828 Quantifying perivascular immune cells in the stroma of human triple negative breast tumors using deep learning spatial analytics

BackgroundThe spatial organization and density of the immune infiltrate in the tumor microenvironment, referred to as immune contexture, can yield information relevant to prognosis and prediction of response to immunotherapy in cancer. Specifically, a distinct subset of tumor-associated macrophages (TAMs) accumulate around blood vessels where they stimulate tumor angiogenesis and limit tumor responses to frontline anti-cancer therapies like irradiation and chemotherapy.MethodsIn this study we leveraged the NeoGenomics MultiOmyx Multiplex Immunofluorescence platform alongside artificial intelligence (AI) based quantitative image analysis. This AI platform was ultimately used to investigate the distribution of perivascular (PV) TAMs, CD4+ and CD8+ T cells, and CD4+FOXP3+ regulatory T cells (Tregs) of 40 human triple negative breast carcinomas (TNBCs), and how this changed following neoadjuvant chemotherapy. During the multiplexing phase, eleven rounds of paired antibody staining were performed in sequence on tumor sections. After each round of staining, high resolution images were captured for regions of interests (ROIs) selected by a pathologist. We used AI models to segment and classify cells for each biomarker and classify regions as tumor cell islands (TCIs) or stroma. First, each nucleus was segmented out using a convolutional neural network combined with watershed thresholding on the DAPI (diamidino-2-phenylindole) immunofluorescent image. From the resulting nuclear segmentation mask, a pixel dilation on cells classified as non-tumor was employed to generate a cellular segmentation mask. A list of neighbours within a specified distance for each cell was generated by radially expanding from the cellular segmentation mask. Finally, cell neighbour information was combined with the marker expression information to quantify the cell clusters of interest.ResultsWe discovered that in the PV areas, up to 30% of PD1-LAG3-CD3+CD8+ T cells formed direct contact with both CD163+TIM3+ TAMs and CD4+FOXP3+ Tregs. Furthermore, these immune cell triads preferentially accumulated in the PV stroma regions. It is likely that close interaction with immunosuppressive TAMs and Tregs would supress the function of T cells as they enter the PV region to reach the TCIs.ConclusionsUsing an advanced analytics platform, we invented a new method to quantify clusters of cells within various regions of a tumor section. Using this platform, we detected specific immune cell triads, the frequency and location of which could correlate with the efficacy of T-cell based immunotherapies in TNBC. These analyses will enable further investigation of numerous complex cell interactions in TMEs.

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602 STING agonist-based treatment promotes vascular normalization and tertiary lymphoid structure formation in the therapeutic melanoma microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0602 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A637-A637

Author(s):

Manoj Chelvanambi ◽

Ronald Fecek ◽

Jennifer Taylor ◽

Walter Storkus

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Cell ◽

Ex Vivo ◽

In Vitro Assays ◽

Type I ◽

Vascular Normalization ◽

S 100 ◽

Transcriptional Changes

BackgroundThe degree of immune infiltration in tumors, especially CD8+ T cells, greatly impacts patient disease course and response to interventional immunotherapy. Hence, enhancement of TIL prevalence is a preferred clinical endpoint, one that may be achieved via administration of agents that normalize the tumor vasculature (VN) leading to improved immune cell recruitment and/or that induce the development of local tertiary lymphoid structures (TLS) within the tumor microenvironment (TME).MethodsLow-dose STING agonist ADU S-100 (5 μg/mouse) was delivered intratumorally to established s.c. B16.F10 melanomas on days 10, 14 and 17 post-tumor inoculation under an IACUC-approved protocol. Treated and control, untreated tumors were isolated at various time points to assess transcriptional changes associated with VN and TLS formation via qPCR, with corollary immune cell composition changes determined using flow cytometry and immunofluorescence microscopy. In vitro assays were performed on CD11c+ BMDCs treated with 2.5 μg/mL ADU S-100 (vs PBS control) and associated transcriptional changes analyzed via qPCR or profiled using DNA microarrays. For TCRβ-CDR3 analyses, CDR3 was sequenced from gDNA isolated from enzymatically digested tumors and splenocytes.ResultsWe report that activation of STING within the TME leads to slowed melanoma growth in association with increased production of angiostatic factors including Tnfsf15 (Vegi), Cxcl10 and Angpt1, and TLS inducing factors including Ccl19, Ccl21, Lta, Ltb and Tnfsf14 (Light). Therapeutic responses from intratumoral STING activation were characterized by increased vascular normalization (VN), enhanced tumor infiltration by CD8+ T cells and CD11c+ DCs and local TLS neo-genesis, all of which were dependent on host expression of STING. Consistent with a central role for DC in TLS formation, ex vivo ADU S-100-activated mCD11c+ DCs also exhibited upregulated expression of TLS promoting factors including lymphotoxin-α (LTA), IL-36, inflammatory chemokines and type I interferons. TLS formation was associated with the development of a therapeutic TIL TCR repertoire enriched in T cell clonotypes uniquely detected within the tumor but not the peripheral circulation in support or local T cell cross-priming within the TME.ConclusionsOur data support the premise that i.t. delivery of STING agonist promotes a pro-inflammatory TME in support of VN and TLS formation, leading to the local expansion of unique TIL repertoire in association with superior anti-melanoma efficacy.

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TAMI-07. THE IMMUNE MICROENVIRONMENT IN LOWER GRADE GLIOMAS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.896 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii214-ii214

Author(s):

Anupam Kumar ◽

Katharine Chen ◽

Claudia Petritsch ◽

Theodore Nicolaides ◽

Mariarita Santi-Vicini ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Cd8 T Cells ◽

Molecular Mechanisms ◽

T Regulatory Cells ◽

Immune Cell ◽

T Cell Response ◽

Cytotoxic T Cell ◽

Lower Grade ◽

Functional Relationships

Abstract The determinants of the tumor-associated immune response in brain tumors are poorly understood. Using tumor samples from two molecularly distinct subtypes of lower grade glioma, MAPK-driven glioma with biallelic inactivation of CDKN2A (n=30) and IDH-mutant, 1p/19q-intact astrocytoma (n=29), we demonstrate qualitative and quantitative differences in the tumor-associated immune response and we investigate the molecular mechanisms involved. Histologically the MAPK-driven gliomas were comprised of pleomorphic xanthoastrocytoma (PXA) (n=11) and anaplastic PXA (n=19). Seven patients had paired samples from two sequential surgeries. Immune cell populations and their activity were determined by quantitative multiplex immunostaining and Digital Spatial Profiling and gene expression was analyzed by Nanostring. Functional studies were performed using established cell lines and two new patient-derived lines from MAPK-driven LGGs. MAPK-driven tumors exhibited an increased number of CD8+ T cells and tumor-associated microglial/macrophage (TAMs), including CD163+ TAMs, as compared to IDH-mutant astrocytoma. In contrast, IDH-mutant tumors had increased FOXP3+ immunosuppressive T regulatory cells. Transcriptional and protein level analyses in MAPK-driven tumors suggested an active cytotoxic T cell response with robust expression of granzyme B, present on 27% of CD8+ T cells, increased MHC class I expression, and altered cytokine profiles. Interestingly, MAPK-driven tumors also had increased expression of immunosuppressive molecules, including CXCR4, PD-L1, and VEGFA. Expression differences for cell surface and secreted proteins were confirmed in patient-derived tumor lines and functional relationships between altered chemokine expression and immune cell infiltration was investigated. Our data provide novel insights into the immune contexture of MAPK driven LGGs and suggest MAPK driven gliomas with biallelic inactivation of CDKN2A may be particularly vulnerable to immunotherapeutic modulation

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Tumor morphology and location associate with immune cell composition in pleomorphic sarcoma

Cancer Immunology Immunotherapy ◽

10.1007/s00262-021-02935-2 ◽

2021 ◽

Author(s):

Rosanna L. Wustrack ◽

Evans Shao ◽

Joey Sheridan ◽

Melissa Zimel ◽

Soo-Jin Cho ◽

...

Keyword(s):

T Cells ◽

Tumor Microenvironment ◽

Cd8 T Cells ◽

Tumor Size ◽

Immune Cell ◽

Human Cancer ◽

Soft Tissue Sarcomas ◽

Inverse Correlation ◽

Anatomical Location ◽

Pleomorphic Sarcoma

Abstract Background Soft-tissue sarcomas (STS) are a rare group of mesenchymal malignancies that account for approximately 1% of adult human cancer. Undifferentiated pleomorphic sarcoma (UPS) is one of the most common subtypes of adult STS. Clinical stratification of UPS patients has not evolved for decades and continues to rely on tumor-centric metrics including tumor size and depth. Our understanding of how the tumor microenvironment correlates to these clinicopathologic parameters remains limited. Methods Here, we performed single-cell flow cytometric immune-based profiling of 15 freshly resected UPS tumors and integrated this analysis with clinical, histopathologic, and outcomes data using both a prospective and retrospective cohort of UPS patients. Results We uncovered a correlation between physiologic and anatomic properties of UPS tumors and the composition of immune cells in the tumor microenvironment. Specifically, we identified an inverse correlation between tumor-infiltrating CD8 + T cells and UPS tumor size; and a positive correlation between tumor-infiltrating CD8 + T cells and overall survival. Moreover, we demonstrate an association between anatomical location (deep or superficial) and frequency of CD4 + PD1hi infiltrating T cells in UPS tumors. Conclusions Our study provides an immune-based analysis of the tumor microenvironment in UPS patients and describes the different composition of tumor infiltrating lymphocytes based on size and tumor depth.

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Immune classification of clear cell renal cell carcinoma

Scientific Reports ◽

10.1038/s41598-021-83767-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sumeyye Su ◽

Shaya Akbarinejad ◽

Leili Shahriyari

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Renal Cell ◽

Cd4 T Cells ◽

Immune Cell ◽

Clear Cell ◽

P Value ◽

Cell Type ◽

Primary Tumors ◽

Immune Cell Type

AbstractSince the outcome of treatments, particularly immunotherapeutic interventions, depends on the tumor immune micro-environment (TIM), several experimental and computational tools such as flow cytometry, immunohistochemistry, and digital cytometry have been developed and utilized to classify TIM variations. In this project, we identify immune pattern of clear cell renal cell carcinomas (ccRCC) by estimating the percentage of each immune cell type in 526 renal tumors using the new powerful technique of digital cytometry. The results, which are in agreement with the results of a large-scale mass cytometry analysis, show that the most frequent immune cell types in ccRCC tumors are CD8+ T-cells, macrophages, and CD4+ T-cells. Saliently, unsupervised clustering of ccRCC primary tumors based on their relative number of immune cells indicates the existence of four distinct groups of ccRCC tumors. Tumors in the first group consist of approximately the same numbers of macrophages and CD8+ T-cells and and a slightly smaller number of CD4+ T cells than CD8+ T cells, while tumors in the second group have a significantly high number of macrophages compared to any other immune cell type (P-value $$<0.01$$ < 0.01 ). The third group of ccRCC tumors have a significantly higher number of CD8+ T-cells than any other immune cell type (P-value $$<0.01$$ < 0.01 ), while tumors in the group 4 have approximately the same numbers of macrophages and CD4+ T-cells and a significantly smaller number of CD8+ T-cells than CD4+ T-cells (P-value $$<0.01$$ < 0.01 ). Moreover, there is a high positive correlation between the expression levels of IFNG and PDCD1 and the percentage of CD8+ T-cells, and higher stage and grade of tumors have a substantially higher percentage of CD8+ T-cells. Furthermore, the primary tumors of patients, who are tumor free at the last time of follow up, have a significantly higher percentage of mast cells (P-value $$<0.01$$ < 0.01 ) compared to the patients with tumors for all groups of tumors except group 3.

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Suppression of tumor-associated neutrophils by lorlatinib attenuates pancreatic cancer growth and improves treatment with immune checkpoint blockade

Nature Communications ◽

10.1038/s41467-021-23731-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sebastian R. Nielsen ◽

Jan E. Strøbech ◽

Edward R. Horton ◽

Rene Jackstadt ◽

Anu Laitala ◽

...

Keyword(s):

Cd8 T Cells ◽

Immune Cell ◽

Immune Checkpoint Blockade ◽

Ductal Adenocarcinoma ◽

Cancer Growth ◽

Cancer Therapies ◽

Cell Type ◽

Clinical Prognosis ◽

Tumor Associated Neutrophils ◽

Poor Clinical Prognosis

AbstractPancreatic ductal adenocarcinoma (PDAC) patients have a 5-year survival rate of only 8% largely due to late diagnosis and insufficient therapeutic options. Neutrophils are among the most abundant immune cell type within the PDAC tumor microenvironment (TME), and are associated with a poor clinical prognosis. However, despite recent advances in understanding neutrophil biology in cancer, therapies targeting tumor-associated neutrophils are lacking. Here, we demonstrate, using pre-clinical mouse models of PDAC, that lorlatinib attenuates PDAC progression by suppressing neutrophil development and mobilization, and by modulating tumor-promoting neutrophil functions within the TME. When combined, lorlatinib also improves the response to anti-PD-1 blockade resulting in more activated CD8 + T cells in PDAC tumors. In summary, this study identifies an effect of lorlatinib in modulating tumor-associated neutrophils, and demonstrates the potential of lorlatinib to treat PDAC.

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Peripheral Levels of EPCs, T Cells, and Macrophages’ as Possible Predictive Biomarkers of Acute Myocardial Infarction

10.21203/rs.3.rs-107073/v1 ◽

2020 ◽

Author(s):

Amankeldi Salybekov ◽

Katsuaki Sakai ◽

Makoto Natsumeda ◽

Kosit Vorateera ◽

Shuzo Kobayashi ◽

...

Keyword(s):

Myocardial Infarction ◽

Acute Myocardial Infarction ◽

T Cells ◽

Cd8 T Cells ◽

Immune Cell ◽

Temporal Dynamics ◽

Predictive Biomarkers ◽

Mortality And Morbidity ◽

Patients With Diabetes ◽

Functionally Impaired

Abstract Acute myocardial infarction (AMI), with a very relevant global disease burden, remains the major mortality and morbidity cause among all cardiovascular diseases. Patient prognosis is strictly dependent on early diagnosis and the adoption of adequate interventions. AMI diagnosis requires constant optimization, particularly considering the individuals at higher risk (or more vulnerable to worse outcomes) such as patients with diabetes mellitus and atherosclerosis. Herein, we investigated the levels of peripheral blood EPCs and immune cell-subsets from myeloid and lymphoid lineages, as well as their temporal dynamics, in the quest for new prognostic biomarkers of AMI. We collected blood from 18 hospitalized patients (days 3 and 7 after AMI onset) and 16 healthy volunteers, and resolved their circulating PBMC populations via flow cytometry. Overall, our data demonstrate a significant decrease in peripheral EPCs and CD8+ T cells, three days following an AMI. EPCs appear to be functionally impaired in AMI patients, and their circulating numbers associate with cardiac vessel lesions. Furthermore, CD8+ T cells (and even M1-macrophages) in the periphery, in combination with the classical laboratory determinations, may serve as high accuracy biomarkers of AMI, potentially aiding to prevent worse AMI outcomes.

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GDF15 expression in metastatic colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.3_suppl.116 ◽

2021 ◽

Vol 39 (3_suppl) ◽

pp. 116-116

Author(s):

Priya Jayachandran ◽

Joanne Xiu ◽

Shivani Soni ◽

Richard M. Goldberg ◽

Benjamin Adam Weinberg ◽

...

Keyword(s):

Colon Cancer ◽

T Cells ◽

Nk Cells ◽

Chromatin Remodeling ◽

Cancer Progression ◽

Cd8 T Cells ◽

Immune Cell ◽

Checkpoint Inhibitors ◽

Metastatic Colon Cancer ◽

Genomic Alterations

116 Background: Cachexia affects many cancer patients. Growth differentiation factor-15 (GDF15) is a protein that regulates weight and the stress response of cells. The GDF15 gene encodes a ligand of TGF-beta that triggers cachexia and modulates the progression from tumorigenesis to metastasis. Inhibition of GDF15 with an antibody restored muscle mass and fat in animal models. Serum levels rise in proportion to the progression of colon cancer, predict outcome, and have been correlated with CEA. Methods: We retrospectively reviewed 7607 CRC tumors profiled by Caris Life Sciences (Phoenix, AZ) from 2019 to 2020. Profiling included whole transcriptome sequencing (RNA-Seq by NovoSeq). Tumor mutational burden, mismatch repair status, and pathway genomic alterations were evaluated. QuantiSEQ was used to assess immune cell infiltration in the tumor microenvironment. Results: GDF15 expression ranged from 0 to 593 transcripts per million (TPM) with median of 30 (IQR = 15.02). There was no association with age, sex, or primary tumor sidedness. MSI-H/dMMR tumors had higher GDF15 expression (median 37 vs 30, p = 0.0004); TMB > = 17 tumors was seen in 5.9% of bottom quartile (Q1) GDF15 expressors and 8.3% of top quartile (Q4). PDL1 IHC positivity was inversely correlated with GDF15 expression (7.1% in Q1 vs. 2.6% in Q4, p < 0.0001). Genomic alterations associated with higher GDF15 expression (Q4 vs Q1) included genes on TGF-B (SMAD2/4), PI3K (PIK3CA, MTOR), chromatin remodeling (ARID1A, KMT2C), DDR (ATM) and Wnt pathway (APC); those inversely associated included MYC CNA and TP53. Q1 tumors had higher CNA of ERBB2 and FGFR1. Relative neutrophils and NK cells in the TME increased from Q1 to Q4 (p < 0.001). There was a decrease in CD8+ T-cells and Treg cells from Q1 to Q4. Conclusions: GDF15 expression correlates with increased dMMR/MSI-H and TMB, but not with PDL1 expression. Mutations and activated pathways associated with GDF15 expression may explain increased cachexia with more aggressive disease. The association with chromatin remodeling may warrant therapies targeting histone modification and epigenetics. The increase in NK cells but decrease in CD8+ T cells in the TME with increasing GDF15 suggests approaches to treatment. Higher CD8+ lymphocyte counts correlate with PFS with immunotherapy. Anti-PD-L1 therapy reinvigorates the killing function of CD8+ T cells. The decrease in CD8+ T cells and PDL1 positivity with rising GDF15 suggests worse outcome and a lack of response to anti-PDL1 therapy. NK cell checkpoint inhibitors, CARs, and an anti-GFRAL antibody are now in clinical trials and might be utilized in high GDF15 cancers. GDF15 is emerging as a target in the treatment of obesity and cachexia and as a prognostic marker in oncology. Understanding its expression in metastatic colon cancer may reveal which patients could benefit from developing anti-GDF15 targeted therapies against cancer progression.

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Immunohistochemical phenotyping of T cells, granulocytes, and phagocytes in the muscle of cancer patients: association with radiologically defined muscle mass and gene expression

Skeletal Muscle ◽

10.1186/s13395-019-0209-y ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Ana Anoveros-Barrera ◽

Amritpal S. Bhullar ◽

Cynthia Stretch ◽

Abha R. Dunichand-Hoedl ◽

Karen J. B. Martins ◽

...

Keyword(s):

Skeletal Muscle ◽

T Cells ◽

Cancer Patients ◽

Muscle Mass ◽

Muscle Fiber ◽

Cd8 T Cells ◽

Immune Cell ◽

Cell Populations ◽

Cd4 Cells ◽

Gene Correlation

Abstract Background Inflammation is a recognized contributor to muscle wasting. Research in injury and myopathy suggests that interactions between the skeletal muscle and immune cells confer a pro-inflammatory environment that influences muscle loss through several mechanisms; however, this has not been explored in the cancer setting. This study investigated the local immune environment of the muscle by identifying the phenotype of immune cell populations in the muscle and their relationship to muscle mass in cancer patients. Methods Intraoperative muscle biopsies were collected from cancer patients (n = 30, 91% gastrointestinal malignancies). Muscle mass was assessed histologically (muscle fiber cross-sectional area, CSA; μm2) and radiologically (lumbar skeletal muscle index, SMI; cm2/m2 by computed tomography, CT). T cells (CD4 and CD8) and granulocytes/phagocytes (CD11b, CD14, and CD15) were assessed by immunohistochemistry. Microarray analysis was conducted in the muscle of a second cancer patient cohort. Results T cells (CD3+), granulocytes/phagocytes (CD11b+), and CD3−CD4+ cells were identified. Muscle fiber CSA (μm2) was positively correlated (Spearman’s r = > 0.45; p = < 0.05) with the total number of T cells, CD4, and CD8 T cells and granulocytes/phagocytes. In addition, patients with the smallest SMI exhibited fewer CD8 T cells within their muscle. Consistent with this, further exploration with gene correlation analyses suggests that the presence of CD8 T cells is negatively associated (Pearson’s r = ≥ 0.5; p = <0.0001) with key genes within muscle catabolic pathways for signaling (ACVR2B), ubiquitin proteasome (FOXO4, TRIM63, FBXO32, MUL1, UBC, UBB, UBE2L3), and apoptosis/autophagy (CASP8, BECN1, ATG13, SIVA1). Conclusion The skeletal muscle immune environment of cancer patients is comprised of immune cell populations from the adaptive and innate immunity. Correlations of T cells, granulocyte/phagocytes, and CD3−CD4+ cells with muscle mass measurements indicate a positive relationship between immune cell numbers and muscle mass status in cancer patients. Further exploration with gene correlation analyses suggests that the presence of CD8 T cells is negatively correlated with components of muscle catabolism.

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