Tumor macrophages are generally considered to be alternatively/M2 activated to induce secretion of pro-angiogenic factors such as VEGF and MMPs. EMMPRIN (CD147, basigin) is overexpressed in many tumor types, and has been shown to induce fibroblasts and endothelial cell expression of MMPs and VEGF. We first show that tumor cell interactions with macrophages resulted in increased expression of EMMPRIN and induction of MMP-9 and VEGF. Human A498 renal carcinoma or MCF-7 breast carcinoma cell lines were co-cultured with the U937 monocytic-like cell line in the presence of TNFα (1 ng/ml). Membranal EMMPRIN expression was increased in the co-cultures (by 3–4-folds, p < 0.01), as was the secretion of MMP-9 and VEGF (by 2–5-folds for both MMP-9 and VEGF, p < 0.01), relative to the single cultures with TNFα. Investigating the regulatory mechanisms, we show that EMMPRIN was post-translationally regulated by miR-146a, as no change was observed in the tumoral expression of EMMPRIN mRNA during co-culture, expression of miR-146a was increased and its neutralization by its antagomir inhibited EMMPRIN expression. The secretion of EMMPRIN was also enhanced (by 2–3-folds, p < 0.05, only in the A498 co-culture) via shedding off of the membranal protein by a serine protease that is yet to be identified, as demonstrated by the use of wide range protease inhibitors. Finally, soluble EMMPRIN enhanced monocytic secretion of MMP-9 and VEGF, as inhibition of its expression levels by neutralizing anti-EMMPRIN or siRNA in the tumor cells lead to subsequent decreased induction of these two pro-angiogenic proteins. These results reveal a mechanism whereby tumor cell-macrophage interactions promote angiogenesis via an EMMPRIN-mediated pathway.

Background Fatigue is the most prevalent and debilitating long COVID symptom, however risk factors and pathophysiology of this condition remain unknown. We assessed risk factors for long COVID fatigue and explored its possible pathophysiology. Methods Nested case-control study in a COVID recovery clinic. Individuals with (cases) and without (controls) significant fatigue were included. We performed a multidimensional assessment evaluating various parameters, including pulmonary function tests and cardiopulmonary exercise testing, and implemented multivariable logistic regression to assess risk factors for significant long COVID fatigue. Results Total of 141 individuals were included. Mean age was 47 (SD 13) years; 115 (82%) were recovering from mild COVID-19. Mean time for evaluation was 8 months following COVID-19. Sixty-six (47%) individuals were classified with significant long COVID fatigue. They had significantly higher number of children, lower proportion of hypothyroidism, higher proportion of sore throat during acute illness and long COVID symptoms, and of physical limitation in daily activities. Individuals with fatigue had poorer sleep quality and higher degree of depression. They had significantly lower heart rate [153.52 (22.64) vs 163.52 (18.53), p=0.038] and oxygen consumption per Kg [27.69 (7.52) vs 30.71 (7.52), p=0.036] at peak exercise. The two independent risk factors for fatigue identified in multivariable analysis were peak exercise heart rate (odds ratio [OR] 0.79 per 10 beats/minute, 95% confidence interval [CI] 0.65-0.96, p=0.019); and long COVID memory impairment (OR 3.76, 95% CI 1.57-9.01, p=0.003). Conclusions Long COVID fatigue may be related to autonomic dysfunction, impaired cognition and decreased mood. This may suggest a limbic-vagal pathophysiology. Clinical Trial registration: NCT04851561

Tumors survive and progress by evading killing mechanisms of the immune system, and by generating a tumor microenvironment (TME) that reprograms macrophages in situ to produce factors that support tumor growth, angiogenesis, and metastasis. We have previously shown that by blocking the translation of the enzyme inducible nitric oxide synthase (iNOS), miR-146a-5p inhibits nitric oxide (NO) production in a mouse renal carcinoma cell line (RENCA), thereby endowing RENCA cells with resistance to macrophage-induced cell death. Here, we expand these findings to the mouse colon carcinoma CT26 cell line and demonstrate that neutralizing miR-146a-5p’s activity by transfecting both RENCA and CT26 cells with its antagomir restored iNOS expression and NO production and enhanced susceptibility to macrophage-induced cell death (by 48 and 25%, respectively, p < 0.001). Moreover, miR-146a-5p suppression simultaneously inhibited the expression of the pro-angiogenic protein EMMPRIN (threefolds, p < 0.001), leading to reduced MMP-9 and vascular endothelial growth factor secretion (twofolds and threefolds, respectively, p < 0.05), and reduced angiogenesis, as estimated by in vitro tube formation and scratch assays. When we injected tumors with pro-inflammatory-stimulated RAW 264.7 macrophages together with i.v. injection of the miR-146a-5p antagomir, we found inhibited tumor growth (sixfolds, p < 0.001) and angiogenesis (twofolds, p < 0.01), and increased apoptosis (twofolds, p < 0.01). This combination therapy increased nitrites and reduced TGFβ concentrations in tumor lysates, alleviated immune suppression, and allowed enhanced infiltration of cytotoxic CD8+ T cells. Thus, miR-146a-5p functions as a control switch between angiogenesis and cell death, and its neutralization can manipulate the crosstalk between tumor cells and macrophages and profoundly change the TME. This strategy can be therapeutically utilized in combination with the macrophage therapy approach to induce the immune system to successfully attack the tumor, and should be further explored as a new therapy for the treatment of cancer.

Previously, we have identified a new epitope in EMMPRIN, a multifunctional protein that mediates tumor cell-macrophage interactions and induces both MMP-9 and VEGF. Here, we synthesized this epitope as an octa-branched multiple antigenic peptide (MAP) to vaccinate mice implanted with subcutaneous syngeneic colon (CT26), prostate (TRAMP-C2) or renal (RENCA) cell line carcinomas. Vaccination inhibited, and sometimes regressed, tumor growth in a dose-dependent manner, reaching 94%, 71% and 72% inhibition, respectively, at a 50 μg dose ( < 0.01). Mice with regressed tumors demonstrated immune memory, preventing tumor recurrence upon re-implantation ( < 0.001). When tumor cells were administered through the tail vein to generate lung metastases, vaccination reduced the number of metastatic foci (by 15- and 23-folds, < 0.001), and increased the median survival time by 25% and 53% in RENCA and CT26 metastases, respectively ( < 0.01) relative to scrambled-MAP controls. No significant adverse responses were observed in all experiments. We show that the tumor microenvironment was immune modulated, as vaccination induced production of EMMPRIN-specific antibodies, increased CD8 T cells infiltration and cytotoxicity, alleviated immune suppression by decreasing TGFβ concentrations, reduced angiogenesis and cell proliferation, and enhanced apoptosis. Thus, our successful active peptide vaccination strategy differs from previous, unsuccessful attempts, both in the selected target (the EMMPRIN epitope) and in the use of a modified, MAP configuration, and demonstrates that this may be an efficient approach for the treatment and prevention of some types of cancer.