Various factors in the tumor microenvironment (TME) regulate the expression of PD‐L1 in cancer cells. In TME, mesenchymal stem cells (MSCs) play a crucial role in tumor progression, metastasis, and drug resistance. Emerging evidence suggests that MSCs can modulate the immune‐suppression capacity of TME through the stimulation of PD‐L1 expression in various cancers; nonetheless, their role in the induction of PD‐L1 in breast cancer remained elusive. Here, we assessed the potential of MSCs in the stimulation of PD‐L1 expression in a low PD‐L1 breast cancer cell line and explored its associated cytokine. We assessed the expression of MSCs‐related genes and their correlation with PD‐L1 across 1826 breast cancer patients from the METABRIC cohort. After culturing an ER+/differentiated/low PD‐L1 breast cancer cells with MSCs conditioned‐medium (MSC‐CM) in a microfluidic device, a variety of in‐vitro assays was carried out to determine the role of MSC‐CM in breast cancer cells' phenotype plasticity, invasion, and its effects on induction of PD‐L1 expression. In‐silico analysis showed a positive association between MSCs‐related genes and PD‐L1 expression in various types of breast cancer. Through functional assays, we revealed that MSC‐CM not only prompts a phenotype switch but also stimulates PD‐L1 expression at the protein level through secretion of various cytokines, especially CCL5. Treatment of MSCs with cytokine inhibitor pirfenidone showed a significant reduction in the secretion of CCL5 and consequently, expression of PD‐L1 in breast cancer cells. We concluded that MSCs‐derived CCL5 may act as a PD‐L1 stimulator in breast cancer.

The aim of this study was to assess the effects of pirfenidone (PFD) on promoting epithelial–mesenchymal-transition (EMT) and stemness features in breast carcinoma cells through targeting cancer-associated-fibroblasts (CAFs). Using The Cancer Genome Atlas (TCGA) database, we analyzed the association between stromal index, EMT, and stemness-related genes across 1084 breast cancer patients, identifying positive correlation between YAP1, EMT, and stemness genes in samples with a high-stromal index. We monitored carcinoma cell invasion and spheroid formation co-cultured with CAFs in a 3D microfluidic device, followed by exposing carcinoma cells, spheroids, and CAFs with PFD. We depicted a positive association between the high-stromal index and the expression of EMT and stemness genes. High YAP1 expression in samples correlated with more advanced EMT status and stromal index. Additionally, we found that CAFs promoted spheroid formation and induced the expression of YAP1, VIM, and CD44 in spheroids. Treatment with PFD reduced carcinoma cell migration and decreased the expression of these genes at the protein level. The cytokine profiling showed significant depletion of various EMT- and stemness-regulated cytokines, particularly IL8, CCL17, and TNF-beta. These data highlight the potential application of PFD on inhibiting EMT and stemness in carcinoma cells through the targeting of critical cytokines.

Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), are believed to play key roles in facilitating the metastatic cascade. Metastatic lesions often exhibit a similar epithelial-like state to that of the primary tumour, in particular, by forming carcinoma cell clusters via E-cadherin-mediated junctional complexes. However, the factors enabling mesenchymal-like micrometastatic cells to resume growth and reacquire an epithelial phenotype in the target organ microenvironment remain elusive. In this study, we developed a workflow using image-based cell profiling and machine learning to examine morphological, contextual and molecular states of individual breast carcinoma cells (MDA-MB-231). MDA-MB-231 heterogeneous response to the host organ microenvironment was modelled by substrates with controllable stiffness varying from 0.2kPa (soft tissues) to 64kPa (bone tissues). We identified 3 distinct morphological cell types (morphs) varying from compact round-shaped to flattened irregular-shaped cells with lamellipodia, predominantly populating 2-kPa and >16kPa substrates, respectively. These observations were accompanied by significant changes in E-cadherin and vimentin expression. Furthermore, we demonstrate that the bone-mimicking substrate (64kPa) induced multicellular cluster formation accompanied by E-cadherin cell surface localisation. MDA-MB-231 cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers, and cluster formation on bone-mimicking substrate. Our results suggest that the stiffest microenvironment can induce MET.

Background. A growing number of hepatocellular carcinoma (HCC), and recurrence frequency recently have drawn researchers’ attention to alternative approaches. The concept of differentiation therapies (DT) relies on inducing differentiation in HCC cells in order to inhibit recurrence and metastasis. Hepatocyte nuclear factor 4 alpha (HNF4α) is the key hepatogenesis transcription factor and its upregulation may decrease the invasiveness of cancerous cells by suppressing epithelial-mesenchymal transition (EMT). This study aimed to evaluate the effect of conjugated linoleic acid (CLA) treatment, natural ligand of HNF4α, on the proliferation, migration, and invasion capacities of HCC cells in vitro. Materials and Method. Sk-Hep-1 and Hep-3B cells were treated with different doses of CLA or BIM5078 [1-(2-chloro-5-nitrobenzenesulfonyl)−2-methylbenzimidazole], an HNF4α antagonist. The expression levels of HNF4a and EMT related genes were evaluated and associated to hepatocytic functionalities, migration, and colony formation capacities, as well as to viability and proliferation rate of HCC cells. Results. In both HCC lines, CLA treatment induced HNF4α expression in parallel to significantly decreased EMT marker levels, migration, colony formation capacity, and proliferation rate, whereas BIM5078 treatment resulted in the opposite effects. Moreover, CLA supplementation also upregulated ALB, ZO1, and HNF4α proteins as well as glycogen storage capacity in the treated HCC cells. Conclusion. CLA treatment can induce a remarkable hepatocytic differentiation in HCC cells and attenuates cancerous features. This could be as a result of HNF4a induction and EMT inhibition.

Purpose: Breast cancer is the leading cause of cancer-related death among women worldwide. Conventional chemotherapy is considered a clinical state of the art treatment; however, resistance or recurrence occurs among a considerable portion of these patients. Besides understanding the genomic alterations pattern of tumor cells and their association with drug resistance or response, the development of a reliable tumor models that reflect the major cellular and molecular features of tumors may aid with screening of candidate drugs and identification of appropriate treatment regimens. Here, we developed a simple and low-cost tumor model of breast cancer to screen library of chemotherapy agents in a pre-clinical setting. Methods: we generated and cultured ex-vivo 3D culture of patient-derived tumor spheroids from both pre-treated primary and metastatic tumors using a partial digestion approach in a microfluidic device. We assessed chemotherapy response of the seven patient-derived breast tumor spheroids and expanded evaluation of drug sensitivity through molecular analysis of a small panel of genes. Results: We observed various chemotherapy responses across primary and metastasis tumor samples. Interestingly, we demonstrated response to paclitaxel and doxorubicin and resistance to cisplatin in 2/3 metastatic tumor samples while most of the primary tumor were responsive to chemotherapy. Additionally, the expression of PIK3CA and loss of PTEN were associated to treatment resistance. Conclusion: Our study suggests potential application of microfluidic-based cell culture technology coupled with patient derived tumor spheroids in prediction of treatment response in a personalized manner.

Summary: Carcinoma of Unknown Primary (CUP) is a heterogeneous and metastatic disease where the primary site of origin is undetectable. Currently, chemotherapy is an only state-of-art treatment option for the CUP patient. Employing molecular profiling of the tumour, particularly mutation detection, offers a new treatment for CUP in a personalized fashion. Here, we analyzed mutation and copy number alterations profile of 1,709 CUP samples deposited in GENIE cohort and explored potential druggable mutations. We identified 52 significant mutated genes (SMG) among CUP samples, of which 13 (25%) of SMG were potentially targetable with drugs reproved for the know primary tumour or undergoing clinical trials. The most variants detected were TP53 (43%), KRAS (19.90%), KMT2D (12.60%), and CDKN2A (10.30%). Additionally, the presence of similar variants of TERT promoter in CUP compared to NSCLC samples suggests these mutations may serve as a diagnostic marker for identifying the primary tumour in CUP. Taken together, analyzing mutation profiling of the CUP tumours may open a new way of identifying druggable targets and consequently administrating appropriate treatment in a personalized manner.