Expression Of Immunomodulatory Factors Research Articles

Abstract 4259: A novel human model of lung squamous cell carcinoma to deconvolve the cell-intrinsic effects of key dysregulated pathways

Abstract Tractable, patient relevant models are needed to investigate the essential components of normal cell transformation. We developed a unique in vitro model of early lung squamous cell carcinoma (LUSC) genetic alterations and driver pathway dysregulation using genetically modified primary human bronchial basal cells (hBCs) from three independent donors. The co-operation of ubiquitous and early LUSC alterations (TP53 and CDKN2A loss) and commonly deregulated pathways including squamous differentiation (SOX2), PI3K signaling (PTEN) and the oxidative stress response (KEAP1) was investigated using a combinatorial approach, culminating in eight unique hBC mutants harboring cumulative pathway alterations in a TP53-/-/CDKN2A-/- background. Phenotypes indicative of mutant hBC transformation were investigated using proliferation assays, organotypic cultures and anchorage independent growth assays. Organotypic cultures revealed LUSC pathway alterations to drive dramatic changes in airway histology, ranging from a normal epithelium to severe dysplasia. Furthermore, these analyses confirmed the role of SOX2-overexpression in initiating transformation by inducing squamous cell commitment and anchorage independent growth. We also identified a co-operation of SOX2-overexpression with the oxidative stress response and PI3K pathways to drive more advanced lesions in-vivo and in-vitro. Importantly, we were able to map changes in the expression of immunomodulatory factors with the pathway dysregulation. We demonstrated that this strategy constitute an alternative and patient-relevant system to model LUSC and identify genotype-phenotype correlations of clinical relevance. Citation Format: Julia Ogden, Robert Sellers, Anthony Oojageer, Sudhakar Sahoo, Anshuman Chaturvedi, Carlos Lopez-Garcia. A novel human model of lung squamous cell carcinoma to deconvolve the cell-intrinsic effects of key dysregulated pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4259.

Bushen Antai recipe ameliorates immune microenvironment and maternal-fetal vascularization in STAT3-deficient abortion-prone mice

Ethnopharmacological relevanceSpontaneous abortion (SA) is an intricate disorder affecting women of reproductive age. Previous studies have confirmed the indispensable role of signal transducer and activator of transcription (STAT) 3 in normal pregnancy. Bushen Antai recipe (BAR) is a satisfactory formula commonly used in practice, based on the rationale of traditional Chinese medicine (TCM) for SA. Aim of the studyThe current study explores the potential therapeutic effects and mechanistic insights of BAR in STAT3-deficient abortion-prone mice. Materials and methodsA STAT3-deficient abortion-prone mouse model was developed using intraperitoneal injection of stattic from embryo day (ED) 5.5 to ED9.5 among pregnant females (C57BL/6). We separately administered BAR1 (5.7 g/kg), BAR2 (11.4 g/kg), progesterone (P4), or distilled water at 10 ml/kg/day from ED0.5 until ED10.5. The embryo resorption rate and placenta-uterus structure were observed on ED10.5. The systemic immune status was examined by analyzing the frequency of immunosuppressive myeloid-derived suppressor cells (MDSCs), the ratio of two macrophage (M) subtypes, and the protein expression of associated molecules. Morphological observation, immunohistochemistry, and western blotting were used to evaluate the vascularization conditions at the maternal-fetal interface. ResultsBAR1, BAR2, or P4 treatment exerted remarkable effects in alleviating embryo resorption rate and disordered placental-uterus structure in STAT3-deficient abortion-prone mice. Western blotting indicated the deficiency of phosphorylated STAT3 and two prime target molecules, PR and HIF-1α, at the maternal-fetal interface under STAT3 inhibition. Simultaneously, BAR2 treatment significantly upregulated their expression levels. The systemic immune environment was disrupted, indicated by the reduced serum cytokine concentrations, MDSCs frequency, M2/M1 ratio, and the expression of immunomodulatory factors. Nonetheless, BAR2 or P4 treatment revived the immune tolerance for semi-allogenic embryos by enhancing the immune cells and factors. Besides, the western blot and immunohistochemistry results revealed that BAR2 or P4 treatment upregulated VEGFA/FGF2 and activated ERK/AKT phosphorylation. Therefore, BAR2 or P4 facilitated vascularization at the maternal-fetal interface in STAT3-deficient abortion-prone mice. ConclusionsBAR sustained pregnancy by reviving the systemic immune environment and promoting angiogenesis at the maternal-fetal interface in STAT3-deficient abortion-prone mice.

Ferroptosis Biomarkers for Predicting Prognosis and Immunotherapy Efficacy in Adrenocortical Carcinoma

Numerous studies have suggested that ferroptosis plays an important regulatory role in cancer cell death. Nonetheless, the potential effects of ferroptosis regulators on the prognosis, the expression of immunomodulatory factors in the tumor microenvironment and on the efficacy of immunotherapy in adrenocortical carcinoma (ACC) remain largely unknown. Public ACC datasets were used to investigate the relationship between ferroptosis regulators and prognosis and clinical features. A ferroptosis scoring system was established for individual cases of ACC using principal component analysis algorithms. Hub ferroptosis-related genes involved in immunoregulation and immunotherapy efficacy in ACC were further identified. Twenty ferroptosis regulators were differentially expressed in ACC and 17 ferroptosis regulators were closely related to prognosis in ACC. A ferroptosis scoring system was developed based on ACSL4, FANCD2, and SLC7A1 expression, and the ferroptosis regulators could serve as an independent prognostic factor for ACC. Further analyses indicated that the ferroptosis score integrated with the tumor mutation burden (TMB), and immune-checkpoint gene expression could predict prognosis in ACC. RNA isolation and reverse transcription‑quantitative polymerase chain reaction (RT-qPCR) demonstrated significant differences in the expression levels of ACSL4, FANCD2, and SLC7A1 between ACC and normal tissues. Furthermore, FANCD2 was significantly related to immunotherapy efficacy and prognosis in ACC. Our study demonstrated that ferroptosis was significantly associated with prognosis, clinical characteristics, immune-checkpoint gene expression, and tumor microenvironment immune cell infiltration in ACC. The current study provides comprehensive evidence for further research on ferroptosis regulators in ACC and provides new insight into the epigenetic regulation of the antitumor immune response.

Hypoxia preconditioning elicit differential response in tissue-specific MSCs via immunomodulation and exosomal secretion.

Mesenchymal stromal cells (MSCs) are emerging as an ideal candidate for regenerative medicine. It is known that the culture conditions impact the cellular properties of MSCs and their therapeutic behavior. Moreover, maintenance of MSCs in low oxygen tension for a short duration has shown to be beneficial for MSCs as it is similar to that of their physiological niche. However, the precise mechanism through which hypoxia pre-conditioning affects MSCs is not clear yet. Thus, in this study, we have investigated the effect of hypoxia exposure (1% O2) on tissue-specific MSCs over a period of time under serum-free culture conditions and evaluated the changes in expression of immuno-modulatory molecules and exosome biogenesis and secretion markers. It was observed that all MSCs responded differentially towards hypoxia exposure as indicated by the expression of HIF-1α. Moreover, this short-term exposure did not induce any changes in MSCs cellular morphology, proliferation rate, and surface marker profiling. In addition, we observed an enhancement in the expression of immunomodulatory factors (HLA-G, PGE-2, and IDO) after hypoxia exposure of 12 to 24h in all tissue-specific MSCs. Interestingly, we have also observed the upregulation in exosome secretion that was further corelated to the upregulation of expression of exosome biogenesis and secretion markers (ALIX, TSG101, RAB27a, RAB27b). Though there was a differential response of MSCs where WJ-MSCs and BM-MSCs showed upregulation of these markers at 6-12h of hypoxia pre-conditioning, while AD-MSCs showed similar changes beyond 24h of hypoxia exposure.

Evaluation of the impact of pre-operative stereotactic radiotherapy on the acute changes in histopathologic and immune marker profiles of brain metastases

The unique acute effects of the large fractional doses that characterize stereotactic radiosurgery (SRS) or radiotherapy (SRT), specifically in terms of antitumor immune cellular processes, vascular damage, tumor necrosis, and apoptosis on brain metastasis have yet to be empirically demonstrated. The objective of this study is to provide the first in-human evaluation of the acute biological effects of SRS/SRT in resected brain metastasis. Tumor samples from patients who underwent dose-escalated preoperative SRT followed by resection with available non-irradiated primary tumor tissues were retrieved from our institutional biorepository. All primary tumors and irradiated metastases were evaluated for the following parameters: tumor necrosis, T-cells, natural killer cells, vessel density, vascular endothelial growth factor, and apoptotic factors. Twenty-two patients with irradiated and resected brain metastases and paired non-irradiated primary tumor samples met inclusion criteria. Patients underwent a median preoperative SRT dose of 18 Gy (Range: 15–20 Gy) in 1 fraction, with 3 patients receiving 27–30 Gy in 3–5 fractions, followed by resection within median interval of 67.8 h (R: 18.25–160.61 h). The rate of necrosis was significantly higher in irradiated brain metastases than non-irradiated primary tumors (p < 0.001). Decreases in all immunomodulatory cell populations were found in irradiated metastases compared to primary tumors: CD3 + (p = 0.003), CD4 + (p = 0.01), and CD8 + (p = 0.01). Pre-operative SRT is associated with acute effects such as increased tumor necrosis and differences in expression of immunomodulatory factors, an effect that does not appear to be time dependent, within the limited intervals explored within the context of this analysis.

Extracellular Vesicles from Thapsigargin-Treated Mesenchymal Stem Cells Ameliorated Experimental Colitis via Enhanced Immunomodulatory Properties.

Therapeutic applications of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have attracted considerable attention because of their immunomodulatory properties against immune-mediated, inflammatory diseases. Here, we demonstrated enhanced immunomodulatory properties of EVs secreted from endoplasmic reticulum (ER) stress inducer thapsigargin (TSG)-primed human Wharton’s jelly-derived MSCs (WJ-MSCs). EVs from TSG-primed WJ-MSCs (TSG-EV) showed increased yield and expression of immunomodulatory factors, such as transforming growth factor-β1 (TGFβ), cyclooxygenase-2 (COX2), and especially indoleamine 2,3-dioxygenase (IDO), compared to control EVs. TSG-EV showed a significantly enhanced immunosuppressive effect on human peripheral blood-derived T cell proliferation and Th1 and Th17 differentiation, whereas Treg and M2-type macrophage were enriched compared to a control EV-treated group. Furthermore, TSG-EV substantially mitigated mouse experimental colitis by reducing the inflammatory response and maintaining intestinal barrier integrity. A significant increase of Tregs and M2-type macrophages in colitic colons of a TSG-EV-treated mouse suggests an anti-inflammatory effect of TSG-EV in colitis model, possibly mediated by Treg and macrophage polarization. These data indicate that TSG treatment promoted immunomodulatory properties of EVs from WJ-MSCs, and TSG-EV may provide a new therapeutic approach for treatment of colitis.

Abstract B8: Multiplex quantification of immunomodulatory proteins in tissue and plasma using next-gen targeted MRM mass spectrometry

Abstract Quantifying the many immunomodulatory proteins in the “cancer-immunity cycle” is critical for both patient selection and development of novel immunotherapies. Conventional technologies to measure proteins (IHC, ELISA, Western blot, flow cytometry) are often semiquantitative, not multiplexable, feature poor specificity, and are difficult to develop. Due to these limitations, investigators have turned to mRNA-based measurements to infer protein expression of immunomodulatory factors. However, as has been unequivocally demonstrated for human breast, colorectal, and ovarian cancers (Pubmed IDs: 27251275, 25043054, 27372738), RNA levels can be poor predictors of protein levels or activity. Thus, direct quantification of proteins is desirable. As part of the National Cancer Institute’s Cancer Moonshot, we are developing &amp;gt;300 multiplex assays for quantifying immunomodulatory proteins in tissue and plasma using a next-gen technology for protein quantification, based on targeted multiple reaction monitoring mass spectrometry (MRM-MS). MRM-MS complements traditional immunoassays by enabling highly specific, precise, harmonizable quantification of proteins in biospecimens, even at high multiplex levels. As proof of concept, we will present a novel multiplex assay based on next-gen MRM-MS for quantification of 47 immunomodulatory proteins in plasma and/or tumor tissue. The technology uses immunoaffinity enrichment coupled with MRM-MS (immuno-MRM assay). Assay bioanalytical validation was conducted in accordance with industry standards for use in FFPE tissue and plasma matrices and included determination of limits of detection, linear range of response, intraday and interday repeatability, stability of analytes in sample processing, and characterization of reproducibility of endogenous analyte measurement. The mean linear range was over three orders of magnitude with median intra- and interday CVs less than 10%. The characterized assays are being applied to a panel of tumor specimens from a variety of cancer types to document detection and determine minimum sample requirements. Additional assay panels are under development to provide measurements of up to 350 immunomodulatory proteins. Citation Format: Jeffrey R. Whiteaker, Lei Zhao, Richard G. Ivey, Julia Voytovich, Regine M. Schoenherr, Dongqing Huang, Jacob J. Kennedy, Amanda G. Paulovich. Multiplex quantification of immunomodulatory proteins in tissue and plasma using next-gen targeted MRM mass spectrometry [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B8.

A new approach for immuno-oncology biomarker discovery: High-plex, spatial protein profiling based on NanoString digital quantification.

27 Background: The immune response to cancer is shaped by the abundance and localization of immune cell populations, their activation status, and expression of immunomodulatory factors. To detect proteins at high multiplex with spatial resolution, NanoString optical barcoding technology was used to digitally profile protein expression in formalin fixed paraffin embedded (FFPE) samples and compared to traditional IHC. Methods: NanoString has enabled digital profiling of immuno-oncology protein targets, including immune cell markers and checkpoint proteins. Protein detection is enabled via primary antibodies (Abs) which are attached via a UV cleavable linker to DNA indexing oligos. FFPE samples are stained with a multiplex cocktail of labeled Abs, then DNA oligos are released by UV light exposure. Liberated oligos are then hybridized to optical barcodes for quantitation on a NanoString instrument. This technique enables quantitative, multiplex protein detection over 5 logs of dynamic range. IHC and NanoString spatial protein profiling were performed on alternating sections from blocks of tonsil, melanoma, and colorectal cancer. Sections were fluorescently labeled with panCK, Ki67, and Syto-83 to visualize morphology, and regions of interest (ROI) were selected for profiling using up to 30 oligo-tagged Abs. Results: NanoString counts strongly correlated with quantification of CD3, CD4, CD8, PanCK, Ki67, PD-1, and PD-L1 derived from IHC. To explore limits of detection, ROIs of 1, 2, 4, or 8 cells were profiled. Digital counts were detected above background at the single cell level and linearly correlated with cell count. Furthermore, quantification with two cell lineage markers and double positive cells were demonstrated using a 30-plex Ab cocktail. Conclusions: Multiplex, digital protein profiling with spatial resolution will enable deep characterization of immune responses in tumors. Additionally, single cell profiling enables inter-cellular characterization of variations in immune response. The strong correlation of NanoString data to IHC indicates the feasibility to spatially profile multiple key proteins with minimal consumption of patient tissue.

PD‐1/PDL1 and CD28/CD80 pathways modulate natural killer T cell function to inhibit hepatitis B virus replication

α-Galactosylceramide (α-GalCer)-activated natural killer T (NKT) cells have antiviral properties against hepatitis B virus (HBV). However, α-GalCer activation of NKT cells can induce anergy. We hypothesized that this effect may be overcome by a treatment strategy that includes manipulation of CD28/CD80 costimulatory and PD-1/PDL1 coinhibitory signals of NKT cells, thereby enhancing the anti-HBV effect of α-GalCer. We established a transgenic mouse model of chronic HBV infection and investigated hepatic NKT cell frequencies, functions and expression of immunomodulatory factors. Our results showed that compared with uninfected control mice, hepatic NKT cells from HBV transgenic mice displayed lower frequencies (7.91% vs 16.74%, P<0.05), impaired capabilities to produce interferon (IFN)-γ (5.6% vs 1.4%, P<0.05) and interleukin (IL)-4 (6.8% vs 0.3%, P<0.05), higher expression of PD-1 (9.64% vs 6.36%, P<0.05) and lower expression of CD28 (5.05% vs 28.88%, P<0.05). However, when hepatic mononuclear cells (MNCs) were isolated from HBV transgenic mice, α-GalCer exposure in culture remarkably upregulated both PD-1(+) NKT cells (P<0.05) and CD28(+) NKT cells (P<0.05). Furthermore, when HBV transgenic mice were treated with combination therapies consisting of α-GalCer and anti-PDL1 monoclonal antibody (mAb) and/or anti-CD80/anti-CD28 mAbs, IFN-γ(+) NKT cell frequency was selectively increased (P<0.05) and HBV replication was suppressed; these effects were accompanied by varying degrees and types of liver damage. Surprisingly, activating CD28/CD80 signal in HBV transgenic mice was more effective but caused less liver injury than blocking PD-1/PDL1 signal in modulating αGalCer-activated NKT cell function to inhibit HBV infection. Our findings also show that combined therapy with blocking PD-1/PDL1 and activating CD28/CD80 signal in the presence of aGalCer cannot superimpose the effect of antivirus. α-GalCer combination therapy that modulates the CD28/CD80 pathways of NKT cells may represent a promising approach to inhibit HBV replication in chronically infected patients.

Effect of Arthritic Synovial Fluids on the Expression of Immunomodulatory Factors by Mesenchymal Stem Cells: An Explorative in vitro Study

Background: In diseased joints, the catabolic environment results in progressive joint damage. Mesenchymal stem cells (MSCs) can have immunomodulatory effects by secreting anti-inflammatory factors. To exert these effects, MSCs need to be triggered by pro-inflammatory cytokines. To explore the potential of MSCs as a treatment for diseased joints, we studied the effect of synovial fluid (SF) from donors with different joint diseases and donors without joint pathology on the immunomodulatory capacities of human MSCs in vitro. We hypothesized that SF of diseased joints influences the immunomodulatory effects of MSCs. Materials and Methods: MSCs were cultured in medium with SF of six osteoarthritis (OA) or six rheumatoid arthritis (RA) donors and three donors without joint pathology were used as control. Gene expressions of IL-6, HGF, TNFa, TGFb1, and indoleamine 2,3-dioxygenase (IDO) were analyzed. l-kynurenine concentration in conditioned medium (CM) by MSCs with SF was determined as a measure of IDO activity by MSCs. Furthermore, the effect of CM with SF on proliferation of activated lymphocytes was analyzed. Results: Addition of SF significantly up-regulated the mRNA expression of IL-6 and IDO in MSCs. SF(OA) induced significantly higher expression of IDO than SF(control), although no difference in IDO activity of the MSCs could be shown with a l-kynurenine assay. Medium conditioned by MSCs with SF(OA or RA) suppressed activated lymphocyte proliferation in vitro more than medium conditioned by MSCs without SF or with SF(control). Discussion: SF can influence the expression of genes involved in immunomodulation by MSCs and the effect on lymphocyte proliferation. We found indications for disease-specific differences between SFs but the variation between donors, even within one disease group was high. These data warrant further research to examine the potential application of MSC therapy in arthritic joints.