Tumor Stromal Cells Research Articles

Tumor Purity Coexpressed Genes Related to Immune Microenvironment and Clinical Outcomes of Lung Adenocarcinoma.

Purpose Lung cancer tissue includes tumor tissue, stromal cells, immune cells, and epithelial cells. These nontumor cells dilute the tumor purity in lung cancer tissues. Tumor purity plays an essential role in the immune response to lung cancer. At present, the biological processes related to the purity of lung cancer tumors remains unclear. Methods We measured tumor purity in 486 lung carcinoma tissues from TCGA-LUAD FPKM by using the “estimate” R package. Lung carcinoma tumor mutation burden was calculated by analyzing TCGA single nucleotide polymorphism data. The immune cell proportion was also evacuated via the CIBERSORT method. Lung carcinoma samples with P < 0.05 were considered significant. Based on the tumor purity and lung carcinoma gene matrix, we performed weighted gene coexpression network analysis (WGCNA), and the tumor purity-related module was identified. Then, we analyzed the functions of the factors involved in the module. We screened the coexpressed factors related to clinical outcome and immunophenotype. Finally, expression levels of these factors were measured at tissue and single-cell levels. Results A lung cancer tumor purity correlated coexpression network was determined. Five coexpressed genes (CD4, CD53, EVI2B, PLEK, and SASH3) were identified as tumor purity coexpressed genes that negatively correlated with tumor purity. Because the factors in the coexpression network often participate in similar biological processes, we found that CD4, CD53, EVI2B, PLEK, and SASH3 were most related to positive regulation of cytokine production and interleukin−2 production through functional enrichment. In a clinical phenotype analysis, we found that these five factors can be used as independent prognostic risk factors. We found that these factors were significantly negatively correlated with tumor purity and positively correlated with the immune score in the immunophenotyping analysis. Using GSEA analysis, we found that the antigen processing and presentation pathway were related to the five tumor coexpressed genes mentioned above. SASH3 and CD53 were used to conduct a prognostic model based on the interaction analysis of the Support Vector Machine and the Least Absolute Shrinkage and Selection Operator. SASH3 was verified to be related to CD8A using a single-cell analysis. Conclusion Tumor purity-related coexpression factors in the tumor microenvironment have essential clinical, genomic, and biological significance in lung cancer. These coexpression factors (SASH3 and CD53) can be used to classify tumor purity phenotypes and to predict clinical outcomes.

Identification of key genes in the tumor microenvironment of lung adenocarcinoma.

The tumor microenvironment plays an important role in tumor development and progression, but the role of immune and stromal cells in this environment has not been sufficiently studied. In this study, we aimed to identify key genes associated with the microenvironment of lung adenocarcinoma (LUAD). Raw data for stromal and immune cells in malignant tumors were downloaded from The Cancer Genome Atlas (TCGA). These expression data were used to identify the differentially expressed genes (DEGs) in tissue samples of LUAD with high and low immune scores. A protein-protein interaction (PPI) network based on genes with significant differential expression was constructed. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to functionally annotate putative hub genes. These genes were assessed via Kaplan Meier analysis to determine their correlation with overall survival. In total, we identified 216 DEGs which were correlated with immune and stromal scores, including 30 hub genes which were identified based on the PPI network. Further analysis suggested that the expression levels of 10 of these genes were significantly correlated with overall survival of LUAD patients. These key hub genes included CCR2, CCR5, CD53, CYBB, HCK, IRF8, LCP2, PLEK, PTPRC, and TLR7. Moreover, the expression level of CCR2 was found to have strong prognostic value for LUAD patients. Additionally, high expression of CYBB was also correlated with better survival of patients with LUAD. The results of this study open several new avenues to explore in the treatment of LUAD.

Cancer-associated fibroblasts promote tumor progression by lncRNA-mediated RUNX2/GDF10 signaling in oral squamous cell carcinoma.

Cancer‐associated fibroblasts (CAF) are the most abundant stromal cells in tumor and exert a pro‐tumoral effect in cancer progression. Numerous evidence shows long non‐coding RNA (lncRNA) abnormally regulates gene expression in various cancers. However, little is known about the role of lncRNA in the interaction between CAF and cancer cells. Here, we first identify an uncharacterized lncRNA, LOC100506114, which is significantly upregulated in CAF and is involved in the functional transformation of normal fibroblasts (NF) and CAF. Expression of LOC100506114 enhances the expression of fibroblast activation protein alpha and α‐smooth muscle actin in NF and promotes malignant characteristics of NF and CAF in vivo and in vitro. The profile of gene co‐expression analysis shows that growth differentiation factor 10 (GDF10) is positively correlated with the expression of LOC100506114. CAF promote stromal fibroblast activation and the proliferation and migration of tumor cells by secreting GDF10. Our data demonstrate that lncRNA plays a critical role in the interplay of stromal fibroblasts and tumor cells in oral squamous cell carcinoma.

Exosomal linc-ROR mediates crosstalk between cancer cells and adipocytes to promote tumor growth in pancreatic cancer

Exosomes are emerging as important mediators of the crosstalk between tumor cells and stromal cells in the microenvironment. However, the underlying molecular mechanism of pancreatic cancer (PC)-derived exosomes in the progression of the tumor microenvironment (TME) and crosstalk with adipocytes has not been elucidated. Exosomes isolated from PC cell culture supernatant through ultracentrifugation were rich in long intergenic non-coding ROR (linc-ROR). After constructing PC cell lines with stable linc-ROR knockdown or overexpression via the transfection of short hairpin RNA (shRNA) and pLent-U6-GFP-Puro, direct and indirect coculture systems were established to simulate the interaction between adipocytes and PC cells. Next, the effects of conditioned medium collected from dedifferentiated adipocytes on PC cell proliferation, motility, metastasis, and epithelial-mesenchymal transition (EMT) were evaluated by western blot analysis, colony forming, real-time cell analysis (RTCA), 5-ethynyl-2′-deoxyuridine (EdU), immunofluorescence (IF), Transwell, and wound-healing assays in vitro. Xenograft models were employed to identify whether conditioned medium loaded with interleukin-1β (IL-1β) promoted PC cell growth in vivo. Our results demonstrate that linc-ROR delivery via exosomes represents a brand-new perspective of dedifferentiating adipocytes in the TME of PC, which further induce PC cell EMT via the hypoxia inducible factor 1α (HIF1α)-ZEB1 axis. Moreover, exosomal linc-ROR may become a novel diagnostic marker for PC patients.

Features of treatment rare forms of stromal cell ovarian tumors

Objective: identification and description of clinical and morphological characteristics of rare forms of stromal cell tumors of the ovaries.Materials and methods. Retrospectively processed over 10 thousand case histories of patients with various ovarian tumors who were examined and treated at the N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia and the National Center for Oncology and Hematology of the Ministry of Health of Kyrgyz Republic. In 473 patients were diagnosed stromal cell tumors, among them 3 cases of rare forms of tumors of the stroma of the sex cord were identified: stromal tumor from Sertoli cells, malignant luteoma of the ovary, lipid‑cell tumor from Leydig cells. All patients underwent a planned morphological study of surgical material and immune-histochemical study.Results. The detailed data of clinical observations of rare types of tumors, which was presented in the literature by isolated cases, are presented, the description of which in the literature is presented by isolated cases. The possible effect of clinical and morphological characteristics on the survival of patients with rare forms of stromal cell tumors of the ovaries were analyzed.Conclusions. The necrobiotic changes in the tumor, the degree of malignancy, and the stage of the disease are potential predictors of poor prognosis in patients with rare forms stromal cell tumors of the ovaries which require confirmation in a larger sample of patients.

Single-cell advances in stromal-leukocyte interactions in cancer.

Mesenchymal stromal cells in solid tumors have emerged as important mediators of immune function and response to immunotherapies. As such, comprehensive insights into their biology may reveal new predictors of drug response and new drug targets. While our understanding of mesenchymal biology in cancer is nascent, it is rapidly evolving, driven by advances in single-cell technologies. These studies reveal distinct subclasses of cancer-associated fibroblasts (CAFs) with unique properties for immune regulation and control of leukocyte activity. While these studies have revealed several similarities across distinct types of cancer, they still face key challenges in nomenclature. Single-cell analysis of tumors has also revealed an abundance of perivascular cells with unique biology and associations with immune infiltration. They are often misclassified, likely confounding previous bulk studies, revealing a distinct lineage of cells that remain to be fully characterized. These studies have also shed light on the discrete cell types or transient cell states that shape mesenchymal heterogeneity in tumors, offering insights into new therapeutic strategies to modulate stromal cell differentiation. In this review, we will address how recent advances in single-cell technologies have shaped our understanding of stromal heterogeneity and their coordination of immune responses in cancer.

Identification of collagen genes related to immune infiltration and epithelial-mesenchymal transition in glioma

BackgroundGliomas account for the majority of fatal primary brain tumors, and there is much room for research in the underlying pathogenesis, the multistep progression of glioma, and how to improve survival. In our study, we aimed to identify potential biomarkers or therapeutic targets of glioma and study the mechanism underlying the tumor progression.Methods We downloaded the microarray datasets (GSE43378 and GSE7696) from the Gene Expression Omnibus (GEO) database. Then, we used weighted gene co-expression network analysis (WGCNA) to screen potential biomarkers or therapeutic targets related to the tumor progression. ESTIMATE (Estimation of STromal and Immune cells in MAlignant Tumors using Expression data) algorithm and TIMER (Tumor Immune Estimation Resource) database were used to analyze the correlation between the selected genes and the tumor microenvironment. Real-time reverse transcription polymerase chain reaction was used to measure the selected gene. Transwell and wound healing assays were used to measure the cell migration and invasion capacity. Western blotting was used to test the expression of epithelial-mesenchymal transition (EMT) related markers.ResultsWe identified specific module genes that were positively correlated with the WHO grade but negatively correlated with OS of glioma. Importantly, we identified that 6 collagen genes (COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, and COL5A2) could regulate the immunosuppressive microenvironment of glioma. Moreover, we found that these collagen genes were significantly involved in the EMT process of glioma. Finally, taking COL3A1 as a further research object, the results showed that knockdown of COL3A1 significantly inhibited the migration, invasion, and EMT process of SHG44 and A172 cells.ConclusionsIn summary, our study demonstrated that collagen genes play an important role in regulating the immunosuppressive microenvironment and EMT process of glioma and could serve as potential therapeutic targets for glioma management.

Development of LC-HRMS methods for evaluation of metabolic conversion of 5-fluorocytosine at GDEPT procedure

Gene-directed enzyme/prodrug therapy represents one of the experimental treatment approaches. The system based on conversion of nontoxic prodrug 5-fluorocytosine to chemotherapeutic 5-fluorouracil by cytosine deaminase or fusion cytosine deaminase::uracil phosphoribosyl transferase belongs to the most frequently used. The detailed analysis of 5-fluorocytosine, 5-fluorouracil and its metabolites enables to understand various responses of tumour cells to treatment as well as mechanisms of resistance.A fast, sensitive and accurate methods based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) for the identification and quantification of 5-fluorocytosine, 5-fluorouracil and its major metabolites were developed. Two different hybrid high-resolution mass spectrometers sufficient for study of metabolic pathways were used. The LC-ESI IT-TOF MS method was successfully used for identification of 5-fluorocytosine, 5-fluorouracil and its metabolites in complex biological matrices (mesenchymal stromal cells and tumour cells media) and for confirmation of the metabolic conversion of 5-fluorocytosine even in chemoresistant tumour cells media samples. For quantification, the LC-HESI QExactive MS method was developed and validated. The developed method demonstrated a very good linear range for 5-fluorocytosine from 1 ng/mL to 1000 ng/mL and for its major metabolites from 5 ng/mL to 1000 ng/mL. The limits of detection and limits of quantification ranged from 1.1 to 26 ng/mL and from 3.6 to 87 ng/mL, respectively.Both developed methods confirmed the ability of gene-directed enzyme prodrug therapy to metabolically convert 5-fluorocytosine to 5-fluorouracil and its major metabolites in real samples of tumour cell media and mesenchymal stromal cells.

Correlation between driver mutation and immune microenvironment in colorectal cancer patients.

e15513 Background: Immune checkpoint inhibitor (ICI) therapy has made great achievements, but ICI monotherapies show less effectiveness in colorectal cancer patients. Biomarker exploration have carried out from PD-L1 expression, neo-antigen, gene mutation, etc., but no satisfactory results have been obtained. Methods: Patients, Colorectal cancer patients. Methods, Multi-color immunohistochemistry (multi-IHC) was used to evaluate CD8+ T cells, macrophages and natural killer cell (NK cell) in tumors and tumor stroma. The Shapiro-Wilk method was used to test the normality of the data, and the t-test or Mann-Whitney U test was used according to the test results. A two-sided P &lt; 0.05 was considered a significant difference. Results: The study included 72 colorectal cancer patients, including 26 female (36.7%) and 46 male (63.8%), with a median age of 59.5 (50-67.3). There were 6 patients (8.3%) with BRAF mutation, 43 patients (59.7%) with KRAS mutation, and 56 patients (77.8%) with TP53 mutation. The results of immunohistochemistry showed that, BRAF mutation Vs BRAF wild-type or KRAS mutation Vs KRAS wild-type, the number or proportion of CD8+ T cells, macrophages or NK cells in tumor and tumor stroma were not statistically different. For TP53 mutation Vs TP53 wild-type, the number and proportion of CD8+ T cells or macrophages in tumor and tumor stroma were not statistically different. There was no statistical difference in the number and proportion of NK cells in tumor. But, the median number of NK cells in tumor stroma was 345 Vs 129, p = 0.06, and the proportion of NK cells was 5.2% Vs 1.39%, p = 0.02. Conclusions: There is no significant change in the immune microenvironment of colorectal cancer patients with BRAF mutation and KRAS mutation. There are more NK cells in tumor stroma of colorectal cancer patients with TP3 mutation.

Investigation of immune microenvironment in EGFR mutant NSCLC.

e20517 Background: Immune checkpoint inhibitor therapy has made great achievements in NSCLC, but patients with EGFR mutations have poor efficacy with immunotherapy. Previous studies have explored the expression of PD-L1, neo-antigen, co-mutation and regulatory pathways in EGFR mutate NSCLC. This work compared the immune microenvironment of EGFR mutant and wild-type NSCLC. Methods: Patients: NSCLC. Using multi-color immunohistochemistry (multi-IHC) to evaluate the expression of 2 indicators in tumors and tumor stroma, namely CD8+ T cell and macrophage. Shapiro-Wilk was used for normality test, and t-test or Mann-Whitney U test was used according to the results. Two-sided P &lt; 0.05 was considered a significant difference. Results: The study included 119 NSCLC patients, including 59 women (49.6%) and 60 men (50.4%), with a median age of 57. There were 68 patients (57.1%) with EGFR mutations, 19 patients (16%) with KRAS mutations, and 58 patients (48.7%) with TP53 mutations. multi-IHC results showed that, EGFR mutation Vs EGFR wild type samples, (1) the number and proportion of CD8+ T cells in tumor were not statistically different. The median number of CD8+ T cells in tumor stroma was 231.5 vs 359, p = 0.05 and the proportion of CD8+ T cells was 3.92% vs 5.64%, p = 0.02; (2) The median number of macrophage in tumors was 1522 vs 110, p &lt; 0.01, and the proportion of macrophage cells was 24.93% Vs 1.38%, p &lt; 0.01. The median value of macrophages in tumor stroma was 617.5 Vs 208, p &lt; 0.01, and the proportion of macrophages cells was 10.88% vs 3.03%, p &lt; 0.01. Conclusions: Compared with EGFR wild-type patients, EGFR mutation patients have a lower proportion of CD8+ T cells and a higher proportion of macrophages in the immune microenvironment.

Safety, tolerability and preliminary efficacy of CAN04, a first in class monoclonal antibody against IL1RAP, in combination with nab-paclitaxel and gemcitabine (NG) in subjects with pancreatic cancer.

e16228 Background: Interleukin-1 Receptor Accessory Protein (IL1RAP) is expressed on cancer and stromal cells in many solid tumors. CAN04 is a fully humanized IgG1 targeting IL1RAP blocking IL-1α and β signaling and triggering ADCC. As monotherapy, CAN04 has been administered without reaching MTD up to 15mg/kg. We report initial data from CAN04 in combination with NG in pts with pancreatic cancer (PDAC). Methods: Primary objective was to determine safety and tolerability of CAN04 in combination with standard NG as first line treatment in pts with locally advanced/metastatic PDAC. Secondary objectives included establishing preliminary efficacy. Tumor responses were evaluated according to RECIST/iRECIST. Results: 36 patients (pts) received at least 1 dose of CAN04: median age 62 yrs (46-87), 47% women, 94% stage IV, 8% had received prior adjuvant therapy; 59% ECOG 0, 41% ECOG 1. Dosing was initiated at 5mg/kg, escalated to 7.5mg/kg (n = 8) which was considered above MTD due to hematological toxicity, followed by expansion at 5mg/kg. Infusion related reactions (IRRs) were seen primarily with CAN04 priming dose of 0.5 mg/kg (25% G1, 25% G2, 3% G3). G3 or higher AEs (all/related) were dominated by hematological toxicity and reported in 89%/72% of pts (see table). SAEs were reported in 67% pts (44% G3/G4), in 33% considered related (19% G3/G4). 33% of pts discontinued due to AEs: 4 deterioration of general condition, 2 IRR, 2 bilirubin elevation, 1 bile duct obstruction, 1 hemolytic uremic syndrome, 1 pancreatitis, 1 asthenia, 1 decreased appetite, 1 pneumonitis. There were 3 on treatment deaths: disease progression, gastrointestinal obstruction and cholangitis. For the mITT efficacy analysis (n = 31), 5 pts were excluded: 3 discontinued before receiving NG (2 consent withdrawals after IRRs, tumor related physical deterioration) and 2 did not complete Cycle 1 due to cholangitis and GI obstruction respectively. 12 pts (39%) had confirmed or unconfirmed PR as best response, 8 (26%) more showed SD and 9 (29%) had PD. Six pts underwent PD confirmation: 3 pts had prolonged immune unconfirmed PD (iUPD) by iRECIST and &gt; 50% decreases in CA19-9 and were on treatment for 4.5 (ongoing), 6 and 8 months, respectively. Clinical benefit rate (PR+SD+prolonged iUPD): 74%. At the time of this analysis, 12 pts (38%) are ongoing. Median duration of response is currently 7.5 months (1.8-13.8 months). Conclusions: The clinical benefit rate of CAN04 in combination with NG is encouraging (74%). The G3/G4 neutropenia and febrile neutropenia rate is higher than expected for NG alone. Expansion cohorts at lower CAN04 doses have been initiated. Clinical trial information: NCT03267316. [Table: see text]

Cancer-Associated Fibroblasts and Tumor-Associated Macrophages in Cancer and Cancer Immunotherapy.

Our understanding of the tumor microenvironment (TME), including the interplay between tumor cells, stromal cells, immune cells, and extracellular matrix components, is mandatory for the innovation of new therapeutic approaches in cancer. The cell-cell communication within the TME plays a pivotal role in the evolution and progression of cancer. Cancer-associated fibroblasts (CAF) and tumor-associated macrophages (TAM) are major cell populations in the stroma of all solid tumors and often exert protumorigenic functions; however, the origin and precise functions of CAF and TAM are still incompletely understood. CAF and TAM hold significant potential as therapeutic targets to improve outcomes in oncology when combined with existing therapies. The regulation of CAF/TAM communication and/or their differentiation could be of high impact for improving the future targeted treatment strategies. Nevertheless, there is much scope for research and innovation in this field with regards to the development of novel drugs. In this review, we elaborate on the current knowledge on CAF and TAM in cancer and cancer immunotherapy. Additionally, by focusing on their heterogenous functions in different stages and types of cancer, we explore their role as potential therapeutic targets and highlight certain aspects of their functions that need further research.

Upregulation of miR-206 is a potential diagnostic biomarker in breast cancer

Breast cancer is one of the most common malignancies, and like most cancers, most cases are caused by somatic mutations. Due to estrogen's role in the growth, differentiation, and division of breast and endometrial cancer cells, tamoxifen is used as an estrogen receptor antagonist in breast cancer cells with estrogen receptor (ER +) has a special place, which unfortunately in one-third of the Cases are resisted. This study aimed to investigate the effect of tamoxifen-treated tumor-derived exosomes on the expression pattern of Twist and Bcl-2 oncogenic genes in fibroblast cells. MCF-7 breast cancer cell line and fibroblast cells were purchased and cultured in a complete culture medium. After the appropriate number of cells was reached, they were treated with the appropriate concentration of tamoxifen. Cellular supernatant was then gathered in flasks, and exosomes were extracted from them. After extracting RNA from exosomes and cDNA synthesis, the expression level of miR-206, Twist-1, and Bcl-2 genes were evaluated using the Real-Time PCR method. The electronic microscope results confirmed the correctness of the exosomes isolated from the tumor cell culture medium. It has also been shown that tamoxifen treatment increases the expression of miR-206 in exosomes derived from breast tumor cells. The control group which has been kept untreated induced the expression level of Twist-1 and Bcl-2 genes time-dependently. However, when tamoxifen-treated tumor-derived exosomes treated the target cells, the expression level of oncogenic miRs Twist-1 and Bcl-2 were declined over time. Overall, this study showed that tamoxifen treatment on breast cancer cells could apply its antioncogenic effects on tumor stromal cells, such as fibroblasts, by altering the expression levels of exosomal microRNAs in tumor cells.

Identification of a Prognostic Signature for Ovarian Cancer Based on the Microenvironment Genes.

Background: Ovarian cancer is highly malignant and has a poor prognosis in the advanced stage. Studies have shown that infiltration of tumor microenvironment cells, immune cells and stromal cells has an important impact on the prognosis of cancers. However, the relationship between tumor microenvironment genes and the prognosis of ovarian cancer has not been studied.Methods: Gene expression profiles and SNP data of ovarian cancer were downloaded from the TCGA database. Cluster analysis, WGCNA analysis and univariate survival analysis were used to identify immune microenvironment genes as prognostic signatures for predicting the survival of ovarian cancer patients. External data were used to evaluate the signature. Moreover, the top five significantly correlated genes were evaluated by immunohistochemical staining of ovarian cancer tissues.Results: We systematically analyzed the relationship between ovarian cancer and immune metagenes. Immune metagenes expression were associated with prognosis. In total, we identified 10 genes related to both immunity and prognosis in ovarian cancer according to the expression of immune metagenes. These data reveal that high expression of ETV7 (OS, HR = 1.540, 95% CI 1.023–2.390, p = 0.041), GBP4 (OS, HR = 1.834, 95% CI 1.242–3.055, p = 0.004), CXCL9 (OS, HR = 1.613, 95% CI 1.080 –2.471, p = 0.021), CD3E (OS, HR = 1.590, 95% CI 1.049 –2.459, p = 0.031), and TAP1 (OS, HR = 1.766, 95% CI 1.163 –2.723, p = 0.009) are associated with better prognosis in patients with ovarian cancer.Conclusion: Our study identified 10 immune microenvironment genes related to the prognosis of ovarian cancer. The list of tumor microenvironment-related genes provides new insights into the underlying biological mechanisms driving the tumorigenesis of ovarian cancer.

Bone marrow-derived mesenchymal stem cells inhibit CD8+ T cell immune responses via PD-1/PD-L1 pathway in multiple myeloma.

High expression of the inhibitory receptor programmed cell death ligand 1 (PD-L1) on tumor cells and tumor stromal cells have been found to play a key role in tumor immune evasion in several human malignancies. However, the expression of PD-L1 on bone marrow mesenchymal stem cells (BMSCs) and whether the programmed cell death 1 (PD-1)/PD-L1 signal pathway is involved in the BMSCs versus T cell immune response in multiple myeloma (MM) remains poorly defined. In this study, we explored the expression of PD-L1 on BMSCs from newly diagnosed MM (NDMM) patients and the role of PD-1/PD-L1 pathway in BMSC-mediated regulation of CD8+ T cells. The data showed that the expression of PD-L1 on BMSCs in NDMM patients was significantly increased compared to that in normal controls (NC) (18·81±1·61 versus 2·78±0·70%; P<0·001). Furthermore, the PD-1 expression on CD8+ T cells with NDMM patients was significantly higher than that in normal controls (43·22±2·98 versus 20·71±1·08%; P<0·001). However, there was no significant difference in PD-1 expression of CD4+ T cells and natural killer (NK) cells between the NDMM and NC groups. Additionally, the co-culture assays revealed that BMSCs significantly suppressed CD8+ T cell function. However, the PD-L1 inhibitor effectively reversed BMSC-mediated suppression in CD8+ T cells. We also found that the combination of PD-L1 inhibitor and pomalidomide can further enhance the killing effect of CD8+ T cells on MM cells. In summary, our findings demonstrated that BMSCs in patients with MM may induce apoptosis of CD8+ T cells through the PD-1/PD-L1 axis and inhibit the release of perforin and granzyme B from CD8+ T cells to promote the immune escape of MM.

Prognostic value of tumour microenvironment-related genes by TCGA database in rectal cancer.

Rectal cancer is a common malignant tumour and the progression is highly affected by the tumour microenvironment (TME). This study intended to assess the relationship between TME and prognosis, and explore prognostic genes of rectal cancer. The gene expression profile of rectal cancer was obtained from TCGA and immune/stromal scores were calculated by Estimation of Stromal and Immune cells in Malignant Tumors using Expression data (ESTIMATE) algorithm. The correlation between immune/stromal scores and survival time as well as clinical characteristics were evaluated. Differentially expressed genes (DEGs) were identified according to the stromal/immune scores, and the functional enrichment analyses were conducted to explore functions and pathways of DEGs. The survival analyses were conducted to clarify the DEGs with prognostic value, and the protein‐protein interaction (PPI) network was performed to explore the interrelation of prognostic DEGs. Finally, we validated prognostic DEGs using data from the Gene Expression Omnibus (GEO) database by PrognoScan, and we verified these genes at the protein levels using the Human Protein Atlas (HPA) databases. We downloaded gene expression profiles of 83 rectal cancer patients from The Cancer Genome Atlas (TCGA) database. The Kaplan‐Meier plot demonstrated that low‐immune score was associated with worse clinical outcome (P = .034), metastasis (M1 vs. M0, P = .031) and lymphatic invasion (+ vs. ‐, P < .001). A total of 540 genes were screened as DEGs with 539 up‐regulated genes and 1 down‐regulated gene. In addition, 60 DEGs were identified associated with overall survival. Functional enrichment analyses and PPI networks showed that the DEGs are mainly participated in immune process, and cytokine‐cytokine receptor interaction. Finally, 19 prognostic genes were verified by GSE17536 and GSE17537 from GEO, and five genes (ADAM23, ARHGAP20, ICOS, IRF4, MMRN1) were significantly different in tumour tissues compared with normal tissues at the protein level. In summary, our study demonstrated the associations between TME and prognosis as well as clinical characteristics of rectal cancer. Moreover, we explored and verified microenvironment‐related genes, which may be the potential key prognostic genes of rectal cancer. Further clinical samples and functional studies are needed to validate this finding.

The Story of a Radiologically Undetectable, Testosterone Producing Leydig Cell Tumor

Introduction: Leydig stromal cell tumors are uncommon ovarian tumors that produce testosterone leading to hyperandrogenism. We present a case of a 63 year old lady with significantly elevated testosterone levels that did not have clear ovaries visualized on imaging, but was subsequently found to have a Leydig cell tumor on pathology after ovarian resection.Clinical Case: A 63 year old female with a past medical history of COPD, hypothyroidism, hyperlipidemia, hypertension and uterine fibroids status post hysterectomy and left oophorectomy in 1995 was referred to endocrinology for hirsutism. The patient reported first noticing abnormal hair growth approximately one year prior to presentation having developed increasingly coarse and thick facial hair, abdominal wall hair, and chest hair. On physical examination, she was noted to have coarse hair across her upper lip and chin continuous along the jawline along with fine, dark hair diffusely across her anterior abdomen. Initial laboratory cell evaluation revealed total testosterone 378 ng/dL, free testosterone 53ng/dL, DHEAS 64 ug/dL. Repeat labs drawn three months later confirmed the markedly elevated total testosterone of 362 ng/dL and free testosterone 44.2 ng/dL, concentrations normally seen in males. A CT scan of the abdomen and pelvis was done and did not reveal any masses. In addition, no ovaries were appreciated on imaging. A transvaginal ultrasound also did not reveal any clear ovaries. The patient ended up undergoing a right oophorectomy. Histological examination was consistent with a Leydig cell tumor. Following oophorectomy, her testosterone concentrations normalized (5ng/dL) and hirsutism began to regress.Clinical Lesson: Hyperandrogenism in women is typically classified into non-tumorous and tumorous. The differential for non-tumorous hyperandrogenism includes PCOS, congenital adrenal hyperplasia (CAH), and ovarian hyperthecosis. Tumorous causes include ovarian tumors such as Sertoli-Leydig cell tumors, hilus cell tumors, and theca cell tumors. Adrenal tumors secreting testosterone are extremely rare. Often with these tumors, there is significantly increased testosterone levels (> 140ng/dL) and rapid progression of symptoms. Sex cord stromal tumors account for only 5-8% of all ovarian tumors with Leydig stromal tumors a rare group that accounts for less than 0.1% of all ovarian tumors. The tumors are functional producing testosterone leading to marked hyperandrogenism and virilization. They are also usually benign and unilateral. This patient had hyperandrogenism manifested by hirsutism with markedly elevated testosterone concentrations. In addition, this case is unique in that a CT scan and transvaginal ultrasound did not clearly demonstrate her right ovary. Despite having a normal appearing right ovary during surgery, patient was found to have Leydig cell tumor following histological examination.

Virilization Secondary to an Ovarian Leydig Cell Tumor

A 60-year-old female presented with a three-year history of virilizing symptoms including facial hirsutism and deepening of voice. Her medical history was significant for renal transplantation with immunosuppressive therapy consisting of mycophenolate, cyclosporine, and low-dose prednisone. She was noted to have temporal balding and darkly pigmented terminal hair on the upper lip, cheeks, chin, shoulders, and sternum. Pelvic examination revealed clitoromegaly. Menarche occurred at age 12 with regular menstrual cycles until menopause which occurred at age 50. She had two pregnancies: a miscarriage followed by a successful pregnancy.Labs revealed an elevated total testosterone of 530 ng/dL (< 60 ng/dL), free testosterone 14.8 ng/dL (<0.87 ng/dL), androstenedione 2140 ng/dL (<200 ng/dL), and 17-hydroxyprogesterone 704 ng/dL (<285 ng/dL). LH, FSH, and estradiol were inappropriately normal in this post-menopausal female. Prolactin, TSH, DHEA-S, IGF-1 were within normal limits. Transvaginal ultrasound found a 2 cm hypoechoic right ovarian mass which was confirmed on MRI. MRI also revealed a 5 mm right adrenal nodule. Tumor markers including CA-125, Inhibin A, Inhibin B, HCG, and AFP were within normal limits. Dexamethasone suppression testing did not lower the testosterone level. 17-hydroxyprogesterone level after cosyntropin stimulation testing was 704 ng/dL (<1000 ng/dL). The patient underwent laparoscopic bilateral oophorectomy and salpingectomy, pelvic washout and omental biopsy. Pathology was consistent with a benign Leydig cell tumor. Following oophorectomy there was complete normalization of the total testosterone level (15 ng/dL, n< 60 ng/dL).A thorough history and physical exam is vital in determining the cause of hirsutism. Medications, including over-the-counter and herbal formulations should be carefully reviewed. Although cyclosporine has been associated with hirsutism, patients typically present with vellus hair formation in the affected areas rather than darkly pigmented terminal hair. In this case, hirsutism progressively worsened following menopause and physical examination was significant for virilization. Hirsutism in a combination with virilization is typically neoplastic in nature. Endogenous androgen production can originate from either the adrenal glands or ovaries. In our patient, with workup showing both ovarian and adrenal as potential sources of endogenous androgen production, an adrenal cause was excluded due to a normal DHEA-S level at baseline and a lack of suppression of testosterone after dexamethasone suppression testing. As a result, the source was localized to the ovary. While excessive androgen production resulting in virilization is seen with ovarian tumors, Leydig stromal cell tumors are extremely rare and account for less than 0.1% of all ovarian tumors.

Development and Validation of an Autophagy-Stroma-Based Microenvironment Gene Signature for Risk Stratification in Colorectal Cancer.

BackgroundColorectal cancer is the fourth most common cancer and the second leading cause of cancer-related death in the USA. The aim of this study was to establish a tumor gene signature based on tumor stromal cell and autophagy for predicting the risk of recurrence in patients with colorectal cancer.MethodsWe used “Rtsne” and “xCell” R packages to estimate autophagy and stroma status, respectively. The discovery cohort used microarray gene expression data retrieved from the GSE39582 dataset. The Cox regression model and Least Absolute Shrinkage and Selection Operator (LASSO) were used to identify prognostic genes and to construct an autophagy-stroma-based gene signature. Moreover, external validation was conducted using GSE17538, GSE38832, TCGA database, and patient data obtained from the First Hospital of China Medical University (CMU).ResultsThe LASSO model identified three genes (TNS1, TAGLN, and SFRP4) which were used to develop a risk stratification gene signature. The autophagy-stroma-based gene signature was identified as an independent prognostic factor by multivariate analysis (p = 0.0023). The results were validated in GSE17538 (p=0.0062), GSE38832 (p=0.028), TCGA (p=0.046) database, and patient data obtained from the First Hospital of China Medical University (CMU) (p=0.027).ConclusionWe have established and verified a feasible prognostic model of colorectal cancer based on autophagy and stromal cell characteristics of patients. The model can be used to evaluate recurrence risk of cancer patients, and the hub genes in the model provide potential targets for targeted colorectal cancer treatment.

38P Triple-negative breast cancers with expression of glucocorticoid receptor in immune cells show better prognosis

Glucocorticoid receptor (GR) is shown to have variable frequency of expression in invasive tumors of the breast. Investigation of additional nuclear receptors like GR in receptor negative tumors like triple negative breast cancer (TNBC) may have prognostic and therapeutic significance. Expression of GR was evaluated by immunohistochemistry in 175 tumors of invasive breast cancer with long term follow up. GR Expression was separately evaluated in invasive tumor cells, stromal cells and tumor infiltrating lymphocytes (TIL's). Staining pattern was categorised as positive when more than 1% of the cells stained in each subpopulation of cells. Disease free survival was analysed between GR positive and negative status by Kaplan Meier analysis. Of the 175 tumors, 121 (70%) were ER positive, 53 (30%) were ER negative and 29% (51) were triple negative. 74% (130/175) tumors showed expression of GR in invasive tumor cells while (84%) 147/175 had expression in TIL's. No significant difference in distribution of GR was noted between ER positive and ER negative tumors (78% vs 66%, p-0.1). Of the TNBC's 54% (28/51) and 70% (36/51) showed expression of GR in invasive tumor and TIL's respectively. Overall, GR positive tumors had significant better survival than GR negative tumors (mean survival time of 85 vs 59 months respectively, p-0.04) Contrary to the reports that GR expression in TIL's are associated with immunosuppressive activity in model systems, TNBC's with increased expression of GR in immune cells were associated with better survival (Mean survival time 74 vs 41 months, log rank test- p-0.03). TNBC tumors which were GR negative had higher lymph node metastases (p-0.04) and none of the other clinical features like age, menopausal state, tumor size and grade were different between GR positive and negative tumors within TNBC. Glucocorticoids (GC) are often used to alleviate the adverse symptoms during chemotherapy. Determining the GR status is of importance due to the pro cell survival effect of the glucocorticoids mediated through GR during chemotherapy. Though GC mediated effects on chemotherapy are controversial, our results indicate favourable effects in TNBC.