Metastatic recurrence in colorectal cancer arises from residual EMP1+ cells.

Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years1. Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5+ stem-like tumour cells2-4, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1high cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.

Extracellular vesicles secreted by triple-negative breast cancer stem cells trigger premetastatic niche remodeling and metastatic growth in the lungs.

Tumor secreted extracellular vesicles (EVs) are potent intercellular signaling platforms. They are responsible for the accommodation of the premetastatic niche (PMN) to support cancer cell engraftment and metastatic growth. However, complex cancer cell composition within the tumor increases also the heterogeneity among cancer secreted EVs subsets, a functional diversity that has been poorly explored. This phenomenon is particularly relevant in highly plastic and heterogenous triple-negative breast cancer (TNBC), in which a significant representation of malignant cancer stem cells (CSCs) is displayed. Herein, we selectively isolated and characterized EVs from CSC or differentiated cancer cells (DCC; EVsCSC and EVsDCC , respectively) from the MDA-MB-231 TNBC cell line. Our results showed that EVsCSC and EVsDCC contain distinct bioactive cargos and therefore elicit a differential effect on stromal cells in the TME. Specifically, EVsDCC activated secretory cancer associated fibroblasts (CAFs), triggering IL-6/IL-8 signaling and sustaining CSC phenotype maintenance. Complementarily, EVsCSC promoted the activation of α-SMA+ myofibroblastic CAFs subpopulations and increased the endothelial remodeling, enhancing the invasive potential of TNBC cells in vitro and in vivo. In addition, solely the EVsCSC mediated signaling prompted the transformation of healthy lungs into receptive niches able to support metastatic growth of breast cancer cells.

Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells.

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

CAFs and Cancer Cells Co-Migration in 3D Spheroid Invasion Assay.

In many solid tumors, collective cell invasion prevails over single-cell dissemination strategies. Collective modes of invasion often display specific front/rear cellular organization, where invasive leader cells arise from cancer cell populations or the tumor stroma. Collective invasion involves coordinated cellular movements which require tight mechanical crosstalk through specific combinations of cell-cell interactions and cell-matrix adhesions. Cancer Associated Fibroblasts (CAFs) have been recently reported to drive the dissemination of epithelial cancer cells through ECM remodeling and direct intercellular contact. However, the cooperation between tumor and stromal cells remains poorly understood. Here we present a simple spheroid invasion assay to assess the role of CAFs in the collective migration of epithelial tumor cells. This method enables the characterization of 3D spheroid invasion patterns through live cell fluorescent labeling combined with spinning disc microscopy. When embedded in extracellular matrix, the invasive strands of spheroids can be tracked and leader/follower organization of CAFs and cancer cells can be quantified.

Combined acetyl-11-keto-ß-boswellic acid and radiation treatment inhibited glioblastoma tumor cells.

Glioblastoma multiforme (GBM) is the most common and most aggressive subtype of malignant gliomas. The current standard of care for newly diagnosed GBM patients involves maximal surgical debulking, followed by radiation therapy and temozolomide chemotherapy. Despite the advances in GBM therapy, its outcome remains poor with a median survival of less than two years. This poor outcome is partly due to the ability of GBM tumors to acquire adaptive resistance to therapy and in particular to radiation. One of the mechanisms contributing to GBM tumor progression and resistance is an aberrant activation of NF-ĸB, a family of inducible transcription factors that play a pivotal role in regulation of many immune, inflammatory and carcinogenic responses. Acetyl-11-keto-ß-boswellic acid (AKBA) is a pentacyclic terpenoid extracted from the gum Ayurvedic therapeutic plant Boswellia serrata. AKBA is anti-inflammatory agent that exhibits potent cytotoxic activities against various types of tumors including GBM. One of the mechanisms underlying AKBA anti-tumor activity is its ability to modulate the NF-ĸB signaling pathway. The present study investigated in vitro and in vivo the effect of combining AKBA with ionizing radiation in the treatment of GBM and assessed AKBA anti-tumor activity and radio-enhancing potential. The effect of AKBA and/or radiation on the survival of cultured glioblastoma cancer cells was evaluated by XTT assay. The mode of interaction of treatments tested was calculated using CalcuSyn software. Inducing of apoptosis following AKBA treatment was evaluated using flow cytometry. The effect of combined treatment on the expression of PARP protein was analysed by Western blot assay. Ectopic (subcutaneous) GBM model in nude mice was used for the evaluation of the effect of combined treatment on tumor growth. Immunohistochemical analysis of formalin-fixed paraffin-embedded tumor sections was used to assess treatment-related changes in Ki-67, CD31, p53, Bcl-2 and NF-ĸB-inhibitor IĸB-α. AKBA treatment was found to inhibit the survival of all four tested cell lines in a dose dependent manner. The combined treatment resulted in a more significant inhibitory effect compared to the effect of treatment with radiation alone. A synergistic effect was detected in some of the tested cell lines. Flow cytometric analysis with Annexin V-FITC/PI double staining of AKBA treated cells indicated induction of apoptosis. AKBA apoptotic activity was also confirmed by PARP cleavage detected by Western blot analysis. The combined treatment suppressed tumor growth in vivo compared to no treatment and each treatment alone. Immunohistochemical analysis showed anti-angiogenic and anti-proliferative activity of AKBA in vivo. It also demonstrated a decrease in p53 nuclear staining and in Bcl-2 staining and an increase in IĸB-α staining following AKBA treatment both alone and in combination with radiotherapy. In this study, we demonstrated that AKBA exerts potent anti-proliferative and apoptotic activity, and significantly inhibits both the survival of glioblastoma cells in vitro and the growth of tumors generated by these cells. Combination of AKBA with radiotherapy was found to inhibit factors which involved in cell death regulation, tumor progression and radioresistence, therefore it may serve as a novel approach for GBM patients.

Modified Citrus Pectin as a Potential Sensitizer for Radiotherapy in Prostate Cancer.

BACKGROUND: Radiotherapy is one of the primary therapies for localized prostatic carcinoma. Therefore, there is an emerging need to sensitize prostatic cancer cells to chemotherapy/radiotherapy. Modified citrus pectin (MCP) is an effective inhibitor of galectin-3 (Gal-3), which is correlated with tumor progression, proliferation, angiogenesis, and apoptosis. PURPOSE: This study was directed to evaluate the efficacy of combining ionizing radiation (IR) with MCP on PCa cells. STUDY DESIGN: Effects of treatments on PCa cells survival were evaluated using XTT assay, flow cytometry, and clonogenic survival assay. Expression of selected proteins was estimated using western blotting. Cell motility, migration, and invasion were determined. Contribution of reactive oxygen species production to treatment effects on cell viability was tested. RESULTS: Radiotherapy combined with MCP reduced viability and enhanced radiosensitivity associated with a decrease in Gal-3, cleavage of the precursor of caspase-3, increased expression of the pro-apoptotic protein Bax, and downregulation of DNA repair pathways, poly-ADP-ribose polymerase, and proliferating cell nuclear antigen. MCP significantly reduced the invasive and migratory potential of PCa cells. Combining sodium pyruvate with MCP and IR mitigated the effect on cell viability. CONCLUSION: Our findings demonstrated that MCP sensitized PCa cells to IR by downregulating anti-apoptotic Gal-3, modulating DNA repair pathways, and increasing ROS production. For the first time the correlation between MCP, radiotherapy, and Gal-3 for prostatic cancer treatment was found. In addition, MCP reduced the metastatic properties of PCa cells. These findings provide MCP as a radiosensitizing agent to enhance IR cytotoxicity, overcome radioresistance, and reduce clinical IR dose.