Bettie M. Steinberg

Figure S2 shows that gefitinib has no effect on the size of the primary tumor.

Most patients with osteosarcoma have subclinical pulmonary micrometastases at diagnosis. Mounting evidence suggests that macrophages facilitate metastasis. As the EGFR has been implicated in carcinoma–macrophage cross-talk, in this study, we asked whether gefitinib, an EGFR inhibitor, reduces osteosarcoma invasion and metastatic outgrowth using the K7M2-Balb/c syngeneic murine model. Macrophages enhanced osteosarcoma invasion in vitro, which was suppressed by gefitinib. Oral gefitinib inhibited tumor extravasation in the lung and reduced the size of metastatic foci, resulting in reduced metastatic burden. Gefitinib also altered pulmonary macrophage phenotype, increasing MHCII and decreasing CD206 expression compared with controls. Surprisingly, these effects are mediated through inhibition of macrophage receptor interacting protein kinase 2 (RIPK2), rather than EGFR. Supporting this, lapatinib, a highly specific EGFR inhibitor that does not inhibit RIPK2, had no effect on macrophage-promoted invasion, and RIPK2−/− macrophages failed to promote invasion. The selective RIPK2 inhibitor WEHI-345 blocked tumor cell invasion in vitro and reduced metastatic burden in vivo. In conclusion, our results indicate that gefitinib blocks macrophage-promoted invasion and metastatic extravasation by reprogramming macrophages through inhibition of RIPK2.

Figure S1 shows the reduction in pulmonary macrophages with inhaled clodronate.

Most patients with osteosarcoma have subclinical pulmonary micrometastases at diagnosis. Mounting evidence suggests that macrophages facilitate metastasis. As the EGFR has been implicated in carcinoma–macrophage cross-talk, in this study, we asked whether gefitinib, an EGFR inhibitor, reduces osteosarcoma invasion and metastatic outgrowth using the K7M2-Balb/c syngeneic murine model. Macrophages enhanced osteosarcoma invasion in vitro, which was suppressed by gefitinib. Oral gefitinib inhibited tumor extravasation in the lung and reduced the size of metastatic foci, resulting in reduced metastatic burden. Gefitinib also altered pulmonary macrophage phenotype, increasing MHCII and decreasing CD206 expression compared with controls. Surprisingly, these effects are mediated through inhibition of macrophage receptor interacting protein kinase 2 (RIPK2), rather than EGFR. Supporting this, lapatinib, a highly specific EGFR inhibitor that does not inhibit RIPK2, had no effect on macrophage-promoted invasion, and RIPK2−/− macrophages failed to promote invasion. The selective RIPK2 inhibitor WEHI-345 blocked tumor cell invasion in vitro and reduced metastatic burden in vivo. In conclusion, our results indicate that gefitinib blocks macrophage-promoted invasion and metastatic extravasation by reprogramming macrophages through inhibition of RIPK2.

Figure S2 shows that gefitinib has no effect on the size of the primary tumor.

Figure S1 shows the reduction in pulmonary macrophages with inhaled clodronate.

A live, virtual conference, “Driving Responsible Conduct of Research during a Pandemic,” was held in April 2021, 13 months after the COVID-19 pandemic fundamentally altered the conduct of clinical research across the USA. New York was an early epicenter of the US pandemic, highlighting preexisting problems in clinical research and allowing us to assess lessons learned and to identify best practices for the future. Risks and opportunities were categorized broadly into three areas, protecting the welfare and safety of human subjects, ensuring trust in science and medicine, and implementing efficient, ethical, and compliant clinical research. Analysis of conference proceedings, and recent publications, shows a need for preparedness that is more effective, robust partnerships, and organizational systems and standards to strengthen the ethical and responsible conduct of research.

IL-36γ, a pro-inflammatory member of the IL-1 cytokine superfamily, can be induced and secreted by normal human foreskin keratinocytes (HFKs) in response to pathogenic stimuli, however, the mechanisms underlying the secretion are unknown. In this study, we demonstrate that stimulation with the TLR3 agonist, poly (I:C), led to a delayed secretion of IL-36γ compared to stimulation with the TLR5 agonist, flagellin, despite equal levels of the cytokine (p = 0.006). IL-36γ was shown to be released from HFKs in its inactive, uncleaved form, based on western blotting. Moreover, recombinant IL-36γ in its activated, cleaved form induced endogenous IL-36γ 10-fold (p = 0.004) and CXCL8 five-fold (p = 0.003) over baseline levels compared to unactivated full-length recombinant IL-36γ. The ratio of LC3b-II/LC3b-I was significantly higher in poly(I:C)-treated cells compared to flagellin-treated and unstimulated controls without a change in SQSTM1/p62 after 24 hours of stimulation (p = 0.043). Under fluorescence microscopy, poly(I:C) led to a two-fold increase at eight hours and four-fold increase at 24 hours in accumulated autophagosomes post-stimulation (p = 0.032). In contrast, autophagosomes were unchanged relative to baseline in response to flagellin. Bafilomycin A1 treatment enhanced poly(I:C)-mediated IL-36γ secretion (p = 0.044) while rapamycin led to a noticeable, but non-significant, increase in flagellin-mediated IL-36γ secretion, indicating that interrupting autophagic flux can alter IL-3γ grelease from HFKs. Finally, we show that, compared to clinically normal laryngeal tissue, there were significantly higher levels of LC3b-II in HPV-infected respiratory papilloma tissue, indicating a higher number of autophagosomes; a signature of disrupted autophagic flux.

Most patients with osteosarcoma have subclinical pulmonary micrometastases at diagnosis. Mounting evidence suggests that macrophages facilitate metastasis. As the EGFR has been implicated in carcinoma–macrophage cross-talk, in this study, we asked whether gefitinib, an EGFR inhibitor, reduces osteosarcoma invasion and metastatic outgrowth using the K7M2-Balb/c syngeneic murine model. Macrophages enhanced osteosarcoma invasion in vitro, which was suppressed by gefitinib. Oral gefitinib inhibited tumor extravasation in the lung and reduced the size of metastatic foci, resulting in reduced metastatic burden. Gefitinib also altered pulmonary macrophage phenotype, increasing MHCII and decreasing CD206 expression compared with controls. Surprisingly, these effects are mediated through inhibition of macrophage receptor interacting protein kinase 2 (RIPK2), rather than EGFR. Supporting this, lapatinib, a highly specific EGFR inhibitor that does not inhibit RIPK2, had no effect on macrophage-promoted invasion, and RIPK2−/− macrophages failed to promote invasion. The selective RIPK2 inhibitor WEHI-345 blocked tumor cell invasion in vitro and reduced metastatic burden in vivo. In conclusion, our results indicate that gefitinib blocks macrophage-promoted invasion and metastatic extravasation by reprogramming macrophages through inhibition of RIPK2.

Background: Osteosarcoma is a highly metastatic primary bone tumor that predominantly affects adolescents and young adults. A mainstay of treatment in osteosarcoma is removal of the primary tumor. However, surgical excision itself has been implicated in promoting tumor growth and metastasis, an effect known as surgery-accelerated metastasis. The underlying mechanisms contributing to surgery-accelerated metastasis remain poorly understood, but pro-tumorigenic alterations in macrophage function have been implicated. Methods: The K7M2-BALB/c syngeneic murine model of osteosarcoma was used to study the effect of surgery on metastasis, macrophage phenotype, and overall survival. Pharmacological prevention of surgery-accelerated metastasis was examined utilizing gefitinib, a receptor interacting protein kinase 2 inhibitor previously shown to promote anti-tumor macrophage phenotype. Results: Surgical excision of the primary tumor resulted in increases in lung metastatic surface nodules, overall metastatic burden and number of micrometastatic foci. This post-surgical metastatic enhancement was associated with a shift in macrophage phenotype within the lung to a more pro-tumor state. Treatment with gefitinib prevented tumor-supportive alterations in macrophage phenotype and resulted in reduced metastasis. Removal of the primary tumor coupled with gefitinib treatment resulted in enhanced median and overall survival. Conclusions: Surgery-accelerated metastasis is mediated in part through tumor supportive alterations in macrophage phenotype. Targeted pharmacologic therapies that prevent pro-tumor changes in macrophage phenotype could be utilized perioperatively to mitigate surgery-accelerated metastasis and improve the therapeutic benefits of surgery.