Proliferation, glucose uptake and ROS in A-5RT3 cells were assessed as previously described
Proliferation, glucose uptake and ROS in A-5RT3 cells were assessed as previously described.47, 48, 49 FACS was performed with the Accuri C6 Flow Cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo (Treestar, Ashland, OR, USA). Energy charge determination Energy charge in A-5RT3 cells was determined as previously described.50 Multiple analyte detection MILLIPLEX MAP Human MMP, Human Angiogenesis/Growth Factor and Human Circulating Cancer Biomarker kits were used for multiplexed immunoassay detection of secreted factors in CM (Merck Millipore, Billerica, MA USA). clinical cutaneous squamous cell carcinoma (SCC) biopsies. We further identified a cluster of driver NRs in CAFs as important modifiers of CAF function with profound influence on cancer cell invasiveness, proliferation, drug resistance, energy metabolism and oxidative stress status. Importantly, guided by the NR profile of CAFs, retinoic acid receptor and androgen receptor antagonists were identified for concurrent therapy with cisplatin, resulting in the inhibition of chemoresistance in recurred SCC:CAF xenografts. Our work demonstrates that treatments targeting both the tumor epithelia and the surrounding CAFs can extend the efficacy of conventional chemotherapy. Introduction The tumor microenvironment consists of a variety of stromal cells and a fibrotic matrix that surround and support malignant epithelia.1, 2 The interactions among the various components of the tumor microenvironment, mediated largely by cytokines and growth factors, are significant. Tumor epithelia can change the nature of the microenvironment, and conversely, the microenvironment can affect how a tumor grows and spreads.3, 4 Furthermore, tumor stroma ROC-325 co-evolution further disrupts tissue organization,5, 6 and the resultant loss of organ homeostasis creates a feed-forward reaction permissive to tumor aggressiveness and malignancy.4, 7 Despite this, many conventional cancer treatments are designed around ROC-325 druggable features of tumor epithelia, ignoring the supportive role of stromal cells. The diversity of patient outcomes from such treatments not only suggests that rapid resistance occurs, but also highlights an incomplete understanding of the tumor microenvironment.8, 9 As the most abundant cell population in the tumor stroma, cancer-associated fibroblasts (CAFs) are a potent source of growth factors, extracellular matrix components, matrix remodeling enzymes, inflammatory cytokines and reactive oxygen species (ROS). Hence, CAFs create a microenvironment that promotes proliferation, invasiveness, oxidative stress, aberrant metabolism, immune evasion and therapy resistance of tumors. Although CAFs have been well characterized by their expression of alpha-smooth muscle actin,10 fibroblast (FIB) activation protein,11 platelet-derived growth factor receptors,12 asporin13 and collagen 111,14 the underlying transcriptional programs enabling the pro-oncogenic functions of CAFs remain poorly understood. Moreover, whereas transcription factor signaling nodes control many cellular behaviors, most transcription factors cannot be directly modulated by chemical drugs, and are considered poor pharmacological ROC-325 targets.15, 16 Nuclear hormone receptors (NRs) represent a unique class of transcription factors that regulate gene expression under the strict control of endogenous or synthetic ligands.3, 17 In humans, the 48 known NRs play numerous roles in development, physiology and EM9 pathology. Therefore, ligands of NRs have the potential to modulate the cytokine profile of CAFs, leading to tumor suppression or tumor sensitization to standard chemotherapy. However, the manifestation of NRs in CAFs from squamous cell carcinoma (SCC) tumors is definitely unknown, and their non-redundant tasks in SCC progression and chemoresistance is definitely unclear. As the primary experimental system to explore CAF NR-directed therapy, we defined an NR profile for CAFs from individuals diagnosed with cutaneous SCC. Guided by this ROC-325 manifestation profile, the genetic and pharmacological focusing on of specific driver NRs in CAFs diminished SCC invasiveness, proliferation, drug resistance, energy rate of metabolism and oxidative stress status. Furthermore, main and recurred xenograft tumor growth was attenuated by a combination treatment with NR ligands and cisplatin, in part due to reduced chemoresistance. Our findings suggest that NR-directed ligands that have successfully treated additional pathologies such as swelling, dyslipidemia and diabetes, may be repurposed as concurrent treatments to standard anticancer chemotherapeutics. Results NRs are differentially indicated between CAFs and normal FIBs Paired samples of CAFs and peri-tumoral FIBs from archived SCC biopsies ((right). Expression ideals in CAFs are relative to that in normal FIBs, The 1st column in the heatmap signifies the manifestation of NRs from five different FIB settings. NRs that form heterodimers with retinoid X receptors (RXRs) are labeled in reddish, while those that form homodimers are labeled in blue. ROC-325 Superscript figures distinguish NRs with known ligands (1) from orphan NRs (2). Color scales: green=downregulated, reddish=upregulated. N.D. denotes the gene was not recognized by RT-qPCR. Peroxisome proliferator-activated receptor (PPAR) manifestation was downregulated to the greatest degree in CAFs, followed by the vitamin D receptor.