Journal Article for Review: Apoptosis-induced CXCL5

The article under review examines the acceleration of inflammation and growth of prostate tumor metastasis in bone due to apoptosis-induced CXCL5. Tumor cell death, which can be increased by targeted therapies, causes immense efferocytosis to clear the apoptotic cells. It is thought that this can lead to accelerated tumor growth. The scope of this research article is limited to prostate cancer cells and the proinflammatory cytokine, CKCL5, along with other variants. This article will exclude other ways of metastasis of prostate tumors and focus solely on apoptosis-induced CXCL5.

The general findings from this research concluded that apoptotic cancer cells accelerate the inflammation mediated by the cytokine CXCL5 and bone tumor growth. These findings point to CXCL5 as a potential target for cancer therapies.

Proinflammatory cytokines are induced in macrophages upon apoptotic cancer cell efferocytosis. To investigate apoptotic cancer cell clearance in skeletal tumor progression, in vivo models of skeletal tumor growth and apoptosis-inducible prostate cancer cells were used. The first hypothesis made was that macrophages discriminate between different types of apoptotic cells and conduct a response. Cocultures of macrophages and highly apoptotic cells were analyzed. The activation of cytokines was observed in apoptotic cells and not noncancerous cells. Apoptotic cells showed reduced levels of cytokines compared to macrophages. The next investigation was into whether the inflammatory response induced in the macrophages was a result of the interaction with apoptotic epithelial cells. Only apoptotic RM1 cancer cells could induce an inflammatory response in bone marrow macrophages. An ELISA test, which is an immunological assay commonly used in the detection of antibodies, antigens, proteins, and glycoproteins, was necessary in the evaluation of CXCL1 and CXCL5 proteins. From these results, a response occurred that was dependent on the cell type engulfed and a common inflammatory expression pattern in macrophages efferocytosing apoptotic cancer cells. TRACER technology was used, along with cocultures of apoptotic RM1 or MC4, to evaluate the transcription factor activity in macrophages in response to apoptotic cells. The results showed that both transcription factors activate inflammatory responses and cooperate with the activation of proinflammatory cytokines.

Efferocytosis induces an inflammatory response via activation of Stat3 and NF-?B signaling. The role of efferocytosis in inducing this response commenced with the production of apoptosis-inducible RM1-iC9 from murine RM1 cells, subsequently treated with the dimerizer drug AP20187 (AP) or a controlled vehicle (VEH). A western blot analysis ensued, followed by a series of events beginning with the labeling of cells with CFSE dye and concluding with an analysis by an ImageStream flow cytometer. Double-positive cells are indicative of efferocytic macrophages. AP-treated cells displayed higher internalization relative to VEH, corroborating that efferocytosis amplified with the induction of apoptosis in cancer cells. PCR analysis revealed heightened expression of proinflammatory cytokines CXCL5, CCl5, and Il-6 in efferocytic macrophages. Furthermore, the increase in phosphorylation/activation of NF-?B and Stat3 in macrophages via efferocytosis of apoptotic RM1 cells was explored. The findings proposed the predominant role of canonical signaling, primarily activating transcription factors and altering target gene expression ("What is Canonical Wnt Receptor Signaling Pathway?," n.d.), in the efferocytic inflammatory response. The dependence of Stat3 and NF-?B in the production of inflammatory cytokines was investigated. Inhibitors significantly mitigated proinflammatory cytokine expression in macrophages. Emetine treatment was utilized, inhibiting p-NF-?B, p-Stat3, and efferocytosis. Emetine's effect on efferocytosis could be due to simultaneous blockage of Stat3 and Nf-?B activation. These findings collectively suggest a correlation between efferocytosis and persistent inflammation within the tumor microenvironment via activation of Stat3 and NF-?B signaling in macrophages.

Cancer cell death accelerates tumor progression and increases tumor CXCL5 levels. Efferocytosis of cancer cells influences prostate cancer tumor progression in the bone, as illustrated via an osseous implant vossicle model. A significant increase in CXCL5 was observed in macrophages upon efferocytosis of cancer cells, correlating with prostate cancer progression and metastasis. A significant correlation between tumor weight and CXCL5 concentrations was evident for both VEH- and AP-treated tumor vossicle groups. Flow cytometry was utilized to analyze the immune cell composition of the tumor vossicle groups. The lack of difference in the whole blood count for each group suggests that the results observed in the tumor microenvironment are localized.

CXCL5 acts as a crucial inflammatory-microenvironment cytokine that accelerates tumor progression. CXCL5 was found to increase with cancer cell death and efferocytosis in the tumor microenvironment. Blood count of mice bearing tumor vossicles showed lower concentrations of leukocytes, lymphocytes, and monocytes. This indicates the critical role of CXCL5 produced by the efferocytic macrophages in inducing inflammation, thereby accelerating tumor growth in the bone microenvironment.

Cancer cell death induces accelerated tumor growth and bone destruction, while CXCL5 deficiency hinders tumor progression. The effect of cancer cell death and CXCL5 in tumor progression was analyzed in an intratibial inoculation model. When comparing tumor growth in the bones of WT and CXCL5 deficient mice, both sets were treated with AP to induce cancer cell apoptosis. An H&E tumor section showed reduced tumor areas in CXCL5 deficient mice relative to WT, confirming the proinflammatory role of CXCL5 induced by efferocytosis of apoptotic cancer cells.

Nonclassical phagocytic peripheral blood mononuclear cells and elevated CXCL5 serum levels were associated with human prostate cancer skeletal metastasis. The hypothesis proposed that prostate cancer patients with bone metastasis would have increased circulating phagocytic monocytes due to the presence of circulating tumor cells. A correlating hypothesis suggested that phagocytic monocytes/macrophages would elevate serum levels of the inflammatory CXCL5 cytokine in patients with bone metastasis, due to the clearance of tumor cells both in the circulatory system and in the bone marrow compartment. An ELISA test was conducted to measure three closely related cytokines: CXCL5, CXCL6, and IL-8. Human IL-8 is a proinflammatory cytokine involved in neutrophil activation (“Interleukin-8,” 1989). Higher levels of CXCL5 were detected in the serum of patients with bone metastasis. Conversely, samples from the bone metastasis showed reduced IL-8 concentrations. These findings suggest a predominant role of proinflammatory CXCL5 in prostate cancer bone metastasis, coupled with an increased phagocytic circulating monocytic cell population.

From this investigation of apoptosis-induced CXCL5 in the acceleration of inflammation and growth of prostate tumor metastasis, important findings included chronic inflammation increasing cell stress and tissue damage, and subsequently, inducing apoptotic/necrotic cell death. Efferocytosis, an essential function in this investigation, seems to be fundamental to macrophages and a critical mechanism of homeostasis that helps prevent inflammation (Immunol, 2017). New evidence correlates with these findings that the response of bone marrow macrophages is different when engaged in efferocytosis of prostate cancer cells compared to normal cells. In vivo studies done on breast and colon cancer showed efferocytosis inducing M2 polarization and metastasis. This gives rise to the notion that IL-6 induces M2-like macrophage polarization in human macrophages.

This investigation was not without errors: differential results could occur because treatments used, such as AP, did not equally reach all cancer cells due to large tumor complexity. Cytokine arrays also missed other cytokines present but was not essential because the array did express considerable levels of CXCL5. This was the major contribution to the study and held the most importance from this array. Linking previous in vivo studies of prostate cancer models suggested the activation of certain cells in the bone marrow model that was mediated by IL-6 to accelerate tumor progression. This suggested the importance of IL-6 in human macrophages. CXCL6 is also of importance because it is the human homolog of mouse CXCL5, which is upregulated in prostate cancer tissues. Studies on human monocytes indicate these cells are important in inflammatory diseases. To progress this investigation further, it would be insightful to explore more into the roles of monocytes in metastasis-promoting inflammatory responses.

More human studies should be conducted to link the findings in mice with human orientation. Ultimately, these findings reveal the role dying tumor cells by macrophages have on inflammation in the tumor bone microenvironment, via the use of CXCL5 cytokines, along with other variants to facilitate cancer progression. Through the identification of CXCL5 in enhanced cell death and efferocytosis produced by cancer therapies, designing additional therapies will now be able to take into account the role of cytokines produced by therapies, with the hope of achieving more effective results.

References

1. Horlock, C. (n.d.). Enzyme-linked immunosorbent assay (ELISA). Retrieved from https://www.immunology.org/public-information/bitesized-immunology/experimental-techniques/enzyme-linked-immunosorbent-assay
2. Immunol, J. (2017, February 15). Efferocytosis signaling in the regulation of macrophage inflammatory responses. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5301545/
3. Interleukin-8. (1989, July 1). Retrieved from https://www.uniprot.org/uniprot/P10145
4. What is Canonical Wnt Receptor Signaling Pathway? (n.d.). Retrieved from https://www.mechanobio.info/what-is-mechanosignaling/signaling-pathways/what-is-the-canonical-wnt-receptor-signaling-pathway/"

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