Bulk Cancer Cells Research Articles

Abstract B068: FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models

Abstract The unfavorable prognosis of high-grade serous ovarian cancer, HGSOC, may be driven by a small subset of ovarian cancer stem cells (OCSC), which are endowed with tumor-initiating ability and are intrinsically resistant to cytotoxic treatments. These characteristics are sustained by signals coming from the tumor microenvironment (TME). As a common site of HGSOC presentation is the omentum, we partially recapitulated the architecture of human omentum in vitro using 3D co-cultures of primary human omental fibroblasts and mesothelial cells. Patient-derived HGSOC cells were then integrated in these organotypic models, either as bulk cancer cells or as OCSC from the same patients. After co-culture in this model, malignant cells were re-isolated by FACS sorting and subjected to whole-transcriptome RNA sequencing, comparing them to control cells that have not been in contact with the in vitro TME. Our data revealed transcriptional reprogramming induced by the in vitro TME in HGSOC cells. We found a number of pathways differentially regulated in the bulk population of OC cells vs OCSC, shedding light on the impact of the TME on OC stemness. Moreover, we identified a panel of transcription factors (TFs) specifically altered by the in vitro TME in OCSC. These TF-regulated gene networks represent OCSC vulnerabilities and, hence, potential targets in relapsing tumors. Among these, we focused on FOXM1, a TF with pleiotropic functions in cancer cells, whose activity is altered in 84% of ovarian cancer patients. The FOXM1 pathway was strongly induced by the in vitro TME specifically in OCSC. Mechanistically, the contact with the in vitro TME triggered a FAK/YAP axis which, in turn, accounted for the induction of FOXM1 and its downstream target genes. Moreover, the pharmacological inhibition of FOXM1 significantly impaired OCSC fitness, supporting, on one hand, the key role of TME in orchestrating OC stemness and suggesting, on the other, a new approach for the eradication of OCSC from the omental niche. Overall, our patient-derived organotypic platform offers the opportunity to unravel the impact of the TME on OCSC at the molecular level in experimental models with clinical relevance and can be exploited to design innovative HGSOC-eradicating treatments. Citation Format: Chiara Battistini, Hilary A. Kenny, Valentina Nieddu, Valentina Melocchi, Alessandra Decio, Beatrice Malacrida, Alessia Gatto, Fabrizio Bianchi, Raffaella Giavazzi, Nicoletta Colombo, Frances R. Balkwill, Ernst Lengyel, Ugo Cavallaro. FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B068.

MiRNA on the Battlefield of Cancer: Significance in Cancer Stem Cells, WNT Pathway, and Treatment.

Carcinogenesis is a complex process characterized by intricate changes in organ histology, biochemistry, epigenetics, and genetics. Within this intricate landscape, cancer stem cells (CSCs) have emerged as distinct cell types possessing unique attributes that significantly contribute to the pathogenesis of cancer. The WNT signaling pathway plays a critical role in maintaining somatic stem cell pluripotency. However, in cancer, overexpression of WNT mediators enhances the activity of β-catenin, resulting in phenomena such as recurrence and unfavorable survival outcomes. Notably, CSCs exhibit heightened WNT signaling compared to bulk cancer cells, providing intriguing insights into their functional characteristics. MicroRNAs (miRNAs), as post-transcriptional gene expression regulators, modulate various physiological processes in numerous diseases including cancer. Upregulation or downregulation of miRNAs can affect the production of pro-oncogenic or anti-oncogenic proteins, influencing cellular processes that maintain tissue homeostasis and promote either apoptosis or differentiation, even in cancer cells. In order to understand the dysregulation of miRNAs, it is essential to examine miRNA biogenesis and any possible alterations at each step. The potential of a miRNA as a biomarker in prognosis, diagnosis, and detection is being assessed using technologies such as next-generation sequencing. Extensive research has explored miRNA expression profiles in cancer, leading to their utilization as diagnostic tools and the development of personalized and targeted cancer therapies. This review delves into the role of miRNAs in carcinogenesis in relation to the WNT signaling pathway along with their potential as druggable compounds.

Liposome-enabled bufalin and doxorubicin combination therapy for trastuzumab-resistant breast cancer with a focus on cancer stem cells

Breast cancer stem cells (BCSCs) play a key role in therapeutic resistance in breast cancer treatments and disease recurrence. This study aimed to develop a combination therapy loaded with pH-sensitive liposomes to kill both BCSCs and the okbulk cancer cells using trastuzumab-sensitive and resistant human epidermal growth factor receptor 2 positive (HER2+) breast cancer cell models. The anti-BCSCs effect and cytotoxicity of all-trans retinoic acid, salinomycin, and bufalin alone or in combination with doxorubicin were compared in HER2+ cell line BT-474 and a validated trastuzumab-resistant cell line, BT-474R. The most potent anti-BCSC agent was selected and loaded into a pH-sensitive liposome system. The effects of the liposomal combination on BCSCs and bulk cancer cells were assessed. Compared with BT-474, the aldehyde dehydrogenase positive BCSC population was elevated in BT-474R (3.9 vs. 23.1%). Bufalin was the most potent agent and suppressed tumorigenesis of BCSCs by ∼50%, and showed strong synergism with doxorubicin in both BT-474 and BT-474R cell lines. The liposomal combination of bufalin and doxorubicin significantly reduced the BCSC population size by 85%, and inhibited both tumorigenesis and self-renewal, although it had little effect on the migration and invasiveness. The cytotoxicity against the bulk cancer cells was also enhanced by the liposomal combination than either formulation alone in both cell lines (p < 0.001). The liposomal bufalin and doxorubicin combination therapy may effectively target both BCSCs and bulk cancer cells for a better outcome in trastuzumab-resistant HER2+ breast cancer.

Intratumor Heterogeneity and Treatment Resistance of Solid Tumors with a Focus on Polyploid/Senescent Giant Cancer Cells (PGCCs).

Single cell biology has revealed that solid tumors and tumor-derived cell lines typically contain subpopulations of cancer cells that are readily distinguishable from the bulk of cancer cells by virtue of their enormous size. Such cells with a highly enlarged nucleus, multiple nuclei, and/or multiple micronuclei are often referred to as polyploid giant cancer cells (PGCCs), and may exhibit features of senescence. PGCCs may enter a dormant phase (active sleep) after they are formed, but a subset remain viable, secrete growth promoting factors, and can give rise to therapy resistant and tumor repopulating progeny. Here we will briefly discuss the prevalence and prognostic value of PGCCs across different cancer types, the current understanding of the mechanisms of their formation and fate, and possible reasons why these tumor repopulating "monsters" continue to be ignored in most cancer therapy-related preclinical studies. In addition to PGCCs, other subpopulations of cancer cells within a solid tumor (such as oncogenic caspase 3-activated cancer cells and drug-tolerant persister cancer cells) can also contribute to therapy resistance and pose major challenges to the delivery of cancer therapy.

Nalbuphine Suppresses Leukemia Stem Cells and Acts Synergistically with Chemotherapy Drugs via Inhibiting Ras/Raf/Mek/Erk Pathway.

Retrospective clinical studies have shown that opioids could potentially affect the risk of cancer recurrence and metastasis. Better understanding of the effects of opioids on cancer will help to select the optimal anesthetic regimens to achieve better outcomes in cancer patients. Increasing evidence has shown the direct effects of opioids on bulk cancer cells and cancer stem cells. Opioid such as nalbuphine is approved to control cancer-associated pain but little is known on their possible cancer effects. To assess the biological effects of nalbuphine on acute myeloid leukemia (AML) differentiated and stem/progenitor CD34+ cells. AML CD34+ cells were isolated with colony formation, growth and apoptosis assays performed. Biochemical and immunoblotting analyses were conducted in AML cells exposed to nalbuphine. Nalbuphine at clinically relevant concentrations was active against a panel of AML cell lines with varying IC50. Importantly, nalbuphine augmented the efficacy of cytarabine and daunorubicin in decreasing AML cell viability/ growth. Besides bulk AML cells, we noted that nalbuphine was effective and selective in decreasing viability and colony formation of AML CD34+ cells while sparing normal hematopoietic CD34+ cells. The action of nalbuphine on AML cells is not associated with opioid receptors but via inhibiting Ras/Raf/MEK/ERK signaling pathway. Overexpression of constitutively active Ras partially but significantly reversed the inhibitory effects of nalbuphine on AML cells. Our findings reveal the selective anti-AML activity of nalbuphine and its ability in inhibiting Ras signaling. Our work suggests that nalbuphine may be beneficial for leukemia patients.

Abstract 2444: RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion

Abstract Non-small cell lung cancer (NSCLC), a major subtype of lung cancer, accounts for ~85% of all lung cancer diagnoses. A sub-population of cells within human NSCLC tumors called cancer stem cells (CSCs) have been found to exhibit distinct properties that contribute to factors associated with poor outcomes in NSCLC patients including metastasis, relapse, and therapeutic resistance. We previously performed RNAseq analysis on bulk cancer cells (BCCs) and CSCs from a panel of primary NSCLC tumors and cell lines to identify signaling mechanisms involved in the tumorigenic behavior of NSCLC CSCs. Our analysis revealed that expression of RAB27B, a small GTPase that plays a critical role in extracellular vesicle (EV) formation and secretion, was significantly upregulated in CSCs when compared to BCCs. Interestingly, RAB27B is overexpressed and predicts patient survival in NSCLC. The goal of this study was to assess the role of RAB27B in the tumorigenic behavior of NSCLC CSCs and determine the mechanism by which RAB27B promotes an aggressive stem-cell like phenotype in NSCLC cells an aspect of RAB27B function that has not been investigated in NSCLC. QPCR and immunoblot analysis was used to compare RAB27B mRNA and protein expression in a panel of NSCLC BCCs and CSCs. Two independent shRNA constructs were used to knockdown (KD) RAB27B in NSCLC CSCs. The effects of RAB27B KD on CSC expansion, transformed growth and invasion were analyzed by clonal expansion, soft agar colony formation and Boyden chamber assays, respectively. Xenograft and tail vein mouse models were used to assess the effects of RAB27B KD on CSC tumor growth and lung colonization. EVs were isolated from the conditioned media of BCCs, CSCs and RAB27B KD CSCs using differential ultracentrifugation, and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblotting according to MISEV guidelines. Our analysis revealed that RAB27B is significantly upregulated in NSCLC CSCs compared to BCCs. RAB27B KD cells exhibited a significant decrease in clonal expansion, transformed growth and invasion when compared to non-target shRNA control CSCs. In vivo studies demonstrated that RAB27B is important for the tumorigenicity of CSCs and promotes tumor cell proliferation, and angiogenesis. EVs were positive for EV-specific markers and negative for an intracellular vesicle marker. CSCs secrete significantly more EVs when compared to BCCs and KD of RAB27B in CSCs led to a decrease in the levels of EVs secreted. Furthermore, delivery of CSC-derived, but not BCC-derived EVs to BCCs induced spheroid growth, clonal expansion, and invasion in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating and invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Citation Format: Kayleah M. Meneses, Jennifer A. Lindemann, Prita Pandya, Dania S. Al-Qasrawi, Ryan Argo, Celeste Weems, Danielle J. Beetler, Irene K. Yan, Joy Wolfram, Tushar C. Patel, Verline Justilien. RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion [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 2444.

Abstract 5791: Autophagy &amp; PIM1 kinase inhibition in breast cancer stem cells &amp; bulk cancer cells attenuates breast cancer &amp; metastasis

Abstract Autophagy is a self-degradation process, it is essential for growth of heterogenous of breast cancer cells. Breast tumor heterogeneity majorly comprises bulk cancer cells &amp; cancer stem cells (CSCs). Targeting either CSCs or cancer cells could lead to interconversion between these cells due to EMT/MET transitions. Therefore, it is important to identify a common molecular targets &amp; its mechanism of regulation in these populations for improvement of breast cancer therapy. Here, we utilized a PCR based screen &amp; identified NUMA1 downregulation in FIP200 KO (autophagy gene) cells of three major breast cancer subtypes, FF99 (luminal), N418 (Her2+) &amp; BF3M (basal). Further, NUMA1 KO in these cells reduced tumor formation &amp; CSCs indicating the role of NUMA1 on regulation of CSCs. Therefore, we sorted breast CSCs, ALDH+ and CD29hi CD61+ from WT &amp; NUMA1 KO cells, tail-vein injected nude mice depicted reduced metastatic potential with NUMA1 KO than WT CSCs. Mechanistically, NUMA1 regulation by PIM1 kinase was observed in CSCs but not in bulk cancer cells. Cell viability assays revealed that combination treatment of autophagy &amp; PIM1 inhibitor showed higher efficacy against cancer cells of three breast cancer subtypes than individual treatments alone. Finally, the combination treatment of autophagy inhibitor, Lys05 &amp; PIM1 inhibitor, SMI-4a reduced tumor formation &amp; metastasis of breast cancer. Human tissue microarray results depicted high expression of NUMA1 &amp; PIM1 in different breast cancer subtypes than non- tumor tissues. Our study demonstrated that inhibition of autophagy &amp; PIM1 in bulk cancer cells &amp; CSCs showed attenuation of tumor formation &amp; metastasis, suggesting this combination treatment serves as an important therapeutic approach for treating breast cancer. Citation Format: Kanakaraju Manupati, Mingang Hao, Michael Haas, Syn Kok Yeo, Jun-Lin Guan. Autophagy &amp; PIM1 kinase inhibition in breast cancer stem cells &amp; bulk cancer cells attenuates breast cancer &amp; metastasis [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 5791.

Doxorubicin and erastin co-loaded hydroxyethyl starch-polycaprolactone nanoparticles for synergistic cancer therapy.

Cancer stem cells (CSCs), enabled to self-renew, differentiate, and initiate the bulk tumor, are recognized as the culprit of treatment resistance, metastasis, and recurrence. Simultaneously eradicating CSCs and bulk cancer cells is crucial for successful cancer therapy. Herein, we reported that doxorubicin (Dox) and erastin co-loaded hydroxyethyl starch-polycaprolactone nanoparticles (DEPH NPs) eliminated CSCs and cancer cells by regulating redox status. We found that an excellently synergistic effect existed when Dox and erastin were co-delivered by DEPH NPs. Specifically, erastin could deplete intracellular glutathione (GSH), thereby inhibiting the efflux of intracellular Dox and boosting Dox-induced reactive oxygen species (ROS) to amplify redox imbalance and oxidative stress. The high ROS levels restrained CSCs self-renewal via downregulating Hedgehog pathways, promoted CSCs differentiation, and rendered differentiated cancer cells vulnerable to apoptosis. As such, DEPH NPs significantly eliminated not only cancer cells but more importantly CSCs, contributing to suppressed tumor growth, tumor-initiating capacity, and metastasis, in various tumor models of triple negative breast cancer. This study demonstrates that the combination of Dox and erastin is potent in elimination of both cancer cells and CSCs, and that DEPH NPs represent a promising treatment against CSCs-rich solid tumors.

Paracrine secretion of IL8 by breast cancer stem cells promotes therapeutic resistance and metastasis of the bulk tumor cells

BackgroundBreast tumors consist of heterogeneous cellular subpopulations that differ in molecular properties and functional attributes. Cancer stem cells (CSCs) play pivotal roles in cancer therapeutic failure and metastasis. However, it remains indeterminate how CSCs determine the progression of the bulk cancer cell population.MethodsCo-culture systems in vitro and co-implantation systems in vivo were designed to characterize the interactions between breast cancer stem cells (BCSCs) and bulk cancer cells. RNA sequencing was performed to study the functional and mechanistic implications of the BCSC secretome on bulk cancer cells. A cytokine antibody array was employed to screen the differentially secreted cytokines in the BCSC secretome. Tail vein injection metastatic models and orthotopic xenograft models were applied to study the therapeutic potential of targeting IL8.ResultsWe identified that the BCSC secretome potentiated estrogen receptor (ER) activity in the bulk cancer cell population. The BCSC secretome rendered the bulk cancer cell population resistant to anti-estrogen and CDK4/6 inhibitor therapy; as well as increased the metastatic burden attributable to bulk cancer cells. Screening of the BCSC secretome identified IL8 as a pivotal factor that potentiated ERα activity, endowed tamoxifen resistance and enhanced metastatic burden by regulation of bulk cancer cell behavior. Pharmacological inhibition of IL8 increased the efficacy of fulvestrant and/or palbociclib by reversing tamoxifen resistance and abrogated metastatic burden.ConclusionTaken together, this study delineates the mechanism by which BCSCs determine the therapeutic response and metastasis of bulk cancer cells; and thereby suggests potential therapeutic strategies to ameliorate breast cancer outcomes.-fLEjwWBuA1CFtkWuqXTMGVideo

HES1 promoter activation dynamics reveal the plasticity, stemness and heterogeneity in neuroblastoma cancer stem cells.

Notch signaling and its downstream gene target HES1 play a critical role in regulating and maintaining cancer stem cells (CSCs), similar to as they do during embryonic development. Here, we report a unique subclass of Notch-independent Hes-1 (NIHes-1)-expressing CSCs in neuroblastoma. These CSCs maintain sustained HES1 expression by activation of HES1 promoter region upstream of classical CBF-1 binding sites, thereby completely bypassing Notch receptor-mediated activation. These stem cells have self-renewal ability and potential to generate tumors. Interestingly, we observed that NIHes-1 CSCs could transition to Notch-dependent Hes-1-expressing (NDHes-1) CSCs where HES1 is expressed by Notch receptor-mediated promoter activation. We observed that NDHes-1-expressing CSCs also had the potential to transition to NIHes-1 CSCs and during this coordinated bidirectional transition, both CSCs gave rise to the majority of the bulk cancer cells, which had an inactive HES1 promoter (PIHes-1). A few of these PIHes-1 cells were capable of reverting into a CSC state. These findings explain the existence of a heterogenic mode of HES1 promoter activation within the IMR-32 neuroblastoma cell line and the potential to switch between them. This article has an associated First Person interview with the first authors of the paper.

Green Synthesis of Gold Nanoparticles and Study of Their Inhibitory Effect on Bulk Cancer Cells and Cancer Stem Cells in Breast Carcinoma.

Chemo-resistance from cancer stem cells (CSCs) subpopulation is a current issue in cancer treatment. It is important to select alternative therapies to efficiently eradicate both bulk cancer cells and CSCs. Here, gold nanoparticles (AuNPs) have been selected regarding their biocompatibility, facile and controllable synthesis, potent anti-cancer activity and photothermal conversion performance. We reported a green synthesis of functionalized AuNPs using hyaluronic acid (HA) as a reductant, capping, stabilizing and hydrophilic substance. The resultant AuNPs were spherical-shaped with an average diameter of around 30 nm. These AuNPs displayed improved physico-chemical (yield, stability, photothermal effect) and biological properties (cellular uptake, cytotoxicity and apoptotic effect) against bulk MDA-MB-231 cells, in comparison with other organic anti-cancer drugs. The intensified bioactivity was dependent on a mitochondria-mediated cascade, reflected by the damage in mitochondria, oxidative stress, intensified Caspase 3 activity and increased/decreased expression of certain pro-apoptotic (Bax, P53, Caspase 3)/anti-apoptotic (Bcl-2) genes. Moreover, these AuNPs posed a dramatically improved inhibitory effect in cell viability and self-renewable capacity on CSC subpopulation. All the results were attributed from the nano-scaled structure of AuNPs and combined effect from NIR-induced hyperthermia. In addition, the biocompatible nature of these AuNPs supported them to be a potential candidate in the development of novel chemotherapeutic drugs.

Retraction of "Label-Free Isolation of Low-Adhesion Cells with Stem Properties for Cancer Stem Cell-Specific Drug Evaluation".

Isolating and obtaining sufficient numbers of cancer stem-like cells from bulk cancer cells for cancer stem cells (CSCs)-specific drug evaluation remain challenging due to the rarity and their relative instability in culture. Here, we report a label-free microfluidic method to isolate low-adhesion cells (LACs) with stem properties based on a cell adhesion microfluidic chip (CA-chip) using cell adhesion differences. In this chip, suspended cells are driven by hydrodynamic force to flow through microchannels coated with basement membrane extract (BME). By manipulating the hydrodynamic force, heterogeneous tumor cells can be separated into different adhesive cell subpopulations. LACs flow out first and are collected from the outlet, while high-adhesion cells (HACs) are captured into the chip and can be recovered by washing with a cell dissociation buffer. We found that the isolated LACs showed cancer stem-like properties, which were further utilized as rare CSC candidates to evaluate CSC-specific drugs. The results showed that only CSC-specific drugs had complete inhibition of sphere formation, demonstrating the feasibility of evaluating the specificity of CSC drugs. The label-free method is highly versatile as it employs cell adhesion as a unique phenotypic marker to effectively isolate cancer stem-like cells and offers an opportunity to evaluate on the large scale CSC-specific drugs, making it applicable to the analysis of certain cancer types, especially CSC-specific biomarkers that have not been identified.

Cancer Associated Fibroblast (CAF) Regulation of PDAC Parenchymal (CPC) and CSC Phenotypes Is Modulated by ECM Composition.

Simple SummaryHere, we demonstrate for the first time that ECM composition cooperates with CAFs to jointly regulate/modulate the highly dynamic interactions between the CPC and CSC cell lines and establish a continuum between tumor initiation and progression in primary PDAC tumors. Altogether, these findings propose a scenario in which the ECM composition and the cellular secretome of the CAFs cooperate to jointly regulate both growth and morphology of the CPC and CSC cell lines and, by modulating the highly dynamic interactions between them, establishes a continuum between tumor initiation and progression in primary PDAC tumors.Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of all cancers, having one of the lowest five-year survival rates. One of its hallmarks is a dense desmoplastic stroma consisting in the abnormal accumulation of extracellular matrix (ECM) components, especially Collagen I. This highly fibrotic stroma embeds the bulk cancer (parenchymal) cells (CPCs), cancer stem cells (CSCs) and the main producers of the stromal reaction, the Cancer Associated Fibroblasts (CAFs). Little is known about the role of the acellular ECM in the interplay of the CAFs with the different tumor cell types in determining their phenotypic plasticity and eventual cell fate. Methods: Here, we analyzed the role of ECM collagen I in modulating the effect of CAF-derived signals by incubating PDAC CPCs and CSCs grown on ECM mimicking early (low collagen I levels) and late (high collagen I levels) stage PDAC stroma with conditioned medium from primary cultured CAFs derived from patients with PDAC in a previously described three-dimensional (3D) organotypic model of PDAC. Results: We found that CAFs (1) reduced CPC growth while favoring CSC growth independently of the ECM; (2) increased the invasive capacity of only CPCs on the ECM mimicking the early tumor; and (3) favored vasculogenic mimicry (VM) especially of the CSCs on the ECM mimicking an early tumor. Conclusions: We conclude that the CAFs and acellular stromal components interact to modulate the tumor behaviors of the PDAC CPC and CSC cell types and drive metastatic progression by stimulating the phenotypic characteristics of each tumor cell type that contribute to metastasis.

Abstract 1007: Spatial, temporal and heterogeneous changes in transport phenotype in liver metastasis of breast cancer

Abstract Various heterogeneities in response of metastatic tumors to systemic anti-cancer therapy are major source of therapeutic resistance. Cancer cells from bulk tumors and cancer cell lines are not clonal. A highly heterogeneous tumor microenvironment is known to develop by subclones. Furthermore, metastasis can evolve during the disease progression as well as in response to systemic therapies. These spatial and temporal variations of heterogeneity in tumors can affect blood perfusion and vascular permeability which can determine the level of mass/anti-cancer drugs transport to tumors (we refer to as “transport phenotype”). Insufficient drug transport to tumor in vivo can generate transport-based therapeutic resistance even for agents with proven efficacy against the target cancer cell lines in vitro. Our goal is to understand spatial and temporal heterogeneity in transport of mass/anti-cancer drugs to metastatic tumors to overcome the therapeutic resistance. The study of bulk tumors will provide a mixture of different functions from heterogeneous sets of various clones. We sought to establish single cell-derived clones from a 4T1 parental murine breast cancer cell line in order to dissect and simplify the heterogeneity in polyclonal cell populations in tumors. First, we found heterogeneous accumulation of systemically injected PEGylated liposomal doxorubicin (PLD) among multiple, experimental liver metastases of 4T1 parental (polyclonal) breast cancer in mouse. Intravital Microscopy imaging of liver metastases through a window chamber over time revealed different transport phenotypes, in terms of circulation and diffusion of systemically injected fluorescent tracers around/inside parental (polyclonal) tumors. Furthermore, transport phenotype can be modulated from one to another after PLD therapy, creating possible mechanisms of transport based on drug resistance. When single cell clone-derived metastases were established, a clone dependent difference in transport phenotype was found. Interestingly transport phenotype even in clonal tumors was temporally evolved and increased its heterogeneity. These data suggest that transport phenotype cannot be determined solely by genotype of tumor and can be evolved and more heterogeneous as a function of tumor growth and systemic chemotherapy. Clinical implication of our findings is that transport of therapeutics to tumor and its efficacy may not be predicted by DNA-based assay for clinical decision-making and transport-based therapeutics resistance will eventually occur in every tumor types. Citation Format: Aygul Sadigova, Megumi Kai, Yan Ting Liu, Thao Nguyen, Chihiro Hashimoto, Kenji Yokoi. Spatial, temporal and heterogeneous changes in transport phenotype in liver metastasis of 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 1007.

Antagonizing RARγ Drives Necroptosis of Cancer Stem Cells

There is a need for agents that eliminate cancer stem cells, which sustain cancer and are also largely responsible for disease relapse and metastasis. Conventional chemotherapeutics and radiotherapy are often highly effective against the bulk of cancer cells, which are proliferating, but spare cancer stem cells. Therapeutics that target cancer stem cells may also provide a bona fide cure for cancer. There are two rationales for targeting the retinoic acid receptor (RAR)γ. First, RARγ is expressed selectively within primitive cells. Second, RARγ is a putative oncogene for a number of human cancers, including cases of acute myeloid leukemia, cholangiocarcinoma, and colorectal, renal and hepatocellular carcinomas. Prostate cancer cells depend on active RARγ for their survival. Antagonizing all RARs caused necroptosis of prostate and breast cancer stem cell-like cells, and the cancer stem cells that gave rise to neurospheres from pediatric patients’ primitive neuroectodermal tumors and an astrocytoma. As tested for prostate cancer, antagonizing RARγ was sufficient to drive necroptosis. Achieving cancer-selectively is a longstanding paradigm for developing new treatments. The normal prostate epithelium was less sensitive to the RARγ antagonist and pan-RAR antagonist than prostate cancer cells, and fibroblasts and blood mononuclear cells were insensitive. The RARγ antagonist and pan-RAR antagonist are promising new cancer therapeutics.

Targeting Cancer Stem Cells through Epigenetic Modulation of Interferon Response.

Cancer stem cells (CSCs) are a small subset of cancer cells and are thought to play a critical role in the initiation and maintenance of tumor mass. CSCs exhibit similar hallmarks to normal stem cells, such as self-renewal, differentiation, and homeostasis. In addition, CSCs are equipped with several features so as to evade anticancer mechanisms. Therefore, it is hard to eliminate CSCs by conventional anticancer therapeutics that are effective at clearing bulk cancer cells. Interferons are innate cytokines and are the key players in immune surveillance to respond to invaded pathogens. Interferons are also crucial for adaptive immunity for the killing of specific aliens including cancer cells. However, CSCs usually evolve to escape from interferon-mediated immune surveillance and to shape the niche as a “cold” tumor microenvironment (TME). These CSC characteristics are related to their unique epigenetic regulations that are different from those of normal and bulk cancer cells. In this review, we introduce the roles of epigenetic modifiers, focusing on LSD1, BMI1, G9a, and SETDB1, in contributing to CSC characteristics and discussing the interplay between CSCs and interferon response. We also discuss the emerging strategy for eradicating CSCs by targeting these epigenetic modifiers, which can elevate cytosolic nuclei acids, trigger interferon response, and reshape a “hot” TME for improving cancer immunotherapy. The key epigenetic and immune genes involved in this crosstalk can be used as biomarkers for precision oncology.

Abstract P1-06-04: Spatial, temporal and therapy dependent heterogeneity in transport phenotype of multiple liver metastases in murine breast cancer model

Abstract Introduction: Inter- and intra-tumor heterogeneities in response of tumors to systemic anti-cancer therapy are the major source of therapeutic resistance. Cancer cells from bulk tumors and cancer cell lines are not clonal but are composed of subclones, creating heterogeneous tumor microenvironment. Furthermore, metastasis can evolve during the disease progression as well as in response to systemic therapies. These spatial and temporal heterogeneities in tumors can affect blood perfusion and vascular permeability which determine the transport of various masses and chemotherapeutic (we refer to as “transport phenotype”). Insufficient drug transport to the tumor in vivo can generate transport-based therapeutic resistance even for agents with proven efficacy against the target cells in vitro. Our goal is to understand spatial and temporal heterogeneity in the transport of mass/anti-cancer drugs to metastatic tumors to leverage novel strategies for improving therapeutic efficacy using mouse tumor models. The study of bulk tumors will provide a mixture of different functions from heterogeneous sets of various clones. We sought to establish single cell-derived clones from the parental murine breast cancer cell line to dissect and simplify tumor heterogeneity. Methods: Single cells were isolated via aliquoting 4T1-luc-tomato red cell suspension (polyclonal) in single-cell concentration into 96 well plate. To evaluate the phenotype of the clonal and parental cells, we performed clonogenic assay, proliferation and growth inhibition assay using pegylated liposomal doxorubicin (PLD), and angiogenic protein production assay using membrane array. Among the established clones, we selected 2 clones and parental cells for further experiments. Experimental liver metastases were created by injecting cell suspension into the spleen of BALB/c mice. Then, the window chamber was implanted above the liver of anesthetized mice and fluorescently labeled dextran tracers with different molecular weights were intravenously (iv) injected. To determine transport phenotype, circulation and diffusion of tracers inside/around individual metastasis were imaged and quantified through the window using Intravital Microscopy (IVM) at various time points. In another set of experiment, PLD was iv injected, and change in the transport phenotype was monitored. Results: While parental cells formed heterogeneous colonies, clonal cells created each cell line dependent uniform colonies in petri dish. Also, parental and selected two clonal cells exhibited different growth, response to PLD, and protein production in vitro. IVM imaging of individual liver metastasis over time using different cell lines revealed that relative fluorescent intensity of tracers transported inside/around tumor compared to that in the uninvolved tumor-free liver was more heterogeneous among parental cell derived tumors compared to clonal cells. Interestingly, these “transport phenotypes” evolved and increased their heterogeneity as tumors progressed regardless of cell type. Furthermore, transport phenotype was modulated from one to another after PLD therapy, creating possible mechanisms of transport based on drug resistance. Discussion: These data suggest that transport phenotype cannot be determined solely by genotype of tumors but modulated as a function of tumor growth and therapy response to become more heterogeneous. The intrinsic heterogeneity of transport phenotype and its acquired transformation suggests a more complicated scenario of tumor drug-resistance mechanism. The clinical implication of our findings is that the transport of therapeutics to the tumor and its efficacy may not be predicted by DNA-based assay and transport-based therapeutics resistance will eventually occur in every tumor type. Citation Format: Thao Nguyen, Yan ting Liu, Arturas Ziemys, Shen Haifa, Kenji Yokoi. Spatial, temporal and therapy dependent heterogeneity in transport phenotype of multiple liver metastases in murine breast cancer model [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 P1-06-04.

A Novel In Situ Dendritic Cell Vaccine Triggered by Rose Bengal Enhances Adaptive Antitumour Immunity.

Dendritic cell- (DC-) based vaccination has emerged as a promising antitumour immunotherapy. However, overcoming immune tolerance and immunosuppression in the tumour microenvironment (TME) is still a great challenge. Recent studies have shown that Rose Bengal (RB) can effectively induce immunogenic cell death (ICD) in cancer cells, presenting whole tumour antigens for DC processing and presentation. However, the synergistic antitumour effect of combining intralesional RB with immature DCs (RB-iDCs) remains unclear. In the present study, we investigated whether RB-iDCs have superior antitumour effects compared with either single agent and evaluated the immunological mechanism of RB-iDCs in a murine lung cancer model. The results showed that intralesional RB-iDCs suppressed subcutaneous tumour growth and lung metastasis, which resulted in 100% mouse survival and significantly increased TNF-α production by CD8+ T cells. These effects were closely related to the induction of the expression of distinct ICD hallmarks by RB in both bulk cancer cells and cancer stem cells (CSCs), especially calreticulin (CRT), thus enhancing immune effector cell (i.e., CD4+, CD8+, and memory T cells) infiltration and attenuating the accumulation of immunosuppressive cells (i.e., Tregs, macrophages, and myeloid-derived suppressor cells (MDSCs)) in the TME. This study reveals that the RB-iDC vaccine can synergistically destroy the primary tumour, inhibit distant metastasis, and prevent tumour relapse in a lung cancer mouse model, which provides important preclinical data for the development of a novel combinatorial immunotherapy.

Targeted liposomal doxorubicin/ceramides combinations: The importance to assess the nature of drug interaction beyond bulk tumor cells

One of the major assets of anticancer nanomedicine is the ability to co-deliver drug combinations, as it enables targeting of different cellular populations and/or signaling pathways implicated in tumorigenesis and thus tackling tumor heterogeneity. Moreover, drug resistance can be circumvented, for example, upon co-encapsulation and delivery of doxorubicin and sphingolipids, as ceramides. Herein, the impact of short (C6) and long (C18) alkyl chain length ceramides on the nature of drug interaction, within the scope of combination with doxorubicin, was performed in bulk triple-negative breast cancer (TNBC) cells, as well as on the density of putative cancer stem cells and phenotype, including live single-cell tracking.C6- or C18-ceramide enabled a synergistic drug interaction in all conditions and (bulk) cell lines tested. However, differentiation among these two ceramides was reflected on the migratory potential of cancer cells, particularly significant against the highly motile MDA-MB-231 cells. This effect was supported by the downregulation of the PI3K/Akt pathway enabled by C6-ceramide and in contrast with C18-ceramide. The decrease of the migratory potential enabled by the targeted liposomal combinations is of high relevance in the context of TNBC, due to the underlying metastatic potential.Surprisingly, the nature of the drug interaction assessed at the level of bulk cancer cells revealed to be insufficient to predict whether a drug combination enables a decrease in the percentage of the master regulators of tumor relapse as ALDH+/high putative TNBC cancer stem cells, suggesting, for the first time, that it should be extended further down to this level.

Rational Design of Nanotherapeutics Based on the Five Features Principle for Potent Elimination of Cancer Stem Cells.

Cancer stem cells (CSCs), also known as tumor initiating cells or tumor repopulating cells, which comprise only a small fraction of tumor, have received tremendous attention during the past two decades, as they are considered as the ringleader for initiation and progression of tumors, therapy resistance, metastasis, and recurrence in the clinic. Hence, eradicating CSCs is critical for successful cancer treatment. To that end, various CSC-targeting therapeutic agents have been pursued. However, these CSC-specific drugs are ineffective toward bulk cancer cells. Furthermore, these anti-CSC drugs not only eradicate CSCs but also affect conventional stem cells in normal organs or tissues. By virtue of the enhanced permeability and retention (EPR) effect, nanomaterial drug delivery systems (NDDSs) passively accumulate in tumor tissues, thereby alleviating severe side effects toward normal viscera. NDDSs can be further functionalized with CSC-specific binding molecules to promote targeted drug delivery toward CSCs. Moreover, NDDSs have unique advantages in encapsulating CSC-specific drugs and cytotoxic agents, realizing synchronized killing of CSCs and bulk cancer cells both temporally and spatially. For these reasons, leveraging nanotherapeutic strategies to target CSCs has gained tremendous attention recently.Some ten years ago, we summarized five basic features of efficient nanotherapeutics (the five features principle), which consist of long circulation, tumor accumulation, deep penetration, cellular internalization, and drug release. Based on this design rationale, we constructed several NDDSs, including nanogels with adaptive hydrophobicity, CSC-derived microparticles with tailored softness, and tumor exosome sheathed porous silicon biomimetic nanoparticles, for targeted drug delivery to tumor. To our astonishment, these NDDSs that possess the five basic features achieve decent drug delivery efficiency toward not only bulk tumor cells but more importantly CSCs. Consequently, such nanotherapeutics as-designed based on the five features principle are potent in eradicating CSCs, even with only cytotoxic drugs, for instance, doxorubicin. Furthermore, commercialized nanomedicines, such as Doxil and Abraxane, can be endowed with these five basic features by hyperbaric oxygen therapy and therefore achieve outstanding drug delivery efficiency, potent CSC elimination, and efficient cancer therapy. These studies suggest that intractable CSCs can be tackled with a material-based approach, highlight the critical role of the five features principle in designing effective nanotherapeutics, and pinpoint the significance of drug delivery efficiency in eliminating CSCs and bulk cancer cells.