Model Of Human Breast Cancer Research Articles

Long-term breast cancer response to CDK4/6 inhibition defined by TP53-mediated geroconversion

Inhibition of CDK4/6 kinases has led to improved outcomes in breast cancer. Nevertheless, only a minority of patients experience long-term disease control. Using a large, clinically annotated cohort of patients with metastatic hormone receptor-positive (HR+) breast cancer, we identify TP53 loss (27.6%) and MDM2 amplification (6.4%) to be associated with lack of long-term disease control. Human breast cancer models reveal that p53 loss does not alter CDK4/6 activity or G1 blockade but instead promotes drug-insensitive p130 phosphorylation by CDK2. The persistence of phospho-p130 prevents DREAM complex assembly, enabling cell-cycle re-entry and tumor progression. Inhibitors of CDK2 can overcome p53 loss, leading to geroconversion and manifestation of senescence phenotypes. Complete inhibition of both CDK4/6 and CDK2 kinases appears to be necessary to facilitate long-term response across genomically diverse HR+ breast cancers.

Molecular and Pathobiology of Canine Mammary Tumour: Defining a Translational Model for Human Breast Cancer.

Canine mammary tumours (CMT) have histological, clinicopathological and molecular resemblances to human breast cancer (HBC), positioning them as viable models for studying the human disease. CMT initiation and progression occur spontaneously in immune-competent animals, which challenge the suggested limitations of genetically modified mice, also enabling the evaluation of immunotherapies in canine patients. Dogs have shorter life expectancy compared to humans, and cancer advances more rapidly in this species. This makes it possible to perform studies about the clinical efficacy of new therapeutic modalities in a much shorter time than in human patients. The identification of biomarkers for tumour subtypes, progression and treatment response paves the way for the development of novel therapeutic and diagnostic approaches. This review addresses the similarities between CMT and HBC and the molecular signatures identified in CMT samples that have been explored to date. We proposed a detailed molecular exploration of the CMT stroma using state-of-the-art methods in transcriptomics and proteomics. Using CMT as an analog for HBC not only helps to understand the complexities of the disease, but also to advance comparative oncology to the next level to prove the claim of dogs as a valid translational model.

Exploring the role of microRNAs as diagnostic and prognostic biomarkers in canine mammary tumors.

Canine mammary tumors (CMTs) represent a significant health concern in dogs, with a high incidence among intact female dogs. CMTs are a promising comparative model for human breast cancer, due to sharing several pathophysiological features. Additionally, CMTs have a strong genetic correlation with their human counterpart, including the expression of microRNAs (miRNAs). MiRNAs are a class of non-coding RNAs that play important roles in post-translational regulation of gene expression, being implicated in carcinogenesis, tumor progression, and metastasis. Moreover, miRNAs hold promise as diagnostic, prognostic, and metastatic biomarkers. Understanding the molecular mechanisms underlying CMTs is crucial for improving diagnosis, prognosis, and monitoring of treatments. Herein, we provide a comprehensive overview of the current knowledge on miRNAs in CMTs, highlighting their roles in carcinogenesis and their potential as biomarkers. Additionally, we highlight the current limitations and critically discuss the overarching challenges in this field, emphasizing the need for future research to translate miRNA findings into veterinary clinical practice.

Isophorone-based crystallization-induced-emission sensors detect proteome aggregation in live cells and tissues with breast cancer

BackgroundProtein misfolding and aggregation can lead to various diseases. Recent studies have shed light on the aggregated protein in breast cancer pathology, which suggests that it is crucial to design chemical sensors that visualize protein aggregates in breast cancer, especially in clinical patient-derived samples. However, most reported sensors are constrained in cultured cell lines. ResultsIn this work, we present the development of two isophorone-based crystallization-induced-emission fluorophores for detecting proteome aggregation in breast cancer cell line and tissues biopsied from diseased patients, designated as A1 and A2. These probes exhibited viscosity sensitivity and recovered their fluorescence strongly at crystalline state. Moreover, A1 and A2 exhibit selective binding capacity and strong fluorescence for various aggregated proteins. Utilizing these probes, we detect protein aggregation in stressed breast cancer cells, xenograft mouse model of human breast cancer and clinical patient-derived samples. Notably, the fluorescence intensity of both probes light up in tumor tissues. SignificanceThe synthesized isophorone-based crystallization-induced-emission fluorophores, A1 and A2, enable sensitive detection of protein aggregation in breast cancer cells and tissues. In the future, aggregated proteins are expected to become indicators for early diagnosis and clinical disease monitoring of breast cancer.

Enhanced eMAGE applied to identify genetic factors of nuclear hormone receptor dysfunction via combinatorial gene editing

Technologies that generate precise combinatorial genome modifications are well suited to dissect the polygenic basis of complex phenotypes and engineer synthetic genomes. Genome modifications with engineered nucleases can lead to undesirable repair outcomes through imprecise homology-directed repair, requiring non-cleavable gene editing strategies. Eukaryotic multiplex genome engineering (eMAGE) generates precise combinatorial genome modifications in Saccharomyces cerevisiae without generating DNA breaks or using engineered nucleases. Here, we systematically optimize eMAGE to achieve 90% editing frequency, reduce workflow time, and extend editing distance to 20 kb. We further engineer an inducible dominant negative mismatch repair system, allowing for high-efficiency editing via eMAGE while suppressing the elevated background mutation rate 17-fold resulting from mismatch repair inactivation. We apply these advances to construct a library of cancer-associated mutations in the ligand-binding domains of human estrogen receptor alpha and progesterone receptor to understand their impact on ligand-independent autoactivation. We validate that this yeast model captures autoactivation mutations characterized in human breast cancer models and further leads to the discovery of several previously uncharacterized autoactivating mutations. This work demonstrates the development and optimization of a cleavage-free method of genome editing well suited for applications requiring efficient multiplex editing with minimal background mutations.

Canine Mammary Tumors as a Potential Model for Human Breast Cancer in Comparative Oncology.

Clinical and molecular similarities between canine mammary tumors (CMTs) and human breast cancer (HBC) propel scientists to further study their application in comparative oncology as a model for human breast cancer. In total, 64 canine mammary tumors were selected to study the most common markers, which are applicable for human breast cancer treatment, including estrogen and progesterone receptors (ER and PR), human epidermal growth factor (HER2/neu), Ki67, and cyclooxygenase 2 (Cox2). Immunohistochemistry (IHC) was used to assess the protein expression. The Veterinary Nottingham Prognostic Index (Vet-NPI) was also computed. Moreover, univariate and multivariable Cox proportional hazard analyses were applied to estimate hazard ratios (HRs). The results demonstrated that Ki67 was strongly expressed in the triple-negative tumors, and Ki67 protein expression continuously increased over the increase of Cox2 protein expression (p < 0.001). Further analysis revealed a significant difference among canine mammary subtypes and Vet-NPI, in which triple-negative tumors displayed the highest mean score compared to other subtypes (p < 0.001). In addition, the multivariable analysis revealed that the regional mastectomy procedure (adjusted HR = 2.78 (1.14-6.8)), the triple-negative tumors (adjusted HR = 48.08 (7.74-298.8)), strong Ki67 protein expression group (adjusted HR = 7.88 (2.02-30.68)), and strong Cox2 protein expression group (adjusted HR = 29.35 (5.18-166.4)) demonstrated significantly lower disease-free survival rates compared to other corresponding groups. Overall, canine mammary tumors showed strong similarities to human breast cancer in terms of clinical and molecular aspects; therefore, they could be suggested as a model for human breast cancer in comparative oncology.

Activation of the Anaphase Promoting Complex Restores Impaired Mitotic Progression and Chemosensitivity in Multiple Drug-Resistant Human Breast Cancer.

The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro studies using canine MDR lymphoma cancer cells demonstrate that the Anaphase Promoting Complex (APC) is impaired in MDR cells compared to normal canine control and drug-sensitive cancer cells. Here, we sought to establish whether this phenomena is a generalizable mechanism independent of species, malignancy type, or chemotherapy regime. To test the association of blunted APC activity with MDR cancer behavior, we used matched parental and MDR MCF7 human breast cancer cells, and a patient-derived xenograft (PDX) model of human triple-negative breast cancer. We show that APC activating mechanisms, such as APC subunit 1 (APC1) phosphorylation and CDC27/CDC20 protein associations, are reduced in MCF7 MDR cells when compared to chemo-sensitive matched cell lines. Consistent with impaired APC function in MDR cells, APC substrate proteins failed to be effectively degraded. Similar to our previous observations in canine MDR lymphoma cells, chemical activation of the APC using Mad2 Inhibitor-1 (M2I-1) in MCF7 MDR cells enhanced APC substrate degradation and resensitized MDR cells in vitro to the cytotoxic effects of the alkylating chemotherapeutic agent, doxorubicin (DOX). Using cell cycle arrest/release experiments, we show that mitosis is delayed in MDR cells with elevated substrate levels. When pretreated with M2I-1, MDR cells progress through mitosis at a faster rate that coincides with reduced levels of APC substrates. In our PDX model, mice growing a clinically MDR human triple-negative breast cancer tumor show significantly reduced tumor growth when treated with M2I-1, with evidence of increased DNA damage and apoptosis. Thus, our results strongly support the hypothesis that APC impairment is a driver of aggressive tumor development and that targeting the APC for activation has the potential for meaningful clinical benefits in treating recurrent cases of MDR malignancy.

Pegylated liposome encapsulating docetaxel using microfluidic mixing technique: Process optimization and results in breast cancer models

The development of nanoparticles could help to improve the efficacy/toxicity balance of drugs. This project aimed to develop liposomes and immunoliposomes using microfluidic mixing technology.Various formulation tests were carried out to obtain liposomes that met the established specifications. The liposomes were then characterized in terms of size, polydispersity index (PDI), docetaxel encapsulation rate and lamellarity. Antiproliferative activity was tested in human breast cancer models ranging from near-negative (MDA-MB-231), positive (MDA-MB-453) to HER2 positive. Pharmacokinetic studies were performed in C57BL/6 mice.Numerous batches of liposomes were synthesised using identical molar ratios and by varying the microfluidic parameters TFR, FRR and buffer. All synthesized liposomes have a size < 200 nm, but only Lipo-1, Lipo-6, Lipo-7, Lipo-8 have a PDI < 0.2, which meets our initial requirements. The size of the liposomes was correlated with the total FRR, for a 1:1 FRR the size is 122.2 ± 12.3 nm, whereas for a 1:3 FRR the size obtained is 163.4 ± 34.0 nm (p = 0.019. Three batches of liposomes were obtained with high docetaxel encapsulation rates > 80 %. Furthermore, in vitro studies on breast cancer cell lines demonstrated the efficacy of liposomes obtained by microfluidic mixing technique. These liposomes also showed improved pharmacokinetics compared to free docetaxel, with a longer half-life and higher AUC (3-fold and 3.5-fold increase for the immunoliposome, respectively).This suggests that switching to the microfluidic process will produce batches of liposomes with the same characteristics in terms of in vitro properties and efficacy, as well as the ability to release the encapsulated drug over time in vivo. This time-efficiency of the microfluidic technique is critical, especially in the early stages of development.

Abstract ND12: Discovery of PF-07220060, a potent and selective CDK4 inhibitor

Abstract PF-07220060 was developed based on the clinical need to improve efficacy and safety outcomes in patients treated with currently approved dual CDK4/6 inhibitors, including Palbociclib. Although all dual CDK4/6 inhibitors increase response rates and prolong disease control, they show common dose-limiting hematologic toxicities, foremost neutropenia. This poses challenges to provide these agents at doses necessary to extinguish cell cycling in tumors. Human ER+ breast cancer cells are highly dependent on CDK4 but not CDK6. By contrast, CDK4 is dispensable for human bone marrow derived cells, or cells enriched for neutrophil precursors or hematopoietic stem cells. This is primarily due to the compensatory role of CDK6 in these compartments. These observations prompted us to develop PF-07220060, an aminopyrimidine-based inhibitor, endowed with 20-fold and 4-fold increased selectivity for CDK4 over CDK6 when compared to Palbociclib and Abemaciclib/Ribociclib, respectively. CDK4-over-CDK6 selectivity is explained by G-loop conformational differences between the two kinases, with the G-loop of CDK4 being more flexible, assuming a more open conformation when bound to CDK4 selective versus non-selective inhibitors. PF-07220060 reduces dose-limiting neutropenia in beagle dogs by 15-fold when compared with Palbociclib, consistent with early clinical data obtained with PF-07220060. Higher tolerated plasma concentrations of PF-07220060 versus dual CDK4/6 inhibitors enable increased inhibition of CDK4 kinase activity in tumors. This translates to improved tumor growth inhibition across CDK4-driven models of human breast and prostate cancer at clinically relevant doses. Our work establishes proof-of-concept for CDK4 selective inhibition and introduces a best-in-class CDK4 inhibitor for treatment of HR+ HER2- breast cancer and other CDK4 dependent tumor types. Citation Format: Lars Anders, Gary Gallego. Discovery of PF-07220060, a potent and selective CDK4 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND12.

Abstract 1321: In vivo immune cell programming using mRNA-LNP chimeric antigen receptors

Abstract Introduction: The tumor microenvironment (TME) is a dynamic and multifaceted system that comprises largely myeloid lineage immune cells including monocytes, neutrophils, macrophages, and dendritic cells. Reprogramming the myeloid compartment within the context of cancer therapy presents an attractive strategy to overcome current limitations in solid tumor treatment. We have developed a novel approach that harnesses the natural ability of Myeloid cells to take up mRNA loaded lipid nanoparticles, to reprogram these cells with chimeric antigen receptors (CAR’s) that arm these cells to recognize and kill tumor cells. Methods: Current LNPs technology is sufficient to deliver significant mRNA cargo to numerous cell types, including B cells, T cells and myeloid cells. Leveraging this phenomenon we have developed a new class of chimeric antigen receptors (CARs), using protein biology that imparts targeted immune cell expression and functionality, specifically in Myeloid cells. These CARs harness Immunoreceptor Tyrosine-based Activation Motif (ITAM) signaling adaptors expressed exclusively in immune cells, achieving immune cell-type specific CAR-directed tumor lysis and cytokine responses. With a FcaR I (CD89) based CAR design, we have developed a CAR that is dependent on the common Fc receptor gamma chain, primarily restricted to myeloid cells, for stable expression and function. Results: We have demonstrated specific expression of CD89-based CAR in myeloid cells in mouse and monkey following systemic mRNA/LNP delivery, and in human myeloid cells following in vitro treatment with mRNA/LNPs. Systemic mRNA/LNP delivery of CD89-based CAR has demonstrated robust anti-tumor efficacy against multiple tumor antigens in various xenograft mouse models of human breast cancer, hepatocellular carcinoma, and ovarian cancer. Furthermore, in syngeneic mouse tumor models, robust anti-tumor immune responses, characterized by tumoral infiltration of activated CD8+ T cells, diminished tumor-associated Tregs, systemic enhancement of dendritic cells and anti-tumor IgG production, were observed for treated animals. Conclusions: Taken together, we present CAR designs that achieve expression and function in targeted immune cell populations, capable of eliciting anti-tumor efficacy against multiple target antigens by leveraging innate and adaptive immune responses, and demonstrating a class of novel CAR designs to overcome limitations faced by current in vivo mRNA/LNP delivery strategy. A phase I clinical trial is currently on-going to assess the safety and preliminary efficacy of CD89-based CAR against TROP2+ epithelial malignancies. Citation Format: Shannon Argueta, Franciele K. Melber, Michael Gorgievski, Neha Divangi, Edward Cochran, Thach Chu, Yuxiao Wang, Jian Ding, Daniel Getts. In vivo immune cell programming using mRNA-LNP chimeric antigen receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1321.

Abstract 1869: The AI-powered KisoSeek™ platform identifies functional anti-TROP2 heavy-chain antibodies with potent anti-cancer activity

Abstract Functional heavy-chain antibodies (HCAbs) are promising therapeutic agents for targeted cancer therapy. Since they do not have a light chain, HCAbs have a small antigen-binding domain that can unlock new functionalities by reaching epitopes that are inaccessible to classical antibodies. HCAbs can be obtained by immunizing camelids, but this approach is expensive, time-consuming, and results in a limited antibody repertoire. Previously, we engineered the KisoMouse™- a transgenic mouse with multi-camelid species VHH/VH genes and the complete alpaca D and J gene segments integrated into the mouse IgH locus, along with a functional loss of the heavy chain CH1 domain to eliminate light chain pairing. KisoMouse™ are healthy and generate a robust serum HCAb response against a diverse range of immunogens. Trophoblast cell surface antigen-2 (TROP2) is a membrane protein overexpressed in numerous solid tumor types such as breast, colon, and lung cancers. TROP2 is associated with tumor aggressiveness and decreased survival and has emerged as an attractive therapeutic target. An anti-TROP2 antibody-drug conjugate has been approved for breast and bladder cancer, but challenges related to payload toxicity and resistance highlight the need for alternative therapeutics. We had previously identified an anti-TROP2 HCAb with high potency in in vivo preclinical models of human breast and colorectal cancer. Conventional antibody discovery approaches are resource-intensive and can fail to identify rare antibodies. To overcome such limitations, we developed the KisoSeek™ platform, a proprietary AI-powered toolkit that categorizes antibodies in an immune response based on their structural and biophysical features. With KisoSeek™, we analyzed the entire antibody repertoire generated from KisoMouse™ immunized with TROP2 and rapidly identified HCAbs with similar features as the high potency reference antibody described above. We selected three additional antibodies based on their in vitro TROP2 affinity, competitiveness for the reference antibody’s epitope, and yield in CHO cells. These candidates showed comparable potent efficacy in inducing regression in human breast and colorectal xenograft models, akin to the original reference antibody. In summary, the KisoSeek™ platform identified novel anti-TROP2 HCAbs out of the KisoMouse™ antibody repertoire that were similar to our original reference antibody. This demonstrates how KisoMouse™ and KisoSeek™ are two powerful complementary platforms for novel clinical antibody discovery. By categorizing the entire antibody repertoire and identifying antibodies with similar characteristics as known functional antibodies, KisoSeek™ has the potential to leverage existing discovery efforts to uncover many more rare yet potent therapeutic antibodies - the proverbial needles in a haystack. Citation Format: Sophie E. Cousineau, Amit Subedi, Richard Wargachuk, Juliana The, Xiaowei Wang, Wenyang Hou, Amogh Nair, Wansu Qiu, Alex Zhou, Tiffany Cheng, Morteza Babaie, Benyamin Gojogh, Marinieve Montero, Hiba Zahreddine, Elijus Undzys, Lucy Lai, Gordon Ngan, Luis Da Cruz, David Young. The AI-powered KisoSeek™ platform identifies functional anti-TROP2 heavy-chain antibodies with potent anti-cancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1869.

Development of L-phenylalanine-grafted poly (vinyl alcohol) scaffold for selective capturing of cancer cells

Breast cancer is the most common cancer and the leading cause of cancer death in women. The majority of deaths from breast cancer do not result from the primary tumor itself but from metastasis to other organs in the body. Here, we report the development of a novel polymeric material that selectively traps breast cancer cells. The cancer cell-capturing polymer was synthesized by grafting L-phenylalanine onto a biocompatible poly(vinyl alcohol) (PVA-Phe). L-phenylalanine exhibits high affinity to L-type amino acid transporter 1 (LAT1), which is overexpressed on various cancer cells. We evaluated cancer cell-selective adhesion ability of PVA-Phe using MCF-7, a human hormone receptor-positive breast cancer model cell expressing LAT1. Our results suggest that LAT1 on cancer cells recognizes the L-phenylalanine and that MCF-7 selectively adheres to the synthesized PVA-Phe. For patients at risk of cancer recurrence, we expect that PVA-Phe synthesized in this study could serve to capture the remaining cancer cells when implanted in the body. Together with an appropriate scaffold that functions to attract cancer cells, PVA-Phe may have the potential to completely prevent cancer recurrence.

Dendritic nanomedicine enhances chemo-immunotherapy by disturbing metabolism of cancer-associated fibroblasts for deep penetration and activating function of immune cells

Inefficient drug penetration hurdled by the stroma in the tumor tissue leads to a diminished therapeutic effect for drugs and a reduced infiltration level of immune cells. Herein, we constructed a PEGylated dendritic epirubicin (Epi) prodrug (Epi-P4D) to regulate the metabolism of cancer-associated fibroblasts (CAFs), thus enhancing Epi penetration into both multicellular tumor spheroids (MTSs) and tumor tissues in mouse colon cancer (CT26), mouse breast cancer (4T1) and human breast cancer (MDA-MB-231) models. Enhanced cytotoxicity against CT26 MTSs and remarkable antitumor efficacy of Epi-P4D were ascribed to reduced fibronectin, α-SMA, and collagen secretion. Besides, thinning of the tumor tissue stroma and efficient eradication of tumor cells promoted the immunogenic cell death effect for dendritic cell (DC) maturation and subsequent immune activation, including elevating the CD4+ T cell population, reducing CD4+ and CD8+ T cell hyperactivation and exhaustion, and amplifying the natural killer (NK) cell proportion and effectively activating them. As a result, this dendritic nanomedicine thinned the stroma of tumor tissues to enhance drug penetration and facilitate immune cell infiltration for elevated antitumor efficacy.

The NDV-MLS as an Immunotherapeutic Strategy for Breast Cancer: Proof of Concept in Female Companion Dogs with Spontaneous Mammary Cancer.

The absence of tumor-infiltrating lymphocytes negatively impacts the response to chemotherapy and prognosis in all subtypes of breast cancer. Therapies that stimulate a proinflammatory environment may help improve the response to standard treatments and also to immunotherapies such as checkpoint inhibitors. Newcastle disease virus (NDV) shows oncolytic activity, as well as immune modulating potential, in the treatment of breast cancer in vitro and in vivo; however, its potential to enhance tumor-infiltrating immune cells in breast cancer has yet to be evaluated. Since spontaneous canine mammary tumors represent a translational model of human breast cancer, we conducted this proof-of-concept study, which could provide a rationale for further investigating NDV-MLS as immunotherapy for mammary cancer. Six female companion dogs with spontaneous mammary cancer received a single intravenous and intratumoral injection of oncolytic NDV-MLS. Immune cell infiltrates were evaluated by histology and immunohistochemistry in the stromal, intratumoral, and peritumoral compartments on day 6 after viral administration. Increasing numbers of immune cells were documented post-viral treatment, mainly in the peritumoral compartment, where plasma cells and CD3+ and CD3-/CD79- lymphocytes predominated. Viral administration was well tolerated, with no significant adverse events. These findings support additional research on the use of NDV-MLS immunotherapy for mammary cancer.

Evaluation of engraftment and growth dynamics of orthotopic and heterotopic in vivo models of human breast cancer

Purpose of the study. This work was to assess the engraftment and growth dynamics of breast cancer xenografts during orthotopic and subcutaneous injection using various types of biological material, as well as to develop an adequate model of breast cancer for further research.Materials and methods. We used a disaggregated fragment of a tumor obtained from the patient, a certified breast cancer cell line VT20 – human breast carcinoma; a primary human breast carcinoma cell line. Female immunodeficient mice of the Balb/c Nude line in the amount of 36 animals were used as recipient animals. The subcutaneous and orthotopic models of breast cancer were developed in this project. Tumor growth was observed for 28 days from the moment of injection and tumor nodes were measured 2 times a week until the end of the experiment. Results were assessed using medians and percentiles. The nonparametric Mann-Whitney test was used to assess the significance of differences.Results. The dynamics of the growth of tumor cells when injected into various sites was determined in the process of this work. The most successful in terms of a subcutaneous injection was the injection of tumor cells of the certified VT20 line. By the end of the experiment, the median tumor node of this group was 100.32 mm³. The analysis revealed tumor dynamics with orthotopic injection of tumor material, and the median volume of the tumor node in the group with the passport culture cell VT20 and the primary culture cell reached the same value – 149.22 and 148.25. mm³. It was found that both the cell line and the cell suspension were injected into tumor nodes that reached a significantly larger volume when injected orthotopically.Conclusion. We have obtained a tumor model of breast cancer using various methods of material implantation and with the possibility of further use in testing new pharmacological substances.

Activation of cytotoxic lymphocytes through CD6 enhances killing of cancer cells

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell–cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft mouse models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8 + T cells. In particular, tumor-infiltrating cytotoxic lymphocytes from UMCD6-treated mice expressed higher levels of perforin and were found in higher proportions than those from IgG-treated mice. Moreover, RNA-seq analysis of human NK-92 cells treated with UMCD6 revealed that UMCD6 up-regulates the NKG2D-DAP10 receptor complex, important in NK cell activation, as well as its downstream target PI3K. Our results now describe the phenotypic changes that occur on immune cells upon treatment with UMCD6 and further confirm that the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

The Root Extracts of Valeriana Officinalis L. may Control Programmed Cell Death Pathways in Breast Cancer Cell Line, MCF-7

Natural product-derived phytochemicals are now accepted as promising agents in developing new strategies for cancer treatment and prevention. The root extracts of valerian (Valeriana officinalis L.), which is a supplement widely used for improving circadian rhythm-dependent sleep disorders and insomnia, might be a good candidate in that context. In the present study we hypothesized whether extract of valerian root induce programmed cell death machineries in a human breast cancer model, MCF-7 cells. To test the hypothesis, we treated MCF-7 cells with the extract at different concentrations for 24 h. Giemsa staining was used to evaluate the apoptotic morphology and apoptotic index, and monodancylcadaverine assay was used to determine vacuoles that are associated with autophagic flux. Our results indicated that extracts of the roots of valerian have apototic and autophagic effect on human breast cancer cell MCF-7 in a dose dependent manner. Moreover, the typical cobblestone morphology of the cells was disrupted after the extract treatments and the cells lost contact with each other. This morphological alteration was attributed to anoikis, is a programmed cell death type induced by loss of cell communication with extracellular matrix or neighboring cells. In conclusion, although this study has many methodological shortcomings, our findings suggest that Valeriana officinalis L. might be a potential anti-cancer agent for the treatment of breast cancer.

The Power and Promise of Patient-Derived Xenografts of Human Breast Cancer.

In 2016, a group of researchers engaged in the development of patient-derived xenografts (PDXs) of human breast cancer provided a comprehensive review of the state of the field. In that review, they summarized the clinical problem that PDXs might address, the technical approaches to their generation (including a discussion of host animals and transplant conditions tested), and presented transplantation success (take) rates across groups and across transplantation conditions. At the time, there were just over 500 unique PDX models created by these investigators representing all three clinically defined subtypes (ER+, HER2+, and TNBC). Today, many of these PDX resources have at least doubled in size, and several more PDX development groups now exist, such that there may be well upward of 1000 PDX models of human breast cancer in existence worldwide. They also presented a series of open questions for the field. Many of these questions have been addressed. However, several remain open, or only partially addressed. Herein, we revisit these questions, and recount the progress that has been made in a number of areas with respect to generation, characterization, and use of PDXs in translational research, and re-present questions that remain open. These open questions, and others, are now being addressed not only by individual investigators, but also large, well-funded consortia including the PDXNet program of the National Cancer Institute in the United States, and the EuroPDX Consortium, an organization of PDX developers across Europe. Finally, we discuss the new opportunities in PDX-based research.

Abstract B146: Design and preclinical evaluation of CPL976-MMAE - novel, potent AXL-PD-L1 bispecific antibody conjugated with MMAE in targeted anticancer therapy

Abstract Background: Antibody-drug conjugates (ADCs) combine the specificity of antibodies with the potency of highly cytotoxic agents, reducing the off-target effects and improve effectiveness with more precising delivery of the payload to the tumor site. Strong correlation has been established between AXL and PD-L1 expression levels in many types of cancer. Therefore, targeting both proteins simultaneously ensure high cancer specificity in delivering the toxic payload. We have developed a bispecific antibody, that strongly induces receptor internalization. Our bispecific ADC combines anti-AXL and anti-PD-L1 construct with toxic MMAE (Monomethyl auristatin E, a potent tubulin inhibitor toxin), for better targeting to solid tumor cells and greatly reducing off-target effects. Materials and Methods: CPL976-MMAE was prepared with site-specific conjugation technology using Fc glycan remodeling and click-chemistry. Briefly, after enzymatic trimming of the N-linked glycans in the Fc pocket of antibody, the terminal GlcNAc was extended with 6-N3-GalNAc using GalNAc-transferase, allowing metal-free click conjugation of the MMAE payload. Bispecific antibodies chosen for conjugation were first characterized in vitro for the interaction with the extracellular domain of human AXL and PD-L1 using surface plasmon resonance and flow cytometry. After the conjugation with MMAE, ADCs cytotoxicity, and selectivity were evaluated in the human breast cancer model (MDA-MB-231) with high expression of both targets, AXL and PD-L1, using SytoX and Hoechst. The human embryonic kidney cell line (HEK-293) with minimal expression of targets, was used as a negative control. The efficacy of conjugates were evaluated in xenograft mouse model. Results: Analytical characterization by RP-HPLC confirmed efficient conjugation and homogeneity of the conjugates. Conjugates showed improved cellular uptake, selectivity, and anticancer activity, compared to unconjugated MMAE in the AXL/PD-L1 overexpressing cancer cell lines. Additionally, HEK-293 cells were not affected by conjugates in concentrations applied in the assay with use of the breast cancer cell lines. This implies enhanced safety by reducing off-target side effects compared to free auristatin. The efficacy of the compound was confirmed in xenograft mouse model. Conclusions: We have designed a new, potentially first-in-class anti-AXL/PD-L1 bispecific antibody conjugated with MMAE, which showed robust and selective antitumor effects in human cancer models. Citation Format: Delfina Popiel, Krzysztof Lacek, Anna Jabłońska, Aleksandra Sowińska, Agnieszka Bojko - Matuszek, Damian Kołakowski, Filip Mituła, Sebastian Kwiatkowski, Magdalena Bojko, Tomasz Kornatowski, Beata Kliszcz, Tomasz Banach, Michał Górka, Jerzy Pieczykolan, Maciej Wieczorek, Olga Abramczyk. Design and preclinical evaluation of CPL976-MMAE - novel, potent AXL-PD-L1 bispecific antibody conjugated with MMAE in targeted anticancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B146.

Integration of an LPAR1 Antagonist into Liposomes Enhances Their Internalization and Tumor Accumulation in an Animal Model of Human Metastatic Breast Cancer.

Lysophosphatidic acid receptor 1 (LPAR1) is elevated in breast cancer. The deregulation of LPAR1, including the function and level of expression, is linked to cancer initiation, progression, and metastasis. LPAR1 antagonists, AM095 or Ki16425, may be effective therapeutic molecules, yet their limited water solubility hinders in vivo delivery. In this study, we report on the synthesis of two liposomal formulations incorporating AM095 or Ki16425, embedded within the lipid bilayer, as targeted nanocarriers for metastatic breast cancer (MBC). The data show that the Ki16425 liposomal formulation exhibited a 50% increase in internalization by MBC mouse epithelial cells (4T1) and a 100% increase in tumor accumulation in a mouse model of MBC compared with that of a blank liposomal formulation (control). At the same time, normal mouse epithelial cells (EpH-4Ev) internalized the Ki16425 liposomal formulation 25% lesser than the control formulation. Molecular dynamics simulations show that the integration of AM095 or Ki16425 modified the physical and mechanical properties of the lipid bilayer, making it more flexible in these liposomal formulations compared with liposomes without drug. The incorporation of an LPAR1 antagonist within a liposomal drug delivery system represents a viable therapeutic approach for targeting the LPA-LPAR1 axis, which may hinder the progression of MBC.