The radiation-induced abscopal effect (RIAE) can suppress distal metastatic lesions and elicit a systemic anti-tumor response; however, the underlying mechanisms remain to be fully elucidated. Current research has shown that autophagy promotes the production of IFN-β by regulating mitochondrial DNA (mtDNA), thereby contributing to the modulation of RIAE. Nevertheless, the downstream pathways through which IFN-β influences RIAE require further investigation. In this study, we observed accumulation of an abundance of mtDNA in the cytosol of mammary tumor cells following RT, along with the presence of mitochondrial RNA (mtRNA). These molecules activated the cGAS-STING and RIG-I-MAVS signaling pathways, respectively, thereby synergistically promoting the production of IFN-β and secretion into the extracellular matrix. Subsequently, IFN-β facilitated the polarization of macrophages in distant non-irradiated tumor microenvironment towards the M1 phenotype through activating STAT1. Furthermore, our findings indicate that high linear energy transfer (LET) carbon ions are significantly more effective in inducing the production of IFN-β and promoting macrophage polarization compared to low-LET X-rays. Thus, our findings provide insights into the intricate mechanisms by which mtDNA/RNA and IFN-β mediate RIAE, suggesting that IFN-β could be a promising target for provoking RT immunogenicity in patients with breast cancer and high-LET radiation might effectively elicit RIAE.

We aimed to evaluate the potential effects of Plantago major (PM) to enhance the radiosensitivity of human breast adenocarcinoma cell line (MCF-7) in an animal model.

Seventy-two female Balb/c mice were divided into nine groups (8 mice per group) as follows: MCF-7 breast cancer control group, MCF-7+low dose of PM (1000 mg/kg), MCF-7+high dose of PM (1500 mg/kg), MCF-7+3 Gy superficial X-ray, MCF-7+5 Gy superficial X-ray, MCF-7+1000&1500 mg/kg of PM with 3 and 5 Gy irradiations. For each treatment group, micronuclei in 500 binucleate MCF-7 cells from a minimum of three experiments were counted. The alkaline comet assay was used to calculate % DNA in the tail and % of apoptotic comets. The tumor size in the treated groups (3 mice per group) was assessed at 4- and 8-weeks post-treatment.

The number of total micronuclei and binucleated micronuclei in the PM and/or irradiation treated groups was significantly higher than in the control group (P < 0.05). Irradiation+PM resulted in a significant increase treatment effect compared to the irradiation-only groups (P < 0.01). In addition, the higher PM concentration had a significantly higher number of micronucleated binucleate cells when combined with 5 Gy irradiation (P = 0.022). Irradiation alone or in combination with PM resulted in significant increases in percentages of DNA in tail and apoptotic comet values compared to the PM-only treatment groups (P < 0.02). Combining 5 Gy of irradiation with 1000 mg/kg of PM led to a 26% reduction in tumor size (0.28±0.04 vs. 0.38 ± 0.03) after 4 weeks, and combining 5 Gy of irradiation with 1500 mg/kg of PM resulted in a 40% decrease in tumor volume after 4 weeks (0.21±0.04 vs. 0.35±0.03).

PM extract at both doses demonstrated an antitumor effect on induced MCF-7 breast cancer tumors, with this effect being enhanced when combined with irradiation.

Rectal cancer is a leading cause of cancer-related mortality worldwide. The recurrence of locally advanced rectal cancer (LARC), particularly in cases involving lymph node-positive tumours, remains a critical challenge in rectal cancer management. In this study, a therapeutic strategy, radiation-activated photodynamic therapy (RA-PDT), for the treatment of LARC with lymph node-positive tumours was developed and evaluated.

RA-PDT was achieved by using a lipid-polymer hybrid nanoplatform loaded with verteporfin (VP) and functionalised with folic acid (FA) as a targeting molecule. Upon receiving a single 4 Gy fraction of radiation, VP was effectively activated, generating sufficient reactive oxygen species (ROS) to induce cancer cell death-however surrounding tissue was less affected and was spared. The efficacy of this strategy was assessed through in vitro cytotoxicity studies in HCT116 cells, as well as in orthotopic and subcutaneous mouse models. In vivo lymph node tumour progression was also evaluated.

RA-PDT effectively generated ROS following 4 Gy irradiation and exhibited significant cytotoxicity in HCT116 cells. In vivo, this strategy largely inhibited primary tumour growth in both orthotopic and subcutaneous mouse models while also suppressing lymph node tumour progression. Surrounding tissues were minimally affected, highlighting the precision and safety of this approach.

RA-PDT demonstrates potential as a safe therapeutic strategy for LARC, paving the way for its clinical translation.

This study was supported by the Australian National Health and Medical Research Council (GNT1181889), fellowship award (2019/CDF1013) from Cancer Institute NSW, Australia, the Australian Research Council Centre of Excellence for Nanoscale Biophotonics (CE140100003), UNSW SHARP funding, project grant from National Foundation for Medical Research and Innovation, Australia, International Research Training Program Scholarship (IRTP) from Australian Government, PhD Research Scholar Award from Sydney Vital Translational Cancer Research, and Translational Cancer Research Network PhD Scholarship Top-up award.

Retinoblastoma (RB) is a type of intraocular tumor in childhood with a high lethality rate. N4-acetylcytosine (ac4C) modification is known to regulate multiple cancers, which is mediated by the only known ac4C writer N-Acetyltransferase 10 (NAT10). In this study, the authors aimed to reveal the mechanism of RB progression regulated by ac4C modification.

Phenotypically, dot blot assay and quantitative real-time PCR were used to detect the ac4C levels and NAT10 expression in clinical samples and RB cell lines. Then, NAT10 was knocked down to assess its effect on glycolysis. Mechanically, RNA immunoprecipitation assay, immunofluorescence assay, and dual luciferase report were used to explore the mRNA modified by NAT10-mediated ac4C modification. Mice xenograft model was used to determine the effect of NAT10 on tumor growth in vivo.

The present results demonstrated that the levels of ac4C and NAT10 were increased in cancer tissues and RB cell lines. Furthermore, NAT10 knockdown inhibited the glycolysis in RB cell lines. Moreover, the authors revealed that NAT10 knockdown decreased the ac4C modification and mRNA stability of HK1, while the inhibition of glycolysis by NAT10 knockdown was reversed by HK1 overexpression. Finally, NAT10 knockdown relieved the growth of tumors in mice models.

The authors illustrated that NAT10 plays an important role in the progression of RB by regulating the ac4C modification on HK1 mRNA and affects its stability, which may provide a novel theoretical basis for the treatment of RB.

Immune checkpoint inhibitors, such as anti-PD1, revolutionized melanoma treatment. However, resistance and low response rates remain problems. Our goal was to pinpoint actionable biomarkers of resistance to anti-PD1 treatment and verify therapeutic effectiveness in vivo.

Using receiver operating characteristic (ROC) and survival analysis in a database of 1434 samples, we identified the strongest resistance-associated genes. Inhibitors were evaluated in C57BL/6J mice using wild-type B16-F10, and BRAF, -PTEN, -CDKN2A-mutant YUMM1.7 melanoma cell lines. We investigated the synergistic impact of anti-PD1 therapy and yes-associated protein 1 (YAP1) inhibition by non-photoactivated Verteporfin. Tumour volume was determined at fixed cutoff points, normalized to body weights.

In the anti-PD1-treated melanoma cohort, YAP1 was the strongest druggable candidate overexpressed in non-responder patients (ROC AUC = 0.699, FC = 1.8, P=1.1E-8). The baseline YAP1 expression correlated with worse progression-free survival (HR = 2.51, P=1.2E-6, FDR = 1%), and overall survival (HR = 2.15, P = 1.2E-5, FDR = 1%). In YUMM1.7, combination of Verteporfin plus anti-PD1 reduced tumour size more than anti-PD1 monotherapy (P=0.008), or control (P=0.021). There was no difference between the cohorts in B16-F10 inoculated mice. We found increased expression of YAP1 in YUMM1.7 mice compared to B16-F10. The combination therapy induced a more-immune-inflamed phenotype characterized by increased expression of T cell and M1 macrophage markers.

Verteporfin with anti-PD1 exhibited antitumor potential by promoting a pro-inflammatory tumour microenvironment in melanoma. We believe that YAP1 acts as a master regulator of anti-PD1 resistance.

There is always a quest for newer, more effective chemopreventive agents to prevent and manage oral cancer. A novel nanogel prepared using thiocolchicoside, lauric acid, and chitosan showed promising anticancer activity in KB-1 cell lines. The current manuscript aims to investigate the chemopreventive activity of chitosan nanogel with thiocolchicoside and lauric acid in chemically induced oral carcinogenesis using Wistar rats. Forty-six male Wistar rats were divided into three different groups: group I (control group), group II (cancer induction group), and group III (cancer induction with a chemopreventive agent). Male Wistar rats were given 20 μl/ml 4NQO solutions daily in their drinking water. One group received a daily oral application of CTL nanogel and carcinogen in drinking water. After the 23-week carcinogen treatment, the rats were euthanized; the tongues of the rats were dissected and histopathologically examined. Additionally, RT-PCR was employed to assess the gene expression of various signaling molecules involved in cancer progression like Erk1/2, β-catenin, Ki-67, Cyclin D1, TNF-α, NFκB, COX-2, and RAC1. Wistar rats developed white lesions and growth in the tongue in the cancer induction group. At the same time, the incidence and size of tumors were significantly less in the CTL nanogel-treated group. There was a significant increase in p53, Caspase-3, and Bax expression levels, while Bcl-2 showed a decreased expression in the CTL nanogel-treated group. There was also a significant decrease in the expression of EGFR and VEGFR signaling molecules in the CTL nanogel-treated group (p < 0.05 level). CTL nanogel shows potent chemopreventive efficiency in reducing the occurrence and severity of 4NQO-induced oral cancer in Wistar rats, marking it a promising candidate for further investigation in cancer prevention strategies.

Positron emission tomography (PET) hybrid imaging targeting HER2 requires antibodies labelled with longer half-life isotopes. With a suitable radiation profile, 52Mn coupled with DOTAGA as a bifunctional chelator is a potential candidate. In this study, we investigated the tumor HER2 specificity and the temporal biodistribution of the [52Mn]Mn-DOTAGA(anhydride)-trastuzumab in preclinical models.

PET/MRI and PET/CT were performed on SCID mice bearing orthotopic and ectopic HER2-positive and ectopic HER2-negative tumors at 4, 24, 48, 72, and 120 h post-injection with [52Mn]Mn-DOTAGA(anhydride)-trastuzumab. Melanoma xenografts were included for comparison of specificity.

In vivo biodistribution demonstrated strong contrast in HER2-positive tumors, particularly in orthotopic tumors, where uptake was significantly higher than in the blood pool and other organs from 24 h onwards and consistently higher than in ectopic HER2-positive tumors at all time points. Significantly higher tumor-to-blood and tumor-to-muscle ratios were observed in HER2-positive ectopic tumors compared to HER2-negative tumors but only at 4 and 24 h; the differences were likely due to non-specific binding of the tracer. The ratios for orthotopic HER2-positive tumors were significantly higher than those for ectopic HER2-negative tumors and melanoma at all time points. However, the differences between HER2-positive and HER2-negative tumors decreased at later time points.

These results suggest that [52Mn]Mn-DOTAGA(anhydride)-trastuzumab demonstrates efficient tumor-to-background contrast, emphasize the higher tumor uptake observed in orthotopic tumors, and highlight the influence of tumor environment characteristics on uptake.

Signaling through the β2-adrenergic receptor (β2-AR) mobilizes immune cells during exercise and is implicated in tumor lymphocyte infiltration. We investigated mechanisms governing immune cell mobilization in humans and the role of adrenergic signaling in anti-cancer responses to a murine lymphoma. Human studies included double-blind, placebo-controlled, crossover trials with beta blocker drugs and a phosphodiesterase inhibitor during steady-state and graded exercise. β1 + β2-AR blockade reduced lymphocyte and NK-cell mobilization during steady-state exercise, while β1-AR blockade enhanced the mobilization of NK-cells. Combining a β1-AR antagonist with a phosphodiesterase-4 (PDE4) inhibitor during graded exercise further increased mobilization of CD8 + T-cells, γδ T-cells, and monocytes. Isoproterenol infusion also elevated lymphocyte and NK-cell levels similarly to exercise at 70 % VO2max. Single cell RNA sequencing revealed complex signaling downstream of cAMP that relate to lymphocyte activation and effector function. In murine models of voluntary wheel running, β2-AR signaling and NK-cells were critical for exercise-induced protection against B-cell lymphoma, as β2-AR blockade or NK-cell depletion abrogated these effects. These findings highlight the pivotal role of β2-AR signaling in mobilizing cytotoxic immune cells and protecting against tumor progression through exercise, suggesting potential therapeutic strategies combining exercise with adrenergic modulation to enhance immune responses.

Colorectal cancer (CRC) is one of the most aggressive malignancies of the digestive tract, characterized by aberrant post-transcriptional RNA modifications, including pseudouridine (Ψ). TruB pseudouridine synthase family member 1 (TRUB1) is a key pseudouridine synthase but its role in CRC progression remains unclear.

Public databases and CRC cell lines were analysed to assess TRUB1 expression in CRC. Receiver-operating characteristic (ROC) curve analysis and survival analysis were performed to evaluate the diagnostic and prognostic significance of TRUB1. The impact of TRUB1 on tumor proliferation and Ψ modification was examined in TRUB1-knock-down HCT116 cell lines. Mechanistically, RNA sequencing of control and TRUB1-knock-down HCT116 cells was conducted to identify potential pathways, which were validated by using real-time polymerase chain reaction (PCR), Western blot, and immunofluorescence assays.

TRUB1 was significantly upregulated in CRC tumor tissues and cell lines. ROC analysis showed that TRUB1 had strong diagnostic potential and its overexpression was associated with poorer overall survival in CRC patients. In TRUB1-knock-down HCT116 cells, apoptosis increased and tumor growth slowed in nude mice, with a corresponding increase in apoptosis-related proteins and decreased Ψ modification. Mechanistically, RNA sequencing indicated that tumor necrosis factor α signaling via the nuclear factor kappa B (NFκB) pathway was activated in TRUB1-knock-down HCT116 cells. Further analysis identified Baculoviral inhibitor of apoptosis proteins repeat-containing 3 (BIRC3) as a potential downstream target gene that was regulated by TRUB1 in the NFκB pathway.

TRUB1 serves as a potential biomarker for CRC diagnosis and prognosis, and it can inhibit apoptosis in CRC cells via BIRC3-mediated NFκB signaling.

Various immunotherapeutic strategies are being developed to fight cancer, which is one of the leading causes of mortality. Dendritic cells (DCs), being professional antigen-presenting cells, after efficient manipulation with tumor-associated antigens, can lead to effective T-cell recruitment and activation at the tumor site, resulting in cytotoxic T-cell-mediated cancer cell killing. To circumvent the inefficiencies of ex vivo DC modification and patient infusion, an alternative strategy involving in situ DC activation has been explored here. Here, the vaccine components are tumor lysates, as antigens, and polyinosinic:polycytidylic acid (poly(I:C)), a toll-like receptor-3 (TLR3) agonist, as an adjuvant. Our in vitro studies demonstrate that complexing poly(I:C) with a carrier molecule, chitosan, enhances its stability and accessibility to TLR3 in the DC endosomal membrane. Material-based localized delivery of immunomodulatory factors is known to improve their stability and reduce their off-target side effects. Here, PEGDA-PLL-based macroporous scaffolds allow easy recruitment of host cells, thereby enabling effective interaction between the vaccine components loaded on them and the infiltrating immune cells. The vaccine components present in the scaffold facilitate efficient DC activation and migration, leading to subsequent T-cell activation and antitumor response, as shown by our in vivo studies.

Objective: In previous studies, echogenic liposomes with liquid and gas cores were analyzed as alternative carriers of drug molecules and cavitation nuclei for sonoporation. The possibility of small interfering RNA (si-RNA) encapsulation has also been presented. In this study, the usability of echogenic liposomes as drug carriers and cavitation seeds was evaluated using an in vivo model. Methods: A doxorubicin-loaded echogenic liposome was synthesized as a drug carrier. The size distribution and the number of formed echogenic liposomes were measured. Five comparative in vivo experiments were conducted with and without doxorubicin-loaded echogenic liposomes, and the results were statically analyzed. Results: Sonoporation with doxorubicin-loaded echogenic liposomes at 3.05 W/cm2 of ISPTA ultrasound sonication and 0.98 MHz results in an average tumor volume growth of less than 25% of that following the simple administration of doxorubicin. Considering the p-value between the two groups is approximately 0.03, doxorubicin-loaded echogenic liposomes were effectively applicable as cavitation nuclei for sonoporation. Conclusions: Although further studies are needed to clarify the responses to incident ultrasound fields, the proposed echogenic liposome appears to be a promising alternative cavitation nuclei/carrier for sonoporation.

Boron, a trace element, is involved in various physiological and metabolic processes, and recent studies suggest that boron compounds may have potential in cancer prevention and treatment. In this study, the antiangiogenic effects of a boron compound, borax pentahydrate (BPH), were investigated. Angiogenesis is a tightly regulated biological process responsible for the formation of new blood vessels from existing vasculatures. This process plays a critical role in cancer progression, making it an important target for cancer therapy. Pancreatic and kidney cancers are difficult to treat because they are aggressive and resistant to chemotherapy.

The antiproliferative activity was evaluated using the MTT assay, while antiangiogenic effects were tested through in vitro tube formation assays and in ovo chick chorioallantoic membrane (CAM) assay. The effect of BPH on VEGF levels was determined using Western blot analysis in HUVEC, ACHN, PANC-1 cells. The effect of BPH on tumor angiogenesis was investigated with an in vivo Ehrlich ascites carcinoma model (EAC).

BPH exhibited potent antiproliferative and antiangiogenic activities, inhibiting the proliferation of ACHN, PANC-1, and HUVECs, disrupting endothelial tube formation, and inhibiting vascular formation on the CAM surface in a dose-dependent manner. VEGF levels were significantly decreased in ACHN, PANC-1 and HUVECs. There was also a decrease in VEGF and TGF-β1 levels in BPH-treated tumor groups. In addition, BPH caused a decrease in tumor size.

These findings suggest that BPH may be a new antiangiogenic and antitumoral agent. BPH may contribute to drug development studies targeting angiogenesis-related diseases as a promising new therapeutic agent.

Objectives: Ovarian cancer is a common gynaecological malignancy. Photodynamic therapy (PDT) mediated by 5-aminolaevulinic acid (5-ALA-PDT) is widely used in clinical practice. However, hypoxia may impact the efficacy of this treatment. In the present study, we combined the bioreductively active drug tirapazamine (TPZ) with PDT to explore its potential in enhancing ovarian cancer cell death. Methods: A cell counting kit-8 assay was used to determine cytotoxicity under different intervention conditions. The distribution of protoporphyrin IX, a metabolite of 5-ALA, was observed using in vivo fluorescence imaging. The effect of the combined treatment was assessed by measuring changes in tumour size following the corresponding interventions and by haematoxylin and eosin staining of tumour tissues. Immunohistochemical staining was used to detect the expression levels of relevant proteins. Results: TPZ exhibited no cytotoxicity under normoxic conditions but was activated under hypoxic conditions, inducing cytotoxic effects that were enhanced when combined with PDT. Over time, protoporphyrin IX achieved systemic distribution, and high drug concentrations were maintained within the tumour. The combination therapy suppressed tumour growth, and pathological staining showed that necrotic tumour areas were significantly enlarged after treatment. The enhanced therapeutic effect may be attributable to the inhibition of the hypoxia-inducible factor-1α/vascular endothelial growth factor axis and PI3K/Akt/mTOR pathway. Conclusions: 5-ALA-PDT combined with TPZ can overcome both the hypoxic state of ovarian cancer tissues and the increased hypoxia induced by PDT, thereby inhibiting tumour growth.

Effective therapies for solid tumors, including breast cancers, are hindered by several roadblocks that can be largely attributed to the fibrotic extracellular matrix (ECM). Fibronectin (FN) is a highly upregulated ECM component in the fibrotic tumor stroma and is associated with poor patient prognosis. This study aimed to investigate the therapeutic potential of an anti-fibrotic peptide that specifically targets FN and blocks the fibrillar assembly of FN.

To target FN, we used PEGylated Functional Upstream Domain (PEG-FUD), which binds to the 70 kDa N-terminal region of FN with high affinity, localizes to mammary tumors, and potently inhibits FN assembly in vitro and in vivo. Here, we used the 4T1 tumor model to investigate the efficacy and mechanisms of PEG-FUD to inhibit tumor growth.

Our data demonstrates that PEG-FUD monotherapy reduces tumor growth without systemic toxicity. Analysis of the tumor microenvironment revealed that PEG-FUD effectively inhibited FN matrix assembly within tumors and reduced adhesion-mediated signaling through α5 integrin and FAK leading to enhanced tumor cell death. Notably, signaling through FAK has been associated with resistance mechanisms to doxorubicin (DOX). Therefore, we tested the combination of PEG-FUD and Dox, which significantly reduced tumor growth by 60% compared to vehicle control and 30% compared to Dox monotherapy.

Our findings demonstrate that PEG-FUD significantly modifies the peritumoral ECM of breast cancer, leading to increased tumor cell death, and potentiates the efficacy of conventional breast cancer therapy.

There is overwhelming interest to use actinium-225 ([225Ac]Ac) to develop targeted α therapies. Antibody-drug conjugates (ADC) are highly cytotoxic. Combining [225Ac]Ac with an ADC to develop an antibody-drug radioconjugate [225Ac]Ac-macropa-trastuzumab(T)-PEG6-emtansine (DM1), is expected to be more effective than its ADC (T-PEG6-DM1) against breast cancer.

[89Zr]Zr-p-isothiocyanatobenzyl desferrioxamine (DFO)-T-PEG6-DM1 (imaging) and [225Ac]Ac-macropa-T-PEG6-DM1 (radiotherapy) were developed. Biodistribution and safety evaluations of [225Ac]Ac-macropa-T-PEG6-DM1 were carried out in non-tumor-bearing BALB/c mice. MicroPET imaging and biodistribution were done using [89Zr]Zr-DFO-T-PEG6-DM1, and radiotherapy using [225Ac]Ac-macropa-T-PEG6-DM1 was carried out in athymic BALB/c nude mice bearing trastuzumab-resistant HCC1954 and trastuzumab-DM1 (T-DM1)/trastuzumab-resistant JIMT-1 tumor-bearing mice.

After 7 days of incubation at 37°C, [225Ac]Ac-macropa-T-PEG6-DM1 was stable in both human serum (89.2% ± 0.9%) and PBS (82.8% ± 0.4%). T-PEG6-DM1 (8 mg/kg) and [225Ac]Ac-macropa-T-PEG6-DM1 (3 × 18 kBq) administered separately in non-tumor-bearing mice 10 days apart were well tolerated biochemically and hematologically. Imaging and biodistribution showed high tumor uptake of [89Zr]Zr-DFO-T-PEG6-DM1 in tumor-bearing mice at 120 hours after injection: 38.1% ± 2.8% IA/g (HCC1954) and 14.6% ± 1% IA/g (JIMT-1). In HCC1954 tumor-bearing mice, all treatment groups had complete remission (8/8), indicative of the responsiveness of the xenograft to T-DM1-based treatments, whereas for JIMT-1 xenografts (having 1/8 complete remission) at 23 days after treatment, tumor volumes were 332.1 ± 77.5 vs. 244.6 ± 63 vs. 417.9 ± 62.1 vs. 102.4 ± 18.5 for the saline (negative control), T-DM1 (positive control), T-PEG6-DM1, and [225Ac]Ac-macropa-T-PEG6-DM1, respectively.

[225Ac]Ac-macropa-T-PEG6-DM1 is more potent than ADC against trastuzumab-resistant breast cancer and necessitates clinical translation.

Strategies that selectively bind proteins of interest and target them to the intracellular protein recycling machinery for targeted protein degradation have recently emerged as powerful tools for undruggable targets in biomedical research and the pharmaceutical industry. However, targeting any new protein of interest with current degradation tools requires a laborious case-by-case design for different diseases and cell types, especially for extracellular targets. Here we observe that nanoparticles can mediate specific receptor-independent internalization of a bound protein and further develop a general strategy for degradation of extracellular proteins of interest by making full use of clinically approved components. This extremely flexible strategy aids in targeted protein degradation tool development and provides knowledge for targeted drug therapies and nanomedicine design.

Esophageal squamous cell carcinoma (ESCC) accounts for about 90% of esophageal cancer cases. The lack of effective therapeutic targets makes it difficult to improve the overall survival of patients with ESCC. Reticulon 4 Interacting Protein 1 (RTN4IP1) is a novel mitochondrial oxidoreductase. Here, a notable upregulation of RTN4IP1 is demonstrated, which is associated with poor survival in patients with ESCC. RTN4IP1 depletion impairs cell proliferation and induces apoptosis of ESCC cells. Furthermore, c-Myc regulates RTN4IP1 expression via iron regulatory protein 2 (IRP2) at the post-transcriptional level. Mechanistically, RTN4IP1 mRNA harbors functional iron-responsive elements (IREs) in the 3' UTR, which can be targeted by IRP2, resulting in increased mRNA stability. Finally, RTN4IP1 depletion abrogates amino acid uptake and induces amino acid starvation via downregulation of the amino acid transporters SLC1A5, SLC3A2, and SLC7A5, indicating a possible pathway through which RTN4IP1 contributes to ESCC carcinogenesis and progression. In vivo studies using cell-derived xenograft and patient-derived xenograft mouse models as well as a 4-nitroquinoline 1-oxide-induced ESCC model in esophageal-specific Rtn4ip1 knockout mice demonstrate the essential role of RTN4IP1 in ESCC development. Thus, RTN4IP1 emerges as a key cancer-promoting protein in ESCC, suggesting therapeutic RTN4IP1 suppression as a promising strategy for ESCC treatment.

Solute carrier family 25 member 1 (SLC25A1) affects lipid metabolism and energy regulation in multiple types of tumor cell, affecting their proliferation and survival. To the best of our knowledge, however, the impact of SLC25A1 on the proliferation and survival of esophageal squamous cell carcinoma (ESCC) cells has yet to be explored. Here, SLC25A1 expression was detected in ESCC tissues and cell lines. SLC25A1 was silenced or blocked by lentivirus transfection or 2‑[(4‑chloro‑3‑nitrophenyl)sulfonylamino]benzoic acid in ESCC cells. To evaluate the impact of SLC25A1 on in vivo and in vitro proliferation, invasion and migration of ESCC cells, Cell Counting‑Kit, wound healing, colony formation, Transwell, EdU, flow cytometry, tumor xenograft in nude mice, lipid metabolism and energy metabolism detection assays were performed. Reverse transcription‑quantitative PCR and western blot analysis were performed to determine expression of downstream molecules and pathway proteins following the silencing and blockade of SLC25A1. SLC25A1 was significantly overexpressed in ESCC tissue and cell lines. The targeted silencing of SLC25A1 or inhibition of its protein led to a significant decrease in proliferative, invasive and migratory capabilities of ESCC cells, accompanied by increased apoptosis. Additionally, silencing of the SLC25A1 gene significantly inhibited xenograft tumor growth in vivo. The present results indicate that knockdown or blockade of SLC25A1 can significantly impede the malignant biological behavior of ESCC.

The goal of precision oncology is to find effective therapeutics for every patient. Through the inclusion of emerging therapeutics in a high-throughput drug screening platform, our functional genomics pipeline inverts the common paradigm to identify patient populations that are likely to benefit from novel therapeutic strategies.

Utilizing drug screening data across a panel of 46 cancer cell lines from 11 tumor lineages, we identified an ovarian cancer-specific sensitivity to the first-in-class CRL4 inhibitors KH-4-43 and 33-11. CRL4 (i.e., Cullin-4 RING E3 ubiquitin ligase) is known to be dysregulated in a variety of cancer contexts, making it an attractive therapeutic target. Unlike proteasome inhibitors that are associated with broad toxicity, CRL4 inhibition offers the potential for tumor-specific effects.

We observed that CRL4 inhibition negatively regulates core gene signatures that are upregulated in ovarian tumors and significantly slowed tumor growth as compared to the standard of care, cisplatin, in OVCAR8 xenografts. Building on this, we performed combination drug screening in conjunction with proteomic and transcriptomic profiling to identify ways to improve the antitumor effects of CRL4 inhibition in ovarian cancer models. CRL4 inhibition consistently resulted in activation of the mitogen-activated protein kinase (MAPK) signaling cascade at both the transcriptomic and protein levels, suggesting that survival signaling is induced in response to CRL4 inhibition. These observations were concordant with the results of the combination drug screens in seven ovarian cancer cell lines that showed CRL4 inhibition cooperates with MEK inhibition. Preclinical studies in OVCAR8 and A2780 xenografts confirmed the therapeutic potential of the combination of KH-4-43 and trametinib, which extended overall survival and slowed tumor progression relative to either single agent or the standard of care.

Together, these data demonstrate the prospective utility of functional modeling pipelines for therapeutic development and underscore the clinical potential of CRL4 inhibition in the ovarian cancer context.

A precision medicine pipeline identifies ovarian cancer sensitivity to CRL4 inhibitors. CRL4 inhibition induces activation of MAPK signalling as identified by RNA sequencing, proteomics, and phosphoproteomics. Inhibitor combinations that target both CRL4 and this CRL4 inhibitor-induced survival signalling enhance ovarian cancer sensitivity to treatment.

Mitochondrial targeting in gliomas represents a novel therapeutic strategy with significant potential to enhance drug sensitivity by effectively killing glioma cells at the mitochondrial level. In this study, we developed artificial mitochondria derived from mitochondrial membrane-based nanovesicles, enabling precise mitochondrial targeting of doxorubicin (Dox) to selectively eradicate cancer cells by amplifying multiple cell death pathways. It was found that Dox-encapsulating mitochondria-based nanovesicles (DOX-MitoNVs) exhibited an extraordinary ability to penetrate the blood-brain barrier (BBB), specifically targeting gliomas. By targeting mitochondria instead of locating at the nucleus, DOX-MitoNVs not only amplified Dox mediated apoptosis effects through the overloading of intracellular Ca2+ but also intensified ferroptosis by generating reactive oxygen species (ROS). Furthermore, DOX-MitoNVs demonstrated a significant ability to modulate the tumor immune microenvironment, thereby inducing pronounced immunogenic cell death (ICD) effects. In summary, it presents a novel therapeutic strategy utilizing DOX-MitoNVs for precise mitochondrial targeting in gliomas, enhancing drug sensitivity, inducing multiple cell death pathways, and modulating the tumor immune microenvironment to promote immunogenic cell death. STATEMENT OF SIGNIFICANCE: Mitochondrial targeting in gliomas is a promising therapeutic strategy that enhances drug sensitivity by exploiting glioma cells' mitochondrial vulnerabilities. We engineered mitochondrial membrane-based nanovesicles as artificial mitochondria for precise mitochondrial targeting of Dox. This approach facilitates selective cancer cell eradication and amplifies multiple cell death pathways alongside immunogenic chemotherapy. Notably, DOX-MitoNVs effectively cross the BBB and specifically target gliomas. By focusing on mitochondria, Dox induces apoptosis and intensifies ferroptosis through ROS generation. Additionally, DOX-MitoNVs can transform the tumor immune microenvironment, promoting ICD. Overall, DOX-MitoNVs offer a promising platform for enhanced glioma therapy.

Colorectal carcinomas (CRCs) are seldom eradicated by cytotoxic chemotherapy. Cancer cells with stem-like functional properties, often referred to as "cancer stem cells" (CSCs), display preferential resistance to several anti-tumor agents used in cancer chemotherapy, but the molecular mechanisms underpinning their selective survival remain only partially understood.

In this study, we used Transcription Factor Target Genes (TFTG) enrichment analysis to identify transcriptional regulators (activators or repressors) that undergo preferential activation by chemotherapy in CRC cells with a "bottom-of-the-crypt" phenotype (EPCAM+/CD44+/CD166+; CSC-enriched) as compared to CRC cells with a "top-of-the-crypt" phenotype (EPCAM+/CD44neg/CD166neg; CSC-depleted). The two cell populations were purified in parallel by fluorescence-activated cell sorting (FACS) from a patient-derived xenograft (PDX) line representative of a moderately differentiated human CRC, following in vivo chemotherapy with irinotecan (CPT-11). The transcriptional regulators identified as differentially activated were tested for differential expression in normal vs. cancer tissues, and in cell populations enriched in stem/progenitor cell-types as compared to differentiated lineages (goblet cells, enterocytes) in the mouse colon epithelium. Finally, the top candidate was tested for mechanistic contribution to drug-resistance by selective down-regulation using short-hairpin RNAs (shRNAs).

Our analysis identified E2F4 and TFDP1, two core components of the DREAM transcriptional repression complex, as transcriptional modulators preferentially activated by irinotecan in EPCAM+/CD44+/CD166+ as compared to EPCAM+/CD44neg/CD166neg cancer cells. The expression levels of both genes (E2F4, TFDP1) were found up-regulated in CRCs as compared to human normal colon tissues, and in a sub-population of mouse colon epithelial cells enriched in stem/progenitor elements (Epcam+/Cd44+/Cd66alow/Kitneg) as compared to other sub-populations enriched in either goblet cells (Epcam+/Cd44+/Cd66alow/Kit+) or enterocytes (Epcam+/Cd44neg/Cd66ahigh). Most importantly, E2F4 down-regulation using shRNAs dramatically enhanced the sensitivity of human CRCs to in vivo treatment with irinotecan, across three independent PDX models.

Our data identified E2F4 and the DREAM repressor complex as critical regulators of human CRC resistance to irinotecan, and as candidate targets for the development of chemo-sensitizing agents.

Bladder cancer is one of the most common malignancies of the urinary system. Despite significant advances in diagnosis and treatment, the compromised therapeutic effect of chemotherapeutic agents, such as Oxaliplatin (OXA), remains a major clinical challenge. Thus, a combination therapy is required to enhance the OXA's therapeutic effectiveness and improve patient outcomes.

The thin film hydration method was used to prepare the liposomes. Drug encapsulation efficiency and loading capacity were determined to investigate the advantages of the SRT3025-loaded cell membrane hybrid liposomes (3025@ML). Bladder cancer cell lines T24 and 5637 were cultured in McCoy's 5 A and RPMI 1640 medium, respectively. The Cell Counting Kit-8 assay was used to determine the cell viability by treating cells with a medium containing either the vehicle solution (control), the cell membrane hybrid liposomes (ML), 3025@ML, or compound 3 K. The antiproliferative activities were investigated after treating cells with OXA + 3025@ML and compound 3 K + OXA. Cell death and apoptosis were quantified by trypan blue and Annexin V-APC/PI apoptosis assay after treating cells with control, OXA, OXA + 3025@ML, and 3025@ML. Western blot analysis was performed after treating cells with 3025@ML, OXA, 3 K, 3025@ML + OXA, and 3 K + OXA to determine the protein levels of pyruvate kinase M2 (PKM2) and fatty acid synthase (FASN), etc. RESULTS: The present study demonstrated that 3025@ML enhances the chemotherapeutic effect of OXA. 3025@ML + OXA treated T24 and 5637 cells showed that combination therapy significantly reduced cell viability and increased cell death rate. Flow cytometry analysis showed that the combination of 3025@ML and OXA significantly increased the percentage of apoptotic cells in T24 cells. 3025@ML and compound 3 K reduced the levels of FASN in T24 and 5637 cells and increased the anti-tumor activity of OXA. Mechanistic studies showed that 3025@ML inhibited the PI3K/AKT/mTOR signaling pathway and reduced the expression of key metabolic regulators PKM2 and FASN. Furthermore, this study demonstrated that targeting lipid metabolism and inhibiting FASN can effectively overcome the compromised therapeutic effect of OXA.

The study demonstrated that 3025@ML significantly enhances the anti-tumor activity of OXA. This novel drug delivery system inhibits key metabolic pathways, which increase DNA damage and tumor cell apoptosis. The results indicate that 3025@ML is a promising therapeutic strategy for overcoming OXA's compromised therapeutic effect and potentially improving cancer treatment outcomes.

A series of semisynthetic noscapine-urea congeners (7a-7h) as potential tubulin-binding agents are being developed by integrating a urea pharmacophore at the C-9 position of the noscapine scaffold. Their binding affinity to tubulin was predicted through molecular docking, molecular dynamics (MD) simulations, and the MM-PBSA approach. These molecules were subsequently chemically synthesized and assessed using breast cancer cell lines (MCF-7 and MDA-MB-231) and normal human embryonic kidney cells (HEK). Both the docking score and the predicted binding free energy (ΔGbind,pred) revealed that urea congeners had a stronger affinity towards tubulin than noscapine and effectively inhibited the proliferation of all cancer cell types without affecting normal healthy cells. The results indicated that compound 7g exhibited the most promise and was chosen for further studies. Moreover, MDA-MB-231 cells treated with 7g at its IC50 concentration showed morphological changes such as membrane blebbing, fragmented nuclei, and the presence of apoptotic bodies. Apoptosis induction was further confirmed by flow cytometry. Moreover, the tubulin binding assay revealed a greater binding affinity with an equilibrium dissociation constant (KD) of 42 ± 2.4 μM for compound 7g. The number of MCF-7 cells engrafted as breast tumors in nude mice was found to be reduced significantly without any adverse effects. Noscapine is already in clinical trials, but the urea noscapine congener offers an advantage because of its increased potency without impacting the nontoxic profile of noscapine.

The high mortality rate of breast cancer motivates researchers to search for effective treatments. Due to their ability to simulate human conditions, xenograft models such as CDX (Cell line-Derived Xenografts) and PDX (Patient-Derived Xenografts) have gained popularity in pre-clinical research. The choice of xenograft technique is influenced by the type of tumor employed, particularly in more aggressive tumor models like TNBC with metastases. Subcutaneous or orthotopic implantation may influence tumor engraftment rates and the applicability of the models for drug testing. To optimize xenograft models and support the development of breast cancer drugs, selecting a suitable transplantation technique is essential to attaining the best results.

This scoping review used PRISMA-Scr methodology to summarize findings from eleven articles published between 2012 and 2024 on pre-clinical trials related to xenograft models for breast cancer considering PDX began traction after 2010. Using specific criteria, the review included studies from electronic platforms. The inclusion criteria ensured relevant English sources were available in full text, while the exclusion criteria eliminated certain types of articles and inadequately comprehensive studies.

Subcutaneous and orthotopic implantation are critical methods for xenograft models in cancer research. Subcutaneous implantation is less invasive and more manageable but does not fully mimic the tumor's natural environment. Orthotopic implantation accurately mimic the migration, invasion, and molecular characteristics of the original tumor, although the procedure is more complex and requires specialized techniques. The specific research objectives determine their choice, the need for accurate tumor replication, and the testing convenience.

Orthotopic implantation is the preferable method for developing PDX and CDX models of breast cancer because it closely mimics the tumor microenvironment and metastatic behavior, yielding clinically relevant results for drug testing. Subcutaneous implantation may result in higher engraftment rates, but it cannot accurately represent the complexity of tumors.

We propose a model of combination treatment of Lewis lung carcinoma (LLC) in C57BL/6 mice that includes tumor resection and chemotherapy. A single injection of 5×106 LLC cells into the right lateral subcostal region caused the growth of the primary tumor and its metastasis to the lung. For reducing metastasis and mortality after resection, the primary tumor should be removed with subcutaneous fat on day 8 after inoculation. Antitumor and antimetastatic effects and reduced mortality were achieved by intraperitoneal injection of carboplatin (63.3 mg/kg) and paclitaxel (13.3 mg/kg); chemotherapy was administered twice. The combination of the two approaches increased the survival: the antitumor and antimetastatic effects were observed in 60% of mice with LLC relative to resection alone or treatment with cytostatics alone. However, chemoresistance was formed in 40% of mice, the values of the tumor growth inhibition index and the metastasis inhibition index decreased. This was accompanied by the appearance of cancer stem cells in the circulation with the potential for the formation of spheroids in vitro. The presented model (combination of tumor resection and chemotherapy) can be used in the development of new approaches to improve treatment efficiency in patients with lung cancer.

Human-derived tumor models are essential for preclinical development of new anticancer drug entities. Generating animal models bearing tumors of human origin, such as patient-derived or cell line-derived xenograft tumors, is dependent on immunodeficient strains. Tumor-bearing immunodeficient mice are susceptible to developing unwanted disorders primarily irrelevant to the tumor nature; and if get involved with such disorders, reliability of the study results will be undermined, inevitably confounding the research in general. Therefore, a rigorous health surveillance and clinical monitoring system, along with the establishment of a strictly controlled barrier facility to maintain a pathogen-free state, are mandatory. Even if all pathogen control and biosafety measures are followed, there are various noninfectious disorders capable of causing tissue and multiorgan damage in immunodeficient animals. Therefore, the researchers should be aware of sentinel signs to carefully monitor and impartially report them. This review discusses clinical signs of common unwanted disorders in experimental immunodeficient mice, and how to examine and report them.

Postoperative radiotherapy remains the gold standard for malignant glioma treatment. Clinical limitations, including tumor growth between surgery and radiotherapy and the emergence of radioresistance, reduce treatment effectiveness and result in local disease progression. This study aimed to develop a local drug delivery system to inhibit tumor growth before radiotherapy and enhance the subsequent anticancer effects of limited-dose radiotherapy. We developed a compound of carboplatin-loaded hydrogel (CPH) incorporated with carboplatin-loaded calcium carbonate (CPCC) to enable two-stage (peritumoral and intracellular) release of carboplatin to initially inhibit tumor growth and to synergize with limited-dose radiation (10 Gy in a single fraction) to eliminate malignant glioma (ALTS1C1 cells) in a C57BL/6 mouse subcutaneous tumor model. The doses of carboplatin in CPH and CPCC treatments were 150 μL (carboplatin concentration of 5 mg/mL) and 15 mg (carboplatin concentration of 4.1 μg/mg), respectively. Mice receiving the combination of CPH-CPCC treatment and limited-dose radiation exhibited significantly reduced tumor growth volume compared to those receiving double-dose radiation alone. Furthermore, combining CPH-CPCC treatment with limited-dose radiation resulted in significantly longer progression-free survival than combining CPH treatment with limited-dose radiation. Local CPH-CPCC delivery synergized effectively with limited-dose radiation to eliminate mouse glioma, offering a promising solution for overcoming clinical limitations.

Overexpression of aspartic proteases, as cathepsin D, is an independent marker of poor prognosis in breast cancer, correlated with the incidence of clinical metastasis. We aimed to find if HIV-1 aspartic protease (PR) can play a similar role. Murine adenocarcinoma 4T1luc2 cells were transduced with lentivirus encoding inactivated drug-resistant PR, generating subclones PR20.1 and PR20.2. Subclones were assessed for production of reactive oxygen species (ROS), expression of epithelial-mesenchymal transition (EMT) factors, and in vitro migratory activity in the presence or absence of antioxidant N-acetyl cysteine and protease inhibitors. Tumorigenic activity was evaluated by implanting cells into BALB/c mice and following tumor growth by calipering and bioluminescence imaging in vivo, and metastases, by organ imaging ex vivo. Both subclones expressed PR mRNA, and PR20.2, also the protein detected by Western blotting. PR did not induce production of ROS, and had no direct effect on cell migration rate, however, treatment with inhibitors of drug-resistant PR suppressed the migratory activity of both subclones. Furthermore, expression of N-cadherin and Vimentin in PR20.2 cells and their migration were enhanced by antioxidant treatment. Sensitivity of in vitro migration to protease inhibitors and to antioxidant, known to restore PR activity, related the effects to the enzymatic activity of PR. In vivo, PR20.2 cells demonstrated higher tumorigenic and metastatic activity than PR20.1 or parental cells. Thus, HIV-1 protease expressed in breast cancer cells determines their migration in vitro and metastatic activity in vivo. This effect may aggravate clinical course of cancers in people living with HIV-1.

Cancer-related cognitive impairment (CRCI) is prevalent among cancer patients. A critical disparity in the CRCI field is that most pre-clinical studies have been conducted on young cancer-free male rodents, although CRCI predominantly affects breast cancer and ovarian cancer women survivors. Since oxidative stress is widely implicated in the development of CRCI, we developed an ovarian cancer xenograft rat model of CRCI in Cr:NIH-RNU female rats to examine whether administration of the antioxidant N-acetylcysteine (NAC) prevents cisplatin-induced CRCI without altering its anti-cancer efficacy. In vitro, delayed treatment with NAC (10 h) following cisplatin treatment in the human ovarian cancer cell line SKOV3.ip1 did not decrease cisplatin's anti-cancer efficacy while mitigating hippocampal dendritic branching damage and neuronal apoptosis. Rats received subcutaneous and intraperitoneal implantation of SKOV3.ip1 cells. Rats received one cisplatin (5 mg/kg) injection every two weeks for a total of four cycles, with or without NAC (250 mg/kg/day), given for five consecutive days during cisplatin treatment. NAC was administered 10 h after cisplatin, based on our in vitro data. Cognitive testing was performed six to seven weeks after treatment cessation. In vivo, cognitive impairments were observed in tumor-bearing rats in the vehicle and cisplatin-treatment groups, while delayed NAC prevented cognitive impairments. Delayed NAC administration did not affect cisplatin-induced tumor volume reduction. Our study supports using NAC to mitigate cisplatin-induced CRCI through the novel development of an ovarian cancer rodent model. This study highlights the importance of developing clinically relevant tumor-bearing models to elucidate the underlying mechanisms associated with CRCI, which will aid in identifying potential therapeutic agents for preventing CRCI.

Glioma is a prevalent brain malignancy associated with poor prognosis. Although chemotherapy serves as the primary treatment for brain tumors, its effectiveness is hindered by the limited ability of drugs to traverse the blood-brain barrier (BBB) and the development of drug resistance linked to tumor hypoxia. Herein, we report the creation of hybrid camouflaged multifunctional nanovesicles comprising membranes of tumor C6 cells (mT) and bacterial outer membrane vesicles (OMVs) and co-loaded with manganese dioxide nanoparticles (MnO2 NPs) and doxorubicin (DOX) to synergistically enhance the chemotherapy/chemodynamic therapy (CDT) of glioma. Owing to OMV-mediated BBB penetration and mT-inherited tumor-homing properties, MnO2-DOX@mT/OMVs can penetrate the BBB and enhance the tumor cell-specific uptake of DOX via "proton sponge effect"-mediated lysosomal escape. This enhances the apoptotic effect induced by DOX and minimizing DOX-associated cardiotoxicity by facilitating the accumulation of DOX at the tumor site. Furthermore, the MnO2 NPs in MnO2-DOX@mT/OMVs can generate potent CDT by accelerating the Fenton-like reaction with DOX-generated H2O2 and achieving glutathione (GSH)-depletion-induced glutathione peroxidase 4 (GPX4) inactivation. These results showed that MnO2-DOX@mT/OMVs, designed for brain tumor targeting, significantly inhibited tumor growth and exhibited favorable biological safety. This innovative approach offers the augmentation of anticancer treatment efficacy via a potential combination of chemotherapy and CDT.

Macrophage efferocytosis (clearance of apoptotic cells) is crucial for tissue homeostasis and wound repair, where macrophages secrete factors that promote resolution of inflammation and regenerative signalling. This study examined the role of efferocytic macrophage-associated CCL2 secretion, its influence on mesenchymal stem/progenitor cell (MSPC) chemotaxis, and in vivo cell recruitment using Ccr2-/- (KO) mice with disrupted CCL2 receptor signalling in two regenerative models: ossicle implants and ulnar stress fractures. Single cell RNA sequencing and PCR validation indicated that efferocytosis of various apoptotic cells at bone injury sites (osteoblasts, pre-osteoblasts, MSPC) upregulated CCL2. CCL2 gradients enhanced MSPC migration through type I collagen matrices. In vivo, MSPC (LepR+) infiltration was significantly reduced while macrophage (F4/80+) infiltration increased in KO ossicle implants versus WT. In ulnar stress fractures, micro-CT revealed increased mineralized callus incidence in CCR2 KO male mice 5 days post injury (dpi) versus WT. By 7-dpi callus fractional bone volume, trabecular thickness, and bone mineral density were increased versus WT. Immunohistochemistry of mice 5-dpi confirmed an increase in callus area (including soft tissue); however, the percent of osteoprogenitors (%Osx+) within the callus was not different. These findings suggest that CCL2 differentially impacts MSPC recruitment depending on bone wound healing model.

Carcinogenesis results from the sequential acquisition of oncogenic mutations that convert normal cells into invasive, metastasizing cancer cells. Colorectal cancer exemplifies this process through its well-described adenoma-carcinoma sequence, modeled previously using clustered regularly interspaced short palindromic repeats (CRISPR) to induce four consecutive mutations in wild-type human gut organoids. Here, we demonstrate that long-term culture of mismatch-repair-deficient organoids allows the selection of spontaneous oncogenic mutations through the sequential withdrawal of Wnt agonists, epidermal growth factor (EGF) agonists and the bone morphogenetic protein (BMP) antagonist Noggin, while TP53 mutations were selected through the addition of Nutlin-3. Thus, organoids sequentially acquired mutations in AXIN1 and AXIN2 (Wnt pathway), TP53, ACVR2A and BMPR2 (BMP pathway) and NRAS (EGF pathway), gaining complete independence from stem cell niche factors. Quadruple-pathway (Wnt, EGF receptor, p53 and BMP) mutant organoids formed solid tumors upon xenotransplantation. This demonstrates that carcinogenesis can be recapitulated in a DNA repair-mutant background through in vitro selection that targets four consecutive cancer pathways.