Murine Orthotopic Breast Cancer Model Research Articles

Novel C-terminal heat shock protein 90 inhibitors target breast cancer stem cells and block migration, self-renewal, and epithelial-mesenchymal transition.

In patients with triple‐negative breast cancer (TNBC), evidence suggests that tumor‐initiating cells (TIC) have stem cell‐like properties, leading to invasion and metastasis. HSP90 plays a critical role in the conformational maintenance of many client proteins in TIC development. Therefore, we hypothesize that the novel C‐terminal HSP90 inhibitors KU711 and KU758 can target TIC and represent a promising strategy for overcoming metastasis. Human breast cancer cells (MDA‐MB‐468LN, MDA‐MB‐231) treated with the HSP90 inhibitors KU711, KU758, and 17‐AAG showed a 50–80% decrease in TIC markers CD44 and aldehyde dehydrogenase (P < 0.01) as assessed by flow cytometry. A decrease in sphere formation, which was used to assess self‐renewal, was observed after the treatment of TNBC cells starting at 2.5 µm KU711 and 0.31 µm KU758. KU compounds also blocked the invasion and migration of TNBC cells in a dose‐dependent manner. The knockdown of HSP90 clients was observed without any change in prosurvival HSP70 levels. In vivo, in a murine orthotopic breast cancer model, treatment with KU758 and KU711 yielded an approximately twofold and a fourfold reduction in tumor volumes versus control, respectively, without demonstrated toxicity. In conclusion, C‐terminal HSP90 inhibitors are potent novel therapeutics against TNBC in vitro and in vivo as they target TICs and block invasion, EMT transition, and self‐renewal.

A novel anti-HER2 anthracycline-based antibody-drug conjugate induces adaptive anti-tumor immunity and potentiates PD-1 blockade in breast cancer

Increasing evidence suggests that antibody-drug conjugates (ADCs) can enhance anti-tumor immunity and improve clinical outcome. Here, we elucidate the therapeutic efficacy and immune-mediated mechanisms of a novel HER2-targeting ADC bearing a potent anthracycline derivate as payload (T-PNU) in a human HER2-expressing syngeneic breast cancer model resistant to trastuzumab and ado-trastuzumab emtansine. Mechanistically, the anthracycline component of the novel ADC induced immunogenic cell death leading to exposure and secretion of danger-associated molecular signals. RNA sequencing derived immunogenomic signatures and TCRβ clonotype analysis of tumor-infiltrating lymphocytes revealed a prominent role of the adaptive immune system in the regulation of T-PNU mediated anti-cancer activity. Depletion of CD8 T cells severely reduced T-PNU efficacy, thus confirming the role of cytotoxic T cells as drivers of the T-PNU mediated anti-tumor immune response. Furthermore, T-PNU therapy promoted immunological memory formation in tumor-bearing animals protecting those from tumor rechallenge. Finally, the combination of T-PNU and checkpoint inhibition, such as α-PD1, significantly enhanced tumor eradication following the treatment. In summary, a novel PNU-armed, HER2-targeting ADC elicited long-lasting immune protection in a murine orthotopic breast cancer model resistant to other HER2-directed therapies. Our findings delineate the therapeutic potential of this novel ADC payload and support its clinical development for breast cancer patients and potentially other HER2 expressing malignancies.

Inflammatory peroxidases promote breast cancer progression in mice via regulation of the tumour microenvironment.

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, well known for their antimicrobial activity, are released in high quantities by infiltrating immune cells in breast cancer. However, the functional importance of their presence within the tumour microenvironment is unclear. We have recently described a new role for peroxidases as key regulators of fibroblast and endothelial cell functionality. In the present study, we investigate for the first time, the ability of peroxidases to promote breast cancer development and progression. Using the 4T1 syngeneic murine orthotopic breast cancer model, we examined whether increased levels of peroxidases in developing mammary tumours influences primary tumour growth and metastasis. We showed that MPO and EPO stimulation increased mammary tumour growth and enhanced lung metastases, effects that were associated with reduced tumour necrosis, increased collagen deposition and neo-vascularisation within the primary tumour. Invitro, peroxidase treatment, robustly stimulated human mammary fibroblast migration and collagen typeI and typeVI secretion. Mechanistically, peroxidases induced the transcription of pro-tumorigenic and metastatic MMP1, MMP3 and COX-2 genes. Taken together, these findings identify peroxidases as key contributors to cancer progression by augmenting pro-tumorigenic collagen production and angiogenesis. Importantly, this identifies inflammatory peroxidases as therapeutic targets in breast cancer therapy.

Treatment with the vascular disruptive agent OXi4503 induces an immediate and widespread epithelial to mesenchymal transition in the surviving tumor

Epithelial to mesenchymal transition (EMT) is considered an important mechanism in tumor resistance to drug treatments; however, in vivo observation of this process has been limited. In this study we demonstrated an immediate and widespread EMT involving all surviving tumor cells following treatment of a mouse model of colorectal liver metastases with the vascular disruptive agent OXi4503. EMT was characterized by significant downregulation of E-cadherin, relocation and nuclear accumulation of β-catenin as well as significant upregulation of ZEB1 and vimentin. Concomitantly, significant temporal upregulation in hypoxia and the pro-angiogenic growth factors hypoxia-inducible factor 1-alpha, hepatocyte growth factor, vascular endothelial growth factor and transforming growth factor-beta were seen within the surviving tumor. The process of EMT was transient and by 5 days after treatment tumor cell reversion to epithelial morphology was evident. This reversal, termed mesenchymal to epithelial transition (MET) is a process implicated in the development of new metastases but has not been observed in vivo histologically. Similar EMT changes were observed in response to other antitumor treatments including chemotherapy, thermal ablation, and antiangiogenic treatments in our mouse colorectal metastasis model and in a murine orthotopic breast cancer model after OXi4503 treatment. These results suggest that EMT may be an early mechanism adopted by tumors in response to injury and hypoxic stress, such that inhibition of EMT in combination with other therapies could play a significant role in future cancer therapy.Vascular disruptive treatments effectively destroy over 90% of solid tumors with minimal effects on host tissues but a viable rim of cells persists in the tumor periphery that leads to recurrence. An immediate and widespread epithelial to mesenchymal transition (EMT) occurs within the viable rim after treatment that may be responsible for this resistance to treatment. Targeting EMT in combination with vascular disruptive agents or other therapies in the clinic may improve treatment outcomes.

Evaluation of new bi-functional terpolymeric nanoparticles for simultaneous in vivo optical imaging and chemotherapy of breast cancer.

Successful development of a nanoparticulate system for cancer chemotherapy requires detailed knowledge of its biodistribution, clearance and anti-tumour efficacy in vivo. Herein we developed new bi-functional nanoparticles for simultaneous in vivo optical imaging and delivery of the anticancer drug doxorubicin (Dox) for enhanced chemotherapy. Two types of nanoparticles were synthesized, namely preformed nanoparticles (PF-NPs) and self-assembled nanoparticles (SA-NPs). The PF-NPs were prepared by cross-linking graft polymerization of methacrylic acid and polysorbate 80 with starch (PMAA-PS 80-g-St) and then loading the particles with Dox. The SA-NPs were formed upon addition of Dox to non-cross-linked PMAA-PS 80-g-St. A near infrared fluorescent probe was conjugated with the PMAA unit of the nanoparticles. The biodistribution, tumour targeting and pharmacokinetics of the Dox-loaded nanoparticles in mice were determined by in vivo/ex vivo fluorescence imaging and ex vivo fluorescence microscopy. The anti-tumour efficacy of the nanoparticles was investigated using a murine orthotopic breast cancer model. PF-NPs had an average hydrodynamic diameter and zeta potential of 137 ± 3nm and -38 ± 1mV, respectively. These values were measured at 62 ± 5nm and -35 ± 5mV for SA-NPs. PF-NPs exhibited a porous morphology while the SA-NPs appeared to have a denser structure. SA-NPs outperformed the PF-NPs in terms of blood circulation, tumour uptake and penetration. PF-NPs and SA-NPs exhibited no systemic toxicity and inhibited tumour growth significantly better than the free Dox solution with SA-NPs being the best, attributable to their excellent tumour uptake and penetration. This work demonstrates the usefulness of these bi-functional nanoparticles as nanotheranostics.

Comparison of the therapeutic efficacy of 188Rhenium-liposomes and liposomal doxorubicin in a 4T1 murine orthotopic breast cancer model

Liposomal doxorubicin (Lipo-DOX) has been widely and successfully used in chemotherapy for breast cancer patients. Since our previous studies found that 188Rhenium (188Re)-N,N-bis (2-mercaptoethyl)-N',N'-diethy-lethylenediamine (BMEDA)-labeled pegylated liposomes (188Re-liposomes) have radiotherapeutic potential in a colon cancer model, and little information is available to make a comparison of the therapeutic efficacy of internal radiotherapy and chemotherapy, this study evaluates the therapeutic efficacy of 188Re-liposomes and Lipo-DOX, in a 4T1 murine orthotopic breast cancer model. MicroSPECT/CT imaging showed that the highest uptake of 188Re-liposomes was found at 24 h after intravenous administration. The results of a bio-distribution assay also demonstrated that the highest uptake of 188Re-liposomes in a tumor was 3.03±0.29 (%ID/g) at 24 h, and that the highest tumor to muscle ratio was approximately 17 at 48 h. According to measurements of body weight and survival rate, the maximum tolerated doses (MTD) of 188Re-liposomes and Lipo-DOX were 37 MBq and 25 mg/kg, respectively. In a study of therapeutic efficacy, mice with 4T1 orthotopic breast tumors that were treated with 188Re-liposomes (4/5 MTD, 29.6 MBq) or Lipo-DOX (4/5 MTD, 20 mg/kg), showed a significant inhibition of tumor growth. In the small tumor model (50 mm3), the lifespan of 4T1 tumor-bearing mice treated with 188Re-liposomes and Lipo-DOX was increased by 21.7 and 169.6%, respectively, compared to those treated with normal saline. In the large tumor model (300 mm3), the lifespan of the 188Re-liposomes and the Lipo-DOX treated group was also increased by 35.2 and 141.2%, respectively. In this study, it was found that Lipo-DOX is better than 188Re-liposomes, for the treatment of 4T1 breast cancer. A further investigation of combined therapy, in a breast cancer model, using 188Re-liposomes and Lipo-Dox, to determine whether a synergistic effect exists, is ongoing in our laboratory.

A multistage nanodelivery system for therapeutic applications and medical imaging: in vivo biodistribution and efficacy study in a murine orthotopic breast cancer model.

Abstract Abstract #2160 The ability to deliver therapeutic compounds specifically to diseased sites is crucial for effectively treating human illnesses. Nanotechnology is emerging as a tool for resolving challenges in the delivery of poorly administrable drugs by transforming them into nanometer-sized particles (nanotherapeutics). The progress thus far gave rise to a large number of nanotherapeutics, but the accomplishment of the original objective, which is to increase drug concentration at target sites, has not been fully realized. A series of biological barriers pose as insurmountable obstacles which limit or completely abolish the ability to selectively deliver a therapeutic agent. We hypothesized that a multi-stage system for systemic delivery (MSDS) could be designed to interact with and successfully overcome sequential biological barriers. We developed a MSDS using biodegradable, biocompatible silicon particles optimally sized and shaped to travel into the blood flow, avoid RES, marginate and adhere to tumor vasculature. The first stage carriers (FSCs) contain nanopores of specific size that can load, carry, release over time, and deliver multiple types of second stage nanoparticles (SSNs). The nanoparticles can be optimized to reach, through vessels fenestrations, the tumor environment and finally release their therapeutic payload into target cells.&amp;#x2028; &amp;#x2028; The ease in chemically modifying the silicon surface permits the attachment of fluorescent and radiolabeled imaging molecules to the FSCs. Fluorescent and magnetic SSNs can be loaded into the pores of the FSCs. Once assembled, the resulting MSDS can be imaged using different in vivo imaging modalities from its administration to its final localization into the body. We further modified our FSCs with PEG molecules to increase their circulation time and with antibodies directed against the VEGFR2 membrane protein to increase the localization of the MSDS in tumor associated vessels. We monitored the biodistribution of all MSDSs and characterized their biocompatibility in the different tissues. Finally, we loaded the pores of the FSCs with a lipidic nanoformulation of the potent anti cancer drug doxorubicin (dox-liposomes and dox-micelles) and followed the efficacy of this treatment. Taken together, these studies provide first time evidence that silicon nanoporous particles can be used as effective carriers for the simultaneous delivery of different nanotherapeutics in vivo. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2160.