Abstract
Background/Aim: Tumor immunology and immunotherapy have been intensely researched recently, especially with the development of immune checkpoint inhibitors (ICIs). However, only a small percentage of patients respond to ICIs, and this response is limited to a minority of cancer types, such as colon cancer, lung cancer, and melanoma. The aim of the present study was to develop an imageable in-vivo-like in vitro system to directly visualize tumor immunology and immunotherapy in real-time, to further understand tumor immunology and develop improved tumor immunotherapy.
Materials and Methods: Lewis lung carcinoma cells labeled with red fluorescent protein (LLC-RFP) and peripheral blood mononuclear cells labeled with green fluorescent protein (PBMCs-GFP), derived from transgenic GFP mice, were cultured in two dimensions (2-D) on plastic or in three dimensions (3-D) in Gelfoam® histoculture, using RPMI-1640 culture medium with 10% fetal bovine serum, and 1% penicillin/streptomycin, and with concanavalin-A.
Results: LLC-RFP cells seeded on Gelfoam® formed glandular-like structures after 2 days of histoculture. PBMCs-GFP were seeded on Gelfoam® with LLC-RFP after one day to establish a co-culture. The PBMCs-GFP co-localized with many of the structures formed by the LLC-RFP cells after one day of seeding PBMCs-GFP, suggesting a very specific interaction necessary for an immunological interaction. In contrast, only a few randomized co-localizations of PBMCs-GFP and the LLC-RFP cells were seen on plastic in 2-D culture.
Conclusion: The present results demonstrate a new 3-D co-culture color-coded system to visualize in real-time the interaction of PBMCs and cancer cells using fluorescence color-coded imaging. Future experiments will test drugs that can stimulate immune reactions between the PBMCs and cancer cells.
- Tumor immunology
- peripheral blood mononuclear cell
- PBMC
- Lewis lung carcinoma
- LLC
- color-coded imaging
- Gelfoam®
- 3-D histoculture
- red fluorescent protein
- RFP
- green fluorescent protein GFP
- GFP transgenic mice
Introduction
Tumor immunology and immunotherapy have been intensely researched recently, with a Nobel Prize awarded to Honjo and Allison for the discovery of immune checkpoints inhibitors (ICIs) comprising anti-PD-1 antibodies to target T-cells and anti-PD-L1 antibodies to target cancer cells (1-3). However, ICI therapy has had only limited clinical success, at least in part due to difficulties in studying the interaction of cancer cells and immune cells in vivo, both for obtaining basic information on cancer immunology as well as to improve cancer immunotherapy.
Leighton pioneered sponge gel histoculture (4, 5), and our laboratory developed sponge gel histoculture to study cancer biology (6) and pioneered the histoculture drug response assay (HDRA) for cancer-drug testing and individualized cancer therapy (7-9). Our laboratory also pioneered in vivo fluorescent-protein imaging (10-14) including color-coded imaging of cancer cell-immune cell interaction (11).
Transgenic green fluorescent protein (GFP)-expressing mice were originally developed by Okabe at Osaka University (15). The mice express GFP in essentially all cell types, including peripheral blood mononuclear cells (PBMCs) that include T-cells that attack cancer cells. In the present study, we demonstrate the establishment of an in vitro 3-D Gelfoam® histoculture model of tumor immunology model using color-coded imaging to visualize the interaction of cancer cells expressing red fluorescent protein (RFP) and immune cells expressing GFP in real-time.
Materials and Methods
Cells. Mouse Lewis lung carcinoma cells were transfected with RFP (LLC-RFP) (16) and cultured in RPMI-1640 (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) with 10% heat-activated fetal bovine serum (GIBCO, Grand Island, NY, USA) and 1% penicillin/streptomycin (GIBCO).
Mice. Transgenic BALB/c mice expressing GFP were obtained from AntiCancer, Inc. (San Diego, CA, USA) (Figure 1). The mice, approximately eight weeks old, were used as the source of PBMC. All mice were bred in a barrier facility with a high efficiency particulate air (HEPA)-filtered rack and kept in standard settings with 12-hour light/dark cycles. The present study was approved by the AntiCancer Inc. Institutional Animal Care and Use Committee protocol outlined in the National Institute of Health Guide for the Care and Use of Animals.
Transgenic green fluorescent protein (GFP) expressing BALB/c mouse. The image was captured using the Analytik Jena UVP Biospectrum Advanced system. Please see the Materials and Methods for details.
Isolation of peripheral blood mononuclear cells (PBMCs) expressing GFP. Blood was obtained from the GFP mouse’s cheek. PBMCs were isolated using density gradient centrifugation with HISTOPAQUE®-1083 (Sigma-Aldrich, St. Louis, MO, USA). PMBCs were cultured in RPMI-1640 with 2 μl/ml concanavalin A (Invitrogen, Carlsbad, CA, USA).
Gelfoam® histoculture. Gelfoam® (Pfizer, New York, NY, USA) was placed in 6-well tissue culture plates with a sterile procedure. PPMI-1640 medium was placed in the wells and incubated at 37°C with 5% CO2 allowing Gelfoam® to absorb the medium. LLC-RFP cells (1.0×106) were seeded on the Gelfoam®. Isolated PBMCs (1.0×106) were seeded in the medium with Concanavalin-A one day after seeding LLC-RFP cells in the medium. Some of the LLC-RFP cells and PBMCs-GFP placed on the Gelfoam® histoculture seeded directly on the plastic.
Imaging. A UVP Biospectrum Advanced system (Analytik Jena, Jena, Germany) was used for macro imaging. Imaging of mice was performed using an anesthetic solution of 25% ketamine (100 mg/ml), 19% xylazine (100 mg/ml), and 6% acepromazine maleate (10 mg/ml) in phosphate-buffered saline (PBS) intramuscularly. Cells in Gelfoam® histoculture were examined under an Olympus IX71 fluorescence microscope (×200) (Olympus Corp., Tokyo, Japan).
Results
Formation of glandular structures by LLC-RFP cells on Gelfoam®. Two days after seeding on Gelfoam®, LLC-RFP cells were observed microscopically to form glandular-like structures (Figure 2).
Lewis lung carcinoma cells expressing red fluorescent protein (LLC-RFP) on Gelfoam®. RFP fluorescence was detected on Gelfoam®. one day after seeding (A). LLC-RFP cells formed glandular-like structures two days after seeding (B). Scale bar: 250 μm. Please see the Materials and Methods for details.
Targeting of LLC-RFP cells by PBMCs-GFP on Gelfoam®. One day after LLC-RFP cells were seeded on the Gelfoam®, PBMCs-GFP derived from transgenic GFP mice were seeded on the Gelfoam®. One day after PBMCs-GFP were seeded on the Gelfoam®, they co-localized with LLC-RFP cells in the LLC-RFP structures on Gelfoam®. The next day, more extensive LLC-RFP cell glandular-like structures were formed with more co-localization by PBMCs-GFP with LLC-RFP cells (Figure 3).
One day after peripheral blood mononuclear cells (PBMCs) derived from transgenic green fluorescent mouse (PBMCs-GFP) were seeded on Gelfoam®., they co-localized with many of the Lewis lung carcinoma cells expressing red fluorescent protein (LLC-RFP) in glandular-like structures on Gelfoam®. (A). The next day, more extensive glandular-like structures of LLC-RFP cells were formed with more extensive co-localization with PBMCs-GFP (B) 2B and 3A are the same microscopic field. Scale bar: 250 μm. Please see the Materials and Methods for details.
Only a few randomized co-localizations of PBMCs-GFP with LLC-RFP cells occurred on plastic. Two days after seeding PBMCs-GFP, and after rinsing with PBS to remove non-attached cells, only a few randomized co-localizations of PBMCs-GFP with LLC-RFP cells were observed on plastic dishes where LLC-RFP cells did not form glandular-like structures (Figure 4).
Two days after seeding on plastic dishes peripheral blood mononuclear cells expressing green fluorescent protein (PBMCs-GFP) to establish co-cultures with Lewis lung carcinoma cells expressing red fluorescent protein (LLC-RFP), only a few randomized co-localizations of PBMCs-GFP with LLC-RFP were observed on plastic with respect to the LLC-RFP cells. Scale bar: 250 μm. Please see the Materials and Methods for details.
Discussion
The present study showed that LLC-RFP cells formed distinct glandular-like structures on Gelfoam® but not plastic. It also showed that one day after co-culture of PBMCs-GFP on the Gelfoam® with LLC-RFP cells, the PBMCs-GFP co-localized with many of the LLC-RFP glandular-like structures. In contrast on plastic, LLC-RFP cells did not form structures and only a few PBMCs-GFP co-localized with LLC-RFP cells.
Thus the 3-D Gelfoam® histoculture in-vivo-like in vitro model has been established to study the interaction of cancer cells and immune cells to better understand the basic biology of tumor immunology and interaction and to develop improved cancer immunotherapy. In the future, co-cultures of cancer cells and immune cells can be established from individual patients to optimize their immunotherapy.
Conclusion
The new 3-D co-culture color-coded tumor immunology system allows real-time visualization of the interaction of PBMCs and cancer cells by fluorescence color-coded imaging in vitro. The system can be utilized for the study of cancer immunotherapy and to develop improved immunotherapy for all cancer types.
Acknowledgements
This paper is dedicated to the memory of A.R. Moossa, MD, Professor Philip Miles, Sun Lee, MD, Richard W. Erbe, MD, Professor Milton Plesur, Gordon H. Sato, PhD, John W. Littlefield, MD, Professor Li Jiaxi, Masaki Kitajima, MD, Shigeo Yagi, PhD, Jack Geller, MD, Joseph R. Bertino, MD, J.A.R. Mead PhD, John Mendelsohn, MD, Professor Sheldon Penman, Professor John R. Raper and Joseph Leighton, MD.
Footnotes
Authors’ Contributions
KM performed experiments. KM and RMH wrote the article. JR, YA, BMK, JSK, MV, YT, and KN critically reviewed this article.
Conflicts of Interest
The Authors declare that there are no competing interests in relation to this study.
Funding
The Robert M. Hoffman Foundation for Cancer Research funded the present study.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received April 7, 2025.
- Revision received May 5, 2025.
- Accepted May 6, 2025.
- Copyright © 2025 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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