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Patient demographic and tumor pathology

Image cytometry infiltration results by layer

Image cytometry methods overview

Purpose:This clinical trial combined pembrolizumab and vorinostat in recurrent/metastatic squamous cell carcinomas of the head and neck (HN), and salivary gland cancer (SGC).Patients and Methods:Patients with progressing incurable HN and SGC, Eastern Cooperative Oncology Group (ECOG) ≤1, no prior immunotherapy, RECIST1.1 measurable disease, and normal organ function were eligible. Pembrolizumab 200 mg was given intravenous every 21 days, and vorinostat 400 mg given orally 5 days on and 2 days off during each 21-day cycle. Primary endpoints were safety and objective response rates.Results:From November 2015 to August 2017, 25 patients with HN and 25 SGC were enrolled. Median age was 61 (range, 33–86) years, 39 (78%) were male, 21 (62%) were never smokers, and 27 (54%) had ECOG 0. In HN, 13 (52%) were p16+ oropharynx. Most common SGC histologies were adenoid cystic 12 (48%), acinic cell 3 (12%), and mucoepidermoid 3 (12%). Adverse events (AEs) in all patients were: 27 (54%) with grade ≥ 1 and 18 (36%) with grade ≥ 3. The most common AEs in all patients were renal insufficiency in seven, (14%), fatigue in six, (12%), and nausea in three (6%). There were three (12%) deaths on study. Responses in HN were complete response (CR) 0, partial response (PR) eight (32%), and stable disease (SD) five (20%). Efficacy in SGCs was CR 0, PR four (16%) in one lymphoepithelioma-like carcinoma, two acinic cell, one adenoid cystic, and SD 14 (56%). In the HN group, median follow-up (mFUP) was 12.6 months, median overall survival (mOS) was 12.6 months, and median progression-free survival (mPFS) was 4.5 months. In SGC, mFUP was 13.1 months, mOS was 14.0 months, and mPFS was 6.9 months.Conclusions:This combination demonstrated activity in HN, with fewer responses in SGC. Toxicities were higher than reported with pembrolizumab alone.

Supplement Figure 1: Synergistic cytotoxic effect of pembrolizumab combined with vorinostat in HNSCC tissue slice culture. HNSCC slices (250 �m) were cultured and treated with 20 �g/ml of isotype control antibody IgG4 or pembrolizumab, with or without vorinostat (20 �M, 100 �M) for 6 days. Graph shows the percentage of cleaved-caspase-3+ cells. Unpaired student's tests, P-values are as follows: *p < 0.05, **P < 0.01, ***P< 0.001.

Live microscopy analysis of CD8+ T cell distribution in slice cultures

PDA tumor slice culture platform utilized for multimodal analysis before and after ex vivo immune modulation

TCR overlap analysis

Purpose:Pancreatic ductal adenocarcinoma (PDA) is rarely cured, and single-agent immune checkpoint inhibition has not demonstrated clinical benefit despite the presence of large numbers of CD8+ T cells. We hypothesized that tumor-infiltrating CD8+ T cells harbor latent antitumor activity that can be reactivated using combination immunotherapy.Experimental Design:Preserved human PDA specimens were analyzed using multiplex IHC (mIHC) and T-cell receptor (TCR) sequencing. Fresh tumor was treated in organotypic slice culture to test the effects of combination PD-1 and CXCR4 blockade. Slices were analyzed using IHC, flow cytometry, and live fluorescent microscopy to assess tumor kill, in addition to T-cell expansion and mobilization.Results:mIHC demonstrated fewer CD8+ T cells in juxtatumoral stroma containing carcinoma cells than in stroma devoid of them. Using TCR sequencing, we found clonal expansion in each tumor; high-frequency clones had multiple DNA rearrangements coding for the same amino acid binding sequence, which suggests response to common tumor antigens. Treatment of fresh human PDA slices with combination PD-1 and CXCR4 blockade led to increased tumor cell death concomitant with lymphocyte expansion. Live microscopy after combination therapy demonstrated CD8+ T-cell migration into the juxtatumoral compartment and rapid increase in tumor cell apoptosis.Conclusions:Endogenous tumor-reactive T cells are present within the human PDA tumor microenvironment and can be reactivated by combined blockade of PD-1 and CXCR4. This provides a new basis for the rational selection of combination immunotherapy for PDA.See related commentary by Medina and Miller, p. 3747

Combination immunotherapy tumor slice culture experiment replicates

Supplementary Video 2 from Regulation of Microtubule-Dependent Protein Transport by the TSC2/Mammalian Target of Rapamycin Pathway

Supplementary Figure 3 from Regulation of Microtubule-Dependent Protein Transport by the TSC2/Mammalian Target of Rapamycin Pathway

Supplementary Figure 2 from Regulation of Microtubule-Dependent Protein Transport by the TSC2/Mammalian Target of Rapamycin Pathway

Supplementary Video 1 from Regulation of Microtubule-Dependent Protein Transport by the TSC2/Mammalian Target of Rapamycin Pathway

Supplementary Video 3 from Regulation of Microtubule-Dependent Protein Transport by the TSC2/Mammalian Target of Rapamycin Pathway

Supplementary Figure 1 from Regulation of Microtubule-Dependent Protein Transport by the TSC2/Mammalian Target of Rapamycin Pathway

Protein transport plays a critical role in the interaction of the cell with its environment. Recent studies have identified TSC1 and TSC2, two tumor suppressor genes involved in tuberous sclerosis complex, as regulators of the mammalian target of rapamycin (mTOR) pathway. Cells deficient in TSC1 or TSC2 possess high levels of Rheb-GTP resulting in constitutive mTOR activation. We have shown previously that the TSC1/TSC2 complex is involved in post-Golgi transport of VSVG and caveolin-1 in mammalian cells. Here, we show that modulation of mTOR activity affects caveolin-1 localization and that this effect is independent of p70S6K. Tsc1- and Tsc2-null cells exhibit abnormal caveolin-1 localization that is accompanied by disorganized microtubules in the subcortical region. Analyses of green fluorescent protein-EB1 and tubulin in live mutant cells suggest a failure of the plus-ends to sense cortical signals and to halt microtubule growth. Down-regulation of CLIP-170, a putative mTOR substrate with microtubule-binding properties, rescued the abnormal microtubule arrangement and caveolin-1 localization in Tsc2−/− cells. Together, these findings highlight a novel role of the TSC2/mTOR pathway in regulating microtubule-dependent protein transport. (Cancer Res 2006; 66(10): 5258-69)

ESCRT-III family proteins form composite polymers that deform and cut membrane tubes in the context of a wide range of cell biological processes across the tree of life. In reconstituted systems, sequential changes in the composition of ESCRT-III polymers induced by the AAA–adenosine triphosphatase Vps4 have been shown to remodel membranes. However, it is not known how composite ESCRT-III polymers are organized and remodeled in space and time in a cellular context. Taking advantage of the relative simplicity of the ESCRT-III–dependent division system in Sulfolobus acidocaldarius , one of the closest experimentally tractable prokaryotic relatives of eukaryotes, we use super-resolution microscopy, electron microscopy, and computational modeling to show how CdvB/CdvB1/CdvB2 proteins form a precisely patterned composite ESCRT-III division ring, which undergoes stepwise Vps4-dependent disassembly and contracts to cut cells into two. These observations lead us to suggest sequential changes in a patterned composite polymer as a general mechanism of ESCRT-III–dependent membrane remodeling.

Although it can promote effector T cell function, the summative effect of interleukin-10 (IL-10) in the tumor microenvironment (TME) appears to be suppressive; therefore, blocking this critical regulatory cytokine has therapeutic potential to enhance antitumor immune function. As macrophages efficiently localize to the TME, we hypothesized that they could be used as a delivery vehicle for drugs designed to block this pathway. To test our hypothesis, we created and evaluated genetically engineered macrophages (GEMs) that produce an IL-10-blocking antibody (aIL-10). Healthy donor human peripheral blood mononuclear cells were differentiated and transduced with a novel lentivirus (LV) encoding BT-063, a humanized aIL-10 antibody. The efficacy of aIL-10 GEMs was assessed in human gastrointestinal tumor slice culture models developed from resected specimens of pancreatic ductal adenocarcinoma primary tumors and colorectal cancer liver metastases. LV transduction led to sustained production of BT-063 by aIL-10 GEMs for at least 21 days. Transduction did not alter GEM phenotype as evaluated by flow cytometry, but ⍺IL-10 GEMs produced measurable quantities of BT-063 in the TME that was associated with an approximately 5-fold higher rate of tumor cell apoptosis than control.

ESCRT-III family proteins form composite polymers that deform and cut membrane tubes in the context of a wide range of cell biological processes across the tree of life. In reconstituted systems sequential changes in the composition of ESCRT-III polymers induced by the AAA ATPase Vps4 have been shown to remodel membranes. However, it is not known how composite ESCRT-III polymers are organised and remodelled in space and time in cells. Here, taking advantage of the relative simplicity of the ESCRT-III-dependent division system in Sulfolobus acidocaldarius, one of the closest experimentally tractable prokaryotic relative of eukaryotes, we use super-resolution microscopy and computational modelling to show how CdvB/CdvB1/CdvB2 proteins form a precisely patterned composite ESCRT-III division ring which undergoes stepwise Vps4-dependent disassembly and contracts to cut cells into two. These observations lead us to suggest sequential changes in a patterned composite polymer as a general mechanism of ESCRT-III-dependent membrane remodelling.

Microbial nanowires are fascinating biological structures that allow bacteria to transport electrons over micrometers for reduction of extracellular substrates. It was recently established that the nanowires of both Shewanella and Geobacter are made of multi-heme proteins; but, while Shewanella employs the 20-heme protein complex MtrCAB, Geobacter uses a redox polymer made of the hexa-heme protein OmcS, begging the question as to which protein architecture is more efficient in terms of long-range electron transfer. Using a multiscale computational approach we find that OmcS supports electron flows about an order of magnitude higher than MtrCAB due to larger heme-heme electronic couplings and better insulation of hemes from the solvent. We show that heme side chains are an essential structural element in both protein complexes, accelerating rate-limiting electron tunnelling steps up to 1000-fold. Our results imply that the alternating stacked/T-shaped heme arrangement present in both protein complexes may be an evolutionarily convergent design principle permitting efficient electron transfer over very long distances.

Dysfunction of glucokinase (GCK) caused by mutations in the GCK gene is the main cause of maturity-onset diabetes of the young type-2 (MODY2, also known as GCK-MODY), which is usually present in adolescence or young adulthood. MODY2 is characterized by mild, stable fasting hyperglycemia that presents at birth, usually 5.4-8.3 mmol L

Significance Cell division is an essential requirement for life. Division requires mechanical forces, often exerted by protein assemblies from the cell interior, that split a single cell into two. Using coarse-grained computer simulations and live cell imaging we define a distinct cell division mechanism—based on the forces generated by the supercoiling of an elastic filament as it disassembles. Our analysis suggests that such a mechanism could explain ESCRT-III–dependent division in Sulfolobus cells, based on the similarity of the dynamics of division obtained in simulations to those observed using live cell imaging. In this way our study furthers our understanding of the physical mechanisms used to reshape cells across evolution and identifies additional design principles for a minimal division machinery., Living systems propagate by undergoing rounds of cell growth and division. Cell division is at heart a physical process that requires mechanical forces, usually exerted by assemblies of cytoskeletal polymers. Here we developed a physical model for the ESCRT-III–mediated division of archaeal cells, which despite their structural simplicity share machinery and evolutionary origins with eukaryotes. By comparing the dynamics of simulations with data collected from live cell imaging experiments, we propose that this branch of life uses a previously unidentified division mechanism. Active changes in the curvature of elastic cytoskeletal filaments can lead to filament perversions and supercoiling, to drive ring constriction and deform the overlying membrane. Abscission is then completed following filament disassembly. The model was also used to explore how different adenosine triphosphate (ATP)-driven processes that govern the way the structure of the filament is changed likely impact the robustness and symmetry of the resulting division. Comparisons between midcell constriction dynamics in simulations and experiments reveal a good agreement with the process when changes in curvature are implemented at random positions along the filament, supporting this as a possible mechanism of ESCRT-III–dependent division in this system. Beyond archaea, this study pinpoints a general mechanism of cytokinesis based on dynamic coupling between a coiling filament and the membrane.

Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen of Johne’s disease (paratuberculosis), which mainly causes chronic infectious granulomatous enteritis in ruminants and has brought huge economic losses to animal husbandry. As a specific intracellular pathogen, when MAP invades the body, it is internalized by macrophages where it is able to replicate by inhibition of the phagosome maturation, escaping the host immune system and surviving, which leads to the spread of the disease. More recent studies have shown that circRNA is involved in many pathological and physiological processes of the body as the molecular sponge of miRNA, the scaffold of RNA binding protein and having the characteristic of being able to translate into protein. In this study, the mRNA and circRNA expression profiles of MAP-infected bovine monocyte-macrophages and uninfected bovine cells were analyzed by high-throughput sequencing. A total of 618 differentially expressed mRNA were screened out, including 322 upregulated mRNA and 296 downregulated mRNA. In addition, the analysis of circRNA differential expression profile showed 39 differentially expressed genes including 12 upregulated and 27 downregulated genes. Moreover, differential genes belonging to cytokine activity, chemokine activity, inflammatory reaction, apoptosis, and other functional groups related to macrophage immune response were significantly enriched in Gene Ontology (GO). Multiple signal pathways including NF-κB, MAPK, Toll-like receptor, IL-17, JAK-STAT, and other signaling pathways related to activating macrophage immune response were significantly enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, RT-qPCR technology verified the accuracy of the mRNA sequencing results. In this study, we have obtained the transcriptome information of mRNA and circRNA of bovine monocyte-macrophage infected with MAP. These results will provide data support for the further study of mRNA–miRNA–circRNA network and immune escape mechanism of MAP and will enrich the knowledge of the molecular immune mechanisms of Johne’s disease as well.

Purpose: This clinical trial combined pembrolizumab and vorinostat in recurrent/metastatic squamous cell carcinomas of the head and neck (HN), and salivary gland cancer (SGC). Patients and Methods: Patients with progressing incurable HN and SGC, Eastern Cooperative Oncology Group (ECOG) ≤1, no prior immunotherapy, RECIST1.1 measurable disease, and normal organ function were eligible. Pembrolizumab 200 mg was given intravenous every 21 days, and vorinostat 400 mg given orally 5 days on and 2 days off during each 21-day cycle. Primary endpoints were safety and objective response rates. Results: From November 2015 to August 2017, 25 patients with HN and 25 SGC were enrolled. Median age was 61 (range, 33–86) years, 39 (78%) were male, 21 (62%) were never smokers, and 27 (54%) had ECOG 0. In HN, 13 (52%) were p16+ oropharynx. Most common SGC histologies were adenoid cystic 12 (48%), acinic cell 3 (12%), and mucoepidermoid 3 (12%). Adverse events (AEs) in all patients were: 27 (54%) with grade ≥ 1 and 18 (36%) with grade ≥ 3. The most common AEs in all patients were renal insufficiency in seven, (14%), fatigue in six, (12%), and nausea in three (6%). There were three (12%) deaths on study. Responses in HN were complete response (CR) 0, partial response (PR) eight (32%), and stable disease (SD) five (20%). Efficacy in SGCs was CR 0, PR four (16%) in one lymphoepithelioma-like carcinoma, two acinic cell, one adenoid cystic, and SD 14 (56%). In the HN group, median follow-up (mFUP) was 12.6 months, median overall survival (mOS) was 12.6 months, and median progression-free survival (mPFS) was 4.5 months. In SGC, mFUP was 13.1 months, mOS was 14.0 months, and mPFS was 6.9 months. Conclusions: This combination demonstrated activity in HN, with fewer responses in SGC. Toxicities were higher than reported with pembrolizumab alone.

The present study evaluated the antibacterial activity and mode of action of fibrauretine on Escherichia coli (E. coli) and Staphylococcus aureus, and synergistic effect with kanamycin against multi-drug resistant E. coli. The fibrauretine exhibited inhibitory effect on the growth of the tested bacteria with minimum inhibitory concentration (MIC) and minimum bactericidal concentration of 2.5–5 and 5–10 mg/ml, respectively. Morphological changes of cell microstructure were observed after adding fibrauretine at MIC. The mode of action was further confirmed by measuring release of 260-nm absorbing materials and extracellular potassium ions. Checkerboard dilution test suggested that fibrauretine exhibited synergistic activity when combined with kanamycin (FICI ranging from 0.5625 to 0.625). Our results indicated that fibrauretine exerted synergistic effect with kanamycin and its antibacterial mode of action mainly attributed to disruption of cell membrane integrity.

Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 μm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme-heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis. We observed rates of heme-to-heme electron transfer on the order of 109 s-1 (3.7-4.3 A edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are amongst the highest reported for ground state electron transfer in biology. Yet, some fall 2-3 orders of magnitude below the Moser-Dutton ruler because electron transfer at these short distances is through-space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met ligated heme creates an electron sink that stabilizes the charge separated state on the 100 microsecond time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies.

ObjectiveProgrammed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures.DesignWe created organotypic slice cultures from human CRLM (n=38 patients’ tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CAR-T cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy.ResultsαIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8+T cells without exhaustion transcription changes, and increased human leukocyte antigen - DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CAR-T cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CAR-T cell activation and CAR-T cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CAR-T function.ConclusionNeutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CAR-T cells.

Background Schmid‐type metaphyseal chondrodysplasia (SMCD) is a rare autosomal dominant skeletal dysplasia caused by heterozygous mutations in COL10A1, the gene which encodes collagen type X alpha 1 chain. However, its genotype–phenotype relationship has not been fully determined. Subjects and Methods The proband is a 2‐year‐old boy, born of non‐consanguineous Chinese parents. We conducted a systematic analysis of the clinical and radiological characteristics and a follow‐up study of the proband. Whole‐exome sequencing was applied for the genetic analysis, together with bioinformatic analysis of predicted consequences of the identified variant. A homotrimer model was built to visualize the affected region and predict possible outcomes of this variant. Furthermore, a literature review and genotype–phenotype analysis were performed by online searching all cases with SMCD. Results A novel heterozygous variant (NM_000493.4: c.1863_1866delAATG, NP_000484.2: p.(Met622 Thrfs*54)) was identified in COL10A1 gene in the affected child. And it was predicted to be pathogenic by in silico analysis. Protein modeling revealed that the variant was located in the NC1 domain, which was predicted to produce truncated collagen and impair the trimerization of collagen type X alpha 1 chain and combination with molecules in the matrix. Moreover, genotype–phenotype correlation analysis demonstrated that patients with truncating variants or variants in NC1 domain often presented earlier onset and severer symptoms compared with those with non‐truncating or variants in non‐NC1 domains. Conclusion The NC1 domain of COL10A1 was proved to be the hotspot region underlying SMCD, patients with variants in NC1 domain were more likely to present severer manifestations at an earlier age., This study characterized a novel COL10A1 variant (c.1863_1866delAATG, p.M622 Tfs*54) associated with Schmid type metaphyseal chondrodysplasia in a two‐year‐old Chinese patient. The novel variant was predicted to impair the trimerization of collagen X (α1) and combination with molecules in the matrix by in silico analysis. The NC1 domain of COL10A1 was a key region underlying SMCD, patients with NC1 mutations tend to present severer manifestations at earlier age.

Rationale: The Schaaf-Yang syndrome (SYS) is an autosomal dominant multi-system genetic disease caused by melanoma antigen L2 (MAGEL2) gene mutations imprinted by mothers and expressed by fathers on the 15q11–15q13 chromosomes in the critical region of Prader-Willi. MAGEL2 is a single exon gene and one of the protein-coding genes of the Prader-Willi domain. MAGEL2 is a matrilineal imprinted gene (i.e., the maternal chromosome is methylated). It is only expressed by unmethylated paternal alleles, and the individual is affected only when the variation occurs on the paternal allele. Patient concerns: We reported a patient with MAGEL2 gene new site mutation who had mild intellectual disability, social fear, small hands and feet, obesity issues, dyskinesia, growth retardation, language lag and sexual development disorder. Diagnosis: Whole-exome sequencing showed a heterozygous variation in the MAGEL2 gene, NM_019066.4:c.1687C > T (p.Q563X) and diagnosed as Schaaf-Yang syndrome. Interventions: Patient was advised to reduce weight, control blood lipids, blood glucose through appropriate strengthening of exercise and diet control in the future. At the same time, the family members were advised to provide mental training to the patient to strengthen the contact and communication with the outside world and improve the autistic symptoms. Because of the patient's bilateral cryptorchidism, it is recommended that the patient should be treated with bilateral cryptorchidism reduction fixation. Outcomes: After a follow-up of the patient for 2 months, the patient is still walking unsteadily and requires an auxiliary reference material to walk normally. There is no significant change in height compared to before, and the weight has dropped by about 2 kg in the past 2 months. The symptoms of autism have improved slightly. The patient is willing to communicate with outsiders; his intelligence has not improved significantly, and his academic performance in school is still at the middle and lower levels. Lessons: The pathogenesis of SYS is complex, involving multiple pathways such as Leptin-POMC, MAGEL2-USP7-TRIM27 complex and oxytocin. Our study has also found that certain fatal phenotypes such as respiratory distress have a high incidence at individual sites, and early detection and timely intervention may prolong the life span of patients. Therefore, for patients in whom SYS is highly suspected, gene detection should be carried out as soon as possible.

It was recently suggested that cytochrome c operates in an ergodicity-breaking regime characterized by unusually large energy gap thermal fluctuations and associated reorganization free energies for heme oxidation of up to 3.0 eV. The large fluctuations were reported to lower activation free energy for oxidation of the heme cofactor by almost a factor of 2 compared to the case where ergodicity is maintained. Our group has recently investigated this claim computationally at several levels of theory and found no evidence for such large energy gap fluctuations. Here we address the points of our earlier work that have raised criticism and we also extend our previous investigation by considering a simple linear polarizability model for cytochrome c oxidation. We find very consistent results among all our computational approaches, ranging from classical molecular dynamics, to the linear polarizability model to QM(PMM)/MM to full QM(DFT)/MM electrostatic emdedding. None of them support the notion of very large energy gap fluctuations or ergodicity breaking. The deviation between our simulations and the ones reported in [

Background: Colorectal cancer liver metastasis (CRLM) causes major morbidity and mortality. Improved systemic therapies are crucial. We previously reported that macrophages infiltrated CRLM and expressed the immunosuppressive cytokine interleukin-10 (IL-10). In patient-derived CRLM tumor slice cultures, we found that a neutralizing antibody against IL-10 (anti-IL-10) caused tumor apoptosis. Here, we investigated the efficacy and immune-dependent mechanisms of IL-10 blockade in a larger cohort of CRLM patients. Methods: Tumor specimens were obtained from consenting patients at the time of surgery and cut into 250mm-thick tumor slice cultures as previously described. Tumor slices were treated with control or neutralizing antibodies against programmed cell death protein 1 (PD-1, EH12.1), IL-10 (JES3-9D7), IL-10 receptor alpha (IL-10RA, 3F9), major histocompatibility complex (MHC) class I (G46-2.6) or class II (Tu39). After 4-6 days of treatment, tumor apoptosis was evaluated by cleaved caspase-3 (CC3) immunohistochemistry (IHC) or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Tumors were evaluated by IHC and in situ hybridization (ISH) for immune markers. Student's t-test, paired t-test, or 1-way ANOVA was used as indicated. p < 0.05 was defined as significant. Results: We generated tumor slice cultures from 34 unique CRLM patients, 76% (26/34) of whom received preoperative chemotherapy, and all of whom had either microsatellite stable tumors (76%, 26/34) or unknown microsatellite status. Anti-PD-1 did not generate tumor apoptosis (n = 4 patients' tumors). In contrast, anti-IL-10 caused nearly 2-fold increased tumor apoptosis compared with control, in the majority of 34 CRLM patients' tumors evaluated (median 50.1% versus 27.4% apoptotic cells, p < 0.0001). IL-10 receptor blockade generated a similar but non-significant increase in apoptosis. Tumors treated with anti-IL-10 demonstrated increased frequency of CD8+ T cells (median 17.8% versus 7.0% of total cells, n = 5 patients' tumors, p = 0.02) and a non-significant increase in activated PD-1+CD3+ T cells. IL-10 blockade also increased tumor IFNG expression (median 2.9 versus 2.3 counts per total cell number, n = 6 patients' tumors, p < 0.05). Macrophage frequency did not increase, but MHC class II expression nearly doubled (median 19.5% versus 11.5% of total cells, n = 5 patients' tumors, p = 0.02). To test which immune cells were required for anti-IL-10 effect, we treated human CRLM slices with anti-IL-10 +/- blocking antibodies against MHC class I or II. In the 3 of 4 patients' tumors that responded to anti-IL-10, the effect was nearly completely reversed by aMHC-I or aMHC-II (p = 0.0005). Conclusion: IL-10 is a dominant and reversible mechanism of immune evasion in human CRLM. IL-10 blockade nearly doubled tumor apoptosis in a heterogeneous cohort of CRLM patients' tumors, through a mechanism that required intact CD8+ T cell and antigen-presenting cell function. IL-10 is a compelling immunotherapeutic target for CRLM. Citation Format: Teresa S. Kim, Kevin Sullivan, Xiuyun Jiang, Cynthia Hsu, Kevin Labadie, Karan Kohli, Heidi Kenerson, Sara Daniel, Arezou Abbasi, Raymond Yeung, Venu Pillarisetty. Interleukin-10 is a dominant and reversible mechanism of immune evasion in human colorectal cancer liver metastasis [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P037.

Introduction Wilms' tumor (WT), the most common childhood tumor, occurs in sporadic or familial forms. Recent findings reported that abnormal expression in microRNA (miRNA) suggests an important role of miRNAs during WT progress. MiRNAs are endogenous short-chain noncoding RNAs, which have been reported as key biomarkers for detecting tumor onset and progression. However, the functional role of miR-1180 in WT has remained unknown. Materials and methods MTT and clonogenic survival assays were used to detect WT cell proliferation. Flow cytometry Annexin V-FITC was used to measure apoptosis. In addition, proteins expressions in the cells were determined by Western blotting. Results In the present study, we demonstrated that miR-1180 is upregulated in WT when compared with adjacent tissues by quantitative reverse-transcription polymerase chain reaction. In addition, the inhibition of miR-1180 induced apoptosis in SK-NEP-1 cell line in vitro. Moreover, luciferase reporter assay showed that p73 protein was the target of miR-1180, which was confirmed by the results of Western blotting. Finally, in vivo data indicated that the tumor growth in mice was significantly inhibited by miR-1180 inhibitor. Conclusion Our results indicate that miR-1180 might serve as a therapeutic target for future WT therapy.

Therapies using T cells that are programmed to express chimeric antigen receptors (CAR T cells) consistently produce positive results in patients with hematologic malignancies. However, CAR T cell treatments are less effective in solid tumors for several reasons. First, lymphocytes do not efficiently target CAR T cells; second, solid tumors create an immunosuppressive microenvironment that inactivates T cell responses; and third, solid cancers are typified by phenotypic diversity and thus include cells that do not express proteins targeted by the engineered receptors, enabling the formation of escape variants that elude CAR T cell targeting. Here, we have tested implantable biopolymer devices that deliver CAR T cells directly to the surfaces of solid tumors, thereby exposing them to high concentrations of immune cells for a substantial time period. In immunocompetent orthotopic mouse models of pancreatic cancer and melanoma, we found that CAR T cells can migrate from biopolymer scaffolds and eradicate tumors more effectively than does systemic delivery of the same cells. We have also demonstrated that codelivery of stimulator of IFN genes (STING) agonists stimulates immune responses to eliminate tumor cells that are not recognized by the adoptively transferred lymphocytes. Thus, these devices may improve the effectiveness of CAR T cell therapy in solid tumors and help protect against the emergence of escape variants.