Osteosarcoma-Induced Pain Is Mediated by Glial Cell Activation in the Spinal Dorsal Horn, but Not Capsaicin-Sensitive Nociceptive Neurons: A Complex Functional and Morphological Characterization in Mice.

Bone cancer and its related chronic pain are huge clinical problems since the available drugs are often ineffective or cannot be used long term due to a broad range of side effects. The mechanisms, mediators and targets need to be identified to determine potential novel therapies. Here, we characterize a mouse bone cancer model induced by intratibial injection of K7M2 osteosarcoma cells using an integrative approach and investigate the role of capsaicin-sensitive peptidergic sensory nerves. The mechanical pain threshold was assessed by dynamic plantar aesthesiometry, limb loading by dynamic weight bearing, spontaneous pain-related behaviors via observation, knee diameter with a digital caliper, and structural changes by micro-CT and glia cell activation by immunohistochemistry in BALB/c mice of both sexes. Capsaicin-sensitive peptidergic sensory neurons were defunctionalized by systemic pretreatment with a high dose of the transient receptor potential vanilloid 1 (TRPV1) agonist resiniferatoxin (RTX). During the 14- and 28-day experiments, weight bearing on the affected limb and the paw mechanonociceptive thresholds significantly decreased, demonstrating secondary mechanical hyperalgesia. Signs of spontaneous pain and osteoplastic bone remodeling were detected both in male and female mice without any sex differences. Microglia activation was shown by the increased ionized calcium-binding adapter molecule 1 (Iba1) immunopositivity on day 14 and astrocyte activation by the enhanced glial fibrillary acidic protein (GFAP)-positive cell density on day 28 in the ipsilateral spinal dorsal horn. Interestingly, defunctionalization of the capsaicin-sensitive afferents representing approximately 2/3 of the nociceptive fibers did not alter any functional parameters. Here, we provide the first complex functional and morphological characterization of the K7M2 mouse osteosarcoma model. Bone-cancer-related chronic pain and hyperalgesia are likely to be mediated by central sensitization involving neuroinflammation via glial cell activation in the spinal dorsal horn, but not the capsaicin-sensitive sensory neuronal system.

Transient Receptor Potential Ankyrin 1 Ion Channel Is Expressed in Osteosarcoma and Its Activation Reduces Viability.

Osteosarcoma is a highly malignant, painful cancer with poor treatment opportunities and a bad prognosis. Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are non-selective cation channels that have been of great interest in cancer, as their expression is increased in some malignancies. In our study we aim to characterize the expression and functionality of the TRPA1 and TRPV1 channels in human and mouse osteosarcoma tissues and in a mouse cell line. TRPA1/Trpa1 and TRPV1/Trpv1 mRNA expressions were demonstrated by PCR gel electrophoresis and RNAscope in situ hybridization. The function of these channels was confirmed by their radioactive 45Ca2+ uptake in response to the TRPA1 agonist, Allyl-isothiocyanate (AITC), and TRPV1 agonist, capsaicin, in K7M2 cells. An ATP-based K2M7 cell viability luminescence assay was used to determine cell viability after AITC or capsaicin treatments. Both TRPA1/Trpa1 and TRPV1/Trpv1 were expressed similarly in human and mouse osteosarcoma tissues, while Trpa1 transcripts were more abundantly present in K7M2 cells. TRPA1 activation with 200 µM AITC induced a significant 45Ca2+ influx into K7M2 cells, and the antagonist attenuated this effect. In accordance with the lower Trpv1 expression, capsaicin induced a moderate 45Ca2+ uptake, which did not reach the level of statistical significance. Both AITC and capsaicin significantly reduced K7M2 cell viability, demonstrating EC50 values of 22 µM and 74 µM. The viability-decreasing effect of AITC was significantly but only partially antagonized by HC-030031, but the action of capsaicin was not affected by the TRPV1 antagonist capsazepine. We provide here the first data on the functional expression of the TRPA1 and TRPV1 ion channels in osteosarcoma, suggesting novel diagnostic and/or therapeutic perspectives.

IL-1 Mediates Chronic Stress-Induced Hyperalgesia Accompanied by Microglia and Astroglia Morphological Changes in Pain-Related Brain Regions in Mice.

Chronic stress causes several pain conditions including fibromyalgia. Its pathophysiological mechanisms are unknown, and the therapy is unresolved. Since the involvement of interleukin-1 (IL-1) has been described in stress and inflammatory pain but no data are available regarding stress-induced pain, we studied its role in a chronic restraint stress (CRS) mouse model. Female and male C57Bl/6J wild-type (WT) and IL-1αß-deficient (knock-out: IL-1 KO) mice were exposed to 6 h of immobilization/day for 4 weeks. Mechanonociception, cold tolerance, behavioral alterations, relative thymus/adrenal gland weights, microglia ionized calcium-binding adaptor molecule 1 (IBA1) and astrocyte glial fibrillary acidic protein (GFAP) integrated density, number and morphological transformation in pain-related brain regions were determined. CRS induced 15-20% mechanical hyperalgesia after 2 weeks in WT mice in both sexes, which was significantly reduced in female but not in male IL-1 KOs. Increased IBA1+ integrated density in the central nucleus of amygdala, primary somatosensory cortex hind limb representation part, hippocampus cornu ammonis area 3 (CA3) and periaqueductal gray matter (PAG) was present, accompanied by a cell number increase in IBA1+ microglia in stressed female WTs but not in IL-1 KOs. CRS induced morphological changes of GFAP+ astrocytes in WT but not in KO mice. Stress evoked cold hypersensitivity in the stressed animals. Anxiety and depression-like behaviors, thymus and adrenal gland weight changes were detectable in all groups after 2 but not 4 weeks of CRS due to adaptation. Thus, IL-1 mediates chronic stress-induced hyperalgesia in female mice, without other major behavioral alterations, suggesting the analgesic potentials of IL-1 in blocking drugs in stress-related pain syndromes.

Desensitization of Capsaicin-Sensitive Afferents Accelerates Early Tumor Growth via Increased Vascular Leakage in a Murine Model of Triple Negative Breast Cancer.

There is growing interest in the role of nerve-driven mechanisms in tumorigenesis and tumor growth. Capsaicin-sensitive afferents have been previously shown to possess antitumoral and immune-regulatory properties, the mechanism of which is currently poorly understood. In this study, we have assessed the role of these terminals in the triple negative 4T1 orthotopic mouse model of breast cancer. The ultrapotent capsaicin-analogue resiniferatoxin (RTX) was used for the selective, systemic desensitization of capsaicin-sensitive afferents. Growth and viability of orthotopically implanted 4T1 tumors were measured by caliper, in vivo MRI, and bioluminescence imaging, while tumor vascularity and protease enzyme activity were assessed using fluorescent in vivo imaging. The levels of the neuropeptides Calcitonin Gene-Related Peptide (CGRP), Substance P (SP), and somatostatin were measured from tumor tissue homogenates using radioimmunoassay, while tumor structure and peritumoral inflammation were evaluated by conventional use of CD31, CD45 and CD3 immunohistology. RTX-pretreated mice demonstrated facilitated tumor growth in the early phase measured using a caliper, which was coupled with increased tumor vascular leakage demonstrated using fluorescent vascular imaging. The tumor size difference dissipated by day seven. The MRI tumor volume was similar, while the intratumoral protease enzyme activity measured by fluorescence imaging was also comparable in RTX-pretreated and non-pretreated animals. Tumor viability or immunohistopathological profile was measured using CD3, CD31, and CD45 stains and did not differ significantly from the non-pretreated control group. Intratumoral somatostatin, CGRP, and SP levels were similar in both groups. Our results underscore the beneficial, antitumoral properties of capsaicin sensitive nerve terminals in this aggressive model of breast cancer, which is presumed to be due to the inhibition of tumor vascular bed disruption. The absence of any difference in intratumoral neuropeptide levels indicates non-neural sources playing a substantial part in their expression.