This minireview covers the chromatographic methods utilized in acyl-CoA detection, but with greater emphasis on LC-MS methods due their advantages over preceding approaches. In general, the review summarizes the types of mass spectrometers, sample preparation approaches for different biological matrices, and the LC conditions for potential biomarker applications.

The Cxcl12/Cxcr4 signaling axis has been shown to promote metastasis in multiple mouse models of breast carcinoma and to be associated with increased metastatic risk in humans. Indeed, prior studies have specifically linked Cxcl12/Cxcr4 to breast cancer cell seeding, homing, survival and proliferation at future metastatic sites, due to the aberrant Cxcl12 expression in these sites (e.g. lung, liver and bone marrow). Interestingly however, the precise mechanism via which Cxcr4+ breast cancer cells escape the primary tumors in the first place (which also highly express Cxcl12), remains poorly understood. By using a novel methodology for quantifying chemotactic gradients using fixed tissue multichannel immunofluorescence (mIF), here, we demonstrate in mouse primary breast tumors that Cxcl12 gradients are concentrically expressed around cancer cell intravasation sites, known as Tumor Microenvironment of Metastasis (TMEM) doorways. Via distance analysis algorithms using mIF, we also demonstrate that TMEM-mediated Cxcl12 gradients contextually associate with Cxcr4+ breast cancer cells migrating towards the underlying TMEM doorways. As such, pharmacological inhibition of the Cxcl12/Cxcr4 pathway significantly abrogates the translocation of Cxcr4+ cancer cells to TMEM doorways, suppressing TMEM-mediated metastatic dissemination. However, targeted elimination of the Cxcr4+ gene specifically from breast cancer cells, paradoxically results in a suboptimal response, thus suggesting the existence of a bypass or compensatory mechanism. Previously, it was shown that Cxcr4+ tumor-associated macrophages (TAMs) support the invasive and migratory properties of tumor cells utilizing TMEM doorways. We thus theorized that, in the absence of Cxcr4 expression in tumor cells, the accompanying Cxcr4+ TAMs may still “read” TMEM-generated Cxcl12 chemotactic gradients. Indeed, clodronate-mediated TAM depletion results in the significant suppression of Cxcr4+ cancer cell translocation to TMEM doorways and their subsequent dissemination to the peripheral circulation and future metastatic sites. Finally, we used a variety of stromal and immune cell lineage markers to identify the precise source of TMEM-generated Cxcl12 gradients in mouse primary breast cancers. Despite that blood vessels (irrespective of presence of TMEM doorways) were primarily lined by Pdgfrb+ stromal cells with basal Cxcl12 expression, TMEM-generated Cxcl12 gradients were specifically linked to a subset of Cxcl12+Iba1+ perivascular TAMs. Pharmacological inhibition of Pdgfrb depletes Pdgfrb+Cxcl12+ stromal cells, but does not significantly affect Cxcl12/Cxcr4- mediated translocation of Cxcr4+ tumor cells to TMEM doorways. Overall, our data support a new paradigm for the implication of the Cxcl12/Cxcr4 axis during the early stages of the metastatic cascade, and propose a new avenue for rationalized antimetastatic treatments for breast cancer. Citation Format: Maria K. Lagou, Luis G. Rivera, Camille E. Duran, Joseph Burt, Xiaoming Chen, Yu Lin, Robert Eddy, Allison S. Harney, David Entenberg, John S. Condeelis, Maja H. Oktay, George S. Karagiannis. An emerging paradigm of Cxcl12/Cxcr4 involvement in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3124.

Previous studies have reported sex differences in irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) patients, including differences in visceral pain perception. Despite this, sex differences in behavioral manifestations of visceral pain and underlying pathology of the gastrointestinal tract have been largely understudied in preclinical research. In this study, we evaluated potential sex differences in spontaneous visceral nociceptive responses, referred abdominal hypersensitivity, disease progression and bowel pathology in mouse models of acute and persistent colon inflammation. Our experiments show that females exhibit more visceral nociceptive responses and referred abdominal hypersensitivity than males in the context of acute but not persistent colon inflammation. We further demonstrate that, following acute and persistent colon inflammation, visceral pain-related behavioral responses in females and males are distinct, with increases in licking of the abdomen only observed in females and increases in abdominal contractions only seen in males. During persistent colon inflammation, males exhibit worse disease progression than females, which is manifested as worse physical appearance and higher weight loss. However, no measurable sex differences were observed in persistent inflammation-induced bowel pathology, stool consistency or fecal blood. Overall, our findings demonstrate that visceral pain-related behaviors and disease progression in the context of acute and persistent colon inflammation are sex-dependent, highlighting the importance of considering sex as a biological variable in future mechanistic studies of visceral pain as well as in the development of diagnostics and therapeutic options for chronic gastrointestinal diseases.

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