Synergistic Viro-chemoimmunotherapy in Breast Cancer Enabled by Bioengineered Immunostimulatory Exosomes and Dual-Targeted Coxsackievirus B3

Breast cancer’s immunosuppressive environment hinders effective immunotherapy, but oncolytic viruses hold promise for addressing this challenge by targeting tumor cells and altering the microenvironment. Yet, neutralizing antibodies and immune clearance impede their clinical utility. This study explored microRNA-modified coxsackievirus B3 (miR-CVB3), an innovative oncolytic virus, and its potential in breast cancer treatment. It investigated miR-CVB3’s impact on immune-related proteins and utilized exosomes as both protective shields and delivery carriers. Results demonstrated miR-CVB3’s capacity to reshape immune-related protein profiles toward a more immunostimulatory state and enhance exosome-mediated immune cell activation. Notably, cancer cell-released exosomes encapsulating miR-CVB3 (ExomiR-CVB3) maintained its antitumor cytotoxicity and bolstered its immunostimulatory effects. Moreover, ExomiR-CVB3 shielded miR-CVB3 from neutralizing antibodies and rapid immune clearance when it was systemically administered. Building on these findings, ExomiR-CVB3 was engineered with the AS1411 aptamer and doxorubicin (ExomiR-CVB3/DoxApt), enhancing therapeutic efficacy. This notable approach, combining genomic modification, aptamer surface decoration, and doxorubicin addition, demonstrated safe delivery of CVB3 to cancer cells. Comprehensive in vitro and in vivo analyses revealed selective breast cancer cell targeting, cell death induction, and significant immune cell infiltration within the tumor microenvironment while sparing healthy organs. In summary, this study highlights ExomiR-CVB3/DoxApt as a pioneering breast cancer treatment strategy adaptable for diverse cancer types, offering a potent and versatile approach to reshaping cancer immunotherapy

CVB3 RNA-dependent RNA polymerase 3D is ubiquitinated.

<p>HeLa cells were infected with CVB3 or sham-infected with PBS for 7 h, Cell lysates were collected and immuoprecipitated (IP) with a monoclonal anti-ubiquitin antibody. Protein-ubiquitin conjugates were detected by immunoblots (IB) using a polyclonal anti-3D^pol antibody. Immunoblot for antibody IgG was shown as loading controls. Similar results were observed in three independent experiments.</p

Proteasome inhibitors decrease coxsackieviral RNA expression, viral protein synthesis and viral progeny release in HeLa cells.

<p>HeLa cells were sham-infected with PBS or infected with CVB3 in the presence or absence of MG132 or lactacystin. (A). Seven hours post-infection (pi), positive-stranded viral RNA was determined by <i>in situ</i> hybridization using anti-sense riboprobes for CVB3 (red). Cell nuclei were counterstained with hematoxylin (blue). (B). Cell lysates were collected at 7 h pi and immunoblotted with anti-VP1 and anti-β-actin (loading control) antibodies. (C). Medium was collected from CVB3-infected cells at 16 h pi and virus titer was determined by plaque assays. The data shown are mean±SE (standard errors) from three independent experiments. ^# p<0.001 as compared to CVB3 infection without treatment. (D). Cell viability assay was performed at 16 h pi by the MTS assay which measures mitochondrial function (mean±SE, n = 3). One hundred percent survival was defined as the level of MTS in sham-infected cells in the absence of MG132. ^# p<0.001 as compared to CVB3 infection only without MG132 treatment.</p

DUB inhibition further enhances the inhibitory effect of proteasome inhibitors on CVB3 replication.

<p>HeLa cells were infected with CVB3 or sham-infected with PBS, UCH L1 inhibitor, UCH L3 inhibitor and proteasome inhibitor MG132 were added 1 h pi as indicated. Seven hours pi, cell lysates and supernatant were collected for immunoblotting and plaque assay, respectively. (A). Immunoblot was performed using anti-VP1 and anti-β-actin (loading control) antibodies. Similar results were observed in two independent experiments. (B). Virus titer was measured by plaque assay (Mean±SE, n = 4). Results represent data from three independent experiments. ^# p<0.001 as compared to CVB3 infection only without treatment; ^& p<0.01 as compared to MG132 treatment alone.</p

Knockdown of ubiquitin expression by siRNA reduces CVB3 replication.

<p>HeLa cells were transiently transfected with the ubiquitin siRNA or a scramble control siRNA. Twenty-four hours post-transfection, HeLa cells were infected with CVB3 or sham-infected with PBS. Cell lysates were collected at the indicated timepoints. (A). Immunoblot was performed with anti-ubiquitin and anti-β-actin (loading control) antibodies. (B). Supernatants of infected cells were collected at 7 h pi to measure CVB3 progeny virion release by plaque assay (Mean±SE, n = 4). Results represent data from three independent experiments. * p<0.05 as compared to virus titers in scramble siRNA-transfected cells.</p

CVB3 infection results in increased protein polyubiquitination and decreased free ubiquitin.

<p>(A). HeLa cells were infected with CVB3 or sham-infected with PBS. At different timepoints after viral infection, cell lysates were collected and immunoblotted with anti-ubiquitin, anti-VP1 and anti-β-actin (loading control) antibodies as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002585#s4" target="_blank">Material and Methods</a>”. Protein levels of protein-ubiquitin conjugates (molecular weight starting from 82.2 kDa to approximately 230 kDa) and free ubiquitin were quantitated by densitometric analysis using NIH ImageJ program and normalized to the sham infection, which was arbitrarily set to a value of 1.0. Similar results were observed in two independent experiments. (B). Statistical analysis of protein-ubiquitin conjugates at 3 h, 5 h and 7 h after CVB3 infection. The data represent mean±SE of five different experiments. * p<0.05; ^& p<0.01 as compared to protein expression in sham infection.</p

A proposed model for UPS regulation of CVB3 replication (See text).

<p>Abbreviation: CVB3, coxsackievirus B3; Ub, ubiquitin; DUBs, deubiquitinating enzymes; siRNA, small-interfering RNA; 3D^pol, coxsackievirus RNA-dependent RNA polymerase 3D.</p

Oxygen-Self-Produced Nanoplatform for Relieving Hypoxia and Breaking Resistance to Sonodynamic Treatment of Pancreatic Cancer

Hypoxia as one characteristic hallmark of solid tumors has been demonstrated to be involved in cancer metastasis and progression, induce severe resistance to oxygen-dependent therapies, and hamper the transportation of theranostic agents. To address these issues, an oxygen-self-produced sonodynamic therapy (SDT) nanoplatform involving a modified fluorocarbon (FC)-chain-mediated oxygen delivery protocol has been established to realize highly efficient SDT against hypoxic pancreatic cancer. In this nanoplatform, mesopores and FC chains of FC-chain-functionalized hollow mesoporous organosilica nanoparticle carriers can provide sufficient storage capacity and binding sites for sonosensitizers (IR780) and oxygen, respectively. <i>In vitro</i> and <i>in vivo</i> experiments demonstrate the nanoplatform involving this distinctive oxygen delivery protocol indeed breaks the hypoxia-specific transportation barriers, supplies sufficient oxygen to hypoxic PANC-1 cells especially upon exposure to ultrasound irradiation, and relieves hypoxia. Consequently, hypoxia-induced resistance to SDT is inhibited and sufficient highly reactive oxygen species (ROS) are produced to kill PANC-1 cells and shrink hypoxic PANC-1 pancreatic cancer. This distinctive FC-chain-mediated oxygen delivery method provides an avenue to hypoxia oxygenation and holds great potential in mitigating hypoxia-induced resistance to those oxygen-depleted therapies, <i>e</i>.<i>g</i>., photodynamic therapy, radiotherapy, and chemotherapy

Data_Sheet_2_Nxhl Controls Angiogenesis by Targeting VE-PTP Through Interaction With Nucleolin.zip

Precise regulation of angiogenesis is required for organ development, wound repair, and tumor progression. Here, we identified a novel gene, nxhl (New XingHuo light), that is conserved in vertebrates and that plays a crucial role in vascular integrity and angiogenesis. Bioinformatic analysis uncovered its essential roles in development based on co-expression with several key developmental genes. Knockdown of nxhl in zebrafish causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels and decreased sprouting in the caudal vein plexus. The nxhl gene also affects human endothelial cell behavior in vitro. We found that nxhl functions in part by targeting VE-PTP through interaction with NCL (nucleolin). Loss of ptprb (a VE-PTP ortholo) in zebrafish resulted in defects similar to nxhl knockdown. Moreover, nxhl deficiency attenuates tumor invasion and proteins (including VE-PTP and NCL) associated with angiogenesis and EMT. These findings illustrate that nxhl can regulate angiogenesis via a novel nxhl–NCL–VE-PTP axis, providing a new therapeutic target for modulating vascular formation and function, especially for cancer treatment.</p

Data_Sheet_1_Nxhl Controls Angiogenesis by Targeting VE-PTP Through Interaction With Nucleolin.docx

Precise regulation of angiogenesis is required for organ development, wound repair, and tumor progression. Here, we identified a novel gene, nxhl (New XingHuo light), that is conserved in vertebrates and that plays a crucial role in vascular integrity and angiogenesis. Bioinformatic analysis uncovered its essential roles in development based on co-expression with several key developmental genes. Knockdown of nxhl in zebrafish causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels and decreased sprouting in the caudal vein plexus. The nxhl gene also affects human endothelial cell behavior in vitro. We found that nxhl functions in part by targeting VE-PTP through interaction with NCL (nucleolin). Loss of ptprb (a VE-PTP ortholo) in zebrafish resulted in defects similar to nxhl knockdown. Moreover, nxhl deficiency attenuates tumor invasion and proteins (including VE-PTP and NCL) associated with angiogenesis and EMT. These findings illustrate that nxhl can regulate angiogenesis via a novel nxhl–NCL–VE-PTP axis, providing a new therapeutic target for modulating vascular formation and function, especially for cancer treatment.</p