Tumor-microenvironment Interactions Research Articles

Proteomes of Extracellular Vesicles From Pancreatic Cancer Cells and Cancer-Associated Fibroblasts.

Extracellular vesicles (EVs) are lipid bound vesicles secreted by cells into the extracellular environment. Studies have implicated EVs in cell proliferation, epithelial-mesenchymal transition, metastasis, angiogenesis, and mediating the interaction of tumor cells and microenvironment. A systematic characterization of EVs from pancreatic cancer cells and cancer-associated fibroblasts (CAFs) would be valuable for studying the roles of EV proteins in pancreatic tumorigenesis. Proteomic and functional analyses were applied to characterize the proteomes of EVs released from 5 pancreatic cancer lines, 2 CAF cell lines, and a normal pancreatic epithelial cell line (HPDE). More than 1400 nonredundant proteins were identified in each EV derived from the cell lines. The majority of the proteins identified in the EVs from the cancer cells, CAFs, and HPDE were detected in all 3 groups, highly enriched in the biological processes of vesicle-mediated transport and exocytosis. Protein networks relevant to pancreatic tumorigenesis, including epithelial-mesenchymal transition, complement, and coagulation components, were significantly enriched in the EVs from cancer cells or CAFs. These findings support the roles of EVs as a potential mediator in transmitting epithelial-mesenchymal transition signals and complement response in the tumor microenvironment and possibly contributing to coagulation defects related to cancer development.

ROR2 can function independent of tyrosine kinase activity to promote ovarian cancer metastasis (264)

ROR2 can function independent of tyrosine kinase activity to promote ovarian cancer metastasis (264)

γδ T Cells in the Tumor Microenvironment-Interactions With Other Immune Cells.

A growing number of studies have shown that γδ T cells play a pivotal role in mediating the clearance of tumors and pathogen-infected cells with their potent cytotoxic, cytolytic, and unique immune-modulating functions. Unlike the more abundant αβ T cells, γδ T cells can recognize a broad range of tumors and infected cells without the requirement of antigen presentation via major histocompatibility complex (MHC) molecules. Our group has recently demonstrated parts of the mechanisms of T-cell receptor (TCR)-dependent activation of Vγ9Vδ2+ T cells by tumors following the presentation of phosphoantigens, intermediates of the mevalonate pathway. This process is mediated through the B7 immunoglobulin family-like butyrophilin 2A1 (BTN2A1) and BTN3A1 complexes. Such recognition results in activation, a robust immunosurveillance process, and elicits rapid γδ T-cell immune responses. These include targeted cell killing, and the ability to produce copious quantities of cytokines and chemokines to exert immune-modulating properties and to interact with other immune cells. This immune cell network includes αβ T cells, B cells, dendritic cells, macrophages, monocytes, natural killer cells, and neutrophils, hence heavily influencing the outcome of immune responses. This key role in orchestrating immune cells and their natural tropism for tumor microenvironment makes γδ T cells an attractive target for cancer immunotherapy. Here, we review the current understanding of these important interactions and highlight the implications of the crosstalk between γδ T cells and other immune cells in the context of anti-tumor immunity.

Humanized Ovarian Cancer Patient-Derived Xenografts for Improved Preclinical Evaluation of Immunotherapies.

Simple SummaryEpithelial ovarian cancer (EOC) is a heterogenous disease and new combination therapies are employed to improve treatment, decrease disease recurrence, and avoid the development of treatment resistance. Immunotherapy has been suggested to boost the immune system and improve the prognosis of EOC patients. However, overall low response rates and missing reliable biomarkers to stratify patients to their best-suited personalized treatment regime hinder the successful implementation. Our aim is to advance the development and characterization of humanized patient-derived xenograft models aiding to unravel the function and interaction of the unique tumor microenvironment and the immune system in EOC. These developed and clinically relevant humanized models of EOC have the potential to test various immune cell-targeting combination therapies and identify mechanisms in heterogenous EOC cohorts to ultimately allow patient stratification.High-grade serous ovarian cancer (HGSOC) has poor prognosis and new treatment modalities are needed. Immunotherapy, with checkpoint inhibitors, have demonstrated limited impact. To evaluate the suitability for immunotherapeutics, contextualized preclinical models are required to secure meaningful clinical translation. Therefore, we developed and characterized humanized patient-derived xenograft (hu PDX) murine models of HGSOC, which were established by orthotopic implantation of tumor cell suspensions and intravenous injection of CD34+ cells isolated from umbilical cord blood samples. The developing human immune system in NSG and NSGS mice was followed longitudinally by flow cytometry and characterized by mass cytometry with a panel of 34 surface markers. Molecular imaging of tumor burden, survival analysis, and characterization of tumor-infiltrating immune cells was performed to assess the treatment response to anti-PD-1 (nivolumab) monotherapy. Successful generation of hu PDX models was achieved. Mice treated with nivolumab showed a decrease in tumor burden, however no significant survival benefit was identified when compared to untreated controls. No correlation was seen between PD-L1 expression and CD8 T cell infiltration and response parameters. As the characterization showed an immune infiltration of predominantly myeloid cells, similar to what is observed in HGSOC patients, the models may have the potential to evaluate the importance of myeloid cell immunomodulation as well.

Hodgkin Lymphoma: Biology and Differential Diagnostic Problem.

Hodgkin lymphomas (HLs) are lymphoid neoplasms that are morphologically defined as being composed of dysplastic cells, namely, Hodgkin and Reed–Sternberg cells, in a reactive inflammatory background. The biological nature of HLs has long been unclear; however, our understanding of HL-related genetics and tumor microenvironment interactions is rapidly expanding. For example, cell surface overexpression of programmed cell death 1 ligand 1 (CD274/PD-L1) is now considered a defining feature of an HL subset, and targeting such immune checkpoint molecules is a promising therapeutic option. Still, HLs comprise multiple disease subtypes, and some HL features may overlap with its morphological mimics, posing challenging diagnostic and therapeutic problems. In this review, we summarize the recent advances in understanding the biology of HLs, and discuss approaches to differentiating HL and its mimics.

Myeloid Derived Suppressor Cells Migrate in Response to Flow and Lymphatic Endothelial Cell Interaction in the Breast Tumor Microenvironment.

Simple SummaryMyeloid-derived suppressor cells (MDSCs) are a subset of immune cells that contribute to a pro-tumorigenic microenvironment, promoting immunosuppression in multiple tumors. In breast cancer, they have been mostly studied in pre-clinical mouse models. These models allow for examination of the development of pre-metastatic niches and immune infiltration at the primary tumor site, but are difficult to use to extricate complex intercellular signaling events and MDSC migration. Here, we describe the development of a breast tumor microenvironment model that includes primary-derived mouse MDSCs. We use this model to identify interactions that drive MDSC migration through both biophysical and cellular signaling events within the tumor microenvironment.At the site of the tumor, myeloid derived suppressor cells (MDSCs) infiltrate and interact with elements of the tumor microenvironment in complex ways. Within the invading tumor, MDSCs are exposed to interstitial fluid flow (IFF) that exists within the chronic inflammatory tumor microenvironment at the tumor–lymphatic interface. As drivers of cell migration and invasion, the link between interstitial fluid flow, lymphatics, and MDSCs have not been clearly established. Here, we hypothesized that interstitial fluid flow and cells within the breast tumor microenvironment modulate migration of MDSCs. We developed a novel 3D model to mimic the breast tumor microenvironment and incorporated MDSCs harvested from 4T1-tumor bearing mice. Using live imaging, we found that sorted GR1+ splenocytes had reduced chemotactic index compared to the unsorted population, but their speed and displacement were similar. Using our adapted tissue culture insert assay, we show that interstitial fluid flow promotes MDSC invasion, regardless of absence or presence of tumor cells. Coordinating with lymphatic endothelial cells, interstitial fluid flow further enhanced invasion of MDSCs in the presence of 4T1 cells. We also show that VEGFR3 inhibition reduced both MDSC and 4T1 flow response. Together, these findings indicate a key role of interstitial fluid flow in MDSC migration as well as describe a tool to explore the immune microenvironment in breast cancer.

Abstract 6108: In vivo CRISPR screens identified dual function of MEN1-MLL1 in regulating tumor-microenvironment interactions

Abstract INTRODUCTION: CRISPR functional genomic screens have been widely adopted to identify essential genes and potential drug targets in cell line models. However, it is well known that cell line models studied in vitro do not fully capture the biology in patient tumors due to the lack of the tumor microenvironment. The primary objective of this study was to perform functional genomic screens in in vivo models to identify clinically relevant epigenetic vulnerabilities. METHODS: We designed an EpiDrug sgRNA library that target 357 epigenetic regulators in human, and applied this targeted sgRNA library for essentiality screen in 2D cell culture and 3D xenograft models. We subsequently selected targets that can only be identified in vivo for validation. We also investigated the underlying mechanism of the in vivo specific phenotype caused by target knockouts. Finally, we explored the clinical significance of treating selected target with a small molecule inhibitor. RESULTS: We identified MEN1 as the top hit that confers differential essentialities between in vitro and in vivo models. Knockout of MEN1 has no impact on cell proliferation in 2D cell culture but profoundly promotes tumor growth in human lung and colorectal adenocarcinoma cell line derived xenograft models. In syngeneic colon cancer model CT26, knockout of Men1 resulted in faster tumor growth in immune deficient mice, but reduced tumor growth in immune competent mice. Mechanistically, knockout of MEN1/Men1 can cause the redistribution of its interaction partner MLL1/Mll1, a histone methyl transferase, to repeat regions in chromatin and stimulate the expression of double stranded RNA. This resulted in viral mimicry response, which activates a group of genes involved in cytokine-cytokine receptor interaction. Single-cell RNA-seq and CyTOF analysis revealed that activation of the cytokines induces tumor promoting neutrophil and tumor suppressing CD8+ T cell infiltration in immunodeficient and immunocompetent mice, respectively. Using patient data from TCGA, we identified that the expression level of MEN1 is negatively correlated with neutrophil and CD8+ T cell tumor infiltration in most cancer types including lung and colorectal adenocarcinoma. A small molecular inhibitor targeting Men1-Mll1 interaction dramatically reduced CT26 tumor growth in immunocompetent mice, but its phenotype was reversed by CD8 neutralizing antibody. Finally, we demonstrated the strong synergy of the inhibitor with anti-PD-L1 antibody. CONCLUSION: Our study demonstrated the utility of in vivo CRISPR screen in identifying therapeutic targets that modulate tumor-microenvironment interactions, and identified MEN1 as a promising therapeutic targets alone or in combination with immunotherapy. Citation Format: Peiran Su, Yin Liu, Ming-Sound Tsao, Housheng H. He. In vivo CRISPR screens identified dual function of MEN1-MLL1 in regulating tumor-microenvironment interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6108.

Abstract 5077: Proteomics analysis of extracellular vesicles from pancreatic cancer cells and cancer associated fibroblasts

Abstract Extracellular vesicles (EVs) are comprised of lipid bound vesicles secreted by cells into the extracellular environment with various roles in cell-to-cell communication. Recent studies have implicated EVs in cell proliferation, epithelial-mesenchymal transition (EMT), metastasis, angiogenesis, and mediating the interaction of tumor cells and tumor microenvironment. To systematically characterize the EVs associated with pancreatic cancer, we performed proteomic and functional analyses on the protein contents of EVs released from pancreatic cancer lines, CAF cell lines and a normal pancreatic epithelial cell line. More than 1400 non-redundant proteins were identified in each EV proteome derived from these cell lines. The common EV proteins identified in these cell lines were enriched in biological processes such as vesicle-mediated transport and exocytosis. Protein networks relevant to pancreatic tumorigenesis, such as of EMT, complement and coagulation components, were significantly enriched in the EVs from cancer cells or CAFs in comparison to normal pancreatic epithelial cells. These findings support the roles of EVs as a potential mediator in transmitting EMT signals and complement response in the tumor microenvironment and possibly contributing to coagulation defects related to cancer development. Citation Format: Sharon Pan, Lisa A. Lai, Diane M. Simeone, David W. Dawson, Yuanqing Yan, Tatjana Crnogorac-Jurcevic, Ru Chen, Teresa A. Brentnall. Proteomics analysis of extracellular vesicles from pancreatic cancer cells and cancer associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5077.

Abstract 3831: Utilizing decellularized triple-negative breast cancer patient-derived tumor models as drug testing platforms

Abstract Triple-negative breast cancer (TNBC) is an aggressive, heterogenous disease that lacks approved targeted therapeutics. Due to the complexity of tumor microenvironment (TME) interactions in TNBC, it is crucial to identify therapeutic targets that regulate the extracellular matrix (ECM). Traditional 2D TNBC models do not accurately mimic the cell-matrix interactions and structural matrix proteins that are key in maintaining the unique TME in individual patient tumors. Here, we introduce our decellularized TNBC patient-derived xenograft (PDX) models to address this gap in knowledge. Utilizing our decellularized models, we can investigate the unique ECM composition and cell-matrix interactions in PDX models from understudied patients with diverse clinical presentations. We can also use our decellularized tumor scaffolds as drug testing models. We utilize our tissue decellularization method on PDX tumor scaffolds derived from two of our established TNBC PDX models (TU-BcX-56S and TU-BcX-4QX). Decellularization of tumor scaffolds was confirmed by DNA quantification and histological analysis (H&E, Masson’s Trichrome, and Movat’s staining). MDA-MB-231 cells expressing GFP and luciferase were seeded onto scaffolds and treated with 100nM of Paclitaxel, a microtubule-stabilizing cytotoxic chemotherapeutic, or DMSO for 48 hours. Concordantly, cells in 2D culture were treated with 100nM of Paclitaxel or DMSO for 48 hours. Using the IVIS imaging system, we measured the bioluminescence of cells on our seeded scaffolds before treatment and at 24 and 48 hours after treatment. Cells in 2D culture showed a significant decrease in bioluminescent signal (p-value < 0.05) in the Paclitaxel group compared to the DMSO group at 48 hours. Cells seeded on TU-BcX-56S scaffold also showed a significant decrease in bioluminescent signal (p-value < 0.05) in the Paclitaxel group compared to the DMSO group at 48 hours. Interestingly, cells seeded on TU-BcX-4QX scaffold showed a decrease in bioluminescence in both the treatment and control groups at 24 and 48 hours, albeit there was no significant difference. Using qPCR, we investigated scaffold regulation of ECM gene composition in our treatment and control groups. These results demonstrate that the unique ECM composition and architecture of these scaffolds are important drivers of cell behavior. Here, we demonstrate that our decellularized PDX tumor scaffold models can be used as drug testing platforms. Furthermore, our models recapitulate key structural matrix proteins in the TNBC TME, can be used to investigate ECM onco-architecture and composition, and can be used in therapeutic discovery research. Citation Format: Maryl Wright, Khoa Nguyen, Elizabeth Martin, Melyssa Bratton, Bridgette Collins-Burow, Matthew Burow. Utilizing decellularized triple-negative breast cancer patient-derived tumor models as drug testing platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3831.

Increased neutrophil infiltration as a body-wide effect in pancreatic cancer development.

Increased neutrophil infiltration as a body-wide effect in pancreatic cancer development.

SNS-101, a highly selective monoclonal antibody against the active form of VISTA, demonstrates significantly reduced cytokine release.

e14504 Background: Active cancer immunotherapeutics frequently give rise to immune-mediated adverse events (AEs), including cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). On-target, off-tumor activation of myeloid lineage cells (e.g. monocytes) has been implicated as a factor in the generation of these immune-mediated AEs. VISTA (V-domain Ig suppressor of T-cell activation) is an immune checkpoint, which is highly expressed on myeloid cells, and binds PSGL-1 on T-cells, but only when ‘activated’ by protonation at low pH (̃pH 6). Inhibition of the VISTA:PSGL-1 interaction in the acidic tumor microenvironment has been shown to be efficacious in multiple syngeneic murine tumor models. However, antibodies binding to VISTA at physiological pH 7.4 have significant potential to induce dose-limiting toxicities such as CRS and/or ICANS and be prematurely eliminated from circulation through targeted-mediated drug disposition (TMDD), reducing the likelihood of reaching efficacious drug occupancy levels. Among several non-pH-selective antibodies in clinical development, JNJ-61610588 (now CI-8993) induced dose-limiting on-target CRS at subtherapeutic dose levels and exhibited TMDD. To mitigate potential CRS and prevent TMDD, we developed SNS-101, a highly selective monoclonal IgG1 antibody for “active” VISTA, which inhibits the critical interaction with PSGL-1. Methods: Using flow cytometry at physiological pH, we compared SNS-101 with clinical stage non-pH-selective anti-VISTA antibodies and examined binding to VISTA-positive cell populations in human PBMCs as well as in human CD34+ cord blood cells reconstituted BRGSF-HIS mice (humanized BRGSF-HIS mice), which develop both human lymphoid and myeloid compartments. Furthermore, we performed in vitro and in vivo CRS studies in a HUVEC/PBMC co-culture system and humanized BRGSF-HIS mice, respectively. Results: Under physiological pH, non-pH-selective antibodies bound to monocytes, neutrophils and NK cells whereas SNS-101 did not exhibit any significant interactions. CRS assays indicate that the magnitude of induced cytokine levels was significantly higher with non-pH-selective antibodies compared to SNS-101. Conclusions: We assessed the binding profile of SNS-101 vs. non-pH-selective VISTA antibodies. Our results demonstrate that SNS-101 does not bind to VISTA-positive cells in circulating blood. In addition, in vitro and in vivo CRS studies suggest that SNS-101 has a lower risk of inducing CRS compared to non-pH selective anti-VISTA antibodies, alleviating liabilities previously associated with anti-VISTA antibodies. IND-enabling studies, including pharmacokinetic and toxicology studies, are ongoing.

Understanding Tricky Cellular and Molecular Interactions in Pancreatic Tumor Microenvironment: New Food for Thought.

Pancreatic ductal adenocarcinoma (PDAC) represents 90% of all pancreatic cancer cases and shows a high mortality rate among all solid tumors. PDAC is often associated with poor prognosis, due to the late diagnosis that leads to metastasis development, and limited efficacy of available treatments. The tumor microenvironment (TME) represents a reliable source of novel targets for therapy, and even if many of the biological interactions among stromal, immune, and cancer cells that populate the TME have been studied, much more needs to be clarified. The great limitation in the efficacy of current standard chemoterapy is due to both the dense fibrotic inaccessible TME barrier surrounding cancer cells and the immunological evolution from a tumor-suppressor to an immunosuppressive environment. Nevertheless, combinatorial therapies may prove more effective at overcoming resistance mechanisms and achieving tumor cell killing. To achieve this result, a deeper understanding of the pathological mechanisms driving tumor progression and immune escape is required in order to design rationale-based therapeutic strategies. This review aims to summarize the present knowledge about cellular interactions in the TME, with much attention on immunosuppressive functioning and a specific focus on extracellular matrix (ECM) contribution.

Investigating Cutaneous Squamous Cell Carcinoma in vitro and in vivo: Novel 3D Tools and Animal Models.

Cutaneous Squamous Cell Carcinoma (cSCC) represents the second most common type of skin cancer, which incidence is continuously increasing worldwide. Given its high frequency, cSCC represents a major public health problem. Therefore, to provide the best patients’ care, it is necessary having a detailed understanding of the molecular processes underlying cSCC development, progression, and invasion. Extensive efforts have been made in developing new models allowing to study the molecular pathogenesis of solid tumors, including cSCC tumors. Traditionally, in vitro studies were performed with cells grown in a two-dimensional context, which, however, does not represent the complexity of tumor in vivo. In the recent years, new in vitro models have been developed aiming to mimic the three-dimensionality (3D) of the tumor, allowing the evaluation of tumor cell-cell and tumor-microenvironment interaction in an in vivo-like setting. These models include spheroids, organotypic cultures, skin reconstructs and organoids. Although 3D models demonstrate high potential to enhance the overall knowledge in cancer research, they lack systemic components which may be solved only by using animal models. Zebrafish is emerging as an alternative xenotransplant model in cancer research, offering a high-throughput approach for drug screening and real-time in vivo imaging to study cell invasion. Moreover, several categories of mouse models were developed for pre-clinical purpose, including xeno- and syngeneic transplantation models, autochthonous models of chemically or UV-induced skin squamous carcinogenesis, and genetically engineered mouse models (GEMMs) of cSCC. These models have been instrumental in examining the molecular mechanisms of cSCC and drug response in an in vivo setting. The present review proposes an overview of in vitro, particularly 3D, and in vivo models and their application in cutaneous SCC research.

Monensin and its analogues show anti‐glioblastoma activity in an organoid model of cancer

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Despite numerous clinical trials, the standard of care therapy has remained unchanged for the last decade. One of the main problems contributing to the limited effective treatment options and poor overall survival is the lack of models which can reliably recapitulate tumor heterogeneity. Organoids are 3D self‐organized structures which mimic tumor architecture, microenvironment, and cellular interactions which makes them an improved model for anti‐cancer drug discovery.Monensin (MON) is a polyether ionophore antibiotic characterized by wide range of biological properties including anti‐cancer activity. In order to identify more potent compounds based on the scaffold of MON, we investigated the anti‐GBM activity of 14 novel esters and urethanes of MON. In 3D mini‐ring cell viability assays, we identified seven analogues (IC50 = 91.5 ± 54.4 ‐ 291.7 ± 68.8 nM) more potent towards GBM than the parental MON (IC50 = 612.6 ± 184.4 nM). These analogues induced DNA fragmentation in an organoid model of GBM, suggestive of apoptotic cell death. Furthermore, the most potent analog, compound 1, significantly reduced GBM cell migration, induced PARP cleavage and degradation, increased ɣH2AX signaling and increased expression of the autophagy marker LCII. To investigate the activity of these novel compounds in a tumor microenvironment, we have developed a host:tumor hybrid 3D organoid system. For host tissue, we generated human cerebral organoids (COs) from hiPSCs. The COs displayed multiple neural rosettes with a proliferative zone of neural stem cells (Nestin+), neurons (TUJ1+), primitive ventricular system (SOX2+/Ki67+), intermediate zone (TBR2+) and cortical plate (MAP2+). These findings suggest the level of differentiation and development of our COs as equivalent to early stage human fetal brain. We then co‐cultured RFP‐labeled U87MG cells with fully formed COs. After establishing U87MG tumor formation, hybrid organoids were treated with MON or compound 1. Compound 1 significantly reduced U87MG tumor size after four days of treatment. Our findings highlight the therapeutic potential of MON analogues towards GBM and support further research and clinical development of these compounds.

Single-Cell RNA Sequencing Analysis for Oncogenic Mechanisms Underlying Oral Squamous Cell Carcinoma Carcinogenesis with Candida albicans Infection.

Oral squamous cell carcinoma (OSCC) carcinogenesis involves heterogeneous tumor cells, and the tumor microenvironment (TME) is highly complex with many different cell types. Cancer cell–TME interactions are crucial in OSCC progression. Candida albicans (C. albicans)—frequently pre-sent in the oral potentially malignant disorder (OPMD) lesions and OSCC tissues—promotes malignant transformation. The aim of the study is to verify the mechanisms underlying OSCC car-cinogenesis with C. albicans infection and identify the biomarker for the early detection of OSCC and as the treatment target. The single-cell RNA sequencing analysis (scRNA-seq) was performed to explore the cell subtypes in normal oral mucosa, OPMD, and OSCC tissues. The cell composi-tion changes and oncogenic mechanisms underlying OSCC carcinogenesis with C. albicans infec-tion were investigated. Gene Set Variation Analysis (GSVA) was used to survey the mechanisms underlying OSCC carcinogenesis with and without C. albicans infection. The results revealed spe-cific cell clusters contributing to OSCC carcinogenesis with and without C. albicans infection. The major mechanisms involved in OSCC carcinogenesis without C. albicans infection are the IL2/STAT5, TNFα/NFκB, and TGFβ signaling pathways, whereas those involved in OSCC carcinogenesis with C. albicans infection are the KRAS signaling pathway and E2F target down-stream genes. Finally, stratifin (SFN) was validated to be a specific biomarker of OSCC with C. albicans infection. Thus, the detailed mechanism underlying OSCC carcinogenesis with C. albicans infection was determined and identified the treatment biomarker with potential precision medicine applications.

Roles and new Insights of Macrophages in the Tumor Microenvironment of Thyroid Cancer.

Although most thyroid cancers have a good and predictable prognosis, the anaplastic, medullary, and refractory thyroid cancers still prone to recurrence and metastasis, resulting in poor prognosis. Although a number of newly developed targeted therapies have begun to be indicated for the above types of thyroid cancer in recent years, their ability to improve overall survival remain hindered by low efficacy. As the largest component of immune cells in tumor microenvironment, tumor-associated macrophages play a key role in the invasion and metastasis of thyroid cancer. There is much evidence that the immune system, tumor microenvironment and cancer stem cell interactions may revolutionize traditional therapeutic directions. Tumor-associated macrophages have been extensively studied in a variety of tumors, however, research on the relationship between thyroid cancer and macrophages is still insufficient. In this review, we summarize the functions of tumor-associated macrophages in different types of thyroid cancer, their cytokines or chemokines effect on thyroid cancer and the mechanisms that promote tumor proliferation and migration. In addition, we discuss the mechanisms by which tumor-associated macrophages maintain the stemness of thyroid cancer and potential strategies for targeting tumor-associated macrophages to treat thyroid cancer.

The immune microenvironment shapes transcriptional and genetic heterogeneity in chronic lymphocytic leukemia

AbstractIn chronic lymphocytic leukemia (CLL), B-cell receptor signaling, tumor–microenvironment interactions, and somatic mutations drive disease progression. To better understand the intersection between the microenvironment and molecular events in CLL pathogenesis, we integrated bulk transcriptome profiling of paired peripheral blood (PB) and lymph node (LN) samples from 34 patients. Oncogenic processes were upregulated in LN compared with PB and in immunoglobulin heavy-chain variable (IGHV) region unmutated compared with mutated cases. Single-cell RNA sequencing (scRNA-seq) distinguished 3 major cell states: quiescent, activated, and proliferating. The activated subpopulation comprised only 2.2% to 4.3% of the total tumor bulk in LN samples. RNA velocity analysis found that CLL cell fate in LN is unidirectional, starts in the proliferating state, transitions to the activated state, and ends in the quiescent state. A 10-gene signature derived from activated tumor cells was associated with inferior treatment-free survival (TFS) and positively correlated with the proportion of activated CD4+ memory T cells and M2 macrophages in LN. Whole exome sequencing (WES) of paired PB and LN samples showed subclonal expansion in LN in approximately half of the patients. Since mouse models have implicated activation-induced cytidine deaminase in mutagenesis, we compared AICDA expression between cases with and without clonal evolution but did not find a difference. In contrast, the presence of a T-cell inflamed microenvironment in LN was associated with clonal stability. In summary, a distinct minor tumor subpopulation underlies CLL pathogenesis and drives the clinical outcome. Clonal trajectories are shaped by the LN milieu, where T-cell immunity may contribute to suppressing clonal outgrowth. The clinical study is registered at clinicaltrials.gov as NCT00923507.

In situ imaging for tumor microbiome interactions via imaging mass cytometry on single‐cell level

Co-detection of multiplex cancer subtypes and bacteria subtypes in situ is crucial for understanding tumor microbiome interactions in tumor microenvironment. Current standard techniques such as immunohistochemical staining and immunofluorescence staining are limited for their multiplicity. Simultaneously visualizing detailed cell subtypes and bacteria distribution across the same pathological section remains a major technical challenge. Herein, we developed a rapid semi-quantitative method for in situ imaging of bacteria and multiplex cell phenotypes on the same solid tumor tissue sections. We designed a panel of antibody probes labeled with mass tags, namely prokaryotic and eukaryotic cell hybrid probes for in situ imaging (PEHPSI). For application demonstration, PEHPSI stained two bacteria subtypes (lipopolysaccharides (LPS) for Gram-negative bacteria and lipoteichoic acid (LTA) for Gram-positive bacteria) simultaneously with four types of immune cells (leukocytes, CD8 + T-cells, B-cells and macrophages) and four breast cancer subtypes (classified by a panel of 12 human proteins) on the same tissue section. We unveiled that breast cancer cells are commonly enriched with Gram-negative bacteria and almost absent of Gram-positive bacteria, regardless of the cancer subtypes (triple-negative breast cancer [TNBC], HER2+, Luminal A and Luminal B). Further analysis revealed that on the single-cell level, Gram-negative bacteria have a significant correlation with CD8 + T-cells only in HER2+ breast cancer, while PKCD, ER, PR and Ki67 are correlated with Gram-negative bacteria in the other three subtypes of breast cancers. On the cell population level, in TNBC, CD19 expression intensity is up-regulated by approximately 25% in bacteria-enriched cells, while for HER2+, Luminal A and Luminal B breast cancers, the intensity of biomarkers associated with the malignancy, metastasis and proliferation of cancer cells (PKCD, ISG15 and IFI6) is down-regulated by 29%-38%. The flexible and expandable PEHPSI system permits intuitive multiplex co-visualization of bacteria and mammalian cells, which facilitates future research on tumor microbiome and tumor pathogenesis.

Role and regulation of autophagy in cancer

Autophagy is an intracellular self-degradative mechanism which responds to cellular conditions like stress or starvation and plays a key role in regulating cell metabolism, energy homeostasis, starvation adaptation, development and cell death. Numerous studies have stipulated the participation of autophagy in cancer, but the role of autophagy either as tumor suppressor or tumor promoter is not clearly understood. However, mechanisms by which autophagy promotes cancer involves a diverse range of modifications of autophagy associated proteins such as ATGs, Beclin-1, mTOR, p53, KRAS etc. and autophagy pathways like mTOR, PI3K, MAPK, EGFR, HIF and NFκB. Furthermore, several researches have highlighted a context-dependent, cell type and stage-dependent regulation of autophagy in cancer. Alongside this, the interaction between tumor cells and their microenvironment including hypoxia has a great potential in modulating autophagy response in favour to substantiate cancer cell metabolism, self-proliferation and metastasis. In this review article, we highlight the mechanism of autophagy and their contribution to cancer cell proliferation and development. In addition, we discuss about tumor microenvironment interaction and their consequence on selective autophagy pathways and the involvement of autophagy in various tumor types and their therapeutic interventions concentrated on exploiting autophagy as a potential target to improve cancer therapy.

Image-Based Quantification of Gold Nanoparticle Uptake and Localization in 3D Tumor Models to Inform Radiosensitization Schedule.

Gold nanoparticles (GNPs) have shown particular promise as radiosensitizing agents and as complementary drug delivery agents to improve therapeutic index in cancer treatment. Optimal implementation, however, depends critically on the localization of GNPs at the time of irradiation, which, in turn, depends on their size, shape, and chemical functionalization, as well as organism-level pharmacokinetics and interactions with the tumor microenvironment. Here, we use in vitro 3D cultures of A549 lung carcinoma cells, which recapitulate interaction with extracellular matrix (ECM) components, combined with quantitative fluorescence imaging to study how time-dependent localization of ultrasmall GNPs in tumors and ECM impacts the degree of damage enhancement to tumor cells. Confocal imaging of fluorescence-labeled GNPs in 3D culture reveals that nanoparticles are initially embedded in ECM and only gradually accumulate in cancer cells over multiple days. Furthermore, the timing of GNP redistribution from ECM to cellular compartments directly impacts efficacy, with major damage enhancement when irradiation is performed after GNPs have accumulated significantly in 3D tumor nodules. These results underscore the importance of the timing and scheduling in treatment planning to ensure optimal radiosensitization, as well as the necessity of studying these effects in model systems that recapitulate elements of tumor microenvironment interaction.