Changes In Tumor Microenvironment Research Articles

Bioinformatics Analysis of Tumor-Associated Macrophages, Vascular Endothelial Growth Factor and Microvascular Density in Endometrial Carcinomas

Objective: The aim of our study was to investigate the expression of tumor-associated macrophage (TAMs), Vascular Endothelial Growth Factor (VEGF) and regional microvascular density (MVD) in different parts of the endometrial carcinoma based on bioinformatics. Methods: Immunohistochemistry assay were used to detect the density of TAMs in endometrial cancer nest, tumor stroma, and infiltrating marginal and its relationship with clinicopathological parameters, VEGF and MVD. Results: To compare with normal tissue, the TAMs in tumor stroma, cancer nest and infiltrating marginal were significantly higher. The density of TAMs in infiltrating marginal was correlated to the FIGO stage, histological grade, myometrial invasion and also lymph node metastasis in endometrial cancer; while the TAMs in tumor stroma is not related to the FIGO stage. Positive correlation between the density of TAMs in tumor stroma, infiltrating marginal and VEGF and MVD in endometrial cancer was found. Conclusion: The change of tumor microenvironment caused by increased density of TAMs promote tumor microangiogenesis and aggravate the progression of endometrial cancer.

The dynamic change of cancer genome and T-cell receptor repertoire of an early gastric cancer cohort.

e16553 Background: The dynamics of the cancer genome and T-cell receptor (TCR) repertoire are largely unclear during the development of gastric cancer, especially in the early stage. In this study, we studied the characteristics of genome and tumor microenvironment changes chronologically in 15 early gastric cancer (EGC) patients and illustrated their influence on carcinogenesis. Methods: 75 gastric tissues including precancerous lesions, cancer lesions and paired reference gastric samples, and 29 peripheral blood samples were obtained chronologically from 15 patients. All the lesions were categorized into 3 groups according to its pathology type (group1: inflammation or intestinal metaplasia, group 2: low-grade dysplasia (LGD), group 3: EGC). Deep sequencing of whole-exome and CDR3 chain of TCR of each sample was performed. Tumor mutation burden (TMB), TCR repertoire diversity and clonality were evaluated based on the sequencing data. Results: Heterogeneity existed in cancer genome and TCR repertoire among different patients. During carcinogenesis, TMB increased at first and then decreased, reaching its peak at LGD, which was different from what we have known from other advanced cancer. A detailed analysis showed that only part of the driver gene mutations maintained in this process. Compared with T cells infiltrated in reference samples, tumor-infiltrating T cells shared more clones with peripheral blood T cells. Diversity and clonality of TCR repertoire didn’t show significant change with the development of cancer (Shannon index: group 1 = 6.32, group 2 = 6.06, group 3 = 6.09, p = 0.836; Clonality: group 1 = 0.19, group 2 = 0.19, group 3 = 0.17, p = 0.374). Some CDR3 clones appeared in early-stage and maintained during the carcinogenesis process. Conclusions: Our research is the first to analyze the cancer genome and TCR repertoire changes from both the spatial and temporal perspectives during the development of early gastric cancer. Somatic mutations appeared in the very early stage of gastric cancer and may induce the immune microenvironment changes in gastric mucosa.

Cell cycLe inhibitiON to target the EVolution of urOthelial cancer (CLONEVO): A single-arm, open-label window-of-opportunity trial of neoadjuvant abemaciclib in platinum-ineligible muscle invasive bladder cancer patients.

TPS5096 Background: The standard of care for clinically localized muscle-invasive bladder cancer (MIBC) is neoadjuvant platinum-based combination chemotherapy followed by radical cystectomy (RC). Up to 40% of patients (pts) are ineligible to receive cisplatin and proceed to RC without any neoadjuvant therapy. We and others have demonstrated enrichment of molecular alterations in cell cycle genes in MIBC, including copy number losses of CDKN2A in 41% of pts. Abemaciclib is a unique CDK4/6 inhibitor with single agent activity and a target kinome distinct from other CDK4/6 inhibitors. We have demonstrated that CRISPR knockout of CDKN2A increases susceptibility to abemaciclib in bladder cancer cell lines. Beyond tumor-intrinsic effects, abemaciclib also modulates the tumor microenvironment (TME) via upregulating human endogenous retroviral elements and increasing T cell infiltration. Methods: Cell cycLe inhibitiON to target the EVolution of urOthelial cancer (CLONEVO) is a single arm, window-of-opportunity trial of neoadjuvant abemaciclib which will evaluate tumor cell and TME changes in response to abemaciclib. Enrolled pts must be ineligible for platinum-based neoadjuvant therapy for resectable MIBC. Pts receive abemaciclib (200 mg BID PO) for 4 weeks prior to RC. Tumor tissue collected via transurethral resection of bladder tumor (TURBT) and residual tumor at RC undergo single cell RNA sequencing and whole-exome sequencing. Patient-derived organoids and xenografts are generated for a co-clinical trial of abemaciclib alone or in combination. The primary endpoint is the measurement of changes in cell cycle dynamics. Secondary objectives are assessment of toxicity via NCI CTCAE v 5.0 and pathologic downstaging of MIBC. We will perform targeted sequencing of a panel of cell cycle genes in serial plasma and urine cell free DNA to evaluate changes in the variant allele fractions of somatic alterations. The novel design of this trial allows dynamic in vivo assessment of tumor changes and creates a new paradigm for studying tumor evolution in real time. Clinical trials information: NCT03837821. Clinical trial information: NCT03837821 .

Combination of Chinese and western medicine to prevent and reverse resistance of cancer cells to anticancer drugs

Cancer is a common and complex disease with various genetic and environmental risks. Chemotherapy and targeted therapy are one of the main therapeutic approaches for the treatment of advanced cancer patients. However, prolonged patient treatment often provokes drug resistance phenomena that render the therapeutic treatment highly ineffective. Therefore, the development of novel and effective treatment strategies to overcome drug resistance is urgent for cancer therapy. A variety of factors contribute to drug resistance, including tumor cell heterogeneity, drug efflux, drug inactivation, changes in tumor microenvironment, DNA damage repair disorders, epigenetic alterations in cancer cells and so on. However, how to circumvent drug resistance to improve anticancer efficacy remains to be determined. Currently, the most important approach for overcoming drug resistance is drug combination therapeutic regimen, however, it is easy to induce new drug resistance or even multi-drug resistance (MDR) which is defined as the resistance of cancer cells to multiple anticancer drugs with different structures and mechanisms of action. Whereas, the “multi-components and multi-targets” manifestations of Traditional Chinese Medicine (TCM) make them an attractive direction for developing new adjuvant therapeutics to reverse drug resistance. In recent years, several compounds derived from TCM have been reported to augment sensitivity or overcome resistance of anticancer therapeutic drugs, including elemene, bufalin, curcumin, magnolol, honokiol, resveratrol, quercetin, and so on. Thus, the combination of TCM and conventional anticancer drugs will hopefully provide a novel strategy for enhancing the effects of anticancer strategies and overcoming their resistance in the treatment of cancer patients. In the current paper, we review the molecular mechanisms of drug resistance and the therapeutic strategies. We also discuss the current basic research and clinical studies in overcoming drug resistance from Traditional Chinese Medicine. Finally, we focus the integrated traditional Chinese and Western medicine as a promising new therapeutic approach to enhance the effects of anticancer strategies or overcome their resistance in the treatment of cancer patients.

Inhibigens, personal neoantigens that drive suppressive T cell responses, abrogate protection of therapeutic anti-tumor vaccines

Abstract Therapeutic cancer vaccine efficacy is reliant on tumor-specific neoantigens. Empirical methods to identify relevant neoantigens may overcome poor predictive values of current in silico approaches. The ATLAS™ platform is a personalized bioassay that uses high-throughput screening of autologous APCs and T cells against a patient’s mutanome to identify neoantigens that are characterized as stimulatory or inhibitory (inhibigens) based on changes in T cell cytokine secretion. In a B16F10 murine model, therapeutic vaccination with ATLAS-identified neoantigens resulted in arrested tumor growth with durable immune responses that protected mice from re-challenge. Mice that were vaccinated with an inhibigen and neoantigen combination had significantly abrogated tumor protection relative to controls. IFNγ ELISpot analysis revealed that inhibigen vaccination suppressed all antigen-specific immune responses, suggesting that inhibigens can be immunodominant. Inhibigen vaccinated mice had multiple tumor microenvironment changes including reduced T cell infiltration, altered CD8+/CD4+ T cell ratios and increased inhibitory marker expression relative to controls. These results indicate that the presence of inhibigens in an otherwise protective vaccine can alter the tumor microenvironment and abolish T cell-mediated protection. Epitope mapping of neoantigens and inhibigens and tetramer design for differential gene signature analysis of inhibigen-specific T cells is being performed. These data suggest that identification and exclusion of inhibigens from cancer vaccines is critical to prevent unintended pro-tumor responses.

Permissive State of EMT: The Role of Immune Cell Compartment.

The Epithelial to Mesenchymal Transition (EMT) type 3 is a reversible dynamic process recognized as a major determinant of the metastatic event, although many questions regarding its role throughout this process remain unanswered. The ability of cancer cells to migrate and colonize distant organs is a key aspect of tumor progression and evolution, requiring constant tumor cells and tumor microenvironment (TME) changes, as well as constant changes affecting the cross-talk between the two aforementioned compartments. Alterations affecting tumor cells, such as transcription factors, trans-membrane receptors, chromatin remodeling complexes and metabolic pathways, leading to the disappearance of the epithelial phenotype and concomitant gaining of the undifferentiated mesenchymal phenotype are undoubtedly major players of the EMT process. However, several lines of evidence point out toward a more critical role of TME composition in creating an “EMT-permissive state.” The “EMT-permissive state” consists in changes affecting physical and biochemical properties (i.e., stiffness and/or hypoxia) as well as changes of the TME cellular component (i.e., immune-cell, blood vessel, lymphatic vessels, fibroblasts, and fat cells) that favor and induce the epithelial mesenchymal transition. In this mini review, we will discuss the role of the tumor microenvironment cellular component that are involved in supporting the EMT, with particular emphasis on the immune-inflammatory cells component.

Perineural Invasion and Postoperative Adjuvant Chemotherapy Efficacy in Patients With Gastric Cancer.

Purpose: There is currently a lack of validated predictors for adjuvant chemotherapy efficacy in patients with gastric cancer (GC). Perineural invasion (PNI) is the process of neoplastic invasion of the nerves, accompanied by tumor microenvironment (TME) changes. TME can determine treatment outcome while the impact of PNI on chemotherapy efficacy remains unknown in GC. We investigated the association between PNI and the efficacy of postoperative adjuvant chemotherapy in patients with resected GC.Methods: Patients who underwent radical resection of stage IB-III GC with or without fluoropyrimidine (FU)-based adjuvant chemotherapy were retrospectively selected from two separate patient cohorts. PNI was confirmed with S100 immunohistochemistry (IHC). Tumor hypoxia and activity of selected pathways were quantified by mRNA-based signature scoring based on publicly available data. A hypoxia biomarker, ERO1A, and a FU resistance biomarker, thymidylate synthase (TS), were assessed by IHC, respectively.Results: Two cohorts included 223 and 599 patients, respectively. Adjuvant chemotherapy significantly improved overall survival (OS) and disease-free survival (DFS) in PNI-positive but not in PNI-negative patients, which was not impacted by stages. Multivariate models showed that adjuvant chemotherapy was an independent predictor for OS and DFS in PNI-positive patients in both cohorts. For TME, PNI-negative tumors were more hypoxic than were PNI-positive tumors, and displayed relative up-regulation of signaling along the pathways that are important in FU metabolism or resistance. Expressions of ERO1A and TS significantly decreased in PNI-positive compared to PNI-negative tumors.Conclusions: PNI might help predict adjuvant chemotherapy benefit in patients with resected GC. Validation in prospective studies is required. Novel treatment strategies need to be developed in PNI-negative patients.

Cell cycLe inhibitiON to target the EVolution of urOthelial cancer (CLONEVO): A single-arm, open-label window-of-opportunity trial of neoadjuvant abemaciclib in platinum-ineligible muscle invasive bladder cancer patients.

TPS606 Background: The standard of care for clinically localized muscle-invasive bladder cancer (MIBC) is neoadjuvant platinum-based combination chemotherapy followed by radical cystectomy (RC). Up to 40% of patients (pts) are ineligible to receive cisplatin and proceed to RC without any neoadjuvant therapy. We and others have demonstrated enrichment of molecular alterations in cell cycle genes in MIBC, including copy number losses of CDKN2A in 41% of pts. Abemaciclib is a unique CDK4/6 inhibitor with single agent activity and a target kinome distinct from other CDK4/6 inhibitors. We have demonstrated that CRISPR knockout of CDKN2A increases susceptibility to abemaciclib in bladder cancer cell lines. Beyond tumor-intrinsic effects, abemaciclib also modulates the tumor microenvironment (TME) via upregulating human endogenous retroviral elements and increasing T cell infiltration. Methods: Cell cycLe inhibitiON to target the EVolution of urOthelial cancer (CLONEVO) is a single arm, window-of-opportunity trial of neoadjuvant abemaciclib which will evaluate tumor cell and TME changes in response to abemaciclib. Enrolled pts must be ineligible for platinum-based neoadjuvant therapy for resectable MIBC. Pts receive abemaciclib (200 mg BID PO) for 4 weeks prior to RC. Tumor tissue collected via transurethral resection of bladder tumor (TURBT) and residual tumor at RC undergo single cell RNA sequencing and whole-exome sequencing. Patient-derived organoids and xenografts are generated for a co-clinical trial of abemaciclib alone or in combination. The primary endpoint is the measurement of changes in cell cycle dynamics. Secondary objectives are assessment of toxicity via NCI CTCAE v 5.0 and pathologic downstaging of MIBC. We will perform targeted sequencing of a panel of cell cycle genes in serial plasma and urine cell free DNA to evaluate changes in the variant allele fractions of somatic alterations. The novel design of this trial allows dynamic in vivo assessment of tumor changes and creates a new paradigm for studying tumor evolution in real time. Clinical trial information: NCT03837821.

A Comprehensive Picture of Extracellular Vesicles and Their Contents. Molecular Transfer to Cancer Cells.

Critical processes such as growth, invasion, and metastasis of cancer cells are sustained via bidirectional cell-to-cell communication in tissue complex environments. Such communication involves the secretion of soluble factors by stromal cells and/or cancer cells within the tumor microenvironment (TME). Both stromal and cancer cells have been shown to export bilayer nanoparticles: encapsulated regulatory molecules that contribute to cell-to-cell communication. These nanoparticles are known as extracellular vesicles (EVs) being classified into exosomes, microvesicles, and apoptotic bodies. EVs carry a vast repertoire of molecules such as oncoproteins and oncopeptides, DNA fragments from parental to target cells, RNA species (mRNAs, microRNAs, and long non-coding RNA), and lipids, initiating phenotypic changes in TME. According to their specific cargo, EVs have crucial roles in several early and late processes associated with tumor development and metastasis. Emerging evidence suggests that EVs are being investigated for their implication in early cancer detection, monitoring cancer progression and chemotherapeutic response, and more relevant, the development of novel targeted therapeutics. In this study, we provide a comprehensive understanding of the biophysical properties and physiological functions of EVs, their implications in TME, and highlight the applicability of EVs for the development of cancer diagnostics and therapeutics.

Anti-tumor immune response varies among individuals: A gene expression profiling of mouse melanoma

Melanoma is amongst the most aggressive malignant tumors. The purpose of this study is to detect the tumor microenvironment systematically using multi-omics analyses and to propose strategies for precision medicine. Multiple factors of tumor microenvironment contribute to the drug resistance and immune surveillance failure. Here we analyzed genome mutations and characterized the immune state of tumor microenvironments in mouse melanoma by whole exome sequencing (WES) and RNA sequencing (RNA-Seq) approaches. Somatic mutation analysis revealed 35.1% novel mutations in mouse tumors when compared with B16F10 cell line, provided a basis for multi-site sequencing for accurate neoantigen selection. Mutation cluster, gene expression comparison, and gene ontology (GO) analyses by R packages proved DNA repair damage, inflammation, slower cell division, and metabolic change in tumor microenvironment. Further analyses of T-cell receptor (TCR) sequences, immune signaling pathway activation, tumor infiltrated immune cells and chemokine expression revealed extensive difference of antitumor immune response among individuals. Our study revealed the characteristics of tumor microenvironment with mouse melanoma model, suggested the need of comprehensive genome mutations and personal immune state analyses for cancer precision medicine.

Progress and current status of molecule-targeted therapy and drug resistance in gastric cancer

Gastric cancer is one of the most common malignant tumors in the world. In China, its morbidity and mortality are second only to lung cancer. Chemotherapy combined with targeted therapy brings survival benefits to patients with advanced gastric cancer. Targets for targeted therapy of gastric cancer include human epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor (VEGF), mammalian target of rapamycin (mTOR) and Claudin 18.2 (CLDN 18.2). The main challenge of tumor molecule-targeted drugs is resistance. The main mechanisms of drug resistance include tumor establishment of compensatory signaling pathways, target protein changes, tumor microenvironment changes, tumor heterogeneity and tumor adaptation to targeted drugs. The combined action of multiple drug resistance mechanisms promotes the development of targeted drug resistance. In order to attract the attention of researchers, this paper reviews the mechanisms of drug resistance in gastric cancer-targeted therapy. In addition, the research status of drug resistance in molecule-targeted therapy of gastric cancer is summarized. It is of great clinical significance to explore the drug resistance mechanisms of targeted drugs and reverse drug resistance in gastric cancer. Last, the future development of molecule-targeted therapy is prospected.

Mechanistic Insights into Chemoresistance Mediated by Oncogenic Viruses in Lymphomas.

Viral lymphomagenesis induced by infection with oncogenic viruses, such as Kaposi’s sarcoma associated herpesvirus (KSHV), Epstein–Barr virus (EBV) and human T-cell leukemia virus (HTLV-1), represents a group of aggressive malignancies with a diverse range of pathological features. Combined chemotherapy remains the standard of care for these virus-associated lymphomas; however, frequent chemoresistance is a barrier to achieving successful long-term disease-free survival. There is increasing evidence that indicates virus-associated lymphomas display more resistance to cytotoxic chemotherapeutic agents than that observed in solid tumors. Although the tumor microenvironment and genetic changes, such as key oncogene mutations, are closely related to chemoresistance, some studies demonstrate that the components of oncogenic viruses themselves play pivotal roles in the multidrug chemoresistance of lymphoma cells. In this review, we summarize recent advances in the understanding of the mechanisms through which oncogenic viruses mediate lymphoma cell chemoresistance, with a particular focus on KSHV and EBV, two major oncogenic viruses. We also discuss the current challenges to overcome these obstacles in the treatment of virus-associated lymphomas.

Inducing Tumor Suppressive Microenvironments through Genome Edited CD47\u2212/\u2212 Syngeneic Cell Vaccination

Tumors can escape from the immune system by overexpressing CD47 and other checkpoint blockades. CD47 is expressed ubiquitously by all cells in the body, posing an obstacle for CD47 blocking treatments due to their systemic toxicity. We performed a study to determine how the tumor microenvironment changes after vaccination with genome edited CD47−/− syngeneic tumor cells. We discovered that inactivated CD47-depleted mouse melanoma cells can protect mice from melanoma. Our animal study indicated that 33% of vaccinated mice remained tumor-free, and 100% of mice had 5-fold reduced growth rates. The characterization of immunomodulatory effects of the vaccine revealed a highly anti-tumorigenic and homogenous microenvironment after vaccination. We observed consistently that in the tumors that failed to respond to vaccines, there were reduced natural killer cells, elevated regulatory T cells, M2-type macrophages, and high PD-L1 expression in these cells. These observations suggested that the tumor microenvironments became more suppressive to tumor growth after vaccination, suggesting a potential new immunotherapy for solid tumors.

Glutamine deprivation counteracts hypoxia-induced chemoresistance

The microenvironment of solid tumors is a key determinant of therapy efficacy. The co-occurrence of oxygen and nutrient deprivation is a common phenomenon of the tumor microenvironment and associated with treatment resistance. Cholangiocarcinoma (CCA) is characterized by a very poor prognosis and pronounced chemoresistance. A better understanding of the underlying molecular mechanisms is urgently needed to improve therapy strategies against CCA. We sought to investigate the importance of the conditionally essential amino acid glutamine, a centrally important nutrient for a variety of solid tumors, for CCA. Glutamine levels were strongly decreased in CCA samples and the growth of established human CCA cell lines was highly dependent on glutamine. Using gradual reduction of external glutamine, we generated derivatives of CCA cell lines which were able to grow without external glutamine (termed glutamine-depleted (GD)). To analyze the effects of coincident oxygen and glutamine deprivation, GD cells were treated with cisplatin or gemcitabine under normoxia and hypoxia. Strikingly, the well-established phenomenon of hypoxia-induced chemoresistance was completely reversed in GD cells. In order to better understand the underlying mechanisms, we focused on the oncogene c-Myc. The combination of cisplatin and hypoxia led to sustained c-Myc protein expression in wildtype cells. In contrast, c-Myc expression was reduced in response to the combinatorial treatment in GD cells, suggesting a functional importance of c-Myc in the process of hypoxia-induced chemoresistance. In summary, these findings indicate that the mechanisms driving adaption to tumor microenvironmental changes and their relevance for the response to therapy are more complex than expected.

Radiation-Induced Changes in Tumor Vessels and Microenvironment Contribute to Therapeutic Resistance in Glioblastoma.

Glioblastoma (GBM) is a largely fatal and highly angiogenic malignancy with a median patient survival of just over 1 year with radiotherapy (RT). The effects of RT on GBM remain unclear, although increasing evidence suggests that RT-induced alterations in the brain microenvironment affect the recurrence and aggressiveness of GBM. Glioma stem cells (GSCs) in GBM are resistant to conventional therapies, including RT. This study aimed to investigate the effect of radiation on tumor growth and the GSC microenvironment in a mouse model of glioma. To evaluate the growth-inhibitory effects of ionizing radiation on GSCs, tumor volume was measured via anatomical magnetic resonance imaging (MRI) after the intracranial injection of 1 × 104 human patient-derived GSCs (83NS cells), which exhibit marked radioresistance. When a tumor mass of ~5 mm3 was detected in each animal, 10 Gy of cranial irradiation was administered. Tumor progression was observed in the orthotopic xenografted GSC tumor (primary tumor) from a detectable tumor mass (5 mm3) to a lethal tumor mass (78 mm3) in ~7 d in the non-irradiated group. In the RT group, tumor growth was halted for almost 2 weeks after administering 10 Gy cranial irradiation, with tumor growth resuming thereafter and eventually approaching a lethal mass (56 mm3) 21 d after radiation. Radiation therapy yielded good therapeutic effects, with a 2-fold increase in GSC glioma survival; however, tumor relapse after RT resulted in higher mortality for the mice with a smaller tumor volume (p = 0.029) than the non-irradiated tumor-bearing mice. Moreover, tumor regrowth after IR resulted in different phenotypes associated with glioma aggressiveness compared with the non-irradiated mice; the apparent diffusion coefficient by diffusion MRI decreased significantly (p < 0.05, 0 Gy vs. 10 Gy) alongside decreased angiogenesis, abnormal vascular dilatation, and upregulated CD34, VWF, AQP1, and AQP4 expression in the tumor. These findings demonstrate that radiation affects GSCs in GBM, potentially resulting in therapeutic resistance by changing the tumor microenvironment. Thus, the results of this study suggest potential therapeutic targets for overcoming the resistance of GBMs to RT.

Phase II Study of the Combination of Daratumumab, Ixazomib, Pomalidomide, and Dexamethasone As Salvage Therapy in Relapsed/Refractory Multiple Myeloma: Results of a Safety Run-in Analysis

Background: Triplet combination strategies have widely been accepted as the standard of care for the management of multiple myeloma due to improved outcomes as compared to doublets. The combination of daratumumab, pomalidomide and dexamethasone (DPd) has previously demonstrated deep and durable responses, including high rates of MRD negativity, in a heavily pretreated patient population. Quadruplet regimens offer an opportunity to further improve upon these results. We report preliminary findings from an ongoing phase 2 multicenter trial of the addition of ixazomib to the combination of DPd in patients with relapsed/refractory multiple myeloma. The primary objective is to determine overall response rate and the safety and tolerability of this novel regimen. Key secondary endpoints include PFS, OS and MRD negativity rates. Methods: Eligible patients may have received ≥1 and ≤3 prior lines of therapy, have had no prior exposure to daratumumab or ixazomib, and may have not progressed on prior pomalidomide. Patients receive daratumumab 16mg/kg IV weekly x 8 doses, biweekly x 8 doses, then monthly, pomalidomide 4mg PO days 1-21/28, ixazomib 4mg PO days 1,8,15 every 28 days, and dexamethasone 40mg PO weekly. Patients continue on therapy until disease progression, intolerability or preference. Dose-limiting toxicities (DLT) were defined as grade 3-4 hematologic adverse events (AE) or any AE that required a dose modification of pomalidomide or ixazomib at the lowest dose levels on a dose de-escalation plan. An interim safety review was performed after the first 6 patients were enrolled and completed the DLT observation period, which is the first cycle (28 days) since the start of a new dose level of pomalidomide and/or ixazomib. Results: At the time of this analysis, six patients have been enrolled and treated, and completed the DLT observation period. Patients had a median age of 62 (range 52-65) and median number of 2 prior lines of therapy (range 1-2). All patients were refractory to lenalidomide and pomalidomide-naïve. Common adverse events (AEs) included neutropenia, thrombocytopenia, GI upset, and upper respiratory infection. Grade 3-4 AEs were predominantly hematologic including neutropenia and thrombocytopenia, but also included grade 3 hypertension in 1 patient, and grade 3 hypophosphatemia, grade 4 hypokalemia, and grade 3 small bowel infection in 1 patient. No IRR &gt; grade 2 occurred with daratumumab administration. No DLTs occurred in the first six patients in the safety run-in. The overall response rate of the cohort is 100% with 3 patients achieving a stringent complete response (CR), and 3 patients achieving a very good partial response (VGPR) after a median of 7 cycles of treatment. One patient discontinued therapy due to influenza A, the other five remain on therapy. Minimal residual disease assessments are being performed by EuroFlow for patients in VGPR or better due to concern for daratumumab interference. Pharmacodynamic changes in patients' tumor microenvironments were established by custom panel mass cytometry to include T-cell memory and activated subpopulations, B-cell content, NK-cell subpopulations as well as MDSCs, Tregs and T-exhaustive markers, monocytes and dendritic cells. Conclusion: The quadruplet regimen DIPd in patients with relapsed/refractory myeloma is a well-tolerated combination and has shown early safety in an initial safety run-in analysis. Enrollment continues in an expansion cohort to assess efficacy at multiple sites with the University of California Hematologic Malignancies Consortium. Figure Disclosures Costello: Takeda: Honoraria, Research Funding; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

A proteomic clock for malignant gliomas: The role of the environment in tumorigenesis at the presymptomatic stage.

Malignant gliomas remain incurable with a poor prognosis despite of aggressive treatment. We have been studying the development of brain tumors in a glioma rat model, where rats develop brain tumors after prenatal exposure to ethylnitrosourea (ENU), and there is a sizable interval between when the first pathological changes are noted and tumors become detectable with MRI. Our aim to define a molecular timeline through proteomic profiling of the cerebrospinal fluid (CSF) such that brain tumor commitment can be revealed earlier than at the presymptomatic stage. A comparative proteomic approach was applied to profile CSF collected serially either before, at and after the time MRI becomes positive. Elastic net (EN) based models were developed to infer the timeline of normal or tumor development respectively, mirroring a chronology of precisely timed, “clocked”, adaptations. These CSF changes were later quantified by longitudinal entropy analyses of the EN predictive metric. False discovery rates (FDR) were computed to control the expected proportion of the EN models that are due to multiple hypothesis testing. Our ENU rat brain tumor dating EN model indicated that protein content in CSF is programmed even before tumor MRI detection. The findings of the precisely timed CSF tumor microenvironment changes at presymptomatic stages, deviation from the normal development timeline, may provide the groundwork for the understanding of adaptation of the brain environment in tumorigenesis to devise effective brain tumor management strategies.

Fluorescence In Situ Hybridization (FISH) Detection of Viral Nucleic Acids in Oncolytic H-1 Parvovirus-Treated Human Brain Tumors.

Fluorescence in situ hybridization (FISH) is a specific, sensitive, accurate, and reliable technique widely applied in both research and clinic. Here we describe the detailed protocol of a FISH method established by us to serve the scientific purposes of the first oncolytic parvovirus clinical trial (ParvOryx01). This trial was launched in Germany in 2011. After trial completion in 2015, results were published in Molecular Therapy in 2017. The primary purpose of the trial was to evaluate the safety of an oncolytic parvovirus, H-1PV (ParvOryx), in recurrent glioblastoma patients. In addition, the efficiency of H-1PV tumor targeting after intratumoral or systemic virus administration was assessed by FISH detection of viral nucleic acids (genomic single-stranded DNA, mRNA and parvovirus double-stranded replicative forms) in formalin-fixed paraffin-embedded glioblastoma tissues resected at day 10 after ParvOryx treatment. The FISH method allowed the detection-for the first time in humans-of H-1PV replication markers in brain tumors of parvovirus-treated patients. A protocol combining mRNA FISH with simultaneous immunofluorescent staining for tumor and tumor microenvironment markers was also developed and is described here, in order to better characterize H-1PV cellular targets and H-1PV treatment-associated tumor microenvironment changes.

Electroporation-induced changes in tumor vasculature and microenvironment can promote the delivery and increase the efficacy of sorafenib nanoparticles

Blood vessels, the extracellular space, and the cell membrane represent physiologic barriers to nanoparticle-based drug delivery for cancer therapy. We demonstrate that electroporation (EP) can assist in the delivery of dye stabilized sorafenib nanoparticles (SFB-IR783) by increasing the permeability of endothelial monolayers, improving diffusion through the extracellular space in tumorspheres, and by disrupting plasma membrane function in cancer cells. These changes occur in a dose-dependent fashion, increasing proportionally with electric field strength. Cell death from irreversible electroporation (IRE) was observed to contribute to the persistent transport of SFB-IR783 through these physiologic barriers. In a model of mice bearing bilateral xenograft HCT116 colorectal tumors, treatment with EP resulted in the immediate and increased uptake of SFB-IR783 when compared with the untreated contralateral tumor. The uptake of SFB-IR783 was independent of direct transfection of cells through EP and was mediated by changes in vascular permeability and extracellular diffusion. The combination of EP and SFB-IR783 was observed to result in 40% reduction in mean tumor diameter when compared with sham treatment (p < .05) at the time of sacrifice, which was not observed in cohorts treated with EP alone or SFB-IR783 alone. Treatment of tumor with EP can augment the uptake and increase the efficacy of nanoparticle therapy.

6-Gingerol stabilized the p-VEGFR2/VE-cadherin/\u03b2-catenin/actin complex promotes microvessel normalization and suppresses tumor progression

BackgroundAnti-angiogenic therapies demonstrate anti-tumor effects by decreasing blood supply to tumors and inhibiting tumor growth. However, anti-angiogenic therapy may leads to changes in tumor microenvironment and increased invasiveness of tumor cells, which in turn promotes distant metastasis and increased drug resistance.MethodsThe CO-IP assays, N-STORM and cytoskeleton analysis were used to confirm the mechanism that p-VEGFR2/VE-cadherin/β-catenin/actin complex regulates vascular remodeling and improves the tumor microenvironment. 6-gingerol (6G), the major bioactive component in ginger, stabilized this complex by enhancing the binding of VEGFa to VEGFR2 with non-pathway dependent. Biacore, pull down and molecular docking were employed to confirm the interaction between 6G and VEGFR2 and enhancement of VEGFa binding to VEGFR2.ResultsHere, we report that microvascular structural entropy (MSE) may be a prognostic factor in several tumor types and have potential as a biomarker in the clinic. 6G regulates the structural organization of the microvascular bed to decrease MSE via the p-VEGFR2/VE-cadherin/β-catenin/actin complex and inhibit tumor progression. 6G promotes the normalization of tumor vessels, improves the tumor microenvironment and decreases MSE, facilitating the delivery of chemotherapeutic agents into the tumor core and thereby reducing tumor growth and metastasis.ConclusionsThis study demonstrated the importance of vascular normalization in tumor therapy and elucidated the mechanism of action of ginger, a medicinal compound that has been used in China since ancient times.