Single-cell dissection of Merkel cell carcinoma heterogeneity unveils transcriptomic plasticity and therapeutic vulnerabilities.

Merkel cell carcinoma (MCC), a rare but aggressive skin cancer, remains a challenge in the era of precision medicine. Immune checkpoint inhibitors (ICIs), the only approved therapy for advanced MCC, are impeded by high primary and acquired resistance. Hence, we dissect transcriptomic heterogeneity at single-cell resolution in a panel of patient tumors, revealing phenotypic plasticity in a subset of treatment-naive MCC. The tumor cells in a "mesenchymal-like" state are endowed with an inflamed phenotype that portends a better ICI response. This observation is also validated in the largest whole transcriptomic dataset available from MCC patient tumors. In contrast, ICI-resistant tumors predominantly express neuroepithelial markers in a well-differentiated state with "immune-cold" landscape. Importantly, a subtle shift to "mesenchymal-like" state reverts copanlisib resistance in primary MCC cells, highlighting potential strategies in patient stratification for therapeutics to harness tumor cell plasticity, augment treatment efficacy, and avert resistance.

Salmonella Typhimurium Infection Reduces the Ascorbic Acid Uptake in the Intestine.

Salmonella Typhimurium infection of the gastrointestinal tract leads to damage that compromises the integrity of the intestinal epithelium and results in enterocolitis and inflammation. Salmonella infection promotes the expression of inflammasome NLRP3, leading to activation and release of proinflammatory cytokines such as IL-1ß, and the infected host often displays altered nutrient levels. To date, the effect of Salmonella infection and proinflammatory cytokine IL-1ß on the intestinal uptake of ascorbic acid (AA) is unknown. Our results revealed a marked decrease in the rate of AA uptake in mouse jejunum infected with Salmonella wild type (WT). However, the nonpathogenic mutant (Δ invA Δ spiB) strain did not affect AA uptake. The decrease in AA uptake due to Salmonella WT infection is accompanied by significantly lower expression of mouse (m)SVCT1 protein, mRNA, and hnRNA levels. NLRP3 and IL-1ß expression levels were markedly increased in Salmonella-infected mouse jejunum. IL-1ß-exposed Caco-2 cells displayed marked inhibition in AA uptake and significantly decreased hSVCT1 expression at both protein and mRNA levels. Furthermore, the activity of the SLC23A1 promoter was significantly inhibited by IL-1ß exposure. In addition, GRHPR (a known SVCT1 interactor) protein and mRNA expression levels were significantly reduced in Salmonella-infected mouse jejunum. These results indicate that Salmonella infection inhibits AA absorption in mouse jejunum and IL-1ß-exposed Caco-2 cells. The observed inhibitory effect may partially be mediated through transcriptional mechanisms.

Spatiotemporal Alterations in Gait in Humanized Transgenic Sickle Mice.

Sickle cell disease (SCD) is a hemoglobinopathy affecting multiple organs and featuring acute and chronic pain. Purkinje cell damage and hyperalgesia have been demonstrated in transgenic sickle mice. Purkinje cells are associated with movement and neural function which may influence pain. We hypothesized that Purkinje cell damage and/or chronic pain burden provoke compensatory gait changes in sickle mice. We found that Purkinje cells undergoe increased apoptosis as shown by caspase-3 activation. Using an automated gait measurement system, MouseWalker, we characterized spatiotemporal gait characteristics of humanized transgenic BERK sickle mice in comparison to control mice. Sickle mice showed alteration in stance instability and dynamic gait parameters (walking speed, stance duration, swing duration and specific swing indices). Differences in stance instability may reflect motor dysfunction due to damaged Purkinje cells. Alterations in diagonal and all stance indices indicative of hesitation during walking may originate from motor dysfunction and/or arise from fear and/or anticipation of movement-evoked pain. We also demonstrate that stance duration, diagonal swing indices and all stance indices correlate with both mechanical and deep tissue hyperalgesia, while stance instability correlates with only deep tissue hyperalgesia. Therefore, objective analysis of gait in SCD may provide insights into neurological impairment and pain states.

NUCB2/nesfatin-1 - Inhibitory effects on food intake, body weight and metabolism.

Since its discovery in 2006 by Oh-I and colleagues, NUCB2/nesfatin-1 encoded by nucleobindin-2 (NUCB2) has drawn sustained attention as reflected in over 500 publications. Among those, more than half focused on the alterations of food intake, body weight and metabolism (glucose, fat) induced by nesfatin-1 and/or NUCB2/nesfatin-1. In the current review we discuss the existing literature focusing on NUCB2/nesfatin-1's influence on food intake, body weight and glucose as well as fat metabolism and highlight gaps in knowledge.

Tamoxifen-induced, intestinal-specific deletion of Slc5a6 in adult mice leads to spontaneous inflammation: involvement of NF-κB, NLRP3, and gut microbiota.

The sodium-dependent multivitamin transporter (SMVT; SLC5A6) is involved in intestinal absorption of vitamin B7 (biotin). We have previously shown that mice with an embryonic intestinal-specific SMVT knockout (KO) develop biotin deficiency and severe spontaneous intestinal inflammation in addition to growth retardation, developmental delays, and death within the first 6-7 wk of life. The profound morbidity and mortality associated with the SMVT-KO has limited our ability to further characterize the intestinal inflammation and other sequelae of this deletion in adult mice with a mature gut microbiota. To overcome this limitation, we generated an intestine-specific, tamoxifen-inducible, conditional SMVT-KO (SMVT-icKO). Our results showed that adult SMVT-icKO mice have reduced body weight, biotin deficiency, shorter colonic length, and bloody diarrhea compared with age- and sex-matched control littermates. All SMVT-icKO mice also developed spontaneous intestinal inflammation associated with induction of calprotectin (S100a8/S100a9), proinflammatory cytokines (IL-1ß, TNF-α, IFN-γ, and IL-6), and an increase in intestinal permeability. Additionally, the intestines of SMVT-icKO showed activation of the NF-κB pathway and the nucleotide-binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) inflammasome. Notably, administration of broad-spectrum antibiotics reduced lethality and led to normalization of intestinal inflammation, proinflammatory cytokines, altered mucosal integrity, and reduced expression of the NLRP3 inflammasome. Overall, these findings support our conclusion that the biotin transport pathway plays an important role in the maintenance of intestinal homeostasis, and that NF-κB and the NLRP3 inflammasome, as well as gut microbiota, drive the development of intestinal inflammation when SMVT is absent.NEW & NOTEWORTHY This study demonstrates that deletion of the intestinal biotin uptake system in adult mice leads to the development of spontaneous gut inflammation and that luminal microbiota plays a role in its development.

Biotin and pantothenic acid oversupplementation to conditional SLC5A6 KO mice prevents the development of intestinal mucosal abnormalities and growth defects.

Intestinal absorption of the water-soluble vitamins biotin and pantothenic acid is carrier mediated and involves the sodium-dependent multivitamin transporter (SMVT; product of the SLC5A6 gene). We recently observed that intestinal-specific (conditional) knockout of the mouse Slc5a6 gene (SMVT-cKO) is associated with growth retardation, the development of spontaneous and severe inflammation, abnormal histology in the large intestine, altered gut permeability, and early death. Our aim in this study was to examine the possibility that biotin and pantothenic acid oversupplementation (BPS) of the SMVT-cKO mice could reverse the above-described abnormalities. BPS was provided in the drinking water to mice before conception, to dams during pregnancy and lactation, and to the SMVT-cKO mice throughout their life. Our findings showed that such a regimen prevents early death, as well as normalizes the growth rate, intestinal integrity, pathology, and inflammation in SMVT-cKO mice. These findings provide clear evidence for a role for biotin and/or pantothenic acid in the maintenance of normal intestinal integrity and health.

Biotin Deficiency Induces Th1- and Th17-Mediated Proinflammatory Responses in Human CD4+ T Lymphocytes via Activation of the mTOR Signaling Pathway.

Biotin (vitamin B7) is essential for human health because of its involvement, as a cofactor, in a variety of critical cellular metabolic reactions. Previous studies have shown that biotin deficiency enhances inflammation, and certain chronic inflammatory diseases are associated with biotin deficiency; however, the mechanisms that mediate the association between biotin status and inflammation are not well understood. In this study, we examined the effect of biotin deficiency on human CD4+ T cell responses to determine their role in biotin deficiency-associated inflammation. Our investigations revealed that anti-CD3/CD28-stimulated CD4+ T cells cultured in biotin-deficient medium secreted significantly enhanced levels of the proinflammatory cytokines IFN-γ, TNF, and IL-17. Expression of the transcription factors T-bet and RORγt was increased, whereas Foxp3 expression was decreased, in biotin-deficient CD4+ T cells. The percentage of T regulatory cells was also decreased under biotin-deficient condition. A similar increase in T-bet, RORγt, and proinflammatory cytokine levels, as well as a decrease in Foxp3, was observed in inguinal lymph nodes of mice fed a biotin-deficient diet relative to pair-fed controls. Furthermore, differentiation of CD4+ T cells toward Th1 and Th17 cells was also enhanced. In vitro and in vivo investigations indicated that the increased inflammatory response was due to enhanced activation of the mammalian target of rapamycin signaling pathway in biotin-deficient CD4+ T cells. In summary, these results demonstrate that biotin deficiency enhances the inflammatory responses in CD4+ T cells, which may contribute to inflammation associated with biotin deficiency.

Prostate cancer diagnosis using epigenetic biomarkers, 3D high-content imaging and probabilistic cell-by-cell classifiers.

BACKGROUND: Prostate cancer (PCa) management can benefit from novel concepts/biomarkers for reducing the current 20-30% chance of false-negative diagnosis with standard histopathology of biopsied tissue. METHOD: We explored the potential of selected epigenetic markers in combination with validated histopathological markers, 3D high-content imaging, cell-by-cell analysis, and probabilistic classification in generating novel detailed maps of biomarker heterogeneity in patient tissues, and PCa diagnosis. We used consecutive biopsies/radical prostatectomies from five patients for building a database of ∼140,000 analyzed cells across all tissue compartments and for model development; and from five patients and the two well-characterized HPrEpiC primary and LNCaP cancer cell types for model validation. RESULTS: Principal component analysis presented highest covariability for the four biomarkers 4',6-diamidino-2-phenylindole, 5-methylcytosine, 5-hydroxymethylcytosine, and alpha-methylacyl-CoA racemase in the epithelial tissue compartment. The panel also showed best performance in discriminating between normal and cancer-like cells in prostate tissues with a sensitivity and specificity of 85%, correctly classified 87% of HPrEpiC as healthy and 99% of LNCaP cells as cancer-like, identified a majority of aberrant cells within histopathologically benign tissues at baseline diagnosis of patients that were later diagnosed with adenocarcinoma. Using k-nearest neighbor classifier with cells from an initial patient biopsy, the biomarkers were able to predict cancer stage and grade of prostatic tissue that occurred at later prostatectomy with 79% accuracy. CONCLUSION: Our approach showed favorable diagnostic values to identify the portion and pathological category of aberrant cells in a small subset of sampled tissue cells, correlating with the degree of malignancy beyond baseline.

Role of the sodium-dependent multivitamin transporter (SMVT) in the maintenance of intestinal mucosal integrity.

Utilizing a conditional (intestinal-specific) knockout (cKO) mouse model, we have recently shown that the sodium-dependent multivitamin transporter (SMVT) (SLC5A6) is the only biotin uptake system that operates in the gut and that its deletion leads to biotin deficiency. Unexpectedly, we also observed that all SMVT-cKO mice develop chronic active inflammation, especially in the cecum. Our aim here was to examine the role of SMVT in the maintenance of intestinal mucosal integrity [permeability and expression of tight junction (TJ) proteins]. Our results showed that knocking out the mouse intestinal SMVT is associated with a significant increase in gut permeability and with changes in the level of expression of TJ proteins. To determine whether these changes are related to the state of biotin deficiency that develops in SMVT-cKO mice, we induced (by dietary means) biotin deficiency in wild-type mice and examined its effect on the above-mentioned parameters. The results showed that dietary-induced biotin deficiency leads to a similar development of chronic active inflammation in the cecum with an increase in the level of expression of proinflammatory cytokines, as well as an increase in intestinal permeability and changes in the level of expression of TJ proteins. We also examined the effect of chronic biotin deficiency on permeability and expression of TJ proteins in confluent intestinal epithelial Caco-2 monolayers but observed no changes in these parameters. These results show that the intestinal SMVT plays an important role in the maintenance of normal mucosal integrity, most likely via its role in providing biotin to different cells of the gut mucosa.

Conditional (intestinal-specific) knockout of the riboflavin transporter-3 (RFVT-3) impairs riboflavin absorption.

Riboflavin (RF) is indispensable for normal cell metabolism, proliferation, and growth. The RFVT-3 protein (product of the Slc52a3 gene) is expressed in the gut with the expression being restricted to the apical membrane domain of the polarized intestinal epithelial cells. The relative contribution of RFVT-3 to total carrier-mediated RF uptake in the native intestine, however, is not clear. We addressed this issue in the current investigation using a conditional (intestinal-specific) RFVT-3 knockout (cKO) mouse model developed by the Cre/Lox approach. All RFVT-3 cKO mice were found to be RF deficient and showed a significant growth and development retardation; also, nearly two-thirds of them died prematurely between the age of 6 and 12 wk. In vivo (intestinal and colonic loops) and in vitro (native isolated intestinal epithelial cells) uptake studies showed a severe inhibition in carrier-mediated RF uptake in the cKO mice compared with control littermates. We also observed a significant increase in the level of expression of oxidative stress-responsive genes in the intestine of the cKO mice compared with control littermates. Supplementation of the RFVT-3 cKO mice with pharmacological doses of RF led to a complete correction of the growth retardation and to normalization in the level of expression of the oxidative stress-responsive genes in the gut. These results show, for the first time, that the RFVT-3 system is the main transporter involved in carrier-mediated RF uptake in the native mouse small and large intestine, and that its dysfunction impairs normal RF body homeostasis.

Changes in tumor-antigen expression profile as human small-cell lung cancers progress.

OBJECTIVE: Our group has previously observed that in patients with small-cell lung cancers (SCLCs), the expression of a tumor antigen, glioma big potassium (gBK) ion channel, is higher at the time of death than when the cancer is first treated by surgical resection. This study aimed to determine whether this dichotomy was common in other potential lung tumor antigens by examining the same patient samples using our more extensive profile analysis of tumor-antigen precursor protein (TAPP). We then tested the hypothesis that therapeutic intervention may inadvertently cause this increased gBK production. METHODS: SCLC samples (eight surgical resections and three autopsy samples) and three control lungs were examined by quantitative real-time polymerase chain reaction for 42 potential TAPPs that represent potential T-cell-mediated immunological targets. RESULTS: Twenty-two TAPP mRNAs displayed the same profile as gBK, i.e., more mRNAs were expressed at autopsy than in their surgical counterparts. B-cyclin and mouse double minute 2, human homolog of P53-binding protein were elevated in both autopsy and surgical specimens above the normal-lung controls. When HTB119 cells were incubated with doxorubicin, gBK was strongly induced, as confirmed by intracellular flow cytometry with a gBK-specific antibody. CONCLUSION: Our findings suggested that more immunological targets became available as the tumor responded to chemotherapy and proceeded toward its terminal stages.

Small cell lung cancer cells express the late stage gBK tumor antigen: a possible immunotarget for the terminal disease.

Big Potassium (BK) ion channels have several splice variants. One splice variant initially described within human glioma cells is called the glioma BK channel (gBK). Using a gBK-specific antibody, we detected gBK within three human small cell lung cancer (SCLC) lines. Electrophysiology revealed that functional membrane channels were found on the SCLC cells. Prolonged exposure to BK channel activators caused the SCLC cells to swell within 20 minutes and resulted in their death within five hours. Transduction of BK-negative HEK cells with gBK produced functional gBK channels. Quantitative RT-PCR analysis using primers specific for gBK, but not with a lung-specific marker, Sox11, confirmed that advanced, late-stage human SCLC tissues strongly expressed gBK mRNA. Normal human lung tissue and early, lower stage SCLC resected tissues very weakly expressed this transcript. Immunofluorescence using the anti-gBK antibody confirmed that SCLC cells taken at the time of the autopsy intensely displayed this protein. gBK may represent a late-stage marker for SCLC. HLA-A*0201 restricted human CTL were generated in vitro using gBK peptide pulsed dendritic cells. The exposure of SCLC cells to interferon-γ (IFN-γ) increased the expression of HLA; these treated cells were killed by the CTL better than non-IFN-γ treated cells even though the IFN-γ treated SCLC cells displayed diminished gBK protein expression. Prolonged incubation with recombinant IFN-γ slowed the in vitro growth and prevented transmigration of the SCLC cells, suggesting IFN-γ might inhibit tumor growth in vivo. Immunotherapy targeting gBK might impede advancement to the terminal stage of SCLC via two pathways.

HCA519/TPX2: a potential T-cell tumor-associated antigen for human hepatocellular carcinoma.

BACKGROUND: Immunotherapy for human hepatocellular cancer (HCC) is slowly making progress towards treating these fatal cancers. The identification of new antigens can improve this approach. We describe a possible new antigen, hepatocellular carcinoma-associated antigen-519/targeting protein for Xklp-2 (HCA519/TPX2), for HCC that might be beneficial for T-cell specific HCC immunotherapy. METHODS: HCC was studied for the expression for 15 tumor-associated antigens considered useful for immunotherapy within three HCC cell lines (HepG2, Hep3B, and PLC/PRF/5), lymphocytes, non-cancerous livers, and clinical HCC. The expression of tumor antigenic precursor proteins (TAPPs) messenger RNA was first screened by reverse transcriptase quantitative real-time polymerase chain reaction. RESULTS: Four antigens (alpha fetoprotein, aspartyl/asparaginyl ß-hydroxylase, glypican-3 and HCA519/TPX2) proved to be the best expressed TAPPs within the HCC specimens by molecular analyses. HCA519/TPX2 was detected by intracellular cell flow cytometry within HCC cell lines by using a specific antibody towards this TAPP. This antibody also detected the protein within primary HCCs. We synthesized two HCA519/TPX2 peptides (HCA519464-472 and HCA519351-359) which can bind to human leukocyte antigen (HLA)-A*0201. Dendritic cells pulsed with these peptides stimulated cytolytic T lymphocytes (CTLs). These killer T-cells lysed HLA-A*0201+ T2 cells exogenously loaded with the correct specific peptide. The CTLs killed HepG2 (HLA-A2+ and HCA519+), but not the Hep3B and PLC/PRF/5 cell lines, which are HCA519+ but HLA-A2-negative. In silico analysis reveals that HCA519/TPX2 has the inherent ability to bind to a very wide variety of HLA antigens. CONCLUSION: HCA519/TPX2 is a viable immunotarget that should be further investigated within HCC patients.

Therapeutic role of niacin in the prevention and regression of hepatic steatosis in rat model of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD), a leading cause of liver damage, comprises a spectrum of liver abnormalities including the early fat deposition in the liver (hepatic steatosis) and advanced nonalcoholic steatohepatitis. Niacin decreases plasma triglycerides, but its effect on hepatic steatosis is elusive. To examine the effect of niacin on steatosis, rats were fed either a rodent normal chow, chow containing high fat (HF), or HF containing 0.5% or 1.0% niacin in the diet for 4 wk. For regression studies, rats were first fed the HF diet for 6 wk to induce hepatic steatosis and were then treated with niacin (0.5% in the diet) while on the HF diet for 6 wk. The findings indicated that inclusion of niacin at 0.5% and 1.0% doses in the HF diet significantly decreased liver fat content, liver weight, hepatic oxidative products, and prevented hepatic steatosis. Niacin treatment to rats with preexisting hepatic steatosis induced by the HF diet significantly regressed steatosis. Niacin had no effect on the mRNA expression of fatty acid synthesis or oxidation genes (including sterol-regulatory element-binding protein 1, acetyl-CoA carboxylase 1, fatty acid synthase, and carnitine palmitoyltransferase 1) but significantly inhibited mRNA levels, protein expression, and activity of diacylglycerol acyltrasferase 2, a key enzyme in triglyceride synthesis. These novel findings suggest that niacin effectively prevents and causes the regression of experimental hepatic steatosis. Approved niacin formulation(s) for other indications or niacin analogs may offer a very cost-effective opportunity for the clinical development of niacin for treating NAFLD and fatty liver disease.

The ghrelin activating enzyme ghrelin-O-acyltransferase (GOAT) is present in human plasma and expressed dependent on body mass index.

Ghrelin is the only known peripherally produced and centrally acting peptide hormone stimulating food intake. The acylation of ghrelin is essential for binding to its receptor. Recently, the ghrelin activating enzyme ghrelin-O-acyltransferase (GOAT) was identified in mice, rats and humans. In addition to gastric mucosal expression, GOAT was also detected in the circulation of rodents and its expression was dependent on metabolic status. We investigated whether GOAT is also present in human plasma and whether expression levels are affected under different conditions of body weight. Normal weight, anorexic and obese subjects with body mass index (BMI) 30-40, 40-50 and >50 were recruited (n=9/group). In overnight fasted subjects GOAT protein expression was assessed by Western blot and ghrelin measured by ELISA. GOAT protein was detectable in human plasma. Anorexic patients showed reduced GOAT protein levels (-42%, p<0.01) whereas obese patients with BMI>50 had increased concentrations (+34%) compared to normal weight controls. Ghrelin levels were higher in anorexic patients compared to all other groups (+62-78%, p<0.001). Plasma GOAT protein expression showed a positive correlation with BMI (r=0.71, p<0.001) and a negative correlation with ghrelin (r=-0.60, p<0.001). Summarized, GOAT is also present in human plasma and GOAT protein levels depend on the metabolic environment with decreased levels in anorexic and increased levels in morbidly obese patients. These data may indicate that GOAT counteracts the adaptive changes of ghrelin observed under these conditions and ultimately contributes to the development or maintenance of anorexia and obesity as it is the only enzyme acylating ghrelin.

Ghrelin and NUCB2/nesfatin-1 are expressed in the same gastric cell and differentially correlated with body mass index in obese subjects.

The orexigenic peptide ghrelin and the anorexigenic peptide nesfatin-1 are expressed by the same endocrine cell of the rat stomach, the X/A-like cell. However, data in humans are lacking, especially under conditions of obesity. We collected gastric tissue of obese patients undergoing sleeve gastrectomy and investigated the expression of nesfatin-1 and ghrelin in the gastric oxyntic mucosa by immunofluorescence. Nesfatin-1 immunoreactivity was detected in the human oxyntic mucosa in cells with an endocrine phenotype. A major portion of nesfatin-1 immunoreactive cells (78 %) co-localized with ghrelin indicating the occurrence in human X/A-like cells. In patients with very high body mass index (BMI 55-65 kg/m(2)), the number of nesfatin-1 immunoreactive cells/low-power field was significantly higher than in obese patients with lower BMI (40-50 kg/m(2), 118 ± 10 vs. 82 ± 11, p < 0.05). On the other hand, the number of ghrelin immunoreactive cells was significantly reduced in obese patients with higher compared to lower BMI (96 ± 12 vs. 204 ± 21, p < 0.01). Also the ghrelin-acylating enzyme ghrelin-O-acyltransferase decreased with increasing BMI. In conclusion, nesfatin-1 immunoreactivity is also co-localized with ghrelin in human gastric X/A-like cells giving rise to a dual role of this cell type with differential effects on stimulation and inhibition of appetite dependent on the peptide released. The expression of these two peptides is differentially regulated under obese conditions with an increase of nesfatin-1 and a decrease of ghrelin immunoreactivity with rising BMI pointing towards an adaptive change of expression that may counteract further body weight increase.

Conditional knockout of the Slc5a6 gene in mouse intestine impairs biotin absorption.

The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.

Lung cancer: a classic example of tumor escape and progression while providing opportunities for immunological intervention.

Lung cancers remain one of the most common and deadly cancers in the world today (12.5% of newly diagnosed cancers) despite current advances in chemo- and radiation therapies. Often, by the time these tumors are diagnosed, they have already metastasized. These tumors demonstrate the classic hallmarks of cancer in that they have advanced defensive strategies allowing them to escape various standard oncological treatments. Immunotherapy is making inroads towards effectively treating other fatal cancers, such as melanoma, glioblastoma multiforme, and castrate-resistant prostate cancers. This paper will cover the escape mechanisms of bronchogenic lung cancer that must be overcome before they can be successfully treated. We also review the history of immunotherapy directed towards lung cancers.