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Figure S1. Efficacy of pemetrexed in Lewis Lung carcinoma model and metabolomic analysis of MC38 tumor bearing mice treated with 50 mg/kg of pemetrexed Figure S2. T cell inflamed phenotype induced by pemetrexed is modestly reduced in tumors upon combination with cisplatin Figure S3. Combination Effects of pemetrexed -/+ anti-PD-L1 in MC38, LLC and Colon26 tumor models Figure S4 (A) Mice bearing Colon26 tumors were treated starting 10 days after tumor implantation with pemetrexed (50 mg/kg, 5 days on, 2 days off, IP) and/or anti-PD-L1 (αPD-L1) (500 ug/mouse, weekly IP), and tumors were isolated after 14 days of treatment and single cell suspensions were subjected to flow cytometry analysis. FACS plots show representative data of intra-tumor CD45+ tumor-infiltrating lymphocyte (TIL) T cell (CD3+) and Myeloid cell (CD11b+) subsets frequencies; and percentage of Ki67+ CD4+ T effector cells based on the gating scheme indicated. (B) Spleens, TDLNs and tumors were isolated and antigen specific T cell responses were measured by IFNγ ELISpot, gp70 Tetramer staining (Tet) and Intracellular cytokine staining. (Left) CD8 T cells isolated from pooled spleens and TDLNs from individual mice were cultured alone (control) with irrelevant BALB/c tumor EMT6 or cognate Colon26 tumor cells overnight and number of IFNγ producing T cells was measured by ELISpot. (Middle) Freshly isolated CD8+ T cells from spleens and TDLNs were assessed for % of gp70 Tet+ cells. (Right) Freshly isolated tumors single cell suspensions were stimulated with PMA/ phorbol myristate acetate (PMA) and ionomycin with monensin for 4 hrs and the % of TNF-alpha positive and gp70 Tet+ CD8 T cells were measured from the Live CD45+, CD3+ T cells by FACS. Figure S5. Concordance between QuantiGene and nCounter gene expression data for in vitro activated T cells treated in the presence or absence of pemetrexed (related to Figure 6F). Supplementary Table 1. Ingenuity pathway analysis (IPA) of MC38 tumors treated with pemetrexed or paclitaxel with or without carboplatin Supplementary Table 2: IPA results for Colon26 experiment in Figure 4 sorted by functional class. Pemetrexed monotherapy in this experiment at this timepoint did not have enough differentially expressed genes to generate IPA results.

Purpose:Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non–small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown.Experimental Design:Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis.Results:Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell–intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1.Conclusions:Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell–intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade.See related commentary by Buque et al., p. 6890

Background The aim of this study was to scrutinize changes of islet β-cell function in Chinese elderly patients with type 2 diabetes mellitus (T2DM) after insulin versus oral anti-diabetic (OAD) treatments. Methods In this prospective study, islet β-cell function was determined by oral glucose tolerance test-derived fasting C-peptide, maximal C-peptide, C-peptide area under the curve (AUC c-pep ) and fasting c-pep /fasting glu , maximal c-pep /maximal glu , AUC c-pep /AUC glu . Differences in β-cell function indices between baseline and follow-up were compared among insulin monotherapy, OAD monotherapy and combined therapies. Results From 2009 to 2017, 419 elderly patients (men: 51.1%) with T2DM (insulin monotherapy, n=105; OAD monotherapy, n=172; insulin + OAD, n=142) had undergone baseline and follow-up β-cell function assessment after a mean observation of 4.4 (range, 3.0 - 7.2) years. Overall, islet β-cell function indices were decreased in all the three groups: insulin (fasting C-peptide: p=0.002; maximal C-peptide: p=0.005; fasting c-pep /fasting glu : p=0.015; maximal c-pep /maximal glu : p=0.031), OAD (fasting C-peptide: p=0.026; maximal C-peptide: p=0.013; fasting c-pep /fasting glu : p=0.025; maximal c-pep /maximal glu : p=0.029), insulin + OAD (fasting C-peptide: p

Additional file 1. Appendix S1: Details of ICD codes for diabetes mellitus and for diabetic vascular complications used in this study. Appendix S2: Flow chart of countries selection. Appendix S3: Available years (grey) for diabetes microvascular complication deaths, by country. Appendix S4: Available years (grey) for total diabetes deaths, by country. Appendix S5: Available years (grey) for midyear population, by country. Appendix S6: Description of joinpoint regression model. Appendix S7: Crude and age-standardized proportions and rates of diabetes microvascular complication related deaths, by country. Appendix S8: Crude and age-standardized odd ratios of proportions and rates compared to overall, by country. Appendix S9: Crude and age-standardized proportions from 2000 to 2016, by sex. Appendix S10: Crude age specific proportions from 2000 to 2016, by sex. Appendix S11: Crude and age-standardized proportions from 2000 to 2016, by region. Appendix S12: Crude and age-standardized rates from 2000 to 2016, by sex. Appendix S13: Crude age specific rates from 2000 to 2016, by sex. Appendix S14: Crude and age-standardized rates from 2000 to 2016, by region. Appendix S15: Age-standardized odd ratio of rates compared to year 2000, by different subgroups. Appendix S16: Age-standardized odd ratio of proportions compared to year 2000, by different subgroups. Appendix S17: Country code and corresponding country name.

Purpose: Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non–small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown. Experimental Design: Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis. Results: Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell–intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1. Conclusions: Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell–intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade. See related commentary by Buque et al., p. 6890

Objectives: HOXA5 has been identified as a biomarker in pathogenesis of several cancers, such as non-small cell lung cancer (NSCLC) and breast cancer cells. The role has not been explored in cervical squamous cell carcinoma (CSCC). Methods: Tissues of 120 cases with CSCC and 30 controls with chronic cervicitis were constructed from our archived surgical pathology files and staining with HOXA5. Additional antibodies to E-cadherin and β-catenin were stained for comparison. For each marker, low expression was defined as staining score 0 to 3 points, whereas high expression referred to 4 points and above. Fifty-four patients in this research with cervical cancer were followed up for prognostic assessment. Result: HOXA5 had high expression in chronic cervicitis and low in CSCC (P=0.004). The positivity rates of HOXA5 in patients without muscular layer invasion (MLI) and lymphatic invasion (LI) was higher than that in metastasis (113 vs. 17; 117 vs. 3). Consistently, low expression of HOXA5 was more common in poorly differentiated carcinoma, CSCC subjects without MLI and LI. Expression of E-cadherin and β-catenin was parallel with the expression of HOXA5. Additionally, patients with higher expression of HOXA5 had much more favorable prognosis than those with lower expression among follow up of the 54 patients. Conclusion: In parallel with E-cadherin and β-catenin, low expression of HOXA5 was more common in CSCC patients with poor differentiation, without MLI and LI, among those which showed poor prognosis.

Rationale: Latent autoimmune diabetes in adults (LADA) refers to an autoimmune disorder characterized with detectable islets antibodies in the early diagnosis and increased autoimmune beta-cell failure progression. Notably, this kind of diabetes seems to be confused with other phenotypic diabetes. Patient concerns: A young woman suffered an emotional exhaustion-induced LADA, showing asthenia, polydipsia, polyuria, and visible weight loss. The patient emotionally ended a 14-year romantic relationship, leading to the emotional flooding. Diagnoses: The data from physical examination and laboratory tests exhibited as follows: glutamic acid decarboxylase antibody (GADA) = 63.83 U/mL, the fasting blood glucose (FBG) = 13.3 mmol/L, and glycated haemoglobin (HbA1c) = 10.9%. According to levels of GADA, the patient was diagnosed as LADA. Interventions: The patient was clinically treated with insulin for 3-month. Then, running, diet-control, and emotional treatment were combined, such as the patient started a new relationship. Outcomes: An emotional recovery initiated from a new romantic relationship and a baby, showing normal levels of GAD65 (27.007 IU/mL) and FBG (5.46) mmol/L. Lessons: The emotional exhaustion might play a significant role in induction of LADA. It is important that individuals should maintain optimism, cheer, and a positive attitude.

Combination strategies leveraging chemotherapy and immunotherapy have held the promise as a method to improve benefit to cancer patients. However, most chemotherapies have detrimental effects on immune homeostasis and do not induce immunogenic cell death. The positive phase III trial of pemetrexed/carboplatin with the PD-1 antibody pembrolizumab in NSCLC (Keynote-189) lead to the first chemotherapy/immunotherapy combination ever approved. While this suggests a positive interaction between pemetrexed-based chemotherapy and PD-1 therapy, the underlying mechanism is unknown. Therefore, it is important to understand the role of pemetrexed in modulating antitumor immune response to assure application of this therapy to the appropriate patients. To characterize the effects of pemetrexed on intra-tumor immune response, murine tumor models which were sensitive to pemetrexed and known to be sensitive to PD-L1, were treated with pemetrexed with or without carboplatin, or anti-mouse PD-L1. In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intra-tumor leukocytes that was accompanied by immune-related gene expression changes indicative of enhanced T cell infiltration and/or activation. Gene expression induced by pemetrexed was largely unaffected by carboplatin. Treatment of both MC38 and Colon26 tumor cells in vitro with pemetrexed induced release of HMGB1, indicative of immunogenic cell death. Although proliferation of primary human T cells was slightly reduced by pemetrexed, at clinically relevant concentrations, treatment lead to an enhanced T cell activation phenotype, including upregulation of multiple interferon gamma-induced genes, and increased mitochondrial respiration. This correlated with improved antigen specific in vitro cytotoxic activity of OT-1 TCR transgenic CD8 T cells when treated with pemetrexed during priming with OVA peptide. Treatment with pemetrexed and PD-L1 demonstrated a combination benefit compared to either monotherapy in both tumor models. Pathway Analysis of gene expression data revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell-mediated immunity during the combination. This correlated with upregulation of MHC-I & II on monocytes, macrophages and tumor cells, suggesting increased immune priming. Accordingly, treatment with S1P1R antagonist (FTY720, preventing T cell LN egress) after initiation of therapy resulted in a loss of combination efficacy. This data suggests that pemetrexed promotes intra-tumor T cell-mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells. The combination of these effects results in enhanced T cell function leading to an improved anti-tumor efficacy in combination with PD-L1 antibody Citation Format: David A. Schaer, Nelusha Amaladas, Zhao Hai Lu, Erik Rasmussen, Andreas Sonyi, Darin Chin, Andrew Capen, Yanxia Li, Catalina Meyer, Bonita Jones, Xiaodong Hong, Shuang Luo, Carmine Carpenito, Kenneth Roth, Alexander Nikolayev, Bo Tan, Manisha Brahmachary, Krishna Chodavarapu, Frank Charles Dorsey, Jason Manro, Thompson Doman, Greg Donoho, David Surguladze, Gerald Hall, Sandaruwan Geeganage, Michael Kalos, Ruslan Novosiadly. The folate pathway inhibitor pemetrexed pleiotropically enhances effects of cancer immunotherapy via immunogenic tumor cell death and T cell-intrinsic mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3945.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD+, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD+ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic. Background: NAMPT catalyzes the rate-limiting reaction in converting nicotinamide to NAD+ in cancers. Results: NAMPT inhibition attenuates glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step, resulting in perturbing metabolic pathways related to glycolysis. Conclusion: The metabolic basis of NAMPT inhibition is the attenuation of glycolysis by reducing NAD+ available to glyceraldehyde 3-phosphate dehydrogenase. Significance: This study sheds new light on how NAMPT regulates cancer metabolism.

Background: Recently, pemetrexed and carboplatin in combination with PD–1 antibody (pembrolizumab) demonstrated markedly improved clinical outcome in NSCLC patients (KEYNOTE–021G trial) suggesting a positive interaction between pemetrexed–based chemotherapy and immunotherapy. However, the role of pemetrexed in modulating antitumor immune response is largely unknown. The objective of this study was to characterize the effects of pemetrexed on intra–tumor immune response in monotherapy and combination with carboplatin or PD–1 pathway blockade in preclinical models. Methods: Mice bearing syngeneic MC38 or Colon26 tumors were treated with pemetrexed with or without carboplatin or anti–mouse PD–L1 antibody (178G7). Immune cell subsets and immune–related changes in mouse tumor tissue and T cells were assessed by flow cytometry and gene expression analysis (Quantigene Plex and nanoString nCounter assays). Effects of pemetrexed on immunogenic cell death in tumor cells and mitochondrial respiration in T cells were evaluated by HMGB1 ELISA and Agilent Seahorse XF analysis, respectively. Results: In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intratumoral leukocytes and total and cycling (Ki67+) T cells accompanied by immune–related gene expression changes indicative of enhanced antigen presentation, T cell infiltration and/or activation and down–modulation of the myeloid cell compartment. Immune gene expression signature induced by pemetrexed was largely unaffected by carboplatin. In both MC38 and Colon26 tumor cells, in vitro treatment with pemetrexed induced a robust release of HMGB1 indicative of immunogenic cell death. Proliferation of primary human T cells stimulated with CD3/CD28 was inhibited by pemetrexed in a dose–dependent manner. At clinically relevant concentrations pemetrexed also enhanced T cell activation phenotype exemplified by an increased frequency of CD137+, GITR+ and PD–L1+ T cells as well as upregulation of multiple interferon gamma–induced genes, and increased mitochondrial respiration. In both MC38 and Colon26 models, treatment with pemetrexed and 178G7 demonstrated a combination benefit compared to either monotherapy, and nCounter profiling of Colon26 tumors followed by Ingenuity Pathway Analysis revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell–mediated immunity might underlie this combination effect. Conclusions: Pemetrexed promotes intra–tumor T cell–mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells leading to an enhanced anti–tumor efficacy in combination with PD–L1 antibody. Citation Format: Ruslan Novosiadly, David Schaer, Nelusha Amaladas, Erik Rasmussen, Zhao Hai Lu, Andreas Sonyi, Carmine Carpenito, Yanxia Li, Shuang Luo, Andrew Capen, Catalina Meyer, Xiaodong Huang, Jason Manro, Gregory Donoho, Thompson Doman, Gerald Hall, Sandaruwan Geeganage, Michael Kalos. Pemetrexed enhances anti-tumor efficacy of PD1 pathway blockade by promoting intra tumor immune response via immunogenic tumor cell death and T cell intrinsic mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4549.

Supplemental digital content is available in the text, Gastrointestinal (GI) disorders often manifest similar symptoms with overlapping clinical diagnosis and unmet medical needs. Traditional Chinese medicine (TCM) has history-proven benefits for GI diseases; albeit language barrier prevents Western readers from accessing the original reports in Chinese. The TCM formula Si-Ni-San (SNS) consists of 4 herbs targeting on homeostatic disturbances characterized by “reflux” and “irritable” problems. Here we used SNS as a therapeutic tool to explore the common mechanisms of pathogenesis in non-neoplastic GI diseases. Data sources from PUBMED, Chinese National Knowledge Infrastructure, and Wanfang databases were searched for clinical trials. Comparisons were SNS as intervention and Western conventional medicine as control, which treat patients with upper GI disorders (gastroesophageal reflux disease, peptic ulcer, chronic gastritis, duodenogastric reflux), lower GI diseases (irritable bowel syndrome, ulcerative colitis), and functional dyspepsia. Participants and studies in accordance with the Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement were eligible. We used the Jadad scale to assess methodological qualities, the fixed or random-effect model to evaluate therapeutic efficacy, and the funnel plots to explore publication bias. Outcome was clinical efficacy defined by symptom relief with normal GI endoscopy, radiology, and pathology. We included 83 studies involving 7762 participants: 1708 versus 1397 of the upper GI disorders in 34 studies, 901 versus 768 of the lower GI diseases in 19 studies, 1641 versus 1348 of functional dyspepsia in 30 studies, and 328 versus 287 of relapse rate in 8 studies. Six studies had a Jadad score >2 points and the rest were