CyTOF Analyses Research Articles

P0025 The effect of very small superparamagnetic nanoparticles on human blood cells as a diagnostic tool in Inflammatory Bowel Disease

Abstract Background The field of application of organic or inorganic nanoparticles (1-100 nm size) is extensive and involves their use in medicine. This includes using nanoparticles in human beings or animals (life-stock) which holds advantages but also bears risks. Therefore, nanoparticles used in diagnostics and therapy should be inert not affecting tissues or immune cells. Additionally, nanoparticles should not aggregate to form clusters exceeding the size of 100 nm or accumulate in sensitive structures like bone marrow or brain for long-term deposition. We developed in-house very small superparamagnetic iron oxide nanoparticles (VSOP) with a size of 7 nm that were successfully used in preclinical magnetic resonance imaging (MRI) for the detection of intestinal inflammation and sclerosis. This study elucidates the effect of nanoparticles on human blood cells, including monocytes as well as monocyte-derived macrophages, in vitro as a first step toward clinical application in inflammatory bowel disease (IBD). Methods Whole blood of healthy donors and patients suffering from IBD as well as monocytes and monocyte-derived macrophages were treated with VSOP in vitro and analysed for changes in their transcriptome, phenotype and function. For transcriptome analysis, RNA sequencing was performed. Cell abundance, activation and proliferation were measured by mass cytometry. Migratory behavior of monocytes was analyzed by live microscopy. Cell metabolism was detected by Seahorse assay. Results RNA sequencing of monocytes showed that the most significantly regulated gene is encoding for the transferrin receptor (Tcfr). As a response to VSOP treatment and VSOP uptake thereby increasing the levels of intracellular iron, Tcfr is probably downregulated to restrict further iron uptake. While RNA sequencing of whole blood showed only significant differences in non-coding genes (C3orf86P, ENSG00000278996, ENSG00000259002), CyTOF analyses revealed that VSOP-treated monocytes are neither activated nor showing increased proliferation. Also, the migratory behavior is not influenced by VSOP uptake. Additionally, VSOP uptake resulted in an enhanced oxygen consumption rate and extracellular acidification rate. In comparison to the uptake of Latex beads, these increased rates are rather attributed to phagocytosis. Conclusion Analyses of our data with PBMC, monocytes and monocyte-derived macrophages suggest, that treatment with VSOP has no major affect on phenotype and function of immune cells. Therefore, VSOP are a promising tool for the early detection of IBD through MRI.

Macrophage PET imaging in mouse models of cardiovascular disease and cancer with an apolipoprotein-inspired radiotracer

Macrophages are key inflammatory mediators in many pathological conditions, including cardiovascular disease (CVD) and cancer, the leading causes of morbidity and mortality worldwide. This makes macrophage burden a valuable diagnostic marker and several strategies to monitor these cells have been reported. However, such strategies are often high-priced, non-specific, invasive, and/or not quantitative. Here, we developed a positron emission tomography (PET) radiotracer based on apolipoprotein A1 (ApoA1), the main protein component of high-density lipoprotein (HDL), which has an inherent affinity for macrophages. We radiolabeled an ApoA1-mimetic peptide (mA1) with zirconium-89 (89Zr) to generate a lipoprotein-avid PET probe (89Zr-mA1). We first characterized 89Zr-mA1’s affinity for lipoproteins in vitro by size exclusion chromatography. To study 89Zr-mA1’s in vivo behavior and interaction with endogenous lipoproteins, we performed extensive studies in wildtype C57BL/6 and Apoe-/- hypercholesterolemic mice. Subsequently, we used in vivo PET imaging to study macrophages in melanoma and myocardial infarction using mouse models. The tracer’s cell specificity was assessed by histology and mass cytometry (CyTOF). Our data show that 89Zr-mA1 associates with lipoproteins in vitro. This is in line with our in vivo experiments, in which we observed longer 89Zr-mA1 circulation times in hypercholesterolemic mice compared to C57BL/6 controls. 89Zr-mA1 displayed a tissue distribution profile similar to ApoA1 and HDL, with high kidney and liver uptake as well as substantial signal in the bone marrow and spleen. The tracer also accumulated in tumors of melanoma-bearing mice and in the ischemic myocardium of infarcted animals. In these sites, CyTOF analyses revealed that natZr-mA1 was predominantly taken up by macrophages. Our results demonstrate that 89Zr-mA1 associates with lipoproteins and hence accumulates in macrophages in vivo. 89Zr-mA1’s high uptake in these cells makes it a promising radiotracer for non-invasively and quantitatively studying conditions characterized by marked changes in macrophage burden.

Abstract 2459: ADT-030 is a novel dual-acting RAS/β-catenin inhibitor that potentiates antitumor immunity

Abstract ADT-030 is a novel indene identified from a screening campaign and chemically optimized for drug-like properties that acts as a dual inhibitor of RAS and β-catenin signaling. Here we demonstrate that ADT-030 binds RAS with high affinity and inhibits MAPK/AKT signaling with high potency and selectivity in KRAS mutant cells to inhibit proliferation and induce apoptosis. ADT-030 also inhibits phosphodiesterase 10A (PDE10) that is overexpressed in cancer cells to selectively activate cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling, which induces phosphorylation and degradation of the oncogenic pool of β-catenin. ADT-030 exhibited cytotoxicity to a variety of murine cancer cell lines in vitro, and inhibited tumor growth in syngeneic immunocompetent mice when orally administered. The antitumor activity of ADT-030 was diminished in mice that were immunodeficient or depleted of CD8+ T cells, indicating a critical role of the host immune system. In a metastatic breast tumor mouse model (4T1), ADT-030 not only delayed the growth of the primary tumor but also reduced tumor colonization in the lungs, leading to increased survival. CyTOF and scRNAseq analyses provided a plethora of information revealing the impact of ADT-030 on the immune milieu in the tumor microenvironment. Notably, ADT-030 selectively induced apoptosis of myeloid-derived suppressor cells (MDSCs) while CD8+ T cells were not affected. Consequently, ADT-030 treatment resulted in a marked reduction of MDSCs, which was accompanied by increased tumor infiltration of effector CD8+ T cells. ADT-030 treatment of cancer cells led to immunogenic cell death and enhanced dendritic cell activation. Furthermore, the ability of ADT-030 to augment the antitumor activity of immune checkpoint blockade therapy underlines its potential as a desirable immune-potentiating agent to improve the outcome of cancer immunotherapy. ADT-030 represents a development candidate ready for IND-enabling safety assessment. Citation Format: Md Yeashin Gazi, Ogacheko Okoko, Xin Wang, Caitlin Brandle, Xi Chen, Khalda Fadlalla, Adam Keeton, Yulia Maxuitenko, Catherine Hedrick, Huidong Shi, Gary Piazza, Gang Zhou. ADT-030 is a novel dual-acting RAS/β-catenin inhibitor that potentiates antitumor immunity [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 2459.

Nicotinamide mononucleotide impacts HIV-1 infection by modulating immune activation in T lymphocytes and humanized mice

HIV-1-associated immune activation drives CD4+ T cell depletion and the development of acquired immunodeficiency syndrome. We aimed to determine the role of nicotinamide mononucleotide (NMN), the direct precursor of nicotinamide adenine dinucleotide (NAD) co-enzyme, in CD4+ T cell modulation during HIV-1 infection. We examined HIV-1 integrated DNA or transcribed RNA, intracellular p24 protein, and T cell activation markers in CD4+ T cells including invitro HIV-1-infected cells, reactivated patient-derived cells, and in HIV-1-infected humanized mice, under NMN treatment. RNA-seq and CyTOF analyses were used for investigating the effect of NMN on CD4+ T cells. We found that NMN increased the intracellular NAD amount, resulting in suppressed HIV-1 p24 production and proliferation in infected CD4+ T cells, especially in activated CD25+CD4+ T cells. NMN also inhibited CD25 expression on reactivated resting CD4+ T cells derived from cART-treated people living with HIV-1 (PLWH). In HIV-1-infected humanized mice, the frequency of CD4+ T cells was reconstituted significantly by combined cART and NMN treatment as compared with cART or NMN alone, which correlated with suppressed hyperactivation of CD4+ T cells. Our results highlight the suppressive role of NMN in CD4+ T cell activation during HIV-1 infection. It warrants future clinical investigation of NMN as a potential treatment in combination with cART in PLWH. This work was supported by the Hong Kong Research Grants Council Theme-Based Research Scheme (T11-706/18-N), University Research Committee of The University of Hong Kong, the Collaborative Research with GeneHarbor (Hong Kong) Biotechnologies Limited and National Key R&D Program of China (Grant2021YFC2301900).

Tumor-Myeloid Cell Crosstalk Pathways Associated with Abscopal Responses in Breast Cancer

To identify predictive biomarkers for combined radiotherapy (RT) and immune checkpoint inhibitor (ICI) therapy, which can induce systemic anti-tumor immune responses (i.e., "abscopal" responses) in breast cancer. Four Trp53-/- Balb/c breast cancer syngeneic allograft models with low tumor mutational burden and resistance to dual ICI (anti-PD1 and anti-CTLA4, BioXcell) were used. Two lines exhibited abscopal responses to RT (8Gyx3) plus ICI combination therapy (i.e., "abscopal models"), whereas the other two did not (i.e., "non-abscopal models"). We performed spatial transcriptomics analysis (GeoMx whole transcriptome assay) of pan-cytokeratin positive (panCK+) tumor and tumor-adjacent CD45+ immune cell segments in orthotopically implanted tumors 10 days after initiating treatment with IgG control, RT alone, ICI alone, or RT/ICI combination (N>5 per group). Genes selectively induced by RT/ICI in panCK+ and CD45+ segments in abscopal models and were not induced in non-abscopal models were identified using two-tailed t-tests and FDR correction for multiple testing (FDR<5%). In vitro analyses of RT-induced secreted inflammatory signaling were conducted by exposing supernatant from irradiated (8Gy) tumor cells to a RAW264.7 macrophage cell line expressing an interferon-inducible luciferase reporter construct, followed by luciferase signal quantification (Invivogen). Hierarchical clustering of immune-related genes in CD45+ immune segments from breast cancer models with abscopal responses to RT/ICI revealed global differences in tumor-adjacent immune profiles at baseline and after RT/ICI treatment. Abscopal responsive breast cancer models at baseline showed an enrichment of myeloid cell subtypes expressing complement protein C1q, Ccl8, and Csf1r in tumor-adjacent CD45+ immune cells, and CD8+ T cells expressing Ccl5. 10 days after treatment with RT/ICI, tumor-adjacent CD45+ immune cells in abscopal models were enriched in Cxcl10, Irf7, and FcgRIV, whereas these genes were not induced in non-abscopal models. CyTOF analyses confirmed the induction of inflamed professional antigen presenting cells (CD11c+CD80+IA/IE+) by RT/ICI in abscopal models. Within panCK+ tumor segments, RT/ICI in abscopal models selectively induced Isg15 and Zbp1, both of which are involved in secreted inflammatory signaling. Consistently, RT-induced secreted inflammatory signaling from tumor cells to macrophages evaluated in vitro was significantly greater in abscopal models compared to non-abscopal models. Breast cancer models with abscopal responses to RT/ICI show increased Isg15- and Zbp1-dependent secreted inflammatory crosstalk that activates Cxcl10 and Irf7 expression in tumor-adjacent myeloid cells, which warrants further investigation as a potential predictive biomarker for combined RT/ICI therapy in breast cancer.

A transcriptional response to replication stress selectively expands a subset of Brca2-mutant mammary epithelial cells

Germline BRCA2 mutation carriers frequently develop luminal-like breast cancers, but it remains unclear how BRCA2 mutations affect mammary epithelial subpopulations. Here, we report that monoallelic Brca2mut/WT mammary organoids subjected to replication stress activate a transcriptional response that selectively expands Brca2mut/WT luminal cells lacking hormone receptor expression (HR-). While CyTOF analyses reveal comparable epithelial compositions among wildtype and Brca2mut/WT mammary glands, Brca2mut/WT HR- luminal cells exhibit greater organoid formation and preferentially survive and expand under replication stress. ScRNA-seq analysis corroborates the expansion of HR- luminal cells which express elevated transcript levels of Tetraspanin-8 (Tspan8) and Thrsp, plus pathways implicated in replication stress survival including Type I interferon responses. Notably, CRISPR/Cas9-mediated deletion of Tspan8 or Thrsp prevents Brca2mut/WT HR- luminal cell expansion. Our findings indicate that Brca2mut/WT cells activate a transcriptional response after replication stress that preferentially favours outgrowth of HR- luminal cells through the expression of interferon-responsive and mammary alveolar genes.

Characterization of papillary and clear cell renal cell carcinoma through imaging mass cytometry reveals distinct immunologic profiles.

To characterize and further compare the immune cell populations of the tumor microenvironment (TME) in both clear cell and papillary renal cell carcinoma (RCC) using heavy metal-labeled antibodies in a multiplexed imaging approach (imaging mass cytometry). Formalin-fixed paraffin-embedded (FFPE) baseline tumor tissues from metastatic patients with clear cell renal cell carcinoma (ccRCC) and papillary renal cell carcinoma (pRCC) were retrospectively requisitioned from an institutional biorepository. Pretreated FFPE samples from 33 RCC patients (10 ccRCC, 23 pRCC) were accessioned and stained for imaging mass cytometry (IMC) analysis. Clinical characteristics were curated from an institutional RCC database. FFPE samples were prepared and stained with heavy metal-conjugated antibodies for IMC. An 11-marker panel of tumor stromal and immune markers was used to assess and quantify cellular relationships in TME compartments. To validate our time-of-flight (CyTOF) analysis, we cross-validated findings with The Cancer Genome Atlas Program (TCGA) analysis and utilized the CIBERSORTx tool to examine the abundance of main immune cell types in pRCC and ccRCC patients. Patients with ccRCC had a longer median overall survival than did those with pRCC (67.7 vs 26.8 mo, respectively). Significant differences were identified in the proportion of CD4+ T cells between disease subtypes (ccRCC 14.1%, pRCC 7.0%, p<0.01). Further, the pRCC cohort had significantly more PanCK+ tumor cells than did the ccRCC cohort (24.3% vs 9.5%, respectively, p<0.01). There were no significant differences in macrophage composition (CD68+) between cohorts. Our results demonstrated a significant correlation between the CyTOF and TCGA analyses, specifically validating that ccRCC patients exhibit higher levels of CD4+ T cells (ccRCC 17.60%, pRCC 15.7%, p<0.01) and CD8+ T cells (ccRCC 17.83%, pRCC 11.15%, p<0.01). The limitation of our CyTOF analysis was the large proportion of cells that were deemed non-characterizable. Our findings emphasize the need to investigate the TME in distinct RCC histological subtypes. We observed a more immune infiltrative phenotype in the TME of the ccRCC cohort than in the pRCC cohort, where a tumor-rich phenotype was noted. As practical predictive biomarkers remain elusive across all subtypes of RCC, further studies are warranted to analyze the biomarker potential of such TME classifications.

A Phase I study of Milademetan (DS3032b) in combination with low dose cytarabine with or without venetoclax in acute myeloid leukemia: Clinical safety, efficacy, and correlative analysis

In TP53 wild-type acute myeloid leukemia (AML), inhibition of MDM2 can enhance p53 protein expression and potentiate leukemic cell apoptosis. MDM2 inhibitor (MDM2i) monotherapy in AML has shown modest responses in clinical trials but combining options of MDM2i with other potent AML-directed agents like cytarabine and venetoclax could improve its efficacy. We conducted a phase I clinical trial (NCT03634228) to study the safety and efficacy of milademetan (an MDM2i) with low-dose cytarabine (LDAC)±venetoclax in adult patients with relapsed refractory (R/R) or newly diagnosed (ND; unfit) TP53 wild-type AML and performed comprehensive CyTOF analyses to interrogate multiple signaling pathways, the p53-MDM2 axis and the interplay between pro/anti-apoptotic molecules to identify factors that determine response and resistance to therapy. Sixteen patients (14 R/R, 2 N/D treated secondary AML) at a median age of 70 years (range, 23–80 years) were treated in this trial. Two patients (13%) achieved an overall response (complete remission with incomplete hematological recovery). Median cycles on trial were 1 (range 1–7) and at a median follow-up of 11 months, no patients remained on active therapy. Gastrointestinal toxicity was significant and dose-limiting (50% of patients ≥ grade 3). Single-cell proteomic analysis of the leukemia compartment revealed therapy-induced proteomic alterations and potential mechanisms of adaptive response to the MDM2i combination. The response was associated with immune cell abundance and induced the proteomic profiles of leukemia cells to disrupt survival pathways and significantly reduced MCL1 and YTHDF2 to potentiate leukemic cell death. The combination of milademetan, LDAC±venetoclax led to only modest responses with recognizable gastrointestinal toxicity. Treatment-induced reduction of MCL1 and YTHDF2 in an immune-rich milieu correlate with treatment response.

Abstract 4600: Phenotypic &amp; functional diversity of tumor associated neutrophils in murine breast tumor models

Abstract Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNA-Seq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. We performed scRNA-Seq and CyTOF analyses of the myeloid cell populations of tumors and spleens from metastatic 4T1 and non-invasive EMT6 tumor-bearing mice. Tumors and spleens from 4T1 tumor-bearing mice exhibited a marked expansion of myeloid cell subsets that are characterized by the expression of immunosuppressive as well as progenitor markers. On the contrary, indicated tissues from EMT6 mice were enriched in NK cells, T and B lymphocytes and they were lacking immunosuppressive myeloid cell subsets. Furthermore, we identified a distinct differentiation pattern of immature myeloid cell subsets from neutrophil progenitors (NP) in 4T1 tumor-bearing mice. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Hasan Korkaya, Elayne Benson, Fulya Koksalar Alkan, Justin Wilson, Tulshi Patel, Hilmi K. Alkan, Virginia McEvoy, Nika Shekastehband, Nate Francois, Huidong Shi, Catherine C. Hedrick. Phenotypic &amp; functional diversity of tumor associated neutrophils in murine breast tumor models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4600.

Abstract P2-21-09: Detailed analyses of pro-metastatic and anti-metastatic microenvironments of murine breast tumors in syngeneic mice

Abstract Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNA-Seq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. We performed scRNA-Seq and CyTOF analyses of the myeloid cell populations of tumors and spleens from metastatic 4T1 and non-invasive EMT6 tumor-bearing mice. Tumors and spleens from 4T1 tumor-bearing mice exhibited a marked expansion of myeloid cell subsets that are characterized by the expression of immunosuppressive as well as progenitor markers. On the contrary, indicated tissues from EMT6 mice were enriched in NK cells, T and B lymphocytes and they were lacking immunosuppressive myeloid cell subsets. Furthermore, we identified a distinct differentiation pattern of immature myeloid cell subsets from neutrophil progenitors (NP) in 4T1 tumor-bearing mice. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Fulya Alkan, Justin D. Wilson, Nika Shekastehband, Catherine HEDRICK, Alicia Arnold, Roni Bollag, Huidong Shi, Hasan Korkaya. Detailed analyses of pro-metastatic and anti-metastatic microenvironments of murine breast tumors in syngeneic mice [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-09.

Epigenetic Signatures Discriminate Patients With Primary Sclerosing Cholangitis and Ulcerative Colitis From Patients With Ulcerative Colitis.

BackgroundPrimary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease affecting the intra- and extrahepatic bile ducts, and is strongly associated with ulcerative colitis (UC). In this study, we explored the peripheral blood DNA methylome and its immune cell composition in patients with PSC-UC, UC, and healthy controls (HC) with the aim to develop a predictive assay in distinguishing patients with PSC-UC from those with UC alone.MethodsThe peripheral blood DNA methylome of male patients with PSC and concomitant UC, UC and HCs was profiled using the Illumina HumanMethylation Infinium EPIC BeadChip (850K) array. Differentially methylated CpG position (DMP) and region (DMR) analyses were performed alongside gradient boosting classification analyses to discern PSC-UC from UC patients. As observed differences in the DNA methylome could be the result of differences in cellular populations, we additionally employed mass cytometry (CyTOF) to characterize the immune cell compositions.ResultsGenome wide methylation analysis did not reveal large differences between PSC-UC and UC patients nor HCs. Nonetheless, using gradient boosting we were capable of discerning PSC-UC from UC with an area under the receiver operator curve (AUROC) of 0.80. Four CpG sites annotated to the NINJ2 gene were found to strongly contribute to the predictive performance. While CyTOF analyses corroborated the largely similar blood cell composition among patients with PSC-UC, UC and HC, a higher abundance of myeloid cells was observed in UC compared to PSC-UC patients.ConclusionDNA methylation enables discerning PSC-UC from UC patients, with a potential for biomarker development.

Abstract P2-14-14: Durvalumab and tremelimumab before surgery in patients with hormone receptor positive, HER2 negative stage II-III breast cancer

Abstract Background: The checkpoint inhibitors atezolizumab (anti-PD-L1) and pembrolizumab (anti-PD-1) are FDA approved for the treatment of patients with PD-L1 positive, metastatic triple negative breast cancer in combination with chemotherapy. The response rates to checkpoint blockade in hormone receptor positive, HER2 negative metastatic breast cancer have been less encouraging (RR 12% for pembrolizumab in KEYNOTE-028 and RR 2.8% for avelumab in the phase 1b trial JAVELIN). We conducted a feasibility trial and enrolled patients with Stage II-III hormone receptor positive, HER2 negative breast cancer to treatment with 2 cycles of durvalumab (anti-PD-L1) plus tremelimumab (anti-CTLA-4) prior to standard neoadjuvant chemotherapy and breast surgery.Methods: Eligible patients were treated with durvalumab at a dose of 1500 mg and tremelimumab at a dose of 75 mg, both administered intravenously, on days 1 and 28. Pre- and post-treatment tumor biopsies and blood samples were available for 5 out of 8 patients and were analyzed by CyTOF, IHC, and NanoString. Patients then received standard neoadjuvant chemotherapy prior to breast surgery. The target enrollment was 20 patients. Results: After 8 patients were enrolled and treated on protocol, the trial was stopped early due to toxicity, slow accrual, and delays in patients receiving standard neoadjuvant chemotherapy. The median age of the patients was 55 (range 39-66) and all 8 patients were women. All patients had ER/PR positive, HER2 negative invasive ductal carcinoma (2 with lobular features, 1 with focal mucinous features) and all patients had clinical Stage II disease. Five patients received both cycles of durvalumab/tremelimumab and the remaining 3 patients only received the first cycle. One patient discontinued therapy out of concern for progression, though repeat breast and lymph node biopsies were benign and she went on to have a pathologic complete response after standard neoadjuvant chemotherapy. The remaining 2 patients discontinued treatment after the first cycle due to Grade 3 colitis (Patient #1) and Grade 3 thyroiditis/adrenal insufficiency (Patient #8). Both patients required treatment with steroids and experienced delays in receiving standard therapy due to these adverse events (AEs). The other AEs reported were all Grade 1 or 2. A post-durvalumab/tremelimumab breast ultrasound was performed in 7 of 8 patients and the percentage change in volume of each patient’s primary breast mass was: -55%, +104% (biopsy benign), +65%, -30%, +90%, -8%, and -53%. Patient #1 (colitis) had chemotherapy administered adjuvantly and Patient #8 (thyroiditis, adrenal insufficiency) declined chemotherapy. Only 1 of 8 patients had a pathologic complete response at the time of surgery. IHC, CYTOF, and gene expression analyses showed an increase in immune cell subsets in tumor stroma post neoadjuvant immunotherapy. Conclusions: We conducted a feasibility trial of neoadjuvant durvalumab plus tremelimumab administered prior to standard neoadjuvant chemotherapy in patients with hormone receptor positive/HER2 negative Stage II or III breast cancer. The trial was stopped early after 2 of 8 patients experienced Grade 3 immune-related adverse events (colitis in 1 patient and thyroiditis/adrenal insufficiency in another patient). Citation Format: Haven R. Garber, Sreyashi Basu, Sonali Jindal, Akshara Singareeka Raghavendra, Lumarie Santiago, Beatriz E. Adrada, Padmanee Sharma, James P. Allison, Jennifer Litton. Durvalumab and tremelimumab before surgery in patients with hormone receptor positive, HER2 negative stage II-III breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-14-14.

The Polygenic Map of Keloid Fibroblasts Reveals Fibrosis-Associated Gene Alterations in Inflammation and Immune Responses.

Due to many inconsistencies in differentially expressed genes (DEGs) related to genomic expression changes during keloid formation and a lack of satisfactory prevention and treatment methods for this disease, the critical biomarkers related to inflammation and the immune response affecting keloid formation should be systematically clarified. Normal skin/keloid scar tissue-derived fibroblast genome expression data sets were obtained from the Gene Expression Omnibus (GEO) and ArrayExpress databases. Hub genes have a high degree of connectivity and gene function aggregation in the integration network. The hub DEGs were screened by gene-related protein–protein interactions (PPIs), and their biological processes and signaling pathways were annotated to identify critical biomarkers. Finally, eighty-one hub DEGs were selected for further analysis, and some noteworthy signaling pathways and genes were found to be closely related to keloid fibrosis. For example, IL17RA is involved in IL-17 signal transduction, TIMP2 and MMP14 activate extracellular matrix metalloproteinases, and TNC, ITGB2, and ITGA4 interact with cell surface integrins. Furthermore, changes in local immune cell activity in keloid tissue were detected by DEG expression, immune cell infiltration, and mass CyTOF analyses. The results showed that CD4+ T cells, CD8+ T cells and NK cells were abnormal in keloid tissue compared with normal skin tissue. These findings not only support the key roles of fibrosis-related pathways, immune cells and critical genes in the pathogenesis of keloids but also expand our understanding of targets that may be useful for the treatment of fibrotic diseases.

Systemic high-dose dexamethasone treatment may modulate the efficacy of intratumoral viral oncolytic immunotherapy in glioblastoma models

BackgroundIntratumoral viral oncolytic immunotherapy is a promising new approach for the treatment of a variety of solid cancers. CAN-2409 is a replication-deficient adenovirus that delivers herpes simplex virus thymidine kinase...

Analysis of the Single-Cell Heterogeneity of Adenocarcinoma Cell Lines and the Investigation of Intratumor Heterogeneity Reveals the Expression of Transmembrane Protein 45A (TMEM45A) in Lung Adenocarcinoma Cancer Patients.

Simple SummaryNon-small cell lung cancer (NSCLC) is one of the main causes of cancer-related deaths worldwide. Intratumoral heterogeneity (ITH) is responsible for the majority of difficulties encountered in the treatment of lung-cancer patients. Therefore, the heterogeneity of NSCLC cell lines and primary lung adenocarcinoma was investigated by single-cell mass cytometry (CyTOF). Human NSCLC adenocarcinoma cells A549, H1975, and H1650 were studied at single-cell resolution for the expression pattern of 13 markers: GLUT1, MCT4, CA9, TMEM45A, CD66, CD274, CD24, CD326, pan-keratin, TRA-1-60, galectin-3, galectin-1, and EGFR. The intra- and inter-cell-line heterogeneity of A549, H1975, and H1650 cells were demonstrated through hypoxic modeling. Additionally, human primary lung adenocarcinoma, and non-involved healthy lung tissue were homogenized to prepare a single-cell suspension for CyTOF analysis. The single-cell heterogeneity was confirmed using unsupervised viSNE and FlowSOM analysis. Our results also show, for the first time, that TMEM45A is expressed in lung adenocarcinoma. Intratumoral heterogeneity (ITH) is responsible for the majority of difficulties encountered in the treatment of lung-cancer patients. Therefore, the heterogeneity of NSCLC cell lines and primary lung adenocarcinoma was investigated by single-cell mass cytometry (CyTOF). First, we studied the single-cell heterogeneity of frequent NSCLC adenocarcinoma models, such as A549, H1975, and H1650. The intra- and inter-cell-line single-cell heterogeneity is represented in the expression patterns of 13 markers—namely GLUT1, MCT4, CA9, TMEM45A, CD66, CD274 (PD-L1), CD24, CD326 (EpCAM), pan-keratin, TRA-1-60, galectin-3, galectin-1, and EGFR. The qRT-PCR and CyTOF analyses revealed that a hypoxic microenvironment and altered metabolism may influence cell-line heterogeneity. Additionally, human primary lung adenocarcinoma and non-involved healthy lung tissue biopsies were homogenized to prepare a single-cell suspension for CyTOF analysis. The CyTOF showed the ITH of human primary lung adenocarcinoma for 14 markers; particularly, the higher expressions of GLUT1, MCT4, CA9, TMEM45A, and CD66 were associated with the lung-tumor tissue. Our single-cell results are the first to demonstrate TMEM45A expression in human lung adenocarcinoma, which was verified by immunohistochemistry.

IL-7 expands lymphocyte populations and enhances immune responses to sipuleucel-T in patients with metastatic castration-resistant prostate cancer (mCRPC)

BackgroundSipuleucel-T (sip-T) is a Food and Drug Administration (FDA)-approved autologous cellular immunotherapy for metastatic castration-resistant prostate cancer (mCRPC). We hypothesized that combining sip-T with interleukin (IL)-7, a homeostatic cytokine that...

Ruxolitinib or Dasatinib in Combination with Chemotherapy for Patients with Relapsed/Refractory Philadelphia (Ph)-like Acute Lymphoblastic Leukemia: A Phase I-II Trial

Ruxolitinib or Dasatinib in Combination with Chemotherapy for Patients with Relapsed/Refractory Philadelphia (Ph)-like Acute Lymphoblastic Leukemia: A Phase I-II Trial

Neonatal Osteomacs and Bone Marrow Macrophages Differ in Phenotypic Marker Expression and Function.

Osteomacs (OM) are specialized bone-resident macrophages that are a component of the hematopoietic niche and support bone formation. Also located in the niche are a second subset of macrophages, namely bone marrow-derived macrophages (BM Mφ). We previously reported that a subpopulation of OM co-express both CD166 and CSF1R, the receptor for macrophage colony-stimulating factor (MCSF), and that OM form more bone-resorbing osteoclasts than BM Mφ. Reported here are single-cell quantitative RT-PCR (qRT-PCR), mass cytometry (CyTOF), and marker-specific functional studies that further identify differences between OM and BM Mφ from neonatal C57Bl/6 mice. Although OM express higher levels of CSF1R and MCSF, they do not respond to MCSF-induced proliferation, in contrast to BM Mφ. Moreover, receptor activator of NF-κB ligand (RANKL), without the addition of MCSF, was sufficient to induce osteoclast formation in OM but not BM Mφ cultures. OM express higher levels of CD166 than BM Mφ, and we found that osteoclast formation by CD166-/- OM was reduced compared with wild-type (WT) OM, whereas CD166-/- BM Mφ showed enhanced osteoclast formation. CD110/c-Mpl, the receptor for thrombopoietin (TPO), was also higher in OM, but TPO did not alter OM-derived osteoclast formation, whereas TPO stimulated BM Mφ osteoclast formation. CyTOF analyses demonstrated OM uniquely co-express CD86 and CD206, markers of M1 and M2 polarized macrophages, respectively. OM performed equivalent phagocytosis in response to LPS or IL-4/IL-10, which induce polarization to M1 and M2 subtypes, respectively, whereas BM Mφ were less competent at phagocytosis when polarized to the M2 subtype. Moreover, in contrast to BM Mφ, LPS treatment of OM led to the upregulation of CD80, an M1 marker, as well as IL-10 and IL-6, known anti-inflammatory cytokines. Overall, these data reveal that OM and BM Mφ are distinct subgroups of macrophages, whose phenotypic and functional differences in proliferation, phagocytosis, and osteoclast formation may contribute physiological specificity during health and disease. © 2021 American Society for Bone and Mineral Research (ASBMR).

The use and limitations of single-cell mass cytometry for studying human microglia function.

Microglia, the resident innate immune cells of the central nervous system (CNS), play an important role in brain development and homoeostasis, as well as in neuroinflammatory, neurodegenerative and psychiatric diseases. Studies in animal models have been used to determine the origin and development of microglia, and how these cells alter their transcriptional and phenotypic signatures during CNS pathology. However, little is known about their human counterparts. Recent studies in human brain samples have harnessed the power of multiplexed single‐cell technologies such as single‐cell RNA sequencing (scRNA‐seq) and mass cytometry (cytometry by time‐of‐flight [CyTOF]) to provide a comprehensive molecular view of human microglia in healthy and diseased brains. CyTOF is a powerful tool to study high‐dimensional protein expression of human microglia (huMG) at the single‐cell level. This technology widens the possibilities of high‐throughput quantification (of over 60 targeted molecules) at a single‐cell resolution. CyTOF can be combined with scRNA‐seq for comprehensive analysis, as it allows single‐cell analysis of post‐translational modifications of proteins, which provides insights into cell signalling dynamics in targeted cells. In addition, imaging mass cytometry (IMC) has recently become commercially available, and will be useful for analysing multiple cell types in human brain sections. IMC leverages mass spectrometry to acquire spatial data of cell–cell interactions on tissue sections, using (theoretically) over 40 markers at the same time. In this review, we summarise recent studies of huMG using CyTOF and IMC analyses. The uses and limitations as well as future directions of these technologies are discussed.

Immune biomarkers link air pollution exposure to blood pressure in adolescents

BackgroundChildhood exposure to air pollution contributes to cardiovascular disease in adulthood. Immune and oxidative stress disturbances might mediate the effects of air pollution on the cardiovascular system, but the underlying mechanisms are poorly understood in adolescents. Therefore, we aimed to identify immune biomarkers linking air pollution exposure and blood pressure levels in adolescents.MethodsWe randomly recruited 100 adolescents (mean age, 16 years) from Fresno, California. Using central-site data, spatial-temporal modeling, and distance weighting exposures to the participant’s home, we estimated average pollutant levels [particulate matter (PM), polyaromatic hydrocarbons (PAH), ozone (O3), carbon monoxide (CO) and nitrogen oxides (NOx)]. We collected blood samples and vital signs on health visits. Using proteomic platforms, we quantitated markers of inflammation, oxidative stress, coagulation, and endothelial function. Immune cellular characterization was performed via mass cytometry (CyTOF). We investigated associations between pollutant levels, cytokines, immune cell types, and blood pressure (BP) using partial least squares (PLS) and linear regression, while adjusting for important confounders.ResultsUsing PLS, biomarkers explaining most of the variance in air pollution exposure included markers of oxidative stress (GDF-15 and myeloperoxidase), acute inflammation (C-reactive protein), hemostasis (ADAMTS, D-dimer) and immune cell types such as monocytes. Most of these biomarkers were independently associated with the air pollution levels in fully adjusted regression models. In CyTOF analyses, monocytes were enriched in participants with the highest versus the lowest PM2.5 exposure. In both PLS and linear regression, diastolic BP was independently associated with PM2.5, NO, NO2, CO and PAH456 pollution levels (P ≤ 0.009). Moreover, monocyte levels were independently related to both air pollution and diastolic BP levels (P ≤ 0.010). In in vitro cell assays, plasma of participants with high PM2.5 exposure induced endothelial dysfunction as evaluated by eNOS and ICAM-1 expression and tube formation.ConclusionsFor the first time in adolescents, we found that ambient air pollution levels were associated with oxidative stress, acute inflammation, altered hemostasis, endothelial dysfunction, monocyte enrichment and diastolic blood pressure. Our findings provide new insights on pollution-related immunological and cardiovascular disturbances and advocate preventative measures of air pollution exposure.