Immuno-PET Monitoring of CD8+ T Cell Infiltration Post ICOS Agonist Antibody Treatment Alone and in Combination with PD-1 Blocking Antibody Using a 89Zr Anti-CD8+ Mouse Minibody in EMT6 Syngeneic Tumor Mouse.

PURPOSE: The presence and functional competence of intratumoral CD8+ T cells is often a barometer for successful immunotherapeutic responses in cancer. Despite this understanding and the extensive number of clinical-stage immunotherapies focused on potentiation (co-stimulation) or rescue (checkpoint blockade) of CD8+ T cell antitumor activity, dynamic biomarker strategies are often lacking. To help fill this gap, immuno-PET nuclear imaging has emerged as a powerful tool for in vivo molecular imaging of antibody targeting. Here, we took advantage of immuno-PET imaging using 89Zr-IAB42M1-14, anti-mouse CD8 minibody, to characterize CD8+ T-cell tumor infiltration dynamics following ICOS (inducible T-cell co-stimulator) agonist antibody treatment alone and in combination with PD-1 blocking antibody in a model of mammary carcinoma. PROCEDURES: Female BALB/c mice with established EMT6 tumors received 10 µg, IP of either IgG control antibodies, ICOS agonist monotherapy, or ICOS/PD-1 combination therapy on days 0, 3, 5, 7, 9, 10, or 14. Imaging was performed at 24 and 48 h post IV dose of 89Zr IAB42M1-14. In addition to 89Zr-IAB42M1-14 uptake in tumor and tumor-draining lymph node (TDLN), 3D radiomic features were extracted from PET/CT images to identify treatment effects. Imaging mass cytometry (IMC) and immunohistochemistry (IHC) was performed at end of study. RESULTS: 89Zr-IAB42M1-14 uptake in the tumor was observed by day 11 and was preceded by an increase in the TDLN as early as day 4. The spatial distribution of 89Zr-IAB42M1-14 was more uniform in the drug treated vs. control tumors, which had spatially distinct tracer uptake in the periphery relative to the core of the tumor. IMC analysis showed an increased percentage of cytotoxic T cells in the ICOS monotherapy and ICOS/PD-1 combination group compared to IgG controls. Additionally, temporal radiomics analysis demonstrated early predictiveness of imaging features. CONCLUSION: To our knowledge, this is the first detailed description of the use of a novel immune-PET imaging technique to assess the kinetics of CD8+ T-cell infiltration into tumor and lymphoid tissues following ICOS agonist and PD-1 blocking antibody therapy. By demonstrating the capacity for increased spatial and temporal resolution of CD8+ T-cell infiltration across tumors and lymphoid tissues, these observations underscore the widespread potential clinical utility of non-invasive PET imaging for T-cell-based immunotherapy in cancer.

Activating Inducible T-cell Costimulator Yields Antitumor Activity Alone and in Combination with Anti-PD-1 Checkpoint Blockade.

In recent years, there has been considerable interest in mAb-based induction of costimulatory receptor signaling as an approach to combat cancer. However, promising nonclinical data have yet to translate to a meaningful clinical benefit. Inducible T-cell costimulator (ICOS) is a costimulatory receptor important for immune responses. Using a novel clinical-stage anti-ICOS immunoglobulin G4 mAb (feladilimab), which induces but does not deplete ICOS+ T cells and their rodent analogs, we provide an end-to-end evaluation of the antitumor potential of antibody-mediated ICOS costimulation alone and in combination with programmed cell death protein 1 (PD-1) blockade. We demonstrate, consistently, that ICOS is expressed in a range of cancers, and its induction can stimulate growth of antitumor reactive T cells. Furthermore, feladilimab, alone and with a PD-1 inhibitor, induced antitumor activity in mouse and humanized tumor models. In addition to nonclinical evaluation, we present three patient case studies from a first-time-in-human, phase I, open-label, dose-escalation and dose-expansion clinical trial (INDUCE-1; ClinicalTrials.gov: NCT02723955), evaluating feladilimab alone and in combination with pembrolizumab in patients with advanced solid tumors. Preliminary data showing clinical benefit in patients with cancer treated with feladilimab alone or in combination with pembrolizumab was reported previously; with example cases described here. Additional work is needed to further validate the translation to the clinic, which includes identifying select patient populations that will benefit from this therapeutic approach, and randomized data with survival endpoints to illustrate its potential, similar to that shown with CTLA-4 and PD-1 blocking antibodies. Significance: Stimulation of the T-cell activation marker ICOS with the anti-ICOS agonist mAb feladilimab, alone and in combination with PD-1 inhibition, induces antitumor activity across nonclinical models as well as select patients with advanced solid tumors.

Tumor-immune profiling of murine syngeneic tumor models as a framework to guide mechanistic studies and predict therapy response in distinct tumor microenvironments.

Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. Despite their widespread use, a comprehensive view of their tumor-immune compositions and their relevance to human tumors has only begun to emerge. We propose each model possesses a unique tumor-immune infiltrate profile that can be probed with immunotherapies to inform on anti-tumor mechanisms and treatment strategies in human tumors with similar profiles. In support of this endeavor, we characterized the tumor microenvironment of four commonly used models and demonstrate they encompass a range of immunogenicities, from highly immune infiltrated RENCA tumors to poorly infiltrated B16F10 tumors. Tumor cell lines for each model exhibit different intrinsic factors in vitro that likely influence immune infiltration upon subcutaneous implantation. Similarly, solid tumors in vivo for each model are unique, each enriched in distinct features ranging from pathogen response elements to antigen presentation machinery. As RENCA tumors progress in size, all major T cell populations diminish while myeloid-derived suppressor cells become more enriched, possibly driving immune suppression and tumor progression. In CT26 tumors, CD8 T cells paradoxically increase in density yet are restrained as tumor volume increases. Finally, immunotherapy treatment across these different tumor-immune landscapes segregate into responders and non-responders based on features partially dependent on pre-existing immune infiltrates. Overall, these studies provide an important resource to enhance our translation of syngeneic models to human tumors. Future mechanistic studies paired with this resource will help identify responsive patient populations and improve strategies where immunotherapies are predicted to be ineffective.

Hypoxia and the unfolded protein response.

Tumor hypoxia refers to the development of regions within solid tumors in which the oxygen concentration is lower (0-3%) compared to that in most normal tissues (4-9%) (Vaupel and Hockel, 2000). Considerable experimental and clinical evidence exists supporting the notion that hypoxia fundamentally alters the physiology of the tumor towards a more aggressive phenotype (Hockel and Vaupel, 2001). Therefore, delineating the mechanisms by which hypoxia affects tumor physiology at the cellular and molecular levels will be crucial for a better understanding of tumor development and metastasis and for designing better antitumor modalities.

Activating transcription factor 4 is translationally regulated by hypoxic stress.

Hypoxic stress results in a rapid and sustained inhibition of protein synthesis that is at least partially mediated by eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation by the endoplasmic reticulum (ER) kinase PERK. Here we show through microarray analysis of polysome-bound RNA in aerobic and hypoxic HeLa cells that a subset of transcripts are preferentially translated during hypoxia, including activating transcription factor 4 (ATF4), an important mediator of the unfolded protein response. Changes in mRNA translation during the unfolded protein response are mediated by PERK phosphorylation of the translation initiation factor eIF2alpha at Ser-51. Similarly, PERK is activated and is responsible for translational regulation under hypoxic conditions, while inducing the translation of ATF4. The overexpression of a C-terminal fragment of GADD34 that constitutively dephosphorylates eIF2alpha was able to attenuate the phosphorylation of eIF2alpha and severely inhibit the induction of ATF4 in response to hypoxic stress. These studies demonstrate the essential role of ATF4 in the response to hypoxic stress, define the pathway for its induction, and reveal that GADD34, a target of ATF4 activation, negatively regulates the eIF2alpha-mediated inhibition of translation. Taken with the concomitant induction of additional ER-resident proteins identified by our microarray analysis, this study suggests an important integrated response between ER signaling and the cellular adaptation to hypoxic stress.

Expression of ECRG4, a novel esophageal cancer-related gene, downregulated by CpG island hypermethylation in human esophageal squamous cell carcinoma.

AIM: To study the mechanisms responsible for inactivation of a novel esophageal cancer related gene 4 (ECRG4) in esophageal squamous cell carcinoma (ESCC). METHODS: A pair of primers was designed to amplify a 220 bp fragment, which contains 16 CpG sites in the core promoter region of the ECRG 4 gene. PCR products of bisulfite-modified CpG islands were analyzed by denaturing high-performance liquid chromatography (DHPLC), which were confirmed by DNA sequencing. The methylation status of ECRG 4 promoter in 20 cases of esophageal cancer and the adjacent normal tissues, 5 human tumor cell lines (esophageal cancer cell line-NEC, EC109, EC9706; gastric cancer cell line- GLC; human embryo kidney cell line-Hek293) and 2 normal esophagus tissues were detected. The expression level of the ECRG 4 gene in these samples was examined by RT-PCR. RESULTS: The expression level of ECRG 4 gene was varied. Of 20 esophageal cancer tissues, nine were unexpressed, six were lowly expressed and five were highly expressed compared with the adjacent tissues and the 2 normal esophageal epithelia. In addition, 4 out of the 5 human cell lines were also unexpressed. A high frequency of methylation was revealed in 12 (8 unexpressed and 4 lowly expressed) of the 15 (80 %) downregulated cancer tissues and 3 of the 4 unexpressed cell lines. No methylation peak was observed in the two highly expressed normal esophageal epithelia and the methylation frequency was low (3/20) among the 20 cases in the highly expressed adjacent tissues. The methylation status of the samples was consistent with the result of DNA sequencing. CONCLUSION: These results indicate that the inactivation of ECRG 4 gene by hypermethylation is a frequent molecular event in ESCC and may be involved in the carcinogenesis of this cancer.

Using yeast two-hybrid system to identify ECRG2 associated proteins and their possible interactions with ECRG2 gene.

AIM: To identify esophageal cancer related gene2 (ECRG2) associated proteins and their possible interactions with ECRG2 gene. METHODS: In the yeast forward two-hybrid system, ECRG2 was fused with the DNA-binding domain (DBD) of Gal4 and human fetal liver cDNA library was fused with the transcriptional activation domain (AD) of Gal4. We performed a high-stringency scale procedure to screen ECRG2 against human fetal liver cDNA library and characterized positives by sequence analysis. RESULTS: We found the following 9 putatively associated proteins. They were metallothionein2A, metallothionein1H, metallothionein1G, ferritin, erythrocyte membrane protein band4.2, mitochondrial ribosomal protein S12, hypothetical protein FLJ10101, and a novel gene whose cDNA was found to have no strong homology to any other previously characterized gene whose DDBJ/EMBL/GenBank accession number is AF422192 mapped to human chromosome 14q31. CONCLUSION: MT, a potential interaction partner for ECRG2, might be involved in the regulation of cell proliferation and apoptosis, and in various physiological processes. Determination of a reliability score for each single protein-protein interaction, especially interaction of ECRG2 and MT, permits the assignment of ECRG2 and unannotated proteins to biological pathways. A further understanding of the association between ECRG2 and MT should facilitate the functions of ECRG2 gene.

Using Lab On-line to Clone and Identify the Esophageal Cancer Related Gene 4.

Using Internet as platform, databases as materials and software as tools to assemble a lab on-line is revolutionizing in bioscience research. The major works of lab on-line are cloning, identification, localization of genes, and the structural and functional analysis of proteins. In this report, the esophageal cancer related gene 4(ECRG-4)(accession number AF325503) was successfully isolated. The 97 bp ECRG-4 EST was initiallyusedto fish the human EST databases. Five pieces of ESTs showed strong homology to it, and they were assembled to one 772 bp cDNA sequence by DNASTAR software. Then the 447 bp full open reading frame of ECRG-4 was determined by ORF FINDER to encode 148 amino acids. Sequence of ECRG-4 did not reveal remarkable similarity to the known sequences in a homology analysis with the public database of GenBank, showing that it is a new gene. Homology analysis of protein coding by ECRG-4 showed a 31% homology with mouse IgG V region. ECRG-4 gene is expressed in normal esophagus, bladder and brain tissues, but its expression was significantly down-regulated in prostate tumors and tumor cell lines. ECRG-4 gene was located in 2q14.1--4.3 by HTGS and STS, and was conformed by radiation hybrid (RH) method. We propose that this purely lab on-line cloning approach can be used by modestly sized laboratories to rapidly and accurately characterize human genes without wasting too much money and time.

ER stress-mediated autophagy promotes Myc-dependent transformation and tumor growth.

The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc-induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism that promotes cellular transformation and tumorigenesis. The accumulation of unfolded proteins in the ER initiates a cellular stress program termed the unfolded protein response (UPR) to support cell survival. Analysis of spontaneous mouse and human lymphomas demonstrated significantly higher levels of UPR activation compared with normal tissues. Using multiple genetic models, we demonstrated that c-Myc and N-Myc activated the PERK/eIF2α/ATF4 arm of the UPR, leading to increased cell survival via the induction of cytoprotective autophagy. Inhibition of PERK significantly reduced Myc-induced autophagy, colony formation, and tumor formation. Moreover, pharmacologic or genetic inhibition of autophagy resulted in increased Myc-dependent apoptosis. Mechanistically, we demonstrated an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute (L24+/-) mutant, which resulted in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we showed that Myc-induced UPR activation was reversed. Our findings establish a role for UPR as an enhancer of c-Myc-induced transformation and suggest that UPR inhibition may be particularly effective against malignancies characterized by c-Myc overexpression.

Molecular cloning and characterization of a novel esophageal cancer related gene.

We previously identified four novel cDNA fragments related to human esophageal cancer. One of the fragments was named esophageal cancer related gene 2 (ECRG2). We report here the molecular cloning, sequencing, and expression of the ECRG2 gene. The ECRG2 cDNA comprises a 258 bp nucleotide sequence which encodes for 85 amino acids with a predicted molecular weight of 9.2 kDa. Analysis of the protein sequence reveals the presence at the N terminus of a signal peptide followed by 56 amino acids with a significant degree of sequence similarity with the conserved Kazal domain which characterizes the serine protease inhibitor family. Pulse-chase experiments showed that ECRG2 protein was detected in both cell lysates and culture medium, indicating that the ECRG2 protein was extracellularly secreted after the post-translational cleavage. In vitro uPA/plasmin activity analysis showed the secreted ECRG2 protein inhibited the uPA/plasmin activity, indicating that ECRG2 may be a novel serine protease inhibitor. Northern blot analysis revealed the presence of the major band corresponding to a size of 569 kb throughout the fetal skin, thymus, esophagus, brain, lung, heart, stomach, liver, spleen, colon, kidney, testis, muscle, cholecyst tissues and adult esophageal mucosa, brain, thyroid tissue and mouth epithelia. However, ECRG2 gene was significantly down-regulated in primary esophageal cancer tissues. Taken together, these results indicate that ECRG2 is a novel member of the Kazal-type serine protease inhibitor family and may function as a tumor suppressor gene regulating the protease cascades during carcinogenesis and migration/invasion of esophageal cancer.

ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth.

Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF-1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2alpha or by a dominant-negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities.

ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma, interacts directly with metallothionein 2A and links to apoptosis.

Esophageal cancer related gene 2 (ECRG2) is a novel candidate of the tumor suppressor gene identified from human esophagus. To study the biological role of the ECRG2 gene, we performed a GAL4-based yeast two-hybrid screening of a human fetal liver cDNA library. Using the ECRG2 cDNA as bait, we identified nine putative clones as associated proteins. The interaction of ECRG2 and metallothionein 2A (MT2A) was confirmed by glutathione S-transferase pull-down assays in vitro and co-immunoprecipitation experiments in vivo. ECRG2 co-localized with MT2A mostly to nuclei and slightly to cytoplasm, as shown by confocal microscopy. Transfection of ECRG2 gene inhibited cell proliferation and induced apoptosis in esophageal cancer cells. In the co-transfection of ECRG2 and MT2A assays, cell proliferation was inhibited and apoptosis was slightly induced compared with control groups. When we used antisense MT2A to interdict the effect of MT2A, the inhibition of cell proliferation and induction of apoptosis were significantly enhanced. When we used antisense ECRG2 to interdict the effect of ECRG2 in the group of Bel7402 cells co-transfected with ECRG2 and MT2A, the inhibition of cell proliferation and induction of apoptosis disappeared. The results provide evidence for ECRG2 in esophageal cancer cells acting as a bifunctional protein associated with the regulation of cell proliferation and induction of apoptosis. ECRG2 might reduce the function of MT2A on the regulation of cell proliferation and induction of apoptosis. The physical interaction of ECRG2 and MT2A may play an important role in the carcinogenesis of esophageal cancer.

Gastrin regulates the heparin-binding epidermal-like growth factor promoter via a PKC/EGFR-dependent mechanism.

Gastrin is a known growth/differentiation factor for the gastric mucosa. Its effects are likely mediated by the induction of heparin-binding epidermal-like growth factor (HB-EGF), a member of the EGF family of growth factors that is expressed by gastric parietal cells. In this study, we investigated the regulation of the HB-EGF promoter by gastrin in a human gastric cancer cell line. Serial human HB-EGF promoter-luciferase reporter deletion constructs and heterologous promoter constructs were transfected into AGS-E cells and stimulated with gastrin (10(-7) M) with or without various signal transduction inhibitors. EMSA were also performed. Gastrin stimulation resulted in a fivefold increase in HB-EGF-luciferase activity. The cis-acting element mediating gastrin responsiveness was mapped to the -69 to -58 region of the HB-EGF promoter. Gastrin stimulation was PKC dependent and at least partially mediated by activation of the EGF receptor.

Short tandem repeat polymorphism in a novel esophageal cancer-related gene (ECRG2) implicates susceptibility to esophageal cancer in Chinese population.

We have previously cloned and identified a novel esophageal cancer related gene 2 (ECRG2; GenBank Accession Number AF268198), which is down-regulated in esophageal squamous cell carcinoma (ESCC) and involved in the induction of the apoptosis in esophageal cancer cell lines. In the present study, we have found a short tandem repeat (STR) polymorphism in the noncoding region of the exon 4 of the ECRG2 gene by using PCR-denaturing high-performance liquid chromatography (DHPLC). Three STR genotypes, TCA3/TCA3, TCA3/TCA4 and TCA4/TCA4 were revealed and confirmed by DNA sequencing analysis. A total of 661 objects including 228 patients with ESCC and 373 normal controls were analyzed to investigate the impact of this ECRG2 STR polymorphism on risk of ESCC in case-control studies. Genotypes were determined in 231 controls and 162 cases from Beijing, which is a low risk area of ESCC, and in 142 controls and 126 cases from Linxian, a well-known high-risk area of ESCC. In both of the Beijing and Linxian population, subjects who carried the TCA3/TCA3 genotype were at an increased risk of ESCC compared to those carrying the TCA4/TCA4 genotype, with the adjusted odds ratios (ORs) being 2.05 [95% confidence interval (CI), 1.02-4.06] for the subjects from Beijing and 4.40 (95% CI, 1.93-10.01) for the subjects from Linxian. Furthermore, comparison of the genotype distributions among other cancer sites might suggest that risk of the ECRG2 STR polymorphism might be specific to the esophagus. These findings indicate for the first time that the ECRG2 STR is a genetic susceptibility factor for ESCC and the TCA3/TCA3 allele might play a role in the development of this cancer.