3D Tumorsphere Research Articles

127 (PB117) - Using patient-derived xenografts as sources of 3D tumor sphere cultures to study autologous tumor-infiltrating lymphocytes in metastatic uveal melanoma

Introduction: Uveal melanoma (UM) is a rare form of melanoma, but the most common primary malignancy of the eye. 50% of patients with UM develop metastasis, mainly to the liver, with a median survival of less than a year. Immune checkpoint inhibitors are markedly less effective in patients with the uveal form as compared to the cutaneous form of melanoma. Adoptive cell therapy (ACT) has not been extensively studied clinically or preclinically in UM. There has been a lack of robust ex vivo screening models and very few patient-derived xenograft (PDX) mouse models.

Regional gain and global loss of 5-hydroxymethylcytosine coexist in genitourinary cancers and regulate different oncogenic pathways

BackgroundDNA 5-hydroxymethylcytosine (5hmC) is produced by dynamic 5mC oxidation process contributing to tissue specification, and loss of 5hmC has been reported in multiple cancers including genitourinary cancers. However, 5hmC is also cell-type specific, and its variability may exist between differentiated tumor cells and cancer stem cells. Thus, cancer-associated changes in 5hmC may be contributed by distinct sets of tumor cells within the tumor tissues.ResultsHere, we applied a sensitive immunoprecipitation-based method (hMeDIP-seq) to analyze 5hmC changes during genitourinary carcinogenesis (including prostate, urothelial and kidney). We confirmed the tissue-specific distribution of 5hmC in genitourinary tissues and identified regional gain and global loss of 5hmC coexisting in genitourinary cancers. The genes with gain of 5hmC during tumorigenesis were functionally enriched in regulating stemness and hypoxia, whereas were associated with poor clinical prognosis irrespective of their differences in tumor type. We identified that gain of 5hmC occurred in soft fibrin gel-induced 3D tumor spheres with a tumor-repopulating phenotype in two prostate cancer cell lines, 22RV1 and PC3, compared with conventional two-dimensional (2D) rigid dishes. Then, we defined a malignant signature derived from the differentially hydroxymethylated regions affected genes of cancer stem-like cells, which could predict a worse clinical outcome and identified phenotypically malignant populations of cells from prostate cancer tumors. Notably, an oxidation-resistant vitamin C derivative, ascorbyl phosphate magnesium, restored 5hmC and killed the cancer stem cell-like cells leading to apoptosis in prostate cancer cell lines.ConclusionsCollectively, our study dissects the regional gain of 5hmC in maintaining cancer stem-like cells and related to poor prognosis, which provides proof of concept for an epigenetic differentiation therapy with vitamin C by 5hmC reprogramming.Graphic

Abstract 5346: WAVE3 phosphorylation promotes chemoresistance and cancer stemness in triple negative breast cancer through downstream of β-catenin oncogenic signaling

Abstract Acquisition of the metastatic phenotype is responsible for the death of ~90% of breast cancer (BC) patients. Metastatic BC is the 2nd leading cause of cancer-related deaths in women in the United States, annually accounting for more than 40,100 deaths and 266,000 new cases of invasive BC. Amongst individual BC subtypes, those classified as TNBCs are especially lethal due to their highly metastatic behavior and propensity to recur rapidly. As a group, TNBCs lack expression of hormone receptors (ER-α and PR) and ErbB2/HER2, which has prevented the development of FDA-approved targeted drug therapies that are effective against this BC subtype. Likewise, recurrent TNBCs frequently acquire resistance to standard-of-care chemotherapeutic agents (e.g., doxorubicin, cyclophosphamides, and taxanes) through mechanisms that remain incompletely understood. We established Wiskott-Aldrich Syndrome Protein-3 (WAVE3) as a novel promoter of TNBC development and metastatic progression. Our recently published studies have shown that WAVE3 phosphorylation regulates the interplay between PI3K, TGF-β, and EGF signaling pathways. In this study, we show that WAVE3 phosphorylation promotes chemotherapy-resistance and cancer stemness in TNBC, both in vitro and in in vivo mouse models. CRISPR/Cas9-mediated inactivation of WAVE3 resulted in inhibition of 2D growth and 3D tumorspheres’ invasion of TNBC cells in vitro, as well as tumor growth and metastasis in vivo. In addition, while re-expression of phospho-active WAVE3 in the WAVE3-deficient TNBC cells restored the oncogenic activity of WAVE3, re-expression of phospho-mutant WAVE3 or the pharmacologic inhibition of WAVE3 phosphorylation did not. Further studies revealed that dual blocking WAVE3 expression or phosphorylation with chemotherapy treatment inhibited the activity and expression of β-catenin. Importantly, a combination of WAVE3-deficiency and chemotherapy suppressed the oncogenic behavior of chemo-resistant TNBC cells, both in vitro and in vivo. This study suggests that a targeted therapeutic strategy against WAVE3 could be effective for the treatment of chemoresistant TNBC tumors. Citation Format: Wei Wang, Priyanka Rana, Akram Alkrekshi, Vesna Markovic, Khalid Sossey-Alaoui. WAVE3 phosphorylation promotes chemoresistance and cancer stemness in triple negative breast cancer through downstream of β-catenin oncogenic signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5346.

Abstract 5986: WAVE2/EGF signaling and miR29 regulates WAVE2 to promote tumor growth and metastasis in triple negative breast cancer

Abstract Breast cancer is the most frequently diagnosed malignancy in women and is one of the leading causes of death due to cancer invasion, metastasis, and resistance to therapies. Among its variants, triple-negative breast cancer (TNBC) is considered the most aggressive due to its early age of diagnosis, invasive and metastatic properties with poor prognosis. TNBC is a type of breast cancer that lacks expression of the receptors that are commonly found in this type of cancer (estrogen, progesterone, and HER2), which makes it a more difficult target for treatment. Cancer invasiveness and metastasis are known to be promoted by increased cell motility and upregulation of the Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE) - WAVE1, WAVE2, and WAVE3. While the contribution of WAVE2 to cancer progression is well documented, the WAVE2-mediated regulation of TNBC oncogenic properties is still under investigated. In this study, we show that WAVE2 plays a significant role in TNBC development, progression, and metastasis. To investigate this notion, we used CRISPR/Cas9 gene editing to successfully inhibit WAVE2 expression in several TNBC cell lines. In vitro, loss of WAVE2 expression inhibited 3D tumorsphere formation and invasion of TNBC cells. In vivo, in mouse models for TNBC, loss of WAVE2 inhibited tumor growth and metastasis. Mechanistically, we show that the WAVE2-mediated modulation of the oncogenic behavior of TNBC cells is in part due to its phosphorylation downstream of EGF. At the post transcriptional level, we found WAVE2 expression to be regulated by miR29. Together, our findings identify a novel post-translational WAVE2/EGF oncogenic signaling axis, along with a post-transcriptional regulatory axis downstream of miR29, which together regulate the oncogenic activity of WAVE2 in TNBC and open a new therapeutic strategy for the treatment of TNBC. Citation Format: Priyanka S. Rana, Wei Wang, Akram Alkrekshi, Vesna Markovic, Khalid Sossey-Alaoui. WAVE2/EGF signaling and miR29 regulates WAVE2 to promote tumor growth and metastasis in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5986.

A New Liposomal Formulation of Hydrogenated Anacardic Acid to Improve Activities Against Cancer Stem Cells

Anacardic acid (AA) is a natural active ingredient that accounts for 65% of the liquid extract from the shell of the cashew nut. Due to the stronger cytotoxic activity of hydrogenated AA (HAA) against NTERA-2 cancer stem cells (CSCs) than AA itself, HAA was co-conjugated with CD133 monoclonal antibody (mAb^CD133) into nanoliposomal particles (AMC). This nanoliposomal complex is expected to improved HAA activities against CSCs based on the targeting capacity of mAb^CD133 toward CD133, a typical CSCs’ surface marker. AMC was manufactured with a mean size of 100.9 nm, a zeta potential of −40.7 mV, and a PDI of 0.283. We report a 100% encapsulation efficiency of HAA into liposomes and a 90.7% conjugation efficiency with mAb^CD133. The penetration of AMC into NTERA-2 CSCs after 2 h was 83.7%. The AMC complex inhibited NTERA-2 growth with an IC50 (inhibition concentration at 50%) value of 75.83 ± 6.70 µM, showing the targeting ability and lower toxicity (IC50 > 100 µM) on healthy cells. The AMC nanoparticles also demonstrated significant potential apoptotic induction by activating caspase 3 activity by up to 2.57 and 2.06 folds compared to that of the negative control at 20 and 4 µM, respectively. This induction was significant improvement in comparison with that of unconjugated HAA ( P < .05). AMC presented a clear effect on the solid structure of NTERA-2 spheroids and significantly suppressed the proliferation of CSCs in the 3D tumorspheres with an IC50 = 64.25 ± 3.15 µM, compared to the free form with an IC50 = 82.22 ± 0.65 µM ( P < .05). Therefore, this nanoliposomal complex exhibits promising capacities as an effective material against NTERA-2 CSCs.

Targeted Therapy of Lung Adenocarcinoma by the Nanoplatform Based on Milk Exosomes Loaded with Paclitaxel.

Lung cancer is the most common cancer throughout the world. Currently, most lung cancer therapies are still limited by serious side effects caused. This paper reports a biocompatible drug delivery system that utilizes milk-derived exosomes to deliver paclitaxel to treat lung adenocarcinoma. First, milk-derived exosomes were modified with integrin αVβ₃, αVβ5-binding peptide iRGD so that they could successfully target lung adenocarcinoma cells. Then, iRGD modified exosomes were loaded with paclitaxel (PAC) via electroporation and used for tumor therapy. These modified exosomes proved effective in killing lung adenocarcinoma cells, and the exosome-based nanoplatform showed no obvious toxicity to normal cells. Further more, the exosome-based nanoplatform could effectively penetrate the interior of the 3D tumor sphere, reaching more tumor cells and demonstrating that it is a promising tool for lung adenocarcinoma therapy.

Abstract P4-01-09: The WAVE3 phosphorylation/β-catenin oncogenic signaling axis promotes chemoresistance and cancer stemness in triple negative breast cancer

Abstract Acquisition of the metastatic phenotype is responsible for the death of ~90% of breast cancer (BC) patients. In fact, metastatic BC is the 2nd leading cause of cancer-related deaths in women in the United States, annually accounting for more than 40,100 deaths and 266,000 new cases of invasive BC. Amongst individual BC subtypes, those classified as TNBCs are especially lethal due to their highly metastatic behavior and propensity to recur rapidly. As a group, TNBCs lack expression of hormone receptors (ER-α and PR) and ErbB2/HER2, which has prevented the development of FDA-approved targeted drug therapies that are effective against this BC subtype. Likewise, recurrent TNBCs frequently acquire resistance to standard-of-care chemotherapeutic agents (e.g., doxorubicin, cyclophosphamides, and taxanes) through mechanisms that remain incompletely understood. We established Wiskott-Aldrich Syndrome Protein-3 (WAVE3) as a novel promoter of TNBC development and metastatic progression. Our recently published studies have shown that WAVE3 phosphorylation regulates the interplay between PI3K, TGF-β, and EGF signaling pathways. In this study, we show that WAVE3 phosphorylation promotes chemotherapy-resistance and cancer stemness in TNBC, both in vitro and in in vivo mouse models. CRISPR/Cas9-mediated inactivation of WAVE3 resulted in inhibition of 2D growth and 3D tumorspheres’ invasion of TNBC cells in vitro, as well as tumor growth and metastasis in vivo. In addition, while re-expression of phospho-active WAVE3 in the WAVE3-deficient TNBC cells restored the oncogenic activity of WAVE3, re-expression of phospho-mutant WAVE3 or the pharmacologic inhibition of WAVE3 phosphorylation did not. Further studies revealed that dual blocking WAVE3 expression or phosphorylation with chemotherapy treatment inhibited the activity and expression of β-catenin. Importantly, a combination of WAVE3-deficiency and chemotherapy suppressed the oncogenic behavior of chemo-resistant TNBC cells, both in vitro and in vivo. This study suggests that a targeted therapeutic strategy against WAVE3 could be effective for the treatment of chemoresistant TNBC tumors. Citation Format: Wei Wang, Priyanka Rana, Akram Alkrekshi, Vesna Markovic, Khalid Sossey-Alaoui. The WAVE3 phosphorylation/β-catenin oncogenic signaling axis promotes chemoresistance and cancer stemness in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-09.

Plasma induced reactive oxygen species‐dependent cytotoxicity in glioblastoma 3D tumourspheres

AbstractThe aim of this study was to determine the effects of a pin‐to‐plate cold atmospheric plasma (CAP) on U‐251 MG three‐dimensional (3D) glioblastoma spheroids under different conditions. 3D tumorspheres showed higher resistance to the CAP treatment compared to 2D monolayer cells. A single CAP treatment was able to induce cytotoxicity, while multiple CAP treatments augmented this effect. CAP was also able to induce cytotoxicity throughout the tumoursphere, and we identified that reactive oxygen species (ROS) plays a major role, while H2O2 plays a partial role in CAP‐induced cytotoxicity in tumourspheres. We conclude that ROS‐dependent cytotoxicity is induced uniformly throughout glioblastoma and epidermoid tumourspheres by direct CAP treatment.

Identification of a cytisine-based EED-EZH2 protein-protein interaction inhibitor preventing metastasis in triple-negative breast cancer cells

Enhancer of zeste homolog 2 (EZH2) is activated in breast cancer, particularly in triple-negative breast cancer (TNBC), and is critical for cell invasion. It interacts with embryonic ectoderm development (EED) in maintaining cancer stem cells (CSC) and epithelial-mesenchymal transition (EMT) properties, hence promoting CSC metastasis. Because the association of EZH2 with EED promotes the catalytic activity of EZH2, inhibiting the EED-EZH2 interaction is a potential therapeutic strategy for treating EZH2-dependent cancer. Although several EED-EZH2 protein-protein interaction (PPI) inhibitors have been developed, few target EED. Here, we identified that a cytisine derivative compound (1) potently binds EED, thus blocking the EED-EZH2 PPI. Compound 1 was found to inhibit cell proliferation and suppress the growth of 3D tumor spheres of TNBC cells. Moreover, by reversing EMT and decreasing the ratio of CSCs, the compound inhibited TNBC metastasis and invasion ability. Therefore, targeting EED to disrupt the EED-EZH2 PPI may provide a new approach for treating TNBC metastasis. To our knowledge, compound 1 is the first cytisine-based EED-EZH2 PPI inhibitor preventing metastasis in TNBC cells. This study may provide a new avenue for the development of more efficacious EED-EZH2 PPI inhibitors in TNBC treatment.

Patient-derived xenograft culture-transplant system for investigation of human breast cancer metastasis

Metastasis is a fatal disease where research progress has been hindered by a lack of authentic experimental models. Here, we develop a 3D tumor sphere culture-transplant system that facilitates the growth and engineering of patient-derived xenograft (PDX) tumor cells for functional metastasis assays in vivo. Orthotopic transplantation and RNA sequencing (RNA-seq) analyses show that PDX tumor spheres maintain tumorigenic potential, and the molecular marker and global transcriptome signatures of native tumor cells. Tumor spheres display robust capacity for lentiviral engineering and dissemination in spontaneous and experimental metastasis assays in vivo. Inhibition of pathways previously reported to attenuate metastasis also inhibit metastasis after sphere culture, validating our approach for authentic investigations of metastasis. Finally, we demonstrate a new role for the metabolic enzyme NME1 in promoting breast cancer metastasis, providing proof-of-principle that our culture-transplant system can be used for authentic propagation and engineering of patient tumor cells for functional studies of metastasis.

Targeting Cancer Stem Cell Markers or Pathways: A Potential Therapeutic Strategy for Oral Cancer Treatment.

Cancer stem cells (CSCs) are a small subset of cancer cells with stem cell-like properties, self-renewal potential, and differentiation capacity into multiple cell types. Critical genetic alterations or aberrantly activated signaling pathways associated with drug resistance and recurrence have been observed in multiple types of CSCs. In this context, CSCs are considered to be responsible for tumor initiation, growth, progression, therapeutic resistance, and metastasis. Therefore, to effectively eradicate CSCs, tremendous efforts have been devoted to identify specific target molecules that play a critical role in regulating their distinct functions and to develop novel therapeutics, such as proteins, monoclonal antibodies, selective small molecule inhibitors, and small antisense RNA (asRNA) drugs. Similar to other CSC types, oral CSCs can be characterized by certain pluripotency-associated markers, and oral CSCs can also survive and form 3D tumor spheres in suspension culture conditions. These oral CSC-targeting therapeutics selectively suppress specific surface markers or key signaling components and subsequently inhibit the stem-like properties of oral CSCs. A large number of new therapeutic candidates have been tested, and some products are currently in the pre-clinical or clinical development phase. In the present study, we review new oral CSC-targeted therapeutic strategies and discuss the various specific CSC surface markers and key signaling components involved in the stem-like properties, growth, drug resistance, and tumorigenicity of oral CSCs.

Multicomponent thermosensitive lipid complexes enhance desmoplastic tumor therapy through boosting anti-angiogenesis and synergistic strategy

Currently, the chemotherapy drugs-loaded thermosensitive liposomes have not shown an over standard of clinical effects compared to preclinical trials. In addition to the limiting factors of clinical trial design and heating device, abnormal angiogenesis in desmoplastic tumor is a key factor for unexpected clinical efficacy. Malformed tumor vasculature may result in reduced vascular transport and the heterogeneous distribution of thermosensitive liposomes in tumor. Here, we report an anti-angiogenesis strategy through hypoxia-inducible factors (HIF)-1α-vascular endothelial growth factor (VEGF) axis based on icaritin and coix seed oil dual loaded multicomponent thermosensitive lipid complexes (IC-ML). IC-ML could downregulate the HIF-1α expression in HepG2 cells with a synergetic antitumor effect. In addition, HepG2 + LX-2 cells co-cultured 3D tumor spheres administered IC-ML showed the strongest penetration and inhibition of growth. Accordingly, IC-ML displayed improved tumor penetration and superior synergistic antitumor efficacy with HIF-1α-VEGF downregulation in vivo under mild hyperthermia. The improvement of antitumor efficacy of IC-ML comes from the anti-angiogenesis strategy and comprehensive tumor microenvironment remodeling, including depletion of cancer-associated fibroblasts as well as inhibition of M2-type tumor associated macrophage infiltration in desmoplastic tumor. This study proposes a novel multicomponent synergistic antitumor strategy to improve the therapeutic potential of thermosensitive lipid complexes for hepatocellular carcinoma.

Identification of PARP-1 in cancer stem cells of gastrointestinal cancers: A preliminary study

Advanced-stage gastrointestinal tumors have high mortality due to chemotherapy limitations. One of the causes of treatment failure is the presence of cancer stem cells (CSCs), which show resistance mechanisms against DNA damage, such as poly (adenosine diphosphate-ribose) polymerase 1 (PARP-1). However, little is known about the relevance of PARP-1 in these tumor cells. Our purpose is to analyze the expression of PARP-1 in cancer cells and CSCs from gastrointestinal tumors, its relationship with the DNA damage repair process and its modulation by cytotoxic and PARP-1 inhibitors. We used pancreatic, liver and colon cancer cell lines and isolated CSCs using Aldefluor technology to analyze PARP-1 expression. In addition, we examined the effect of classic cytotoxic drugs (Doxorubicin, Gemcitabine, Irinotecan and 5-Fluorouracil) and a PARP-1 inhibitor (Olaparib) in cultured cells and 3D tumorspheres. We demonstrated that PARP-1 is highly expressed in pancreatic, liver and colon tumor cells and that this expression was significantly higher in cell populations with CSC characteristics. In addition, Doxorubicin and Gemcitabine increased their cytotoxic effect when administered simultaneously with Olaparib, decreasing the formation of 3D tumorspheres. Our findings suggest that PARP-1 is a common and relevant resistance mechanism in CSCs from gastrointestinal tumors and that the use of PARP-1 inhibitors may be an adjuvant therapy to increase apoptosis in this type of cells which are responsible to cancer recurrence and metastasis.

Identification of potential therapeutic target of naringenin in breast cancer stem cells inhibition by bioinformatics and in vitro studies

Cancer therapy is a strategic measure in inhibiting breast cancer stem cell (BCSC) pathways. Naringenin, a citrus flavonoid, was found to increase breast cancer cells’ sensitivity to chemotherapeutic agents. Bioinformatics study and 3D tumorsphere in vitro modeling in breast cancer (mammosphere) were used in this study, which aims to explore the potential therapeutic targets of naringenin (PTTNs) in inhibiting BCSCs. Bioinformatic analyses identified direct target proteins (DTPs), indirect target proteins (ITPs), naringenin-mediated proteins (NMPs), BCSC regulatory genes, and PTTNs. The PTTNs were further analyzed for gene ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein–protein interaction (PPI) networks, and hub protein selection. Mammospheres were cultured in serum-free media. The effects of naringenin were measured by MTT-based cytotoxicity, mammosphere forming potential (MFP), colony formation, scratch wound-healing assay, and flow cytometry-based cell cycle analyses and apoptosis assays. Gene expression analysis was performed using real-time quantitative polymerase chain reaction (q-RT PCR). Bioinformatics analysis revealed p53 and estrogen receptor alpha (ERα) as PTTNs, and KEGG pathway enrichment analysis revealed that TGF-ß and Wnt/ß-catenin pathways are regulated by PTTNs. Naringenin demonstrated cytotoxicity and inhibited mammosphere and colony formation, migration, and epithelial to mesenchymal transition in the mammosphere. The mRNA of tumor suppressors P53 and ERα were downregulated in the mammosphere, but were significantly upregulated upon naringenin treatment. By modulating the P53 and ERα mRNA, naringenin has the potential of inhibiting BCSCs. Further studies on the molecular mechanism and formulation of naringenin in BCSCs would be beneficial for its development as a BCSC-targeting drug.

Mimetics of extra virgin olive oil phenols with anti-cancer stem cell activity.

The extra virgin olive oil (EVOO) dihydroxy-phenol oleacein is a natural inhibitor of multiple metabolic and epigenetic enzymes capable of suppressing the functional traits of cancer stem cells (CSC). Here, we used a natural product-inspired drug discovery approach to identify new compounds that phenotypically mimic the anti-CSC activity of oleacein. We coupled 3D quantitative structure-activity relationship-based virtual profiling with phenotypic analysis using 3D tumorsphere formation as a gold standard for assessing the presence of CSC. Among the top 20 computationally-predicted oleacein mimetics, four fulfilled the phenotypic endpoint of specifically suppressing the tumorsphere-initiating capacity of CSC, in the absence of significant cytotoxicity against differentiated cancer cells growing in 2D cultures in the same low micromolar concentration range. Of these, 3,4-dihydrophenetyl butyrate –a lipophilic ester conjugate of the hydroxytyrosol moiety of oleacein– and (E)-N-allyl-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide) –an inhibitor of Trypanosoma cruzi triosephosphate isomerase– were also highly effective at significantly reducing the proportion of aldehyde dehydrogenase (ALDH)-positive CSC-like proliferating cells. Preservation of the mTOR/DNMT binding mode of oleacein was dispensable for suppression of the ALDH+-CSC functional phenotype in hydroxytyrosol-unrelated mimetics. The anti-CSC chemistry of complex EVOO phenols such as oleacein can be phenocopied through the use of mimetics capturing its physico-chemical properties.

Doxorubicin Improves Cancer Cell Targeting by Filamentous Phage Gene Delivery Vectors.

Merging targeted systemic gene delivery and systemic chemotherapy against cancer, chemovirotherapy, has the potential to improve chemotherapy and gene therapy treatments and overcome cancer resistance. We introduced a bacteriophage (phage) vector, named human adeno-associated virus (AAV)/phage or AAVP, for the systemic targeting of therapeutic genes to cancer. The vector was designed as a hybrid between a recombinant adeno-associated virus genome (rAAV) and a filamentous phage capsid. To achieve tumor targeting, we displayed on the phage capsid the double-cyclic CDCRGDCFC (RGD4C) ligand that binds the alpha-V/beta-3 (αvβ3) integrin receptor. Here, we investigated a combination of doxorubicin chemotherapeutic drug and targeted gene delivery by the RGD4C/AAVP vector. Firstly, we showed that doxorubicin boosts transgene expression from the RGD4C/AAVP in two-dimensional (2D) cell cultures and three-dimensional (3D) tumor spheres established from human and murine cancer cells, while preserving selective gene delivery by RGD4C/AAVP. Next, we confirmed that doxorubicin does not increase vector attachment to cancer cells nor vector cell entry. In contrast, doxorubicin may alter the intracellular trafficking of the vector by facilitating nuclear accumulation of the RGD4C/AAVP genome through destabilization of the nuclear membrane. Finally, a combination of doxorubicin and RGD4C/AAVP-targeted suicide gene therapy exerts a synergistic effect to destroy human and murine tumor cells in 2D and 3D tumor sphere settings.

Anti-tumor and immune modulating activity of T cell induced tumor-targeting effectors (TITE)

Adoptive transfer of Bispecific antibody Armed activated T cells (BATs) showed promising anti-tumor activity in clinical trials in solid tumors. The cytotoxic activity of BATs occurs upon engagement with tumor cells via the bispecific antibody (BiAb) bridge, which stimulates BATs to release cytotoxic molecules, cytokines, chemokines, and other signaling molecules extracellularly. We hypothesized that the release of BATs Induced Tumor-Targeting Effectors (TITE) by this complex interaction of T cells, bispecific antibody, and tumor cells may serve as a potent anti-tumor and immune-activating immunotherapeutic approach. In a 3D tumorsphere model, TITE showed potent cytotoxic activity against multiple breast cancer cell lines compared to control conditioned media (CM): Tumor-CM (T-CM), BATs-CM (B-CM), BiAb Armed PBMC-CM (BAP-CM) or PBMC-CM (P-CM). Multiplex cytokine analysis showed high levels of Th1 cytokines and chemokines; phospho-protein signaling array data suggest that the prominent JAK1/STAT1 pathway may be responsible for the induction and release of Th1 cytokines/chemokines in TITE. In xenograft breast cancer models, IV injections of 10× concentrated TITE (3×/week for 3weeks; 150μl TITE/injection) was able to inhibit tumor growth significantly (ICR/scid, p < 0.003; NSG p < 0.008) compared to the control mice. We tested the key components of the TITE for immune activating and anti-tumor activity individually and in combinations, the combination of IFN-γ, TNF-α and MIP-1β recapitulates the key activities of the TITE. In summary, master mix of active components of BATs-Tumor complex-derived TITE can provide a clinically controllable cell-free platform to target various tumor types regardless of the heterogeneous nature of the tumor cells and mutational tumor.

Abstract 6084: Wave3 tyrosine phosphorylation activates the invasion-metastasis cascade of triple negative breast cancer tumors through the maintenance of the cancer stem cell niche

Abstract The acquisition of metastatic phenotypes is responsible for the death of ~90% of breast cancer (BC) patients. In fact, metastatic BC is the 2nd leading cause of cancer-related deaths in women in the United States, annually accounting for more than 40,100 deaths and 266,000 new cases of invasive BC. Typically, metastases are incurable and result in a median survival of only 1.5 to 3 years for BC patients. Clinically, ~30% of BC patients diagnosed with early-stage, noninvasive disease will ultimately progress to late-stage, metastatic disease, an event that severely limits treatment options and associates with dismal clinical outcomes. This problem is exacerbated by the fact the BCs are heterogeneous and comprised of at least 5 genetically distinct subtypes. Amongst individual BC subtypes, those classified as TNBCs are especially lethal due to their highly metastatic behavior and propensity to recur rapidly. As a group, TNBCs lack expression of hormone receptors (ER-α and PR) and ErbB2/HER2, which has prevented the development of FDA-approved targeted drug therapies effective against this BC subtype. Likewise, recurrent TNBCs frequently acquire resistance to standard-of-care chemotherapeutic agents (e.g., doxorubicin, cyclophosphamides, and taxanes) through mechanisms that remain incompletely understood. We established Wiskott-Aldrich Syndrome Protein-3 (WAVE3) as a novel promoter of TNBC development and metastatic progression. Our previously published studies have shown that phosphorylation of WAVE3 at specific tyrosine residues by c-Abl non-receptor tyrosine kinase is required for cancer cell migration and invasion. In this study, we show that WAVE3 phosphorylation is required for the activation of the invasion-metastasis cascade of TNBCs, both in vitro and in in vivo mouse models. Genetic inactivation of WAVE3 via CRISPR/Cas9 gene editing suppressed migration, invasion and the formation of 3D tumorspheres of TNBC cells, in vitro, as well as tumor growth and metastasis in vivo. While re-expression of phospho-active WAVE3 in the WAVE3-deficient TNBC cells restored the oncogenic activity of WAVE3, re-expression of phospho-mutant WAVE3 or the pharmacologic inhibition of WAVE3 phosphorylation did not. Further studies revealed that phosphorylation of WAVE3 was required for the activation of several hallmarks of cancer through the upregulation of survival signaling pathways. Blocking WAVE3 expression or phosphorylation inhibited epithelial-mesenchymal transition (EMT) as well as disrupted the breast cancer stem cell (BCSC) niche by inhibiting expression of BCSC-specific markers. Finally, loss of WAVE3 phosphorylation was sufficient to resensitize TNBC cells to standard of care chemotherapy-induced apoptosis and cell death through the targeting of the BCSCs. Together, our data support the targeting of WAVE3 as a novel therapeutic strategy to treat metastasis in patients with TNBC tumors by specifically targeting BCSCs. Citation Format: Wei Wang, Urna Kansakar, Vesna Markovic, Khalid Sossey-Alaoui. Wave3 tyrosine phosphorylation activates the invasion-metastasis cascade of triple negative breast cancer tumors through the maintenance of the cancer stem cell niche [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6084.

Deferiprone (DFP) Targets Cancer Stem Cell (CSC) Propagation by Inhibiting Mitochondrial Metabolism and Inducing ROS Production.

Deferiprone (DFP), also known as Ferriprox, is an FDA-approved, orally active, iron chelator that is currently used clinically for the treatment of iron-overload, especially in thalassaemia major. As iron is a critical factor in Fe-S cluster assembly that is absolutely required for the metabolic function of mitochondria, we hypothesized that DFP treatment could be used to selectively target mitochondria in cancer stem cells (CSCs). For this purpose, we used two ER(+) human breast cancer cell lines, namely MCF7 and T47D cells, as model systems. More specifically, a 3D tumorsphere assay was employed as a functional readout of CSC activity which measures anchorage-independent growth under low attachment conditions. Here, we show that DFP dose dependently inhibited the propagation of CSCs, with an IC-50 of ~100 nM for MCF7 and an IC-50 of ~0.5 to 1 μM for T47D cells, making DFP one the most potent FDA-approved drugs that we and others have thus far identified for targeting CSCs. Mechanistically, we show that high concentrations of DFP metabolically targeted both mitochondrial oxygen consumption (OCR) and glycolysis (extracellular acidification rates (ECAR)) in MCF7 and T47D cell monolayers. Most importantly, we demonstrate that DFP also induced a generalized increase in reactive oxygen species (ROS) and mitochondrial superoxide production, and its effects reverted in the presence of N-acetyl-cysteine (NAC). Therefore, we propose that DFP is a new candidate therapeutic for drug repurposing and for Phase II clinical trials aimed at eradicating CSCs.

Cancer Stem Cell Target Labeling and Efficient Growth Inhibition of CD133 and PD-L1 Monoclonal Antibodies Double Conjugated with Luminescent Rare-Earth Tb3+ Nanorods

Rare-earth nanomaterials are being widely applied in medicine as cytotoxicity agents, in radiation and photodynamic therapy, as drug carriers, and in biosensing and bioimaging technology. Terbium (Tb), a rare-earth element belonging to the lanthanides, has a long luminescent lifetime, large stock displacement, narrow spectral width, and biofriendly probes. In cancer therapy, cancer stem cell (CSC)-targeted treatment is receiving considerable attention due to these cells’ harmful characteristics. However, CSCs remain barely understood. Therefore, to effectively label and inhibit the growth of CSCs, we produced a nanocomplex in which TbPO4·H2O nanorods were double conjugated with CD133 and PD-L1 monoclonal antibodies. The Tb3+ nanomaterials were created in the presence of a soft template (polyethylene glycol 2000). The obtained nanomaterial TbPO4·H2O was hexagonal crystal and nanorod in shape, 40–80 nm in diameter, and 300–800 nm in length. The nanorods were further surfaced through tetraethyl orthosilicate hydrolysis and functionalized with amino silane. Finally, the glutaraldehyde-activated Tb3+ nanorods were conjugated with CD133 monoclonal antibody and PD-L1 monoclonal antibody on the surface to obtain the nanocomplex TbPO4·H2O@silica-NH2+mAb^CD133+mAb^PD-L1 (TMC). The formed nanocomplex was able to efficiently and specifically label NTERA-2 cells, a highly expressed CD133 and PD-L1 CSC cell line. The conjugate also demonstrated promising anti-CSC activity by significant inhibition (58.50%) of the growth of 3D tumor spheres of NTERA-2 cells (p &lt; 0.05).