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B1-integrin binding and lysosome targeting aptamer homes to triple-negative breast cancer in mice (in vivo images)

ABSTRACT: 2 nmol of GreenB1-FAM or RND-FAM were injected i.v. (n=3 in each group). After one hour of circulation time, mice were anaesthetised, sacrificed, perfused, and tumour, heart, liver, lung, and kidney were collected. Organs were flash-frozen in liquid nitrogen and kept at -80 °C until further use. Organs were sectioned at 30 um and fixed in methanol (equilibrated at -20 °C) for 2 minutes. 5%BSA in PBS with 0.05% Tween-20 was applied to tissue sections for 30 min at room temperature to block non-specific antibody binding. GreenB1-FAM or RND-FAM was detected using a 1:200 dilution of rabbit anti-fluorescein antibody (#A-11090, ThermoFisher) at room temperature for one hour, followed by 1:400 dilution of secondary anti-rabbit AlexaFluor555 labelled antibody (#A32794, ThermoFisher) at room temperature for one hour. Staining with secondary antibody alone was used as a control. Five fields of view were imaged from all organs from all mice. Both primary and secondary antibody labelled, and secondary antibody alone were imaged. For statistical analysis, mean pixel intensity from primary and secondary antibody labelled organ sections were divided by mean pixel intensity from the corresponding organ from the same animal when stained with secondary antibody alone. Nuclei were stained using Hoechst 33342 (#62249, ThermoFisher) solution at 20 uM concentration. Statistical analysis was performed in Prism 9.3.1 (GraphPad) using 2-way ANOVA followed by Šidák’s multiple comparison test.

ORGANISM(S): Mus musculus (mouse)

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PROVIDER: S-BIAD483 | bioimages |

REPOSITORIES: bioimages

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Project description:PurposeStemming from a myriad of genetic and epigenetic alterations, triple-negative breast cancer (TNBC) is tied to poor clinical outcomes and aspires for individualized therapies. Here we investigated the therapeutic potential of co-inhibiting integrin-dependent signaling pathway and BRD4, a transcriptional and epigenetic mediator, for TNBC.MethodsTwo independent patient cohorts were subjected to bioinformatic and IHC examination for clinical association of candidate cancer drivers. The efficacy and biological bases for co-targeting these drivers were interrogated using cancer cell lines, a protein kinase array, chemical inhibitors, RNAi/CRISPR/Cas9 approaches, and a 4 T1-Balb/c xenograft model.ResultsWe found that amplification of the chromosome 8q24 region occurred in nearly 20% of TNBC tumors, and that it coincided with co-upregulation or amplification of c-Myc and FAK, a key effector of integrin-dependent signaling. This co-upregulation at the mRNA or protein level correlated with a poor patient survival (p < 0.0109 or p < 0.0402, respectively). Furthermore, we found that 14 TNBC cell lines exhibited high vulnerabilities to the combination of JQ1 and VS-6063, potent pharmacological antagonists of the BRD4/c-Myc and integrin/FAK-dependent pathways, respectively. We also observed a cooperative inhibitory effect of JQ1 and VS-6063 on tumor growth and infiltration of Ly6G+ myeloid-derived suppressor cells in vivo. Finally, we found that JQ1 and VS-6063 cooperatively induced apoptotic cell death by altering XIAP, Bcl2/Bcl-xl and Bim levels, impairing c-Src/p130Cas-, PI3K/Akt- and RelA-associated signaling, and were linked to EMT-inducing transcription factor Snail- and Slug-dependent regulation.ConclusionBased on our results, we conclude that the BRD4/c-Myc- and integrin/FAK-dependent pathways act in concert to promote breast cancer cell survival and poor clinical outcomes. As such, they represent promising targets for a synthetic lethal-type of therapy against TNBC.

Project description:Selective delivery of chemotherapy to the tumor site while sparing healthy cells and tissues is an attractive approach for cancer treatment. Carriers such as peptides can facilitate selective tumor targeting and payload delivery. Peptides with specific affinity for the overexpressed cell-surface receptors in cancer cells are conjugated to chemotherapy to afford peptide-drug conjugates (PDCs) that show selective uptake by cancer cells. Using a 10-mer linear peptide (WxEAAYQrFL) called 18-4 that targets and binds breast cancer cells, we designed a peptide 18-4-doxorubicin (Dox) conjugate with high specific toxicity toward triple-negative breast cancer (TNBC) MDA-MB-231 cells and 30-fold lower toxicity to normal breast MCF10A epithelial cells. Here, we elucidate the in vivo activity of this potent and tumor-selective peptide 18-4-Dox conjugate in mice bearing orthotopic MDA-MB-231 tumors. Mice treated with four weekly injections of the conjugate showed significantly lower tumor volumes compared to mice treated with free Dox at an equivalent Dox dose. Immunohistochemical (IHC) analysis of mice tissues revealed that treatment with a low dose of PDC (2.5 mg/kg of Dox equiv) reduced the expression of proliferation markers (PCNA and Ki-67) and increased apoptosis (evidenced by increased caspase-3 expression). At the same dose of free Dox (2.5 mg/kg), the expression of these markers was similar to that of saline treatment. Accordingly, significantly more Dox accumulated in tumors of conjugate-treated mice (7-fold) compared to the Dox-treated mice, while lower levels of Dox were observed in the liver, heart, and lungs of peptide-Dox conjugate-treated mice (up to 3-fold less) than Dox-treated mice. The IHC analysis of keratin 1 (K1), the receptor for peptide 18-4, revealed K1 upregulation in tumors and low levels in normal mammary fat pad and liver tissues from mice, suggesting preferential uptake of PDCs by TNBC to be K1 receptor-mediated. Taken together, our data support the use of a PDC approach to deliver chemotherapy selectively to the TNBC to inhibit tumor growth.

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B1-integrin binding and lysosome targeting aptamer homes to triple-negative breast cancer in mice (in vivo images)

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