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Nonlinear response to cancer nanotherapy due to macrophage interactions revealed by mathematical modeling and evaluated in a murine model via CRISPR-modulated macrophage polarization

Abstract

Tumor-associated macrophages (TAMs) have been shown to both aid and hinder tumor growth, with patient outcomes potentially hinging on the proportion of M1, pro-inflammatory/growth-inhibiting, to M2, growth-supporting, phenotypes. Strategies to stimulate tumor regression by promoting polarization to M1 are a novel approach that harnesses the immune system to enhance therapeutic outcomes, including chemotherapy. We recently found that nanotherapy with mesoporous particles loaded with albumin-bound paclitaxel (MSV-nab-PTX) promotes macrophage polarization towards M1 in breast cancer liver metastases (BCLM). However, it remains unclear to what extent tumor regression can be maximized based on modulation of the macrophage phenotype, especially for poorly perfused tumors such as BCLM. Here, for the first time, a CRISPR system is employed to permanently modulate macrophage polarization in a controlled in vitro setting. This enables the design of 3D co-culture experiments mimicking the BCLM hypovascularized environment with various ratios of polarized macrophages. We implement a mathematical framework to evaluate nanoparticle-mediated chemotherapy in conjunction with TAM polarization. The response is predicted to be not linearly dependent on the M1:M2 ratio. To investigate this phenomenon, the response is simulated via the model for a variety of M1:M2 ratios. The modeling indicates that polarization to an all-M1 population may be less effective than a combination of both M1 and M2. Experimental results with the CRISPR system confirm this model-driven hypothesis. Altogether, this study indicates that response to nanoparticle-mediated chemotherapy targeting poorly perfused tumors may benefit from a fine-tuned M1:M2 ratio that maintains both phenotypes in the tumor microenvironment during treatment.

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Abbreviations

AAMP:

Agent affecting macrophage polarization

BCA:

Bicinchoninic acid

BCLM:

Breast cancer liver metastasis

BSA:

Bovine serum albumin

CCD:

Charge-coupled device

cDNA:

Complementary DNA

CRISPR:

Clustered regularly interspaced short palindromic repeats

crRNA:

CRISPR RNA

DAPI:

4′,6-Diamidino-2-phenylindole

DNA:

Deoxyribonucleic acid

ECL:

Enhanced chemiluminescence

FBS:

Fetal bovine serum

gRNA:

Guide RNA

HMW:

High molecular weight

HRP:

Horseradish peroxidase

mAb:

Monoclonal antibody

MEM:

Minimum essential medium

miRNA:

Micro-RNA

mRNA:

Messenger RNA

MRI:

Magnetic resonance imaging

MSV-nab-PTX:

Mesoporous particles loaded with nab-PTX

mTOR:

Mammalian target of rapamycin

nab-PTX:

Albumin-bound paclitaxel

NEAA:

Non-essential amino acids

OCT:

Optimal cutting temperature

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

PTX:

Paclitaxel

PVDF:

Polyvinylidene fluoride

qPCR:

Quantitative PCR

RICTOR:

Rapamycin-insensitive companion of mTOR

RIPA:

Radioimmunoprecipitation assay

RNA:

Ribonucleic acid

sgRNA:

Single-guide RNA

siRNA:

Small interfering RNA

TAM:

Tumor-associated macrophage

TME:

Tumor microenvironment

TBST:

Tris-buffered saline with Tween-20

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Funding

Leonard acknowledges Houston Methodist Research Institute Department of Nanomedicine Innovative Grant Award and METAvivor Foundation Early Career Investigator Award. Leonard and Godin gratefully acknowledge funding from George and Angelina Kostas Research Center for Cardiovascular Nanomedicine Grant. Frieboes acknowledges partial support by the National Institutes of Health/National Cancer Institute Grant R15CA203605.

Author information

Study conception and design: FL, LC, BG, and HF; experiments: FL, AH, BG, DS, EC, and CZ; CRISPR liposome system development: FL; mathematical model implementation and testing: LC and HF; data collection and analysis: FL, LC, AH, BG, HF, DS, EC, and CZ; manuscript preparation and revision: FL, LC, BG, and HF.

Correspondence to Biana Godin or Hermann B. Frieboes.

Ethics declarations

Conflict of interest

The authors have no conflicts to disclose.

Ethical approval and ethical standards

In vivo mouse studies were performed in accordance with the Houston Methodist Research Institute Institutional Animal Care and Use Committee (IACUC—approval number: AUP-0617–0020). The animal research was conducted in full compliance with federal, state, and local regulations and institutional policies.

Animal source

Balb/c mice (6–8 weeks, females) were purchased from Jackson laboratory for all of the animal experiments in this study.

Cell line authentication

4T1 mouse breast cancer cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA), which tests and authenticates the cells in its collection.

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Leonard, F., Curtis, L.T., Hamed, A.R. et al. Nonlinear response to cancer nanotherapy due to macrophage interactions revealed by mathematical modeling and evaluated in a murine model via CRISPR-modulated macrophage polarization. Cancer Immunol Immunother (2020). https://doi.org/10.1007/s00262-020-02504-z

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Keywords

  • Cancer immunotherapy
  • Macrophage polarization
  • Nanotherapy
  • Breast cancer liver metastases
  • Mathematical modeling
  • computational simulation