Transcriptomic intratumor heterogeneity of breast cancer patient-derived organoids may reflect the unique biological features of the tumor of origin.

BACKGROUND: The intratumor heterogeneity (ITH) of cancer cells plays an important role in breast cancer resistance and recurrence. To develop better therapeutic strategies, it is necessary to understand the molecular mechanisms underlying ITH and their functional significance. Patient-derived organoids (PDOs) have recently been utilized in cancer research. They can also be used to study ITH as cancer cell diversity is thought to be maintained within the organoid line. However, no reports investigated intratumor transcriptomic heterogeneity in organoids derived from patients with breast cancer. This study aimed to investigate transcriptomic ITH in breast cancer PDOs. METHODS: We established PDO lines from ten patients with breast cancer and performed single-cell transcriptomic analysis. First, we clustered cancer cells for each PDO using the Seurat package. Then, we defined and compared the cluster-specific gene signature (ClustGS) corresponding to each cell cluster in each PDO. RESULTS: Cancer cells were clustered into 3-6 cell populations with distinct cellular states in each PDO line. We identified 38 clusters with ClustGS in 10 PDO lines and used Jaccard similarity index to compare the similarity of these signatures. We found that 29 signatures could be categorized into 7 shared meta-ClustGSs, such as those related to the cell cycle or epithelial-mesenchymal transition, and 9 signatures were unique to single PDO lines. These unique cell populations appeared to represent the characteristics of the original tumors derived from patients. CONCLUSIONS: We confirmed the existence of transcriptomic ITH in breast cancer PDOs. Some cellular states were commonly observed in multiple PDOs, whereas others were specific to single PDO lines. The combination of these shared and unique cellular states formed the ITH of each PDO.

Signaling adaptor protein Crk is involved in malignant feature of pancreatic cancer associated with phosphorylation of c-Met.

Signaling adaptor protein Crk has been shown to play an important role in various human cancers. Crk links tyrosine kinases and guanine nucleotide exchange factors (GEFs) such as C3G and Dock180 to activate small G-proteins Rap and Rac, respectively. In pancreatic cancer, various molecular targeted therapies have provided no significant therapeutic benefit for the patients so far due to constitutive activation of KRAS by frequent KRAS mutation. Therefore, the establishment of novel molecular targeted therapy in KRAS-independent manner is required. Here, we investigated a potential of Crk as a therapeutic target in pancreatic cancer. Immunohistochemistry on human pancreatic cancer specimens revealed that the patients with high expression of Crk had a worse prognosis than those with low expression. We established Crk-knockdown pancreatic cancer cells by siRNA using PANC-1, AsPC-1, and MIA PaCa-2 cells, which showed decreased cell proliferation, invasion, and adhesion. In Crk-knockdown pancreatic cancer cells, the decrease of c-Met phosphorylation was observed. In the orthotopic xenograft model, Crk depletion prolonged survival of mice significantly. Thus, signaling adaptor protein Crk is involved in malignant potential of pancreatic cancer associated with decrease of c-Met phosphorylation, and Crk can be considered to be a potential therapeutic molecular target.

Novel rapid immunohistochemistry using an alternating current electric field identifies Rac and Cdc42 activation in human colon cancer FFPE tissues.

It is important to determine the activation status of Rac and Cdc42 in cancer tissues for the prediction of metastasis and patient prognosis. However, it has been impossible to detect their spatial activation on formalin-fixed paraffin embedded (FFPE) surgical specimens thus far. Here, we established a novel detection technique for activated Rac/Cdc42 in human colon cancer FFPE tissues by using a p21-activated kinase (PAK)-Rac binding domain (RBD) detection probe fused with glutathione S-transferase (GST), designated GST-PAK-RBD, and novel rapid-immunohistochemistry (R-IHC) systems using noncontact alterating-current electric field mixing, although there is a technical limitation in that it may not distinguish between Rac members and Cdc42. In 50 cases of colon cancer, various activation patterns of Rac/Cdc42 were observed, which were designated plasma membrane, cytoplasm, mixed pattern, and polarized distribution. The activity was striking in the invasive fronts of tumors and significantly correlated with tumor invasion properties evaluated by TNM classification. Of note, in tissue microarray (TMA) samples, 29 of 33 cases demonstrated higher Rac1/Cdc42 activity in the tumor area than the corresponding normal mucosa. In addition, positive correlations were detected between Rac/Cdc42 activity and clinicopathological factors such as venous and lymphatic vessel invasion. These results suggest that understanding Rac and Cdc42 activations in cancer tissues would be valuable as an option for molecular therapy as personalized medicine.

Involvement of BMP and Wnt Signals Leadingto Epithelial-Mesenchymal Transition in Colon Adenocarcinoma with Heterotopic Ossification.

Here we present the case of a 73-year-old male with rectal adenocarcinoma with heterotopic ossification (HO). Cancer-associated HO in the digestive system is rare. Thus, the precise mechanism and clinicopathological significance of HO have not yet been defined. To clarify the molecular mechanisms of HO, we analyzed the expression levels of signaling molecules related to epithelial-mesenchymal transition (EMT) that lead to ossification in the tumor cells discriminating the ossified area (HO-area) and non-ossified area (non-HO area). Expression levels of BMP4 were elevated in both areas, whereas BMP2 was specifically increased in the HO-area by qPCR. EMT-related molecules such as Snail and Slug were especially higher in the HO-area. By immunohistochemistry, the expression of Smad4, nuclear staining of ß-catenin, and the phosphorylated form of GSK-3ß were detectable in both areas, and GSK-3ß was highly phosphorylated in the HO-area. The tumor growth rate was extremely high, with the Ki-67 labeling index at 90%. In the HO-area, osteoblasts with alkaline phosphatase expression were distributed surrounding the tumor cells. This is the first demonstration of the involvement of EMT in HO of colon cancer through BMP/SMAD and WNT/ß-catenin signaling pathways, which are especially prominent in the HO-area leading to the osteogenic property.

Rapid reprogramming of tumour cells into cancer stem cells on double-network hydrogels.

Cancer recurrence can arise owing to rare circulating cancer stem cells (CSCs) that are resistant to chemotherapies and radiotherapies. Here, we show that a double-network hydrogel can rapidly reprogramme differentiated cancer cells into CSCs. Spheroids expressing elevated levels of the stemness genes Sox2, Oct3/4 and Nanog formed within 24 h of seeding the gel with cells from any of six human cancer cell lines or with brain cancer cells resected from patients with glioblastoma. Human brain cancer cells cultured on the double-network hydrogel and intracranially injected in immunodeficient mice led to higher tumorigenicity than brain cancer cells cultured on single-network gels. We also show that the double-network gel induced the phosphorylation of tyrosine kinases, that gel-induced CSCs from primary brain cancer cells were eradicated by an inhibitor of the platelet-derived growth factor receptor, and that calcium channel receptors and the protein osteopontin were essential for the regulation of gel-mediated induction of stemness in brain cancer cells.