(F-234) Location matters: Distinct patterns of macrophage activation and tumor promotion in bioengineered colorectal tumor models

Introduction:: Macrophages are an abundant cell type in the tumor microenvironment, controlling metastasis, angiogenesis and promoting tumor growth. However, macrophage activation exists on a spectrum, with conventional wisdom associating pro-tumoral M2-like activation to tumor growth and progression. In fact, in colorectal cancer (CRC), the increased presence of M1-like inflammatory macrophages is associated with good prognosis, while increased M2-like macrophages with poor prognosis. However, curiously in CRC patient biospecimen, M1-like macrophages are typically associated with the invasive tumor front, while M2-like macrophages are interspersed within the bulk tumor. Previously, our own work demonstrated that cancer cells heavily activate macrophages into an M2-like phenotype when macrophages were integrated with cancer cells within an engineered tumor spheroid. Here, we tested the hypothesis that the location of the macrophages within engineered cancer/macrophage heterospheroids dictates the activation of macrophages, and subsequent tumor progression.


Materials and Methods:: A patient-derived xenograft line PDX-1 was used in this study to model tumor cells, along with THP-1 monocyte-derived macrophages. CRC monospheroids (100 cells) or CRC/macrophage heterospheroids (100 PDX-1 CRC cells:100 THP-1 macrophages) were generated on a hanging drop array. To generate Integrated Heterospheroids. CRC cells and macrophages were mixed prior to seeding on the hanging drop arrays. To generate External Heterospheroids, CRC spheroids were first formed and allowed to aggregate for 24hrs, at which time macrophages were added to the periphery of pre-formed CRC spheroids. Following 7 days in heterospheroid culture, each seeding condition was evaluated for: i) macrophage localization (CD68 immunofluorescence staining), ii) macrophage polarization (M1: CD86; M2: CD163) and cancer stem cell emergence (CD133; flow cytometry), ii) ECM protein (collagen, tenascin-C; ELISA), and iii) chemotherapy resistance (5FU, oxaliplatin; MTS viability assay).

Results, Conclusions, and Discussions:: Results: Integrated Heterospheroids generated with mixed CRCs and macrophages maintained this integration over the course of 7 days in spheroid culture, with CD68 fluorescence throughout the spheroid (Fig. 1A). External Heterospheroids demonstrated a preferential localization of a majority of macrophages on the outer surface of spheroids (Fig. 1B). Most macrophages present in both types of heterospheroids demonstrated CD163 expression, indicating pro-tumoral activation. Interestingly, External Heterospheroids also robustly maintained a CD86⁺ M1-like macrophage population compared to integrated macrophages (Fig. 1C). Despite the M1 (anti-tumoral) activation induced in External Heterospheroids with macrophages localized to the periphery, they were 2.5X more chemoresistant to 5-fluorouracil treatment. This chemoresistance was associated with an increased enrichment of CD133⁺ cancer stem cells with the inclusion of macrophages in both types of heterospheroids, regardless of macrophage location, which aligns with our previously published work (Fig. 1E). This indicated that the emergence of cancer stem cells alone could not explain the increased chemoresistance driven by macrophages at the tumor front. Lastly, we assessed if increased collagen deposition might explain the chemoresistant phenotype, but noted that collagen I was significantly decreased in heterospheroids compared to monospheroids. This was likely due to the documented ability of M2-like macrophages to digest collagen I, and the absence of mesenchymal stroma in these engineered models. Interestingly, associated with the External Heterospheroids we noted an increased presence of tenascin-C, a matrisomal protein and an emerging biomarker for CRC chemoresistance and metastasis.



Conclusions and Discussion: Our work collectively demonstrates that the localization of macrophages in CRC tumors alters not only macrophage activation itself, but also has different functional consequences for CRC tumor progression. Our future studies will explore the active mechanisms that allow colorectal tumors to utilize both pro-inflammatory and pro-tumoral macrophages toward chemoresistance and tumor progression.


Acknowledgements (Optional): : This work was supported by the CPRIT Award RP210108, NIH R37CA269224, and the Texas A&M University Department of Biomedical Engineering.

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