Cancer Models Research Articles

Synthesis and in vitro cytotoxicity of benzoxazole-based PPARα/γ antagonists in colorectal cancer cell lines.

A series of benzoxazole-based amides and sulfonamides were synthesized and evaluated for their human peroxisome proliferator-activated receptor (PPAR)α and PPARγ activity. All tested compounds showed a dual antagonist profile on both PPAR subtypes; based on transactivation results, seven compounds were selected to test their in vitro antiproliferative activity in a panel of eight cancer cell lines with different expression rates of PPARα and PPARγ. 3f was identified as the most cytotoxic compound, with higher potency in the colorectal cancer cell lines HT-29 and HCT116. Compound 3f induced a concentration-dependent activation of caspases and cell-cycle arrest in both colorectal cancer models. Docking experiments were also performed to shed light on the putative binding mode of this novel class of dual PPARα/γ antagonists.

Biosurfactant-amphiphilized hyaluronic acid: A dual self-assembly anticancer nanoconjugate and drug vector for synergistic chemotherapy

Novel amphiphilic nanoconjugates of hyaluronic acid (HA), 50 kDa (HA50) and 100 kDa (HA100), and the lipopeptide biosurfactant surfactin (SF) were developed for potential anticancer applications. Physicochemical characterization indicated the formation of an ester conjugate (HA: SF molar ratio 1: 40) with the HA50-SF derivative exhibiting higher degree of substitution, hydrolytic stability, and surface activity. Self-assembly resulted in nanomicelles with smaller size and greater negative charge relative to SF micelles. Biological data demonstrated distinct anticancer activity of HA50-SF which displayed greater synergistic cytotoxicity and selectivity for MDA-MB 231 and MCF-7 breast cancer cells alongside greater modulation of apoptosis-related biomarkers leading to apoptosis. As bioactive vector for chemotherapeutic agents, the selected HA50-SF nanoconjugate efficiently (70 %) entrapped berberine (BER) producing a sustained release BER-HA50-SF synergistic anticancer nanoformulation. Lactoferrin (Lf) coating for dual HA/Lf targeting endowed Lf/BER-HA50-SF with significantly greater selectivity for both cell lines. A murine Ehrlich breast cancer model provided evidence for the efficacy and safety of Lf/BER-HA50-SF via tumoral, histological, immunohistochemical, molecular and systemic toxicity assessments. Thus, HA-SF nanoconjugates integrating the HA and SF properties and biofunctionalties present a novel biopolymer-biosurfactant platform of benefit to oncology nanomedicine and possibly other applications.

Advances in CAR-NK cell therapy for lung cancer: is it a better choice in the future?

Lung cancer remains one of the leading causes of cancer-related mortality worldwide necessitating the development of innovative therapeutic strategies. Chimeric antigen receptor (CAR) natural killer (NK) cell therapy represents a promising advancement in the field of oncology offering a novel approach to target and eliminate tumor cells with high specificity and reduced risk of immune-related adverse effects. This paper reviews the mechanism, potential targets, and recent advances in CAR-NK cell therapy for lung cancer, including the design and engineering of CAR-NK cells, preclinical studies, and the outcomes of early-phase clinical trials. We highlight the unique advantages of using NK cells, such as their innate ability to recognize and kill cancer cells and their reduced potential for inducing graft-versus-host disease (GvHD) and cytokine release syndrome (CRS) compared to CAR T-cell therapies. Results from recent studies demonstrate significant antitumor activity in lung cancer models with improved targeting and persistence of CAR-NK cells observed in vitro and in vivo. Finally, we discuss the challenges in optimizing CAR-NK cell therapies, including the potential resistance mechanisms. The paper concludes with an outlook on the future directions of CAR-NK cell research and its implications for lung cancer treatment emphasizing the importance of continued innovation and collaboration in the field.

Glycyrrhiza Glabra Extract Modulates Type 1 T Helper (TH1) and Regulatory T Cell-Related Immune Responses in an Animal Model of Breast Cancer.

It is well-known that TH1 and Treg cells exert anti- and pro-tumorigenic activity, respectively. Thus, TH1 cell suppression together with Treg cell hyperactivation contribute to tumor development. Glycyrrhiza glabra (G. glabra) has various immunomodulatory and anti-tumorigenic properties. To explore the impacts of G. glabra extract on different parameters related to TH1 and Treg cells using a breast cancer (BC) model. Four groups of Balb/C mice bearing 4T1 cell-induced BC were treated intraperitoneally with either saline or G. glabra extract at dosages of 50, 100 and 150 mg/kg (G. glabra-50, G. glabra-100, and G. glabra-150, respectively). After sacrificing animals on day 26, the frequency of splenic TH1 and Treg cells, the levels of serum IFN-γ, TGF-β, and IL-12, and intra-tumoral expressions of granzyme-B, T-bet, and FOXP3 were assessed. Compared to untreated tumor control (UTC) group, treatment with G. glabra-50, G. glabra-100, or G. glabra-150 increased the survival rate, percentage of TH1 cells, and T-bet expression. Conversely, they reduced the percentage of Treg cells, and serum TGF-β levels. In comparison to the UTC group, treatment with G. glabra-50 and G. glabra-150 increased the serum IL-12 levels. Treatment with G. glabra-100 and G. glabra-150 boosted granzyme-B expression. Treatment with G. glabra-150 elevated IFN-γ levels, while treatment with G. glabra-50 decreased the FOXP3 expression. IL-12 levels were higher in mice treated with G. glabra-150 compared to those treated with G. glabra-100. Treatment of mice with BC using G. glabra extract improved survival rate, reduced tumor growth, and modulated T cell-mediated immune responses.

Prognostic prediction model of colorectal cancer based on preoperative serum tumor markers.

Preoperative serum tumor markers not only play a role in the auxiliary diagnosis and postoperative monitoring in colorectal cancer (CRC), but also have been found to have potential prognostic value. To analyze whether preoperative serum tumor markers, including carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9), affect the prognosis of CRC. This was a retrospective study conducted in a single center. Patients with nonmetastatic CRC who underwent initial surgery between January 2011 and January 2020 were enrolled and divided into development site and validation site groups at a ratio of 7:3. The independent prognostic factors were screened by Cox regression analysis, and finally, a prognostic nomogram model was established. The newly developed model was tested by internal validation. Eventually, 3526 postoperative patients with nonmetastatic CRC were included in the study. There were 2473 patients at the development site and 1056 patients at the validation site. Age (P < 0.01, HR = 1.042, 95%CI = 1.033-1.051), tumor node metastasis (TNM) classification (P < 0.01, HR = 1.938, 95%CI = 1.665-2.255), preoperative CEA (P = 0.001, HR = 1.393, 95%CI = 1.137-1.707) and CA19-9 (P < 0.01, HR = 1.948, 95%CI = 1.614-2.438) levels were considered independent prognostic factors for patients with nonmetastatic CRC and were used as variables in the nomogram model. The areas under the curve of the development and validation sites were 0.655 and 0.658, respectively. The calibration plot also showed the significant performance of the newly established nomogram. We successfully constructed a nomogram model based on age, TNM stage, preoperative CEA, and CA19-9 levels to evaluate the overall survival of patients with nonmetastatic CRC.

Perinatal thymic-derived CD8αβ-expressing γδ T cells are innate IFN-γ producers that expand in IL-7R-STAT5B-driven neoplasms.

The contribution of γδ T cells to immune responses is associated with rapid secretion of interferon-γ (IFN-γ). Here, we show a perinatal thymic wave of innate IFN-γ-producing γδ T cells that express CD8αβ heterodimers and expand in preclinical models of infection and cancer. Optimal CD8αβ+ γδ T cell development is directed by low T cell receptor signaling and through provision of interleukin (IL)-4 and IL-7. This population is pathologically relevant as overactive, or constitutive, IL-7R-STAT5B signaling promotes a supraphysiological accumulation of CD8αβ+ γδ T cells in the thymus and peripheral lymphoid organs in two mouse models of T cell neoplasia. Likewise, CD8αβ+ γδ T cells define a distinct subset of human T cell acute lymphoblastic leukemia pediatric patients. This work characterizes the normal and malignant development of CD8αβ+ γδ T cells that are enriched in early life and contribute to innate IFN-γ responses to infection and cancer.

C5aR1 inhibition reprograms tumor associated macrophages and reverses PARP inhibitor resistance in breast cancer

Although Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have been approved in multiple diseases, including BRCA1/2 mutant breast cancer, responses are usually transient requiring the deployment of combination therapies for optimal efficacy. Here we thus explore mechanisms underlying sensitivity and resistance to PARPi using two intrinsically PARPi sensitive (T22) and resistant (T127) syngeneic murine breast cancer models in female mice. We demonstrate that tumor associated macrophages (TAM) potentially contribute to the differential sensitivity to PARPi. By single-cell RNA-sequencing, we identify a TAM_C3 cluster, expressing genes implicated in anti-inflammatory activity, that is enriched in PARPi resistant T127 tumors and markedly decreased by PARPi in T22 tumors. Rps19/C5aR1 signaling is selectively elevated in TAM_C3. C5aR1 inhibition or transferring C5aR1hi cells increases and decreases PARPi sensitivity, respectively. High C5aR1 levels in human breast cancers are associated with poor responses to immune checkpoint blockade. Thus, targeting C5aR1 may selectively deplete pro-tumoral macrophages and engender sensitivity to PARPi and potentially other therapies.

Multifunctional elastin-like polypeptide nanocarriers for efficient miRNA delivery in cancer therapy.

The exogenous delivery of miRNA to mimic and restore miRNA-34a activity in various cancer models holds significant promise in cancer treatment. Nevertheless, its effectiveness is often impeded by challenges, including a short half-life, propensity for off-target accumulation, susceptibility to inactivation by blood-based enzymes, concerns regarding patient safety, and the substantial cost associated with scaling up. As a means of overcoming these barriers, we propose the development of miRNA-loaded Tat-A86 nanoparticles by virtue of Tat-A86's ability to shield the loaded agent from external environmental factors, reducing degradation and inactivation, while enhancing circulation time and targeted accumulation. Genetically engineered Tat-A86, featuring 16 copies of the interleukin-4 receptor (IL-4R)-binding peptide (AP1), Tat for tumor penetration, and an elastin-like polypeptide (ELP) for presenting target ligands and ensuring stability, served as the basis for this delivery system. Comparative groups, including Tat-E60 and A86, were employed to discern differences in binding and penetration. The designed ELP-based nanoparticle Tat-A86 effectively condensed miRNA, forming stable nanocomplexes under physiological conditions. The miRNA/Tat-A86 formulation bound specifically to tumor cells and facilitated stable miRNA delivery into them, effectively inhibiting tumor growth. The efficacy of miRNA/Tat-A86 was further evaluated using three-dimensional spheroids of lewis lung carcinoma (LLC) as in vitro model and LLC tumor-bearing mice as an in vivo model. It was found that miRNA/Tat-A86 facilitates effective cell killing by markedly improving miRNA penetration, leading to a substantial reduction in the size of LLC spheroids. Compared to other controls, Tat-A86 demonstrated superior efficacy in suppressing the growth of 3D cellular aggregates. Moreover, at equivalent doses, miRNA-34a delivered by Tat-A86 inhibited the growth of LLC cells in allograft mice. Overall, these studies demonstrate that Tat-A86 nanoparticles can deliver miRNA systemically, overcoming the basic hurdles impeding miRNA delivery by facilitating both miRNA uptake and stability, ultimately leading to improved therapeutic effects.

NeMu: a comprehensive pipeline for accurate reconstruction of neutral mutation spectra from evolutionary data.

The recognized importance of mutational spectra in molecular evolution is yet to be fully exploited beyond human cancer studies and model organisms. The wealth of intraspecific polymorphism data in the GenBank repository, covering a broad spectrum of genes and species, presents an untapped opportunity for detailed mutational spectrum analysis. Existing methods fall short by ignoring intermediate substitutions on the inner branches of phylogenetic trees and lacking the capability for cross-species mutational comparisons. To address these challenges, we present the NeMu pipeline, available at https://nemu-pipeline.com, a tool grounded in phylogenetic principles designed to provide comprehensive and scalable analysis of mutational spectra. Utilizing extensive sequence data from numerous available genome projects, NeMu rapidly and accurately reconstructs the neutral mutational spectrum. This tool, facilitating the reconstruction of gene- and species-specific mutational spectra, contributes to a deeper understanding of evolutionary mechanisms across the broad spectrum of known species.

Safety and feasibility concerns of radiotherapy in the presence of subcutaneous implantable cardioverter-defibrillator

Abstract Introduction Radiotherapy (RT) in patients with cardiac implantable electronic devices (CIED) is performed increasingly more frequently. Currently, there is no evidence, whether RT in patients with subcutaneous implantable cardioverter-defibrillator (sICD) is feasible and safe. Purpose To perform the first in-vitro analyses using a model of left lung cancer treated with stereotactic body radiotherapy (SBRT) with the presence of an sICD. Methods Three functional sICDs explanted from patients following the heart transplant underwent RT. In one device (No º3), the lead had to be cut during explantation at ~ 3 cm from the tip. A computed tomography-based SBRT plan was calculated for a left lung tumor (located 3.5 cm from the sICD). Three fractions of 18 Gy with a nominal energy of 6 MV were delivered, and the maximum dose calculated for the sICD was 3.5 Gy. Each sICD and lead were placed on an anthropomorphic phantom in the nominal location. The intervals between fractions were 7 days. The next step was a direct irradiation of the sICD with an open-field RT with 25 fractions of 2 Gy, then 2 of 20 Gy, 1 of 40 Gy, and a final fraction of 100 Gy. Before and after each fraction, the devices were interrogated, and to verify whether RT affects the devices in the possibly least optimistic clinical scenario, high-voltage therapies were turned on for each fraction and off for the intervals between them. Results A major depletion in the battery percentage (from 88% to 16%) between the first and second fraction of RT in device No. 2 was observed, while in two other devices, major reductions in the device battery longevity were observed after the third fraction of RT (from 38% to 14% and from 60% to 15%, respectively). The estimated doses for sICDs in those stages of RT were respectively 1.2 Gy, 3.5 Gy, and 3.5 Gy. Due to a significant drop of the battery longevity in device No 2º, only two remaining sICD underwent the open-field RT. During the direct irradiation, an unannounced shock was delivered during the interrogation directly after the 14th fraction of 2 Gy, in a device with a macroscopically intact lead. An external interference was demonstrated, which was interpreted as an arrhythmia in the ventricular fibrillation zone. The complete device reset occurred in both devices after having received approximately 195 Gy. Conclusions The presented findings strongly indicate an association between RT and the malfunction of the sICD occurring in the anthropomorphic phantom model of left lung tumor, as well as the risk of inappropriate shocks during the open-beam direct radiation on the devices.Battery longevities before and after RT

Single-Cell RNA Sequencing Analysis Reveals Metabolic Changes in Epithelial Glycosphingolipids and Establishes a Prognostic Risk Model for Pancreatic Cancer.

Metabolic reprogramming serves as a distinctive feature of cancer, impacting proliferation and metastasis, with aberrant glycosphingolipid expression playing a crucial role in malignancy. Nevertheless, limited research has investigated the connection between glycosphingolipid metabolism and pancreatic cancer. This study utilized a single-cell sequencing dataset to analyze the cell composition in pancreatic cancer tissues and quantified single-cell metabolism using a newly developed computational pipeline called scMetabolism. A gene signature developed from the differential expressed genes (DEGs), related to epithelial cell glycosphingolipid metabolism, was established to forecast patient survival, immune response, mutation status, and reaction to chemotherapy with pancreatic adenocarcinoma (PAAD). The single-cell sequencing analysis revealed a significant increase in epithelial cell proportions in PAAD, with high glycosphingolipid metabolism occurring in the cancerous tissue. A six-gene signature prognostic model based on abnormal epithelial glycosphingolipid metabolism was created and confirmed using publicly available databases. Patients with PAAD were divided into high- and low-risk categories according to the median risk score, with those in the high-risk group demonstrating a more unfavorable survival outcome in all three cohorts, with higher rates of gene mutations (e.g., KRAS, CDKN2A), increased levels of immunosuppressive cells (macrophages, Th2 cells, regulatory T cells), and heightened sensitivity to Acetalax and Selumetinlb. Abnormal metabolism of glycosphingolipids in epithelial cells may promote the development of PAAD. A model utilizing a gene signature associated with epithelial glycosphingolipids metabolism has been established, serving as a valuable indicator for the prognostic stratification of patients with PAAD.

Synergistic effects of Smac mimetic APG-1387 with anti-PD-1 antibody are attributed to increased CD3 + NK1.1 + cell recruitment secondary to induction of cytokines from tumor cells

BackgroundImmune checkpoint inhibitors are approved for the treatment of various tumors, but the response rate is not satisfactory in certain malignancies. Inhibitor of apoptosis proteins (IAP) ubiquitin-E3 ligase activity is involved in the regulation of immune responses. APG-1387 is a novel second mitochondria-derived activator of caspase (Smac) mimetic IAP inhibitor. The aim of this study was to explore the synergistic effect of APG-1387 when combined with anti-PD-1 antibody in a preclinical setting.MethodsWe utilized syngeneic mouse models of ovarian cancer (ID8), colon cancer (MC38), malignant melanoma (B16), and liver cancer (Hepa1-6) to assess the combination effect of APG-1387 and anti-PD-1 antibody, including immune-related factors, tumor growth, and survival. MSD V-PLEX validated assays were used to measure in vitro and in vivo cytokine release.ResultsIn ID8 ovarian cancer and MC38 colon cancer models, APG-1387 and anti-PD1 antibody had synergistic antitumor effects. In the MC38 model, the combination of APG-1387 and anti-PD-1 antibody significantly inhibited tumor growth (P < 0.0001) and increased the survival rate of tumor-bearing animals (P < 0.001). Moreover, we found that APG-1387 upregulated tumor-infiltrating CD3 + NK1.1 + cells by nearly 2-fold, by promoting tumor cell secretion of IL-12. Blocking IL-12 secretion abrogated the synergistic effects of APG-1387 and anti-PD-1 antibody in both MC38 and ID8 models.ConclusionsAPG-1387 has the potential to turn “cold tumors” into hot ones by recruiting more CD3 + NK1.1 + cells into certain tumors. Based on these and other data, the safety and therapeutic effect of this combination will be investigated in a phase 1/2 trial in patients with advanced solid tumors or hematologic malignancies (NCT03386526).Graphic abstract

Defining three ferroptosis-based molecular subtypes and developing a prognostic risk model for high-grade serous ovarian cancer.

As a newly defined regulated cell death, ferroptosis is a potential biomarker in ovarian cancer (OV). However, its underlying mechanism in tumor microenvironment (TME) and clinical prediction significance in OV remained to be elucidated. The transcriptome data of high-grade serous OV from The Cancer Genome Atlas (TCGA) database were downloaded. Molecular subtypes were classified based on ferroptosis-correlated genes from the FerrDb database by performing consensus clustering analysis. The associations between the subtypes and clinicopathologic characteristics, mutation, regulatory pathways and immune landscape were assessed. A ferroptosis-related prognostic model was constructed and verified using International Cancer Genome Consortium (ICGC) cohort and GSE70769. Three molecular subtypes of OV were defined. Patients in subtype C3 tended to have the most favorable prognosis, while subtype C1 showing more mesenchymal cells, increased immune infiltration of Macrophages_M2, lower tumor purity, and epithelial-to-mesenchymal transition (EMT) features had the poorest prognosis. A ferroptosis-related risk model was constructed using 8 genes (PDP1, FCGBP, EPHA4, GAS1, SLC7A11, BLOC1S1, SPOCK2, and CXCL9) and manifested a strong prediction performance. High-risk patients had enriched EMT pathways, more Macrophages_M2, less plasma cells and CD8 cell infiltration, greater tendency of immune escape and worse prognosis. The risk score has negatively correlated relation with LAG3, TIGIT, CTLA4, IDO1, CD27, ICOS, and IL2RB but positively correlated with PVR, CD276, and CD28. Moreover, low-risk patients were more sensitive to Cisplatin and Gefitinib, Gemcitabine. Our results could improve the understanding of ferroptosis in OV, providing promising insights for the clinical targeted therapy for the cancer.

P.127 Quality of life (QoL) using EORTC QoL-C30 and BN20 among patients who underwent brain tumor resection in a tertiary hospital in Saudi Arabia

Background: Quality of life (QoL) is the awareness of individuals’ well-being in life in physical, personal, mental and social wellbeing and needs to be addressed in brain tumor patients. Methods: A retrospective study conducted in 2017 in a single academic center that included patients diagnosed with brain tumors in a 10 year period. The assessment of the QoL was done using the European Organization for Research and Treatment of Cancer (EORTC), a standardized model (QLQ-C30) that assess several domains (Global Health, Physical function, Role functioning, Emotional Functioning, Cognitive functioning, social functioning and symptoms domain) and Brain cancer model (BN20) to assess symptoms to evaluate all aspects of wellbeing. Results: The total number of patients included in this study is 76 patients with no gender predilection. The most common brain tumor was meningioma by 40% followed by glioma/ others. More than half of the brain tumor patients had a WHO grade I (65%), intermediate grading grade II (15%) and higher grading grade III/IV (20%). The scales and measurements of functioning in life were low in all types of brain tumors. Conclusions: Quality of life in brain tumor patients seemed poor regardless of the type. Further prospective studies are needed to assess QoL worldwide.

2-(4-Nitrophenyl)isothiazol-3(2H)-one: A Promising Selective Agent against Hepatocellular Carcinoma Cells.

Liver cancer ranks among the most prevalent malignancies globally and stands as a leading cause of cancer-related mortality. Numerous isothiazolone derivatives and analogues have been synthesized and investigated for their potential as anticancer agents; however, limited data exist regarding their efficacy against liver cancer. In the present study, two nitrophenyl-isothiazolones, the 5-benzoyl-2-(4-nitrophenyl)isothiazol-3(2H)-one (IsoA) and the 2-(4-nitrophenyl)isothiazol-3(2H)-one (IsoB), were preliminarily investigated for their cytotoxicity against hepatoma human (Huh7) cells as a liver cancer model and Immortalized Human Hepatocytes (IHHs) as a model of non-cancerous hepatocytes. IsoB, derived from IsoA after removal of the benzoyl moiety, demonstrated the highest cytotoxic effect against Huh7 cells with CC50 values of 19.3 μΜ at 24 h, 16.4 μΜ at 48 h, and 16.2 μΜ at 72 h of incubation, respectively. IsoB also exhibited selective toxicity against the liver cancerous Huh7 cells compared to IHH cells, reinforcing its role as a potent and selective anticancer agent. Remarkably, the cytotoxicity of IsoB was higher when compared with the standard chemotherapeutical agent 5-fluorouracil (5-FU), which also failed to exhibit higher toxicity against the liver cancerous cell lines. Moreover, IsoB-treated Huh7 cells presented a noteworthy reduction in mitochondrial membrane potential (ΔΨm) after 48 and 72 h, while mitochondrial superoxide levels showed an increase after 24 h of incubation. The molecular mechanism of the IsoB cytotoxic effect was also investigated using RT-qPCR, revealing an apoptosis-mediated cell death along with tumor suppressor TP53 overexpression and key-oncogene MYCN downregulation.

Restoring adiponectin via rosiglitazone ameliorates tissue wasting in mice with lung cancer.

To investigate systemic regulators of the cancer-associated cachexia syndrome (CACS) in a pre-clinical model for lung cancer with the goal to identify therapeutic targets for tissue wasting. Using the Kras/Lkb1 (KL) mouse model, we found that CACS is associated with white adipose tissue (WAT) dysfunction that directly affects skeletal muscle homeostasis. WAT transcriptomes showed evidence of reduced adipogenesis, and, in agreement, we found low levels of circulating adiponectin. To preserve adipogenesis and restore adiponectin levels, we treated mice with the PPAR-γ agonist, rosiglitazone. Rosiglitazone treatment increased serum adiponectin levels, delayed weight loss, and preserved skeletal muscle and adipose tissue mass, as compared to vehicle-treated mice. The preservation of muscle mass with rosiglitazone was associated with increases in AMPK and AKT activity. Similarly, activation of the adiponectin receptors in muscle cells increased AMPK activity, anabolic signaling, and protein synthesis. Our data suggest that PPAR-γ agonists may be a useful adjuvant therapy to preserve tissue mass in lung cancer.

Study of metal organic framework with siRNA for overcoming matrix barrierin breast cancer

Objective: This study aimed to develop a new delivery strategy that utilized metal organic framework (MOF) loaded with small-interfering RNA (siRNA) targeting ITGAV to overcome tumor matrix barrier, and thus enhance drug penetration and immune accessibility in breast cancer. Methods: MOF@siITGAV particles were constructed and characterized. The uptake of MOF@siITGAV in breast cancer cell line 4T1 was observed by the cellular uptake assay. The toxicity of MOF@siITGAV was detected by cell counting kit 8 (CCK-8). The blank control group, naked siITGAV group and MOF@siITGAV group were set. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the expressions of ITGAV. The level of transforming growth factor β1 (TGF-β1) in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA). The penetration of MOF@siITGAV in 4T1 cells was tested by constructing 3D spheroids. Mouse models of triple negative breast cancer were established. The effect of MOF@siITGAV on the growth of transplanted tumors and main organs was verified. Imminohistochemical (IHC) was used to test the expression of collagen and CD8. Results: MOF@siITGAV particles were constructed with sizes of (198.0±3.3) nm and zeta potential of -(20.2±0.4) mV. MOF@siITGAV could be engulfed by 4T1 cells and triggered to release siRNA. Compared to the blank control group, the expression of ITGAV in the MOF@siITGAV group [(46.5±11.3)%] and the naked siITGAV group [(109.9±19.0)%] was lower. TGF-β1 in the cell culture medium of the blank control group, naked siITGAV group, and MOF@siITGAV group was (474.5±34.4) pg/ml, (437.2±16.5) pg/ml, and (388.4±14.4) pg/ml, respectively. MOF@siITGAV could better penetrate into 4T1 spheroids and exhibit no obvious toxicity. The cell viability was (99.7±3.5)%, (98.2±5.2)%, (97.3±6.6)%, (92.1±8.1)%, and (92.4±4.1)%, respectively, after MOF@siITGAV treatment with the concentration of 0, 10, 20, 40, 80, and 160 μg/ml, respectively, for 24 h. The tumor growth in the MOF@siITGAV group was suppressed significantly. After 15-day treatment, the tumor volume of the MOF@siITGAV group was (135.3±41.9) mm3, smaller than that of the blank control group [(691.1±193.0) mm3] (P=0.025). The expression of collagen and the number of CD8 positive cells of the MOF@siITGAV group were lower than those of the other two groups. No significant abnormalities were observed in the main organs of mice. Conclusions: Targeting the integrinαv on the surface of cancer cells could destroy extracellular matrix, improve drug delivery, and increase immune infiltration.

Spatial and functional targeting of intratumoral Tregs reverses CD8+ T cell exhaustion and promotes cancer immunotherapy.

Intratumoral regulatory T cells (Tregs) are key mediators of cancer immunotherapy resistance, including anti-PD-(L)1 immune checkpoint blockade (ICB). The mechanisms driving Treg infiltration into the tumor microenvironment (TME) and the consequence on CD8+ T cell exhaustion remains elusive. Herein, we report that heat shock protein gp96 (GRP94) is indispensable for Treg tumor infiltration, primarily through gp96's roles in chaperoning integrins. Among various gp96-dependent integrins, we found that only LFA-1 (αL integrin) but not αV, CD103 (αE) or β7 integrin was required for Treg tumor homing. Loss of Treg infiltration into the TME by genetically deleting gp96/LFA-1 potently induces rejection of multiple ICB-resistant murine cancer models in a CD8+ T cell-dependent manner without loss of self-tolerance. Moreover, gp96 deletion impeded Treg activation primarily by suppressing IL-2/STAT5 signaling, which also contributes to tumor regression. By competing for intratumoral IL-2, Tregs prevent activation of CD8+ tumor-infiltrating lymphocytes (TILs), drive TOX induction and induce bona fide CD8+ T cell exhaustion. By contrast, Treg ablation leads to striking CD8+ T cell activation without TOX induction, demonstrating clear uncoupling of the two processes. Our study reveals that the gp96/LFA-1 axis plays a fundamental role in Treg biology and suggests that Treg-specific gp96/LFA-1 targeting represents a valuable strategy for cancer immunotherapy without inflicting autoinflammatory conditions.

How Much Do You Fuse? A Comparison of Cell Fusion Assays in a Breast Cancer Model.

Cell fusion is a biological process that is crucial for the development and homeostasis of different tissues, but it is also pathophysiologically associated with tumor progression and malignancy. The investigation of cell fusion processes is difficult because there is no standardized marker. Many studies therefore use different systems to observe and quantify cell fusion in vitro and in vivo. The comparability of the results must be critically questioned, because both the experimental procedure and the assays differ between studies. The comparability of the fluorescence-based fluorescence double reporter (FDR) and dual split protein (DSP) assay was investigated as part of this study, in which general conditions were kept largely constant. In order to be able to induce both a high and a low cell fusion rate, M13SV1 breast epithelial cells were modified with regard to the expression level of the fusogenic protein Syncytin-1 and its receptor ASCT2 and were co-cultivated for 72 h with different breast cancer cell lines. A high number of fused cells was found in co-cultures with Syncytin-1-overexpressing M13SV1 cells, but differences between the assays were also observed. This shows that the quantification of cell fusion events in particular is highly dependent on the assay selected, but the influence of fusogenic proteins can be visualized very well.

Retraction: Combination effect of cold atmospheric plasma with green synthesized zero-valent iron nanoparticles in the treatment of melanoma cancer model.

Retraction: Combination effect of cold atmospheric plasma with green synthesized zero-valent iron nanoparticles in the treatment of melanoma cancer model.