Probes for CCL2

Intratumoral immune cells are crucial for tumor control and antitumor responses during immunotherapy. Immune cell trafficking into tumors is mediated by binding of specific immune cell receptors to chemokines, a class of secreted chemotactic cytokines. To broadly characterize chemokine expression and function in melanoma, we used multiplexed mass cytometry-based imaging of protein markers and RNA transcripts to analyze the chemokine landscape and immune infiltration in metastatic melanoma samples. Tumors that lacked immune infiltration were devoid of most of the profiled chemokines and exhibited low levels of antigen presentation and markers of inflammation. Infiltrated tumors were characterized by expression of multiple chemokines. CXCL9 and CXCL10 were often localized in patches associated with dysfunctional T cells expressing the B lymphocyte chemoattractant CXCL13. In tumors with B cells but no B cell follicles, T cells were the sole source of CXCL13, suggesting that T cells play a role in B cell recruitment and potentially in B cell follicle formation. B cell patches and follicles were also enriched with TCF7+ naïve-like T cells, a cell type that is predictive of response to immune checkpoint blockade. Our data highlight the strength of targeted RNA and protein codetection to analyze tumor immune microenvironments based on chemokine expression and suggest that the formation of tertiary lymphoid structures may be accompanied by naïve and naïve-like T cell recruitment, which may contribute to antitumor activity.

Purpose: The synovial joint exhibits extraordinary biotribological properties allowing the articular cartilage layers to slide past each other at very low friction even under local pressures of up to 18 MPa (~180 atm). Articular cartilage is exquisitely mechanical sensitive. Compressive mechanical load contributes to articular cartilage homeostasis; however, overuse or destabilizing the joint increases surface shear stress, which promotes cartilage degradation. Our previous Results show that shear stress, induced by joint destabilization, regulates a number of inflammatory genes 6h post surgery, including Mmp3, Il1b, Arg1, Ccl2, and Il6. Immobilizing the joint by prolonged anesthesia or sciatic neurectomy abrogates the regulation of inflammatory genes and prevents development of OA. In this study, we use RNA Scope to identify which cells of the cartilage are activated by surface shear after joint destabilisation, and test whether this is modifiable by injection of a biocompatible phospholipid-based lubricant. Methods: Destabilization of the medial meniscus (DMM) or sham surgery was performed on the right knee of 10-week-old male C57BL/6 mice. 30 ml of lubricant (PMPC: poly(methacryloylphosphsphorylcholine)-functionalized lipid vesicles) or vehicle control (PBS) solution was injected in the joint two days before and at the time of surgery. Cartilage from naïve (no surgery) and DMM-operated knees of four mice per data point was collected by microdissection for bulk mRNA extraction. Expression levels of selected genes including shear-responsive genes Il1b and Mmp3 were tested by RT-PCR using TaqMan Low Density Arrays (TLDA) microfluidic cards. In addition, whole joints were collected and processed following the standard protocol for RNAscope (Advanced Cell Diagnostics). Coronal sections in the middle of the joints were sliced by a cryostat. Consecutive sections were used for Safranin O staining and RNAscope to identify anatomical tissues and detect the expression of genes of interest. Gene expression signals were collated from 11 stacks by confocal microscopy (Zeiss Confocal 880) focusing on the medial tibia cartilage, and were quantified by counting individual mRNA dots in the sham, DMM, vehicle and lubricant groups. Results: We observed the upregulation of injury-responsive genes Il1b, Mmp3, Ccl2, Adamts 4, Nos2, and Timp1 in the articular cartilage of DMM operated joints compared to Naïve (non-operated) animals. The injection of the lubricant in the joint significantly suppressed the expression of shear-responsive genes Il1b and Mmp3 after DMM, but did not influence the increase of other injury-induced inflammatory genes, such as Timp1, Adamts 4, Ccl2, Nos2. For RNAscope, focusing on Mmp3 expression, the number of Mmp3 positive cells increased two-fold in the DMM-vehicle group compared with the sham-vehicle group. Most of Mmp3 signal was expressed in the superficial region of the cartilage. DMM-PMPC groups showed a reduced number of Mmp3 positive cells compared with DMM-vehicle, with levels similar to sham-vehicle and sham-PMPC groups. Conclusions: Our data demonstrate that shear stress-induced inflammatory genes are regulated in the superficial layer of cartilage after joint destabilisation and can be suppressed by joint injection of a biocompatible engineered lubricant. As these lubricants have long retention times in the joint (data not presented), we believe that they may provide a potential novel therapeutic strategy for preventing the development of post-trauma OA. These studies are underway