Tumor-associated tertiary lymphoid structures (TA-TLS) are associated with enhanced patient survival and responsiveness to cancer therapies, but the mechanisms underlying their development are unknown. We show here that TA-TLS development in murine melanoma is orchestrated by cancer-associated fibroblasts (CAF) with characteristics of lymphoid tissue organizer cells that are induced by tumor necrosis factor receptor signaling. CAF organization into reticular networks is mediated by CD8 T cells, while CAF accumulation and TA-TLS expansion depend on CXCL13-mediated recruitment of B cells expressing lymphotoxin-a 1 b 2 . Some of these elements are also overrepresented in human TA-TLS. Additionally, we demonstrate that immunotherapy induces more and larger TA-TLS that are more often organized with discrete T and B cell zones, and that TA-TLS presence, number, and size are correlated with reduced tumor size and overall response to checkpoint immunotherapy. This work provides a platform for manipulating TA-TLS development as a cancer immunotherapy strategy.

Cancer cells (relative to normal cells) demonstrate alterations in oxidative metabolism characterized by increased steady-state levels of reactive oxygen species [i.e. hydrogen peroxide, H 2 O 2 ] that may be compensated for by increased glucose metabolism but the therapeutic significance of these observations is unknown. In the current study, inhibitors of glucose [i.e., 2-deoxy-D-glucose, 2DG] and hydroperoxide [i.e., L-buthionine-S, R-sulfoximine, BSO] metabolism were utilized in combination with a chemotherapeutic agent paclitaxel [PTX], thought to induce oxidative stress, to treat breast cancer cells. 2DG+PTX were found to be more toxic than either agent alone in T47D and MDA-MB231 human breast cancer cells, but not in normal human fibroblasts or normal human mammary epithelial cells. Increases in parameters indicative of oxidative stress, including steadystate levels of H 2 O 2 , total glutathione, and glutathione disulfide accompanied the enhanced toxicity of 2DG+PTX in cancer cells. Antioxidants, including N-acetyl-cysteine [NAC], polyethylene glycolconjugated catalase [PEG-CAT] and superoxide dismutase [PEG-SOD], inhibited the toxicity of 2DG+PTX and suppressed parameters indicative of oxidative stress in cancer cells, while inhibition of glutathione synthesis using BSO further sensitized breast cancer cells to 2DG+PTX. These results show that combining inhibitors of glucose [2DG] and hydroperoxide [BSO] metabolism with PTX selectively (relative to normal cells) enhances breast cancer cell killing via H 2 O 2 -induced metabolic oxidative stress, and suggests that this biochemical rationale may be effectively utilized to treat breast cancers.

Object The primary goal of this study was to review the immediate postoperative neurological function in patients surgically treated for symptomatic cervical spine disease without intraoperative neurophysiological monitoring. The secondary goal was to assess the economic impact of intraoperative monitoring (IOM) in this patient population. Methods This study is a retrospective review of 720 consecutively treated patients who underwent cervical spine procedures. The patients were identified and the data were collected by individuals who were not involved in their care. Results A total of 1534 cervical spine levels were treated in 720 patients using anterior, posterior, and combined (360°) approaches. Myelopathy was present preoperatively in 308 patients. There were 185 patients with increased signal intensity within the spinal cord on preoperative T2-weighted MR images, of whom 43 patients had no clinical evidence of myelopathy. Three patients (0.4%) exhibited a new neurological deficit postoperatively. Of these patients, 1 had a preoperative diagnosis of radiculopathy, while the other 2 were treated for myelopathy. The new postoperative deficits completely resolved in all 3 patients and did not require additional treatment. The Current Procedural Terminology (CPT) codes for IOM during cervical decompression include 95925 and 95926 for somatosensory evoked potential monitoring of the upper and lower extremities, respectively, as well as 95928 and 95929 for motor evoked potential monitoring of the upper and lower extremities. In addition to the charge for the baseline [monitoring] study, patients are charged hourly for ongoing electrophysiology testing and monitoring using the CPT code 95920. Based on these codes and assuming an average of 4 hours of monitoring time per surgical case, the savings realized in this group of patients was estimated to be $1,024,754. Conclusions With the continuing increase in health care costs, it is our responsibility as providers to minimize expenses when possible. This should be accomplished without compromising the quality of care to patients. This study demonstrates that decompression and reconstruction for symptomatic cervical spine disease without IOM may reduce the cost of treatment without adversely impacting patient safety.

CD8 T-cell infiltration of metastatic melanoma may be a useful biomarker for prediction of prognosis and response to therapy. The heterogeneous distribution of CD8 T cells within a single tumor, and across different tumors within a single patient, may complicate quantification of infiltration. However, the impact of heterogeneity has not been quantified sufficiently. To address this, we have assessed intratumoral heterogeneity of CD8 T-cell counts, as well as intertumoral heterogeneity for synchronous and metachronous metastases. In a tissue microarray containing 189 melanoma metastases from 147 patients, the density of CD8 T cells per sample was determined by immunohistochemistry. The mean density and coefficient of variation were calculated for each tumor and the rates of discordant values were determined. CD8 counts varied widely among different core samples of the same tumors (average coefficient of variation=0.77, 95% confidence interval: 0.70-0.85), with discordance occurring in 40% of tumors. CD8 densities were similar among pairs of simultaneous tumors; however, significant changes in CD8 densities were observed among 35 pairs of metachronous tumors. CD8 T-cell density is not well represented by a single 1 mm diameter sample. Differences in CD8 T-cell counts, observed in clinical trials, from pretreatment to post-treatment specimens may be explained by the spatial and temporal heterogeneity of CD8 distribution, especially if the assessed samples are small (i.e. 1 mm). A sufficiently large biopsy of one of several synchronous tumors may be representative of CD8 T-cell infiltration of a patient's disease.

Objective: To understand role of barrier molecules in melanomas. Background:We have reported poor patient survival and low immune infiltration of melanomas that overexpress a set of genes that include filaggrin (FLG), dystonin (DST), junction plakoglobin (JUP), and plakophilin-3 (PKP3), and are involved in cell-cell adhesions. We hypothesized that these associations are causal, either by interfering with immune cell infiltration or by enhancing melanoma cell growth.Methods: FLG and DSTwere knocked out by CRISPR/Cas9 in human DM93 and murine B16-F1 melanoma cells. PKP3 and JUP were overexpressed in murine B16-AAD and human VMM39