Regulatory miRNAs in cancer cell recovery from therapy exposure and its implications as a novel therapeutic strategy for preventing disease recurrence.

The use of small-molecule kinase inhibitors is a promising therapeutic strategy for the management of breast cancer. Positive responses to these agents, however, are often transient, and acquired resistance arises in the course of weeks to months. Recent studies have demonstrated that phenotypic plasticity in cancer cell populations can provide adaptive resistance to small-molecule kinase inhibitors, whereby cancer cells transition to drug-tolerant phenotypic states, reliant on compensatory survival and proliferative signaling pathways. Drug combinations and sequences can prevent this adaptive resistance to targeted therapy, however, nominating effective drug pairs is challenging. In this study, we seek to identify such drug combinations through first identifying single agent therapeutics that reduce phenotypic heterogeneity and enrich distinct cell-states with common pathway reliance. To do so, we focus on phenotypic heterogeneity in tumor-cell lineage-state; using immunofluorescent staining against markers of the luminal, basal, and mesenchymal lineages, we combine high-throughput drug screening with high-content imaging and pursue small-molecule inhibitors that reduce phenotypic heterogeneity and promote the accumulation of particular lineage-states in residual cell populations. We observe pronounced lineage-state heterogeneity in Triple-Negative tumors and Basal-Like breast cancer cell lines, and find that the lineage-state distributions are greatly influenced by numerous therapeutics. MEK and PI3K/mTOR inhibitors in particular induce robust time- and dose-dependent alterations is lineage-state distribution in residual cell populations, selecting for a cell population enriched in, or depleted of a basal lineage-state, respectively. Through gene expression profiling and master-regulator analysis of active transcriptional states in the residual cell populations, we are able to identify compensatory-signaling pathways. We demonstrate that combining MEK and PI3K/mTOR inhibitors with agents targeting these compensatory pathways induces synergistic antiproliferative effects. Citation Format: Tyler Risom, Ellen Langer, Juha Rantala, Mariano Alvarez, Katie Johnson-Camacho, Carl Pelz, Nicholas Wang, Paul Spellman, Andrea Califano, Joe Gray, Rosalie Sears. Overcoming phenotypic heterogeneity and plasticity in basal-like breast cancer through targeting adaptive pathway use. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B52.

Introduction: Circulating tumor cells (CTCs) is a promising tool for disease monitoring and for better-targeted therapies for patients with disseminating tumors. However, isolation of CTCs is challenging due to their scarcity, variation in size, morphology, and expression profile. The lack of a universal marker impairs reliable detection and characterization of CTCs. We are using acoustophoresis, ultrasound standing wave radiation forces to enable label free separation of cancer cells from white blood cells (WBC). The cell separation is based on intrinsic cells properties such as size, density and compressibility. Materials and Methods: The acoustic setup is a glass/silicon microchip connected to a pressure driven system. The system is temperature controlled and has a pre-alignment step for optimal separation performance. The separation system can process samples up to 10 mL, with a processing speed of 1 mL in 13 minutes without compromising the cell separation capacity. If required the separation step can be complemented with a subsequent concentration channel, which allows at least 20x concentration of the sample, by extracting the cells in a smaller liquid volume compared to the input sample. The prostate cancer cell line DU145 and the breast cancer cell line MCF7 spiked in red blood cell (RBC) lysed blood from healthy donors, are used to evaluate the model system performance for cancer cell separation. Results and Discussion: The acoustic separation model system is flexible and can be amended to suit different requirements and conditions. The cancer cell recovery after acoustic separation of blood samples spiked with 50 DU145 cells per mL is approximately 80% with a WBC contamination level of less than 0.3%. At this performance level there is a 100x cancer cell enrichment in the collected cancer cell fraction compared to input. However, by changing parameters such as the voltage setting and sample flow rate it is possible to regulate the cancer cell recovery and WBC contamination levels after acoustophoresis. Cancer cell recovery well above 90% is possible with slightly higher WBC contamination levels. If high cancer cell purity is of importance, a 1000x cancer cell enrichment can be achieved at the cost of slightly lower cancer cell recovery. RBC lysing is required before acoustic separation, due to cell crowding in the separation channel when the cell concentration is too high. We have identified eosinophils as the major contaminant in our enriched cancer cell fraction. They constitute 85% of the contaminating WBCs. The eosinophils have similar acoustic properties as the smaller cancer cells, and can therefore not be completely isolated from the cancer cells by acoustophoresis. This work demonstrates the flexibility with acoustophoresis that allows for integration of three previously established units: pre-alignment, sorting and concentrating, onto the same chip. It also serves as a proof of concept for in line rare cell label free sorting and isolation. Citation Format: Cecilia Magnusson, Andreas Lenshof, Per Augustsson, Maria Antfolk, Thomas Laurell, Hans Lilja. Label-free separation and concentration of cancer cells by acoustophoresis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1697.

The 2019 SLAS Technology Ten: Translating Life Sciences Innovation.

Introduction: The main cause of death for cancer patients is the development of metastasis. These arise mainly due to irresponsiveness of cancer cells to the administered therapy, which then fails to eliminate all cancer cells present in the patient. To overcome this problem, it is essential to understand which mechanisms are involved in the lack of treatment response. We are investigating how the tumour microenvironment (TME) affects the response of cancer cells to chemotherapy (CTX) and how it can be modulated to improve the outcome of patients to therapy. Materials and Methods: Co-culture of chemotherapy-treated breast cancer cell lines with primary fibroblasts isolated from breast cancer patients was performed to investigate if fibroblasts affect the response of tumour cells to commonly used agents, such as epirubicin and paclitaxel. Recovery of cells was assessed using colony formation assays (CFA) and cell cycle profiling by EdU and the FUCCI system. To further explore the complex crosstalk between cancer cells and fibroblasts in the context of CTX, gene expression analysis of both cell types was done using next generation sequencing. Validation and evaluation of the biological impact of the identified pathways was done using RT-qPCR, western-blot and perturbation experiments. Lastly, publicly available datasets for breast cancer were used to investigate the clinical relevance of our findings. Results and Discussion: We show that cancer cells utilize paracrine signalling with stromal fibroblasts to drive their recovery after treatment withdrawal. Cell cycle analysis and RNA-sequencing revealed an increase in cell cycle re-entry of CTX-treated cancer cells in co-culture with fibroblasts. In addition, we have successfully shown that treated cancer cells upregulate an important secreted factor that modulates fibroblasts into a pro-tumorigenic state. Moreover, analysis of human breast carcinomas supported the proposed role of the identified factor since its expression is inversely correlated with recurrence free survival (RFS). Moreover, expression of the gene signature identified in stromal fibroblasts in co-culture with CTX-treated cancer cells was equally associated with higher recurrence rates and a worse outcome in breast cancer patients. Conclusion: CTX-induced secretory profile of cancer cells orchestrates the reprogramming of stromal fibroblasts into a pro-tumorigenic state, which drives the expansion of cancer cells. Our study unravels a novel paracrine communication between cancer cells and stromal fibroblasts that ultimately results in the escape of malignant cells to treatment, highlighting the importance of the TME in drug response. Targeting of this axis could potentially improve the outcome of breast cancer patients to CTX treatment. Citation Format: Ana Maia, Zuguang Gu, André Koch, Mireia Berdiel-Acer, Rainer Will, Matthias Schlesner, Stefan Wiemann. Paracrine signalling with stromal fibroblasts drives recovery of cancer cells after chemotherapy treatment [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-36.

Circulating tumor cells (CTCs) are widely known as useful biomarkers in the liquid biopsies of cancer patients. Recently, in addition to counting the number of CTCs, genetic analysis of CTCs provides critical insights into cancer metastases and add detailed clinical information that enhances patient care. It is widely known that CTCs are genetically heterogeneous, the molecular characterization of CTCs should be performed at the single-cell level. In fact, several studies have identified inter- and intra-patient heterogeneity in the mutational status of CTCs in metastatic breast and lung cancers. These studies also reported that CTCs related to drug resistance are novel therapeutic targets. Thus, genetic analysis of single CTCs has the potential to be widely applicable in personalized medicine and drug discovery. However, CTCs are extremely rare cells that only 1-100 cells are contained in 1 ml of blood, so the recovery of such extremely rare CTCs from the peripheral blood is technically challenging. So far, we have developed a CTC recovery system using a microcavity array (MCA), which demonstrates highly efficient recovery of cancer cells based on differences in cell size and deformability. Furthermore, we have proposed a novel cell manipulation method through the visualization of single cells through hydrogel encapsulation for subsequent genetic analysis of single CTCs. Subsequently, recovered CTCs can be subjected to a hydrogel-encapsulation for the following genetic analysis of single cells. Clinical studies indicated that the genetic analysis of CTCs from lung, pancreatic and gastric cancer patients was successfully achieved. However, this hydrogel encapsulation has limitations in throughput. In this study, we demonstrate a high-throughput single-cell analysis for CTCs using a multiple single-cell encapsulation system with a digital micromirror device (DMD). The novel multiple single-cell encapsulation systemwas composed of two optical systems: the wide-field fluorescence imaging system and the photopolymerization system equipped with DMD. The wide-field fluorescence imaging system using a CMOS sensor was designed to visualize 2D fluorescence imaging of the entire MCA (6.0 × 6.0 mm2). We evaluated the sensitivity of fluorescence detection of the wide-field fluorescence imaging system using the calibration slide. The limit of detection of the wide-field fluorescence imaging system was 2.1 × 102 molecules-Cy3/μm2, which is comparable to conventional fluorescence microscopy (1.0 × 102 molecules-Cy3/μm2). Generally, the expression of cellular marker protein, such as cytokeratin, was estimated to be 1.3 × 103 molecules/μm2. Therefore, our proposed system had enough sensitivity for the detection of antibody-stained CTCs. Furthermore, this wide-field fluorescence imaging system for CTC detection enables the rapid visualization of all stained cells within the field of the MCA via one-shot imaging. This rapid detection method also allows for the rapid completion of single-CTC manipulation. The photopolymerization system for single-cell encapsulation was controlled by the DMD. The curing light (λmax = 365 nm) modulated by the DMD was projected onto an MCA for hydrogel generation, and single-cell isolation by hydrogel-photopolymerization was examined at the optimized conditions. After recovery of cancer cells from whole blood onto MCA and staining by the fluorescent-labeled antibodies, cancer cells were identified by the wide-field imaging system. Then light irradiation (3 sec) was performed by the photopolymerization system. To minimize the area of the hydrogel reserved for a single cell, we newly designed the convex-type hydrogel. In the spike-in experiment, single-cell isolation rates were 97.6% (41/42) for NCI-H1975 cells (non-small cell lung cancer cell) and 94.4% (51/54) for NCI-N87 (gastric cancer cell) that were comparable with our previous study using fluorescence microscopy. With this proposed system, more than 50 single cells were encapsulated simultaneously within 1 min and isolated with an efficiency. Furthermore, single cells were successfully isolated from adjacent cells on the same microcavity without any contamination. Single cancer cell was partially encapsulated on the hydrogel, and the partial encapsulation allows us to subject them to whole genome amplification (WGA). Light irradiation and photopolymerizaion did not affect the quality of the amplified WGA products for single-cell genomic analyses. We are currently investigating single cell genetic analysis of metastatic cancer patients, and this developed multiple single-cell encapsulation system will improve the high throughput single-cell genetic analysis of CTCs in clinical samples. Our system provides an attractive application for other targets such as adherent cells, tissue samples, and microorganisms, in addition to widespread use in the isolation of CTCs for liquid biopsy.

Introduction: One of the major burdens in the treatment of epithelial ovarian cancer (EOC) is the high percentage of recurrence characterized by chemoresistance. The biology underlying the tumor’s high capacity for recurrence has not been elucidated. Tumors are made of a heterogeneous cell population consisting of both cancer and non-cancer cells. New data suggest that even the cancer cell population is heterogeneous and contains a small subset of cells, the cancer stem cells (CSC), which constitute a reservoir that can self-renew and therefore maintain the tumor. CSC can divide and expand their pool and also differentiate into non-CSC, which constitute the bulk of the tumor. Contrary to CSC, the non-CSC are rapidly dividing and therefore sensitive to therapies, which target highly proliferative cells. In the present study we identified and characterized the CSC of EOC. Methods: EOC cells were isolated from malignant ovarian cancer ascities and solid tumors (n=80). Marker expression was determined using Flow Cytometry, Western Blots and Immunocytochemistry. Xenograft nude mice model was used for tumor growth by injecting cancer cells either s.c. or i.p. Isolation of CD44+ population was done using FACS. Cancer cells were maintained in culture as previously described {Kamsteeg, 2003 #358} Results: CSC were identified in EOC cells isolated form ascites and solid tumors with the following characteristics: 1) cellular markers: CD44+, MyD88+, constitutive NFκB activity and cytokine and chemokine production, high capacity for repair, chemoresistance to conventional chemotherapies, resistance to TNFα-mediated apoptosis, capacity to form spheroids in suspension, and unique microRNA phenotype; 2) tumor formation in animals: 100% CD44+ cells formed tumors which contained 10% CD44+ and 90% CD44-negative cells. Re-injection of isolated CD44+ cells from previous engraftment was able to again recapitulate the original tumor phenotype. Isolation and in vitro treatment of CD44+ cells from fresh samples showed resistance to carboplatin and paclitaxel. In contrast, the sorted CD44-negative cell population from the same sample/patient was chemosensitive. Conclusion: Present chemotherapy modalities eliminate the bulk of the tumor but it leaves a core of cancer cells with high capacity for repair and renewal. CSC corresponds to the core of malignant cells that promotes recurrence and chemoresistance. Identification of these cells represents the first step in the development of therapeutic modalities that can eliminate not only the bulk of the tumor but its source. Prevention of recurrence will be achieved only with therapies targeting this cell population.

Background: Isolation of rare circulating tumor cells (CTCs) has emerged as an increasingly important non-invasive tool in the pursuit for better-targeted therapies for patients with disseminating tumors. In the present study we aimed to exploit ultrasound standing wave technology in a microchip setting, (acoustophoresis), to separate spiked epithelial prostate tumor cells in blood samples. Acoustophoresis is a non-contact and label free separation technique, which enriches cancer cells independent of EpCAM and other antigen expressions. Methods: The separation device is a silicon/glass microfluidic channel with trifurcation inlets and outlets. A piezoceramic actuates the separation channel at λ/2 resonance, creating a force on suspended cells directed towards the center of the channel. To achieve sufficiently high selectivity, we have developed a temperature stabilized acoustophoresis microchannel into which we have incorporated a novel cell pre-alignment channel, which orders the cells before entering an acoustophoresis cell separation channel. The pre-alignment provides vastly improved separation performance as compared to conventional acoustophoresis. Prostate cancer cell lines (DU145, LNCaP and PC3) spiked in red blood cell lysed blood were processed in the acoustophoresis chip. Results: To demonstrate the resolution of the device for separation based on intrinsic acoustophysical properties and mobility, a population of polystyrene microspheres of non-overlapping sizes 5 and 7 μm in diameter was dissected into two fractions that each displayed 99% purity. Bead data allow for calibration of the system to achieve stable and repeatable cell separation. Three different prostate cancer cell lines were used as a model system to address the expected diversity of CTCs in the peripheral blood of metastatic prostate cancer patients. At present the acoustic separation with pre-alignment allows cancer cell recovery up to 97% (±1.0), with a sample cancer cell purity of 98% (±0.25) for PFA fixed cells. Higher recovery rates are achievable but at the expense of lower sample purities. For non-fixed cells recovery was 84% (±3.2) when the cancer cell purity in the collected sample was 93% (±2.7). The acoustic separation method is innoxious and does not affect cell viability, cell proliferation, Androgen receptor function or PSA secretion. It is therefore an interesting alternative to current available techniques that require cell fixation. Conclusion: Acoustophoresis with cell pre-alignment allows for recovery of cancer cells independent of antigen expression. Enrichment of cancer cells in blood with acoustophoresis is a promising method for future non-invasive molecular interrogation of metastatic cancers. Citation Format: Cecilia Magnusson, Per Augustsson, Thomas Laurell, Hans Lilja. Label-free prostate cancer cell enrichment in blood using microfluidic acoustic wave technology [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C39.

Enumeration of circulating tumor cells (CTCs) predicts overall survival and treatment response in metastatic cancer, but as many commercialized assays isolate CTCs positive for epithelial cell markers alone, CTCs with little or no epithelial cell adhesion molecule (EpCAM) expression stay undetected. Therefore, CTC enrichment and isolation by label-free methods based on biophysical rather than biochemical properties could provide a more representative spectrum of CTCs. Here, we report on a clinical-scale automated acoustic microfluidic platform processing 5 mL of erythrocyte-depleted paraformaldehyde (PFA)-fixed blood (diluted 1:2) at a flow rate of 75 μL/min, recovering 43/50 (86 ± 2.3%) breast cancer cell line cells (MCF7), with 0.11% cancer cell purity and 162-fold enrichment in close to 2 h based on intrinsic biophysical cell properties. Adjustments of the voltage settings aimed at higher cancer cell purity in the central outlet provided 0.72% cancer cell purity and 1445-fold enrichment that resulted in 62 ± 8.7% cancer cell recovery. Similar rates of cancer-cell recovery, cancer-cell purity, and fold-enrichment were seen with both prostate cancer (DU145, PC3) and breast cancer (MCF7) cell line cells. We identified eosinophil granulocytes as the predominant white blood cell (WBC) contaminant (85%) in the enriched cancer-cell fraction. Processing of viable cancer cells in erythrocyte-depleted blood provided slightly reduced results as to fixed cells (77% cancer cells in the enriched cancer cell fraction, with 0.2% WBC contamination). We demonstrate feasibility of enriching either PFA-fixed or viable cancer cells with a clinical-scale acoustic microfluidic platform that can be adjusted to meet requirements for either high cancer-cell recovery or higher purity and can process 5 mL blood samples in close to 2 h.

There is intense search for disease biomarkers that can reliably detect lethal prostate cancer (PCa) at early stage, monitor response to treatment, detect disease recurrence, and predict survival. Enrichment, enumeration, and transcriptional profiling of rare circulating tumor cells (CTCs) can predict survival of patients with disseminated tumors, and may provide clinical benefit in predicting treatment responses of targeted therapies. We exploit ultrasound radiation forces to enable label free diversion of cancer cells from nucleated blood cells in a temperature-controlled, microfluidic continuous flow system (acoustophoresis). A third generation, standing-wave ultrasound microchip (silicon/glass) incorporated a cell pre-alignment channel, and a microchannel with a trifurcated inlet and outlet. The system is driven by compressed air (500 mBar) to mitigate and minimize continuous flow-fluctuations and provide more reproducible and reliable discrimination of tumor cells from nucleated blood cells compared to previous syringe driven setups, and allow us to process clinically relevant sample volumes of blood (5-10 mL). We fixed, spiked, and processed 50 or more PCa cells per mL in undiluted white blood cells (WBC) in the acoustic microchip. The separation system is very flexible and the ratio between cancer cell recovery and cell contamination can be tuned by changing the sample flow rates or the acoustic energy. A consistent and reproducible recovery of cancer cells above 90% with less than 0.4% contamination of WBC was provided using flow rates of 100 l/min. We also found that about 90% of the contaminating WBCs were granulocytes, whereas the vast majority of lymphocytes were reliably diverted. Our results were consistent using different PCa cell lines (DU145, LNCaP, PC3) along with the breast cancer cell line (MCF7). The isolation of cancer cells by acoustophoresis is a label free method that consistently enables high recovery of cancer cells independent of the expression of surface antigens. This allows for separation of EpCAM negative populations of CTCs, mesenchymal cells along with cancer stem cells, which can be identified with subsequent qRT-PCR analysis. In acoustophoresis, cells are separated according to size, density and compressibility (dV/dp), where size is the major factor. Using non-fixed samples, we can separate viable from dead cells, since dead cells display significantly different acoustic properties to their live counterpart. Acoustophoresis is a gentle separation method and does not affect cell viability, allowing post isolation ex vivo cultivation of CTCs. This allows functional assays to test CTCs ability to form metastasis. It is therefore a promising method for future non-invasive molecular interrogation of metastatic cancers and the characterization of CTCs will relate CTCs to disease outcome and predict for sensitivity to treatment and match patient to drug response. Citation Format: Cecilia Magnusson, Per Augustsson, Benedikta Haflidadottir, Andreas Lenshof, Yvonne Ceder, Thomas Laurell, Hans Lilja. Label free prostate cancer cell isolation from blood by acoustic standing wave technology - acoustophoresis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3077. doi:10.1158/1538-7445.AM2014-3077

Fast and Label-Free Isolation of Circulating Tumor Cells from Blood: From a Research Microfluidic Platform to an Automated Fluidic Instrument, VTX-1 Liquid Biopsy System.

Cancer is an evolutionary disease driven by molecular alterations in cancer cells and concomitant tumor microenvironments. Unfortunately, cancer cells often evolve into aggressive tumors that ultimately evade treatment. Thus, in order to improve clinical outcomes, there is an urgent need to define mechanisms by which cancer cells evolve. Recent multi-region sequencing studies, including our own, have inferred phylogenetic evolution across distinct lesions collected from patients. While these studies analyzed the extent of intratumoral heterogeneity across tumor types, the molecular determinants of cancer evolution remain unclear. For example, it is challenging to precisely quantify the adaptive dynamics of a cancer cell lineage before, during, and after a selective pressure. Moreover, tumor microenvironments tend to be spatially and temporally heterogeneous, which complicates evolutionary analyses of cancer cells within these microenvironments. To address these challenges in resolving the evolutionary dynamics of cancer cells, our current work combined bioreactor culturing, longitudinal sampling, single cell sequencing, and metabolomics. Cancer cell lines were selected to represent diverse hematological and solid tumor types, including leukemia, lymphoma, myeloma, colorectal, retinoblastoma, and lung cancers. For four weeks, we consistently maintained multiple environmental parameters of each cancer cell population including temperature, pH, oxygen, and agitation. All cancer cell populations were initiated with the same seeding density in identical media. Since we aimed to quantify growth patterns and to define mechanisms by which cancer cells adapt to nutrient starvation, we allowed each cancer cell population to alter cell density as well as metabolite consumption over the course of the experiment. Every 48 hours, cells and media were collected and preserved to establish a “fossil record” for analysis, and cell density and viability were measured. We found that all cancer cell populations demonstrated exponential growth, plateau and death phases over the course of these experiments, and that each cell line exhibited its own characteristic growth pattern and carrying capacity (range 125 - 250 million cancer cells) despite all cell lines having been grown in the same environmental condition. Moreover, these growth patterns were highly concordant among independently maintained populations. Given our environmental controls, these results suggest that the cancer cell population growth patterns we observed reflected cell-intrinsic features. To explore transcriptional dynamics, we used single cell RNA sequencing to analyze longitudinal samples of the cancer cells. We found that transcriptional subclones emerged over the course of the experiment with altered gene expression profiles, including in genes with functions related to cancer cell metabolism such as biosynthesis, stress responses, and nutrient uptake, indicating putative mechanisms by which the cancer cells were adapting to an increasingly stringent environment. To further define environmental constituents, we analyzed longitudinal media samples that were collected at each timepoint with the cancer cells. Multiple metabolites were consumed within the first seven days of culture, including amino acids, vitamins, and nucleotides. Strikingly, we also observed metabolites that were secreted into the media over the course of the experiment, including nucleotides and signaling molecules. These metabolite patterns were consistent with the concomitant gene expression changes of the cancer cells. Overall, our results showed that the cancer cells were simultaneously adapting to and remodeling their environment rather than solely depleting nutrients. Defining such dynamics, especially in the context of fluctuating environmental conditions, will be essential for mechanistic studies of cancer evolution. Citation Format: Alvin P. Makohon-Moore. Transcriptional and metabolic dynamics of cancer cells under nutrient deprivation. [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 NG08.

Selective Cancer Targeting via Aberrant Behavior of Cancer Cell-associated Glucocorticoid Receptor

Cancer microenvironment is complex and consists of various immune cells. There is evidence for mast cell (MC) infiltration of tumors, but their role thereof is poorly understood. In this study, we explored the effects of mast cell and their mediators on the growth of hematological cancer cells. The affect is demonstrated using RBL-2H3 MCs, and YAC-1, EL4 and L1210 as hematological cancer cell lines. Direct contact with MCs or stimulation by their mediators caused growth inhibition of YAC-1 cells, growth enhancement of EL4 cells and no change in growth of L1210 cells. This effect was confirmed by cancer cell recovery, cell viability, mitochondrial health, and cell cycle analysis. MCs showed mediator release in direct contact with tumor cells. MC mediators' treatment to YAC-1 and EL4 yielded exactly opposite modulations of survival markers, Survivin and COX-2 and apoptosis markers, Caspase-3, Bcl-2, in the two cell lines. Histamine being an important MC mediator, effect of histamine on cell recovery, survival markers and expression of various histamine receptors and their modulation in cancer cells was studied. Again, YAC-1 and EL4 cells showed contrary histamine receptor expression modulation in response to MC mediators. Histamine receptor antagonist co-treatment with MC mediators to the cancer cells suggested a major involvement of H2 and H4 receptor in growth inhibition in YAC-1 cells, and contribution of H1, H2, and H4 receptors in cell growth enhancement in EL4 cells. L1210 showed changes in the histamine receptors' expression but no effect on treatment with receptor antagonists. It can be concluded that anti-cancerous action of MCs or their mediators may include direct growth inhibition, but their role may differ depending on the tumor.

Epithelial-mesenchymal heterogeneity implies that cells within the same tumor can exhibit different phenotypes—epithelial, mesenchymal, or one or more hybrid epithelial-mesenchymal phenotypes. This behavior has been reported across cancer types, both in vitro and in vivo, and implicated in multiple processes associated with metastatic aggressiveness including immune evasion, collective dissemination of tumor cells, and emergence of cancer cell subpopulations with stem cell-like properties. However, the ability of a population of cancer cells to generate, maintain, and propagate this heterogeneity has remained a mystifying feature. Here, we used a computational modeling approach to show that epithelial-mesenchymal heterogeneity can emerge from the noise in the partitioning of biomolecules (such as RNAs and proteins) among daughter cells during the division of a cancer cell. Our model captures the experimentally observed temporal changes in the fractions of different phenotypes in a population of murine prostate cancer cells, and describes the hysteresis in the population-level dynamics of epithelial-mesenchymal plasticity. The model is further able to predict how factors known to promote a hybrid epithelial-mesenchymal phenotype can alter the phenotypic composition of a population. Finally, we used the model to probe the implications of phenotypic heterogeneity and plasticity for different therapeutic regimens and found that co-targeting of epithelial and mesenchymal cells is likely to be the most effective strategy for restricting tumor growth. By connecting the dynamics of an intracellular circuit to the phenotypic composition of a population, our study serves as a first step towards understanding the generation and maintenance of non-genetic heterogeneity in a population of cancer cells, and towards the therapeutic targeting of phenotypic heterogeneity and plasticity in cancer cell populations.

Reducing WBC background in cancer cell separation products by negative acoustic contrast particle immuno-acoustophoresis