Optimization of an in vitro 3D method for studying modulators of the mmunosuppressive tumor microenvironment for enhancing the response to immunotherapy
Publication Date
July 2026
Document Type
Abstract
Start Date
24-7-2026 9:00 AM
End Date
24-7-2026 3:00 PM
Description
Purpose: This study aims to establish physiologically relevant in vitro models of immunosuppressive tumor microenvironment (TME) for multiple myeloma (MM) and triplenegative breast cancer (BC) using three-dimensional (3D) multicellular coculture systems incorporating immunosuppressive and inflammatory myeloid cells, including M2-polarized THP1 macrophages and low-density neutrophils (LDNs) with RPMI-8226 (MM) or MDA-MB-231 cells (BC). These models will provide a platform to investigate the mechanisms by which myeloid cells promote tumor-associated immune suppression and to evaluate strategies that overcome myeloid-driven immunosuppression, thereby enhancing the efficacy of cancer immunotherapies. Methods: Differentiated THP1 into macrophages using phorbol 12-myristate 13-acetate (PMA) were polarized into immunosuppressive-like phenotype (M2) with recombinant human IL4 and IL13 cytokines and cancer cells-derived conditioned media (CC-CM). Primary LDNs from peripheral blood were stimulated with visceral adipose tissue-conditioned media (VAT-CM). Fluorescent labeled myeloid M2-THP1 and LDNs. Activation and M2 markers (CD206, CD163, PD-L1, B7-H4, HLA-DR) were evaluated by flow cytometry on alive cells. Collagen-embedded 3D spheroids from MDA-MB-231 and RPMI-8226 cells were formed in ultra-low-attachment (ULA) 96-round-well-plates and cocultured with different ratios and treatments of myeloid cells. Spheroid size over time and infiltration of myeloid cells was tracked using the Live-Cell Imaging Incucyte SX3. Findings: Coculture with M2-THP1 macrophages enhanced the growth of 3D spheroids generated from RPMI-8226, with the greatest increase observed when macrophages were exposed to tumor cell-conditioned medium, compared with spheroids cultured alone or with M2polarized THP1 macrophages in the absence of conditioned medium. Similarly, MDA-MB-231 spheroids exhibited increased size when cocultured with M2-polarized THP1 macrophages, regardless of the presence of conditioned medium, relative to tumor spheroids cultured alone. Notably, coculture with VAT-CM-activated LDNs produced a marked acceleration of MDA-MB231 spheroid growth over time. This tumor-promoting effect was selective for malignant cells, as VAT-CM-activated LDNs did not alter the growth of spheroids generated from the non-malignant mammary epithelial cell line 184A1. Conclusion: We were able to replicate an immunosuppressive and inflammatory TME to evaluate tumor growth in vitro. Further optimization is fundamental for evaluating the effect of therapeutic modulation of the myeloid phenotype on spheroid growth and response of cellular immunotherapy.
Recommended Citation
Pritchett, Amyri, "Optimization of an in vitro 3D method for studying modulators of the mmunosuppressive tumor microenvironment for enhancing the response to immunotherapy" (2026). Summer Research Internship Program. 23.
https://digitalscholar.lsuhsc.edu/srip/2026/undergrad/23
Optimization of an in vitro 3D method for studying modulators of the mmunosuppressive tumor microenvironment for enhancing the response to immunotherapy
Purpose: This study aims to establish physiologically relevant in vitro models of immunosuppressive tumor microenvironment (TME) for multiple myeloma (MM) and triplenegative breast cancer (BC) using three-dimensional (3D) multicellular coculture systems incorporating immunosuppressive and inflammatory myeloid cells, including M2-polarized THP1 macrophages and low-density neutrophils (LDNs) with RPMI-8226 (MM) or MDA-MB-231 cells (BC). These models will provide a platform to investigate the mechanisms by which myeloid cells promote tumor-associated immune suppression and to evaluate strategies that overcome myeloid-driven immunosuppression, thereby enhancing the efficacy of cancer immunotherapies. Methods: Differentiated THP1 into macrophages using phorbol 12-myristate 13-acetate (PMA) were polarized into immunosuppressive-like phenotype (M2) with recombinant human IL4 and IL13 cytokines and cancer cells-derived conditioned media (CC-CM). Primary LDNs from peripheral blood were stimulated with visceral adipose tissue-conditioned media (VAT-CM). Fluorescent labeled myeloid M2-THP1 and LDNs. Activation and M2 markers (CD206, CD163, PD-L1, B7-H4, HLA-DR) were evaluated by flow cytometry on alive cells. Collagen-embedded 3D spheroids from MDA-MB-231 and RPMI-8226 cells were formed in ultra-low-attachment (ULA) 96-round-well-plates and cocultured with different ratios and treatments of myeloid cells. Spheroid size over time and infiltration of myeloid cells was tracked using the Live-Cell Imaging Incucyte SX3. Findings: Coculture with M2-THP1 macrophages enhanced the growth of 3D spheroids generated from RPMI-8226, with the greatest increase observed when macrophages were exposed to tumor cell-conditioned medium, compared with spheroids cultured alone or with M2polarized THP1 macrophages in the absence of conditioned medium. Similarly, MDA-MB-231 spheroids exhibited increased size when cocultured with M2-polarized THP1 macrophages, regardless of the presence of conditioned medium, relative to tumor spheroids cultured alone. Notably, coculture with VAT-CM-activated LDNs produced a marked acceleration of MDA-MB231 spheroid growth over time. This tumor-promoting effect was selective for malignant cells, as VAT-CM-activated LDNs did not alter the growth of spheroids generated from the non-malignant mammary epithelial cell line 184A1. Conclusion: We were able to replicate an immunosuppressive and inflammatory TME to evaluate tumor growth in vitro. Further optimization is fundamental for evaluating the effect of therapeutic modulation of the myeloid phenotype on spheroid growth and response of cellular immunotherapy.
Comments
Mentor: Dr. Maria Sanchez-Pino, Interdisciplinary Oncology