Orthotopic Breast Cancer Mouse Model Research Articles

Iron-Tannin Coating Reduces Clearance and Increases Tumor Colonization of Systemically Delivered Bacteria.

Engineered bacteria offer a novel approach to targeted cancer therapy, but challenges remain in delivering enough bacteria safely for effective treatment. Previous efforts have used either a native or synthetic coating to achieve better control over the half-life of bacteria in the body but have limitations in delivery or versatility. In this work, we optimized and evaluated a synthetic coating for probiotic Escherichia coli Nissle 1917 to increase its half-life in blood and thereby increase the bioavailability of intravenous doses of bacteria to colonize and treat tumors. Using a simple one-pot chemical process, we coated bacteria with iron and tannic acid (FeTA) to form a temporary adhesive protective coating surrounding the bacterial cell surface. The iron to tannic acid ratio of the coating was optimized for intravenous use, and FeTA-coated bacteria of several different genetic backgrounds showed 15-fold higher survival in blood survival assays for up to 4 hours. We found that the FeTA coating reduced both complement-mediated bacterial killing and phagocyte-mediated bacterial killing in vitro. As a result, systemic delivery of attenuated bacteria had up to 60% colonization efficiency of FeTA-coated bacteria in an orthotopic breast cancer mouse model compared to 10% for the non-coated control, all the while maintaining a two-fold decrease in weight loss of attenuated bacteria compared to wild-type. Altogether, we show that an optimized FeTA coating significantly extends the half-life and colonization efficiency of engineered bacteria, overcoming a key limitation of their application in cancer therapy.

Mechanotransduction-Piloted Whole-Cell Vaccines for Spatiotemporal Modulation of Postoperative Antitumor Immunity.

Whole tumor cell vaccines hold promise by presenting a broader spectrum of autologous-origin tumor antigens to combat postoperative recurrence and metastasis. However, challenges such as intractable adjuvant modification and obscure interactions with antigen-presenting cells in the postoperative microenvironment impede their translation into effective personalized immunotherapies. In this study, we propose cancer vaccines derived from manganese oxide-immobilized resected tumor cells, featuring whole tumor antigens and adjustable stiffness to modulate interactions with antigen-presenting cells in the postoperative microenvironment. These vaccines effectively stimulate dendritic cell phagocytosis and function through sequential stiffness-mediated mechanotransduction and interferon signaling. We evaluated their efficacy using an orthotopic triple-negative breast cancer mouse model and found that combining the vaccines with radiotherapy effectively inhibits postoperative tumor recurrence and metastasis. Our study underscores the potential of utilizing mechanotransduced adjuvants alongside directly inactivated whole-cell vaccines as a universal solution for preventing postoperative tumor recurrence.

Regulating Manganese-Site Electronic Structure via Reconstituting Nitrogen Coordination for Efficient Non-Oxygen-Dependent Sonocatalytic Therapy against Orthotopic Breast Cancer.

Sonocatalytic therapy (SCT) has emerged as a promising noninvasive modality for tumor treatment but is hindered by the insufficient generation of ultrasound-induced reactive oxygen species (ROS) and the hypoxic tumor microenvironments. Herein, we fabricated a carbon nanoframe-confined N-coordinated manganese single-atom sonocatalyst with a five-coordinated structure (MnN5 SA/CNF) using a phthalocyanine-mediated pyrolysis strategy. The precise coordination structure was identified by synchrotron X-ray absorption fine structure analyses. The MnN5 SA/CNF exhibits superior and nonoxygen-dependent sonocatalytic activity owing to the optimized coordination structure and cavitation effect enhanced by defects. Additionally, density functional theory studies reveal that the five-coordination structure downshifts the d-band center of Mn from -0.547 to -0.829 eV and enhances the desorption capacity for oxygen-containing intermediates, thus accelerating the catalytic process. Finally, the as-synthesized MnN5 SA/CNF demonstrates a significantly enhanced antitumor effect through mitochondrial apoptosis in an orthotopic breast cancer mouse model. This work explores the potential of SAzymes-supported biomedical interventions by leveraging enzymatic activity with sonocatalytic properties.

A Novel Method for the Early Detection of Single Circulating, Metastatic and Self-Seeding Cancer Cells in Orthotopic Breast Cancer Mouse Models.

Metastasis is the main cause of cancer-related deaths, but efficient targeted therapies against metastasis are still missing. Major gaps exist in our understanding of the metastatic cascade, as existing methods cannot combine sensitivity, robustness, and practicality to dissect cancer progression. Addressing this issue requires improved strategies to distinguish early metastatic colonization from metastatic outgrowth. Luciferase-labelled MDA-MB-231, MCF7, and 4T1 breast cancer cells were spiked into samples from tumour-naïve mice to establish the limit of detection for disseminated tumour cells. Luciferase-labelled breast cancer cells (±unlabelled cancer-associated fibroblasts; CAFs) were orthotopically implanted in immunocompromised mice. An ex vivo luciferase assay was used to quantify tumour cell dissemination. In vitro luciferase assay confirmed a linear and positive correlation between cancer cell numbers and the bioluminescence detected at single cell level in blood, brain, lung, liver, and mammary fat pad samples. Remarkably, single luciferase-labelled cancer cells were detectable in all of these sites, as the bioluminescence quantified in the analysed samples was substantially higher than background levels. Ex vivo, circulating tumour cells, metastasis, and tumour self-seeding were detected in all samples from animals implanted with highly metastatic luciferase-labelled MDA-MB-231 cells. In turn, detection of poorly metastatic luciferase-labelled MCF7 cells was scarce but significantly enhanced upon co-implantation with CAFs as early as 20 days after the experiment was initiated. These results demonstrate the feasibility of using an ultrasensitive luciferase-based method to dissect the mechanisms of early metastatic colonization to improving the development of antimetastatic therapies.

Abstract 1638: Regulation of spindle pole integrity by the MASTL-ENSA-aurora a pathway during mitosis

Abstract The preservation of spindle pole integrity is crucial for proper spindle assembly and chromosome segregation in mitosis, yet the precise mechanisms governing spindle pole integrity remain elusive. During mitosis, phosphorylated Ensa (p-ENSA) localizes to spindle poles, and the inhibition of ENSA leads to a range of mitotic defects, including misaligned chromosomes, multipolar spindles, asymmetric bipolar spindles, and centrosome aberrations, resulting in a delayed mitotic progression. Remarkably, the mitotic delay induced by ENSA inhibition is alleviated upon depletion of PP2A-B55α, but spindle pole defects persist. Significantly, we have observed an interaction between ENSA and Aurora A during mitosis, and the inhibition of ENSA diminishes Aurora A expression at the mitotic spindle poles. Intriguingly, the injection of MKI-2-sensitized tumors is associated with heightened chromosomal instability and downregulation of the MASTL-ENSA-Aurora A pathway in an orthotopic breast cancer mouse model. These findings offer new insights into the regulation of spindle pole integrity through the MASTL-ENSA-Aurora A pathway during mitosis, emphasizing the pivotal role of ENSA in recruiting Aurora A to the spindle pole, independently of PP2A-B55α. Citation Format: Seul Kim, Kyoungho Jun, Ye-Hyun Kim, Jae Sung Kim. Regulation of spindle pole integrity by the MASTL-ENSA-aurora a pathway during mitosis [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 1638.

Abstract 4278: Role of WNK1 in extracellular matrix modification in breast cancer

Abstract Introduction: Breast cancer (BCa) is the most common cancer and the second leading cause of cancer related deaths in women in the United States. Metastatic dissemination of cancer cells remains the leading cause of mortality in BCa. Therefore, determination of the mechanisms that promote metastasis is important for development of new therapeutic strategies. The extracellular matrix (ECM), which is the main non-cellular component of the tumor microenvironment (TME), plays an important role in BCa progression and metastasis. The cancer-associated fibroblasts (CAFs) are the principal cell types that together with BCa cells drive the modification of ECM during cancer development. Lysine deficient receptor protein kinase 1 (WNK1), a widely expressed serine threonine kinase that regulates blood pressure and electrolyte balance in the body, and is highly overexpressed in BCa microenvironment. The aim of the present study was to investigate the role of WNK1 in modification of ECM in BCa. Methods: The clinical relevance of WNK1 overexpression in BCa was determined by immunohistochemically staining human BCa sections, analyzing data from publicly available databases and creating orthotopic mouse models of BCa using 4T1 mouse BCa cells followed by treatment of the tumor bearing mice with WNK1 inhibitors, WNK-IN-11 and WNK463 (10 mg/kg , oral for 10 days). In vitro assays were conducted using 4T1 cells and mouse embryonic fibroblasts (MEFs) to determine the regulatory role of WNK1 on cancer cells and fibroblasts. Results and Conclusion: Our results indicated that WNK1 is strongly expressed in BCa cells and fibroblasts, the two most abundant cell types in the BCa microenvironment. Furthermore, WNK1 expression strongly correlated with expressions of matrix regulatory and cross-linking genes. Our results demonstrated that WNK1 inhibitors significantly reduced orthotopic 4T1 growth and metastasis. WNK1 inhibition also significantly inhibited collagen deposition in tumor tissues, which is a negative prognostic indicator in BCa. In vitro proliferation and migration assays using 4T1 cells and MEFs identified that WNK1 inhibitors (1-100nM) can significantly inhibit the proliferation and migration of 4T1 cells and MEFs. Results from co-culture studies with 4T1 cells and MEFs further indicated that WNK1 inhibition in MEFs could significantly reduce the invasive properties of 4T1 cells. Taken together, our results suggest that blocking the actions of WNK1 in BCa may be a promising therapeutic approach. Citation Format: Prabhat Suman, Sooraj Kakkat, Suhas S. Patil, Veronica Ramirez, Elba A. Turbat-Herrera, Seema Singh, Chandrani Sarkar, Debanjan Chakroborty. Role of WNK1 in extracellular matrix modification in breast cancer [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 4278.

Yeast-derived nanocarriers enable binary reprogramming macrophages for cancer immunotherapy

Tumor-associated macrophages (TAMs) play an important role in promoting tumor growth and metastasis, thus reprogramming TAMs to modulate the tumor immune microenvironment is a promising tumor treatment stratagem. Here, we present novel yeast-derived nanocarriers (YNCs) as IPI-549 delivery system for binary reprogramming of TAMs to induce anticancer immunity. The nanocarriers were fabricated from yeast cell walls with immune-oncology agent IPI-549 loading and coated with a shielding polymer for prolonged blood circulation. After intravenous injection, these YNCs could accumulate at the tumor site and be phagocytosed by TAMs via the Dectin-1 pathway to induce macrophage polarization towards M1 phenotype. In combination with IPI-549, the YNC system has efficiently inhibited tumor growth and modulated the tumor immune microenvironment through macrophage phenotypic alteration and increase of T cell-mediated antitumor immunity, which was demonstrated in a 4T1 orthotopic breast cancer mouse model. All these features indicate the promising values of our YNC delivery system in clinical antitumor applications.

Inducible caspase 9-mediated suicide gene therapy using AAV6 vectors in a murine model of breast cancer

Breast carcinoma has one of the highest incidence rates (11.7%), with significant clinical heterogeneity. Although conventional chemotherapy and surgical resection are the current standard of care, the resistance and recurrence, after these interventions, necessitate alternate therapeutic approaches. Cancer gene therapy for breast cancer with the suicide gene is an attractive option due to their directed delivery into the tumor. In this study, we have developed a novel treatment strategy against breast cancer with recombinant adeno-associated virus (AAV) serotype 6 vectors carrying a suicide gene, inducible Caspase 9 (iCasp9). Upon treatment with AAV6-iCasp9 vectors and the chemical inducer of dimerizer, AP20187, the viability of murine breast cancer cells (4T1) was significantly reduced to ∼40%-60% (mock control 100%). Following intratumoral delivery of AAV6-iCasp9 vectors in an orthotopic breast cancer mouse model, we observed a significant increase in iCasp9 transgene expression and a significant reduction in tumor growth rate. At the molecular level, immunohistochemical analysis demonstrated subsequent activation of the effector caspase 3 and cellular death. These data highlight the potential of AAV6-iCasp9-based suicide gene therapy for aggressive breast cancer in patients.

The inhibitory effect of Gremlin-2 on adipogenesis suppresses breast cancer cell growth and metastasis

BackgroundGremlin-1 (GREM1) and Gremlin-2 (GREM2) are bone morphogenetic protein antagonists that play important roles in organogenesis, tissue differentiation, and tissue homeostasis. Although GREM1 has been reported to be involved in promoting various cancers, little has been reported about effects of GREM2 on cancer. Recently, it has been reported that GREM2 can inhibit adipogenesis in adipose-derived stromal/stem cells. However, as an inhibitor of adipogenesis, the role of GREM2 in cancer progression is not well understood yet.MethodsPre-adipocyte 3T3-L1 cells overexpressing mock or Grem2 were established using a lentiviral transduction system and differentiated into adipocytes-mock and adipocytes-Grem2, respectively. To investigate the effect of adipocyte-Grem2 on breast cancer cells, we analyzed the proliferative and invasion abilities of spheroids using a 3D co-culture system of breast cancer cells and adipocytes or conditioned medium (CM) of adipocytes. An orthotopic breast cancer mouse model was used to examine the role of adipocytes-Grem2 in breast cancer progression.ResultsGrem2 overexpression suppressed adipogenesis of 3T3-L1 cells. Proliferative and invasion abilities of spheroids formed by co-culturing MTV/TM-011 breast cancer cells and adipocytes-Grem2 were significantly reduced compared to those of spheroids formed by co-culturing MTV/TM-011 cells and adipocytes-mock. Compared to adipocytes-mock, adipocytes-Grem2 showed decreased mRNA expression of several adipokines, notably IL-6. The concentration of IL-6 in the CM of these cells was also decreased. Proliferative and invasive abilities of breast cancer cells reduced by adipocytes-Grem2 were restored by IL-6 treatment. Expression levels of vimentin, slug, and twist1 in breast cancer cells were decreased by treatment with CM of adipocytes-Grem2 but increased by IL-6 treatment. In orthotopic breast cancer mouse model, mice injected with both MTV/TM-011 cells and adipocytes-Grem2 showed smaller primary tumors and lower lung metastasis than controls. However, IL-6 administration increased both the size of primary tumor and the number of metastatic lung lesions, which were reduced by adipocytes-Grem2.ConclusionsOur study suggests that GREM2 overexpression in adipocytes can inhibit adipogenesis, reduce the expression and secretion of several adipokines, including IL-6, and ultimately inhibit breast cancer progression.

Ambient Light Resistant Shortwave Infrared Fluorescence Imaging for Preclinical Tumor Delineation via the pH Low-Insertion Peptide Conjugated to Indocyanine Green.

Shortwave infrared (900-1,700 nm) fluorescence imaging (SWIRFI) has shown significant advantages over visible (400-650 nm) and near-infrared (700-900 nm) fluorescence imaging (reduced autofluorescence, improved contrast, tissue resolution, and depth sensitivity). However, there is a major lag in the clinical translation of preclinical SWIRFI systems and targeted SWIRFI probes. Methods: We preclinically show that the pH low-insertion peptide conjugated to indocyanine green (pHLIP ICG), currently in clinical trials, is an excellent candidate for cancer-targeted SWIRFI. Results: pHLIP ICG SWIRFI achieved picomolar sensitivity (0.4 nM) with binary and unambiguous tumor screening and resection up to 96 h after injection in an orthotopic breast cancer mouse model. SWIRFI tumor screening and resection had ambient light resistance (possible without gating or filtering) with outstanding signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) values at exposures from 10 to 0.1 ms. These SNR and CNR values were also found for the extended emission of pHLIP ICG invivo (>1,100 nm, 300 ms). Conclusion: SWIRFI sensitivity and ambient light resistance enabled continued tracer clearance tracking with unparalleled SNR and CNR values at video rates for tumor delineation (achieving a tumor-to-muscle ratio above 20). In total, we provide a direct precedent for the democratic translation of an ambient light resistant SWIRFI and pHLIP ICG ecosystem, which can instantly improve tumor resection.

Delivery of miR-200c-3p Using Tumor-Targeted Mesoporous Silica Nanoparticles for Breast Cancer Therapy.

Despite advances in breast cancer treatment, it remains the leading cause of cancer-related death in women worldwide. In this context, microRNAs have emerged as potential therapeutic targets but still present some limitations for in vivo applications. Particularly, miR-200c-3p is a well-known tumor suppressor microRNA that inhibits tumor progression and metastasis in breast cancer through downregulating ZEB1 and ZEB2. Based on the above, we describe the design and validation of a nanodevice using mesoporous silica nanoparticles for miR-200c-3p delivery for breast cancer treatment. We demonstrate the biocompatibility of the synthesized nanodevices as well as their ability to escape from endosomes/lysosomes and inhibit tumorigenesis, invasion, migration, and proliferation of tumor cells in vitro. Moreover, tumor targeting and effective delivery of miR-200c-3p from the nanoparticles in vivo are confirmed in an orthotopic breast cancer mouse model, and the therapeutic efficacy is also evidenced by a decrease in tumor size and lung metastasis, while showing no signs of toxicity. Overall, our results provide evidence that miR-200c-3p-loaded nanoparticles are a potential strategy for breast cancer therapy and a safe and effective system for tumor-targeted delivery of microRNAs.

Modulation of Autophagy Direction to Enhance Antitumor Effect of Endoplasmic-Reticulum-Targeted Therapy: Left or Right?

Strategies that induce dysfunction in the endoplasmic reticulum (ER) hold great promise for anticancer therapy, but remain unsatisfactory due to the compensatory autophagy induction after ER disruption. Moreover, as autophagy can either promote or suppress cell survival, which direction of autophagy better suits ER-targeting therapy remains controversial. Here, a targeted nanosystem is constructed, which efficiently escorts anticancer therapeutics into the ER, triggering substantial ER stress and autophagy. Concurrently, an autophagy enhancer or inhibitor is combined into the same nanoparticle, and their impacts on ER-related activities are compared. In the orthotopic breast cancer mouse model, the autophagy enhancer increases the antimetastasis effect of ER-targeting therapy and suppresses over 90% of cancer metastasis, while the autophagy inhibitor has a bare effect. Mechanism studies reveal that further enhancing autophagy accelerates central protein snail family transcriptional repressor 1 (SNAI1) degradation, suppressing downstream epithelial-mesenchymal transition, while inhibiting autophagy does the opposite. With the same trend, ER-targeting therapy combined with an autophagy enhancer provokes stronger immune response and tumor inhibition than the autophagy inhibitor. Mechanism studies reveal that the autophagy enhancer elevates Ca2+ release from the ER and functions as a cascade amplifier of ER dysfunction, which accelerates Ca2+ release, resulting in immunogenic cell death (ICD) induction and eventually triggering immune responses. Together, ER-targeting therapy benefits from the autophagy-enhancing strategy more than the autophagy-inhibiting strategy for antitumor and antimetastasis treatment.

Cryo-shocked cancer cell microgels for tumor postoperative combination immunotherapy and tissue regeneration

Prevention of recurrence/metastasis and tissue regeneration are critical for post-surgery treatment of malignant tumors. Here, to address these needs, a novel type of microgel co-loading cryo-shocked cancer cells, immunoadjuvant, and immune checkpoint inhibitor is presented by microfluidic electrospray technology and liquid nitrogen treatment. Owing to the encapsulation of cryo-shocked cancer cells and immunoadjuvant, the microgels can recruit dendritic cells and activate them in situ, and evoke a robust immune response. Moreover, with the combination of the immune checkpoint inhibitor, the antitumor immune response is further enhanced by inhibiting the interaction of PD1 and PDL1. With this, the excellent anti-recurrence and anti-metastasis efficacy of the microgels are demonstrated in an orthotopic breast cancer mouse model. Besides, because of the excellent biocompatibility and appropriate degradation performance, the microgels can provide support for normal cell adhesion and growth, which is beneficial to tissue reconstruction. These properties indicate the great value of the cryo-shocked cancer cell microgels for efficient tumor postoperative combination immunotherapy and tissue regeneration.

Smart Telomerase-Gated DNA cage for precise siRNA release in cancer cells

Oligonucleotide nanoparticles (ONPs) have been utilized as a small interfering RNA (siRNA) delivery system because of their multiple advantages. However, the release of siRNA by ONPs inner cells mainly relies on acidic environments and high concentrations of ATP. These conditions do not allow the specific release of siRNA in tumor cells, which would prevent the side effects that result from the release of siRNA in normal cells. In this study, a prism oligonucleotide nanoparticle (pONP)-based delivery system was designed, which targeted with telomeric primers (TS) that can be extended by cancer-specific telomerase for specifically releasing siRNA inside telomerase-positive cancer cells. In vitro tests confirmed that this cage facilitated the release of siRNA specifically into telomerase-positive HeLa and MDA-MB-231 tumor cells. The release was so precise that almost no siRNA was released into telomerase-negative cells. In vivo tests also confirmed that this cage could release sibcl-2 specifically in tumor cells in an MDA-MB-231 orthotopic breast cancer mouse model. Moreover, the cage has a long half-life (e.g., 2.83 h in FBS) in the circulatory system and protects the cargo from nuclease degradation; thus, the released sibcl-2 effectively silences the translation of target cancer-related mRNA (bcl-2), similar to the sibcl-2 delivered by Lipo6000 in the telomerase-positive environment of cancer cells. pONP/TS/siRNA is readily prepared using a simple sequence design together with one-step self-assembly. This nanoparticle cage is a promising delivery method for precise siRNA release into targeted telomerase-positive cancer cells.

Abstract 6392: Combination of pulsed electric field, immunotherapy and cisplatin significantly prolongs survival in an orthotopic breast cancer mouse model

Abstract Introduction: Pulsed Electric Field (PEF) therapy is a novel approach to treat cancer by providing focal tumor ablation while sparing sensitive structures within the tumor micro-environment. Previous studies have demonstrated that PEF induces immunogenic cell death by activating inflammatory signaling pathways and stimulation of antigen presenting cells withing the tumor micro-environment. PEF ablation provides a tumor focused in situ vaccination strategy that combines with checkpoint blockade. We hypothesized that PEF ablation should synergize with chemotherapy and anti-PD1 in a model where checkpoint inhibitors are inactive. Experimental Design: To test this hypothesis, we utilized a highly aggressive syngeneic orthotopic triple negative breast cancer 4T1 mouse model that is non-responsive to anti-PD1 and only marginally responsive to cisplatin to determine if this combination treatment could prolong survival. Thirty-two Balb/c mice were inoculated in the mammary fat pad with 200,000 4T1 cells. Once tumors were established (5mm in diameter), animals were randomized to the following groups: (A) Sham/IgG control (n=8; needle inserted but no PEF), (B) PEF only (n=8), (C) cisplatin + anti-PD-1 (n=8) and (D) PEF + cisplatin + anti PD-1 (n=8). Tumors in groups B and D were treated once with PEF. Anti-PD1 and isotype matched control IgG was administered once per week (200μg, i.p injections) starting on the day of PEF (Group D) for four weeks. Cisplatin was administered (2mg/kg, i.v) once per week for four weeks starting on the day of PEF (d=0) for four weeks. All the mammary fat pad tumors were resected when they achieved a tumor volume of ~200 mm3 to recapitulate post-resection metastatic relapse (See Schematic 1). Results and Conclusions: All mice in the Sham-control IgG group needed to be euthanized by day 43 due to metastases in multiple organs including lungs, bones, liver and brain. All mice in the cisplatin + anti PD-1 group had to be euthanized by day 74. Interestingly, all groups that were treated with PEF survived much longer. On day 90, 28% of mice in the PEF alone group were alive and 43% were alive in the PEF + anti-PD1 + Cisplatin (see Figure 1). The rank-log p-value for the Kaplan-Meier survival curve was statistically significant. In conclusion, our results demonstrate that PEF treatment in combination with anti-PD1 and Cisplatin significantly prolongs survival in a highly aggressive triple negative breast tumor mouse model that is non-responsive to immunotherapy. These findings support evaluation of PEF in cancer patients that are receiving combination of checkpoint blockade and chemotherapy. Citation Format: Chiara Pastori, Mukta Wagh, Ebtesam Nafie, Fatima Murad, Mohit Trikha, Robert Neal. Combination of pulsed electric field, immunotherapy and cisplatin significantly prolongs survival in an orthotopic breast cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6392.

Abstract 459: Targeting the non-junctional tumorigenic function of connexin43 in cancer cell plasticity

Abstract Tumors are highly heterogeneous and dynamic, with both cancer cells and cancer stem cells (CSCs) displaying a high degree of plasticity contributing to metastasis and tumor recurrence following treatment. Epithelial-mesenchymal transition (EMT) enables cancer cells to acquire migratory and invasive characteristics, access the circulatory system, and metastasize. Moreover, EMT is associated with therapeutic resistance and CSC generation. Our previous research demonstrated a redistribution of the gap junction protein connexin43 (Cx43) from the plasma membrane to the cytosol during EMT. Using super-resolution STochastic Optical Reconstruction Microscopy (STORM), we have recently identified increased interaction of such intracellular Cx43 with microtubules during EMT and in primary glioblastoma (GBM) CSCs (GSCs). We further report a tumorigenic function for cytosolic Cx43/microtubule complexing in cancer cell and CSC proliferation, migration, and survival. Cx43 localization and activity is regulated by multiple sites for protein-protein interaction within the Cx43 carboxy-terminus (CT), including a tubulin binding domain. We employed a Cx43 mimetic peptide named JM2 (juxtamembrane 2) encompassing the microtubule binding sequence of the Cx43 CT. Following administration, JM2 efficiently and specifically disrupts the interaction of Cx43 with microtubules, inhibits GSC-derived neurosphere formation in vitro, and patient GBM-derived organoid growth ex vivo. Using an orthotopic GBM patient derived xenograft mouse model, we demonstrate in vivo that JM2 significantly decreases the number of GSCs within brain tumors, inhibits the formation of highly invasive GBM tumors, and increases mouse survival in combination with the chemotherapy alkylating agent temozolomide. Further examining the role of Cx43/microtubule complexing in the context of metastatic disease, we observed JM2 also decreases mesenchymal marker expression and inhibits cell migration and invasion in EMT models. Using an in vivo orthotopic breast cancer mouse model we find that intratumoral administration of JM2 prevents metastasis, significantly decreasing cancer cell lung colonization. Our findings validate targeting Cx43 complexing with microtubules, with JM2, as a novel therapeutic approach for cancer treatment through inhibition of EMT-associated increases in invasion and metastasis, and elimination of CSCs. Modulation of non-junctional Cx43 functions therefore represents a viable therapeutic approach to target cellular plasticity with broad therapeutic implications for many cancers. Citation Format: Samy Lamouille, Christina E. Wheeler, Stacie E. Deaver, Michael J. Zeitz, Zhi Sheng, Robert G. Gourdie, James W. Smyth. Targeting the non-junctional tumorigenic function of connexin43 in cancer cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 459.

Jiawei Yanghe Decoction suppresses breast cancer by regulating immune responses via JAK2/STAT3 signaling pathway

Ethnopharmacological relevanceJiawei Yanghe Decoction (JWYHD) is a widely used traditional Chinese medicine prescription in the clinical setting for the treatment of autoimmune diseases. Many studies showed that JWYHD has anti-tumor activities in cell and animal models. However, the anti-breast cancer effects of JWYHD and the underlying mechanisms of action remain unknown. Aim of studyThis study aimed to determine the anti-breast cancer effect and reveal the underlying mechanisms of action in vivo, in vitro and in silico. Materials and methodsOrthotopic xenograft breast cancer mouse model and inflammatory zebrafish model were used to observe the anti-tumor effect and immune cell regulation of JWYHD. Moreover, the anti-inflammatory effect of JWYHD were evaluated by the expression of RAW 264.7 cells. JWYHD active ingredients were obtained by UPLC-MS/MS and potential targets were screened by network pharmacology. The therapeutic targets and signaling pathways predicted by computer were assessed by Western blot, real-time PCR (RT-PCR), immunohistochemistry (IHC) staining, and Enzyme-linked immunosorbent assays (ELISA) to explore the therapeutic mechanism of JWYHD against breast cancer. At last, Colivelin and Stattic were used to explore the effect of JWYHD on JAK2/STAT3 pathway. ResultsJWYHD significantly decreased the tumor growth in a dose-dependent manner in the orthotopic xenograft breast cancer mouse model. Flow cytometry and IHC results indicated that JWYHD decreased the expressions of M2 macrophages and Treg while increasing M1 macrophages. Meanwhile, ELISA and Western blot results showed a decrease in IL-1β, IL-6, TNFα, PTGS2 and VEGFα in tumor tissue of JWYHD groups. The results were also verified in LPS-induced RAW264.7 cells and zebrafish inflammatory models. TUNEL assay and IHC results showed that JWYHD significantly induced apoptosis. Seventy-two major compounds in JWYHD were identified by UPLC-MS/MS and Network pharmacology. It was found that the significant binding affinity of JWYHD to TNFα, PTGS2, EGFR, STAT3, VEGFα and their expressions were inhibited by JWYHD. IHC and Western blot analysis showed that JWYHD could decrease the expression of JAK2/STAT3 pathway. Furthermore, Colivelin could reverse the decrease effect of JWYHD in vitro. ConclusionJWYHD exerts a significant anti-tumor effect mainly by inhibiting inflammation, activating immune responses and inducing apoptosis via the JAK2/STAT3 signaling pathway. Our findings provide strong pharmacological evidence for the clinical application of JWYHD in the management of breast cancer.

Plexin-B3 expression stimulates MET signaling, breast cancer stem cell specification, and lung metastasis

Intratumoral hypoxia is a microenvironmental feature that promotes breast cancer progression and is associated with cancer mortality. Plexin B3 (PLXNB3) is highly expressed in estrogen receptor-negative breast cancer, but the underlying mechanisms and consequences have not been thoroughly investigated. Here, we report that PLXNB3 expression is increased in response to hypoxia and that PLXNB3 is a direct target gene of hypoxia-inducible factor 1 (HIF-1) in human breast cancer cells. PLXNB3 expression is correlated with HIF-1α immunohistochemistry, breast cancer grade and stage, and patient mortality. Mechanistically, PLXNB3 is required for hypoxia-induced MET/SRC/focal adhesion kinase (FAK) and MET/SRC/STAT3/NANOG signaling as well as hypoxia-induced breast cancer cell migration, invasion, and cancer stem cell specification. PLXNB3 knockdown impairs tumor formation and lung metastasis in orthotopic breast cancer mouse models.

Discovery of New 1,4,6-Trisubstituted-1H-pyrazolo[3,4-b]pyridines with Anti-Tumor Efficacy in Mouse Model of Breast Cancer

Purine analogues are important therapeutic tools due to their affinity to enzymes or receptors that are involved in critical biological processes. In this study, new 1,4,6-trisubstituted pyrazolo[3,4-b]pyridines were designed and synthesized, and their cytotoxic potential was been studied. The new derivatives were prepared through suitable arylhydrazines, and upon successive conversion first to aminopyrazoles, they were converted then to 1,6-disubstituted pyrazolo[3,4-b]pyridine-4-ones; this served as the starting point for the synthesis of the target compounds. The cytotoxic activity of the derivatives was evaluated against several human and murine cancer cell lines. Substantial structure activity relationships (SARs) could be extracted, mainly concerning the 4-alkylaminoethyl ethers, which showed potent in vitro antiproliferative activity in the low μM level (0.75-4.15 μΜ) without affecting the proliferation of normal cells. The most potent analogues underwent in vivo evaluation and were found to inhibit tumor growth in vivo in an orthotopic breast cancer mouse model. The novel compounds exhibited no systemic toxicity; they affected only the implanted tumors and did not interfere with the immune system of the animals. Our results revealed a very potent novel compound which could be an ideal lead for the discovery of promising anti-tumor agents, and could also be further explored for combination treatments with immunotherapeutic drugs.

Peritumoral scaffold neutralizes tumor pH for chemotherapy sensitization and metastasis inhibition.

The abnormal metabolism of rapidly growing tumors can create an acidic tumor microenvironment (TME) that renders cancer cells resistant to chemotherapy and further facilitates endothelial-to-mesenchymal transition (EMT) progress to promote metastasis. Here, we developed a combination strategy consisting of (1) peritumorally injected scaffold that alleviates TME acidosis, and (2) intravenously injected nanoparticles that delivers anti-cancer agents to tumor. Concurrent treatment with these two drug delivery systems profoundly delayed the growth of primary tumor and reduced the spontaneous metastasis to lung in an orthotopic breast cancer mouse model. Mechanism studies both in vitro and in vivo further revealed that neutralization of TME pH by the hydrogel scaffold sensitized cancer cells to nanoparticle-based chemotherapy, thereby strengthening the cytotoxicity against tumor growth; In parallel, reversal of tumor acidity downregulated various pro-metastatic proteins intratumorally to block the EMT progress, thereby reducing the metastatic potential of cancer cells. This work provided proof-of-concept demonstration that chemotherapy sensitization and EMT suppression could be synchronized by the modulation of TME pH, which may be potentially beneficial for simultaneous inhibition of tumor growth and cancer metastasis.