Patient-Specific Vascularized Tumor Model: Blocking TAM Recruitment with Multispecific Antibodies Targeting CCR2 and CSF-1R

Abstract

Tumor-associated inflammation drives cancer progression and therapy resistance, with the infiltration of monocyte-derived tumor-associated macrophages (TAMs) associated with poor prognosis in diverse cancers. Targeting TAMs holds potential against solid tumors, but effective immunotherapies require testing on immunocompetent human models prior to clinical trials. Here, we develop an in vitro model of microvascular networks that incorporates tumor spheroids or patient tissues. By perfusing the vasculature with human monocytes, we investigate monocyte trafficking into the tumor and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via TAM-produced CCL7 and CCL2, mediated by CSF-1R. Additionally, we assess a novel multispecific antibody targeting CCR2, CSF-1R, and neutralizing TGF-β, referred to as CSF1R/CCR2/TGF-β Ab, on monocytes and macrophages using our 3D models. This antibody repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and effectively blocks monocyte migration. Finally, we show that the CSF1R/CCR2/TGF-β Ab inhibits monocyte recruitment in patient-specific vascularized tumor models. Overall, this vascularized tumor model offers valuable insights into monocyte recruitment and enables functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment (TME).

Figure 1:. Monocyte recruitment assays using perfusable vascularized tumor models.

A) Illustration of meniscus trap within a microfluidic chip for integrative vascular bed (iVas) creation. The blue dye solution stays entrapped within the central channel but not inside the central hole. It is sandwiched between 2 media channels due to capillary force caused by the microfluidic device architecture. B) Step-by-step fabrication of the vascularized tumor model and monocyte recruitment assays: seeding of endothelial cells (ECs) and fibroblasts (FBs), insertion of tri-culture tumor spheroids (tumor cells- FBs-MØs, referred to as TFM), and perfusion of monocytes through the vascular networks. C) Perfusability of vasculatures surrounding a tumor spheroid. D–F) Collapsed z-stacks of vasculature beds having the central hole filled with collagen/fibrin mix containing a tumor spheroid TFM from MDA-MB-231 cells (231 TFM, Fig. 1D) in suspension or control device without a tumor spheroid (Ctrl, Fig. 1E) or a tumor spheroid from MDA-MB-231 cells containing only TCs and FBs (TF, Fig. 1F), imaged on days 1, 2, and 3. Scale bars: 500 μm for the left original and 100 μm for the zoomed-in right image. G–I) Graphs showing the total number of monocytes inside the vasculature (luminal, Fig. 1G), total monocytes outside the vasculature but not inside the central hole (extravasated, not recruited, Fig. 1H), and those migrating into the central well (recruited, Fig. 1I) normalized by the total number of monocytes in the 3×3mm region of interest (ROI) in 231 TFM or Ctrl devices. J) Comparison of recruited monocyte percentages in Ctrl, 231 TFM and 231 TF devices on day 2. Each point represents an independent device and statistical significance is obtained with two-way ANOVA and Šídák multiple comparison test (for G–I) and one-way ANOVA and Tukey post-hoc test for J; *, P < 0.05 **<0.01, ***<0.001.

Figure 2:. Macrophages (MØs) in 231 TFM tri-culture tumor spheroids primarily contribute to the secretion of CCL2 and CCL7, and this process is regulated by CSF-1R.

A) Illustration of protocol for generating and analyzing MØs in both 3 or 2 dimensional (3D or 2D) formats and the composition of drug treatments. Ai) TFM tri-culture is dissociated into single cells and sorted by FACS to isolate TCs, fibroblasts and MØs. Media are collected on day 2 for either Luminex^® or MSD assay analyses. Aii) CSF1R/CCR2/TGF-β Ab multispecific Ab drugs used to treat tumor-associated macrophages (TAMs). Aiii) Treatment of MØs on standard well plate (2D) with tumor cell culture media to obtain TCM MØs and characterization using qPCR or MSD. B) Gene expression of CCL2 (Bi), CCL7 (Bii), CCL8 (Biii), and CCL13 (Biv) normalized to GAPDH in sorted TCs, MØs, and FBs. The results indicate that sorted MØs express higher levels of these monocyte chemotactic proteins compared to TCs and FBs. C) Relative gene expression of different M1 and M2 markers in MØs isolated from 231_TFM_spheroids(231_TFM_ MØs) and 468_TFM_spheroids (231_TFM_ MØs), control M0, M1 (IFNγ), M2 MØs (IL4, IL10), as well as 231_TFM_MØs treated with various CSF-1R targeted drugs (CSF1R/CCR2/TGF-β Ab, CSF-1R Ab, and BLZ-945). D) Expression of CCL2 (Di) and CCL7 (Dii) in different MØ phenotypes cultured in a 2D format. E) CCL2 secretion by 231 TFM, 468 TFM, 549 TFM, 427 TFM and H2009 TFM spheroids. F) Quantification of cytokine concentration in tumor spheroids’ pooled cell culture media in log₁₀(x+1). Each point represents a biological repeat and statistical significance is obtained with ANOVA and Tukey post-hoc test for B and Dunnett’ comparison to reference for D and E; *, P < 0.05 **<0.01, ***<0.001, ****<0.0001.

Figure 3:. Characterization of effects of endothelial monolayers, MØs, and drugs on monocyte migration using unidirectional microfluidic chemotaxis assays.

A) Schematic representation of the different experimental conditions. B) Comparison of devices with or without an EC monolayer Bi) Pooled monocyte positions in 15 gel regions within 3 devices 2 days after seeding showing that the presence of endothelial cells increases monocyte migration inside the fibrin gel. Bii) Percentage of monocytes migrating from one side of the device to the other. C) Comparison of devices with a 231-TCM treated macrophage-conditioned media (MCM) gradient or MCM on both sides of the gel channels or control devices. Ci) Image of monocytes inside gel channel in different conditions above after transmigration from a media channel on day 2. Cii) Percentage of migrating monocytes in control device without MCM or device having MCM gradient or MCM on both sides of the gel channel. D) Chemotaxis coefficient of monocytes in the no-chemoattractant control device, devices having M0 MØs, M0 MØs cultured in MDA-MB-231 tumor-conditioned media (231_TCM_MØ), devices with 231_TCM_MØ treated with CCR2 antagonist, anti-CCR2 Ab, and CSF1R/CCR2/TGF-β Ab. Di) Representative images of monocyte migration on day 2. Dii) Chemotaxis coefficients. Scale bars: 500 μm. The statistical test(s) used in Bii is a Mann Whitney test, and in Cii and D are one-way ANOVA with post-hoc Tukey tests with multiple comparisons; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001. Dots represent different ROIs of several devices.

Figure 4:. Monocyte recruitment by tumor spheroids made from various cell lines with or without CSF1R/CCR2/TGF-β Ab.

A) Panels of overlap z-stack images of tumor spheroids within the central well of a vascular bed. From left to right and top to bottom: confocal images of i) a control device without spheroid, devices with TFM tumor spheroids from ii) MDA-MB-231, iii) A-549, iv) H2009, v) MDA-MB-468 TCs, vi) 231 TF spheroid, vii) a device containing 231 TFM spheroid treated with CSF1R/CCR2/TGF-β Ab, devices with A-427 TFM spheroid treated with viii) IgG or ix) CSF1R/CCR2/TGF-β Ab. NSCLC cell lines are not labeled. B) Comparison of the percentage of monocytes recruited to the central well of the devices containing different tumor cell lines. C) Comparison of monocyte recruitment in 231 TFM devices with CSF1R/CCR2/TGF-β Ab treatment, without treatment, and 231 TF devices. D) Comparison of monocyte recruitment in A-427 devices under IgG and CSF1R/CCR2/TGF-β Ab treatments. Significance tested using one-way ANOVA with post-hoc Tukey test with multiple comparisons; *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Each dot represents an independent device. E) Heatmap of cytokines in media of 231 TFM spheroid under CSF-1R Ab and CSF1R/CCR2/TGF-β Ab treatments compared to no-treatment control samples. Fold change was relative to no-treatment control. Log₁₀(Fold change) is shown.

Figure 5:. Monocyte recruitment from vasculature by patient tissues.

A) Recruitment by medium and large tissues from the same patient. B) Recruitment percentages corresponding to A (n=7,5,4, respectively). C) Different patients’ samples causing either no or active recruitment. D) Monocyte recruitment by various patients’ S1 fragments. Active recruitment samples recruit significantly more monocytes than the controls (n=9, 3,1,3,1,3,3,5, respectively). Patient samples differ in size, leading to variations in the number of devices generated. Significance was tested using one-way ANOVA with Tukey’s test for B or Dunnett’s test with multiple comparisons compared to the control and t-test for the comparison between patients 1 and 3 samples for C; *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Figure 6:. Immunotherapy screening on vascularized non-small cell cancer models using ex-vivo patient-derived organotypic tumor spheroids (PDOTS).

A) Staining of different markers of TCs (EpCAM, pan CK), fibroblasts (Vimentin), immune cells (CD45) and MØs (CD68). B) Cytokine concentration (Log₁₀ pg/ml) of cell culture media of different tumor models. C) Z-stack images showing recruitment of monocytes by a responsive PDOTS that causes active monocyte recruitment, under various treatments: IgG, CCR2 Ab, CSF-1R Ab and CSF1R/CCR2/TGF-β Ab on day 2. D) Percentage of monocytes recruited by PDOTS in the ROI of images in C. Significance tested using one-way ANOVA with Dunnett’s test compared to PDOTS IgG; *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Each dot represents an independent device.

Figure 7:. Proposed mechanism of monocyte recruitment by tumor cells.

TCs secrete cytokines, including M-CSF, which plays a role in repolarizing local MØs into a tumor-associated macrophage (TAM) phenotype. These TAMs, characterized by the expression of CD163, then express autocrine M-CSF along with cytokines such as CCL2 and CCL7. Upon extravasation, monocytes are activated by endothelial cells, leading to increased motility and expression of the macrophage marker CD206. These activated monocytes respond to chemotactic signals released by TAMs and migrate into the tumor microenvironment. This process contributes to the establishment of a vicious cycle of myeloid recruitment supporting tumor cell growth and facilitating later metastasis.