Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication.We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps.The experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles.This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.
Most colorectal cancer (CRC) patients present with a microsatellite-stable phenotype, rendering them resistant to immune checkpoint inhibitors (ICIs). Among the contributors to ICI resistance, tumor-derived extracellular vesicles (TEVs) have emerged as critical players. Previously we demonstrated that autologous transfer of TEVs without miR-424 can induce tumor antigen-specific immune responses in CRC models. Therefore, we postulated that allogeneic TEVs, modified to lack miR-424 and derived from an MC38 cells, could induce CD8
Abstract Immunotherapies are used as adjuvant therapies for cancers after surgical resection or multiple lines of chemotherapies and or targeted therapies. Tumor-draining lymph nodes (TdLNs) are usually the first sites of metastasis; therefore, they are routinely resected for diagnostic and/ or treatment proposes. However, knowledge of how traditional cancer treatments affect immunotherapies is still very limited. Here, we present data from multiple mouse models that mimic different conditions demonstrating that TdLNs are critical for anti-tumor immunity initiation by involving tumor antigen-specific T cell priming. However, the development of immunosuppression in TdLNs and dissemination of tumor antigen-specific T cells make TdLNs less important in late-stage diseases. Removal of TdLNs concurrent with primary tumor resection did not affect immune checkpoint blockade response for localized secondary tumors. In another arm, we studied whether the timing of chemotherapy and immunotherapy in combination would affect treatment response. Using 5-fluorouracil (5-FU) cytotoxic chemotherapy as induction therapy, then followed sequentially by immune checkpoint blockade as maintenance treatment, showed better responses than adding immune checkpoint blockades concurrently with 5-FU. Immune profiling of tumors revealed that using 5-FU as an induction treatment increased tumor visibility to the immune cells, decreased immunosuppressive cells in the tumor microenvironment, and limited chemotherapy-induced T cell depletion. Collectively, our study shows the impact of TdLNs and traditional cytotoxic cancer treatment on immunotherapy response and provides essential considerations for designing successful immunotherapy strategies in complex clinical conditions.
Pancreatic cancer stromal microenvironment is considered to be the major reason for failure of conventional and targeted therapy for this disease. The desmoplastic stroma, comprising mainly collagen and glycosaminoglycans like hyaluronan (HA), is responsible for compression of vasculature in the tumor resulting in impaired drug delivery and poor prognosis. Minnelide, a water-soluble prodrug of triptolide currently in phase I clinical trial, has been very effective in multiple animal models of pancreatic cancer. However, whether Minnelide will have efficacious delivery into the tumor despite the desmoplastic stroma has not been evaluated before.Patient tumor-derived xenografts (PDX) and spontaneous pancreatic cancer mice were treated with 0.42 and 0.21 mg/kg body weight for 30 days. Stromal components were determined by IHC and ELISA-based assays. Vascular functionality and drug delivery to the tumor were assessed following treatment with Minnelide.Our current study shows that treatment with Minnelide resulted in reduction of ECM components like HA and collagen in the pancreatic cancer stroma of both the spontaneous KPC mice as well as in patient tumor xenografts. Furthermore, treatment with Minnelide improved functional vasculature in the tumors resulting in four times more functional vessels in the treated animals compared with untreated animals. Consistent with this observation, Minnelide also resulted in increased drug delivery into the tumor compared with untreated animals. Along with this, Minnelide also decreased viability of the stromal cells along with the tumor cells in pancreatic adenocarcinoma.In conclusion, these results are extremely promising as they indicate that Minnelide, along with having anticancer effects is also able to deplete stroma in pancreatic tumors, which makes it an effective therapy for pancreatic cancer.
Use of the autophagy-related markers beclin-1 (BECN1) and microtubule-associated protein light chain 3B (LC3B) as prognostic markers has been extensively investigated in various kinds of cancers. However, their prognostic roles are still controversial and not firmly validated. We systematically reviewed the evidence from various studies concerning the relationship between BECN1 and LC3B expression in cancers and overall survival (OS)/disease-free survival (DFS) to elucidate this issue. PubMed, Embase, Cochrane Library, and China National Knowledge Infrastructure (CNKI) were searched in July 2013 (then updated in April 2014) to identify eligible cohort studies that reported associations between BECN1 or LC3B expression and OS/DFS in cancer patients. Combined hazard ratios (HRs) with 95 % confidence intervals (95 % CIs) were pooled using fixed-effects or random-effects models according to heterogeneity in different groups. A total of 23 studies in distinct cancers were eligible for systematic review and meta-analysis. Our pooled results identified that a high expression of BECN1 is associated with favorable OS in gastric cancer (HR = 0.49, 95 % CI = 0.34-0.72) and lymphoma (HR = 0.25, 95 % CI = 0.11-0.57), whereas a high expression of LC3B predicts adverse OS in breast cancer (HR = 1.98, 95 % CI = 1.25-3.13). This systematic review and meta-analysis indicated that the autophagy-related marker BECN1 might be a predictive factor of favorable prognosis in gastric cancer, breast cancer, and lymphoma and LC3B might predict unfavorable prognosis of breast cancer. Nevertheless, due to the limited number and retrospective design of the original studies, more powerful prospective cohorts are required to verify these conclusions.
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