Development of an in vitro model system to study tricky-to-target tumor microenvironment cells

From the Gujral Lab, Human Biology Division

Our increasing understanding of how cancer cells grow has highlighted the importance of the surrounding tumor microenvironment in supporting or restricting this growth. Part of this environment includes tumor associated macrophages (TAMs), which can promote tumor progression, metastasis and resistance to therapies. While targeting TAMs seems like a promising therapeutic approach, the complexity of the tumor microenvironment has made it challenging to study these tumor-promoting cells. A recent paper from the lab of Dr. Taran Gujral, part of the Human Biology Division, aimed to develop a physiologically relevant system to study tumor associated macrophages. This study by postdoctoral researcher Dr. Nao Nishida-Aoki (now a lecturer at Waseda University) published in Cancer Research, recapitulated TAM protumoral activities in an in vitro system and uncovered TAM signaling pathways that can be pharmacologically targeted to suppress tumor growth in breast cancer mouse models.

Macrophages are important immune cells which produce distinct functional phenotypes in reaction to specific signals in the microenvironment, a process known as macrophage polarization. Dr. Nishida-Aoki explains that “while studying cellular interactions in tumor microenvironment, I became interested in macrophages that drastically alter their phenotypes from anti-tumoral to pro-tumoral traits.” After finding published macrophage polarization protocols were not effective in her system, Dr. Nishida-Aoki wondered if cancer conditioned medium, which she had used extensively in her previous work, may do the trick. “I got a leftover tumor tissue from another project and collected conditioned medium from it. I was thrilled when I saw elongated cells for the first time,” Dr. Nishida-Aoki exclaims. Using this conditioned medium, which contained factors that would normally be secreted from growing tumor cells and signal to nearby macrophages, she developed a physiologically relevant system to polarize TAMs in vitro. Dr. Nishida-Aoki then demonstrated that applying this cultured medium polarized TAMs and induced characteristic anti-inflammatory gene expression and an elongated cell phenotype. Additionally, when co-cultured with cancer cells, the polarized TAMs were able to promote cancer cell proliferation, angiogenesis and impede T-cell infiltration, further validating this model. The authors next used this model to identify potential drugs that could inhibit the protumoral behavior of TAMs. They focused on inhibitors of kinases, critical proteins in cell signaling networks, and sought to identify compounds that inhibited TAM polarization, using the elongation phenotype of polarized macrophages as a read out. They sought to identify kinase inhibitors that prevented TAM cell elongation when treated with conditioned cancer cell medium to polarize them. One of the top hits that inhibited TAM polarization was BMS-794833, a drug known to inhibit c-MET and VEGFR2. To the authors’ satisfaction, this kinase inhibitor was able to suppress tumor growth in breast cancer mouse models.

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The authors then asked mechanistically how this c-MET and VEGFR2 inhibitor suppresses tumor growth. Surprisingly, they found that the TAM-inhibitory function of this compound does not involve c-MET or VEGFR2 inhibition, but rather a range of other targets including SRC kinases, focal adhesion kinases (FAK) and p38 MAPKs. They also observed that targeting a single pathway had little to no effect on TAM polarization. Instead, this tumor suppression was only achieved through the multi-signaling pathway targeting by BMS-794833. Dr. Nishida-Aoki states that “TAM [polarization] has a very robust signaling network and is very tolerant against interventions. Because immune regulation is critical for an individual’s health, TAMs have lots of surrogate pathways that can easily overcome genetic and chemical interventions. BMS-794833 was effective on TAM polarization because it inhibits several signaling pathways simultaneously.” Currently there are no approved therapies that target TAM polarization, but Dr. Nishida-Aoki predicts that “TAM-targeting drugs could be used as either single or combination therapies with the current cancer therapies to enhance their efficacy. Considering that TAMs comprise 30-50% mass in tumor, I think TAM targeting therapy would have drastic effects.” Collectively, this work establishes a physiologically relevant model to study TAM polarization while highlighting the complex regulation of signaling during TAM polarization and the need for multi-target approaches to block the protumoral function of TAMs.

This work was supported by the Breast Cancer Research Foundation, the American Cancer Society, the Fred Hutch Interdisciplinary Training Grant in Cancer Research and the Japan Society for the Promotion of Science Overseas Research Fellowship.

UW/Fred Hutch Cancer Consortium member Taran Gujral contributed to this research.

Nishida-Aoki N, Gujral TS. Polypharmacologic Reprogramming of Tumor-Associated Macrophages toward an Inflammatory Phenotype. Cancer Res. 2022 Feb 1;82(3):433-446. doi: 10.1158/0008-5472.CAN-21-1428. Epub 2021 Dec 13. PMID: 34903600; PMCID: PMC8847322.