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Metastatic pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid tumours despite the use of multi-agent conventional chemotherapy regimens. Such poor outcomes have fuelled ongoing efforts to exploit the tumour microenvironment (TME) for therapy, but strategies aimed at deconstructing the surrounding desmoplastic stroma and targeting the immunosuppressive pathways have largely failed. In fact, evidence has now shown that the stroma is multi-faceted, which illustrates the complexity of exploring features of the TME as isolated targets. In this Review, we describe ways in which the PDAC microenvironment has been targeted and note the current understanding of the clinical outcomes that have unexpectedly contradicted preclinical observations. We also consider the more sophisticated therapeutic strategies under active investigation — multi-modal treatment approaches and exploitation of biologically integrated targets — which aim to remodel the TME against PDAC.Important research efforts are being made to develop therapeutic strategies targeting the tumour microenvironment in pancreatic adenocarcinoma. The authors of this Review describe the apparent contradiction between preclinical studies and clinical outcomes observed to date, presenting more sophisticated strategies under active investigation.

Development of cancer therapeutics requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Three‐dimensional (3‐D) in vitro models have therefore been investigated for drug screening. In this study, we have developed a novel in vitro model in which multicellular aggregates, or spheroids, were incorporated into 3‐D porous scaffolds. Drug resistance assays showed that spheroid‐seeded scaffolds have much higher drug resistance than monolayer cultures, spheroids on flat substrates, or scaffolds seeded with dispersed cells. Furthermore, spheroid‐seeded scaffolds demonstrated higher lactate production leading to acidosis, and higher expression of angiogenic factors. These data suggest that the spheroid‐seeded 3‐D scaffolds might serve as a useful in vitro system for screening cancer therapeutics. (Cancer Sci 2010; 101: 2637–2643)

(1) Background: We studied how the generation position of air anions in a closed-type plant factory affects the growth characteristics of lettuce and identified the optimal position. (2) Methods: We used LEDs (red/green/blue = 8:1:1) as a light source and set the temperature and RH of the plant factory to 20 ± 2 ℃ and 50% ± 5%, respectively. We grew lettuce under three air anion conditions—sideward, upward, and downward—and compared the growth characteristics to those of a control grown without air anions. We measured the growth characteristics of the lettuce at 3 and 4 weeks after sowing, and the measurement items were shoot fresh weight (FW) and dry weight (DW); leaf area (LA), length (LL), and width (LW); SPAD; antioxidant capacity; and total phenol content. (3) Results: At 4 weeks, FW in the downward treatment condition was 25.3% higher than in the control, and DW showed a similar difference. LA was about 1943.94 cm²/plant in the downward treatment condition, which was about 15.5% higher than in the control. (4) Conclusions: We conclude that air anion generation has a positive effect on lettuce growth, and the optimal generation position for air anions is downward.

Concomitant inhibition of vascular endothelial growth factor (VEGF) and programmed cell death protein 1 (PD-1) or its ligand PD-L1 is a standard of care for patients with advanced hepatocellular carcinoma (HCC), but only a minority of patients respond, and responses are usually transient. Understanding the effects of therapies on the tumor microenvironment (TME) can provide insights into mechanisms of therapeutic resistance. 14 patients with HCC were treated with the combination of cabozantinib and nivolumab through the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center. Among them, 12 patients (5 responders + 7 non-responders) underwent successful margin negative resection and are subjects to tissue microarray (TMA) construction containing 37 representative tumor region cores. Using the TMAs, we performed imaging mass cytometry (IMC) with a panel of 27-cell lineage and functional markers. All multiplexed images were then segmented to generate a single-cell dataset that enables (1) tumor-immune compartment analysis and (2) cell community analysis based on graph-embedding methodology. Results from these hierarchies are merged into response-associated biological process patterns. Image processing on 37 multiplexed-images discriminated 59,453 cells and was then clustered into 17 cell types. Compartment analysis showed that at immune-tumor boundaries from NR, PD-L1 level on tumor cells is significantly higher than remote regions; however, Granzyme B expression shows the opposite pattern. We also identify that the close proximity of CD8 T cells to arginase 1 (Arg1 ) macrophages, rather than CD4 T cells, is a salient feature of the TME in non-responders. Furthermore, cell community analysis extracted 8 types of cell-cell interaction networks termed cellular communities (CCs). We observed that in non-responders, macrophage-enriched CC (MCC) and lymphocyte-enriched CC (LCC) strongly communicate with tumor CC, whereas in responders, such communications were undermined by the engagement between MCC and LCC. These results demonstrate the feasibility of a novel application of multiplexed image analysis that is broadly applicable to quantitative analysis of pathology specimens in immuno-oncology and provides further evidence that CD163 Arg1 macrophages may be a therapeutic target in HCC. The results also provide critical information for the development of mechanistic quantitative systems pharmacology models aimed at predicting outcomes of clinical trials.

BackgroundThe prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. Natural Killer (NK) cells can elicit an antileukemic response via a repertoire of activating receptors that bind AML surface ligands. NK-cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity and interleukin (IL)-15 to enhance their persistence.MethodsWe characterized in detail NK-cell populations expressing a panel of AML (CD123)-specific CARs and/or IL-15 in vitro and in AML xenograft models.ResultsCARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ Chimeric Antigen Receptor (CAR)-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK-cell persistence. Transgenic expression of secretory interleukin (sIL)-15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK-cell subsets. 2B4.ζ/sIL-15 CAR and sIL-15 NK cells maintained an overall activated NK-cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK-cell groups. In vivo, 2B4.ζ/sIL-15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL-15 CAR and sIL-15 NK cells induced lethal toxicity in a second model.ConclusionTransgenic expression of CD123-CARs and sIL-15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early-phase clinical testing.

Background Low absolute lymphocyte count (ALC) has previously been established as a marker of poor prognosis in multiple cancer types. There is growing evidence that ALC may also be associated with response to immunotherapy. This study explores whether response to PD1 inhibitors in recurrent and/or metastatic head and neck squamous cell carcinoma (R/M HNSCC) is associated with pretreatment ALC. Methods Thirty-four R/M HNSCC patients who received either nivolumab or pembrolizumab between January 2014 and May 2018 at Johns Hopkins were identified retrospectively. Pretreatment blood counts in patients with and without clinical benefit from PD1 inhibitors were compared. Time-to-progression analyses were performed by dichotomizing the study cohort with the threshold of ALC 600 cells/μl, which is approximately 1.5 standard deviations away from treatment-naïve baseline mean. Results Patients with lower ALC appeared to have significantly less clinical benefit from anti-PD1 therapy. Those patients with pretreatment ALC < 600 cells/μl also had shorter PFS than patients with pretreatment ALC ≥ 600 cells/μl (median PFS 60 days vs. 141 days, p  < 0.05). These results were consistent with multivariate proportional hazards analyses demonstrating significant association with progression. These observations were further supported by an expansion cohort analysis incorporating additional fourteen R/M HNSCC patients who received other checkpoint immunotherapy regimens at our institution. Conclusions This study for the first time demonstrates that pretreatment ALC is significantly associated with response to PD1 inhibitors in R/M HNSCC patients.

Pancreatic ductal adenocarcinoma (PDA) is characterized by low cytotoxic lymphocytes, abundant immune-suppressive cells, and resistance to immune checkpoint inhibitors (ICI). Preclinical PDA models showed the HDAC inhibitor entinostat reduced myeloid cell immunosuppression, sensitizing tumors to ICI therapy. This phase II study combined entinostat with nivolumab (PD1 inhibitor) in patients with advanced PDA (NCT03250273). Patients received entinostat 5 mg orally once weekly for 14-day lead-in, followed by entinostat and nivolumab. The primary endpoint was the objective response rate (ORR) by RECIST v1.1. Secondary endpoints included safety, duration of response, progression free-survival and overall survival. Between November 2017 and November 2020, 27 evaluable patients were enrolled. Three showed partial responses (11% ORR, 95% CI, 2.4%-29.2%) with a median response duration of 10.2 months. Median progression-free survival (PFS) and overall survival (OS) were, respectively, 1.89 (95% CI, 1.381-2.301) and 2.729 (95% CI, 1.841-5.622) months. Grade ≥3 treatment-related adverse events occurred in 19 patients (63%), including decreased lymphocyte count, anemia, hypoalbuminemia, and hyponatremia. As exploratory analysis, peripheral and tumor immune profiles changes were assessed using CyTOF, mIHC, and RNA-seq. Entinostat increased dendritic cell activation and maturation. Gene expression analysis revealed an enrichment in inflammatory response pathways with combination treatment. Although the primary endpoint was not met, entinostat and nivolumab showed durable responses in a small subset of PDA patients. Myeloid cell immunomodulation supported the preclinical hypothesis, providing a basis for future combinatorial therapies to enhance clinical benefits in PDA. Responses to immune checkpoint inhibitors in patients with pancreatic ductal adenocarcinoma (PDA) remain low and alternative combinatorial approaches are warranted. Here the authors report the results of a phase 2 clinical trial of entinostat (histone deacetylases inhibitor) and nivolumab (anti-PD-1 inhibitor) in patients with metastatic PDA.

Most new cancer diagnoses occur in patients over the age of 65. The composition and function of the immune system changes with age, but how the aged immune system affects responses to immune checkpoint inhibitor (ICI) cancer therapies remains incompletely understood. Here, using multiplex cytokine assay and high-parameter mass cytometry, we analyze prospectively collected blood samples from 104 cancer patients receiving ICIs. We find aged patients ( ≥ 65-years-old; n  = 54) derive similar clinical outcomes as younger patients ( n  = 50). However, aged, compared to young, patients have divergent immune phenotypes at baseline that persist during ICI therapy, including diminished cytokine responses, reduced pools of naïve T cells with increased relative expression of immune checkpoint molecules, and more robust effector T cell expansion in responders compared to non-responders. Our study provides insights into age-stratified mechanisms of ICI effects while also implying the utility of age-tailored immunotherapeutic approaches. The aging immune system is characterized by changes in immune cell frequencies and functionality. Here the authors report dynamics of age-related divergence of circulating immune responses in patients with solid tumors treated with immune checkpoint inhibitors

Background The majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed at the metastatic stage, and standard therapies have limited activity with a dismal 5-year survival rate of only 8%. The liver and lung are the most common sites of PDAC metastasis, and each have been differentially associated with prognoses and responses to systemic therapies. A deeper understanding of the molecular and cellular landscape within the tumor microenvironment (TME) metastasis at these different sites is critical to informing future therapeutic strategies against metastatic PDAC. Results By leveraging combined mass cytometry, immunohistochemistry, and RNA sequencing, we identify key regulatory pathways that distinguish the liver and lung TMEs in a preclinical mouse model of metastatic PDAC. We demonstrate that the lung TME generally exhibits higher levels of immune infiltration, immune activation, and pro-immune signaling pathways, whereas multiple immune-suppressive pathways are emphasized in the liver TME. We then perform further validation of these preclinical findings in paired human lung and liver metastatic samples using immunohistochemistry from PDAC rapid autopsy specimens. Finally, in silico validation with transfer learning between our mouse model and TCGA datasets further demonstrates that many of the site-associated features are detectable even in the context of different primary tumors. Conclusions Determining the distinctive immune-suppressive features in multiple liver and lung TME datasets provides further insight into the tissue specificity of molecular and cellular pathways, suggesting a potential mechanism underlying the discordant clinical responses that are often observed in metastatic diseases.

Both epidemiologic and cellular studies in the context of autoimmune diseases have established that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a key regulator of T cell receptor (TCR) signaling. However, its mechanism of action in tumors and its translatability as a target for cancer immunotherapy have not been established. Here we show that a germline variant of PTPN22, rs2476601, portended a lower likelihood of cancer in patients. PTPN22 expression was also associated with markers of immune regulation in multiple cancer types. In mice, lack of PTPN22 augmented antitumor activity with greater infiltration and activation of macrophages, natural killer (NK) cells, and T cells. Notably, we generated a novel small molecule inhibitor of PTPN22, named L-1, that phenocopied the antitumor effects seen in genotypic PTPN22 knockout. PTPN22 inhibition promoted activation of CD8+ T cells and macrophage subpopulations toward MHC-II expressing M1-like phenotypes, both of which were necessary for successful antitumor efficacy. Increased PD1-PDL1 axis in the setting of PTPN22 inhibition could be further leveraged with PD1 inhibition to augment antitumor effects. Similarly, cancer patients with the rs2476601 variant responded significantly better to checkpoint inhibitor immunotherapy. Our findings suggest that PTPN22 is a druggable systemic target for cancer immunotherapy.