Circulating neutrophils and tumor-associated myeloid cells function as a powerful biomarker for response to chemoradiation in locally advanced cervical cancer.

Purpose: The immune system's role in mediating the cytotoxic effects of chemoradiotherapy remains not completely understood. The integration of immunotherapies into treatment will require insight into features and timing of the immune microenvironment associated with treatment response. Here, we investigated the role of circulating neutrophils and tumor-associated myeloid cells (TSAMs) as potential agents and biomarkers for disease-related outcomes in locally advanced cervical cancer (LACC). Material and Methods: Hematologic parameters for two LACC patient cohorts, a retrospective clinical and a prospective translational cohort, were obtained at baseline, weekly during chemoradiotherapy for the retrospective cohort, biweekly during chemoradiotherapy for the prospective cohort, and at the first follow-up visit for both cohorts (mean 14.7 weeks, range 8.1-25.1 weeks for the prospective cohort and 5.3 weeks with a range of 2.7-9.0 weeks for the retrospective cohort). In both cohorts, baseline as well as mean and lowest on-treatment values for platelets, hemoglobin, absolute neutrophil count (ANC), and absolute lymphocyte count (ALC) were analyzed for correlations with disease-related outcomes. In the prospective cohort, circulating myeloid cells were isolated from peripheral blood mononuclear cells (PBMCs), and TSAMs were isolated from tumor tissue via a novel serial cytobrush sampling assay. The samples were analyzed by flow cytometry. Results: In both cohorts, the only hematologic parameter significantly associated with survival was elevated on-treatment mean ANC (mANC), which was associated with lower local failure-free and overall survival rates in the retrospective and prospective cohorts, respectively. mANC was not associated with a difference in distant metastases. CD11b+CD11c- TSAMs, which act as a surrogate marker for intratumoral neutrophils, steadily decreased during the course of chemoRT and nadier'd at week 5 of treatment. Conversely, circulating myeloid cells identified from PBMCs steadily increased through week 5 of treatment. Regression analysis confirmed an inverse relationship between circulating myeloid cells and TSAMs at this time point. Conclusions: These findings identify on-treatment mean neutrophil count as a predictor of disease-related outcomes, suggest that neutrophils contribute to chemoradiation treatment resistance, and demonstrate the importance of techniques to measure intratumoral immune activity.

Immune environment and antigen specificity of the T cell receptor repertoire of malignant ascites in ovarian cancer.

We evaluated the association of disease outcome with T cell immune-related characteristics and T cell receptor (TCR) repertoire in malignant ascites from patients with high-grade epithelial ovarian cancer. Ascitic fluid samples were collected from 47 high-grade epithelial ovarian cancer patients and analyzed using flow cytometry and TCR sequencing to characterize the complementarity determining region 3 TCR ß-chain. TCR functions were analyzed using the McPAS-TCR and VDJ databases. TCR clustering was implemented using Grouping of Lymphocyte Interactions by Paratope Hotspots software. Patients with poor prognosis had ascites characterized by an increased ratio of CD8+ T cells to regulatory T cells, which correlated with an increased productive frequency of the top 100 clones and decreased productive entropy. TCRs enriched in patients with an excellent or good prognosis were more likely to recognize cancer antigens and contained more TCR reads predicted to recognize epithelial ovarian cancer antigens. In addition, a TCR motif that is predicted to bind the TP53 neoantigen was identified, and this motif was enriched in patients with an excellent or good prognosis. Ascitic fluid in high-grade epithelial ovarian cancer patients with an excellent or good prognosis is enriched with TCRs that may recognize ovarian cancer-specific neoantigens, including mutated TP53 and TEAD1. These results suggest that an effective antigen-specific immune response in ascites is vital for a good outcome in high-grade epithelial ovarian cancer.

Expansion of Candidate HPV-Specific T Cells in the Tumor Microenvironment during Chemoradiotherapy Is Prognostic in HPV16+ Cancers.

Human papillomavirus (HPV) infection causes 600,000 new cancers worldwide each year. HPV-related cancers express the oncogenic proteins E6 and E7, which could serve as tumor-specific antigens. It is not known whether immunity to E6 and E7 evolves during chemoradiotherapy or affects survival. Using T cells from 2 HPV16+ patients, we conducted functional T-cell assays to identify candidate HPV-specific T cells and common T-cell receptor motifs, which we then analyzed across 86 patients with HPV-related cancers. The HPV-specific clones and E7-related T-cell receptor motifs expanded in the tumor microenvironment over the course of treatment, whereas non-HPV-specific T cells did not. In HPV16+ patients, improved recurrence-free survival was associated with HPV-responsive T-cell expansion during chemoradiotherapy.

Extracellular vesicles derived from ascitic fluid enhance growth and migration of ovarian cancer cells.

Ovarian cancer is associated with a high mortality rate due to diagnosis at advanced stages. Dissemination often occurs intraperitoneally within the ascites fluid. The microenvironment can support dissemination through several mechanisms. One potential ascites factor which may mediate dissemination are EVs or extracellular vesicles that can carry information in the form of miRNAs, proteins, lipids, and act as mediators of cellular communication. We present our observations on EVs isolated from ascitic supernatants from patients diagnosed with high grade serous ovarian carcinoma in augmenting motility, growth, and migration towards omental fat. MicroRNA profiling of EVs from malignant ascitic supernatant demonstrates high expression of miR 200c-3p, miR18a-5p, miR1246, and miR1290 and low expression of miR 100- 5p as compared to EVs isolated from benign ascitic supernatant. The migration of ovarian cancer spheroids towards omental fat is enhanced in the presence of malignant ascitic EVs. Gene expression of these cells showed increased expression of ZBED2, ZBTB20, ABCC3, UHMK1, and low expression of Transgelin and MARCKS. We present evidence that ovarian ascitic EVs increase the growth of ovarian cancer spheroids through miRNAs.

Effect of Antibiotics on Gut and Vaginal Microbiomes Associated with Cervical Cancer Development in Mice.

Antibiotics affect microbial diversity in the gut, leading to dysbiosis and impaired immunity. However, the impact of antibiotics on microbial communities at other sites, such as vagina is less understood. It is also not clear whether changes induced by antibiotics in both microbiomes affect the development of cervical cancer. In this study, we utilized the murine model to evaluate these questions. We show that oral application of broad-spectrum antibiotics in mice changed not only diversity, but composition and sharing of gut and vaginal microbiomes in mice and influenced cervical cancer development in an orthotopic tumor model. Antibiotics decreased richness and diversity indexes in the gut but increased them in the vagina. Some beneficial taxa, such as Bacteroides, Ruminococcaceae, and Lachnospiraceae increased their abundance in the vagina while other pathogenic species, such as Proteobacteria, were decreased. As a result of the changes, mice with greater richness and diversity of the vaginal microbiome after antibiotics exposure were less likely developed tumors. No association between richness and diversity of the gut microbiome and tumor development was identified.

Microbial Diversity and Composition Is Associated with Patient-Reported Toxicity during Chemoradiation Therapy for Cervical Cancer.

PURPOSE: Patients receiving pelvic radiation for cervical cancer experience high rates of acute gastrointestinal (GI) toxicity. The association of changes in the gut microbiome with bowel toxicity from radiation is not well characterized. METHODS AND MATERIALS: Thirty-five patients undergoing definitive chemoradiation therapy (CRT) underwent longitudinal sampling (baseline and weeks 1, 3, and 5) of the gut microbiome and prospective assessment of patient-reported GI toxicity. DNA was isolated from stool obtained at rectal examination and analyzed with 16S rRNA sequencing. GI toxicity was assessed with the Expanded Prostate Cancer Index Composite instrument to evaluate frequency, urgency, and discomfort associated with bowel function. Shannon diversity index was used to characterize alpha (within sample) diversity. Weighted UniFrac principle coordinates analysis was used to compare beta (between sample) diversity between samples using permutational multivariate analysis of variance. Linear discriminant analysis effect size highlighted microbial features that best distinguish categorized patient samples. RESULTS: Gut microbiome diversity continuously decreased over the course of CRT, with the largest decrease at week 5. Expanded Prostate Cancer Index Composite bowel function scores also declined over the course of treatment, reflecting increased symptom burden. At all individual time points, higher diversity of the gut microbiome was linearly correlated with better patient-reported GI function, but baseline diversity was not predictive of eventual outcome. Patients with high toxicity demonstrated different compositional changes during CRT in addition to compositional differences in Clostridia species. CONCLUSIONS: Over time, increased radiation toxicity is associated with decreased gut microbiome diversity. Baseline diversity is not predictive of end-of-treatment bowel toxicity, but composition may identify patients at risk for developing high toxicity.

Stromal Cells Derived from Visceral and Obese Adipose Tissue Promote Growth of Ovarian Cancers.

Obesity, and in particular visceral obesity, has been associated with an increased risk of developing cancers as well as higher rates of mortality following diagnosis. The impact of obesity on adipose-derived stromal cells (ASC), which contribute to the formation of tumor stroma, is unknown. Here we hypothesized that visceral source and diet-induced obesity (DIO) changes the ASC phenotype, contributing to the tumor promoting effects of obesity. We found that ASC isolated from subcutaneous (SC-ASC) and visceral (V-ASC) white adipose tissue(WAT) of lean(Le) and obese(Ob) mice exhibited similar mesenchymal cell surface markers expression, and had comparable effects on ovarian cancer cell proliferation and migration. Obese and visceral derived ASC proliferated slower and exhibited impaired differentiation into adipocytes and osteocytes in vitro as compared to ASC derived from subcutaneous WAT of lean mice. Intraperitoneal co-injection of ovarian cancer cells with obese or visceral derived ASC, but not lean SC-ASC, increased growth of intraperitoneal ID8 tumors as compared to controls. Obese and V-ASC increased stromal infiltration of inflammatory cells, including CD3+ T cells and F4/80+ macrophages. Obese and visceral derived ASC, but not lean SC-ASC, increased expression of chemotactic factors IL-6, MIP-2, and MCP-1 when cultured with tumor cells. Overall, these results demonstrate that obese and V-ASC have a unique phenotype, with more limited proliferation and differentiation capacity but enhanced expression of chemotactic factors in response to malignant cells which support infiltration of inflammatory cells and support tumor growth and dissemination.

Human omental-derived adipose stem cells increase ovarian cancer proliferation, migration, and chemoresistance.

OBJECTIVES: Adipose tissue contains a population of multipotent adipose stem cells (ASCs) that form tumor stroma and can promote tumor progression. Given the high rate of ovarian cancer metastasis to the omental adipose, we hypothesized that omental-derived ASC may contribute to ovarian cancer growth and dissemination. MATERIALS AND METHODS: We isolated ASCs from the omentum of three patients with ovarian cancer, with (O-ASC4, O-ASC5) and without (O-ASC1) omental metastasis. BM-MSCs, SQ-ASCs, O-ASCs were characterized with gene expression arrays and metabolic analysis. Stromal cells effects on ovarian cancer cells proliferation, chemoresistance and radiation resistance was evaluated using co-culture assays with luciferase-labeled human ovarian cancer cell lines. Transwell migration assays were performed with conditioned media from O-ASCs and control cell lines. SKOV3 cells were intraperitionally injected with or without O-ASC1 to track in-vivo engraftment. RESULTS: O-ASCs significantly promoted in vitro proliferation, migration chemotherapy and radiation response of ovarian cancer cell lines. O-ASC4 had more marked effects on migration and chemotherapy response on OVCA 429 and OVCA 433 cells than O-ASC1. Analysis of microarray data revealed that O-ASC4 and O-ASC5 have similar gene expression profiles, in contrast to O-ASC1, which was more similar to BM-MSCs and subcutaneous ASCs in hierarchical clustering. Human O-ASCs were detected in the stroma of human ovarian cancer murine xenografts but not uninvolved ovaries. CONCLUSIONS: ASCs derived from the human omentum can promote ovarian cancer proliferation, migration, chemoresistance and radiation resistance in-vitro. Furthermore, clinical O-ASCs isolates demonstrate heterogenous effects on ovarian cancer in-vitro.

The antihelmintic drug pyrvinium pamoate targets aggressive breast cancer.

WNT signaling plays a key role in the self-renewal of tumor initiation cells (TICs). In this study, we used pyrvinium pamoate (PP), an FDA-approved antihelmintic drug that inhibits WNT signaling, to test whether pharmacologic inhibition of WNT signaling can specifically target TICs of aggressive breast cancer cells. SUM-149, an inflammatory breast cancer cell line, and SUM-159, a metaplastic basal-type breast cancer cell line, were used in these studies. We found that PP inhibited primary and secondary mammosphere formation of cancer cells at nanomolar concentrations, at least 10 times less than the dose needed to have a toxic effect on cancer cells. A comparable mammosphere formation IC50 dose to that observed in cancer cell lines was obtained using malignant pleural effusion samples from patients with IBC. A decrease in activity of the TIC surrogate aldehyde dehydrogenase was observed in PP-treated cells, and inhibition of WNT signaling by PP was associated with down-regulation of a panel of markers associated with epithelial-mesenchymal transition. In vivo, intratumoral injection was associated with tumor necrosis, and intraperitoneal injection into mice with tumor xenografts caused significant tumor growth delay and a trend toward decreased lung metastasis. In in vitro mammosphere-based and monolayer-based clonogenic assays, we found that PP radiosensitized cells in monolayer culture but not mammosphere culture. These findings suggest WNT signaling inhibition may be a feasible strategy for targeting aggressive breast cancer. Investigation and modification of the bioavailability and toxicity profile of systemic PP are warranted.

Cellular differentiation in three-dimensional lung cell cultures.

INTRODUCTION: Aspects of human biology that are not sufficiently addressed by current cell culture models are cellular differentiation and three-dimensional (3-D) structural organization. A model that more closely associates the presence and biology of organelles to molecular expressions relevant to these organelles may provide evidence of cellular differentiation and the beginning steps in the construction of a 3-D architecture. The development of a new model--3-D cell cultures--may ultimately provide a better understanding of lung biology and pathobiology. PURPOSE: The purpose of this study was to develop both traditional monolayer and 3-D cell cultures of a known and well documented normal lung cell line and then to determine similarities and differences between these cultures in terms of differentiation and molecular marker expression. RESULTS: Electron microscopy identified presence of lipid inclusion, microvilli, extra cellular matrix, and tight junctions in the 3-D cultures; differentiation not seen in ML cultures. The degree of differentiation determined by immunohistochemistry when the cell line was grown as ML or 3-D cultures shows that ultra-structure and marker expressions were more representative of control tissue than when cells were grown in 3-D than as MLs. METHODS: An immortalized cell line was grown as a traditional monolayer (ML) in culture flasks and as 3-D cultures in rotating walled vessels and incubated under identical conditions. Comparison for presence of differentiation and marker expression between these cultures and control tissue collected from surgical patient specimens was studied. Electron microscopy for identification of ultra structures, and immunohistochemistry (ZO-1, EMA, ICAM-1, villin, tubulin, CK 18, VWF, Collagen IV and human mucin) for phenotypic comparisons between cells in ML and 3-D cell cultures was conducted. SUMMARY: The development of 3-D cell cultures will provide for a new and more powerful tool in the study of lung biology and pathobiology.