with cerebellar development. From E20 on, two populations of VEGFR-3-expressing cells can be clearly distinguished in the developing cerebellum: a population of differentiating and postmitotic neurons and the Bergmann glia. VEGFR-3 expression in neurons occurred during the period of neuronal differentiation, and increased with maturation. In particular, the expression of VEGFR-3 mRNA revealed different temporal patterns in different neuronal populations. Neurons generated early, Purkinje cells, and deep nuclear neurons expressed VEGFR-3 mRNA during late embryonic stages, whereas VEGFR-3 transcription in local interneurons appeared by P14 with weaker expression. In addition, Bergmann glia expressed VEGFR-3 throughout cerebellar maturation into adulthood. However, receptor expression was absent in the progenitors in the external granular layer and during further migration. The results of this study suggest that VEGFR-3 has even broader functions than previously thought, regulating both developmental processes and adult neuronal function in the cerebellum.

Assessing tumor heterogeneity: integrating tissue and circulating tumor DNA (ctDNA) analysis in the era of immuno-oncology - blood TMB is not the same as tissue TMB.

Tissue tumor mutational burden (tTMB) is calculated to aid in cancer treatment selection. High tTMB predicts a favorable response to immunotherapy in patients with non-small cell lung cancer. Blood TMB (bTMB) from circulating tumor DNA is reported to have similar predictive power and has been proposed as an alternative to tTMB. Across many studies not only are tTMB and bTMB not concordant but also as reported previously by our group predict conflicting outcomes. This implies that bTMB is not a substitute for tTMB, but rather a composite index that may encompass tumor heterogeneity. Here, we provide a thorough overview of the predictive power of TMB, discuss the use of tumor heterogeneity alongside TMB to predict treatment response and review several methods of tumor heterogeneity assessment. Furthermore, we propose a hypothetical method of estimating tumor heterogeneity and touch on its clinical implications.

Differential regulation of vascular endothelial growth factor-C and its receptor in the rat hippocampus following transient forebrain ischemia.

We investigated the changes in the expression of vascular endothelial growth factor-C (VEGF-C) and its receptor, VEGFR-3, in the rat hippocampus following transient forebrain ischemia. The expression profiles of VEGF-C and VEGFR-3 were very similar in the control hippocampi, where both genes were constitutively expressed in neurons in the pyramidal cell and granule cell layers. The spatiotemporal expression pattern of VEGF-C was similar to that of VEGFR-3 in the ischemic hippocampus, and in the CA1 and dentate hilar regions both VEGF-C and VEGFR-3 were strongly expressed in activated glial cells rather than in neurons. Most of the activated glial cells expressing both genes were reactive astrocytes, although some were a subpopulation of brain macrophages. In the dentate gyrus, however, VEGFR-3 expression was transiently increased in the innermost layer of granule cells on days 7-10 after reperfusion, coinciding with an increase in polysialylated neural cell adhesion molecule staining--a marker for immature neurons. These data suggest that VEGF-C may be involved in glial reaction via paracrine or autocrine mechanisms in the ischemic brain and may carry out specific roles in adult hippocampal neurogenesis during ischemic insults.

Induction of suppressor of cytokine signaling-3 in astrocytes of the rat hippocampus following transient forebrain ischemia.

We investigated the spatiotemporal expression of suppressor of cytokine signaling-3 (SOCS-3) in the rat hippocampus following transient forebrain ischemia using in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Messenger RNA for SOCS-3 was constitutively expressed in neurons of the pyramidal cell and granule cell layers in control animals; however, significant induction was detected in reactive astrocytes preferentially located in the CA1 and the dentate hilar regions of the ischemic hippocampus. SOCS-3 mRNA was induced within 3 days of ischemia and maintained for more than 2 weeks. The in situ hybridization data agreed with the semiquantitative RT-PCR analysis. These results demonstrate SOCS-3 induction occurs in reactive astrocytes of the post-ischemic hippocampus, suggesting that SOCS-3 is involved in regulating the astroglial reaction to an ischemic insult.

Thermal stability of Cu and Cu2O nanoparticles in a polyimide film.

Nanoparticles of Cu or Cu oxide dispersed in a polyimide (PI) film were fabricated by reaction of polyamic acid with a thin Cu film during imidization. In this paper, the thermal stability of the Cu or Cu oxide nanoparticles was investigated under various atmospheres. The PI/nanoparticle composites were heat-treated at 140 degrees C and 250 degrees C in air, N2, Ar, and 5% H2 atmospheres. Nanoparticles in the PI film were characterized by UV-VIS spectroscopy and transmission electron microscopy. The optical absorption peaks originating from Cu or Cu2O nanoparticles were changed by heat-treatment in different atmospheres. When Cu nanoparticles were oxidized by heat-treatment in air, the surface plasmon resonance (SPR) peak originating from the Cu nanoparticles disappeared. The quantum confined absorption peak of Cu2O was not affected by heat-treatment in N2 or Ar. Cu2O nanoparticles were reduced by heat-treatment at 250 degrees C in 5% H2 atmosphere and a new SPR peak appeared. Our results show that Cu nanoparticles are easily oxidized and highly dense Cu nanoparticles can be formed by reducing Cu2O nanoparticles.

Upregulation of vascular endothelial growth factor receptors Flt-1 and Flk-1 following acute spinal cord contusion in rats.

To investigate the possible role of vascular endothelial growth factor (VEGF) in the injured spinal cord, we analyzed the distribution and time course of the two tyrosine kinase receptors for VEGF, Flt-1 and Flk-1, in the rat spinal cord following contusion injury using a weight-drop impactor. The semi-quantitative RT-PCR analysis of Flt-1 and Flk-1 in the spinal cord showed slight upregulation of these receptors following spinal cord injury. Although mRNAs for Flt-1 and Flk-1 were constitutively expressed in neurons, vascular endothelial cells, and some astrocytes in laminectomy control rats, their upregulation was induced in association with microglia/macrophages and reactive astrocytes in the vicinity of the lesion within 1 day in rats with a contusion injury and persisted for at least 14 days. The spatiotemporal expression of Flt-1 in the contused spinal cord mirrored that of Flk-1 expression. In the early phase of spinal cord injury, upregulation of Flt-1 and Flk-1 mRNA occurred in microglia/macrophages that infiltrated the lesion. In addition, the expression of both receptors increased progressively in reactive astrocytes within the vicinity of the lesion, predominately in the white matter, and almost all reactive astrocytes coexpressed Flt-1 or Flk-1 and nestin. These results suggest that VEGF may be involved in the inflammatory response and the astroglial reaction to contusion injuries of the spinal cord via specific VEGF receptors.

Upregulation of vascular endothelial growth factor receptors Flt-1 and Flk-1 in rat hippocampus after transient forebrain ischemia.

This study characterizes the distribution of the two tyrosine kinase receptors for vascular endothelial growth factor (VEGF), Flt-1 and Flk-1, in the rat hippocampus following transient forebrain ischemia. The semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of Flt-1 and Flk-1 in hippocampal CA1 showed upregulation of these receptors following ischemic injury. Expression of Flt-1 and Flk-1 mRNA was restricted to neurons in the pyramidal cell and granule cell layers in control animals; however, upregulation was detected in activated glial cells and in the vascular endothelial cells rather than in neurons, in ischemic hippocampi. Most of the activated glial cells expressing Flt-1 and Flk-1 were reactive astrocytes, although some were microglial cells. The spatiotemporal expression of Flt-1 in the ischemic hippocampus mirrored that of Flk-1 expression. Expression of mRNA for both receptors was induced after 12 h, appeared to be increased progressively until 3 days when the highest expression was reached, and was sustained for more than 2 weeks. Flt-1 and Flk-1 immunoreactivity in the ischemic hippocampus matched the mRNA induction patterns except for a somewhat delayed onset. These data suggest that VEGF may be involved in the glial response via specific VEGF receptors in the rat hippocampus following transient forebrain ischemia.

Transient microglial and prolonged astroglial upregulation of osteopontin following transient forebrain ischemia in rats.

Osteopontin (OPN) is an adhesive glycoprotein linked to a variety of pathophysiological processes, with neuroprotective properties in ischemic injury. We examined the postischemic expression and localization of OPN in the rat brain after transient forebrain ischemia. The semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that OPN expression in the hippocampal CA1 region was biphasic, peaking at day 3 after reperfusion and again between days 14 and 28. The two phases of OPN induction occurred in a time- and cell-dependent manner in the ischemic hippocampus. OPN mRNA expression in activated microglia was first induced 1 day after reperfusion, reached a peak at 3 days, and returned to basal levels by 7 days. In contrast, OPN expression in reactive astrocytes was first induced by 10 days after reperfusion in the hippocampal CA1. Astroglial OPN expression further increased, reaching a peak at day 14 and was maintained up to day 28, the latest time point we examined. OPN immunoreactivity in the ischemic hippocampus matched the mRNA induction patterns. OPN protein was first localized in the astroglial cytoplasm and later in the extracellular matrix of the hippocampal CA1. The temporal and cellular patterns of OPN induction in the ischemic hippocampus suggest a multifunctional capacity in the pathogenesis of ischemic injury, with the increased OPN production and secretion by reactive astrocytes being involved in subsequent tissue repair and reorganization.

Expression of vascular endothelial growth factor receptors Flt-1 and Flk-1 in embryonic rat forebrain.

To define better the putative targets of vascular endothelial growth factor (VEGF) in the developing brain we have examined the ontogeny of the two VEGF tyrosine kinase receptors, Flt-1 and Flk-1, in embryonic rat forebrain. Semiquantitative reverse transcriptase-polymerase chain reaction and immunoblot analysis showed expression of both receptors in the forebrain at all embryonic ages studied. Messenger RNAs for Flt-1 and Flk-1 appeared along most of the ventricular zone of the lateral ventricle as early as embryonic day (E) 13. Messages gradually became restricted to a limited ventricular zone at E20. Expression of VEGF receptors was also observed in the cerebral cortex, hippocampus and thalamic nuclei. In the cortex, expression of mRNA for both receptors was detected in the cortical plate around E15, and became relatively weak and restricted to the deeper layers of the cortical plate at E20. These data suggest that VEGF may contribute to early developmental processes including the proliferation, differentiation and maturation of specific neuronal populations via specific VEGF receptors in the developing rat forebrain.

Upregulation of vascular endothelial growth factor receptor-3 in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis.

We investigated the spatiotemporal expression of vascular endothelial growth factor receptor-3 (VEGFR-3) in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. VEGFR-3 mRNA and protein were constitutively expressed in gray matter neurons and in a few white matter astrocytes. Induction of VEGFR-3 occurred predominantly in perivascular infiltrated macrophages in the spinal cord white matter during the inductive phase of EAE. VEGFR-3 expression was also induced in activated microglial cells in the gray and white matter, mainly in the peak phase. In addition, reactive astrocytes in the white matter, but not in the gray matter, expressed VEGFR-3 as disease severity increased. These data suggest that VEGFR-3 is involved in the recruitment of monocytic macrophages and in glial reactions during EAE.

Expression and cellular localization of inducible nitric oxide synthase in lipopolysaccharide-treated rat kidneys.

Although inducible nitric oxide synthase (iNOS) is known to play significant roles in the kidney, its renal localization has long been controversial. To resolve this issue, the authors identified iNOS-positive cell types in rat kidneys using double immunohistochemistry and confirmed iNOS positivity using enzyme histochemistry with NADPH-diaphorase (NADPH-d) and in situ RT-PCR. Adult male Sprague-Dawley rats were injected intraperitoneally with lipopolysaccharide (LPS) or saline as a control and sacrificed at various time intervals after injection. Quantitative real-time reverse transcriptase polymerase chain reaction showed that iNOS was not expressed in control kidneys but was induced in LPS-treated kidneys. iNOS immunostaining was strongest 6 to 18 hr after injection and decreased gradually to control levels by day 7. Double immunohistochemistry and NADPH-d revealed that iNOS expression was induced in the interstitial cells, glomerular parietal epithelial cells, the proximal part of the short-looped descending thin limb, the upper and middle papillary parts of the long-looped descending thin limb, some inner medullary collecting duct cells, and almost all calyceal and papillary epithelial cells. The present study determines the precise localization of iNOS in LPS-treated rat kidneys and provides an important morphological basis for examining the roles of iNOS in sepsis-induced acute kidney injury.

Expression of ciliary neurotrophic factor and its receptor in experimental obstructive nephropathy.

Ciliary neurotrophic factor (CNTF) is well known as a growth/survival factor of neuronal tissue. We investigated the expression of CNTF and its specific receptor alpha (CNTFRα) in a unilateral ureteral obstruction (UUO) model. Complete UUO was produced by left ureteral ligation in Sprague-Dawley rats. The animals were sacrificed on days 1, 3, 5, 7, 14, 21, and 28 after UUO. The kidneys were fixed, and processed for both immunohistochemistry and in situ hybridization. CNTF immunoreactivity in sham-operated kidneys was observed only in the descending thin limb (DTL) of the loop of Henle. In UUO kidneys, CNTF expression was induced in the S3 segment (S3s) of the proximal tubule from day 1, and progressively expanded into the entire S3s and a part of the convoluted proximal tubules, distal tubules (DT), and glomerular parietal epithelium up to day 7. Upregulated CNTF expression was maintained to day 28. From day 14, the inner medullary collecting duct showed weak CNTF immunoreactivity. The CNTFRα mRNA hybridization signal in sham-operated kidneys was weakly detected in the DTL, DT, medullary thick ascending limb, and in a few S3s cells. After UUO, CNTFRα mRNA expression increased progressively in both the renal cortex and the medulla up to day 7 and increased expression was maintained until day 28. The results suggest that the S3s may be the principal induction site for CNTF in response to renal injury, and that CNTF may play a role in chronic renal injury.

Distribution of vascular endothelial growth factor receptor-3/Flt4 mRNA in adult rat central nervous system.

Vascular endothelial growth factor receptor (VEGFR)-3/Flt4 binds VEGF-C and VEGF-D with high affinity. It has been suggested to be involved in neurogenesis and adult neuronal function. However, little is known about the localization of VEGFR-3 in the adult central nervous system (CNS). The present study presents, to our knowledge, the first detailed mapping of VEGFR-3 mRNA expression in adult rat brain and spinal cord by using in situ hybridization and reverse transcription-polymerase chain reaction analysis (RT-PCR). Varying VEGFR-3 expression intensity was detected in functionally diverse nuclei, with the highest levels in the mitral cells of the olfactory bulb, piriform cortex, anterodorsal thalamic nucleus, several nuclei of the hypothalamus, and the brainstem cranial nerve nuclei. VEGFR-3 mRNA was abundantly expressed in the ventral motor neurons of the spinal cord and in some circumventricular organs such as the median eminence and the area postrema. Moreover, the locus coeruleus and some of the nuclei of the reticular formation showed moderate-to-high hybridization signals. VEGFR-3 expression appeared to be localized mostly within neurons, but weak labeling was also found in some astrocytes. In particular, VEGFR-3 was highly expressed in ependymal cells of the ventral third ventricle and the median eminence, which were co-labeled with vimentin but not with glial fibrillary acidic protein, suggesting that these cells are tanycytes. RT-PCR analysis revealed similar levels of VEGFR-3 expression in all regions of the adult rat CNS. The specific but widespread distribution of VEGFR-3 mRNA in the adult rat CNS suggests that VEGFR-3 functions more broadly than expected, regulating adult neuronal function playing important roles in tanycyte function.

Expression of calponin in periglomerular myofibroblasts of rat kidney with experimental chronic injuries.

Our previous research demonstrated that calponin-immunoreactivity was localized in myofibroblasts of the periglomerular region of human kidney specimens obtained at the time of transplantation from organ recipients. In the present study we examined calponin expression in two chronic nephropathy models, puromycin aminonucleoside (PAN) nephropathy and subtotal nephrectomy (SNx), to investigate the role of calponin in chronic renal injury. Male Sprague-Dawley rats were used, and both nephropathy models were established at 1, 2, 4, and 8 weeks after surgery. There were no periglomerular calponin-positive cells in sham, PAN 1 and 2 week, and SNx 1, 2, and 4 week groups. In SNx 8 week and PAN 4 and 8 week groups, only a few glomeruli with periglomerular calponin-reactivity, which covered half or a very small part of the periglomerular space, were observed. All glomeruli with periglomerular calponin-reactivity showed sclerotic changes, especially thickening of parietal epithelial cells (PECs). In conjunction with our previous report, this data represents the first documentation of the expression of calponin in renal myofibroblasts. We suggest that interactions between PECs and calponin-positive myofibroblasts may play a key role in the late stage of glomerulosclerosis.

Induction of vascular endothelial growth factor receptor-3 mRNA in glial cells following focal cerebral ischemia in rats.

To identify whether vascular endothelial growth factor receptor (VEGFR)-3, a receptor for VEGF-C and VEGF-D, is involved in pathophysiology of stroke, we investigated the spatiotemporal regulation of VEGFR-3 mRNA after transient focal cerebral ischemia. Most of the increase in VEGFR-3 expression in the ischemic core could be attributed to brain macrophages, whereas VEGFR-3 in the peri-infarct penumbra region was predominantly expressed in reactive astrocytes. A subpopulation of VEGFR-3-expressing brain macrophages was positive for NG2 proteoglycan and showed proliferative activity. In addition, in vitro model of stroke revealed no significant induction of VEGFR-3 in activated microglial cells, indicating that infiltrating exogenous macrophages expressed VEGFR-3 after focal ischemia. These data suggest that VEGFR-3 may be involved in the glial reaction and possibly in the recruitment of monocytic macrophages during ischemic insults.

Enhanced expression of vascular endothelial growth factor receptor-3 in the subventricular zone of stroke-lesioned rats.

Vascular endothelial growth factor receptor (VEGFR)-3, a receptor for VEGF-C and VEGF-D, has recently been proposed to be involved in adult hippocampal neurogenesis in response to cerebral ischemia. To identify whether VEGFR-3 is involved in poststroke neurogenesis, we investigated the temporal regulation of VEGFR-3 mRNA expression in the subventricular zone (SVZ) of rats with transient focal cerebral ischemia by in situ hybridization analysis, and identified the phenotypes of cells expressing VEGFR-3 by double- and triple-labeling techniques. In sham-operated rats, hybridization signals for VEGFR-3 mRNA were evident at a weaker intensity in the SVZ of the lateral ventricle. VEGFR-3 was transiently increased in the dorsolateral SVZ of the infarcted hemisphere on days 3-7 after reperfusion. Almost all VEGFR-3-expressing cells in the ipsilateral SVZ were colabeled with glial fibrillary acidic protein and the neural progenitor marker nestin, and were highly proliferative. In addition, a subset of VEGFR-3-labeled cells in the ipsilateral SVZ expressed the immature neuronal marker, polysialic acid-neural cell adhesion molecule. These data indicate that VEGFR-3 is upregulated in SVZ astrocytes and immature neurons after focal ischemia, suggesting that VEGFR-3 might mediate the adult neurogenesis after ischemic stroke.

Spatial and temporal changes of osteopontin in oxygen-glucose-deprived hippocampal slice cultures.

We have examined the temporal changes and cellular localization of osteopontin (OPN) mRNA and protein in organotypic hippocampal slice cultures subjected to ischemia-like oxygen-glucose deprivation (OGD). The sequential induction pattern response consisted of neuronal and microglial OPN upregulation, followed by a later extended phase of expression in reactive astrocytes. OPN immunoreactivity after OGD matched the mRNA induction patterns. Activated microglia revealed OPN staining in focal deposits, whereas neurons and reactive astrocytes showed perinuclear staining with a punctate cytosolic pattern of OPN, typical of secreted proteins. These data demonstrated that the temporal and cellular patterns of OPN induction in reactive glial cells in this in vitro model closely correlated with that in the in vivo model, suggesting that OPN has a multifunctional role in the pathogenesis of ischemic injury.